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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7131838/

Prevention of Infectious Diseases in Athletes

The sports medicine physician may face challenging issues regarding infectious diseases when dealing with teams or highly competitive athletes who have difficulties taking time off to recover. One must treat the individual sick athlete and take the necessary precautions to contain the spread of communicable disease to the surrounding team, staff, relatives, and other contacts. This article reviews preventive strategies for infectious disease in athletes, including immunization recommendations and prophylaxis guidelines, improvements in personal hygiene and prevention of spread of infectious organisms by direct contact, insect-borne disease precautions, and prevention of sexually transmitted diseases. A special emphasis on immunizations focuses on pertussis, influenza, and meningococcal prophylaxis. Public Health, Infectious Diseases, and Sports Public health plays an insidious role in our everyday lives to keep individuals safe from communicable diseases, accidents, environmental concerns, and countless other dangers. Programs are most successful when a disease no longer becomes a worry for the population through active prevention strategies. Much of our understanding comes from history and from recognizing the imminent dangers so we can prevent diseases from occurring the next time. Medical reports from major athletic events provide examples of issues that can be encountered by the sports medicine physician. Mass-gathering events, such as the Olympics, highlight some of the concerns that can occur with sports and infectious diseases. International athletes compete together from countries with different endemic microorganisms and variable health care practices. For example, an outbreak of measles occurred in a Special Olympics event in St. Paul, Minnesota. The point of infection was suspected to be a track and field athlete from Argentina, resulting in measles infections in 16 individuals from 7 different states [3] . An outbreak of influenza that occurred during the 1988 Calgary Olympics was believed to have possibly affected the performances of some athletes [4] . The pneumococcal vaccine was recommended to athletes before competing in the 1992 Barcelona Olympics because of resistant Streptococcus pneumoniae strains endemic in Spain [5] . Medical reporting and surveillance of infections are extremely important to attempt to contain spread of disease. At the 1996 Atlanta Olympics, a priority of surveillance was to identify unusual presentations and infectious disease outbreaks to actively implement same-day medical and public health interventions [6] . It is an important responsibility of physicians to report specific infectious diseases, especially if an outbreak is suspected ( Box 1 ) [7] . Health professionals should contact their local public health officer to determine whether other cases are occurring and what precautions need to be taken in the event of a serious outbreak. In the past few years, infections such as severe acute respiratory syndrome [8] have required the need of quarantine to help control the spread of these dangerous diseases. Health care professionals should report all clinically significant adverse events following immunization to the Vaccine Adverse Events Reporting System [9] . Box 1 Infectious diseases designated as notifiable at the national level during 2004 Acquired immunodeficiency syndrome (AIDS) Anthrax Botulism Brucellosis Chancrodi Chlamydia trachomatis, genital infection Cholera Coccidioidomycosis Cryptosproidiososis Cryptosporidiosis Cyclosporiasis Diphtheria Ehrlichiosis Human granulocytic Human monocytic Human, other or unspecified agent Encephalitis/meningitis, arboviral California serogroup Eastern equine Powassan St. Louis Western equine West Nile Enterohemorrhagic Escherichia coli (EHEC) EHEC O157:H7 EHEC Shiga toxin-positive, serogroup non-O157 EHEC Shiga toxin-positive, not serogrouped Giardiasis Gonorrhea Haemophilus influenzae , invasive disease Hansen disease (leprosy) Hantavirus pulmonary syndrome Hemolytic uremic syndrome, postdiarrheal Hepatitis A, viral, acute Hepatitis B, viral, acute Hepatitis B, viral, chronic Hepatitis B, perinatal infection Hepatitis C, acute Hepatitis C, virus infection (past or present) Human immunodeficiency virus (HIV) infection Adult (age ≥13 years) Pediatric (age 65 years 6. Residents of nursing homes or chronic-care facilities 7. Health care workers 8. Travelers to influenza-endemic areas Adapted from Smith NM, Bresee JS, Shay DK, et al. Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2006;55(RR-10):1–42. Inactivated influenza vaccine is generally appropriate for all populations requiring influenza vaccine. Three influenza vaccines were available in the United States for the 2006 to 2007 season: Fluzone (manufactured by Sanofi-Pasteur); Fluvirin (manufactured by Novartis); and Fluarix (manufactured by GlaxoSmithKline). The typical dose is 0.5 mL administered intramuscularly, usually in the deltoid muscle. Live, attenuated influenza vaccine (LAIV) is approved for use in healthy, nonpregnant individuals aged 5 to 49 years. The LAIV is administered by way of a nasal spray once in each nostril (FluMist, manufactured by MedImmune). Individuals who have a hypersensitivity or anaphylactic reaction to components of the flu vaccine or to eggs should not be vaccinated [9] . Adults reported having a 19% reduction in severe febrile illnesses after LAIV compared with placebo [38] . Side effects from LAIV increased in adults within 7 days of immunization compared with placebo and consisted mainly of nasal congestion (44.5% versus 27.1%) and sore throat (27.8% versus 17.1%), which lasted, on average, 2 days. Less common complaints were tiredness, cough, and chills. There was no significant difference in the number of mild febrile illnesses between immunization and placebo groups [39] . Injections can be scheduled to occur at the optimum time during the athlete's competitive schedule to minimize concern about side effects. When inactivated influenza vaccine shortages occurred in previous years, the vaccine was recommended for high-risk groups as priority; however, the general recommendation now is to offer the immunization annually to anyone who wishes to reduce the likelihood of being ill with influenza or transmitting the virus if they should become infected [9] . Although this policy cannot be directly translated into a benefit for the athlete, depending on the level of athlete, the use of the LAIV may also be beneficial to prevent lost time from sport. Influenza vaccine has been suggested for competitive athletes and essential personnel, especially before international events occurring during the influenza season [4] , [40] . Secondary prevention Treatment with antiviral medications can reduce the duration of uncomplicated influenza A and B illness by approximately 1 day when administered within 2 days of illness onset [41] , [42] . Recommended antiviral treatment should be given for 5 days [9] . Four antiviral agents are currently available: amantadine, rimantadine, zanamivir, and oseltamivir [9] . The influenza A virus, however, has become resistant to amantadine and rimantadine, which are presently not recommended to be used as first-line drugs [43] . Zanamivir (Ralenza, dry powder taken by orally inhaled route) and oseltamivir (Tamiflu, capsule or oral suspension) are neuraminidase inhibitors and can be used to treat patients and to control influenza outbreaks in closed settings. Although typically used in nursing homes, an outbreak in a dormitory may require chemoprophylaxis [9] . There are limited data to suggest that serious complications from influenza, such as lower respiratory tract infections, may be reduced [44] . The use of antiviral medications for prophylaxis of influenza is unclear and is not yet recommended for routine seasonal control [45] . The use of oseltamivir, however, has been recommended in specific cases, especially if there is high risk of spread such as household contacts and if individuals have not been immunized [46] . Oseltamivir was used to treat 36 of 188 patients, including 13 athletes during the 2002 Salt Lake City Winter Olympics, with medications given to close contacts, which was believed to limit the spread of influenza [47] . Clinical history and physical examination are still the mainstays for diagnosing influenza. Rapid swab tests are available and take approximately 30 minutes to detect the influenza virus. The tests are less sensitive (72%–95%) and specific (76%–86%) than the traditional viral cultures [48] . They have moderate sensitivities for influenza antigens and are more likely to produce false negative rather than false positive results [48] , [49] . Direct and indirect fluorescent antibody staining tests are also available, but they are ordered more in hospitals because they take 2 to 4 hours to obtain results [49] . Viral cultures are still the "gold standard" for confirming the presence of influenza and identifying the strains and subtypes [9] . Meningitis Neisseria meningitides is a serious concern, especially for the adolescent and college populations. An alarming trend during the 1970s demonstrated an increase in meningitis deaths in college students, with living in dormitories being a risk factor. The disease can be spread by asymptomatic carriers. Students living in dormitories were 9 to 23 times more likely of getting infected than those living in other types of accommodations [50] . Freshmen who lived in dormitories had an elevated risk of meningococcal disease (odds ratio, 3.6; 95% confidence interval, 1.6–8.5; P = .003) compared with other college students [51] . Aside from the risk of death, 11% to 19% of survivors of meningitis have serious sequelae such as neurologic disability, limb loss, and hearing loss [50] . Primary prevention Routine vaccination with meningococcal vaccine is recommended for college freshmen living in dormitories and for other populations at increased risk. The CDC Advisory Committee on Immunization Practices recommends routine vaccination of young adolescents (11–12 years old) with meningococcal vaccine (MCV4) at the preadolescent health care visit [50] . Therefore, sports medicine physicians may be faced with higher frequency of checking for meningococcal immunization status for high school and college athletes. A tetravalent conjugate vaccine (Menactra, Sanofi Pasteur) is available against Neisseria meningitidis isolates A, C, Y, and W-135 in a 0.5-mL single-dose vial. Over the age of 11 years, 75% of the meningococcal infections are caused by strains C, Y, or W-135 (CDC, unpublished data, 2004) [50] . Another vaccine, Menomune (Aventis Pasteur Limited), has been licensed since 1981 and has a similar immunogenicity profile to Menactra and is delivered subcutaneously as a 0.5-mL dose. Menactra and Menomune have serum bactericidal protection ranging from 89.4% and 94.4% for strain W-135 and 73.5% and 79.4% for strain Y, respectively [50] . Revaccination may be necessary for individuals at high risk after 5 years [52] , [53] . Common side effects with MCV4 were local pain in just over 50% of patients, followed by swelling, induration, and redness in approximately 10.8% to 17.1%. Fever was reported in 5.1% of children 18 years old or younger and in 1.5% of adults [50] . Secondary prevention Close contacts are at high risk and should be treated with chemoprophylaxis ideally within 24 hours of identifying the index patient [50] . The goal of treatment is to reduce nasopharyngeal carriage of N meningitidis . After more than 14 days after the onset of illness in the index patient, chemoprophylaxis is not necessary [50] . A single dose of ciprofloxacin (500 mg orally) or ceftriaxone (250 mg by intramuscular injection), or rifampin (600 mg twice a day for 2 days) is recommended for adults. Children between 1 month and 18 years old may take rifampin (10 mg/kg every 12 hours for 2 days), or ceftriaxone (125 mg intramuscularly) if younger than 15 years [50] . One dose of azithromycin (500 mg) was also shown to eradicate N meningitidis and may represent another treatment option [54] . Human Papillomavirus Human papillomavirus (HPV) is associated with 99% of cervical cancers and anogenital, head and neck, and nonmelanoma skin cancers. It is an STD and can be diagnosed by abnormal cervical cell changes seen on Pap smear [55] . This is a common infection, especially in sexually active adolescents and university students [56] . Primary prevention Primary prevention is now possible with two new vaccines: a bivalent vaccine against HPV types 16 and 18 and a quadravalent vaccine against types 6, 11, 16, and 18. The vaccines have a three-dose schedule: 0, 1, and 6 months (bivalent vaccine) and 0, 2, and 6 months (quadravalent vaccine). At 4.5 years, the bivalent vaccine was effective for producing a persistent antibody response against HPV 16 and 18, with more than 98% seropositivity and 96.9% effectiveness (95% confidence interval, 81.3–99.9) in reducing the number of reported abnormalities on Pap smear, colposcopy, or both [57] . Routine vaccination with three doses of quadrivalent HPV vaccine is recommended for girls 11 to 12 years old but can be started in girls as young as 9 years. Girls and women aged 13 to 26 years who have not been vaccinated previously or who have not completed the full vaccine series are recommended to receive a catch-up series. The vaccine is intended to be administered before potential exposure to HPV through sexual contact [58] . Secondary prevention Secondary prevention involves checking the affected individual's partners for signs of genital warts and other STDs. Regular cervical screen is recommended. Use of condoms and education on spread is important. HPV infection persists for life; however, the degree and duration of contagiousness is yet unknown [59] . Travel Immunizations Athletes traveling need to consider the endemic diseases in the geographic location where they are competing. They should be aware of risks of acquiring common diseases, their accommodations (urban versus rural), local foods, and customs. Immunizations should ideally be planned 4 or more months in advance to allow for adequate time to administer vaccines ( Table 3 ). There are many resources for information about prevention of infectious diseases for travelers ( Table 4 ). Table 3 Recommended immunizations for travelers to developing countries a Length of travel Immunizations Brief, 3 mo Review and complete age-appropriate childhood schedule (see text for details) + + + DTaP, poliovirus, pneumococcal, and Haemophilus influenzae type b vaccines may be given at 4-wk intervals if necessary to complete the recommended schedule before departure Measles: 2 additional doses given if younger than 12 mo of age at first dose Varicella Hepatitis B b Yellow fever c + + + Hepatitis A d + + + Typhoid fever e ± + + Meningococcal disease f ± ± ± Rabies g ± + + Japanese encephalitis h ± ± + + = recommended; ± = consider. From American Academy of Pediatrics. International travel. In: Pickering LK, editor. Red book: 2006 report of the committee on infectious diseases. 27th edition. Elk Grove Village (IL): American Academy of Pediatrics; 2006. p. 99; with permission from the American Academy of Pediatrics. a See disease-specific chapters in Section 3 of the AAP Red Book for details: [ Red Book: 2006 report of the Committee of Infectious Diseases . 27th edition. Elk Grove Village, (IL) American Academy of Pediatrics; 2006]. For further sources of information, see text. b If insufficient time to complete 6-month primary series, accelerated series can be given (see text for details). c For regions with endemic infection. d Indicated for travelers to areas with intermediate or high endemic rates of HAV infection. e Indicated for travelers who will consume food and liquids in areas of poor sanitation. f Recommended for regions of Africa with endemic infection and during local epidemics, and required for travel to Saudi Arabia for the Hajj. g Indicated for people with high risk of animal exposure (especially to dogs) and for travelers to countries with endemic infection. h For regions with endemic infection. For high-risk activities in areas experiencing outbreaks, vaccine is recommended, even for brief travel. Table 4 More common tick-borne diseases Tick-borne disease Organism Common vector (geographic area) Rocky Mountain spotted fever Rickettsia rickettsii Dog tick, Dermacentor variabilis (central, Pacific coastal, and eastern US) Rocky Mountain wood tick, Dermacentor andersoni (western US) Human monocytotropic ehrlichiosis Ehrlichia chaffeensis Lone Star tick (south central US in Maryland, Arkansas, Tennessee, Oklahoma, and Missouri) [60] Human granulocytotropic anaplasmosis Anaplasma phagocytophilum Blacklegged tick, Ixodes scapularis (north central US and New England) Ixodes pacificus (California) [75] Lyme disease Borrelia burgdorferi Ixodes scapularis (eastern US in New England and mid-Atlantic states and Midwest US in Wisconsin and Minnesota) Ixodes pacificus (west in northern California) Babesiosis Parasite, Babesia microti Ixodes scapularis (northeast of the US) Abbreviation: US, United States. Measles, Mumps, and Rubella Although MMR vaccines have been administered for many decades and incidences of disease are presently low [7] , the diseases can still occur in the adult population. Between 1986 and 1989, 6% of the measles cases occurred in college students [19] . Enzyme-linked immunosorbent assay tests for antibodies to MMR are available to detect for immunization status [19] . In a series of 256 students, 53 (21%) were found to be seronegative to measles alone, 13 (5%) were seronegative to rubella alone, and 5 (2%) were seronegative to measles and rubella. Eighty-six percent of the individuals seronegative to measles had previously received a dose of measles vaccine. Following a second injection, conversion to seropositive status rose to 97% and 100% for measles and rubella, respectively. These data support the need for a two-dose vaccine schedule [19] . Pneumococcal Vaccine Pneumococcal vaccine is administered to prevent Streptococcus pneumoniae infections. A conjugate heptavalent is given to during the first 2 years of life. A polysaccharide vaccine is provided to high-risk individuals older than 2 years against 23 types of Streptococcus pneumonia that account for 90% of invasive disease [20] . High-risk groups include patients who have asplenia, sickle cell disease, diabetes mellitus, cirrhosis, immunocompromised states, chronic cardiac or pulmonary disease, or age 65 years or older. Immunity following vaccination is successful for periods of 5 to 10 years, requiring booster injections [20] . Hepatitis B Hepatitis B is a blood-borne virus transmitted through sexual contact and parenteral exposure to blood and blood components [14] . Hepatitis B has a greater risk for transmission in sports than HIV. The risk of HIV transmission is estimated to between 1 in 1 million games and 1 in 85 million games [14] , [21] . The risk arises if bleeding and skin exudates from an infected individual come into contact with open wounds in another athlete, particularly during contact and collision sports. There are no confirmed cases of spread of HIV through sports [14] ; however, 5 out of 10 high school sumo wrestlers at one club developed hepatitis B [22] . Another case series reported on 11 of 65 American football players who developed hepatitis B over a period of 19 months [23] . Contact through open wounds, cuts, and abrasions were the suspected routes of transmission. Primary prevention Although hepatitis A is a considered immunization in athletes who are traveling to endemic areas, routine vaccination for hepatitis B is recommended for all individuals after birth using single or combination vaccines [24] . A three-dose immunization schedule is typically used after 18 years of age, with injections at 0 months, 1 month, and 6 months, although there is an optional four-dose schedule [25] . The licensed vaccines have had 90% to 95% efficacy of preventing hepatitis B, with immunity lasting 15 years or longer [25] . Immunizations for hepatitis B should be checked during the preparticipation physical examination, and catch-up immunizations recommended to the individual (see Table 2 ). If individuals are uncertain about their immunization status, serologic testing for antibody to hepatitis B surface antigen can determine immunity. Secondary prevention When athletes are known to be infected with hepatitis B, secondary prevention includes education on personal hygiene, appropriate management of open wounds, proper use of protective equipment, safe sex practices using a condom, and avoidance of intravenous blood transmission (eg, through needle sharing and illicit drug use). Primary prevention Although hepatitis A is a considered immunization in athletes who are traveling to endemic areas, routine vaccination for hepatitis B is recommended for all individuals after birth using single or combination vaccines [24] . A three-dose immunization schedule is typically used after 18 years of age, with injections at 0 months, 1 month, and 6 months, although there is an optional four-dose schedule [25] . The licensed vaccines have had 90% to 95% efficacy of preventing hepatitis B, with immunity lasting 15 years or longer [25] . Immunizations for hepatitis B should be checked during the preparticipation physical examination, and catch-up immunizations recommended to the individual (see Table 2 ). If individuals are uncertain about their immunization status, serologic testing for antibody to hepatitis B surface antigen can determine immunity. Secondary prevention When athletes are known to be infected with hepatitis B, secondary prevention includes education on personal hygiene, appropriate management of open wounds, proper use of protective equipment, safe sex practices using a condom, and avoidance of intravenous blood transmission (eg, through needle sharing and illicit drug use). Pertussis, Tetanus, and Polio Bordetella pertussis , which is responsible for whooping cough, is a gram-negative coccobacillus transmitted by way of airborne droplets [26] . Although tetanus and polio have been controlled well with the use of vaccines [7] , the rate of pertussis cases has been increasing in adolescents and adults despite routine immunizations [27] . Most cases occur in patients 10 years or older [28] . The infection is most concerning for infants because immunity is not complete until older ages. The spread to infants is typically from adults. Pertussis usually presents with nonspecific upper respiratory tract infection symptoms for 1 to 2 weeks (catarrhal stage), after which the paroxysmal and sometimes uncontrollable cough develops [26] . The cough is not necessarily always followed by the classic "whooping" sound, and pertussis should be considered with any persistent, prolonged cough. Primary prevention The whole-cell pertussis vaccine is estimated to be approximately 85% effective [29] . This vaccine is still recommended for use in the routine immunization of young children; however, the immunity provided begins to decline at 4 to 12 years following vaccination, which makes adolescents and adults susceptible [27] . Rare adverse reactions from the vaccine include hypotonic, hyporesponsive episodes, high fever, seizures, and anaphylaxis [26] . Two acellular vaccines have been introduced that are as effective as whole-cell vaccines and have fewer adverse reactions [30] . These vaccines are combined with tetanus toxoid and reduced diphtheria toxoid (DTaP). The Centers for Disease Control and Prevention (CDC) recommends use of these DTaP boosters rather than the tetanus-diptheria (Td) booster starting after 11 to 12 years of age [31] . Secondary prevention For pertussis, individuals are most contagious during the first 1 to 2 weeks during the catarrhal stage but should be considered contagious until 3 weeks after the paroxysmal stage ends or after taking antibiotics for 5 days [32] . Diagnosis of pertussis infection is best performed through polymerase chain reaction assay (sensitivity, 94%; specificity, 97%) or through direct fluorescent antibody testing (sensitivity, 52%; specificity, 98%). Nasal swab cultures (sensitivity, 15%; specificity, 100%) are routinely performed; however, they have high false negative rates and take 7 to 12 days to yield results [33] . Physicians in the United States are legally required to report cases of pertussis to state public health departments [26] . It is estimated that 80% of susceptible household contacts will be infected after close contact [26] . Antibiotic prophylaxis is recommended for close contacts of persons who have pertussis to prevent outbreaks [34] . Preferred drugs are azithromycin for 5 days, clarithromycin for 7 days, or trimethoprim-sulfamethoxazole or erythromycin for 14 days, which are similar for prophylaxis and treatment [34] . Primary prevention The whole-cell pertussis vaccine is estimated to be approximately 85% effective [29] . This vaccine is still recommended for use in the routine immunization of young children; however, the immunity provided begins to decline at 4 to 12 years following vaccination, which makes adolescents and adults susceptible [27] . Rare adverse reactions from the vaccine include hypotonic, hyporesponsive episodes, high fever, seizures, and anaphylaxis [26] . Two acellular vaccines have been introduced that are as effective as whole-cell vaccines and have fewer adverse reactions [30] . These vaccines are combined with tetanus toxoid and reduced diphtheria toxoid (DTaP). The Centers for Disease Control and Prevention (CDC) recommends use of these DTaP boosters rather than the tetanus-diptheria (Td) booster starting after 11 to 12 years of age [31] . Secondary prevention For pertussis, individuals are most contagious during the first 1 to 2 weeks during the catarrhal stage but should be considered contagious until 3 weeks after the paroxysmal stage ends or after taking antibiotics for 5 days [32] . Diagnosis of pertussis infection is best performed through polymerase chain reaction assay (sensitivity, 94%; specificity, 97%) or through direct fluorescent antibody testing (sensitivity, 52%; specificity, 98%). Nasal swab cultures (sensitivity, 15%; specificity, 100%) are routinely performed; however, they have high false negative rates and take 7 to 12 days to yield results [33] . Physicians in the United States are legally required to report cases of pertussis to state public health departments [26] . It is estimated that 80% of susceptible household contacts will be infected after close contact [26] . Antibiotic prophylaxis is recommended for close contacts of persons who have pertussis to prevent outbreaks [34] . Preferred drugs are azithromycin for 5 days, clarithromycin for 7 days, or trimethoprim-sulfamethoxazole or erythromycin for 14 days, which are similar for prophylaxis and treatment [34] . Influenza Influenza presents with constitutional symptoms of fever, chills, malaise, fatigue, and myalgia in addition to upper respiratory tract symptoms of a sore throat, cough, and rhinitis. Rarely, more serious conditions can occur, including encephalopathy, transverse myelitis, myocarditis, and pericarditis [9] . Immunogenicity is determined by hemaglutinins and neuraminidases on the virus surface. Antigenic drift can occur that can mutate the virus into different strains. Transmission occurs by way of respiratory droplets. The virus has an incubation period of usually 2 days (range, 1–4 days), and adults are infectious from the day before symptoms begin to approximately 5 days after the illness starts [9] . Symptoms usually last a week, although less likely, symptoms can last longer than 2 weeks. These symptoms can be very disruptive for treatment and challenging for the athlete to keep training and competing. A case series of 81 students, mostly healthy adolescents at a ski school in Austria, reported a severe outbreak of influenza A, leading to an attack rate of 49%, with 69% becoming ill within 2 days of the outbreak. Two students were hospitalized with pneumonia and 1 died [35] . Primary prevention Influenza vaccines contain strains of antigenically equivalent strains of influenza similar to those annually recommended: influenza A (H3N2), influenza A (H1N1), and a B virus. Depending on the emergence and spread of new strains, other virus strains can be added to update the vaccine [9] . The efficacy of influenza vaccine is approximately 70% to 90% for individuals under age 65 years [36] . Vaccination for influenza should occur in the fall (October or November), at the beginning of the flu season ( Box 2 ) [9] . Antibodies develop approximately 2 weeks after vaccination [9] , [37] . Box 2 Indications for influenza vaccine 1. Adults and children with chronic disorders of the cardiorespiratory system, including asthma 2. Adults and children who have chronic disease which may require regular medical follow-up or hospitalization, including immunodeficiency 3. Young children aged 6–23 months 4. Children aged 6 months to 18 years on long-term aspirin therapy and at risk for Reye's syndrome 5. Persons aged >65 years 6. Residents of nursing homes or chronic-care facilities 7. Health care workers 8. Travelers to influenza-endemic areas Adapted from Smith NM, Bresee JS, Shay DK, et al. Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2006;55(RR-10):1–42. Inactivated influenza vaccine is generally appropriate for all populations requiring influenza vaccine. Three influenza vaccines were available in the United States for the 2006 to 2007 season: Fluzone (manufactured by Sanofi-Pasteur); Fluvirin (manufactured by Novartis); and Fluarix (manufactured by GlaxoSmithKline). The typical dose is 0.5 mL administered intramuscularly, usually in the deltoid muscle. Live, attenuated influenza vaccine (LAIV) is approved for use in healthy, nonpregnant individuals aged 5 to 49 years. The LAIV is administered by way of a nasal spray once in each nostril (FluMist, manufactured by MedImmune). Individuals who have a hypersensitivity or anaphylactic reaction to components of the flu vaccine or to eggs should not be vaccinated [9] . Adults reported having a 19% reduction in severe febrile illnesses after LAIV compared with placebo [38] . Side effects from LAIV increased in adults within 7 days of immunization compared with placebo and consisted mainly of nasal congestion (44.5% versus 27.1%) and sore throat (27.8% versus 17.1%), which lasted, on average, 2 days. Less common complaints were tiredness, cough, and chills. There was no significant difference in the number of mild febrile illnesses between immunization and placebo groups [39] . Injections can be scheduled to occur at the optimum time during the athlete's competitive schedule to minimize concern about side effects. When inactivated influenza vaccine shortages occurred in previous years, the vaccine was recommended for high-risk groups as priority; however, the general recommendation now is to offer the immunization annually to anyone who wishes to reduce the likelihood of being ill with influenza or transmitting the virus if they should become infected [9] . Although this policy cannot be directly translated into a benefit for the athlete, depending on the level of athlete, the use of the LAIV may also be beneficial to prevent lost time from sport. Influenza vaccine has been suggested for competitive athletes and essential personnel, especially before international events occurring during the influenza season [4] , [40] . Secondary prevention Treatment with antiviral medications can reduce the duration of uncomplicated influenza A and B illness by approximately 1 day when administered within 2 days of illness onset [41] , [42] . Recommended antiviral treatment should be given for 5 days [9] . Four antiviral agents are currently available: amantadine, rimantadine, zanamivir, and oseltamivir [9] . The influenza A virus, however, has become resistant to amantadine and rimantadine, which are presently not recommended to be used as first-line drugs [43] . Zanamivir (Ralenza, dry powder taken by orally inhaled route) and oseltamivir (Tamiflu, capsule or oral suspension) are neuraminidase inhibitors and can be used to treat patients and to control influenza outbreaks in closed settings. Although typically used in nursing homes, an outbreak in a dormitory may require chemoprophylaxis [9] . There are limited data to suggest that serious complications from influenza, such as lower respiratory tract infections, may be reduced [44] . The use of antiviral medications for prophylaxis of influenza is unclear and is not yet recommended for routine seasonal control [45] . The use of oseltamivir, however, has been recommended in specific cases, especially if there is high risk of spread such as household contacts and if individuals have not been immunized [46] . Oseltamivir was used to treat 36 of 188 patients, including 13 athletes during the 2002 Salt Lake City Winter Olympics, with medications given to close contacts, which was believed to limit the spread of influenza [47] . Clinical history and physical examination are still the mainstays for diagnosing influenza. Rapid swab tests are available and take approximately 30 minutes to detect the influenza virus. The tests are less sensitive (72%–95%) and specific (76%–86%) than the traditional viral cultures [48] . They have moderate sensitivities for influenza antigens and are more likely to produce false negative rather than false positive results [48] , [49] . Direct and indirect fluorescent antibody staining tests are also available, but they are ordered more in hospitals because they take 2 to 4 hours to obtain results [49] . Viral cultures are still the "gold standard" for confirming the presence of influenza and identifying the strains and subtypes [9] . Primary prevention Influenza vaccines contain strains of antigenically equivalent strains of influenza similar to those annually recommended: influenza A (H3N2), influenza A (H1N1), and a B virus. Depending on the emergence and spread of new strains, other virus strains can be added to update the vaccine [9] . The efficacy of influenza vaccine is approximately 70% to 90% for individuals under age 65 years [36] . Vaccination for influenza should occur in the fall (October or November), at the beginning of the flu season ( Box 2 ) [9] . Antibodies develop approximately 2 weeks after vaccination [9] , [37] . Box 2 Indications for influenza vaccine 1. Adults and children with chronic disorders of the cardiorespiratory system, including asthma 2. Adults and children who have chronic disease which may require regular medical follow-up or hospitalization, including immunodeficiency 3. Young children aged 6–23 months 4. Children aged 6 months to 18 years on long-term aspirin therapy and at risk for Reye's syndrome 5. Persons aged >65 years 6. Residents of nursing homes or chronic-care facilities 7. Health care workers 8. Travelers to influenza-endemic areas Adapted from Smith NM, Bresee JS, Shay DK, et al. Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2006;55(RR-10):1–42. Inactivated influenza vaccine is generally appropriate for all populations requiring influenza vaccine. Three influenza vaccines were available in the United States for the 2006 to 2007 season: Fluzone (manufactured by Sanofi-Pasteur); Fluvirin (manufactured by Novartis); and Fluarix (manufactured by GlaxoSmithKline). The typical dose is 0.5 mL administered intramuscularly, usually in the deltoid muscle. Live, attenuated influenza vaccine (LAIV) is approved for use in healthy, nonpregnant individuals aged 5 to 49 years. The LAIV is administered by way of a nasal spray once in each nostril (FluMist, manufactured by MedImmune). Individuals who have a hypersensitivity or anaphylactic reaction to components of the flu vaccine or to eggs should not be vaccinated [9] . Adults reported having a 19% reduction in severe febrile illnesses after LAIV compared with placebo [38] . Side effects from LAIV increased in adults within 7 days of immunization compared with placebo and consisted mainly of nasal congestion (44.5% versus 27.1%) and sore throat (27.8% versus 17.1%), which lasted, on average, 2 days. Less common complaints were tiredness, cough, and chills. There was no significant difference in the number of mild febrile illnesses between immunization and placebo groups [39] . Injections can be scheduled to occur at the optimum time during the athlete's competitive schedule to minimize concern about side effects. When inactivated influenza vaccine shortages occurred in previous years, the vaccine was recommended for high-risk groups as priority; however, the general recommendation now is to offer the immunization annually to anyone who wishes to reduce the likelihood of being ill with influenza or transmitting the virus if they should become infected [9] . Although this policy cannot be directly translated into a benefit for the athlete, depending on the level of athlete, the use of the LAIV may also be beneficial to prevent lost time from sport. Influenza vaccine has been suggested for competitive athletes and essential personnel, especially before international events occurring during the influenza season [4] , [40] . Secondary prevention Treatment with antiviral medications can reduce the duration of uncomplicated influenza A and B illness by approximately 1 day when administered within 2 days of illness onset [41] , [42] . Recommended antiviral treatment should be given for 5 days [9] . Four antiviral agents are currently available: amantadine, rimantadine, zanamivir, and oseltamivir [9] . The influenza A virus, however, has become resistant to amantadine and rimantadine, which are presently not recommended to be used as first-line drugs [43] . Zanamivir (Ralenza, dry powder taken by orally inhaled route) and oseltamivir (Tamiflu, capsule or oral suspension) are neuraminidase inhibitors and can be used to treat patients and to control influenza outbreaks in closed settings. Although typically used in nursing homes, an outbreak in a dormitory may require chemoprophylaxis [9] . There are limited data to suggest that serious complications from influenza, such as lower respiratory tract infections, may be reduced [44] . The use of antiviral medications for prophylaxis of influenza is unclear and is not yet recommended for routine seasonal control [45] . The use of oseltamivir, however, has been recommended in specific cases, especially if there is high risk of spread such as household contacts and if individuals have not been immunized [46] . Oseltamivir was used to treat 36 of 188 patients, including 13 athletes during the 2002 Salt Lake City Winter Olympics, with medications given to close contacts, which was believed to limit the spread of influenza [47] . Clinical history and physical examination are still the mainstays for diagnosing influenza. Rapid swab tests are available and take approximately 30 minutes to detect the influenza virus. The tests are less sensitive (72%–95%) and specific (76%–86%) than the traditional viral cultures [48] . They have moderate sensitivities for influenza antigens and are more likely to produce false negative rather than false positive results [48] , [49] . Direct and indirect fluorescent antibody staining tests are also available, but they are ordered more in hospitals because they take 2 to 4 hours to obtain results [49] . Viral cultures are still the "gold standard" for confirming the presence of influenza and identifying the strains and subtypes [9] . Meningitis Neisseria meningitides is a serious concern, especially for the adolescent and college populations. An alarming trend during the 1970s demonstrated an increase in meningitis deaths in college students, with living in dormitories being a risk factor. The disease can be spread by asymptomatic carriers. Students living in dormitories were 9 to 23 times more likely of getting infected than those living in other types of accommodations [50] . Freshmen who lived in dormitories had an elevated risk of meningococcal disease (odds ratio, 3.6; 95% confidence interval, 1.6–8.5; P = .003) compared with other college students [51] . Aside from the risk of death, 11% to 19% of survivors of meningitis have serious sequelae such as neurologic disability, limb loss, and hearing loss [50] . Primary prevention Routine vaccination with meningococcal vaccine is recommended for college freshmen living in dormitories and for other populations at increased risk. The CDC Advisory Committee on Immunization Practices recommends routine vaccination of young adolescents (11–12 years old) with meningococcal vaccine (MCV4) at the preadolescent health care visit [50] . Therefore, sports medicine physicians may be faced with higher frequency of checking for meningococcal immunization status for high school and college athletes. A tetravalent conjugate vaccine (Menactra, Sanofi Pasteur) is available against Neisseria meningitidis isolates A, C, Y, and W-135 in a 0.5-mL single-dose vial. Over the age of 11 years, 75% of the meningococcal infections are caused by strains C, Y, or W-135 (CDC, unpublished data, 2004) [50] . Another vaccine, Menomune (Aventis Pasteur Limited), has been licensed since 1981 and has a similar immunogenicity profile to Menactra and is delivered subcutaneously as a 0.5-mL dose. Menactra and Menomune have serum bactericidal protection ranging from 89.4% and 94.4% for strain W-135 and 73.5% and 79.4% for strain Y, respectively [50] . Revaccination may be necessary for individuals at high risk after 5 years [52] , [53] . Common side effects with MCV4 were local pain in just over 50% of patients, followed by swelling, induration, and redness in approximately 10.8% to 17.1%. Fever was reported in 5.1% of children 18 years old or younger and in 1.5% of adults [50] . Secondary prevention Close contacts are at high risk and should be treated with chemoprophylaxis ideally within 24 hours of identifying the index patient [50] . The goal of treatment is to reduce nasopharyngeal carriage of N meningitidis . After more than 14 days after the onset of illness in the index patient, chemoprophylaxis is not necessary [50] . A single dose of ciprofloxacin (500 mg orally) or ceftriaxone (250 mg by intramuscular injection), or rifampin (600 mg twice a day for 2 days) is recommended for adults. Children between 1 month and 18 years old may take rifampin (10 mg/kg every 12 hours for 2 days), or ceftriaxone (125 mg intramuscularly) if younger than 15 years [50] . One dose of azithromycin (500 mg) was also shown to eradicate N meningitidis and may represent another treatment option [54] . Primary prevention Routine vaccination with meningococcal vaccine is recommended for college freshmen living in dormitories and for other populations at increased risk. The CDC Advisory Committee on Immunization Practices recommends routine vaccination of young adolescents (11–12 years old) with meningococcal vaccine (MCV4) at the preadolescent health care visit [50] . Therefore, sports medicine physicians may be faced with higher frequency of checking for meningococcal immunization status for high school and college athletes. A tetravalent conjugate vaccine (Menactra, Sanofi Pasteur) is available against Neisseria meningitidis isolates A, C, Y, and W-135 in a 0.5-mL single-dose vial. Over the age of 11 years, 75% of the meningococcal infections are caused by strains C, Y, or W-135 (CDC, unpublished data, 2004) [50] . Another vaccine, Menomune (Aventis Pasteur Limited), has been licensed since 1981 and has a similar immunogenicity profile to Menactra and is delivered subcutaneously as a 0.5-mL dose. Menactra and Menomune have serum bactericidal protection ranging from 89.4% and 94.4% for strain W-135 and 73.5% and 79.4% for strain Y, respectively [50] . Revaccination may be necessary for individuals at high risk after 5 years [52] , [53] . Common side effects with MCV4 were local pain in just over 50% of patients, followed by swelling, induration, and redness in approximately 10.8% to 17.1%. Fever was reported in 5.1% of children 18 years old or younger and in 1.5% of adults [50] . Secondary prevention Close contacts are at high risk and should be treated with chemoprophylaxis ideally within 24 hours of identifying the index patient [50] . The goal of treatment is to reduce nasopharyngeal carriage of N meningitidis . After more than 14 days after the onset of illness in the index patient, chemoprophylaxis is not necessary [50] . A single dose of ciprofloxacin (500 mg orally) or ceftriaxone (250 mg by intramuscular injection), or rifampin (600 mg twice a day for 2 days) is recommended for adults. Children between 1 month and 18 years old may take rifampin (10 mg/kg every 12 hours for 2 days), or ceftriaxone (125 mg intramuscularly) if younger than 15 years [50] . One dose of azithromycin (500 mg) was also shown to eradicate N meningitidis and may represent another treatment option [54] . Human Papillomavirus Human papillomavirus (HPV) is associated with 99% of cervical cancers and anogenital, head and neck, and nonmelanoma skin cancers. It is an STD and can be diagnosed by abnormal cervical cell changes seen on Pap smear [55] . This is a common infection, especially in sexually active adolescents and university students [56] . Primary prevention Primary prevention is now possible with two new vaccines: a bivalent vaccine against HPV types 16 and 18 and a quadravalent vaccine against types 6, 11, 16, and 18. The vaccines have a three-dose schedule: 0, 1, and 6 months (bivalent vaccine) and 0, 2, and 6 months (quadravalent vaccine). At 4.5 years, the bivalent vaccine was effective for producing a persistent antibody response against HPV 16 and 18, with more than 98% seropositivity and 96.9% effectiveness (95% confidence interval, 81.3–99.9) in reducing the number of reported abnormalities on Pap smear, colposcopy, or both [57] . Routine vaccination with three doses of quadrivalent HPV vaccine is recommended for girls 11 to 12 years old but can be started in girls as young as 9 years. Girls and women aged 13 to 26 years who have not been vaccinated previously or who have not completed the full vaccine series are recommended to receive a catch-up series. The vaccine is intended to be administered before potential exposure to HPV through sexual contact [58] . Secondary prevention Secondary prevention involves checking the affected individual's partners for signs of genital warts and other STDs. Regular cervical screen is recommended. Use of condoms and education on spread is important. HPV infection persists for life; however, the degree and duration of contagiousness is yet unknown [59] . Primary prevention Primary prevention is now possible with two new vaccines: a bivalent vaccine against HPV types 16 and 18 and a quadravalent vaccine against types 6, 11, 16, and 18. The vaccines have a three-dose schedule: 0, 1, and 6 months (bivalent vaccine) and 0, 2, and 6 months (quadravalent vaccine). At 4.5 years, the bivalent vaccine was effective for producing a persistent antibody response against HPV 16 and 18, with more than 98% seropositivity and 96.9% effectiveness (95% confidence interval, 81.3–99.9) in reducing the number of reported abnormalities on Pap smear, colposcopy, or both [57] . Routine vaccination with three doses of quadrivalent HPV vaccine is recommended for girls 11 to 12 years old but can be started in girls as young as 9 years. Girls and women aged 13 to 26 years who have not been vaccinated previously or who have not completed the full vaccine series are recommended to receive a catch-up series. The vaccine is intended to be administered before potential exposure to HPV through sexual contact [58] . Secondary prevention Secondary prevention involves checking the affected individual's partners for signs of genital warts and other STDs. Regular cervical screen is recommended. Use of condoms and education on spread is important. HPV infection persists for life; however, the degree and duration of contagiousness is yet unknown [59] . Travel Immunizations Athletes traveling need to consider the endemic diseases in the geographic location where they are competing. They should be aware of risks of acquiring common diseases, their accommodations (urban versus rural), local foods, and customs. Immunizations should ideally be planned 4 or more months in advance to allow for adequate time to administer vaccines ( Table 3 ). There are many resources for information about prevention of infectious diseases for travelers ( Table 4 ). Table 3 Recommended immunizations for travelers to developing countries a Length of travel Immunizations Brief, 3 mo Review and complete age-appropriate childhood schedule (see text for details) + + + DTaP, poliovirus, pneumococcal, and Haemophilus influenzae type b vaccines may be given at 4-wk intervals if necessary to complete the recommended schedule before departure Measles: 2 additional doses given if younger than 12 mo of age at first dose Varicella Hepatitis B b Yellow fever c + + + Hepatitis A d + + + Typhoid fever e ± + + Meningococcal disease f ± ± ± Rabies g ± + + Japanese encephalitis h ± ± + + = recommended; ± = consider. From American Academy of Pediatrics. International travel. In: Pickering LK, editor. Red book: 2006 report of the committee on infectious diseases. 27th edition. Elk Grove Village (IL): American Academy of Pediatrics; 2006. p. 99; with permission from the American Academy of Pediatrics. a See disease-specific chapters in Section 3 of the AAP Red Book for details: [ Red Book: 2006 report of the Committee of Infectious Diseases . 27th edition. Elk Grove Village, (IL) American Academy of Pediatrics; 2006]. For further sources of information, see text. b If insufficient time to complete 6-month primary series, accelerated series can be given (see text for details). c For regions with endemic infection. d Indicated for travelers to areas with intermediate or high endemic rates of HAV infection. e Indicated for travelers who will consume food and liquids in areas of poor sanitation. f Recommended for regions of Africa with endemic infection and during local epidemics, and required for travel to Saudi Arabia for the Hajj. g Indicated for people with high risk of animal exposure (especially to dogs) and for travelers to countries with endemic infection. h For regions with endemic infection. For high-risk activities in areas experiencing outbreaks, vaccine is recommended, even for brief travel. Table 4 More common tick-borne diseases Tick-borne disease Organism Common vector (geographic area) Rocky Mountain spotted fever Rickettsia rickettsii Dog tick, Dermacentor variabilis (central, Pacific coastal, and eastern US) Rocky Mountain wood tick, Dermacentor andersoni (western US) Human monocytotropic ehrlichiosis Ehrlichia chaffeensis Lone Star tick (south central US in Maryland, Arkansas, Tennessee, Oklahoma, and Missouri) [60] Human granulocytotropic anaplasmosis Anaplasma phagocytophilum Blacklegged tick, Ixodes scapularis (north central US and New England) Ixodes pacificus (California) [75] Lyme disease Borrelia burgdorferi Ixodes scapularis (eastern US in New England and mid-Atlantic states and Midwest US in Wisconsin and Minnesota) Ixodes pacificus (west in northern California) Babesiosis Parasite, Babesia microti Ixodes scapularis (northeast of the US) Abbreviation: US, United States. Bug-Borne Disease Prevention Mosquito-Borne Disease A number of arthropods, such as mosquitoes and ticks, can transmit diseases. Mosquito-vector diseases include West Nile virus, yellow fever virus, and dengue virus. West Nile virus, a flavivirus, has demonstrated a seasonally endemic epidemiology with geographic variation in the United States, especially in California, Arizona, and Colorado [7] , [61] . This disease typically presents between July and October, although cases have presented between April and December. The prevention of mosquito bites is the cornerstone of prevention. An athlete in an endemic area should wear an insect repellant such as deet ( N , N -diethyl-m-toluamide), picaridin (KBR-3023), or oil of lemon eucalyptus ( p -menthane-3,8 diol). Deet and permethin may be applied to the clothing [62] . If a sunscreen is used concomitantly, the insect repellant should be applied on top of this and removed at the end of the day. Long-sleeved shirts that are tucked into long pants are also useful. Tick-Borne Disease Tick-borne diseases include rickettsial diseases, Lyme disease, babesiosis, tick-borne relapsing fever, and occasionally, tularemia and Q fever ( Table 5 ). Certain athletes who participate in rural outdoor activities are more susceptible to tick bites. These sports include cross-country running, training in multiple sports in rural areas, and recreational outdoor sports such as fishing and hiking. Children are more at risk to tick bites. Table 5 Suggested resources for preventing infections Topic Web site Vaccines licensed for immunization and distribution in the United States www.fda.gov/cber/vaccine/licvacc.htm http://www.vaccineinformation.org/ How to store and handle vaccines www.cdc.gov/nip/menus/vaccines.htm#Storage Adult immunization schedule http://www.cdc.gov/nip/recs/adult-schedule.htm Travel information www.cdc.gov/travel www.who.int/ith Children and adolescents immunization schedule http://www.cdc.gov/nip/recs/child-schedule.htm HIV position statements http://www.fims.org/ (International Federation of Sports Medicine) http://www.casm-acms.org/forms/statements/HIVEng.pdf (Canadian Academy of Sport Medicine) http://www.amssm.org/hiv.html (American Medical Society for Sports Medicine and the American Orthopaedic Society for Sports Medicine) Morbidity and Mortality Weekly Reports www.cdc.gov/mmwr Primer for physicians for preventing food-borne illnesses http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5002a1.htm Web sites accessed November 3, 2006. Three more common rickettsial illnesses are Rocky Mountain spotted fever, human monocytotropic ehrlichiosis, and human granulocytotropic anaplasmosis [60] . The infectious organisms responsible for these illnesses maintain their lifecycles in mammals and ticks. Their prevalence reflects the geographic locations and the seasonality of the tick abundance. Their season is usually from April to September, but they can present throughout the year. Newer rickettsial diseases are emerging. These potentially lethal diseases are difficult to diagnose because they often mimic viral syndromes. As many as 60% to 75% of patients are initially misdiagnosed [63] , [64] . With Rocky Mountain spotted fever, more than 50% of cases are reported in the five states of North Carolina, South Carolina, Tennessee, Oklahoma, and Arkansas [65] . The presentation most often manifests as a sudden febrile illness with headache, myalgia, and a maculopapular rash that spreads in a centripetal pattern. Rickettsia rickettsii has a predilection for endothelial cells and can cause a diffuse vasculitis and an untreated mortality of 10%. The diagnosis is based on clinical presentation, with epidemiologic, geographic, and seasonal considerations. Laboratory testing may be supportive with thrombocytopenia and mild liver enzyme elevation. Serologic testing is supportive only on a delayed basis with acute and convalescent titers. Human monocytotropic ehrlichiosis and human granulocytotropic anaplasmosis can also present with acute headache, fever, and myalgia. Laboratory evaluation often demonstrates leukopenia, thrombocytopenia, and transaminase elevation. Common tick-borne illnesses that have been reported in the northeast United States are Lyme disease and babesiosis, which are transmitted by the tick Ixodes scapularis [66] . Babesiosis can cause a febrile illness and possibly life-threatening anemia and thrombocytopenia. Lyme disease is a rickettsial disease caused by Borrelia burgdorferi. As such, concurrent disease may be caused by the same tick bite (see Table 4 ). Tick-bite prevention There are no proven vaccines for these tick-borne illnesses, but all are preventable by careful vigilance and protection. The key to prevention is to understand the regional epidemiology and seasonality of the diseases. Vaccination for Lyme disease (LYMErix) was originally approved; however, the manufacturer took the vaccine off the market due to declining sales. There was a 49% efficacy after two doses and a 76% efficacy after three doses [67] ; however, the protection diminished after 2 years. Ticks thrive in a wooded environment and at the edge of woods with surrounding high vegetation. Ticks are uncommon in well-mowed lawns. Relative tick-free zones can be created by placing wood chips or gravel around recreational areas to separate the woods [68] . Other landscape management tips include removing clippings and leaves, keeping stone walls clean of leaves, and restricting the use of groundcover, such as pachysandra, where pets and children may play. Widening woodland trails andkeeping in the center of the trail while walking may be helpful. When traveling in wooded areas, light-colored clothing is helpful to identify the tick. Long pants tucked into tightly woven socks and closed shoes minimize exposure. Deet at 10% to 25% should be applied to the skin. Permethrin may be applied only to the clothing. Clothes should be removed and cleaned and dried after exposure. The clothes dryer is effective in killing ticks. One should carefully check for ticks in the nymphal phase that may be the size of a pin head. Careful inspection should be done of the hair, ears, axilla, belly button, and legs. Children and pets should also be checked. It is also important to monitor pets that may travel in the woods and return indoors. The technique of tick removal is critical. Tweezers with fine tips should be used close to the skin and pulled directly away. Squeezing the body may allow contamination of the disease into the host [69] . Lyme disease is not contracted until at least 24 hours of tick adherence [70] ; however, ehrlichiosis may transmit in less than 24 hours. Preventive antibiotics are generally not indicated because less than 5% of bites are Lyme infected, especially with a flat tick. After a high-risk exposure (when the tick has been engaged for more than 24 hours and is engorged), a single dose of 200 mg of doxycycline is believed to be effective [71] . Mosquito-Borne Disease A number of arthropods, such as mosquitoes and ticks, can transmit diseases. Mosquito-vector diseases include West Nile virus, yellow fever virus, and dengue virus. West Nile virus, a flavivirus, has demonstrated a seasonally endemic epidemiology with geographic variation in the United States, especially in California, Arizona, and Colorado [7] , [61] . This disease typically presents between July and October, although cases have presented between April and December. The prevention of mosquito bites is the cornerstone of prevention. An athlete in an endemic area should wear an insect repellant such as deet ( N , N -diethyl-m-toluamide), picaridin (KBR-3023), or oil of lemon eucalyptus ( p -menthane-3,8 diol). Deet and permethin may be applied to the clothing [62] . If a sunscreen is used concomitantly, the insect repellant should be applied on top of this and removed at the end of the day. Long-sleeved shirts that are tucked into long pants are also useful. Tick-Borne Disease Tick-borne diseases include rickettsial diseases, Lyme disease, babesiosis, tick-borne relapsing fever, and occasionally, tularemia and Q fever ( Table 5 ). Certain athletes who participate in rural outdoor activities are more susceptible to tick bites. These sports include cross-country running, training in multiple sports in rural areas, and recreational outdoor sports such as fishing and hiking. Children are more at risk to tick bites. Table 5 Suggested resources for preventing infections Topic Web site Vaccines licensed for immunization and distribution in the United States www.fda.gov/cber/vaccine/licvacc.htm http://www.vaccineinformation.org/ How to store and handle vaccines www.cdc.gov/nip/menus/vaccines.htm#Storage Adult immunization schedule http://www.cdc.gov/nip/recs/adult-schedule.htm Travel information www.cdc.gov/travel www.who.int/ith Children and adolescents immunization schedule http://www.cdc.gov/nip/recs/child-schedule.htm HIV position statements http://www.fims.org/ (International Federation of Sports Medicine) http://www.casm-acms.org/forms/statements/HIVEng.pdf (Canadian Academy of Sport Medicine) http://www.amssm.org/hiv.html (American Medical Society for Sports Medicine and the American Orthopaedic Society for Sports Medicine) Morbidity and Mortality Weekly Reports www.cdc.gov/mmwr Primer for physicians for preventing food-borne illnesses http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5002a1.htm Web sites accessed November 3, 2006. Three more common rickettsial illnesses are Rocky Mountain spotted fever, human monocytotropic ehrlichiosis, and human granulocytotropic anaplasmosis [60] . The infectious organisms responsible for these illnesses maintain their lifecycles in mammals and ticks. Their prevalence reflects the geographic locations and the seasonality of the tick abundance. Their season is usually from April to September, but they can present throughout the year. Newer rickettsial diseases are emerging. These potentially lethal diseases are difficult to diagnose because they often mimic viral syndromes. As many as 60% to 75% of patients are initially misdiagnosed [63] , [64] . With Rocky Mountain spotted fever, more than 50% of cases are reported in the five states of North Carolina, South Carolina, Tennessee, Oklahoma, and Arkansas [65] . The presentation most often manifests as a sudden febrile illness with headache, myalgia, and a maculopapular rash that spreads in a centripetal pattern. Rickettsia rickettsii has a predilection for endothelial cells and can cause a diffuse vasculitis and an untreated mortality of 10%. The diagnosis is based on clinical presentation, with epidemiologic, geographic, and seasonal considerations. Laboratory testing may be supportive with thrombocytopenia and mild liver enzyme elevation. Serologic testing is supportive only on a delayed basis with acute and convalescent titers. Human monocytotropic ehrlichiosis and human granulocytotropic anaplasmosis can also present with acute headache, fever, and myalgia. Laboratory evaluation often demonstrates leukopenia, thrombocytopenia, and transaminase elevation. Common tick-borne illnesses that have been reported in the northeast United States are Lyme disease and babesiosis, which are transmitted by the tick Ixodes scapularis [66] . Babesiosis can cause a febrile illness and possibly life-threatening anemia and thrombocytopenia. Lyme disease is a rickettsial disease caused by Borrelia burgdorferi. As such, concurrent disease may be caused by the same tick bite (see Table 4 ). Tick-bite prevention There are no proven vaccines for these tick-borne illnesses, but all are preventable by careful vigilance and protection. The key to prevention is to understand the regional epidemiology and seasonality of the diseases. Vaccination for Lyme disease (LYMErix) was originally approved; however, the manufacturer took the vaccine off the market due to declining sales. There was a 49% efficacy after two doses and a 76% efficacy after three doses [67] ; however, the protection diminished after 2 years. Ticks thrive in a wooded environment and at the edge of woods with surrounding high vegetation. Ticks are uncommon in well-mowed lawns. Relative tick-free zones can be created by placing wood chips or gravel around recreational areas to separate the woods [68] . Other landscape management tips include removing clippings and leaves, keeping stone walls clean of leaves, and restricting the use of groundcover, such as pachysandra, where pets and children may play. Widening woodland trails andkeeping in the center of the trail while walking may be helpful. When traveling in wooded areas, light-colored clothing is helpful to identify the tick. Long pants tucked into tightly woven socks and closed shoes minimize exposure. Deet at 10% to 25% should be applied to the skin. Permethrin may be applied only to the clothing. Clothes should be removed and cleaned and dried after exposure. The clothes dryer is effective in killing ticks. One should carefully check for ticks in the nymphal phase that may be the size of a pin head. Careful inspection should be done of the hair, ears, axilla, belly button, and legs. Children and pets should also be checked. It is also important to monitor pets that may travel in the woods and return indoors. The technique of tick removal is critical. Tweezers with fine tips should be used close to the skin and pulled directly away. Squeezing the body may allow contamination of the disease into the host [69] . Lyme disease is not contracted until at least 24 hours of tick adherence [70] ; however, ehrlichiosis may transmit in less than 24 hours. Preventive antibiotics are generally not indicated because less than 5% of bites are Lyme infected, especially with a flat tick. After a high-risk exposure (when the tick has been engaged for more than 24 hours and is engorged), a single dose of 200 mg of doxycycline is believed to be effective [71] . Tick-bite prevention There are no proven vaccines for these tick-borne illnesses, but all are preventable by careful vigilance and protection. The key to prevention is to understand the regional epidemiology and seasonality of the diseases. Vaccination for Lyme disease (LYMErix) was originally approved; however, the manufacturer took the vaccine off the market due to declining sales. There was a 49% efficacy after two doses and a 76% efficacy after three doses [67] ; however, the protection diminished after 2 years. Ticks thrive in a wooded environment and at the edge of woods with surrounding high vegetation. Ticks are uncommon in well-mowed lawns. Relative tick-free zones can be created by placing wood chips or gravel around recreational areas to separate the woods [68] . Other landscape management tips include removing clippings and leaves, keeping stone walls clean of leaves, and restricting the use of groundcover, such as pachysandra, where pets and children may play. Widening woodland trails andkeeping in the center of the trail while walking may be helpful. When traveling in wooded areas, light-colored clothing is helpful to identify the tick. Long pants tucked into tightly woven socks and closed shoes minimize exposure. Deet at 10% to 25% should be applied to the skin. Permethrin may be applied only to the clothing. Clothes should be removed and cleaned and dried after exposure. The clothes dryer is effective in killing ticks. One should carefully check for ticks in the nymphal phase that may be the size of a pin head. Careful inspection should be done of the hair, ears, axilla, belly button, and legs. Children and pets should also be checked. It is also important to monitor pets that may travel in the woods and return indoors. The technique of tick removal is critical. Tweezers with fine tips should be used close to the skin and pulled directly away. Squeezing the body may allow contamination of the disease into the host [69] . Lyme disease is not contracted until at least 24 hours of tick adherence [70] ; however, ehrlichiosis may transmit in less than 24 hours. Preventive antibiotics are generally not indicated because less than 5% of bites are Lyme infected, especially with a flat tick. After a high-risk exposure (when the tick has been engaged for more than 24 hours and is engorged), a single dose of 200 mg of doxycycline is believed to be effective [71] . Hygiene Precautions and Infection Control Personal Hygiene Most infectious diseases are spread from contact with the microorganism directly or indirectly from the infected individual. Athletes frequently interact with teammates, coaches, athletic trainers, and physicians and share equipment, water bottles, towels, and supplies. This interaction is particularly a concern, with the recent outbreaks of methicillin-resistent Staphylococcus aureus (MRSA) infections among sports teams [72] , [73] . Three categories of potential risk factors for spreading infection have been suggested: "sharing" (sharing soap/towels/water bottles with teammates), "skin injury" (cuts, abrasions), and "close contact" (locker adjacent to infected teammate, living on-campus) [74] . Good personal hygiene can help reduce colonization of bacteria. Bacterial counts can range from 5000 to 5 million colony-forming units per square centimeter on the hands [75] . Universal body fluid precautions—for example, using disposable gloves when examining the oral cavity or wounds and frequent hand washing—can reduce the risk of infection. MRSA is transmitted from an infected patient to the gloves of a health care worker in approximately 17% (9%–25%). Physicians, in particular, have a low compliance for using gloves and washing their hands [76] . Proper surgical hand washing is recommended to be 15 to 30 seconds with soap, a 30-second rinse with water, followed by complete drying with a towel. The use of rinses and gels with concentrations of 50% to 95% alcohol take 15 seconds to use and are effective at killing organisms [75] . The use of chlorhexidine soap has been useful for reducing MRSA infections. Viruses and bacteria can exist on equipment. MRSA was found in the taping gel and whirlpool in the training facilities of a professional football team [72] . Using diluted bleach (1 part bleach in 9 parts water) to cleanse training areas and equipment is recommended [8] . Routine cleaning schedules for shared equipment should be established and recorded. For upper respiratory tract infections, isolation of those who have had close contact with someone who has a confirmed or suspected infection, especially those who have active symptoms such as persistent fever and cough, is an effective and practical method of avoiding contact [8] . Any athlete who has a scratch, abrasion, or laceration or who has potentially infectious skin lesions such as vesicular or weeping skin lesions should be removed from play until the area can be securely covered with occlusive bandages or dressings to prevent leakage of blood or serous fluid [77] . Uniforms with fresh blood should be removed and replaced immediately after stopping any bleeding. Bleach diluted with tap water in a 1:10 ratio can be used to wash equipment that has had contact with blood or body fluid. Body substance precautions should be taken by health care professionals at all times when treating open wounds. Prevention of Methicillin-Resistant Staphylococcus Aureus One type of bacteria that has become more common in the hospital and a community-acquired infection is MRSA. Although contact sports such as wrestling and football have been commonly associated with MRSA spread, this infection has also been discovered in minimal-contact sports such as fencing [78] . Three factors are associated with MRSA spread in sports. First, even with sports that have minimal contact, there are often abrasions and chaffing from clothing and hot environments. Second, equipment is often shared and there is potential for transmission of bacteria. Third, many sports have sufficient skin-to-skin contact to transmit organisms. Subsequently, health care providers should strongly encourage good overall and hand hygiene in addition to covering all wounds and limiting shared equipment. It is crucial to have an ample supply of soap and water and alcohol-based hand cleansers. Athletes, staff, and coaches should be educated in proper first aid for wounds, in recognition of wounds that are potentially infected, and in seeking medical attention for lesions that have concerning signs, especially large pustules or boils. Prevention of Fungal Rashes Athlete's foot, tinea pedis, is a common ailment not only during the hot summer months but also during the winter months with indoor sports. A number of prevention items include washing feet daily; drying between the toes; wearing cotton, nonsynthetic socks; wearing bathing shoes in public showers; and wearing sandals in warmer weather. Jock itch, tinea cruris, is best prevented by showering immediately after athletic endeavors and wearing cotton briefs. A good talc powder may be used for prevention of athlete's foot and jock itch. Ring worm, tinea corporis, is best prevented by avoiding contact. Contact athletes such as wrestlers should not participate until any lesions have cleared or can be safely and effectively covered. Personal Hygiene Most infectious diseases are spread from contact with the microorganism directly or indirectly from the infected individual. Athletes frequently interact with teammates, coaches, athletic trainers, and physicians and share equipment, water bottles, towels, and supplies. This interaction is particularly a concern, with the recent outbreaks of methicillin-resistent Staphylococcus aureus (MRSA) infections among sports teams [72] , [73] . Three categories of potential risk factors for spreading infection have been suggested: "sharing" (sharing soap/towels/water bottles with teammates), "skin injury" (cuts, abrasions), and "close contact" (locker adjacent to infected teammate, living on-campus) [74] . Good personal hygiene can help reduce colonization of bacteria. Bacterial counts can range from 5000 to 5 million colony-forming units per square centimeter on the hands [75] . Universal body fluid precautions—for example, using disposable gloves when examining the oral cavity or wounds and frequent hand washing—can reduce the risk of infection. MRSA is transmitted from an infected patient to the gloves of a health care worker in approximately 17% (9%–25%). Physicians, in particular, have a low compliance for using gloves and washing their hands [76] . Proper surgical hand washing is recommended to be 15 to 30 seconds with soap, a 30-second rinse with water, followed by complete drying with a towel. The use of rinses and gels with concentrations of 50% to 95% alcohol take 15 seconds to use and are effective at killing organisms [75] . The use of chlorhexidine soap has been useful for reducing MRSA infections. Viruses and bacteria can exist on equipment. MRSA was found in the taping gel and whirlpool in the training facilities of a professional football team [72] . Using diluted bleach (1 part bleach in 9 parts water) to cleanse training areas and equipment is recommended [8] . Routine cleaning schedules for shared equipment should be established and recorded. For upper respiratory tract infections, isolation of those who have had close contact with someone who has a confirmed or suspected infection, especially those who have active symptoms such as persistent fever and cough, is an effective and practical method of avoiding contact [8] . Any athlete who has a scratch, abrasion, or laceration or who has potentially infectious skin lesions such as vesicular or weeping skin lesions should be removed from play until the area can be securely covered with occlusive bandages or dressings to prevent leakage of blood or serous fluid [77] . Uniforms with fresh blood should be removed and replaced immediately after stopping any bleeding. Bleach diluted with tap water in a 1:10 ratio can be used to wash equipment that has had contact with blood or body fluid. Body substance precautions should be taken by health care professionals at all times when treating open wounds. Prevention of Methicillin-Resistant Staphylococcus Aureus One type of bacteria that has become more common in the hospital and a community-acquired infection is MRSA. Although contact sports such as wrestling and football have been commonly associated with MRSA spread, this infection has also been discovered in minimal-contact sports such as fencing [78] . Three factors are associated with MRSA spread in sports. First, even with sports that have minimal contact, there are often abrasions and chaffing from clothing and hot environments. Second, equipment is often shared and there is potential for transmission of bacteria. Third, many sports have sufficient skin-to-skin contact to transmit organisms. Subsequently, health care providers should strongly encourage good overall and hand hygiene in addition to covering all wounds and limiting shared equipment. It is crucial to have an ample supply of soap and water and alcohol-based hand cleansers. Athletes, staff, and coaches should be educated in proper first aid for wounds, in recognition of wounds that are potentially infected, and in seeking medical attention for lesions that have concerning signs, especially large pustules or boils. Prevention of Fungal Rashes Athlete's foot, tinea pedis, is a common ailment not only during the hot summer months but also during the winter months with indoor sports. A number of prevention items include washing feet daily; drying between the toes; wearing cotton, nonsynthetic socks; wearing bathing shoes in public showers; and wearing sandals in warmer weather. Jock itch, tinea cruris, is best prevented by showering immediately after athletic endeavors and wearing cotton briefs. A good talc powder may be used for prevention of athlete's foot and jock itch. Ring worm, tinea corporis, is best prevented by avoiding contact. Contact athletes such as wrestlers should not participate until any lesions have cleared or can be safely and effectively covered. Sexually Transmitted Disease Prevention Athletes may manifest risk-taking behavior and subsequently be at increased risk for STDs [2] . The preparticipation examination affords the opportunity for the clinician to address these concerns. The CDC has emphasized the five intervention strategies [79] , which include education on sexual behavior, identification of asymptomatic individuals, diagnosis and treatment of infected individuals, counseling of sexual partners of persons who have an STD, and pre-exposure vaccination when applicable. Individuals at risk should be questioned about partners regarding number and same or opposite sex. Information about the type of sexual activity, the use of protection, and history of previous STDs should also be identified. Preventive measures for an STD include abstinence if an individual or partner is actively infected and undergoing treatment. Pre-exposure prophylaxis is relevant in several situations. Hepatitis B vaccine is recommended in all individuals potentially exposed to STDs. Hepatitis A vaccine is recommended for all men who have sex with men or users of illicit drugs (injectable and noninjectable). For girls and women aged 9 to 26 years, the new quadrivalent vaccine for HPV types 6, 11,16, and 18 is recommended due to the higher associated risk of cervical cancer. Most condoms are made of latex and are quite effective in STD prevention. In one study of partners of HIV-infected individuals, partners were 80% less likely to seroconvert than those who did not use condoms [80] . The male condom can also reduce the transmission of gonorrhea, chlamydiosis, and trichomiasis [81] . There may be some added protection against herpes simplex virus 2 and a 70% reduction of HPV transmission [82] , [83] . When an individual is allergic to latex, certain polyurethane condoms are likely just as effective, although they may break more readily. Conversely, natural-membrane condoms such as lambskin are too porous to be used for STD prevention. Only water-based lubricants should be used with latex condoms because oil bases will weaken the latex. The female condom is a double-ringed polyurethane sheath that is used vaginally and during anal receptive intercourse that is effective in limited trials in preventing HIV/STDs [84] , [85] . Spermicides and nonbarrier contraception have no role in STD prevention. Finally, providers should encourage patients who have STDs to notify their partners. Often, this notification is pursued by the public health department. In the event of exposure to HIV by sexual exposure or needle stick, HIV prophylaxis is often undertaken and should be immediate. Prophylactic treatment usually involves the hospital infectious disease division to determine the best combination therapy. Summary Education is paramount in public health and in the prevention of infectious diseases. Athletes are a high-risk population often due to their increased exposure to different people and environments and, sometimes, their outgoing lifestyle behaviors. Primary prevention can be promoted through accurate immunizations; appropriate, planned health maintenance; good hygiene practices; and behavior modification to minimize high-risk activities. Secondary prevention can be achieved through vigilant surveillance for reportable illnesses, proper education and containment for reducing spread if an illness occurs, and timely prophylaxis with medications and immunizations in certain cases.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8051409/

The role of Namibia Field Epidemiology and Laboratory Training Programme in strengthening the public health workforce in Namibia, 2012–2019

Namibia faces a critical shortage of skilled public health workers to perform emergency response operations, preparedness activities and real-time surveillance. The Namibia Field Epidemiology and Laboratory Training Programme (NamFELTP) increases the number of skilled public health professionals and strengthens the public health system in Namibia. We describe the NamFELTP during its first 7 years, assess its impact on the public health workforce and provide recommendations to further strengthen the workforce. We reviewed disease outbreak investigations and response reports, field projects and epidemiological investigations conducted during 2012–2019. The data were analysed using descriptive methods such as frequencies and rates. Maps representing the geographical distribution of NamFELTP workforce were produced using QGIS software V.3.2. There were no formally trained field epidemiologists working in Namibia before the NamFELTP. In its 7 years of operation, the programme graduated 189 field epidemiologists, of which 28 have completed the Advanced FELTP. The graduates increased epidemiological capacity for surveillance and response in Namibia at the national and provincial levels, and enhanced epidemiologist-led outbreak responses on 35 occasions, including responses to outbreaks of human and zoonotic diseases. Trainees analysed data from 51 surveillance systems and completed 31 epidemiological studies. The NamFELTP improved outcomes in the Namibia's public health systems; including functional and robust public health surveillance systems that timely and effectively respond to public health emergencies. However, the current epidemiological capacity is insufficient and there is a need to continue training and mentorship to fill key leadership and strategic roles in the public health system. Introduction Namibia is a large, sparsely populated sub-Saharan African country with 13 regions and a population of 2.5 million. 1 Namibia lacks an adequate healthcare workforce, and the health system is mostly clinically focused with very few public health experts. The critical shortage of skilled public health workers includes epidemiologists, biostatisticians and other quantitative scientists with skills in data collection and analysis. 2 A primary goal of field epidemiology is to guide, as quickly as possible, the processes of selecting and implementing effective and appropriate interventions to lessen or prevent illness or death when such problems arise. 3 To address the deficit of trained public health staff, in 2012, the Namibia Ministry of Health and Social Services (MOHSS), in partnership with the US Centers for Disease Control and Prevention (US-CDC), established the Namibia Field Epidemiology and Laboratory Training Programme (NamFELTP). The programme is modelled after the US-CDC Epidemic Intelligence Service (EIS) which has trained public health workers in over 70 countries to respond to outbreaks of infectious diseases to minimise impact and prevent national or international spread. 4 5 In Namibia, trainees address high prevalence infectious diseases (eg, HIV and tuberculosis), zoonotic diseases (eg, anthrax) and non-communicable diseases (NCDs) such as cardiovascular disease, cancer, diabetes and hypertension. 6–8 Namibia requires an epidemiologically trained public health workforce to help with emergency response operations, preparedness and real-time surveillance and reporting, as well as to manage the public health laboratory system. Globally, the Field Epidemiology Training Programme (FETP) is structured into three tiers ( figure 1 ). Each tier addresses a need to improve surveillance, epidemiology, response and scientific communication skills of public health workers. The front-line tier is a 3-month training that focuses on strengthening the surveillance system at the district level along with improving the participants' skills within the context of their current job responsibilities. The intermediate tier is a 9-month training that trains mid-level epidemiologists and focuses on strengthening the surveillance system at the regional or provincial level. The advanced tier is a 2-year training that prepares trainees for leadership positions within the public health system. The NamFELTP was introduced to enhance epidemiology skills at various levels, that is at the district, regional and national to strengthen the public health system of Namibia. Figure 1 Field Epidemiology Training Programme pyramid. 32 At the inception of the programme, the MoHSS had no field epidemiologists or any staff with a related discipline. There was no domestic training programme in field epidemiology. Field epidemiology functions were performed on an ad hoc basis by clinicians (doctors and nurses) who had no or limited training. We describe NamFELTP in its first 7 years, assess its impact on the public health workforce, and provide recommendations for improving the programme and public health system. NamFETP programme description The NamFELTP is hosted by the MoHSS and implements the programme with the University of Namibia (UNAM) School of Public Health. Trainees of the 2-year advanced programme matriculate in an MSc Applied Field Epidemiology degree programme in UNAM, while those that graduate from the 3-month front-line programme receive a certificate of participation. Trainees come from MoHSS, Agriculture, Water and Forestry (MAWF); and Defence, and Namibia Institute of Pathology to ensure a multidisciplinary and a one-health approach towards public health preparedness and response. The one-health concept is a collaborative, multisectoral and transdisciplinary approach with the goal of achieving optimal health outcomes by recognising the interconnection between human health, animal health and their shared environment. 9 Using a one-health framework where humans, animals and the environment are viewed as interconnected, the NamFELTP recruited people across these related fields and trained them to jointly respond to public health emergencies. The selection process for the front-line tier is done though nominations of qualified participants from their various ministries and agencies. The UNAM admission process selects candidates for the advanced programme. A Resident Advisor with expertise in field epidemiology led the advanced programme during 2014–2019, with support from CDC, UNAM faculty and external consultants. NamFELTP modified the standard US-CDC EIS training materials to incorporate the local context for the trainees. The curriculum consists of 25% didactic classroom sessions and 75% field placement as in other FELTPs. 10 11 Didactic training modules cover principles of field epidemiology, data management, biostatistics, public health surveillance, epidemiology of priority public health conditions and NCDs, leadership and management, advanced epidemiological methods, preventive effectiveness (health economics), scientific communication, mentorship and research methods. The field placement outputs are outbreak investigation and response, data quality audits, secondary data analysis of surveillance data, surveillance system evaluation and epidemiological studies. The trainees conduct these field placement activities in their assigned districts and regions by collecting the relevant data, analysing and producing field output reports under the supervision of their mentors with the guidance of the resident advisor. The mentor to resident mentee ratio in the advance programme is one mentor to three mentees while that of the front line is a one mentor to five or six mentees. Trainees are expected to present or disseminate their findings at international scientific conferences as part of their graduation requirements. Approach This is a description of NamFELTP from its inception in January 2012 through January 2019. NamFELTP implemented two of the three FELTP tiers; front line started in 2012 and was previously called the short course, and Advanced FELTP started in 2014 ( figure 1 ). All cohorts enrolled within the 7 years' period were reviewed. We conducted records review and described the programme implementation and evaluated the programme deliverables. Evaluation of programme deliverables Disease outbreak investigations and response reports were reviewed to determine the number and types of outbreaks investigated. We reviewed surveillance data analysis reports and surveillance system evaluation reports to determine any changes in key surveillance indicators such as data quality and timeliness of reporting. Epidemiological investigations and other field projects conducted were also reviewed to determine their impact on the health system. We assessed the trainee's presentation of their field project outputs made at international scientific conferences and also the current positions of the graduates as a result of the training acquired. Data were analysed and summarised into frequencies, proportions and rates, and presented in tables. A map representing the geographical distribution of the NamFELTP workforce was produced using QGIS software V.3.2. Per the International Health Regulation (IHR) standard of having one trained epidemiologist per 200 000 population, 12 13 we calculated the number of Advanced level trainees trained per 200 000 population by region using the 2018 regional population estimates. 14 Enrolment of trainees into the front-line FELTP In total, NamFELTP trained 161 front-line health workers from all 13 regions and all 34 districts of Namibia with no drop-outs ( figure 2 ). Health professionals trained in the Front-line Programme were primarily female (63%) and included medical, veterinary, laboratory and environmental health practitioners ( table 1 ). A majority were healthcare workers (71%) and environmental health practitioners (24%) with few animal health workers (5%). The capitol region of Khomas has the highest number (20). In Namibia, the Front-line Programme trained 5–20 staff per region. Figure 2 Number and location of advanced and frontl-ine FELTP graduates by region, Namibia, 2020. 33 FELTP, Field Epidemiology and Laboratory Training Programmes. Table 1 Characteristics of front-line and advanced FELTP trainees in Namibia, 2012–2019 Characteristics Population No enrolled (%) No enrolled per 200 000 population No completed (%) No completed per 200 000 population Front-lineFELTP Sex Male – 59 (37) 59 (37) Female – 102 (63) 102 (63) Professional category Healthcare workers (medical doctors, nurses and medical technologist) – 114 (71) 114 (71) Environmental health practitioners – 38 (24) 38 (24) Animal health workers (veterinarians and veterinary technicians) – 8 (5) 8 (5) Other (statistician) – 1 (0.6) 1 (0.6) Region Khomas 447 600 20 (12) 8.9 20 (12) 8.9 Hardap 90 300 7 (4) 15.5 7 (4) 15.5 Karas 89 200 5 (3) 11.2 5 (3) 11.2 Kavango 243 900 16 (10) 13.1 16 (10) 13.1 Kunene 102 500 9 (6) 17.6 9 (6) 17.6 Ohangwena 260 200 15 (9) 11.5 15 (9) 11.5 Erongo 195 700 15 (9) 15.3 15 (9) 15.3 Omaheke 75 700 5 (3) 13.2 5 (3) 13.2 Omusati 252 700 14 (9) 11.1 14 (9) 11.1 Oshana 194 600 13 (8) 13.4 13 (8) 13.4 Oshikoto 200 700 12 (7) 12.0 12 (7) 12.0 Otjozondjupa 158 200 11 (7) 13.9 11 (7) 13.9 Zambezi 102 300 7 (4) 13.7 7 (4) 13.7 National level 2 413 600 12 (7) – 12 (7) – Total 161 161 Advanced feltp Sex Female – 29 (72) 22 (79) Male – 11 (28) 6 (21) Professional categories Healthcare workers (medical doctors, nurses and medical technologist) – 25 (62) 19 (68) Environmental health practitioners – 8 (20) 4 (14) Animal health workers (veterinarians and veterinary technicians) – 6 (15) 5 (18) Others (demographer) – 1 (3) Region Khomas 447 600 18 (45) 8 10 (36) 4.5 Hardap 90 300 4 (10) 8.9 4 (14) 8.9 Karas 89 200 2 (5) 4.5 2 (7) 4.5 Kavango 243 900 4 (10) 3.3 2 (7) 1.6 Kunene 102 500 2 (5) 3.9 2 (7) 3.9 Ohangwena 260 200 4 (10) 3.1 3 (1) 2.3 Erongo 195 700 1 (3) 1 1 (4) 1 Omaheke 75 700 0 (0) 0 0 (0) 0 Omusati 252 700 0 (0) 0 0 (0) 0 Oshana 194 600 4 (10) 4.1 4 (14) 4.1 Oshikoto 200 700 0 (0) 0 0 (0) 0 Otjozondjupa 158 200 0 (0) 0 0 (0) 0 Zambezi 102 300 1 (3) 2 0 (0) 0 Total 2 413 600 40 28 FELTP, Field Epidemiology and Laboratory Training Programmes. Enrolment of trainees into the advanced FELTP A total of 40 trainees from 9 regions have enrolled in the Advanced FELTP ( figure 2 ). The WHO-IHR standard for number of trained epidemiologists is one field epidemiologist per 200 000 population. For Namibia nationally, that will require 13 trained epidemiologists. Namibia's Advanced FELTP programme enrolled six cohorts during 2014–2019 (range: 5–9 trainees per cohort) and graduated 28. The last two cohorts have 11 trainees in training. The majority of NamFELTP trainees were females. NamFELTP trainees were healthcare workers, primarily nurses, as well as three medical doctors (enrolled in the 2017 and 2019 cohorts), followed by environmental health practitioners, animal health workers and a demographer ( table 1 ). The highest proportion, 45% (18/40) were from the Khomas region, which is the capital region, location of the UNAM and national government ministries, including MoHSS and MAWF headquarters. This is logical; most of the public health workforce in the country is in the Khomas region. Other regions have enrolled trainees in an ad hoc fashion based on the interest of trainee and willingness of regional leadership to release staff for training. Regional distribution of FELTP trainees Both the front-line and advanced FELTP trainees are centralised within the Khomas region which is the national capital. All regions have front-line trainees; however, for the advanced programme, four regions (Omaheke, Omusati, Oshikoto and Otjozondjupa) have not enrolled any trainees ( figure 2 ). Disease outbreak investigation and response NamFELTP advanced trainees investigated 35 disease outbreaks in 5 years. Outbreaks dealt with cholera, measles, rubella, neonatal tetanus, malaria, schistosomiasis 15 and hepatitis E, and zoonotic diseases such as rabies in domestic dogs, foot and mouth disease in cattle, anthrax among wildlife and domestic animals (cattle and goats), and Crimean-Congo haemorrhagic fever (CCHF) in humans ( table 2 ). CCHF, a zoonotic viral disease caused by tick bites, is endemic in Namibia. This is an important disease because it affects numerous agricultural workers. Three outbreaks were reported during 2017—2018 with four cases among agricultural workers, including two fatalities. NamFELTP alumni and trainees participated in all three CCHF outbreak investigations and provided health education, identified and traced contacts of infected persons, conducted environmental assessments and administered acaricide to animals to kill ticks in agricultural areas. Table 2 Outbreak investigations, surveillance data analyses and evaluations conducted in Namibia by advanced NamFELTP trainees, 2014–2019 Characteristic No Percentage Outbreak investigations and response Zoonotic diseases (foot and mouth, rabies, anthrax, CCHF, Rift Valley fever) 13 37 Vaccine preventable diseases (measles, rubella, chickenpox, tetanus, meningitis) 10 29 Food-waterborne diseases (cholera, schistosomiasis, acute watery diarrhoea, hepatitis E virus) 8 23 Malaria 3 9 H1N1 influenza 1 3 Subtotal 35 100 Surveillance data analyses and surveillance system evaluation TB/HIV 17 33 Vaccine preventable disease (measles, rubella, chicken pox, tetanus, meningitis) 11 22 Zoonotic diseases (foot and mouth, rabies, brucellosis, bovine spongiform encephalitis) 7 14 Food-waterborne diseases (cholera, schistosomiasis, acute watery diarrhoea) 5 10 Malaria 5 10 Maternal and Neonatal Health 3 6 Others (accident and injuries, mental health, clubfoot) 3 6 Subtotal 51 100 Epidemiological studies Non-communicable diseases (hypertension, diabetes, breast, cervical and prostate cancers) 8 26 Zoonotic diseases (rabies, porcine cysticercosis, foot and mouth) 6 19 Others (food safety, patient referral patterns, hepatitis E virus) 4 13 Maternal and child health (adverse pregnancy outcomes, neonatal mortality, stillbirth and childhood undermalnutrition) 4 13 TB 4 13 Vaccine preventable diseases (measles, hepatitis B) 2 7 HIV 2 7 Malaria 1 3 Subtotal 31 100 Total 117 NamFELTP, Namibia Field Epidemiology and Laboratory Training Programme; TB, tuberculosis. In October 2017, the Ministry of Environment and Tourism reported a suspected anthrax outbreak in the Kavango region where hippopotamus and buffalo carcasses were characteristic of anthrax. During October—December 2017, 155 hippopotamuses, 86 buffalos and 2 impalas died. The response was conducted using a one-health approach recognising common threats to animal and human health. 16 NamFELTP trainees determined how many humans were exposed to anthrax, administered postexposure prophylaxis (PEP) and delivered health education to community members and healthcare workers. NamFELTP trainees and alumni identified 142 exposed persons who handled or ate hippopotamus meat and provided PEP. No subsequent human cases of anthrax during this outbreak in wildlife, suggesting success of the timely and effective public health intervention. NamFELTP trainees also confirmed multiple suspected measles outbreaks as rubella which lead to the introduction of the rubella vaccination on the country. Surveillance data analysis and surveillance system evaluation and other field projects Trainees conducted 51 surveillance data analyses and surveillance system evaluations and 31 epidemiological studies on priority public health conditions and other disease programmes ( table 2 ). The surveillance data analysis and surveillance system evaluations showed enhanced data quality and improved timeliness in reporting of surveillance data over the period. The planned epidemiological studies have resulted in policy changes such as the rabies serosurvey among vaccinated domestic dogs showed lower immunogenicity among dogs who were vaccinated only once a year and this resulted in a twice yearly antirabies vaccination. Trainees of Advanced FELTP have lead or coauthored three peer-review manuscripts. 15–17 NamFELTP graduates currently hold key positions in MoHSS and MAWF. These include Epidemiology Division head at MoHSS and Chief Veterinary Epidemiologist at MAWF. Attendance at international scientific conferences Training Programmes in Epidemiology and Public Health Interventions Network (TEPHINET) is a global network of FETPs, while African Field Epidemiology Network (AFENET) is a networking alliance of African Field Epidemiology and Laboratory Training Programmes (FELTPs). The major scientific conferences for FELTP programmes are TEPHINET Global Scientific Conference and AFENET Regional Scientific Conference. FELTP trainees present their scientific work at these international conferences as part of field placement competencies to demonstrate their ability to present at international fora. Since 2012, NamFELTP trainees have attended and presented at six International Conferences, including two TEPHINET and three AFENET conferences. NamFELTP's 35 trainees presented 59 abstracts, including 17 (29%) oral and 42 (71%) posters, with three award winning poster presentations in 2016, 2017 and 2018. Programme impact Local capacity is crucial for effective and timely public health responses. District health officers are often the first people called during a public health emergency and are prioritised for front-line FETP training. 18 NamFELTP training resulted in producing a significant increase in skilled workforce, 161 front-line field epidemiologists and 28 Advanced field epidemiologists, as compared with zero at the inception of the training programme. This achievement has resulted in timely detection of outbreaks. The improved epidemiological capacity at district and regional levels enhances data quality and timeliness of reporting. Detecting and responding to disease outbreaks at lower levels in a timely manner helps prevent spread of outbreak. After the Ebola outbreak in 2014, West African countries including Liberia, Côte d'Ivoire, Senegal, and Togo implemented front-line FELTP to increase and improve their epidemiological capacity at the lower levels to enhance data quality and timeliness of reporting. 18 19 Strengthening the public health workforce in low-income and middle- income countries is crucial in ensuring that countries have the needed capacity to prepare and respond to public health emergencies. Advanced NamFELTP trained medical, veterinary, laboratory and environmental health practitioners in a comprehensive approach to surveillance and outbreak response within the one-health approach. This one-health approach embedded in the NamFELTP yielded a remarkable response to major zoonotic disease outbreaks such as recurring CCHF outbreaks and the Anthrax outbreak among hippopotamus in Bwabwata National Park, Namibia. 16 However, only three physicians (7.5%) have been enrolled, in contrast to other FELTP programmes where many trainees are physicians. A possible reason for low physician enrolment is the need for physicians to deliver clinical services since training for medical doctors within Namibia began in 2010; prior to that date all physicians trained outside the country. 20 21 Enrolling more physician into the programme is crucial to ensure the programme viability. According to the Global FETP Roadmap, evidence justifying the target of 1 trained field epidemiologist/200 000 population is thin and serves as a general guide to the sufficient number of field epidemiologists in a country. A few countries use different metrics such as one field epidemiologist per district or per national programme and can accommodate their demographics, health system and geopolitical structures, and population needs (eg, percent rural vs urban population). 22 In the case of Namibia with a population of 2.5 million, although 28 Advanced FETP trainees have matriculated, this number and geographic placement of epidemiologists is insufficient to fill key leadership and strategic roles in the country's public health system for several reasons including inadequate number of epidemiologist for the huge land mass, the need of several epidemiologist to manage the varied disease programmes at the national level and limited expertise for the control of emerging and re-emerging disease considering the countries disease profile. Expanding the programme to include provincial/regional staff and other relevant agencies by giving quota to these regions and sectors will be critical to ensure a well-distributed workforce to respond adequately to public health emergencies. NamFELTP remains the country's premier public health training programme and graduates must support public health efforts beyond field epidemiology. In many cases, NamFELTP graduates return to clinical healthcare after their training rather than contributing to the public health sector because there is currently no position as epidemiologists in the Human Resource staff establishment of the Public Service Commission for Ministry of Health, hence no public health career path for the graduates. Because of these limitations, there are no full-time employees in MoHSS positions classified as epidemiologists. This situation is similar to other African countries as indicated in the third Ministerial Round Table at the seventh AFENET Scientific Conference in Maputo, Mozambique in 2018, where only three countries (Liberia, Tanzania and Zimbabwe) out of 12 African countries evaluated had a formal policy on career path for field epidemiology graduates. 23 Four regions in Namibia have no trained field epidemiologists. Some graduates left government service to work with private and non-governmental organisations. The critical shortage of available public health workers, especially those with specialised training in field epidemiology, diminishes preparedness and response throughout the country. Moreover, Namibia covers a vast landmass with a dispersed, low-density population. There is a need for field epidemiologists to work in remote geographical areas to detect disease outbreaks at their origin rather than relying on a disproportionate number of Advanced graduates located in the Khomas region. Surveillance systems and responses to public health events have improved because of NamFELTP. Trainees and alumni have responded in a timely manner to 35 outbreak investigations. Prompt public health response by the FELTP during the 2017 anthrax outbreak among wildlife in Bwabwata National Park, 24 led to zero human cases compared with similar outbreaks among hippopotamuses in Zambia 25 26 and Zimbabwe 27 which had multiple human cases and deaths. The inclusion of rubella vaccination as part of nationwide childhood routine immunisations in 2017 28 was a result of the multiple confirmed rubella outbreaks investigated. NamFELTP trainees conduct secondary surveillance data analyses periodically to determine disease trends and to evaluate control and prevention measures to support planning and decision making. Surveillance system evaluations identify gaps in the surveillance system and provide recommendations to strengthen the surveillance system. As in other FELTP programmes, such epidemiological analyses and evaluations help improve surveillance system for both human and animal health. 29 30 A rabies seroprevalence survey conducted among dogs in the Ohangwena region identified lower immunogenicity of rabies in dogs who were vaccinated once a year compared with twice a year. 31 These data led to increasing national rabies vaccination campaigns to twice a year. Conclusion In summary, NamFELTP trained 161 front-line and 28 advanced graduates who are contributing to the human and animal health systems. NamFELTP has greatly improved critical outcomes in the public health system, including enhancing public health surveillance systems to respond effectively and rapidly to public health emergencies from previously where there was a weak surveillance system, no surveillance system evaluation and data analysis conducted, and no capacity to respond to public health emergencies in an effective and timely manner. CDC, WHO, AFENET and TEPHINET have contributed funds and technical assistance for NamFELTP. Government of the Republic of Namibia (GRN) ownership is key for the success and sustainability of the programme. Despite the crucial role and functions that field epidemiologists and other public health workers play, there are no posts for epidemiologists within the GRN system. Consequently, it is necessary for the Public Service Commission to adapt job descriptions and requirements to establish epidemiologist positions. These steps can provide Namibia with the skilled epidemiological workforce it requires for sustained health security. The training and mentorship in NamFELTP should continue to fill key leadership and strategic roles in the public health system. We recommend that the MoHSS and MAWF purposefully recruit trainees from the four regions with no trainees for the advanced programme to ensure equitable distribution of public health workforce for effective and timely response to public health threats. Data availability statement Data are available upon reasonable request. Ethics statements Patient consent for publication Not required. Ethics approval This article is a document review of the NamFELTP programme efforts in field epidemiology and does not include human subjects. Programme documents required no ethical approval to access; however, administrative approval was obtained from the MoHSS. Confidentiality of past and current NamFELTP trainees was maintained. Patient consent for publication Not required. Ethics approval This article is a document review of the NamFELTP programme efforts in field epidemiology and does not include human subjects. Programme documents required no ethical approval to access; however, administrative approval was obtained from the MoHSS. Confidentiality of past and current NamFELTP trainees was maintained.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824258/

Genome Sequence of Bacillus anthracis Strain Stendal, Isolated from an Anthrax Outbreak in Cattle in Germany

In July 2012, an anthrax outbreak occurred among cattle in northern Germany resulting in ten losses. Here, we report the draft genome sequence of Bacillus anthracis strain Stendal, isolated from one of the diseased cows. GENOME ANNOUNCEMENT The zoonotic disease anthrax caused by Bacillus anthracis is a rare disease in Germany, with only three reported outbreaks between 2009 and the end of 2015 ( 1 , 2 ). An outbreak early in July 2012 occurred among livestock in the county of Stendal (federal state of Saxony-Anhalt) resulting in livestock loss of 10 out of 55 cows ( 2 ). As a consequence, 50 people received postexposure prophylaxis with antibiotic therapy, and the affected pasture was decontaminated with formalin. This outbreak received considerable media attention because one of the diseased cows had died at the shores of the river Elbe and was swept away by the current. Strain Stendal 1 was isolated from blood culture of one of the diseased cows, and the presence of both virulence plasmids pXO1 and pXO2 of B. anthracis was confirmed by real-time PCR assays ( 3 , 4 ). Canonical single-nucleotide polymorphism (canSNP)–based genotyping ( 5 ) revealed that the outbreak strain belonged to the branch A.Br.001/002, which has previously been isolated in Germany and its neighboring countries. Indeed, it appears that this canSNP-genotype of B. anthracis is predominant in Germany, Belgium, the Netherlands, Denmark, and Norway ( 6 ). Whole-genome shotgun (WGS) sequencing of B. anthracis Stendal was performed by Ion Torrent sequencing technology (Ion Torrent Systems Inc., USA). For the WGS library, 1,705,145 reads with a total of 460 Mb were generated. Bowtie-2 ( 7 ) was used for mapping to the Ames Ancestor chromosome, plasmids pXO1 and pXO2 (NC_007530.2, NC_007322.2, AE017335.3), and the sequence was amended by multilocus variable-number of tandem-repeat analysis data according to ( 8 ), where necessary. The G+C content was calculated using an in-house Python script. The total length of the genome shotgun sequence of B. anthracis Stendal was 5,227,419 bp with a 63-fold coverage for the chromosome (137-fold for pXO1 and 103-fold for pXO2), and the mean G+C content was 35.5%. For initial annotation, the assembled contigs were submitted to the RAST annotation pipeline ( 9 ). The B. anthracis Stendal draft genome encodes 5,639 putative coding sequences. Annotation of the genome identified 11 copies of genes for the 16S rRNA, the 5S rRNA, and the 23S rRNA; 61 tRNA loci were identified. The B. anthracis Stendal genome represents a third major genotype, A.Br.001/002, of this bacterium from rare outbreaks in Germany in this millennium. The other two were strain UR-1, canSNP-group A.Br.008/011, from a diseased heroin consumer in Bavaria ( 10 ), and strain BF-1, canSNP-group B.Br.001/002, from a dead cow of the Bavarian Alps region ( 1 ). Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession numbers CP014177 (pXO1), CP014178 (pXO2), and CP014179 (chromosome). The versions described in this paper are the first versions. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession numbers CP014177 (pXO1), CP014178 (pXO2), and CP014179 (chromosome). The versions described in this paper are the first versions.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10975237/

State-of-the-Art Review on Inhalable Lipid and Polymer Nanocarriers: Design and Development Perspectives

Nowadays, the interest in research towards the local administration of drugs via the inhalation route is growing as it enables the direct targeting of the lung tissue, at the same time reducing systemic side effects. This is of great significance in the era of nucleic acid therapeutics and personalized medicine for the local treatment of severe lung diseases. However, the success of any inhalation therapy is driven by a delicate interplay of factors, such as the physiochemical profile of the payload, formulation, inhalation device, aerodynamic properties, and interaction with the lung fluids. The development of drug delivery systems tailored to the needs of this administration route is central to its success and to revolutionize the treatment of respiratory diseases. With this review, we aim to provide an up-to-date overview of advances in the development of nanoparticulate carriers for drug delivery to the lung tissue, with special regard concerning lipid and polymer-based nanocarriers (NCs). Starting from the biological barriers that the anatomical structure of the lung imposes, and that need to be overcome, the current strategies to achieve efficient lung delivery and the best support for the success of NCs for inhalation are highlighted. 1. Introduction The pulmonary route is currently regarded as the administration route of choice for the local treatment of severe lung diseases since: (i) it directly acts on the target and (ii) achieves high local drug availability, while limiting systemic toxicity. Meanwhile, lungs may represent the port of entry for the needle-free systemic administration of macromolecules as well as for small molecules, to obtain the rapid onset of action, low metabolism and high bioavailability [ 1 ]. Nevertheless, some disadvantages still exist and are mainly related to the need of opportunely engineered inhalable drug particles able to maximize the amount of drug deposited and/or absorbed in the lung, while shielding its unfavorable interactions with the pulmonary environment. Although carrier-free drug particles raise fewer safety concerns for inhalation, biocompatible materials, such as lipids and biodegradable polymers, may provide the efficient protection and delivery of a variety of therapeutics, spanning from antibiotics, headed for resistant biofilm bacteria, and gene materials, directed inside airway epithelial cells. Thus, a growing interest has been especially devoted to the application of nanoparticulate carriers for inhalation. Recent advances in materials, particle engineering techniques, and inhalation devices enabled the formulation of nanocarriers (NCs) for the pulmonary delivery of drugs with an improved ability to land in the lungs, to overcome lung barriers and to fulfill specific therapeutic needs [ 2 , 3 ]. Indeed, the peculiar properties of NCs, including their small size, confer them with the special ability to surpass the mucus barrier lining lung epithelium and to gain access to the cell target. Based on the constituents more widely used, the carriers can be classified as organic (i.e., polymers, lipids) and inorganic (i.e., metals, quantum dots, calcium phosphate, oxide, and silica) [ 4 ]. Despite being promising, the design and development of NCs for inhalation is not straightforward. The efficient lung deposition of inhaled drugs is a very complex task, resulting from the interplay of several factors. It depends not only on NCs' size and morphology, but also on the inhalation device, the breathing pattern, and even the lung anatomy of the individual [ 5 , 6 ]. Acknowledging that efficient deposition in the lungs is an essential prerequisite for successful pulmonary drug delivery, there is also an increasing need to control what happens after the drug particles have landed. After deposition in the lung, the success of any inhalation therapy will strongly depend upon (i) drug permeation through airway mucus; (ii) the drug interaction with the cell target; and (iii) macrophage clearance escape. The drug's capacity to infiltrate bacterial biofilm is especially crucial in the context of antimicrobials [ 7 ]. The aim of this review is to provide an overview on recent advances in developing NCs for drug inhalation. After a brief description of the main factors governing the deposition of drugs in the lungs and their fate after landing, the advantages of engineered NCs for inhalation are described. Then, an update on inhalable biocompatible lipids and polymeric and hybrid NCs is presented. Despite their intriguing properties, some of the most studied inorganic carriers for pulmonary applications have been associated with the largest records of pulmonary toxicity. For this reason, they will be not the object of this review [ 8 ]. An exhaustive review on the topic highlighting the potential for the future prospective of inorganic materials for inhalation are reported [ 8 , 9 ]. 2. Factors Governing Drug Deposition in the Lung A better understanding of the mechanisms and factors determining aerosol deposition is of upmost importance to provide for the maximum and reproducible amounts of drug deposited in the desired region of the lungs. As well known, three primary mechanisms account for drug deposition along the respiratory tract, namely inertial impaction, gravitational sedimentation, and Brownian diffusion [ 10 ]. They are complemented by electrostatic attractions and particle interception (in case of elongated particles) and are likely affected by the air flow turbulence. The particle mass median aerodynamic diameter (MMAD), resulting from the size, density, and shape of the particle, critically influences the mechanism and the site of drug deposition [ 11 , 12 ]. In detail, particles with an MMAD larger than 10 µm are mainly deposited by inertial impaction in the extra-thoracic region and in the bifurcations. Indeed, particles become more and more inert with their growing size and their ability to follow the respiratory flow is reduced proportionally to the flow rate. If pharmaceutical aerosols penetrate the small conducting airways, the dominant deposition mechanism is gravitational sedimentation (i.e., particles fall under gravity onto the airway walls). Both impaction and sedimentation cause the deposition of particles larger than 3–5 µm in the smaller airways (i.e., bronchus, bronchioles) before reaching the alveoli. Differently, particles with an MMAD in the 1–2 µm range will likely deposit into the capillary-rich alveolar airspaces, which represent the target for systemic drug delivery through the lungs. Here, deposition is mainly influenced by Brownian diffusion, which dominates for particles with diameters of less than 1 µm. Particles with an MMAD between 0.1 and 1 μm, such as NCs, are mostly exhaled, even though ultrafine particles (lower than 100 nm) may paradoxically deposit in the respiratory tract taking advantage of their random Brownian motion. Inhalation devices significantly influence the pattern of drug deposition within the lungs [ 10 ]. The increasing research interest towards inhaled therapeutics has recently led to tremendous innovations in designing inhalation devices able to ensure a high aerosolization performance, consistent therapeutic efficacy, and satisfactory patient adherence to treatment [ 13 , 14 ]. Vibrating mesh and software technologies have resulted in nebulizers having highly accurate dosing and portability. On the other hand, advanced particle engineering techniques harnessed dry powder inhalers (DPIs) for also delivering high-dose drugs, such as antibiotics. Though new propellant systems attempt to improve the performance of aerosols delivered by pressurised metered dose inhalers (pMDIs), drug-propellant incompatibility and delivering high-dose drugs are still important technical constraints. Detailed reviews have recently summarized all the advantages and challenges of the different inhalation devices [ 13 , 15 ]. Inhalation flow and the velocity at which aerosol particles are emitted from the device and travel through the airways also strongly impact pulmonary deposition patterns [ 15 , 16 ]. As a rule, faster inhalation increases the inertial impaction of aerosols in the oropharynx and in bifurcations in the large central airways, whereas slower inhalation results in more peripheral deposition patterns. Nevertheless, when using a low-resistance passive DPI, slow inhalation may be insufficient to disaggregate the inhaled powder particles, and can therefore limit lung deposition. Thus, lung deposition from DPIs paradoxically increases as inspiratory flow increases. Overall, an optimal design strategy for inhaled drugs should consider both the inhalation device and the inhalation flow rate [ 16 , 17 ]. 3. Overcoming Lung Barriers through Tailored Nanocarriers Predicting the fate of inhaled drugs after landing in the lung is key to formulation development, but it still represents a relatively complex issue. An in-depth knowledge of the barriers imposed by the route of administration appears mandatory to improve the lung availability of drugs and, thus, the therapeutic outcome. The lung barriers can be grouped into two main categories: the non-cellular and cellular barriers ( Figure 1 ) [ 18 , 19 , 20 ]. Lung-lining fluids, such as airway mucus and lung surfactant, represent the main non-cellular barriers. They strongly affect the behavior of drug particles in the lung determining their solubility, diffusion, permeation, and, in so doing, drug bioavailability. Underneath the lung-lining fluids, the human airway epithelium represents the main cell barrier to drug transport towards its intracellular target and systemic absorption. Cell barriers also comprise the immune system's cells, such as macrophages, which can uptake inhaled drug particles, reducing their availability and effectiveness in the lung [ 21 ]. For some respiratory diseases, other barriers need to be considered, such as the bacterial biofilm in lung infections. In this case, the effectiveness of inhaled antimicrobials can be dramatically reduced by the interactions with the component of the biofilm, such as polysaccharides and proteins, leading to the drug resistance mechanism (i.e., antibiotic resistance) [ 22 , 23 ]. The success of NCs for drug inhalation depends upon the possibility of tailoring the composition, size, surface charge, and morphology of the particle to overcome the barrier imposed by the lung and to gain access to the specific target [ 15 ]. 3.1. Non-Cellular Barriers The human lung epithelium is covered by the lung-lining fluid (LLF), a primary and heterogeneous constituent of the pulmonary host defense system. The two essential elements of the LLF are the airway surface liquid (ASL), a mucus gel–aqueous sol complex lining conductive airways, and the alveolar subphase fluid (AVSF), at the alveolar level. Airway mucus is one of the most investigated non-cellular barriers affecting in situ permanence and the extent of absorption of the inhaled drug particles [ 24 ]. Highly cross-linked mucin chains create a dense porous structure, with the thickness and porosity being variable for the lung area and pathological conditions. Two major mechanisms may stop particles from readily diffusing through mucus gel, which are "size filtering" through the mucus meshes and "interaction filtering" [ 24 ]. Insoluble particles might be filtered by the mucus gel layer if bigger than the mesh-spacing of the mucin network, or establish hydrophobic, electrostatic, and/or hydrogen bonding interactions with the negatively charged mucin chains. Trapped particles are moved toward the pharynx, and ultimately to the gastrointestinal tract, by the upward movement of mucus generated by the cilia beating (i.e., mucociliary clearance) [ 25 ]. NCs' properties, including their size, charge, and hydrophobicity, are crucial in determining drug diffusion through mucus [ 26 , 27 ]. The pore size of the mucus gel layer ranges from 100 to 200 nm, suggesting that only NCs in this size range could potentially penetrate. Nevertheless, significant healthy-to-diseased and/or patient-to-patient variations are reported. It is nowadays acknowledged that a sufficiently hydrophilic and uncharged surface may minimize the adhesive interactions between mucin and the NC, thus improving their mucus-penetrating ability [ 26 , 28 ]. The pulmonary fluid layer reduces in thickness throughout the airways, forming single droplets on top of the limited ciliated cells of the lower bronchi and an extremely thin layer of surfactant in the alveoli. The pulmonary surfactant layer prevents alveolar collapse during expiration and is composed of approximately 90% lipids, mainly dipalmitoylphosphatidylcholine (DPPC), and 10% proteins (i.e., surfactant protein A, B, C, and D) [ 29 ]. Upon contact with the surfactant, larger sized particles are displaced from the airspace to the hypo-phase due to wetting forces, a phenomenon that probably also occurs with nano-sized particles [ 30 ]. In the hypo-phase, the particles may interact with surfactant proteins or may be taken up by alveolar macrophages [ 30 , 31 ]. Many of the literature findings suggest that, depending on their composition and size, inhaled particles may also interfere with the function of the pulmonary surfactant, thus hindering its physiological and essential role in the lung [ 23 , 24 ]. This issue should be properly considered when designing novel inhaled nanomedicines [ 32 , 33 ]. 3.2. Lung Epithelial Barrier The interactions of inhaled particles with the human airway epithelial barrier also play a crucial role in determining the availability of drugs in the lung. The lungs are made by many different types of cells, of which the main type are epithelial cells. The thickness and the properties of the lung's epithelial cell layer vary from a columnar cell monolayer with a thickness of 60 µm in the up airways (i.e., bronchi) to a broad cell monolayer 0.2 µm thick in the alveoli. The alveolar epithelial layer separates the lumen airspace from the pulmonary aqueous interstitial compartment, which is composed by different elements, such as the lymphatic vessel, collagen, and fiber [ 5 , 34 , 35 ]. The uptake of the inhaled drug by the lung's epithelial cells is influenced by the lung physiopathology [ 34 ]. As a matter of fact, while lipophilic drugs are thought to be rapidly absorbed by passive transcellular diffusion through epithelial cells, small hydrophilic compounds likely diffuse across the epithelium through aqueous pores in intercellular gap junctions [ 36 ]. Tight junctions, efflux proteins, and cellular enzymes play an important role as barriers in the absorption process as well [ 36 , 37 ]. Of note, tight junctions, mainly located on the apical side of the cell layer, are influenced by pathological conditions [ 36 ]. Indeed, the epithelial barrier function may decrease due to the disruption of tight junctions in asthma and chronic obstructive pulmonary diseases (COPD) [ 38 , 39 ], while their function may be enhanced in cystic fibrosis (CF) airway epithelial cells [ 40 , 41 ]. To improve drug accumulation at the cell level, different formulation approaches can be pursued. Drug interactions with cells can be regulated by simply using carriers with adequate size and surface properties, such as charge, hydrophilicity, and a shielding cloud [ 42 , 43 ]. However, efficient drug delivery to the final intracellular target is still challenging [ 44 , 45 ]. This is the case of emerging nucleic acid-based therapeutics (DNA, siRNA, and oligonucleotides), which demand for adequately engineered nanoparticulate systems, comprising not only of biodegradable polymers able to compact, to protect, and to release the entrapped nucleic acid, but also a biomimetic shell containing agents able to facilitate its endo-lysosomal escape (e.g., fusogenic peptides or lipids, endosome destabilizing polymers) [ 45 , 46 ]. The decoration of the carrier surface with different functional motifs can also be attempted to build up actively targeted constructs [ 47 , 48 , 49 ]. In the light of these findings, advanced in vitro models are paramount to design and to develop effective inhaled drugs. During recent years, many cell culture models of nasal, bronchial, and alveolar barriers have been developed, varying from 2D monolayer cultures to advanced 3D co-cultures, with the aim to better resemble what happens in vivo and to provide further insight into cellular responses or interactions with inhaled drug particles [ 50 , 51 , 52 , 53 , 54 ]. The direct aerosolization of the drug formulation on the cells through appropriately designed exposure systems, such as the VITROCELL ® Cloud or the PreciseInhale ® , is feasible to further increase the significance of the biological results [ 50 , 55 ]. Finally, lung-on-chips are emerging to emulate both the morphological features and biological functionality of the airway barrier with the ability to integrate respiratory motions and ensuing tissue strains [ 54 , 56 , 57 ]. 3.3. Macrophage-Mediated Clearance Macrophages are a type of lung-resident immune cells, designed to eliminate any foreign material that reaches the pulmonary environment [ 58 , 59 , 60 ]. They represent a unique cell population in the peripheral lungs that promptly responds to any airborne irritant or microbe [ 58 ]. Drug interactions with macrophages and the subsequent uptake can be enhanced or reduced by adequately tuning the properties of inhaled drug particles [ 61 ]. The size of particulate carrier systems significantly influences particle–macrophage interactions, as widely discussed in the literature [ 62 , 63 ]. Particles with a 1–5 µm size are taken up by alveolar macrophages, both in vitro and in vivo, to a greater extent compared to particles that are smaller or larger [ 64 ]. Also, the mechanism of particle uptake changes as a function of the carrier size. Micron-sized inhaled particles (1–5 µm) are taken up by alveolar macrophages mainly via active phagocytosis, whereas this is unlikely for nanosized particle [ 63 ]. Depending on their size, NCs enter alveolar macrophages by pathways other than phagocytosis: while NCs bigger than 0.2 µm are probably internalized via pinocytosis, smaller carriers (less than 150 nm) can be internalized via calveolae (50–100 nm) or clathrin-mediated (100–120 nm) uptake [ 63 , 65 ]. There is clear evidence also of the importance of the shape in governing particle uptake by alveolar macrophages [ 63 ]. When particles are internalized, the macrophage membrane spreads around the engulfed particle, and the progression of internalization is dependent on the contact angle between the particle and the membrane [ 61 ]. It was successfully demonstrated that rod-shaped nanostructured particles can be internalized with high efficiency (90% of uptake in 48 h) without a cytotoxic effect [ 66 ]. In addition, for aspherical particles, the orientation of the particle is crucial. The local shape of the particle at the point of cell attachment, rather than the overall shape, determines cell internalization. For example, if an ellipse-shaped particle is used, a macrophage attached to the pointed end of the ellipse internalizes the NCs in less time than the same attached to a flat region. Spherical particles, in contrast, are symmetrical and thus can be internalized from any point of attachment [ 67 , 68 ]. Similarly, shape-switching elongated particles were quickly engulfed by macrophages once the particles became spherical in shape [ 69 ]. Besides particle size and shape, stiffness is emerging as a design parameter for modulating the interaction of NCs with phagocytic cells [ 70 , 71 ]. As a rule, the increased mechanical robustness and overall stiffness of particles leads to increased phagocytosis. Inspired by blood cell behavior, deformable discoidal polymeric nano-constructs have been especially designed to minimize sequestration by phagocytic cells [ 70 , 72 ]. Tailoring nano-constructs' softness has been demonstrated to be crucial for modulating phagocytic cell sequestration [ 73 ]. By three different shapes (circular, elliptical, and quadrangular), two characteristic sizes, and a Young's modulus varying over two orders of magnitude (from 100 kPa to 10 MPa), professional phagocytic cells were observed to engulf more avidly rigid nano-constructs as compared to soft ones [ 73 ]. 3.4. Bacterial Biofilm In the case of lung infections, the effectiveness of inhaled antimicrobials can be seriously limited by the biofilm-producing capacity of some bacteria. Bacterial biofilm is a community of bacteria that is embedded in a self-produced matrix, the extracellular polymeric substance (EPS), composed of polysaccharides, secreted proteins, and extracellular DNAs [ 74 ]. Biofilm formation boosts antimicrobial resistance through different mechanisms, such as the sequestration and limited drug diffusion and the increase in antimicrobial efflux pump expression [ 75 ]. In particular, the EPS may establish electrostatic/hydrophobic interactions with the antimicrobial drugs, thus limiting drug diffusion towards bacteria and consequent antimicrobial activity [ 75 ]. Drug delivery through engineered NCs has been demonstrated to be a promising approach to assist drug diffusion across biofilm towards the bacterial target. Again, the achievement of this goal strongly depends on particle size and surface properties [ 19 ]. The results achieved on different biofilm bacteria highlighted how the size of the EPS meshes can make the difference, with a size cut-off for the optimal penetration of polymeric NCs into biofilm clusters, independently of bacteria, of around 100–130 nm [ 76 ]. Reduced but effective diffusion across a P. aeruginosa biofilm was found in vitro for liposomes with a diameter of 200 and 300 nm, while no diffusion was revealed for liposomes with size of 1 µm [ 77 ]. Although particle size plays a role in governing particle diffusion through the biofilm, the complex composition of the EPS matrix results also in several electrostatic/hydrophobic interactions between the NC and biofilm. Thus, the engineering of the NC surface represents another crucial step in the design of the inhaled particulate system. Again, surface charge and hydrophilicity play a pivotal role. As a general rule, while negatively charged particles may interact with positively charged polysaccharides, imparting a positive charge may improve the particle's interaction with the negative components of the matrix, such as alginates, proteins, and DNA [ 76 ]. In fact, the mobility of NCs into the biofilm appears increased for particles with a neutral surface, as in the case of particles with polyethylene glycol (PEG) surface modification (PEGylation) [ 76 , 78 ]. Nevertheless, conflicting data are reported in the literature. Positively charged quantum dots are able to penetrate and diffuse across a bacterial biofilm faster and more efficiently than negative and neutral ones [ 79 , 80 ]. Furthermore, some examples of cationic particles that were efficiently diffused and distributed into the bacterial biofilm are reported in the literature [ 79 , 80 ]. In some cases, positively-charged NCs showed enhanced distribution in the bacterial biofilm and consequent improved antimicrobial activity when compared to negatively charged NCs [ 81 ]. Notably, the surface charge may result also in a different location inside the biofilm matrix after diffusion. Actually, charged NCs can be localized close to the bacteria membrane [ 76 ]. The differential localization can be attributed to the hydrophilic nature of the NC surface, and more specifically, the higher hydrophobicity of the particle surface may enhance NC colocalization with bacterial cells within the biofilm [ 82 ]. Based on this principle, different attempts were pursued to provide an NC surface switch to adapt the NC properties to the target (i.e., mucus before and bacterial biofilm after). This is the case of environment-adaptive NCs developed using d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) [ 83 , 84 ]. After PEG-assisted mucus penetration, the enzymatic cleavage of PEG chains generates a lipophilic surface that allows the anchoring of the NC to the biofilm, where it serves as a depot for the prolonged exposure of bacteria to antibiotics [ 83 ]. 3.1. Non-Cellular Barriers The human lung epithelium is covered by the lung-lining fluid (LLF), a primary and heterogeneous constituent of the pulmonary host defense system. The two essential elements of the LLF are the airway surface liquid (ASL), a mucus gel–aqueous sol complex lining conductive airways, and the alveolar subphase fluid (AVSF), at the alveolar level. Airway mucus is one of the most investigated non-cellular barriers affecting in situ permanence and the extent of absorption of the inhaled drug particles [ 24 ]. Highly cross-linked mucin chains create a dense porous structure, with the thickness and porosity being variable for the lung area and pathological conditions. Two major mechanisms may stop particles from readily diffusing through mucus gel, which are "size filtering" through the mucus meshes and "interaction filtering" [ 24 ]. Insoluble particles might be filtered by the mucus gel layer if bigger than the mesh-spacing of the mucin network, or establish hydrophobic, electrostatic, and/or hydrogen bonding interactions with the negatively charged mucin chains. Trapped particles are moved toward the pharynx, and ultimately to the gastrointestinal tract, by the upward movement of mucus generated by the cilia beating (i.e., mucociliary clearance) [ 25 ]. NCs' properties, including their size, charge, and hydrophobicity, are crucial in determining drug diffusion through mucus [ 26 , 27 ]. The pore size of the mucus gel layer ranges from 100 to 200 nm, suggesting that only NCs in this size range could potentially penetrate. Nevertheless, significant healthy-to-diseased and/or patient-to-patient variations are reported. It is nowadays acknowledged that a sufficiently hydrophilic and uncharged surface may minimize the adhesive interactions between mucin and the NC, thus improving their mucus-penetrating ability [ 26 , 28 ]. The pulmonary fluid layer reduces in thickness throughout the airways, forming single droplets on top of the limited ciliated cells of the lower bronchi and an extremely thin layer of surfactant in the alveoli. The pulmonary surfactant layer prevents alveolar collapse during expiration and is composed of approximately 90% lipids, mainly dipalmitoylphosphatidylcholine (DPPC), and 10% proteins (i.e., surfactant protein A, B, C, and D) [ 29 ]. Upon contact with the surfactant, larger sized particles are displaced from the airspace to the hypo-phase due to wetting forces, a phenomenon that probably also occurs with nano-sized particles [ 30 ]. In the hypo-phase, the particles may interact with surfactant proteins or may be taken up by alveolar macrophages [ 30 , 31 ]. Many of the literature findings suggest that, depending on their composition and size, inhaled particles may also interfere with the function of the pulmonary surfactant, thus hindering its physiological and essential role in the lung [ 23 , 24 ]. This issue should be properly considered when designing novel inhaled nanomedicines [ 32 , 33 ]. 3.2. Lung Epithelial Barrier The interactions of inhaled particles with the human airway epithelial barrier also play a crucial role in determining the availability of drugs in the lung. The lungs are made by many different types of cells, of which the main type are epithelial cells. The thickness and the properties of the lung's epithelial cell layer vary from a columnar cell monolayer with a thickness of 60 µm in the up airways (i.e., bronchi) to a broad cell monolayer 0.2 µm thick in the alveoli. The alveolar epithelial layer separates the lumen airspace from the pulmonary aqueous interstitial compartment, which is composed by different elements, such as the lymphatic vessel, collagen, and fiber [ 5 , 34 , 35 ]. The uptake of the inhaled drug by the lung's epithelial cells is influenced by the lung physiopathology [ 34 ]. As a matter of fact, while lipophilic drugs are thought to be rapidly absorbed by passive transcellular diffusion through epithelial cells, small hydrophilic compounds likely diffuse across the epithelium through aqueous pores in intercellular gap junctions [ 36 ]. Tight junctions, efflux proteins, and cellular enzymes play an important role as barriers in the absorption process as well [ 36 , 37 ]. Of note, tight junctions, mainly located on the apical side of the cell layer, are influenced by pathological conditions [ 36 ]. Indeed, the epithelial barrier function may decrease due to the disruption of tight junctions in asthma and chronic obstructive pulmonary diseases (COPD) [ 38 , 39 ], while their function may be enhanced in cystic fibrosis (CF) airway epithelial cells [ 40 , 41 ]. To improve drug accumulation at the cell level, different formulation approaches can be pursued. Drug interactions with cells can be regulated by simply using carriers with adequate size and surface properties, such as charge, hydrophilicity, and a shielding cloud [ 42 , 43 ]. However, efficient drug delivery to the final intracellular target is still challenging [ 44 , 45 ]. This is the case of emerging nucleic acid-based therapeutics (DNA, siRNA, and oligonucleotides), which demand for adequately engineered nanoparticulate systems, comprising not only of biodegradable polymers able to compact, to protect, and to release the entrapped nucleic acid, but also a biomimetic shell containing agents able to facilitate its endo-lysosomal escape (e.g., fusogenic peptides or lipids, endosome destabilizing polymers) [ 45 , 46 ]. The decoration of the carrier surface with different functional motifs can also be attempted to build up actively targeted constructs [ 47 , 48 , 49 ]. In the light of these findings, advanced in vitro models are paramount to design and to develop effective inhaled drugs. During recent years, many cell culture models of nasal, bronchial, and alveolar barriers have been developed, varying from 2D monolayer cultures to advanced 3D co-cultures, with the aim to better resemble what happens in vivo and to provide further insight into cellular responses or interactions with inhaled drug particles [ 50 , 51 , 52 , 53 , 54 ]. The direct aerosolization of the drug formulation on the cells through appropriately designed exposure systems, such as the VITROCELL ® Cloud or the PreciseInhale ® , is feasible to further increase the significance of the biological results [ 50 , 55 ]. Finally, lung-on-chips are emerging to emulate both the morphological features and biological functionality of the airway barrier with the ability to integrate respiratory motions and ensuing tissue strains [ 54 , 56 , 57 ]. 3.3. Macrophage-Mediated Clearance Macrophages are a type of lung-resident immune cells, designed to eliminate any foreign material that reaches the pulmonary environment [ 58 , 59 , 60 ]. They represent a unique cell population in the peripheral lungs that promptly responds to any airborne irritant or microbe [ 58 ]. Drug interactions with macrophages and the subsequent uptake can be enhanced or reduced by adequately tuning the properties of inhaled drug particles [ 61 ]. The size of particulate carrier systems significantly influences particle–macrophage interactions, as widely discussed in the literature [ 62 , 63 ]. Particles with a 1–5 µm size are taken up by alveolar macrophages, both in vitro and in vivo, to a greater extent compared to particles that are smaller or larger [ 64 ]. Also, the mechanism of particle uptake changes as a function of the carrier size. Micron-sized inhaled particles (1–5 µm) are taken up by alveolar macrophages mainly via active phagocytosis, whereas this is unlikely for nanosized particle [ 63 ]. Depending on their size, NCs enter alveolar macrophages by pathways other than phagocytosis: while NCs bigger than 0.2 µm are probably internalized via pinocytosis, smaller carriers (less than 150 nm) can be internalized via calveolae (50–100 nm) or clathrin-mediated (100–120 nm) uptake [ 63 , 65 ]. There is clear evidence also of the importance of the shape in governing particle uptake by alveolar macrophages [ 63 ]. When particles are internalized, the macrophage membrane spreads around the engulfed particle, and the progression of internalization is dependent on the contact angle between the particle and the membrane [ 61 ]. It was successfully demonstrated that rod-shaped nanostructured particles can be internalized with high efficiency (90% of uptake in 48 h) without a cytotoxic effect [ 66 ]. In addition, for aspherical particles, the orientation of the particle is crucial. The local shape of the particle at the point of cell attachment, rather than the overall shape, determines cell internalization. For example, if an ellipse-shaped particle is used, a macrophage attached to the pointed end of the ellipse internalizes the NCs in less time than the same attached to a flat region. Spherical particles, in contrast, are symmetrical and thus can be internalized from any point of attachment [ 67 , 68 ]. Similarly, shape-switching elongated particles were quickly engulfed by macrophages once the particles became spherical in shape [ 69 ]. Besides particle size and shape, stiffness is emerging as a design parameter for modulating the interaction of NCs with phagocytic cells [ 70 , 71 ]. As a rule, the increased mechanical robustness and overall stiffness of particles leads to increased phagocytosis. Inspired by blood cell behavior, deformable discoidal polymeric nano-constructs have been especially designed to minimize sequestration by phagocytic cells [ 70 , 72 ]. Tailoring nano-constructs' softness has been demonstrated to be crucial for modulating phagocytic cell sequestration [ 73 ]. By three different shapes (circular, elliptical, and quadrangular), two characteristic sizes, and a Young's modulus varying over two orders of magnitude (from 100 kPa to 10 MPa), professional phagocytic cells were observed to engulf more avidly rigid nano-constructs as compared to soft ones [ 73 ]. 3.4. Bacterial Biofilm In the case of lung infections, the effectiveness of inhaled antimicrobials can be seriously limited by the biofilm-producing capacity of some bacteria. Bacterial biofilm is a community of bacteria that is embedded in a self-produced matrix, the extracellular polymeric substance (EPS), composed of polysaccharides, secreted proteins, and extracellular DNAs [ 74 ]. Biofilm formation boosts antimicrobial resistance through different mechanisms, such as the sequestration and limited drug diffusion and the increase in antimicrobial efflux pump expression [ 75 ]. In particular, the EPS may establish electrostatic/hydrophobic interactions with the antimicrobial drugs, thus limiting drug diffusion towards bacteria and consequent antimicrobial activity [ 75 ]. Drug delivery through engineered NCs has been demonstrated to be a promising approach to assist drug diffusion across biofilm towards the bacterial target. Again, the achievement of this goal strongly depends on particle size and surface properties [ 19 ]. The results achieved on different biofilm bacteria highlighted how the size of the EPS meshes can make the difference, with a size cut-off for the optimal penetration of polymeric NCs into biofilm clusters, independently of bacteria, of around 100–130 nm [ 76 ]. Reduced but effective diffusion across a P. aeruginosa biofilm was found in vitro for liposomes with a diameter of 200 and 300 nm, while no diffusion was revealed for liposomes with size of 1 µm [ 77 ]. Although particle size plays a role in governing particle diffusion through the biofilm, the complex composition of the EPS matrix results also in several electrostatic/hydrophobic interactions between the NC and biofilm. Thus, the engineering of the NC surface represents another crucial step in the design of the inhaled particulate system. Again, surface charge and hydrophilicity play a pivotal role. As a general rule, while negatively charged particles may interact with positively charged polysaccharides, imparting a positive charge may improve the particle's interaction with the negative components of the matrix, such as alginates, proteins, and DNA [ 76 ]. In fact, the mobility of NCs into the biofilm appears increased for particles with a neutral surface, as in the case of particles with polyethylene glycol (PEG) surface modification (PEGylation) [ 76 , 78 ]. Nevertheless, conflicting data are reported in the literature. Positively charged quantum dots are able to penetrate and diffuse across a bacterial biofilm faster and more efficiently than negative and neutral ones [ 79 , 80 ]. Furthermore, some examples of cationic particles that were efficiently diffused and distributed into the bacterial biofilm are reported in the literature [ 79 , 80 ]. In some cases, positively-charged NCs showed enhanced distribution in the bacterial biofilm and consequent improved antimicrobial activity when compared to negatively charged NCs [ 81 ]. Notably, the surface charge may result also in a different location inside the biofilm matrix after diffusion. Actually, charged NCs can be localized close to the bacteria membrane [ 76 ]. The differential localization can be attributed to the hydrophilic nature of the NC surface, and more specifically, the higher hydrophobicity of the particle surface may enhance NC colocalization with bacterial cells within the biofilm [ 82 ]. Based on this principle, different attempts were pursued to provide an NC surface switch to adapt the NC properties to the target (i.e., mucus before and bacterial biofilm after). This is the case of environment-adaptive NCs developed using d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) [ 83 , 84 ]. After PEG-assisted mucus penetration, the enzymatic cleavage of PEG chains generates a lipophilic surface that allows the anchoring of the NC to the biofilm, where it serves as a depot for the prolonged exposure of bacteria to antibiotics [ 83 ]. 4. Lipid-Based Nanocarriers for Lung Administration: State-of-the-Art In the field of nanomedicine, lipid-based delivery systems are certainly the most investigated delivery platforms and arguably the most successful one [ 85 , 86 , 87 ]. Their growth overtime has been slow but steady and punctuated by several important milestones. The first big success has been the approval of Doxil back in 1995. Then, in 2018, the approval of Onpratto by the US FDA as a non-viral gene therapy approach became a watershed in the pharmaceutical research field providing the validation that clinically effective non-viral nucleic acid therapeutics can be successfully developed [ 88 , 89 ]. Finally, the claim of the most promising drug delivery system was undeniably endorsed in 2020 when mRNA COVID-19 vaccines were developed and approved, even if issued through an emergency use authorization [ 90 ]. The key of the success of lipid-based delivery systems is the combination of the high drug loading capacity of both hydrophilic and hydrophobic drugs, as well as the possibility of being easily engineered to yield a desired size, surface charge, composition, and morphology just by modifying the phospholipid composition [ 91 , 92 , 93 ]. This last aspect is especially appealing when physical, biochemical, and cellular barriers hinder drug transport to its target inside the human body, as for the inhalation route. To this regard, another advantage of lipid-based NCs for inhalation lies in the fact that the most commonly employed lipid materials (i.e., phospholipids and cholesterol) constitute a significant portion of the naturally occurring pulmonary surfactant, which is the first barrier to get in contact with the inhaled particles [ 32 ]. Historically, liposomes were suggested as surfactants in patients with respiratory distress and, recently, a mixture of phospholipids has been commercialized for prophylaxis against distress symptoms in neonates (Survanta, AbbVie) [ 29 ]. Liposomal formulations may improve tolerability, increase compliance by reducing the dosing frequency, enhance the penetration of biofilms, and support the treatment of intracellular infections [ 94 ]. Furthermore, the hydrophobic nature of lipids, especially of neutral lipids, reduces the absorption of the ubiquitous vapor onto particles during inhalation, limiting aggregation and adhesion phenomena [ 95 ]. If it is true that the pinnacle of the development of a pharmaceutical product is when it enters clinical evaluation and demonstrates a meaningful benefit to patients, the approval in 2018 of amikacin liposomes for inhalation (Arikayce) is the ultimate proof that lipid-based NCs might also be a successful pharmaceutical tool for pulmonary administration [ 96 ], opening the path towards novel lipid-based inhaled treatments. In general, lipid-based delivery systems include lipoplexes that electrostatically self-assemble with negatively charged nucleic acids, liposomes composed of a phospholipids bilayer, and solid-lipid nanoparticles (SLNs) with a solid lipid core matrix enclosed in a lipid monolayer ( Figure 2 ) [ 97 , 98 ]. Among lipid-based carriers of interest in inhalation therapy, both research and industry attention has been focused on liposomes as drug carriers for the encapsulation of small-molecule drugs or large proteins. Meanwhile, lipoplexes and LNPs are commonly used for the encapsulation of large cargoes such as proteins and nucleic acids (e.g., DNA, mRNA, siRNA, etc.) [ 99 , 100 ]. The main in vitro/ex vivo/in vivo findings achieved with lipid-based carriers for drug delivery to the lungs are reported in Table 1 . 4.1. Liposomes Liposomes can be considered as the first generation of lipid NCs, and they made their successful entry into the market in 1995 with the approval of the PEGylated liposomal formulation Doxil ® . Since then, liposomes have been successfully investigated as a strategy to formulate a wide spectrum of pharmaceuticals (i.e., anticancer, antimicrobial, and anaesthetic agents, and vaccines) not only for parenteral delivery, but also for oral, pulmonary, or topical delivery [ 111 ]. In particular, inhaled liposome-encapsulated drugs represent a very promising strategy for application in cancer and CF therapy [ 112 , 113 ]. ARIKAYCE ® is the first and only liposome suspension for inhalation approved by the FDA to treat lung diseases caused by the Mycobacterium avium complex (MAC), a type of nontuberculous mycobacteria (NTM). Amikacin (AMK) is entrapped in liposomes (0.2–0.3 µm) composed of neutral, biocompatible lipids (i.e., DPPC and cholesterol), and ARIKAYCE has been developed for administration via an electronic nebulizer (eFlow ® ). The path that leads to approval has not been straightforward and has seen, all at once, the exploitation of several indications that, up to now, have not known the same fortunate ending [ 111 ]. From a general technological standpoint, liposome design is highly versatile, since single lipid blocks can be assembled in order to tune physicochemical properties and, consequently, optimize interactions with the lung environment, mucus, biofilm matrix, and bacterial cell surface [ 112 ]. In the specific case of CF, several studies have been conducted in order to better correlate the liposome composition with the in vivo performance, in term of stability, drug entrapment efficiency, drug release, as well as the ability to interact with the biological environment (i.e., different strain of P. aeruginosa ), demonstrating how the composition plays a crucial role in the carrier design [ 114 , 115 ]. Furthermore, the therapeutic efficacy has been extensively investigated and the safety, pharmacokinetic advantage, and therapeutic effect of liposomes has been demonstrated in preclinical in vitro and in vivo models for antibiotics, such as tobramycin [ 116 ] as well as for chemotherapeutics molecules such as doxorubicin, gemcitabine, and paclitaxel [ 117 , 118 , 119 ]. In all cases, inhaled liposomes increase the drug retention, thus enhancing the therapeutic activity while simultaneously reducing the extra pulmonary side effects. Conversely, the systemic administration of liposomes resulted in a short residence time in the blood due to elimination via the reticuloendothelial system, which strongly limits their therapeutic application. With an approach analogue to Arikayce, Aradigm developed liposome-based formulations for the lung delivery of ciprofloxacin: Pulmaquin ® (ARD-3150) to treat Infections in Non-Cystic Fibrosis Bronchiectasis (NCFB), Lipoquin ® (ARD-3100) to treat infections in CF patients, and, more recently, ARD-1100 for the local treatment and prevention of inhalation anthrax. Pulmaquin ® is a simple 1:1 mixture of Lipoquin ® (50 mg/mL) and free ciprofloxacin (20 mg/mL). In a Phase 2b study (ORBIT-2 and ORBIT-1), it showed superior performance as compared to Lipoquin ® alone. Therefore, Pulmaquin ® progressed into Phase 3 clinical trials in BE [ 120 ]. Unfortunately, at the end of two different phase 3 studies (ORBIT-3 and ORBIT 4), the efficacy of the inhaled ciprofloxacin agents in the treatment of patients with NCFB was controversial. Further research was required by the FDA, though Savara Inc. discontinued the work on Pulmaquin ® in December 2020 [ 121 , 122 ]. Novel inhalable and controlled release powder formulations of ciprofloxacin nanocrystals inside liposomes (CNL) were recently developed [ 123 , 124 , 125 , 126 , 127 ]. Though current data on the efficacy of inhaled liposomal antibiotics are quite encouraging, the use of inhaled liposomes is, in general, challenged by their well-established physical and chemical instability in aqueous dispersions for long-term storage, often causing vesicle aggregation, drug leakage, phospholipid hydrolysis, and/or oxidation and vesicle fragmentation during aerosolization via nebulizers [ 112 ]. To address these limitations, many methods have been investigated. For instance, the design of specially customized vibrating mesh nebulizers with larger mesh apertures that could have a less disruptive effect has been taken into account [ 128 ]. It was also shown how the composition could play a key role and that the use of cholesterol-enriched dipalmitoyl phosphatidylcholine or surface modifications could improve stability during nebulization [ 129 , 130 ]. Last but not least, liposomal dry powders for inhalation (e.g., lyophilizing, spray drying, and supercritical fluid technology) have been developed, showing suited features for lung deposition [ 131 , 132 ]. 4.2. Solid Lipid Nanoparticles (SLNs) Solid lipid nanoparticles (SLNs) have been investigated as a viable alternative to liposomes for drug and gene delivery to the lung. SLNs are characterized by a hydrophobic core assuring the ability to encapsulate both hydrophilic and hydrophobic therapeutics while remaining solid at 37 °C, assuring stability in vivo [ 122 ]. Compared with liposomes, SLNs offer improved physical stability before and after nebulization [ 104 , 133 ], a controlled release that can be modulated according to the environmental pH [ 105 ], and the easy industrial scale-up of the production techniques [ 134 ]. Moreover, the SLNs efficacy has been demonstrated in vivo upon nebulization [ 105 , 133 ]. On the other hand, low drug loading and drug expulsion during storage are the main disadvantages [ 135 ]. Nanostructured Lipid Carriers (NLCs) can be considered a "second generation" of SLN, and consist, at room and body temperature, of a liquid lipid matrix surrounded by a solid lipid shell. NLCs were developed to overcome the limitations faced by the SLNs, thus they are generally characterized by a higher encapsulation efficiency and finer control of drug release, and simple and inexpensive production on a large scale. In particular, the production aspect is a great advantage compared to liposomes [ 136 , 137 ]. SLNs and NLCs have been studied as a drug delivery system for various applications and for different administration routes [ 138 , 139 ]. With special regard to the lung administration, several advantages have been shown. Their small size helps the delivery and deposition to the lower respiratory tract with a prolonged residence time thanks to the ability to escape the evade clearance operated by alveolar macrophage. Furthermore, thanks to the lipophilic nature, they have shown optimal bio-adhesive properties [ 140 ]. Promising preclinical studies have been shown for the encapsulation of many drugs, such as anti-inflammatory [ 141 , 142 ], antibiotics [ 143 , 144 ], chemotherapeutic [ 145 ], and gene delivery, also in combination therapy [ 146 , 147 ]. In most cases, the SLNs and NLCs are formulated to be delivered through aerosolization, but also some dry powder for inhalation have been developed [ 106 ]. 4.3. Nucleic Acid Delivery through Engineered Lipid Nanocarriers In recent years, the use of lipid NCs encapsulating nucleic acids (NA) for the treatment of severe lung diseases has been gaining increasing attention. To this purpose, cationic lipid nanoparticles, or "lipoplexes", and lipid nanoparticles (LNPs) are likely the most interesting ones [ 122 , 148 ]. Over the past few decades, cationic lipid nanoparticles have been the gold standard for NA delivery, taking advantage of the electrostatic interactions between negatively charged NAs and a cationic lipid, thus obtaining the so-called "lipoplex", able to facilitate the interaction with the negatively charged cell membrane [ 149 ]. Felgner et al. [ 150 ] were the first to demonstrate the feasibility of the development of a lipid-based carrier by using a non-natural cationic lipid [N-[1-(2,3-dioleyloxy)propyl]-N,N,N trimethylammonium chloride (DOTMA)] to deliver plasmid DNA into eukaryotic cells lines. Since then, other cationic lipids commonly used in the production of lipoplexes have been 1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP), or the more advanced 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), which is especially used to improve the in vivo delivery. Unfortunately, lipoplexes bear the potential for inducing dose-dependent cellular toxicity [ 151 ]. Another important limitation to develop successful lipoplexes is the possible interaction between cation-charged lipids and the negatively charged region present in the CF mucus, resulting in the disassembling of the drug delivery systems. The inclusion of a third component on the surface, such as a PEG layer, has been proposed to stabilize the lipoplexes, demonstrating good mucus-penetrating properties [ 152 ]. The most ambitious nonviral clinical trial to date, involving cationic lipids, was conducted by Alton and colleagues. A CF transmembrane conductance regulator (CFTR) plasmid (pGM169) was formulated with Genzyme lipid 67 (GL67). The incorporation of small amounts of DMPE-PEG5000 enabled the preparation of concentrated lipoplexes with an optimal cationic lipid:pDNA ratio of 0.75:1 for aerosolization. Thus, a phase I clinical trial was initiated in 2008 followed by phase II clinical trials by the UK CF consortium ( www.cfgenetherapy.org.uk , accessed on 26 February 2024). Patients received the nebulized lipoplex once per month for 1 year. Lung function was modestly stabilized in some individuals, and no significant adverse effects were observed. However, despite these encouraging results, the approach was not enough to achieve clear phenotypic correction [ 153 , 154 ]. In recent years, LNPs have emerged as a promising platform for RNA delivery and have shed light by resolving the inherent instability issues of naked RNA, thereby enhancing the therapeutic potency. LNPs consisting of ionizable lipids, helper lipids, cholesterol, and poly(ethylene glycol)-anchored lipids can stably enclose RNA and help them release into the cells' cytosol [ 155 ]. The approach that is leading the path to overcome all the limitations imposed by cationic lipids is the substitution of the quaternary ammonium head with a titratable moiety which produces an ionizable lipid. Pieter Cullis' research group has been the first to exploit the potential associated with the use of an ionizable lipid in order to deliver nucleic acids, and their studies have opened the way to, first, the approval of Onpattro ® in 2018 and then mRNA vaccines for COVID-19. Having an ionizable excipient offers the possibility of changing the charge status according to the environmental pH, and in this way, it is possible to maximize the interaction with the nucleic acid during the production phase, have a stable complex in the bloodstream (or at physiological pH), and finally, offer an effective escaping solution once the vector is inside the acidic pH of the endosome thanks to the re-ionization of the amino lipid component and the formation of electrostatic and hydrophobic interactions between disassembled lipids and the endosomal membrane [ 156 , 157 , 158 ]. Onpattro ® approval represents a milestone to many extents. From a therapeutical point of view, it gives new hope to the hATTR patients who can now count on an approach able to stop the progression of the disease. From an siRNA development point of view, Onpattro ® is not only the first non-viral vector that made it to the market, but it also opened a new perspective for the development of a more universal option to solve the endosomal escape problem. Furthermore, we cannot forget that based on the proof of concept provided by Onpattro ® technology, the COVID-19 vaccines were developed and were, for the first time, administrated on a large scale [ 159 ]. LNPs' ability to deliver mRNA to the inside of cells is not only limited to vaccination, but has versatile applications such as treating genetic disorders [ 160 , 161 ]. Recently, LNPs were employed in a clinical trial to deliver Cas9 mRNA and guide RNA for editing the gene causing transthyretin amyloidosis [ 162 ]. The therapeutic application of the LNP platform is restricted mostly for hepatic diseases because LNPs innately accumulate in the liver when administered systemically, which significantly limits its access to other organs [ 88 ]. However, investigations into applying LNPs to deliver inhaled therapeutics to the lungs are underway [ 122 ]. In fact, even if recent studies have shown that modulating the nanoparticle surface charge permits the systemically administered LNPs to reach the lungs [ 27 , 163 , 164 , 165 ], the focused delivery of nucleic acids-based therapeutics to the lungs via the inhalation route still represents the most promising approach to treat severe lung diseases, providing stronger control over the induction of off-target effects. The designing criteria of LNPs are under evaluation in order to adapt LNPs to inhalation [ 97 , 109 ]. Encouraging results have already been shown in terms of aerodynamic properties for deposition to the lower respiratory tract, with good stability upon nebulization [ 97 , 109 ] and in vivo activity through intratracheal administration [ 110 ]. Moreover, the feasibility of engineering LNP-based powders by spray drying was recently demonstrated [ 137 , 166 ]. Optimized spray-dried LNPs penetrated the lung mucus layer and maintained bioactivity resulting in >90% protein downregulation with a confirmed safety profile in a lung adenocarcinoma cell line. Furthermore, the spray-dried LNPs successfully achieved up to 50% gene silencing of the house keeping gene GAPDH in ex vivo human precision-cut lung slices without inducing any toxic effect, as shown by the cytokine levels [ 109 ]. 4.1. Liposomes Liposomes can be considered as the first generation of lipid NCs, and they made their successful entry into the market in 1995 with the approval of the PEGylated liposomal formulation Doxil ® . Since then, liposomes have been successfully investigated as a strategy to formulate a wide spectrum of pharmaceuticals (i.e., anticancer, antimicrobial, and anaesthetic agents, and vaccines) not only for parenteral delivery, but also for oral, pulmonary, or topical delivery [ 111 ]. In particular, inhaled liposome-encapsulated drugs represent a very promising strategy for application in cancer and CF therapy [ 112 , 113 ]. ARIKAYCE ® is the first and only liposome suspension for inhalation approved by the FDA to treat lung diseases caused by the Mycobacterium avium complex (MAC), a type of nontuberculous mycobacteria (NTM). Amikacin (AMK) is entrapped in liposomes (0.2–0.3 µm) composed of neutral, biocompatible lipids (i.e., DPPC and cholesterol), and ARIKAYCE has been developed for administration via an electronic nebulizer (eFlow ® ). The path that leads to approval has not been straightforward and has seen, all at once, the exploitation of several indications that, up to now, have not known the same fortunate ending [ 111 ]. From a general technological standpoint, liposome design is highly versatile, since single lipid blocks can be assembled in order to tune physicochemical properties and, consequently, optimize interactions with the lung environment, mucus, biofilm matrix, and bacterial cell surface [ 112 ]. In the specific case of CF, several studies have been conducted in order to better correlate the liposome composition with the in vivo performance, in term of stability, drug entrapment efficiency, drug release, as well as the ability to interact with the biological environment (i.e., different strain of P. aeruginosa ), demonstrating how the composition plays a crucial role in the carrier design [ 114 , 115 ]. Furthermore, the therapeutic efficacy has been extensively investigated and the safety, pharmacokinetic advantage, and therapeutic effect of liposomes has been demonstrated in preclinical in vitro and in vivo models for antibiotics, such as tobramycin [ 116 ] as well as for chemotherapeutics molecules such as doxorubicin, gemcitabine, and paclitaxel [ 117 , 118 , 119 ]. In all cases, inhaled liposomes increase the drug retention, thus enhancing the therapeutic activity while simultaneously reducing the extra pulmonary side effects. Conversely, the systemic administration of liposomes resulted in a short residence time in the blood due to elimination via the reticuloendothelial system, which strongly limits their therapeutic application. With an approach analogue to Arikayce, Aradigm developed liposome-based formulations for the lung delivery of ciprofloxacin: Pulmaquin ® (ARD-3150) to treat Infections in Non-Cystic Fibrosis Bronchiectasis (NCFB), Lipoquin ® (ARD-3100) to treat infections in CF patients, and, more recently, ARD-1100 for the local treatment and prevention of inhalation anthrax. Pulmaquin ® is a simple 1:1 mixture of Lipoquin ® (50 mg/mL) and free ciprofloxacin (20 mg/mL). In a Phase 2b study (ORBIT-2 and ORBIT-1), it showed superior performance as compared to Lipoquin ® alone. Therefore, Pulmaquin ® progressed into Phase 3 clinical trials in BE [ 120 ]. Unfortunately, at the end of two different phase 3 studies (ORBIT-3 and ORBIT 4), the efficacy of the inhaled ciprofloxacin agents in the treatment of patients with NCFB was controversial. Further research was required by the FDA, though Savara Inc. discontinued the work on Pulmaquin ® in December 2020 [ 121 , 122 ]. Novel inhalable and controlled release powder formulations of ciprofloxacin nanocrystals inside liposomes (CNL) were recently developed [ 123 , 124 , 125 , 126 , 127 ]. Though current data on the efficacy of inhaled liposomal antibiotics are quite encouraging, the use of inhaled liposomes is, in general, challenged by their well-established physical and chemical instability in aqueous dispersions for long-term storage, often causing vesicle aggregation, drug leakage, phospholipid hydrolysis, and/or oxidation and vesicle fragmentation during aerosolization via nebulizers [ 112 ]. To address these limitations, many methods have been investigated. For instance, the design of specially customized vibrating mesh nebulizers with larger mesh apertures that could have a less disruptive effect has been taken into account [ 128 ]. It was also shown how the composition could play a key role and that the use of cholesterol-enriched dipalmitoyl phosphatidylcholine or surface modifications could improve stability during nebulization [ 129 , 130 ]. Last but not least, liposomal dry powders for inhalation (e.g., lyophilizing, spray drying, and supercritical fluid technology) have been developed, showing suited features for lung deposition [ 131 , 132 ]. 4.2. Solid Lipid Nanoparticles (SLNs) Solid lipid nanoparticles (SLNs) have been investigated as a viable alternative to liposomes for drug and gene delivery to the lung. SLNs are characterized by a hydrophobic core assuring the ability to encapsulate both hydrophilic and hydrophobic therapeutics while remaining solid at 37 °C, assuring stability in vivo [ 122 ]. Compared with liposomes, SLNs offer improved physical stability before and after nebulization [ 104 , 133 ], a controlled release that can be modulated according to the environmental pH [ 105 ], and the easy industrial scale-up of the production techniques [ 134 ]. Moreover, the SLNs efficacy has been demonstrated in vivo upon nebulization [ 105 , 133 ]. On the other hand, low drug loading and drug expulsion during storage are the main disadvantages [ 135 ]. Nanostructured Lipid Carriers (NLCs) can be considered a "second generation" of SLN, and consist, at room and body temperature, of a liquid lipid matrix surrounded by a solid lipid shell. NLCs were developed to overcome the limitations faced by the SLNs, thus they are generally characterized by a higher encapsulation efficiency and finer control of drug release, and simple and inexpensive production on a large scale. In particular, the production aspect is a great advantage compared to liposomes [ 136 , 137 ]. SLNs and NLCs have been studied as a drug delivery system for various applications and for different administration routes [ 138 , 139 ]. With special regard to the lung administration, several advantages have been shown. Their small size helps the delivery and deposition to the lower respiratory tract with a prolonged residence time thanks to the ability to escape the evade clearance operated by alveolar macrophage. Furthermore, thanks to the lipophilic nature, they have shown optimal bio-adhesive properties [ 140 ]. Promising preclinical studies have been shown for the encapsulation of many drugs, such as anti-inflammatory [ 141 , 142 ], antibiotics [ 143 , 144 ], chemotherapeutic [ 145 ], and gene delivery, also in combination therapy [ 146 , 147 ]. In most cases, the SLNs and NLCs are formulated to be delivered through aerosolization, but also some dry powder for inhalation have been developed [ 106 ]. 4.3. Nucleic Acid Delivery through Engineered Lipid Nanocarriers In recent years, the use of lipid NCs encapsulating nucleic acids (NA) for the treatment of severe lung diseases has been gaining increasing attention. To this purpose, cationic lipid nanoparticles, or "lipoplexes", and lipid nanoparticles (LNPs) are likely the most interesting ones [ 122 , 148 ]. Over the past few decades, cationic lipid nanoparticles have been the gold standard for NA delivery, taking advantage of the electrostatic interactions between negatively charged NAs and a cationic lipid, thus obtaining the so-called "lipoplex", able to facilitate the interaction with the negatively charged cell membrane [ 149 ]. Felgner et al. [ 150 ] were the first to demonstrate the feasibility of the development of a lipid-based carrier by using a non-natural cationic lipid [N-[1-(2,3-dioleyloxy)propyl]-N,N,N trimethylammonium chloride (DOTMA)] to deliver plasmid DNA into eukaryotic cells lines. Since then, other cationic lipids commonly used in the production of lipoplexes have been 1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP), or the more advanced 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), which is especially used to improve the in vivo delivery. Unfortunately, lipoplexes bear the potential for inducing dose-dependent cellular toxicity [ 151 ]. Another important limitation to develop successful lipoplexes is the possible interaction between cation-charged lipids and the negatively charged region present in the CF mucus, resulting in the disassembling of the drug delivery systems. The inclusion of a third component on the surface, such as a PEG layer, has been proposed to stabilize the lipoplexes, demonstrating good mucus-penetrating properties [ 152 ]. The most ambitious nonviral clinical trial to date, involving cationic lipids, was conducted by Alton and colleagues. A CF transmembrane conductance regulator (CFTR) plasmid (pGM169) was formulated with Genzyme lipid 67 (GL67). The incorporation of small amounts of DMPE-PEG5000 enabled the preparation of concentrated lipoplexes with an optimal cationic lipid:pDNA ratio of 0.75:1 for aerosolization. Thus, a phase I clinical trial was initiated in 2008 followed by phase II clinical trials by the UK CF consortium ( www.cfgenetherapy.org.uk , accessed on 26 February 2024). Patients received the nebulized lipoplex once per month for 1 year. Lung function was modestly stabilized in some individuals, and no significant adverse effects were observed. However, despite these encouraging results, the approach was not enough to achieve clear phenotypic correction [ 153 , 154 ]. In recent years, LNPs have emerged as a promising platform for RNA delivery and have shed light by resolving the inherent instability issues of naked RNA, thereby enhancing the therapeutic potency. LNPs consisting of ionizable lipids, helper lipids, cholesterol, and poly(ethylene glycol)-anchored lipids can stably enclose RNA and help them release into the cells' cytosol [ 155 ]. The approach that is leading the path to overcome all the limitations imposed by cationic lipids is the substitution of the quaternary ammonium head with a titratable moiety which produces an ionizable lipid. Pieter Cullis' research group has been the first to exploit the potential associated with the use of an ionizable lipid in order to deliver nucleic acids, and their studies have opened the way to, first, the approval of Onpattro ® in 2018 and then mRNA vaccines for COVID-19. Having an ionizable excipient offers the possibility of changing the charge status according to the environmental pH, and in this way, it is possible to maximize the interaction with the nucleic acid during the production phase, have a stable complex in the bloodstream (or at physiological pH), and finally, offer an effective escaping solution once the vector is inside the acidic pH of the endosome thanks to the re-ionization of the amino lipid component and the formation of electrostatic and hydrophobic interactions between disassembled lipids and the endosomal membrane [ 156 , 157 , 158 ]. Onpattro ® approval represents a milestone to many extents. From a therapeutical point of view, it gives new hope to the hATTR patients who can now count on an approach able to stop the progression of the disease. From an siRNA development point of view, Onpattro ® is not only the first non-viral vector that made it to the market, but it also opened a new perspective for the development of a more universal option to solve the endosomal escape problem. Furthermore, we cannot forget that based on the proof of concept provided by Onpattro ® technology, the COVID-19 vaccines were developed and were, for the first time, administrated on a large scale [ 159 ]. LNPs' ability to deliver mRNA to the inside of cells is not only limited to vaccination, but has versatile applications such as treating genetic disorders [ 160 , 161 ]. Recently, LNPs were employed in a clinical trial to deliver Cas9 mRNA and guide RNA for editing the gene causing transthyretin amyloidosis [ 162 ]. The therapeutic application of the LNP platform is restricted mostly for hepatic diseases because LNPs innately accumulate in the liver when administered systemically, which significantly limits its access to other organs [ 88 ]. However, investigations into applying LNPs to deliver inhaled therapeutics to the lungs are underway [ 122 ]. In fact, even if recent studies have shown that modulating the nanoparticle surface charge permits the systemically administered LNPs to reach the lungs [ 27 , 163 , 164 , 165 ], the focused delivery of nucleic acids-based therapeutics to the lungs via the inhalation route still represents the most promising approach to treat severe lung diseases, providing stronger control over the induction of off-target effects. The designing criteria of LNPs are under evaluation in order to adapt LNPs to inhalation [ 97 , 109 ]. Encouraging results have already been shown in terms of aerodynamic properties for deposition to the lower respiratory tract, with good stability upon nebulization [ 97 , 109 ] and in vivo activity through intratracheal administration [ 110 ]. Moreover, the feasibility of engineering LNP-based powders by spray drying was recently demonstrated [ 137 , 166 ]. Optimized spray-dried LNPs penetrated the lung mucus layer and maintained bioactivity resulting in >90% protein downregulation with a confirmed safety profile in a lung adenocarcinoma cell line. Furthermore, the spray-dried LNPs successfully achieved up to 50% gene silencing of the house keeping gene GAPDH in ex vivo human precision-cut lung slices without inducing any toxic effect, as shown by the cytokine levels [ 109 ]. 5. Polymer-Based Nanocarriers for Lung Administration: State-of-the-Art In the last decade, polymeric NCs have gained considerable interest regarding their pulmonary delivery applications due to their unique properties [ 79 , 167 , 168 , 169 ]. They represent a well-established platform for the encapsulation and delivery of a multitude of therapeutic molecules due to their versatility in polymer physiochemical properties as well as the variety of available production techniques, which can be selected in view of the specific drug and intended application [ 6 , 170 , 171 ]. The most employed polymers in NC design and development are biocompatible, biodegradable, and capable of the sustaining the release of the encapsulated drug without the relevant side effects or cytotoxicity. Furthermore, recent studies underline how polymeric NCs may assist drug diffusion across the lung barriers, which are paramount for efficient drug delivery in the lungs [ 79 , 172 ]. To date, several materials have been studied to achieve optimal polymer NCs for inhalation. Depending on the polymer properties and the adopted production technique, various polymeric NCs can be obtained, such as nanogels, nanospheres, and polyplexes ( Figure 3 ) [ 2 , 173 ]. According to their nature, the systems can be broadly classified in natural and synthetic polymer-based NCs. 5.1. Natural Polymer-Based Nanocarriers Natural polymers appear to be very interesting materials in NC production thanks to their biocompatibility, low toxicity, and biodegradability [ 174 ]. Furthermore, the techniques usually employed in the production of natural polymer-based NCs (i.e., crosslinking gelation) are very gentle and characterized by low shear forces, thus they are ideal for the encapsulation of unstable molecules [ 175 , 176 ]. The main in vitro/in vivo findings achieved with natural polymer NCs for drug delivery to the lungs are reported in Table 2 . Between the natural materials for pulmonary delivery, albumin is one of the most studied polymers due to its low antigenicity, low toxicity, biocompatibility, biodegradability, low costs, and abundance. Serum albumin is the most abundant protein in the plasma, and it is characterized by a high affinity with different molecules; thus, as a nanocarrier, it has been chosen to incorporate a variety of active compounds [ 177 , 178 , 179 ]. pharmaceutics-16-00347-t002_Table 2 Table 2 Main in vitro/in vivo findings achieved with natural polymeric NCs for the pulmonary delivery of drugs. Polymer Encapsulated Molecule In Vitro Model In Vivo Model Main Findings Ref. Albumin Tacrolimus - Intratracheal administration in bleomycin-induced pulmonary fibrosis mouse Anti-fibrotic effect significantly higher than intraperitoneal administration [ 180 ] Albumin - Macrophages derived from BALB/C mice Oropharyngeal aspiration in male BALB/C mice High in vivo biocompatibility with mild inflammation at highest dose tested. Slower clearance. No accumulation in major organs [ 174 ] HSA Benzothiazinone (BTZ043) Murine bone marrow-derived macrophages infected with M. tuberculosis Intranasal instillation in old female C3HeB/FeJ mice infected with M. tuberculosis Enhanced efficacy in vitro compared to the free drug; reduced bacterial load in vivo [ 181 ] TRAIL-HSA Doxorubicin Apoptotic and cytotoxicity activity on H226 cell line (human lung squamous carcinoma cell line) Insufflation of nanoparticle dispersion in mouse bearing H226 cell-induced metastatic tumors Synergistic apoptotic activity and anti-tumor efficacy in vitro and in vivo [ 182 ] BSA siRNA Cellular uptake and cytotoxicity on A549 cell line; gene-silencing on KRAS G12S mutant A459 cells line - Low cytotoxicity with enhanced cellular uptake. High knock-down efficiency in vitro [ 183 ] CS Influenza vaccine Cytokines secretion in porcine monocyte-derived dendritic cells Intranasal nebulization in pigs Augmented cross-reactive T and B lymphocytes response [ 184 ] CS Bedaquiline Cytotoxicity profile on macrophage cell line Inhalation of freeze-dried nanoparticles in rats Low acute and chronic toxicity in vivo [ 185 ] CS Salmon Calcitonin - Intratracheal administration in rats Higher absorption and deposition in deep lung [ 186 ] CS Prothionamide - Intratracheal administration of dry powder containing nanoparticles in rats Prolonged drug persistence in lungs [ 187 ] CS-HA Gallium (III) Human epithelial bronchial cells (16HBE14o-) and P. aeruginosa Intratracheal administration of dry powder containing nanoparticles in rats Improved accumulation of drug in lung tissue and high tolerability in vivo [ 188 ] CS-PVA Magnolol Cytotoxicity profile on cells A549 cell line - Enhanced lung deposition with high cell viability [ 189 ] ALG_CS-DNase Tobramycin Antimicrobial activity on CF sputum sample and P. aeruginosa strain (PA01) Injection of nanoparticles dispersion in Galleria melonella Increased penetration across CF sputum and enhanced anti-pseudomonal activity in vitro and in vivo [ 190 ] ALG-CS/Tween80 Rifampicin and ascorbic acid Antibacterial activity on Mycobacterium Tuberculosis (M. tb.); cytotoxicity on kidney epithelial cells - Increased antibacterial activity Low cytotoxicity on kidney epithelial cell lines [ 191 ] List of Abbreviations: CS: Chitosan; BSA: Bovine-serum albumin; PVA: Poly(vinyl alcohol); siRNA: small interfering RNA; ALG: alginate; GCS: glycol chitosan; TGA: thioglycolic acid; HSA: human serum albumin; TRAIL: tumor necrosis factor (TNF)-related apoptosis-inducing ligand. In recent years, albumin NCs have gained considerable research attention as a drug delivery system owing to the approval by the FDA of nanoparticle albumin-bound (NAB) paclitaxel (Abraxane ® ) in the treatment of metastatic breast cancer (2005), advanced/metastatic non-small cell lung cancer (2012), and metastatic pancreatic cancer (2013). Inspired by the success of Abraxane ® , albumin-based NCs have stimulated interest also for inhalation [ 174 , 181 , 182 , 192 , 193 ]. The first in vivo proof-of-concept study on the lung biocompatibility and biodistribution of inhaled albumin NCs was performed by Woods et al. [ 174 ]. The results showed the absence of a significant inflammatory response in mice after the single pulmonary administration of bovine serum albumin (BSA) NCs as compared to the control BSA solution. Meanwhile, SPECT/CT imaging and post-mortem organ biodistribution studies demonstrated that lung tissue accumulation up to 48 h was significantly higher for BSA NCs compared with the control BSA solution. The absence of major NCs accumulation in secondary organs, and likely of related side effects, was further encouraging. In view of the proven efficacy of serum albumin NCs for cancer therapy [ 177 ], their potential for the direct delivery of anti-cancer drugs by inhalation has been explored [ 182 ]. Briefly, human serum albumin (HSA) NCs were loaded with doxorubicin and modified with the apoptotic protein TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) to maximize specificity for lung cancer cells. The resulting TRAIL/Dox HSA-NCs exhibited a robust anti-tumor effect after pulmonary administration in a xenograft mouse model [ 182 ]. Due to the presence of charged amino acids able to electrostatically interact with charged molecules, albumin raised awareness also for the delivery of nucleic acid therapeutics. With this idea in mind, Merkel and co-workers developed BSA NCs for the delivery of siRNA targeting the KRAS G12S mutation, the most frequent mutation in human cancers. The study demonstrated that BSA NCs could protect the siRNA payload against RNases, enable in vitro transport to A549 cells, and mediate significant sequence-specific KRAS knockdown, resulting in the reduced cell growth of siRNA-transfected lung cancer cells [ 183 ]. A relevant issue in the development of inhalable NCs based on albumin is represented by unfavorable mucus/carrier interactions. In fact, albumin may establish quite strong interactions with mucus components (i.e., an NC/mucin electrostatic interaction or covalent binding to mucin), which could be mitigated incorporating (e.g., cyclodextrin) or surface-engineering (e.g., PEGylation) polymers in NCs' design [ 194 , 195 ]. The mucoadhesivity of albumin NCs prompted the exploration of their potential for local rather than systemic delivery through the lungs. Along this line, J. Seo and co-workers demonstrated the superior efficacy of inhaled tacrolimus-bound albumin NCs in mice with bleomycin-induced pulmonary fibrosis as compared to an intraperitoneal tacrolimus solution [ 196 ]. When conceiving these systems for pulmonary delivery, the results of Papay et al. are also relevant, as they successfully developed lactose-based dry powders for inhalation embedding apigenin-loaded albumin NCs, likely useful for the local treatment of lung injury in asthmatic conditions [ 197 ]. Among natural polymers, chitosan (CS) has been widely investigated for pulmonary drug delivery [ 198 ]. The positive charge of CS is a critical attribute to improve the absorption by opening the tight junctions of the lung epithelium [ 199 , 200 ]. Furthermore, CS charges are responsible for the interaction with mucus components, and thus for the mucoadhesive effect, and the amount of charges could be tuned through the deacetylation grade [ 201 ]. In recent years, numerous studies have indicated CS-based NCs as promising inhalable delivery systems [ 202 ], both for systemic and local delivery. CS NCs are usually prepared by the gelation technique, coacervation, nanoprecipitation, and the reverse micellar method [ 203 ]. They are effective in protecting, controlling the release, and promoting drug absorption through lung epithelia when administered both as NC dispersion and embedded into inert carrier microparticles (Nano-embedded Microparticles—NEM) [ 186 , 189 , 204 ]. One of the most studied and interesting applications of CS is the development of drug delivery systems (DDS) for the pulmonary administration of antimicrobial drugs [ 185 , 205 ]. In fact, CS itself shows antibacterial effects likely ascribable to the electrostatic interactions between its protonated amino groups and the phosphoryl groups and lipopolysaccharides of bacterial cell membranes [ 206 , 207 , 208 ]. The consequent membrane perturbation and release of the cell content may result in an increased therapeutic effect of the drug cargo [ 209 ]. Furthermore, the affinity of CS with alveolar macrophages, due to the electrostatic interactions between the positively charged polymer and the negatively charged sialic acid on the cell membranes [ 210 ], boosted the development of CS-based delivery systems for the local treatment of tuberculosis. Different drugs have been encapsulated into CS or CS-derivative NCs with increased anti-tubercular activity with respect to the free drugs [ 185 , 211 , 212 ]. The mucoadhesive tendency of CS (mostly due to its chemical structure) is both an advantage and a disadvantage and has solicited the researchers' attention. In this direction, even if inhalable CS NCs were shown to be effective in different in vivo applications [ 188 , 201 , 213 ], it was recently highlighted how CS-NCs may be entrapped in the superficial mucus layer, which is quickly cleared, and are not expected to reach the underlying airway epithelium [ 214 , 215 ]. As a matter of fact, the current trend is based on the development of NCs modified on the surface with inert polymers, which can improve the particle mobility into the mucus gel layer [ 26 , 216 , 217 ]. Another issue is the in vivo elimination of inhaled CS NCs. Though CS NCs display an acceptable safety profile and low chronic toxicity [ 185 ], no study has conclusively demonstrated the complete biodegradation or elimination of CS NCs in vivo. Nevertheless, this aspect has been reviewed to help shed light on this point [ 218 ]. 5.2. Synthetic Polymer-Based Nanocarriers The pulmonary route has some distinctive features that significantly limit the panel of synthetic materials available for inhalation. Biodegradable aliphatic polyesters, such as poly (lactic-co-glycolic acid) (PLGA) or Poly(N-isopropyl acrylamide)(PNIPAM), are the most exploited polymers because of their biodegradability, biocompatibility, and versatility [ 2 , 219 , 220 , 221 ]. The properties of the polymer, such as the grade of hydrophobicity and the degradation rate, and those of the resulting NCs, such as the particle size, surface properties, and drug encapsulation/release, can be easily tuned in order to optimize the affinity and the compatibility of the delivery system with the lung environment. The main in vitro/in vivo findings achieved with synthetic polymer NCs for drug delivery to the lungs are reported in Table 3 . As reported in Table 2 , PLGA is among the polymers that have been extensively explored for the development of inhalable NCs. If PLGA-based NCs are considered promising in terms of drug encapsulation, protection, controlled release, and aerodynamic properties, these systems appear not always efficient in crossing cellular/extracellular pulmonary barriers. As a consequence, the examples of inhalable PLGA NCs reported in the literature are often engineered at the surface with hydrophilic polymers, which can shield nanocarrier interactions with lung-lining fluids, thus promoting drug transport to the target [ 222 , 223 , 224 , 231 , 232 , 233 ]. Polyethylene glycol (PEG) is the most widely exploited polymer to achieve mucoinert PLGA-based NCs. In fact, the PEGylation leads to the formation of a hydrophilic shell and neutral surface charge that prevents hydrophobic or electrostatic NC interactions with the mucus gel layer [ 26 , 234 , 235 ]. Surface properties can be further tuned by changing the PEG molecular weight and its density on the particle surface [ 234 , 235 ]. The transport of PEGylated NCs to the inner layer of the mucus likely increases their retention in the lungs by reducing the clearance mechanism and facilitating macrophages' escape [ 26 , 214 , 230 ]. The effectiveness of PEGylation in the pulmonary drug delivery of PLGA NCs was extensively explored and promising results have been achieved in both in vitro and in vivo models in different therapeutic applications, such as antimicrobial and anticancer therapy [ 236 , 237 ]. Alternative polymers for the functionalization of NCs able to assist their penetration through mucus have been investigated. These include poly(2-oxazolines), polysarcosine, zwitterionic polymers (polybetaines), proteolytic enzymes, and poly(vinyl alcohol) (PVA) [ 28 ]. Between them, PVA is the most employed emulsifier in polymeric nano- and microparticles and it is able to provide uniform particulate systems with low polydispersity. Recently, we have developed PLGA NCs engineered with PVA for the delivery of an antimicrobial peptide, Esculentin, to the lung [ 224 ]. We have found that the PVA shell can assist the particles transport across the mucus layer, which is very low for the naked antimicrobial peptide. Furthermore, the NCs can control the release of the encapsulated molecule, providing a prolonged antimicrobial effect on Pseudomonas aeruginosa both in vitro, on bacteria culture, and after pulmonary administration as NC dispersion in an acute infected murine model in vivo. Although the PVA is generally considered a mucoadhesive polymer, the ability of PVA to improve the particle diffusion across the mucus was found to be related to the grade of hydrolysis, the molecular weight, and the density of the particle shell. In particular, the non-covalent shell of partially hydrolized PVA, with a hydrolysis degree between 75 and 95%, were found to improve the PLA NCs' mobility in mucus [ 238 ]. Despite the efficacy in overcoming the mucus barrier, NC engineering with hydrophilic polymers seems to not be the best strategy in cellular target-based therapies, such as gene delivery, in which the NCs need to be designed for cell penetration [ 239 ]. An early exploited strategy to achieve mucus-penetrating particles able to improve the drug cell uptake is represented by the hybrid lipid-polymer NCs [ 55 , 172 , 240 ]. Hybrid NCs consisting of a PLGA core and a lipid shell of dipalmitoylphosphatidylcholine (DPPC) have been developed in our labs for the pulmonary delivery of siRNA. Thanks to its ability to form a shell around the polymer core, DPPC was chosen to improve NC compatibility and tolerance in the lung environment. Furthermore, it has been demonstrated that a DPPC shell can provide mucoinert and muco-diffusive properties to the particulate system while improving the drug cell uptake, crucial in siRNA therapies [ 46 , 74 , 163 , 230 ]. This strategy appears very promising in gene delivery, in which different lipids were used with success for the development of inhalable hybrid NCs [ 240 , 241 ]. In order to assist the gene uptake, particle surface engineering is often associated with the encapsulation of non-viral vectors as cationic polymers. One of the most employed in gene delivery is polyethylenimine (PEI), which can electrostatically interact with nucleic acids forming complexes (polyplexes), improving the encapsulation and release from polymeric NCs and facilitating gene cellular internalization [ 55 , 230 ]. A recent study investigated the impact of various biomimetic endogenous pulmonary phospholipids on the in vivo behavior of PLGA NCs. The study revealed that surface engineering with neutral phospholipids, such as DPPC and dipalmitoylphosphatidylamine (DPPE), resulted in a reduction in the uptake of NCs by alveolar macrophages, while the use of negatively charged phospholipids, such as dipalmitoylphos-phatidylserine (DPPS) and dipalmitoylphosphatidylglycerol (DPPG), enhanced the uptake. Regardless of the charge, phospholipid surface engineering increased the uptake of NCs by A549 cells and drug retention in the entire lung. DPPC, DPPE, and DPPG facilitated drug retention in BALF, whereas DPPS facilitated drug absorption in lung tissue. However, no impact of phospholipids on drug tissue distribution was observed [ 242 ]. Concerning the particle size, NCs with an hydrodynamic diameter of 100 nm showed significantly higher lung retention and tissue adsorption than NCs with a higher diameter (300, 800, and 2000 nm); therefore, NCs with a small size are the most effective for pulmonary drug delivery, especially for local lung disease therapy [ 243 ]. In order to conceive mucus-penetrating and long-residence DDS for pulmonary administration, macrophage uptake must be considered. Sometimes, being a further limit in drug pulmonary administration, it becomes a desired effect in specific macrophage-target therapy. In fact, the selective and controlled internalization of PLGA-based NCs loaded with anti-infective drugs by alveolar macrophages is under investigation for targeted antimicrobial therapy [ 7 , 183 , 210 ]. This is the case of antitubercular therapy, in which the target is Mycobacterium tuberculosis located in the alveolar macrophages (host cells) [ 244 ]. Unmodified PLGA NCs, which are usually inappropriate for pulmonary delivery due to their fast elimination by macrophage clearance, have been successfully developed as inhalable systems for the delivery of three frontline antitubercular drugs [ 245 ]. In the attempt to improve the NC uptake in macrophages and thus increase the drug concentration at the site of action, appropriate surface modifications of PLGA NCs have been explored. A recent study underlined the higher effectiveness of PLGA nanocapsules, modified with CS, in killing intracellular Staphylococcusaureus and Micobacterum abscessus with the same dose of the drug in its free form. The CS confers to NCs with a positively charged surface which is able to increase the uptake in macrophages and improve the drug effect [ 223 ]. Surface engineering the NC with CS has been exploited in different applications, such as lung infections and pulmonary fibrosis. As a matter of fact, despite the controversial discussion about the higher effectiveness of muco-inert particles with respect to the mucoadhesive ones, CS-modified NCs are able to improve the drug in vivo bioavailability after inhalation [ 180 , 222 ]. The CS mucoadhesive effect can increase the NC residence time in the lung and, thanks to the opening effect on the tight junction, can increase the drug absorption, leading to high systemic bioavailability. Furthermore, thanks to the antimicrobial effect, CS has gained great interest in antimicrobial therapies in association with PLGA in order to achieve NCs able to control drug encapsulation and release (PLGA core) while tuning the particle/infected lung environment interactions (CS coating). One of the main barriers in bacterial infections in the lung is represented by the bacteria biofilm, which some Gram (-) bacteria can produce. In particular, the ability of CS-covered PLGA NCs to effectively penetrate the bacteria biofilm of Pseudomonas aerugionosa , providing an antimicrobial depot in situ and to enhance drug activity for a longer time, has been demonstrated [ 246 , 247 ]. Just like the lipid nanoparticles mentioned earlier, numerous polymeric nucleic acid carriers form electrostatic interactions to bind their payload. These carriers are classified into two types—the polyelectrolyte complex (polyplex) and the polyplex micelle (micelleplex). The micelleplex formation typically incorporates hydrophobic groups to stabilize the polyplexes, which would otherwise rely solely on electrostatic interactions for their structural stability [ 248 ]. The formation of both poly- and micelleplexes depends on electrostatic interactions, which are established via protonable amine groups. As a result, polymeric materials with polyamine groups, such as polyethyleneimine (PEI), polylysine (PLL), or poly-(amidoamine) PAMAM, have been extensively studied for nucleic acid delivery [ 249 ]. For instance, PEI-based polyplexes have demonstrated significant potential in pulmonary delivery, and their ease of modification enables the attachment of shielding agents, targeting moieties, or lytic peptides to enhance their efficacy [ 250 , 251 , 252 ]. However, due to the inherent toxicity often associated with polyamines, it became clear that alternatives are required to develop safe and efficient NCs using polyplex systems. Poly(beta-amino esters) (PBAEs) emerged as a popular alternative due to their favorable toxicity profile. Several studies have demonstrated impressive results with inhaled PBAE formulations in efficiently condensing mRNA, facilitating intracellular uptake and releasing the gene cargo at the cytosol level to allow the translation of the encoded protein [ 252 ]. Furthermore, poloxamines have also been shown to effectively deliver RNA and DNA cargos to the lungs in CF treatment [ 212 ]. The developed NCs have demonstrated that they are able to facilitate the long-term restoration of CFTR in CFBE-delF cells and CF mice with a favorable safety profile. Despite the great potential of pulmonary nanotherapies, no products based on polymeric NCs, neither natural nor synthetic, are approved for human use. The complete evaluation of NC toxicity in vivo after aerosolization remains a significant challenge. Numerous are the results achieved in recent years, but an additional effort is needed to assess the safety of inhalable nanoformulations. 5.1. Natural Polymer-Based Nanocarriers Natural polymers appear to be very interesting materials in NC production thanks to their biocompatibility, low toxicity, and biodegradability [ 174 ]. Furthermore, the techniques usually employed in the production of natural polymer-based NCs (i.e., crosslinking gelation) are very gentle and characterized by low shear forces, thus they are ideal for the encapsulation of unstable molecules [ 175 , 176 ]. The main in vitro/in vivo findings achieved with natural polymer NCs for drug delivery to the lungs are reported in Table 2 . Between the natural materials for pulmonary delivery, albumin is one of the most studied polymers due to its low antigenicity, low toxicity, biocompatibility, biodegradability, low costs, and abundance. Serum albumin is the most abundant protein in the plasma, and it is characterized by a high affinity with different molecules; thus, as a nanocarrier, it has been chosen to incorporate a variety of active compounds [ 177 , 178 , 179 ]. pharmaceutics-16-00347-t002_Table 2 Table 2 Main in vitro/in vivo findings achieved with natural polymeric NCs for the pulmonary delivery of drugs. Polymer Encapsulated Molecule In Vitro Model In Vivo Model Main Findings Ref. Albumin Tacrolimus - Intratracheal administration in bleomycin-induced pulmonary fibrosis mouse Anti-fibrotic effect significantly higher than intraperitoneal administration [ 180 ] Albumin - Macrophages derived from BALB/C mice Oropharyngeal aspiration in male BALB/C mice High in vivo biocompatibility with mild inflammation at highest dose tested. Slower clearance. No accumulation in major organs [ 174 ] HSA Benzothiazinone (BTZ043) Murine bone marrow-derived macrophages infected with M. tuberculosis Intranasal instillation in old female C3HeB/FeJ mice infected with M. tuberculosis Enhanced efficacy in vitro compared to the free drug; reduced bacterial load in vivo [ 181 ] TRAIL-HSA Doxorubicin Apoptotic and cytotoxicity activity on H226 cell line (human lung squamous carcinoma cell line) Insufflation of nanoparticle dispersion in mouse bearing H226 cell-induced metastatic tumors Synergistic apoptotic activity and anti-tumor efficacy in vitro and in vivo [ 182 ] BSA siRNA Cellular uptake and cytotoxicity on A549 cell line; gene-silencing on KRAS G12S mutant A459 cells line - Low cytotoxicity with enhanced cellular uptake. High knock-down efficiency in vitro [ 183 ] CS Influenza vaccine Cytokines secretion in porcine monocyte-derived dendritic cells Intranasal nebulization in pigs Augmented cross-reactive T and B lymphocytes response [ 184 ] CS Bedaquiline Cytotoxicity profile on macrophage cell line Inhalation of freeze-dried nanoparticles in rats Low acute and chronic toxicity in vivo [ 185 ] CS Salmon Calcitonin - Intratracheal administration in rats Higher absorption and deposition in deep lung [ 186 ] CS Prothionamide - Intratracheal administration of dry powder containing nanoparticles in rats Prolonged drug persistence in lungs [ 187 ] CS-HA Gallium (III) Human epithelial bronchial cells (16HBE14o-) and P. aeruginosa Intratracheal administration of dry powder containing nanoparticles in rats Improved accumulation of drug in lung tissue and high tolerability in vivo [ 188 ] CS-PVA Magnolol Cytotoxicity profile on cells A549 cell line - Enhanced lung deposition with high cell viability [ 189 ] ALG_CS-DNase Tobramycin Antimicrobial activity on CF sputum sample and P. aeruginosa strain (PA01) Injection of nanoparticles dispersion in Galleria melonella Increased penetration across CF sputum and enhanced anti-pseudomonal activity in vitro and in vivo [ 190 ] ALG-CS/Tween80 Rifampicin and ascorbic acid Antibacterial activity on Mycobacterium Tuberculosis (M. tb.); cytotoxicity on kidney epithelial cells - Increased antibacterial activity Low cytotoxicity on kidney epithelial cell lines [ 191 ] List of Abbreviations: CS: Chitosan; BSA: Bovine-serum albumin; PVA: Poly(vinyl alcohol); siRNA: small interfering RNA; ALG: alginate; GCS: glycol chitosan; TGA: thioglycolic acid; HSA: human serum albumin; TRAIL: tumor necrosis factor (TNF)-related apoptosis-inducing ligand. In recent years, albumin NCs have gained considerable research attention as a drug delivery system owing to the approval by the FDA of nanoparticle albumin-bound (NAB) paclitaxel (Abraxane ® ) in the treatment of metastatic breast cancer (2005), advanced/metastatic non-small cell lung cancer (2012), and metastatic pancreatic cancer (2013). Inspired by the success of Abraxane ® , albumin-based NCs have stimulated interest also for inhalation [ 174 , 181 , 182 , 192 , 193 ]. The first in vivo proof-of-concept study on the lung biocompatibility and biodistribution of inhaled albumin NCs was performed by Woods et al. [ 174 ]. The results showed the absence of a significant inflammatory response in mice after the single pulmonary administration of bovine serum albumin (BSA) NCs as compared to the control BSA solution. Meanwhile, SPECT/CT imaging and post-mortem organ biodistribution studies demonstrated that lung tissue accumulation up to 48 h was significantly higher for BSA NCs compared with the control BSA solution. The absence of major NCs accumulation in secondary organs, and likely of related side effects, was further encouraging. In view of the proven efficacy of serum albumin NCs for cancer therapy [ 177 ], their potential for the direct delivery of anti-cancer drugs by inhalation has been explored [ 182 ]. Briefly, human serum albumin (HSA) NCs were loaded with doxorubicin and modified with the apoptotic protein TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) to maximize specificity for lung cancer cells. The resulting TRAIL/Dox HSA-NCs exhibited a robust anti-tumor effect after pulmonary administration in a xenograft mouse model [ 182 ]. Due to the presence of charged amino acids able to electrostatically interact with charged molecules, albumin raised awareness also for the delivery of nucleic acid therapeutics. With this idea in mind, Merkel and co-workers developed BSA NCs for the delivery of siRNA targeting the KRAS G12S mutation, the most frequent mutation in human cancers. The study demonstrated that BSA NCs could protect the siRNA payload against RNases, enable in vitro transport to A549 cells, and mediate significant sequence-specific KRAS knockdown, resulting in the reduced cell growth of siRNA-transfected lung cancer cells [ 183 ]. A relevant issue in the development of inhalable NCs based on albumin is represented by unfavorable mucus/carrier interactions. In fact, albumin may establish quite strong interactions with mucus components (i.e., an NC/mucin electrostatic interaction or covalent binding to mucin), which could be mitigated incorporating (e.g., cyclodextrin) or surface-engineering (e.g., PEGylation) polymers in NCs' design [ 194 , 195 ]. The mucoadhesivity of albumin NCs prompted the exploration of their potential for local rather than systemic delivery through the lungs. Along this line, J. Seo and co-workers demonstrated the superior efficacy of inhaled tacrolimus-bound albumin NCs in mice with bleomycin-induced pulmonary fibrosis as compared to an intraperitoneal tacrolimus solution [ 196 ]. When conceiving these systems for pulmonary delivery, the results of Papay et al. are also relevant, as they successfully developed lactose-based dry powders for inhalation embedding apigenin-loaded albumin NCs, likely useful for the local treatment of lung injury in asthmatic conditions [ 197 ]. Among natural polymers, chitosan (CS) has been widely investigated for pulmonary drug delivery [ 198 ]. The positive charge of CS is a critical attribute to improve the absorption by opening the tight junctions of the lung epithelium [ 199 , 200 ]. Furthermore, CS charges are responsible for the interaction with mucus components, and thus for the mucoadhesive effect, and the amount of charges could be tuned through the deacetylation grade [ 201 ]. In recent years, numerous studies have indicated CS-based NCs as promising inhalable delivery systems [ 202 ], both for systemic and local delivery. CS NCs are usually prepared by the gelation technique, coacervation, nanoprecipitation, and the reverse micellar method [ 203 ]. They are effective in protecting, controlling the release, and promoting drug absorption through lung epithelia when administered both as NC dispersion and embedded into inert carrier microparticles (Nano-embedded Microparticles—NEM) [ 186 , 189 , 204 ]. One of the most studied and interesting applications of CS is the development of drug delivery systems (DDS) for the pulmonary administration of antimicrobial drugs [ 185 , 205 ]. In fact, CS itself shows antibacterial effects likely ascribable to the electrostatic interactions between its protonated amino groups and the phosphoryl groups and lipopolysaccharides of bacterial cell membranes [ 206 , 207 , 208 ]. The consequent membrane perturbation and release of the cell content may result in an increased therapeutic effect of the drug cargo [ 209 ]. Furthermore, the affinity of CS with alveolar macrophages, due to the electrostatic interactions between the positively charged polymer and the negatively charged sialic acid on the cell membranes [ 210 ], boosted the development of CS-based delivery systems for the local treatment of tuberculosis. Different drugs have been encapsulated into CS or CS-derivative NCs with increased anti-tubercular activity with respect to the free drugs [ 185 , 211 , 212 ]. The mucoadhesive tendency of CS (mostly due to its chemical structure) is both an advantage and a disadvantage and has solicited the researchers' attention. In this direction, even if inhalable CS NCs were shown to be effective in different in vivo applications [ 188 , 201 , 213 ], it was recently highlighted how CS-NCs may be entrapped in the superficial mucus layer, which is quickly cleared, and are not expected to reach the underlying airway epithelium [ 214 , 215 ]. As a matter of fact, the current trend is based on the development of NCs modified on the surface with inert polymers, which can improve the particle mobility into the mucus gel layer [ 26 , 216 , 217 ]. Another issue is the in vivo elimination of inhaled CS NCs. Though CS NCs display an acceptable safety profile and low chronic toxicity [ 185 ], no study has conclusively demonstrated the complete biodegradation or elimination of CS NCs in vivo. Nevertheless, this aspect has been reviewed to help shed light on this point [ 218 ]. 5.2. Synthetic Polymer-Based Nanocarriers The pulmonary route has some distinctive features that significantly limit the panel of synthetic materials available for inhalation. Biodegradable aliphatic polyesters, such as poly (lactic-co-glycolic acid) (PLGA) or Poly(N-isopropyl acrylamide)(PNIPAM), are the most exploited polymers because of their biodegradability, biocompatibility, and versatility [ 2 , 219 , 220 , 221 ]. The properties of the polymer, such as the grade of hydrophobicity and the degradation rate, and those of the resulting NCs, such as the particle size, surface properties, and drug encapsulation/release, can be easily tuned in order to optimize the affinity and the compatibility of the delivery system with the lung environment. The main in vitro/in vivo findings achieved with synthetic polymer NCs for drug delivery to the lungs are reported in Table 3 . As reported in Table 2 , PLGA is among the polymers that have been extensively explored for the development of inhalable NCs. If PLGA-based NCs are considered promising in terms of drug encapsulation, protection, controlled release, and aerodynamic properties, these systems appear not always efficient in crossing cellular/extracellular pulmonary barriers. As a consequence, the examples of inhalable PLGA NCs reported in the literature are often engineered at the surface with hydrophilic polymers, which can shield nanocarrier interactions with lung-lining fluids, thus promoting drug transport to the target [ 222 , 223 , 224 , 231 , 232 , 233 ]. Polyethylene glycol (PEG) is the most widely exploited polymer to achieve mucoinert PLGA-based NCs. In fact, the PEGylation leads to the formation of a hydrophilic shell and neutral surface charge that prevents hydrophobic or electrostatic NC interactions with the mucus gel layer [ 26 , 234 , 235 ]. Surface properties can be further tuned by changing the PEG molecular weight and its density on the particle surface [ 234 , 235 ]. The transport of PEGylated NCs to the inner layer of the mucus likely increases their retention in the lungs by reducing the clearance mechanism and facilitating macrophages' escape [ 26 , 214 , 230 ]. The effectiveness of PEGylation in the pulmonary drug delivery of PLGA NCs was extensively explored and promising results have been achieved in both in vitro and in vivo models in different therapeutic applications, such as antimicrobial and anticancer therapy [ 236 , 237 ]. Alternative polymers for the functionalization of NCs able to assist their penetration through mucus have been investigated. These include poly(2-oxazolines), polysarcosine, zwitterionic polymers (polybetaines), proteolytic enzymes, and poly(vinyl alcohol) (PVA) [ 28 ]. Between them, PVA is the most employed emulsifier in polymeric nano- and microparticles and it is able to provide uniform particulate systems with low polydispersity. Recently, we have developed PLGA NCs engineered with PVA for the delivery of an antimicrobial peptide, Esculentin, to the lung [ 224 ]. We have found that the PVA shell can assist the particles transport across the mucus layer, which is very low for the naked antimicrobial peptide. Furthermore, the NCs can control the release of the encapsulated molecule, providing a prolonged antimicrobial effect on Pseudomonas aeruginosa both in vitro, on bacteria culture, and after pulmonary administration as NC dispersion in an acute infected murine model in vivo. Although the PVA is generally considered a mucoadhesive polymer, the ability of PVA to improve the particle diffusion across the mucus was found to be related to the grade of hydrolysis, the molecular weight, and the density of the particle shell. In particular, the non-covalent shell of partially hydrolized PVA, with a hydrolysis degree between 75 and 95%, were found to improve the PLA NCs' mobility in mucus [ 238 ]. Despite the efficacy in overcoming the mucus barrier, NC engineering with hydrophilic polymers seems to not be the best strategy in cellular target-based therapies, such as gene delivery, in which the NCs need to be designed for cell penetration [ 239 ]. An early exploited strategy to achieve mucus-penetrating particles able to improve the drug cell uptake is represented by the hybrid lipid-polymer NCs [ 55 , 172 , 240 ]. Hybrid NCs consisting of a PLGA core and a lipid shell of dipalmitoylphosphatidylcholine (DPPC) have been developed in our labs for the pulmonary delivery of siRNA. Thanks to its ability to form a shell around the polymer core, DPPC was chosen to improve NC compatibility and tolerance in the lung environment. Furthermore, it has been demonstrated that a DPPC shell can provide mucoinert and muco-diffusive properties to the particulate system while improving the drug cell uptake, crucial in siRNA therapies [ 46 , 74 , 163 , 230 ]. This strategy appears very promising in gene delivery, in which different lipids were used with success for the development of inhalable hybrid NCs [ 240 , 241 ]. In order to assist the gene uptake, particle surface engineering is often associated with the encapsulation of non-viral vectors as cationic polymers. One of the most employed in gene delivery is polyethylenimine (PEI), which can electrostatically interact with nucleic acids forming complexes (polyplexes), improving the encapsulation and release from polymeric NCs and facilitating gene cellular internalization [ 55 , 230 ]. A recent study investigated the impact of various biomimetic endogenous pulmonary phospholipids on the in vivo behavior of PLGA NCs. The study revealed that surface engineering with neutral phospholipids, such as DPPC and dipalmitoylphosphatidylamine (DPPE), resulted in a reduction in the uptake of NCs by alveolar macrophages, while the use of negatively charged phospholipids, such as dipalmitoylphos-phatidylserine (DPPS) and dipalmitoylphosphatidylglycerol (DPPG), enhanced the uptake. Regardless of the charge, phospholipid surface engineering increased the uptake of NCs by A549 cells and drug retention in the entire lung. DPPC, DPPE, and DPPG facilitated drug retention in BALF, whereas DPPS facilitated drug absorption in lung tissue. However, no impact of phospholipids on drug tissue distribution was observed [ 242 ]. Concerning the particle size, NCs with an hydrodynamic diameter of 100 nm showed significantly higher lung retention and tissue adsorption than NCs with a higher diameter (300, 800, and 2000 nm); therefore, NCs with a small size are the most effective for pulmonary drug delivery, especially for local lung disease therapy [ 243 ]. In order to conceive mucus-penetrating and long-residence DDS for pulmonary administration, macrophage uptake must be considered. Sometimes, being a further limit in drug pulmonary administration, it becomes a desired effect in specific macrophage-target therapy. In fact, the selective and controlled internalization of PLGA-based NCs loaded with anti-infective drugs by alveolar macrophages is under investigation for targeted antimicrobial therapy [ 7 , 183 , 210 ]. This is the case of antitubercular therapy, in which the target is Mycobacterium tuberculosis located in the alveolar macrophages (host cells) [ 244 ]. Unmodified PLGA NCs, which are usually inappropriate for pulmonary delivery due to their fast elimination by macrophage clearance, have been successfully developed as inhalable systems for the delivery of three frontline antitubercular drugs [ 245 ]. In the attempt to improve the NC uptake in macrophages and thus increase the drug concentration at the site of action, appropriate surface modifications of PLGA NCs have been explored. A recent study underlined the higher effectiveness of PLGA nanocapsules, modified with CS, in killing intracellular Staphylococcusaureus and Micobacterum abscessus with the same dose of the drug in its free form. The CS confers to NCs with a positively charged surface which is able to increase the uptake in macrophages and improve the drug effect [ 223 ]. Surface engineering the NC with CS has been exploited in different applications, such as lung infections and pulmonary fibrosis. As a matter of fact, despite the controversial discussion about the higher effectiveness of muco-inert particles with respect to the mucoadhesive ones, CS-modified NCs are able to improve the drug in vivo bioavailability after inhalation [ 180 , 222 ]. The CS mucoadhesive effect can increase the NC residence time in the lung and, thanks to the opening effect on the tight junction, can increase the drug absorption, leading to high systemic bioavailability. Furthermore, thanks to the antimicrobial effect, CS has gained great interest in antimicrobial therapies in association with PLGA in order to achieve NCs able to control drug encapsulation and release (PLGA core) while tuning the particle/infected lung environment interactions (CS coating). One of the main barriers in bacterial infections in the lung is represented by the bacteria biofilm, which some Gram (-) bacteria can produce. In particular, the ability of CS-covered PLGA NCs to effectively penetrate the bacteria biofilm of Pseudomonas aerugionosa , providing an antimicrobial depot in situ and to enhance drug activity for a longer time, has been demonstrated [ 246 , 247 ]. Just like the lipid nanoparticles mentioned earlier, numerous polymeric nucleic acid carriers form electrostatic interactions to bind their payload. These carriers are classified into two types—the polyelectrolyte complex (polyplex) and the polyplex micelle (micelleplex). The micelleplex formation typically incorporates hydrophobic groups to stabilize the polyplexes, which would otherwise rely solely on electrostatic interactions for their structural stability [ 248 ]. The formation of both poly- and micelleplexes depends on electrostatic interactions, which are established via protonable amine groups. As a result, polymeric materials with polyamine groups, such as polyethyleneimine (PEI), polylysine (PLL), or poly-(amidoamine) PAMAM, have been extensively studied for nucleic acid delivery [ 249 ]. For instance, PEI-based polyplexes have demonstrated significant potential in pulmonary delivery, and their ease of modification enables the attachment of shielding agents, targeting moieties, or lytic peptides to enhance their efficacy [ 250 , 251 , 252 ]. However, due to the inherent toxicity often associated with polyamines, it became clear that alternatives are required to develop safe and efficient NCs using polyplex systems. Poly(beta-amino esters) (PBAEs) emerged as a popular alternative due to their favorable toxicity profile. Several studies have demonstrated impressive results with inhaled PBAE formulations in efficiently condensing mRNA, facilitating intracellular uptake and releasing the gene cargo at the cytosol level to allow the translation of the encoded protein [ 252 ]. Furthermore, poloxamines have also been shown to effectively deliver RNA and DNA cargos to the lungs in CF treatment [ 212 ]. The developed NCs have demonstrated that they are able to facilitate the long-term restoration of CFTR in CFBE-delF cells and CF mice with a favorable safety profile. Despite the great potential of pulmonary nanotherapies, no products based on polymeric NCs, neither natural nor synthetic, are approved for human use. The complete evaluation of NC toxicity in vivo after aerosolization remains a significant challenge. Numerous are the results achieved in recent years, but an additional effort is needed to assess the safety of inhalable nanoformulations. 6. Harnessing Nanocarriers for Inhalation: From Liquid Aerosols to Dry Powders In order to square the circle of particle deposition in the deep airways, an approach that is gaining success is the development of nano-embedded microparticles (NEM). NEM are usually produced by spray drying or freeze drying and consist of nanocomposite particles obtained by the inclusion of drug-loaded NCs within microparticles made of an inert material (i.e., lactose, mannitol) [ 227 , 253 ]. The underlying concept is that, once the inert carrier reaches the deep lung and dissolves in the lung-lining fluid, the primary NCs are released to exert their action. The addition of this second level of complexity is expected to add a number of advantages to those already arising from the presence of an NC system, for instance, all the advantages arising from the micro-sized particles, such as better flow and aerosolization properties resulting in the engineering of a potentiated system for local therapy [ 16 , 254 ]. Furthermore, through a careful selection of the carrier material, it is also possible to further functionalize the delivery system. For instance, we have shown how hydroxypropyl beta cyclodextrin (HPβCD) can contribute to the inhibition of the biofilm activity in lung infections triggered by P. aeruginosa . This effect, based on previous evidence, can be explained by the entrapment of N-acyl homoserine lactones, involved in bacterial quorum sensing inside the CD cavity [ 84 , 255 ]. In another work, the addition of mannitol, thanks to its innate mucolytic activity, assists the released NCs in the navigation of the biofilm barrier, one of the biggest challenges in the development of CF therapies for lung administration [ 254 , 256 ]. Of note, to realize the full potential of this approach is necessary to have a thorough understanding of the physiological barrier and of the modifications that happen in pathologic conditions [ 172 ]. From a translational perspective, if the right attention is devoted to the process parameters, a powder can be manufactured which is ready to use as an MDI or DPI formulation featuring long-term stability, often even at room temperature [ 109 , 257 ]. However, a drawback that needs to be considered is the unavoidable dilution caused by the additional excipient that impacts the concentration of the active pharmaceutical ingredient (API), leading to a higher dose due to the diluted drug content, potentially raising concerns about tolerance as well [ 258 ]. This underscores the significance of carefully selecting the most suitable excipient. Some authors suggest that the choice of an excipient should go beyond serving as a stabilizer; it should also function as a shell former during the spray-drying process to optimize drug loading in the formulation [ 257 ]. 7. Patents on "Inhalable Nanocarriers" The rising interest in the application of NCs in inhalable therapy has promoted the patent applications. The most relevant patents in this field are reported in Table 4 , as identified by searching on the Espace Patent search web site ( https://worldwide.espacenet.com , accessed on 23 January 2024) "Inhalable nanocarriers". 8. Conclusions and Perspectives NCs have emerged as a promising strategy for drug delivery to the lungs, showcasing their potential in overcoming biological barriers, such as mucus and, in the context of pulmonary infections, the bacterial biofilms. The unique properties of NCs, including their size, surface charge, and tailored surface functionalities, enable them to navigate through the complex pulmonary environment. Their nanoscale dimensions facilitate penetration through mucus layers, while surface modifications can enhance interactions with specific cell target. Moreover, NCs offer controlled drug release profiles, improving drug efficacy and minimizing potential side effects. These characteristics make NCs a valuable strategy for targeted drug delivery to the lungs, particularly in respiratory diseases where overcoming lung barriers is pivotal for therapeutic success. The unique properties of NCs allow for the targeted delivery of drugs to the lungs, improved drug diffusion across the lung barriers, and the controlled release of drugs. However, several challenges still need to be addressed to fully realize the potential of NC-based therapies for pulmonary drug delivery. One major challenge is the potential toxicity of NCs to lung cells, which requires the careful evaluation and optimization of the NC properties. Additionally, there is a need for further research to optimize the NC formulation, stability, and pharmacokinetics in vivo. The use of advanced techniques for particle production and characterization can help to appropriately outline the fate and behavior of NCs in the lungs. Despite these challenges, NC-based pulmonary drug delivery has the potential to revolutionize the treatment of respiratory diseases, including gene therapy, lung infections, and rare diseases (i.e., CF). Continued research and development in this field can lead to the development of more effective and targeted therapies with fewer side effects, improving patient outcomes and quality of life.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2478701/

Development of a Rapid and Sensitive Immunoassay for Detection and Subsequent Recovery of Bacillus anthracis Spores in Environmental Samples

Bacillusanthracis is considered a major threat as an agent of bioterrorism. B. anthracis spores are readily dispersed as aerosols, are very persistent, and are resistant to normal disinfection treatments. Immunoassays have been developed to rapidly detect B. anthracis spores at high concentrations. However, detection of B. anthracis spores at lower concentrations is problematic due to the fact that closely related Bacillus species (e.g., B. thuringiensis ) can cross react with anti- B. anthracis antibodies, resulting in false positive detections. Subsequent polymerase chain reaction (PCR) analysis is required to differentiate virulent strains. We report here on a protocol for the rapid, sensitive detection of B. anthracis spore using the Integrating Waveguide Biosensor followed by a method for the rapid release and germination of immunocaptured spores. A detection limit of ca. 10 3 spores was achieved by incubating spores simultaneously with capture and detection antibodies ('liquid-phase" assay) prior to capture on capillary tubes/waveguides. Subsequent incubation with BHI broth directly in capillary tubes allowed for rapid germination, outgrowth, and release of spores, resulting in vegetative cells for PCR analysis.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10376142/

Cancer Drug Delivery Systems Using Bacterial Toxin Translocation Mechanisms

Recent advances in targeted cancer therapy hold great promise for both research and clinical applications and push the boundaries in finding new treatments for various currently incurable cancers. However, these therapies require specific cell-targeting mechanisms for the efficient delivery of drug cargo across the cell membrane to reach intracellular targets and avoid diffusion to unwanted tissues. Traditional drug delivery systems suffer from a limited ability to travel across the barriers posed by cell membranes and, therefore, there is a need for high doses, which are associated with adverse reactions and safety concerns. Bacterial toxins have evolved naturally to specifically target cell subtypes via their receptor binding module, penetrating the cell membrane efficiently through the membrane translocation process and then successfully delivering the toxic cargo into the host cytosol. They have, thus, been harnessed for the delivery of various drugs. In this review, we focus on bacterial toxin translocation mechanisms and recent progress in the targeted delivery systems of cancer therapy drugs that have been inspired by the receptor binding and membrane translocation processes of the anthrax toxin protective antigen, diphtheria toxin, and Pseudomonas exotoxin A. We also discuss the challenges and limitations of these studies that should be addressed before bacterial toxin-based drug delivery systems can become a viable new generation of drug delivery approaches in clinical translation. 1. Introduction Bacterial toxins are virulence factors that harm specific host cells by inhibiting cell growth and inducing cell death to favor bacterial infections that cause diseases in humans and animals [ 1 , 2 , 3 ]. Many bacterial toxins exert their toxic effects by targeting specific types of cells, entering the cells, and then interrupting key host intracellular cell signaling pathways [ 4 ]. The function of these bacterial toxins depends on their highly modular and efficient subdomains that can act as guided membrane translocation machinery; this includes the receptor binding domain, the translocation domain, as well as the catalytic domain. The receptor binding domain specifically targets host cell surface receptors and even host cell membranes, which enable the toxins to target various cell types, including neurons and immune cells [ 4 , 5 , 6 ]. The translocation domain confers the ability of toxins to become absorbed by the host cells. Additionally, the catalytic domain directly modulates host signaling pathways to inhibit host cell growth and even kill the host cells. The translocation domains of bacterial toxins, in particular, are an evolutionally powerful machine that can overcome the lipid bilayer barrier to deliver cargo into the host cells [ 7 , 8 ]. The overall translocation domains of bacterial toxins can be mainly divided into two classes, depending on the beta-sheet or the alpha-helix membrane integration elements. The former class of toxins is represented by the anthrax toxin, and the latter class is represented by the diphtheria toxin and botulinum neurotoxin [ 9 , 10 , 11 , 12 ]. The process by which bacterial toxins overcome the membrane barrier and achieve cargo delivery is an intricate and intriguing process involving a comprehensive and sequential series of events [ 4 ]. Understanding the precise molecular events during the membrane translocation of bacterial toxins is crucial for deciphering the cargo delivery process and reprogramming bacterial toxin translocation for various medical purposes, including targeted cancer drug delivery. Cancer is one of the leading causes of human death worldwide each year and is characterized by abnormal growth and uncontrollable expansion of cells. Despite great improvements in the treatment of cancer, it is still one of the top diseases that threaten human health [ 13 ]. It is still difficult to target and treat certain types of cancers because it is especially challenging to target and deliver the drugs to certain cancer cell types [ 14 ]. Bacterial toxins are a naturally evolved protein machinery that can target and deliver a toxic cargo to disrupt specific types of cells. Thus, harnessing the cell-specific transmembrane delivery properties of bacterial toxins to treat cancer is a promising strategy for the intracellular delivery of various drugs. Bacterial and plant toxins attached with cell-specific targeting monoclonal antibodies have been developed to kill cancer cells. These antibody-toxin bi-functional molecules are called immunotoxins (ITs), which are composed of antibodies that are produced by immune systems linked to toxins [ 15 , 16 ]. Several bacterial toxin-based immunotoxin cancer drugs have been approved, and more immunotoxin prodrugs are now under clinical trials [ 17 , 18 , 19 ]. In this review, we mainly focus on recent progress in the membrane translocation mechanisms of the anthrax toxin protective antigen (PA), diphtheria toxin, and Pseudomonas exotoxin A and the related cancer-targeting immunotoxins that are inspired by their receptor binding and membrane translocation processes. 2. Anthrax Toxin PA-Based Cancer Drug Delivery 2.1. Anthrax Toxin Anthrax toxin is the major virulence factor for Bacillus anthracis and is the causative agent of the severe disease called anthrax. It is a binary toxin that consists of the receptor-binding and translocation machinery protective antigen (PA) plus enzymatic executor factors, which are referred to as the lethal factor (LF) and edema factor (EF) ( Figure 1 ) [ 20 ]. The mechanism by which anthrax toxins exert their toxic effect on the host cell involves a series of sequential steps, which are summarized as follows: 1. Anthrax toxin PA specifically targets host cell membrane proteins called anthrax toxin receptor 1 (ANTXR1) as well as anthrax toxin receptor 2 (ANTXR2) [ 21 , 22 , 23 ]. Then, the 83 kDa PA monomer (PA83) is cleaved by the cell surface furin family protease to form an active form 63 kDa PA monomer (PA63). 2. Furin protease cleavage and PA63 oligomerization provide interfaces for LF and EF binding and create the pre-channel for LF and EF's further translocation. The PA63 heptamer is a more prevalent oligomerization state than the HA63 octamer on the host cell surface, even though the PA63 heptamer is less stable and more prone to form a premature channel than the PA63 octamer under physiologic temperatures and pH conditions. One explanation is that the host extracellular PA receptor drives the PA oligomerization and stabilizes the PA63 pentamer [ 24 , 25 ]. 3. The anthrax toxin complexes then become endocytosed by the host clathrin-mediated pathway. 4. Endosome acidification triggers the membrane insertion as well as the anthrax PA channel formation, which mediates the transmembrane delivery of LF and EF. 5. After endosome translocation, refolded anthrax toxin LF becomes a protein endoprotease that cleaves the N-terminal fragment of mitogen-activated protein kinase kinases (MAPKKs) and deactivates these kinases, leading to altered downstream signaling and cell apoptosis. Anthrax toxin EF is a calmodulin and Ca 2+ -dependent adenylyl cyclase. Refolded EF catalyzes the conversion of ATP to cAMP and induces the accumulation of intracellular cAMP, which can lead to impaired water homeostasis and edema [ 26 ]. 2.2. Anthrax Translocation Mechanisms As the membrane translocation module of the anthrax toxin, PA mediates the delivery of LF and EF through the membrane barrier into the host cytosol. LF and EF bind to the oligomeric HA63 pre-channel, forming the "flowers-in-vase" conformation, where the flowers correspond to the LF or EF cargo and the vase corresponds to the oligomeric HA ( Figure 2 a) [ 27 ]. The anthrax toxin complex hijacks the endocytosis process and enters the endosome, which then becomes acidified. The PA pre-channel is then triggered by the endosome's low pH to form a membrane-inserted pore structure that contains an ion-conductive channel for the cargo transport ( Figure 2 b) [ 9 ]. According to the cryo-EM structure of the PA channel, the pore architecture of PA is a mushroom-like object with a 7.5 nm long and 12.5 nm diameter cap and a stem that is 10.5 nm long and 2.7 nm in diameter. During channel formation, the PA domain 2 disordered 2β2-2β3 loops together with the flanking loops generating a long β barrel that inserts into the membrane and forms a channel that is embedded in the lipid bilayer. This transmembrane channel only allows the translocation of unfolded LF or EF [ 9 , 28 ]. MOLE toolkit analysis for the characterization of channel macromolecular structures shows that the PA channel could be divided into four parts from the top to the bottom: α clamp containing mouth, Φ clamp, negatively charged throat, and the tube ( Figure 2 b). The translocation of the cargo starts from the channel mouth near the α clamp, a hydrophobic groove created by two nearby protomers to nonspecifically bind to the cargo protein α helix. The narrowest part of the channel is the Φ clamp, with a diameter of 6 angstroms formed by the Phenylalanine 427 (Phe427) residues contributed by each PA protomer, which is just large enough to pass through the unfolded α-helix but not large enough to accommodate the well-folded protein [ 9 ]. Since the cargo should be unfolded to proceed through the Φ clamp, highly stable cargo is not able to be translocated efficiently [ 4 , 29 ]. The 19 F NMR study, with the site-specific labeling of the Phe427 residues with p-fluorophenylalanine (pF-Phe427), shows that pF-Phe427 is intrinsically dynamic in the pre-channel state and even more dynamic in the channel state. Such dynamic behavior of the Φ clamp could provide flexibility and room for unfolded polypeptide chain movement during cargo translocation [ 30 ]. The mouth on the top and the tube on the bottom are the opening of the channel, while the Φ clamp seals the channel to ensure that it is impermeable to small molecules before and during the cargo translocation. In contrast to the largely hydrophilic inner surface of the channel, the outer surface of the PA channel is largely hydrophobic, which could contribute to the binding of the hydrophobic lipid bilayer and stabilize the transmembrane channel [ 9 ]. PA63-bound LF or EF unfolding is induced by endosome acidification [ 31 ]. The N-terminus of PA63-bound LF or EF then enter the PA channel and initiate the entry of cargo into the PA channel [ 20 ]. In the presence of a pH gradient or membrane potential, the PA channel serves as an active transporter and moves the cargo to further the N-to-C translocation [ 28 , 31 ]. A charged state-dependent Brownian-ratchet mechanism, with the help of molecular chaperones in concert with the translocation process, leads to successful and efficient transmembrane cargo delivery [ 32 , 33 ]. Acidic amino acid residues within a PA cavity are mostly protonated and positively charged. Once exposed to the cytosol, the cargo residues are more negatively charged. Since the inner cavity of the channel is negatively charged, the negatively charged residues translocated out of the channel could not move back due to the electrostatic repulsion force, thus ensuring the unidirectional movement of the translocating cargo [ 9 , 29 ]. 2.3. Anthrax Toxin PA-Based Drug Delivery for Cancer Therapy The anthrax toxin tripartite system is versatile for the drug delivery of enzymatic moieties into cells. In 1992, Naveen Arora and Stephen H. Leppla et al. first reported that Pseudomonas exotoxin A ADP-ribosylation domain and the LF fusion protein could be delivered into the cytosol of mammalian cells by anthrax PA. This discovery opened a new frontier with regard to the use of anthrax toxin PA as a drug delivery system for various non-native cargoes [ 34 ]. Later discoveries have showed that the N-terminal sequences of PA initiate the translocation, and the N-terminal sequences of LF (LFn) are required to deliver the peptide into the cytosol [ 20 , 35 ]. Thus, various cancer cell-killing cargoes can fuse with LFn, and then be guided by LFn to translocate through the PA channel into the cytosol ( Table 1 ). Native PA mostly targets cells that express ANTXR1 and ANTXR2. To alter the targeting of these cells, PA domain 4 can be mutated (mPA) to ablate the binding to native receptors and then become fused with EGF (mPA-EGF) to target cancer cells that express the EGF receptor [ 36 ]. The conjugated mPA-EGF triggered apoptosis in EGFR-expressing bladder cancer cells within about three minutes of toxin exposure time. Additionally, upon mPA-EGF treatment, decreases in the tumor mass were consistently observed in six tested dogs with a treatment-resistant bladder. In tumor-free mice and dogs, mPA-EGF induced no toxicity [ 37 ]. Additionally, PA could be engineered to fuse with a HER2 high-affinity affibody (mPA-ZHER2) to deliver various cytocidal effectors into trastuzumab-resistant HER2-positive tumor cells and induce cell death [ 38 ]. To further decrease this toxin's off-target effects, the high-specificity tumor-targeting of anthrax-based drug delivery is required. Numerous proteases that enable tumor invasion and metastases are highly expressed in cancer cells and can be utilized for the cell-specific activation of anticancer pro-drugs. The furin cleavage site of PA could, thus, be mutated to sequences cleaved by proteases (such as matrix metalloproteinase and urokinase) that are highly expressed in target tumor cells [ 39 , 40 , 41 ]. Such an approach could synergize with cell-specific targeting moieties to further reduce non-specific toxicity to healthy cells and decrease the off-target adverse effects [ 42 ]. bioengineering-10-00813-t001_Table 1 Table 1 Anthrax toxin PA-based cancer prodrugs. Toxin/Toxin Fragment Targeting Moiety Target Cancer Cells or Diseases Obtained Outcome References C-terminus of PA c-Myc c-Myc-specific hybridoma cell line Mouse macrophages and c-Myc-specific hybridoma cell killing [ 43 ] Mutant PA (PA N682A D683A) EGF EGFR positive Human A431 epidermoid carcinoma cells Enzymatic effector proteins transported into A431 carcinoma cells [ 36 ] Mutant PA (mPA) HER2 Affibody HER2 positive breast cancer cell lines Specific killing of HER2 positive breast cancer cell lines; no off-target killing of HER2-negative cells [ 38 ] Zymogen activation PA ANTXR1/2 Ovarian tumor cell lines Selective killing of ovarian tumor cells; inhibition of ovarian tumor growth in preclinical xenograft models [ 42 ] 2.1. Anthrax Toxin Anthrax toxin is the major virulence factor for Bacillus anthracis and is the causative agent of the severe disease called anthrax. It is a binary toxin that consists of the receptor-binding and translocation machinery protective antigen (PA) plus enzymatic executor factors, which are referred to as the lethal factor (LF) and edema factor (EF) ( Figure 1 ) [ 20 ]. The mechanism by which anthrax toxins exert their toxic effect on the host cell involves a series of sequential steps, which are summarized as follows: 1. Anthrax toxin PA specifically targets host cell membrane proteins called anthrax toxin receptor 1 (ANTXR1) as well as anthrax toxin receptor 2 (ANTXR2) [ 21 , 22 , 23 ]. Then, the 83 kDa PA monomer (PA83) is cleaved by the cell surface furin family protease to form an active form 63 kDa PA monomer (PA63). 2. Furin protease cleavage and PA63 oligomerization provide interfaces for LF and EF binding and create the pre-channel for LF and EF's further translocation. The PA63 heptamer is a more prevalent oligomerization state than the HA63 octamer on the host cell surface, even though the PA63 heptamer is less stable and more prone to form a premature channel than the PA63 octamer under physiologic temperatures and pH conditions. One explanation is that the host extracellular PA receptor drives the PA oligomerization and stabilizes the PA63 pentamer [ 24 , 25 ]. 3. The anthrax toxin complexes then become endocytosed by the host clathrin-mediated pathway. 4. Endosome acidification triggers the membrane insertion as well as the anthrax PA channel formation, which mediates the transmembrane delivery of LF and EF. 5. After endosome translocation, refolded anthrax toxin LF becomes a protein endoprotease that cleaves the N-terminal fragment of mitogen-activated protein kinase kinases (MAPKKs) and deactivates these kinases, leading to altered downstream signaling and cell apoptosis. Anthrax toxin EF is a calmodulin and Ca 2+ -dependent adenylyl cyclase. Refolded EF catalyzes the conversion of ATP to cAMP and induces the accumulation of intracellular cAMP, which can lead to impaired water homeostasis and edema [ 26 ]. 2.2. Anthrax Translocation Mechanisms As the membrane translocation module of the anthrax toxin, PA mediates the delivery of LF and EF through the membrane barrier into the host cytosol. LF and EF bind to the oligomeric HA63 pre-channel, forming the "flowers-in-vase" conformation, where the flowers correspond to the LF or EF cargo and the vase corresponds to the oligomeric HA ( Figure 2 a) [ 27 ]. The anthrax toxin complex hijacks the endocytosis process and enters the endosome, which then becomes acidified. The PA pre-channel is then triggered by the endosome's low pH to form a membrane-inserted pore structure that contains an ion-conductive channel for the cargo transport ( Figure 2 b) [ 9 ]. According to the cryo-EM structure of the PA channel, the pore architecture of PA is a mushroom-like object with a 7.5 nm long and 12.5 nm diameter cap and a stem that is 10.5 nm long and 2.7 nm in diameter. During channel formation, the PA domain 2 disordered 2β2-2β3 loops together with the flanking loops generating a long β barrel that inserts into the membrane and forms a channel that is embedded in the lipid bilayer. This transmembrane channel only allows the translocation of unfolded LF or EF [ 9 , 28 ]. MOLE toolkit analysis for the characterization of channel macromolecular structures shows that the PA channel could be divided into four parts from the top to the bottom: α clamp containing mouth, Φ clamp, negatively charged throat, and the tube ( Figure 2 b). The translocation of the cargo starts from the channel mouth near the α clamp, a hydrophobic groove created by two nearby protomers to nonspecifically bind to the cargo protein α helix. The narrowest part of the channel is the Φ clamp, with a diameter of 6 angstroms formed by the Phenylalanine 427 (Phe427) residues contributed by each PA protomer, which is just large enough to pass through the unfolded α-helix but not large enough to accommodate the well-folded protein [ 9 ]. Since the cargo should be unfolded to proceed through the Φ clamp, highly stable cargo is not able to be translocated efficiently [ 4 , 29 ]. The 19 F NMR study, with the site-specific labeling of the Phe427 residues with p-fluorophenylalanine (pF-Phe427), shows that pF-Phe427 is intrinsically dynamic in the pre-channel state and even more dynamic in the channel state. Such dynamic behavior of the Φ clamp could provide flexibility and room for unfolded polypeptide chain movement during cargo translocation [ 30 ]. The mouth on the top and the tube on the bottom are the opening of the channel, while the Φ clamp seals the channel to ensure that it is impermeable to small molecules before and during the cargo translocation. In contrast to the largely hydrophilic inner surface of the channel, the outer surface of the PA channel is largely hydrophobic, which could contribute to the binding of the hydrophobic lipid bilayer and stabilize the transmembrane channel [ 9 ]. PA63-bound LF or EF unfolding is induced by endosome acidification [ 31 ]. The N-terminus of PA63-bound LF or EF then enter the PA channel and initiate the entry of cargo into the PA channel [ 20 ]. In the presence of a pH gradient or membrane potential, the PA channel serves as an active transporter and moves the cargo to further the N-to-C translocation [ 28 , 31 ]. A charged state-dependent Brownian-ratchet mechanism, with the help of molecular chaperones in concert with the translocation process, leads to successful and efficient transmembrane cargo delivery [ 32 , 33 ]. Acidic amino acid residues within a PA cavity are mostly protonated and positively charged. Once exposed to the cytosol, the cargo residues are more negatively charged. Since the inner cavity of the channel is negatively charged, the negatively charged residues translocated out of the channel could not move back due to the electrostatic repulsion force, thus ensuring the unidirectional movement of the translocating cargo [ 9 , 29 ]. 2.3. Anthrax Toxin PA-Based Drug Delivery for Cancer Therapy The anthrax toxin tripartite system is versatile for the drug delivery of enzymatic moieties into cells. In 1992, Naveen Arora and Stephen H. Leppla et al. first reported that Pseudomonas exotoxin A ADP-ribosylation domain and the LF fusion protein could be delivered into the cytosol of mammalian cells by anthrax PA. This discovery opened a new frontier with regard to the use of anthrax toxin PA as a drug delivery system for various non-native cargoes [ 34 ]. Later discoveries have showed that the N-terminal sequences of PA initiate the translocation, and the N-terminal sequences of LF (LFn) are required to deliver the peptide into the cytosol [ 20 , 35 ]. Thus, various cancer cell-killing cargoes can fuse with LFn, and then be guided by LFn to translocate through the PA channel into the cytosol ( Table 1 ). Native PA mostly targets cells that express ANTXR1 and ANTXR2. To alter the targeting of these cells, PA domain 4 can be mutated (mPA) to ablate the binding to native receptors and then become fused with EGF (mPA-EGF) to target cancer cells that express the EGF receptor [ 36 ]. The conjugated mPA-EGF triggered apoptosis in EGFR-expressing bladder cancer cells within about three minutes of toxin exposure time. Additionally, upon mPA-EGF treatment, decreases in the tumor mass were consistently observed in six tested dogs with a treatment-resistant bladder. In tumor-free mice and dogs, mPA-EGF induced no toxicity [ 37 ]. Additionally, PA could be engineered to fuse with a HER2 high-affinity affibody (mPA-ZHER2) to deliver various cytocidal effectors into trastuzumab-resistant HER2-positive tumor cells and induce cell death [ 38 ]. To further decrease this toxin's off-target effects, the high-specificity tumor-targeting of anthrax-based drug delivery is required. Numerous proteases that enable tumor invasion and metastases are highly expressed in cancer cells and can be utilized for the cell-specific activation of anticancer pro-drugs. The furin cleavage site of PA could, thus, be mutated to sequences cleaved by proteases (such as matrix metalloproteinase and urokinase) that are highly expressed in target tumor cells [ 39 , 40 , 41 ]. Such an approach could synergize with cell-specific targeting moieties to further reduce non-specific toxicity to healthy cells and decrease the off-target adverse effects [ 42 ]. bioengineering-10-00813-t001_Table 1 Table 1 Anthrax toxin PA-based cancer prodrugs. Toxin/Toxin Fragment Targeting Moiety Target Cancer Cells or Diseases Obtained Outcome References C-terminus of PA c-Myc c-Myc-specific hybridoma cell line Mouse macrophages and c-Myc-specific hybridoma cell killing [ 43 ] Mutant PA (PA N682A D683A) EGF EGFR positive Human A431 epidermoid carcinoma cells Enzymatic effector proteins transported into A431 carcinoma cells [ 36 ] Mutant PA (mPA) HER2 Affibody HER2 positive breast cancer cell lines Specific killing of HER2 positive breast cancer cell lines; no off-target killing of HER2-negative cells [ 38 ] Zymogen activation PA ANTXR1/2 Ovarian tumor cell lines Selective killing of ovarian tumor cells; inhibition of ovarian tumor growth in preclinical xenograft models [ 42 ] 3. Diphtheria Toxin Translocation Domain-Based Cancer Drug Delivery 3.1. Diphtheria Toxin and Its Mechanism of Translocation The Diphtheria toxin (DT) is a highly potent single-chain diphtheria-causing toxin that is produced by Corynebacterium diphtheriae with a lysogenic beta phage [ 44 , 45 ]. It is a short AB-type toxin that consists of a catalytic A subunit plus the receptor-binding and membrane translocation B subunit. The crystal structure of the Diphtheria toxin reveals a Y-shaped architecture with a cytotoxic enzymatic domain (A domain), a receptor-binding domain (B domain) on top, and the translocation domain (T domain) on the bottom [ 11 ]. The B domain first binds to the host cell receptor heparin-binding EGF-like growth factor (HB-EGF) and then becomes endocytosed by the host endocytosis pathway into an endosome. Then, the endosome's low pH facilitates the structural rearrangement of the T domain as well as the membrane translocation of the A domain into the cytosol ( Figure 3 ). Once there, the A domain refolds and targets eEF-2 through the addition of ADP-ribose, which subsequently inhibits protein synthesis and leads to cell death [ 46 , 47 , 48 ]. The T domain of DT is mainly composed of a helical architecture [ 11 ]. The acidic environment within the endosome induces the partial unfolding of the T domain and the formation of a molten globule. During translocation, the T domain is triggered by the endosome acidic pH, and a loop in between helix 8 and helix 9 initiates the endosome membrane interaction and insertion of the T domain upon the protonation of the residues glutamic acid 349 (Glu349) and aspartic acid 352 (Asp352). In addition, the proline 345 (Pro345) at the end of helix 8 is also critical for mediating the membrane insertion of the T domain [ 49 , 50 , 51 , 52 ]. At least two hydrophobic helical segments are then inserted into the endosome membrane to form the channel for A domain translocation. This is referred to as the "double dagger" model for DT translocation. The helical "double dagger" motifs (the T domain hydrophobic helices 5–9) are very conserved [ 11 , 51 , 53 ]. 3.2. Diphtheria Toxin T Domain-Based Drug Delivery Compared with other bacterial toxins, diphtheria toxin is a readily expressed and extremely potent toxin that has minimal adverse effects on humans; it is thus widely used to selectively treat various cancers. Replacing the B domain with various cancer antigen-targeting antibodies or growth factors can successfully achieve tumor cell-specific targeting and tumor cell killing ( Table 2 ). For example, interleukin-2 (IL-2) is an important immunomodulatory cytokine, mainly produced by CD4-positive (CD4+) T cells, and thus can be utilized to target some tumor cells that overexpress interleukin-2 receptor (IL-2R). A diphtheria toxin in which the B domain is truncated (DAB486) was fused with IL-2 to form a recombinant protein called DAB486IL-2 [ 54 , 55 ]. A subsequent shorter version of the recombinant protein DAB389IL-2 showed reduced immunogenicity and an increased half-life of the recombinant protein [ 56 ]. In a cell toxicity assay, DAB389IL-2 showed at least 100 times lower half maximal inhibitory concentrations (IC50s) to hematopoietic tumor cells expressing high affinity IL-2R than cells expressing low-affinity IL-2R. Success in clinical trials for the treatment of persistent and recurrent cutaneous T-cell lymphoma (CTCL) led to the FDA approval of DAB389IL-2 (denileukin diftitox or ONTAK TM ) in 2008. However, ONTAK TM suffered from production issues due to its E. coli expression system. It also had a severe side effect of vascular leak syndrome and was thus discontinued in 2014. The following studies show that ONTAK TM from diphtheria toxin-resistant yeast or C.diphtheria expression systems have higher activity and purity than that from E.coli [ 46 , 57 ]. In addition, vascular leaks can be reduced by mutated versions of immunotoxins [ 58 , 59 ]. Similarly, since IL3-R is highly expressed in blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, DT388IL-3 was developed to selectively kill IL3-R overexpressing dendritic cell neoplasm cells [ 17 , 60 ]. The clinical trial results on patients with BPDCN have shown major responses, including complete response (CR) and partial response (PR), which has led to the FDA approval of DAB388IL-3 under the commercial name of Tagraxofusp TM in 2018 [ 19 ]. 3.1. Diphtheria Toxin and Its Mechanism of Translocation The Diphtheria toxin (DT) is a highly potent single-chain diphtheria-causing toxin that is produced by Corynebacterium diphtheriae with a lysogenic beta phage [ 44 , 45 ]. It is a short AB-type toxin that consists of a catalytic A subunit plus the receptor-binding and membrane translocation B subunit. The crystal structure of the Diphtheria toxin reveals a Y-shaped architecture with a cytotoxic enzymatic domain (A domain), a receptor-binding domain (B domain) on top, and the translocation domain (T domain) on the bottom [ 11 ]. The B domain first binds to the host cell receptor heparin-binding EGF-like growth factor (HB-EGF) and then becomes endocytosed by the host endocytosis pathway into an endosome. Then, the endosome's low pH facilitates the structural rearrangement of the T domain as well as the membrane translocation of the A domain into the cytosol ( Figure 3 ). Once there, the A domain refolds and targets eEF-2 through the addition of ADP-ribose, which subsequently inhibits protein synthesis and leads to cell death [ 46 , 47 , 48 ]. The T domain of DT is mainly composed of a helical architecture [ 11 ]. The acidic environment within the endosome induces the partial unfolding of the T domain and the formation of a molten globule. During translocation, the T domain is triggered by the endosome acidic pH, and a loop in between helix 8 and helix 9 initiates the endosome membrane interaction and insertion of the T domain upon the protonation of the residues glutamic acid 349 (Glu349) and aspartic acid 352 (Asp352). In addition, the proline 345 (Pro345) at the end of helix 8 is also critical for mediating the membrane insertion of the T domain [ 49 , 50 , 51 , 52 ]. At least two hydrophobic helical segments are then inserted into the endosome membrane to form the channel for A domain translocation. This is referred to as the "double dagger" model for DT translocation. The helical "double dagger" motifs (the T domain hydrophobic helices 5–9) are very conserved [ 11 , 51 , 53 ]. 3.2. Diphtheria Toxin T Domain-Based Drug Delivery Compared with other bacterial toxins, diphtheria toxin is a readily expressed and extremely potent toxin that has minimal adverse effects on humans; it is thus widely used to selectively treat various cancers. Replacing the B domain with various cancer antigen-targeting antibodies or growth factors can successfully achieve tumor cell-specific targeting and tumor cell killing ( Table 2 ). For example, interleukin-2 (IL-2) is an important immunomodulatory cytokine, mainly produced by CD4-positive (CD4+) T cells, and thus can be utilized to target some tumor cells that overexpress interleukin-2 receptor (IL-2R). A diphtheria toxin in which the B domain is truncated (DAB486) was fused with IL-2 to form a recombinant protein called DAB486IL-2 [ 54 , 55 ]. A subsequent shorter version of the recombinant protein DAB389IL-2 showed reduced immunogenicity and an increased half-life of the recombinant protein [ 56 ]. In a cell toxicity assay, DAB389IL-2 showed at least 100 times lower half maximal inhibitory concentrations (IC50s) to hematopoietic tumor cells expressing high affinity IL-2R than cells expressing low-affinity IL-2R. Success in clinical trials for the treatment of persistent and recurrent cutaneous T-cell lymphoma (CTCL) led to the FDA approval of DAB389IL-2 (denileukin diftitox or ONTAK TM ) in 2008. However, ONTAK TM suffered from production issues due to its E. coli expression system. It also had a severe side effect of vascular leak syndrome and was thus discontinued in 2014. The following studies show that ONTAK TM from diphtheria toxin-resistant yeast or C.diphtheria expression systems have higher activity and purity than that from E.coli [ 46 , 57 ]. In addition, vascular leaks can be reduced by mutated versions of immunotoxins [ 58 , 59 ]. Similarly, since IL3-R is highly expressed in blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells, DT388IL-3 was developed to selectively kill IL3-R overexpressing dendritic cell neoplasm cells [ 17 , 60 ]. The clinical trial results on patients with BPDCN have shown major responses, including complete response (CR) and partial response (PR), which has led to the FDA approval of DAB388IL-3 under the commercial name of Tagraxofusp TM in 2018 [ 19 ]. 4. Pseudomonas Exotoxin A Translocation Domain-Based Cancer Drug Delivery 4.1. Pseudomonas Exotoxin A and Its Translocation Mechanism Pseudomonas exotoxin A (PE) is a highly potent toxin that is secreted by Pseudomonas aeruginosa . It is a single-chain multidomain AB toxin made up of an enzymatic A fragment and a cell-binding B fragment. The B fragment of PE specifically binds to the host cell receptor LRP1 (low-density lipoprotein receptor-related protein 1, or α2-macroglobulin), and then this toxin is subsequently internalized by clathrin-coated vesicles-mediated endocytosis. After furin cleavage and protein disulfide isomerase reduction, the cleaved PE fragment (in the late endosome) reaches the trans-Golgi network via the Rab9-regulated pathway and then the ER by KDEL-receptor pathway in a retrograde manner [ 67 , 68 , 69 , 70 , 71 , 72 ]. Alternatively, receptor-bound PE, with the help of the detergent-resistant membrane microdomain (lipid rafts) and caveolae-mediated endocytosis, hijacks the lipid-dependent sorting pathway to reach ER directly. Then PE utilizes the conserved cellular quality control ER-associated protein degradation pathway to move into the cytosol [ 73 ]. Once translocated, the catalytic A fragment subsequently inhibits the function of eukaryotic elongation factor-2 (eEF-2), which is critical for host protein synthesis through its ADP-ribosyltransferase activity using NAD+. This mechanism is very similar to that used by the Diphtheria toxin [ 74 ]. 4.2. Pseudomonas Exotoxin-Based Cancer Drug Delivery As one of the most potent bacterial toxins, PE-based immunotoxins for cancer treatment have also attracted intensive investigation and gained remarkable success. To minimize the protein size and reduce immune clearance, PE40 and PE38 have been created by removing the native receptor binding domain of PE. Then the truncated versions of the PE were linked to various targeting moieties such as antibodies, antibody fragments, or ligands ( Table 3 ) [ 75 ]. As a successful example, Moxetumomab pasudotox (FDA approval: 2018) is a recombinant protein of PE38 that is fused with the disulfide stabilized variable fragment (dsFv) of the monoclonal antibody RFB4 against CD22. Since CD22 is an inhibitory BCR (B-cell receptor) co-receptor that is highly expressed in malignant B cells such as hairy cell leukemia (HCL), Moxetumomab pasudotox showed high specificity as well as high toxicity toward HCL tumor cells [ 76 ]. In addition, with an improved version of the original RFB4 antibody with higher CD22 affinity and the improvement of the Moxetumomab production process, Moxetumomab pasudotox showed remarkably enhanced IT activity, higher HCL efficacy and reduced toxicity in clinical trials [ 77 ]. In late 2018, Moxetumomab pasudotox (Lumoxiti TM ) was approved by the US FDA as a treatment for adult patients with HCL refractory to prior systemic chemotherapy [ 18 ]. Moxetumomab pasudotox was approved by the European Medicines Agency (EMA) for HCL treatment in December 2020. However, Moxetumomab pasudotox still shows adverse effects such as capillary leakage syndrome and decreased renal function. Such side effects are mostly due to the non-specific targeting of Moxetumomab pasudotox to normal cells. Efforts have been made to generate less immunogenetic versions of IT mutants with less binding to normal cells [ 58 , 78 ]. Besides Moxetumomab pasudotox, PE has been fused with interleukin 13 or antibodies targeting CD326 (EpCAM), EGFR, and mesothelin for the treatment of various types of tumors. These ITs are still in clinical trials or have been discontinued due to either severe side effects or low efficacy. Given the FDA and EMA-approved Moxetumomab pasudotox for HCL treatment and continuous efforts to reduce the immunogenicity and off-targeting of PE-ITs, PE-based ITs are still a promising field for targeted cancer therapies [ 79 , 80 , 81 ]. 4.1. Pseudomonas Exotoxin A and Its Translocation Mechanism Pseudomonas exotoxin A (PE) is a highly potent toxin that is secreted by Pseudomonas aeruginosa . It is a single-chain multidomain AB toxin made up of an enzymatic A fragment and a cell-binding B fragment. The B fragment of PE specifically binds to the host cell receptor LRP1 (low-density lipoprotein receptor-related protein 1, or α2-macroglobulin), and then this toxin is subsequently internalized by clathrin-coated vesicles-mediated endocytosis. After furin cleavage and protein disulfide isomerase reduction, the cleaved PE fragment (in the late endosome) reaches the trans-Golgi network via the Rab9-regulated pathway and then the ER by KDEL-receptor pathway in a retrograde manner [ 67 , 68 , 69 , 70 , 71 , 72 ]. Alternatively, receptor-bound PE, with the help of the detergent-resistant membrane microdomain (lipid rafts) and caveolae-mediated endocytosis, hijacks the lipid-dependent sorting pathway to reach ER directly. Then PE utilizes the conserved cellular quality control ER-associated protein degradation pathway to move into the cytosol [ 73 ]. Once translocated, the catalytic A fragment subsequently inhibits the function of eukaryotic elongation factor-2 (eEF-2), which is critical for host protein synthesis through its ADP-ribosyltransferase activity using NAD+. This mechanism is very similar to that used by the Diphtheria toxin [ 74 ]. 4.2. Pseudomonas Exotoxin-Based Cancer Drug Delivery As one of the most potent bacterial toxins, PE-based immunotoxins for cancer treatment have also attracted intensive investigation and gained remarkable success. To minimize the protein size and reduce immune clearance, PE40 and PE38 have been created by removing the native receptor binding domain of PE. Then the truncated versions of the PE were linked to various targeting moieties such as antibodies, antibody fragments, or ligands ( Table 3 ) [ 75 ]. As a successful example, Moxetumomab pasudotox (FDA approval: 2018) is a recombinant protein of PE38 that is fused with the disulfide stabilized variable fragment (dsFv) of the monoclonal antibody RFB4 against CD22. Since CD22 is an inhibitory BCR (B-cell receptor) co-receptor that is highly expressed in malignant B cells such as hairy cell leukemia (HCL), Moxetumomab pasudotox showed high specificity as well as high toxicity toward HCL tumor cells [ 76 ]. In addition, with an improved version of the original RFB4 antibody with higher CD22 affinity and the improvement of the Moxetumomab production process, Moxetumomab pasudotox showed remarkably enhanced IT activity, higher HCL efficacy and reduced toxicity in clinical trials [ 77 ]. In late 2018, Moxetumomab pasudotox (Lumoxiti TM ) was approved by the US FDA as a treatment for adult patients with HCL refractory to prior systemic chemotherapy [ 18 ]. Moxetumomab pasudotox was approved by the European Medicines Agency (EMA) for HCL treatment in December 2020. However, Moxetumomab pasudotox still shows adverse effects such as capillary leakage syndrome and decreased renal function. Such side effects are mostly due to the non-specific targeting of Moxetumomab pasudotox to normal cells. Efforts have been made to generate less immunogenetic versions of IT mutants with less binding to normal cells [ 58 , 78 ]. Besides Moxetumomab pasudotox, PE has been fused with interleukin 13 or antibodies targeting CD326 (EpCAM), EGFR, and mesothelin for the treatment of various types of tumors. These ITs are still in clinical trials or have been discontinued due to either severe side effects or low efficacy. Given the FDA and EMA-approved Moxetumomab pasudotox for HCL treatment and continuous efforts to reduce the immunogenicity and off-targeting of PE-ITs, PE-based ITs are still a promising field for targeted cancer therapies [ 79 , 80 , 81 ]. 5. Discussion Intracellular proteins and signaling pathways represent vast drug targets, yet the cell membrane is a formidable barrier that prevents drugs from reaching their intracellular targets. Various drug delivery approaches are now being developed and optimized to overcome this challenge, including adeno-associated virus vectors, lipid nanoparticles, toxin proteins, endosymbiotic bacterial extracellular contractile injection systems (eCISs), and homologs of capsid protein-based platforms [ 85 , 86 , 87 ]. Among these, bacterial toxins have evolved by nature to efficiently penetrate the cell membrane and successfully deliver effector proteins into the host cytosol. Compared with systemic delivery systems, bacterial toxin-based targeted delivery systems are poised to minimize the off-target accumulation of drugs and thus have lower side effects. In recent decades, various bacterial toxin-based anti-cancer drugs have been designed and developed for targeted cancer therapy. Numerous tumor cell targeting moieties have also been optimized to increase targeting specificity and avoid general systemic diffusion. Among them, tamed Anthrax PA, Diphtheria toxin, and PE-based immunotoxins have been demonstrated to specifically deliver toxic cargoes into cells efficiently and cure previously hard-to-treat cancers. Nonetheless, the off-target effects remain a concern in current bacterial toxin-based therapies. A common off-target side effect is the capillary leak syndrome. When a toxin is administered intravenously, it enters the tissue from the capillary bed and can nonspecifically kill capillary endothelial cells. Thus, plasma fluid often leaks from the damaged capillary bed into nearby viscera, causing hypotension and fluid retention. Such off-target toxicity can be managed conservatively with hydration and steroids in the hope that the capillary leak syndrome is short-lived and can be controlled [ 59 , 60 , 78 , 88 ]. However, improved targeting moieties, with minimal off-target binding to reduce capillary leak syndrome, are still urgently needed. A second problem of current bacterial toxin-based targeted delivery systems is the low efficiency of translocation during transmembrane cargo delivery. Currently, we know few details about the translocation of most bacterial toxins due to their dynamic nature, drastic structural rearrangements, as well as the involvement of the lipid bilayer environment. This highlights the need to study these mechanisms further. Another problem of the current bacterial toxin-based delivery systems is the immune clearance of the drug. Because bacterial toxins are exogenous antigens, the immune systems of patients can recognize the toxin and neutralize it before it enters the targeting cells, which can significantly reduce the efficacy of these immunotoxins. Even though various mutants have been designed to reduce immunogenicity based on the study of the B cell and T cell epitopes as well as human neutralizing antibodies, advances in our understanding of immunology could help to design de-immunogenized versions of the bacterial toxins so that they have less clearance by anti-drug antibodies [ 89 , 90 , 91 , 92 ]. 6. Future Directions Capillary leak syndrome is one of the leading adverse effects of immunotoxin therapeutics. Although it can be partially controlled by proper medical management, it is still the dominant dose-limiting factor of ITs [ 78 , 88 ]. Capillary leak syndrome is initiated by the binding and damage to human endothelial cells by ITs. Previous studies have shown that toxin consensus structural motifs (x)D(y) are exposed to toxin surfaces and affect cell-cell interactions and damage endothelial cells, where x could be amino acid L, I, G, or V, and y could be amino acid V, L, or S. For example, the Diphtheria toxin A subunit contains two VDS motifs, while the PE38 toxin fragment has one GDV and two GDL motifs. The deletion or mutation of these (x)D(y) structural motifs without compromising IT efficacy is a successful approach in decreasing human endothelial cell damage and the resulting capillary leak syndrome [ 58 , 59 , 60 ]. Additionally, other motifs are involved in the nonspecific cell binding and the associated side effect of Its [ 78 ]. To gain a comprehensive understanding of the underlying etiology and origins of these side effects, it is crucial to identify targeted human cell lines and employ human models to mimic the capillary leak syndrome. Further studies involving cell surface toxin receptor screening, as well as toxin binding motif identification and modulation, could allow for the development of an improved version of ITs with reduced adverse effects stemming from non-specific binding [ 3 , 6 ]. Another major hurdle for IT-based drug delivery is the relatively low translocation efficiency across cellular membranes, as a significant portion of toxin molecules fail to reach the cytosol [ 93 ]. A mechanistic understanding of the bacterial toxin translocation process, especially the interplay between the toxin and lipid membrane during translocation, is crucial for realizing the potential of bacterial toxin-based immunotoxins. To unveil the structural and functional dynamics of bacterial toxins during translocation, high-resolution single-particle cryo-Electron Microscopy, single-molecule fluorescence resonance energy transfer (FRET), and electrophysiology, in combination with liposome and nanodisc lipid bilayer systems are needed to determine the high-resolution of structures and measure the functional dynamics of toxin translocation intermediates in detergents and a native-like lipid environment embedded in nanodiscs. Studying these structures could advance our comprehensive understanding of the spatial and temporal patterns of the protein cargo transmembrane delivery process of these toxins at the single-molecule level [ 10 , 94 , 95 ]. This could also contribute to the engineering and optimization of bacterial toxin translocation domains that can deliver cancer drugs into the sub-cellular compartment with enhanced efficacy of delivery [ 5 ]. Such an understanding could even establish a solid foundation to further design and engineer novel and programmable drug delivery systems for various intracellular protein-targeting drugs based on this naturally evolved and delicate protein delivery machinery [ 85 ]. The current ITs mainly utilize native toxic cargo to achieve cancer cell killing. Due to the highly modular nature of ITs, it is relatively easy to replace native toxic cargoes with other cargoes to finetune the intracellular pathways and cell-killing effects [ 4 ]. Phage-assisted evolution is another powerful approach to evolve toxin cargoes into enzymes with reprogrammed specificity. As a successful example, botulinum neurotoxin has been evolved by phage-assisted evolution to cleave the phosphatase and tensin homolog but not its native substrate in neurons [ 96 ]. It can be used to fine-tune the toxicity of the cargo and modulate vast intracellular cancer pathways to achieve precision cancer medicine. Since bacterial toxin-based targeted delivery systems can specifically target cancer cells and kill them, such systems, with new antigenic targets, optimized translocation domains, fine-tuned toxic cargoes, and reduced off-target toxicity and immunogenicity, hold great promise to push the boundaries in developing novel treatments of cancers that remain incurable. The combination of immunotoxins with chimeric antigen receptor T cells (CAR-T), immune checkpoint blockade therapy, as well as anticancer nanoparticles can also create novel treatment opportunities for synergistic and superior anticancer outcomes [ 60 , 97 , 98 , 99 , 100 ].

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9958956/

Bacteriophage T4 Head: Structure, Assembly, and Genome Packaging

Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures and mechanisms. Recent discoveries on the T4 head (capsid) structure, portal vertex, and genome packaging add a significant body of new literature to phage biology. Head structures in unexpanded and expanded conformations show dramatic domain movements, structural remodeling, and a ~70% increase in inner volume while creating high-affinity binding sites for the outer decoration proteins Soc and Hoc. Small changes in intercapsomer interactions modulate angles between capsomer planes, leading to profound alterations in head length. The in situ cryo-EM structure of the symmetry-mismatched portal vertex shows the remarkable structural morphing of local regions of the portal protein, allowing similar interactions with the capsid protein in different structural environments. Conformational changes in these interactions trigger the structural remodeling of capsid protein subunits surrounding the portal vertex, which propagate as a wave of expansion throughout the capsid. A second symmetry mismatch is created when a pentameric packaging motor assembles at the outer "clip" domains of the dodecameric portal vertex. The single-molecule dynamics of the packaging machine suggests a continuous burst mechanism in which the motor subunits adjusted to the shape of the DNA fire ATP hydrolysis, generating speeds as high as 2000 bp/s. 1. Introduction One of us (Rao) was closely associated with Lindsay Black for 4 decades. The following are a few words from him on this background: my journey with Lindsay began on 7 December 1980, on a blistering cold winter night in Baltimore. It was fitting that I came to Lindsay's lab "cold", knowing little about bacteriophage T4 or phages in general. I was a biochemist by training, worked on fungal amylases for my PhD at the Indian Institute of Science, Bangalore, and took the first flight to US the day after submitting my thesis. Lindsay picked me up at the airport and drove me in his ancient stick-shift Volkswagen Beetle and dropped me off in a dorm room across his laboratory at the University of Maryland Medical School, Baltimore. So it began. For the next 9 years, Lindsay's trust, extraordinary patience, unique insights, and bedrock principle of empowering his students and postdocs fueled my passion for T4 phage and led to our first biochemical papers on DNA packaging [ 1 , 2 ]. We continued sharing our interests over the next 30-plus years and cowrote several review articles on T4 head structure, assembly, and genome packaging [ 3 , 4 , 5 ]. What follows here is an update of these reviews, focusing on the most significant recent advancements and discoveries of these shared interests. 2. Architecture of T4 Head The T4 and T4-like bacteriophages belong to Straboviridae family and are ubiquitously distributed on Earth [ 6 , 7 ]. They occupy nearly all environmental niches that are often quite hostile, such as the guts of mammals and humans, and sewage waters. Phage T4 infecting the Escherichia coli bacterium has long served as an extraordinary model to elucidate the basic mechanisms of molecular biology. Its particular similarities with herpes viruses [ 8 ] make it a compelling model to tease out the mechanisms of virus assembly and genome packaging, and to develop as a platform for drug discovery, vaccine design, and other biotechnology and biomedical applications [ 9 , 10 , 11 ]. The basic features of T4 virion include a large elongated (prolate) icosahedral head containing 155 hexameric capsomers made of the major capsid protein gp23* and 11 pentameric vertices made of the minor capsid protein gp24* ( Figure 1 A,B). The 12th vertex is a unique dodecameric portal vertex made of gp20 through which the genome enters and exists the capsid. T4 has a 140 nm long contractile tail, which terminates with a complex multiprotein hexagonal baseplate to which six ~160 nm long kinked tail fibers are attached. The T4 head, tail, and fibers assemble by separate pathways and then join to form the infectious virion [ 12 , 13 ]. 2.1. Capsid Shell The structure and dimensions of the phage T4 prolate capsid shell are displayed in Figure 2 A. The head is elongated along its fivefold axis and has a length of 120 nm and a width of 86 nm [ 14 , 15 ]. The head encapsidates ~171 kbp linear double-stranded genomic DNA (~2–3% more than the unit-length genome). The major capsid protein, gp23*, is organized into a hexagonal lattice characterized by the triangulation numbers T end = 13 laevo for the end caps and T mid = 20 for the elongated midsection [ 14 ]. Gp23* is the cleaved form of gp23 from which the 65 N-terminal residues are removed during capsid maturation by a prohead protease. The prolate shell contains 930 subunits, or 155 hexameric capsomers of gp23* [ 14 , 16 , 17 ]. Like the major capsid proteins of other tailed phages, gp23* subunits [ 15 , 18 ] have a polypeptide fold similar to that of the bacteriophage HK97 capsid protein [ 19 ] ( Figure 2 B). This fold is characterized by the wedge-shaped axial (A) domain located near the capsomer axis and the peripheral (P) domain, forming the capsomer's periphery [ 19 ]. T4 gp23* has an additional 60-residue globular insertion (I) domain, which makes characteristic bumps on the capsid surface [ 15 , 18 , 20 , 21 ]. This I domain is connected to the rest of the structure via long linkers, which are analogous to the elongated E loop in the HK97 fold [ 19 ]. The I domain is involved in extensive intra-capsomer interactions [ 15 , 18 ]. In the gp23* capsomers, the I domain sits on top of a neighboring subunit belonging to the same hexameric capsomer, thus greatly reinforcing the capsomer structure. The I domain linkers are also involved in stabilizing interactions with the subunits of the same capsomer and with neighboring capsomers. Although extra domains inserted into the HK97 fold were also observed in the capsid proteins of other phages, such as phi29 and P22 [ 22 , 23 , 24 , 25 ], the T4 I domain inserted into the E loop and resting on a neighboring subunit from the same capsomer is a unique feature of T4-like phages. In the mature capsid, the gp23* protein contains an extended N-arm in its N-terminal region, like the major capsid protein of HK97. In addition, gp23* contains an unusual 25-residue N-terminal N-fist ( Figure 2 B) structure that interacts with four subunits: two from the same capsomer and two from an adjacent capsomer [ 18 ]. The intercapsomer binding is reinforced by attractive electrostatic forces between the P domains of gp23* subunits in different capsomers. Specifically, the negatively charged small helix from one P domain interacts with a positively charged β-sheet from an adjacent P domain [ 15 , 18 ]. These electrostatic interactions occur near the quasi-threefold axes that relate adjacent capsomers. Electrostatic interactions between the similar regions of the capsid proteins were observed in other phages and are conserved largely in the immature (unexpanded) and mature (expanded) capsid structures (see below). Consequently, a large network of extensive intra- and intercapsomer interactions form, generating a stable capsid structure capable of withstanding the substantial internal pressure (~25 atm) induced by the tightly packed genomic DNA [ 26 , 27 ]. Further, 11 of the 12 vertices of the capsid are occupied by pentamers of the gp24* protein, the cleaved form of gp24 lacking the first 10 residues removed by prohead protease during maturation. The high-resolution structures of the T4 capsid show that the gp24* structure is quite similar to gp23* [ 15 , 18 , 20 ] ( Figure 2 C), although the sequence identity between these two proteins is only ~20%. Thus, contrary to other well-studied phages, in which the major capsid protein occupies the pentameric vertices, T4 has evolved a separate gp24* protein specifically tailored to make the vertices, regions of the capsid where the curvature is the highest. The network of interaction observed between gp24* and neighboring subunits is similar to that observed for gp23*, though the gp24* structure is adapted for the vertex environment [ 15 , 18 ]. The gp24 protein is essential for phage viability. However, point mutations that bypass the gp24 requirement were found in gene 23 [ 16 , 28 ]. These mutations allow gp23 to substitute for gp24 in the vertices when gp24 is absent. Some of the gp24 bypass mutations sites are in the A domain of gp23, in the interface between adjacent subunits of the same capsomer. These mutations probably alter the gp23 structure and allow it to make pentamers (in addition to the usual hexamers) that now occupy the pentameric vertices. The vertex protein gp24 was probably derived from gp23 by gene duplication, followed by sequence divergence and optimization to adjust to its specific role of making stable pentameric vertices. On the other hand, the wild-type major capsid protein gp23, while optimized for hexamer assembly, probably lost its ability to assemble stable pentameric vertices during evolution, but this feature can be restored by the gp24 bypass mutations. 2.2. Portal In addition to the 11 pentameric vertices occupied by gp24*, the T4 capsid, like most icosahedral phages and herpes viruses, has a unique portal vertex that creates a platform for attaching the "neck" and tail. This vertex is occupied by a dodecamer of the portal protein, gp20, which initiates the capsid assembly [ 4 , 16 , 29 ] and also creates the binding site for the DNA-packaging motor [ 30 , 31 ] (see below). The structure of the E. coli -expressed gp20 protein [ 32 ] and of the same within the capsid vertex [ 33 ] have been determined through cryo-EM to near-atomic resolution. The 12 gp20 subunits form a flying-saucer-shape oligomer with a central channel ( Figure 3 A–C) that serves as a conduit for DNA packaging into the capsid during head assembly and as an exit during infection. The gp20 subunits have a fold similar to that of the portal proteins of other phages and herpesviruses [ 34 , 35 , 36 ], indicating their common evolutionary origin. The gp20 portal subunit can be subdivided into clip, stem, wing, and crown domains/regions ( Figure 3 D). The clip region is exposed outside the capsid shell and is involved in interactions with different proteins during virion assembly, namely the DNA-packaging motor protein, gp17, for genome packaging [ 31 ] and, later, the dodecameric neck protein, gp13, which through an interaction with the hexameric gp14 [ 37 ] seal the portal vertex after headful genome packaging. The assembled neck creates a binding site for the docking of the phage tail that is independently assembled. The gp20 clip region contains two positively charged residues near the channel entrance that might be involved in capturing the genomic DNA end at the initiation of the packaging process. The stem, wing, and crown regions are inside the capsid. In the full capsid, the crown and wing regions interact with genomic DNA, while the wing region also interacts with the major capsid protein capsomers surrounding the portal. The central stem domain containing two long antiparallel helices is negatively charged, while the small region in the clip domain near the channel entrance is positively charged. The modeling of a B-DNA helix into the portal channel shows that possible contacts between the portal protein and the DNA are confined to three polypeptide loops, separated from each other by approximately one helical turn of DNA [ 32 ]. The first "tunnel" loop connects the stem and the wing region, the second "channel" loop connects the clip domain with the stem helix α7, and the third "inner clip" loop is at the end of the clip domain ( Figure 3 D). Through conformational changes in the portal, some of these loops may constrict or expand the central channel. Thus, the portal may act a molecular valve that controls the flow of DNA into the capsid during packaging and out of the capsid during ejection [ 34 ]. Because the portal protein 12-mer is surrounded by the fivefold-symmetric gp23* capsid shell, there is a symmetry mismatch between the portal and the capsid, and each of the 12 portal subunits faces different regions of the gp23* shell. The structure of the symmetry-mismatched portal–capsid interface was resolved to near-atomic resolution in the asymmetric cryo-EM reconstruction, which included the portal protein and the five surrounding gp23* capsomers. The reconstruction showed the remarkable structural morphing of the portal to compensate for the symmetry mismatch [ 33 ]. Namely, the flexible components of the portal protein, in the periphery of its wing region, display significant conformational differences among the 12 portal subunits, whereas the gp23* shell surrounding the portal strictly obeys the fivefold symmetry and does not show any significant conformational changes induced by the portal [ 33 ]. The flexible portal components showing large structural variations include the N-terminal "whisker" Met1-Leu6, the "hairpin" Arg185-Glu204, and the "loop" Asp209-Lys227 ( Figure 3 E–G), all of which are parts of the portal wing. The cryo-EM reconstruction showed that, due to the the portal's flexibility and structural adaptation, similar interactions between different portal subunits and the surrounding capsid protein molecules repeatedly occur [ 33 ]. For instance, the N-terminal whiskers of portal subunits 1 ( p ) and 6 ( p. + 5 ) interact with the two fivefold-symmetry-related regions of the gp23* shell and form potential methionine-metal clusters with gp23* molecules ( Figure 3 H,I). Furthermore, similar salt bridges occur between the fivefold-symmetry-related regions of the gp23* shell and the portal subunits whose numbers have the p , p. + 5 , and p. + 7 relationship [ 33 ]. Owing to the 12-fold symmetry of the portal and the fivefold symmetry of the gp23* shell, portal subunits obeying this p , p. + 5 , and p. + 7 relationship encounter similar gp23* environments, which differ by either a +6° or a −6° rotation. The cryo-EM structure shows that the flexible components of the portal protein morph to compensate for these environmental differences and reach similar interaction partners [ 33 ]. The cryo-EM structure also showed that the portal and capsid axes are slightly misaligned, resulting in a 0.9° tilt of the portal with respect to the capsid [ 33 ]. This portal tilt results in favorable hydrophobic interactions of some portal subunits with the neighboring gp23* molecules. The portal tilt axis was found to be roughly parallel to the line connecting the two potential methionine-metal clusters formed by the N-terminal methionine of subunits 1 and 6 of the portal with two methionines and one histidine of the neighboring gp23* subunits, though the coordinating metal atom has not been identified. From the structural disposition, it appears that these clusters serve as anchors attaching the portal to the capsid and might be important for regulating the portal–capsid interactions during assembly, capsid expansion, and genome packaging. Consistently, genetic and biochemical studies showed that the length of the portal N-terminal whisker is critical for the phage viability. Shortening the six-amino-acid whisker by one or two amino acids did not affect phage viability, whereas three or four amino acid deletions resulted in lethality and failure to correctly assemble the capsids [ 33 ]. The shortened whisker presumably disrupted the potential methionine-metal clusters and was unable to properly interact with the capsid protein subunits. 2.3. Decoration Proteins The T4 head has two decoration proteins, Hoc (highly immunogenic outer capsid protein) and Soc (small outer capsid protein), that bind to the capsid surface during the late stage of capsid assembly [ 16 , 38 , 39 , 40 ]. The wild-type prolate capsid has 155 binding sites for Hoc, one per gp23* capsomer, and 870 binding sites for Soc, one per gp23* subunit, except for the gp23* subunits interacting with the gp24* subunits at the pentameric vertices ( Figure 1 B–D). One Hoc subunit attaches to the center of each gp23* hexameric capsomer. The elongated fiber-like Hoc molecule consists of four domains, where the C-terminal domain is responsible for the capsid attachment [ 39 , 41 ]. The three N-terminal domains of Hoc exposed to the solvent have immunoglobulin (Ig)-like folds [ 39 ] ( Figure 1 C). Because Hoc monomers bind to the centers of gp23* hexamers, each Hoc molecule can randomly bind in one out of six possible orientations related by the hexamer axis. This leads to an enormous number of combinations of different orientations in which 155 Hoc molecules can bind to their sites on the capsid surface. This in turn leads to diverse Hoc orientation patterns exposed on different T4 particles. The Hoc protein is nonessential under laboratory conditions and has only a marginal effect on capsid stability. However, the Ig-like domains of Hoc probably help the phage to bind to different surfaces [ 39 , 42 , 43 , 44 ]. Biochemical experiments showed that the expressed Hoc protein can bind to the E. coli surface [ 39 ]. Therefore, Hoc may be beneficial to the phage in that it may help the virion to stay attached to the cell while the tail fibers search for their receptors. In addition, Hoc might allow the virions to attach to bacterial cells and use them as vehicles to travel to different locations [ 39 ]. Additionally, because E. coli and T4 populate the human gut, Hoc may help the phage to interact with molecules abundant on the surfaces of cells in the gut environment. A recent study found that a Hoc mutation (Asp246 to Asn) caused altered phage binding to fucosylated mucin glycans and provided the mutant phage a competitive fitness advantage over the wild-type phage in the gut-on-a-chip mucosal environment [ 45 ]. The tadpole-shape Soc molecules bind to the capsid surface at the interfaces between adjacent gp23* capsomers and clamp the capsomers [ 18 , 38 ] ( Figure 4 ; Figure 1 C). The Soc structure is different from the decoration proteins of other phages that bind to intercapsomer interfaces [ 46 , 47 ]. Although Soc protein is not essential for phage assembly, it reinforces the capsid shell and stabilizes it against extremes of pH (above pH 10.6) and temperature [ 38 , 40 ]. Interestingly, the high-resolution icosahedral structure of the empty isometric head [ 18 ] showed that the Soc binding sites are not equally occupied. In the isometric head, the occupancies varied from ~0.4 to ~0.6 and correlated with the size of the angle between the planes of adjacent hexameric capsomers clamped by Soc. The largest Soc occupancies and the largest intercapsomer angles were observed near capsid vertices where there is the largest deviation from a planar hexagonal array. Therefore, Soc molecules prefer to bind and reinforce the capsid near the vertices, where there is probably the greatest strain in the gp23* hexagonal lattice. Both Hoc and Soc exhibit exquisite specificity to T4 capsid and nanomolar binding affinity. As these are nonessential for phage infection, Hoc and Soc have been extensively used as adapters to efficiently display pathogen epitopes, antigens, and large complexes at high density on the capsid surface. Lindsay's laboratory and Rao's laboratory, as well as other laboratories, have used this platform to design vaccines against a number of bacterial and viral diseases [ 9 , 48 , 49 ]. When administered to animals, the antigen-decorated phage nanoparticles induce robust and broad immune responses that include neutralizing antibodies, T cell responses, and mucosal responses. Immunized mice, rats, rabbits, and macaques were completely protected against challenges with lethal doses of infectious disease agents such as Bacillus anthracis (anthrax), Yersinia pestis (plague), SARS-CoV-2 (COVID-19), and H1N1 influenza A (flu) [ 50 , 51 , 52 ]. It appears that the surface architecture of the T4 phage capsid mimics the pathogen-associated molecular patterns (PAMPs), triggering strong immune responses by the host. In addition to the delivery of vaccines, Hoc and Soc have also been used to display targeting molecules that help deliver genes packaged in T4 heads to human cells, which in future could be developed as gene therapy devices to treat various genetic diseases [ 10 ]. 2.1. Capsid Shell The structure and dimensions of the phage T4 prolate capsid shell are displayed in Figure 2 A. The head is elongated along its fivefold axis and has a length of 120 nm and a width of 86 nm [ 14 , 15 ]. The head encapsidates ~171 kbp linear double-stranded genomic DNA (~2–3% more than the unit-length genome). The major capsid protein, gp23*, is organized into a hexagonal lattice characterized by the triangulation numbers T end = 13 laevo for the end caps and T mid = 20 for the elongated midsection [ 14 ]. Gp23* is the cleaved form of gp23 from which the 65 N-terminal residues are removed during capsid maturation by a prohead protease. The prolate shell contains 930 subunits, or 155 hexameric capsomers of gp23* [ 14 , 16 , 17 ]. Like the major capsid proteins of other tailed phages, gp23* subunits [ 15 , 18 ] have a polypeptide fold similar to that of the bacteriophage HK97 capsid protein [ 19 ] ( Figure 2 B). This fold is characterized by the wedge-shaped axial (A) domain located near the capsomer axis and the peripheral (P) domain, forming the capsomer's periphery [ 19 ]. T4 gp23* has an additional 60-residue globular insertion (I) domain, which makes characteristic bumps on the capsid surface [ 15 , 18 , 20 , 21 ]. This I domain is connected to the rest of the structure via long linkers, which are analogous to the elongated E loop in the HK97 fold [ 19 ]. The I domain is involved in extensive intra-capsomer interactions [ 15 , 18 ]. In the gp23* capsomers, the I domain sits on top of a neighboring subunit belonging to the same hexameric capsomer, thus greatly reinforcing the capsomer structure. The I domain linkers are also involved in stabilizing interactions with the subunits of the same capsomer and with neighboring capsomers. Although extra domains inserted into the HK97 fold were also observed in the capsid proteins of other phages, such as phi29 and P22 [ 22 , 23 , 24 , 25 ], the T4 I domain inserted into the E loop and resting on a neighboring subunit from the same capsomer is a unique feature of T4-like phages. In the mature capsid, the gp23* protein contains an extended N-arm in its N-terminal region, like the major capsid protein of HK97. In addition, gp23* contains an unusual 25-residue N-terminal N-fist ( Figure 2 B) structure that interacts with four subunits: two from the same capsomer and two from an adjacent capsomer [ 18 ]. The intercapsomer binding is reinforced by attractive electrostatic forces between the P domains of gp23* subunits in different capsomers. Specifically, the negatively charged small helix from one P domain interacts with a positively charged β-sheet from an adjacent P domain [ 15 , 18 ]. These electrostatic interactions occur near the quasi-threefold axes that relate adjacent capsomers. Electrostatic interactions between the similar regions of the capsid proteins were observed in other phages and are conserved largely in the immature (unexpanded) and mature (expanded) capsid structures (see below). Consequently, a large network of extensive intra- and intercapsomer interactions form, generating a stable capsid structure capable of withstanding the substantial internal pressure (~25 atm) induced by the tightly packed genomic DNA [ 26 , 27 ]. Further, 11 of the 12 vertices of the capsid are occupied by pentamers of the gp24* protein, the cleaved form of gp24 lacking the first 10 residues removed by prohead protease during maturation. The high-resolution structures of the T4 capsid show that the gp24* structure is quite similar to gp23* [ 15 , 18 , 20 ] ( Figure 2 C), although the sequence identity between these two proteins is only ~20%. Thus, contrary to other well-studied phages, in which the major capsid protein occupies the pentameric vertices, T4 has evolved a separate gp24* protein specifically tailored to make the vertices, regions of the capsid where the curvature is the highest. The network of interaction observed between gp24* and neighboring subunits is similar to that observed for gp23*, though the gp24* structure is adapted for the vertex environment [ 15 , 18 ]. The gp24 protein is essential for phage viability. However, point mutations that bypass the gp24 requirement were found in gene 23 [ 16 , 28 ]. These mutations allow gp23 to substitute for gp24 in the vertices when gp24 is absent. Some of the gp24 bypass mutations sites are in the A domain of gp23, in the interface between adjacent subunits of the same capsomer. These mutations probably alter the gp23 structure and allow it to make pentamers (in addition to the usual hexamers) that now occupy the pentameric vertices. The vertex protein gp24 was probably derived from gp23 by gene duplication, followed by sequence divergence and optimization to adjust to its specific role of making stable pentameric vertices. On the other hand, the wild-type major capsid protein gp23, while optimized for hexamer assembly, probably lost its ability to assemble stable pentameric vertices during evolution, but this feature can be restored by the gp24 bypass mutations. 2.2. Portal In addition to the 11 pentameric vertices occupied by gp24*, the T4 capsid, like most icosahedral phages and herpes viruses, has a unique portal vertex that creates a platform for attaching the "neck" and tail. This vertex is occupied by a dodecamer of the portal protein, gp20, which initiates the capsid assembly [ 4 , 16 , 29 ] and also creates the binding site for the DNA-packaging motor [ 30 , 31 ] (see below). The structure of the E. coli -expressed gp20 protein [ 32 ] and of the same within the capsid vertex [ 33 ] have been determined through cryo-EM to near-atomic resolution. The 12 gp20 subunits form a flying-saucer-shape oligomer with a central channel ( Figure 3 A–C) that serves as a conduit for DNA packaging into the capsid during head assembly and as an exit during infection. The gp20 subunits have a fold similar to that of the portal proteins of other phages and herpesviruses [ 34 , 35 , 36 ], indicating their common evolutionary origin. The gp20 portal subunit can be subdivided into clip, stem, wing, and crown domains/regions ( Figure 3 D). The clip region is exposed outside the capsid shell and is involved in interactions with different proteins during virion assembly, namely the DNA-packaging motor protein, gp17, for genome packaging [ 31 ] and, later, the dodecameric neck protein, gp13, which through an interaction with the hexameric gp14 [ 37 ] seal the portal vertex after headful genome packaging. The assembled neck creates a binding site for the docking of the phage tail that is independently assembled. The gp20 clip region contains two positively charged residues near the channel entrance that might be involved in capturing the genomic DNA end at the initiation of the packaging process. The stem, wing, and crown regions are inside the capsid. In the full capsid, the crown and wing regions interact with genomic DNA, while the wing region also interacts with the major capsid protein capsomers surrounding the portal. The central stem domain containing two long antiparallel helices is negatively charged, while the small region in the clip domain near the channel entrance is positively charged. The modeling of a B-DNA helix into the portal channel shows that possible contacts between the portal protein and the DNA are confined to three polypeptide loops, separated from each other by approximately one helical turn of DNA [ 32 ]. The first "tunnel" loop connects the stem and the wing region, the second "channel" loop connects the clip domain with the stem helix α7, and the third "inner clip" loop is at the end of the clip domain ( Figure 3 D). Through conformational changes in the portal, some of these loops may constrict or expand the central channel. Thus, the portal may act a molecular valve that controls the flow of DNA into the capsid during packaging and out of the capsid during ejection [ 34 ]. Because the portal protein 12-mer is surrounded by the fivefold-symmetric gp23* capsid shell, there is a symmetry mismatch between the portal and the capsid, and each of the 12 portal subunits faces different regions of the gp23* shell. The structure of the symmetry-mismatched portal–capsid interface was resolved to near-atomic resolution in the asymmetric cryo-EM reconstruction, which included the portal protein and the five surrounding gp23* capsomers. The reconstruction showed the remarkable structural morphing of the portal to compensate for the symmetry mismatch [ 33 ]. Namely, the flexible components of the portal protein, in the periphery of its wing region, display significant conformational differences among the 12 portal subunits, whereas the gp23* shell surrounding the portal strictly obeys the fivefold symmetry and does not show any significant conformational changes induced by the portal [ 33 ]. The flexible portal components showing large structural variations include the N-terminal "whisker" Met1-Leu6, the "hairpin" Arg185-Glu204, and the "loop" Asp209-Lys227 ( Figure 3 E–G), all of which are parts of the portal wing. The cryo-EM reconstruction showed that, due to the the portal's flexibility and structural adaptation, similar interactions between different portal subunits and the surrounding capsid protein molecules repeatedly occur [ 33 ]. For instance, the N-terminal whiskers of portal subunits 1 ( p ) and 6 ( p. + 5 ) interact with the two fivefold-symmetry-related regions of the gp23* shell and form potential methionine-metal clusters with gp23* molecules ( Figure 3 H,I). Furthermore, similar salt bridges occur between the fivefold-symmetry-related regions of the gp23* shell and the portal subunits whose numbers have the p , p. + 5 , and p. + 7 relationship [ 33 ]. Owing to the 12-fold symmetry of the portal and the fivefold symmetry of the gp23* shell, portal subunits obeying this p , p. + 5 , and p. + 7 relationship encounter similar gp23* environments, which differ by either a +6° or a −6° rotation. The cryo-EM structure shows that the flexible components of the portal protein morph to compensate for these environmental differences and reach similar interaction partners [ 33 ]. The cryo-EM structure also showed that the portal and capsid axes are slightly misaligned, resulting in a 0.9° tilt of the portal with respect to the capsid [ 33 ]. This portal tilt results in favorable hydrophobic interactions of some portal subunits with the neighboring gp23* molecules. The portal tilt axis was found to be roughly parallel to the line connecting the two potential methionine-metal clusters formed by the N-terminal methionine of subunits 1 and 6 of the portal with two methionines and one histidine of the neighboring gp23* subunits, though the coordinating metal atom has not been identified. From the structural disposition, it appears that these clusters serve as anchors attaching the portal to the capsid and might be important for regulating the portal–capsid interactions during assembly, capsid expansion, and genome packaging. Consistently, genetic and biochemical studies showed that the length of the portal N-terminal whisker is critical for the phage viability. Shortening the six-amino-acid whisker by one or two amino acids did not affect phage viability, whereas three or four amino acid deletions resulted in lethality and failure to correctly assemble the capsids [ 33 ]. The shortened whisker presumably disrupted the potential methionine-metal clusters and was unable to properly interact with the capsid protein subunits. 2.3. Decoration Proteins The T4 head has two decoration proteins, Hoc (highly immunogenic outer capsid protein) and Soc (small outer capsid protein), that bind to the capsid surface during the late stage of capsid assembly [ 16 , 38 , 39 , 40 ]. The wild-type prolate capsid has 155 binding sites for Hoc, one per gp23* capsomer, and 870 binding sites for Soc, one per gp23* subunit, except for the gp23* subunits interacting with the gp24* subunits at the pentameric vertices ( Figure 1 B–D). One Hoc subunit attaches to the center of each gp23* hexameric capsomer. The elongated fiber-like Hoc molecule consists of four domains, where the C-terminal domain is responsible for the capsid attachment [ 39 , 41 ]. The three N-terminal domains of Hoc exposed to the solvent have immunoglobulin (Ig)-like folds [ 39 ] ( Figure 1 C). Because Hoc monomers bind to the centers of gp23* hexamers, each Hoc molecule can randomly bind in one out of six possible orientations related by the hexamer axis. This leads to an enormous number of combinations of different orientations in which 155 Hoc molecules can bind to their sites on the capsid surface. This in turn leads to diverse Hoc orientation patterns exposed on different T4 particles. The Hoc protein is nonessential under laboratory conditions and has only a marginal effect on capsid stability. However, the Ig-like domains of Hoc probably help the phage to bind to different surfaces [ 39 , 42 , 43 , 44 ]. Biochemical experiments showed that the expressed Hoc protein can bind to the E. coli surface [ 39 ]. Therefore, Hoc may be beneficial to the phage in that it may help the virion to stay attached to the cell while the tail fibers search for their receptors. In addition, Hoc might allow the virions to attach to bacterial cells and use them as vehicles to travel to different locations [ 39 ]. Additionally, because E. coli and T4 populate the human gut, Hoc may help the phage to interact with molecules abundant on the surfaces of cells in the gut environment. A recent study found that a Hoc mutation (Asp246 to Asn) caused altered phage binding to fucosylated mucin glycans and provided the mutant phage a competitive fitness advantage over the wild-type phage in the gut-on-a-chip mucosal environment [ 45 ]. The tadpole-shape Soc molecules bind to the capsid surface at the interfaces between adjacent gp23* capsomers and clamp the capsomers [ 18 , 38 ] ( Figure 4 ; Figure 1 C). The Soc structure is different from the decoration proteins of other phages that bind to intercapsomer interfaces [ 46 , 47 ]. Although Soc protein is not essential for phage assembly, it reinforces the capsid shell and stabilizes it against extremes of pH (above pH 10.6) and temperature [ 38 , 40 ]. Interestingly, the high-resolution icosahedral structure of the empty isometric head [ 18 ] showed that the Soc binding sites are not equally occupied. In the isometric head, the occupancies varied from ~0.4 to ~0.6 and correlated with the size of the angle between the planes of adjacent hexameric capsomers clamped by Soc. The largest Soc occupancies and the largest intercapsomer angles were observed near capsid vertices where there is the largest deviation from a planar hexagonal array. Therefore, Soc molecules prefer to bind and reinforce the capsid near the vertices, where there is probably the greatest strain in the gp23* hexagonal lattice. Both Hoc and Soc exhibit exquisite specificity to T4 capsid and nanomolar binding affinity. As these are nonessential for phage infection, Hoc and Soc have been extensively used as adapters to efficiently display pathogen epitopes, antigens, and large complexes at high density on the capsid surface. Lindsay's laboratory and Rao's laboratory, as well as other laboratories, have used this platform to design vaccines against a number of bacterial and viral diseases [ 9 , 48 , 49 ]. When administered to animals, the antigen-decorated phage nanoparticles induce robust and broad immune responses that include neutralizing antibodies, T cell responses, and mucosal responses. Immunized mice, rats, rabbits, and macaques were completely protected against challenges with lethal doses of infectious disease agents such as Bacillus anthracis (anthrax), Yersinia pestis (plague), SARS-CoV-2 (COVID-19), and H1N1 influenza A (flu) [ 50 , 51 , 52 ]. It appears that the surface architecture of the T4 phage capsid mimics the pathogen-associated molecular patterns (PAMPs), triggering strong immune responses by the host. In addition to the delivery of vaccines, Hoc and Soc have also been used to display targeting molecules that help deliver genes packaged in T4 heads to human cells, which in future could be developed as gene therapy devices to treat various genetic diseases [ 10 ]. 3. Head Assembly 3.1. Assembly Pathway and Maturation Like other tailed dsDNA phages and herpesviruses, T4 assembles its capsid via the formation of a DNA-free proteinaceous precursor, called procapsid or prohead ( Figure 5 A) [ 4 , 16 , 53 ]. The prohead formation is initiated by the gp20 portal protein dodecamer, attached to the inner membrane of the E. coli cell. The assembly of the membrane-bound portal initiator depends on a viral chaperone, gp40, and it can be crosslinked to host membrane proteins Tig, DnaK, and YidC [ 54 ]. It appears likely that DnaK transports the protein to the membrane, while YidC may function as a membrane-associated chaperone allowing the binding of gp20 to the surface of the lipid bilayer until the prohead assembly has completed (Quinten and Kuhn, personal communication). The portal protein nucleates the assembly of the inner scaffolding core and the gp23 capsid protein shell surrounding the core. The inner core is composed of the major core protein, gp22 (~580 copies); the prohead protease, gp21 (~55 copies); internal proteins IPI (~360 copies), IPII (~360 copies), and IPIII (~370 copies); gp67 (~340 copies); gp68 (~240 copies); and gpAlt (~40 copies) [ 16 , 17 ]. The IPI, IPII, IPIII, gp68, and gpAlt proteins are not essential for assembly. However, IPI is beneficial for the phage in that it is injected into the host, along with the T4 genome, and serves as an inhibitor of the GmrSD endonuclease, thus protecting the phage DNA from restriction [ 55 , 56 ]. The gpAlt protein ADP-ribosylates the host RNA polymerase and increases its affinity for the early T4 promoters, thus leading to the preferential transcription of the early T4 genes [ 57 ]. The mechanisms that localize certain proteins in the scaffolding core remain mysterious, and the structure of the core remains unresolved. Interactions between the core proteins lining the scaffold and the regions of the major capsid protein that line the capsid interior would be clearly essential for coassembling the capsid and the core. Lindsay and colleagues discovered that a ~10 aa N-terminal capsid-targeting sequence (CTS) is conserved in IPII and IPIII, but not in other core proteins [ 58 ]. This CTS sequence when attached to foreign proteins such as the green fluorescent protein (GFP) and expressed during T4 infection localized these proteins in the core structure [ 59 ]. Upon completion of the icosahedral prohead assembly, the gp21 protease becomes active and starts to digest the inner scaffolding core. The protease is pentameric, with a shape of a starfish, where the catalytic centers are in the starfish arms [ 60 ]. The X-ray structure of gp21 in the preactive form shows that the N-terminal region of the protein blocks its catalytic center, indicating that the activation mechanism involves the self-cleavage of nine N-terminal residues. Biochemical studies of T4 mutants [ 61 ] have suggested that the protease activation is triggered by the attachment of the gp24 protein to the prohead vertices. Together with the pentameric nature of gp21, these studies suggest that in the prohead core, the gp21 pentamers are initially located near the pentameric vertices. Later, during the core degradation, the protease can diffuse to the interior of the prohead and digest the core and capsid proteins. The active gp21 protease degrades gp22, gp67, and gp68 into small peptides and cleaves off small N-terminal peptides from the core proteins IPI, IPII, IPIII, and gpAlt [ 16 , 17 ]. The peptides presumably escape from the prohead through openings in the prohead shell, liberating space for packaging the phage genome. In addition, the protease removes the 65-residue N-terminal region of the major capsid protein gp23 and the 10-residue N-terminal peptide of the vertex protein gp24, producing gp23* and gp24*, respectively. The protease pentamers also degrade each other, apparently leaving about one pentamer in the capsid [ 17 , 60 ]. Following the maturation cleavages, the "empty" prohead is released from the cell membrane, and the clip domain of the portal becomes accessible for the assembly of the DNA-packaging motor protein as a pentamer. The pentameric packaging motor translocates genomic DNA into the capsid through the portal channel fueled by ATP hydrolysis [ 31 ]. During packaging, the capsid expands and increases its inner volume by an additional 70% [ 15 ]. The expansion is accompanied by profound conformational changes in the gp23* shell, and this expansion creates binding sites for Hoc and Soc. Genome packaging continues until the head becomes full (~1.02 to 1.03 of the unit-length T4 genome is packaged), and a signal is sent through the portal to the gp17 motor, triggering packaging termination. Next, gp17 cuts the packaged DNA from the rest of the DNA concatemer by using its nuclease activity and departs from the portal vertex [ 2 , 62 ]. The exposed portal clip domains now interact with 12 gp13 subunits, to which six gp14 subunits attach [ 37 ]. The (gp13) 12 -(gp14) 6 complex seals the portal vertex of the DNA-full head and creates the binding site for the docking of the phage tail. 3.2. Expansion Although the T4 head has been studied for many decades, the structure of the unexpanded gp23* shell has remained unresolved because the prohead particles are fragile and can spontaneously expand in vitro and in vivo [ 1 ], with no DNA being packaged. The difficulty of producing large amounts of unexpanded prohead particles for structural study has recently been overcome by the pre-expression of the gp20 portal protein in E. coli cells and the infection of these cells with a T4 mutant deficient in the portal protein, the DNA-packaging motor, and the neck and tail assembly ( 10am13am17am20am ) [ 15 ]. The empty proheads isolated from these infections did not expand, although their inner scaffolding core was completely degraded by the gp21 protease, and their gp23 and gp24 proteins underwent normal maturation cleavages. This suggests that the portal protein plays an important role in triggering the capsid expansion. Fang et al. [ 15 ] suggested that the pre-expressed portal protein is unable to trigger expansion because when the portal is expressed in the absence of its interacting partners (gp40, gp23, and scaffolding core proteins), it might assemble in a conformation that is different from that in a natural T4 infection. The 5.1 à -resolution cryo-EM structure of the unexpanded prohead was determined ( Figure 5 A–C), and its comparison with the 3.4 à -resolution structure of the mature capsid [ 15 ] revealed dramatic conformational changes during capsid expansion and stabilization. Overall, after the expansion, the capsid length increases from ~950 à to ~1200 à , and the width increases from ~700 à to ~860 à ( Figure 5 A), while the capsid wall becomes thinner. Consequently, the capsid volume increases by an additional ~70%, which is essential to accommodate the complete viral genome. In the unexpanded prohead, each hexameric gp23* capsomer adjacent to a pentameric vertex is skewed into two gp23* trimers, roughly related by a twofold axis. However, these gp23* capsomers become almost sixfold symmetric after expansion. In the unexpanded prohead, adjacent capsomers are bound to each other mainly near the quasi-threefold axes of the capsid shell via hydrophobic interactions between the three adjacent P-loops and by electrostatic interactions between the short, negatively charged α-helix and a positively charged β-strand region from adjacent P domains. These interactions are largely preserved after the capsid expansion, indicating their importance for maintaining capsid integrity. Moreover, the structures of these regions near the quasi-threefold axes also remain relatively unchanged, indicating that they act as anchor points around which the capsid subunits rotate and twist during the expansion process. During expansion, the disordered N-terminal regions of gp23* and gp24* migrate from the prohead interior to the outer capsid surface and form ordered structures, N-arms, and N-fists (in case of gp23*), which form extensive interactions with the neighboring subunits, stabilizing the expanded shell. Furthermore, the gp23* protein has an unusual A-loop ( Figure 5 D), which plays a critical role in A domain–A domain interactions and the stabilization of hexameric capsomers in the unexpanded prohead. However, in the expanded structure, this loop folds back to the bottom of its own A domain and makes much fewer intracapsomer contacts. In both the unexpanded and expanded capsids, the I domain of gp23* sits on top of an adjacent subunit from the same capsomer. However, the binding interfaces between the I domain and the adjacent subunit are very different in the unexpanded and expanded structures. After expansion, the interactions between the I domain and the adjacent subunit become more extensive, and the binding interface area increases from ~460 à 2 to ~650 à 2 . The I domain linkers, connecting the I domain to the rest on the structure, are partially disordered in the unexpanded structure but become ordered and form intercapsomer interactions stabilizing the capsid shell. In addition, these linkers, together with N-fists, create binding sites for the Soc protein, which further reinforces the expanded capsid. All these conformational changes occurring during expansion result in a substantial increase in capsid stability, which is necessary to endure the pressure imposed by the tightly packed DNA. Capsid expansion is probably triggered at the portal vertex and then propagates as a "wave" through the entire capsid structure [ 15 ]. The expanded head structure shows that the N-terminal methionines of portal subunits 1 and 6 coordinate with Met98, Met284, and His282 from adjacent gp23* molecules to form potential metal-binding clusters ( Figure 5 E). These clusters probably anchor the portal to the capsid shell. An analysis of the unexpanded shell structure suggests that these clusters are likely also present in the unexpanded prohead, although the portal appears to be in a more dynamic state. Furthermore, the composition of the gp23* molecules that form the clusters is different. The Met98 residue of gp23* in the cluster is replaced by Met444, belonging to a neighboring gp23* subunit ( Figure 5 E), rather than that of the same subunit in the expanded head ( Figure 3 H,I). This represents an expansion-associated conformational switch in the capsid subunits at the portal–capsid interface, which probably acts as a trigger for expansion [ 15 ]. Additional observations from independent studies are consistent with the portal as the epicenter of expansion: (i) capsids assembled using a pre-expressed portal protein did not spontaneously expand, contrary to the capsids produced during phage infection [ 15 ], (ii) mutant heads in which some of the portal protein subunits were replaced by a recombinantly expressed portal–GFP fusion were found to remain in the unexpanded conformation [ 63 ], (iii) electron micrographs of giant heads showed some particles in which expansion was interrupted halfway, where the expansion front was perpendicular to the capsid axes [ 64 , 65 ]. These points lead to the conclusion that expansion is polar and propagates as a wave from the portal vertex. How does the conformational switch occur at the portal–capsid interface? As the genome becomes encapsidated, the internal pressure exerted by the packaged DNA would push down on the portal dodecamer. There is evidence that the first packaged DNA end may be clamped at the portal vertex [ 66 , 67 ]. Given that the portal is anchored to the capsid via the metal-binding clusters, this would cause a pull on gp23* molecules, inducing conformational changes. Consequently, the remodeling of the metal-binding clusters would occur, leading to unexpanded-to-expanded transitions in gp23* subunits anchored to the portal. These conformational transitions would then trigger energetically favorable conformational changes in their neighbors, causing a "domino effect". Thus, the expansion wave initiated at the symmetry-mismatched portal–capsid interface would propagate through the portal-proximal icosahedral cap and then through the capsid midsection to the distal cap [ 15 ]. 3.3. Length Control The wild-type T4 head has an elongated midsection, characterized by the triangulation number T mid = 20, the length of which is strictly controlled during assembly. However, several single-point mutations that alter the capsid length and result in mixtures of isometric (icosahedral), intermediate, prolate, and giant heads were found in gene 23 [ 68 , 69 , 70 ] ( Figure 5 F). For example, a single Ala275-to-Thr substitution results in the production of ~80% of the isometric heads, together with ~20% of the wild-type and intermediate-length heads. This mutation maps to the short negatively charged helix of the P domain involved in electrostatic interactions with the adjacent capsomer. A mutant phage containing this mutation was used to produce isometric particles for the high-resolution icosahedral cryo-EM reconstruction of the capsid [ 18 ]. Generally, the length-changing mutations cluster near the quasi-threefold axes that relate adjacent hexameric capsomers [ 15 , 18 ] ( Figure 5 F). These mutations probably affect the intercapsomer interactions and modulate angles between adjacent gp23 capsomers, which, in turn, result in an altered capsid length. An analysis of the intercapsomer angles in the prohead [ 15 ] shows that small angles (2–5°) occur more frequently in the midsection than in the caps. Mutations affecting intercapsomer interactions can make certain intercapsomer angles more or less favorable. For example, if a mutation makes small intercapsomer angles more favorable, the midsection would further elongate during assembly, generating "giant" heads. If, on the other hand, a mutation makes small angles less favorable, the gp23 capsomers would assemble into isometric heads (having shorter midsection). Thus, small changes in the intercapsomer interactions may lead to profound shifts in viral capsid morphology and volume. Because a single amino acid substitution can switch the capsid morphology from prolate to isometric (and vice versa), it is tempting to speculate [ 15 ] that T4 phage originally might have had an icosahedral capsid, but a small change in the intercapsomer contacts changed it to a prolate capsid that could package ~50 kbp more DNA, providing space for extra genes that conferred survival advantages. 3.1. Assembly Pathway and Maturation Like other tailed dsDNA phages and herpesviruses, T4 assembles its capsid via the formation of a DNA-free proteinaceous precursor, called procapsid or prohead ( Figure 5 A) [ 4 , 16 , 53 ]. The prohead formation is initiated by the gp20 portal protein dodecamer, attached to the inner membrane of the E. coli cell. The assembly of the membrane-bound portal initiator depends on a viral chaperone, gp40, and it can be crosslinked to host membrane proteins Tig, DnaK, and YidC [ 54 ]. It appears likely that DnaK transports the protein to the membrane, while YidC may function as a membrane-associated chaperone allowing the binding of gp20 to the surface of the lipid bilayer until the prohead assembly has completed (Quinten and Kuhn, personal communication). The portal protein nucleates the assembly of the inner scaffolding core and the gp23 capsid protein shell surrounding the core. The inner core is composed of the major core protein, gp22 (~580 copies); the prohead protease, gp21 (~55 copies); internal proteins IPI (~360 copies), IPII (~360 copies), and IPIII (~370 copies); gp67 (~340 copies); gp68 (~240 copies); and gpAlt (~40 copies) [ 16 , 17 ]. The IPI, IPII, IPIII, gp68, and gpAlt proteins are not essential for assembly. However, IPI is beneficial for the phage in that it is injected into the host, along with the T4 genome, and serves as an inhibitor of the GmrSD endonuclease, thus protecting the phage DNA from restriction [ 55 , 56 ]. The gpAlt protein ADP-ribosylates the host RNA polymerase and increases its affinity for the early T4 promoters, thus leading to the preferential transcription of the early T4 genes [ 57 ]. The mechanisms that localize certain proteins in the scaffolding core remain mysterious, and the structure of the core remains unresolved. Interactions between the core proteins lining the scaffold and the regions of the major capsid protein that line the capsid interior would be clearly essential for coassembling the capsid and the core. Lindsay and colleagues discovered that a ~10 aa N-terminal capsid-targeting sequence (CTS) is conserved in IPII and IPIII, but not in other core proteins [ 58 ]. This CTS sequence when attached to foreign proteins such as the green fluorescent protein (GFP) and expressed during T4 infection localized these proteins in the core structure [ 59 ]. Upon completion of the icosahedral prohead assembly, the gp21 protease becomes active and starts to digest the inner scaffolding core. The protease is pentameric, with a shape of a starfish, where the catalytic centers are in the starfish arms [ 60 ]. The X-ray structure of gp21 in the preactive form shows that the N-terminal region of the protein blocks its catalytic center, indicating that the activation mechanism involves the self-cleavage of nine N-terminal residues. Biochemical studies of T4 mutants [ 61 ] have suggested that the protease activation is triggered by the attachment of the gp24 protein to the prohead vertices. Together with the pentameric nature of gp21, these studies suggest that in the prohead core, the gp21 pentamers are initially located near the pentameric vertices. Later, during the core degradation, the protease can diffuse to the interior of the prohead and digest the core and capsid proteins. The active gp21 protease degrades gp22, gp67, and gp68 into small peptides and cleaves off small N-terminal peptides from the core proteins IPI, IPII, IPIII, and gpAlt [ 16 , 17 ]. The peptides presumably escape from the prohead through openings in the prohead shell, liberating space for packaging the phage genome. In addition, the protease removes the 65-residue N-terminal region of the major capsid protein gp23 and the 10-residue N-terminal peptide of the vertex protein gp24, producing gp23* and gp24*, respectively. The protease pentamers also degrade each other, apparently leaving about one pentamer in the capsid [ 17 , 60 ]. Following the maturation cleavages, the "empty" prohead is released from the cell membrane, and the clip domain of the portal becomes accessible for the assembly of the DNA-packaging motor protein as a pentamer. The pentameric packaging motor translocates genomic DNA into the capsid through the portal channel fueled by ATP hydrolysis [ 31 ]. During packaging, the capsid expands and increases its inner volume by an additional 70% [ 15 ]. The expansion is accompanied by profound conformational changes in the gp23* shell, and this expansion creates binding sites for Hoc and Soc. Genome packaging continues until the head becomes full (~1.02 to 1.03 of the unit-length T4 genome is packaged), and a signal is sent through the portal to the gp17 motor, triggering packaging termination. Next, gp17 cuts the packaged DNA from the rest of the DNA concatemer by using its nuclease activity and departs from the portal vertex [ 2 , 62 ]. The exposed portal clip domains now interact with 12 gp13 subunits, to which six gp14 subunits attach [ 37 ]. The (gp13) 12 -(gp14) 6 complex seals the portal vertex of the DNA-full head and creates the binding site for the docking of the phage tail. 3.2. Expansion Although the T4 head has been studied for many decades, the structure of the unexpanded gp23* shell has remained unresolved because the prohead particles are fragile and can spontaneously expand in vitro and in vivo [ 1 ], with no DNA being packaged. The difficulty of producing large amounts of unexpanded prohead particles for structural study has recently been overcome by the pre-expression of the gp20 portal protein in E. coli cells and the infection of these cells with a T4 mutant deficient in the portal protein, the DNA-packaging motor, and the neck and tail assembly ( 10am13am17am20am ) [ 15 ]. The empty proheads isolated from these infections did not expand, although their inner scaffolding core was completely degraded by the gp21 protease, and their gp23 and gp24 proteins underwent normal maturation cleavages. This suggests that the portal protein plays an important role in triggering the capsid expansion. Fang et al. [ 15 ] suggested that the pre-expressed portal protein is unable to trigger expansion because when the portal is expressed in the absence of its interacting partners (gp40, gp23, and scaffolding core proteins), it might assemble in a conformation that is different from that in a natural T4 infection. The 5.1 à -resolution cryo-EM structure of the unexpanded prohead was determined ( Figure 5 A–C), and its comparison with the 3.4 à -resolution structure of the mature capsid [ 15 ] revealed dramatic conformational changes during capsid expansion and stabilization. Overall, after the expansion, the capsid length increases from ~950 à to ~1200 à , and the width increases from ~700 à to ~860 à ( Figure 5 A), while the capsid wall becomes thinner. Consequently, the capsid volume increases by an additional ~70%, which is essential to accommodate the complete viral genome. In the unexpanded prohead, each hexameric gp23* capsomer adjacent to a pentameric vertex is skewed into two gp23* trimers, roughly related by a twofold axis. However, these gp23* capsomers become almost sixfold symmetric after expansion. In the unexpanded prohead, adjacent capsomers are bound to each other mainly near the quasi-threefold axes of the capsid shell via hydrophobic interactions between the three adjacent P-loops and by electrostatic interactions between the short, negatively charged α-helix and a positively charged β-strand region from adjacent P domains. These interactions are largely preserved after the capsid expansion, indicating their importance for maintaining capsid integrity. Moreover, the structures of these regions near the quasi-threefold axes also remain relatively unchanged, indicating that they act as anchor points around which the capsid subunits rotate and twist during the expansion process. During expansion, the disordered N-terminal regions of gp23* and gp24* migrate from the prohead interior to the outer capsid surface and form ordered structures, N-arms, and N-fists (in case of gp23*), which form extensive interactions with the neighboring subunits, stabilizing the expanded shell. Furthermore, the gp23* protein has an unusual A-loop ( Figure 5 D), which plays a critical role in A domain–A domain interactions and the stabilization of hexameric capsomers in the unexpanded prohead. However, in the expanded structure, this loop folds back to the bottom of its own A domain and makes much fewer intracapsomer contacts. In both the unexpanded and expanded capsids, the I domain of gp23* sits on top of an adjacent subunit from the same capsomer. However, the binding interfaces between the I domain and the adjacent subunit are very different in the unexpanded and expanded structures. After expansion, the interactions between the I domain and the adjacent subunit become more extensive, and the binding interface area increases from ~460 à 2 to ~650 à 2 . The I domain linkers, connecting the I domain to the rest on the structure, are partially disordered in the unexpanded structure but become ordered and form intercapsomer interactions stabilizing the capsid shell. In addition, these linkers, together with N-fists, create binding sites for the Soc protein, which further reinforces the expanded capsid. All these conformational changes occurring during expansion result in a substantial increase in capsid stability, which is necessary to endure the pressure imposed by the tightly packed DNA. Capsid expansion is probably triggered at the portal vertex and then propagates as a "wave" through the entire capsid structure [ 15 ]. The expanded head structure shows that the N-terminal methionines of portal subunits 1 and 6 coordinate with Met98, Met284, and His282 from adjacent gp23* molecules to form potential metal-binding clusters ( Figure 5 E). These clusters probably anchor the portal to the capsid shell. An analysis of the unexpanded shell structure suggests that these clusters are likely also present in the unexpanded prohead, although the portal appears to be in a more dynamic state. Furthermore, the composition of the gp23* molecules that form the clusters is different. The Met98 residue of gp23* in the cluster is replaced by Met444, belonging to a neighboring gp23* subunit ( Figure 5 E), rather than that of the same subunit in the expanded head ( Figure 3 H,I). This represents an expansion-associated conformational switch in the capsid subunits at the portal–capsid interface, which probably acts as a trigger for expansion [ 15 ]. Additional observations from independent studies are consistent with the portal as the epicenter of expansion: (i) capsids assembled using a pre-expressed portal protein did not spontaneously expand, contrary to the capsids produced during phage infection [ 15 ], (ii) mutant heads in which some of the portal protein subunits were replaced by a recombinantly expressed portal–GFP fusion were found to remain in the unexpanded conformation [ 63 ], (iii) electron micrographs of giant heads showed some particles in which expansion was interrupted halfway, where the expansion front was perpendicular to the capsid axes [ 64 , 65 ]. These points lead to the conclusion that expansion is polar and propagates as a wave from the portal vertex. How does the conformational switch occur at the portal–capsid interface? As the genome becomes encapsidated, the internal pressure exerted by the packaged DNA would push down on the portal dodecamer. There is evidence that the first packaged DNA end may be clamped at the portal vertex [ 66 , 67 ]. Given that the portal is anchored to the capsid via the metal-binding clusters, this would cause a pull on gp23* molecules, inducing conformational changes. Consequently, the remodeling of the metal-binding clusters would occur, leading to unexpanded-to-expanded transitions in gp23* subunits anchored to the portal. These conformational transitions would then trigger energetically favorable conformational changes in their neighbors, causing a "domino effect". Thus, the expansion wave initiated at the symmetry-mismatched portal–capsid interface would propagate through the portal-proximal icosahedral cap and then through the capsid midsection to the distal cap [ 15 ]. 3.3. Length Control The wild-type T4 head has an elongated midsection, characterized by the triangulation number T mid = 20, the length of which is strictly controlled during assembly. However, several single-point mutations that alter the capsid length and result in mixtures of isometric (icosahedral), intermediate, prolate, and giant heads were found in gene 23 [ 68 , 69 , 70 ] ( Figure 5 F). For example, a single Ala275-to-Thr substitution results in the production of ~80% of the isometric heads, together with ~20% of the wild-type and intermediate-length heads. This mutation maps to the short negatively charged helix of the P domain involved in electrostatic interactions with the adjacent capsomer. A mutant phage containing this mutation was used to produce isometric particles for the high-resolution icosahedral cryo-EM reconstruction of the capsid [ 18 ]. Generally, the length-changing mutations cluster near the quasi-threefold axes that relate adjacent hexameric capsomers [ 15 , 18 ] ( Figure 5 F). These mutations probably affect the intercapsomer interactions and modulate angles between adjacent gp23 capsomers, which, in turn, result in an altered capsid length. An analysis of the intercapsomer angles in the prohead [ 15 ] shows that small angles (2–5°) occur more frequently in the midsection than in the caps. Mutations affecting intercapsomer interactions can make certain intercapsomer angles more or less favorable. For example, if a mutation makes small intercapsomer angles more favorable, the midsection would further elongate during assembly, generating "giant" heads. If, on the other hand, a mutation makes small angles less favorable, the gp23 capsomers would assemble into isometric heads (having shorter midsection). Thus, small changes in the intercapsomer interactions may lead to profound shifts in viral capsid morphology and volume. Because a single amino acid substitution can switch the capsid morphology from prolate to isometric (and vice versa), it is tempting to speculate [ 15 ] that T4 phage originally might have had an icosahedral capsid, but a small change in the intercapsomer contacts changed it to a prolate capsid that could package ~50 kbp more DNA, providing space for extra genes that conferred survival advantages. 4. Genome Packaging Genome packaging in phages and viruses is carried out by a powerful packaging machine that generates up to 80–100 pN of force [ 30 , 71 , 72 , 73 , 74 , 75 ]. Such high forces are essential to compact the dsDNA inside the capsid to near-crystalline density as an ordered condensate, against forces that oppose order, bending, and electrostatic repulsion. The T4 packaging machine consists of three key components: a portal (gp20), a motor (gp17), and a regulator (gp16) [ 3 , 4 ]. The motor, an oligomer ring formed by gp17, is central to the energetic translocation mechanism, while the gp20 portal and the gp16 regulator play supporting, yet critical, roles in the packaging mechanism and in generating the fully packaged head. The gp17 motor protein and the gp16 regulator protein are referred to as "terminases" (TerL for gp17 or large terminase and TerS for gp16 or small terminase) because these proteins, in addition to their roles in packaging, form a hetero-oligomeric complex that makes cuts in the concatemeric phage genome to generate the termini [ 5 , 76 , 77 ]. After the first cut, the terminase-DNA complex docks on the portal, presumably orienting one of the cleaved ends into the portal channel and initiating DNA translocation into the capsid. The T4 TerS-TerL terminase complex is unstable in vitro [ 2 ], and the structure of the complex is unknown. The dynamic interactions and the remodeling of the complex during genome cleavage and genome translocation are also poorly understood. However, the structural and functional aspects of individual TerS and TerL proteins have been well characterized. 4.1. TerS The 18 kDa small terminase, TerS. is dispensable in vitro but essential in vivo for DNA packaging [ 2 , 78 ] ( Figure 6 A). Chain-terminating mutations in gene 16 lead to lethality and accumulate mostly empty proheads and some partially filled heads. Previous mutational analyses suggested that gp16 might be important for viral genome recognition and packaging initiation [ 79 , 80 ], whereas biochemical studies suggest that gp16 acts as a regulator of gp17 and motor functions [ 81 , 82 ]. The overall structure of phage T4 TerS and other small terminases is conserved. It consists of three domains: a central oligomerization domain that forms the core, an N-terminal domain containing a helix-turn-helix motif involved in potential DNA binding, and a C-terminal domain that might be involved in gp17-ATPase stimulation [ 82 ]. The latter also appears to determine the specificity of the interaction with gp17. Swapping this region between phages T4 and RB49 switches the ATPase stimulation specificity from T4 gp17 to RB49 gp17 and vice versa [ 81 , 83 ]. The electron microscopy of purified gp16 shows oligomers of single rings and side-by-side double rings [ 79 , 83 ], and mass spectrometry revealed that the single and double rings correspond to 11-mers and 22-mers, respectively [ 84 ]. Sequence analyses predicted coiled–coil motifs in gp16 and other phage TerS sequences, consistent with their propensity to form stable oligomers [ 85 ]. Oligomerization occurs through these coiled–coil interactions between neighboring subunits. Mutations that perturb these interactions cause defects in oligomerization [ 85 ]. The X-ray structure of the central oligomerization domain of the T4-related phage 44rr2 TerS has been determined [ 86 ] ( Figure 6 A). Consistent with the mass spectrometry data, the structure showed 11-mers and 12-mers that were stabilized by extensive hydrophobic and electrostatic interactions between the two long helices of the central domain, as predicted by biochemical analyses. These helices form a tightly packed interface generating a cone-like structure that has a height of about 40 à and an inner diameter of 32 à (11-mer) to 37 à (12-mer) at the wider end and 24 à (11-mer) to 27 à (12-mer) at the narrower end. The function of the oligomerization domain appears to be to display the N-terminal DNA-binding helix-turn-helix domains around the ring structure [ 87 ]. Such an arrangement would allow for the wrapping of phage genomic DNA around the TerS ring. An interaction with gp17 through its C-terminal domain would lead to the formation of TerS-TerL complex that then proceeds with DNA cleavage and insertion of DNA end into the portal channel to initiate packaging. Though models have been proposed in which DNA passes through the TerS channel and may even be threaded during DNA translocation, the evidence is not consistent with such models [ 88 , 89 ]. Mutations of residues that line the channel, including partial or complete deletion of one of the channel helices, do not lead to loss of DNA binding in vitro or cause lethality in vivo. A favorite model of Lindsay, referred to as the "synapsis" model, was based on a large amount of genetic data from his laboratory, beginning with the thesis work of his graduate student Du Gong Wu [ 90 ], that implicated a link between recombination and packaging initiation. At the center of the synapsis model is the oligomeric gp16, as double or multiple rings that recognize putative pac sites in the viral genome. Interactions between the rings and the pac sites would generate a higher order TerS-genome complex in which the concatemeric viral genome is aligned as a "bundle" of unit-length genomes, similar to chromosome segregation in higher organisms [ 91 ]. This structure would then be resolved into individual genomes by packaging into capsids. However, there are no defined pac sites demonstrated in T4 genome, as was documented in the classic pac phages such as SPP1 and P22. Lindsay's work identified putative pac sites in g16 and g19 on the basis of evidence that these sites enhanced site-specific recombination in a gp16-dependent manner [ 92 ]. The synoptic complex is thought to facilitate interactions with TerL, and the TerS-TreL-DNA ternary holoterminase complex thus formed cuts DNA and attaches the end to the prohead to initiate the processive packaging of the concatemeric genome. In this model, the rings are predicted to be helical lock washers rather than flat, closed ring structures [ 93 ]. Such complexes might be difficult to crystallize, but cryo-EM might be able to generate the structures in the future, which would fill an important gap in understanding the mechanism of the initiation of phage genome packaging. Another key function of gp16, discovered rather unexpectedly, was that it stimulates the gp17-ATPase activity by >50-fold [ 78 ]. Such a stimulation was also observed in many other phages, in subsequent studies [ 94 , 95 ]. Hence, it is a common function of small terminases, probably linked to ATP-powered translocation. This is also consistent with the gp16 stimulation of in vitro DNA-packaging using crude mutant-infected extracts that also contain the DNA replication/transcription/recombination proteins. This system, in many respects, mimics the in vivo packaging of viral genomes [ 2 , 78 ]. However, the TerS gp16 is not required in a defined in vitro packaging assay consisting of only two purified components; proheads and gp17. On the other hand, gp16 inhibits packaging in this defined system [ 96 , 97 ]. Gp16 stimulates gp17-nuclease activity in vivo, and it does so in vitro in the presence of ATP, but it inhibits nuclease when ATP is absent [ 62 , 81 ]. Furthermore, gp16 inhibits the binding of gp17 N-terminal domain to DNA [ 98 ]. Both the N- and C-domains of gp16 are required for ATPase stimulation and nuclease inhibition, and the maximum activity was observed at a ratio of one gp16 oligomer to one gp17 [ 81 ]. These results are compelling, implicating communication between the gp17 ATPase and nuclease domains, which is modulated by the nucleotide-binding state and the interaction with gp16 TerS. A communication track through which signals might be transmitted has been proposed based on structure and molecular dynamics simulations [ 62 ]. The gp16 TerS also contains an ATP binding site with broad nucleotide specificity [ 79 , 81 ], but it lacks the canonical ATP binding signatures such as Walker A and Walker B [ 83 ]. However, curiously, nucleotide binding was not correlated with gp17-ATPase stimulation or gp17-nuclease inhibition. Therefore, the exact function(s) of gp16′s ATP binding is unknown. Taken together, the above observations suggest that gp16 regulates the DNA-packaging machine and its components. Although its primary function might be the recognition of the viral genome, it also modulates the ATPase, nuclease, and translocase activities of gp17 for efficient packaging initiation and DNA translocation. The overall model, therefore, is that a packaging initiation complex consisting of gp16, gp17, and DNA, forms potentially at preferred sites (putative pac sites) on the concatemeric viral genome. Gp17 then makes a cut and inserts one of the ends into the portal channel while itself assembling as an oligomeric motor and together forming the packaging machine (complex of motor, portal, and DNA). The gp17 ATPase of the motor is then stimulated by gp16, causing the firing of the ATPases in rapid succession, "jump-starting" the DNA-packaging machine (cranking the engine) [ 86 , 99 ]. 4.2. TerL The 70 kDa TerL, the large terminase subunit, is the motor protein of the T4 DNA-packaging machine [ 2 ] ( Figure 6 B). TerL consists of two domains: an N-terminal ATPase domain and a C-terminal nuclease domain. The N-terminal domain encodes the canonical ATPase signatures, including the Walker A and Walker B motifs, and the catalytic carboxylate [ 83 ]. The C-terminal domain contains two potential DNA-binding grooves and a nuclease active formed by a catalytic metal cluster containing conserved aspartic and glutamic acid residues coordinating with Mg [ 100 ]. 4.2.1. ATPase Extensive biochemical studies have established that gp17 alone, in the absence of gp16, is sufficient to efficiently package DNA in vitro [ 2 ]. However, its ATPase activity in the absence of DNA packaging is weak (K cat = ~1–2 ATPs hydrolyzed per gp17 molecule/min). The small terminase gp16 stimulate the gp17 ATPase, by as much as 50–100 fold [ 78 , 95 ]. Although gp16 stimulation might be essential for packaging initiation in vivo, it is not so for the same in vitro in the presence of excess gp17. Furthermore, during active packaging, conformational transitions in the packaging machine stimulate the gp17 ATPases to fire in a continuous burst (see below). Bioinformatic analyses predicted the catalytic residues of the ATPase center in the N-terminal domain of gp17 and other large terminases [ 83 ]. Extensive molecular genetics and biochemical analyses demonstrated that these predicted catalytic residues are essential for the ATPase and DNA-packaging activities [ 101 ]. Even highly conservative substitutions in these catalytic signatures, such as changing an aspartic acid to glutamic acid and vice versa, resulted in the complete loss of DNA packaging. These data for the first time provided compelling evidence that this ATPase center provides energy for powering DNA translocation [ 102 , 103 ]. One of the ATPase mutants in which the Asp-Glu residues corresponding to the Walker B and catalytic carboxylate motifs were switched to Glu-Asp showed tighter binding to ATP, although the mutant protein failed to hydrolyze ATP [ 102 ]. Remarkably, the mutant ATPase domain readily formed crystals in ATP-bound, ADP-bound, and apo (nucleotide-free) states and the atomic structures were determined up to ~1.8 à resolution [ 104 ]. The ATPase domain is a relatively flat structure with two subdomains ( Figure 6 B): a large subdomain I (NsubI) with the classic nucleotide-binding Rossmann fold and a smaller subdomain II (NsubII) forming a cleft in which ATP binds. All the previously predicted catalytic residues by biochemical and genetic analyses were found to be in the ATP binding pocket, forming a network of interactions with bound ATP. The catalytic pocket also contained a cis-arginine finger, which was also predicted by genetic analyses and was proposed to trigger βγ-phosphoanhydride bond cleavage. Additionally, the structure showed an adenine binding motif, an ATPase coupling motif (Motif III), and a loop near the adenine binding motif that exhibited significant movement in response to ATP hydrolysis. It is reasonable to consider that all of these components might be directly involved in the ATP energy transduction mechanism. 4.2.2. Nuclease Early studies established that when gp17 is overexpressed in E. coli , it exhibited sequence-nonspecific endonuclease activity, similar to that found associated with the purified gp17 in vitro, apparently producing blunt ends [ 105 , 106 ]. Similar activities have since been demonstrated in many of the well-characterized pac phage TerL homologs [ 107 , 108 , 109 , 110 ]. Biochemical and structural studies suggest that this activity probably makes packaging initiation and headful termination cuts in vivo during phage infection. A histidine-rich site was identified in the C-terminal domain of gp17 by random mutagenesis and selection of mutants that are deficient in nuclease activity [ 111 ]. Sequence alignments and extensive site-directed mutagenesis of this region mapped a cluster of aspartic acid and glutamic acid residues that are conserved in all phage TerLs, and they are essential for DNA cleavage, as determined by the nuclease assays [ 112 ]. In contrast to the ATPase mutants that lost the gp16-stimulated ATPase activity, these mutants retained the ATPase activity but lost the DNA cleavage activity. The mutants could, however, package DNA in vitro if the substrate is an already-cut linear DNA, but they failed to package circular DNA because it required cutting to generate an end. The X-ray structures of the C-terminal nuclease domain of T4 gp17 and its homolog from the T4-family phage RB49, which has ~72% sequence identity, have been determined [ 31 , 113 ] ( Figure 6 B). The nuclease domain, unlike the ATPase domain, has a more globular structure with an RNAse H fold containing antiparallel β-strands, similar to that found in resolvases, RNase Hs, and integrases. Importantly, the structure showed all the predicted catalytic residues: Asp401, Glu458, and Asp542 forming a catalytic triad and coordinating with a Mg ion. In addition, the structure showed a putative DNA-binding groove lined with a number of basic residues where the acidic catalytic metal center was buried at one end of this groove. Together, these constitute the nuclease cleavage center of the gp17 large terminase. 4.2.3. Translocase DNA translocation requires both the ATPase and nuclease domains as part of the full-length gp17. Again, the Glu-Asp mutant (ED mutant) of the full-length gp17 readily crystallized, and the structure was determined to 2.8 à resolution ( Figure 6 B) [ 31 ]. No significant conformational changes were observed in the N- and C-domain structures of the full-length gp17 when these were superimposed with the individually crystallized domains, as described above. However, the full-length TerL structure exhibited unique features. It contains a flexible "hinge" or "linker" domain that connects the ATPase and nuclease domains, which is essential for DNA packaging. In the absence of this hinge, the individual ATPase and nuclease domains retained the respective functions but completely lost the DNA translocation activity [ 100 ]. Additionally, the N- and C-domains share a >1000 square à complementary surface area consisting of five charged pairs and hydrophobic patches at the interface [ 31 ] ( Figure 6 C). There is also a bound phosphate ion associated with the C-domain in the crystal structure. When a B-form DNA was docked with one of its phosphates superimposed on the bound phosphate, guided by shape and charge complementarity, the DNA aligned with a number of basic residues, forming what appeared to be a shallow DNA groove. This groove was predicted to be involved in DNA translocation. Thus, it appears that the C-domain of phage T4 TerL has two DNA grooves on different faces of the structure, one that aligns with the nuclease catalytic site that is involved in DNA cutting and another that aligns with the DNA in the motor channel that is involved in DNA translocation. The structural features of TerL suggest that gp17's nuclease might be tightly controlled by ATP, as was observed in in vitro studies. The nuclease is active at the initiation and termination steps of DNA packaging, but not during translocation, when the nuclease groove would not be in contact with the DNA. This suggests that there is a mechanism in which the headful nuclease is regulated by a "communication track" that consists of residues from subdomain II, hinge, and a β-hairpin that relay conformational signals between the ATPase and the nuclease centers [ 62 ]. When the DNA is being actively translocated, gp17 would be in the nuclease-inactive state, and furthermore, its oligomeric motor structure (see below) would not allow the formation of an antiparallel dimer that is essential to simultaneously make the cuts in both the DNA strands. 4.3. Packaging Motor A functional packaging motor complex is assembled at the portal vertex by simply mixing purified proheads and gp17. This complex, in a bulk in vitro assay, can package the 171 kb phage T4 DNA, or any linear DNA [ 96 , 97 ]. If short DNA molecules are added as the DNA substrate, the motor packages multiple molecules in succession, one molecule after another [ 114 ]. This can lead to head filling when large plasmid DNA molecules are used (~30 Kb), but with shorter DNAs, mostly partially filled heads are produced [ 99 , 115 ]. Although the unexpanded prohead is the likely precursor for DNA packaging in vivo, the expanded prohead, the partially-full head, the once-filled and emptied head, or even the nearly full head can efficiently package DNA in the in vitro assay. In fact, surprisingly, even the virion's packaged DNA can be emptied, and the emptied phage head can be repackaged with DNA again and again [ 1 , 116 , 117 ]. Lindsay and his collaborators developed a novel fluorescence correlation spectroscopy assay [ 115 ] to analyze packaging in real time at the single-molecule level. The kinetics of the translocation of a 100 bp DNA labeled with rhodamine (R6G) was analyzed by determining the decrease in the diffusion coefficient of the label as the DNA is translocated and confined inside the 120 × 86 nm capsid. Furthermore, fluorescence resonance energy transfer (FRET) occurred between the DNA label and the packaged GFP molecules, which further confirmed the ATP-powered DNA translocation and packaging of multiple DNA molecules into the same capsid [ 115 ]. Additionally, these studies determined that the ends of the translocated DNA were 8–9 nm apart, suggesting that the DNA might be translocated as a loop, with one end likely fixed near the portal [ 66 ]. The above points are consistent with the measurements made using the dual optical tweezers system in which the packaging motor complexes, where gp17 is assembled on the prohead portal, are physically tethered to a microsphere coated with capsid protein antibody and the biotinylated DNA is tethered to another microsphere coated with streptavidin [ 118 ]. When the microspheres are brought together into near contact, the motor captures the DNA end and begins translocation in the presence of ATP. This system allowed for the recording of single packaging events in real time and the detailed analysis of the dynamics of the T4 packaging process [ 118 ]. These data showed that the T4 motor generates forces of at least ~60 pN, a power density of ~5000 kW/m 3 , and a rate as high as >2000 bp/s, among the highest recorded to date. The T4 motor does slip and pause during translocation, but these are relatively short and infrequent. When these do occur, the motor recovers and recaptures DNA, continuing translocation. The high rate of translocation observed with the phage T4 motor is consistent with the need to package its 171 kb size viral genome in about 5 min. The power exerted by the T4 motor is extraordinary, as evident when external loads were applied. At an external load of 40 pN, the T4 motor translocates at a remarkable speed of ~380 bp/s, and at a load of 60 pN, the motor can still translocate at a rate of ~100 bp/s. When scaled up to a macromotor, the T4 motor is approximately twice as powerful as a typical automobile engine. The speed of the packaging motor, and its mechanism and control, are of significant interest because it is tied to genome length and viral fitness. Phages such as T4 that package a large genome must compete with phages with smaller genomes by being able to package the genome in approximately the same amount of time. Hence, T4 would need a faster motor. Molecular genetic studies provided evidence that the T4 ATPase center encodes a speed controller consisting of appropriately placed hydrophobic residues in the microenvironment of the catalytic glutamate [ 119 ]. It appears that by controlling the rate of activation of the "lytic water" molecule by the catalytic glutamate, the rate of ATP hydrolysis and the motor speed are controlled. Mutations that reduced the hydrophobicity or introduced polar functional groups into this microenvironment resulted in a reduced packaging rate or even a loss of function. These mutants also show frequent pausing and "unpackaging", a phenomenon involving the release of packaged DNA when the ATP concentration is limiting. The T4 motor, when ATP is limited, instead of having greatly reduced packaging rate, as was observed in the phi29 motor [ 120 , 121 ], it frequently pauses, causing unpackaging [ 122 ]. This does reduce the average speed of the motor but only slightly. Apparently, when the motor encounters an apo subunit (lacking bound ATP), which frequently happens under limiting ATP concentrations, it pauses, which results in the loosening of its grip on DNA. The DNA–motor interactions become misaligned and the packaged DNA is slowly released. When the subunit recaptures ATP, the motor subunits adjust and realign with the DNA, forming a tight grip, and translocation is restored. These results are consistent with the tight DNA gripping observed when the motor is bound to ATP analogs, but weaker gripping is observed when bound to ADP or AMP, and essentially no gripping in the apo state [ 117 ]. The cryo-EM structure of the T4 packaging machine is consistent with the single-molecule studies ( Figure 6 B). It shows five molecules of gp17 bound to the clip domains of the dodecameric portal vertex. The pentamer stoichiometry of the motor creates a second symmetry mismatch at the portal vertex. Two rings of density are seen associated with the portal [ 31 ], with a flat upper ring resembling the shape of the ATPase domain and a lower ring of globular domains resembling the nuclease domain. The portal-interacting residues appear to reside in a helix-loop-helix region that is associated with the hinge domain. A peptide corresponding to the helix-loop-helix sequence inhibits in vitro DNA packaging [ 123 ]. The portal and the motor form a channel, and the putative translocation groove in the C-domain faces the channel. The X-ray structures of ATPase and nuclease domains fit into the cryo-EM density. However, the resolution of the cryo-EM structure is low, ~30 à , which is not sufficient to resolve the structural details. Attempts thus far to improve the resolution have not been successful, in part owing to the symmetry mismatch of portal–motor interactions and the inherently dynamic nature of the motor subunits. The pentamer stoichiometry of the T4 packaging motor has been confirmed by single-molecule fluorescence studies by using total internal reflection fluorescence microscopy (TIRF) [ 124 ]. By assembling packaging machines using Cy-3-labeled gp17, the number of subunits in individual machines that actively translocated Cy-5 DNA oligonucleotides have been counted. These measurements gave a value of five subunits per packaging motor. However, the orientation of the motor with respect to the portal has not been verified. Lindsay's studies using FRET measurements of packaging machines containing GFP-labeled portal protein and red-fluorescent ReAsH-labeled gp17 showed that the C-terminus of gp17 is closer to the portal than the N-terminus [ 125 ]. This predicted an orientation opposite to that predicted by the cryo-EM structure. A high-resolution structure of the packaging machine is therefore desperately needed to resolve the orientation as well as to define the interactions between the portal and the motor. 4.4. Packaging Dynamics and Mechanism 4.4.1. Electrostatic Force Generation Of several initial models proposed to explain the mechanism of viral DNA translocation, the portal rotation model [ 126 ] has gained the most attention. In this model, portal and DNA act analogous to a nut and bolt, respectively. The unique symmetry-mismatched portal vertex consisting of the fivefold-symmetric capsid and 12-fold-symmetric portal creates asymmetric, flexible interactions between these two structures. These enable the directional rotation of the portal (nut), powered by ATP hydrolysis, causing the translocation of the DNA (bolt) into the capsid. However, the first X-ray structures of portals determined from phages phi29 and SPP1 did not reveal such a nut-bolt type architecture, although the structures are thought to be basically consistent with the portal rotation model. Thus, newer and more-detailed rotation-incorporating models such as the rotation-compression-relaxation model [ 127 ], the electrostatic gripping model [ 128 ], and the molecular lever model [ 129 ] were proposed. To directly test these models, Lindsay's laboratory constructed GFP fusions to either the N- or C-terminal end of the T4 portal protein and demonstrated that up to ~one-half of the dodecamer positions can be occupied with the fusion proteins without any loss of prohead function. As compared to the wild-type, portals containing C-terminal GFP fusions but not N-terminal GFP fusions [ 125 ] lock the proheads in an unexpanded conformation unless the terminase packages DNA, suggesting that the portal plays a key role in controlling prohead expansion. This has been confirmed by recent studies that showed that the assembly of the portal dodecamer in the absence of other head assembly components locks the portal in a different conformation that stabilizes the unexpanded state of the head [ 15 ]. Fusion to GFP is not required. Expansion, however, is required to protect the packaged DNA from DNAse because the unexpanded heads are leaky, as demonstrated by FCS [ 63 ]. Importantly, the retention of DNA packaging by the GFP-modified portals is inconsistent with the portal rotation model in that rotation would require that the bulky C-terminal GFP fusion proteins rotate without encountering any clashes. Lindsay has also designed a more direct test by tethering the portal to the capsid through Hoc interactions [ 130 ]. As described above, Hoc binding sites appear in the expanded heads following capsid expansion. By taking advantage of this feature, unexpanded Hoc-minus proheads were prepared by replacing some of the portal subunits with N-terminal Hoc-portal fusion proteins. The proheads were then expanded in vitro at a low salt concentration to expose the Hoc binding sites, allowing the portal-fused Hoc to bind to the center of the nearest hexon. This would lead to tethering of one to five portal subunits to the capsid through Hoc bridges, as indicated by the protection of Hoc from proteolysis. These head particles are able to package DNA in vitro. Thus, both the genetic and biochemical approaches strongly suggested that the portal rotation could not be the mechanism for packaging [ 130 ]. This conclusion was further supported by single-molecule fluorescence measurements in Bustamante's laboratory, which showed with 99% certainty that the phage phi29 portal subunits failed to show rotation [ 131 ]. Lindsay's approaches and experimental designs were therefore critical to finally put the portal rotation to rest that narrowed down the plausible packaging models. A second class of packaging models was proposed, in which the terminase not only provides the energy but also serves as a molecular motor that couples the ATP energy to the active translocation of DNA. In a specific model [ 132 ], ATP-hydrolysis-driven conformational changes in the terminase domains cause changes in the DNA-binding affinities of the motor subunits, resulting in the binding and releasing of DNA. These would lead to the inchworm-type linear translocation of DNA, reminiscent of the mechanisms proposed for helicases. The sequence alignments of gp17 and numerous large terminases identified an ATPase coupling motif (also known as Motif III) that is commonly present in helicases and translocases [ 132 ]. Mutations in the coupling motif lead to binding and hydrolysis of one ATP, but the ATPase does not turn over in a catalytic manner, resulting in the loss of both ATPase and DNA-packaging activities. The cryo-EM and X-ray structures ( Figure 6 ) are consistent with this model and further refine it by postulating a more detailed, structure-based, electrostatic-force-driven packaging mechanism [ 31 ]. The pentameric T4 packaging machine can be considered analogous to an automobile with a five-cylinder engine containing the following components: an "engine", or the ATPase center in NsubI; a "wheel", or the C-domain translocation groove that moves DNA; a "transmission" NsubII domain that couples the engine to the wheel via a flexible hinge; an arginine finger "spark plug" that fires the ATPase; and an "alternator", charged pairs that generate electrostatic force by alternating between relaxed and tensed states that is then converted to mechanical movement of DNA ( Figure 6 C). The nuclease groove faces away from the translocating DNA and is activated when packaging is completed. In the cryo-EM structure, the two lobes (domains) of the motor are separated ("relaxed" or "extended" state), whereas in the X-ray structure, the domains are in close contact ("tensed" or "compact" state) [ 31 ] ( Figure 6 C). In the compact state, the NsubII of ATPase is rotated by 6° degrees and the C-domain is pulled upwards by 7 à , equivalent to 2 bp. The arginine finger between NsubI and NsubII is positioned toward the βγ phosphates of the modeled ATP, and the ion pairs are aligned. In the extended conformational state (cryo-EM structure), the hinge is extended. The binding of DNA to the translocation groove and of ATP to NsubI locks the motor in translocation mode and brings the arginine finger into position, firing ATP hydrolysis. The repulsion between the negatively charged ADP(3-) and Pi(3-) drive them apart, causing NsubII to rotate by 6° degrees, aligning the charge pairs and the complimentary surfaces between the N- and C-domains. This generates electrostatic force, attracting the C-domain-DNA complex and causing ~7 à upward movement resulting in the compact conformational state (X-ray structure). Thus, ~2 bp of DNA are translocated into the capsid in one cycle ( Figure 6 C). Product release and the loss of six negative charges causes NsubII to rotate back to the original position, misaligning the ion pairs and returning the C-domain to the relaxed state. The translocation of 2 bp would bring the translocation groove of the adjacent subunit into alignment with the backbone phosphates. DNA is then handed over to the next subunit by the matching motor and DNA symmetries. Thus, ATPase catalysis induces conformational changes, which generate electrostatic force, causing the directional motion of DNA into capsid. The pentameric motor translocates 10 bp (one turn of the helix) when all five gp17 subunits fire in succession. There is evidence for this electrostatic-force-driven translocation mechanism. Single-molecule optical tweezer measurements have shown that mutations in the charged pairs at the N- and C-domain interacting surfaces result in an impairment of force generation, a reduction in motor velocity, and an increased frequency of pausing and slipping [ 133 , 134 ]. For instance, when the charge of one of the pairs was reversed, the motor velocity dropped to zero when a 60 pN external force was applied, whereas the wild-type motor still packaged at a rate of ~100 bp/s. Furthermore, molecular dynamics simulations have shown that the measured impairments correlated with the free-energy differences computed between the extended and compact conformational states, according to the changes made to the ion pairs at the interface. 4.4.2. DNA Structural Transitions While Lindsay was in favor of this model and while his results were in agreement with it, he believed that the DNA was not translocated by a simple linear motion. His experiments with modified DNA substrates indicated a torsion-compression mechanism in which the portal grips the DNA while a power stroke is applied by the above conformational changes in the large terminase motor [ 135 ]. The DNA structure becomes compressed in the translocation channel between the portal and the ATPase motor, and releasing the grip would lead to DNA movement into prohead. The presence of nicks in 2000 bp/s, ~eight times faster than the phi29 motor [ 118 ]. The phi29 motor, on the other hand, takes a pause (dwell) after each burst cycle to reload the motor with ATP [ 73 ]. These periodic dwells that follow bursts in each cycle slow down the motor. This model is consistent with recent studies in which flexible coordination of the T4 motor was observed [ 124 ], as opposed to strict coordination of the phi29 motor. Mutant T4 motors were assembled consisting of a mixture of wild-type and Cy-3-labeled, ATPase-defective (dead) gp17 subunits. Single motors containing a defined number of dead subunits (0, 1, 2, etc) were selected by counting the number of Cy-3 labels. Engagement of individual motors with Cy-5 labeled DNA and their encapsidation behaviors were then examined in real time [ 124 ]. These measurements showed that the T4 motor can tolerate one, two, or even three dead subunits, although the defective motors spend less time in the packaging mode and are less efficient in encapsidating the oligonucleotide substrates. Whenever an inactive subunit encounters DNA, unable to hydrolyze ATP, the mutant subunit pauses and undergoes microslips such that the DNA grip is adjusted and realigned such that another wild-type subunit takes over and restarts ATPase firing and DNA translocation. These micropauses and microslips occur at fast timescales and could not be resolved by TIRF, though they are reflected in reduced packaging velocity and encapsidation efficiency. Furthermore, on some occasions, the pauses are long and lead to unpackaging, as has been observed when one or more motor subunits are in the apo state when ATP concentration is limiting [ 122 ]. Thus, the continuous burst model is overall consistent with a large number of structural, molecular, and single-molecule data from T4 and phi29 motors. The evolution of a flexible T4 motor with a continuous burst might allow phage T4 to package its 171 kb DNA in about the same amount of time as the phi29 motor takes to package its 19 kb DNA. In essence, it appears that there might be two classes of packaging motors: strictly coordinated, slow motors (phi-29 type), and flexible, fast motors (T4-type). However, the underlying basic translocation mechanism might be well conserved in phages and viruses. The former type is coordinated at the whole motor level whereas the latter type is controlled at the individual subunit level such that the basic tasks of translocation, namely ATP loading, DNA gripping, and ATP hydrolysis, occur without strict dependence on its neighbor. While this mechanism might lead to more-frequent pauses and slips owing to a lack of tight coordination, its ability to readjust the DNA grip or skip subunits when needed would allow T4 phage to package fast and to more easily overcome obstacles encountered when packaging a highly metabolically active, recombinogenic, and branched concatemeric genomic DNA. 4.1. TerS The 18 kDa small terminase, TerS. is dispensable in vitro but essential in vivo for DNA packaging [ 2 , 78 ] ( Figure 6 A). Chain-terminating mutations in gene 16 lead to lethality and accumulate mostly empty proheads and some partially filled heads. Previous mutational analyses suggested that gp16 might be important for viral genome recognition and packaging initiation [ 79 , 80 ], whereas biochemical studies suggest that gp16 acts as a regulator of gp17 and motor functions [ 81 , 82 ]. The overall structure of phage T4 TerS and other small terminases is conserved. It consists of three domains: a central oligomerization domain that forms the core, an N-terminal domain containing a helix-turn-helix motif involved in potential DNA binding, and a C-terminal domain that might be involved in gp17-ATPase stimulation [ 82 ]. The latter also appears to determine the specificity of the interaction with gp17. Swapping this region between phages T4 and RB49 switches the ATPase stimulation specificity from T4 gp17 to RB49 gp17 and vice versa [ 81 , 83 ]. The electron microscopy of purified gp16 shows oligomers of single rings and side-by-side double rings [ 79 , 83 ], and mass spectrometry revealed that the single and double rings correspond to 11-mers and 22-mers, respectively [ 84 ]. Sequence analyses predicted coiled–coil motifs in gp16 and other phage TerS sequences, consistent with their propensity to form stable oligomers [ 85 ]. Oligomerization occurs through these coiled–coil interactions between neighboring subunits. Mutations that perturb these interactions cause defects in oligomerization [ 85 ]. The X-ray structure of the central oligomerization domain of the T4-related phage 44rr2 TerS has been determined [ 86 ] ( Figure 6 A). Consistent with the mass spectrometry data, the structure showed 11-mers and 12-mers that were stabilized by extensive hydrophobic and electrostatic interactions between the two long helices of the central domain, as predicted by biochemical analyses. These helices form a tightly packed interface generating a cone-like structure that has a height of about 40 à and an inner diameter of 32 à (11-mer) to 37 à (12-mer) at the wider end and 24 à (11-mer) to 27 à (12-mer) at the narrower end. The function of the oligomerization domain appears to be to display the N-terminal DNA-binding helix-turn-helix domains around the ring structure [ 87 ]. Such an arrangement would allow for the wrapping of phage genomic DNA around the TerS ring. An interaction with gp17 through its C-terminal domain would lead to the formation of TerS-TerL complex that then proceeds with DNA cleavage and insertion of DNA end into the portal channel to initiate packaging. Though models have been proposed in which DNA passes through the TerS channel and may even be threaded during DNA translocation, the evidence is not consistent with such models [ 88 , 89 ]. Mutations of residues that line the channel, including partial or complete deletion of one of the channel helices, do not lead to loss of DNA binding in vitro or cause lethality in vivo. A favorite model of Lindsay, referred to as the "synapsis" model, was based on a large amount of genetic data from his laboratory, beginning with the thesis work of his graduate student Du Gong Wu [ 90 ], that implicated a link between recombination and packaging initiation. At the center of the synapsis model is the oligomeric gp16, as double or multiple rings that recognize putative pac sites in the viral genome. Interactions between the rings and the pac sites would generate a higher order TerS-genome complex in which the concatemeric viral genome is aligned as a "bundle" of unit-length genomes, similar to chromosome segregation in higher organisms [ 91 ]. This structure would then be resolved into individual genomes by packaging into capsids. However, there are no defined pac sites demonstrated in T4 genome, as was documented in the classic pac phages such as SPP1 and P22. Lindsay's work identified putative pac sites in g16 and g19 on the basis of evidence that these sites enhanced site-specific recombination in a gp16-dependent manner [ 92 ]. The synoptic complex is thought to facilitate interactions with TerL, and the TerS-TreL-DNA ternary holoterminase complex thus formed cuts DNA and attaches the end to the prohead to initiate the processive packaging of the concatemeric genome. In this model, the rings are predicted to be helical lock washers rather than flat, closed ring structures [ 93 ]. Such complexes might be difficult to crystallize, but cryo-EM might be able to generate the structures in the future, which would fill an important gap in understanding the mechanism of the initiation of phage genome packaging. Another key function of gp16, discovered rather unexpectedly, was that it stimulates the gp17-ATPase activity by >50-fold [ 78 ]. Such a stimulation was also observed in many other phages, in subsequent studies [ 94 , 95 ]. Hence, it is a common function of small terminases, probably linked to ATP-powered translocation. This is also consistent with the gp16 stimulation of in vitro DNA-packaging using crude mutant-infected extracts that also contain the DNA replication/transcription/recombination proteins. This system, in many respects, mimics the in vivo packaging of viral genomes [ 2 , 78 ]. However, the TerS gp16 is not required in a defined in vitro packaging assay consisting of only two purified components; proheads and gp17. On the other hand, gp16 inhibits packaging in this defined system [ 96 , 97 ]. Gp16 stimulates gp17-nuclease activity in vivo, and it does so in vitro in the presence of ATP, but it inhibits nuclease when ATP is absent [ 62 , 81 ]. Furthermore, gp16 inhibits the binding of gp17 N-terminal domain to DNA [ 98 ]. Both the N- and C-domains of gp16 are required for ATPase stimulation and nuclease inhibition, and the maximum activity was observed at a ratio of one gp16 oligomer to one gp17 [ 81 ]. These results are compelling, implicating communication between the gp17 ATPase and nuclease domains, which is modulated by the nucleotide-binding state and the interaction with gp16 TerS. A communication track through which signals might be transmitted has been proposed based on structure and molecular dynamics simulations [ 62 ]. The gp16 TerS also contains an ATP binding site with broad nucleotide specificity [ 79 , 81 ], but it lacks the canonical ATP binding signatures such as Walker A and Walker B [ 83 ]. However, curiously, nucleotide binding was not correlated with gp17-ATPase stimulation or gp17-nuclease inhibition. Therefore, the exact function(s) of gp16′s ATP binding is unknown. Taken together, the above observations suggest that gp16 regulates the DNA-packaging machine and its components. Although its primary function might be the recognition of the viral genome, it also modulates the ATPase, nuclease, and translocase activities of gp17 for efficient packaging initiation and DNA translocation. The overall model, therefore, is that a packaging initiation complex consisting of gp16, gp17, and DNA, forms potentially at preferred sites (putative pac sites) on the concatemeric viral genome. Gp17 then makes a cut and inserts one of the ends into the portal channel while itself assembling as an oligomeric motor and together forming the packaging machine (complex of motor, portal, and DNA). The gp17 ATPase of the motor is then stimulated by gp16, causing the firing of the ATPases in rapid succession, "jump-starting" the DNA-packaging machine (cranking the engine) [ 86 , 99 ]. 4.2. TerL The 70 kDa TerL, the large terminase subunit, is the motor protein of the T4 DNA-packaging machine [ 2 ] ( Figure 6 B). TerL consists of two domains: an N-terminal ATPase domain and a C-terminal nuclease domain. The N-terminal domain encodes the canonical ATPase signatures, including the Walker A and Walker B motifs, and the catalytic carboxylate [ 83 ]. The C-terminal domain contains two potential DNA-binding grooves and a nuclease active formed by a catalytic metal cluster containing conserved aspartic and glutamic acid residues coordinating with Mg [ 100 ]. 4.2.1. ATPase Extensive biochemical studies have established that gp17 alone, in the absence of gp16, is sufficient to efficiently package DNA in vitro [ 2 ]. However, its ATPase activity in the absence of DNA packaging is weak (K cat = ~1–2 ATPs hydrolyzed per gp17 molecule/min). The small terminase gp16 stimulate the gp17 ATPase, by as much as 50–100 fold [ 78 , 95 ]. Although gp16 stimulation might be essential for packaging initiation in vivo, it is not so for the same in vitro in the presence of excess gp17. Furthermore, during active packaging, conformational transitions in the packaging machine stimulate the gp17 ATPases to fire in a continuous burst (see below). Bioinformatic analyses predicted the catalytic residues of the ATPase center in the N-terminal domain of gp17 and other large terminases [ 83 ]. Extensive molecular genetics and biochemical analyses demonstrated that these predicted catalytic residues are essential for the ATPase and DNA-packaging activities [ 101 ]. Even highly conservative substitutions in these catalytic signatures, such as changing an aspartic acid to glutamic acid and vice versa, resulted in the complete loss of DNA packaging. These data for the first time provided compelling evidence that this ATPase center provides energy for powering DNA translocation [ 102 , 103 ]. One of the ATPase mutants in which the Asp-Glu residues corresponding to the Walker B and catalytic carboxylate motifs were switched to Glu-Asp showed tighter binding to ATP, although the mutant protein failed to hydrolyze ATP [ 102 ]. Remarkably, the mutant ATPase domain readily formed crystals in ATP-bound, ADP-bound, and apo (nucleotide-free) states and the atomic structures were determined up to ~1.8 à resolution [ 104 ]. The ATPase domain is a relatively flat structure with two subdomains ( Figure 6 B): a large subdomain I (NsubI) with the classic nucleotide-binding Rossmann fold and a smaller subdomain II (NsubII) forming a cleft in which ATP binds. All the previously predicted catalytic residues by biochemical and genetic analyses were found to be in the ATP binding pocket, forming a network of interactions with bound ATP. The catalytic pocket also contained a cis-arginine finger, which was also predicted by genetic analyses and was proposed to trigger βγ-phosphoanhydride bond cleavage. Additionally, the structure showed an adenine binding motif, an ATPase coupling motif (Motif III), and a loop near the adenine binding motif that exhibited significant movement in response to ATP hydrolysis. It is reasonable to consider that all of these components might be directly involved in the ATP energy transduction mechanism. 4.2.2. Nuclease Early studies established that when gp17 is overexpressed in E. coli , it exhibited sequence-nonspecific endonuclease activity, similar to that found associated with the purified gp17 in vitro, apparently producing blunt ends [ 105 , 106 ]. Similar activities have since been demonstrated in many of the well-characterized pac phage TerL homologs [ 107 , 108 , 109 , 110 ]. Biochemical and structural studies suggest that this activity probably makes packaging initiation and headful termination cuts in vivo during phage infection. A histidine-rich site was identified in the C-terminal domain of gp17 by random mutagenesis and selection of mutants that are deficient in nuclease activity [ 111 ]. Sequence alignments and extensive site-directed mutagenesis of this region mapped a cluster of aspartic acid and glutamic acid residues that are conserved in all phage TerLs, and they are essential for DNA cleavage, as determined by the nuclease assays [ 112 ]. In contrast to the ATPase mutants that lost the gp16-stimulated ATPase activity, these mutants retained the ATPase activity but lost the DNA cleavage activity. The mutants could, however, package DNA in vitro if the substrate is an already-cut linear DNA, but they failed to package circular DNA because it required cutting to generate an end. The X-ray structures of the C-terminal nuclease domain of T4 gp17 and its homolog from the T4-family phage RB49, which has ~72% sequence identity, have been determined [ 31 , 113 ] ( Figure 6 B). The nuclease domain, unlike the ATPase domain, has a more globular structure with an RNAse H fold containing antiparallel β-strands, similar to that found in resolvases, RNase Hs, and integrases. Importantly, the structure showed all the predicted catalytic residues: Asp401, Glu458, and Asp542 forming a catalytic triad and coordinating with a Mg ion. In addition, the structure showed a putative DNA-binding groove lined with a number of basic residues where the acidic catalytic metal center was buried at one end of this groove. Together, these constitute the nuclease cleavage center of the gp17 large terminase. 4.2.3. Translocase DNA translocation requires both the ATPase and nuclease domains as part of the full-length gp17. Again, the Glu-Asp mutant (ED mutant) of the full-length gp17 readily crystallized, and the structure was determined to 2.8 à resolution ( Figure 6 B) [ 31 ]. No significant conformational changes were observed in the N- and C-domain structures of the full-length gp17 when these were superimposed with the individually crystallized domains, as described above. However, the full-length TerL structure exhibited unique features. It contains a flexible "hinge" or "linker" domain that connects the ATPase and nuclease domains, which is essential for DNA packaging. In the absence of this hinge, the individual ATPase and nuclease domains retained the respective functions but completely lost the DNA translocation activity [ 100 ]. Additionally, the N- and C-domains share a >1000 square à complementary surface area consisting of five charged pairs and hydrophobic patches at the interface [ 31 ] ( Figure 6 C). There is also a bound phosphate ion associated with the C-domain in the crystal structure. When a B-form DNA was docked with one of its phosphates superimposed on the bound phosphate, guided by shape and charge complementarity, the DNA aligned with a number of basic residues, forming what appeared to be a shallow DNA groove. This groove was predicted to be involved in DNA translocation. Thus, it appears that the C-domain of phage T4 TerL has two DNA grooves on different faces of the structure, one that aligns with the nuclease catalytic site that is involved in DNA cutting and another that aligns with the DNA in the motor channel that is involved in DNA translocation. The structural features of TerL suggest that gp17's nuclease might be tightly controlled by ATP, as was observed in in vitro studies. The nuclease is active at the initiation and termination steps of DNA packaging, but not during translocation, when the nuclease groove would not be in contact with the DNA. This suggests that there is a mechanism in which the headful nuclease is regulated by a "communication track" that consists of residues from subdomain II, hinge, and a β-hairpin that relay conformational signals between the ATPase and the nuclease centers [ 62 ]. When the DNA is being actively translocated, gp17 would be in the nuclease-inactive state, and furthermore, its oligomeric motor structure (see below) would not allow the formation of an antiparallel dimer that is essential to simultaneously make the cuts in both the DNA strands. 4.2.1. ATPase Extensive biochemical studies have established that gp17 alone, in the absence of gp16, is sufficient to efficiently package DNA in vitro [ 2 ]. However, its ATPase activity in the absence of DNA packaging is weak (K cat = ~1–2 ATPs hydrolyzed per gp17 molecule/min). The small terminase gp16 stimulate the gp17 ATPase, by as much as 50–100 fold [ 78 , 95 ]. Although gp16 stimulation might be essential for packaging initiation in vivo, it is not so for the same in vitro in the presence of excess gp17. Furthermore, during active packaging, conformational transitions in the packaging machine stimulate the gp17 ATPases to fire in a continuous burst (see below). Bioinformatic analyses predicted the catalytic residues of the ATPase center in the N-terminal domain of gp17 and other large terminases [ 83 ]. Extensive molecular genetics and biochemical analyses demonstrated that these predicted catalytic residues are essential for the ATPase and DNA-packaging activities [ 101 ]. Even highly conservative substitutions in these catalytic signatures, such as changing an aspartic acid to glutamic acid and vice versa, resulted in the complete loss of DNA packaging. These data for the first time provided compelling evidence that this ATPase center provides energy for powering DNA translocation [ 102 , 103 ]. One of the ATPase mutants in which the Asp-Glu residues corresponding to the Walker B and catalytic carboxylate motifs were switched to Glu-Asp showed tighter binding to ATP, although the mutant protein failed to hydrolyze ATP [ 102 ]. Remarkably, the mutant ATPase domain readily formed crystals in ATP-bound, ADP-bound, and apo (nucleotide-free) states and the atomic structures were determined up to ~1.8 à resolution [ 104 ]. The ATPase domain is a relatively flat structure with two subdomains ( Figure 6 B): a large subdomain I (NsubI) with the classic nucleotide-binding Rossmann fold and a smaller subdomain II (NsubII) forming a cleft in which ATP binds. All the previously predicted catalytic residues by biochemical and genetic analyses were found to be in the ATP binding pocket, forming a network of interactions with bound ATP. The catalytic pocket also contained a cis-arginine finger, which was also predicted by genetic analyses and was proposed to trigger βγ-phosphoanhydride bond cleavage. Additionally, the structure showed an adenine binding motif, an ATPase coupling motif (Motif III), and a loop near the adenine binding motif that exhibited significant movement in response to ATP hydrolysis. It is reasonable to consider that all of these components might be directly involved in the ATP energy transduction mechanism. 4.2.2. Nuclease Early studies established that when gp17 is overexpressed in E. coli , it exhibited sequence-nonspecific endonuclease activity, similar to that found associated with the purified gp17 in vitro, apparently producing blunt ends [ 105 , 106 ]. Similar activities have since been demonstrated in many of the well-characterized pac phage TerL homologs [ 107 , 108 , 109 , 110 ]. Biochemical and structural studies suggest that this activity probably makes packaging initiation and headful termination cuts in vivo during phage infection. A histidine-rich site was identified in the C-terminal domain of gp17 by random mutagenesis and selection of mutants that are deficient in nuclease activity [ 111 ]. Sequence alignments and extensive site-directed mutagenesis of this region mapped a cluster of aspartic acid and glutamic acid residues that are conserved in all phage TerLs, and they are essential for DNA cleavage, as determined by the nuclease assays [ 112 ]. In contrast to the ATPase mutants that lost the gp16-stimulated ATPase activity, these mutants retained the ATPase activity but lost the DNA cleavage activity. The mutants could, however, package DNA in vitro if the substrate is an already-cut linear DNA, but they failed to package circular DNA because it required cutting to generate an end. The X-ray structures of the C-terminal nuclease domain of T4 gp17 and its homolog from the T4-family phage RB49, which has ~72% sequence identity, have been determined [ 31 , 113 ] ( Figure 6 B). The nuclease domain, unlike the ATPase domain, has a more globular structure with an RNAse H fold containing antiparallel β-strands, similar to that found in resolvases, RNase Hs, and integrases. Importantly, the structure showed all the predicted catalytic residues: Asp401, Glu458, and Asp542 forming a catalytic triad and coordinating with a Mg ion. In addition, the structure showed a putative DNA-binding groove lined with a number of basic residues where the acidic catalytic metal center was buried at one end of this groove. Together, these constitute the nuclease cleavage center of the gp17 large terminase. 4.2.3. Translocase DNA translocation requires both the ATPase and nuclease domains as part of the full-length gp17. Again, the Glu-Asp mutant (ED mutant) of the full-length gp17 readily crystallized, and the structure was determined to 2.8 à resolution ( Figure 6 B) [ 31 ]. No significant conformational changes were observed in the N- and C-domain structures of the full-length gp17 when these were superimposed with the individually crystallized domains, as described above. However, the full-length TerL structure exhibited unique features. It contains a flexible "hinge" or "linker" domain that connects the ATPase and nuclease domains, which is essential for DNA packaging. In the absence of this hinge, the individual ATPase and nuclease domains retained the respective functions but completely lost the DNA translocation activity [ 100 ]. Additionally, the N- and C-domains share a >1000 square à complementary surface area consisting of five charged pairs and hydrophobic patches at the interface [ 31 ] ( Figure 6 C). There is also a bound phosphate ion associated with the C-domain in the crystal structure. When a B-form DNA was docked with one of its phosphates superimposed on the bound phosphate, guided by shape and charge complementarity, the DNA aligned with a number of basic residues, forming what appeared to be a shallow DNA groove. This groove was predicted to be involved in DNA translocation. Thus, it appears that the C-domain of phage T4 TerL has two DNA grooves on different faces of the structure, one that aligns with the nuclease catalytic site that is involved in DNA cutting and another that aligns with the DNA in the motor channel that is involved in DNA translocation. The structural features of TerL suggest that gp17's nuclease might be tightly controlled by ATP, as was observed in in vitro studies. The nuclease is active at the initiation and termination steps of DNA packaging, but not during translocation, when the nuclease groove would not be in contact with the DNA. This suggests that there is a mechanism in which the headful nuclease is regulated by a "communication track" that consists of residues from subdomain II, hinge, and a β-hairpin that relay conformational signals between the ATPase and the nuclease centers [ 62 ]. When the DNA is being actively translocated, gp17 would be in the nuclease-inactive state, and furthermore, its oligomeric motor structure (see below) would not allow the formation of an antiparallel dimer that is essential to simultaneously make the cuts in both the DNA strands. 4.3. Packaging Motor A functional packaging motor complex is assembled at the portal vertex by simply mixing purified proheads and gp17. This complex, in a bulk in vitro assay, can package the 171 kb phage T4 DNA, or any linear DNA [ 96 , 97 ]. If short DNA molecules are added as the DNA substrate, the motor packages multiple molecules in succession, one molecule after another [ 114 ]. This can lead to head filling when large plasmid DNA molecules are used (~30 Kb), but with shorter DNAs, mostly partially filled heads are produced [ 99 , 115 ]. Although the unexpanded prohead is the likely precursor for DNA packaging in vivo, the expanded prohead, the partially-full head, the once-filled and emptied head, or even the nearly full head can efficiently package DNA in the in vitro assay. In fact, surprisingly, even the virion's packaged DNA can be emptied, and the emptied phage head can be repackaged with DNA again and again [ 1 , 116 , 117 ]. Lindsay and his collaborators developed a novel fluorescence correlation spectroscopy assay [ 115 ] to analyze packaging in real time at the single-molecule level. The kinetics of the translocation of a 100 bp DNA labeled with rhodamine (R6G) was analyzed by determining the decrease in the diffusion coefficient of the label as the DNA is translocated and confined inside the 120 × 86 nm capsid. Furthermore, fluorescence resonance energy transfer (FRET) occurred between the DNA label and the packaged GFP molecules, which further confirmed the ATP-powered DNA translocation and packaging of multiple DNA molecules into the same capsid [ 115 ]. Additionally, these studies determined that the ends of the translocated DNA were 8–9 nm apart, suggesting that the DNA might be translocated as a loop, with one end likely fixed near the portal [ 66 ]. The above points are consistent with the measurements made using the dual optical tweezers system in which the packaging motor complexes, where gp17 is assembled on the prohead portal, are physically tethered to a microsphere coated with capsid protein antibody and the biotinylated DNA is tethered to another microsphere coated with streptavidin [ 118 ]. When the microspheres are brought together into near contact, the motor captures the DNA end and begins translocation in the presence of ATP. This system allowed for the recording of single packaging events in real time and the detailed analysis of the dynamics of the T4 packaging process [ 118 ]. These data showed that the T4 motor generates forces of at least ~60 pN, a power density of ~5000 kW/m 3 , and a rate as high as >2000 bp/s, among the highest recorded to date. The T4 motor does slip and pause during translocation, but these are relatively short and infrequent. When these do occur, the motor recovers and recaptures DNA, continuing translocation. The high rate of translocation observed with the phage T4 motor is consistent with the need to package its 171 kb size viral genome in about 5 min. The power exerted by the T4 motor is extraordinary, as evident when external loads were applied. At an external load of 40 pN, the T4 motor translocates at a remarkable speed of ~380 bp/s, and at a load of 60 pN, the motor can still translocate at a rate of ~100 bp/s. When scaled up to a macromotor, the T4 motor is approximately twice as powerful as a typical automobile engine. The speed of the packaging motor, and its mechanism and control, are of significant interest because it is tied to genome length and viral fitness. Phages such as T4 that package a large genome must compete with phages with smaller genomes by being able to package the genome in approximately the same amount of time. Hence, T4 would need a faster motor. Molecular genetic studies provided evidence that the T4 ATPase center encodes a speed controller consisting of appropriately placed hydrophobic residues in the microenvironment of the catalytic glutamate [ 119 ]. It appears that by controlling the rate of activation of the "lytic water" molecule by the catalytic glutamate, the rate of ATP hydrolysis and the motor speed are controlled. Mutations that reduced the hydrophobicity or introduced polar functional groups into this microenvironment resulted in a reduced packaging rate or even a loss of function. These mutants also show frequent pausing and "unpackaging", a phenomenon involving the release of packaged DNA when the ATP concentration is limiting. The T4 motor, when ATP is limited, instead of having greatly reduced packaging rate, as was observed in the phi29 motor [ 120 , 121 ], it frequently pauses, causing unpackaging [ 122 ]. This does reduce the average speed of the motor but only slightly. Apparently, when the motor encounters an apo subunit (lacking bound ATP), which frequently happens under limiting ATP concentrations, it pauses, which results in the loosening of its grip on DNA. The DNA–motor interactions become misaligned and the packaged DNA is slowly released. When the subunit recaptures ATP, the motor subunits adjust and realign with the DNA, forming a tight grip, and translocation is restored. These results are consistent with the tight DNA gripping observed when the motor is bound to ATP analogs, but weaker gripping is observed when bound to ADP or AMP, and essentially no gripping in the apo state [ 117 ]. The cryo-EM structure of the T4 packaging machine is consistent with the single-molecule studies ( Figure 6 B). It shows five molecules of gp17 bound to the clip domains of the dodecameric portal vertex. The pentamer stoichiometry of the motor creates a second symmetry mismatch at the portal vertex. Two rings of density are seen associated with the portal [ 31 ], with a flat upper ring resembling the shape of the ATPase domain and a lower ring of globular domains resembling the nuclease domain. The portal-interacting residues appear to reside in a helix-loop-helix region that is associated with the hinge domain. A peptide corresponding to the helix-loop-helix sequence inhibits in vitro DNA packaging [ 123 ]. The portal and the motor form a channel, and the putative translocation groove in the C-domain faces the channel. The X-ray structures of ATPase and nuclease domains fit into the cryo-EM density. However, the resolution of the cryo-EM structure is low, ~30 à , which is not sufficient to resolve the structural details. Attempts thus far to improve the resolution have not been successful, in part owing to the symmetry mismatch of portal–motor interactions and the inherently dynamic nature of the motor subunits. The pentamer stoichiometry of the T4 packaging motor has been confirmed by single-molecule fluorescence studies by using total internal reflection fluorescence microscopy (TIRF) [ 124 ]. By assembling packaging machines using Cy-3-labeled gp17, the number of subunits in individual machines that actively translocated Cy-5 DNA oligonucleotides have been counted. These measurements gave a value of five subunits per packaging motor. However, the orientation of the motor with respect to the portal has not been verified. Lindsay's studies using FRET measurements of packaging machines containing GFP-labeled portal protein and red-fluorescent ReAsH-labeled gp17 showed that the C-terminus of gp17 is closer to the portal than the N-terminus [ 125 ]. This predicted an orientation opposite to that predicted by the cryo-EM structure. A high-resolution structure of the packaging machine is therefore desperately needed to resolve the orientation as well as to define the interactions between the portal and the motor. 4.4. Packaging Dynamics and Mechanism 4.4.1. Electrostatic Force Generation Of several initial models proposed to explain the mechanism of viral DNA translocation, the portal rotation model [ 126 ] has gained the most attention. In this model, portal and DNA act analogous to a nut and bolt, respectively. The unique symmetry-mismatched portal vertex consisting of the fivefold-symmetric capsid and 12-fold-symmetric portal creates asymmetric, flexible interactions between these two structures. These enable the directional rotation of the portal (nut), powered by ATP hydrolysis, causing the translocation of the DNA (bolt) into the capsid. However, the first X-ray structures of portals determined from phages phi29 and SPP1 did not reveal such a nut-bolt type architecture, although the structures are thought to be basically consistent with the portal rotation model. Thus, newer and more-detailed rotation-incorporating models such as the rotation-compression-relaxation model [ 127 ], the electrostatic gripping model [ 128 ], and the molecular lever model [ 129 ] were proposed. To directly test these models, Lindsay's laboratory constructed GFP fusions to either the N- or C-terminal end of the T4 portal protein and demonstrated that up to ~one-half of the dodecamer positions can be occupied with the fusion proteins without any loss of prohead function. As compared to the wild-type, portals containing C-terminal GFP fusions but not N-terminal GFP fusions [ 125 ] lock the proheads in an unexpanded conformation unless the terminase packages DNA, suggesting that the portal plays a key role in controlling prohead expansion. This has been confirmed by recent studies that showed that the assembly of the portal dodecamer in the absence of other head assembly components locks the portal in a different conformation that stabilizes the unexpanded state of the head [ 15 ]. Fusion to GFP is not required. Expansion, however, is required to protect the packaged DNA from DNAse because the unexpanded heads are leaky, as demonstrated by FCS [ 63 ]. Importantly, the retention of DNA packaging by the GFP-modified portals is inconsistent with the portal rotation model in that rotation would require that the bulky C-terminal GFP fusion proteins rotate without encountering any clashes. Lindsay has also designed a more direct test by tethering the portal to the capsid through Hoc interactions [ 130 ]. As described above, Hoc binding sites appear in the expanded heads following capsid expansion. By taking advantage of this feature, unexpanded Hoc-minus proheads were prepared by replacing some of the portal subunits with N-terminal Hoc-portal fusion proteins. The proheads were then expanded in vitro at a low salt concentration to expose the Hoc binding sites, allowing the portal-fused Hoc to bind to the center of the nearest hexon. This would lead to tethering of one to five portal subunits to the capsid through Hoc bridges, as indicated by the protection of Hoc from proteolysis. These head particles are able to package DNA in vitro. Thus, both the genetic and biochemical approaches strongly suggested that the portal rotation could not be the mechanism for packaging [ 130 ]. This conclusion was further supported by single-molecule fluorescence measurements in Bustamante's laboratory, which showed with 99% certainty that the phage phi29 portal subunits failed to show rotation [ 131 ]. Lindsay's approaches and experimental designs were therefore critical to finally put the portal rotation to rest that narrowed down the plausible packaging models. A second class of packaging models was proposed, in which the terminase not only provides the energy but also serves as a molecular motor that couples the ATP energy to the active translocation of DNA. In a specific model [ 132 ], ATP-hydrolysis-driven conformational changes in the terminase domains cause changes in the DNA-binding affinities of the motor subunits, resulting in the binding and releasing of DNA. These would lead to the inchworm-type linear translocation of DNA, reminiscent of the mechanisms proposed for helicases. The sequence alignments of gp17 and numerous large terminases identified an ATPase coupling motif (also known as Motif III) that is commonly present in helicases and translocases [ 132 ]. Mutations in the coupling motif lead to binding and hydrolysis of one ATP, but the ATPase does not turn over in a catalytic manner, resulting in the loss of both ATPase and DNA-packaging activities. The cryo-EM and X-ray structures ( Figure 6 ) are consistent with this model and further refine it by postulating a more detailed, structure-based, electrostatic-force-driven packaging mechanism [ 31 ]. The pentameric T4 packaging machine can be considered analogous to an automobile with a five-cylinder engine containing the following components: an "engine", or the ATPase center in NsubI; a "wheel", or the C-domain translocation groove that moves DNA; a "transmission" NsubII domain that couples the engine to the wheel via a flexible hinge; an arginine finger "spark plug" that fires the ATPase; and an "alternator", charged pairs that generate electrostatic force by alternating between relaxed and tensed states that is then converted to mechanical movement of DNA ( Figure 6 C). The nuclease groove faces away from the translocating DNA and is activated when packaging is completed. In the cryo-EM structure, the two lobes (domains) of the motor are separated ("relaxed" or "extended" state), whereas in the X-ray structure, the domains are in close contact ("tensed" or "compact" state) [ 31 ] ( Figure 6 C). In the compact state, the NsubII of ATPase is rotated by 6° degrees and the C-domain is pulled upwards by 7 à , equivalent to 2 bp. The arginine finger between NsubI and NsubII is positioned toward the βγ phosphates of the modeled ATP, and the ion pairs are aligned. In the extended conformational state (cryo-EM structure), the hinge is extended. The binding of DNA to the translocation groove and of ATP to NsubI locks the motor in translocation mode and brings the arginine finger into position, firing ATP hydrolysis. The repulsion between the negatively charged ADP(3-) and Pi(3-) drive them apart, causing NsubII to rotate by 6° degrees, aligning the charge pairs and the complimentary surfaces between the N- and C-domains. This generates electrostatic force, attracting the C-domain-DNA complex and causing ~7 à upward movement resulting in the compact conformational state (X-ray structure). Thus, ~2 bp of DNA are translocated into the capsid in one cycle ( Figure 6 C). Product release and the loss of six negative charges causes NsubII to rotate back to the original position, misaligning the ion pairs and returning the C-domain to the relaxed state. The translocation of 2 bp would bring the translocation groove of the adjacent subunit into alignment with the backbone phosphates. DNA is then handed over to the next subunit by the matching motor and DNA symmetries. Thus, ATPase catalysis induces conformational changes, which generate electrostatic force, causing the directional motion of DNA into capsid. The pentameric motor translocates 10 bp (one turn of the helix) when all five gp17 subunits fire in succession. There is evidence for this electrostatic-force-driven translocation mechanism. Single-molecule optical tweezer measurements have shown that mutations in the charged pairs at the N- and C-domain interacting surfaces result in an impairment of force generation, a reduction in motor velocity, and an increased frequency of pausing and slipping [ 133 , 134 ]. For instance, when the charge of one of the pairs was reversed, the motor velocity dropped to zero when a 60 pN external force was applied, whereas the wild-type motor still packaged at a rate of ~100 bp/s. Furthermore, molecular dynamics simulations have shown that the measured impairments correlated with the free-energy differences computed between the extended and compact conformational states, according to the changes made to the ion pairs at the interface. 4.4.2. DNA Structural Transitions While Lindsay was in favor of this model and while his results were in agreement with it, he believed that the DNA was not translocated by a simple linear motion. His experiments with modified DNA substrates indicated a torsion-compression mechanism in which the portal grips the DNA while a power stroke is applied by the above conformational changes in the large terminase motor [ 135 ]. The DNA structure becomes compressed in the translocation channel between the portal and the ATPase motor, and releasing the grip would lead to DNA movement into prohead. The presence of nicks in 2000 bp/s, ~eight times faster than the phi29 motor [ 118 ]. The phi29 motor, on the other hand, takes a pause (dwell) after each burst cycle to reload the motor with ATP [ 73 ]. These periodic dwells that follow bursts in each cycle slow down the motor. This model is consistent with recent studies in which flexible coordination of the T4 motor was observed [ 124 ], as opposed to strict coordination of the phi29 motor. Mutant T4 motors were assembled consisting of a mixture of wild-type and Cy-3-labeled, ATPase-defective (dead) gp17 subunits. Single motors containing a defined number of dead subunits (0, 1, 2, etc) were selected by counting the number of Cy-3 labels. Engagement of individual motors with Cy-5 labeled DNA and their encapsidation behaviors were then examined in real time [ 124 ]. These measurements showed that the T4 motor can tolerate one, two, or even three dead subunits, although the defective motors spend less time in the packaging mode and are less efficient in encapsidating the oligonucleotide substrates. Whenever an inactive subunit encounters DNA, unable to hydrolyze ATP, the mutant subunit pauses and undergoes microslips such that the DNA grip is adjusted and realigned such that another wild-type subunit takes over and restarts ATPase firing and DNA translocation. These micropauses and microslips occur at fast timescales and could not be resolved by TIRF, though they are reflected in reduced packaging velocity and encapsidation efficiency. Furthermore, on some occasions, the pauses are long and lead to unpackaging, as has been observed when one or more motor subunits are in the apo state when ATP concentration is limiting [ 122 ]. Thus, the continuous burst model is overall consistent with a large number of structural, molecular, and single-molecule data from T4 and phi29 motors. The evolution of a flexible T4 motor with a continuous burst might allow phage T4 to package its 171 kb DNA in about the same amount of time as the phi29 motor takes to package its 19 kb DNA. In essence, it appears that there might be two classes of packaging motors: strictly coordinated, slow motors (phi-29 type), and flexible, fast motors (T4-type). However, the underlying basic translocation mechanism might be well conserved in phages and viruses. The former type is coordinated at the whole motor level whereas the latter type is controlled at the individual subunit level such that the basic tasks of translocation, namely ATP loading, DNA gripping, and ATP hydrolysis, occur without strict dependence on its neighbor. While this mechanism might lead to more-frequent pauses and slips owing to a lack of tight coordination, its ability to readjust the DNA grip or skip subunits when needed would allow T4 phage to package fast and to more easily overcome obstacles encountered when packaging a highly metabolically active, recombinogenic, and branched concatemeric genomic DNA. 4.4.1. Electrostatic Force Generation Of several initial models proposed to explain the mechanism of viral DNA translocation, the portal rotation model [ 126 ] has gained the most attention. In this model, portal and DNA act analogous to a nut and bolt, respectively. The unique symmetry-mismatched portal vertex consisting of the fivefold-symmetric capsid and 12-fold-symmetric portal creates asymmetric, flexible interactions between these two structures. These enable the directional rotation of the portal (nut), powered by ATP hydrolysis, causing the translocation of the DNA (bolt) into the capsid. However, the first X-ray structures of portals determined from phages phi29 and SPP1 did not reveal such a nut-bolt type architecture, although the structures are thought to be basically consistent with the portal rotation model. Thus, newer and more-detailed rotation-incorporating models such as the rotation-compression-relaxation model [ 127 ], the electrostatic gripping model [ 128 ], and the molecular lever model [ 129 ] were proposed. To directly test these models, Lindsay's laboratory constructed GFP fusions to either the N- or C-terminal end of the T4 portal protein and demonstrated that up to ~one-half of the dodecamer positions can be occupied with the fusion proteins without any loss of prohead function. As compared to the wild-type, portals containing C-terminal GFP fusions but not N-terminal GFP fusions [ 125 ] lock the proheads in an unexpanded conformation unless the terminase packages DNA, suggesting that the portal plays a key role in controlling prohead expansion. This has been confirmed by recent studies that showed that the assembly of the portal dodecamer in the absence of other head assembly components locks the portal in a different conformation that stabilizes the unexpanded state of the head [ 15 ]. Fusion to GFP is not required. Expansion, however, is required to protect the packaged DNA from DNAse because the unexpanded heads are leaky, as demonstrated by FCS [ 63 ]. Importantly, the retention of DNA packaging by the GFP-modified portals is inconsistent with the portal rotation model in that rotation would require that the bulky C-terminal GFP fusion proteins rotate without encountering any clashes. Lindsay has also designed a more direct test by tethering the portal to the capsid through Hoc interactions [ 130 ]. As described above, Hoc binding sites appear in the expanded heads following capsid expansion. By taking advantage of this feature, unexpanded Hoc-minus proheads were prepared by replacing some of the portal subunits with N-terminal Hoc-portal fusion proteins. The proheads were then expanded in vitro at a low salt concentration to expose the Hoc binding sites, allowing the portal-fused Hoc to bind to the center of the nearest hexon. This would lead to tethering of one to five portal subunits to the capsid through Hoc bridges, as indicated by the protection of Hoc from proteolysis. These head particles are able to package DNA in vitro. Thus, both the genetic and biochemical approaches strongly suggested that the portal rotation could not be the mechanism for packaging [ 130 ]. This conclusion was further supported by single-molecule fluorescence measurements in Bustamante's laboratory, which showed with 99% certainty that the phage phi29 portal subunits failed to show rotation [ 131 ]. Lindsay's approaches and experimental designs were therefore critical to finally put the portal rotation to rest that narrowed down the plausible packaging models. A second class of packaging models was proposed, in which the terminase not only provides the energy but also serves as a molecular motor that couples the ATP energy to the active translocation of DNA. In a specific model [ 132 ], ATP-hydrolysis-driven conformational changes in the terminase domains cause changes in the DNA-binding affinities of the motor subunits, resulting in the binding and releasing of DNA. These would lead to the inchworm-type linear translocation of DNA, reminiscent of the mechanisms proposed for helicases. The sequence alignments of gp17 and numerous large terminases identified an ATPase coupling motif (also known as Motif III) that is commonly present in helicases and translocases [ 132 ]. Mutations in the coupling motif lead to binding and hydrolysis of one ATP, but the ATPase does not turn over in a catalytic manner, resulting in the loss of both ATPase and DNA-packaging activities. The cryo-EM and X-ray structures ( Figure 6 ) are consistent with this model and further refine it by postulating a more detailed, structure-based, electrostatic-force-driven packaging mechanism [ 31 ]. The pentameric T4 packaging machine can be considered analogous to an automobile with a five-cylinder engine containing the following components: an "engine", or the ATPase center in NsubI; a "wheel", or the C-domain translocation groove that moves DNA; a "transmission" NsubII domain that couples the engine to the wheel via a flexible hinge; an arginine finger "spark plug" that fires the ATPase; and an "alternator", charged pairs that generate electrostatic force by alternating between relaxed and tensed states that is then converted to mechanical movement of DNA ( Figure 6 C). The nuclease groove faces away from the translocating DNA and is activated when packaging is completed. In the cryo-EM structure, the two lobes (domains) of the motor are separated ("relaxed" or "extended" state), whereas in the X-ray structure, the domains are in close contact ("tensed" or "compact" state) [ 31 ] ( Figure 6 C). In the compact state, the NsubII of ATPase is rotated by 6° degrees and the C-domain is pulled upwards by 7 à , equivalent to 2 bp. The arginine finger between NsubI and NsubII is positioned toward the βγ phosphates of the modeled ATP, and the ion pairs are aligned. In the extended conformational state (cryo-EM structure), the hinge is extended. The binding of DNA to the translocation groove and of ATP to NsubI locks the motor in translocation mode and brings the arginine finger into position, firing ATP hydrolysis. The repulsion between the negatively charged ADP(3-) and Pi(3-) drive them apart, causing NsubII to rotate by 6° degrees, aligning the charge pairs and the complimentary surfaces between the N- and C-domains. This generates electrostatic force, attracting the C-domain-DNA complex and causing ~7 à upward movement resulting in the compact conformational state (X-ray structure). Thus, ~2 bp of DNA are translocated into the capsid in one cycle ( Figure 6 C). Product release and the loss of six negative charges causes NsubII to rotate back to the original position, misaligning the ion pairs and returning the C-domain to the relaxed state. The translocation of 2 bp would bring the translocation groove of the adjacent subunit into alignment with the backbone phosphates. DNA is then handed over to the next subunit by the matching motor and DNA symmetries. Thus, ATPase catalysis induces conformational changes, which generate electrostatic force, causing the directional motion of DNA into capsid. The pentameric motor translocates 10 bp (one turn of the helix) when all five gp17 subunits fire in succession. There is evidence for this electrostatic-force-driven translocation mechanism. Single-molecule optical tweezer measurements have shown that mutations in the charged pairs at the N- and C-domain interacting surfaces result in an impairment of force generation, a reduction in motor velocity, and an increased frequency of pausing and slipping [ 133 , 134 ]. For instance, when the charge of one of the pairs was reversed, the motor velocity dropped to zero when a 60 pN external force was applied, whereas the wild-type motor still packaged at a rate of ~100 bp/s. Furthermore, molecular dynamics simulations have shown that the measured impairments correlated with the free-energy differences computed between the extended and compact conformational states, according to the changes made to the ion pairs at the interface. 4.4.2. DNA Structural Transitions While Lindsay was in favor of this model and while his results were in agreement with it, he believed that the DNA was not translocated by a simple linear motion. His experiments with modified DNA substrates indicated a torsion-compression mechanism in which the portal grips the DNA while a power stroke is applied by the above conformational changes in the large terminase motor [ 135 ]. The DNA structure becomes compressed in the translocation channel between the portal and the ATPase motor, and releasing the grip would lead to DNA movement into prohead. The presence of nicks in 2000 bp/s, ~eight times faster than the phi29 motor [ 118 ]. The phi29 motor, on the other hand, takes a pause (dwell) after each burst cycle to reload the motor with ATP [ 73 ]. These periodic dwells that follow bursts in each cycle slow down the motor. This model is consistent with recent studies in which flexible coordination of the T4 motor was observed [ 124 ], as opposed to strict coordination of the phi29 motor. Mutant T4 motors were assembled consisting of a mixture of wild-type and Cy-3-labeled, ATPase-defective (dead) gp17 subunits. Single motors containing a defined number of dead subunits (0, 1, 2, etc) were selected by counting the number of Cy-3 labels. Engagement of individual motors with Cy-5 labeled DNA and their encapsidation behaviors were then examined in real time [ 124 ]. These measurements showed that the T4 motor can tolerate one, two, or even three dead subunits, although the defective motors spend less time in the packaging mode and are less efficient in encapsidating the oligonucleotide substrates. Whenever an inactive subunit encounters DNA, unable to hydrolyze ATP, the mutant subunit pauses and undergoes microslips such that the DNA grip is adjusted and realigned such that another wild-type subunit takes over and restarts ATPase firing and DNA translocation. These micropauses and microslips occur at fast timescales and could not be resolved by TIRF, though they are reflected in reduced packaging velocity and encapsidation efficiency. Furthermore, on some occasions, the pauses are long and lead to unpackaging, as has been observed when one or more motor subunits are in the apo state when ATP concentration is limiting [ 122 ]. Thus, the continuous burst model is overall consistent with a large number of structural, molecular, and single-molecule data from T4 and phi29 motors. The evolution of a flexible T4 motor with a continuous burst might allow phage T4 to package its 171 kb DNA in about the same amount of time as the phi29 motor takes to package its 19 kb DNA. In essence, it appears that there might be two classes of packaging motors: strictly coordinated, slow motors (phi-29 type), and flexible, fast motors (T4-type). However, the underlying basic translocation mechanism might be well conserved in phages and viruses. The former type is coordinated at the whole motor level whereas the latter type is controlled at the individual subunit level such that the basic tasks of translocation, namely ATP loading, DNA gripping, and ATP hydrolysis, occur without strict dependence on its neighbor. While this mechanism might lead to more-frequent pauses and slips owing to a lack of tight coordination, its ability to readjust the DNA grip or skip subunits when needed would allow T4 phage to package fast and to more easily overcome obstacles encountered when packaging a highly metabolically active, recombinogenic, and branched concatemeric genomic DNA. 5. Perspective It has been 42 years since Lindsay and one of us (Rao) began exploring the T4 packaging machinery, inspired by his deep interest in this problem. Substantial progress has been made over these years. The structures of most of the components have been determined, and their biochemical functions and genetic phenotypes have been well characterized. Single-molecule assay systems have been developed that uncovered the dynamics of individual motors in real time. The mechanism is now narrowed to basically one model, and its details are emerging. Some of the basic knowledge is being translated to vaccines and gene therapies. Lindsay's contributions span the entire spectrum of this fascinating phage biology, using the "beautiful" T4 as a model specimen. While Lindsay's contributions were numerous and broad, the one question that received his most attention was how DNA might be actively participating in the packaging mechanism, a question that hardly received anyone else's attention. On the basis of his early genetic data, Lindsay proposed a DNA supercoiling model for packaging in 1978 [ 144 ] and continuously refined it over the years as his approaches became more and more precise and sophisticated. The field has learned a great deal from his creative and out-of-the box ideas, and he passed on his unique perspectives to numerous students, research fellows, collaborators, and colleagues all over the world. Sharing a bench with Lindsay for 9 years was a major highlight of my career. Through natural osmosis, I received a bit of his passion, creativity, and dedication, which continue to serve as sources of inspiration for me. The particular delight that Lindsay took in working on the bench, virtually every single day like a graduate student, inspires me and many others who had the privilege and good fortune to be part of his research program. Perhaps the best way to honor Lindsay's memory is to continue to strive to tease out the packaging mechanism, particularly the DNA structural transitions, at the highest resolution possible. While we have a good plausible model, it is still speculative. The emerging cryo-EM and single-molecule biophysics approaches, combined with the classic genetic and biochemical approaches, now provide us with powerful new opportunities to examine the packaging machine in action. We might be able to resolve the DNA structural and motor domain movements and transitions at near-atomic resolution. Equally fascinating is how the translocation mechanism is intimately connected to genome compaction inside the shell and how it seamlessly unravels and flows into a new host during infection, which is nothing short of a "miracle".

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2094061/

Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA)

We have developed a new method for identifying specific single- or double-stranded DNA sequences called nicking endonuclease signal amplification (NESA). A probe and target DNA anneal to create a restriction site that is recognized by a strand-specific endonuclease that cleaves the probe into two pieces leaving the target DNA intact. The target DNA can then act as a template for fresh probe and the process of hybridization, cleavage and dissociation repeats. Laser-induced fluorescence coupled with capillary electrophoresis was used to measure the probe cleavage products. The reaction is rapid; full cleavage of probe occurs within one minute under ideal conditions. The reaction is specific since it requires complete complementarity between the oligonucleotide and the template at the restriction site and sufficient complementarity overall to allow hybridization. We show that both Bacillus subtilis and B. anthracis genomic DNA can be detected and specifically differentiated from DNA of other Bacillus species. When combined with multiple displacement amplification, detection of a single copy target from less than 30 cfu is possible. This method should be applicable whenever there is a requirement to detect a specific DNA sequence. Other applications include SNP analysis and genotyping. The reaction is inherently simple to multiplex and is amenable to automation. INTRODUCTION Hybridization provides the basis for specific nucleotide sequence detection in a number of techniques commonly used in molecular biology. These include microarrays, polymerase chain reaction (PCR) ( 1 , 2 ), Southern blotting ( 3 ), rolling circle amplification ( 4 ) and many others ( 5 ). All hybridization-based methods require small oligonucleotides, primers or probes, to recognize specific sequences in target DNA and specifically hybridize to these target regions as part of the detection process. Discrimination of such primers or probes between identical and related DNA sequences requires precise control of oligonucleotide T m , hybridization temperature and salt concentration. Hybridization-based methods are commonly used to identify organisms present in environmental or biological samples. All have both strengths and weaknesses. Microarrays, for example, are very specific because multiple probes can be used in parallel to interrogate different regions of a genome ( 6 , 7 ). However, microarrays are not the most sensitive technique with reported limits of detection of around 1000 to 2000 cells ( 7 , 8 ). PCR on the other hand is both specific and highly sensitive (LODs of 10 or fewer organisms) but it is inhibited by contaminants commonly found in both environmental and biological samples. As a result, stringent isolation and purification pre-processing procedures are required to avoid false negatives ( 7 , 9 , 10 ). We have developed a hybridization-based nucleic acid detection method that is specific and, when combined with multiple displacement amplification, is sensitive and tolerant to contaminants commonly found in biological samples ( 10 ). Restriction enzymes classically recognize a double-stranded DNA-binding site, and cleave each strand of the DNA using two independent catalytic cleavage centers ( 11 ). In contrast, nicking endonucleases such as Nt.BstNBI ( 12 ), only cut one strand. Nt.BstNBI is a naturally occurring nicking endonuclease that only cleaves one strand due to its inability to form dimers. New England BioLabs engineered the nicking endonuclease Nt.AlwI by creating a chimeric protein, which consists of the DNA-binding domain and catalytic center of AlwI fused to the defective dimerization domain of Nt.BstNBI ( 13 ). We have used the single-strand cleavage activities of Nt.Alw1 to develop a sensitive method for detecting the presence of unique DNA sequences that contain a nicking endonuclease recognition site. The presence of a restriction site within the probe increases specificity since hybridization alone is not enough for enzyme recognition. Instead, an exact sequence match is usually required [see ref. ( 14 ) and New England BioLabs catalog for details on DNA binding and the effects of buffer solutions on DNA cleavage specificity]. In this system, the probe binds to its target and is cleaved into two pieces that then dissociate from the target. More full-length probe then binds to the target, and the process continues until either all the probe oligonucleotides are cleaved or the enzyme is no longer active. We call this reaction nicking endonuclease signal amplification (NESA) since multiple probes are cleaved per target DNA molecule. MATERIALS AND METHODS Oligonucleotides, genomic DNA and nicking enzyme All oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA, USA). Bsub 3 (5′ 6-FAM™/TTT GGA TCG TTT CAA AGA GAG), Bsub 6 (5′ 6-FAM™/CCG GAT CTG AGG TAA CGA TGT) and BA1 (5′ 6-FAM™/AGG ATC GAA TAA GAG GTC CTT CAT) are 5′-fluorescently labeled oligonucleotide probes that were purified by ion exchange HPLC. They are identical to bases 1335052–1335072 and 431471–431491 of the Bacillus subtilis strain 168 genome ( 15 ) and bases 4610697–4610674 of the B. anthracis Ames strain respectively. Bsub 3c and Bsub 6c are the non-fluorescently labeled complementary oligonucleotides of Bsub 3 (CTC TCT TTG AAA CGA TCC AAA) and Bsub 6 (ACA TCG TTA CCT CAG ATC CGG) and were purified by standard desalting methods. Five oligonucleotide size standards (5, 17, 20, 30 and 50 bases) were synthesized with hexachlorofluorescein (HEX™) on their 5′ ends. Genomic DNA for B. subtilis, B. cereus, B. thuringiensis and B. anthracis was obtained from Dr Kevin O'Connell of the U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD. The nicking enzyme, Nt.Alw1, was obtained from New England BioLabs (Ipswich, MA, USA). Nt.Alw1 (10 units = 0.09 pmol) has a specific activity of 1.8 × 10 6 U/mg (Richard Grandoni, personal communication) and a reported turnover rate of 120 cleavage events per hour per pmol ( 13 ). Whole genome amplification Genomic DNA was amplified by multiple displacement amplification (MDA) ( 16 ). MDA uses Phi29 polymerase and random hexamers to uniformly amplify the entire genome of an organism with little to no amplification bias ( 17 ). MDA was performed using the REPLI-g kit from Qiagen. Ten nanograms of genomic DNA from various Bacillus species were amplified according to the manufacturer's instructions. Amplified samples were stored at −20°C. MDA DNA was quantified using the PicoGreen assay using the manufacturer's protocol (Invitrogen), and specificity of amplification by PCR analysis. The following primers were used: B. subtilis 5′-TGATCTTAGTTGCCAGCATTCAGTT, 5′-TCTGTCCATTGTAGCACGTGTGTAG; B. anthracis , 5′-GAGAAAGATGAGTAAAAAACAACAA, 5′-CATTTGTGCTTTGAATGCTAG. Bacillus subtilis genomic and MDA DNA were compared using qPCR. qPCR was performed on a BioRad iCycler iQ System using the Sybr Green assay with the following conditions: 95°C for 3 min, 95°C for 30 s, 55°C for 30 s, 72°C for 30 s with all but the 3′ at 95°C step repeated 40 times. The reactions contained 25 pmol of the primers (above), Sybr Green Supermix (BioRad) and target DNA (1 pg to 10 ng) in a total volume of 50 µl. MDA DNA had a slightly higher allele frequency than genomic DNA (1 fmol) when using WGA genomic DNA. Maximum cleavage was ∼15% using 0.4 fmol of genomic target (1 µg). This is more than sufficient for adequate identification since cleavage levels of as little as 1% are well over background. One of the possible uses of NESA is to detect and identify bacteria present in a crude sample. To determine the limits of detection of NESA in such an assay, we used serial dilutions of a log phase culture of B. subtilis . One aliquot of each dilution was plated on LB agar plates to determine the number of colony forming units (cfu) present, and the other aliquot underwent MDA, NESA and CE detection without any prior sample clean up. As shown in Figure 5 , we can detect a single copy target from less than 30 cfu from unpurified sample. Figure 5. Limit of detection using bacterial cells. A log phase culture of B. subtilis was serially diluted and 1 µl of each dilution was used to determine colony forming units by growth overnight on LB plates. Another 1 µl aliquot from each of these dilutions was amplified directly with multiple displacement amplification, without sample clean up, then underwent NESA analysis with both Bsub 3 and Bsub 6 probes. Specificity of NESA The genomic targets of Bsub 3 and Bsub 6 were chosen with little regard to specificity, although both probes represent single copy regions of the genome as their exact sequences are not present elsewhere in the genome. To determine the specificity of these probes, WGA DNA from a panel of Bacillus species was screened using our standard NESA ( Figure 6 ). Bsub 6 recognizes B. subtilis with no significant activity against B. anthracis, B. cereus and B. thuringiensis . Bsub 3, in contrast, recognizes B. subtilis, B. anthracis and B. cereus. To show that it is possible to generate other species-specific probes, we synthesized a probe against B. anthracis (BA1, Methods section) and tested it against closely and distantly related organisms. BA1 recognized B. anthracis but had no activity at all against B. cereus, B. subtilis, B. thuringiensis , mouse and human DNA (data not shown). Figure 6. Probe specificity. Standard NESA reactions were performed using 0.2 fmol of genomic DNA from various Bacillus species. No cut probe was detected in the absence of added DNA or using the Bsub 6 probe with B. anthracis, B. cereus or B thuringiensis genomic DNA. *, significantly different ( t -test) from the no added DNA control ( P 1 fmol) when using WGA genomic DNA. Maximum cleavage was ∼15% using 0.4 fmol of genomic target (1 µg). This is more than sufficient for adequate identification since cleavage levels of as little as 1% are well over background. One of the possible uses of NESA is to detect and identify bacteria present in a crude sample. To determine the limits of detection of NESA in such an assay, we used serial dilutions of a log phase culture of B. subtilis . One aliquot of each dilution was plated on LB agar plates to determine the number of colony forming units (cfu) present, and the other aliquot underwent MDA, NESA and CE detection without any prior sample clean up. As shown in Figure 5 , we can detect a single copy target from less than 30 cfu from unpurified sample. Figure 5. Limit of detection using bacterial cells. A log phase culture of B. subtilis was serially diluted and 1 µl of each dilution was used to determine colony forming units by growth overnight on LB plates. Another 1 µl aliquot from each of these dilutions was amplified directly with multiple displacement amplification, without sample clean up, then underwent NESA analysis with both Bsub 3 and Bsub 6 probes. Specificity of NESA The genomic targets of Bsub 3 and Bsub 6 were chosen with little regard to specificity, although both probes represent single copy regions of the genome as their exact sequences are not present elsewhere in the genome. To determine the specificity of these probes, WGA DNA from a panel of Bacillus species was screened using our standard NESA ( Figure 6 ). Bsub 6 recognizes B. subtilis with no significant activity against B. anthracis, B. cereus and B. thuringiensis . Bsub 3, in contrast, recognizes B. subtilis, B. anthracis and B. cereus. To show that it is possible to generate other species-specific probes, we synthesized a probe against B. anthracis (BA1, Methods section) and tested it against closely and distantly related organisms. BA1 recognized B. anthracis but had no activity at all against B. cereus, B. subtilis, B. thuringiensis , mouse and human DNA (data not shown). Figure 6. Probe specificity. Standard NESA reactions were performed using 0.2 fmol of genomic DNA from various Bacillus species. No cut probe was detected in the absence of added DNA or using the Bsub 6 probe with B. anthracis, B. cereus or B thuringiensis genomic DNA. *, significantly different ( t -test) from the no added DNA control ( P < 0.005). Multiplex assays Theoretically, the NESA reaction can be multiplexed based on oligonucleotide size and by using oligonucleotides labeled with different fluors. In Figure 7 A, we performed a multiplex assay using both Bsub 3 and Bsub 6 (FAM-labeled) probes and their complements. When cleaved in NESA, Bsub 3 and Bsub 6 migrated indistinguishably from each other on CE even though they are 12 and 11 bases long respectively. In order to increase the separation of the two probes, two new FAM-labeled probes were synthesized that had 2 additional bases (Ts) on the 5′ end. These probes are clearly distinguishable from the original Bsub 3 and Bsub 6 probes ( Figure 7 B and C). We demonstrated the ability to multiplex using probes labeled with different fluors by synthesizing an additional two probes, HEX-labeled Bsub 3 and HEX-labeled Bsub 6 ( Figure 7 D–F). We expected that the FAM- and HEX-labeled Bsub 3 probes would migrate at the same position and that they could be distinguished by their spectral characteristics, a routine function of the ABI3130 that we use for capillary electrophoresis. However, in this experiment, the use of alternate dyes in itself altered the mobility of the fragments so that the cleaved fragments of FAM-Bsub 3 and HEX-Bsub 3 could be easily distinguished both with oligonucleotide complements ( Figure 7 D) and with B. subtilis MDA DNA ( Figure 7 F). Figure 7. Multiplex assays. Standard NESA reactions were set up using either oligonucleotide complements ( A–E ) or 500 ng B. subtilis MDA DNA. Peaks labeled 3 and 6 are derived from FAM-labeled probes Bsub 3 and Bsub 6, respectively (blue lines). Peaks labeled 3T and 6T are derived from FAM-labeled Bsub 3 and Bsub 6 probes with two additional nucleotides (T 2 ) at the 5′ end. Peaks labeled 3H and 6H are HEX-labeled Bsub 3 and Bsub 6 probes, respectively (green lines). The positions of the 5 and 17 base hex-labeled standards are shown. The cleaved probes migrate at positions that cannot be determined solely by their length. The apparent sizes of the cleaved probes are: FAM-Bsub 3, 13.4 bases; FAM-Bsub 6, 13.6 bases; FAM-Bsub 3T2, 14.6 bases; FAM-Bsub 6T2, 14.4 bases; HEX-Bsub 3 bases, 14.5; HEX-BSub 6, 15.1 bases. DISCUSSION The ability to detect specific DNA sequences is a common requirement of many techniques in molecular biology. A method of identifying specific DNA sequences that relies on the single-strand specific nucleolytic activity of a nicking endonuclease has been developed. NESA is rapid and sensitive, detecting a single copy target from less than 30 cfu of unpurified cellular material in less than 1 h. We have also demonstrated that the technique can be used to develop species-specific probes capable of discriminating between closely related organisms. NESA is extremely rapid, cleaving over 90% of the probe in lesser than a minute with 10-fold less enzyme than substrate (probe). Nt.AlwI has been reported to have a turnover rate of approximately two cleavage events per minute in an experiment that was performed at the recommended incubation temperature of 37°C and on a substrate of supercoiled, circular DNA ( 13 ). Under our conditions, an incubation temperature of 58°C with a linear oligonucleotide substrate, we have achieved turnover rates upwards of 10 cleavage events per minute ( Figure 3 A). Preliminary experiments with 100-fold lesser enzyme than substrate indicate turnover rates as high as 16 cleavage events per minute (data not shown). The increased turnover rate in NESA compared to the supercoil relaxation assay could be simply due to the increased temperature (58°C for NESA versus 37°C) or it could be due to the destruction of the substrate in NESA (at 58°C the products dissociate) but not in the supercoil relaxation assay. NESA requires a single-strand DNA target and we have shown that robust signals can be generated from both short oligonucleotides and longer, more complex genomic DNA. In reactions containing more than 10 fmol of oligonucleotide target DNA, we routinely obtain near complete cleavage of the probe. At lower oligonucleotide target levels or with genomic targets, we get lower levels of probe cleavage. Even so, we reliably achieve 5–15% cleavage of the input probe using WGA genomic DNA as target. Decreased rates of probe cleavage occur with low levels of both genomic and oligonucleotide targets ( Figures 3 and 4 ), therefore the phenomenon appears not to be specific to the type of target DNA. We know that this effect is not due to loss of enzyme activity since Nt.AlwI preincubated at 58°C for 1 h is as active as fresh enzyme. Similarly, it is not due to insufficient enzyme since the addition of more enzyme halfway through the reaction does not increase the signal. In the case of WGA genomic DNA, there is an indication that the lower rates may be due, at least partially, to probe access issues since sonication of the genomic DNA can increase the signal up to 25% (data not shown). Figure 4. Sensitivity using genomic targets. Standard NESA reactions were set up using B. subtilis WGA DNA. ( A) Reactions contained 0.2 fmol WGA DNA and were incubated at 58°C for the indicated times. ( B) NESA reactions containing the indicated amounts of MDA DNA were run for 60 min at 58°C. The 10-fold increase in sensitivity with genomic DNA compared to oligonucleotide target was unexpected since genomic DNA is not only more complex but also contains many Nt.AlwI sites outside of the target sequence that, if re-annealed, could compete for enzyme. It does not appear to be due to the presence of multiple targets within the genomic DNA because, based upon sequence analysis, the probes were designed to single copy regions of the B. subtilis genome. One possibility is that the longer DNA allows the nicking endonuclease to search for recognition sites in 1D space ( 14 ), rather than by 3D diffusion as with short oligos. Another possibility is that the products of the reaction, cut probe pieces, bind transiently to the oligonucleotide target and thus, compete with full-length probe for the target sites. This should be less of a problem with complex DNA since there are plenty of sites for the probe pieces to transiently bind other than the actual target sites. Therefore, the target sites remain available for binding with the full-length probe. A final possibility is that the single-stranded regions on either side of probe hybridized to genomic DNA increase cleavage efficiency. This seems unlikely, however, as single-stranded regions next to a restriction enzyme binding site have not been shown to contribute to cleavage efficiency. Indeed, we have found that even the length of a double stranded 5′ extension has little effect on the cleavage reaction. For instance, Bsub 3 has 3 bp 5′ of the restriction enzyme recognition site while Bsub 6 has a 2 bp overhang, yet they have similar sensitivities on both genomic and oligo targets. In addition, we have found that probes with only 1 bp 5′ of the recognition site work well. NESA can be used as a method to detect specific organisms based on their DNA sequence. One of the common problems with current detection methods is the requirement to purify the DNA away from contaminants prior to analysis. When combined with multiple displacement amplification (MDA), NESA can be used to detect specific sequences from crudely prepared samples ( Figure 5 ). MDA has the ability to amplify DNA from an unpurified sample because it is tolerant of the contaminants commonly found in biological or environmental samples ( 10 , 17 ). Preliminary studies indicate that MDA can amplify DNA from unpurified environmental samples in cases where real-time PCR fails (data not shown). While NESA already exhibits excellent sensitivity, detecting a single DNA target in less than 30 cfu ( Figure 5 ), further increases in sensitivity are possible by optimization of the capillary electrophoresis step. The CE step currently requires a 100-fold dilution of the reaction. Therefore, of the 1 pmol of probe that was in the reaction, only 10 fmol is interrogated by the CE system. If the reaction can be modified such that the 1:100 dilution is not required, a 100-fold increase in sensitivity can be gained. The dilution step serves two purposes. First it decreases the ionic strength of the buffer allowing more efficient electrokinetic injection. Second, it reduces the level of fluorescent oligo that would otherwise overload the capillary. In reactions where increased sensitivity is required, most of the fluorescent signal comes from uncut probe. In preliminary experiments, we have had some success in removing full-length probe by incorporating biotin into the fluorescent probe and then using streptavidin conjugated to magnetic beads to remove full-length probe following NESA. The specificity of NESA is in part due to the requirement of the target sequence to contain a nicking endonuclease target site. We have found that with Nt.AlwI probes a mismatch in any one of the 5 bp that constitute the recognition sequence completely inhibits cleavage (unpublished data). However, we do not know if impurities can give rise to star activity ( 18 ). The ideal length of the probe is not known, but presumably it is partially a function of the T m of the full-length probe and the T m of the two products. That is, the T m differential has to be sufficient to allow initial hybridization of the probe followed by subsequent dissociation of both probe pieces. The probe also requires a certain length to retain its uniqueness in terms of base-pair composition, and thus its specificity for the sequence to which it was designed. Furthermore, the thermostability of the nicking enzyme must be taken into account because the enzyme becomes denatured at temperatures upwards of 60°C. Therefore, probes cannot have a T m much greater than 60°C because specificity would have to be forfeited in order to maintain enzyme function. We analyzed two probes at an incubation temperature of 58°C, Bsub 3 and Bsub 6. Bsub 6 had a T m of 56.3°C and was specific for B. subtilis while Bsub 3 had a lower T m of 50.6°C and was more promiscuous detecting both B. subtilis, B. anthracis and B. cereus . Clearly, specificity is not merely a function of probe T m as illustrated by these two examples. Instead, it is likely a combination of parameters, most notably T m and sequence composition, that influence specificity. NESA is also highly amenable to multiplexing since multiple sequences can be detected in a single reaction. Multiplexing by both probe size and fluorescent color is possible ( Figure 7 ). For size discrimination, the position of the restriction site within the probe can be varied ( Figure 7 ) or the fluorescent label can be placed at either the 5′ or 3′ end (data not shown). For color discrimination, we have shown that both FAM- and HEX-labeled probes can be used. Theoretically any fluorescent dye can be used that can be used to modify the oligonucleotide and that can be discriminated from the other dyes used in the reaction. This of course depends on the spectral characteristics of the dye and the capabilities of the capillary electrophoresis instrument used. We have found that the length of the probes is not a good predictor of their migration on CE; probes of the same length can separate while probes of different lengths can migrate together. In addition the fluor used can alter the migration pattern. While we focused on Nt.Alw1, there are a number of other naturally occurring or designed nicking endonucleases that have been described ( 19 , 20 and references within). However, only a few are available commercially, mainly from New England BioLabs ( 19 ). We have successfully tested six enzymes [Nb.BbvCI ( 21 ), Nb.BsmI, Nb.BsrDI, Nt.AlwI ( 13 ), Nt.BbvCI ( 21 ), Nt.BstNBI ( 12 )] in NESA but we have yet to perform a careful analysis of their relative effectiveness. Nb.BsmI and Nb.BsrDI are particularly interesting in that they are active up to at least 65°C and so should allow the development of longer, more specific probes. Nb.BbvCI and Nt.BbvCI recognize the same 7 bp DNA sequence but cleave opposite strands. The 7-bp recognition site increases specificity of the probes but also reduces the number of regions of DNA that can be targeted (Nt.AlwI recognizes 5 bp). NESA, although designed to detect DNA sequences, theoretically can be used to detect RNA sequences as well, as long as the RNA can be converted to cDNA. In some applications, such as rapid detection of RNA viruses, the ability to detect RNA directly would be beneficial. We have not tried to use NESA with RNA targets but there is some evidence in the literature which suggests that detection of DNA/RNA hybrids by wild type ( 22 ) or genetically modified restriction enzymes ( 23 ) may be possible. In summary, the utility of NESA was demonstrated for detection of specific DNA sequences. Several occasions can be contemplated where NESA may represent the method of choice for detection of DNA sequences. First, NESA can be used as an assay in the detection of specific organisms. In medical diagnostics, NESA can be used as an assay for bacterial and viral agents, which affect both human and animal health. Since NESA is amenable to high-throughput automation, it can be used to combat bioterrorism in stand-alone devices that test aerosol samples for agents such as B. anthracis , the causative agent of anthrax. Of course with this assay as with all other DNA-based assays, terrorists could modify the target sequences rendering the detection system ineffective. The use of multiple probes and keeping targets secret makes this countermeasure less likely. In ecological studies, NESA would be useful in species and/or strain analysis. Genomic analysis is another area of research where it may be useful. NESA can distinguish between closely related DNA sequences and detect the presence of single nucleotide mutations/polymorphisms. In an experiment, where we introduced point mutations into oligonucleotide targets at each base-pair position, we found that a single mutation at any of the 5′ positions in the enzyme recognition site is sufficient to completely eliminate the signal (data not shown).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4997813/

Systematic review of potential health risks posed by pharmaceutical, occupational and consumer exposures to metallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide and its soluble salts

Aluminum (Al) is a ubiquitous substance encountered both naturally (as the third most abundant element) and intentionally (used in water, foods, pharmaceuticals, and vaccines); it is also present in ambient and occupational airborne particulates. Existing data underscore the importance of Al physical and chemical forms in relation to its uptake, accumulation, and systemic bioavailability. The present review represents a systematic examination of the peer-reviewed literature on the adverse health effects of Al materials published since a previous critical evaluation compiled by Krewski et al. (2007) . Challenges encountered in carrying out the present review reflected the experimental use of different physical and chemical Al forms, different routes of administration, and different target organs in relation to the magnitude, frequency, and duration of exposure. Wide variations in diet can result in Al intakes that are often higher than the World Health Organization provisional tolerable weekly intake (PTWI), which is based on studies with Al citrate. Comparing daily dietary Al exposures on the basis of "total Al" assumes that gastrointestinal bioavailability for all dietary Al forms is equivalent to that for Al citrate, an approach that requires validation. Current occupational exposure limits (OELs) for identical Al substances vary as much as 15-fold. The toxicity of different Al forms depends in large measure on their physical behavior and relative solubility in water. The toxicity of soluble Al forms depends upon the delivered dose of Al +3 to target tissues. Trivalent Al reacts with water to produce bidentate superoxide coordination spheres [Al(O 2 )(H 2 O 4 ) +2 and Al(H 2 O) 6 +3 ] that after complexation with O 2 •− , generate Al superoxides [Al(O 2 • )](H 2 O 5 )] +2 . Semireduced AlO 2 • radicals deplete mitochondrial Fe and promote generation of H 2 O 2 , O 2 •− and OH • . Thus, it is the Al +3 -induced formation of oxygen radicals that accounts for the oxidative damage that leads to intrinsic apoptosis. In contrast, the toxicity of the insoluble Al oxides depends primarily on their behavior as particulates. Aluminum has been held responsible for human morbidity and mortality, but there is no consistent and convincing evidence to associate the Al found in food and drinking water at the doses and chemical forms presently consumed by people living in North America and Western Europe with increased risk for Alzheimer's disease (AD). Neither is there clear evidence to show use of Al-containing underarm antiperspirants or cosmetics increases the risk of AD or breast cancer. Metallic Al, its oxides, and common Al salts have not been shown to be either genotoxic or carcinogenic. Aluminum exposures during neonatal and pediatric parenteral nutrition (PN) can impair bone mineralization and delay neurological development. Adverse effects to vaccines with Al adjuvants have occurred; however, recent controlled trials found that the immunologic response to certain vaccines with Al adjuvants was no greater, and in some cases less than, that after identical vaccination without Al adjuvants. The scientific literature on the adverse health effects of Al is extensive. Health risk assessments for Al must take into account individual co-factors (e.g., age, renal function, diet, gastric pH). Conclusions from the current review point to the need for refinement of the PTWI, reduction of Al contamination in PN solutions, justification for routine addition of Al to vaccines, and harmonization of OELs for Al substances.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396676/

Novel Anti-Inflammatory Approaches for Cystic Fibrosis Lung Disease: Identification of Molecular Targets and Design of Innovative Therapies

Cystic fibrosis (CF) is the most common genetic disorder among Caucasians, estimated to affect more than 70,000 people in the world. Severe and persistent bronchial inflammation and chronic bacterial infection, along with airway mucus obstruction, are hallmarks of CF lung disease and participate in its progression. Anti-inflammatory therapies are, therefore, of particular interest for CF lung disease. Furthermore, a better understanding of the molecular mechanisms involved in airway infection and inflammation in CF has led to the development of new therapeutic approaches that are currently under evaluation by clinical trials. These new strategies dedicated to CF inflammation are designed to treat different dysregulated aspects such as oxidative stress, cytokine secretion, and the targeting of dysregulated pathways. In this review, we summarize the current understanding of the cellular and molecular mechanisms that contribute to abnormal lung inflammation in CF, as well as the new anti-inflammatory strategies proposed to CF patients by exploring novel molecular targets and novel drug approaches. Introduction Cystic fibrosis (CF) is the most common lethal monogenic disorder in Caucasians estimated to affect one out of 2.500-4.000 newborns. It is caused by a Cystic Fibrosis Transmembrane conductance Regulator ( CFTR ) gene mutation, which encodes a chloride channel expressed at the apical membrane of the epithelial cells ( Riordan et al., 1989 ). CF is a multi-system disease that affects the respiratory tract, intestines, pancreas, genital tract, the hepatobiliary system, and exocrine glands, leading to diverse pathology ranges and clinical problems ( Elborn, 2016 ). While most patients have multiple organ alterations, the leading causes of both morbidity and mortality in more than 90% of patients remain chronic progressive pulmonary disease and respiratory failure ( Elborn, 2016 ). In CF patients, the lack of CFTR chloride channel activity leads to progressive pulmonary obstruction associated with critical and constant neutrophil-dominated endobronchial inflammation and overwhelming bacterial infection ( Figure 1 ). On a pulmonary level, scientists developed many new symptomatic therapies with either anti-inflammatory properties, antibiotics, or molecules improving mucociliary clearance (mucolytics) in order to treat inflammation, infection, or mucus abnormalities ( Figure 2A ). The discovery of these new drugs was made possible by the accumulation of knowledge in these three areas. After the discovery of CFTR, researchers aimed for the development of therapies that can correct the disease's origin. Their work mainly focused on infection, rather than on anti-inflammatory drugs or mucus abnormalities. The proportion of published articles on infection is more than 70% compared to those published on inflammation or mucus. This proportion reaches more than 80% when focusing on publications on antibiotics compared to those on anti-inflammatory drugs and mucolytics ( Figures 2B, D ). In the allocation of priorities, the anti-inflammatory drugs have been, for long, the "poor relatives" in basic research compared to the modulators of CFTR activity. Figure 1 Progression of CF pathophysiology in bronchial epithelial cells. In healthy airways, sodium (Na + ) absorption and chloride (Cl − ) secretion control hydration of the airway surface layer (ASL). In CF airways, impaired Cl − secretion due to the CFTR absence or loss of function leads to unregulated Na + absorption and result in inadequate hydration of ASL, causing mucociliary clearance and bacterial killing impairment. As a result, mucus obstructs the lung airways and provides a nidus for bacterial infection and inflammation. The bacteria adhere to the surface and continue to grow, ultimately forming a biofilm. The inflammation of the CF lung is characterized by exaggerated secretion of pro-inflammatory cytokines by the airway epithelial cells, leading to the infiltration of polymorphonuclear neutrophils that release reactive oxygen species (ROS) and proteases. Neutrophil released elastase in the CF airway secretions correlates with lung function deterioration and respiratory exacerbations. The acidification of the ASL and the increase of its salt concentration, along with the increase of proteases levels, have been shown to impair the bactericidal activity of numbers of anti-microbial peptides (AMPs). Figure 2 (A) The proportion of publications published in Pubmed ( https://www.ncbi.nlm.nih.gov/pubmed ) by years about "anti-inflammatory," "mucolytic," and "antibiotic" in combination with "cystic fibrosis" compared to the total number of publications in CF. (B) The proportion of publications published in Pubmed by years about "anti-inflammatory," "mucolytic," and "antibiotic" in CF. (C) The proportion of publications published in Pubmed by years about "mucus," "infection," and "inflammation" in combination with "cystic fibrosis" compared to the total number of publications in CF. (D) The proportion of publications published in Pubmed by years about "mucus," "infection," and "inflammation" in CF. These drug modulators targeting CFTR are designed to reestablish, at least partially, the CFTR expression, and improve its activity. So far, many of these treatments got through to the market, and these therapies are upgrading patients' life quality through short- and long-term improvements in clinical outcomes ( Lopes-Pacheco, 2019 ). Despite this, the main treatments remain symptomatic, focusing on different dysregulated clinical manifestations observed in CF patients (pancreatic insufficiency, intestinal malabsorption, and lung deterioration). However, their use is limited by insufficient basic scientific knowledge ( Figure 2C ), which has reduced the number of medicinal products currently on the market ( Lopes-Pacheco, 2019 ). A deeper understanding of the natural evolution of CF pathology brought about new treatment tactics in order to improve pulmonary functions and increase life expectancy. CFTR chloride channel is also involved in the regulation of other channels such as the epithelial sodium channel (ENaC). Other channels are directly or indirectly linked to CFTR, such as the calcium-activated chloride channels ANO1 (also called TMEM16a) ( Benedetto et al., 2017 ) ( Figure 3 ). Therefore a deregulated CFTR activity leads to an abnormal mucus composition and alteration of the airway surface liquid (ASL) hydration that could participate in the inflammatory process in CF airways ( Puthia et al., 2020 ). Recent publications have also highlighted that a loss of CFTR-mediated bicarbonate secretion and pH acidification impairs airways host defense by increasing mucus viscosity and reducing bacteria-killing ( Shah et al., 2016 ). Current studies have established that the CFTR function is not restricted to ion transport regulation. Results have suggested a significant role of CFTR as a surface receptor for the internalization of Pseudomonas aeruginosa ( P. aeruginosa ) via endocytosis and consequent bacteria removal from the airway ( Pier, 2000 ). In the CF airways, the permanent presence of bacteria might participate in the inflammatory process contributing to a vicious cycle between airway mucus obstruction, chronic infection, and exaggerated inflammation ( Figure 4 ). Nowadays, it remains unclear how and why this vicious cycle is initiated, even though different elements suggest that different inflammatory pathways are deregulated in CF airways independently from infection ( Bardin et al., 2019 ). However, mucus alterations could be one of the triggers of this process. Mucins tethering to the apical bronchial surfaces lead to acidification of ASL, thus reducing the anti-bacterial properties of CF airways ( Song et al., 2006 ; Quinton, 2008 ; Adam et al., 2015 ). Figure 3 Schematic representation of ion transports in the cystic fibrosis airway. (A) In healthy airways, Na + absorption, and CFTR and ANO1 Cl − secretion regulate the hydration of the airway surface layer (ASL). Wild-type CFTR downregulates ENaC and participates in the activity of the ANO1 channel. (B) In CF airways epithelial cells, the lack of a functional CFTR channel reduces Cl − secretion and causes Na + hyperabsorption leading to ASL dehydration, which favors mucostasis. Figure 4 Interrelation between the main dysregulated aspects in the airway of Cystic Fibrosis patients. CFTR mutations affect inflammation, mucus properties, and infection. These different aspects are very intertwined, and treating one of these elements will have consequences on the other two. Finally, it is essential to bear in mind that mucus alteration, infection, and inflammation are elements that are very carefully intertwined and difficult to separate in the process of an inflammatory response ( Figure 4 ). Multiple hypotheses explain the early events leading to the CF lung pathophysiology progression ( Jacquot et al., 2008a ; Esther et al., 2019 ). Pathophysiology in CF Airways Inflammation Although inflammation is a natural and protective process resulting from aggression, it plays a major role in CF lung pathology and progression. Inflammation was initially recorded by the Roman encyclopedist Aulus Cornelius Celsus in the 1 st century A.D. by some typical characteristic signs of inflammation as heat (calor), pain (dolor), redness (rubor), and swelling (tumor). Chronic and exaggerated inflammation in people with CF causes damages to lung tissues that can eventually lead to respiratory failure ( Cantin et al., 2015 ). Many recent results show that bronchial epithelial cells play a significant role in the progression of the disease. In addition to being a physical barrier, epithelial cells secrete many inflammatory factors such as cytokines, eicosanoids, enzymes, and adhesion molecules ( Roesch et al., 2018 ). This CF airway inflammation is characterized by an excessive production of interleukin (IL)-8 secreted by airway epithelial cells, and the presence of large numbers of neutrophils and macrophages among other inflammatory cells ( Hubeau et al., 2001 ). However, it is not the only pro-inflammatory cytokine enhanced. In the airways of CF patients, TNF-α, IL-1β, IL-6, IL-8, IL-33, GM-CSF, and G-CSF are increased, also other molecules also play a major role such as the pro-inflammatory metabolites of arachidonic acid metabolism. Very recent results have highlighted the central role of other cytokines such as IL-17 ( Roesch et al., 2018 ). In CF, the infiltration of inflammatory cells across the epithelium into the lumen can be very deleterious to epithelia and, as a consequence, requires robust regulation. Numerous works have tried to identify targets and strategies to reduce the exaggerated immune response that causes chronic inflammation without affecting the natural defenses against infection ( Muhlebach and Noah, 2002 ). It is unclear whether the inflammation is a direct consequence of the cftr mutation or whether it is a consequence of infection and mucus accumulation. We do not know the contribution of infection to airway inflammation, but it must act as a catalyst and becomes self-perpetuating. Different studies have demonstrated the direct implication of the CFTR protein in this process mainly in the lung but also in extra-pulmonary tissues as the intestine or pancreas ( Raia et al., 2000 ; Cohen and Prince, 2012 ; Stoltz et al., 2015 ; Bardin et al., 2019 ). Even before symptom onset, pulmonary inflammation and infection are often present in CF patients ( Muhlebach and Noah, 2002 ). Although which comes first has been uncertain, this aspect is well reviewed in the article from Stoltz ( Armstrong et al., 1995 ; Khan et al., 1995 ; Nixon et al., 2002 ; Stoltz et al., 2015 ). Moreover, new models lacking CFTR, including pigs, ferrets, and rat manifest inflammatory features typically observed with CF even in absence of infection ( Rogers et al., 2008 ; Sun et al., 2010 ; Tuggle et al., 2014 ). For example, airways of CF piglets show no evidence of inflammation during the first hours after birth ( Stoltz et al., 2010 ). Evidence has also demonstrated that non-infected human CF airway graft is in a pro-inflammatory state ( Tirouvanziam et al., 2000 ; Tirouvanziam et al., 2002 ; Perez et al., 2007 ; Cantin et al., 2015 ). These data are reinforced by in vitro experiments using specific CFTR inhibitor. For example, Perez et al. have shown that Inh-172 treatment conducted in significant increase in IL-8 secretion in basal but also in response to P. aeruginosa infection ( Perez et al., 2007 ). All these data support the hypothesis that mutations in cftr gene make epithelial cells intrinsically more pro-inflammatory compared with healthy cells ( Perez et al., 2007 ; Cantin et al., 2015 ), which, once infection is introduced, sets the stage for mucosal damage and chronic airway infection ( Tirouvanziam et al., 2000 ). Although the link between CFTR deficiency and host inflammatory response remains unclear, this aspect has long been recognized as a central pathological feature, and consequently, an important therapeutic target. Some have hypothesized that in CF, the unfolded proteins accumulation on the endoplasmic reticulum induced a proteinopathy responsible for inflammation, impaired trafficking, altered metabolism, cholesterol, and lipids accumulation, and impaired autophagy at the cellular level. Some have speculated that chloride dysregulation participated in a stress-inducing ionic imbalance in the airway, with the implication of calcium activation, which could induce an inflammatory state ( Ribeiro et al., 2005 ; Tabary et al., 2006a ). New hypotheses have emerged with the direct activation of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome and can be a key regulator of CF inflammation and a promising target ( McElvaney et al., 2019 ; Jarosz-Griffiths et al., 2020 ). However, since the appearance of high throughput sequencing, many studies have attempted to study the deregulated mechanisms, but the heterogeneity of samples and data makes analysis difficult. A meta-analysis of the different studies has summarized all this data ( Ideozu et al., 2019 ). To summarize, many proteins are dysregulated, including gene from signal transduction (PI3K/Akt/mTOR signaling pathway) and immune system (NFκB and MAP kinase pathways), but this method is more relevant to highlight the consequence than the cause of the inflammatory dysregulation. A very recent article has confirmed the implication of NLRP3 inflammation activation due to the alteration of electrolyte homeostasis induced by the over-activation of β-ENac channel in CF ( Scambler et al., 2019 ). Furthermore, different authors showed more than 15 years ago that there is a deregulation of lipid metabolism in CF with an imbalance between pro-inflammatory metabolites of arachidonic acid metabolism and pro-resolving mediators form eicosanoid pathway ( Freedman et al., 2004 ; Karp et al., 2004 ; Serhan, 2017 ; Roesch et al., 2018 ). Ceramide (CER) is an airway component composed of fatty acid and sphingosine that may alter the CF inflammatory response. CER is present in the cells membrane and when in contact with a specific stimulus, like a bacterial infection, CER in transmembrane signaling processes to help regulate cellular responses to infection by activating the inflammation processes. This could be an interesting alternative to treat CF inflammatory dysregulation by inhibiting CER synthesis ( Mingione et al., 2020 ). Although there is no consensus regarding the regulation of CER in CF cells currently, even if more recent data have demonstrated their implication on the progression of CF lung disease ( Horati et al., 2020 ; Mingione et al., 2020 ). Consequently, these results have led to the proposal that upregulated inflammation is related to the molecular defect of CF with a strong implication of nuclear factor kappa B (NFκB) or mitogen-activated protein (MAP) kinase pathways with other transcription factors including NFAT, NF-IL6, AP1 and AP2 ( Tabary et al., 1999 ; Tabary et al., 2003 ; Muselet-Charlier et al., 2007 ). More recently, different articles have also associated microRNA (miRNA) dysregulation to CF inflammation ( Fabbri et al., 2014 ; Bardin et al., 2018a ; Bardin et al., 2019 ). How the lack of CFTR expression in ionocytes, ciliated, and submucosal gland epithelial cells of the respiratory tract, boosts pulmonary inflammation is still partially comprehended. Different authors have also highlighted the central role of neutrophil in CF airway inflammation, and many believed that bronchiectasis results from the proteolytic and oxidative damage induced by these cells. Longitudinal data from the Australian Respiratory Early Surveillance Team for Cystic Fibrosis demonstrated that neutrophil elastase activity at 3 months of age was a predictor of bronchiectasis at 12 months and 3 years ( Wijker et al., 2020 ). The central role of neutrophils and its genesis has been extensively review by Nichols et al. and Perrem et al. ( Nichols and Chmiel, 2015 ; Perrem and Ratjen, 2019 ). Understanding the initial host defense defects in CF airways could suggest new prevention strategies and treatments, the means to assess disease status and efficacy of therapeutics ( Stoltz et al., 2015 ). Several mechanisms are suggested to explain in what way CF basal inflammation promotes subsequent bacterial infection. One possible explanation is that serine protease, released by activated neutrophils, degrades innate immune mechanisms, including anti-microbial peptides (AMP), participates in secondary infection, and to this vicious cycle. The molecular mechanisms relating to abnormal CFTR chloride function in airway epithelial cells to excessive lung neutrophilic inflammation have not yet been fully clarified even if extensive works have already been published ( Taggart et al., 2000 ; Tabary et al., 2006b ). Decreased neutrophil apoptosis and the high secretion of IL-8 by epithelial cells are contributing factors. In 2016, researchers discovered the leukocyte adhesion deficiency IV (LAD-IV), which is a defect in monocyte integrin activation in CF patients. The study showed that CFTR mutations could lead to a monocyte-specific adhesion deficiency (~80%) and impairment in transmigration into the alveolar space, which could explain the extreme infiltration of neutrophil since monocytes play a crucial part in inflammation and its resolution. Thus, failing to recruit monocytes in CF patients' lungs may explain the excessive production of cytokines, the impaired inflammation resolution, and pathogen capture impairment ( Sorio et al., 2016 ). The continuous driven recruitment of neutrophils and other immune cells and their implication in non-resolving inflammation have been already discussed in different reviews ( Cantin et al., 2015 ; Nichols and Chmiel, 2015 ; Roesch et al., 2018 ). Whether CFTR dysfunction causes directly or indirectly, a more important predisposition to infection and whether the inflammation occurs separately from the infection has yet to be determined. The development of new anti-inflammatory strategies in CF remains limited due to the limited researches in this area compared to infection ( Figure 2D ). Bacterial Infection Respiratory infections in CF occur from childhood. In progressive lung diseases like CF, typical pathogens ( P. aeruginosa, Streptococcus aureus, Burkholderia cepacia, Achromobacter xylosoxidans ) colonize the airways ( Palser et al., 2019 ). More than 50% of children diagnosed at birth have shown positive P. aeruginosa cultures by five years of age ( Palser et al., 2019 ). If P. aeruginosa is neither spontaneously cleared nor eradicated with antibiotic therapy, the CF lung environment facilitates the infection. The presence of pathogens triggers inflammatory processes in the airways contributing to the destruction of the cell barrier. Since inflammation is a natural process of defense and the eradication of pathogens, limiting it too much or for a long term could be counterproductive. For this reason, antibiotics are more frequently recommended than anti-inflammatory drugs in CF lung disease treatment and could indirectly serve to diminish airway inflammation ( Oermann et al., 1999 ). The anti-inflammatory drugs that could alter the natural defense of the lung are only prescribed during exacerbations. Constant development and ideal usage of new anti-microbial compounds are vital for improving the CF patients survival chance and quality of life ( Waters and Smyth, 2015 ). As a result of long-term antibiotic treatment, the decrease in infection and inflammation is associated with lung function improvements and pulmonary exacerbations reduction ( Waters, 2018 ). In a normal situation, the airways can defend themselves by forming a physical barrier between the outside and the inside. Also, the lung is capable of secreting cytokines that will allow the recruitment of inflammatory cells, but it is also capable of secreting anti-bacterial molecules. Thus, many natural AMPs, contained in the airways, are part of the innate immune response to the airway defense ( Hancock et al., 2016 ). AMPs exhibit microbicidal activities on a broad spectrum of microbes, but bacteria appear to be the most targeted pathogens ( Scott and Hancock, 2000 ; Zasloff, 2002 ). AMPs can kill bacteria rapidly in a few minutes. If most of the AMPs kill targeted pathogens via an electrostatic action on their membranes, some of them kill by more sophisticated mechanisms such as the IIA secretory phospholipase A2 (sPLA2-IIA) which kills bacteria through selective hydrolysis of their membrane phospholipids ( Van Hensbergen et al., 2020 ), or by interfering with intracellular targets in bacteria ( Geitani et al., 2019 ; Wang et al., 2019 ). Except for very few examples, little is known about the specificity of AMPs toward Gram-positive vs. Gram-negative bacteria. The sPLA2-IIA is one of the rare AMPs that target Gram-positive bacteria that exerts its bactericidal effect at much lower concentrations than other molecules [For details, see the review ( Van Hensbergen et al., 2020 )]. AMPs represent an essential part of the host defenses against infections and also as a potential therapeutic tool, as has been shown in infections animal models ( Morrison et al., 2002 ; Piris-Gimenez et al., 2005 ). This effect was also supported by studies in patients with infectious diseases showing that altered AMP expression and/or gene polymorphisms were associated with increased infections ( Rivas-Santiago et al., 2009 ). On the other hand, unfavorable circumstances for AMPs actions as abnormal salt concentration or acidification, and inactivation by proteases, in ASL of CF patients ( Figure 1 ), have been shown to inactivate AMPs bactericidal functions which may explain increased airway infections ( Bals et al., 2001 ; Lecaille et al., 2016 ; Simonin et al., 2019 ). Normalizing ASL pH by inhibition of the persistent proton secretion, mediated by ATPase H + /K + transporting non-gastric alpha2 subunit (ATP12A), might enhance innate airway defense in CF newborns during the onset of S. aureus infection. A recent study showed that the hydrophobic N-terminal domain of Cg- BigDef1 (a big defensin from oysters) exhibits salt-stable interactions with bacterial membranes opening the doors to eventual drug developments when physiological salt concentrations inhibit the anti-microbial activity of β-defensins such as in CF disease ( Loth et al., 2019 ). In parallel to their anti-microbial functions, several AMPs have been shown to play immuno-modulatory roles, in particular by interacting with the inflammatory reaction produced by host cells. Several studies have shown that AMPs can target host cells involved in innate immunity and modulated their production of inflammatory mediators, including cytokines. Although it is not always easy to dissociate these actions as most AMPs exhibit both functions, depending on their concentrations, the host cell targets, and the environments. However, AMPs have been shown to impair the inflammatory reaction induced by invading pathogens by different mechanisms ( Masera et al., 1996 ; Finlay and Hancock, 2004 ; McInturff et al., 2005 ). The anti-inflammatory potential of AMPs correlates with their capability of attracting and recruiting neutrophils and other inflammatory cells. They may also have direct or indirect effects on their maturation, differentiation, degranulation, or apoptosis ( Lai and Gallo, 2009 ). AMPs also act by blocking neutrophils apoptosis, therefore prolonging their lifetime, and ultimately their phagocytic functions ( Nagaoka et al., 2012 ). AMPs can also potentiate the effects of inflammatory cells such as macrophages while limiting other tissue damage ( Brook et al., 2016 ). Mucus Alteration In healthy people, ASL is a thin liquid film covering the airways and participating in mucociliary clearance and airways desiccation ( Figure 1 ). Historically, studies suggested that different secretory cells (goblet cells, submucosal glands cells, and serous cells) contribute to ASL production ( Tarran et al., 2001 ). The recent finding of the airway "ionocyte" could similarly result in a revised understanding of ASL production ( Plasschaert et al., 2018 ). This group has identified by RNA sequencing all the RNAs present inside airway cells and by a new method, called pulse-seq, has discovered this scarce cell type. They created the term "ionocytes" due to the cell's likeness to ionocytes in charge of regulating ion transport and hydration in the fish gills and frog skin. In the airway, ASL consists of two main layers: 1) the apical layer consisting of a water-based polymeric mucus, and 2) a periciliary layer (PCL) that bathes the epithelium ( Atanasova and Reznikov, 2019 ). Normal mucus is made of 97% water and 3% proteins, lipids, and salt. The mucus gel layer acts as a physical barrier to prevent most pathogens from accessing the cells ( Button et al., 2012 ). The mucus hydration and the mucin concentration dramatically affects its viscoelastic properties, which, in turn, determines how effectively it is cleared from the distal airways toward the trachea by ciliary action and cough ( Fahy and Dickey, 2010 ). The commonly accepted explanation for airway disease in CF is the "low volume" hypothesis. A reduced volume of the periciliary fluid layer (PCL) causes failure of mucociliary clearance, the 'lungs' innate defense mechanism. In addition to having altered physical properties, the mucus composition is modified and will participate in the CF pathophysiology by altering host defense proteins ( Henderson et al., 2014 ). An increase in mucin secretion and an abnormal composition of mucus are implied by the formation of endobronchial mucus plaques and plugs. Mucus present in bronchia becomes the primary site of airflow obstruction, and subsequently for chronic infection, and persistent inflammation leading to early small airways disease succeeded by bronchiectasis development. Increased mucus and airway obstruction are hallmark features of multiple respiratory diseases and contribute, especially in CF, to a complicated, inflammatory process ( Puthia et al., 2020 ). A chronic cycle of infection and inflammation could be initiated, resulting in airways structural integrity damages and leading to bronchiectasis development ( Chalmers et al., 2017 ). More recent studies from Esther et al. have shown that the increase of mucus burden and inflammatory markers without infection suggest that mucolytic therapies could serve as preventive therapy for CF lung pathology ( Esther et al., 2019 ). More, mucus composition and properties also depend on the levels of mucin production by epithelial cells that can be increased by bacteria suggesting a complex role of inflammation, infection, and mucus, especially in CF pathology ( Mohamed et al., 2012 ). The up-regulation of airway mucin genes by inflammatory/immune response mediators at the transcriptional and/or posttranscriptional level is one of the major contributors to mucin overproduction. The MUC5AC gene is transcriptionally up-regulated by several inflammatory mediators, including LPS, IL-9, neutrophil elastase, TNF-α, and IL-1β ( Song et al., 2003 ). IL-8-induced binding of RNA-binding proteins to the 3-untranslated region of MUC5AC is a potential mechanism for regulating MUC5AC gene expression at the posttranscriptional level ( Bautista et al., 2009 ). Several studies have shown that PMA induces a matrix metalloprotease-mediated release of transforming growth factor-( Shao et al., 2003 ). Eicosanoids mediate inflammation and mucus secretion in chronic pulmonary inflammatory diseases ( Garcia-Verdugo et al., 2012 ). Some studies in the field have shown a substantial increase of eicosanoid levels, including PGE2 and LTB4 in CF airways ( Bautista et al., 2009 ) and CF bronchial epithelial cells (BECs) stimulated by LPS from P. aeruginosa ( Medjane et al., 2005 ). On the other hand, this bacterium stimulates mucus production through the induction of several mucins such as MUC5AC and MUC2 both in cultured BECs and in a mouse model of lung infection by P. aeruginosa . This induction mainly involves the stimulation of BECs by flagellin through the TLR5 and Naip pathways and is accompanied by the secretion of IL-8 by BECs, which amplify mucus production ( Mohamed et al., 2012 ). Thus, we can suggest that in CF airways, mucus abnormalities offer a niche that favors bacterial infections, which in turn amplify mucus accumulation via a vicious circle that can participate in the exacerbation of the severity of CF disease. This amplification can occur either directly via virulence factors (such as flagellin and LPS) of infecting bacteria or via cytokines and eicosanoids produced by CF airways during infection. Proteases and Lipids Imbalance Current studies on mucolytic agents therapy used in CF have been demonstrated to increase markedly neutrophil elastase (NE) activity in CF sputum. Serine proteases, including NE, cathepsin G, and proteinase 3, are the three most major proteases found in the CF lung. These proteases are not only secreted by BECs, but also by monocytes, lymphocytes, granulocytes, and, more importantly, neutrophils ( Pelaia et al., 2004 ; Hunt et al., 2020 ). Different approaches have exposed their participation in intracellular and extracellular activities, including inflammation, tissue remodeling, mucin expression, bacterial killing, and neutrophil chemotaxis. NE, a significant product of neutrophils granule degranulation, is extensively studied in CF and is implicated in cleavage and inactivation of CFTR protein ( Chalmers et al., 2017 ). Besides, NE also upregulates IL-8 and participates in activating cysteinyl cathepsins and matrix metalloproteases. In the CF airway, different articles have described the protease and anti-protease imbalance, which could be explained by two different mechanisms ( Galli et al., 2012 ; Causer et al., 2020 ). Firstly, CFTR is also a transporter of glutathione (GSH), a protease that is the main non-enzymatic antioxidant present in the ASL ( Rahman and MacNee, 2000 ). Antioxidants are an essential protective response to tissue injury and occur mainly in an inflammatory environment. An absence of GSH in the extracellular medium disequilibrates this balance and induces an oxidative environment. This environment is intended to fight bacteria and viruses that may be present. The goal of this process is to break up and eliminate the injured tissues and, thus, promote tissue repair for the inflammatory process resolution. When this natural response arises in an uncontrolled way, the outcome is extreme tissue damage that could induce chronic inflammation, as observed in CF ( Figure 1 ). During inflammation, reactive oxygen species (ROS) such as the superoxide anion are liberated by phagocytes and are thought to be the main cause of tissue damage. In CF, the presence of numerous inflammatory cells that release many oxidants will have a significant role in the deregulation of the pro- and anti-inflammatory balance. Lung cells are vulnerable to the damaging effects of ROS and release inflammatory mediators, thereby amplifying lung inflammation. ROS are extraordinarily reactive, and when produced near the cell membranes, they diminish intracellular GSH and cause lipid peroxidation, which may harshly disrupt its function and may lead to cell death or DNA damage in alveolar epithelial cells. So, when ROS production increases, the redox balance of the airways is altered, and this can lead to bronchial hyperactivity and to further inflammation and participates in CF co-morbidity. GSH is a sulfhydryl containing tripeptide (L-γ-glutamyl-L-cysteinyl-glycine) that scavenges oxidants and could, therefore, participate in the control of the inflammatory process by reducing oxidative stress ( Rahman and MacNee, 2000 ; Ehre et al., 2019 ). Therefore, a CFTR deficiency leads to an increased accumulation of intracellular GSH in the epithelial lining fluid compared with plasma. Secondly, different dysregulated parameters such as infection, inflammation, and hypoxia, increase the free radicals derived from oxygen and nitrogen. This pro-oxidative environment may directly exert its effects by activating transcription factors such as NFκB and MAP kinase pathways responsible for the coordinated expression of numerous genes involved in inflammation, cell death, proliferation, as well as cytoprotection and antioxidant defenses ( Pelaia et al., 2004 ). CFTR-deficient tracheal epithelial cells are characterized by high GSH levels that decrease the intracellular content of ceramide (CER). CER deficiency occurring in CF seems to be responsible for the increased activation of the pro-inflammatory transcriptional nuclear factor NFκB that, in turn, is responsible for the abnormally high inflammatory response in CF respiratory epithelial cells ( Vilela et al., 2006 ; Aureli et al., 2016 ). An increasing number of studies indicate that sphingolipids play an important regulatory role in CF concerning pulmonary inflammation. In different models, it has been shown that de novo sphingolipid synthesis is an inflammation responsive pathway. It is enhanced by inflammatory mediators, both at transcriptional and enzyme activity level, and the accumulation of its metabolite CER potentiates inflammation in a vicious circle ( Caretti et al., 2014 ). Sphingosine-1-phosphate (S1P), generated in the nucleus by phosphorylation of SphK2 ((Sphingosine Kinase 2), modulates HDAC (histone deacetylases) activity either by direct binding or through activation of nuclear ROS, and, regulates cell cycle and pro-inflammatory gene expression ( Fu et al., 2018 ). The accumulation of CER causes Cftr - deficient mice to suffer from constitutive age-dependent pulmonary inflammation, death of respiratory epithelial cells, deposits of DNA in bronchi, and high susceptibility to severe P. aeruginosa infections ( Teichgräber et al., 2008 ). Aggregates accrual, formed by misfolded mutant CFTR and a miscellaneous of sequestered proteins within, induces inflammation and oxidative stress, impairing proteins and lipids transport, and consequently inflammatory statement ( Mingione et al., 2020 ). Inflammation Although inflammation is a natural and protective process resulting from aggression, it plays a major role in CF lung pathology and progression. Inflammation was initially recorded by the Roman encyclopedist Aulus Cornelius Celsus in the 1 st century A.D. by some typical characteristic signs of inflammation as heat (calor), pain (dolor), redness (rubor), and swelling (tumor). Chronic and exaggerated inflammation in people with CF causes damages to lung tissues that can eventually lead to respiratory failure ( Cantin et al., 2015 ). Many recent results show that bronchial epithelial cells play a significant role in the progression of the disease. In addition to being a physical barrier, epithelial cells secrete many inflammatory factors such as cytokines, eicosanoids, enzymes, and adhesion molecules ( Roesch et al., 2018 ). This CF airway inflammation is characterized by an excessive production of interleukin (IL)-8 secreted by airway epithelial cells, and the presence of large numbers of neutrophils and macrophages among other inflammatory cells ( Hubeau et al., 2001 ). However, it is not the only pro-inflammatory cytokine enhanced. In the airways of CF patients, TNF-α, IL-1β, IL-6, IL-8, IL-33, GM-CSF, and G-CSF are increased, also other molecules also play a major role such as the pro-inflammatory metabolites of arachidonic acid metabolism. Very recent results have highlighted the central role of other cytokines such as IL-17 ( Roesch et al., 2018 ). In CF, the infiltration of inflammatory cells across the epithelium into the lumen can be very deleterious to epithelia and, as a consequence, requires robust regulation. Numerous works have tried to identify targets and strategies to reduce the exaggerated immune response that causes chronic inflammation without affecting the natural defenses against infection ( Muhlebach and Noah, 2002 ). It is unclear whether the inflammation is a direct consequence of the cftr mutation or whether it is a consequence of infection and mucus accumulation. We do not know the contribution of infection to airway inflammation, but it must act as a catalyst and becomes self-perpetuating. Different studies have demonstrated the direct implication of the CFTR protein in this process mainly in the lung but also in extra-pulmonary tissues as the intestine or pancreas ( Raia et al., 2000 ; Cohen and Prince, 2012 ; Stoltz et al., 2015 ; Bardin et al., 2019 ). Even before symptom onset, pulmonary inflammation and infection are often present in CF patients ( Muhlebach and Noah, 2002 ). Although which comes first has been uncertain, this aspect is well reviewed in the article from Stoltz ( Armstrong et al., 1995 ; Khan et al., 1995 ; Nixon et al., 2002 ; Stoltz et al., 2015 ). Moreover, new models lacking CFTR, including pigs, ferrets, and rat manifest inflammatory features typically observed with CF even in absence of infection ( Rogers et al., 2008 ; Sun et al., 2010 ; Tuggle et al., 2014 ). For example, airways of CF piglets show no evidence of inflammation during the first hours after birth ( Stoltz et al., 2010 ). Evidence has also demonstrated that non-infected human CF airway graft is in a pro-inflammatory state ( Tirouvanziam et al., 2000 ; Tirouvanziam et al., 2002 ; Perez et al., 2007 ; Cantin et al., 2015 ). These data are reinforced by in vitro experiments using specific CFTR inhibitor. For example, Perez et al. have shown that Inh-172 treatment conducted in significant increase in IL-8 secretion in basal but also in response to P. aeruginosa infection ( Perez et al., 2007 ). All these data support the hypothesis that mutations in cftr gene make epithelial cells intrinsically more pro-inflammatory compared with healthy cells ( Perez et al., 2007 ; Cantin et al., 2015 ), which, once infection is introduced, sets the stage for mucosal damage and chronic airway infection ( Tirouvanziam et al., 2000 ). Although the link between CFTR deficiency and host inflammatory response remains unclear, this aspect has long been recognized as a central pathological feature, and consequently, an important therapeutic target. Some have hypothesized that in CF, the unfolded proteins accumulation on the endoplasmic reticulum induced a proteinopathy responsible for inflammation, impaired trafficking, altered metabolism, cholesterol, and lipids accumulation, and impaired autophagy at the cellular level. Some have speculated that chloride dysregulation participated in a stress-inducing ionic imbalance in the airway, with the implication of calcium activation, which could induce an inflammatory state ( Ribeiro et al., 2005 ; Tabary et al., 2006a ). New hypotheses have emerged with the direct activation of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome and can be a key regulator of CF inflammation and a promising target ( McElvaney et al., 2019 ; Jarosz-Griffiths et al., 2020 ). However, since the appearance of high throughput sequencing, many studies have attempted to study the deregulated mechanisms, but the heterogeneity of samples and data makes analysis difficult. A meta-analysis of the different studies has summarized all this data ( Ideozu et al., 2019 ). To summarize, many proteins are dysregulated, including gene from signal transduction (PI3K/Akt/mTOR signaling pathway) and immune system (NFκB and MAP kinase pathways), but this method is more relevant to highlight the consequence than the cause of the inflammatory dysregulation. A very recent article has confirmed the implication of NLRP3 inflammation activation due to the alteration of electrolyte homeostasis induced by the over-activation of β-ENac channel in CF ( Scambler et al., 2019 ). Furthermore, different authors showed more than 15 years ago that there is a deregulation of lipid metabolism in CF with an imbalance between pro-inflammatory metabolites of arachidonic acid metabolism and pro-resolving mediators form eicosanoid pathway ( Freedman et al., 2004 ; Karp et al., 2004 ; Serhan, 2017 ; Roesch et al., 2018 ). Ceramide (CER) is an airway component composed of fatty acid and sphingosine that may alter the CF inflammatory response. CER is present in the cells membrane and when in contact with a specific stimulus, like a bacterial infection, CER in transmembrane signaling processes to help regulate cellular responses to infection by activating the inflammation processes. This could be an interesting alternative to treat CF inflammatory dysregulation by inhibiting CER synthesis ( Mingione et al., 2020 ). Although there is no consensus regarding the regulation of CER in CF cells currently, even if more recent data have demonstrated their implication on the progression of CF lung disease ( Horati et al., 2020 ; Mingione et al., 2020 ). Consequently, these results have led to the proposal that upregulated inflammation is related to the molecular defect of CF with a strong implication of nuclear factor kappa B (NFκB) or mitogen-activated protein (MAP) kinase pathways with other transcription factors including NFAT, NF-IL6, AP1 and AP2 ( Tabary et al., 1999 ; Tabary et al., 2003 ; Muselet-Charlier et al., 2007 ). More recently, different articles have also associated microRNA (miRNA) dysregulation to CF inflammation ( Fabbri et al., 2014 ; Bardin et al., 2018a ; Bardin et al., 2019 ). How the lack of CFTR expression in ionocytes, ciliated, and submucosal gland epithelial cells of the respiratory tract, boosts pulmonary inflammation is still partially comprehended. Different authors have also highlighted the central role of neutrophil in CF airway inflammation, and many believed that bronchiectasis results from the proteolytic and oxidative damage induced by these cells. Longitudinal data from the Australian Respiratory Early Surveillance Team for Cystic Fibrosis demonstrated that neutrophil elastase activity at 3 months of age was a predictor of bronchiectasis at 12 months and 3 years ( Wijker et al., 2020 ). The central role of neutrophils and its genesis has been extensively review by Nichols et al. and Perrem et al. ( Nichols and Chmiel, 2015 ; Perrem and Ratjen, 2019 ). Understanding the initial host defense defects in CF airways could suggest new prevention strategies and treatments, the means to assess disease status and efficacy of therapeutics ( Stoltz et al., 2015 ). Several mechanisms are suggested to explain in what way CF basal inflammation promotes subsequent bacterial infection. One possible explanation is that serine protease, released by activated neutrophils, degrades innate immune mechanisms, including anti-microbial peptides (AMP), participates in secondary infection, and to this vicious cycle. The molecular mechanisms relating to abnormal CFTR chloride function in airway epithelial cells to excessive lung neutrophilic inflammation have not yet been fully clarified even if extensive works have already been published ( Taggart et al., 2000 ; Tabary et al., 2006b ). Decreased neutrophil apoptosis and the high secretion of IL-8 by epithelial cells are contributing factors. In 2016, researchers discovered the leukocyte adhesion deficiency IV (LAD-IV), which is a defect in monocyte integrin activation in CF patients. The study showed that CFTR mutations could lead to a monocyte-specific adhesion deficiency (~80%) and impairment in transmigration into the alveolar space, which could explain the extreme infiltration of neutrophil since monocytes play a crucial part in inflammation and its resolution. Thus, failing to recruit monocytes in CF patients' lungs may explain the excessive production of cytokines, the impaired inflammation resolution, and pathogen capture impairment ( Sorio et al., 2016 ). The continuous driven recruitment of neutrophils and other immune cells and their implication in non-resolving inflammation have been already discussed in different reviews ( Cantin et al., 2015 ; Nichols and Chmiel, 2015 ; Roesch et al., 2018 ). Whether CFTR dysfunction causes directly or indirectly, a more important predisposition to infection and whether the inflammation occurs separately from the infection has yet to be determined. The development of new anti-inflammatory strategies in CF remains limited due to the limited researches in this area compared to infection ( Figure 2D ). Bacterial Infection Respiratory infections in CF occur from childhood. In progressive lung diseases like CF, typical pathogens ( P. aeruginosa, Streptococcus aureus, Burkholderia cepacia, Achromobacter xylosoxidans ) colonize the airways ( Palser et al., 2019 ). More than 50% of children diagnosed at birth have shown positive P. aeruginosa cultures by five years of age ( Palser et al., 2019 ). If P. aeruginosa is neither spontaneously cleared nor eradicated with antibiotic therapy, the CF lung environment facilitates the infection. The presence of pathogens triggers inflammatory processes in the airways contributing to the destruction of the cell barrier. Since inflammation is a natural process of defense and the eradication of pathogens, limiting it too much or for a long term could be counterproductive. For this reason, antibiotics are more frequently recommended than anti-inflammatory drugs in CF lung disease treatment and could indirectly serve to diminish airway inflammation ( Oermann et al., 1999 ). The anti-inflammatory drugs that could alter the natural defense of the lung are only prescribed during exacerbations. Constant development and ideal usage of new anti-microbial compounds are vital for improving the CF patients survival chance and quality of life ( Waters and Smyth, 2015 ). As a result of long-term antibiotic treatment, the decrease in infection and inflammation is associated with lung function improvements and pulmonary exacerbations reduction ( Waters, 2018 ). In a normal situation, the airways can defend themselves by forming a physical barrier between the outside and the inside. Also, the lung is capable of secreting cytokines that will allow the recruitment of inflammatory cells, but it is also capable of secreting anti-bacterial molecules. Thus, many natural AMPs, contained in the airways, are part of the innate immune response to the airway defense ( Hancock et al., 2016 ). AMPs exhibit microbicidal activities on a broad spectrum of microbes, but bacteria appear to be the most targeted pathogens ( Scott and Hancock, 2000 ; Zasloff, 2002 ). AMPs can kill bacteria rapidly in a few minutes. If most of the AMPs kill targeted pathogens via an electrostatic action on their membranes, some of them kill by more sophisticated mechanisms such as the IIA secretory phospholipase A2 (sPLA2-IIA) which kills bacteria through selective hydrolysis of their membrane phospholipids ( Van Hensbergen et al., 2020 ), or by interfering with intracellular targets in bacteria ( Geitani et al., 2019 ; Wang et al., 2019 ). Except for very few examples, little is known about the specificity of AMPs toward Gram-positive vs. Gram-negative bacteria. The sPLA2-IIA is one of the rare AMPs that target Gram-positive bacteria that exerts its bactericidal effect at much lower concentrations than other molecules [For details, see the review ( Van Hensbergen et al., 2020 )]. AMPs represent an essential part of the host defenses against infections and also as a potential therapeutic tool, as has been shown in infections animal models ( Morrison et al., 2002 ; Piris-Gimenez et al., 2005 ). This effect was also supported by studies in patients with infectious diseases showing that altered AMP expression and/or gene polymorphisms were associated with increased infections ( Rivas-Santiago et al., 2009 ). On the other hand, unfavorable circumstances for AMPs actions as abnormal salt concentration or acidification, and inactivation by proteases, in ASL of CF patients ( Figure 1 ), have been shown to inactivate AMPs bactericidal functions which may explain increased airway infections ( Bals et al., 2001 ; Lecaille et al., 2016 ; Simonin et al., 2019 ). Normalizing ASL pH by inhibition of the persistent proton secretion, mediated by ATPase H + /K + transporting non-gastric alpha2 subunit (ATP12A), might enhance innate airway defense in CF newborns during the onset of S. aureus infection. A recent study showed that the hydrophobic N-terminal domain of Cg- BigDef1 (a big defensin from oysters) exhibits salt-stable interactions with bacterial membranes opening the doors to eventual drug developments when physiological salt concentrations inhibit the anti-microbial activity of β-defensins such as in CF disease ( Loth et al., 2019 ). In parallel to their anti-microbial functions, several AMPs have been shown to play immuno-modulatory roles, in particular by interacting with the inflammatory reaction produced by host cells. Several studies have shown that AMPs can target host cells involved in innate immunity and modulated their production of inflammatory mediators, including cytokines. Although it is not always easy to dissociate these actions as most AMPs exhibit both functions, depending on their concentrations, the host cell targets, and the environments. However, AMPs have been shown to impair the inflammatory reaction induced by invading pathogens by different mechanisms ( Masera et al., 1996 ; Finlay and Hancock, 2004 ; McInturff et al., 2005 ). The anti-inflammatory potential of AMPs correlates with their capability of attracting and recruiting neutrophils and other inflammatory cells. They may also have direct or indirect effects on their maturation, differentiation, degranulation, or apoptosis ( Lai and Gallo, 2009 ). AMPs also act by blocking neutrophils apoptosis, therefore prolonging their lifetime, and ultimately their phagocytic functions ( Nagaoka et al., 2012 ). AMPs can also potentiate the effects of inflammatory cells such as macrophages while limiting other tissue damage ( Brook et al., 2016 ). Mucus Alteration In healthy people, ASL is a thin liquid film covering the airways and participating in mucociliary clearance and airways desiccation ( Figure 1 ). Historically, studies suggested that different secretory cells (goblet cells, submucosal glands cells, and serous cells) contribute to ASL production ( Tarran et al., 2001 ). The recent finding of the airway "ionocyte" could similarly result in a revised understanding of ASL production ( Plasschaert et al., 2018 ). This group has identified by RNA sequencing all the RNAs present inside airway cells and by a new method, called pulse-seq, has discovered this scarce cell type. They created the term "ionocytes" due to the cell's likeness to ionocytes in charge of regulating ion transport and hydration in the fish gills and frog skin. In the airway, ASL consists of two main layers: 1) the apical layer consisting of a water-based polymeric mucus, and 2) a periciliary layer (PCL) that bathes the epithelium ( Atanasova and Reznikov, 2019 ). Normal mucus is made of 97% water and 3% proteins, lipids, and salt. The mucus gel layer acts as a physical barrier to prevent most pathogens from accessing the cells ( Button et al., 2012 ). The mucus hydration and the mucin concentration dramatically affects its viscoelastic properties, which, in turn, determines how effectively it is cleared from the distal airways toward the trachea by ciliary action and cough ( Fahy and Dickey, 2010 ). The commonly accepted explanation for airway disease in CF is the "low volume" hypothesis. A reduced volume of the periciliary fluid layer (PCL) causes failure of mucociliary clearance, the 'lungs' innate defense mechanism. In addition to having altered physical properties, the mucus composition is modified and will participate in the CF pathophysiology by altering host defense proteins ( Henderson et al., 2014 ). An increase in mucin secretion and an abnormal composition of mucus are implied by the formation of endobronchial mucus plaques and plugs. Mucus present in bronchia becomes the primary site of airflow obstruction, and subsequently for chronic infection, and persistent inflammation leading to early small airways disease succeeded by bronchiectasis development. Increased mucus and airway obstruction are hallmark features of multiple respiratory diseases and contribute, especially in CF, to a complicated, inflammatory process ( Puthia et al., 2020 ). A chronic cycle of infection and inflammation could be initiated, resulting in airways structural integrity damages and leading to bronchiectasis development ( Chalmers et al., 2017 ). More recent studies from Esther et al. have shown that the increase of mucus burden and inflammatory markers without infection suggest that mucolytic therapies could serve as preventive therapy for CF lung pathology ( Esther et al., 2019 ). More, mucus composition and properties also depend on the levels of mucin production by epithelial cells that can be increased by bacteria suggesting a complex role of inflammation, infection, and mucus, especially in CF pathology ( Mohamed et al., 2012 ). The up-regulation of airway mucin genes by inflammatory/immune response mediators at the transcriptional and/or posttranscriptional level is one of the major contributors to mucin overproduction. The MUC5AC gene is transcriptionally up-regulated by several inflammatory mediators, including LPS, IL-9, neutrophil elastase, TNF-α, and IL-1β ( Song et al., 2003 ). IL-8-induced binding of RNA-binding proteins to the 3-untranslated region of MUC5AC is a potential mechanism for regulating MUC5AC gene expression at the posttranscriptional level ( Bautista et al., 2009 ). Several studies have shown that PMA induces a matrix metalloprotease-mediated release of transforming growth factor-( Shao et al., 2003 ). Eicosanoids mediate inflammation and mucus secretion in chronic pulmonary inflammatory diseases ( Garcia-Verdugo et al., 2012 ). Some studies in the field have shown a substantial increase of eicosanoid levels, including PGE2 and LTB4 in CF airways ( Bautista et al., 2009 ) and CF bronchial epithelial cells (BECs) stimulated by LPS from P. aeruginosa ( Medjane et al., 2005 ). On the other hand, this bacterium stimulates mucus production through the induction of several mucins such as MUC5AC and MUC2 both in cultured BECs and in a mouse model of lung infection by P. aeruginosa . This induction mainly involves the stimulation of BECs by flagellin through the TLR5 and Naip pathways and is accompanied by the secretion of IL-8 by BECs, which amplify mucus production ( Mohamed et al., 2012 ). Thus, we can suggest that in CF airways, mucus abnormalities offer a niche that favors bacterial infections, which in turn amplify mucus accumulation via a vicious circle that can participate in the exacerbation of the severity of CF disease. This amplification can occur either directly via virulence factors (such as flagellin and LPS) of infecting bacteria or via cytokines and eicosanoids produced by CF airways during infection. Proteases and Lipids Imbalance Current studies on mucolytic agents therapy used in CF have been demonstrated to increase markedly neutrophil elastase (NE) activity in CF sputum. Serine proteases, including NE, cathepsin G, and proteinase 3, are the three most major proteases found in the CF lung. These proteases are not only secreted by BECs, but also by monocytes, lymphocytes, granulocytes, and, more importantly, neutrophils ( Pelaia et al., 2004 ; Hunt et al., 2020 ). Different approaches have exposed their participation in intracellular and extracellular activities, including inflammation, tissue remodeling, mucin expression, bacterial killing, and neutrophil chemotaxis. NE, a significant product of neutrophils granule degranulation, is extensively studied in CF and is implicated in cleavage and inactivation of CFTR protein ( Chalmers et al., 2017 ). Besides, NE also upregulates IL-8 and participates in activating cysteinyl cathepsins and matrix metalloproteases. In the CF airway, different articles have described the protease and anti-protease imbalance, which could be explained by two different mechanisms ( Galli et al., 2012 ; Causer et al., 2020 ). Firstly, CFTR is also a transporter of glutathione (GSH), a protease that is the main non-enzymatic antioxidant present in the ASL ( Rahman and MacNee, 2000 ). Antioxidants are an essential protective response to tissue injury and occur mainly in an inflammatory environment. An absence of GSH in the extracellular medium disequilibrates this balance and induces an oxidative environment. This environment is intended to fight bacteria and viruses that may be present. The goal of this process is to break up and eliminate the injured tissues and, thus, promote tissue repair for the inflammatory process resolution. When this natural response arises in an uncontrolled way, the outcome is extreme tissue damage that could induce chronic inflammation, as observed in CF ( Figure 1 ). During inflammation, reactive oxygen species (ROS) such as the superoxide anion are liberated by phagocytes and are thought to be the main cause of tissue damage. In CF, the presence of numerous inflammatory cells that release many oxidants will have a significant role in the deregulation of the pro- and anti-inflammatory balance. Lung cells are vulnerable to the damaging effects of ROS and release inflammatory mediators, thereby amplifying lung inflammation. ROS are extraordinarily reactive, and when produced near the cell membranes, they diminish intracellular GSH and cause lipid peroxidation, which may harshly disrupt its function and may lead to cell death or DNA damage in alveolar epithelial cells. So, when ROS production increases, the redox balance of the airways is altered, and this can lead to bronchial hyperactivity and to further inflammation and participates in CF co-morbidity. GSH is a sulfhydryl containing tripeptide (L-γ-glutamyl-L-cysteinyl-glycine) that scavenges oxidants and could, therefore, participate in the control of the inflammatory process by reducing oxidative stress ( Rahman and MacNee, 2000 ; Ehre et al., 2019 ). Therefore, a CFTR deficiency leads to an increased accumulation of intracellular GSH in the epithelial lining fluid compared with plasma. Secondly, different dysregulated parameters such as infection, inflammation, and hypoxia, increase the free radicals derived from oxygen and nitrogen. This pro-oxidative environment may directly exert its effects by activating transcription factors such as NFκB and MAP kinase pathways responsible for the coordinated expression of numerous genes involved in inflammation, cell death, proliferation, as well as cytoprotection and antioxidant defenses ( Pelaia et al., 2004 ). CFTR-deficient tracheal epithelial cells are characterized by high GSH levels that decrease the intracellular content of ceramide (CER). CER deficiency occurring in CF seems to be responsible for the increased activation of the pro-inflammatory transcriptional nuclear factor NFκB that, in turn, is responsible for the abnormally high inflammatory response in CF respiratory epithelial cells ( Vilela et al., 2006 ; Aureli et al., 2016 ). An increasing number of studies indicate that sphingolipids play an important regulatory role in CF concerning pulmonary inflammation. In different models, it has been shown that de novo sphingolipid synthesis is an inflammation responsive pathway. It is enhanced by inflammatory mediators, both at transcriptional and enzyme activity level, and the accumulation of its metabolite CER potentiates inflammation in a vicious circle ( Caretti et al., 2014 ). Sphingosine-1-phosphate (S1P), generated in the nucleus by phosphorylation of SphK2 ((Sphingosine Kinase 2), modulates HDAC (histone deacetylases) activity either by direct binding or through activation of nuclear ROS, and, regulates cell cycle and pro-inflammatory gene expression ( Fu et al., 2018 ). The accumulation of CER causes Cftr - deficient mice to suffer from constitutive age-dependent pulmonary inflammation, death of respiratory epithelial cells, deposits of DNA in bronchi, and high susceptibility to severe P. aeruginosa infections ( Teichgräber et al., 2008 ). Aggregates accrual, formed by misfolded mutant CFTR and a miscellaneous of sequestered proteins within, induces inflammation and oxidative stress, impairing proteins and lipids transport, and consequently inflammatory statement ( Mingione et al., 2020 ). History of "Classical" Anti-Inflammatory Drugs A better understanding of the molecular mechanisms involved in inflammation has led to the development of new anti-inflammatory therapeutic strategies. In CF, the intertwining of inflammation, infection, and airway mucus obstruction complicates therapeutic approaches. Thus, anti-inflammatory treatments, combined with antibiotic therapies and airway clearance techniques, play an essential role in patient care, particularly during periods of exacerbations and hospitalization. Steroid Anti-Inflammatory Treatments Glucocorticoids (GC), a class of corticosteroids (CS), are potent anti-inflammatory molecules frequently applied in the treatment of "inflammatory" pulmonary diseases. GC target many of the proteins involved in inflammation, including IL-1β and IL-8 and NFκB and activator protein (AP-1) ( Tabary et al., 1998 ; Barnes, 2006 ). Until recently, CS were the main anti-inflammatory CF treatments and were mainly used during exacerbations through inhaled or oral administrations ( Balfour-Lynn and Welch, 2014 ; Lands and Stanojevic, 2019 ). Since the first Prednisone clinical trials ( Auerbach et al., 1985 ), oral CS have been shown to diminish the lung inflammation and reduce the development of the pathology in CF patients. However, the use of CS is still controversial in the CF context due to medium- and long-term use. The side effects include growth impairment, cataract formation, glucose intolerance, and osteoporosis ( Balfour-Lynn and Welch, 2014 ). Nonetheless, oral CS are promptly used during an exacerbation to decrease inflammation in CF lungs. Even though the use of GC in CF is common, the signaling pathways remain partially described. Interestingly, we have published that the NFκB signaling pathway was significantly involved and refractory to the action of GC in glandular epithelial cells ( Tabary et al., 1998 ). Moreover, we have confirmed these results in airway neutrophils from CF patients ( Corvol et al., 2003 ). Even though inhaled CS have a better safety profile, their efficacy has not yet been demonstrated ( Balfour-Lynn and Welch, 2014 ). The inhaled steroids withdrawal impact was established in a multicentric randomized, double-blind placebo-controlled trial, including CF children and adults ( Balfour-Lynn et al., 2006 ). This study failed to show any beneficial effect of inhaled CS in CF patients treated for six months. Finally, GC remain interesting molecules, especially during exacerbations, as they significantly reduce inflammation. However, their use in CF can only be limited to specific cases. Non-Steroid Anti-Inflammatory Treatments As GC have significant side effects, alternative molecules have been proposed. For a few years, Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), has emerged and was proposed to the CF patients as a GC alternative. Most of NSAID (such as Aspirin) are known to block cyclo-oxygenase (COX) enzymes that produce prostaglandins from free arachidonic acid ( Kurumbail et al., 1996 ). Ibuprofen, discovered by Stewart Adams laboratory in 1961, was sold initially as Brufen to treat rheumatoid arthritis ( Balfour-Lynn et al., 2006 ; Halford et al., 2012 ). In CF, Ibuprofen acts directly on neutrophil activation, inhibiting their mobility and recruitment in the airways ( Konstan et al., 2003 ). High-dose of Ibuprofen can reduce the development of CF patients' lung disease, especially in children ( Lands and Stanojevic, 2007 ; Lands and Stanojevic, 2019 ). A meta-analysis from a current update of a regular review has been published on the Cochrane database ( Lands and Stanojevic, 2019 ). Multiple unwanted effects were a matter of concern due to the high doses usage, which has limited the Ibuprofen use in CF. Recent results have described that obvious benefits of Ibuprofen therapy outbalance the low risk of gastrointestinal bleeding, although long-term safety results are limited. In low doses, some shreds of evidence indicate that Ibuprofen may cause inflammation ( Lands and Stanojevic, 2019 ). Nonetheless, these outcomes are still a subject of debate among scientists who suspect the inappropriate use of Ibuprofen for CF patients ( Lands and Stanojevic, 2016 ). The association of Ibuprofen with infections is more complicated in that it confers risk in some situations but benefits in others, therefore its usage might require close monitoring ( Varrassi et al., 2020 ). Macrolides Among the most exciting new anti-inflammatory drug treatments established in the last few years in the CF context the macrolides ( Southern et al., 2012 ). Macrolides were discovered in 1952 and were initially isolated from cultures of Streptomyces erythraea . The frequently used macrolides have 14 (Clarithromycin, Erythromycin, and Roxithromycin) or 15 (Azithromycin) atoms attached to their macrocyclic rings and were named macrolides in regards to the presence of macrocyclic lactone ring. Macrolides are interesting original antibiotics because of their double action of not only reducing infections but also reducing inflammation. The macrolides were used as antibiotics to treat different infectious diseases, including numerous airway pathology as pneumonia, CF, bronchitis, pharyngitis ( Zalewska-Kaszubska and Gorska, 2001 ). Surprisingly, in 1987, a Japanese group has reported a spectacular effect in panbronchiolitis patients' lifespan when treated with Erythromycin antibiotic ( Kudoh et al., 1987 ). This pathology is a typical and representative disease of chronic respiratory tract infection in Japan, characterized by chronic inflammation localized predominantly in the respiratory bronchioles with inflammatory cells such as monocytes, macrophages, neutrophils and, T lymphocytes. The molecule showing the most interesting effects in CF patients is Azithromycin, with an improvement of lung parameters, a decrease of P. aeruginosa infection, and hospitalization duration ( Clement et al., 2006 ; Saiman et al., 2010 ; Nichols et al., 2020 ). Prolonged use of small dose Azithromycin was related to a beneficial impact on lung disease expression, well ahead of P. aeruginosa infection. A metanalysis of these researches proved substantial improvement or maintenance of the forced expiratory volume in one second (FEV1, a measure of lung function) and forced vital capacity (FVC) in treated patients vs. controls after 12 months of therapy. Even though there was no decline in the intravenous antibiotic therapy necessity or the hospitalization duration of any of these studies, a positive effect on the restoration of Cl - efflux in CF has also been shown ( Saint-Criq et al., 2011 ). Moreover, some scientists demonstrated that macrolides operate by limiting pro-inflammatory cytokines and provoking direct alterations in the neutrophils function ( Equi et al., 2002 ; Southern and Barker, 2004 ; Haydar et al., 2019 ). However, they failed to reduce the inflammation in BECs in CF patients ( Saint-Criq et al., 2012 ). One recently published article has demonstrated that Azithromycin could modify the M2 phenotype macrophage and, therefore, indirectly modify the inflammatory process by inhibiting NFκB activation by increasing IKKβ expression in J774 murine macrophages ( Haydar et al., 2019 ). However, some macrolides, such as Clarithromycin, can induce neutrophil extracellular trap (NET) generation, a mechanism implicated in innate immunity and some inflammatory processes. NETosis is a mechanism by which neutrophils extrude their DNA and protein contents to form NET, including AMPs. The physiology and the formation of the NET have been extensively described in the review from Ravindran et al. (2019) . In the fetal stage and early childhood, neutrophilic inflammation in the peri-bronchial regions is present in CF patients who have mucus excess and obstructive secretions but no persistent bacterial infections. Various microbial components like inflammatory cytokines, lipid mediators, and extracellular DNA found in CF patients induce NET formation ( Henke and Ratjen, 2007 ). In CF airways, neutrophils are recruited to the airway upon infection and exacerbate the disease by producing NETs, which can increase mucus viscosity and consequently participate in the airway obstruction. The excess of NETs and their cytotoxic components, associated with hypervisquous mucus, exacerbate CF NET produced by Clarithromycin and inhibit Acinetobacter baumannii infection by acting on its growth and biofilm formation in an LL-37-dependent manner ( Konstantinidis et al., 2016 ; Khan et al., 2019 ). Clarithromycin also enhances the antibacterial defense of fibroblasts and improves their wound healing capacity through the upregulation of LL-37 on NET structures ( Arampatzioglou et al., 2018 ). Although Azithromycin and Chloramphenicol show that neutrophils pretreatment with these macrolides decreases the NETs release. Moreover, Azithromycin showed a concentration-dependent effect on respiratory burst in neutrophils, whereas Chloramphenicol did not affect degranulation, apoptosis or respiratory burst. So, these antibiotics modulate the ability of neutrophils to release NETs influencing human innate immunity ( Bystrzycka et al., 2017 ). The macrolide immunomodulatory role depends on the macrolide used and the pathology involved. As a final point, conventional anti-inflammatory treatments for CF are limited and have not been explicitly developed for this pathology, and could induce counterproductive effects. Research in this field is still limited compared to antibiotics, but despite this, new molecules or strategies are being evaluated. Steroid Anti-Inflammatory Treatments Glucocorticoids (GC), a class of corticosteroids (CS), are potent anti-inflammatory molecules frequently applied in the treatment of "inflammatory" pulmonary diseases. GC target many of the proteins involved in inflammation, including IL-1β and IL-8 and NFκB and activator protein (AP-1) ( Tabary et al., 1998 ; Barnes, 2006 ). Until recently, CS were the main anti-inflammatory CF treatments and were mainly used during exacerbations through inhaled or oral administrations ( Balfour-Lynn and Welch, 2014 ; Lands and Stanojevic, 2019 ). Since the first Prednisone clinical trials ( Auerbach et al., 1985 ), oral CS have been shown to diminish the lung inflammation and reduce the development of the pathology in CF patients. However, the use of CS is still controversial in the CF context due to medium- and long-term use. The side effects include growth impairment, cataract formation, glucose intolerance, and osteoporosis ( Balfour-Lynn and Welch, 2014 ). Nonetheless, oral CS are promptly used during an exacerbation to decrease inflammation in CF lungs. Even though the use of GC in CF is common, the signaling pathways remain partially described. Interestingly, we have published that the NFκB signaling pathway was significantly involved and refractory to the action of GC in glandular epithelial cells ( Tabary et al., 1998 ). Moreover, we have confirmed these results in airway neutrophils from CF patients ( Corvol et al., 2003 ). Even though inhaled CS have a better safety profile, their efficacy has not yet been demonstrated ( Balfour-Lynn and Welch, 2014 ). The inhaled steroids withdrawal impact was established in a multicentric randomized, double-blind placebo-controlled trial, including CF children and adults ( Balfour-Lynn et al., 2006 ). This study failed to show any beneficial effect of inhaled CS in CF patients treated for six months. Finally, GC remain interesting molecules, especially during exacerbations, as they significantly reduce inflammation. However, their use in CF can only be limited to specific cases. Non-Steroid Anti-Inflammatory Treatments As GC have significant side effects, alternative molecules have been proposed. For a few years, Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), has emerged and was proposed to the CF patients as a GC alternative. Most of NSAID (such as Aspirin) are known to block cyclo-oxygenase (COX) enzymes that produce prostaglandins from free arachidonic acid ( Kurumbail et al., 1996 ). Ibuprofen, discovered by Stewart Adams laboratory in 1961, was sold initially as Brufen to treat rheumatoid arthritis ( Balfour-Lynn et al., 2006 ; Halford et al., 2012 ). In CF, Ibuprofen acts directly on neutrophil activation, inhibiting their mobility and recruitment in the airways ( Konstan et al., 2003 ). High-dose of Ibuprofen can reduce the development of CF patients' lung disease, especially in children ( Lands and Stanojevic, 2007 ; Lands and Stanojevic, 2019 ). A meta-analysis from a current update of a regular review has been published on the Cochrane database ( Lands and Stanojevic, 2019 ). Multiple unwanted effects were a matter of concern due to the high doses usage, which has limited the Ibuprofen use in CF. Recent results have described that obvious benefits of Ibuprofen therapy outbalance the low risk of gastrointestinal bleeding, although long-term safety results are limited. In low doses, some shreds of evidence indicate that Ibuprofen may cause inflammation ( Lands and Stanojevic, 2019 ). Nonetheless, these outcomes are still a subject of debate among scientists who suspect the inappropriate use of Ibuprofen for CF patients ( Lands and Stanojevic, 2016 ). The association of Ibuprofen with infections is more complicated in that it confers risk in some situations but benefits in others, therefore its usage might require close monitoring ( Varrassi et al., 2020 ). Macrolides Among the most exciting new anti-inflammatory drug treatments established in the last few years in the CF context the macrolides ( Southern et al., 2012 ). Macrolides were discovered in 1952 and were initially isolated from cultures of Streptomyces erythraea . The frequently used macrolides have 14 (Clarithromycin, Erythromycin, and Roxithromycin) or 15 (Azithromycin) atoms attached to their macrocyclic rings and were named macrolides in regards to the presence of macrocyclic lactone ring. Macrolides are interesting original antibiotics because of their double action of not only reducing infections but also reducing inflammation. The macrolides were used as antibiotics to treat different infectious diseases, including numerous airway pathology as pneumonia, CF, bronchitis, pharyngitis ( Zalewska-Kaszubska and Gorska, 2001 ). Surprisingly, in 1987, a Japanese group has reported a spectacular effect in panbronchiolitis patients' lifespan when treated with Erythromycin antibiotic ( Kudoh et al., 1987 ). This pathology is a typical and representative disease of chronic respiratory tract infection in Japan, characterized by chronic inflammation localized predominantly in the respiratory bronchioles with inflammatory cells such as monocytes, macrophages, neutrophils and, T lymphocytes. The molecule showing the most interesting effects in CF patients is Azithromycin, with an improvement of lung parameters, a decrease of P. aeruginosa infection, and hospitalization duration ( Clement et al., 2006 ; Saiman et al., 2010 ; Nichols et al., 2020 ). Prolonged use of small dose Azithromycin was related to a beneficial impact on lung disease expression, well ahead of P. aeruginosa infection. A metanalysis of these researches proved substantial improvement or maintenance of the forced expiratory volume in one second (FEV1, a measure of lung function) and forced vital capacity (FVC) in treated patients vs. controls after 12 months of therapy. Even though there was no decline in the intravenous antibiotic therapy necessity or the hospitalization duration of any of these studies, a positive effect on the restoration of Cl - efflux in CF has also been shown ( Saint-Criq et al., 2011 ). Moreover, some scientists demonstrated that macrolides operate by limiting pro-inflammatory cytokines and provoking direct alterations in the neutrophils function ( Equi et al., 2002 ; Southern and Barker, 2004 ; Haydar et al., 2019 ). However, they failed to reduce the inflammation in BECs in CF patients ( Saint-Criq et al., 2012 ). One recently published article has demonstrated that Azithromycin could modify the M2 phenotype macrophage and, therefore, indirectly modify the inflammatory process by inhibiting NFκB activation by increasing IKKβ expression in J774 murine macrophages ( Haydar et al., 2019 ). However, some macrolides, such as Clarithromycin, can induce neutrophil extracellular trap (NET) generation, a mechanism implicated in innate immunity and some inflammatory processes. NETosis is a mechanism by which neutrophils extrude their DNA and protein contents to form NET, including AMPs. The physiology and the formation of the NET have been extensively described in the review from Ravindran et al. (2019) . In the fetal stage and early childhood, neutrophilic inflammation in the peri-bronchial regions is present in CF patients who have mucus excess and obstructive secretions but no persistent bacterial infections. Various microbial components like inflammatory cytokines, lipid mediators, and extracellular DNA found in CF patients induce NET formation ( Henke and Ratjen, 2007 ). In CF airways, neutrophils are recruited to the airway upon infection and exacerbate the disease by producing NETs, which can increase mucus viscosity and consequently participate in the airway obstruction. The excess of NETs and their cytotoxic components, associated with hypervisquous mucus, exacerbate CF NET produced by Clarithromycin and inhibit Acinetobacter baumannii infection by acting on its growth and biofilm formation in an LL-37-dependent manner ( Konstantinidis et al., 2016 ; Khan et al., 2019 ). Clarithromycin also enhances the antibacterial defense of fibroblasts and improves their wound healing capacity through the upregulation of LL-37 on NET structures ( Arampatzioglou et al., 2018 ). Although Azithromycin and Chloramphenicol show that neutrophils pretreatment with these macrolides decreases the NETs release. Moreover, Azithromycin showed a concentration-dependent effect on respiratory burst in neutrophils, whereas Chloramphenicol did not affect degranulation, apoptosis or respiratory burst. So, these antibiotics modulate the ability of neutrophils to release NETs influencing human innate immunity ( Bystrzycka et al., 2017 ). The macrolide immunomodulatory role depends on the macrolide used and the pathology involved. As a final point, conventional anti-inflammatory treatments for CF are limited and have not been explicitly developed for this pathology, and could induce counterproductive effects. Research in this field is still limited compared to antibiotics, but despite this, new molecules or strategies are being evaluated. Novel Anti-Inflammatory Approaches Better insight into the pathways involved has led to the development of new therapeutic approaches that are currently being evaluated under cell experiments or clinical trials. These new strategies aiming at the CF inflammation are designed to treat different dysregulated aspects such as channel modulators, oxidative stress, cytokines secretion, lung remodeling, and the regulation of dysregulated pathways. New Channel Modulators CFTR Channel The discovery of the CFTR gene in 1989 resulted in insights on how CFTR mutations induce CF pathology and encouraged many researchers to develop new drugs or strategies to correct the mutation or increase the protein activity ( Riordan et al., 1989 ). Genetic therapy using adeno-associated virus (AAV) or other strategies aiming to correct the CFTR gene was very promising because CF is a monogenic disease. Nonetheless, the subsequent realization tempered expectations because the airways are well defended and are not absorptive surfaces. The natural barrier of mucus considerably impairs gene transfer into the lungs, and the epithelium renewing necessitates numerous administrations. For these reasons, only one study has demonstrated a significant but moderate effect on CF patients. Thus, further optimizations or other strategies are needed and in progress ( Alton et al., 2015 ; Alton et al., 2017 ). These data provided the grounds for pharmacologic modulations of chloride transport, by targeting mutant CFTR and/or alternative ion channels as anoctamin-1 (ANO1) that can compensate for CFTR malfunction. This excitement has now proven to be warranted because numerous new therapies approved by the FDA or EMA are now either in the pipeline or available for CF patients ( Figure 5 ). This finding contributes to the innovation of genetic disease pharmacotherapy with Vertex Pharmaceuticals as a leader in the CF research field. Fundamental CF research has set the stage for a better molecular understanding of CFTR mutations by supplying structural pieces of information to design new approaches for the pharmacology dynamic even if the different drugs proposed were obtained by high-throughput screening ( Callebaut et al., 2017 ). Till date, two CFTR-directed molecule classes have been developed: "potentiator" compounds increasing mutated CFTR activity at the cell surface and, "corrector" drugs improving altered protein processing and trafficking to the cell surface ( Wainwright et al., 2015 ; Rowe et al., 2017 ; Davies et al., 2018 ) ( Figure 6 ). The first generation of the compounds has either been limited to a few patients with specific mutations (Ivacaftor) or was addressed to a larger group (Orkambi) and demonstrated moderate effects in CF ( Mayer, 2016 ). For this reason, the U.K. National Institute for Health and Care Excellence (NICE) issued a draft guidance against recommending Orkambi. Recently, the FDA has approved an auspicious combination of molecules (Elexacaftor–Tezacaftor–Ivacaftor called Trikafta) to restore the function of p.Phe508del CFTR protein in CF patients even if patients had a single p.Phe508del allele. The combination of drugs relative to the control resulted in a percentage of predicted FEV1 that was more than 14 points higher and a rate of pulmonary exacerbations that was 60% lower through 24 weeks of treatment ( Keating et al., 2018 ; Middleton et al., 2019 ). Unfortunately, a few pieces of information are available for the inflammatory aspects of these treatments. Although, recent evidence showed that the inflammation and lung status hampers these medications and can hinder their effects. Only one article has demonstrated that CFTR modulators can reduce excessive pro-inflammatory response following LPS (lipopolysaccharide) stimulation of CF monocytes ( Jarosz-Griffiths et al., 2020 ). Moreover, in this article, the authors have also demonstrated that IL-8, IL-1β, and TNF-α (Tumor necrosis factor-α) decreased significantly in the serum of CF patients treated with Ivacaftor and Tezacaftor treatment. It is not known whether the observed effects are due to the restoration of Cl - efflux, GSH (glutathione), or CFTR protein interactions present at the membrane. Figure 5 List of the different categories of drugs under development or clinical trials in the context of CF (adapted from https://www.cff.org/Trials/pipeline/ ). Figure 6 Description of the different classes of CFTR mutations related to the different therapeutic proposed in the literature. I—ynthesis defect, II—processing defect, III—channel gating defect, IV—channel conductance defect, V—reduced CFTR production, VI—defect of stability; ER, endoplasmic reticulum. Other new classes of mutation are in development, such as CFTR amplifiers, CFTR stabilizers, and read-through agents ( Figure 6 ). CFTR amplifiers upregulate the expression, and indirectly, the activity of mutant CFTR. PTI-428 and PTI-CH are the two amplifiers who seem promising in pre-clinical and clinical studies. PTI-428 can enhance lung function in CF patients receiving Orkambi with no significant adverse effects. CFTR stabilizer as Cavosonstat inhibits the enzyme that is involved in regulating how much CFTR protein is present at the cell surface ( Donaldson et al., 2017 ). It could potentially increase the benefits of other medications that target the CFTR function. Read-through drugs can help the ribosome skip over the early stop sequence in order to read the mRNA remaining information and generate CFTR protein. These therapies may be of interest to class I mutations where there is no production of mRNA or CFTR protein. Ataluren was developed as a potential treatment for these mutations, but its development was terminated due to failed clinical trial outcomes ( Shoseyov et al., 2016 ). This approach needs to be completed in the future evaluation of CF trials to understand the effects better and investigate the mechanism complex. It can be assumed that earlier treatment using these drugs may avoid structural damages and give rise to more efficient and prolonged results. We can imagine that the improvement of various dysregulated parameters will have long-term effects on the inflammation present in CF patients, even if indirectly. A recent article has highlighted that by Tobramycin or the AMP, 6K-F17 could restore the effects of Orkambi on p.Phe508del-CFTR protein, suggesting a significant role of infection in the CF pathology ( Laselva et al., 2020 ). Furthermore, using this approach, they have demonstrated that the active AMP can down-regulate the expression of pro-inflammatory cytokines like IL-8, IL-6, and TNF-α in p.Phe508del-CFTR human BECs ( Laselva et al., 2020 ). Some exciting improvements in chloride efflux have been demonstrated using S ildenafil, a phosphodiesterase type 5 (PDE5) inhibitor. This drug recues p.Phe508del-CFTR trafficking in vitro experiments and decreases sputum elastase activity and, consequently, the inflammatory process ( Lubamba et al., 2011 ; Taylor-Cousar et al., 2015 ). In parallel to Vertex's studies, many other companies are interested in similar approaches to develop CFTR modulators that either restore the CFTR protein to the membrane or activate it ( Figure 5 ). This research work has been essential over the last ten years, and many other molecules are currently being evaluated and at a different stage. More recently, another promising strategy has been proposed to modulate post-transcriptionally activity of CFTR regulated by acting through miRNA. Distinct groups have proved that wild-type and mutated p.Phe508del human CFTR is regulated by miR-101-3p, miR-145-5p, miR-223-3p, miR-494-3p,and miR-509-3p ( Glasgow et al., 2018 ). The approaches to inhibit the effect of these miRNAs have demonstrated an increase in CFTR protein expression and activity in BECs ( De Santi et al., 2020 ). This approach is exciting, but further researches are needed to understand the subtility of this regulation better. ENaC Channel Since CFTR negatively regulates the activity of the ENaC sodium channel, different strategies have been proposed to decrease its activity. The first proposed molecule was Amiloride, which acts as a potassium-sparing diuretic, showing some benefit in both animal studies and clinical trials. Unfortunately, its efficacy was limited due to its short half-life ( Zhou et al., 2008 ). This approach was repeated with the use of a new ENaC blocker called AZD5634 from AstraZeneca and BI1265162 from Boehringer Ingelheim. A phase Ib study and a phase II study to test, respectively, the safety and effectiveness of AZD5634 and BI 1265162 are underway in CF adults. Nowadays, a more recent and exciting approach, using aerosol antisense oligonucleotide (ASO) targeting ENaC mRNA (Ionis ENAC 2.5Rx), has demonstrated some interesting and impressive results on mice by restoring inflammation and inhibiting ENaC activity ( Crosby et al., 2017 ). A first clinical study with this therapy is currently ongoing. In the same way, Arrowhead asks to open a phase I/II trial into inhaled small interference RNA (iRNA) therapy. The drug, called ARO-ENaC, is an investigational RNA therapy designed to lower the production of the epithelial sodium channel alpha subunit (αENaC) in the lungs of CF patients. ARO-ENaC is an iRNA molecule intending to block the production of ENaC channels. It works by targeting and destroying the αENaC mRNA molecules, which are genetic messengers that carry the necessary information for making αENaC proteins and consequently ENaC activity. ANO1 Channel Since functional CFTR rescue remains limited, with mutation-dependent effects, alternative strategies have been suggested to compensate for the CFTR deficiency and were proposed as a potential CF therapeutic target. Such a strategy was the stimulation of calcium-activated chloride channels (CaCCs) such as the Anoctamin 1 channel (ANO1) ( Figure 3 ). In the nineties, Knowles et al. have discovered that adenosine '5'-triphosphate and uridine-5'-triphosphate stimulated Cl - secretion in both standards and CF respiratory epithelial, offering a potential by-pass mechanism for defective CFTR ( Knowles et al., 1991 ). These activators transduce a signal through P2Y2 receptors that lead to the release of intracellular calcium and activate the CaCCs. An analog called Denufosol was developed. Different studies have demonstrated that this drug can increase Cl - secretion through a CaCC, inhibit sodium absorption via the epithelial sodium channel called ENaC, and stimulate epithelial ciliary beat frequency ( Accurso et al., 2011 ). Based on these data, 'Denufosol' clinical trials begun in 2001 using a wet nebulization direct airway delivery approach. Unfortunately, the last phase III had failed to demonstrate any benefit, and the project was dropped, but the idea of developing this approach remained ( Moss, 2013 ). At the time of this study, CaCCs were poorly known. Their identity remained elusive for over 20 years until 2008 ( Nilius and Droogmans, 2003 ; Caputo et al., 2008 ; Schroeder et al., 2008 ; Yang et al., 2008 ). When ANO1, the principal CaCC present in the airways, was identified in 2008, it allowed for more targeted approaches. Attractively, ANO1 channel has, at the apical membrane of epithelial cells, the same expression pattern as CFTR channels, and this protein was shown to be essential in the activity of CFTR as a chloride channel ( Benedetto et al., 2017 ; Benedetto et al., 2019 ). Besides, ANO1 is implicated in HCO 3 different permeability, proliferation, wound healing, inflammation, and its expression decreased in CF patients ( Veit et al., 2012 ; Jung et al., 2013 ; Ruffin et al., 2013 ). Moreover, a recent article highlighted that ANO1 inhibition decreased ASL height. The authors have also demonstrated that ANO1 is not required for MUC5AC expression, the main protein of the mucus ( Simoes et al., 2019 ). For this reason, a novel ANO1 potentiator was developed (ETX001), and airway epithelial function and mucus transport were evaluated in the human cells and animal models. This approach confirmed previous results and demonstrated that this drug could increase epithelial fluid secretion and enhance mucus clearance ( Danahay et al., 2020 ). Recently, our group has proposed a particular strategy using an ASO specific to ANO1 to reestablish ANO1 expression in the context of CF. This strategy "hijacks" the miRNA regulatory system and allows highly targeted effects. We have demonstrated that ASO-ANO1 could be used to inhibit the fixation of miR-9 on ANO1 mRNA by a target site blocker, and consequently to activate the alternative chloride channel to compensate CFTR Cl - deficiency regardless of the mutation ( Sonneville et al., 2017 ). We have also shown that with this strategy, we can improve tissue repair on cell lines but also on CF primary patient cells. We have likewise demonstrated that with this approach, we can activate mucociliary clearance on primary cells but also CF mice. Although we have not studied the effects of ANO1 modulation of inflammation, preliminary studies have already shown that activating ANO1 limits the secretion of IL-8 ( Veit et al., 2012 ). Novel Anti-Cytokines Approaches A pathophysiology pulmonary characteristic of CF is a severe neutrophil accumulation, which is correlated with high levels of pro-inflammatory cytokines (IL-8, IL-6, TNF-α), and low levels of anti-inflammatory mediators like IL-10 ( Jacquot et al., 2008b ). For numerous years, different approaches, as curcumin or vitamin D, have been proposed to limit IL-8 secretion and neutrophils influx ( Gaggar et al., 2011 ; Olszowiec-Chlebna et al., 2019 ). Some pre-clinical data have demonstrated that using antibodies, like antibodies directed against intercellular adhesion molecule (ICAM)-1 and IL-8, could be a promising target. The most advanced therapy using SB-656933, an oral CXCR2 antagonist, was already tested in CF patients and has demonstrated along with safety some exciting results in the modulation of airway inflammation ( Moss et al., 2013 ). However, another study using SCH527123 (MK-7123, Navarixin), a CXCR1/2 antagonist, was also attempted in chronic obstructive pulmonary disease (COPD) but was abandoned because of a severe decline in neutrophil number ( Rennard et al., 2015 ). By contrast, a phase II clinical trial has already been carried out in patients with ulcerative colitis and demonstrated inhibition of ozone-induced airway inflammation in humans ( Lazaar et al., 2011 ). Numerous other modulators of cytokines in the context of CF have been proposed, but only in vitro experiments have been performed ( Lampronti et al., 2017 ; De Fenza et al., 2019 ). Cytokine modulation shows that cytokines have a significant role in limiting infections, although these approaches are confusing. A recent publication has highlighted the role of an IL-1 signaling pathway in sterile neutrophilic inflammation and mucus hypersecretion and has suggested that treatment with IL-1 receptor antagonist as Anakinra could be promising to prevent lung inflammation ( Balazs and Mall, 2019 ). The possibility of increasing gene expression and protein activity by the use of ASO has become more and more promising in the last years. However, long-term efficacy, safe delivery, and side effects of long-term treatment must be evaluated in order to be applied in patients with CF ( Bardin et al., 2018b ; Vencken et al., 2019 ). Fabbri et al. have developed this original concept by modulating the IL-8 expression by increasing miR-93 in BECs during P. aeruginosa infection ( Fabbri et al., 2014 ). More recent results have highlighted that other miRNA involved in CF pathology, like miR-199a-3p or miR-636, could be targeted to control the CF lung inflammatory process ( Bardin et al., 2018a ; Bardin et al., 2019 ). Other interesting approaches have been performed to modulate the cascade of inflammation targeting NFκB activity by using, for example, Angelicin derived from different angiosperms or Sulindac, an NSAID ( Rocca et al., 2016 ). Unfortunately, these approaches are not specific, and the risk of side effects remains high. New Development in Antibiotic Approaches "Synthetic" Antibiotics In CF, antibiotics are utilized for various applications, such as initial infection prevention, eradication (for early infection), control (for chronic infection), and finally, pulmonary exacerbations treatment. The antibiotics are given in three different primary ways: oral, inhalation, or intravenous. The choice of antibiotics depends on the nature of the pathogen to be eliminated, the age of the patient, and the nature of other pathogens present such as H. influenza , S. aureus , or P. aeruginosa infections. P. aeruginosa is an opportunistic Gram-negative pathogen and is one of the main reasons for morbidity and mortality in CF and immunosuppressed patients. In order to eradicate new P. aeruginosa infections, antibiotic regimens are now a care standard around the world. Different groups assessed the effectiveness of inhaled Tobramycin, Aztreonam, and Colistin as well as oral Ciprofloxacin in eradicating new P. aeruginosa infection ( Waters, 2018 ; Pang et al., 2019 ), although P. aeruginosa eradication is now much more challenging as a result of its impressive capability to resist antibiotics. These organisms become embedded in an exopolysaccharide biofilm, which protects the organism from phagocytosis and reduces the efficacy of anti-microbial drugs ( Doring, 2010 ). Once this change has occurred, the mucoid P. aeruginosa could acquire multi-drug resistance, and this bacterium is virtually impossible to eradicate ( Southern et al., 2012 ). If the P. aeruginosa infection cannot be cleared, the affected person is faced with an increased treatment burden, accelerated decline in lung function, increased symptom severity, and increased mortality ( Nixon et al., 2002 ). Recently, there has been a growing number of "new" antibiotics, of different classes and formulations, for pulmonary infection treatments in CF patients ( Waters and Smyth, 2015 ). In order to limit toxicity and reduce side effects while directly targeting the lungs, many studies took an interest in using aerosols as a method of administration. In this frame of mind, Levofloxacin was developed for CF patients to target P. aeruginosa infections ( Chirgwin et al., 2019 ; Epps et al., 2019 ). This drug, derived from the fluoroquinolone family, inhibits topoisomerases, which is essential for the synthesis of bacterial DNA. In the same way, inhaled Zitreonam is now available to treat P. aeruginosa infections in CF patients. Although its aerosolized formulation was proven to be beneficial, the formulation for intravenous injections induces significant lung inflammation, which has limited its use. Another example of the existing improvement of drugs is Tobramycin, presented as a dry powder. Inhaled tobramycin provides, in less than 5 minutes, a rapid action directly at the site of the lung infection. In order to increase the efficacy of P. aeruginosa eradication and have a less often resistance development in comparison to the existing "classical" antibiotics, recent P. aeruginosa suggested treatment is the use of a combination of antibiotics and the development of new ones. Also, they can be associated with an alternative strategy such as EDTA (Respirion) or inhaled glycopolymer (SNP113). Thus, a new carbapenem antibiotic called Doripenem has been developed with wide spectrum activity against bacteria through bacterial cell wall synthesis inhibition. Different authors have shown in vitro that this molecule has more significant activity than other antibiotics of the same family on strains isolated from CF patients ( Traczewski and Brown, 2006 ; Riera et al., 2011 ). A clinical phase III study showed that patients infected with P. aeruginosa and treated with Doripenem had higher recovery rates in comparison to Imipenem-treated patients but, no clinical trial with CF patients is in progress ( Chastre et al., 2008 ). In the same way, Plazomicin (a semisynthetic aminoglycoside) and POL7001 (a protein epitope mimetic) came out as an interesting strategy against P. aeruginosa ( Cigana et al., 2016 ). These drugs have demonstrated in vitro some exciting effects on the multidrug-resistant P. aeruginosa isolated from CF patients ( Cigana et al., 2016 ). "Natural" Approaches For many years an original approach using bacteriophages has been advanced. Bacteriophages were discovered in 1915 and can kill bacteria by causing lysis ( Summers, 2001 ). Bacteriophage therapy was applied extensively in the 1930s and 1940s before antibiotics, and it is still being used in Eastern Europe. Nevertheless, after antibiotics became broadly accessible, phage therapy was renounced in Western countries. Many phages can target P. aeruginosa and have demonstrated some exciting effects on mice by decreasing the bacteria burden in the lungs or preventing infection ( Morello et al., 2011 ). Even if clinical studies have shown relative effectiveness, treatments using phages remain negligible so far. Various reasons have limited the treatments with bacteriophages. The idea of introducing a living organism into the body is difficult to accept and remains an important psychological barrier. Moreover, early tests showed that the preparation generated impurities and that these preparations were not very stable ( Morello et al., 2011 ). Although the use of phages in combination with quorum sensing inhibitors seems interesting, this approach remains marginal ( Pang et al., 2019 ), and only a phase Ib/II trial is planned to test the safety and tolerability of AP-PA02 in adults with CF. AP-PA02 is a type of phage intended to control P. aeruginosa infections in CF patients. In in vitro studies, AP-PA02 can kill more than 80% of P. aeruginosa strains from CF people, and some first results are encouraging ( Law et al., 2019 ). Another "natural strategy" is inhaled nitric oxide (NO) for which an initial phase II study is underway. NO is a gas derived from nitrogen with anti-microbial properties. Some in vivo studies have validated this approach to eradicate the infection and to decrease mucus viscoelastic ( Rouillard et al., 2020 ). In the late 1970s, various studies showed that iron played an essential role in bacterial growth and was involved in particular in DNA replication, energy production, and pathogen-host interaction ( Payne and Finkelstein, 1978 ). Recent results demonstrated that the iron content of human sputum is considerably high in CF, which facilitates chronic infections in the lungs of CF patients ( Reid et al., 2007 ). These observations resulted in the development of novel therapeutic strategies in order to limit the amount of iron present in the airways. Gallium is a compound that shares the same properties with iron. It has demonstrated in vitro and in vivo anti-Pseudomonas properties ( Tovar-García et al., 2020 ). The FDA has already approved the intravenous administration of Gallium. Clinical studies, in phase II for intravenous and a phase I for an inhaled strategy, are ongoing to evaluate its efficiency in treating P. aeruginosa infections in CF patients ( Tovar-García et al., 2020 ). During the last decades, AMPs naturally emerged as a potential therapy to cure infections with antibiotic resistance, in CF included. Treatments of bacterial infections by antibiotics result in a worldwide spread of dissemination of antibiotic resistance, both in the community and clinical settings. Besides, the development of new antibiotics is costly and time-consuming. It is hence of great importance to note that AMPs can treat methicillin-resistant S. aureus and multidrug-resistant P. aeruginosa that are resistant to conventional antibiotics ( Geitani et al., 2019 ). Studies showed that treatments of antibiotic-resistant bacterial strains with AMPs were associated with almost no induced resistance to AMPs, which may encourage their use as potential replacement therapy for antibiotics. AMPs can exert anti-inflammatory actions either by suppressing the production of pro-inflammatory cytokines or by stimulating that of anti-inflammatory cytokines by host cells ( Figure 7 ). Cathelicidin LL-37 (one of the most studied AMPs) enhances the production of the anti-inflammatory cytokine IL-1R by the human peripheral blood-derived mononuclear cells and macrophages ( Choi et al., 2014 ), and similar results were observed with LL-37 and beta-defensin-3 (hBD-3) ( Mookherjee et al., 2009 ; Smithrithee et al., 2015 ). Besides their direct actions on host cells involved in the initiation/modulation of inflammation, a number of AMPs, such as LL-37, Magainin-2, and bactericidal-permeability-increasing (BPI), can neutralize the activity of bacterial toxins such as LPS, thus participating in maintaining a balance between pro- and anti-inflammatory cytokines ( Sun and Shang, 2015 ; Skovbakke and Franzyk, 2017 ). Figure 7 General mechanisms by which AMPs exert anti-inflammatory actions on host cells. AMPs can bind to bacterial virulence factors such as LPS or LTA and prevent their interactions with host cells. AMPs are also able to interfere with host cell signaling pathways involved in the inflammatory reaction. The overall consequence is that AMPs reduce the production of inflammatory mediators by these cells that may help in the resolution of inflammation. Most of the reported studies in the field have focused on the roles of AMPs in the modulation of cytokine production. However, cytokines are only the tip of the iceberg in the inflammatory process, and other mediators of inflammation, such as eicosanoids, deserve to be investigated to identify their relative role in the modulation of inflammation by AMPs. Indeed, studies have reported that AMPs such as LL-37 modulates the production of eicosanoids, including leukotriene B4 (LTB4) and thromboxane A2 (TXA2) by macrophages ( Agier et al., 2015 ). TXB2 and LTB4 are metabolites of arachidonic acid conversion by COX and lipoxygenase (LOX), respectively, and known to induce platelet aggregation and neutrophils recruitment at the site of infection ( Yeung and Holinstat, 2011 ). It has been shown that LL-37 AMP blocks the expression of pro-inflammatory pathways involved, such as NF-κB in the presence of LPS ( Agier et al., 2015 ). However, further studies are awaited to decipher the importance of the AMPs/eicosanoids network in the inflammatory reaction and potential implication in inflammatory diseases such as COPD, asthma, and CF. Similar anti-inflammatory effects were observed with WALK11.3 (an AMP with amphipathic helical conformation) in the mouse alveolar macrophage cell line RAW264.7 ( Shim et al., 2015 ). They revealed the ability of this peptide to inhibit the expression of several inflammatory mediators, including NO, COX-derived metabolites, IL-1β, IL-6, interferon (IFN)-β, and TNF-α ( Figure 7 ). The chicken cathelicidin-2 (CATH-2), the known ortholog of the human LL-37, has been shown to reduce inflammation in parallel to its anti-microbial activity against P. aeruginosa -resistant strains from CF patients ( Banaschewski et al., 2017 ). The ability of CATH-2 to downregulate inflammation occurred through the anti-microbial-independent process, as this down-regulation was observed by silencing the inflammatory response that arises from killed bacteria. It is now clear that AMPs play a key role in host defense toward infectious by invading pathogens and represent a potential therapeutic tool to control infections by antibiotic-resistant bacterial strains. They also have the potential to protect the host from harmful inflammation that may result from these infections. Drug design and structure-relationship studies will greatly improve our knowledge of AMPs and the relative importance of their bactericidal vs anti-inflammatory functions, which will be of great help to optimize their potential therapeutic use in disease characterized by both chronic infection and inflammation such as CF. All these data suggested that AMPs could be useful for clinical applications in the view of the protective function against pathogens. A series of clinical trials have started mostly in the pediatric population, and some compounds have been used as topical treatments but not known in the CF context. Different AMPs are under evaluation for the treatment of acute skin infection as Bralicidin, Omiganan, LTC109 (phase II clinical trial), or Pexiganam (phase III clinical trial). Other strategies and applications are currently under study. For example, in sepsis, Talactoferrin was tested by systemic injection in phase II clinical study ( Guntupalli et al., 2013 ). Initial results showed a significant decrease in mortality after 28 days of treatment. However, phase II/III oral Talactoferrin was stopped for problems of safety and efficacy ( Vincent et al., 2015 ). In the case of meningococcemia, rBPI21 pre-clinical trial has demonstrated some anti-bacterial and anti-LPS effects. Encouraging results led to the initiation of a phase III study in children with severe meningococcal sepsis ( Giroir et al., 2001 ). The study outcome showed a reduction in complications with a shorter hospitalization also suggests the possibility to treat with rBPI21 other patients, including CF. The therapeutic applications of P. aeruginosa have been summarized in a recent publication ( Magrone et al., 2018 ). An alternative therapeutic pathway for the use of AMPs has been envisaged by indirectly promoting their expression through the use of natural compounds. Several compounds have been identified as the use of Apigenin to enhance the expression and activity of β-3 defensin and cathelicidin in mice ( Hou et al., 2013 ). Similar effects have been observed with vitamin D on in vitro studies to increase β-2 defensins and LL-37 on keratinocytes ( Kim et al., 2009 ). The use of natural or synthetic antibiotics can have a significant influence on the emergence of new pathogens. It is well established now that microbiota composition and dynamic impact the host immunity, health, and diseases ( Belkaid and Hand, 2014 ). However, a new concept is now progressively emerging, suggesting that the innate immune response of the host can also modulate, at least in part via AMPs, the microbiota composition. For example, recent studies reported the involvement of sPLA2-IIA in the selection of species in pathologies characterized by polymicrobial infections such as CF. P. aeruginosa is known to progressively colonize CF airways to become the dominant pathogen at later stages of CF. This pathogen induces the production by CF airways of sPLA2-IIA, which in turn eradicate S. aureus , therefore helping in its gradual elimination from CF airways and its substitution by P. aeruginosa ( Pernet et al., 2014 ). This effect is mostly due to the intrinsic resistance of P. aeruginosa and high susceptibility of S. aureus to sPLA2-IIA, respectively. Finally, it emerges that AMPs represent valid substitutes of antibiotics when a condition of antibiotic resistance is established. Alternative Strategies Anti-Proteases CF "anti-protease therapies" can be separated into two separate groups of drugs: some to increase anti-protease and some to inhibit protease expression. CFTR is an essential apical GSH transporter in the lung, and can indirectly participate in the inflammatory process by reducing oxidative stress. Evidence supporting the occurrence of oxidative stress in CF is established and extensively described ( Galli et al., 2012 ; Causer et al., 2020 ). Some interesting works have demonstrated that oxidative stress could suppress CFTR expression ( Cantin et al., 2006 ). Oxidative stress has a major role in the development of lung pathology in CF children and will, in addition to having a role in lung remodeling, have a role in the pulmonary microbiota ( Shi et al., 2019 ). A recent metanalysis has positively correlated the expression of antioxidants with body mass index and lung function in CF ( Causer et al., 2020 ). The malabsorption of nutrients with antioxidants properties in CF, participate in the imbalance in favor of oxidative stress and disrupt redox signaling, and, finally, molecular damages even if some data appears to be conflicting ( Shamseer et al., 2010 ; Siwamogsatham et al., 2014 ). Therefore, multiple studies have been carried out to check the anti-protease supplementation in CF ( Galli et al., 2012 ). Some studies have focused on especially serine proteases via two distinct administration routes: aerosolized and intravenously ( McKelvey et al., 2020 ). In CF, exocrine pancreatic insufficiency and reduced bile acids induce critical antioxidants malabsorption, including carotenoids (β-carotene), tocopherols (vitamin E), coenzyme Q10, and selenium. Supplementation of antioxidant micronutrients (vitamin E, C, D, β-carotene, and selenium) may, therefore, potentially help maintain an oxidant-antioxidant balance, and this aspect has been extensively reviewed ( Sagel et al., 2011 ; Ciofu et al., 2019 ). In the same approach, LAU-7b, an oral drug, is a derived form related to vitamin A. This compound can reduce the lung inflammatory response of CF people. In parallel, a phase II clinical study to test the effectiveness and safety of LAU-7b in CF patients is underway ( Lands and Stanojevic, 2016 ). LAU-7b, also called, Fenretidine, work to increase docosahexaenoic acid (DHA) and consequently CER concentration. Some authors supported that the decrease of CER concentration contributes to the persistent bacterial infection and the constitutive MAP kinases and NFκB activation ( Guilbault et al., 2008 ; Guilbault et al., 2009 ). Human α-1 antitrypsin (A1AT) is still the most studied drug by far. Different clinical trials were already achieved. An inhaled α1-proteinase inhibitor is known to reduce NE burden in some patients with CF. A phase I in non-CF bronchiectasis and an IIa clinical study with purified A1AT products given through inhalation in CF subjects were just finalized and have demonstrated safety and efficacy ( Gaggar et al., 2016 ; Watz et al., 2019 ). In the conclusion of the second study, the daily α-1 hydrophobic chromatography process delivered for three weeks was safe, well-tolerated, and effective in raising the α1-PI levels in the sputum of subjects with CF. However, the effects were transient and difficult to predict due to the proteases' variability in CF patients' lungs. The administration by airway routeway effectively increased the concentration of A1AT in sputum. The current study was not powered to assess changes in FEV1 or biomarkers in sputum, and further clinical are needed. In parallel, A1AT gene therapy is emerging. Some recent data have demonstrated encouraging results in the inhibition of miRNA, which targets the A1AT gene called SERPINA1 ( Hunt et al., 2020 ). This strategy aims to by-pass protein regulation systems of the most abundant inhibitor of NE in the airways. It is an alternative to the delivery of recombinant by using miRNA-targeted therapies. It was found that dual miRNA and adeno-associated viral (AAV)-based therapy engendered the long-term knockdown of circulating Z-A1AT and could be a new strategy in CF ( Mueller et al., 2012 ). This approach was fully described in a review published ( Hunt et al., 2020 ). The other approach is to directly activate SERPINA1 using gene therapy by using viral vectors like retrovirus or adenovirus, but numerous side effects have been observed ( Gregory et al., 2011 ). Their use remains challenging, especially in the CF field. Another strategy proposed is to use serine protease inhibitors such as secretory leukoprotease inhibitor (SLPI) which act locally to maintain a protease/anti-protease balance, thereby preventing protease-mediated tissue destruction. SLPI is a well-characterized member of the trapping gene family of proteins and is produced by respiratory tract epithelial cells and phagocytic neutrophils. Different approaches have been proposed to increase the anti-protease activity by nebulizing SLPI, but the efficacy is currently being evaluated alone or in association with other strategies ( McElvaney et al., 1993 ; Quabius et al., 2017 ). Currently, novel protease inhibitor drugs, which have promising interest in the CF context, are in development (DX-890, AZD9668, POL6014, Grifols T6006-201) in order to improve their resistance against inactivation. Promoting tissue repair represents another strategy by focusing on the proteins involved. Matrix metalloproteinases (MMP) are a group of distinct metalloendopeptidase enzymes that regulate various inflammatory and repair processes. They are either secreted or anchored to the cell surface, and therefore their activity is directed against membrane proteins or extracellular proteins, including inflammatory mediators. In CF patients, different articles have demonstrated that MMP is upregulated in the sputum of patients and is related to tissue damage ( Delacourt et al., 1995 ; Gaggar et al., 2011 ). Various pro-inflammatory cytokines induce them at the transcription level. They might include the activation of a diverse group of intracellular signaling pathways (such as p38 MAPK or ERK 1/2 MAPK), causing the activation of nuclear signaling factors like AP1, NFκB, and STAT (signal transducer and activator of transcription). Activation of MMP can be induced by proteases or oxidants and are controlled by tissue inhibitor of metalloproteases (TIMP). There have been increasing interests in modulating MMP activity to enhance disease outcomes, and different clinical studies are in progress with promising effects in CF. A phase II study with Andecaliximab/GS-5745 in CF adults is in progress and was tolerated in patients with ulcerative colitis or Crohn's disease, and could be an exciting approach to control pulmonary degradation. The approaches using protease inhibitors are very varied, and many studies are still in progress. Although these therapies have been shown to improve patients' health outcomes, they can only be considered in combination with other therapeutic targets. Eicosanoids Pathway Alterations in the metabolism of fatty acids present in membrane lipids may have an essential role in the inflammatory CF pulmonary disease. The arachidonic acid (AA): docosahexaenoic acid (DHA) ratio in blood serum, pulmonary airways, and rectal biopsies are increased in CF patients with either pancreatic sufficiency or pancreatic insufficiency, as compared with healthy control subjects ( Freedman et al., 2004 ). AA is stored in cell membranes and is released from membrane lipids by various PLA2 proteins. Some interesting studies have highlighted the implication of sPLA2 in the pathogenicity of CF mice showing that reduced CFTR expression increased cytosolic PLA2α (cPLA2α) activity. A review has summarized the state of the art of fatty acid metabolism in CF ( Strandvik, 2010 ). These effects improved mucus secretion and accumulation in airway epithelia independent of CFTR chloride transport function ( Medjane et al., 2005 ; Dif et al., 2010 ). Therefore, cPLA2α has been proposed as an appropriate new target for therapeutic intervention in CF ( Dif et al., 2010 ). Small lipid mediators were produced in the course of inflammation resolution and generated varied responses, which are cell types and tissue specific. A large number of these molecules modulate inflammation processes and provide essential functions in chemoattraction, aggregation, and degranulation of inflammatory cells. They are also implicated in tissue and vascular permeability, bronchoconstriction, and mucus production. Some of the lipid mediators include lipoxins (LX), resolvins, protectins, and maresins, which are generated by the activity of lipoxygenases lipoxin A4 (LXA4). Interestingly, inhibitors of the 12R-lipoxygenase have demonstrated an essential role in mucin expression. The inhibitors decreased MUC5AC mucin expression by the inhibition of the ERK/SP1 dependent mechanism ( Garcia-Verdugo et al., 2012 ). LXA4 has been described as a significant signal for the inflammation resolution and is generated at a low level in the CF patients' lungs. LXA4 and RvD1 activate a GPCR termed ALX/FPR2. This pro-resolving receptor is recognized by annexin A1, an endogenous anti-inflammatory peptide. A recent article provides evidence that the miR-181b, overexpressed in CF cells, may be considered as a new strategy to decrease the anti-inflammatory process in CF via the normalization of the expression receptor-dependent LXA4 ( Pierdomenico et al., 2017 ). The LXA4 inhalation consequences have been examined in a pilot study of asthmatic and healthy adult subjects. The drug was well-tolerated, and no harmful effect was observed ( Christie et al., 1992 ). Some impressive results were observed in the topical treatment of infantile eczema ( Wu et al., 2013 ). Together with data showing beneficial actions of LXA4 in the CF context, these results highlight additional studies to check whether the upregulation of the lipidic mediators' pathway can be considered as an appropriate tactic to fight inflammation in CF patients ( Higgins et al., 2015 ). Similarly, the LTB4 produced by resting BECs has been proposed as a target. Inflammatory stimuli increase the production of LTB4 and might also contribute to progressive pulmonary destruction in CF. Bronchial epithelial LTB4 acts as a potent chemoattractant for neutrophils via the cell surface integrins upregulation. When these cells are activated and present at the site of inflammation, they can also participate in the secretion of LTB4. LTB4 synthesis includes lipid peroxidation by 5-lipoxygenase, and produce numerous ROS, and consequently, pro-inflammatory activation. A clinical trial with Montelukast (BIIL 284), a leukotriene receptor agonist, counting a small number of patients, has provided contentious results in CF patients. This therapy has demonstrated a notable decrease in serum eosinophil cationic protein levels and eosinophils without any significant improvement in FEV1, and FEF25–75%. Also, this strategy has shown a significant decrease in cough, serum, and sputum levels of eosinophil cationic protein and IL-8 chemokine. Moreover, an increase in serum and sputum levels of IL-10 has been observed. The trial was stopped early due to a significant increase in the risk of severe pulmonary events in patients receiving the active drug ( Schmitt-Grohe and Zielen, 2005 ). A more recent drug, Acebilustat (CTX-4430), has been evaluated in CF patients. This drug has shown anti-inflammatory activity via the LTA4 hydrolase inhibition and LTB4 modulation. In two-phase I clinical trials, Acebilustat decreased the production of LTB4 and pro-inflammatory cytokines in healthy volunteers and CF patients, and in phase II, optimal dose and duration were identified for future studies ( Elborn et al., 2017 ; Elborn et al., 2018 ). Cannabinoid-Derived Drug Ajulemic acid (JBT-101, Lenabasum) is a cannabinoid-derived molecule that preferably binds to the active CB2 receptor and is non-psychoactive. In some pre-clinical trials done on human lung cells obtained from CF patients, it was shown that Lenabasum stopped the production of both TNF-α and IL-6, two crucial pro-inflammatory cytokines that trigger inflammation. In phase I and II clinical trials, this drug demonstrated favorable safety and tolerability. Recently, a group has also shown significant efficacy in mice models of inflammation and fibrosis ( Burstein, 2018 ). Therefore, phase II was initiated. It will be used to test safety, tolerability, pharmacokinetics, and efficacy of JBT-101 in 70 subjects ≥ 18 and < 65 years of age with documented CF. Treatment of CF patients with Lenabasum twice daily has been able to decrease the number of acute lung exacerbations as well as a reduction of inflammatory cells and mediators present in the sputum. A new clinical trial is undergoing and seeks to enroll more than 400 CF patients over numerous clinical sites. Mucus Therapies In the lungs, the abnormal production of mucus has been assumed to participate actively in the early CF pathogenesis ( Ehre et al., 2014 ). For many years, researchers and clinicians have been trying to understand the origin of mucus abnormalities and found mucoactive drugs molecules to control CF bronchial obstruction. Mucoactive drugs are regularly used as a therapeutic option and are defined by their activity as mucolytics, expectorants, and cough facilitating drug. The expectorants, such as hypertonic solution (HSS), increase the ASL layer and decrease mucus adhesiveness. Mucolytics, such as both N-acetylcysteine (NAC) and recombinant human DNase (rhDNase), reduce sputum viscosity. Medications such as inhaled mannitol, rhDNase (Dornase), and hypertonic HSS have proven efficacy in CF and indirectly reduced inflammation in airways of CF patients ( Tarrant et al., 2017 ). The low volume hypothesis would estimate that approaches increasing the ASL height will increase mucociliary clearance, and consequently reduce lung infection. In order to increase the ASL height and fluidity, an HSS (3 to 7% NaCl) has been proposed to treat CFTR deficiency for better mucociliary clearance. Recently, Wark & McDonald have performed a meta-analysis of 17 different clinical trials of HSS and concluded that, after four weeks, a small enhancement in the lung function was observed but was not sustained at 48 weeks. HSS might also have a little impact on improving life quality in adults ( Wark and McDonald, 2018 ). New clinical trials are in progress in order to establish who may benefit most and whether this benefit is sustained in the longer term ( https://www.cff.org/Trials/Finder ). In the same manner, a meta-analysis was performed with mannitol, which is a naturally occurring sugar alcohol. When inhaled mannitol creates a change in the osmotic gradient. It leads to water movement into the CF airway hydrating the ASL, and enhancing mucociliary clearance. In the different studies, there was no evidence showing that the mannitol treatment for over six months is related to an enhancement of lung function in CF patients compared to control ( Nevitt et al., 2018 ). Recently, different groups have observed expression, biochemical and biophysical alterations of the mucous present in the airways of CF patients ( Rhim et al., 2001 ). More, they observed that abnormal glycosylation of the airway mucins is associated with bacterial infection and inflammation. The effects of altered host mucin glycosylation affect P. aeruginosa adhesion and so pathogenicity. A review from Ventalakrishan et al. has extensively described this feature ( Venkatakrishnan et al., 2013 ). Different therapeutic approaches have been proposed to correct this observation by using, for example, mannose-biding lectin, which recognizes bacterial glycoconjugates and participates in an effective defense against pathogens ( Moller-Kristensen et al., 2006 ). Another strategy used in CF is to disrupt the high DNA content present in the airway mucus of CF patients. DNA is a polyanion compound responsible for the viscosity and adhesiveness of the pulmonary secretions. DNA release and accumulation in ASL occur as a result of tissue destruction caused by inflammatory cells on bacteria and epithelial cells. The strategy is to use a recombinant human deoxyribonuclease I (rhDNase), an enzyme that selectively cleaves DNA, hence decreasing mucus viscosity ( Puchelle et al., 1995 ). Nebulized rhDNase hydrolyzes extracellular DNA within the mucus and transforms it from an adhesive gel into a liquid form of fluid through dilution within minutes. In contrast to mannitol or HSS, rhDNase has shown some significant effects on the improvement of lung function of CF patients and is considered as an effective treatment for the liquefaction of viscous mucus in CF. However, individual responses are unpredictable ( Yang and Montgomery, 2018 ). The only approved reducing agent for human use is N-acetylcysteine (NAC), a well-known antioxidant GSH drug. This drug ameliorates the redox imbalance in neutrophils present in the blood and inhibits their recruitment in the airways of CF patients ( Tirouvanziam et al., 2006 ). NAC is also used in CF as an aerosolized mucus solution to break down disulfide bonds between mucin proteins in order to fluidify mucus ( Duijvestijn and Brand, 1999 ). Some evidence demonstrated that NAC has excellent anti-bacterial properties, the capacity to intervene with biofilm formation and, to disturb the adherence of respiratory pathogens to respiratory epithelial cells ( Blasi et al., 2016 ). In CF patients, NAC has been proven to be safe at large doses with negligible interaction with other drugs. NAC was investigated in CF despite its partial effectiveness as an inhaled mucolytic agent because the extremely oxidizing CF airway environment consumes aerosolized antioxidants quickly ( Tirouvanziam et al., 2006 ; Cantin et al., 2007 ). Finally, inhaled NAC is being used as a mucolytic drug in CF for several decades, although the positive results remain limited. Newer agents targeting other components of CF mucus are currently in development or clinical trials (NAC 40630) and exhibit an exciting effect on mucus ( Blasi et al., 2016 ). Another original approach is undergoing with OligoG CF-5/20. OligoG is an alginate oligosaccharide derived from natural seaweed. It is administrated using a dry powder inhaler and also developed as a liquid to use with a nebulizer. Studies have shown that this dry power drug is capable of reducing the mucus thickness in the lungs. In addition, this drug enhances the efficiency of antibiotics and may facilitate mucus clearance in CF patients. The drug could detach CF mucus by calcium chelation ( Ermund et al., 2017 ). Initiated in 2018, phase II includes more than 120 patients from European and Australian sites. It aims to determine the optimal dose of OligoG and to describe long-term safety and efficacy, with FEV1 as a primary endpoint. Recently, numerous articles have been published to describe new regulation mechanisms of the different proteins present in the mucus and especially on mucins expressed in the airways. The epigenetic regulation role of MUC5AC and MUC5B, the main mucins expressed in the airways, has been thoroughly researched in COPD and have highlighted the implication of methylation and miRNA. Different specific therapies are in progress to modulate the miRNA, and new treatment ways are in progress in CF ( Bardin et al., 2018b ). New Channel Modulators CFTR Channel The discovery of the CFTR gene in 1989 resulted in insights on how CFTR mutations induce CF pathology and encouraged many researchers to develop new drugs or strategies to correct the mutation or increase the protein activity ( Riordan et al., 1989 ). Genetic therapy using adeno-associated virus (AAV) or other strategies aiming to correct the CFTR gene was very promising because CF is a monogenic disease. Nonetheless, the subsequent realization tempered expectations because the airways are well defended and are not absorptive surfaces. The natural barrier of mucus considerably impairs gene transfer into the lungs, and the epithelium renewing necessitates numerous administrations. For these reasons, only one study has demonstrated a significant but moderate effect on CF patients. Thus, further optimizations or other strategies are needed and in progress ( Alton et al., 2015 ; Alton et al., 2017 ). These data provided the grounds for pharmacologic modulations of chloride transport, by targeting mutant CFTR and/or alternative ion channels as anoctamin-1 (ANO1) that can compensate for CFTR malfunction. This excitement has now proven to be warranted because numerous new therapies approved by the FDA or EMA are now either in the pipeline or available for CF patients ( Figure 5 ). This finding contributes to the innovation of genetic disease pharmacotherapy with Vertex Pharmaceuticals as a leader in the CF research field. Fundamental CF research has set the stage for a better molecular understanding of CFTR mutations by supplying structural pieces of information to design new approaches for the pharmacology dynamic even if the different drugs proposed were obtained by high-throughput screening ( Callebaut et al., 2017 ). Till date, two CFTR-directed molecule classes have been developed: "potentiator" compounds increasing mutated CFTR activity at the cell surface and, "corrector" drugs improving altered protein processing and trafficking to the cell surface ( Wainwright et al., 2015 ; Rowe et al., 2017 ; Davies et al., 2018 ) ( Figure 6 ). The first generation of the compounds has either been limited to a few patients with specific mutations (Ivacaftor) or was addressed to a larger group (Orkambi) and demonstrated moderate effects in CF ( Mayer, 2016 ). For this reason, the U.K. National Institute for Health and Care Excellence (NICE) issued a draft guidance against recommending Orkambi. Recently, the FDA has approved an auspicious combination of molecules (Elexacaftor–Tezacaftor–Ivacaftor called Trikafta) to restore the function of p.Phe508del CFTR protein in CF patients even if patients had a single p.Phe508del allele. The combination of drugs relative to the control resulted in a percentage of predicted FEV1 that was more than 14 points higher and a rate of pulmonary exacerbations that was 60% lower through 24 weeks of treatment ( Keating et al., 2018 ; Middleton et al., 2019 ). Unfortunately, a few pieces of information are available for the inflammatory aspects of these treatments. Although, recent evidence showed that the inflammation and lung status hampers these medications and can hinder their effects. Only one article has demonstrated that CFTR modulators can reduce excessive pro-inflammatory response following LPS (lipopolysaccharide) stimulation of CF monocytes ( Jarosz-Griffiths et al., 2020 ). Moreover, in this article, the authors have also demonstrated that IL-8, IL-1β, and TNF-α (Tumor necrosis factor-α) decreased significantly in the serum of CF patients treated with Ivacaftor and Tezacaftor treatment. It is not known whether the observed effects are due to the restoration of Cl - efflux, GSH (glutathione), or CFTR protein interactions present at the membrane. Figure 5 List of the different categories of drugs under development or clinical trials in the context of CF (adapted from https://www.cff.org/Trials/pipeline/ ). Figure 6 Description of the different classes of CFTR mutations related to the different therapeutic proposed in the literature. I—ynthesis defect, II—processing defect, III—channel gating defect, IV—channel conductance defect, V—reduced CFTR production, VI—defect of stability; ER, endoplasmic reticulum. Other new classes of mutation are in development, such as CFTR amplifiers, CFTR stabilizers, and read-through agents ( Figure 6 ). CFTR amplifiers upregulate the expression, and indirectly, the activity of mutant CFTR. PTI-428 and PTI-CH are the two amplifiers who seem promising in pre-clinical and clinical studies. PTI-428 can enhance lung function in CF patients receiving Orkambi with no significant adverse effects. CFTR stabilizer as Cavosonstat inhibits the enzyme that is involved in regulating how much CFTR protein is present at the cell surface ( Donaldson et al., 2017 ). It could potentially increase the benefits of other medications that target the CFTR function. Read-through drugs can help the ribosome skip over the early stop sequence in order to read the mRNA remaining information and generate CFTR protein. These therapies may be of interest to class I mutations where there is no production of mRNA or CFTR protein. Ataluren was developed as a potential treatment for these mutations, but its development was terminated due to failed clinical trial outcomes ( Shoseyov et al., 2016 ). This approach needs to be completed in the future evaluation of CF trials to understand the effects better and investigate the mechanism complex. It can be assumed that earlier treatment using these drugs may avoid structural damages and give rise to more efficient and prolonged results. We can imagine that the improvement of various dysregulated parameters will have long-term effects on the inflammation present in CF patients, even if indirectly. A recent article has highlighted that by Tobramycin or the AMP, 6K-F17 could restore the effects of Orkambi on p.Phe508del-CFTR protein, suggesting a significant role of infection in the CF pathology ( Laselva et al., 2020 ). Furthermore, using this approach, they have demonstrated that the active AMP can down-regulate the expression of pro-inflammatory cytokines like IL-8, IL-6, and TNF-α in p.Phe508del-CFTR human BECs ( Laselva et al., 2020 ). Some exciting improvements in chloride efflux have been demonstrated using S ildenafil, a phosphodiesterase type 5 (PDE5) inhibitor. This drug recues p.Phe508del-CFTR trafficking in vitro experiments and decreases sputum elastase activity and, consequently, the inflammatory process ( Lubamba et al., 2011 ; Taylor-Cousar et al., 2015 ). In parallel to Vertex's studies, many other companies are interested in similar approaches to develop CFTR modulators that either restore the CFTR protein to the membrane or activate it ( Figure 5 ). This research work has been essential over the last ten years, and many other molecules are currently being evaluated and at a different stage. More recently, another promising strategy has been proposed to modulate post-transcriptionally activity of CFTR regulated by acting through miRNA. Distinct groups have proved that wild-type and mutated p.Phe508del human CFTR is regulated by miR-101-3p, miR-145-5p, miR-223-3p, miR-494-3p,and miR-509-3p ( Glasgow et al., 2018 ). The approaches to inhibit the effect of these miRNAs have demonstrated an increase in CFTR protein expression and activity in BECs ( De Santi et al., 2020 ). This approach is exciting, but further researches are needed to understand the subtility of this regulation better. ENaC Channel Since CFTR negatively regulates the activity of the ENaC sodium channel, different strategies have been proposed to decrease its activity. The first proposed molecule was Amiloride, which acts as a potassium-sparing diuretic, showing some benefit in both animal studies and clinical trials. Unfortunately, its efficacy was limited due to its short half-life ( Zhou et al., 2008 ). This approach was repeated with the use of a new ENaC blocker called AZD5634 from AstraZeneca and BI1265162 from Boehringer Ingelheim. A phase Ib study and a phase II study to test, respectively, the safety and effectiveness of AZD5634 and BI 1265162 are underway in CF adults. Nowadays, a more recent and exciting approach, using aerosol antisense oligonucleotide (ASO) targeting ENaC mRNA (Ionis ENAC 2.5Rx), has demonstrated some interesting and impressive results on mice by restoring inflammation and inhibiting ENaC activity ( Crosby et al., 2017 ). A first clinical study with this therapy is currently ongoing. In the same way, Arrowhead asks to open a phase I/II trial into inhaled small interference RNA (iRNA) therapy. The drug, called ARO-ENaC, is an investigational RNA therapy designed to lower the production of the epithelial sodium channel alpha subunit (αENaC) in the lungs of CF patients. ARO-ENaC is an iRNA molecule intending to block the production of ENaC channels. It works by targeting and destroying the αENaC mRNA molecules, which are genetic messengers that carry the necessary information for making αENaC proteins and consequently ENaC activity. ANO1 Channel Since functional CFTR rescue remains limited, with mutation-dependent effects, alternative strategies have been suggested to compensate for the CFTR deficiency and were proposed as a potential CF therapeutic target. Such a strategy was the stimulation of calcium-activated chloride channels (CaCCs) such as the Anoctamin 1 channel (ANO1) ( Figure 3 ). In the nineties, Knowles et al. have discovered that adenosine '5'-triphosphate and uridine-5'-triphosphate stimulated Cl - secretion in both standards and CF respiratory epithelial, offering a potential by-pass mechanism for defective CFTR ( Knowles et al., 1991 ). These activators transduce a signal through P2Y2 receptors that lead to the release of intracellular calcium and activate the CaCCs. An analog called Denufosol was developed. Different studies have demonstrated that this drug can increase Cl - secretion through a CaCC, inhibit sodium absorption via the epithelial sodium channel called ENaC, and stimulate epithelial ciliary beat frequency ( Accurso et al., 2011 ). Based on these data, 'Denufosol' clinical trials begun in 2001 using a wet nebulization direct airway delivery approach. Unfortunately, the last phase III had failed to demonstrate any benefit, and the project was dropped, but the idea of developing this approach remained ( Moss, 2013 ). At the time of this study, CaCCs were poorly known. Their identity remained elusive for over 20 years until 2008 ( Nilius and Droogmans, 2003 ; Caputo et al., 2008 ; Schroeder et al., 2008 ; Yang et al., 2008 ). When ANO1, the principal CaCC present in the airways, was identified in 2008, it allowed for more targeted approaches. Attractively, ANO1 channel has, at the apical membrane of epithelial cells, the same expression pattern as CFTR channels, and this protein was shown to be essential in the activity of CFTR as a chloride channel ( Benedetto et al., 2017 ; Benedetto et al., 2019 ). Besides, ANO1 is implicated in HCO 3 different permeability, proliferation, wound healing, inflammation, and its expression decreased in CF patients ( Veit et al., 2012 ; Jung et al., 2013 ; Ruffin et al., 2013 ). Moreover, a recent article highlighted that ANO1 inhibition decreased ASL height. The authors have also demonstrated that ANO1 is not required for MUC5AC expression, the main protein of the mucus ( Simoes et al., 2019 ). For this reason, a novel ANO1 potentiator was developed (ETX001), and airway epithelial function and mucus transport were evaluated in the human cells and animal models. This approach confirmed previous results and demonstrated that this drug could increase epithelial fluid secretion and enhance mucus clearance ( Danahay et al., 2020 ). Recently, our group has proposed a particular strategy using an ASO specific to ANO1 to reestablish ANO1 expression in the context of CF. This strategy "hijacks" the miRNA regulatory system and allows highly targeted effects. We have demonstrated that ASO-ANO1 could be used to inhibit the fixation of miR-9 on ANO1 mRNA by a target site blocker, and consequently to activate the alternative chloride channel to compensate CFTR Cl - deficiency regardless of the mutation ( Sonneville et al., 2017 ). We have also shown that with this strategy, we can improve tissue repair on cell lines but also on CF primary patient cells. We have likewise demonstrated that with this approach, we can activate mucociliary clearance on primary cells but also CF mice. Although we have not studied the effects of ANO1 modulation of inflammation, preliminary studies have already shown that activating ANO1 limits the secretion of IL-8 ( Veit et al., 2012 ). CFTR Channel The discovery of the CFTR gene in 1989 resulted in insights on how CFTR mutations induce CF pathology and encouraged many researchers to develop new drugs or strategies to correct the mutation or increase the protein activity ( Riordan et al., 1989 ). Genetic therapy using adeno-associated virus (AAV) or other strategies aiming to correct the CFTR gene was very promising because CF is a monogenic disease. Nonetheless, the subsequent realization tempered expectations because the airways are well defended and are not absorptive surfaces. The natural barrier of mucus considerably impairs gene transfer into the lungs, and the epithelium renewing necessitates numerous administrations. For these reasons, only one study has demonstrated a significant but moderate effect on CF patients. Thus, further optimizations or other strategies are needed and in progress ( Alton et al., 2015 ; Alton et al., 2017 ). These data provided the grounds for pharmacologic modulations of chloride transport, by targeting mutant CFTR and/or alternative ion channels as anoctamin-1 (ANO1) that can compensate for CFTR malfunction. This excitement has now proven to be warranted because numerous new therapies approved by the FDA or EMA are now either in the pipeline or available for CF patients ( Figure 5 ). This finding contributes to the innovation of genetic disease pharmacotherapy with Vertex Pharmaceuticals as a leader in the CF research field. Fundamental CF research has set the stage for a better molecular understanding of CFTR mutations by supplying structural pieces of information to design new approaches for the pharmacology dynamic even if the different drugs proposed were obtained by high-throughput screening ( Callebaut et al., 2017 ). Till date, two CFTR-directed molecule classes have been developed: "potentiator" compounds increasing mutated CFTR activity at the cell surface and, "corrector" drugs improving altered protein processing and trafficking to the cell surface ( Wainwright et al., 2015 ; Rowe et al., 2017 ; Davies et al., 2018 ) ( Figure 6 ). The first generation of the compounds has either been limited to a few patients with specific mutations (Ivacaftor) or was addressed to a larger group (Orkambi) and demonstrated moderate effects in CF ( Mayer, 2016 ). For this reason, the U.K. National Institute for Health and Care Excellence (NICE) issued a draft guidance against recommending Orkambi. Recently, the FDA has approved an auspicious combination of molecules (Elexacaftor–Tezacaftor–Ivacaftor called Trikafta) to restore the function of p.Phe508del CFTR protein in CF patients even if patients had a single p.Phe508del allele. The combination of drugs relative to the control resulted in a percentage of predicted FEV1 that was more than 14 points higher and a rate of pulmonary exacerbations that was 60% lower through 24 weeks of treatment ( Keating et al., 2018 ; Middleton et al., 2019 ). Unfortunately, a few pieces of information are available for the inflammatory aspects of these treatments. Although, recent evidence showed that the inflammation and lung status hampers these medications and can hinder their effects. Only one article has demonstrated that CFTR modulators can reduce excessive pro-inflammatory response following LPS (lipopolysaccharide) stimulation of CF monocytes ( Jarosz-Griffiths et al., 2020 ). Moreover, in this article, the authors have also demonstrated that IL-8, IL-1β, and TNF-α (Tumor necrosis factor-α) decreased significantly in the serum of CF patients treated with Ivacaftor and Tezacaftor treatment. It is not known whether the observed effects are due to the restoration of Cl - efflux, GSH (glutathione), or CFTR protein interactions present at the membrane. Figure 5 List of the different categories of drugs under development or clinical trials in the context of CF (adapted from https://www.cff.org/Trials/pipeline/ ). Figure 6 Description of the different classes of CFTR mutations related to the different therapeutic proposed in the literature. I—ynthesis defect, II—processing defect, III—channel gating defect, IV—channel conductance defect, V—reduced CFTR production, VI—defect of stability; ER, endoplasmic reticulum. Other new classes of mutation are in development, such as CFTR amplifiers, CFTR stabilizers, and read-through agents ( Figure 6 ). CFTR amplifiers upregulate the expression, and indirectly, the activity of mutant CFTR. PTI-428 and PTI-CH are the two amplifiers who seem promising in pre-clinical and clinical studies. PTI-428 can enhance lung function in CF patients receiving Orkambi with no significant adverse effects. CFTR stabilizer as Cavosonstat inhibits the enzyme that is involved in regulating how much CFTR protein is present at the cell surface ( Donaldson et al., 2017 ). It could potentially increase the benefits of other medications that target the CFTR function. Read-through drugs can help the ribosome skip over the early stop sequence in order to read the mRNA remaining information and generate CFTR protein. These therapies may be of interest to class I mutations where there is no production of mRNA or CFTR protein. Ataluren was developed as a potential treatment for these mutations, but its development was terminated due to failed clinical trial outcomes ( Shoseyov et al., 2016 ). This approach needs to be completed in the future evaluation of CF trials to understand the effects better and investigate the mechanism complex. It can be assumed that earlier treatment using these drugs may avoid structural damages and give rise to more efficient and prolonged results. We can imagine that the improvement of various dysregulated parameters will have long-term effects on the inflammation present in CF patients, even if indirectly. A recent article has highlighted that by Tobramycin or the AMP, 6K-F17 could restore the effects of Orkambi on p.Phe508del-CFTR protein, suggesting a significant role of infection in the CF pathology ( Laselva et al., 2020 ). Furthermore, using this approach, they have demonstrated that the active AMP can down-regulate the expression of pro-inflammatory cytokines like IL-8, IL-6, and TNF-α in p.Phe508del-CFTR human BECs ( Laselva et al., 2020 ). Some exciting improvements in chloride efflux have been demonstrated using S ildenafil, a phosphodiesterase type 5 (PDE5) inhibitor. This drug recues p.Phe508del-CFTR trafficking in vitro experiments and decreases sputum elastase activity and, consequently, the inflammatory process ( Lubamba et al., 2011 ; Taylor-Cousar et al., 2015 ). In parallel to Vertex's studies, many other companies are interested in similar approaches to develop CFTR modulators that either restore the CFTR protein to the membrane or activate it ( Figure 5 ). This research work has been essential over the last ten years, and many other molecules are currently being evaluated and at a different stage. More recently, another promising strategy has been proposed to modulate post-transcriptionally activity of CFTR regulated by acting through miRNA. Distinct groups have proved that wild-type and mutated p.Phe508del human CFTR is regulated by miR-101-3p, miR-145-5p, miR-223-3p, miR-494-3p,and miR-509-3p ( Glasgow et al., 2018 ). The approaches to inhibit the effect of these miRNAs have demonstrated an increase in CFTR protein expression and activity in BECs ( De Santi et al., 2020 ). This approach is exciting, but further researches are needed to understand the subtility of this regulation better. ENaC Channel Since CFTR negatively regulates the activity of the ENaC sodium channel, different strategies have been proposed to decrease its activity. The first proposed molecule was Amiloride, which acts as a potassium-sparing diuretic, showing some benefit in both animal studies and clinical trials. Unfortunately, its efficacy was limited due to its short half-life ( Zhou et al., 2008 ). This approach was repeated with the use of a new ENaC blocker called AZD5634 from AstraZeneca and BI1265162 from Boehringer Ingelheim. A phase Ib study and a phase II study to test, respectively, the safety and effectiveness of AZD5634 and BI 1265162 are underway in CF adults. Nowadays, a more recent and exciting approach, using aerosol antisense oligonucleotide (ASO) targeting ENaC mRNA (Ionis ENAC 2.5Rx), has demonstrated some interesting and impressive results on mice by restoring inflammation and inhibiting ENaC activity ( Crosby et al., 2017 ). A first clinical study with this therapy is currently ongoing. In the same way, Arrowhead asks to open a phase I/II trial into inhaled small interference RNA (iRNA) therapy. The drug, called ARO-ENaC, is an investigational RNA therapy designed to lower the production of the epithelial sodium channel alpha subunit (αENaC) in the lungs of CF patients. ARO-ENaC is an iRNA molecule intending to block the production of ENaC channels. It works by targeting and destroying the αENaC mRNA molecules, which are genetic messengers that carry the necessary information for making αENaC proteins and consequently ENaC activity. ANO1 Channel Since functional CFTR rescue remains limited, with mutation-dependent effects, alternative strategies have been suggested to compensate for the CFTR deficiency and were proposed as a potential CF therapeutic target. Such a strategy was the stimulation of calcium-activated chloride channels (CaCCs) such as the Anoctamin 1 channel (ANO1) ( Figure 3 ). In the nineties, Knowles et al. have discovered that adenosine '5'-triphosphate and uridine-5'-triphosphate stimulated Cl - secretion in both standards and CF respiratory epithelial, offering a potential by-pass mechanism for defective CFTR ( Knowles et al., 1991 ). These activators transduce a signal through P2Y2 receptors that lead to the release of intracellular calcium and activate the CaCCs. An analog called Denufosol was developed. Different studies have demonstrated that this drug can increase Cl - secretion through a CaCC, inhibit sodium absorption via the epithelial sodium channel called ENaC, and stimulate epithelial ciliary beat frequency ( Accurso et al., 2011 ). Based on these data, 'Denufosol' clinical trials begun in 2001 using a wet nebulization direct airway delivery approach. Unfortunately, the last phase III had failed to demonstrate any benefit, and the project was dropped, but the idea of developing this approach remained ( Moss, 2013 ). At the time of this study, CaCCs were poorly known. Their identity remained elusive for over 20 years until 2008 ( Nilius and Droogmans, 2003 ; Caputo et al., 2008 ; Schroeder et al., 2008 ; Yang et al., 2008 ). When ANO1, the principal CaCC present in the airways, was identified in 2008, it allowed for more targeted approaches. Attractively, ANO1 channel has, at the apical membrane of epithelial cells, the same expression pattern as CFTR channels, and this protein was shown to be essential in the activity of CFTR as a chloride channel ( Benedetto et al., 2017 ; Benedetto et al., 2019 ). Besides, ANO1 is implicated in HCO 3 different permeability, proliferation, wound healing, inflammation, and its expression decreased in CF patients ( Veit et al., 2012 ; Jung et al., 2013 ; Ruffin et al., 2013 ). Moreover, a recent article highlighted that ANO1 inhibition decreased ASL height. The authors have also demonstrated that ANO1 is not required for MUC5AC expression, the main protein of the mucus ( Simoes et al., 2019 ). For this reason, a novel ANO1 potentiator was developed (ETX001), and airway epithelial function and mucus transport were evaluated in the human cells and animal models. This approach confirmed previous results and demonstrated that this drug could increase epithelial fluid secretion and enhance mucus clearance ( Danahay et al., 2020 ). Recently, our group has proposed a particular strategy using an ASO specific to ANO1 to reestablish ANO1 expression in the context of CF. This strategy "hijacks" the miRNA regulatory system and allows highly targeted effects. We have demonstrated that ASO-ANO1 could be used to inhibit the fixation of miR-9 on ANO1 mRNA by a target site blocker, and consequently to activate the alternative chloride channel to compensate CFTR Cl - deficiency regardless of the mutation ( Sonneville et al., 2017 ). We have also shown that with this strategy, we can improve tissue repair on cell lines but also on CF primary patient cells. We have likewise demonstrated that with this approach, we can activate mucociliary clearance on primary cells but also CF mice. Although we have not studied the effects of ANO1 modulation of inflammation, preliminary studies have already shown that activating ANO1 limits the secretion of IL-8 ( Veit et al., 2012 ). Novel Anti-Cytokines Approaches A pathophysiology pulmonary characteristic of CF is a severe neutrophil accumulation, which is correlated with high levels of pro-inflammatory cytokines (IL-8, IL-6, TNF-α), and low levels of anti-inflammatory mediators like IL-10 ( Jacquot et al., 2008b ). For numerous years, different approaches, as curcumin or vitamin D, have been proposed to limit IL-8 secretion and neutrophils influx ( Gaggar et al., 2011 ; Olszowiec-Chlebna et al., 2019 ). Some pre-clinical data have demonstrated that using antibodies, like antibodies directed against intercellular adhesion molecule (ICAM)-1 and IL-8, could be a promising target. The most advanced therapy using SB-656933, an oral CXCR2 antagonist, was already tested in CF patients and has demonstrated along with safety some exciting results in the modulation of airway inflammation ( Moss et al., 2013 ). However, another study using SCH527123 (MK-7123, Navarixin), a CXCR1/2 antagonist, was also attempted in chronic obstructive pulmonary disease (COPD) but was abandoned because of a severe decline in neutrophil number ( Rennard et al., 2015 ). By contrast, a phase II clinical trial has already been carried out in patients with ulcerative colitis and demonstrated inhibition of ozone-induced airway inflammation in humans ( Lazaar et al., 2011 ). Numerous other modulators of cytokines in the context of CF have been proposed, but only in vitro experiments have been performed ( Lampronti et al., 2017 ; De Fenza et al., 2019 ). Cytokine modulation shows that cytokines have a significant role in limiting infections, although these approaches are confusing. A recent publication has highlighted the role of an IL-1 signaling pathway in sterile neutrophilic inflammation and mucus hypersecretion and has suggested that treatment with IL-1 receptor antagonist as Anakinra could be promising to prevent lung inflammation ( Balazs and Mall, 2019 ). The possibility of increasing gene expression and protein activity by the use of ASO has become more and more promising in the last years. However, long-term efficacy, safe delivery, and side effects of long-term treatment must be evaluated in order to be applied in patients with CF ( Bardin et al., 2018b ; Vencken et al., 2019 ). Fabbri et al. have developed this original concept by modulating the IL-8 expression by increasing miR-93 in BECs during P. aeruginosa infection ( Fabbri et al., 2014 ). More recent results have highlighted that other miRNA involved in CF pathology, like miR-199a-3p or miR-636, could be targeted to control the CF lung inflammatory process ( Bardin et al., 2018a ; Bardin et al., 2019 ). Other interesting approaches have been performed to modulate the cascade of inflammation targeting NFκB activity by using, for example, Angelicin derived from different angiosperms or Sulindac, an NSAID ( Rocca et al., 2016 ). Unfortunately, these approaches are not specific, and the risk of side effects remains high. New Development in Antibiotic Approaches "Synthetic" Antibiotics In CF, antibiotics are utilized for various applications, such as initial infection prevention, eradication (for early infection), control (for chronic infection), and finally, pulmonary exacerbations treatment. The antibiotics are given in three different primary ways: oral, inhalation, or intravenous. The choice of antibiotics depends on the nature of the pathogen to be eliminated, the age of the patient, and the nature of other pathogens present such as H. influenza , S. aureus , or P. aeruginosa infections. P. aeruginosa is an opportunistic Gram-negative pathogen and is one of the main reasons for morbidity and mortality in CF and immunosuppressed patients. In order to eradicate new P. aeruginosa infections, antibiotic regimens are now a care standard around the world. Different groups assessed the effectiveness of inhaled Tobramycin, Aztreonam, and Colistin as well as oral Ciprofloxacin in eradicating new P. aeruginosa infection ( Waters, 2018 ; Pang et al., 2019 ), although P. aeruginosa eradication is now much more challenging as a result of its impressive capability to resist antibiotics. These organisms become embedded in an exopolysaccharide biofilm, which protects the organism from phagocytosis and reduces the efficacy of anti-microbial drugs ( Doring, 2010 ). Once this change has occurred, the mucoid P. aeruginosa could acquire multi-drug resistance, and this bacterium is virtually impossible to eradicate ( Southern et al., 2012 ). If the P. aeruginosa infection cannot be cleared, the affected person is faced with an increased treatment burden, accelerated decline in lung function, increased symptom severity, and increased mortality ( Nixon et al., 2002 ). Recently, there has been a growing number of "new" antibiotics, of different classes and formulations, for pulmonary infection treatments in CF patients ( Waters and Smyth, 2015 ). In order to limit toxicity and reduce side effects while directly targeting the lungs, many studies took an interest in using aerosols as a method of administration. In this frame of mind, Levofloxacin was developed for CF patients to target P. aeruginosa infections ( Chirgwin et al., 2019 ; Epps et al., 2019 ). This drug, derived from the fluoroquinolone family, inhibits topoisomerases, which is essential for the synthesis of bacterial DNA. In the same way, inhaled Zitreonam is now available to treat P. aeruginosa infections in CF patients. Although its aerosolized formulation was proven to be beneficial, the formulation for intravenous injections induces significant lung inflammation, which has limited its use. Another example of the existing improvement of drugs is Tobramycin, presented as a dry powder. Inhaled tobramycin provides, in less than 5 minutes, a rapid action directly at the site of the lung infection. In order to increase the efficacy of P. aeruginosa eradication and have a less often resistance development in comparison to the existing "classical" antibiotics, recent P. aeruginosa suggested treatment is the use of a combination of antibiotics and the development of new ones. Also, they can be associated with an alternative strategy such as EDTA (Respirion) or inhaled glycopolymer (SNP113). Thus, a new carbapenem antibiotic called Doripenem has been developed with wide spectrum activity against bacteria through bacterial cell wall synthesis inhibition. Different authors have shown in vitro that this molecule has more significant activity than other antibiotics of the same family on strains isolated from CF patients ( Traczewski and Brown, 2006 ; Riera et al., 2011 ). A clinical phase III study showed that patients infected with P. aeruginosa and treated with Doripenem had higher recovery rates in comparison to Imipenem-treated patients but, no clinical trial with CF patients is in progress ( Chastre et al., 2008 ). In the same way, Plazomicin (a semisynthetic aminoglycoside) and POL7001 (a protein epitope mimetic) came out as an interesting strategy against P. aeruginosa ( Cigana et al., 2016 ). These drugs have demonstrated in vitro some exciting effects on the multidrug-resistant P. aeruginosa isolated from CF patients ( Cigana et al., 2016 ). "Natural" Approaches For many years an original approach using bacteriophages has been advanced. Bacteriophages were discovered in 1915 and can kill bacteria by causing lysis ( Summers, 2001 ). Bacteriophage therapy was applied extensively in the 1930s and 1940s before antibiotics, and it is still being used in Eastern Europe. Nevertheless, after antibiotics became broadly accessible, phage therapy was renounced in Western countries. Many phages can target P. aeruginosa and have demonstrated some exciting effects on mice by decreasing the bacteria burden in the lungs or preventing infection ( Morello et al., 2011 ). Even if clinical studies have shown relative effectiveness, treatments using phages remain negligible so far. Various reasons have limited the treatments with bacteriophages. The idea of introducing a living organism into the body is difficult to accept and remains an important psychological barrier. Moreover, early tests showed that the preparation generated impurities and that these preparations were not very stable ( Morello et al., 2011 ). Although the use of phages in combination with quorum sensing inhibitors seems interesting, this approach remains marginal ( Pang et al., 2019 ), and only a phase Ib/II trial is planned to test the safety and tolerability of AP-PA02 in adults with CF. AP-PA02 is a type of phage intended to control P. aeruginosa infections in CF patients. In in vitro studies, AP-PA02 can kill more than 80% of P. aeruginosa strains from CF people, and some first results are encouraging ( Law et al., 2019 ). Another "natural strategy" is inhaled nitric oxide (NO) for which an initial phase II study is underway. NO is a gas derived from nitrogen with anti-microbial properties. Some in vivo studies have validated this approach to eradicate the infection and to decrease mucus viscoelastic ( Rouillard et al., 2020 ). In the late 1970s, various studies showed that iron played an essential role in bacterial growth and was involved in particular in DNA replication, energy production, and pathogen-host interaction ( Payne and Finkelstein, 1978 ). Recent results demonstrated that the iron content of human sputum is considerably high in CF, which facilitates chronic infections in the lungs of CF patients ( Reid et al., 2007 ). These observations resulted in the development of novel therapeutic strategies in order to limit the amount of iron present in the airways. Gallium is a compound that shares the same properties with iron. It has demonstrated in vitro and in vivo anti-Pseudomonas properties ( Tovar-García et al., 2020 ). The FDA has already approved the intravenous administration of Gallium. Clinical studies, in phase II for intravenous and a phase I for an inhaled strategy, are ongoing to evaluate its efficiency in treating P. aeruginosa infections in CF patients ( Tovar-García et al., 2020 ). During the last decades, AMPs naturally emerged as a potential therapy to cure infections with antibiotic resistance, in CF included. Treatments of bacterial infections by antibiotics result in a worldwide spread of dissemination of antibiotic resistance, both in the community and clinical settings. Besides, the development of new antibiotics is costly and time-consuming. It is hence of great importance to note that AMPs can treat methicillin-resistant S. aureus and multidrug-resistant P. aeruginosa that are resistant to conventional antibiotics ( Geitani et al., 2019 ). Studies showed that treatments of antibiotic-resistant bacterial strains with AMPs were associated with almost no induced resistance to AMPs, which may encourage their use as potential replacement therapy for antibiotics. AMPs can exert anti-inflammatory actions either by suppressing the production of pro-inflammatory cytokines or by stimulating that of anti-inflammatory cytokines by host cells ( Figure 7 ). Cathelicidin LL-37 (one of the most studied AMPs) enhances the production of the anti-inflammatory cytokine IL-1R by the human peripheral blood-derived mononuclear cells and macrophages ( Choi et al., 2014 ), and similar results were observed with LL-37 and beta-defensin-3 (hBD-3) ( Mookherjee et al., 2009 ; Smithrithee et al., 2015 ). Besides their direct actions on host cells involved in the initiation/modulation of inflammation, a number of AMPs, such as LL-37, Magainin-2, and bactericidal-permeability-increasing (BPI), can neutralize the activity of bacterial toxins such as LPS, thus participating in maintaining a balance between pro- and anti-inflammatory cytokines ( Sun and Shang, 2015 ; Skovbakke and Franzyk, 2017 ). Figure 7 General mechanisms by which AMPs exert anti-inflammatory actions on host cells. AMPs can bind to bacterial virulence factors such as LPS or LTA and prevent their interactions with host cells. AMPs are also able to interfere with host cell signaling pathways involved in the inflammatory reaction. The overall consequence is that AMPs reduce the production of inflammatory mediators by these cells that may help in the resolution of inflammation. Most of the reported studies in the field have focused on the roles of AMPs in the modulation of cytokine production. However, cytokines are only the tip of the iceberg in the inflammatory process, and other mediators of inflammation, such as eicosanoids, deserve to be investigated to identify their relative role in the modulation of inflammation by AMPs. Indeed, studies have reported that AMPs such as LL-37 modulates the production of eicosanoids, including leukotriene B4 (LTB4) and thromboxane A2 (TXA2) by macrophages ( Agier et al., 2015 ). TXB2 and LTB4 are metabolites of arachidonic acid conversion by COX and lipoxygenase (LOX), respectively, and known to induce platelet aggregation and neutrophils recruitment at the site of infection ( Yeung and Holinstat, 2011 ). It has been shown that LL-37 AMP blocks the expression of pro-inflammatory pathways involved, such as NF-κB in the presence of LPS ( Agier et al., 2015 ). However, further studies are awaited to decipher the importance of the AMPs/eicosanoids network in the inflammatory reaction and potential implication in inflammatory diseases such as COPD, asthma, and CF. Similar anti-inflammatory effects were observed with WALK11.3 (an AMP with amphipathic helical conformation) in the mouse alveolar macrophage cell line RAW264.7 ( Shim et al., 2015 ). They revealed the ability of this peptide to inhibit the expression of several inflammatory mediators, including NO, COX-derived metabolites, IL-1β, IL-6, interferon (IFN)-β, and TNF-α ( Figure 7 ). The chicken cathelicidin-2 (CATH-2), the known ortholog of the human LL-37, has been shown to reduce inflammation in parallel to its anti-microbial activity against P. aeruginosa -resistant strains from CF patients ( Banaschewski et al., 2017 ). The ability of CATH-2 to downregulate inflammation occurred through the anti-microbial-independent process, as this down-regulation was observed by silencing the inflammatory response that arises from killed bacteria. It is now clear that AMPs play a key role in host defense toward infectious by invading pathogens and represent a potential therapeutic tool to control infections by antibiotic-resistant bacterial strains. They also have the potential to protect the host from harmful inflammation that may result from these infections. Drug design and structure-relationship studies will greatly improve our knowledge of AMPs and the relative importance of their bactericidal vs anti-inflammatory functions, which will be of great help to optimize their potential therapeutic use in disease characterized by both chronic infection and inflammation such as CF. All these data suggested that AMPs could be useful for clinical applications in the view of the protective function against pathogens. A series of clinical trials have started mostly in the pediatric population, and some compounds have been used as topical treatments but not known in the CF context. Different AMPs are under evaluation for the treatment of acute skin infection as Bralicidin, Omiganan, LTC109 (phase II clinical trial), or Pexiganam (phase III clinical trial). Other strategies and applications are currently under study. For example, in sepsis, Talactoferrin was tested by systemic injection in phase II clinical study ( Guntupalli et al., 2013 ). Initial results showed a significant decrease in mortality after 28 days of treatment. However, phase II/III oral Talactoferrin was stopped for problems of safety and efficacy ( Vincent et al., 2015 ). In the case of meningococcemia, rBPI21 pre-clinical trial has demonstrated some anti-bacterial and anti-LPS effects. Encouraging results led to the initiation of a phase III study in children with severe meningococcal sepsis ( Giroir et al., 2001 ). The study outcome showed a reduction in complications with a shorter hospitalization also suggests the possibility to treat with rBPI21 other patients, including CF. The therapeutic applications of P. aeruginosa have been summarized in a recent publication ( Magrone et al., 2018 ). An alternative therapeutic pathway for the use of AMPs has been envisaged by indirectly promoting their expression through the use of natural compounds. Several compounds have been identified as the use of Apigenin to enhance the expression and activity of β-3 defensin and cathelicidin in mice ( Hou et al., 2013 ). Similar effects have been observed with vitamin D on in vitro studies to increase β-2 defensins and LL-37 on keratinocytes ( Kim et al., 2009 ). The use of natural or synthetic antibiotics can have a significant influence on the emergence of new pathogens. It is well established now that microbiota composition and dynamic impact the host immunity, health, and diseases ( Belkaid and Hand, 2014 ). However, a new concept is now progressively emerging, suggesting that the innate immune response of the host can also modulate, at least in part via AMPs, the microbiota composition. For example, recent studies reported the involvement of sPLA2-IIA in the selection of species in pathologies characterized by polymicrobial infections such as CF. P. aeruginosa is known to progressively colonize CF airways to become the dominant pathogen at later stages of CF. This pathogen induces the production by CF airways of sPLA2-IIA, which in turn eradicate S. aureus , therefore helping in its gradual elimination from CF airways and its substitution by P. aeruginosa ( Pernet et al., 2014 ). This effect is mostly due to the intrinsic resistance of P. aeruginosa and high susceptibility of S. aureus to sPLA2-IIA, respectively. Finally, it emerges that AMPs represent valid substitutes of antibiotics when a condition of antibiotic resistance is established. "Synthetic" Antibiotics In CF, antibiotics are utilized for various applications, such as initial infection prevention, eradication (for early infection), control (for chronic infection), and finally, pulmonary exacerbations treatment. The antibiotics are given in three different primary ways: oral, inhalation, or intravenous. The choice of antibiotics depends on the nature of the pathogen to be eliminated, the age of the patient, and the nature of other pathogens present such as H. influenza , S. aureus , or P. aeruginosa infections. P. aeruginosa is an opportunistic Gram-negative pathogen and is one of the main reasons for morbidity and mortality in CF and immunosuppressed patients. In order to eradicate new P. aeruginosa infections, antibiotic regimens are now a care standard around the world. Different groups assessed the effectiveness of inhaled Tobramycin, Aztreonam, and Colistin as well as oral Ciprofloxacin in eradicating new P. aeruginosa infection ( Waters, 2018 ; Pang et al., 2019 ), although P. aeruginosa eradication is now much more challenging as a result of its impressive capability to resist antibiotics. These organisms become embedded in an exopolysaccharide biofilm, which protects the organism from phagocytosis and reduces the efficacy of anti-microbial drugs ( Doring, 2010 ). Once this change has occurred, the mucoid P. aeruginosa could acquire multi-drug resistance, and this bacterium is virtually impossible to eradicate ( Southern et al., 2012 ). If the P. aeruginosa infection cannot be cleared, the affected person is faced with an increased treatment burden, accelerated decline in lung function, increased symptom severity, and increased mortality ( Nixon et al., 2002 ). Recently, there has been a growing number of "new" antibiotics, of different classes and formulations, for pulmonary infection treatments in CF patients ( Waters and Smyth, 2015 ). In order to limit toxicity and reduce side effects while directly targeting the lungs, many studies took an interest in using aerosols as a method of administration. In this frame of mind, Levofloxacin was developed for CF patients to target P. aeruginosa infections ( Chirgwin et al., 2019 ; Epps et al., 2019 ). This drug, derived from the fluoroquinolone family, inhibits topoisomerases, which is essential for the synthesis of bacterial DNA. In the same way, inhaled Zitreonam is now available to treat P. aeruginosa infections in CF patients. Although its aerosolized formulation was proven to be beneficial, the formulation for intravenous injections induces significant lung inflammation, which has limited its use. Another example of the existing improvement of drugs is Tobramycin, presented as a dry powder. Inhaled tobramycin provides, in less than 5 minutes, a rapid action directly at the site of the lung infection. In order to increase the efficacy of P. aeruginosa eradication and have a less often resistance development in comparison to the existing "classical" antibiotics, recent P. aeruginosa suggested treatment is the use of a combination of antibiotics and the development of new ones. Also, they can be associated with an alternative strategy such as EDTA (Respirion) or inhaled glycopolymer (SNP113). Thus, a new carbapenem antibiotic called Doripenem has been developed with wide spectrum activity against bacteria through bacterial cell wall synthesis inhibition. Different authors have shown in vitro that this molecule has more significant activity than other antibiotics of the same family on strains isolated from CF patients ( Traczewski and Brown, 2006 ; Riera et al., 2011 ). A clinical phase III study showed that patients infected with P. aeruginosa and treated with Doripenem had higher recovery rates in comparison to Imipenem-treated patients but, no clinical trial with CF patients is in progress ( Chastre et al., 2008 ). In the same way, Plazomicin (a semisynthetic aminoglycoside) and POL7001 (a protein epitope mimetic) came out as an interesting strategy against P. aeruginosa ( Cigana et al., 2016 ). These drugs have demonstrated in vitro some exciting effects on the multidrug-resistant P. aeruginosa isolated from CF patients ( Cigana et al., 2016 ). "Natural" Approaches For many years an original approach using bacteriophages has been advanced. Bacteriophages were discovered in 1915 and can kill bacteria by causing lysis ( Summers, 2001 ). Bacteriophage therapy was applied extensively in the 1930s and 1940s before antibiotics, and it is still being used in Eastern Europe. Nevertheless, after antibiotics became broadly accessible, phage therapy was renounced in Western countries. Many phages can target P. aeruginosa and have demonstrated some exciting effects on mice by decreasing the bacteria burden in the lungs or preventing infection ( Morello et al., 2011 ). Even if clinical studies have shown relative effectiveness, treatments using phages remain negligible so far. Various reasons have limited the treatments with bacteriophages. The idea of introducing a living organism into the body is difficult to accept and remains an important psychological barrier. Moreover, early tests showed that the preparation generated impurities and that these preparations were not very stable ( Morello et al., 2011 ). Although the use of phages in combination with quorum sensing inhibitors seems interesting, this approach remains marginal ( Pang et al., 2019 ), and only a phase Ib/II trial is planned to test the safety and tolerability of AP-PA02 in adults with CF. AP-PA02 is a type of phage intended to control P. aeruginosa infections in CF patients. In in vitro studies, AP-PA02 can kill more than 80% of P. aeruginosa strains from CF people, and some first results are encouraging ( Law et al., 2019 ). Another "natural strategy" is inhaled nitric oxide (NO) for which an initial phase II study is underway. NO is a gas derived from nitrogen with anti-microbial properties. Some in vivo studies have validated this approach to eradicate the infection and to decrease mucus viscoelastic ( Rouillard et al., 2020 ). In the late 1970s, various studies showed that iron played an essential role in bacterial growth and was involved in particular in DNA replication, energy production, and pathogen-host interaction ( Payne and Finkelstein, 1978 ). Recent results demonstrated that the iron content of human sputum is considerably high in CF, which facilitates chronic infections in the lungs of CF patients ( Reid et al., 2007 ). These observations resulted in the development of novel therapeutic strategies in order to limit the amount of iron present in the airways. Gallium is a compound that shares the same properties with iron. It has demonstrated in vitro and in vivo anti-Pseudomonas properties ( Tovar-García et al., 2020 ). The FDA has already approved the intravenous administration of Gallium. Clinical studies, in phase II for intravenous and a phase I for an inhaled strategy, are ongoing to evaluate its efficiency in treating P. aeruginosa infections in CF patients ( Tovar-García et al., 2020 ). During the last decades, AMPs naturally emerged as a potential therapy to cure infections with antibiotic resistance, in CF included. Treatments of bacterial infections by antibiotics result in a worldwide spread of dissemination of antibiotic resistance, both in the community and clinical settings. Besides, the development of new antibiotics is costly and time-consuming. It is hence of great importance to note that AMPs can treat methicillin-resistant S. aureus and multidrug-resistant P. aeruginosa that are resistant to conventional antibiotics ( Geitani et al., 2019 ). Studies showed that treatments of antibiotic-resistant bacterial strains with AMPs were associated with almost no induced resistance to AMPs, which may encourage their use as potential replacement therapy for antibiotics. AMPs can exert anti-inflammatory actions either by suppressing the production of pro-inflammatory cytokines or by stimulating that of anti-inflammatory cytokines by host cells ( Figure 7 ). Cathelicidin LL-37 (one of the most studied AMPs) enhances the production of the anti-inflammatory cytokine IL-1R by the human peripheral blood-derived mononuclear cells and macrophages ( Choi et al., 2014 ), and similar results were observed with LL-37 and beta-defensin-3 (hBD-3) ( Mookherjee et al., 2009 ; Smithrithee et al., 2015 ). Besides their direct actions on host cells involved in the initiation/modulation of inflammation, a number of AMPs, such as LL-37, Magainin-2, and bactericidal-permeability-increasing (BPI), can neutralize the activity of bacterial toxins such as LPS, thus participating in maintaining a balance between pro- and anti-inflammatory cytokines ( Sun and Shang, 2015 ; Skovbakke and Franzyk, 2017 ). Figure 7 General mechanisms by which AMPs exert anti-inflammatory actions on host cells. AMPs can bind to bacterial virulence factors such as LPS or LTA and prevent their interactions with host cells. AMPs are also able to interfere with host cell signaling pathways involved in the inflammatory reaction. The overall consequence is that AMPs reduce the production of inflammatory mediators by these cells that may help in the resolution of inflammation. Most of the reported studies in the field have focused on the roles of AMPs in the modulation of cytokine production. However, cytokines are only the tip of the iceberg in the inflammatory process, and other mediators of inflammation, such as eicosanoids, deserve to be investigated to identify their relative role in the modulation of inflammation by AMPs. Indeed, studies have reported that AMPs such as LL-37 modulates the production of eicosanoids, including leukotriene B4 (LTB4) and thromboxane A2 (TXA2) by macrophages ( Agier et al., 2015 ). TXB2 and LTB4 are metabolites of arachidonic acid conversion by COX and lipoxygenase (LOX), respectively, and known to induce platelet aggregation and neutrophils recruitment at the site of infection ( Yeung and Holinstat, 2011 ). It has been shown that LL-37 AMP blocks the expression of pro-inflammatory pathways involved, such as NF-κB in the presence of LPS ( Agier et al., 2015 ). However, further studies are awaited to decipher the importance of the AMPs/eicosanoids network in the inflammatory reaction and potential implication in inflammatory diseases such as COPD, asthma, and CF. Similar anti-inflammatory effects were observed with WALK11.3 (an AMP with amphipathic helical conformation) in the mouse alveolar macrophage cell line RAW264.7 ( Shim et al., 2015 ). They revealed the ability of this peptide to inhibit the expression of several inflammatory mediators, including NO, COX-derived metabolites, IL-1β, IL-6, interferon (IFN)-β, and TNF-α ( Figure 7 ). The chicken cathelicidin-2 (CATH-2), the known ortholog of the human LL-37, has been shown to reduce inflammation in parallel to its anti-microbial activity against P. aeruginosa -resistant strains from CF patients ( Banaschewski et al., 2017 ). The ability of CATH-2 to downregulate inflammation occurred through the anti-microbial-independent process, as this down-regulation was observed by silencing the inflammatory response that arises from killed bacteria. It is now clear that AMPs play a key role in host defense toward infectious by invading pathogens and represent a potential therapeutic tool to control infections by antibiotic-resistant bacterial strains. They also have the potential to protect the host from harmful inflammation that may result from these infections. Drug design and structure-relationship studies will greatly improve our knowledge of AMPs and the relative importance of their bactericidal vs anti-inflammatory functions, which will be of great help to optimize their potential therapeutic use in disease characterized by both chronic infection and inflammation such as CF. All these data suggested that AMPs could be useful for clinical applications in the view of the protective function against pathogens. A series of clinical trials have started mostly in the pediatric population, and some compounds have been used as topical treatments but not known in the CF context. Different AMPs are under evaluation for the treatment of acute skin infection as Bralicidin, Omiganan, LTC109 (phase II clinical trial), or Pexiganam (phase III clinical trial). Other strategies and applications are currently under study. For example, in sepsis, Talactoferrin was tested by systemic injection in phase II clinical study ( Guntupalli et al., 2013 ). Initial results showed a significant decrease in mortality after 28 days of treatment. However, phase II/III oral Talactoferrin was stopped for problems of safety and efficacy ( Vincent et al., 2015 ). In the case of meningococcemia, rBPI21 pre-clinical trial has demonstrated some anti-bacterial and anti-LPS effects. Encouraging results led to the initiation of a phase III study in children with severe meningococcal sepsis ( Giroir et al., 2001 ). The study outcome showed a reduction in complications with a shorter hospitalization also suggests the possibility to treat with rBPI21 other patients, including CF. The therapeutic applications of P. aeruginosa have been summarized in a recent publication ( Magrone et al., 2018 ). An alternative therapeutic pathway for the use of AMPs has been envisaged by indirectly promoting their expression through the use of natural compounds. Several compounds have been identified as the use of Apigenin to enhance the expression and activity of β-3 defensin and cathelicidin in mice ( Hou et al., 2013 ). Similar effects have been observed with vitamin D on in vitro studies to increase β-2 defensins and LL-37 on keratinocytes ( Kim et al., 2009 ). The use of natural or synthetic antibiotics can have a significant influence on the emergence of new pathogens. It is well established now that microbiota composition and dynamic impact the host immunity, health, and diseases ( Belkaid and Hand, 2014 ). However, a new concept is now progressively emerging, suggesting that the innate immune response of the host can also modulate, at least in part via AMPs, the microbiota composition. For example, recent studies reported the involvement of sPLA2-IIA in the selection of species in pathologies characterized by polymicrobial infections such as CF. P. aeruginosa is known to progressively colonize CF airways to become the dominant pathogen at later stages of CF. This pathogen induces the production by CF airways of sPLA2-IIA, which in turn eradicate S. aureus , therefore helping in its gradual elimination from CF airways and its substitution by P. aeruginosa ( Pernet et al., 2014 ). This effect is mostly due to the intrinsic resistance of P. aeruginosa and high susceptibility of S. aureus to sPLA2-IIA, respectively. Finally, it emerges that AMPs represent valid substitutes of antibiotics when a condition of antibiotic resistance is established. Alternative Strategies Anti-Proteases CF "anti-protease therapies" can be separated into two separate groups of drugs: some to increase anti-protease and some to inhibit protease expression. CFTR is an essential apical GSH transporter in the lung, and can indirectly participate in the inflammatory process by reducing oxidative stress. Evidence supporting the occurrence of oxidative stress in CF is established and extensively described ( Galli et al., 2012 ; Causer et al., 2020 ). Some interesting works have demonstrated that oxidative stress could suppress CFTR expression ( Cantin et al., 2006 ). Oxidative stress has a major role in the development of lung pathology in CF children and will, in addition to having a role in lung remodeling, have a role in the pulmonary microbiota ( Shi et al., 2019 ). A recent metanalysis has positively correlated the expression of antioxidants with body mass index and lung function in CF ( Causer et al., 2020 ). The malabsorption of nutrients with antioxidants properties in CF, participate in the imbalance in favor of oxidative stress and disrupt redox signaling, and, finally, molecular damages even if some data appears to be conflicting ( Shamseer et al., 2010 ; Siwamogsatham et al., 2014 ). Therefore, multiple studies have been carried out to check the anti-protease supplementation in CF ( Galli et al., 2012 ). Some studies have focused on especially serine proteases via two distinct administration routes: aerosolized and intravenously ( McKelvey et al., 2020 ). In CF, exocrine pancreatic insufficiency and reduced bile acids induce critical antioxidants malabsorption, including carotenoids (β-carotene), tocopherols (vitamin E), coenzyme Q10, and selenium. Supplementation of antioxidant micronutrients (vitamin E, C, D, β-carotene, and selenium) may, therefore, potentially help maintain an oxidant-antioxidant balance, and this aspect has been extensively reviewed ( Sagel et al., 2011 ; Ciofu et al., 2019 ). In the same approach, LAU-7b, an oral drug, is a derived form related to vitamin A. This compound can reduce the lung inflammatory response of CF people. In parallel, a phase II clinical study to test the effectiveness and safety of LAU-7b in CF patients is underway ( Lands and Stanojevic, 2016 ). LAU-7b, also called, Fenretidine, work to increase docosahexaenoic acid (DHA) and consequently CER concentration. Some authors supported that the decrease of CER concentration contributes to the persistent bacterial infection and the constitutive MAP kinases and NFκB activation ( Guilbault et al., 2008 ; Guilbault et al., 2009 ). Human α-1 antitrypsin (A1AT) is still the most studied drug by far. Different clinical trials were already achieved. An inhaled α1-proteinase inhibitor is known to reduce NE burden in some patients with CF. A phase I in non-CF bronchiectasis and an IIa clinical study with purified A1AT products given through inhalation in CF subjects were just finalized and have demonstrated safety and efficacy ( Gaggar et al., 2016 ; Watz et al., 2019 ). In the conclusion of the second study, the daily α-1 hydrophobic chromatography process delivered for three weeks was safe, well-tolerated, and effective in raising the α1-PI levels in the sputum of subjects with CF. However, the effects were transient and difficult to predict due to the proteases' variability in CF patients' lungs. The administration by airway routeway effectively increased the concentration of A1AT in sputum. The current study was not powered to assess changes in FEV1 or biomarkers in sputum, and further clinical are needed. In parallel, A1AT gene therapy is emerging. Some recent data have demonstrated encouraging results in the inhibition of miRNA, which targets the A1AT gene called SERPINA1 ( Hunt et al., 2020 ). This strategy aims to by-pass protein regulation systems of the most abundant inhibitor of NE in the airways. It is an alternative to the delivery of recombinant by using miRNA-targeted therapies. It was found that dual miRNA and adeno-associated viral (AAV)-based therapy engendered the long-term knockdown of circulating Z-A1AT and could be a new strategy in CF ( Mueller et al., 2012 ). This approach was fully described in a review published ( Hunt et al., 2020 ). The other approach is to directly activate SERPINA1 using gene therapy by using viral vectors like retrovirus or adenovirus, but numerous side effects have been observed ( Gregory et al., 2011 ). Their use remains challenging, especially in the CF field. Another strategy proposed is to use serine protease inhibitors such as secretory leukoprotease inhibitor (SLPI) which act locally to maintain a protease/anti-protease balance, thereby preventing protease-mediated tissue destruction. SLPI is a well-characterized member of the trapping gene family of proteins and is produced by respiratory tract epithelial cells and phagocytic neutrophils. Different approaches have been proposed to increase the anti-protease activity by nebulizing SLPI, but the efficacy is currently being evaluated alone or in association with other strategies ( McElvaney et al., 1993 ; Quabius et al., 2017 ). Currently, novel protease inhibitor drugs, which have promising interest in the CF context, are in development (DX-890, AZD9668, POL6014, Grifols T6006-201) in order to improve their resistance against inactivation. Promoting tissue repair represents another strategy by focusing on the proteins involved. Matrix metalloproteinases (MMP) are a group of distinct metalloendopeptidase enzymes that regulate various inflammatory and repair processes. They are either secreted or anchored to the cell surface, and therefore their activity is directed against membrane proteins or extracellular proteins, including inflammatory mediators. In CF patients, different articles have demonstrated that MMP is upregulated in the sputum of patients and is related to tissue damage ( Delacourt et al., 1995 ; Gaggar et al., 2011 ). Various pro-inflammatory cytokines induce them at the transcription level. They might include the activation of a diverse group of intracellular signaling pathways (such as p38 MAPK or ERK 1/2 MAPK), causing the activation of nuclear signaling factors like AP1, NFκB, and STAT (signal transducer and activator of transcription). Activation of MMP can be induced by proteases or oxidants and are controlled by tissue inhibitor of metalloproteases (TIMP). There have been increasing interests in modulating MMP activity to enhance disease outcomes, and different clinical studies are in progress with promising effects in CF. A phase II study with Andecaliximab/GS-5745 in CF adults is in progress and was tolerated in patients with ulcerative colitis or Crohn's disease, and could be an exciting approach to control pulmonary degradation. The approaches using protease inhibitors are very varied, and many studies are still in progress. Although these therapies have been shown to improve patients' health outcomes, they can only be considered in combination with other therapeutic targets. Eicosanoids Pathway Alterations in the metabolism of fatty acids present in membrane lipids may have an essential role in the inflammatory CF pulmonary disease. The arachidonic acid (AA): docosahexaenoic acid (DHA) ratio in blood serum, pulmonary airways, and rectal biopsies are increased in CF patients with either pancreatic sufficiency or pancreatic insufficiency, as compared with healthy control subjects ( Freedman et al., 2004 ). AA is stored in cell membranes and is released from membrane lipids by various PLA2 proteins. Some interesting studies have highlighted the implication of sPLA2 in the pathogenicity of CF mice showing that reduced CFTR expression increased cytosolic PLA2α (cPLA2α) activity. A review has summarized the state of the art of fatty acid metabolism in CF ( Strandvik, 2010 ). These effects improved mucus secretion and accumulation in airway epithelia independent of CFTR chloride transport function ( Medjane et al., 2005 ; Dif et al., 2010 ). Therefore, cPLA2α has been proposed as an appropriate new target for therapeutic intervention in CF ( Dif et al., 2010 ). Small lipid mediators were produced in the course of inflammation resolution and generated varied responses, which are cell types and tissue specific. A large number of these molecules modulate inflammation processes and provide essential functions in chemoattraction, aggregation, and degranulation of inflammatory cells. They are also implicated in tissue and vascular permeability, bronchoconstriction, and mucus production. Some of the lipid mediators include lipoxins (LX), resolvins, protectins, and maresins, which are generated by the activity of lipoxygenases lipoxin A4 (LXA4). Interestingly, inhibitors of the 12R-lipoxygenase have demonstrated an essential role in mucin expression. The inhibitors decreased MUC5AC mucin expression by the inhibition of the ERK/SP1 dependent mechanism ( Garcia-Verdugo et al., 2012 ). LXA4 has been described as a significant signal for the inflammation resolution and is generated at a low level in the CF patients' lungs. LXA4 and RvD1 activate a GPCR termed ALX/FPR2. This pro-resolving receptor is recognized by annexin A1, an endogenous anti-inflammatory peptide. A recent article provides evidence that the miR-181b, overexpressed in CF cells, may be considered as a new strategy to decrease the anti-inflammatory process in CF via the normalization of the expression receptor-dependent LXA4 ( Pierdomenico et al., 2017 ). The LXA4 inhalation consequences have been examined in a pilot study of asthmatic and healthy adult subjects. The drug was well-tolerated, and no harmful effect was observed ( Christie et al., 1992 ). Some impressive results were observed in the topical treatment of infantile eczema ( Wu et al., 2013 ). Together with data showing beneficial actions of LXA4 in the CF context, these results highlight additional studies to check whether the upregulation of the lipidic mediators' pathway can be considered as an appropriate tactic to fight inflammation in CF patients ( Higgins et al., 2015 ). Similarly, the LTB4 produced by resting BECs has been proposed as a target. Inflammatory stimuli increase the production of LTB4 and might also contribute to progressive pulmonary destruction in CF. Bronchial epithelial LTB4 acts as a potent chemoattractant for neutrophils via the cell surface integrins upregulation. When these cells are activated and present at the site of inflammation, they can also participate in the secretion of LTB4. LTB4 synthesis includes lipid peroxidation by 5-lipoxygenase, and produce numerous ROS, and consequently, pro-inflammatory activation. A clinical trial with Montelukast (BIIL 284), a leukotriene receptor agonist, counting a small number of patients, has provided contentious results in CF patients. This therapy has demonstrated a notable decrease in serum eosinophil cationic protein levels and eosinophils without any significant improvement in FEV1, and FEF25–75%. Also, this strategy has shown a significant decrease in cough, serum, and sputum levels of eosinophil cationic protein and IL-8 chemokine. Moreover, an increase in serum and sputum levels of IL-10 has been observed. The trial was stopped early due to a significant increase in the risk of severe pulmonary events in patients receiving the active drug ( Schmitt-Grohe and Zielen, 2005 ). A more recent drug, Acebilustat (CTX-4430), has been evaluated in CF patients. This drug has shown anti-inflammatory activity via the LTA4 hydrolase inhibition and LTB4 modulation. In two-phase I clinical trials, Acebilustat decreased the production of LTB4 and pro-inflammatory cytokines in healthy volunteers and CF patients, and in phase II, optimal dose and duration were identified for future studies ( Elborn et al., 2017 ; Elborn et al., 2018 ). Cannabinoid-Derived Drug Ajulemic acid (JBT-101, Lenabasum) is a cannabinoid-derived molecule that preferably binds to the active CB2 receptor and is non-psychoactive. In some pre-clinical trials done on human lung cells obtained from CF patients, it was shown that Lenabasum stopped the production of both TNF-α and IL-6, two crucial pro-inflammatory cytokines that trigger inflammation. In phase I and II clinical trials, this drug demonstrated favorable safety and tolerability. Recently, a group has also shown significant efficacy in mice models of inflammation and fibrosis ( Burstein, 2018 ). Therefore, phase II was initiated. It will be used to test safety, tolerability, pharmacokinetics, and efficacy of JBT-101 in 70 subjects ≥ 18 and < 65 years of age with documented CF. Treatment of CF patients with Lenabasum twice daily has been able to decrease the number of acute lung exacerbations as well as a reduction of inflammatory cells and mediators present in the sputum. A new clinical trial is undergoing and seeks to enroll more than 400 CF patients over numerous clinical sites. Anti-Proteases CF "anti-protease therapies" can be separated into two separate groups of drugs: some to increase anti-protease and some to inhibit protease expression. CFTR is an essential apical GSH transporter in the lung, and can indirectly participate in the inflammatory process by reducing oxidative stress. Evidence supporting the occurrence of oxidative stress in CF is established and extensively described ( Galli et al., 2012 ; Causer et al., 2020 ). Some interesting works have demonstrated that oxidative stress could suppress CFTR expression ( Cantin et al., 2006 ). Oxidative stress has a major role in the development of lung pathology in CF children and will, in addition to having a role in lung remodeling, have a role in the pulmonary microbiota ( Shi et al., 2019 ). A recent metanalysis has positively correlated the expression of antioxidants with body mass index and lung function in CF ( Causer et al., 2020 ). The malabsorption of nutrients with antioxidants properties in CF, participate in the imbalance in favor of oxidative stress and disrupt redox signaling, and, finally, molecular damages even if some data appears to be conflicting ( Shamseer et al., 2010 ; Siwamogsatham et al., 2014 ). Therefore, multiple studies have been carried out to check the anti-protease supplementation in CF ( Galli et al., 2012 ). Some studies have focused on especially serine proteases via two distinct administration routes: aerosolized and intravenously ( McKelvey et al., 2020 ). In CF, exocrine pancreatic insufficiency and reduced bile acids induce critical antioxidants malabsorption, including carotenoids (β-carotene), tocopherols (vitamin E), coenzyme Q10, and selenium. Supplementation of antioxidant micronutrients (vitamin E, C, D, β-carotene, and selenium) may, therefore, potentially help maintain an oxidant-antioxidant balance, and this aspect has been extensively reviewed ( Sagel et al., 2011 ; Ciofu et al., 2019 ). In the same approach, LAU-7b, an oral drug, is a derived form related to vitamin A. This compound can reduce the lung inflammatory response of CF people. In parallel, a phase II clinical study to test the effectiveness and safety of LAU-7b in CF patients is underway ( Lands and Stanojevic, 2016 ). LAU-7b, also called, Fenretidine, work to increase docosahexaenoic acid (DHA) and consequently CER concentration. Some authors supported that the decrease of CER concentration contributes to the persistent bacterial infection and the constitutive MAP kinases and NFκB activation ( Guilbault et al., 2008 ; Guilbault et al., 2009 ). Human α-1 antitrypsin (A1AT) is still the most studied drug by far. Different clinical trials were already achieved. An inhaled α1-proteinase inhibitor is known to reduce NE burden in some patients with CF. A phase I in non-CF bronchiectasis and an IIa clinical study with purified A1AT products given through inhalation in CF subjects were just finalized and have demonstrated safety and efficacy ( Gaggar et al., 2016 ; Watz et al., 2019 ). In the conclusion of the second study, the daily α-1 hydrophobic chromatography process delivered for three weeks was safe, well-tolerated, and effective in raising the α1-PI levels in the sputum of subjects with CF. However, the effects were transient and difficult to predict due to the proteases' variability in CF patients' lungs. The administration by airway routeway effectively increased the concentration of A1AT in sputum. The current study was not powered to assess changes in FEV1 or biomarkers in sputum, and further clinical are needed. In parallel, A1AT gene therapy is emerging. Some recent data have demonstrated encouraging results in the inhibition of miRNA, which targets the A1AT gene called SERPINA1 ( Hunt et al., 2020 ). This strategy aims to by-pass protein regulation systems of the most abundant inhibitor of NE in the airways. It is an alternative to the delivery of recombinant by using miRNA-targeted therapies. It was found that dual miRNA and adeno-associated viral (AAV)-based therapy engendered the long-term knockdown of circulating Z-A1AT and could be a new strategy in CF ( Mueller et al., 2012 ). This approach was fully described in a review published ( Hunt et al., 2020 ). The other approach is to directly activate SERPINA1 using gene therapy by using viral vectors like retrovirus or adenovirus, but numerous side effects have been observed ( Gregory et al., 2011 ). Their use remains challenging, especially in the CF field. Another strategy proposed is to use serine protease inhibitors such as secretory leukoprotease inhibitor (SLPI) which act locally to maintain a protease/anti-protease balance, thereby preventing protease-mediated tissue destruction. SLPI is a well-characterized member of the trapping gene family of proteins and is produced by respiratory tract epithelial cells and phagocytic neutrophils. Different approaches have been proposed to increase the anti-protease activity by nebulizing SLPI, but the efficacy is currently being evaluated alone or in association with other strategies ( McElvaney et al., 1993 ; Quabius et al., 2017 ). Currently, novel protease inhibitor drugs, which have promising interest in the CF context, are in development (DX-890, AZD9668, POL6014, Grifols T6006-201) in order to improve their resistance against inactivation. Promoting tissue repair represents another strategy by focusing on the proteins involved. Matrix metalloproteinases (MMP) are a group of distinct metalloendopeptidase enzymes that regulate various inflammatory and repair processes. They are either secreted or anchored to the cell surface, and therefore their activity is directed against membrane proteins or extracellular proteins, including inflammatory mediators. In CF patients, different articles have demonstrated that MMP is upregulated in the sputum of patients and is related to tissue damage ( Delacourt et al., 1995 ; Gaggar et al., 2011 ). Various pro-inflammatory cytokines induce them at the transcription level. They might include the activation of a diverse group of intracellular signaling pathways (such as p38 MAPK or ERK 1/2 MAPK), causing the activation of nuclear signaling factors like AP1, NFκB, and STAT (signal transducer and activator of transcription). Activation of MMP can be induced by proteases or oxidants and are controlled by tissue inhibitor of metalloproteases (TIMP). There have been increasing interests in modulating MMP activity to enhance disease outcomes, and different clinical studies are in progress with promising effects in CF. A phase II study with Andecaliximab/GS-5745 in CF adults is in progress and was tolerated in patients with ulcerative colitis or Crohn's disease, and could be an exciting approach to control pulmonary degradation. The approaches using protease inhibitors are very varied, and many studies are still in progress. Although these therapies have been shown to improve patients' health outcomes, they can only be considered in combination with other therapeutic targets. Eicosanoids Pathway Alterations in the metabolism of fatty acids present in membrane lipids may have an essential role in the inflammatory CF pulmonary disease. The arachidonic acid (AA): docosahexaenoic acid (DHA) ratio in blood serum, pulmonary airways, and rectal biopsies are increased in CF patients with either pancreatic sufficiency or pancreatic insufficiency, as compared with healthy control subjects ( Freedman et al., 2004 ). AA is stored in cell membranes and is released from membrane lipids by various PLA2 proteins. Some interesting studies have highlighted the implication of sPLA2 in the pathogenicity of CF mice showing that reduced CFTR expression increased cytosolic PLA2α (cPLA2α) activity. A review has summarized the state of the art of fatty acid metabolism in CF ( Strandvik, 2010 ). These effects improved mucus secretion and accumulation in airway epithelia independent of CFTR chloride transport function ( Medjane et al., 2005 ; Dif et al., 2010 ). Therefore, cPLA2α has been proposed as an appropriate new target for therapeutic intervention in CF ( Dif et al., 2010 ). Small lipid mediators were produced in the course of inflammation resolution and generated varied responses, which are cell types and tissue specific. A large number of these molecules modulate inflammation processes and provide essential functions in chemoattraction, aggregation, and degranulation of inflammatory cells. They are also implicated in tissue and vascular permeability, bronchoconstriction, and mucus production. Some of the lipid mediators include lipoxins (LX), resolvins, protectins, and maresins, which are generated by the activity of lipoxygenases lipoxin A4 (LXA4). Interestingly, inhibitors of the 12R-lipoxygenase have demonstrated an essential role in mucin expression. The inhibitors decreased MUC5AC mucin expression by the inhibition of the ERK/SP1 dependent mechanism ( Garcia-Verdugo et al., 2012 ). LXA4 has been described as a significant signal for the inflammation resolution and is generated at a low level in the CF patients' lungs. LXA4 and RvD1 activate a GPCR termed ALX/FPR2. This pro-resolving receptor is recognized by annexin A1, an endogenous anti-inflammatory peptide. A recent article provides evidence that the miR-181b, overexpressed in CF cells, may be considered as a new strategy to decrease the anti-inflammatory process in CF via the normalization of the expression receptor-dependent LXA4 ( Pierdomenico et al., 2017 ). The LXA4 inhalation consequences have been examined in a pilot study of asthmatic and healthy adult subjects. The drug was well-tolerated, and no harmful effect was observed ( Christie et al., 1992 ). Some impressive results were observed in the topical treatment of infantile eczema ( Wu et al., 2013 ). Together with data showing beneficial actions of LXA4 in the CF context, these results highlight additional studies to check whether the upregulation of the lipidic mediators' pathway can be considered as an appropriate tactic to fight inflammation in CF patients ( Higgins et al., 2015 ). Similarly, the LTB4 produced by resting BECs has been proposed as a target. Inflammatory stimuli increase the production of LTB4 and might also contribute to progressive pulmonary destruction in CF. Bronchial epithelial LTB4 acts as a potent chemoattractant for neutrophils via the cell surface integrins upregulation. When these cells are activated and present at the site of inflammation, they can also participate in the secretion of LTB4. LTB4 synthesis includes lipid peroxidation by 5-lipoxygenase, and produce numerous ROS, and consequently, pro-inflammatory activation. A clinical trial with Montelukast (BIIL 284), a leukotriene receptor agonist, counting a small number of patients, has provided contentious results in CF patients. This therapy has demonstrated a notable decrease in serum eosinophil cationic protein levels and eosinophils without any significant improvement in FEV1, and FEF25–75%. Also, this strategy has shown a significant decrease in cough, serum, and sputum levels of eosinophil cationic protein and IL-8 chemokine. Moreover, an increase in serum and sputum levels of IL-10 has been observed. The trial was stopped early due to a significant increase in the risk of severe pulmonary events in patients receiving the active drug ( Schmitt-Grohe and Zielen, 2005 ). A more recent drug, Acebilustat (CTX-4430), has been evaluated in CF patients. This drug has shown anti-inflammatory activity via the LTA4 hydrolase inhibition and LTB4 modulation. In two-phase I clinical trials, Acebilustat decreased the production of LTB4 and pro-inflammatory cytokines in healthy volunteers and CF patients, and in phase II, optimal dose and duration were identified for future studies ( Elborn et al., 2017 ; Elborn et al., 2018 ). Cannabinoid-Derived Drug Ajulemic acid (JBT-101, Lenabasum) is a cannabinoid-derived molecule that preferably binds to the active CB2 receptor and is non-psychoactive. In some pre-clinical trials done on human lung cells obtained from CF patients, it was shown that Lenabasum stopped the production of both TNF-α and IL-6, two crucial pro-inflammatory cytokines that trigger inflammation. In phase I and II clinical trials, this drug demonstrated favorable safety and tolerability. Recently, a group has also shown significant efficacy in mice models of inflammation and fibrosis ( Burstein, 2018 ). Therefore, phase II was initiated. It will be used to test safety, tolerability, pharmacokinetics, and efficacy of JBT-101 in 70 subjects ≥ 18 and < 65 years of age with documented CF. Treatment of CF patients with Lenabasum twice daily has been able to decrease the number of acute lung exacerbations as well as a reduction of inflammatory cells and mediators present in the sputum. A new clinical trial is undergoing and seeks to enroll more than 400 CF patients over numerous clinical sites. Mucus Therapies In the lungs, the abnormal production of mucus has been assumed to participate actively in the early CF pathogenesis ( Ehre et al., 2014 ). For many years, researchers and clinicians have been trying to understand the origin of mucus abnormalities and found mucoactive drugs molecules to control CF bronchial obstruction. Mucoactive drugs are regularly used as a therapeutic option and are defined by their activity as mucolytics, expectorants, and cough facilitating drug. The expectorants, such as hypertonic solution (HSS), increase the ASL layer and decrease mucus adhesiveness. Mucolytics, such as both N-acetylcysteine (NAC) and recombinant human DNase (rhDNase), reduce sputum viscosity. Medications such as inhaled mannitol, rhDNase (Dornase), and hypertonic HSS have proven efficacy in CF and indirectly reduced inflammation in airways of CF patients ( Tarrant et al., 2017 ). The low volume hypothesis would estimate that approaches increasing the ASL height will increase mucociliary clearance, and consequently reduce lung infection. In order to increase the ASL height and fluidity, an HSS (3 to 7% NaCl) has been proposed to treat CFTR deficiency for better mucociliary clearance. Recently, Wark & McDonald have performed a meta-analysis of 17 different clinical trials of HSS and concluded that, after four weeks, a small enhancement in the lung function was observed but was not sustained at 48 weeks. HSS might also have a little impact on improving life quality in adults ( Wark and McDonald, 2018 ). New clinical trials are in progress in order to establish who may benefit most and whether this benefit is sustained in the longer term ( https://www.cff.org/Trials/Finder ). In the same manner, a meta-analysis was performed with mannitol, which is a naturally occurring sugar alcohol. When inhaled mannitol creates a change in the osmotic gradient. It leads to water movement into the CF airway hydrating the ASL, and enhancing mucociliary clearance. In the different studies, there was no evidence showing that the mannitol treatment for over six months is related to an enhancement of lung function in CF patients compared to control ( Nevitt et al., 2018 ). Recently, different groups have observed expression, biochemical and biophysical alterations of the mucous present in the airways of CF patients ( Rhim et al., 2001 ). More, they observed that abnormal glycosylation of the airway mucins is associated with bacterial infection and inflammation. The effects of altered host mucin glycosylation affect P. aeruginosa adhesion and so pathogenicity. A review from Ventalakrishan et al. has extensively described this feature ( Venkatakrishnan et al., 2013 ). Different therapeutic approaches have been proposed to correct this observation by using, for example, mannose-biding lectin, which recognizes bacterial glycoconjugates and participates in an effective defense against pathogens ( Moller-Kristensen et al., 2006 ). Another strategy used in CF is to disrupt the high DNA content present in the airway mucus of CF patients. DNA is a polyanion compound responsible for the viscosity and adhesiveness of the pulmonary secretions. DNA release and accumulation in ASL occur as a result of tissue destruction caused by inflammatory cells on bacteria and epithelial cells. The strategy is to use a recombinant human deoxyribonuclease I (rhDNase), an enzyme that selectively cleaves DNA, hence decreasing mucus viscosity ( Puchelle et al., 1995 ). Nebulized rhDNase hydrolyzes extracellular DNA within the mucus and transforms it from an adhesive gel into a liquid form of fluid through dilution within minutes. In contrast to mannitol or HSS, rhDNase has shown some significant effects on the improvement of lung function of CF patients and is considered as an effective treatment for the liquefaction of viscous mucus in CF. However, individual responses are unpredictable ( Yang and Montgomery, 2018 ). The only approved reducing agent for human use is N-acetylcysteine (NAC), a well-known antioxidant GSH drug. This drug ameliorates the redox imbalance in neutrophils present in the blood and inhibits their recruitment in the airways of CF patients ( Tirouvanziam et al., 2006 ). NAC is also used in CF as an aerosolized mucus solution to break down disulfide bonds between mucin proteins in order to fluidify mucus ( Duijvestijn and Brand, 1999 ). Some evidence demonstrated that NAC has excellent anti-bacterial properties, the capacity to intervene with biofilm formation and, to disturb the adherence of respiratory pathogens to respiratory epithelial cells ( Blasi et al., 2016 ). In CF patients, NAC has been proven to be safe at large doses with negligible interaction with other drugs. NAC was investigated in CF despite its partial effectiveness as an inhaled mucolytic agent because the extremely oxidizing CF airway environment consumes aerosolized antioxidants quickly ( Tirouvanziam et al., 2006 ; Cantin et al., 2007 ). Finally, inhaled NAC is being used as a mucolytic drug in CF for several decades, although the positive results remain limited. Newer agents targeting other components of CF mucus are currently in development or clinical trials (NAC 40630) and exhibit an exciting effect on mucus ( Blasi et al., 2016 ). Another original approach is undergoing with OligoG CF-5/20. OligoG is an alginate oligosaccharide derived from natural seaweed. It is administrated using a dry powder inhaler and also developed as a liquid to use with a nebulizer. Studies have shown that this dry power drug is capable of reducing the mucus thickness in the lungs. In addition, this drug enhances the efficiency of antibiotics and may facilitate mucus clearance in CF patients. The drug could detach CF mucus by calcium chelation ( Ermund et al., 2017 ). Initiated in 2018, phase II includes more than 120 patients from European and Australian sites. It aims to determine the optimal dose of OligoG and to describe long-term safety and efficacy, with FEV1 as a primary endpoint. Recently, numerous articles have been published to describe new regulation mechanisms of the different proteins present in the mucus and especially on mucins expressed in the airways. The epigenetic regulation role of MUC5AC and MUC5B, the main mucins expressed in the airways, has been thoroughly researched in COPD and have highlighted the implication of methylation and miRNA. Different specific therapies are in progress to modulate the miRNA, and new treatment ways are in progress in CF ( Bardin et al., 2018b ). Conclusion Although current anti-inflammatory drugs (corticosteroids and Ibuprofen) in CF patients have shown little effectiveness, the creation and improvement of new anti-inflammatory drugs for CF lungs has been overlooked for a long time. In the last decade, most of the research fields in CF therapy, have focused mainly on the discovery of new CFTR activators. Despite this, basic researches that are now in the evaluation phase have shown that new approaches could be very promising in resolving efficiently the CF lungs' ongoing inflammatory vicious cycle. However, treatment complexity is challenging. Currently available treatments offered to CF patients certainly help reduce inflammation, but in indirect and non-specific pathways, by targeting the viscosity of the mucus, reducing infection, or activating Cl - efflux. As the traditional approaches have shown their limitations, it seems essential to us that original work should continue in order to identify innovative approaches that would be more specific. The identification of critical druggable molecular targets to decrease inflammation is still an unsatisfied demand that needs numerous additional researches. Author Contributions All authors: CM, PB, ZX, HC, LT, and OT have written and reviewed the manuscript. Funding This review was funded in part by grants from Inserm, Sorbonne Université, Faculté des Sciences, Institut Pasteur, and the non-profit organization Vaincre la Mucoviscidose. CM received a Ph.D. grant from Vaincre la Mucoviscidose. ZX was founded by Yangzhou University. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589096/

Evidence for abnormal cytokine expression in Gulf War Illness: A preliminary analysis of daily immune monitoring data

Background Gulf War Illness (GWI) is a clinically heterogeneous chronic condition that affects many veterans of the 1990–1991 Persian Gulf War. One of the most prevalent and debilitating symptoms of GWI is abnormal fatigue. The mechanisms underlying GWI generally, and fatigue symptoms specifically, have yet to be conclusively identified, although immune system abnormalities are suspected to be involved. The first goal of this immune monitoring study was to determine if GWI is associated with higher absolute levels and daily variability of pro-inflammatory immune factors. The second goal was to explore the relationship between day-to-day immune marker fluctuations and daily self-reported fatigue severity. Methods We recruited veterans with GWI and healthy veteran control (HV) participants to provide self-reported fatigue severity data and blood samples, over 25 consecutive days. We profiled inflammatory processes by using a longitudinal, daily immune-monitoring approach. For each day, serum cytokine and chemokine concentrations were determined using multiplex assays. Results Seven veterans with GWI and eight healthy veteran control (HV) participants completed the study protocol. We found that GWI was associated with higher variability in the expression of eotaxin-1 (p 1:80, erythrocyte sedimentation rate (ESR) >60 mm/h, positive rheumatoid factor, C-reactive protein over 1.0 mg/L, or clinically abnormal results of a complete blood count test according to the testing laboratory's reference ranges. Study design We used an observational longitudinal design to test the role of immune function in GWI. The 42-day study began with the screening session during which participants were provided with the data collection device and instructed in its use. The study consisted of a two-week baseline symptom reporting phase, followed by 25 consecutive days of symptom reporting and venous blood draws, and ended with a three day follow-up phase during which participants reported their daily symptoms. Participants could miss up to 2 days consecutively under special circumstances, in which case additional days were added to the end so that 25 days total were assessed. Questionnaire data collection Baseline questionnaires used for screening purposes included the Kansas Gulf War Veterans Health Project Questionnaire [ 23 ] and the Hospital Anxiety and Depression Scale [ 46 ]. Daily GWI symptoms were scored by the participants on 0–100 visual analog scales (VAS), using commercial survey software (Dooblo SurveyToGo, Kefar Sava, Israel) on an android-based device. Fatigue, the primary dependent variable, was assessed by asking, "How fatigued have you felt today?" The VAS was anchored on the left by "No fatigue at all" and on the right by "Severe fatigue". Similar 101-point VAS were used to collect information about other symptoms in the GWI diagnostic criteria, but were not analyzed in this study. The daily self-report measures were completed by the participants in the afternoon or evening. Immune data collection During the 25-day immune monitoring phase, blood was collected by trained phlebotomists or research nurses at the Stanford Clinical and Translational Research Unit (CTRU) or a mobile phlebotomy service (Health Exams Inc). Venous blood was generally collected from the cubital fossa using a 23-gauge butterfly needle into two 4 cc serum separating tubes. The venipuncture site was rotated daily to minimize participant discomfort and to maintain local vein integrity. Obtained blood samples were coagulated at room temperature for 30 min, centrifuged at 350 g for 15 min, and the serum layer was then aliquotted into vials for storage at −80 °C. Phlebotomy visits were scheduled within a two-hour window for each participant, to minimize the effect of diurnal variation on cytokine concentrations [ 48 , 49 ]. Blood pressure, heart rate, and body temperature were also collected during each visit to monitor for signs of emerging acute illness. Concentrations of cytokines and chemokines in serum samples were determined by Myriad RBM, Inc. using a standardized protocol. In short, Tecan EVO® robots were used to combine an aliquot of each sample with the capture microspheres and incubated for one hour at room temperature. Multiplexed cocktails of biotinylated reporter antibodies were then added robotically, thoroughly mixed, and incubated for an additional hour at room temperature. Multiplexes were developed using an excess of streptavidin-phycoerythrin solution which was thoroughly mixed into each multiplex and incubated for one hour at room temperature. The volume of each multiplexed reaction was reduced by vacuum filtration and the volume increased by dilution into matrix buffer for analysis. Analysis was performed in a Luminex instrument and the resulting data stream was interpreted using proprietary PlateReader data analysis software. Assays were run in high density multiplexed panels and the least detectable dose (LDD) was determined as the mean +3 standard deviations of 20 sample diluent readings. An eight ( n = 8) point standard curve was used to obtain quantitative measurements for each sample. Standards and quality controls were run on each plate with a replicate of each standard positioned in the first and last column of the plate. Quality controls were run in duplicate along different points of the curve to ensure both accuracy and precision for each analyte. The analytes included in the assays and their LDD were: Brain-Derived Neurotrophic Factor (BDNF) [0.027 ng/mL], Eotaxin-1/CCL11 [67 pg/mL], Factor VII [3.3 ng/mL], Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) [14 pg/mL], Intercellular Adhesion Molecule (ICAM)-1 3.3 [ng/mL], Interferon (IF)-γ [1.6 pg/mL], IL-1α [1.5 pg/mL], IL-1β [2.2 pg/mL], IL-1Ra [156 pg/mL], IL-2 [5.9 pg/mL], IL-3 [1.6 pg/mL], IL-4 [9.4 pg/mL], IL-5 [2.7 pg/mL], IL-6 [1.6 pg/mL], IL-7 [8.2 pg/mL], IL-8/CXCL8 [1.3 pg/mL], IL-10 [3.3 pg/mL], IL-12p40 [95 pg/mL], IL-12p70 [34 pg/mL], IL-15 [0.19 ng/mL], IL-17 [1.6 pg/mL], IL-18 [6.7 pg/mL], IL-23 [0.66 ng/mL], Macrophage Inflammatory Protein (MIP)-1α/CCL3 [18 pg/mL], MIP-1β/CCL4 [16 pg/mL], Matrix Metalloproteinase (MMP)-3 [26 pg/mL], MMP-9 [12 ng/mL], Monocyte Chemotactic Protein (MCP)-1/CCL2 [27 pg/mL], SCF [65 pg/mL], Tumor Necrosis Factor (TNF)-α [13 pg/mL], TNF-β [3.0 pg/mL], and VEGF [20 pg/mL]. Concentrations of leptin were determined using human leptin radioimmunoassay kits (Millipore) at the Metabolism Core at the Nutrition Obesity Research Center at the University of Alabama in Birmingham. The LDD of the leptin assays was 0.97 ng/mL and the inter-assay and intra-assay CVs were 4.18 % and 5.32 %, respectively. Data analyses Survey data were collected from the Dooblo SurveyToGo studio software and merged with the main database independently by two investigators, and compared to ensure accuracy. All data analyses were performed using SPSS Statistics for Windows v21.0 (Armonk, NY: IBM Corp). To examine serum inflammatory biomarker differences between GWI and HV, we tested group differences in the levels of cytokines over time. Group differences in cytokine concentrations were tested with generalized estimating equations (GEE). GEEs properly account for repeated measures within participants, and therefore allow for more precise estimates of individual- and group-means. GEEs were conducted in a univariate fashion. The participant identification number was used as a subject nesting variable, and the study day as the repeated measures index. Participant type (GWI or HV) was entered as the between-subjects factor. Differences in between-group cytokine variability over the course of the immune monitoring period were also tested. Day-to-day cytokine fluctuations were used to calculate coefficients of variation (CVs) for each analyte, nested by individual. CV differences between the GWI and HV groups were tested with independent samples t-tests. To test whether daily fatigue in GWI is related to fluctuations in cytokines, we used linear mixed models (LMM). LMMs can properly model effects with repeated measures, as can GEEs, but can also allow for intercepts and slopes to vary for each individual participant, by treating the participant as a random factor. Fatigue and cytokine data were person-centered (z-score-transformed) and temporally smoothed using a three-day moving average to improve pattern detection [ 50 , 51 ]. Because only half of the HV individuals had measurable levels of fatigue, these analyses were performed on the GWI individuals only. For all applicable analyses, we determined significance using an α = 0.05 false-discovery rate to adjust for the 93 planned comparisons, yielding a p = 0.0098 threshold significance. For the cytokine that was most significantly associated with fatigue at the group-level, we also performed visual analyses of the relationship between each individual participant's cytokine concentrations and fatigue symptoms over time. Participant recruitment and consent Study procedures were conducted as approved by the Institutional Review Board at Stanford University. All participants provided written informed consent including consent for publication of data. Participants were recruited through radio advertisements broadcast in the San Francisco Bay Area, advertisements on Craigslist, online support groups, and advertisements at the Veterans Affairs Palo Alto Health Care System. Participant inclusion and exclusion criteria were initially determined following a phone pre-screening interview. Males between the ages of 39 and 65 were considered for this study. Potential participants were excluded for current use of opioid medications, significant psychological comorbidities, current involvement in litigation or worker's compensation claims, current use of blood thinning medications, or current regular use of any anti-inflammatory medication (such as aspirin, ibuprofen, or naproxen) which may have confounded the inflammatory data. Participants were also required to provide 25 consecutive daily blood draws, either at Stanford University or through a mobile phlebotomy service (Health Exams Inc., Burlingame, CA). Secondary screening was conducted in person at the Stanford Adult and Pediatric Pain Laboratory. During this appointment, detailed participant demographic and medical history information were collected. Individuals were admitted into one of two study groups, GWI or HV. Participants included in the GWI group met the Kansas Gulf War Illness case definition criteria [ 23 ]. In brief, these criteria restrict the diagnosis to veterans who were deployed to the Persian Gulf and subsequently reported moderate to severe symptoms in three out of six symptom categories: fatigue or sleep problems; pain symptoms; neurologic, cognitive or mood symptoms; gastrointestinal symptoms; respiratory symptoms; and skin symptoms [ 23 ]. Participants in the HV group were required to have been deployed during the Gulf War, to be free of any current major medical diagnoses, and to not report daily pain or fatigue at the time of the assessment. Exclusionary criteria for all participants included a depression subscale score of ≥16 on the Hospital Anxiety and Depression Scale (HADS) [ 46 ], a score of ≥50 on the Military Post Traumatic Stress Score [ 47 ], or current use of opioid analgesics or anti-inflammatories. Individuals were also excluded if screening blood tests showed abnormal values of thyroid hormone, an ANA ratio >1:80, erythrocyte sedimentation rate (ESR) >60 mm/h, positive rheumatoid factor, C-reactive protein over 1.0 mg/L, or clinically abnormal results of a complete blood count test according to the testing laboratory's reference ranges. Study design We used an observational longitudinal design to test the role of immune function in GWI. The 42-day study began with the screening session during which participants were provided with the data collection device and instructed in its use. The study consisted of a two-week baseline symptom reporting phase, followed by 25 consecutive days of symptom reporting and venous blood draws, and ended with a three day follow-up phase during which participants reported their daily symptoms. Participants could miss up to 2 days consecutively under special circumstances, in which case additional days were added to the end so that 25 days total were assessed. Questionnaire data collection Baseline questionnaires used for screening purposes included the Kansas Gulf War Veterans Health Project Questionnaire [ 23 ] and the Hospital Anxiety and Depression Scale [ 46 ]. Daily GWI symptoms were scored by the participants on 0–100 visual analog scales (VAS), using commercial survey software (Dooblo SurveyToGo, Kefar Sava, Israel) on an android-based device. Fatigue, the primary dependent variable, was assessed by asking, "How fatigued have you felt today?" The VAS was anchored on the left by "No fatigue at all" and on the right by "Severe fatigue". Similar 101-point VAS were used to collect information about other symptoms in the GWI diagnostic criteria, but were not analyzed in this study. The daily self-report measures were completed by the participants in the afternoon or evening. Immune data collection During the 25-day immune monitoring phase, blood was collected by trained phlebotomists or research nurses at the Stanford Clinical and Translational Research Unit (CTRU) or a mobile phlebotomy service (Health Exams Inc). Venous blood was generally collected from the cubital fossa using a 23-gauge butterfly needle into two 4 cc serum separating tubes. The venipuncture site was rotated daily to minimize participant discomfort and to maintain local vein integrity. Obtained blood samples were coagulated at room temperature for 30 min, centrifuged at 350 g for 15 min, and the serum layer was then aliquotted into vials for storage at −80 °C. Phlebotomy visits were scheduled within a two-hour window for each participant, to minimize the effect of diurnal variation on cytokine concentrations [ 48 , 49 ]. Blood pressure, heart rate, and body temperature were also collected during each visit to monitor for signs of emerging acute illness. Concentrations of cytokines and chemokines in serum samples were determined by Myriad RBM, Inc. using a standardized protocol. In short, Tecan EVO® robots were used to combine an aliquot of each sample with the capture microspheres and incubated for one hour at room temperature. Multiplexed cocktails of biotinylated reporter antibodies were then added robotically, thoroughly mixed, and incubated for an additional hour at room temperature. Multiplexes were developed using an excess of streptavidin-phycoerythrin solution which was thoroughly mixed into each multiplex and incubated for one hour at room temperature. The volume of each multiplexed reaction was reduced by vacuum filtration and the volume increased by dilution into matrix buffer for analysis. Analysis was performed in a Luminex instrument and the resulting data stream was interpreted using proprietary PlateReader data analysis software. Assays were run in high density multiplexed panels and the least detectable dose (LDD) was determined as the mean +3 standard deviations of 20 sample diluent readings. An eight ( n = 8) point standard curve was used to obtain quantitative measurements for each sample. Standards and quality controls were run on each plate with a replicate of each standard positioned in the first and last column of the plate. Quality controls were run in duplicate along different points of the curve to ensure both accuracy and precision for each analyte. The analytes included in the assays and their LDD were: Brain-Derived Neurotrophic Factor (BDNF) [0.027 ng/mL], Eotaxin-1/CCL11 [67 pg/mL], Factor VII [3.3 ng/mL], Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) [14 pg/mL], Intercellular Adhesion Molecule (ICAM)-1 3.3 [ng/mL], Interferon (IF)-γ [1.6 pg/mL], IL-1α [1.5 pg/mL], IL-1β [2.2 pg/mL], IL-1Ra [156 pg/mL], IL-2 [5.9 pg/mL], IL-3 [1.6 pg/mL], IL-4 [9.4 pg/mL], IL-5 [2.7 pg/mL], IL-6 [1.6 pg/mL], IL-7 [8.2 pg/mL], IL-8/CXCL8 [1.3 pg/mL], IL-10 [3.3 pg/mL], IL-12p40 [95 pg/mL], IL-12p70 [34 pg/mL], IL-15 [0.19 ng/mL], IL-17 [1.6 pg/mL], IL-18 [6.7 pg/mL], IL-23 [0.66 ng/mL], Macrophage Inflammatory Protein (MIP)-1α/CCL3 [18 pg/mL], MIP-1β/CCL4 [16 pg/mL], Matrix Metalloproteinase (MMP)-3 [26 pg/mL], MMP-9 [12 ng/mL], Monocyte Chemotactic Protein (MCP)-1/CCL2 [27 pg/mL], SCF [65 pg/mL], Tumor Necrosis Factor (TNF)-α [13 pg/mL], TNF-β [3.0 pg/mL], and VEGF [20 pg/mL]. Concentrations of leptin were determined using human leptin radioimmunoassay kits (Millipore) at the Metabolism Core at the Nutrition Obesity Research Center at the University of Alabama in Birmingham. The LDD of the leptin assays was 0.97 ng/mL and the inter-assay and intra-assay CVs were 4.18 % and 5.32 %, respectively. Data analyses Survey data were collected from the Dooblo SurveyToGo studio software and merged with the main database independently by two investigators, and compared to ensure accuracy. All data analyses were performed using SPSS Statistics for Windows v21.0 (Armonk, NY: IBM Corp). To examine serum inflammatory biomarker differences between GWI and HV, we tested group differences in the levels of cytokines over time. Group differences in cytokine concentrations were tested with generalized estimating equations (GEE). GEEs properly account for repeated measures within participants, and therefore allow for more precise estimates of individual- and group-means. GEEs were conducted in a univariate fashion. The participant identification number was used as a subject nesting variable, and the study day as the repeated measures index. Participant type (GWI or HV) was entered as the between-subjects factor. Differences in between-group cytokine variability over the course of the immune monitoring period were also tested. Day-to-day cytokine fluctuations were used to calculate coefficients of variation (CVs) for each analyte, nested by individual. CV differences between the GWI and HV groups were tested with independent samples t-tests. To test whether daily fatigue in GWI is related to fluctuations in cytokines, we used linear mixed models (LMM). LMMs can properly model effects with repeated measures, as can GEEs, but can also allow for intercepts and slopes to vary for each individual participant, by treating the participant as a random factor. Fatigue and cytokine data were person-centered (z-score-transformed) and temporally smoothed using a three-day moving average to improve pattern detection [ 50 , 51 ]. Because only half of the HV individuals had measurable levels of fatigue, these analyses were performed on the GWI individuals only. For all applicable analyses, we determined significance using an α = 0.05 false-discovery rate to adjust for the 93 planned comparisons, yielding a p = 0.0098 threshold significance. For the cytokine that was most significantly associated with fatigue at the group-level, we also performed visual analyses of the relationship between each individual participant's cytokine concentrations and fatigue symptoms over time.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1595399/

Transcriptional Profiling of the Bacillus anthracis Life Cycle In Vitro and an Implied Model for Regulation of Spore Formation †

The life cycle of Bacillus anthracis includes both vegetative and endospore morphologies which alternate based on nutrient availability, and there is considerable evidence indicating that the ability of this organism to cause anthrax depends on its ability to progress through this life cycle in a regulated manner. Here we report the use of a custom B. anthracis GeneChip in defining the gene expression patterns that occur throughout the entire life cycle in vitro. Nearly 5,000 genes were expressed in five distinct waves of transcription as the bacteria progressed from germination through sporulation, and we identified a specific set of functions represented within each wave. We also used these data to define the temporal expression of the spore proteome, and in doing so we have demonstrated that much of the spore's protein content is not synthesized de novo during sporulation but rather is packaged from preexisting stocks. We explored several potential mechanisms by which the cell could control which proteins are packaged into the developing spore, and our analyses were most consistent with a model in which B. anthracis regulates the composition of the spore proteome based on protein stability. This study is by far the most comprehensive survey yet of the B. anthracis life cycle and serves as a useful resource in defining the growth-phase-dependent expression patterns of each gene. Additionally, the data and accompanying bioinformatics analyses suggest a model for sporulation that has broad implications for B. anthracis biology and offer new possibilities for microbial forensics and detection.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7120534/

Drivers of Emerging Zoonotic Infectious Diseases

This chapter discusses drivers of emerging infectious diseases (EID) of humans that have an origin in other vertebrate animals (zoonoses). This is a broad topic, worthy of a book in its own right. This chapter will therefore provide only an overview of key concepts of drivers of the emergence of zoonotic diseases, and particularly infectious diseases with a major disease burden in humans. As the authors mainly work in Asia, the focus of this chapter is Asia, but many of the lessons learned in this region are likely to apply elsewhere. More than 60 % of the world population live in Asia, a region with some of the fastest developing economies in the world. Yet, despite tremendous advances, infectious diseases still remain a major burden for the human population in Asia. Of the estimated 2.1 million deaths in children aged less than 5 years in Southeast Asia in 2010, 47 % are attributable to infectious causes (Liu et al., Lancet 379:2151–2161, 2012). As such, Asia is both vulnerable to imported EIDs and a global focus of major social and environmental change that may facilitate the emergence and dissemination of new pathogens. However, it would be too simplistic to present the extensive changes in Asia as inevitably increasing the risk of EIDs. Some aspects of socio-economic change might serve to reduce the overall risk of infectious disease emergence, but all ecosystem changes have the potential to provide new opportunities for microorganisms to spill-over into human populations. The Concept of Emergence Emerging infectious diseases are often perceived to be 'novel' pathogens of humans, but EIDs are more broadly defined as diseases that are increasing in their incidence, geographic or host species range, or their impact (due for instance to the acquisition of resistance to antimicrobial drugs or new virulence factors) (Fauci and Morens 2012 ). This encompasses both newly recognised pathogens and known pathogens that are 're-emerging' (Box 1 ). The development and introduction over time of new, more sensitive technologies for identifying microbial diversity, such as molecular methods to identify elements of the microbial genome, coupled with geographical heterogeneities in the availability of these technologies, make it difficult to accurately assess the rate at which new infectious diseases emerge. The difficulty is further compounded by changes over time in the ease of publishing articles in the bio-medical literature. Attempts have nevertheless been made, and 335 emerging infectious disease events were identified between 1940 and 2004, an average of five per year, with a peak in the 1980s associated with the emergence of HIV/AIDs and its associated opportunistic infections (Jones et al. 2008 ). New human pathogens must come from somewhere, and unsurprisingly they most often arise from animals; with which we share genes, physiology, microorganisms, and environments. Around 60 % of current human infectious diseases are zoonotic, and those that exclusively infect humans were probably shared with other animal species at some time in the past (e.g. measles, mumps, dengue, pertussis, hepatitis B). In addition, around 60 % of recent emerging infectious diseases of humans have arisen from animals, with an estimated 72 % of these having their origin in wildlife species (Jones et al. 2008 ). The recognition of the shared susceptibility of humans and animals to many diseases has led to the concept of One Health. The infection of humans by the microorganisms of animals is a natural consequence of ecology and evolution (Karesh et al. 2012 ). Microbes are an abundant life form and ecological factors (specifically the environmental living conditions) critically define the niches that they inhabit. Microbes that normally colonise or infect animals are able to 'spill over' to humans when they possess the ability and are given the opportunity to exploit a similar niche (e.g. the gut or bloodstream) of humans. The same is true for microbes of wild animals that spill over to domesticated animals. Once provided this opportunity to infect a new host, a process of Darwinian competition will select those natural variants of the microorganism that are best able to survive and replicate in the new environment. In this way microorganisms adapt to humans, and may become so well adapted as to become exclusive pathogens of humans (Fig. 2.1 ) (Wolfe et al. 2007 ). Fig. 2.1 Five stages of how exclusive animal pathogens can adapt to and infect humans (Wolfe et al. 2007 ). Stage 1: The pathogen is still exclusively infecting animals. Stage 2: The pathogen has been transmitted from animals to humans under natural conditions but not yet from human to human. Stage 3: There is limited transmission from animals to humans and between humans. These are often severe and lethal diseases due to for instance filoviruses (e.g. Ebola). Stage 4: Animal disease that have sustained transmission between humans (e.g. influenza). Stage 5: A microbe that exclusively infects humans (e.g. measles, syphilis) Box 1. Categories of Infectious Diseases (Fauci, NEJM, 2012) Established infectious diseases : endemic diseases that have been around for a sufficient amount of time to allow for a relatively stable and predictable level of morbidity and mortality. Examples are common diarrheal pathogens, drug-susceptible malaria, tuberculosis, helminthic and other parasitic diseases; Newly emerging infectious diseases : diseases that recently have been detected in the human host for the first time. Nipah virus, severe acute respiratory syndrome virus (SARS), human metapneumovirus (hMPV), and new influenza subtypes (swine H1N1, avian H5N1, or avian H7N9) are examples in this category. Often RNA viruses causing respiratory diseases are in this category. Re - emerging infectious diseases : Diseases in this category can be subclassified as follows: Diseases that appear either in new regions, as we have seen for West Nile virus (WNV) in the Americas. Already known diseases that have become drug-resistant. Recent examples are drug resistant bacterial infections (penicillin resistant pneumococcal pneumonia, carbapenem resistant hospital acquired infections), drug resistant malaria, and oseltamivir resistant influenza. Already known diseases that reappear after apparent control or elimination, or under unusual circumstances. Examples in category are the deliberate release of anthrax in the USA in 2001, or the reappearance of dengue on Florida, USA. Box 1. Categories of Infectious Diseases (Fauci, NEJM, 2012) Established infectious diseases : endemic diseases that have been around for a sufficient amount of time to allow for a relatively stable and predictable level of morbidity and mortality. Examples are common diarrheal pathogens, drug-susceptible malaria, tuberculosis, helminthic and other parasitic diseases; Newly emerging infectious diseases : diseases that recently have been detected in the human host for the first time. Nipah virus, severe acute respiratory syndrome virus (SARS), human metapneumovirus (hMPV), and new influenza subtypes (swine H1N1, avian H5N1, or avian H7N9) are examples in this category. Often RNA viruses causing respiratory diseases are in this category. Re - emerging infectious diseases : Diseases in this category can be subclassified as follows: Diseases that appear either in new regions, as we have seen for West Nile virus (WNV) in the Americas. Already known diseases that have become drug-resistant. Recent examples are drug resistant bacterial infections (penicillin resistant pneumococcal pneumonia, carbapenem resistant hospital acquired infections), drug resistant malaria, and oseltamivir resistant influenza. Already known diseases that reappear after apparent control or elimination, or under unusual circumstances. Examples in category are the deliberate release of anthrax in the USA in 2001, or the reappearance of dengue on Florida, USA. Shared Ecologies The conditions or events (the 'drivers') that result in the successful cross–over of an animal microbe into humans are not well characterised, but emergence is often precipitated by changes to ecological or biological systems (Wilcox and Colwell 2005 ). Such changes include altered patterns of contact between wild and domestic animals (e.g. Nipah virus), of direct human and wild animal contact (e.g. HIV, Ebola), and changes in species abundance or diversity (e.g. Hantavirus; Lyme disease). Species diversity, including the diversity of insect vectors and pathogenic microorganisms, increases towards the equator (Guernier et al. 2004 ), and Jones et al. found a correlation between the emergence of zoonotic pathogens and the diversity of mammalian wildlife species (Jones et al. 2008 ). Whilst high animal host and pathogen species diversity may be associated with a high burden of infectious diseases and an increased risk of disease emergence, biodiversity loss may, perhaps counter-intuitively, be associated with increased disease transmission. Biodiversity loss directly disrupts the functioning and stability of ecosystems, producing effects that can extend well beyond the particular lost species (Hooper et al. 2012 ). Changes in species abundance and diversity may favour pathogen amplification and 'spill-over' through a variety of mechanisms, including reduced predation and competition resulting in increased abundance of competent hosts, and the loss of 'buffering species' leading to increased contact between amplifying host species and compatible pathogens (Keesing et al. 2010 ). Tropical regions with a rich pool of existing and potential pathogens that are increasingly connected, but also experiencing high rates of ecosystem disruption and biodiversity loss, may therefore be at a particularly high risk of disease emergence. Human pathogens occasionally re-emerge as a result of dynamics that are beyond the control of humans. For example the Hantavirus outbreaks in the Southwestern U.S. in 1991–1992 and 1997–1998 have been attributed to changes in the abundance of infected rodents following periods of heavy snow and rainfall and vegetation growth leading to abundant production of rodent food. However, many ecological disturbances resulting in an EID seem to originate in the direct actions of humans. A wide range of anthropogenic factors have been linked to infectious disease emergence, including changes in land-use, travel, trade, and demographics. Notably, most of these associations are speculative and supported by little hard data because ecological and biological systems are highly complex and multi-layered (Woolhouse 2011 ). Demonstrating or predicting the impact of particular conditions or events on the functioning of a system is difficult, with further inference of the impact of any changes on the risk of pathogen emergence posing a formidable challenge. Socioeconomic Development and Altered Ecosystems Socioeconomic development is associated with large increases in demand for natural resources. The demand for water, wood, pulp, agricultural land, living space, roads, minerals and power has had an enormous impact on the landscapes of Asia. Deforestation occurred throughout the 1990s and the area of primary forest in Asia has continued to decline (FAO 2012 ). Deforestation, forest fragmentation, and afforestation are all alterations in habitat, which change species composition and the interaction between wild animals, domestic animals, insect vectors and humans, providing new opportunities for microbial transmission and potential emergence. There are well-documented examples of deforestation and forest encroachment resulting in increases in infectious diseases, such as yellow fever, Mayaro, and Chagas disease in the Americas (Saker et al. 2004 ). The clearing of forest and planting of large cacao plantations was linked to the emergence of Oropouche virus in Brazil. In Asia there are already very high pressures on productive land, and the peak in land-use change in Asia has probably passed. Many areas are now in an era of increasing intensification of land productivity. This intensification is driven largely by demographic pressures, which are predicted to result in a 70 % increase in food production by 2050, with decreased consumption of grains and increased demand for meats, fruits and vegetables (FAO 2011a , b ). The increased demand for food, and meat in particular, when combined with demands for natural resources from industry and domestic consumers, and river damming for hydroelectric power, is resulting in a large increase in stress on water resources (FAO 2011b ). The consequences of intensified agricultural production include the depletion and degradation of river and groundwater, reduced soil quality, and biodiversity loss. A direct and predictable effect of reduced access to clean water for low-income families is an increase in the risk of water-washed diseases (diseases that increase when the availability of water for personal hygiene is limited e.g. diarrhoeal and respiratory infections, trachoma), and water-borne diseases such as typhoid and hepatitis E. However, unquantified risks arising from the intensification of agriculture are pollution of freshwater with pesticides and fertilizers, loss of biodiversity, and land abandonment by small-scale farmers. The potential consequences of these changes on the risk of emergence of zoonotic infections have not been assessed. Wildlife Trade Wild animals are an important source of food (bush meat) in some developing countries and bush meat has been implicated in the emergence of HIV, and the spill over of monkeypox, Nipah and Ebola virus (Brashares et al. 2011 ). Whilst the reservoir of the SARS coronavirus is thought to be bats, wild civet cats traded for food are thought to have acted as an intermediate host, transmitting the SARS virus to humans through live animal markets (Li et al. 2005 ). Wild animal products are popular in Asia as traditional medicines, tonics, delicacies, or as symbols of wealth. Although all ten countries in the Association of Southeast Asian Nations (ASEAN) are signatories to the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), Asia continues to host the largest illegal wildlife trade in the world (Rosen and Smith 2010 ). The smuggling of H5N1-infected birds of prey into Europe, the frequent smuggling of bush meat from Africa into the U.S., and the importation into the U.S. of pet rodents infected with monkeypox show that both the legal and illegal trade in wild animals and wild animal products is a potential conduit for the international spread of zoonotic pathogens (Bair-Brake et al. 2013 ; Van Borm et al. 2005 ) Urbanization, Consumer Behavior, and Market Chains Between 2011 and 2050 the world population is expected to increase by 2.3 billion (a 32 % increase), and the increase will be concentrated in urban areas of developing countries (UN-DESA 2012 ). Whilst mega-cities (cities with a population of at least ten million) receive a lot of attention, most urban dwellers live in small cities, with half of the global urban population in 2011 living in cities of less than 500,000 people (UN-DESA 2012 ). This can be perceived positively as cities generally offer better economic opportunities, better educational opportunities, better living conditions, better nutrition, better sanitation, and therefore better health than under-developed rural areas. At the same time it is likely to mean that in many low to middle income countries, health and veterinary infrastructure in rural areas will not improve, or may even deteriorate, adversely affecting the likelihood of the early detection of EID. The demand for food in urban centres will increase and result in livestock and their products being transported over large distances from a wider catchment area, and thereby increase risk of spread and amplification of EID. Urbanisation is one facet of changing human sociocultural systems, which also includes changing consumer demands and dietary habits (Janes et al. 2012 ). The consequence is a spatial concentration of people and animals; not necessarily co-located, but connected through increasingly complex networks of rural and peri-urban farms and markets, distributors, agricultural workers, and consumers. Livestock Production Systems Intensification of Production Due to the increase in global human population and economic development, demand for livestock products has risen dramatically over the last 50 years, with the per capita consumption of meat in developing countries more than tripling since the early 1960s and egg consumption increasing fivefold (FAO 2011a , b ). The increased demand for meat has been met by more intensive and geographically concentrated production of livestock, especially pigs and poultry (Steinfeld et al. 2006 ). Much of this has been through expansion of both the number of small-scale production units and large commercial farms. High-density monoculture of domestic animals is a form of low biodiversity that poses a particular threat for the spread of infectious diseases from farmed animals to humans. Where domesticated animals are a conduit of spread from wild animals to humans, high density livestock production may promote spread of zoonotic diseases. Genetic diversity within an individual host species is important since genetic diversity limits the potential for devastating epidemics (King and Lively 2012 ). The Nipah virus outbreak in Malaysia and Singapore in 1998–1999 is a good example. Once Nipah virus crossed from wild bats to domestic swine, an explosive outbreak in high-density swine farms resulted in widespread exposure of humans and over 250 human cases of encephalitis (Pulliam et al. 2012 ). Other examples where intensified livestock production practices may have led to emergence of a zoonosis include: Streptococcus suis causes severe sepsis and meningitis in humans and is associated with areas of intensive pig production (see Fig. 2.2 ). Risk factors for human infection include swine slaughtering and the eating of undercooked pig products (Wertheim et al. 2009 ). Outbreaks in swine herds of porcine reproductive and respiratory syndrome virus also potentially increases the rate of invasive S. suis infection in swine, which in turn leads to an increased risk of S. suis infection in humans (Hoa et al. 2013 ). Fig. 2.2 Number of swine per km 2 and human Streptococcus suis infections in Vietnam from 2005–2011. Pig density data are based on Vietnam Government Statistics Office data 2006 Highly pathogenic avian influenza A subtype H5N1 crossed-over from wild aquatic birds (the natural reservoir of influenza A viruses) to humans via massive amplification in domestic poultry. The human Q-fever epidemic in the Netherlands during 2007–2010 caused by the bacterium Coxiella burnetii is thought to have arisen when economic drivers led to an increased density of dairy goat farming, which resulted in amplification of Coxiella burnetii prevalence and consequent increased spill- over to humans (Roest et al. 2011 ; Georgiev et al. 2013 ). The classical foodborne diseases such as E. coli , campylobacteriosis and salmonellosis associated with livestock products have been a significant problem in high-income countries for some time (CDC 2011 ; Painter et al. 2013 ). One of the key factors, in particular in the case of Campylobacter , has been the integration of highly intensive poultry production with poultry processing systems facilitating cross-contamination of meat (Moore et al. 2005 ; Nyachuba 2010 ). It is notable that some foodborne pathogens cause subclinical infections in their animal hosts (e.g., Campylobacter in poultry); therefore there is no direct incentive for farmers to control them. In addition, subsequent attribution of the source of foodborne disease in an affected human is notoriously difficult. It is likely that the global incidence of foodborne illness will rise as a result of increased industrial production of poultry in the emerging economies. The situation will be exacerbated by the emergence of antimicrobial resistant pathogens of foodborne pathogens (Sahin et al. 2012 ; Luangtongkum et al. 2009 ; Altekruse and Tollefson 2003 ). Despite the examples above, the intensification of livestock farming often entails more effective separation of domestic and wild animals, improved standards of animal health and welfare, reduced movement and species mixing: all of which reduce the risk of EIDs. Reducing contact between domestic and wild animals, whether the wild animals are in their natural habitat or captive, is a key strategy of the Food and Agriculture Organization (FAO) to reduce risk to human health, and is part of the wider FAO strategy of enhancing 'biosecurity'. Improving biosecurity in farms is a major challenge since a large proportion of farming in Asia is based on small-scale backyard production, and there is often a mix of commercial and backyard farming in any one location. Achieving improvements in biosecurity without adversely affecting the livelihoods of small-scale farmers requires an approach to risk management that is adapted to their socio-economic context. The longer-term vision is to restructure the livestock production sector towards a more commercialised and controlled system, where controls benefit animal health, human health, and commercial profitability. But it has also been recognised that small-scale producers will continue to have a key role in providing food security in developing economies, and in fact their importance may increase due to their more efficient use of natural resources compared with large-scale industrial production (Sjauw-Koen-Fa 2012 ; Quan 2011 ). Intensity and Complexity of Animal Markets Food production has become a complex national, regional and global network of food value chains (Ercsey-Ravasz et al. 2012 ; Dickson-Hoyle and Reenberg 2009 ). Many countries have begun to more tightly regulate live animal trade in the wake of Bovine Spongiform Encephalopathy (BSE), SARS and avian influenza A/H5N1, but complex and poorly regulated food manufacturing and distribution chains still offer ample opportunities for disease outbreaks. In endemic regions, live bird markets are frequently contaminated with highly pathogenic avian influenza A subtype H5N1 (Davis et al. 2010 ; Samaan et al. 2011 ; Wan et al. 2011 ). Contaminated markets can become reservoirs and the source of infection for poultry and humans. Measures to reduce the risk in live animal markets include the separation of species, not allowing animals to remain in the market more than 24 h, not allowing poultry to exit the market alive, improved cleaning and disinfection, and weekly rest-days, when all animals are removed and the market is thoroughly cleaned. The re-emergence of brucellosis, one of the world's most common zoonoses, is thought to be the result of higher risk of transmission through increased within- and between country trade of susceptible livestock and their products (Seleem et al. 2010 ). The development of denser regional road transport networks may be especially important since, compared to air travel, roads offer a more egalitarian form of connectivity that includes animals and goods as well as humans. This provides opportunities for pathogens to disperse beyond their traditional niche by increasing opportunities for informal trade in live animals and their products (Eisenberg et al. 2006 ). Veterinary medical authorities often struggle to enforce compliance with regulations, and it is common practice for farmers and traders to adapt their production and trading behaviors to avoid adverse economic consequences of regulations aimed at controlling zoonotic diseases. As a result, informal trade networks may intensify and re-structure in unpredictable ways during disease outbreaks. Increasing intensification of animal husbandry in Asia may be a trade-off between a lower risk of emergence events, as animals will be 'healthier' and better isolated, but should an EID event occur, an increased risk of massive amplification in large, naïve monocultures. Perhaps the greatest risk arises when a limited number of intensive livestock production units are surrounded by substantial backyard farming with little or no biosecurity, thereby linking extensive and weakly regulated value chains with global food systems. Antibiotics and Other Practices Livestock production practices can have major unintended and unanticipated impacts on the risk of zoonotic infections. Perhaps the most notable example is BSE. The feeding of ruminant-derived meat and bone meal to cattle, and possibly changes in practices for rendering animal tissue, led to an epidemic of BSE in cattle, that was followed by an epidemic of a fatal neurological disease (variant Creutzfeldt-Jacob Disease) in humans (Taylor and Woodgate 2003 ). In contemporary livestock production, the use of antibiotics is a significant concern. Almost 40 % (23/59) of EIDs identified in Asia since 1940 represent the emergence of a new pattern of antimicrobial resistance (Jones et al. 2008 ). Bacteria in some areas show alarmingly high rates of resistance to anti-bacterial agents, and the recent emergence of the New-Delhi Metallo-beta-lactamase-1 (NDM-1) resistance gene, conferring resistance to a last resort antibiotic, and its rapid dissemination to other regions highlights the serious threat to human health of antimicrobial resistance (Khan and Nordmann 2012 ). Antibiotics are used extensively in the livestock and aquaculture sector to treat or prevent infections, or as growth promoters. Non-therapeutic use of antibiotics as growth promoters involves the prolonged administration of sub-therapeutic doses. This practise has a demonstrable effect on the emergence and prevalence of resistant microorganisms in food animals and their environment, as well as resulting in the excretion of antibiotics into the environment, where environmental bacteria may be subject to antibiotic selection pressures (Marshall and Levy 2011 ). Synthetic antibiotics such as quinolones are quite stable in the environment over long periods of time. Heavy metal contamination of animal food may also play a role in selection of antimicrobial resistance. Whilst there remains some debate about the overall impact of these findings on human health, it is clear that the continued use of non-therapeutic antibiotics in an agriculture industry that is rapidly increasing in scale and intensity, has potential for becoming a very real threat through the inability to prevent/cure disease in production animals and the consequences for human food security as well as the transmission, for example, of resistant food-borne bacterial pathogens to humans. Some recent examples concern transmission of methicillin resistant Staphylococcus aureus (MRSA) from pigs to humans and ESBL (extended spectrum beta-lactamase, an important resistance mechanism against most beta-lactam antibiotics) positive E. coli on chicken meat for human consumption (Verkade et al. 2012 ; Kluytmans et al. 2013 ). Swine-associated MRSA is now contributing significantly to invasive disease in patients in the Netherlands (Verkade et al. 2012 ). Antibiotic production and consumption continues to increase, often in an uncontrolled way, accelerating the evolution of antibiotic resistance, which then spreads rapidly across the globe. Since the 1990s few new antibiotics have been developed and we are on the cusp of returning to an era of untreatable infections. The consequences of antibiotic use in animals therefore require better surveillance, research and regulation. Intensification of Production Due to the increase in global human population and economic development, demand for livestock products has risen dramatically over the last 50 years, with the per capita consumption of meat in developing countries more than tripling since the early 1960s and egg consumption increasing fivefold (FAO 2011a , b ). The increased demand for meat has been met by more intensive and geographically concentrated production of livestock, especially pigs and poultry (Steinfeld et al. 2006 ). Much of this has been through expansion of both the number of small-scale production units and large commercial farms. High-density monoculture of domestic animals is a form of low biodiversity that poses a particular threat for the spread of infectious diseases from farmed animals to humans. Where domesticated animals are a conduit of spread from wild animals to humans, high density livestock production may promote spread of zoonotic diseases. Genetic diversity within an individual host species is important since genetic diversity limits the potential for devastating epidemics (King and Lively 2012 ). The Nipah virus outbreak in Malaysia and Singapore in 1998–1999 is a good example. Once Nipah virus crossed from wild bats to domestic swine, an explosive outbreak in high-density swine farms resulted in widespread exposure of humans and over 250 human cases of encephalitis (Pulliam et al. 2012 ). Other examples where intensified livestock production practices may have led to emergence of a zoonosis include: Streptococcus suis causes severe sepsis and meningitis in humans and is associated with areas of intensive pig production (see Fig. 2.2 ). Risk factors for human infection include swine slaughtering and the eating of undercooked pig products (Wertheim et al. 2009 ). Outbreaks in swine herds of porcine reproductive and respiratory syndrome virus also potentially increases the rate of invasive S. suis infection in swine, which in turn leads to an increased risk of S. suis infection in humans (Hoa et al. 2013 ). Fig. 2.2 Number of swine per km 2 and human Streptococcus suis infections in Vietnam from 2005–2011. Pig density data are based on Vietnam Government Statistics Office data 2006 Highly pathogenic avian influenza A subtype H5N1 crossed-over from wild aquatic birds (the natural reservoir of influenza A viruses) to humans via massive amplification in domestic poultry. The human Q-fever epidemic in the Netherlands during 2007–2010 caused by the bacterium Coxiella burnetii is thought to have arisen when economic drivers led to an increased density of dairy goat farming, which resulted in amplification of Coxiella burnetii prevalence and consequent increased spill- over to humans (Roest et al. 2011 ; Georgiev et al. 2013 ). The classical foodborne diseases such as E. coli , campylobacteriosis and salmonellosis associated with livestock products have been a significant problem in high-income countries for some time (CDC 2011 ; Painter et al. 2013 ). One of the key factors, in particular in the case of Campylobacter , has been the integration of highly intensive poultry production with poultry processing systems facilitating cross-contamination of meat (Moore et al. 2005 ; Nyachuba 2010 ). It is notable that some foodborne pathogens cause subclinical infections in their animal hosts (e.g., Campylobacter in poultry); therefore there is no direct incentive for farmers to control them. In addition, subsequent attribution of the source of foodborne disease in an affected human is notoriously difficult. It is likely that the global incidence of foodborne illness will rise as a result of increased industrial production of poultry in the emerging economies. The situation will be exacerbated by the emergence of antimicrobial resistant pathogens of foodborne pathogens (Sahin et al. 2012 ; Luangtongkum et al. 2009 ; Altekruse and Tollefson 2003 ). Despite the examples above, the intensification of livestock farming often entails more effective separation of domestic and wild animals, improved standards of animal health and welfare, reduced movement and species mixing: all of which reduce the risk of EIDs. Reducing contact between domestic and wild animals, whether the wild animals are in their natural habitat or captive, is a key strategy of the Food and Agriculture Organization (FAO) to reduce risk to human health, and is part of the wider FAO strategy of enhancing 'biosecurity'. Improving biosecurity in farms is a major challenge since a large proportion of farming in Asia is based on small-scale backyard production, and there is often a mix of commercial and backyard farming in any one location. Achieving improvements in biosecurity without adversely affecting the livelihoods of small-scale farmers requires an approach to risk management that is adapted to their socio-economic context. The longer-term vision is to restructure the livestock production sector towards a more commercialised and controlled system, where controls benefit animal health, human health, and commercial profitability. But it has also been recognised that small-scale producers will continue to have a key role in providing food security in developing economies, and in fact their importance may increase due to their more efficient use of natural resources compared with large-scale industrial production (Sjauw-Koen-Fa 2012 ; Quan 2011 ). Intensity and Complexity of Animal Markets Food production has become a complex national, regional and global network of food value chains (Ercsey-Ravasz et al. 2012 ; Dickson-Hoyle and Reenberg 2009 ). Many countries have begun to more tightly regulate live animal trade in the wake of Bovine Spongiform Encephalopathy (BSE), SARS and avian influenza A/H5N1, but complex and poorly regulated food manufacturing and distribution chains still offer ample opportunities for disease outbreaks. In endemic regions, live bird markets are frequently contaminated with highly pathogenic avian influenza A subtype H5N1 (Davis et al. 2010 ; Samaan et al. 2011 ; Wan et al. 2011 ). Contaminated markets can become reservoirs and the source of infection for poultry and humans. Measures to reduce the risk in live animal markets include the separation of species, not allowing animals to remain in the market more than 24 h, not allowing poultry to exit the market alive, improved cleaning and disinfection, and weekly rest-days, when all animals are removed and the market is thoroughly cleaned. The re-emergence of brucellosis, one of the world's most common zoonoses, is thought to be the result of higher risk of transmission through increased within- and between country trade of susceptible livestock and their products (Seleem et al. 2010 ). The development of denser regional road transport networks may be especially important since, compared to air travel, roads offer a more egalitarian form of connectivity that includes animals and goods as well as humans. This provides opportunities for pathogens to disperse beyond their traditional niche by increasing opportunities for informal trade in live animals and their products (Eisenberg et al. 2006 ). Veterinary medical authorities often struggle to enforce compliance with regulations, and it is common practice for farmers and traders to adapt their production and trading behaviors to avoid adverse economic consequences of regulations aimed at controlling zoonotic diseases. As a result, informal trade networks may intensify and re-structure in unpredictable ways during disease outbreaks. Increasing intensification of animal husbandry in Asia may be a trade-off between a lower risk of emergence events, as animals will be 'healthier' and better isolated, but should an EID event occur, an increased risk of massive amplification in large, naïve monocultures. Perhaps the greatest risk arises when a limited number of intensive livestock production units are surrounded by substantial backyard farming with little or no biosecurity, thereby linking extensive and weakly regulated value chains with global food systems. Antibiotics and Other Practices Livestock production practices can have major unintended and unanticipated impacts on the risk of zoonotic infections. Perhaps the most notable example is BSE. The feeding of ruminant-derived meat and bone meal to cattle, and possibly changes in practices for rendering animal tissue, led to an epidemic of BSE in cattle, that was followed by an epidemic of a fatal neurological disease (variant Creutzfeldt-Jacob Disease) in humans (Taylor and Woodgate 2003 ). In contemporary livestock production, the use of antibiotics is a significant concern. Almost 40 % (23/59) of EIDs identified in Asia since 1940 represent the emergence of a new pattern of antimicrobial resistance (Jones et al. 2008 ). Bacteria in some areas show alarmingly high rates of resistance to anti-bacterial agents, and the recent emergence of the New-Delhi Metallo-beta-lactamase-1 (NDM-1) resistance gene, conferring resistance to a last resort antibiotic, and its rapid dissemination to other regions highlights the serious threat to human health of antimicrobial resistance (Khan and Nordmann 2012 ). Antibiotics are used extensively in the livestock and aquaculture sector to treat or prevent infections, or as growth promoters. Non-therapeutic use of antibiotics as growth promoters involves the prolonged administration of sub-therapeutic doses. This practise has a demonstrable effect on the emergence and prevalence of resistant microorganisms in food animals and their environment, as well as resulting in the excretion of antibiotics into the environment, where environmental bacteria may be subject to antibiotic selection pressures (Marshall and Levy 2011 ). Synthetic antibiotics such as quinolones are quite stable in the environment over long periods of time. Heavy metal contamination of animal food may also play a role in selection of antimicrobial resistance. Whilst there remains some debate about the overall impact of these findings on human health, it is clear that the continued use of non-therapeutic antibiotics in an agriculture industry that is rapidly increasing in scale and intensity, has potential for becoming a very real threat through the inability to prevent/cure disease in production animals and the consequences for human food security as well as the transmission, for example, of resistant food-borne bacterial pathogens to humans. Some recent examples concern transmission of methicillin resistant Staphylococcus aureus (MRSA) from pigs to humans and ESBL (extended spectrum beta-lactamase, an important resistance mechanism against most beta-lactam antibiotics) positive E. coli on chicken meat for human consumption (Verkade et al. 2012 ; Kluytmans et al. 2013 ). Swine-associated MRSA is now contributing significantly to invasive disease in patients in the Netherlands (Verkade et al. 2012 ). Antibiotic production and consumption continues to increase, often in an uncontrolled way, accelerating the evolution of antibiotic resistance, which then spreads rapidly across the globe. Since the 1990s few new antibiotics have been developed and we are on the cusp of returning to an era of untreatable infections. The consequences of antibiotic use in animals therefore require better surveillance, research and regulation. Conclusions Infectious diseases continue to be an important cause of human and animal morbidity and mortality worldwide. Whilst important health advances (e.g. hygiene and vaccination) have been made, infectious diseases are dynamic and resilient, and continue to challenge local, national and global public health systems (Fauci and Morens 2012 ). The recognition of the linkage between anthropogenic changes, animals, and disease emergence has resulted in repeated calls for a more holistic, interdisciplinary, and integrated approach to the study of infectious diseases. The One Health approach is one initiative aimed at realizing this aspiration. The task ahead is to understand how social, economic, and environmental changes are altering the landscape of infectious disease risks for both animals and humans, and how future emerging risks may be mitigated. A more analytical approach to these emergence events requires characterisation of the attributes of natural and artificial ecological systems before and after disturbance, and the functional relationship between changes in system attributes and pathogen emergence (Woolhouse 2011 ).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861118/

Lay media reporting of monkeypox in Nigeria

Introduction In October 2017, the first confirmed case of a monkeypox outbreak was reported in Bayelsa State, Nigeria, although suspected cases started to be reported in September 2017. 1 It continues to this day, with exported cases reaching the UK, Israel and Singapore. 2 3 Prior to this outbreak, the disease was last reported in Nigeria in 1978. 4 Monkeypox is an orthopox virus, closely related to smallpox, that produces vesicopapular lesions on the skin. Symptoms are usually self-limiting and most people recover within weeks. Severe illness and death usually only occurs among immunosuppressed individuals. 5 When the first cases were announced in Nigeria, media reports exaggerated the symptoms and impact of the outbreak. The outbreak generated front page headlines, with one title describing it as a 'new airborne Ebola'. 6 A European headline (from the Voice of Europe) was ' Horrible Nigerian disease called monkeypox spreads in the United Kingdom for the first time' . The media used unverified pictures of people with skin rashes, to amplify their messages. 7 There may be substantial discrepancies between what the National Public Health Institute responsible for outbreak response coordination aims to communicate during epidemics, and what the media actually disseminates. 8 9 This can exacerbate public uncertainty and distrust. Globally, the response to outbreaks of severe acute respiratory syndrome and avian influenza, releases of anthrax and sarin, and natural disasters such as the South-East Asian tsunami, demonstrated the importance of communication during public health emergencies. 10 Subsequently, particular focus has been placed on strengthening the capacity for risk communication within relevant government institutions. However, such capacity is still evolving in many low/middle-income countries, including Nigeria where event-based surveillance is enhancing the existing indicator-based surveillance system. It uses information from the internet and other channels including phone calls, text messages, Whatsapp messages and others. 11 The Nigeria Centre for Disease Control (NCDC) — Nigeria's National Public Health Institute — uses a proprietary internet crawling system called 'Tatafo', which generates unstructured event-based reports from 350 media sites (webs, newspapers, television, blogs/online media and social media). The system uses keywords including the 41 notifiable diseases as outlined in the Integrated Disease Surveillance and Response guidelines. 12 Searches on this system are automated, but can also be moderated for key conditions. During the peak of the monkeypox outbreak (September to December 2017), 3475 entries generated by the event-based surveillance system mentioned monkeypox. The majority of Tatafo entries came from newspapers, television and blogs. We extracted, reviewed and summarised these lay media reports. How monkeypox was described by the media The majority of articles described governments as their source of information, but only 11% actually cited the source. Outbreak information included number of cases and deaths, newly confirmed cases as well as response activities. The majority of the information on the outbreak from the government was from the NCDC and the Federal Ministry of Health. Others were from State Ministries of Health, State Ministries of Information and government hospitals. One report from Nigeria's Punch newspaper stated, 'It was authoritatively learnt that a medical doctor and 10 persons, who came down with the monkeypox had been quarantined in an isolation centre at the Niger Delta University Teaching Hospital, Okolobiri in the Yenagoa Local Government Area of the state'. 13 However, despite the emphasis on authority by the reporter, no source was cited. The name 'monkeypox' may have lent alarm to descriptions of the outbreak (monkey + pox). Although the more severe Lassa fever virus was circulating in Nigeria at that time, more news reports focused on monkeypox. 14 Several media reports described monkeypox in sensational terms such as 'fatal', 'dreaded', 'small pox like', 'deadly', 'spiritual', 'Ebola-like' or 'rare'. A headline by Nigeria's Pulse news read ' Doctor, 10 others infected as deadly virus breaks out in Bayelsa' . 15 In the Guardian newspaper, a report read ' A new airborne Ebola-like viral disease, referred to as 'Monkey Pox has hit the Niger Delta University Teaching Hospital (NDUTH), Okolobiri in Yenagoa local council of Bayelsa State'. 6 Nigeria's Punch newspaper reported that ' … some church leaders in some states … maintained that the outbreak of the disease was spiritual .' 16 The media did not report that during this period, only two deaths had been reported among 197 suspected cases between September and December 2017, nor that most infected people recovered. 17 Not mentioning these facts may have caused more fear and panic among the public. In addition, many articles used the pictures of a child with an extreme presentation of the disease on the front page. Both traditional news media and new media sources published inaccurate, sensationalised or misleading stories. 18 Such reports can hinder actions to safeguard health. Public health institutes can combat misinformation by frequently updating key facts, available on their official media platforms for clarification. During the monkeypox outbreak, the Nigerian Minister of Health held two press briefings, the Director General of the NCDC made announcements on television and the NCDC provided weekly situation reports. In some cases, there were delays in the publication of these situation reports. The direct communication by the Ministry of Health and NCDC provided an opportunity to get messages to the public without distortion or omission. 19 How the media validated or refuted rumours During the outbreak, there was a rumour that the military was injecting school children with the monkeypox virus. Although none of the articles could verify the source of the rumour, some publications linked it to a military operation to address separatist agitations by a group in the South-East of Nigeria during the period. 20 But in fact, this report refered to a medical 'outreach' focused on other health measures unrelated to the outbreak. This rumour led to the closure of schools, low immunisation rates for other vaccine-preventable diseases during the period, and general widespread panic. This affected trust in the government generally, as an arm of the government was being accused of 'bioterrorism'. 21–24 However, the reaction to the rumour was swift, with statements from the Nigerian Presidency, the Federal Ministries of Information and Health, NCDC, State Governments as well as the spokesperson of the Nigerian Army. Despite this, there was less coverage of the government's reaction to the rumour, compared with the media coverage of the rumour. Communication about personal preventive measures is particularly useful during outbreaks as it empowers the public to take some responsibility for their own health. 20–23 But there were conflicting messages from varying media channels, which may have left the public with a sense of scepticism relating to health prevention messages. Given the size and federal structure of Nigeria, it can be difficult to manage information from various levels of government. For example, several articles showed a lapse in information on whether if there was an outbreak, between one state and the NCDC. And there were discrepancies even among the government sources cited in media reports. Coordination of information preparation and distribution was thus inadequate. It is important that a strong coordination platform for communication is established during outbreaks. Effective media communication requires trust and understanding between public health officials and the media. National Public Health Institutes such as NCDC should maintain an open line of communication with the media at all times, including offering information in easy to understand terms. Conclusion The sensational reporting of the monkeypox outbreak in Nigeria and the use of various forms of media to spread rumours highlights the need to strengthen risk communications capabilities, especially among journalists and health reporters—including training on effective factual reporting of science and health, with limited sensationalism. During disease outbreaks, there is often uncertainty about the facts. 25 It is important that verifiable sources are guided by experts. The media plays a very important role in the dissemination of news pertaining to outbreaks and this can have consequences on how the public reacts. In low/middle-income countries, health reporters and the media should be targeted with increased advocacy and training to improve the messages they distribute.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112011/

Pathogen Surveillance Through Monitoring of Sewer Systems

I INTRODUCTION It is now possible to monitor sewage systems for pathogen occurrence in a community. An epidemiological approach to monitoring sewer systems is especially relevant for an early warning of pathogens used as biological weapons. In many situations, bioterrorist contamination events will result in the pathogen shedding to wastewater before a community level epidemic begins. Detecting the organism early allows the governments to respond on time and eliminate a potential catastrophe. A Monitoring for human pathogens in sewage Monitoring of human pathogens in sewage is possible because they may be excreted in a range of bodily fluids, skin, and hair during active infection ( Feachem et al., 1983 ). All of these materials will find their way into sewage systems during the process of waste elimination (toilet flushing) and cleaning (e.g., bathing, hand washing). In addition to release during active infection, pathogens can be washed into sewage systems from cleaning of indoor (floor washing, kitchen sink use) and outdoor (auto washing, driveway cleaning, storm water collection) facilities. Thus, sewer systems collect pathogens from over a wide area to a common carrier, where they are transported to a central facility for processing. Wastewater presents a time dynamic collection point where many physical, chemical, and biological substances of our society are brought to a central location. Monitoring of centralized wastewater allows detection of intentional, natural, or accidental contamination events. Because of recent bioterrorism concerns in the U.S., routine monitoring is potentially useful since it can result in better preparedness of utilities and the public health response system ( Meinhardt, 2005 ). The qualitative microbial risk assessment (QMRA) framework can be used as a tool to develop and interpret this type of wastewater monitoring system. Because the threat level drives the risk assessment analysis, a monitoring system should be coordinated with findings from modeling studies on the survival and dispersion of contaminants ( Kim et al., 2007 , Romero et al., 2008 , Sinclair et al., 2008 ), the contaminant point of introduction ( Danneels and Finley, 2004 ), the health risk ( Haas et al., 1999 ), and the locations of early warning systems/sensors in wastewater and water treatment systems ( Murray et al., 2004 ). A recent U.S. National Research Council study called for more resilient design/operation of wastewater and drinking water systems ( USNRC, 2007 ) to improve response and recovery from adverse water quality events in collection systems, water distribution systems, and water/wastewater treatment systems. Monitoring programs for pathogens or surrogates could potentially aid in the accomplishment of these goals. The aim of this review of published literature and reports is to assess the feasibility of monitoring sewage systems as an early warning system for the release of pathogens from an intentional, natural, or accidental biological contamination event. We address issues from a QMRA perspective and explore methods to detect and monitor pathogens in wastewater. The review presents our conclusions on: (1) the potential biological agents that might be released into a sewage system, (2) the likely background level of those agents in sewage, (3) laboratory methods and detection, and (4) the probability of detecting select biological agents in sewage. A Monitoring for human pathogens in sewage Monitoring of human pathogens in sewage is possible because they may be excreted in a range of bodily fluids, skin, and hair during active infection ( Feachem et al., 1983 ). All of these materials will find their way into sewage systems during the process of waste elimination (toilet flushing) and cleaning (e.g., bathing, hand washing). In addition to release during active infection, pathogens can be washed into sewage systems from cleaning of indoor (floor washing, kitchen sink use) and outdoor (auto washing, driveway cleaning, storm water collection) facilities. Thus, sewer systems collect pathogens from over a wide area to a common carrier, where they are transported to a central facility for processing. Wastewater presents a time dynamic collection point where many physical, chemical, and biological substances of our society are brought to a central location. Monitoring of centralized wastewater allows detection of intentional, natural, or accidental contamination events. Because of recent bioterrorism concerns in the U.S., routine monitoring is potentially useful since it can result in better preparedness of utilities and the public health response system ( Meinhardt, 2005 ). The qualitative microbial risk assessment (QMRA) framework can be used as a tool to develop and interpret this type of wastewater monitoring system. Because the threat level drives the risk assessment analysis, a monitoring system should be coordinated with findings from modeling studies on the survival and dispersion of contaminants ( Kim et al., 2007 , Romero et al., 2008 , Sinclair et al., 2008 ), the contaminant point of introduction ( Danneels and Finley, 2004 ), the health risk ( Haas et al., 1999 ), and the locations of early warning systems/sensors in wastewater and water treatment systems ( Murray et al., 2004 ). A recent U.S. National Research Council study called for more resilient design/operation of wastewater and drinking water systems ( USNRC, 2007 ) to improve response and recovery from adverse water quality events in collection systems, water distribution systems, and water/wastewater treatment systems. Monitoring programs for pathogens or surrogates could potentially aid in the accomplishment of these goals. The aim of this review of published literature and reports is to assess the feasibility of monitoring sewage systems as an early warning system for the release of pathogens from an intentional, natural, or accidental biological contamination event. We address issues from a QMRA perspective and explore methods to detect and monitor pathogens in wastewater. The review presents our conclusions on: (1) the potential biological agents that might be released into a sewage system, (2) the likely background level of those agents in sewage, (3) laboratory methods and detection, and (4) the probability of detecting select biological agents in sewage. II POTENTIAL BIOLOGICAL AGENTS IN SEWAGE A wide variety of pathogenic organisms pass through municipal waste-water treatment systems. One study found that a single toilet flush containing poliovirus was detectable at a nearby treatment plant for more than 4 days ( Ranta et al., 2001 ). The toilet flush study was designed to replicate the number of virus released from an infected individual. Pathogenic microorganisms can also grow in the host but not produce sickness in the infected host. It is estimated that 50% or less of those individuals infected with enteric viruses or bacteria actually become ill ( Haas et al., 1999 ). In the case of some respiratory pathogens, 90% or more of the persons infected will become ill ( Belshe, 1991 ). During the growth of the organism in the host, the organism will be found in various organs and bodily fluids. Organisms transmitted by the fecal–oral route are usually excreted in large numbers in the feces, since the initial or primary site of replication is in the intestinal tract. However, this does not preclude their replication in other parts of the body. For example, enteroviruses (e.g., poliovirus) will replicate in nerve tissue causing paralytic disease, while Hepatitis A virus will replicate in the liver causing damage there ( Belshe, 1991 ). Respiratory infections are usually the result of replication of the organism in the nose, throat, or lungs. Infection of other organs of the body often leads to the presence of the organisms in the blood and then the urine after their elimination by the kidneys. This explains the occurrence of insect-borne encephalitis viruses and enteric viruses in the urine ( Pichichero et al., 1998 ). Any type of infection ( Fig. 9.1 ) within a community is likely to lead to pathogen excretion in bodily fluids/substances and therefore, transported into the community sewage system. Figure 9.1 Outcomes of exposure to a microbial infection. This review considers biological agents prioritized by the Centers for Disease Control (CDC) as potential biological weapons that could be used by terrorists ( Table 9.1 and 9.2 ). They are listed in three categories (i.e., A, B, and C) of decreasing concern. Category A agents require the most intensive public preparedness efforts due to the potential for mass causalities, public fear, and civil disruption. Category B agents are also moderately easy to spread, but have lower mortality rates. Category C agents do not present a high public health threat, but could emerge as future threats ( Rotz et al., 2002 ). Many other pathogenic agents are present in sewage, but not on the CDC select agent list. Table 9.2 lists some common blood and respiratory agents and emerging pathogens, all of which could potentially be engineered for mass dissemination and detected through monitoring of wastewater. The methods described in this paper apply to many other pathogens and are not limited to those agents listed in Tables 9.1 and 9.2 . Table 9.1 The center for disease control select agents ( Rotz et al., 2002 ) Category A Category B Category C Anthrax Bacillus anthracis Botulism Clostridium botulinum Plague Yersinia pestis Smallpox Variola major Tularemia Francisella tularensis Hemorrhagic fever virus a Arenaviridae Bunyaviridae Filoviridae Flaviviridae Lassa fever Hantavirus Dengue fever Ebola Marburg Brucellosis Brucella abortus Water and Food-borne agents Enteroviruses Poliovirus and Rotavirus Salmonellosis Salmonella Caliciviruses Hepatitis A virus Protozoan parasites Cryptosporidium parvum Giardia lamblia Toxoplasma Microsporidium Glanders Burkholderia mallei Psittacosis Chlamydia psittaci Q fever Coxiella burnetii Typhus fever Rickettsia prowazekii Viral Encephalitis West Nile La Crosse Venezuelan equine encephalitis Japanese encephalitis Nipah virus Tick-borne HFV Crimean-Congo HFV Tick-borne encephalitis viruses Yellow fever Multidrug resistant TB Influenza Other Rickettsias Rabies a Hemorrhagic fever virus (HFV). Table 9.2 Nonenteric pathogens found in sewage and other emerging agents of concern Nonenteric agents Emerging agents Severe Acute Respiratory Syndrome (SARS) Parvoviruses John Cunningham virus (JC Virus) Picobirnaviruses Human Immuno-deficiency Virus (HIV) Enteroviruses types 78–100 Hepatitis B Virus (HBV) Torque teno virus (TTV) A Human pathogens secreted in bodily fluids A literature search was conducted to determine the occurrence of the agents in bodily fluids, feces, skin, and sewage. As indicated in Table 9.3 and a previous publication ( Sinclair et al., 2008 ), many select agents may occur in bodily excretions or secretions even though this may not be their primary site of replication. It would appear that all of the viral agents are excreted in the urine and most of the bacterial agents in the feces or saliva. Since none of the organisms cause enteric infections they have seldom been sought in sewage, however, Bacillus anthracis and Yersina pestis (plague) have been detected in sewage. The source of B . anthracis spores in the sewage was believed to be from an African import tannery operation ( Perone and Gelosa, 1982 ) and presumably not from enteric infections, which would normally result in the presence of spores in the feces. Category B agents differ in that, and they include many enteric pathogens which are excreted in large numbers in the feces ( Table 9.3 ). All of the other agents in this category appear to be excreted in the feces; many of the viral agents are excreted in the urine. No studies were found that report examining sewage for their presence. The Category C viral agents appear to be excreted in the saliva and urine ( Tables 9.3 and 9.4 ). No references for the presence of these agents in sewage could be found. Some typical blood-borne agents such as Hepatitis B virus ( Alter et al., 1977 ) and Human Immuno-Deficiency virus ( Levy, 1989 ) have been detected in sewage by molecular methods ( Table 9.4 ). The coronavirus, which causes Severe Acute Respiratory Disease (SARS), is also excreted in the feces and other bodily fluids such as tears ( Loon et al., 2004 , Wang et al., 2005 ) ( Table 9.4 ). Table 9.3 Select category B and C agents found in human bodily fluids and sewage Agent Category Urine Feces Saliva Sewage Reference C. psittaci B ? Yes a ? ? Midura and Arnon (1976) ; Anderson (1996) ; Smith et al. (2005) C. burnetii B Yes Yes b ? ? Tylewska-Wierzbanowska and Kruszewska (1993) Viral encephalitis B Yes Yes c ? ? Mathur et al. (1995) Nipah virus C Yes ? Yes ? Chua et al., (2002) Rabies C Yes ? Yes ? Wacharapluesadee and Hemachudha (2002) Influenza C ? Yes c Yes ? Buchy et al. (2007) a Nasal. b Semen. c Animals. Table 9.4 Occurrence of other agents of interest in bodily fluids Agents Urine Feces Saliva Sewage Reference Severe Acute Respiratory Syndrome ? Yes Yes ? He et al. (2007) ; Petrich et al. (2006) John Cunningham virus (poliovirus) Yes ? ? Yes Coleman et al. (1980) Human Immuno-deficiency Virus Yes Yes Yes Yes Levy (1989) ; Yolken et al. (1991) Hepatitis B virus ? ? Yes Yes Alter et al. (1977) ; Bancroft et al. (1977) ; Arvanitidou et al. (1998) B Duration of release and concentration in bodily fluids and skin The duration and concentration of pathogens released by a host during the course of an infection varies, with greater numbers being released in more severe infections. After infection, the number of organisms released usually rises rapidly reaching a peak when the symptoms appear in symptomatic infections. This is usually followed with a long decline in the amount of agent released by the host as long as death does not occur. For example, poliovirus appears in the throat and feces 7–10 days before clinical illness (fever) is apparent and may be excreted for more than 30 days after infection ( Fig. 9.2 ). Poliovirus will also be detectable in the blood and urine during the course of infection ( Pichichero et al., 1998 ). Hepatitis A virus appears in the stool of infected individuals 2–3 weeks before clinical illness ( Belshe, 1991 ). Parainfluenza, a virus related to influenza, can be detected in nasal secretions in less than 24 h after infection and up to 2 weeks afterward ( Belshe, 1991 ). In the case of SARS, the virus may still be present in the feces for 37 days after infection ( Holmes, 2003 ). Variola major, the virus that causes smallpox, is released for up to 19 days after infection at concentrations of 10 2 –10 5 per ml of urine ( Table 9.5 ) ( Sarkar et al., 1973 ). In many infections, the greatest concentrations are released during the first few days after the initial infection. Brucella abortus is excreted in concentrations as high as 10 6 per ml of urine for up to 12 weeks ( Table 9.6 ). Marburg virus and flaviviruses are excreted in the urine of animals for 10–12 days. In summary, all of the nonenteric agents of interest (Categories A, B, and C) are released in the host for at least days to weeks in concentrations likely to be detectable in sewage systems ( Table 9.6 ) ( Sinclair et al., 2008 ). Figure 9.2 Occurrence of nonpolio enterovirus in bodily fluids and feces during the course of infection (interpreted from Pichichero et al., 1998). Table 9.5 Concentration of select agents in sewage and duration of agent release after infection of individuals. see Sinclair et al., (2008) for the following category A agents: Variola major, Hantavirus, Marburg virus, and Flavivirus Agent Category Fluid Concentration a Duration Reference C. Botulinum A Feces 10 8 ? Paton et al. (1983) C. Psittaci B Feces 10 2 –10 3 28 days b Takahashi et al. (1988) B. Abortus B Urine 10 2 –10 6 8–12 weeks Bicknell and Bell (1979) ; Carmichael and Joubert (1988) ; Serikawa et al. (1981) Japanese encephalitis B Urine 1–4 3 days Mathur et al. (1995) Enteroviruses B Feces 10 8 –10 12 Weeks to Months Maier et al. (2000) Protozoa B Feces 10 6 –10 7 Weeks to Months Maier et al. (2000) C. Burnetii B Feces 10 3 –10 4 7 days Tylewska-Wierzbanowska and Kruszewska (1993) Influenza C Nasal 10 5 –10 7 5 days to Weeks Belshe (1991) a per "milliliter" of volume or "gram" of solid. b Animals. Table 9.6 Titer of smallpox virus in urine ( Sarkar et al., 1973 ) Days after infection Titer(mL) 3 10 3 –10 5 4 10 2 –10 5 5 10 2 –10 4 6 10 1 –10 4 7 10 1 –10 3 8 10 1 –10 2 10 10 1 –10 2 15 10 1 –10 2 19 10 1 20 0 Most of the existing data on the occurrence and concentration of pathogens was gathered using culture of viable or infective organisms. Molecular methods such as the polymerase chain reaction (PCR) or immuno-chemical methods (enzyme-linked immunoassays or ELISA) can detect both infectious and noninfectious organisms. These molecular techniques can detect concentrations from 1 to 10,000 greater than culture methods because some of the organisms may be inactivated (dead) or may not be able to grow on the selected media (bacteria) or cell culture (used for viruses). In the case of enteric viruses, the ratio of viruses detected by infectivity assay may be 100–50,000 times less than that detected by a molecular method ( Ward et al., 1984 ). This is because cell culture methods have a low efficiency in virus quantification from clinical and environmental samples; however, they do provide a robust measure of viral activity not feasible with molecular methods. Agents causing enteric and respiratory infections are released in large numbers in feces and respiratory secretions ( Table 9.4 ). Many of the enteric viruses such as the enteroviruses and adenoviruses may replicate both in the intestinal and respiratory tract. Using molecular methods the number of enteric viruses detected can approach peak concentrations of 10 12 organisms per gram of stool while protozoa can approach 10 6 -10 7 per gram. Cultivatable enteric bacterial pathogens such as Salmonella may also occur in concentrations as large as 10 11 per gram ( Feachem et al., 1983 ). By infectivity assays, the concentration of respiratory viruses ranges from 10 5 to 10 7 per ml of respiratory secretion. Even blood-borne viruses such as HIV will be found in the feces of infected persons ( Ansari et al., 1992 ) and it appears that many viruses will occur in the urine during infection of the host ( Table 9.6 ), although these excreted viruses may not be infectious. Little information is available on the concentration of pathogenic viruses or bacterial agents of interest in the urine. The total amount of virus released by a person is, of course, also related to the amount of feces, urine, respiratory secretion, and skin that is released by the person. On average, a person excretes between 100 to 400 g of feces and 700–2000 ml of urine per day ( Table 9.7 ). Table 9.7 Factors that affect concentration of the biological agent in sewage Site of replication in the host GI, upper respiratory, nose, skin, internal organs Duration of release from the host Concentration in the source Incidence of disease in the population Water use per capita Season Survival in the sewer system A person with an enteric viral infection may excrete as many as 10 14 viral particles per day and over 10 15 during the course of an infection ( Table 9.8 ). Nonenteric bacterial agents of interest appeared to be released in concentrations from 10° to 10 8 by viability assays ( Boone and Gerba 2007 ). Respiratory pathogens end up in the feces from the swallowing of secretions. Table 9.8 Sources of biological agents in sewers ( Feachem et al., 1983 ) Feces (100–400 g/person/day) Urine (700–2000 ml/day) Skin—from bath and hand washing Saliva, respiratory secretions Blood Food Wash water (kitchen, drains) Storm water a a Some sewer systems are combined with the storm-water collection system. Ecological studies of bovine tuberculosis in badgers introduce the concept of "super-excretors," which maintain the disease and pass infectious organisms in their stool or urine continuously. Super-excretors are individuals who excrete larger numbers than average of a pathogen during an infection. These super-excretors were almost exclusively animals with a progressive infection, which does not resolve and contributed to a higher mortality ( Delahay et al., 2000 ). The occurrence of a similar "super-spreader" was also noted in a clinical epidemiological report of SARS in humans ( Holmes, 2003 ). A Human pathogens secreted in bodily fluids A literature search was conducted to determine the occurrence of the agents in bodily fluids, feces, skin, and sewage. As indicated in Table 9.3 and a previous publication ( Sinclair et al., 2008 ), many select agents may occur in bodily excretions or secretions even though this may not be their primary site of replication. It would appear that all of the viral agents are excreted in the urine and most of the bacterial agents in the feces or saliva. Since none of the organisms cause enteric infections they have seldom been sought in sewage, however, Bacillus anthracis and Yersina pestis (plague) have been detected in sewage. The source of B . anthracis spores in the sewage was believed to be from an African import tannery operation ( Perone and Gelosa, 1982 ) and presumably not from enteric infections, which would normally result in the presence of spores in the feces. Category B agents differ in that, and they include many enteric pathogens which are excreted in large numbers in the feces ( Table 9.3 ). All of the other agents in this category appear to be excreted in the feces; many of the viral agents are excreted in the urine. No studies were found that report examining sewage for their presence. The Category C viral agents appear to be excreted in the saliva and urine ( Tables 9.3 and 9.4 ). No references for the presence of these agents in sewage could be found. Some typical blood-borne agents such as Hepatitis B virus ( Alter et al., 1977 ) and Human Immuno-Deficiency virus ( Levy, 1989 ) have been detected in sewage by molecular methods ( Table 9.4 ). The coronavirus, which causes Severe Acute Respiratory Disease (SARS), is also excreted in the feces and other bodily fluids such as tears ( Loon et al., 2004 , Wang et al., 2005 ) ( Table 9.4 ). Table 9.3 Select category B and C agents found in human bodily fluids and sewage Agent Category Urine Feces Saliva Sewage Reference C. psittaci B ? Yes a ? ? Midura and Arnon (1976) ; Anderson (1996) ; Smith et al. (2005) C. burnetii B Yes Yes b ? ? Tylewska-Wierzbanowska and Kruszewska (1993) Viral encephalitis B Yes Yes c ? ? Mathur et al. (1995) Nipah virus C Yes ? Yes ? Chua et al., (2002) Rabies C Yes ? Yes ? Wacharapluesadee and Hemachudha (2002) Influenza C ? Yes c Yes ? Buchy et al. (2007) a Nasal. b Semen. c Animals. Table 9.4 Occurrence of other agents of interest in bodily fluids Agents Urine Feces Saliva Sewage Reference Severe Acute Respiratory Syndrome ? Yes Yes ? He et al. (2007) ; Petrich et al. (2006) John Cunningham virus (poliovirus) Yes ? ? Yes Coleman et al. (1980) Human Immuno-deficiency Virus Yes Yes Yes Yes Levy (1989) ; Yolken et al. (1991) Hepatitis B virus ? ? Yes Yes Alter et al. (1977) ; Bancroft et al. (1977) ; Arvanitidou et al. (1998) B Duration of release and concentration in bodily fluids and skin The duration and concentration of pathogens released by a host during the course of an infection varies, with greater numbers being released in more severe infections. After infection, the number of organisms released usually rises rapidly reaching a peak when the symptoms appear in symptomatic infections. This is usually followed with a long decline in the amount of agent released by the host as long as death does not occur. For example, poliovirus appears in the throat and feces 7–10 days before clinical illness (fever) is apparent and may be excreted for more than 30 days after infection ( Fig. 9.2 ). Poliovirus will also be detectable in the blood and urine during the course of infection ( Pichichero et al., 1998 ). Hepatitis A virus appears in the stool of infected individuals 2–3 weeks before clinical illness ( Belshe, 1991 ). Parainfluenza, a virus related to influenza, can be detected in nasal secretions in less than 24 h after infection and up to 2 weeks afterward ( Belshe, 1991 ). In the case of SARS, the virus may still be present in the feces for 37 days after infection ( Holmes, 2003 ). Variola major, the virus that causes smallpox, is released for up to 19 days after infection at concentrations of 10 2 –10 5 per ml of urine ( Table 9.5 ) ( Sarkar et al., 1973 ). In many infections, the greatest concentrations are released during the first few days after the initial infection. Brucella abortus is excreted in concentrations as high as 10 6 per ml of urine for up to 12 weeks ( Table 9.6 ). Marburg virus and flaviviruses are excreted in the urine of animals for 10–12 days. In summary, all of the nonenteric agents of interest (Categories A, B, and C) are released in the host for at least days to weeks in concentrations likely to be detectable in sewage systems ( Table 9.6 ) ( Sinclair et al., 2008 ). Figure 9.2 Occurrence of nonpolio enterovirus in bodily fluids and feces during the course of infection (interpreted from Pichichero et al., 1998). Table 9.5 Concentration of select agents in sewage and duration of agent release after infection of individuals. see Sinclair et al., (2008) for the following category A agents: Variola major, Hantavirus, Marburg virus, and Flavivirus Agent Category Fluid Concentration a Duration Reference C. Botulinum A Feces 10 8 ? Paton et al. (1983) C. Psittaci B Feces 10 2 –10 3 28 days b Takahashi et al. (1988) B. Abortus B Urine 10 2 –10 6 8–12 weeks Bicknell and Bell (1979) ; Carmichael and Joubert (1988) ; Serikawa et al. (1981) Japanese encephalitis B Urine 1–4 3 days Mathur et al. (1995) Enteroviruses B Feces 10 8 –10 12 Weeks to Months Maier et al. (2000) Protozoa B Feces 10 6 –10 7 Weeks to Months Maier et al. (2000) C. Burnetii B Feces 10 3 –10 4 7 days Tylewska-Wierzbanowska and Kruszewska (1993) Influenza C Nasal 10 5 –10 7 5 days to Weeks Belshe (1991) a per "milliliter" of volume or "gram" of solid. b Animals. Table 9.6 Titer of smallpox virus in urine ( Sarkar et al., 1973 ) Days after infection Titer(mL) 3 10 3 –10 5 4 10 2 –10 5 5 10 2 –10 4 6 10 1 –10 4 7 10 1 –10 3 8 10 1 –10 2 10 10 1 –10 2 15 10 1 –10 2 19 10 1 20 0 Most of the existing data on the occurrence and concentration of pathogens was gathered using culture of viable or infective organisms. Molecular methods such as the polymerase chain reaction (PCR) or immuno-chemical methods (enzyme-linked immunoassays or ELISA) can detect both infectious and noninfectious organisms. These molecular techniques can detect concentrations from 1 to 10,000 greater than culture methods because some of the organisms may be inactivated (dead) or may not be able to grow on the selected media (bacteria) or cell culture (used for viruses). In the case of enteric viruses, the ratio of viruses detected by infectivity assay may be 100–50,000 times less than that detected by a molecular method ( Ward et al., 1984 ). This is because cell culture methods have a low efficiency in virus quantification from clinical and environmental samples; however, they do provide a robust measure of viral activity not feasible with molecular methods. Agents causing enteric and respiratory infections are released in large numbers in feces and respiratory secretions ( Table 9.4 ). Many of the enteric viruses such as the enteroviruses and adenoviruses may replicate both in the intestinal and respiratory tract. Using molecular methods the number of enteric viruses detected can approach peak concentrations of 10 12 organisms per gram of stool while protozoa can approach 10 6 -10 7 per gram. Cultivatable enteric bacterial pathogens such as Salmonella may also occur in concentrations as large as 10 11 per gram ( Feachem et al., 1983 ). By infectivity assays, the concentration of respiratory viruses ranges from 10 5 to 10 7 per ml of respiratory secretion. Even blood-borne viruses such as HIV will be found in the feces of infected persons ( Ansari et al., 1992 ) and it appears that many viruses will occur in the urine during infection of the host ( Table 9.6 ), although these excreted viruses may not be infectious. Little information is available on the concentration of pathogenic viruses or bacterial agents of interest in the urine. The total amount of virus released by a person is, of course, also related to the amount of feces, urine, respiratory secretion, and skin that is released by the person. On average, a person excretes between 100 to 400 g of feces and 700–2000 ml of urine per day ( Table 9.7 ). Table 9.7 Factors that affect concentration of the biological agent in sewage Site of replication in the host GI, upper respiratory, nose, skin, internal organs Duration of release from the host Concentration in the source Incidence of disease in the population Water use per capita Season Survival in the sewer system A person with an enteric viral infection may excrete as many as 10 14 viral particles per day and over 10 15 during the course of an infection ( Table 9.8 ). Nonenteric bacterial agents of interest appeared to be released in concentrations from 10° to 10 8 by viability assays ( Boone and Gerba 2007 ). Respiratory pathogens end up in the feces from the swallowing of secretions. Table 9.8 Sources of biological agents in sewers ( Feachem et al., 1983 ) Feces (100–400 g/person/day) Urine (700–2000 ml/day) Skin—from bath and hand washing Saliva, respiratory secretions Blood Food Wash water (kitchen, drains) Storm water a a Some sewer systems are combined with the storm-water collection system. Ecological studies of bovine tuberculosis in badgers introduce the concept of "super-excretors," which maintain the disease and pass infectious organisms in their stool or urine continuously. Super-excretors are individuals who excrete larger numbers than average of a pathogen during an infection. These super-excretors were almost exclusively animals with a progressive infection, which does not resolve and contributed to a higher mortality ( Delahay et al., 2000 ). The occurrence of a similar "super-spreader" was also noted in a clinical epidemiological report of SARS in humans ( Holmes, 2003 ). III CONCENTRATION OF BIOLOGICAL AGENTS IN SEWAGE The occurrence and concentration of pathogens in sewage is dependent upon a number of factors listed in Table 9.9 . One of the most important considerations is the amount of pathogen released by a person daily from bodily fluid, feces, skin, and urine. Because one infected individual typically produces at least 100 g of feces per day, a pathogen present at 10 8 per gram will introduce at least 10 10 or more of the pathogen into the sewer system. Logically, pathogens excreted in urine and feces will be released several times during a 24-h period. Enteric and respiratory pathogens are almost always detected in sewage because of the long duration of release from the host during infection, the large concentrations released from the host, and the many infections that are asymptomatic. Table 9.9 Comparative occurrence of enteric agents (category B) in feces and sewage Agent(s) Feces (per gram) Stool a Sewage (100 ml) Enteric viruses (infectivity assay) 10 8 10 10 10 2 Enteric viruses (PCR assay) 10 10 –10 12 10 12 –10 14 10 4 –10 5 Giardia 10 6 10 8 10–10 2 Cryptosporidium 10 6 10 8 0.1-10 2 a 100 g stool (150 g average in the U.S.). Studies have shown that the types and concentration of enteric microorganisms in sewage is directly related to the incidence of disease in the community ( Riordan, 1962 , Sellwood et al., 1981 ). The concentration of enteric pathogens in sewage ranges from 0.1 to 100,000 per ml of sewage ( Table 9.8 ). While many biological agents of interest have been detected in sewage ( Table 9.3 ), the studies are limited and vary by location. IV LABORATORY METHODS AND DETECTION A Detection of pathogens Culture based methods can be used for the detection of pathogens in wastewater, but they may take days to weeks to perform. Alternative molecular methods, such as the PCR, have been successful in detecting bacterial, viral, and protozoan pathogens in sewage without the need for cultivation ( Gilbride et al., 2006 ). These new techniques detect live and dead organisms, have a high sensitivity for wastewater, and can reduce detection time to a few hours ( He and Jiang, 2005 , Holmes, 2003 ). Some promising new wastewater methods use nucleic acid microarrays or antibody/receptor technologies to detect multiple pathogens simultaneously ( Boehm et al., 2007 ). Combining these multiplexed methods with fiberoptic sensors and lab-on-a-chip technology can allow utilities to rapidly screen, identify, and quantify multiple pathogens in real time. Because these technologies rely on PCR DNA techniques, the many interfering substances in raw sewage pose a problem. Without proper sample extraction, the sample analytes are exposed to many varying inhibitors, which can negatively impact the DNA isolation and amplification steps. These methods are also limited by their inability to differentiate between viable and nonviable or nonculturable organisms ( Josephson et al., 1993 ), a vital characteristic when assessing the microbial risk assessment for any given community. Certain methods are in development to automate the sample collection, sample processing, and concentration to separate analytes from inhibitors and deliver a suitable clean sample to a real-time detection microarray technology. These methods use latex beads, carbohydrates, anion exchange resins, or similar substances as part of sample collection and sample processing step ( Straub and Chandler, 2003 ), but no fully automated method has been proposed for wastewater. A biosensor capable of identifying and quantifying a wide group of pathogens is necessary, but future development is needed in the areas of extraction from environmental samples, selection of a suitable target sequence of the pathogen (specificity), detection and differentiation of the signal from interfering sequences (sensitivity), and automation of all processes towards a functional real-time biosensor for wastewater ( Gilbride et al., 2006 ). B Survival of pathogens in sewer systems A principal benefit of wastewater monitoring is that most pathogens of interest are expected to remain viable for at least several days in the sewerage environment ( Table 9.10 ). Enteric and respiratory agents are particularly stable, while data is limited for viral encephalitis agents because transmission in water and other liquid media does not occur naturally. Using molecular methods, survival of the pathogens in the viable form is not necessary for their detection, thus increasing the length of time for which the pathogen may be detected. In the case of select agents, knowing the presence of the organism in the sewer system may be all that is needed to trigger further investigation regardless of viability. Table 9.10 The Helsinki poliovirus experiment ( Ranta et al., 2001 ) Helsinki population = 740,000 Sewage flow 2 × 10 8 l/day Contamination Event 5 × 10 10 TCID 50 Poliovirus vaccine Flushed down toilet in one liter volume 20 km from sewage treatment plant Detection Automatic sampler = 200 ml per 5 × 10 6 liters of sewage flow Four samples pooled per day Concentrated from 400 to 1 ml before assay Result Virus was detected for the next 4 days (cell culture) Peak 24–48 h after flush Virus detected after passage of 800 million liters of sewage pass through system Conclusion Monitoring of sewage could detect 1 infected person in 10,000 Assumes: 10 8 infectious virus excreted by child in 4 days C Lessons learned from poliovirus: Monitoring as an early warning system The benefits of pathogen monitoring in sewage have been recognized for poliovirus for more than 40 years. The relationship between the occurrence of poliovirus in sewage and clinical incidence of disease in a community was first noted in the late 1960s ( Nelson et al., 1967 ). These early detection studies were designed as longitudinal epidemiological investigations to assess the success of polio vaccination campaigns ( Riordan, 1962 ). The results of these studies demonstrated that a definite correlation exists among the isolation of enteroviruses in sewage, and the isolation of viruses in stools, and the number of recognized clinical cases within the community. Using cell culture assay techniques (which measure only infective viruses) and only grab samples (i.e., no steps to concentrate the sample) poliovirus could be detected when only 0.27–0.4% of the population was excreting the virus. It was also demonstrated that small outbreaks and epidemics of enterovirus and adenovirus disease within a community can be predicted by monitoring a community's sewage. Virulent or wildtype (nonvaccine strain) poliovirus type 1 was detected in sewage 9 days before the first clinical case became evident ( Kuwert et al., 1970 ). In an outbreak of Coxsackievirus B5, the virus was detected in the sewage 10 days before clinical cases were positive ( Nelson et al., 1967 ). These studies make it clear that grab samples collected on a regular (weekly or every few days) basis could be used to assess the introduction of a new infectious agent in the community. This approach was later adapted to monitor the success of poliovirus vaccine campaigns internationally ( WHO, 2003 ). To assess the sensitivity of poliovirus monitoring, one study ( Ranta et al., 2001 ) flushed a one-time bolus of 11 containing 2 × 10 10 infective poliovirus type 1 vaccine strain down a toilet 20 km (12 miles) from the sewage plant ( Table 9.11 ). Samples were automatically collected and assayed for the next 4 days. Infectious poliovirus was still detected after 800 million liters had passed through the system. The authors concluded that their monitoring system could detect one infected person in 10,000 residents of the community, assuming that 10 8 infective viruses are excreted by a child over a 4-day period of time. The study showed that pathogens appear to be greatly retarded in sewage systems, where a onetime event resulted in a detection period over 4 days. The pathogen was also easily detected in 200-ml samples for every 5 × 10 6 1 of sewage flow. Table 9.11 Survival time of pathogens in the environment (water, feces, urine, sewage) ( Belanov et al., 1996 , Belshe, 1991 , Mitscherlich and Marth, 1984 , Sinclair et al., 2008 ) Organism Days of survival B. anthracis Weeks to years C. botulinum Weeks Y. pestis Days Variola major (smallpox) Weeks to months F. tularensis 12–60 days Marburg virus (surfaces) 4–5 days Enteric pathogens Days to months B. mallei 28–35 days Psittacosis ( C. psittaci ) Days Q fever ( C. burnetti ) 30–1000 days Typhus fever ( Rickettsia typhi ) Hours to days Influenza ( surfaces ) 3 days Surveillance of poliovirus in sewage has been used by several nations to assess the success of vaccination programs and to identify the potential need for vaccination to prevent outbreaks ( Deshpande et al., 2003 , Manor et al., 1999 , Tambini et al., 1993 ). The World Health Organization has published guidelines for the environmental surveillance of poliovirus circulation ( WHO, 2003 ). These guidelines assume that a single infected person will excrete 10 7 polioviruses per day and that one person infected in 100 could be detected using an infectivity assay without concentrating the sewage. However, if the tested sample is concentrated 100 fold then one infected person among 10,000 could be detected. The Public Health Laboratories of Israel have been conducting an environmental surveillance of sewage on a monthly basis since 1989 ( Manor et al., 1999 ) to assess the spread of the wild type poliovirus strains capable of causing paralytic disease. This was done to determine the success and need for vaccination programs. Between 1989 and 1998, four "silent" separate episodes of wild-type poliovirus circulation were detected when no clinical cases were observed. The study described how surveillance of the sewage is much more effective than surveillance of clinical cases. The greater sensitivity of sewage surveillance was also validated in Mumbai, India where wild type poliovirus was detected 3 months before any clinical cases were observed ( Deshpande et al., 2003 ). D Differentiation of vaccine and virulent strains In the poliovirus surveillance of sewage it is necessary to differentiate between vaccine strains and wild type strains of the virus. In the past this has been accomplished by using different cell lines or incubation conditions to limit the growth of the vaccine strains. However, today this can be accomplished by the use of molecular methods and sequence analysis. Sequences amplified directly from processed sewage samples by PCR using primer pairs specific for the indigenous type 1 genotype could be used to assess its occurrence in the presence of vaccine strains ( Tambini et al., 1993 ). Vaccine strains have unique sequences from wild type strains of pathogens allowing easy differentiation. In addition, sequence analysis of sewage isolations has been shown useful in tracking the spread of wild type poliovirus from one country and community to another ( Deshpande et al., 2003 , Manor et al., 1999 ). This review of poliovirus is offered here as a case study and justification for the use of monitoring additional CDC select biological agents. With current molecular techniques and updated concentration methods, a much greater sensitivity and specificity can be achieved for poliovirus and many other CDC select agents. A Detection of pathogens Culture based methods can be used for the detection of pathogens in wastewater, but they may take days to weeks to perform. Alternative molecular methods, such as the PCR, have been successful in detecting bacterial, viral, and protozoan pathogens in sewage without the need for cultivation ( Gilbride et al., 2006 ). These new techniques detect live and dead organisms, have a high sensitivity for wastewater, and can reduce detection time to a few hours ( He and Jiang, 2005 , Holmes, 2003 ). Some promising new wastewater methods use nucleic acid microarrays or antibody/receptor technologies to detect multiple pathogens simultaneously ( Boehm et al., 2007 ). Combining these multiplexed methods with fiberoptic sensors and lab-on-a-chip technology can allow utilities to rapidly screen, identify, and quantify multiple pathogens in real time. Because these technologies rely on PCR DNA techniques, the many interfering substances in raw sewage pose a problem. Without proper sample extraction, the sample analytes are exposed to many varying inhibitors, which can negatively impact the DNA isolation and amplification steps. These methods are also limited by their inability to differentiate between viable and nonviable or nonculturable organisms ( Josephson et al., 1993 ), a vital characteristic when assessing the microbial risk assessment for any given community. Certain methods are in development to automate the sample collection, sample processing, and concentration to separate analytes from inhibitors and deliver a suitable clean sample to a real-time detection microarray technology. These methods use latex beads, carbohydrates, anion exchange resins, or similar substances as part of sample collection and sample processing step ( Straub and Chandler, 2003 ), but no fully automated method has been proposed for wastewater. A biosensor capable of identifying and quantifying a wide group of pathogens is necessary, but future development is needed in the areas of extraction from environmental samples, selection of a suitable target sequence of the pathogen (specificity), detection and differentiation of the signal from interfering sequences (sensitivity), and automation of all processes towards a functional real-time biosensor for wastewater ( Gilbride et al., 2006 ). B Survival of pathogens in sewer systems A principal benefit of wastewater monitoring is that most pathogens of interest are expected to remain viable for at least several days in the sewerage environment ( Table 9.10 ). Enteric and respiratory agents are particularly stable, while data is limited for viral encephalitis agents because transmission in water and other liquid media does not occur naturally. Using molecular methods, survival of the pathogens in the viable form is not necessary for their detection, thus increasing the length of time for which the pathogen may be detected. In the case of select agents, knowing the presence of the organism in the sewer system may be all that is needed to trigger further investigation regardless of viability. Table 9.10 The Helsinki poliovirus experiment ( Ranta et al., 2001 ) Helsinki population = 740,000 Sewage flow 2 × 10 8 l/day Contamination Event 5 × 10 10 TCID 50 Poliovirus vaccine Flushed down toilet in one liter volume 20 km from sewage treatment plant Detection Automatic sampler = 200 ml per 5 × 10 6 liters of sewage flow Four samples pooled per day Concentrated from 400 to 1 ml before assay Result Virus was detected for the next 4 days (cell culture) Peak 24–48 h after flush Virus detected after passage of 800 million liters of sewage pass through system Conclusion Monitoring of sewage could detect 1 infected person in 10,000 Assumes: 10 8 infectious virus excreted by child in 4 days C Lessons learned from poliovirus: Monitoring as an early warning system The benefits of pathogen monitoring in sewage have been recognized for poliovirus for more than 40 years. The relationship between the occurrence of poliovirus in sewage and clinical incidence of disease in a community was first noted in the late 1960s ( Nelson et al., 1967 ). These early detection studies were designed as longitudinal epidemiological investigations to assess the success of polio vaccination campaigns ( Riordan, 1962 ). The results of these studies demonstrated that a definite correlation exists among the isolation of enteroviruses in sewage, and the isolation of viruses in stools, and the number of recognized clinical cases within the community. Using cell culture assay techniques (which measure only infective viruses) and only grab samples (i.e., no steps to concentrate the sample) poliovirus could be detected when only 0.27–0.4% of the population was excreting the virus. It was also demonstrated that small outbreaks and epidemics of enterovirus and adenovirus disease within a community can be predicted by monitoring a community's sewage. Virulent or wildtype (nonvaccine strain) poliovirus type 1 was detected in sewage 9 days before the first clinical case became evident ( Kuwert et al., 1970 ). In an outbreak of Coxsackievirus B5, the virus was detected in the sewage 10 days before clinical cases were positive ( Nelson et al., 1967 ). These studies make it clear that grab samples collected on a regular (weekly or every few days) basis could be used to assess the introduction of a new infectious agent in the community. This approach was later adapted to monitor the success of poliovirus vaccine campaigns internationally ( WHO, 2003 ). To assess the sensitivity of poliovirus monitoring, one study ( Ranta et al., 2001 ) flushed a one-time bolus of 11 containing 2 × 10 10 infective poliovirus type 1 vaccine strain down a toilet 20 km (12 miles) from the sewage plant ( Table 9.11 ). Samples were automatically collected and assayed for the next 4 days. Infectious poliovirus was still detected after 800 million liters had passed through the system. The authors concluded that their monitoring system could detect one infected person in 10,000 residents of the community, assuming that 10 8 infective viruses are excreted by a child over a 4-day period of time. The study showed that pathogens appear to be greatly retarded in sewage systems, where a onetime event resulted in a detection period over 4 days. The pathogen was also easily detected in 200-ml samples for every 5 × 10 6 1 of sewage flow. Table 9.11 Survival time of pathogens in the environment (water, feces, urine, sewage) ( Belanov et al., 1996 , Belshe, 1991 , Mitscherlich and Marth, 1984 , Sinclair et al., 2008 ) Organism Days of survival B. anthracis Weeks to years C. botulinum Weeks Y. pestis Days Variola major (smallpox) Weeks to months F. tularensis 12–60 days Marburg virus (surfaces) 4–5 days Enteric pathogens Days to months B. mallei 28–35 days Psittacosis ( C. psittaci ) Days Q fever ( C. burnetti ) 30–1000 days Typhus fever ( Rickettsia typhi ) Hours to days Influenza ( surfaces ) 3 days Surveillance of poliovirus in sewage has been used by several nations to assess the success of vaccination programs and to identify the potential need for vaccination to prevent outbreaks ( Deshpande et al., 2003 , Manor et al., 1999 , Tambini et al., 1993 ). The World Health Organization has published guidelines for the environmental surveillance of poliovirus circulation ( WHO, 2003 ). These guidelines assume that a single infected person will excrete 10 7 polioviruses per day and that one person infected in 100 could be detected using an infectivity assay without concentrating the sewage. However, if the tested sample is concentrated 100 fold then one infected person among 10,000 could be detected. The Public Health Laboratories of Israel have been conducting an environmental surveillance of sewage on a monthly basis since 1989 ( Manor et al., 1999 ) to assess the spread of the wild type poliovirus strains capable of causing paralytic disease. This was done to determine the success and need for vaccination programs. Between 1989 and 1998, four "silent" separate episodes of wild-type poliovirus circulation were detected when no clinical cases were observed. The study described how surveillance of the sewage is much more effective than surveillance of clinical cases. The greater sensitivity of sewage surveillance was also validated in Mumbai, India where wild type poliovirus was detected 3 months before any clinical cases were observed ( Deshpande et al., 2003 ). D Differentiation of vaccine and virulent strains In the poliovirus surveillance of sewage it is necessary to differentiate between vaccine strains and wild type strains of the virus. In the past this has been accomplished by using different cell lines or incubation conditions to limit the growth of the vaccine strains. However, today this can be accomplished by the use of molecular methods and sequence analysis. Sequences amplified directly from processed sewage samples by PCR using primer pairs specific for the indigenous type 1 genotype could be used to assess its occurrence in the presence of vaccine strains ( Tambini et al., 1993 ). Vaccine strains have unique sequences from wild type strains of pathogens allowing easy differentiation. In addition, sequence analysis of sewage isolations has been shown useful in tracking the spread of wild type poliovirus from one country and community to another ( Deshpande et al., 2003 , Manor et al., 1999 ). This review of poliovirus is offered here as a case study and justification for the use of monitoring additional CDC select biological agents. With current molecular techniques and updated concentration methods, a much greater sensitivity and specificity can be achieved for poliovirus and many other CDC select agents. V CONCLUSIONS: THE PROBABILITY OF DETECTION Studies with poliovirus demonstrated the feasibility of how monitoring sewage for virulent pathogens can be used to assess the success of vaccine programs. This review identified three important benefits of developing a wastewater monitoring system. Sewage surveillance system has been shown to be more sensitive than reporting of clinical cases of serious illness in a community. It was also demonstrated that pathogens can be greatly retarded in a sewage systems allowing a detection time over many days for a one-time release into a sewage system. Finally, it was shown that infectivity assays have the ability to detect one infected person in 10,000 individuals. Sewage surveillance can detect the presence or increased amount of infections from enteric pathogens excreted in the feces or urine during infection. However, the success of such a surveillance system for nonenteric pathogens has not been demonstrated, although they have been found in sewage. The sensitivity of a sewage surveillance system will depend on several important factors including the amount and duration of the agent released into the sewers, the frequency of monitoring, and the sensitivity of the monitoring method. Nonenteric pathogens are released from the host for a minimum of several days. This has already been demonstrated for HIV, hepatitis B, and Y. pestis (see Tables 9.2 and IV). Given this fact and the expected several day retardation in sewer systems, all or most of the nonenteric category agents will be present in the sewer system if there is an infection in the population served by the sewer system. Based upon the conclusions of the Helsinki experiment, which measured infectious poliovirus ( Table 9.11 ), one individual excreting 10 8 infectious virus per gram of feces for a period of 4 days could be identified in a population of 10,000. If we consider the concentration and amount of infectious agent in the fluid or feces released during infection, this same sensitivity should be achieved with the agents of smallpox, Brucella, botulism and perhaps influenza. Based on existing information in Table 9.5 at least one person in 100 could be detected for most of the agents for which information is available. Because many of the agents take several days to detect by conventional culture methods the preferred detection system would be by a rapid, but highly specific method such as the quantitative real time PCR or other similar molecular detection system. Because PCR can detect both culturable and nonculturable organisms, it can be expected to be more sensitive than methods that have been used in the past for sewage surveillance. Use of PCR should increase sensitivity by as much as 50,000 over cultivation methods ( Ward et al., 1984 ). Also, when using PCR to detect viruses in sewage, a 10-fold loss in sensitivity is likely with current methods. This loss is due to interfering substances present in the sewage, but still leaves a method that may be 5000 times more sensitive than conventional culture methods. Increasing the volume of wastewater that is tested may also increase the sensitivity of current methods. Technology is available ( Hurst and Crawford, 2002 ) which allows for the concentration of bacteria and viruses from up to 10 l of raw sewage. Thus, increasing the volume analyzed from 400 to 4000 ml could increase the sensitivity of detection another 10-fold. Surveillance of pathogens in wastewater has several advantages over aerosol and other monitoring methods. Longer survival times in soil, water, and wastewater ( Sinclair et al., 2008 ) facilitate a retardation of pathogens in sewage which allows a longer sampling window than aerosols where organisms are much more susceptible to factors such as settling, desiccation, and relative humidity. Additionally, wastewater is collected in a central location, allowing monitoring to be defined or subdivided to specific areas. Lastly, wastewater systems can include many pathogens originating in aerosol, surface water, tap water, or fomites as storm-water and watersheds will often flow into sewerage systems. Of course background levels and alert levels of the agents of interest would have to be established. Most agents of interest are likely to occur at one time or another in wastewater or at least have some normal range of background. Further research would be needed to determine what these levels might be and normal variation of concentrations of the agents in wastewater. The types and concentration would be expected to vary by location and the size of the population, area served and type of connections (e.g., the presence of a slaughterhouse may increase the likelihood of finding animal pathogens). In summary, given the potential enhanced sensitivity of molecular methods and current abilities to test larger volumes of all of the CDC select agents of interest (enteric and nonenteric), it is possible to detect if an infected individual enters a monitored population. Although the concentration and duration of release of all of the agents of interest are not known, it is still possible to detect at least one infection in populations of 1000 or more. This monitoring is especially useful when combined with other components of the QMRA framework such as modeling of sewage dispersion, back calculation of contaminant point of introduction, and calculations of the health risk. ACKNOWLEDGMENTS This study was supported by the United States Defense Advanced Research Projects Agency, the Center for Advancing Microbial Risk Assessment funded by the United States Environmental Protection Agency Science to Achieve Results, and the United States Department of Homeland Security University Programs grant number R3236201. Ryan Sinclair was supported through the National Research Council's Research Associate Program with funding from the United Sates Department of Homeland Security.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519269/

Identification and Characterization of Bacillus anthracis Spores by Multiparameter Flow Cytometry ▿

In response to the need for methods that can rapidly detect potentially virulent Bacillus anthracis spores, we developed a two-color flow cytometric assay capable of simultaneously identifying B. anthracis spores and the presence of spore-associated protective antigen, a virulence marker for strains harboring the pXO1 plasmid.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4680858/

A Novel Inhibitor Prevents the Peripheral Neuroparalysis of Botulinum Neurotoxins

Botulinum neurotoxins (BoNTs) form a large class of potent and deadly neurotoxins. Given their growing number, it is of paramount importance to discover novel inhibitors targeting common steps of their intoxication process. Recently, EGA was shown to inhibit the action of bacterial toxins and viruses exhibiting a pH-dependent translocation step in mammalian cells, by interfering with their entry route. As BoNTs act in the cytosol of nerve terminals, the entry into an appropriate compartment wherefrom they translocate the catalytic moiety is essential for toxicity. Herein we propose an optimized procedure to synthesize EGA and we show that, in vitro, it prevents the neurotoxicity of different BoNT serotypes by interfering with their trafficking. Furthermore, in mice, EGA mitigates botulism symptoms induced by BoNT/A and significantly decreases the lethality of BoNT/B and BoNT/D. This opens the possibility of using EGA as a lead compound to develop novel inhibitors of botulinum neurotoxins. Results High yield synthesis of 4-bromobenzaldehyde N-(2,6-dimethylphenyl) semicarbazone (EGA) The reported approach by Jung in 2014 for the preparation of EGA has been adapted and improved to obtain higher yields. The synthesis involves the three steps reported in Fig. 1 . In the first one (i), 2,6-dimethylaniline (1) is allowed to react with phenyl chloroformate to give the corresponding phenylcarbamate (2), which is next subjected to hydrazinolysis to give semicarbazide (indicated as A) (ii). The final step (iii) is the reaction of A with 4-bromobenzaldehyde to form the desired semicarbazone (3, EGA). The procedure described by Jung et al. (2014) involves the isolation of A and rather drastic conditions (acidic solution and high temperature) in the last step, leading to an overall yield of 27%. A much higher overall yield (84%) is obtained by the new procedure that we have devised: 2 is isolated, whereas steps (ii) and (iii) are performed in one-pot without the isolation of A. Furthermore, much milder conditions are used in the last synthetic step. Details are found in the Supplementary information section . EGA prevents the botulinum neurotoxins cleavage of SNARE proteins in cultured neurons The use of cultured cerebellar granular neurons (CGNs) offers a simple and rapid way to screen the efficacy of candidate molecules in inhibiting BoNTs activity. The overnight incubation with 0.3 nM BoNT/A induces the cleavage of SNAP25, as assessed by the appearance in immunofluorescence ( Fig. 2a , middle panel) and western blot ( Fig. 3a , bottom panel) of its truncated form, revealed with a specific antibody. This toxin concentration is sufficient to induce a complete cleavage of its substrate, as evaluated using an antibody recognizing both forms (intact and truncated) of SNAP25 ( Fig. 3a , middle panel, SMI-81). Figure 3b shows that such activity is however inhibited by EGA in a concentration dependent manner, with a maximal effect at 12.5 μM. The inhibitory effect, though substantial, is not complete as a small amount of cleaved SNAP25 is still generated ( Fig. 2a , right panel and Fig. 3b ). Figures 2b and 3c,d show that similar results are obtained with BoNT/B. Notably, to achieve the substantial cleavage of VAMP2, which is normally concentrated at synaptic contacts ( Fig. 2b , left panel), BoNT/B had to be used at a concentration of 5 nM ( Fig. 2b , middle panel). Nevertheless, the pre-treatment with 12.5 μM EGA is sufficient to abrogate its cleavage ( Fig. 2b , right panel). EGA prevents this toxicity in a concentration dependent manner, as shown by the inhibition of VAMP2 cleavage, determined with two different antibodies ( Fig. 3c middle and bottom panels). Interestingly, despite the high amount of toxin used, in this case the effect of EGA, at higher concentration, is complete ( Fig. 3d ). The same set of experiments was replicated using BoNT/D. This serotype is the most potent in rodents 28 and, in CGNs, a minimal concentration (0.025 pM) induces the almost complete cleavage of VAMP2 ( Fig. 2c , middle panel). Similar to what found for BoNT/A, we found that EGA substantially prevents the action of this potent neurotoxin ( Fig. 2c , right panel), and this inhibition is dependent on the amount of the chemical, as estimated in western blot with an antibody specific for the intact form of VAMP2 ( Fig. 3e , bottom panel and Fig. 3f ). Importantly, Figure S1 shows that neurons viability is not significantly affected, even at the highest concentration of EGA used. EGA does not interfere with the four basic steps of BoNTs' mechanism of action The cellular target of EGA is not known and we investigated whether any of the four main steps of the BoNTs' cellular mechanism of action is directly impacted by the action of the drug. The first step of intoxication is the specific binding of BoNTs to peripheral nerve endings followed by their internalization via endocytosis 2 . Given its chemical nature, EGA could in principle intercalate among lipids and alter the properties of the presynaptic membrane, making it less receptive for BoNTs binding. To investigate this possibility, we took advantage of two constructs consisting of the HC domain of BoNT/A and of BoNT/B fused to a fluorescent protein (cpV-HC/A) or tagged by a c-Myc epitope (c-Myc-HC/B), respectively. These chimeras fully maintain the capability of parental BoNTs to bind to the presynaptic membrane of neurons 8 30 and to become endocytosed 12 13 . We found that EGA, used at the concentration which displayed the maximum efficacy in protecting CGNs, does not interfere with the binding and the endocytosis of both BoNT/A and BoNT/B, as assessed by the internalization of their respective derivatives which show the same pattern regardless of drug presence ( Fig. 4a and Figure S2a ). Intriguingly, the two HCs displayed clearly different patterns of staining, suggesting that they may be internalized inside different compartments. Figure 4b and Figure S2b show via western blot analyses the quantitation of the results. We performed this experiment with higher concentrations of HCs to meet the sensitivity requirements of the antibodies used in Western Blot. Consistently, Fig. 4b show that the previous treatment of CGNs with BoNT/D, which cleaves VAMP1/2 thus impairing SVs recycling, significantly decreases the uptake of HC/A, as reported elsewhere 31 32 . At variance, the uptake of HC/B was only partially affected by VAMP1/2 cleavage ( Figure S2b ), leaving open the possibility of a different trafficking of BoNT/B with respect to BoNT/A. Nevertheless, the fact that BoNT/A and BoNT/B use synaptic vesicle proteins as receptors (SV2A/B/C and Synaptotagmin I-II, respectively) strongly suggests that they exploit SVs for their initial step of endocytosis. Accordingly, we decided to test the possible interference of EGA with SVs dynamics using a well-established assay 33 . As shown in Figure S2c , EGA does not affect SVs endocytosis as an antibody specific for the lumenal domain of the synaptic vesicle marker Synaptotagmin I, is internalized at the same extent as controls. On the contrary, if neurons are previously treated with BoNT/D, the uptake of the same antibody is prevented. The quantitation of the result is shown in Figure S2d . Taken together, these results demonstrate that BoNTs binding and internalization through SV cycling are not perturbed by EGA. The BoNT exposure to an intracellular acidic compartment is the next essential step for the neuron intoxication by all BoNTs 2 . Using Lysotracker Red DND-99, a highly sensitive probe of acidic organelles in live cells, we found that EGA does not significantly interfere with the maturation of acidic compartments, both within CGNs cell body and along neurites, where BoNTs act ( Fig. 4c ). At the same time, bafilomycin A1, which prevents BoNTs toxicity by inhibiting the vacuolar-type H + -ATPase proton pump 12 34 35 , completely blocks the acidification of intracellular organelles. This suggests that the essential conditions needed for BoNTs translocation, i.e. an acidic environment, are maintained in the presence of EGA. The final step of the nerve intoxication, the one responsible for neuroparalysis, is the cleavage of SNARE proteins by the L chain. BoNT/A chops off the last 9 amino acids of SNAP25, whereas BoNT/B and BoNT/D cleave at two different sites VAMP1/2. This proteolytic activity can be easily assayed in vitro by using recombinant substrates. As shown in Fig. 4d (left panel), upon reduction of the interchain disulphide bond, BoNT/A cleaves SNAP25, as shown by the shift of its molecular weight in SDS-PAGE (compare lane 1 and 2, upper panels). This activity is however not affected by 12.5 μM EGA ( Fig. 4d , compare lane 2 and 3). The same result was obtained using an antibody specific for the cleaved form of SNAP25 and western blotting as a read out ( Fig. 4d ). Figure S3a and Figure S3b show that similar results were obtained with BoNT/B and BoNT/D, respectively, suggesting that the enzymatic activity of BoNT L chains is not affected by the drug. EGA interferes with BoNTs trafficking within neurons Gillespie et al. (2013) reported that EGA prevents the toxicity of bacterial toxins and viruses by blocking their trafficking from early to late endosomes. Here, we have shown that EGA inhibits BoNTs without interfering with the main events along their mechanism of action. As a consequence, we reasoned that EGA could alter the trafficking of BoNTs after their internalization, possibly preventing them to reach their translocation-competent compartment. If it is the case, EGA should not be capable of inhibiting BoNTs toxicity when their trafficking is bypassed by inducing the entry of the L chain across the plasma membrane of neurons 36 . As this experimental approach strongly depends on the binding to both receptors at the plasma membrane 36 , we could perform this experiment only with BoNT/B, using an established PC12 cell line expressing on the plasma membrane the lumenal domain of Synaptotagmin I, the BoNT/B protein receptor 9 , and with BoNT/D, whose binding domain harbors two ganglioside binding sites 37 , using CGNs 36 . Figure 5a,b show that a low pH jump in the extracellular medium induces the translocation of BoNT/B and BoNT/D L chains across the plasma membrane as evaluated by the cleavage of VAMP2. In agreement with our hypothesis, the same experiment performed in the presence of EGA showed the identical activity of both BoNTs on VAMP2. This suggests that, when these neurotoxins bypass their canonical entry routes, EGA cannot impact on their activity anymore. Taken together the results presented here indicate that EGA prevents the activity of BoNTs by inhibiting their intraneuronal trafficking. EGA interferes with the neuroparalytic activity of BoNT/A, BoNT/B and BoNT/D at the mouse hemidiaphragm assay and in vivo The main aim of the present work was to test the inhibitory capacity of EGA against BoNTs toxicity in vivo . Therefore, after the in vitro approach, we used the mouse hemidiaphragm muscle paralysis model, an ex vivo preparation which represents the standard method to assay the neuroparalytic activity of BoNTs at the neuromuscular junction. In this experimental set up, BoNTs induce a decrease in the twitch capability of the diaphragmatic muscle by exerting its metalloprotease activity within the attached phrenic nerve. This decay is followed over time, and is used to evaluate BoNT potency, but can also be adapted to determine the inhibitory capacity of antitoxins 38 . As shown in Fig. 6(a–c , black traces), BoNT/A, BoNT/B and BoNT/D induce a rapid drop in the twitch capability of the diaphragm muscle. On the other hand, the pre-treatment with 12.5 μM EGA, strongly delays the neuroparalytic activity of the three BoNTs (red traces). This inhibitory effect can be appreciated also by comparing the different parameters reported in Table S1 , and the t 50% values in particular ( Fig. 6d–f ), namely the time needed to halve the muscle twitch capacity, which results greatly increased by the treatment with the drug, and found to be significantly different ( Table S1 ). We then tested the inhibitory effect of EGA in vivo . A wide range of doses from 7.5 mg/kg to 40 mg/kg per day was administered via b.i.d. intraperitoneal injections in mice: even after one week of treatment with this regimen, the drug was well tolerated by mice which did not show any sign of decreased vitality in terms of breathing, eating and drinking nor in terms of motility as compared with vehicle injected controls. The lethality of our preparations of BoNT/A,/B and/D was evaluated in preliminary experiments, and a dose of 0.5 ng/kg (BoNT/A), 0.9 ng/kg (BoNT/B) and 0.045 ng/kg (BoNT/D) was sufficient to progressively induce the classical symptoms of botulism (fur ruffling, sides musculature collapse, generalized weakness, labored breathing) and cause the deadly respiratory failure within 48 hours post injection (black traces of Fig. 6 panels g–i). The red traces of the same figure (panels h and i) show that EGA is particularly efficacious in preventing death from botulism induced by BoNT/B and BoNT/D. Importantly, in those mice that eventually died, the symptoms occurred with delay and were less pronounced. This was the case also for BoNT/A injected mice, where symptoms developed later and were milder, but without a reduced toxin lethality ( Fig. 6g , red trace). High yield synthesis of 4-bromobenzaldehyde N-(2,6-dimethylphenyl) semicarbazone (EGA) The reported approach by Jung in 2014 for the preparation of EGA has been adapted and improved to obtain higher yields. The synthesis involves the three steps reported in Fig. 1 . In the first one (i), 2,6-dimethylaniline (1) is allowed to react with phenyl chloroformate to give the corresponding phenylcarbamate (2), which is next subjected to hydrazinolysis to give semicarbazide (indicated as A) (ii). The final step (iii) is the reaction of A with 4-bromobenzaldehyde to form the desired semicarbazone (3, EGA). The procedure described by Jung et al. (2014) involves the isolation of A and rather drastic conditions (acidic solution and high temperature) in the last step, leading to an overall yield of 27%. A much higher overall yield (84%) is obtained by the new procedure that we have devised: 2 is isolated, whereas steps (ii) and (iii) are performed in one-pot without the isolation of A. Furthermore, much milder conditions are used in the last synthetic step. Details are found in the Supplementary information section . EGA prevents the botulinum neurotoxins cleavage of SNARE proteins in cultured neurons The use of cultured cerebellar granular neurons (CGNs) offers a simple and rapid way to screen the efficacy of candidate molecules in inhibiting BoNTs activity. The overnight incubation with 0.3 nM BoNT/A induces the cleavage of SNAP25, as assessed by the appearance in immunofluorescence ( Fig. 2a , middle panel) and western blot ( Fig. 3a , bottom panel) of its truncated form, revealed with a specific antibody. This toxin concentration is sufficient to induce a complete cleavage of its substrate, as evaluated using an antibody recognizing both forms (intact and truncated) of SNAP25 ( Fig. 3a , middle panel, SMI-81). Figure 3b shows that such activity is however inhibited by EGA in a concentration dependent manner, with a maximal effect at 12.5 μM. The inhibitory effect, though substantial, is not complete as a small amount of cleaved SNAP25 is still generated ( Fig. 2a , right panel and Fig. 3b ). Figures 2b and 3c,d show that similar results are obtained with BoNT/B. Notably, to achieve the substantial cleavage of VAMP2, which is normally concentrated at synaptic contacts ( Fig. 2b , left panel), BoNT/B had to be used at a concentration of 5 nM ( Fig. 2b , middle panel). Nevertheless, the pre-treatment with 12.5 μM EGA is sufficient to abrogate its cleavage ( Fig. 2b , right panel). EGA prevents this toxicity in a concentration dependent manner, as shown by the inhibition of VAMP2 cleavage, determined with two different antibodies ( Fig. 3c middle and bottom panels). Interestingly, despite the high amount of toxin used, in this case the effect of EGA, at higher concentration, is complete ( Fig. 3d ). The same set of experiments was replicated using BoNT/D. This serotype is the most potent in rodents 28 and, in CGNs, a minimal concentration (0.025 pM) induces the almost complete cleavage of VAMP2 ( Fig. 2c , middle panel). Similar to what found for BoNT/A, we found that EGA substantially prevents the action of this potent neurotoxin ( Fig. 2c , right panel), and this inhibition is dependent on the amount of the chemical, as estimated in western blot with an antibody specific for the intact form of VAMP2 ( Fig. 3e , bottom panel and Fig. 3f ). Importantly, Figure S1 shows that neurons viability is not significantly affected, even at the highest concentration of EGA used. EGA does not interfere with the four basic steps of BoNTs' mechanism of action The cellular target of EGA is not known and we investigated whether any of the four main steps of the BoNTs' cellular mechanism of action is directly impacted by the action of the drug. The first step of intoxication is the specific binding of BoNTs to peripheral nerve endings followed by their internalization via endocytosis 2 . Given its chemical nature, EGA could in principle intercalate among lipids and alter the properties of the presynaptic membrane, making it less receptive for BoNTs binding. To investigate this possibility, we took advantage of two constructs consisting of the HC domain of BoNT/A and of BoNT/B fused to a fluorescent protein (cpV-HC/A) or tagged by a c-Myc epitope (c-Myc-HC/B), respectively. These chimeras fully maintain the capability of parental BoNTs to bind to the presynaptic membrane of neurons 8 30 and to become endocytosed 12 13 . We found that EGA, used at the concentration which displayed the maximum efficacy in protecting CGNs, does not interfere with the binding and the endocytosis of both BoNT/A and BoNT/B, as assessed by the internalization of their respective derivatives which show the same pattern regardless of drug presence ( Fig. 4a and Figure S2a ). Intriguingly, the two HCs displayed clearly different patterns of staining, suggesting that they may be internalized inside different compartments. Figure 4b and Figure S2b show via western blot analyses the quantitation of the results. We performed this experiment with higher concentrations of HCs to meet the sensitivity requirements of the antibodies used in Western Blot. Consistently, Fig. 4b show that the previous treatment of CGNs with BoNT/D, which cleaves VAMP1/2 thus impairing SVs recycling, significantly decreases the uptake of HC/A, as reported elsewhere 31 32 . At variance, the uptake of HC/B was only partially affected by VAMP1/2 cleavage ( Figure S2b ), leaving open the possibility of a different trafficking of BoNT/B with respect to BoNT/A. Nevertheless, the fact that BoNT/A and BoNT/B use synaptic vesicle proteins as receptors (SV2A/B/C and Synaptotagmin I-II, respectively) strongly suggests that they exploit SVs for their initial step of endocytosis. Accordingly, we decided to test the possible interference of EGA with SVs dynamics using a well-established assay 33 . As shown in Figure S2c , EGA does not affect SVs endocytosis as an antibody specific for the lumenal domain of the synaptic vesicle marker Synaptotagmin I, is internalized at the same extent as controls. On the contrary, if neurons are previously treated with BoNT/D, the uptake of the same antibody is prevented. The quantitation of the result is shown in Figure S2d . Taken together, these results demonstrate that BoNTs binding and internalization through SV cycling are not perturbed by EGA. The BoNT exposure to an intracellular acidic compartment is the next essential step for the neuron intoxication by all BoNTs 2 . Using Lysotracker Red DND-99, a highly sensitive probe of acidic organelles in live cells, we found that EGA does not significantly interfere with the maturation of acidic compartments, both within CGNs cell body and along neurites, where BoNTs act ( Fig. 4c ). At the same time, bafilomycin A1, which prevents BoNTs toxicity by inhibiting the vacuolar-type H + -ATPase proton pump 12 34 35 , completely blocks the acidification of intracellular organelles. This suggests that the essential conditions needed for BoNTs translocation, i.e. an acidic environment, are maintained in the presence of EGA. The final step of the nerve intoxication, the one responsible for neuroparalysis, is the cleavage of SNARE proteins by the L chain. BoNT/A chops off the last 9 amino acids of SNAP25, whereas BoNT/B and BoNT/D cleave at two different sites VAMP1/2. This proteolytic activity can be easily assayed in vitro by using recombinant substrates. As shown in Fig. 4d (left panel), upon reduction of the interchain disulphide bond, BoNT/A cleaves SNAP25, as shown by the shift of its molecular weight in SDS-PAGE (compare lane 1 and 2, upper panels). This activity is however not affected by 12.5 μM EGA ( Fig. 4d , compare lane 2 and 3). The same result was obtained using an antibody specific for the cleaved form of SNAP25 and western blotting as a read out ( Fig. 4d ). Figure S3a and Figure S3b show that similar results were obtained with BoNT/B and BoNT/D, respectively, suggesting that the enzymatic activity of BoNT L chains is not affected by the drug. EGA interferes with BoNTs trafficking within neurons Gillespie et al. (2013) reported that EGA prevents the toxicity of bacterial toxins and viruses by blocking their trafficking from early to late endosomes. Here, we have shown that EGA inhibits BoNTs without interfering with the main events along their mechanism of action. As a consequence, we reasoned that EGA could alter the trafficking of BoNTs after their internalization, possibly preventing them to reach their translocation-competent compartment. If it is the case, EGA should not be capable of inhibiting BoNTs toxicity when their trafficking is bypassed by inducing the entry of the L chain across the plasma membrane of neurons 36 . As this experimental approach strongly depends on the binding to both receptors at the plasma membrane 36 , we could perform this experiment only with BoNT/B, using an established PC12 cell line expressing on the plasma membrane the lumenal domain of Synaptotagmin I, the BoNT/B protein receptor 9 , and with BoNT/D, whose binding domain harbors two ganglioside binding sites 37 , using CGNs 36 . Figure 5a,b show that a low pH jump in the extracellular medium induces the translocation of BoNT/B and BoNT/D L chains across the plasma membrane as evaluated by the cleavage of VAMP2. In agreement with our hypothesis, the same experiment performed in the presence of EGA showed the identical activity of both BoNTs on VAMP2. This suggests that, when these neurotoxins bypass their canonical entry routes, EGA cannot impact on their activity anymore. Taken together the results presented here indicate that EGA prevents the activity of BoNTs by inhibiting their intraneuronal trafficking. EGA interferes with the neuroparalytic activity of BoNT/A, BoNT/B and BoNT/D at the mouse hemidiaphragm assay and in vivo The main aim of the present work was to test the inhibitory capacity of EGA against BoNTs toxicity in vivo . Therefore, after the in vitro approach, we used the mouse hemidiaphragm muscle paralysis model, an ex vivo preparation which represents the standard method to assay the neuroparalytic activity of BoNTs at the neuromuscular junction. In this experimental set up, BoNTs induce a decrease in the twitch capability of the diaphragmatic muscle by exerting its metalloprotease activity within the attached phrenic nerve. This decay is followed over time, and is used to evaluate BoNT potency, but can also be adapted to determine the inhibitory capacity of antitoxins 38 . As shown in Fig. 6(a–c , black traces), BoNT/A, BoNT/B and BoNT/D induce a rapid drop in the twitch capability of the diaphragm muscle. On the other hand, the pre-treatment with 12.5 μM EGA, strongly delays the neuroparalytic activity of the three BoNTs (red traces). This inhibitory effect can be appreciated also by comparing the different parameters reported in Table S1 , and the t 50% values in particular ( Fig. 6d–f ), namely the time needed to halve the muscle twitch capacity, which results greatly increased by the treatment with the drug, and found to be significantly different ( Table S1 ). We then tested the inhibitory effect of EGA in vivo . A wide range of doses from 7.5 mg/kg to 40 mg/kg per day was administered via b.i.d. intraperitoneal injections in mice: even after one week of treatment with this regimen, the drug was well tolerated by mice which did not show any sign of decreased vitality in terms of breathing, eating and drinking nor in terms of motility as compared with vehicle injected controls. The lethality of our preparations of BoNT/A,/B and/D was evaluated in preliminary experiments, and a dose of 0.5 ng/kg (BoNT/A), 0.9 ng/kg (BoNT/B) and 0.045 ng/kg (BoNT/D) was sufficient to progressively induce the classical symptoms of botulism (fur ruffling, sides musculature collapse, generalized weakness, labored breathing) and cause the deadly respiratory failure within 48 hours post injection (black traces of Fig. 6 panels g–i). The red traces of the same figure (panels h and i) show that EGA is particularly efficacious in preventing death from botulism induced by BoNT/B and BoNT/D. Importantly, in those mice that eventually died, the symptoms occurred with delay and were less pronounced. This was the case also for BoNT/A injected mice, where symptoms developed later and were milder, but without a reduced toxin lethality ( Fig. 6g , red trace). Discussion The main result reported here is simple and very relevant at the same time. EGA is a potent inhibitor of the neuroparalytic activity of botulinum neurotoxins in vitro and in vivo , at doses that cause no apparent toxicity. This result indicates that EGA is the lead of a novel class of inhibitors potentially capable of preventing the activity of BoNTs in humans. This is the more relevant considering that the recent years have witnessed the discovery of a large number of novel BoNTs, with different immunoreactivity 2 39 40 , suggesting the possibility of the identification of BoNT variants that may be poorly neutralized by currently available antisera. This situation calls for the discovery of inhibitors capable of preventing the activity of all BoNTs. Necessarily, these novel inhibitors must be non-toxic to humans and must be effective in vivo . Notwithstanding long efforts of many laboratories, this goal has only partially been achieved 41 . We recently reported on inhibitors of the Thioredoxin reductase–Thioredoxin redox couple that effectively prevent the neuroparalytic activity of all BoNT serotypes without causing toxic effects in mice 19 20 42 Here we add another lead compound with a different mechanism of inhibition. Despite our efforts using primary cultures of neurons and neuromuscular junction preparation, we have not identified the target of EGA, but we did not note toxic effects in mice treated with a dose that largely prevents the action of the three BoNTs used here. We have found that the main steps of BoNTs mechanism of action, i.e. binding, internalization, acidification of intracellular compartment, L chain translocation, disulphide reduction and substrate proteolysis, are not affected by this compound ( Figs 4 and 5 , Figure S2 and Figure S3 ). Notably, the range of concentration that block BoNTs in cultured neurons is the same previously found to inhibit the toxicity of different toxins and viruses in primary and immortalized macrophages. This suggests that, rather than having a direct effect on BoNTs (or on the other pathogens), EGA interferes with an intracellular host target responsible for their trafficking. This conclusion is reinforced by the result showing that EGA had no effect on the translocation of the L chain from the plasma membrane, when the canonical internalization route was bypassed ( Fig. 5 ). All known protein receptors of BoNTs are the lumenal domains of integral proteins of synaptic vesicles which suggests the general conclusion that all BoNTs are endocytosed inside these organelles at nerve terminals. However, the following trafficking of synaptic vesicles is not fully understood, though there is evidence that they may fuse with synaptic endosomes where they are quality controlled and then released to re-enter the synaptic vesicle cycle 43 44 45 46 47 48 . As a consequence, the fact that the three different serotypes considered here are differently protected by EGA, which inhibits the maturation of early endosomes 27 , is an interesting aspect of the current study, because it revives the possibility that different BoNT may be trafficked through different routes inside the nerve terminals. Indeed, the diverse protein receptors of BoNTs may account for distinct fates of each toxin-receptor complexes, which have not yet been determined case by case. An alternative explanation is suggested by the finding that part of BoNT/A may enter terminals independently from SVs endocytosis 31 32 , which is supported by studies showing that BoNTs display toxicity independently of the stimulation of SVs recycling 34 49 50 51 52 53 . The fact that EGA completely inhibits the activity of BoNT/B, although used at a concentration much higher than that of BoNT/A, opens the possibility that the activity of this toxin is dependent on a trafficking through endosomes and does not translocate its catalytic part into the cytosol across the SV membrane. This is a surprising finding which was unexpected on the basis of the knowledge that the SV protein synaptotagmin mediates the entry of BoNT/B 8 9 . However, considering that synaptotagmin can be trafficked through early endosome 54 , the possibility that also BoNT/B may need the passage through this organelle to reach a membrane translocation-competent compartment becomes plausible. It is also in keeping with its slow time course of entry into cultured neurons as compared with other serotypes 34 55 . Moreover, a considerable amount of synaptotagmin molecules remains exposed on the plasma membrane surface, in a steady-state with those recycled through sorting endosomes 56 , which makes possible that BoNT/B forms a toxin-receptor complex on the plasma membrane, rather than within SVs. This fits well with the present findings that: i) the internalization of c-Myc-HC/B was much less affected compared to that of cpV-HC/A, by the pre-treatment with BoNT/D ( Fig. 4b and Figure S2b ) and ii ) the different staining pattern of the BoNT/A and BoNT/B binding domains ( Fig. 4a and Figure S2a ). This possibility is also supported by the in vivo finding that EGA has a remarkable effect against the lethality of BoNT/B and a lower one on BoNT/A ( Fig. 6g,h ). The behavior of BoNT/D in response to inhibition of the endosomal pathway by EGA, in cultured neurons is more similar to that of BoNT/A rather than BoNT/B, as VAMP2 cleavage was not completely prevented ( Figs 2c and 3e,f ). On the other hand, BoNT/D was efficaciously inhibited by EGA in vivo, with an inhibitory profile similar to that of BoNT/B ( Fig. 6i ). The mechanism of BoNT/D binding to neurons is poorly understood and therefore its internalization and trafficking properties are not entirely clear 37 57 , and as a consequence it is even more difficult to envisage how this toxin could be internalized and trafficked. The obtained results clearly show that the observations of cell culture experiments cannot be transferred tout court to in vivo conditions. The present lack of knowledge on the biochemical target of EGA does not prevent research aimed at finding more potent inhibitors of the BoNT neuroparalytic action. Clearly, EGA action is a preventive one, as it cannot affect those L chains that have already translocated in the cytosol. Nevertheless, it can alleviate the symptoms of botulism after diagnosis because a considerable amount of BoNT remains in the general circulation of botulism patient for weeks after the first diagnosis 58 59 60 . Perhaps, more importantly, the present findings are relevant for infant botulism where a continuous entry of BoNT into the general circulation occurs via adsorption of the toxin produced by Clostridia that have colonized the gastrointestinal tract of infants owing to the reduced intestinal flora competing with Clostridia 2 61 . We would like to conclude by pointing out that the search for novel EGA-derived analogues is made simpler by the design of the novel method of synthesis of this compound described here, which provides a much higher yield with respect to the recently described method 29 . This procedure allowed us to rapidly and efficiently synthesize large quantities of EGA, an essential pre-requisite to produce the considerable amount necessary for a possible employment of this or related compounds in humans. Methods Chemical Synthesis Detailed protocol for EGA chemical synthesis is available in Supplementary Information . Botulinum neurotoxin inhibition assay EGA was dissolved in DMSO to prepare a stock solution (12.5 mM). CGNs at 6–8 days in vitro (DIV) were treated for 30 min with the indicated concentrations of EGA in complete culture medium at 37 °C and 5% CO 2 . 0.3 nM BoNT/A, 5 nM BoNT/B or 0.025 pM BoNT/D was added, in the presence of the same concentration of inhibitor, and left for 12 hr at 37 °C and 5% CO 2 . Further details can be found in the Supplementary Information . cpV-HC/A and c-Myc-HC/B binding assay CGNs were treated with EGA 12.5 μM or vehicle (DMSO) in culture medium at 37 °C. After 30 min, for immunocytochemistry experiments, 100 nM cpV-HC/A or c-Myc-HC/B was added in stimulating culture medium (complete culture medium, 57 mM KCl), for 1 hr. The same protocol was used with 250 nM of cpV-HC/A or c-Myc-HC/B but neurons were then lysed and immunoblotted to obtain a quantitative result. Details are in the Supplementary Information . Low pH induced translocation of BoNT/B and BoNT/D across the plasma membrane Experiment was conducted as previously described 36 . Detailed protocol is available in Supplementary Information . Mouse diaphragm and lethality assay All experiments were performed in accordance with the European Communities Council Directive n° 2010/63/UE and approved by the Italian Ministry of Health. Mouse diaphragms were isolated from CD-1 mice weighing about 20–25 g and halved into two contralateral hemi-diaphragms still innervated with the own phrenic nerve, and were treated as described in the Supplemental Experimental Procedures . Lethality assays were performed using Swiss-Webster adult male CD1 mice weighing 26–28 g as described in Supplementary Information . Statistical analysis For all the experiments, data are presented as mean values. Bars indicated the standard deviation. Significance was calculated by Student's t test (unpaired, two-side). *p < 0.05, **p < 0.01, ***p < 0.0001. Only values below 0.05 were considered significant (ns – non significant). Chemical Synthesis Detailed protocol for EGA chemical synthesis is available in Supplementary Information . Botulinum neurotoxin inhibition assay EGA was dissolved in DMSO to prepare a stock solution (12.5 mM). CGNs at 6–8 days in vitro (DIV) were treated for 30 min with the indicated concentrations of EGA in complete culture medium at 37 °C and 5% CO 2 . 0.3 nM BoNT/A, 5 nM BoNT/B or 0.025 pM BoNT/D was added, in the presence of the same concentration of inhibitor, and left for 12 hr at 37 °C and 5% CO 2 . Further details can be found in the Supplementary Information . cpV-HC/A and c-Myc-HC/B binding assay CGNs were treated with EGA 12.5 μM or vehicle (DMSO) in culture medium at 37 °C. After 30 min, for immunocytochemistry experiments, 100 nM cpV-HC/A or c-Myc-HC/B was added in stimulating culture medium (complete culture medium, 57 mM KCl), for 1 hr. The same protocol was used with 250 nM of cpV-HC/A or c-Myc-HC/B but neurons were then lysed and immunoblotted to obtain a quantitative result. Details are in the Supplementary Information . Low pH induced translocation of BoNT/B and BoNT/D across the plasma membrane Experiment was conducted as previously described 36 . Detailed protocol is available in Supplementary Information . Mouse diaphragm and lethality assay All experiments were performed in accordance with the European Communities Council Directive n° 2010/63/UE and approved by the Italian Ministry of Health. Mouse diaphragms were isolated from CD-1 mice weighing about 20–25 g and halved into two contralateral hemi-diaphragms still innervated with the own phrenic nerve, and were treated as described in the Supplemental Experimental Procedures . Lethality assays were performed using Swiss-Webster adult male CD1 mice weighing 26–28 g as described in Supplementary Information . Statistical analysis For all the experiments, data are presented as mean values. Bars indicated the standard deviation. Significance was calculated by Student's t test (unpaired, two-side). *p < 0.05, **p < 0.01, ***p < 0.0001. Only values below 0.05 were considered significant (ns – non significant). Additional Information How to cite this article : Azarnia Tehran, D. et al. A Novel Inhibitor Prevents the Peripheral Neuroparalysis of Botulinum Neurotoxins. Sci. Rep. 5 , 17513; doi: 10.1038/srep17513 (2015). Supplementary Material Supplementary Information

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757978/

Anthrax Lethal Toxin Enhances IκB Kinase Activation and Differentially Regulates Pro-inflammatory Genes in Human Endothelium *

Anthrax lethal toxin (LT) was previously shown to enhance transcriptional activity of NF-κB in tumor necrosis factor-α-activated primary human endothelial cells. Here we show that this LT-mediated increase in NF-κB activation is associated with the enhanced degradation of the inhibitory proteins IκBα and IκBβ but not IκBϵ. Moreover, this was accompanied by enhanced activation of the IκB kinase complex (IKK), which is responsible for targeting IκB proteins for degradation. Importantly, LT enhancement of IκBα degradation was completely blocked by a selective IKKβ inhibitor, whereas IκBβ degradation was attenuated, suggesting a mechanistic link. Consistent with the above data, LT-cotreated cells show elevated phosphorylation of two IKK substrates, IκBα and p65, both of which were blocked by incubation with the IKKβ inhibitor. Consistent with NF-κB activation, LT increased transcription of the NF-κB regulated gene CD40 . Conversely, LT inhibited transcription of another NF-κB-regulated gene, CCL2 . This inhibition was linked to the LT-mediated suppression of another CCL2 -regulating transcription factor, AP-1 (activator protein-1). These data suggest that LT-mediated enhancement of NF-κB is IKK-dependent, but importantly, the net effect of LT on the transcription of proinflammatory genes is driven by the cumulative effect of LT on the particular set of transcription factors that regulate a given promoter. Together, these findings provide new mechanistic insight on how LT may disrupt the host response to anthrax.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125954/

Bioterrorism: Challenges and considerations

Bioterrorism, the deliberate, private use of biological agents to harm and frighten the people of a state or society, is related to the military use of biological, chemical, and nuclear weapons. Attacks with biological agents are among the most insidious and breed the greatest fear. Attacks could go undetected for a long time, potentially exposing a vast number of people, who are unaware of the threat. Dentist's responses to catastrophes have been redefined by bioterrorism. Accurate and substantial information given to the public by credible public health and medical experts can do much to allay their fears and encourage their cooperation and participation in constructive, organized community response efforts. The dental profession could potentially play a significant role in the emergency response to a major bioterrorism attack. Introduction Bioterrorism covers a very broad spectrum of concerns, from catastrophic terrorism with mass casualties, to microevents using low technology but producing civil unrest, disruption, disease, disabilities, and death.[ 1 ] The threat of bioterrorism, long ignored and denied, has heightened over the past few years. We are ill prepared to deal with a terrorist attack that employs biological weapons. As was done in response to the nuclear threat, the medical community should educate the public and policy makers about the threat. In the longer term, we need to be prepared to detect, diagnose, characterize epidemiologically, and respond appropriately to biological weapons use and the threat of new and re-emerging infections. On the immediate horizon, we cannot delay the development and implementation of strategic plans for coping with civilian bioterrorism.[ 2 ] History The use of biological weapons for terror is ancient. Assyrian politicians dumped fungus from rye into their opponents′ wells, giving them fatal ergot poisoning in 650 BC.[ 3 ] Armies besieging a town relied on increased disease among the defending population and threw dead animals into water supplies, to spread it.[ 3 ] Tatars of the 14th century spread bubonic plague by catapulting diseased corpses into towns.[ 3 ] In World War I, United States and Germany developed biological weapons to contaminate animal fodder.[ 3 ] In Cold War, United States and Soviet Union created arsenals of biological agents for use in battle and against civilian populations.[ 3 ] Dr. Anton Dilger worked with cultures of anthrax and glanders, between 1915 and 1916, with the intention of biological sabotage on behalf of the German Government.[ 4 ] Modern bioterrorist incidents In 1984, pseudo-Buddhist Rajneeshee cult distributed Salmonella in restaurants and grocery stores in Oregon to poison civic leaders and gain control of the local Government.[ 3 ] In 1992, Russia had the ability to launch missiles containing weapons-grade small pox. A number of terrorist organizations, including Al-Qaeda, have explored the use of biological agents.[ 3 ] In 1995, Sarin gas was released in a Tokyo subway, by the religious sect Aum Shinrikyo, which immediately killed 12 and hospitalized 5000 people.[ 3 ] In 2001, letters containing anthrax spores were mailed to a television news anchor, US senator, and others, leading to the death of a few people and hospitalization of a few others.[ 3 ] Biological Agents The Center for Disease Control and Prevention ranks the biological agents and diseases that have the potential to be used as weapons into 3 categories.[ 5 6 ] Category A: These agents are characterized by ease of dissemination and transmission of disease with high mortality rate, likelihood of causing public panic and social disruption [ Table 1 ]. Category B: These agents disseminate less easily, have lower morbidity and mortality rate [ Table 2 ]. Category C: Tan viruses comprise this category. These could be used for mass dissemination in the future because of their availability, ease of production, dissemination, and high morbidity and mortality rates. Table 1 Category A biological agents (CDC) Table 2 Category B biological agents (CDC) Role of a dentist in response to a bioterrorism attack Dentistry can contribute valuable assets, both in personnel and in facilities, to the preparation for and in the immediate response to a bioterrorist attack and its aftermath. These assets can make a significant difference in the outcome. In a major bioterrorist attack, the local needs could be massive and immediate. As hospitals become filled, alternate sites for the provision of health care may be required, and dental offices could fill that need.[ 7 ] Preparation before an attack Education of the dental profession regarding the medical and oral manifestations of diseases that may result from a bioterrorist attack will be important. Formal plans for an organized response by dental personnel in case of an attack must be developed, integrated into each community's response plan, and practiced periodically. Dental offices are equipped with potentially useful equipment and supplies and should be prepared to serve as decentralized auxiliary hospitals in case the need arises. Educational programs that provide information about potential biological weapons should be developed and made available to dentists through continuing education courses and to dental students as a part of the dental school curriculum.[ 7 ] Up-to-date sources of information should be developed that can be accessed quickly during an attack and reference materials that can be distributed for use as needed. These quick references should be able to provide dentists with a sufficient level of information concerning the particular agent used in an attack to enable them to respond effectively. Dentists have contact with the general public on a regular basis. Armed with knowledge and connected to scientifically based information sources about agents that may be used in bioterrorism, dentists can educate their patients and correct misinformation that may be circulating throughout the general public. Special training may be needed for risk communication. Dental offices are located throughout any given community and have many of the resources that hospital facilities have: sterilization equipment, air and gas lines, suction equipment, radiology capabilities, instruments, and needles. They may be called on to serve as local "minihospitals" when local hospital facilities become overwhelmed or when the concentration of patients is to be avoided, as in attacks involving contagious agents. Predesignated dental offices may act as stockpiling sites for materials and supplies to be distributed in the event of an attack.[ 7 ] The key to successful preparation for an effective response to a major bioterrorism attack is the development of a response plan that is integrated into each community's disaster response plan and testing it by conducting mock attacks.[ 7 ] Assistance during an attack The assistance that dentists and other dental personnel can provide during the first few days of a significant bioterrorist attack will vary according to the needs of the community and the resources available. These may run the gamut from the packaging of medications in individual doses to providing a major portion of primary medical care in a quarantined area if physicians are unavailable because they have become disabled or have died.[ 7 ] In 1999, the University of Pittsburgh's Center for Biomedicalnone Informatics deployed the first automated bioterrorism detection system, called Real-Time Outbreak Disease Surveillance (RODS). RODS is designed to collect data from many data sources and use them to perform signal detection, that is, to detect a possible bioterrorism event at the earliest possible moment. RODS, and other systems similar to it, collect data from sources, including clinic data, laboratory data, and data from over-the-counter drug sales.[ 8 ] Role of a dentist in response to a bioterrorism attack Dentistry can contribute valuable assets, both in personnel and in facilities, to the preparation for and in the immediate response to a bioterrorist attack and its aftermath. These assets can make a significant difference in the outcome. In a major bioterrorist attack, the local needs could be massive and immediate. As hospitals become filled, alternate sites for the provision of health care may be required, and dental offices could fill that need.[ 7 ] Preparation before an attack Education of the dental profession regarding the medical and oral manifestations of diseases that may result from a bioterrorist attack will be important. Formal plans for an organized response by dental personnel in case of an attack must be developed, integrated into each community's response plan, and practiced periodically. Dental offices are equipped with potentially useful equipment and supplies and should be prepared to serve as decentralized auxiliary hospitals in case the need arises. Educational programs that provide information about potential biological weapons should be developed and made available to dentists through continuing education courses and to dental students as a part of the dental school curriculum.[ 7 ] Up-to-date sources of information should be developed that can be accessed quickly during an attack and reference materials that can be distributed for use as needed. These quick references should be able to provide dentists with a sufficient level of information concerning the particular agent used in an attack to enable them to respond effectively. Dentists have contact with the general public on a regular basis. Armed with knowledge and connected to scientifically based information sources about agents that may be used in bioterrorism, dentists can educate their patients and correct misinformation that may be circulating throughout the general public. Special training may be needed for risk communication. Dental offices are located throughout any given community and have many of the resources that hospital facilities have: sterilization equipment, air and gas lines, suction equipment, radiology capabilities, instruments, and needles. They may be called on to serve as local "minihospitals" when local hospital facilities become overwhelmed or when the concentration of patients is to be avoided, as in attacks involving contagious agents. Predesignated dental offices may act as stockpiling sites for materials and supplies to be distributed in the event of an attack.[ 7 ] The key to successful preparation for an effective response to a major bioterrorism attack is the development of a response plan that is integrated into each community's disaster response plan and testing it by conducting mock attacks.[ 7 ] Assistance during an attack The assistance that dentists and other dental personnel can provide during the first few days of a significant bioterrorist attack will vary according to the needs of the community and the resources available. These may run the gamut from the packaging of medications in individual doses to providing a major portion of primary medical care in a quarantined area if physicians are unavailable because they have become disabled or have died.[ 7 ] In 1999, the University of Pittsburgh's Center for Biomedicalnone Informatics deployed the first automated bioterrorism detection system, called Real-Time Outbreak Disease Surveillance (RODS). RODS is designed to collect data from many data sources and use them to perform signal detection, that is, to detect a possible bioterrorism event at the earliest possible moment. RODS, and other systems similar to it, collect data from sources, including clinic data, laboratory data, and data from over-the-counter drug sales.[ 8 ] Surveillance and Notification Disease surveillance systems are critical not only for the initial detection of an outbreak but also for monitoring the extent and spread of the outbreak and for determining when it is over. Managing a large outbreak would require gathering information from contact tracing and source-of-exposure investigations, as well as information about the availability of critical medicine, medical equipment, and the handling of corpses.[ 9 ] Since there is incubation period before the clinical manifestations of diseases that have been used as weapons in bioterrorist attacks become apparent, the initial recognition that an attack has been perpetrated may be difficult. Dentists can serve as an excellent surveillance resource, as they can detect characteristic intraoral or cutaneous lesions, if they are present and report them to public health authorities. They also may be able to detect unusual patterns of employee absences or patients′ cancelling or missing appointments that are not explainable by recognizable local circumstances. These occurrences may well be a harbinger of serious events about to happen.[ 7 ] Diagnosis and Monitoring Besides assisting in the early identification of the disease or diseases introduced in a bioterrorist attack, dentists can provide individual patient diagnosis by observing the physical and behavioral signs people manifest when the nature of the attack has been determined. Salivary swabs may yield important diagnostic or treatment information and can be collected by dentists for laboratory testing to determine diagnosis when necessary or to monitor treatment progress.[ 7 ] Referral Dentists can refer suspicious cases to the appropriate specialists for confirmation, treatment, or both.[ 7 ] Immunizations In the event that rapid inoculation or vaccination of the public is required to prevent the spread of infection by a biological agent, dentists may be recruited to assist in a mass inoculation program.[ 7 ] Triage Whenever there are a greater number of casualties that the medical care system cannot accommodate or whenever medical care resources are overwhelmed, some system for establishing priorities for treatment must be established. Appropriately trained dentists can fulfill this function, thus freeing up medical professionals to provide definitive care for the large number of patients. This system should be established now, in preparation for potential future attacks.[ 7 ] Medical care augmentation Because of their training and experience, many dentists may be able to augment and assist medical and surgical personnel in providing definitive treatment for victims of bioterrorist attacks. Some of the services dentists may provide include the following: treatment of cranial and facial injuries; providing or assisting in administration of anesthesia; starting intravenous lines; performing appropriate surgery and suturing; assisting in shock management; assisting in stabilizing patients; collecting preantibiotic blood samples; taking medical histories; and providing cardiopulmonary resuscitation.[ 7 ] Referral Dentists can refer suspicious cases to the appropriate specialists for confirmation, treatment, or both.[ 7 ] Immunizations In the event that rapid inoculation or vaccination of the public is required to prevent the spread of infection by a biological agent, dentists may be recruited to assist in a mass inoculation program.[ 7 ] Triage Whenever there are a greater number of casualties that the medical care system cannot accommodate or whenever medical care resources are overwhelmed, some system for establishing priorities for treatment must be established. Appropriately trained dentists can fulfill this function, thus freeing up medical professionals to provide definitive care for the large number of patients. This system should be established now, in preparation for potential future attacks.[ 7 ] Medical care augmentation Because of their training and experience, many dentists may be able to augment and assist medical and surgical personnel in providing definitive treatment for victims of bioterrorist attacks. Some of the services dentists may provide include the following: treatment of cranial and facial injuries; providing or assisting in administration of anesthesia; starting intravenous lines; performing appropriate surgery and suturing; assisting in shock management; assisting in stabilizing patients; collecting preantibiotic blood samples; taking medical histories; and providing cardiopulmonary resuscitation.[ 7 ] Decontamination and Infection Control Dentists and dental auxiliaries are well versed in infection control procedures and can apply their knowledge in reducing the spread of infections—between patients and between patients and caregivers—in mass disasters. The decontamination of casualties, when appropriate, can be accomplished effectively by dental personnel. Dentists who have experience in practicing in a hospital setting may be especially valuable and may be particularly equipped to provide services that require a close working relationship with physicians.[ 7 ] After the initial attack Dentists trained in forensic odontology will work closely with local Disaster Mortuary Operational Response Teams, (DMORTs). Dentists also may provide local surveillance to detect any spreading of disease beyond the original area of attack or re-emergence of infections in the original attack area.[ 7 ] After the initial attack Dentists trained in forensic odontology will work closely with local Disaster Mortuary Operational Response Teams, (DMORTs). Dentists also may provide local surveillance to detect any spreading of disease beyond the original area of attack or re-emergence of infections in the original attack area.[ 7 ] Conclusion Terrorism with biological weapons is likely to remain rare. Because the magnitude of the threat is so difficult to calculate, however, it is sensible to focus on dual-use remedies: pursuing medical countermeasures that will improve public health in general, regardless of whether major biological attacks ever occur. This would include strengthening the international system of monitoring disease outbreaks in humans, animals, and plants and developing better pharmaceutical drugs.[ 10 ] The current public discussion of the threat of biologic terrorism is an opportunity to evaluate our collective capabilities and to assess weaknesses and vulnerabilities. Raising the level of national preparedness will require leadership and action by responsible federal agencies. A thoughtful analysis of the consequences of unpreparedness provides a mandate for action.[ 11 ] For longer-term solutions, the medical community must educate both the public and policy makers about bioterrorism and build a global consensus condemning its use.[ 2 ] Dentists can provide a valuable service to their patients and communities by providing quality information about the potential for attacks, what to watch for, and how to respond appropriately should an attack occur.[ 12 ]

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531067/

A comparative study of international and Chinese public health emergency management from the perspective of knowledge domains mapping

Background At the end of 2019, the outbreak of coronavirus disease 2019 (COVID-19) severely damaged and endangered people's lives. The public health emergency management system in China has played an essential role in handling the response to the outbreak, which has been appreciated by the World Health Organization and some countries. Hence, it is necessary to conduct an overall analysis of the development of the health emergency management system in China. This can provide a reference for scholars to aid in understanding the current situation and to reveal new research topics. Methods We collected 2247 international articles from the Web of Science database and 959 Chinese articles from the China National Knowledge Infrastructure database. Bibliometric and mapping knowledge domain analysis methods were used in this study for temporal distribution analysis, cooperation network analysis, and co-word network analysis. Results The first international article in this field was published in 1991, while the first Chinese article was published in 2005. The research institutions producing these studies mainly existed in universities and health organizations. Developed countries and European countries published the most articles overall, while eastern China published the most articles within China. There were 52 burst words for international articles published from 1999–2018 and 18 burst words for Chinese articles published from 2003–2018. International top-ranked articles according to the number of citations appeared in 2005, 2007, 2009, 2014, 2015, and 2016, while the corresponding Chinese articles appeared in 2003, 2004, 2009, and 2011. Conclusions There are differences in the regional and economic distribution of international and Chinese cooperation networks. International research is often related to timely issues mainly by focusing on emergency preparedness and monitoring of public health events, while China has focused on public health emergencies and their disposition. International research began on terrorism and bioterrorism, followed by disaster planning and emergency preparedness, epidemics, and infectious diseases. China considered severe acute respiratory syndrome as the starting research background and the legal system construction as the research starting point, which was followed by the mechanism, structure, system, and training abroad for public health emergency management. Background At the end of 2019, the outbreak of coronavirus disease 2019 (COVID-19) severely damaged and endangered people's lives. The public health emergency management system in China has played an essential role in handling the response to the outbreak, which has been appreciated by the World Health Organization and some countries. Hence, it is necessary to conduct an overall analysis of the development of the health emergency management system in China. This can provide a reference for scholars to aid in understanding the current situation and to reveal new research topics. Methods We collected 2247 international articles from the Web of Science database and 959 Chinese articles from the China National Knowledge Infrastructure database. Bibliometric and mapping knowledge domain analysis methods were used in this study for temporal distribution analysis, cooperation network analysis, and co-word network analysis. Results The first international article in this field was published in 1991, while the first Chinese article was published in 2005. The research institutions producing these studies mainly existed in universities and health organizations. Developed countries and European countries published the most articles overall, while eastern China published the most articles within China. There were 52 burst words for international articles published from 1999–2018 and 18 burst words for Chinese articles published from 2003–2018. International top-ranked articles according to the number of citations appeared in 2005, 2007, 2009, 2014, 2015, and 2016, while the corresponding Chinese articles appeared in 2003, 2004, 2009, and 2011. Conclusions There are differences in the regional and economic distribution of international and Chinese cooperation networks. International research is often related to timely issues mainly by focusing on emergency preparedness and monitoring of public health events, while China has focused on public health emergencies and their disposition. International research began on terrorism and bioterrorism, followed by disaster planning and emergency preparedness, epidemics, and infectious diseases. China considered severe acute respiratory syndrome as the starting research background and the legal system construction as the research starting point, which was followed by the mechanism, structure, system, and training abroad for public health emergency management. Background Public health emergencies have increased in recent years and have shown a trend of causing considerable damage [ 1 ]. According to the emergency events database (EM-DAT), the most widely used and influential disaster database in the world, the average number of deaths per major public health event was more than 10,000 [ 2 ]. Population growth, urban development, migration, and other issues brought about by globalization have sped up the incidence of public health events, such as epidemics [ 3 , 4 ]. Public health events also propelled the process of emergency management, giving top priority to changes in emergency operations. The outbreak of coronavirus disease 2019 (COVID-19) in China spread rapidly throughout the world in a short time, which illustrated the need to build a resilient health emergency system that can withstand epidemics [ 5 ]. Public health emergency management (PHEM) is a relatively new field that draws on specific sets of knowledge, techniques, and organizing principles found in emergency management [ 6 ]. Specifically, it includes public health emergency planning, organization, leadership, coordination, control, evaluation, prevention, preparation, and response [ 7 ]. For COVID-19, China's PHEM system quickly took the following measures: emergency mobilization measures within the government, lockdown of cities and communities, nationwide medical mobilization, provision of financial support, preferential policies for the medical community and pharmaceutical industry, and the categorical comprehensive publicity to spread prevention and treatment knowledge [ 8 ]. These measures effectively reduced the spread of the disease. Thus, current recommendations are mostly derived from the reported Chinese experience [ 9 ]. Given the weaknesses and deficiencies exposed by the COVID-19 outbreak, people have recognized the need to improve the national PHEM system [ 10 , 11 ]. A growing body of research has studied PHEM from different perspectives, mainly those of institutions, funds, technologies, and laws. The public health emergency was a severe challenge to health institutions such as hospitals, the Centers for Disease Control and Prevention (CDC), and governments [ 7 , 12 ]. The solutions to these challenges were characterized by sustainability, redundancy, and flexibility [ 13 ]. Monetary and technology resources can merge the roles and responsibilities of public health preparedness and emergency management [ 14 ]. Severe deficiencies in legal preparedness can undermine effective responses to public health emergencies [ 15 , 16 ]. These were the essential factors in dealing with public health emergencies. Additionally, many countries took corresponding measures to strengthen the emergency management of public health. For example, the USA established PHEM operations centers either independently in health departments or as a part of the overall command system in the government [ 17 ]. China established the PHEM system from the national level to the local level to be responsible for emergency preparedness and response in 2004 [ 17 ]. In March 2018, the Ministry of Emergency Management of the People's Republic of China was established, which was an integral part of the State Council. Thus, we can see that PHEM is still a timely topic for scholars and governments [ 18 , 19 ]. However, there are still some problems that need to be solved. To the best of our knowledge, there is little evidence about the differences that occurred between international and Chinese PHEM. Moreover, what are the hotspots and trends of PHEM? What are the main research forces of PHEM? It is necessary to sort out the characteristics of the development of PHEM and explore the hotspots of PHEM research. Additionally, we compared international and Chinese research on PHEM. Based on this situation, we reviewed the articles on PHEM that were published over the past nearly 30 years in international and Chinese journals. Then, we used the knowledge map method to reveal the research strengths, frontiers, and development trends in this field. Study conclusions are helpful to draw people's attention to public health emergencies, provide a reference for scholars to understand the current situation and trends of PHEM, and for government departments to formulate guidance strategies. Methods Data sources Data were divided into two categories: international and Chinese data. According to the relevant authoritative research [ 20 ], the database of bibliometric methods should contain complete documents. A considerable amount of literature has shown that the Web of Science (WoS) is the largest comprehensive academic information resource, covering peer-reviewed journals with high impact factors [ 21 – 24 ]. Accordingly, the international data used for our study were collected from the WoS Core Collection, including Science Citation Index Expanded (SCI-E), Science Citation Index Expanded (SSCI), and Arts & Humanities Citation Index (A&HCI) databases. Chinese data were downloaded from the China National Knowledge Infrastructure (CNKI), which had the largest Chinese journal full-text database, including the vast majority of Chinese journals relating to public health management. More importantly, it has become one of the critical basic data sources for bibliometric research in China [ 25 ]. Data retrieval Data obtained by inappropriate literature information retrieval strategies could not accurately reflect the content of the research [ 26 ]. Emergency management is a common term in China that focuses on the occurrence, development, and evolution of emergencies and finding effective ways of responding to them [ 27 ]. However, it was not certified internationally. After consulting the experts from the Chinese CDC who once worked for the World Health Organization (WHO), we learned that the management was refined by preparedness, operation, response, and recovery for an international public health emergency. Additionally, this has been mentioned in articles [ 5 , 28 ]. Based on the above points, the international data retrieval strategies were set as: ((TS = public health) AND (TS = preparedness OR TS = operation OR TS = response OR TS = recovery) AND TS = (emergency)) AND LANGUAGE: (English) AND DOCUMENT TYPES: (Article) Indexes = SCI-EXPANDED, SSCI, A&HCI, Timespan = 1988–2018. When retrieving Chinese data, we choose "public health" and "emergency management" as the theme words, Timespan = 1988–2018. We ran the search query of WoS and CNKI on February 19, 2019. A total of 2759 articles from 1991 to 2018 were retained from WoS, while 999 articles from 2003 to 2018 were retained from CNKI. After discussing the results with the team members, we further selected the articles based on inclusion and exclusion criteria to ensure that all of the data were closely aligned to the study targets. The inclusion criteria were as follows: (1) occurrence, development, and evolution of PHEM; (2) prevention, preparedness, response, operation, and recovery of the PHEM system; (3) planning, organization, leadership, coordination, and control of public health emergencies; and (4) practice and method of PHEM. The exclusion criteria were as follows: (1) health care, medical record management, or disease treatment; (2) guidelines on the action, proceedings paper or book chapter, interviews, summaries of conferences, and patent abstracts. Finally, 2247 international articles and 959 Chinese articles were accepted for the analysis after data filtering and removing duplications. The international articles were downloaded in "plain text" format with a full record and cited references for classification and statistical analysis. The Chinese articles were downloaded in the "Refworks" format. Those downloaded data contained the list of authors, the title of the publication, the abstract, keywords, and so on. Accordingly, we obtained the data for this study. Figure 1 shows the specific search steps. Fig. 1 Retrieval procedure of study data Data analysis tools We used CiteSpace 5.5. R2 and Microsoft Excel 2016 for the data analysis. CiteSpace was a free Java-based application found by Chaomei Chen to analyze the potential knowledge contained in the scientific literature. It has been widely adopted for scientometric analysis in various scientific fields [ 29 ], and has achieved excellent results [ 30 , 31 ]. The parameters of CiteSpace for this study were set as follows: time slicing (1991–2018, 2003–2018, respectively), years per slice (1), term source (all selection), node type (author, institution, keyword, for Chinese and international data; country for international data), selection criteria (top 50), and visualization (cluster view-static, show merged network). The corresponding other settings were selected according to different study questions. Microsoft Excel 2016 was used for temporal distribution and polynomial prediction of the number of articles. It should be noted that the results of the CNKI database made by CiteSpace were presented in Chinese. To make it easier to read, we translated the Chinese results into English. Data analysis strategies This study involved using bibliometric and mapping knowledge domain analysis methods. Bibliometric methods provided an approach to identify the development trends or future research orientations by combining different tools and methods to analyze the articles [ 32 , 33 ]. It allowed researchers to generate information from historical data and indicators, such as keywords, authors, institutes, and countries [ 34 ]. We were mainly engaged in cooperation network analysis (including the network analysis of the authors, institutions, and countries) and co-word network analysis (including keyword co-occurrence network and burst detection analysis) in the mapping knowledge domain analysis. First, we performed a statistical analysis of the temporal distribution of relevant articles. Then, we made a polynomial prediction of the number of articles, fitted the trend line of international and Chinese study, and predicted it for the next 3 years. Cooperation network analysis was used to analyze the contribution to different authors, institutions, and countries in one field. It was obvious that the more an author, a country, or an institution publishes its research findings, the more contributions it will make [ 35 ]. Betweenness centrality is an index to measure the importance of nodes in the network. The purple circle represents documents with betweenness centrality not less than 0.1, which means that the authors, institutions, or countries occupied an essential position in this field [ 20 , 36 ]. Co-word analysis was a content analysis technique that was effective for mapping co-occurrence relationships and the strength of the relationship between a pair of items existing in the same text, revealing the inner construction of a research field [ 24 ]. Analysis of the keyword co-occurrence network was meaningful and valuable for exploring timely topics in a specific knowledge domain [ 37 , 38 ]. In addition, keyword burst detection analysis can clearly grasp articles that receive particular attention from related scientific communities in a certain period. Therefore, the frontiers founded by burst detection analysis can provide researchers with up-to-date information [ 39 , 40 ]. The analysis of the highly cited articles in the field can reflect the development of the discipline in a period, examine timely topics, and supplement the above results to provide a reference for topic selection by scientific researchers [ 41 ]. Data sources Data were divided into two categories: international and Chinese data. According to the relevant authoritative research [ 20 ], the database of bibliometric methods should contain complete documents. A considerable amount of literature has shown that the Web of Science (WoS) is the largest comprehensive academic information resource, covering peer-reviewed journals with high impact factors [ 21 – 24 ]. Accordingly, the international data used for our study were collected from the WoS Core Collection, including Science Citation Index Expanded (SCI-E), Science Citation Index Expanded (SSCI), and Arts & Humanities Citation Index (A&HCI) databases. Chinese data were downloaded from the China National Knowledge Infrastructure (CNKI), which had the largest Chinese journal full-text database, including the vast majority of Chinese journals relating to public health management. More importantly, it has become one of the critical basic data sources for bibliometric research in China [ 25 ]. Data retrieval Data obtained by inappropriate literature information retrieval strategies could not accurately reflect the content of the research [ 26 ]. Emergency management is a common term in China that focuses on the occurrence, development, and evolution of emergencies and finding effective ways of responding to them [ 27 ]. However, it was not certified internationally. After consulting the experts from the Chinese CDC who once worked for the World Health Organization (WHO), we learned that the management was refined by preparedness, operation, response, and recovery for an international public health emergency. Additionally, this has been mentioned in articles [ 5 , 28 ]. Based on the above points, the international data retrieval strategies were set as: ((TS = public health) AND (TS = preparedness OR TS = operation OR TS = response OR TS = recovery) AND TS = (emergency)) AND LANGUAGE: (English) AND DOCUMENT TYPES: (Article) Indexes = SCI-EXPANDED, SSCI, A&HCI, Timespan = 1988–2018. When retrieving Chinese data, we choose "public health" and "emergency management" as the theme words, Timespan = 1988–2018. We ran the search query of WoS and CNKI on February 19, 2019. A total of 2759 articles from 1991 to 2018 were retained from WoS, while 999 articles from 2003 to 2018 were retained from CNKI. After discussing the results with the team members, we further selected the articles based on inclusion and exclusion criteria to ensure that all of the data were closely aligned to the study targets. The inclusion criteria were as follows: (1) occurrence, development, and evolution of PHEM; (2) prevention, preparedness, response, operation, and recovery of the PHEM system; (3) planning, organization, leadership, coordination, and control of public health emergencies; and (4) practice and method of PHEM. The exclusion criteria were as follows: (1) health care, medical record management, or disease treatment; (2) guidelines on the action, proceedings paper or book chapter, interviews, summaries of conferences, and patent abstracts. Finally, 2247 international articles and 959 Chinese articles were accepted for the analysis after data filtering and removing duplications. The international articles were downloaded in "plain text" format with a full record and cited references for classification and statistical analysis. The Chinese articles were downloaded in the "Refworks" format. Those downloaded data contained the list of authors, the title of the publication, the abstract, keywords, and so on. Accordingly, we obtained the data for this study. Figure 1 shows the specific search steps. Fig. 1 Retrieval procedure of study data Data analysis tools We used CiteSpace 5.5. R2 and Microsoft Excel 2016 for the data analysis. CiteSpace was a free Java-based application found by Chaomei Chen to analyze the potential knowledge contained in the scientific literature. It has been widely adopted for scientometric analysis in various scientific fields [ 29 ], and has achieved excellent results [ 30 , 31 ]. The parameters of CiteSpace for this study were set as follows: time slicing (1991–2018, 2003–2018, respectively), years per slice (1), term source (all selection), node type (author, institution, keyword, for Chinese and international data; country for international data), selection criteria (top 50), and visualization (cluster view-static, show merged network). The corresponding other settings were selected according to different study questions. Microsoft Excel 2016 was used for temporal distribution and polynomial prediction of the number of articles. It should be noted that the results of the CNKI database made by CiteSpace were presented in Chinese. To make it easier to read, we translated the Chinese results into English. Data analysis strategies This study involved using bibliometric and mapping knowledge domain analysis methods. Bibliometric methods provided an approach to identify the development trends or future research orientations by combining different tools and methods to analyze the articles [ 32 , 33 ]. It allowed researchers to generate information from historical data and indicators, such as keywords, authors, institutes, and countries [ 34 ]. We were mainly engaged in cooperation network analysis (including the network analysis of the authors, institutions, and countries) and co-word network analysis (including keyword co-occurrence network and burst detection analysis) in the mapping knowledge domain analysis. First, we performed a statistical analysis of the temporal distribution of relevant articles. Then, we made a polynomial prediction of the number of articles, fitted the trend line of international and Chinese study, and predicted it for the next 3 years. Cooperation network analysis was used to analyze the contribution to different authors, institutions, and countries in one field. It was obvious that the more an author, a country, or an institution publishes its research findings, the more contributions it will make [ 35 ]. Betweenness centrality is an index to measure the importance of nodes in the network. The purple circle represents documents with betweenness centrality not less than 0.1, which means that the authors, institutions, or countries occupied an essential position in this field [ 20 , 36 ]. Co-word analysis was a content analysis technique that was effective for mapping co-occurrence relationships and the strength of the relationship between a pair of items existing in the same text, revealing the inner construction of a research field [ 24 ]. Analysis of the keyword co-occurrence network was meaningful and valuable for exploring timely topics in a specific knowledge domain [ 37 , 38 ]. In addition, keyword burst detection analysis can clearly grasp articles that receive particular attention from related scientific communities in a certain period. Therefore, the frontiers founded by burst detection analysis can provide researchers with up-to-date information [ 39 , 40 ]. The analysis of the highly cited articles in the field can reflect the development of the discipline in a period, examine timely topics, and supplement the above results to provide a reference for topic selection by scientific researchers [ 41 ]. Results Temporal distribution analysis Figure 2 shows the first international article on PHEM published in 1991. The number of articles fluctuated slowly over the next few years. After 2001, the number began to increase rapidly. It is worth noting that the number of articles in 2014 was high, reaching 206. It declined over the next 2 years until 2017, when the number of articles reached its peak at 219. The study on PHEM in China started in 2003 and declined slowly until 2005. During 2006–2013, there was a rapid development; many articles appeared in this period. The minimum number, in 2006, was 41, and the maximum was in 2013, reaching an astonishing 96. The average number peaked at 70. The number of articles declined after 2013, reaching 49 in 2018. In contrast, the study of PHEM in China started later than in some other countries, yet a large number of Chinese scholars participated in this study at the beginning. Fig. 2 Temporal distribution and trend of international and Chinese PHEM study After the descriptive analysis of the data, we conducted a polynomial prediction analysis of the number of articles and predicted it for the next 3 years. The trend of chronological distribution of articles related to PHEM in international data can be expressed as follows: y = 0.2790 x 2 + 0.7688 x −7.7708 ( R 2 = 0.9577), while Chinese data was y = −0.0345 x 3 + 1.5079 x 2 −14.142 x + 27.273 ( R 2 = 0.9251). Y indicates the number of articles, and x indicates the years. R 2 > 0.9, indicating a good degree of fit. The chronological distribution of international articles showed a trend of increasing year by year. In 2019–2021, the annual number of articles will exceed that of previous years. At the same time, the trend line of Chinese articles, such as a wave line, and the number of articles will continue to decrease over the next 3 years. In addition, as time goes on, the gap in the number of articles in Chinese and international journals will be gradually increasing. Cooperation network analysis Figure 3 , which shows the international co-author network, shows that there have been many authors writing studies on PHEM, and some had collaborators. The top-ranked item by citation count was Daniel J. Barnett (DANIEL J BARNETT in the figure), with a citation count of 20 (all counts are not shown in the figure). He was followed by Elena Savoia (ELENA SAVOIA) and Lainie Rutkow (LAINIE RUTKOW); both of their citation counts were 17. These were authors who paid great attention to this field and had published the most articles. Most of these authors were from American universities, such as Johns Hopkins University and Harvard University. The top-ranked items by centrality were Frederick M. Brokle (FREDERICK M), Task Force for Pediatric Emergency Mass Critical Care (TASK FORCE PEDIAT EMERGENCY MASS C CA), and James G. Hodge (JAMES G), with a centrality of 0.06 (all centralities are not shown in the figure). However, all three items' centrality was less than 0.1. In Fig. 4 , the top-ranked item for Chinese scholars by citation count was Qunhong Wu with a citation count of 22. She was followed by Yanhua Hao (17), Feng Han (11), Ning (10), Yadong Wang (10), Zheng Kang (8), Ying Liu (6), Jincheng Ma (6), Mingli Jiao (5), and Libo Liang (5). Most of them were teachers at Harbin Medical University. The top-ranked items by centrality were Yanhua Hao and Ning, with a centrality of 0.02. The third one was Qunhong Wu, with a centrality of 0.01. Their nodes' centrality is also less than 0.1. In terms of the quantity and quality of articles, Qunhong Wu and Yanhua Hao were the leaders of PHEM in China. From the co-author network, we can see that many authors internationally and those in China are collaborating on PHEM (Additional file 1 : Table S1, Table S2, and Figure S1). Fig. 3 Co-author network of international database. Different colors represent different time slices. The size of node represents the number of articles by the author. The link strength between two nodes means the collaboration intensity between authors Fig. 4 Co-author network of Chinese database. Different colors represent different time slices. The size of node represents the number of articles by the author. The link strength between two nodes means the collaboration intensity between authors Co-institution Similar to the co-author situation, many institutions have studied PHEM (Figs. 5 and 6 ). The top-ranked item by international citation count was the Centers for Disease Control and Prevention (Ctr Dis Control & Prevent.), with citation counts of 255, which means that the institution publishes the largest number of articles in this field. The second one was Johns Hopkins University (Johns Hopkins Univ.), with citation counts of 92, which means that Johns Hopkins University had the largest number of articles published among the universities in this field. Johns Hopkins was followed by Harvard University (Harvard Univ.), Columbia University (Columbia Univ.), and so on. The above analysis showed that the CDC and universities were the leading institutions to study international PHEM. The centrality ranked item was CDC (0.50). Then, Johns Hopkins University (0.21), Harvard University (0.16), University of Washington (Univ Washington., 0.13), University of Toronto (Univ Toronto., 0.10), University of Pittsburgh (Univ Pittsburgh, 0.10), and Boston University (Boston Univ., 0.10) followed. The centrality of all these nodes was no less than 0.1 with the purple circle, which meant that they were the institutions with higher publication quality and the leading institutions in this field. The top-ranked item by citation count in the Chinese database was the School of Health Management, Harbin Medical University, with a citation count of 20, followed by Shanghai Publishing and Printing College (9), National Health and Family Planning Commission of the People's Republic of China (7), School of Health Administration and Education, Capital Medical University (6), etc. The top-ranked institution by centrality was the School of Health Management, Harbin Medical University, with a centrality of 0.04. The second one was the China National Health Development Research Center, with a centrality of 0.03. Thus, the School of Health Management, Harbin Medical University, is a leader in the field of research in China. In comparison, CDC had the largest publication volume and influence in the international field, while universities instead of health institutions dominated the field of research in China (Additional file 1 : Table S3, Table S4, and Figure S2). Fig. 5 Co-institution network of international database. Different colors represent different time slices. The colors of rings of a circle are corresponding to the year. The diameter of each circle represents the number of the institution's articles. The link strength between two nodes means the intensity of institution cooperation. The purple circles represent the high betweenness centralities Fig. 6 Co-institution network of Chinese database. Different colors represent different time slices. The colors of rings of a circle are corresponding to the year. The diameter of each circle represents the number of the institution's articles. The link strength between two nodes means the intensity of institution cooperation Co-country Table 1 summarizes the top 10 countries in the count and centrality of international PHEM research. We can see that the development of PHEM research differs among countries, and it is mainly centralized in the USA, Canada, England, Australia, People's Republic of China, Switzerland, Italy, Sweden, Japan, and the Netherlands. According to the income levels from the World Bank [ 42 ], these were all high-income countries except China. Geographically, half of them are centralized in Europe. Among these countries, the USA was the most productive, far ahead of the rest in this field, and its centrality was the largest. This showed that in the field of PHEM, the USA carries out the most studies, and their studies were more advanced. Although China ranks fifth on this list, its centrality was only 0.03. Therefore, these results indicate that Chinese scholars could publish some internationally recognized articles in the field, which would offer an advantage in quantity; however, they need to improve their article quality. It is worth noting that although the number of articles from Switzerland (44) and Sweden (34) was much lower than that of the USA, the articles' centrality of these two countries was more than 0.10, which showed that the quality of the articles was still high. Table 1 Top 10 countries in the count and centrality of international database Count Country Centrality Country 1557 USA 0.67 USA 138 Canada 0.32 England 131 England 0.16 Switzerland 114 Australia 0.16 Sweden 95 P. R. China 0.11 Canada 44 Switzerland 0.08 Germany 34 Italy 0.06 Australia 34 Sweden 0.06 India 30 Japan 0.04 Netherlands 27 Netherlands 0.03 P. R. China Abbreviation : P. R. China People's Republic of China Co-word network analysis Keywords co-occurrence network analysis Generally, keywords represent the research hotspots, which represent topics of wide concern for researchers in this field. Figure 7 shows that the top 10 keywords ranked by citation count for the international database were public health (297), preparedness (215), emergency preparedness (191), disaster (187), health (142), emergency (133), care (128), United States (121), bioterrorism (102), and impact (96). The top ten centralities were emergency department (0.22), prevalence (0.18), surveillance (0.17), knowledge (0.15), public health preparedness (0.15), children (0.14), management (0.14), trauma (0.13), simulation (0.13), and policy (0.11). The centrality for all of them was more than 0.10, which meant they had more influence than other keywords. Figure 8 shows that the top 10 keywords of Chinese PHEM research were public health emergencies (394), emergency management (154), health emergency (116), public health (101), emergencies (84), emergency response ability (78), public health events (58), emergency mechanism (33), emergency disposal (28), and public emergencies (27). Nodes with a centrality over 0.1 were health emergency (0.45), public health (0.32), public health emergencies (0.30), emergency management (0.26), emergencies (0.23), public health events (0.13), and emergency response ability (0.12). Comparing the distribution of keywords in international and Chinese countries, we can see that international studies mainly focus on emergency preparedness and monitoring for public health events, while Chinese research mainly focuses on analysis and disposition (Additional file 1 : Table S5, Table S6, and Figure S3). Fig. 7 Keywords co-occurrence network of international database. The colors of crosses of a circle are corresponding to the year. The size of nodes represent research frequency of the keyword Fig. 8 Keywords co-occurrence network of Chinese database. The colors of crosses of a circle are corresponding to the year. The size of nodes represent research frequency of the keyword Burst detection analysis Figures 9 and 10 illustrate the keywords with the strongest citation burst for international and Chinese databases. The international keywords detected 52 burst words, while the Chinese data detected 18 burst words. The research frontier of international PHEM included anthrax, public health, weapon, bioterrorism, accident, trauma, terrorism, emergency response, and so on. Among these words, bioterrorism (28.7902) was the strongest burst keyword during the period between 2002 and 2009. Then, was Ebola (15.6855, 2015–2018), disaster planning (10.2745, 2014–2016), and terrorism (9.9846, 2004–2008). All these findings reflect a greater attention to this study topic and better exemplify the study front in this period. From the perspective of the time development sequence, international research on PHEM began with terrorism and bioterrorism. After that, disaster planning and emergency preparedness became a new research frontier (2004–2014). In recent years (2015–2018), epidemics and infectious diseases have become the focus of study. The research frontier of Chinese PHEM included public health emergencies, United States of America, North America, emergency disposal, united states, sudden events, public health, emergency, emergency response, public emergencies, disease prevention and control agency, evaluate, emergency drill, risk assessment, assessment, health emergency management, health emergency response ability, and the Delphi method. During 2003–2008, Chinese research on PHEM was in its infancy. The main research frontiers were public health emergencies and America. It was mainly at the stage of the formation of the Chinese theory of this field. By learning from the experience of the USA in dealing with health emergencies, scholars began the study of PHEM in China. Then, Chinese scholars began to study the links involved in the disposal of public health emergencies, such as response, organization, evaluation, exercise, and evaluation of PHEM. After 2016, health emergency management, health emergency response ability, and the Delphi method became the new research front. In contrast with Chinese research on PHEM, international research often relates to timely issues, while China focuses on the management procedures (Additional file 1 : Figure S4). Fig. 9 Keywords with the strongest citation bursts of international database. The strength represents the degree of the burst. The begin and end represent the boundaries of the time period of the burst. The blue line is the time interval, the red line segment is the duration of the burst for one keyword Fig. 10 Keywords with the strongest citation bursts of Chinese database. The strength represents the degree of the burst. The begin and end represent the boundaries of the time period of the burst. The blue line is the time interval, the red line segment is the duration of the burst for one keyword To further explain the above research hotspots, the top 8 cited articles are shown in Tables 2 and 3 . The top 8 ranked articles by citation for international databases appeared in 2005, 2007, 2009, 2014, 2015, and 2016. The article Elevated blood lead levels in children associated with the flint drinking water crisis: a spatial analysis of risk and public health response was the most cited (372) international art icle [ 43 ] followed by The 2006 California Heat Wave: Impacts on Hospitalizations and Emergency department visit s [ 44 ]. In terms of the time distribution, the research on international bioterrorism started first [ 45 ], followed by recommendations for health emergency response teams and health incident management [ 46 , 47 ]. In recent years, the causes and disposal of public health events have been the hotspots of international attention [ 43 , 48 – 50 ]. This was basically consistent with the analysis results of the above research keywords. The top 8 Chinese cited articles of PHEM appeared in 2003, 2004, 2009, and 2011. Table 3 shows us that the most frequently cited Chinese article was Emergency logistics [ 51 ], written by Zhongwen Ou, Huiyun Wang, and Dali Jiang et al. with a frequency of an astonishing 473. This was followed by Kaibin Zhong's article Review and prospect: construction of emergency management system in China [ 52 ]. The next three articles, Legislative situation and characteristics of China's emergency law [ 53 ], Legal construction of public emergency response in China: legal construction task proposed by SARS crisis management practice [ 54 ], and The realistic subject of administrative rule of law in public emergency management [ 55 ], mainly researched the legal construction of PHEM in China. They were all published in 2003, proving that China mainly carried out research on the legal construction of PHEM during that time. In other words, PHEM in China starts with legal system construction. In 2004, Chinese scholars began to establish a knowledge domain for health emergency response and disposition. By using extensive literature for reference, Chinese scholars began to research international PHEM in 2011. Table 2 Top 8 cited articles of international database Count Author Title Journal Year 372 Mona Hanna-Attisha, Jenny LaChance, Richard Casey Sadler, et al. Elevated blood lead levels in children associated with the flint drinking water crisis: a spatial analysis of risk and public health response American Journal of Public Health 2016 366 Kim Knowlton, Miriam Rotkin-Ellman, Galatea King, et al. The 2006 California heat wave: impacts on hospitalizations and emergency department visits Environmental Health Perspectives 2009 365 Karin Stettler, Martina Beltramello, Diego A. Espinosa, et al. Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection Science 2016 316 Nathalie Embriaco, Elie Azoulay, Karine Barrau, et al. High level of burnout in intensivists—prevalence and associated factors American Journal of Respiratory and Critical Care Medicine 2007 305 Salim S. Abdool Karim, Gavin J. Churchyard, Quarraisha Abdool Karim, et al. HIV infection and tuberculosis in South Africa: an urgent need to escalate the public health response Lancet 2009 286 Daniel P. Aldrich, Michelle A. Meyer Social Capital and Community Resilience American Behavioral Scientist 2014 238 Lawrence M. Wein, Yifan Liu Analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk Proceedings of the National Academy of Sciences of the United States of America 2005 226 Philip J. Peters, Pamela Pontones, Karen W. Hoover, et al. HIV infection linked to injection use of oxymorphone in Indiana, 2014-2015 The New England Journal Of Medicine 2016 Table 3 Top 8 cited articles of Chinese database Count Author Title Journal Year 473 Zhongwen Ou, Huiyun Wang, Dali Jiang, et al. Emergency logistics Journal of Chongqing University (Natural Science Edition) 2004 177 Kaibin Zhong Review and prospect: construction of emergency management system in China CASS Journal of Political Science 2009 122 Jihong Mo Legislative situation and characteristics of China's emergency law Legal Forum 2003 102 Yuchuan Mo Legal construction of public emergency response in China: legal construction task proposed by SARS crisis management practice Journal of Renmin University of China 2003 94 Yuchuan Mo The realistic subject of administrative rule of law in public emergency management Jurists Review 2003 77 Yifeng Yang, Chenfang Fan, Guangwen Cao Emergency management in immediate response to emergent public health event in China Academic Journal of Second Military Medical University 2004 69 Lexuan Luo, Zhanchun Feng, Jian Zhang Research on the hospital function of response to public health emergency Chinese Hospital Management 2004 65 Liping Fan, Qinghua Zhao The status quo of emergency management system for sudden public health events in America and Japan and its enlightenment Chinese Nursing Research 2011 Temporal distribution analysis Figure 2 shows the first international article on PHEM published in 1991. The number of articles fluctuated slowly over the next few years. After 2001, the number began to increase rapidly. It is worth noting that the number of articles in 2014 was high, reaching 206. It declined over the next 2 years until 2017, when the number of articles reached its peak at 219. The study on PHEM in China started in 2003 and declined slowly until 2005. During 2006–2013, there was a rapid development; many articles appeared in this period. The minimum number, in 2006, was 41, and the maximum was in 2013, reaching an astonishing 96. The average number peaked at 70. The number of articles declined after 2013, reaching 49 in 2018. In contrast, the study of PHEM in China started later than in some other countries, yet a large number of Chinese scholars participated in this study at the beginning. Fig. 2 Temporal distribution and trend of international and Chinese PHEM study After the descriptive analysis of the data, we conducted a polynomial prediction analysis of the number of articles and predicted it for the next 3 years. The trend of chronological distribution of articles related to PHEM in international data can be expressed as follows: y = 0.2790 x 2 + 0.7688 x −7.7708 ( R 2 = 0.9577), while Chinese data was y = −0.0345 x 3 + 1.5079 x 2 −14.142 x + 27.273 ( R 2 = 0.9251). Y indicates the number of articles, and x indicates the years. R 2 > 0.9, indicating a good degree of fit. The chronological distribution of international articles showed a trend of increasing year by year. In 2019–2021, the annual number of articles will exceed that of previous years. At the same time, the trend line of Chinese articles, such as a wave line, and the number of articles will continue to decrease over the next 3 years. In addition, as time goes on, the gap in the number of articles in Chinese and international journals will be gradually increasing. Cooperation network analysis Figure 3 , which shows the international co-author network, shows that there have been many authors writing studies on PHEM, and some had collaborators. The top-ranked item by citation count was Daniel J. Barnett (DANIEL J BARNETT in the figure), with a citation count of 20 (all counts are not shown in the figure). He was followed by Elena Savoia (ELENA SAVOIA) and Lainie Rutkow (LAINIE RUTKOW); both of their citation counts were 17. These were authors who paid great attention to this field and had published the most articles. Most of these authors were from American universities, such as Johns Hopkins University and Harvard University. The top-ranked items by centrality were Frederick M. Brokle (FREDERICK M), Task Force for Pediatric Emergency Mass Critical Care (TASK FORCE PEDIAT EMERGENCY MASS C CA), and James G. Hodge (JAMES G), with a centrality of 0.06 (all centralities are not shown in the figure). However, all three items' centrality was less than 0.1. In Fig. 4 , the top-ranked item for Chinese scholars by citation count was Qunhong Wu with a citation count of 22. She was followed by Yanhua Hao (17), Feng Han (11), Ning (10), Yadong Wang (10), Zheng Kang (8), Ying Liu (6), Jincheng Ma (6), Mingli Jiao (5), and Libo Liang (5). Most of them were teachers at Harbin Medical University. The top-ranked items by centrality were Yanhua Hao and Ning, with a centrality of 0.02. The third one was Qunhong Wu, with a centrality of 0.01. Their nodes' centrality is also less than 0.1. In terms of the quantity and quality of articles, Qunhong Wu and Yanhua Hao were the leaders of PHEM in China. From the co-author network, we can see that many authors internationally and those in China are collaborating on PHEM (Additional file 1 : Table S1, Table S2, and Figure S1). Fig. 3 Co-author network of international database. Different colors represent different time slices. The size of node represents the number of articles by the author. The link strength between two nodes means the collaboration intensity between authors Fig. 4 Co-author network of Chinese database. Different colors represent different time slices. The size of node represents the number of articles by the author. The link strength between two nodes means the collaboration intensity between authors Co-institution Similar to the co-author situation, many institutions have studied PHEM (Figs. 5 and 6 ). The top-ranked item by international citation count was the Centers for Disease Control and Prevention (Ctr Dis Control & Prevent.), with citation counts of 255, which means that the institution publishes the largest number of articles in this field. The second one was Johns Hopkins University (Johns Hopkins Univ.), with citation counts of 92, which means that Johns Hopkins University had the largest number of articles published among the universities in this field. Johns Hopkins was followed by Harvard University (Harvard Univ.), Columbia University (Columbia Univ.), and so on. The above analysis showed that the CDC and universities were the leading institutions to study international PHEM. The centrality ranked item was CDC (0.50). Then, Johns Hopkins University (0.21), Harvard University (0.16), University of Washington (Univ Washington., 0.13), University of Toronto (Univ Toronto., 0.10), University of Pittsburgh (Univ Pittsburgh, 0.10), and Boston University (Boston Univ., 0.10) followed. The centrality of all these nodes was no less than 0.1 with the purple circle, which meant that they were the institutions with higher publication quality and the leading institutions in this field. The top-ranked item by citation count in the Chinese database was the School of Health Management, Harbin Medical University, with a citation count of 20, followed by Shanghai Publishing and Printing College (9), National Health and Family Planning Commission of the People's Republic of China (7), School of Health Administration and Education, Capital Medical University (6), etc. The top-ranked institution by centrality was the School of Health Management, Harbin Medical University, with a centrality of 0.04. The second one was the China National Health Development Research Center, with a centrality of 0.03. Thus, the School of Health Management, Harbin Medical University, is a leader in the field of research in China. In comparison, CDC had the largest publication volume and influence in the international field, while universities instead of health institutions dominated the field of research in China (Additional file 1 : Table S3, Table S4, and Figure S2). Fig. 5 Co-institution network of international database. Different colors represent different time slices. The colors of rings of a circle are corresponding to the year. The diameter of each circle represents the number of the institution's articles. The link strength between two nodes means the intensity of institution cooperation. The purple circles represent the high betweenness centralities Fig. 6 Co-institution network of Chinese database. Different colors represent different time slices. The colors of rings of a circle are corresponding to the year. The diameter of each circle represents the number of the institution's articles. The link strength between two nodes means the intensity of institution cooperation Co-country Table 1 summarizes the top 10 countries in the count and centrality of international PHEM research. We can see that the development of PHEM research differs among countries, and it is mainly centralized in the USA, Canada, England, Australia, People's Republic of China, Switzerland, Italy, Sweden, Japan, and the Netherlands. According to the income levels from the World Bank [ 42 ], these were all high-income countries except China. Geographically, half of them are centralized in Europe. Among these countries, the USA was the most productive, far ahead of the rest in this field, and its centrality was the largest. This showed that in the field of PHEM, the USA carries out the most studies, and their studies were more advanced. Although China ranks fifth on this list, its centrality was only 0.03. Therefore, these results indicate that Chinese scholars could publish some internationally recognized articles in the field, which would offer an advantage in quantity; however, they need to improve their article quality. It is worth noting that although the number of articles from Switzerland (44) and Sweden (34) was much lower than that of the USA, the articles' centrality of these two countries was more than 0.10, which showed that the quality of the articles was still high. Table 1 Top 10 countries in the count and centrality of international database Count Country Centrality Country 1557 USA 0.67 USA 138 Canada 0.32 England 131 England 0.16 Switzerland 114 Australia 0.16 Sweden 95 P. R. China 0.11 Canada 44 Switzerland 0.08 Germany 34 Italy 0.06 Australia 34 Sweden 0.06 India 30 Japan 0.04 Netherlands 27 Netherlands 0.03 P. R. China Abbreviation : P. R. China People's Republic of China Co-word network analysis Keywords co-occurrence network analysis Generally, keywords represent the research hotspots, which represent topics of wide concern for researchers in this field. Figure 7 shows that the top 10 keywords ranked by citation count for the international database were public health (297), preparedness (215), emergency preparedness (191), disaster (187), health (142), emergency (133), care (128), United States (121), bioterrorism (102), and impact (96). The top ten centralities were emergency department (0.22), prevalence (0.18), surveillance (0.17), knowledge (0.15), public health preparedness (0.15), children (0.14), management (0.14), trauma (0.13), simulation (0.13), and policy (0.11). The centrality for all of them was more than 0.10, which meant they had more influence than other keywords. Figure 8 shows that the top 10 keywords of Chinese PHEM research were public health emergencies (394), emergency management (154), health emergency (116), public health (101), emergencies (84), emergency response ability (78), public health events (58), emergency mechanism (33), emergency disposal (28), and public emergencies (27). Nodes with a centrality over 0.1 were health emergency (0.45), public health (0.32), public health emergencies (0.30), emergency management (0.26), emergencies (0.23), public health events (0.13), and emergency response ability (0.12). Comparing the distribution of keywords in international and Chinese countries, we can see that international studies mainly focus on emergency preparedness and monitoring for public health events, while Chinese research mainly focuses on analysis and disposition (Additional file 1 : Table S5, Table S6, and Figure S3). Fig. 7 Keywords co-occurrence network of international database. The colors of crosses of a circle are corresponding to the year. The size of nodes represent research frequency of the keyword Fig. 8 Keywords co-occurrence network of Chinese database. The colors of crosses of a circle are corresponding to the year. The size of nodes represent research frequency of the keyword Burst detection analysis Figures 9 and 10 illustrate the keywords with the strongest citation burst for international and Chinese databases. The international keywords detected 52 burst words, while the Chinese data detected 18 burst words. The research frontier of international PHEM included anthrax, public health, weapon, bioterrorism, accident, trauma, terrorism, emergency response, and so on. Among these words, bioterrorism (28.7902) was the strongest burst keyword during the period between 2002 and 2009. Then, was Ebola (15.6855, 2015–2018), disaster planning (10.2745, 2014–2016), and terrorism (9.9846, 2004–2008). All these findings reflect a greater attention to this study topic and better exemplify the study front in this period. From the perspective of the time development sequence, international research on PHEM began with terrorism and bioterrorism. After that, disaster planning and emergency preparedness became a new research frontier (2004–2014). In recent years (2015–2018), epidemics and infectious diseases have become the focus of study. The research frontier of Chinese PHEM included public health emergencies, United States of America, North America, emergency disposal, united states, sudden events, public health, emergency, emergency response, public emergencies, disease prevention and control agency, evaluate, emergency drill, risk assessment, assessment, health emergency management, health emergency response ability, and the Delphi method. During 2003–2008, Chinese research on PHEM was in its infancy. The main research frontiers were public health emergencies and America. It was mainly at the stage of the formation of the Chinese theory of this field. By learning from the experience of the USA in dealing with health emergencies, scholars began the study of PHEM in China. Then, Chinese scholars began to study the links involved in the disposal of public health emergencies, such as response, organization, evaluation, exercise, and evaluation of PHEM. After 2016, health emergency management, health emergency response ability, and the Delphi method became the new research front. In contrast with Chinese research on PHEM, international research often relates to timely issues, while China focuses on the management procedures (Additional file 1 : Figure S4). Fig. 9 Keywords with the strongest citation bursts of international database. The strength represents the degree of the burst. The begin and end represent the boundaries of the time period of the burst. The blue line is the time interval, the red line segment is the duration of the burst for one keyword Fig. 10 Keywords with the strongest citation bursts of Chinese database. The strength represents the degree of the burst. The begin and end represent the boundaries of the time period of the burst. The blue line is the time interval, the red line segment is the duration of the burst for one keyword To further explain the above research hotspots, the top 8 cited articles are shown in Tables 2 and 3 . The top 8 ranked articles by citation for international databases appeared in 2005, 2007, 2009, 2014, 2015, and 2016. The article Elevated blood lead levels in children associated with the flint drinking water crisis: a spatial analysis of risk and public health response was the most cited (372) international art icle [ 43 ] followed by The 2006 California Heat Wave: Impacts on Hospitalizations and Emergency department visit s [ 44 ]. In terms of the time distribution, the research on international bioterrorism started first [ 45 ], followed by recommendations for health emergency response teams and health incident management [ 46 , 47 ]. In recent years, the causes and disposal of public health events have been the hotspots of international attention [ 43 , 48 – 50 ]. This was basically consistent with the analysis results of the above research keywords. The top 8 Chinese cited articles of PHEM appeared in 2003, 2004, 2009, and 2011. Table 3 shows us that the most frequently cited Chinese article was Emergency logistics [ 51 ], written by Zhongwen Ou, Huiyun Wang, and Dali Jiang et al. with a frequency of an astonishing 473. This was followed by Kaibin Zhong's article Review and prospect: construction of emergency management system in China [ 52 ]. The next three articles, Legislative situation and characteristics of China's emergency law [ 53 ], Legal construction of public emergency response in China: legal construction task proposed by SARS crisis management practice [ 54 ], and The realistic subject of administrative rule of law in public emergency management [ 55 ], mainly researched the legal construction of PHEM in China. They were all published in 2003, proving that China mainly carried out research on the legal construction of PHEM during that time. In other words, PHEM in China starts with legal system construction. In 2004, Chinese scholars began to establish a knowledge domain for health emergency response and disposition. By using extensive literature for reference, Chinese scholars began to research international PHEM in 2011. Table 2 Top 8 cited articles of international database Count Author Title Journal Year 372 Mona Hanna-Attisha, Jenny LaChance, Richard Casey Sadler, et al. Elevated blood lead levels in children associated with the flint drinking water crisis: a spatial analysis of risk and public health response American Journal of Public Health 2016 366 Kim Knowlton, Miriam Rotkin-Ellman, Galatea King, et al. The 2006 California heat wave: impacts on hospitalizations and emergency department visits Environmental Health Perspectives 2009 365 Karin Stettler, Martina Beltramello, Diego A. Espinosa, et al. Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection Science 2016 316 Nathalie Embriaco, Elie Azoulay, Karine Barrau, et al. High level of burnout in intensivists—prevalence and associated factors American Journal of Respiratory and Critical Care Medicine 2007 305 Salim S. Abdool Karim, Gavin J. Churchyard, Quarraisha Abdool Karim, et al. HIV infection and tuberculosis in South Africa: an urgent need to escalate the public health response Lancet 2009 286 Daniel P. Aldrich, Michelle A. Meyer Social Capital and Community Resilience American Behavioral Scientist 2014 238 Lawrence M. Wein, Yifan Liu Analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk Proceedings of the National Academy of Sciences of the United States of America 2005 226 Philip J. Peters, Pamela Pontones, Karen W. Hoover, et al. HIV infection linked to injection use of oxymorphone in Indiana, 2014-2015 The New England Journal Of Medicine 2016 Table 3 Top 8 cited articles of Chinese database Count Author Title Journal Year 473 Zhongwen Ou, Huiyun Wang, Dali Jiang, et al. Emergency logistics Journal of Chongqing University (Natural Science Edition) 2004 177 Kaibin Zhong Review and prospect: construction of emergency management system in China CASS Journal of Political Science 2009 122 Jihong Mo Legislative situation and characteristics of China's emergency law Legal Forum 2003 102 Yuchuan Mo Legal construction of public emergency response in China: legal construction task proposed by SARS crisis management practice Journal of Renmin University of China 2003 94 Yuchuan Mo The realistic subject of administrative rule of law in public emergency management Jurists Review 2003 77 Yifeng Yang, Chenfang Fan, Guangwen Cao Emergency management in immediate response to emergent public health event in China Academic Journal of Second Military Medical University 2004 69 Lexuan Luo, Zhanchun Feng, Jian Zhang Research on the hospital function of response to public health emergency Chinese Hospital Management 2004 65 Liping Fan, Qinghua Zhao The status quo of emergency management system for sudden public health events in America and Japan and its enlightenment Chinese Nursing Research 2011 Keywords co-occurrence network analysis Generally, keywords represent the research hotspots, which represent topics of wide concern for researchers in this field. Figure 7 shows that the top 10 keywords ranked by citation count for the international database were public health (297), preparedness (215), emergency preparedness (191), disaster (187), health (142), emergency (133), care (128), United States (121), bioterrorism (102), and impact (96). The top ten centralities were emergency department (0.22), prevalence (0.18), surveillance (0.17), knowledge (0.15), public health preparedness (0.15), children (0.14), management (0.14), trauma (0.13), simulation (0.13), and policy (0.11). The centrality for all of them was more than 0.10, which meant they had more influence than other keywords. Figure 8 shows that the top 10 keywords of Chinese PHEM research were public health emergencies (394), emergency management (154), health emergency (116), public health (101), emergencies (84), emergency response ability (78), public health events (58), emergency mechanism (33), emergency disposal (28), and public emergencies (27). Nodes with a centrality over 0.1 were health emergency (0.45), public health (0.32), public health emergencies (0.30), emergency management (0.26), emergencies (0.23), public health events (0.13), and emergency response ability (0.12). Comparing the distribution of keywords in international and Chinese countries, we can see that international studies mainly focus on emergency preparedness and monitoring for public health events, while Chinese research mainly focuses on analysis and disposition (Additional file 1 : Table S5, Table S6, and Figure S3). Fig. 7 Keywords co-occurrence network of international database. The colors of crosses of a circle are corresponding to the year. The size of nodes represent research frequency of the keyword Fig. 8 Keywords co-occurrence network of Chinese database. The colors of crosses of a circle are corresponding to the year. The size of nodes represent research frequency of the keyword Burst detection analysis Figures 9 and 10 illustrate the keywords with the strongest citation burst for international and Chinese databases. The international keywords detected 52 burst words, while the Chinese data detected 18 burst words. The research frontier of international PHEM included anthrax, public health, weapon, bioterrorism, accident, trauma, terrorism, emergency response, and so on. Among these words, bioterrorism (28.7902) was the strongest burst keyword during the period between 2002 and 2009. Then, was Ebola (15.6855, 2015–2018), disaster planning (10.2745, 2014–2016), and terrorism (9.9846, 2004–2008). All these findings reflect a greater attention to this study topic and better exemplify the study front in this period. From the perspective of the time development sequence, international research on PHEM began with terrorism and bioterrorism. After that, disaster planning and emergency preparedness became a new research frontier (2004–2014). In recent years (2015–2018), epidemics and infectious diseases have become the focus of study. The research frontier of Chinese PHEM included public health emergencies, United States of America, North America, emergency disposal, united states, sudden events, public health, emergency, emergency response, public emergencies, disease prevention and control agency, evaluate, emergency drill, risk assessment, assessment, health emergency management, health emergency response ability, and the Delphi method. During 2003–2008, Chinese research on PHEM was in its infancy. The main research frontiers were public health emergencies and America. It was mainly at the stage of the formation of the Chinese theory of this field. By learning from the experience of the USA in dealing with health emergencies, scholars began the study of PHEM in China. Then, Chinese scholars began to study the links involved in the disposal of public health emergencies, such as response, organization, evaluation, exercise, and evaluation of PHEM. After 2016, health emergency management, health emergency response ability, and the Delphi method became the new research front. In contrast with Chinese research on PHEM, international research often relates to timely issues, while China focuses on the management procedures (Additional file 1 : Figure S4). Fig. 9 Keywords with the strongest citation bursts of international database. The strength represents the degree of the burst. The begin and end represent the boundaries of the time period of the burst. The blue line is the time interval, the red line segment is the duration of the burst for one keyword Fig. 10 Keywords with the strongest citation bursts of Chinese database. The strength represents the degree of the burst. The begin and end represent the boundaries of the time period of the burst. The blue line is the time interval, the red line segment is the duration of the burst for one keyword To further explain the above research hotspots, the top 8 cited articles are shown in Tables 2 and 3 . The top 8 ranked articles by citation for international databases appeared in 2005, 2007, 2009, 2014, 2015, and 2016. The article Elevated blood lead levels in children associated with the flint drinking water crisis: a spatial analysis of risk and public health response was the most cited (372) international art icle [ 43 ] followed by The 2006 California Heat Wave: Impacts on Hospitalizations and Emergency department visit s [ 44 ]. In terms of the time distribution, the research on international bioterrorism started first [ 45 ], followed by recommendations for health emergency response teams and health incident management [ 46 , 47 ]. In recent years, the causes and disposal of public health events have been the hotspots of international attention [ 43 , 48 – 50 ]. This was basically consistent with the analysis results of the above research keywords. The top 8 Chinese cited articles of PHEM appeared in 2003, 2004, 2009, and 2011. Table 3 shows us that the most frequently cited Chinese article was Emergency logistics [ 51 ], written by Zhongwen Ou, Huiyun Wang, and Dali Jiang et al. with a frequency of an astonishing 473. This was followed by Kaibin Zhong's article Review and prospect: construction of emergency management system in China [ 52 ]. The next three articles, Legislative situation and characteristics of China's emergency law [ 53 ], Legal construction of public emergency response in China: legal construction task proposed by SARS crisis management practice [ 54 ], and The realistic subject of administrative rule of law in public emergency management [ 55 ], mainly researched the legal construction of PHEM in China. They were all published in 2003, proving that China mainly carried out research on the legal construction of PHEM during that time. In other words, PHEM in China starts with legal system construction. In 2004, Chinese scholars began to establish a knowledge domain for health emergency response and disposition. By using extensive literature for reference, Chinese scholars began to research international PHEM in 2011. Table 2 Top 8 cited articles of international database Count Author Title Journal Year 372 Mona Hanna-Attisha, Jenny LaChance, Richard Casey Sadler, et al. Elevated blood lead levels in children associated with the flint drinking water crisis: a spatial analysis of risk and public health response American Journal of Public Health 2016 366 Kim Knowlton, Miriam Rotkin-Ellman, Galatea King, et al. The 2006 California heat wave: impacts on hospitalizations and emergency department visits Environmental Health Perspectives 2009 365 Karin Stettler, Martina Beltramello, Diego A. Espinosa, et al. Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection Science 2016 316 Nathalie Embriaco, Elie Azoulay, Karine Barrau, et al. High level of burnout in intensivists—prevalence and associated factors American Journal of Respiratory and Critical Care Medicine 2007 305 Salim S. Abdool Karim, Gavin J. Churchyard, Quarraisha Abdool Karim, et al. HIV infection and tuberculosis in South Africa: an urgent need to escalate the public health response Lancet 2009 286 Daniel P. Aldrich, Michelle A. Meyer Social Capital and Community Resilience American Behavioral Scientist 2014 238 Lawrence M. Wein, Yifan Liu Analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk Proceedings of the National Academy of Sciences of the United States of America 2005 226 Philip J. Peters, Pamela Pontones, Karen W. Hoover, et al. HIV infection linked to injection use of oxymorphone in Indiana, 2014-2015 The New England Journal Of Medicine 2016 Table 3 Top 8 cited articles of Chinese database Count Author Title Journal Year 473 Zhongwen Ou, Huiyun Wang, Dali Jiang, et al. Emergency logistics Journal of Chongqing University (Natural Science Edition) 2004 177 Kaibin Zhong Review and prospect: construction of emergency management system in China CASS Journal of Political Science 2009 122 Jihong Mo Legislative situation and characteristics of China's emergency law Legal Forum 2003 102 Yuchuan Mo Legal construction of public emergency response in China: legal construction task proposed by SARS crisis management practice Journal of Renmin University of China 2003 94 Yuchuan Mo The realistic subject of administrative rule of law in public emergency management Jurists Review 2003 77 Yifeng Yang, Chenfang Fan, Guangwen Cao Emergency management in immediate response to emergent public health event in China Academic Journal of Second Military Medical University 2004 69 Lexuan Luo, Zhanchun Feng, Jian Zhang Research on the hospital function of response to public health emergency Chinese Hospital Management 2004 65 Liping Fan, Qinghua Zhao The status quo of emergency management system for sudden public health events in America and Japan and its enlightenment Chinese Nursing Research 2011 Discussion International research on PHEM occurred earlier than the Chinese research, and it has been growing over time. This means that international scholars have paid increasing attention to PHEM. In 1991, the first article on PHEM was written by Richard L. Siegel and was titled Code 9: a systematic approach for responding to medical emergencies occurring in and around a hospital [ 56 ]. It mentioned the need for an organized system to respond to such emergencies involving patients, visitors, local community residents, and hospital employees, both inside the hospital and on the grounds surrounding the building. He recommended the establishment of a systematic emergency response system in all health care institutions. Since then, academia has begun to pay attention to emergency management of public health incidents. The number of international articles is increasing gradually, reaching the maximum in 2017, and it is expected to continue to grow in the next 3 years. The development of PHEM in China shows a fluctuating pattern. The occurrence of public health emergencies in the 10 years from 2006 to 2016 showed a general trend of first rising and then slowly declining. It is likely related to the number of significant events that occur in each year [ 57 ]. The severe acute respiratory syndrome (SARS) epidemic in 2003 resulted in significant increases in both the amount of research and articles on emergency management [ 7 ]. The number of articles reached a small climax in 2008. Events such as the Wenchuan earthquake and the southern snow disaster occurred in that year. The maximum was in 2013, with human infection from H7N9, the Ya'an earthquake, and death from a hepatitis B vaccine occurring that year. Moreover, 10 years after the SARS outbreak, some authors compared the development of PHEM in China over the 10-year period. The first Chinese article on PHEM was written by Tiewu Jia and was titled Capacity-building for public health emergency response to disasters (2003) [ 58 ]. This article was published during the epidemic of SARS. In 2003, China did not establish a network and echelon PHEM system. The author combined the development of emergency management, reform of health and epidemic prevention institutions, and discussed the capacity building of public health emergency response. It is helpful for the social function orientation of the disease control center and the improvement of disease prevention ability. Although the number of Chinese articles decreased in the following years, it remained above 48. In summary, the above analysis shows that PHEM is still a timely topic. From the perspective of cooperative networks, we find that there is more cooperation among Chinese authors but less cooperation among authors from different institutions. The cooperation between different research institutions is believed to be highly effective in facilitating high-level and fruitful research, which can also help develop the research field into a more established area [ 59 ]. Therefore, Chinese scholars should strengthen cooperation between different institutions. The research institution focus on PHEM mainly comes from universities and health institutions, while Chinese institutions have regional differences. Reasons include the following: the western region had poor fiscal capacity, a limited personnel size, and an inadequate stockpile in terms of working budget, timely reserves, and prompt delivery [ 60 ]. As a leader in international PHEM, the CDC has begun to help other entities strengthen their capacity, recognition, and technical expertise to strengthen their PHEM capacity [ 61 ]. Additionally, some other health institutions, such as the WHO, have promoted development in this field. In 2005, the 58th World Health Assembly (WHA) adopted the revised International Health Regulations, which instructed the WHO member states to collaboratively confront public health emergencies of global significance [ 5 , 17 , 62 ]. Universities have undertaken the scientific task of PHEM, and they have conducted in-depth research on it in China. The Chinese CDC has carried out more disease prevention and control services, but its scientific research ability is weak. The country network analysis shows obvious differences in regional and economic development levels for PHEM. Those countries with more developed health emergency management systems are the most high-income ones. Geographically, most of these countries are concentrated in Europe, where the numbers of publications and citations are also significantly higher [ 60 ]. The USA, the UK, Japan, and other countries have constantly built and improved their PHEM systems, which have become a comprehensive management network. Co-word analysis of PHEM international research is more complex, extensive, and multidimensional. It reflects some of the major ideas of this research. Based on these ideas, scholars mainly focused on emergency preparedness and monitoring of public health events. From the perspective of Chinese PHEM development, it has gone through a process from theory to preparation, disposition, response, evaluation, organization, and discussion. That is, the main contents of China's health emergency management include the prevention and preparation of health emergencies as well as the key links of disposition, evaluation, and management, system construction, personnel training, and so on. The development of the whole discipline is therefore systematic and clear. The keywords with the strongest citation burst for international research on PHEM started with terrorism and bioterrorism [ 63 ], followed by disaster planning and emergency preparedness. In recent years, epidemics and infectious diseases have become the new research frontier. From the perspective of the whole development context, international research on PHEM has been related to current affairs hotspots, such as terrorism, which may have originated from the 911 incident, and epidemics, which may be related to the epidemic of infectious diseases caused by viruses and bacteria such as the Ebola virus. The study of PHEM in China is a process from theory formation to practice discussion, involving many links of management. During 2003–2008, Chinese scholars focused on health emergency response and disposition. After that, Chinese scholars began to learn more about foreign PHEM models. Some new methods have gradually been applied to Chinese PHEM in recent years. The top-ranked articles by citation for the international knowledge domain of PHEM appeared in 2005, 2007, 2009, 2014, 2015, and 2016. In 2005, Lawrence M. Wein [ 45 ] developed a mathematical model of a cow-to-consumer supply chain to reduce bioterrorism events. Once again, it shows that international emergency management research is based on terrorism and bioterrorism. In 2007, Nathalie Embriaco focused on the working condition of emergency management personnel [ 46 ]. Kim Knowlton [ 44 ] and Salim S. Abdool Karim [ 47 ] mentioned the emergency department. The above three articles are all about the factors involved in health emergency management. The remaining articles analyze the specific events involving the mechanism, response, and recovery [ 43 , 48 – 50 ]. From the above analysis, it can be seen that terrorism, emergency response and health incident management, and the disposition of public health events are the hotspots of international attention. Legislative situation and characteristics of China's emergency law [ 53 ], Legal construction of public emergency response in China: legal construction task proposed by SARS crisis management practice [ 54 ], and The realistic subject of administrative rule of law in public emergency management [ 55 ] were published in 2003. All three articles discussed the problems existing in the construction of the administrative legal system under the background of SARS. After that, three articles were published in 2004, mainly studying the mechanism and structure of PHEM in China. This research proposed the need to establish the emergency response mechanism for PHEM and establish emergency structure construction as soon as possible. In 2009, Kaibin Zhong wrote the article Review and prospect: construction of emergency management system in China [ 52 ]. He elaborated on the core contents of Chinese PHEM construction, including emergency plans, emergency structures, emergency mechanisms, and legal systems. China's PHEM integrates emergency systems, emergency mechanisms, and legal systems in an all-round way, which is characterized by comprehensiveness, institutionalization, openness, and guarantees. In 2011, The status quo of emergency management system for sudden public health events in America and Japan and its enlightenment [ 64 ] was published, showing that China has been learning the theory and experience of PHEM from some advanced countries. From the above analysis, it can be seen that the legal system, mechanism, and structure, system, and learning from abroad are the theoretical guidance for Chinese PHEM in the past 30 years. Admittedly, there are some limitations to this study. First, the conclusions drawn from this study were based on only two large literature retrieval libraries. Other databases, such as Embase and Springer Link, were not studied. Not being able to search all the literature in this field may lead to incomplete retrieval results. Second, CiteSpace has some shortcomings in processing the results of the Chinese database; it cannot translate the result from Chinese into English directly. Third, there is a 1-year or longer time lag between our paper submission and its publication. The database articles may change during this time. Fourth, we conducted a comparison between Chinese and international databases similar to that performed in many other studies. It should be acknowledged that the two databases had different acceptance ratios, and this difference in data sources might lead to bias in the study results. In addition, we categorized English articles focusing on China as being part of the international database and did not analyze them alone. Although only a small part of the total, this may have created some deficiencies. This limitation may constitute an object of future studies, namely, those analyzing the differences between English papers focusing on China vs. Chinese papers. Conclusions In summary, we selected two large retrieval library documents to define the PHEM domain and detected the research status and the trends related to it from 1991 to 2018. According to the analyses, the conclusions are as follows. In the next 3 years, the number of international PHEM articles will continue to increase, while the number of Chinese articles will decline. Chinese scholars show less cooperation among different organizations. There are differences in regional and economic distribution between international and Chinese cooperation networks. China focuses on the east regionally, while developed countries and European countries have a more international focus. International research often relates to timely issues, mainly by focusing on emergency preparedness and monitoring of public health events, while China focuses on public health emergencies and their resolution. The international research on PHEM begins with terrorism and bioterrorism, followed by disaster planning and emergency preparedness, and emerging infectious diseases. China uses SARS as the research background and the legal system construction as the research starting point, which is followed by the mechanism and structure, system, and training abroad. Supplementary information Additional file 1: Table S1. Top 10 authors in the published volume and centrality of international database. Table S2. Top 10 authors in the published volume and centrality of Chinese database. Table S3. Top 10 institutions in the published volume and centrality of international database. Table S4. Top 10 institutions in the published volume and centrality of Chinese database. Table S5. Top 10 keywords ranked by citation counts and centrality of international database. Table S6. Top 10 keywords ranked by citation counts and centrality of Chinese database. Figure S1. Co-author network of Chinese database. Figure S2. Co-institution network of Chinese database. Figure S3. Keyword co-occurrence network of Chinese database. Figure S4. Keywords with the strongest citation bursts of Chinese database. Additional file 1: Table S1. Top 10 authors in the published volume and centrality of international database. Table S2. Top 10 authors in the published volume and centrality of Chinese database. Table S3. Top 10 institutions in the published volume and centrality of international database. Table S4. Top 10 institutions in the published volume and centrality of Chinese database. Table S5. Top 10 keywords ranked by citation counts and centrality of international database. Table S6. Top 10 keywords ranked by citation counts and centrality of Chinese database. Figure S1. Co-author network of Chinese database. Figure S2. Co-institution network of Chinese database. Figure S3. Keyword co-occurrence network of Chinese database. Figure S4. Keywords with the strongest citation bursts of Chinese database. Supplementary information Supplementary information accompanies this paper at 10.1186/s12199-020-00896-z.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088786/

Constructing and transient expression of a gene cassette containing edible vaccine elements and shigellosis, anthrax and cholera recombinant antigens in tomato

Shigella dysenteriae causing shigellosis is one of the diseases that threaten the health of human society in the developing countries. In Shigella, IpaD gene is one of the key pathogenic genes causing strong mucosal immune system reactions. Anthrax disease is caused by Bacillus anthracis . PA protective antigen is one of the subunits in anthrax toxin complex responsible for the transfer of other subunits into the cytosol of host cells. The 20 kDa subunit of PA (PA20) has the property of immunogenicity. CTxB or B subunit of Vibrio cholerae toxin (CT) is a non-toxic protein and has the function to transfer toxic subunit into cytosol of the host cells by binding to GM1 receptor. The aim of this study was to fuse PA20, ipaD and CTxB and transform tomato plants by this cassette in order to produce an oral vaccine against shigellosis, anthrax and cholera. CTxB was used for these two antigens as an immune adjuvant. IpaD and PA20 genes were cloned in pBI121 containing the CTxB gene and Extensin signal peptide. In order to evaluate the transient expression of Shigellosis, Anthrax and Cholera antigens, agro-infiltrated tomato tissues were inoculated with Agrobacterium tumefaciens containing the gene cassette. Cloning was confirmed by PCR, enzymatic digestion and sequencing techniques. Expression of the antigens was examined by SDS-PAGE, dot blot and ELISA. Maturate green fruits demonstrated the highest expression of the recombinant proteins. The first phase of this study was carried out for cloning and expressing of CtxB, ipaD and PA20 antigens in tomato. In the next phase, we aim to analyze the immunogenicity of this vaccine candidate in laboratory animals. Background Shigella is a genus from Escherichia order and the Enterobacteriaceae family that is a small, non-aerobic bacilli form, gram-negative, non-sedentary and non-sporulating bacterium. In terms of biochemical and serological properties Shigella is classified into four different categories including Shigella dysenteriae , Shigella flexneri , Shigella boydii and Shigella sonnei [ 1 ]. The main part of the mechanism of molecular pathogenesis of Shigella is the infection of host intestinal epithelial cells and intracellular survival in the epithelial environment. Pathogenesis controlling factors are coded by both plasmids and chromosomes of bacteria [ 2 , 3 ]. Ipa operon contains ipA, B, C and D genes. The product of these genes are very important antigens, causing the host to show strong immune responses against Shigella. ipaD is a 37 kDa protein that recruits ipaB protein on the cell surface and triggers the invasion. This protein has been located on top of the third type of Shigella secretion system. Bacillus anthracic is a rod-shaped Gram-positive bacterium with sporulation power. It is sedentary and grows rapidly at 37 °C [ 4 ]. Anthrax three main symptoms include skin, respiratory and digestive systems that 95% of cases reported worldwide is skin symptoms [ 5 ]. About 80% of skin type of anthrax is not an important issue and will spontaneously improve within a few weeks [ 6 ]. While 10–20% of cases, if not going to the clinical treatment, will lead to death [ 7 ]. Anthrax toxin consists of three proteins which are non-toxic in the monomeric form and when create a trimeric complex on the surface of mammalian cells, toxic form occurs. Edema factor (EF) and lethal factor (LF) enzymes are transported by the third unit called protective antigen protein (PA) to the cytosol of mammalian cells. EF is an adenylate cyclase causing Edema and impaired immune response to infection when combined with PA and injected to mammals [ 8 , 9 ]. LF is a protease that cuts some of the mitogen-activated protein kinase kinases [ 10 , 11 ]. Association of toxin subunits begins when the PA binds to a cellular receptor called receptor of anthrax toxin, a membrane protein with high expression. Then PA is cleaved into two fragments by a protease from the Furin proteases group. This enzyme removes the 20 kDa fragment of the N-terminal, while carboxyl end of the 63 kDa fragment remains attached to the cell receptors [ 12 ]. Unlike the native PA, the 63 kDa PA is oligomerized and comes in the form of a heptamer ring [ 13 , 14 ]. While native PA remains on the cell surface the heptamer is transported into the cell with endocytosis [ 15 ]. Transferring non-toxic complexes to the low pH of endosomal vesicles causes 63 kDa heptamer to attach to the membrane to form a channel [ 16 , 17 ]. EF and LF passing through the channel to reach the cytosol [ 18 ]. Cholera is a lethal diarrheal disease caused by a Gram-negative bacterium of Vibrio cholerae . The main symptoms of cholera are mainly due to the secretion of cholera toxin called CT. The toxin is a pentameric protein with a molecular weight of 85 kDa composed of two subunits called A and B. The subunit B of cholera toxin (CTxB) with a molecular weight of 11.6 kDa binds to ganglioside GM1 receptor and facilitates the endocytosis of cholera toxin into the host cells. CTxB protein is known as an effective immunogen in the intestinal mucosa. This protein has a variety of applications including usage as an adjuvant in vaccines in which the mucosal immune response is important. It is also known as a suitable vaccine adjuvant for oral and inhalation [ 19 , 20 ]. Tomato is a valuable vegetable in the Middle East and economically is in the second place in the world after potato. Original homeland of tomato is central and South America and more likely the west coast of South America. Tomato belongs to the Solanum Genus. Domestic tomato is Lycopersicum sp. Transgenic plants are a good alternative to the animal cells and prokaryotic expression systems. Transgenic plants are produced with different purposes, such as to obtain higher yield, improved quality and resistance to pests and diseases. Therefore, similarly transgenic plants can be created to express synthetic and medicinal proteins including monoclonal antibodies, antigens, vaccines, therapeutic enzymes, blood proteins, cytokines, growth factors and hormones [ 21 ]. The most important advantage of using transgenic plants is healthy aspect of their products. Transgenic plants are not host to human pathogens. Thus human pathogens such as contaminated products to hepatitis, HIV, carcinogens and bacterial toxins, etc. will not exist in transgenic plants [ 22 ]. Peptide antigens that are expressed in the edible and delicious parts of plants have led to the idea of the synthesis of active edible vaccines [ 23 ]. With this background we aimed to conjugate three pathogenic bacteria antigens to express a conjugated vaccine against three diseases in tomato plants. For this purpose, after cloning of the genes in pBI121 binary expression vector, transient gene expression evaluated by agroinfiltrated tomato leaves and fruit inoculated with Agrobacterium tumefaciens containing the genes construct. Expression of the gene cassette was examined by dot blot and ELISA techniques. Materials and methods Vectors used A plant binary expression vector, pBI121 containing the Extensin signal peptide and CTxB (Fig. 1 ) and a pET28 plasmid containing ipaD and PA20 genes (both dedicated by University of Imam Hussain, Iran) were used in this study. These two plasmids were used for the PCR-based amplification of the three genes and further on for cloning in a single cassette and eventually, agroinfiltration experiments. Fig. 1 Linear and circular map of pBI_Ex-CTxB plasmid Cloning of ipaD A 321 bp fragment of the N-terminal region of ipaD gene was amplified by PCR using the forward and reverse primers (Table 1 ) harboring the restriction sites of Bam HI and Xho I. Digestion of amplified ipaD and pBI_Ex-CTxB vector performed with Bam HI and Xho I (Fermentase). Gel extraction was carried out with GeneAll gel extraction kit. Ligation performed by T4 DNA Ligase (Fermentase). E. coli strain DH5α competent cells prepared [ 24 ] and transformed by the recombinant plasmid. Transgenic colonies selected from the selection medium containing 50 mg/l kanamycin. Individual colonies were picked up and processed for the presence of the ipaD fragments by sequencing and digestion. Table 1 Primers used for amplifying and cloning of PA20 and ipaD . The underlined sequence in PA20 forward primer is relate to Linker and the red colored sequences is related to the added restriction site to the primers Primer Tm Enzyme site Sequence ipaD -F 62 BamH I TG GGATCC CGTACCACCAACC ipaD -R 62 Xho I AG CTCGAG GGTGTAAGAAGACACCGCGTGC PA20 -F 59 BamH I AG GGATCC GAAGTTAAACAGGAGAACCGG PA20 -R 59 BamH I AG GGATCC GGTCCTGGTCCTCTTGAGTTCGAAGATTTTTGTTTTAATT CTGGC Cloning of PA20 A 535 bp fragment of a-1 domain of PA20 was amplified by PCR with the forward and reverse primers (Table 1 ) containing Bam HI restriction sites. The amplified fragment and plasmid were digested by Bam HI. The linearized plasmid was treated with alkaline phosphatase (Fast-AP Fermentase, 1U for 1 µg plasmid) and extracted from gel (GeneAll gel extraction kit). Following transformation of the ligation product, kanamycin-resistant colonies were selected. To confirm the presence of PA20 in pBI_Ex-CTxB-PA20-ipaD vector, sequencing, PCR amplification and restriction digestion were used (Fig. 2 ). Fig. 2 Amplification of ipaD by PCR and digestion of the recombinant plasmid. a 321 bp length of ipaD amplified by PCR and b lane 1 and 2 recombinant plasmid extracted from E. coli after cloning and lane 3 digested recombinant pBI_Ex-CTxB-ipaD plasmid with BamHI and XhoI and separation of ipaD (321 bp) from plasmid Agroinfilteration The recombinant vector was transformed into competent Agrobacterium tumefaciens GV-3101 strain by heat shock [ 25 ]. Kanamycin and rifampin were used to select colonies harboring recombinant plasmid and Agrobacterium (and not other possible bacteria contamination like transformed E. coli ), respectively. A colony of Agrobacterium was first cultured in the LB medium containing 200 µM acetocyringone and 50 mg/l kanamycin overnight and the bacteria was pelleted at 3500 rpm for 10 min at room temperature. Pelleted bacteria were then resuspended in a 100 ml inoculation buffer and remained on the desk for 3–4 h. To evaluate the transient expression of the genes, agro-infiltration was carried out on the two-month-old tomato plant leaves, maturate green and completely red state fruits, grown in the green house. Leaves and fruits were inoculated with Agrobacterium tumefaciens via agro-injection method. In this assay inoculation medium containing 10 mM MgSo4, 10 mM MES and 200 µM acetosyringone (pH adjusted to 5.6) used for injection of Agrobacterium with a syringe. The Agrobacterium solution was injected smoothly with a needleless syringe to leaf and with a needle containing syringe to fruit pericarp tissues. Fortunately, in the injection area, Agrobacterium solution movement inside the leaf and fruit tissue was clearly seen. We tried to inject the parts (about 2 cm in diameter) of the leaf and fruit with Agrobacterium completely until saturated level, that we could not inject anymore. Finally, the areas where the solution completely injected, was carefully marked. For protein extraction assay, the places where Agrobacterium was injected and marked was precisely and carefully sampled to eliminate the non- Agrobacterium injected parts. The inoculated samples were kept in a growth chamber at 26 °C in a 16/8 h dark and light condition for 5–7 days. Protein extraction Inoculated leaves and fruits were sampled and used for protein extraction. The lysis buffer was prepared (Tris–HCL 0.1 M, pH = 8, Ascorbic acid 15 mM, DTT 2 mM and PVP 3%). 1 g of fine powdered samples in liquid nitrogen was poured in 3 ml of lysis buffer and completely vortexed and kept in 4 °C for 1 h. The lysate centrifuged in 4 °C for 20 min at 15,000 rpm. The supernatant retrieved and for every 1 ml supernatant 300 µl glycerol was added and stored in − 80 °C. SDS-PAGE and dot blotting To evaluate the transient expression of antigenes in agro-injected and intact (as control) samples, SDS-PAGE and dot blot analysis were carried out. Anti-ipaD [ 26 ] polyclonal antibodies were used as primary antibody against ipaD and goat Anti-Rabbit HRP conjugated (thermofisher, 65-6120) were used as secondary antibody against FC segment of anti-ipaD antibody. Total proteins were extracted from agro-injected leaves and fruits. Recombinant proteins were purified by Co 2++ column using His-Tag present at the C-terminal of the recombinant protein and analyzed by SDS-PAGE and dot blotting. ELISA To perform an ELISA assay Anti-ipaD Rabbit polyclonal and goat Anti-Rabbit HRP conjugated antibodies were used as primary and secondary antibodies respectively in the presence of TMB substrate. In this experiment the ipaD protein (one of our vaccine peptide) were used to detect indirectly by two antibodies. First, the recombinant protein was coated in ELISA wells and the unbounded sites were blocked by skim milk. Anti-ipaD and further anti-human (anti-FC) antibodies used respectively for detecting the ipaD and providing signal. TMB substrate added to the reaction and after color change, reaction stopped by NaOH. Results were analyzed by ELISA reader (BioTek USA). Vectors used A plant binary expression vector, pBI121 containing the Extensin signal peptide and CTxB (Fig. 1 ) and a pET28 plasmid containing ipaD and PA20 genes (both dedicated by University of Imam Hussain, Iran) were used in this study. These two plasmids were used for the PCR-based amplification of the three genes and further on for cloning in a single cassette and eventually, agroinfiltration experiments. Fig. 1 Linear and circular map of pBI_Ex-CTxB plasmid Cloning of ipaD A 321 bp fragment of the N-terminal region of ipaD gene was amplified by PCR using the forward and reverse primers (Table 1 ) harboring the restriction sites of Bam HI and Xho I. Digestion of amplified ipaD and pBI_Ex-CTxB vector performed with Bam HI and Xho I (Fermentase). Gel extraction was carried out with GeneAll gel extraction kit. Ligation performed by T4 DNA Ligase (Fermentase). E. coli strain DH5α competent cells prepared [ 24 ] and transformed by the recombinant plasmid. Transgenic colonies selected from the selection medium containing 50 mg/l kanamycin. Individual colonies were picked up and processed for the presence of the ipaD fragments by sequencing and digestion. Table 1 Primers used for amplifying and cloning of PA20 and ipaD . The underlined sequence in PA20 forward primer is relate to Linker and the red colored sequences is related to the added restriction site to the primers Primer Tm Enzyme site Sequence ipaD -F 62 BamH I TG GGATCC CGTACCACCAACC ipaD -R 62 Xho I AG CTCGAG GGTGTAAGAAGACACCGCGTGC PA20 -F 59 BamH I AG GGATCC GAAGTTAAACAGGAGAACCGG PA20 -R 59 BamH I AG GGATCC GGTCCTGGTCCTCTTGAGTTCGAAGATTTTTGTTTTAATT CTGGC Cloning of PA20 A 535 bp fragment of a-1 domain of PA20 was amplified by PCR with the forward and reverse primers (Table 1 ) containing Bam HI restriction sites. The amplified fragment and plasmid were digested by Bam HI. The linearized plasmid was treated with alkaline phosphatase (Fast-AP Fermentase, 1U for 1 µg plasmid) and extracted from gel (GeneAll gel extraction kit). Following transformation of the ligation product, kanamycin-resistant colonies were selected. To confirm the presence of PA20 in pBI_Ex-CTxB-PA20-ipaD vector, sequencing, PCR amplification and restriction digestion were used (Fig. 2 ). Fig. 2 Amplification of ipaD by PCR and digestion of the recombinant plasmid. a 321 bp length of ipaD amplified by PCR and b lane 1 and 2 recombinant plasmid extracted from E. coli after cloning and lane 3 digested recombinant pBI_Ex-CTxB-ipaD plasmid with BamHI and XhoI and separation of ipaD (321 bp) from plasmid Agroinfilteration The recombinant vector was transformed into competent Agrobacterium tumefaciens GV-3101 strain by heat shock [ 25 ]. Kanamycin and rifampin were used to select colonies harboring recombinant plasmid and Agrobacterium (and not other possible bacteria contamination like transformed E. coli ), respectively. A colony of Agrobacterium was first cultured in the LB medium containing 200 µM acetocyringone and 50 mg/l kanamycin overnight and the bacteria was pelleted at 3500 rpm for 10 min at room temperature. Pelleted bacteria were then resuspended in a 100 ml inoculation buffer and remained on the desk for 3–4 h. To evaluate the transient expression of the genes, agro-infiltration was carried out on the two-month-old tomato plant leaves, maturate green and completely red state fruits, grown in the green house. Leaves and fruits were inoculated with Agrobacterium tumefaciens via agro-injection method. In this assay inoculation medium containing 10 mM MgSo4, 10 mM MES and 200 µM acetosyringone (pH adjusted to 5.6) used for injection of Agrobacterium with a syringe. The Agrobacterium solution was injected smoothly with a needleless syringe to leaf and with a needle containing syringe to fruit pericarp tissues. Fortunately, in the injection area, Agrobacterium solution movement inside the leaf and fruit tissue was clearly seen. We tried to inject the parts (about 2 cm in diameter) of the leaf and fruit with Agrobacterium completely until saturated level, that we could not inject anymore. Finally, the areas where the solution completely injected, was carefully marked. For protein extraction assay, the places where Agrobacterium was injected and marked was precisely and carefully sampled to eliminate the non- Agrobacterium injected parts. The inoculated samples were kept in a growth chamber at 26 °C in a 16/8 h dark and light condition for 5–7 days. Protein extraction Inoculated leaves and fruits were sampled and used for protein extraction. The lysis buffer was prepared (Tris–HCL 0.1 M, pH = 8, Ascorbic acid 15 mM, DTT 2 mM and PVP 3%). 1 g of fine powdered samples in liquid nitrogen was poured in 3 ml of lysis buffer and completely vortexed and kept in 4 °C for 1 h. The lysate centrifuged in 4 °C for 20 min at 15,000 rpm. The supernatant retrieved and for every 1 ml supernatant 300 µl glycerol was added and stored in − 80 °C. SDS-PAGE and dot blotting To evaluate the transient expression of antigenes in agro-injected and intact (as control) samples, SDS-PAGE and dot blot analysis were carried out. Anti-ipaD [ 26 ] polyclonal antibodies were used as primary antibody against ipaD and goat Anti-Rabbit HRP conjugated (thermofisher, 65-6120) were used as secondary antibody against FC segment of anti-ipaD antibody. Total proteins were extracted from agro-injected leaves and fruits. Recombinant proteins were purified by Co 2++ column using His-Tag present at the C-terminal of the recombinant protein and analyzed by SDS-PAGE and dot blotting. ELISA To perform an ELISA assay Anti-ipaD Rabbit polyclonal and goat Anti-Rabbit HRP conjugated antibodies were used as primary and secondary antibodies respectively in the presence of TMB substrate. In this experiment the ipaD protein (one of our vaccine peptide) were used to detect indirectly by two antibodies. First, the recombinant protein was coated in ELISA wells and the unbounded sites were blocked by skim milk. Anti-ipaD and further anti-human (anti-FC) antibodies used respectively for detecting the ipaD and providing signal. TMB substrate added to the reaction and after color change, reaction stopped by NaOH. Results were analyzed by ELISA reader (BioTek USA). Results Cloning of ipaD A 321 bp fragment of N-terminal of ipaD was amplified using forward and reverse primers by PCR (Table 1 ) and analyzed on a 1% (w/v) agarose gel (Fig. 2 ). Figure 2 b (lane 3) shows the 321 bp fragment related to ipaD that was pulled out from the recombinant pBI_Ex-CTxB-ipaD plasmid by digestion with Bam HI and Xho I restriction enzymes. Cloning of PA20 The PCR-based amplified fragment with the length of 535 bp related to PA20 gene was inserted into pBI_Ex-CTxB-ipaD plasmid between the CTxB and ipaD genes using Bam HI restriction site (Fig. 3 a). In order to prevent self-ligation (due to single digestion), the 5′ phosphate was removed by the alkaline phosphatase treatment. Correct cloning of PA20 in pBI_Ex-CTxB-ipaD was confirmed by sequencing and digestion (Fig. 3 b). Figure 3 b lane 1 shows the result of digestion of the new engineered plasmid (pBI_Ex-CTxB-PA20-ipaD) with Bam HI and the presence of 535 bp fragment ( PA20 ). Since the cloning was carried out with the single digestion, checking the orientation of insertion was necessary. Therefore, plasmids were digested with Hin dIII. There are two Hin dIII restriction sites, one in PA20 and the other in the plasmid backbone. The digestion of the recombinant plasmid led to the appearance of a 1800 bp fragment and the linearized plasmid on the agarose gel. This digestion procedure shows that the fragment had inserted into the plasmid in a correct orientation (Fig. 3 b lane 2). However, digestion of non-recombinant plasmid with Hind III, led only to the conversion of circular plasmid to a linear form (Fig. 3 b lane 3). Fig. 3 Amplification and cloning of PA20 . a 535 bp of PA20 gene that amplified with PCR and b digestion of pBI_Ex-CTxB-PA20-ipaD with Bam HI (lane 1) and Hin DIII (lane 3). In lane 1, 535 bp of PA20 fragment separated from recombinant plasmid. In lane 3, 1800 bp fragment separated from recombinant plasmid because there was two recognition site for Hin DIII, one site in PA20 and another in plasmid. Lane 4, undigested recombinant plasmid and lane 5, 535 bp PA20 amplified with PCR loaded as control In this experiment each steps of cloning were checked and confirmed by PCR. Based on the agarose gel electrophoresis results, all fragments were exactly equal in size to predicted ones (Fig. 4 ; Table 2 ). The results on the agarose gel shows the right assembly of all inserts in the gene cassette. List of primer sequences used in this study were shown in Tables 1 and 2 . Fig. 4 Amplification of different fragments of gene cassette. a 321 bp of ipaD (lane 1), 535 bp 0f PA20 (lane2), b PA20-ipaD 856 bp (lane 1–4), and c 1692 bp of promoter to ipaD (lane1), 1372 bp of promoter to PA20 (lane2) and 535 bp of PA20 as control (lane3) Table 2 The primers used in PCR reaction Primer Fragment to be amplified Size (base pair) ipaD F ipaD 321 ipaD R PA20 F PA20 535 PA20 R PA20 F PA20-ipaD 856 ipaD R CAMV35s F CAMV-Ex-CTxB-PA20 1371 PA20 R CAMV35s F CAMV-Ex-CTxB-PA20-ipaD 1692 ipaD F Agrobacterium transformation Recombinant plasmid was transferred into A. tumefaciens strain GV-3101 by heat shock method (Fig. 5 ). Fig. 5 Agrobacterium tumefaciens colonies containing recombinant plasmid grown in Kanamycin (50 mg/l) and riphampin (50 mg/l) containing medium Transient expression of recombinant protein Greenhouse grown tomato plants were used to inject Agrobacterium into their leaves and fruits (Fig. 6 ). Fig. 6 Injected leaves of tomato plant with Agrobacterium tumefaciens harboring the recombinant plasmid Cloning of ipaD A 321 bp fragment of N-terminal of ipaD was amplified using forward and reverse primers by PCR (Table 1 ) and analyzed on a 1% (w/v) agarose gel (Fig. 2 ). Figure 2 b (lane 3) shows the 321 bp fragment related to ipaD that was pulled out from the recombinant pBI_Ex-CTxB-ipaD plasmid by digestion with Bam HI and Xho I restriction enzymes. Cloning of PA20 The PCR-based amplified fragment with the length of 535 bp related to PA20 gene was inserted into pBI_Ex-CTxB-ipaD plasmid between the CTxB and ipaD genes using Bam HI restriction site (Fig. 3 a). In order to prevent self-ligation (due to single digestion), the 5′ phosphate was removed by the alkaline phosphatase treatment. Correct cloning of PA20 in pBI_Ex-CTxB-ipaD was confirmed by sequencing and digestion (Fig. 3 b). Figure 3 b lane 1 shows the result of digestion of the new engineered plasmid (pBI_Ex-CTxB-PA20-ipaD) with Bam HI and the presence of 535 bp fragment ( PA20 ). Since the cloning was carried out with the single digestion, checking the orientation of insertion was necessary. Therefore, plasmids were digested with Hin dIII. There are two Hin dIII restriction sites, one in PA20 and the other in the plasmid backbone. The digestion of the recombinant plasmid led to the appearance of a 1800 bp fragment and the linearized plasmid on the agarose gel. This digestion procedure shows that the fragment had inserted into the plasmid in a correct orientation (Fig. 3 b lane 2). However, digestion of non-recombinant plasmid with Hind III, led only to the conversion of circular plasmid to a linear form (Fig. 3 b lane 3). Fig. 3 Amplification and cloning of PA20 . a 535 bp of PA20 gene that amplified with PCR and b digestion of pBI_Ex-CTxB-PA20-ipaD with Bam HI (lane 1) and Hin DIII (lane 3). In lane 1, 535 bp of PA20 fragment separated from recombinant plasmid. In lane 3, 1800 bp fragment separated from recombinant plasmid because there was two recognition site for Hin DIII, one site in PA20 and another in plasmid. Lane 4, undigested recombinant plasmid and lane 5, 535 bp PA20 amplified with PCR loaded as control In this experiment each steps of cloning were checked and confirmed by PCR. Based on the agarose gel electrophoresis results, all fragments were exactly equal in size to predicted ones (Fig. 4 ; Table 2 ). The results on the agarose gel shows the right assembly of all inserts in the gene cassette. List of primer sequences used in this study were shown in Tables 1 and 2 . Fig. 4 Amplification of different fragments of gene cassette. a 321 bp of ipaD (lane 1), 535 bp 0f PA20 (lane2), b PA20-ipaD 856 bp (lane 1–4), and c 1692 bp of promoter to ipaD (lane1), 1372 bp of promoter to PA20 (lane2) and 535 bp of PA20 as control (lane3) Table 2 The primers used in PCR reaction Primer Fragment to be amplified Size (base pair) ipaD F ipaD 321 ipaD R PA20 F PA20 535 PA20 R PA20 F PA20-ipaD 856 ipaD R CAMV35s F CAMV-Ex-CTxB-PA20 1371 PA20 R CAMV35s F CAMV-Ex-CTxB-PA20-ipaD 1692 ipaD F Agrobacterium transformation Recombinant plasmid was transferred into A. tumefaciens strain GV-3101 by heat shock method (Fig. 5 ). Fig. 5 Agrobacterium tumefaciens colonies containing recombinant plasmid grown in Kanamycin (50 mg/l) and riphampin (50 mg/l) containing medium Transient expression of recombinant protein Greenhouse grown tomato plants were used to inject Agrobacterium into their leaves and fruits (Fig. 6 ). Fig. 6 Injected leaves of tomato plant with Agrobacterium tumefaciens harboring the recombinant plasmid Recombinant protein analysis Protein extraction Total protein was extracted from the Agrobacterium injected tomato leaves and fruits after five days from the injection (Fig. 7 ). Fig. 7 The method of sampling of inoculated region (pericarp) of tomato fruits for protein extraction SDS-PAGE and dot blotting Total protein was extracted from injected and intact (as control) tissues, then recombinant proteins were purified by Co 2++ column and analyzed by SDS-PAGE. The 50 KD protein on the SDS-Page shows the purified recombinant protein (Fig. 8 ). Figure 9 shows the nitrocellulose paper with the blotted protein. The maximum coloring was detected for F. Green (green fruit). In the red fruit sample, the coloring was lower than the green fruit. This experiment revealed that recombinant protein production rate is much higher in green fruits than leaves and red fruits. Fig. 8 SDS-PAGE of protein. Total protein (lane 1) and recombinant protein purified with Co 2+ column (lane 2) and extracted of tomato fruit, L molecular weight marker Fig. 9 Dot blotting of extracted protein of tomato tissues with anti-ipaD antibody ELISA assay The highest expression (signal) was related to the conjugation of antibody to antigen occurred in tomato green fruit in the 1/100 dilution (Fig. 10 ). Expression of recombinant protein in tomato red fruit and leaves are the same. No signal can observe in the control sample of non-inoculated leaves and fruits (Fig. 10 ). Fig. 10 Data diagram obtained from ELISA reader Protein extraction Total protein was extracted from the Agrobacterium injected tomato leaves and fruits after five days from the injection (Fig. 7 ). Fig. 7 The method of sampling of inoculated region (pericarp) of tomato fruits for protein extraction SDS-PAGE and dot blotting Total protein was extracted from injected and intact (as control) tissues, then recombinant proteins were purified by Co 2++ column and analyzed by SDS-PAGE. The 50 KD protein on the SDS-Page shows the purified recombinant protein (Fig. 8 ). Figure 9 shows the nitrocellulose paper with the blotted protein. The maximum coloring was detected for F. Green (green fruit). In the red fruit sample, the coloring was lower than the green fruit. This experiment revealed that recombinant protein production rate is much higher in green fruits than leaves and red fruits. Fig. 8 SDS-PAGE of protein. Total protein (lane 1) and recombinant protein purified with Co 2+ column (lane 2) and extracted of tomato fruit, L molecular weight marker Fig. 9 Dot blotting of extracted protein of tomato tissues with anti-ipaD antibody ELISA assay The highest expression (signal) was related to the conjugation of antibody to antigen occurred in tomato green fruit in the 1/100 dilution (Fig. 10 ). Expression of recombinant protein in tomato red fruit and leaves are the same. No signal can observe in the control sample of non-inoculated leaves and fruits (Fig. 10 ). Fig. 10 Data diagram obtained from ELISA reader Discussion Undoubtedly vaccination is an integral part of public and individual health of human in the present day. According to the pandemic and epidemic diseases like shigellosis, anthrax and cholera, vaccination against these diseases is essential for their eradication. To produce a vaccine for these diseases, the first step is to produce the antigen. Many studies have shown that plant expression systems are quite suitable for the production of proteins. Edibility, nature friendly and no disadvantages to humans, surpasses plants compared to other expression systems such as bacteria and animal. In the present study, first ipaD and PA20 were conjugated and attached to an antigen called CTxB with adjuvant properties. Plants such as tobacco, tomato, and lettuce have been used as model plants to express various recombinant proteins. Tomato is one of the most consumed vegetables in the world with high biomass per hectare that make it a good option for production of high-scale recombinant proteins or antigens. The reason for using tomato in this study was edibility of its fruit. Undoubtedly, the importance of the prevention of acute bloody diarrhea or shigellosis is not a secret. Strains of Shigella dysentery affect many people every year, especially childrens in developing countries. Here we used ipaD protein that used in various studies and its immunogenicity have been demonstrated. Since Shigella have the ability to attack only to epithelial cells [ 27 ], therefore, one can immunize epithelial system by oral administration of this bacterial antigen. Studies showed that ipaD protein has the immunogenicity property and also the potential of vaccine candidate and evaluated its in-vivo immunogenicity by orally administration [ 28 , 29 ]. Various studies have examined the effect of adjuvant fusion to ipaD. For example, RTB is a plant-derived adjuvant and it was used to promote the immunogenicity of ipaD [ 30 ]. RTB specially delivers the ipaD to the mucosal associated lymph tissues (MALTs). The B subunit of cholera toxin or CTxB is a pentameric, non-toxic unit of cholera toxin responsible for binding of CT to its receptor, GM1. Studies revealed that the CTxA1 subunit of CT is responsible for CT toxicity. Binding of CT to CTxB subunit makes a route that CTxA1 can move via translocation through the central channel of CTxB in the clatrin and caveolin coated vesicles to the cytosol of host cells. Most of the mammalian cells, like red blood cells and leucocytes expresses the GM1 receptor on their surface [ 20 ]. It has been shown that the CTxB acts as an adjuvant for immune system and genetically fusing this protein to an antigen improves the immune response of the host to that antigen [ 20 , 31 ]. Therefore, CTxB can deliver the antigens to MALTs and facilitate its uptake by APC (antigen presenting cells). The second role for CTxB is that it can immunize the host to Vibrio cholerae . The anthrax toxin produced by Bacillus anthracic has three subunits that are not toxic to the host cells. Protective antigen (PA) translocate Edema and lethal factors to host cell cytosol [ 8 , 9 ]. PA is cleaved into two parts, including PA20, by the cell surface proteases. After this cleavage, PA20 releases into the plasma and it seems not to have any roles in disease pathogenesis or bacterial virulence. However, this protein has a diagnostic value for anthrax and can be used to induce immunity against Bacillus anthracis [ 32 ]. PA20 immunogenicity was confirmed through multiple studies in-vivo in laboratory animals [ 33 – 35 ]. Reason et al. showed that more than 62% of antibodies against PA antigen were specific for a-1 domain of PA or PA20 fragment and this suggest the potential of this fragment to develop as a vaccine candidate [ 36 ] even in treatment of cancer due to its capability to induce apoptosis in host cells [ 37 ] We expressed these three mentioned antigens in tomato to achieve an edible vaccine. Various studies in this field have been carried out. Demurtas et al. expressed two corona virus antigens responsible for pandemic sever acute respiratory syndrome or SARS in tobacco [ 38 ]. Chowdhury and Bagasra presented a theory that could express different life cycle antigens of malaria parasite (about 12 antigens) in tomato plant. They introduced tomato as a healthy and natural friendly plant. The suitability of the use of plants for expressing antigens and the production of edible vaccines by plant expression systems shows a bright future [ 39 ]. The first studies that were conducted on potato and tobacco and now other plants such as tomato, banana and lettuce have been put into research on the edible vaccines. Due to the short lifetime for consumption, these plants have a considerable disadvantage. The way suggested is not so complicated; drying and powdering the fresh fruits or plant tissues. In this regard, the tomato fruits expressing a particular antigen could be kept for 1 year without any decrease in the activity of the antigen [ 40 ]. Nowadays, bioinformatics analysis of proteins and antigens became an integrated steps in proteins and antigens identification [ 41 ]. Prior to experimental approaches, in a study using bioinformatics tools, we also, determined the most stable chimeric form of these three antigens (ipaD, PA20 and CTxB) in different eukaryotes and prokaryotes cells [ 42 ]. In the experimental phase of our study, in the first step, a 321 bp fragment of ipaD was inserted into plant expression binary plasmid. The second fragment ( PA20 ) was inserted into the plasmid between ipaD and CTxB . Reverse primer of PA20 included a sequence of 12 nucleotides (proline and glycine) as a linker that was added to the C-terminal of PA20 through PCR amplification. Various investigations have used the proline-glycine linker to form a separator between two fused proteins. This sequence (linker) could be identified as the site of cleavage by the cell surface proteases like furin proteases. Delivering the fused antigens to the host cell surface by CTxB protein, makes the linker accessible to the cell surface proteases. Cleavage of this site by the proteases leads to the separation of PA20 and ipaD antigens. After the cloning procedures, we confirmed the correct sequence of recombinant plasmid by sequencing. We expressed the recombinant protein in tomato different tissues through an agro-infiltration assay. Based on the ELISA results, the expression of recombinant proteins in green fruits was 4 times higher than leaves and red fruits. Two assumptions can be made for this result. First, obviously, tomato fruits are more suitable than leaves for agro-injection by syringe, i.e. greater amounts of Agrobacterium can be injected into its pericarp tissues than leaves. Secondly, green tomato pericarp tissue is likely to be very active in protein biosynthesis. This is due to the fast growth of fruit in the green stage. In the end of fruit biomass production of tomato, development of fruit stops and ripening begins. At the ripening state protein metabolism decreases in fruits. However, our results showed that tomato fruit is a suitable choice for the expression of recombinant proteins. Since the maximum expression of antigens was observed in the green tomato fruits. The noteworthy point is that tomato fruit is consumed in a ripe and red state. the production of recombinant proteins in green fruit or leaves will not be so useful. However, it should be noted that in our gene construct a general CaMV35S promoter was used. Thus, as a suggestion for the specific expression of these proteins, a tomato fruit-specific promoter involved in ethylene biosynthesis can be used, so that these genes are activated and expressed at the time of fruit ripening.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920037/

The Modeling and Forecasting of Carabid Beetle Distribution in Northwestern China

Simple Summary The relationship between species and environment are an important basis for the study of biodiversity. Most researchers have found the distribution of indicator insects such as carabid beetle at the local community scale; however, a few studies on the distribution of indicator insects in grassland in China. Here, we used Generalized Additive Models (GAM) to predict temperate steppe of northwestern China carabid beetle species richness distribution, and to determine the possible underlying causal factors. Predicted values of beetle richness ranged from 3 to 12. The diversity hotspots are located in the southwest, south and southeast of the study area which have moist environment, the carabid beetle is mainly influenced by temperature and precipitation. The results underline the importance of management and conservation strategies for grassland and also provides evidence for assessing beetle diversity in temperature steppe. Abstract Beetles are key insect species in global biodiversity and play a significant role in steppe ecosystems. In the temperate steppe of China, the increasing degeneration of the grasslands threatens beetle species and their habitat. Using Generalized Additive Models (GAMs), we aimed to predict and map beetle richness patterns within the temperate steppe of Ningxia (China). We tested 19 environmental predictors including climate, topography, soil moisture and space as well as vegetation. Climatic variables (temperature, precipitation, soil temperature) consistently appeared among the most important predictors for beetle groups modeled. GAM generated predictive cartography for the study area. Our models explained a significant percentage of the variation in carabid beetle richness (79.8%), carabid beetle richness distribution seems to be mainly influenced by temperature and precipitation. The results have important implications for management and conservation strategies and also provides evidence for assessing and making predictions of beetle diversity across the steppe. Simple Summary The relationship between species and environment are an important basis for the study of biodiversity. Most researchers have found the distribution of indicator insects such as carabid beetle at the local community scale; however, a few studies on the distribution of indicator insects in grassland in China. Here, we used Generalized Additive Models (GAM) to predict temperate steppe of northwestern China carabid beetle species richness distribution, and to determine the possible underlying causal factors. Predicted values of beetle richness ranged from 3 to 12. The diversity hotspots are located in the southwest, south and southeast of the study area which have moist environment, the carabid beetle is mainly influenced by temperature and precipitation. The results underline the importance of management and conservation strategies for grassland and also provides evidence for assessing beetle diversity in temperature steppe. Abstract Beetles are key insect species in global biodiversity and play a significant role in steppe ecosystems. In the temperate steppe of China, the increasing degeneration of the grasslands threatens beetle species and their habitat. Using Generalized Additive Models (GAMs), we aimed to predict and map beetle richness patterns within the temperate steppe of Ningxia (China). We tested 19 environmental predictors including climate, topography, soil moisture and space as well as vegetation. Climatic variables (temperature, precipitation, soil temperature) consistently appeared among the most important predictors for beetle groups modeled. GAM generated predictive cartography for the study area. Our models explained a significant percentage of the variation in carabid beetle richness (79.8%), carabid beetle richness distribution seems to be mainly influenced by temperature and precipitation. The results have important implications for management and conservation strategies and also provides evidence for assessing and making predictions of beetle diversity across the steppe. 1. Introduction The relationship between species and environment has always been a central topic in ecological research, the spatial distribution of species is closely related to environment [ 1 ]. Climate and human activity as a main threat to global biodiversity is increasing, the Global Assessment Report on Biodiversity and Ecosystem Services of IPBES [ 2 ] recorded that one million animal and plant species are facing extinction at present, destroying ecosystem functions and services. Therefore, efficient management tools are urgently needed to protect biodiversity and maintain global ecosystem functioning and services [ 3 , 4 , 5 ]. One management tool is to improve the predictive ability of biodiversity distribution models, such as niche models, species distribution and habitat suitability [ 6 , 7 ]. Studies have shown that a diverse ecosystem characteristic have been predicted from the main environmental drivers, including species distribution, richness, biodiversity value and soil characteristics and also proved models was very valuable to biodiversity research and mostly useful for policy makers [ 8 , 9 ]. Ideally, planning biodiversity conservation should integrate as many taxonomic groups as possible, including indicator insect groups such as the carabid beetles. Carabid beetles are an essential component of global biodiversity and play a vital role in global ecosystems (e.g., indicator, predators) [ 10 , 11 ]. Carabids are commonly used to studies grassland management, as their ecology is well-known [ 12 , 13 ]. Beetles are sensitive to environmental change and perceived good for agriculture, so ecologists and taxonomists have turned to carabid beetles to test ecological research questions, thus beetle currently faced numerous threats [ 14 , 15 ]. The main threats come from the land use and grassland degradation, which leads to the loss and degradation of beetle habitats [ 16 , 17 , 18 ], inducing changes in beetle community composition [ 19 , 20 , 21 ]. The relationships between the environment and beetle communities are complex phenomena. Generalized Additive Models (GAM) can best describe these linear or nonlinear relationships between beetle and environment by using nonparametric smoothing terms [ 22 ]. Hence, based on the advantage of GAM for accommodating nonlinear relationships between variables, GAM is expected to efficiently model the relationship between environmental variables and beetle diversity and provide reliable results. Specifically, in China, grassland vegetation, which accounts for 80% of the steppe, is degrading rapidly due to climate change and human activities that are changing productive steppe into barren land and desert [ 23 ]. The degradation will have a huge impact on steppe biodiversity and there is an urgent need to study the ecology of groups that may serve as an indicator insect. Modelling procedures can be useful tools to provide robust and accurate estimates of current and future distributions, abundance, and the population dynamics of species, and these can be directly applied to conservation and management practices [ 24 ]. Carabid beetles (Coleoptera: Carabidae) represent an abundant and diverse insect group [ 25 , 26 ] and account for an important fraction of total diversity [ 27 , 28 , 29 ]. The vital contributions of this taxonomic group to ecosystem management have been largely recorded, for example, they are used as an index of habitat restoration, land use, degree of urbanization and an indicator of shrub erosion in the steppe [ 13 , 30 , 31 ]. In addition, these species can prey on a large number of pest insects [ 32 ]. Despite beetles having proven ecological benefits and service in grassland ecosystems, their distribution patterns have been poorly recorded [ 33 , 34 ] which currently is endangering the maintenance of their ecological roles. Therefore, it is essential to integrate information about beetle diversity and distribution into grassland sustainable development strategies to improve or at least conserve their biodiversity and ecosystem services in steppe regions. However, field investigations are challenging because of remoteness, inaccessibility of many steppe areas and shortage of staff. Species Distribution models (SDMs) provide a cost-effective tool to overcome these limitations and remotely assess biodiversity over large areas at regular intervals over time [ 35 , 36 ]. SDMs have been widely used to assess distribution and diversity patterns of different organisms [ 37 , 38 , 39 ]. Increasing numbers of studies use SDMs to assess, model, predict or map species' distribution and analyze biodiversity [ 40 , 41 , 42 ]. Here, we used Generalized Additive Models (GAM) to predict and map beetle richness patterns [ 43 ]. In this study, we contribute to model the species richness in unmeasured area to promote grassland management and develop a conservation policy strategy for governments and also to determine the main driving factor of beetle's distribution. Our overall aim is to the conserve beetle biodiversity and maintain their ecosystem services in the grassland regions which form the main ecology in northwestern China. 2. Materials and Methods 2.1. Study Area This study was undertaken in two regions of Ningxia Hui Autonomous Region which represent a temperate steppe ecosystem in northwestern China and comprised between 36° north (N) and 38° N and between 105° east (E) and 108° E. (1) Yanchi region, characterized by a cold, semi-arid continental monsoon-influenced climate, with a mean annual temperature of 5.7 °C and mean annual precipitation of 200 mm [ 44 ]. The soil was of sierozem and the representative vegetation is Agropyron mongolicum , Artemisia desertorum , Lespedez adavurica and Artemisia blepharolepis . (2) Guanyuan region, characterized by a semi-arid continental monsoon-influenced climate, with a mean annual temperature of 7 °C and mean annual precipitation of 400 mm [ 44 ]. The soil was of black thorn and brown and the representative vegetation is Stipa bungeana , Artemisia frigida , Potentilla acaulis and Stipa grandis . 2.2. Beetle Data The beetle data used in this study was from the steppe of northwestern of China which was sampled in 2017, 2018 and 2019; we selected 124 sampling sites and at each sampling site placed at random five pitfall traps (separated by at least five meters from each other), all sampling site were separated by at least 150 m in order to avoid possible autocorrelation. Samples were taken from May to September every year, which allowed us to obtain a good representation of carabid richness. We accounted for number of beetle once a month and take the average of five times for analysis. We divided each study area into 10 × 10 km 2 grid squares in order to discriminate adequately-surveyed grid squares, the value of each 100 km 2 grid, well surveyed were identified and recorded of all species observed ( Figure 1 ). Five pitfall traps (400 mL capacity, 7.5 cm diameter, filled with 40–60 mL of a 2:1:1:20 vinegar, sugar, alcohol and water solution and covered with a suspended opaque plastic roof) were placed at each site and collected three days later. Trapped beetles were stored in 75% ethanol and transported to the laboratory for identification to species level with the aid of a taxonomist expert in carabid beetles (Prof. H. Liang, see Acknowledgments). Analyses were conducted using the pooled data from the average values every year. 2.3. Environmental, Spatial and Climatic Data At each sampling site, we selected a 1 × 1 m quadrat frame (the habitat around each site is very homogeneous) and measured plant dry biomass (PB), cover (PC, %), density (PD), height (PHe), plant richness (PSD), litter dry mass (SL, g/m 2 ), soil moisture (SM, %), bulk density (SBD), and soil temperature (ST), soil organic matter (C), total phosphorus (P), total nitrogen (N), pH value (pH). Above of vegetation and soil were measured once a month. The soil moisture and temperature (underground 10 cm) were measured by a portable soil water potential temperature tester (TRS-II, China). The climate data including the maximum and minimum monthly mean temperature (T, t), the annual mean temperature (TM), and annual precipitation (p) were extracted climatic dataset ( www.worldclim.org , accessed on 1 December 2020). The spatial data (longitude (Lon) and latitude (Lat)) and the geographical data (altitude (Alt)) were measured by GPS (G128BD, China). The information of variables saw Table A1 . 2.4. Data Processing and Statistical Analyses Species activity density was calculated as the number of individuals per square meter; Margalef index was calculated using the formula: ( S − 1 ) ∕ l n N , S is the number of species, where n is the number of collected individuals per square, species richness was expressed as the number of beetle species in a given grid cell. All analyses were performed in R v.4.0.3. 2.5. Model Building A total of 19 potential predictors were preselected. First, the method of variance inflation factor was used to select the most important environmental predictors for each response variable and the largest variable was deleted in turn, that is, the collinear environmental factors were deleted, until all variables were less than 10 from the 'car' package in R (v.4.0.3). This package (vif function) can help us to identify and keep important relevant predictors in our models. Second, the predictor factors were further refined by using the Pearson correlation coefficient to identify highly correlated variables (|r|) > 0.7) and avoid the inclusion of redundant variables in our models. The goodness-factor for the competing functions was measured by an F-ratio with a 5% significance level and the non-significant factors were removed ( Table A2 ). Third, a backward stepwise procedure was used to enter the variables into the model [ 45 ]. The step model was used to detect the lowest AIC value, and the optimal environmental factor was automatically selected. When "none" is at the top, it means the end of model selection. The number of beetle richness as dependent variable in order to remove the non-significant spatial terms. The significant spatial terms ( p 0.7) and avoid the inclusion of redundant variables in our models. The goodness-factor for the competing functions was measured by an F-ratio with a 5% significance level and the non-significant factors were removed ( Table A2 ). Third, a backward stepwise procedure was used to enter the variables into the model [ 45 ]. The step model was used to detect the lowest AIC value, and the optimal environmental factor was automatically selected. When "none" is at the top, it means the end of model selection. The number of beetle richness as dependent variable in order to remove the non-significant spatial terms. The significant spatial terms ( p < 0.05) were retained. The sampling is stratified random sampling. The soil and environmental factors of the unsampled grids were interpolated based on the statistical relationship among the surrounding measured site in each year. To compare different result for GAMs and GLM, an independent dataset was used. The data from 2017 to 2018 for training set and data from 2019 as test set were randomly chosen to evaluate offset between predicted values of the model and the original values. For model validation, we used a correlation coefficient between predictive and real species richness values. The higher the value of the correlation coefficient, the higher the predictive power of the model. 2.6. Model Fitting and Selection Species distributions were modelled with generalized linear model (GLM) and Generalized Additive Models (GAM) in order to seek the best model. GLMs are defined by the response distribution and a link function. The structure is as follows: (1) g ( μ i ) = x i T β where g is the differentiable and monotonic link function, μ i = E ( Y i ) , x i is the explanatory variable for the ith response variable, β is a vector of the parameters. The log-transformation has been found to be fit for many situations and data sources, despite its great generality, the GLM has serious limitations. Generally, AIC is usually used criterion for model selection when GLMs/GAMs are used to estimate species richness [ 46 ]. GAM is an extension of the Generalized Linear Modelling (GLM; [ 47 ]) which uses a link function to establish a relationship between the mean of the response variable and a 'smoothed' function of the explanatory variable [ 48 , 49 ]. GAMs can model highly non-linear and non-monotonic relationships between the response and the set of explanatory variables. GAM has been widely applied in ecological research, as shown by the growing number of published papers incorporating modern regression [ 50 , 51 , 52 , 53 ]. GAM implemented in the mgcv package in R (4.0.3). The most optimum model was selected with the lowest Akaike Information Criterion (AIC) and residual deviance [ 54 ]. The general form is as follows: (2) g ( μ ( Y ) ) = β 0 + f 1 ( x 1 ) + ⋯ + f m ( x m ) where g ( . ) is the connection function; μ ( Y ) is the expected value of the response variable Y ; β 0 is a constant; and f m ( . ) is a smooth function of the explanatory variable x m . Poisson distribution for species richness was used. To avoid data overfitting, the basic dimension was defined as k = 4. In order to improve model performance, the values of the parameters of the GAM algorithm were optimized independently for each model, selecting those that minimized the AIC; this step has been considered as providing an estimate of model reliability. For model assessment, the evidence ratio, AIC and minimized generalized cross validation (GCV) score were applied. The smaller the values of GCV, the better the models fit [ 55 ]. 3. Results 3.1. Population Size The mean number of the carabid beetle (number of beetles in each grid cell) was 39.88 ± 79.4 individuals/km 2 , the mean number of beetle species was 8.92 ± 1.11 individuals/km 2 . On the other hand, species activity density was 0.897 individuals/m 2 and the Margalef index was 8.54. 3.2. Fitted Model Compare to GLM, results for comparing performances are shown in Table 1 . According the AIC criterion results showed that GAMs has a lower score compared to GLM (AIC-GLM = 609.54, AIC-GAM = 598.04). In addition, R 2 (0.774), p -value (<0.001) and correlation coefficient (0.923) also indicated that GAM has a high quality for model performance, GAMs fitted the observed data as much as possible by enabling the smooth effects of the continuous predictors as well as the spatial structure of the data ( Table 1 ). The stepwise algorithm parameters used to develop the models, as well as F value and P estimates are listed in Table 1 . Seven environmental variables: maximum mean temperature, mean annual precipitation, latitude, longitude, plant density, soil bulk density, soil temperature, and PH value were statistically significant after the collinearity analysis of all environmental factors ( Table A3 ). Among the seven environmental variables, the maximum mean temperature (F = 5.336, p < 0.00046), mean annual precipitation (F = 9.031, p < 0.05) and soil temperature (F = 5.336, p < 0.001) had statistically significant effects on species, whereas soil and climatic variables consistently appeared among the most important predictors for the richness of beetle groups modeled ( Table 2 ). General trends seem to be very well identified by the GAM. The GAM parameters used to develop the models, as well as adjust the fit factor (R2), generalized cross validation (GCV) and deviance explained are listed in Table 2 . By comparing the different explanation variable of function of GAM results, selection of model variance explained the largest volume, minimum generalized cross validation, F test ( p ) model of the highest accuracy rate value as the optimal model, in general, when the rate of F test value ( p < 0.05), indicating that explain the response variables affect significantly, if adjust the fitting coefficient (R 2 ) is greater than 0.5, that model has good stability and effectively explains the response variables and explains the relationship between the variables. Among the seven predictor variables, the full model was the best adjusted explaining 79.8% of the variation, the GCV was 0.062 and the adjust the fitting coefficient (R 2 ) was 0.774, our model showed a good predictive performance for beetle richness and the best model was log (species richness) = s(T) + s( p ) + s(ST). Plots of the relationship of predicted richness distribution and environment variables are shown in Figure 2 ; the distribution of beetle richness mainly depends on the maximum mean temperature, mean annual precipitation and soil temperature. The relationship between maximum mean temperatures, mean annual precipitation and spatial distribution of beetle were complex, but it was positively correlated with soil temperature change ( Figure 2 ). 3.3. Predictive Mapping The results from the predictive mapping of the beetle richness at the spatial level are shown in Figure 3 . Predicted values of beetle richness ranged from 3 to 12. Three diversity hotspots are located in the southwest, south and southeast of the study area. The statistics of the coefficient of variation showed that, overall, predictions from individual GAMs of beetle richness at the spatial level were stable. 3.1. Population Size The mean number of the carabid beetle (number of beetles in each grid cell) was 39.88 ± 79.4 individuals/km 2 , the mean number of beetle species was 8.92 ± 1.11 individuals/km 2 . On the other hand, species activity density was 0.897 individuals/m 2 and the Margalef index was 8.54. 3.2. Fitted Model Compare to GLM, results for comparing performances are shown in Table 1 . According the AIC criterion results showed that GAMs has a lower score compared to GLM (AIC-GLM = 609.54, AIC-GAM = 598.04). In addition, R 2 (0.774), p -value (<0.001) and correlation coefficient (0.923) also indicated that GAM has a high quality for model performance, GAMs fitted the observed data as much as possible by enabling the smooth effects of the continuous predictors as well as the spatial structure of the data ( Table 1 ). The stepwise algorithm parameters used to develop the models, as well as F value and P estimates are listed in Table 1 . Seven environmental variables: maximum mean temperature, mean annual precipitation, latitude, longitude, plant density, soil bulk density, soil temperature, and PH value were statistically significant after the collinearity analysis of all environmental factors ( Table A3 ). Among the seven environmental variables, the maximum mean temperature (F = 5.336, p < 0.00046), mean annual precipitation (F = 9.031, p < 0.05) and soil temperature (F = 5.336, p < 0.001) had statistically significant effects on species, whereas soil and climatic variables consistently appeared among the most important predictors for the richness of beetle groups modeled ( Table 2 ). General trends seem to be very well identified by the GAM. The GAM parameters used to develop the models, as well as adjust the fit factor (R2), generalized cross validation (GCV) and deviance explained are listed in Table 2 . By comparing the different explanation variable of function of GAM results, selection of model variance explained the largest volume, minimum generalized cross validation, F test ( p ) model of the highest accuracy rate value as the optimal model, in general, when the rate of F test value ( p < 0.05), indicating that explain the response variables affect significantly, if adjust the fitting coefficient (R 2 ) is greater than 0.5, that model has good stability and effectively explains the response variables and explains the relationship between the variables. Among the seven predictor variables, the full model was the best adjusted explaining 79.8% of the variation, the GCV was 0.062 and the adjust the fitting coefficient (R 2 ) was 0.774, our model showed a good predictive performance for beetle richness and the best model was log (species richness) = s(T) + s( p ) + s(ST). Plots of the relationship of predicted richness distribution and environment variables are shown in Figure 2 ; the distribution of beetle richness mainly depends on the maximum mean temperature, mean annual precipitation and soil temperature. The relationship between maximum mean temperatures, mean annual precipitation and spatial distribution of beetle were complex, but it was positively correlated with soil temperature change ( Figure 2 ). 3.3. Predictive Mapping The results from the predictive mapping of the beetle richness at the spatial level are shown in Figure 3 . Predicted values of beetle richness ranged from 3 to 12. Three diversity hotspots are located in the southwest, south and southeast of the study area. The statistics of the coefficient of variation showed that, overall, predictions from individual GAMs of beetle richness at the spatial level were stable. 4. Discussion SDMs are important ecological tools for conservation planning and management [ 56 ]. The present study demonstrates the efficacy of SDMs to assess the species richness of beetle in grassland. GAM analysis suggested that the three most important factors, which showed the largest effect of the beetle richness, were mean maximum temperature, annual precipitation and soil temperature. The soil temperature changed with the temperature, the result supported the factors determining beetle life cycles include variation in temperature and rainfall [ 57 ]. Our model explained a significant fraction (0.77) of the variation in beetle richness. Our study also provides a potential methodology for conservation of the species groups. The model of distribution of beetle richness helps understanding the relationships between beetles and their environment, and thus is useful for protection and management purposes. GAM used smooth functions to deal with nonlinear relationships between the response variable and explanatory variables, increasing evidence that GAM is likely to be more suitable to estimate the distribution of species richness [ 43 , 58 ]. GAMs showed good performance for species richness estimation in the present study (79.8%), and robustly explain the relationship between the variables and species richness. Our study demonstrated that climatic factors, especially temperature and precipitation are the important environmental factors generating richness patterns of the beetle group. Both temperature and precipitation show a curvilinear relationship with species richness and had a significant effect on beetle species, this result agrees with those obtained in earlier studies of carabid beetles [ 19 ]. Although, the suitable maximum annual temperature value is around 20 °C, the effect may be reflecting the effect of temperatures of the warmest months which can limit the activity of beetles according to their tolerance to desiccation. Because most carabids are active on the ground, their body temperature depends directly on the ambient temperature and it is known that species activity can be stimulated by temperature [ 59 ]. The importance of precipitation can be explained by the free ranging life style of immature larval stages. The amount of precipitation enhances the aboveground vegetation biomass [ 60 ], and vegetation provides food and shelter (from the environment and predators) for the herbivorous species. However, precipitation hinders the survival of some of these species when it exceeds the threshold value. The soil temperature has a significant correlation with carabid beetle richness. Because some species lay eggs in burrows, and others overwinter as larvae or as adults in the soil, the soil temperature can stimulate or hinder species activity. We suggest that lower observed beetle richness may be due to the higher temperature, precipitation, and correlated soil temperature in those areas. Carabid beetles also respond to microhabitat conditions. Carabids perceived microhabitat variation and selected niches accordingly [ 61 ], increasing evidence that management of microhabitats is a key tool for conserving ecosystem function. The Carabid beetle fauna in the steppe ecosystem in Ningxia Hui Autonomous Region has been recorded over ten years, but the beetle information only shows where the entomologist sampled and the composition of beetle [ 62 , 63 ]. In this study, we have shown that with a reasonable sampling distribution, predictive variables for species richness can be derived efficiently from GIS-based data for areas in which species inventories have not yet been conducted, and a reliable forecasted map of species richness may be obtained. The forecasted map can be used to plan and carry out new, targeted studies and regional surveys thus saving on the resources needed for large-scale surveys. It is very expensive and may be impractical to sample all poorly surveyed areas. The forecasted map also can provide an opportunity to manage these habitats and conserved carabid taxa. Carabid beetles live in moist habitats and are excellent model species on research of ecological and conservation theory [ 64 , 65 , 66 ]. The 3rd International Carabidologists' Meeting emphasized that it is needed to concern on the effects of habitat loss and fragmentation on dynamics of beetle population if wise decisions are to be made regarding conservation and land-use [ 67 ]. These beetles readily respond to disturbances and management. Our results show that it is quick and inexpensive to employ forecasting models using simple environmental variables and adequately sampled areas to produce an estimate of the spatial distribution of species richness and obtain reasonable biogeographic patterns. Relating biological data to environmental variables without adding geographic position as a model predictor sometimes overestimates the actual species richness [ 68 , 69 ]. Our results demonstrate that elaborating predictive models using simple environmental variables is quicker and less expensive when based on the concept of adequately sampled areas. Consequently, our model for the species richness of carabid beetle distribution provides a good substitute for information that could not be provided otherwise in the coming years. This information will focus sampling efforts, and also inform management and conservation strategies. 5. Conclusions Our models explained a significant fraction of the variation in beetle richness (79.8%), and predictive mapping of carabid beetle richness at the spatial level helped us to identify important variables determining the richness of beetle species. For the carabid group of beetles, species richness variation was influenced primarily by the climatic factors of maximum temperature and precipitation. If the survival of carabid species is constrained by temperature and precipitation (few species can tolerate high temperature and precipitation), we argue that species richness variation in the steppe of northwestern China is due mainly to the failure of many species to go beyond determined temperature and precipitation range limits. Thus, the regions richest in species are those with a temperature and precipitation compatible with the maintenance of populations.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560737/

Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro

Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro . Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as "respiratory burst", whereas M2-polarization was associated with processes such as "mitosis". Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains , subfamily A , member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for future studies upon the role of macrophage polarization in spontaneous diseases of the dog, a species that has emerging importance for translational research. Introduction Circulating peripheral blood mononuclear cells (PBMCs) play an important role during both the steady state and inflammation. Monocytes, which originate from hematopoietic stem cells, are capable of migrating from the blood into distinct tissues and differentiate into macrophages in order to replenish specific tissue-specific macrophage populations [ 1 ]. Functional diversity and plasticity are hallmarks of macrophages [ 2 , 3 ]. Together they represent a heterogeneous cell population of the mononuclear phagocyte system playing a pivotal role in tissue homeostasis, inflammation, host defense, and tissue repair [ 4 , 5 ]. Depending on the micromilieu, two extremes of macrophage phenotypes have been described following external or endogenous stimulation: "classically" activated M1-macrophages and "alternatively" activated M2-macrophages [ 6 , 7 ]. Classically activated M1-macrophages develop after exposure to pro-inflammatory stimuli such as interferon ɣ (IFNɣ), lipopolysaccharide (LPS), or tumor necrosis factor (TNF). Subsequent to such stimulation, M1-macrophages release pro-inflammatory cytokines, reactive oxygen species (ROS), and nitric oxide (NO) [ 8 ]. Hence, on the functional level, M1-macrophages are characterized by an increased microbicidal, tumoricidal, and antigen presenting capacity [ 2 , 4 , 9 ]. In contrast, M2-macrophages become activated in the presence of interleukin (IL)-4, IL-10, IL-13, glucocorticoids, and transforming growth factor β (TGFβ) leading to enhanced secretion of anti-inflammatory cytokines. Accordingly, M2-macrophages are functionally associated with hypersensitivity, parasite clearance, inflammatory dampening, tissue remodeling, angiogenesis, immunoregulation, and tumor promotion [ 2 , 9 , 10 ]. However, it should be taken into consideration that the M1-/M2–paradigm is a simplified classification, representing only two extremes of phenotypes which do not fully mirror the complexity of the dynamic biological processes behind cell polarization [ 7 ]. Hence, gene expression profiling has been applied as a sophisticated technique to detect the underlying molecular mechanisms following macrophage activation in murine and human cells [ 11 – 14 ]. In fact, macrophage activation by any agonist involves a massive change in gene expression during the transition from one steady state to another [ 5 ]. Notably, current comparative studies strongly support the observation of marked interspecies differences and variability between mice and humans indicating that about 50% of polarization specific markers are selectively expressed in only one of both species [ 13 , 14 ]. Currently, spontaneous diseases in dogs play an established and increasing role as suitable animal models for human disorders including for instance demyelinating central nervous system (CNS) diseases, measles, cancer, and spinal cord injury (SCI) [ 15 – 21 ]. Conclusively, dogs represent a promising so called large animal model in the development of novel therapeutic approaches for naturally occurring diseases. However, despite the essential role of dogs as a translational animal model, there is a considerable lack of knowledge of the role of macrophage polarization in this species, and morphologic, phenotypic, and transcriptomic properties of polarized canine macrophages are enigmatic so far. However, a detailed knowledge of these basic principles doubtlessly represents a prerequisite for envisaged pharmacotherapeutic and cell transplantation studies. Therefore, this in vitro study aimed to (i) characterize morphological differences of polarized canine macrophages, (ii) to test the capability of established murine and human prototypical markers to differentiate canine M1- and M2-macrophages using immunofluorescence, and (iii) to unravel differences in the transcriptome of canine polarized macrophages using microarray technique in order to establish unique gene signatures, which differentiate these polarization states. The presented results provide a highly needed basis for future research upon canine spontaneous diseases such as SCI and distemper leukoencephalitis with a special emphasis upon the so far enigmatic role of macrophage polarization in these diseases. Materials and methods Blood cell isolation Blood samples were collected from a total number of 12 healthy Beagle dogs, which were kept in regulatory approved animal housing facilities of the Departments of Small Animal Medicine and Surgery and the Institute for Parasitology, University of Veterinary Medicine, Hannover, Germany. Blood collection was done by professional veterinarians from non-anesthetized dogs following the regulations of the German Animal Welfare Law and with permission provided by the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany (permission number: AZ33.9-42502-05-13A303). Following a clinical examination of each animal, which included auscultation of the lung and heart, and rectal measurement of the body temperature, the puncture spot was disinfected. A volume of 20 ml blood was collected from the cephalic vein of each dog. Following blood collection, local manual pressure was used to avoid bleeding. No animals were euthanized for the present study. Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation using a routine protocol as described previously [ 22 ]. Briefly, blood samples were diluted 1:3 in phosphate buffered saline (PBS) containing 1% penicillin/streptomycin (Biochrom GmbH, Berlin, Germany) and added onto a gradient with equal volumes of histopaque 1.077 g/ml and 1.119 g/ml (Sigma Aldrich, Taufkirchen, Germany). After 30 minutes of centrifugation, cells were harvested using a Pasteur pipette, washed with PBS and 1% penicillin/streptomycin followed by depletion of erythrocyte contamination by hypotone lysis with distilled water. Subsequently, cells were resuspended in Roswell Park Memorial Institute (RPMI)-medium 1640 (Biochrom GmbH, Berlin, Germany) containing 10% fetal calf serum (PAA, Cölbe, Germany) and 1% penicillin/streptomycin and seeded onto 96½-well plates (ThermoScientific, Waltham, MA, USA) at a density of 0.3 x 10^6 cells/well. After 24 hours of cultivation under standard conditions (5% CO 2 , 37°C), medium was completely removed and cells were washed twice with PBS and 1% penicillin/streptomycin. Cells attached to the base of wells with strong plastic adherence were referred to as monocytes [ 23 – 26 ]. Cell culture and polarization To culture canine macrophages in vitro from blood-derived monocytes, a protocol according to Durafourt et al. (2012) [ 27 ] was used with slight modifications. Briefly, canine monocytes were cultured over 7 days towards M1-/M2-macrophages by stimulation with hematopoietic growth factors over 5 days followed by activation over 2 days with distinct cytokines, purchased from companies that guarantee an endotoxin level below 0.1 EU/μg of protein. In particular, to obtain M1-macrophages, monocytes were treated with 5 ng/ml canine recombinant granulocyte macrophage colony-stimulating factor (GM-CSF; R&D Systems, Minneapolis, MN, USA), 100 ng/ml LPS (Sigma Aldrich, Taufkirchen, Germany), and 20 ng/ml recombinant canine IFNɣ (Kingfisher Biotech Inc., Saint Paul, Minnesota, USA). M2-macrophages were developed by treatment with 25 ng/ml human macrophage colony-stimulating factor (M-CSF, PeproTech Inc., Rocky Hill, NJ, USA) and 20 ng/ml canine recombinant IL-4 (R&D Systems, Minneapolis, MN, USA). Serving as a control, unstimulated monocytes were cultured under the same conditions with medium change every second day (M0-macrophages). After 7 days of cultivation, morphology, phenotype, and transcriptomic changes of M0-, M1-, and M2-macrophages were assessed. Pictures were taken with an inverted fluorescence microscope (Olympus IX-70, Olympus, Optical Co GmbH, Hamburg, Germany). The overall number of cells for each polarization was counted on at least 4 microscopic pictures (200x magnification) of three individual dogs on day 7 in culture and the mean numbers of cells per 200x field were compared using one-factorial ANOVA and post-hoc t tests applying SPSS version 21 for windows (IBM Inc., Chicago, USA). For quantification of cellular morphology, microphotographs were taken at 200x magnification and the number of cells with a distinct morphologic appearance out of at least 100 randomly selected cells was counted for each polarization (M0, M1, M2). Here, 4 distinct morphologic types were defined. In particular, cells with a cellular diameter ≤ 10 μm and absent cytoplasmic projections on the cellular surface were defined as small/roundish cells (morphology 1); cells with a cellular diameter ≥ 10 μm and numerous cytoplasmic processes were defined as amoeboid (morphology 2); cells obtaining an elongated bipolar morphology were classified as spindeloid (morphology 3); and cells possessing a large, round, and flattened morphology with a cellular diameter ≥ 30 μm and > 1 nuclei were classified as multinucleated giant cells (MNGs; morphology 4). The percentage of each of the four different morphologies was calculated in M0-, M1-, and M2-macrophages from 3 animals. A mixed ANOVA with post-hoc alpha adjustment (Tukey-Kramer) and significance level at p≤0.05 was performed using the statistical software programme SPSS version 21 for windows (IBM Inc., Chicago, USA) in order to compare the percentages of each of the morphologies between M0-, M1-, and M2-macrophages. Graphical compilation was done with GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, USA). Scanning electron microscopy M0-, M1-, and M2-macrophages were prepared for representative ultrastructural characterization using scanning electron microscopy as described previously [ 28 ]. Briefly, cells were fixed in 2.5% glutaraldehyde/cacodylate buffer, post-fixed in 1% osmium tetroxide, and dehydrated in a graded series of alcohol. Subsequently, cells were dried under a Critical Point Dryer (E3000, Polaron, London, UK), and sputter-coated with gold, and examined under a digital scanning electron microscope (DSM 940, Zeiss, Oberkochen, Germany). At least 50 cells were photographed for each morphology and evaluated with regard to ultrastructural morphology. Immunocytochemistry Immunolabelling of canine M0-, M1-, and M2-macrophages from 5 individuals was performed using a routine protocol described previously [ 29 , 30 ]. A panel of different literature-based prototype marker antibodies, reported to distinguish between M1- and M2-macrophages was applied ( Table 1 ; [ 4 , 31 ]). Briefly, cells were fixed with paraformaldehyde (PFA, 4%) for 20 minutes at room temperature (RT) and permeabilized with Triton X (0.25%) diluted in PBS (PBST). Non-specific binding was blocked by treatment of cells with bovine serum albumin (BSA, 3%; Sigma Aldrich, Taufkirchen, Germany) and normal goat serum (5%) diluted in PBST for 15 minutes at RT, except for cells intended to be stained with antibodies directed against Fc-receptors, i . e . CD16 and CD32. Subsequently, primary antibodies ( Table 1 ) were added and incubated for 2 hours at RT. All unconjugated primary antibodies were labeled with secondary goat-anti-mouse antibodies coupled to cyanine 3 (Cy3), goat-anti-rat antibodies coupled to Cy2, and goat-anti-rabbit antibodies coupled to Cy3 (all received from Jackson ImmunoResearch Laboratories, Dianova, Hamburg, Germany; dilution 1:200 in PBS), respectively, and incubated for 2 hours at RT. Following appropriate washing steps with PBS and distilled water, nuclei were counterstained with bisbenzimide (H33258, 0.01% in distilled water) for 5 minutes at RT. Species-specific immunoglobulins from mouse, rat, goat, and rabbit diluted according to the immunoglobulin concentration of the primary antibodies served as appropriate negative controls. M0-, M1-, and M2-macrophages were investigated using an inverted fluorescence microscope (Olympus IX-70, Olympus, Optical Co GmbH, Hamburg, Germany). For quantification of immunopositivity, microphotographs were taken at 200x magnification and at least 100 randomly selected cells were counted for each treatment (M0, M1, M2) with respect to the number of immunopositive cells and the total number of cells. Statistical comparison of the percentage of immunopositive cells between M0-, M1-, and M2-macrophages was done using a Kruskal-Wallis-Test and pairwise Mann-Whitney-U-Tests with significance level at p≤0.05 employing the statistical software programme SPSS version 21 for windows, and box plots were depicted using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, USA). 10.1371/journal.pone.0183572.t001 Table 1 List of antibodies used for immunofluorescence. Polarity Antigen Clone Clonality Source Dilution M1 CD16 LNK16 Monoclonal mouse Abcam ‡ 1:20 CD32 AT10 Monoclonal mouse Abcam ‡ 1:10 MHC class II Dog 26 Monoclonal rat Helmholtz Zentrum †1:10 iNOS n.a. Polyclonal rabbit Merck Milipore ᛭ 1:50 LXN n.a. Polyclonal goat Biologo * 1:10 M2 MS4A2 n.a. Polyclonal rabbit Biologo * 1:10 CD163 AM-3K Monoclonal mouse TransGenic Inc. ‖ 1:20 CD206 3.29B1.10 Monoclonal mouse BeckmanCoulter Inc. ¶ 1:20 Arginase-1 n.a. Polyclonal rabbit Sigma Aldrich § 1:125 CD, cluster of differentiation; MHC, major histocompatibility complex; iNOS, inducible nitric oxide synthase, LXN, latexin; n.a. = not applicable ‡Cambridge, UK †kindly provided by Dr. E Kremmer, Institute of Molecular Immunology, Helmholtz Zentrum, München, German Research Center for Environmental Health (GmbH), Munich, Germany ᛭Darmstadt, Germany ‖Kobe, Japan ¶Krefeld, Germany § Taufkirchen, Germany * Kronshagen, Germany Based on the results of the transcriptome investigations (see below), two additional antibodies targeting LXN and MS4A2 ( Table 1 ) were applied on isolated and polarized cells of 3 dogs. Immunofluorescence was performed analogously to the above mentioned methods. For these antibodies, blocking was done with normal horse serum. Donkey-anti-goat and donkey-anti-rabbit antibodies were used as secondary antibodies. Due to the lower n (3 dogs), statistical comparison of the percentage of immunopositive cells between M0-, M1-, and M2-macrophages was done using parametrical tests (one-factorial ANOVA and pairwise t tests) for these both antibodies. RNA isolation, microarray hybridization, and low level analysis For RNA-isolation a separate and analogous in vitro experiment was performed using blood-derived cells from 6 healthy Beagles. Total RNA was isolated from 6 biological replicates of M0-, M1- and M2- macrophages using the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). Quality and integrity of isolated RNA were controlled using the Agilent 6000 RNA Nano Kit and an Agilent Bioanalyzer 2100 (Agilent, Böblingen, Germany). 200 ng RNA of each sample was amplified and biotin-labeled employing the Ovation RNA Amplification Kit V2 and the Encore Biotin Module (NuGen, San Carlos, USA), and hybridized to GeneChip Canine Genome 2.0 arrays (Affymetrix, Santa Clara, USA) in a rotating oven (45°C, 16 hours). Subsequently, arrays were washed and stained with R-phycoerythrin-streptavidin employing Affymetrix GeneChip Fluidics Station 450 (Affymetrix, Santa Clara, USA). For scanning, an Affymetrix GeneChip Scanner 3000 (Affymetrix, Santa Clara, USA) was employed for signal detection. Background adjustment, quantile normalization, and probe-set summarization were performed using the Gene Chip Robust Multichip Average (GC-RMA) algorithm (Bioconductor gcrma for R package, Version 2.3) as previously described [ 32 ]. Raw and processed data sets of the present study are deposited and publically available in the ArrayExpress database (accession number: E-MTAB-5458; http://www.ebi.ac.uk/arrayexpress ). Differentially expressed probe sets Differentially expressed probe sets (DEPs) were detected using the Linear Models for Microarray Data (LIMMA) algorithm, implemented in Babelomics 4.3 ( http://babelomics.bioinfo.cipf.es ; [ 33 ]), with a maximal false discovery rate (FDR) of 5% (q≤0.05) according to Benjamini and Hochberg, followed by post-hoc pairwise comparison of the expression levels of M0-, M1-, and M2-macrophages [ 32 , 34 ]. The fold change (FC) was calculated as the ratio of the inverse-transformed arithmetic means of the log 2 -transformed expression values. Down-regulations are shown as negative reciprocal values [ 34 , 35 ]. Probe sets were annotated with canine gene symbols and gene names according to the Affymetrix annotation file (release 35; 06. October 2014). A statistical significance filter (LIMMA q≤0.05) and a fold change filter (FC≥2.0 or ≤2.0) were employed to identify differentially expressed probe sets (DEPs) [ 32 ]. Differentially expressed genes (DEGs) were defined as probe sets with a unique canine gene symbol annotation [ 36 ]. Functional annotation and hierarchical clustering analysis Functional enrichment analysis of the DEPs for overexpressed Gene Ontology (GO) terms of the biological process category and the Kyoto encyclopedia of genes and genomes (KEGG) was performed using Web-based Gene Set Analysis Toolkit (WebGestalt; http://bioinfo.vanderbilt.edu/webgestalt/ ; [ 37 – 39 ]). In order to detect the genes in the pairwise comparison of M0-, M1-, and M2-macrophages, whose expression was most severely affected, genes with a FC≥50.0 or ≤-50.0 were selected and grouped into biological categories [ 36 ]. Moreover, unsupervised hierarchical clustering analysis of the DEPs was performed on log 2 -transformed data using TM4 MultiExperimentViewer (MeV) with default settings (Euclidean distance and complete linkage) [ 40 ]. The list of DEPs for each of the resulting clusters was functionally analyzed for significant enrichment applying WebGestalt as described above. Correlation-based marker gene selection Based on the transcriptional profile, a subset of probe sets for polarization prediction was selected using Prophet [ 41 ], provided by Babelomics 4.3 [ 33 , 42 ]. Correlation-based feature selection was used to pre-select the informative subset of probe sets and K-nearest-neighbors (KNN) algorithm was used for class prediction with leave-one out error validation [ 42 ]. The informative subset of 369 probe sets derived from Prophet was further analyzed for the polarization prediction strength of each individual probe set in three independent "M0 versus M1 and M2", "M1 versus M0 and M2", and "M2 versus M0 and M1" tests using Signature evaluation tool (SET; [ 42 , 43 ]). The log 2 -transformed expression values of three biomarkers, suggested by Prophet, were additionally statistically compared between M0-, M1-, and M2-macrophages, employing Kruskal-Wallis-Test and subsequent pairwise Mann-Whitney-U-Tests with significance value p≤0.05. Comparative evaluation of canine M1-/M2-genes with established literature-based human and murine orthologous genes All unique gene symbols, which were up-regulated (FC≥2; q≤0.05) in the comparison of M1 vs . M0 and simultaneously up-regulated in the comparison of M1 vs . M2 were defined as M1-exclusive-genes (n = 404). Likewise, all unique gene symbols, which were up-regulated (FC≥ 2; q≤0.05) in the comparison of M2 vs . M0 and simultaneously up-regulated in the comparison of M2 vs . M1 were defined as M2-exclusive-genes (n = 700). The gene sets of these exclusive M1- or M2-associated canine genes were compared with a list of established human and murine genes specifically associated with M1- or M2-macrophages. This list was generated based on peer-reviewed publications [ 4 , 27 , 31 , 44 ] as previously described [ 8 , 45 ]. This list was translated into canine orthologous gene symbols by employing MADgene ( http://cardioserve.nantes.inserm.fr/madtools/madgene/ ; [ 46 ]) and missing orthologous canine official gene symbols were manually added with the help of the web-based "information hyperlinked over proteins" (ihop; http://www.ihop-net.org/UniPub/iHOP/ ; [ 47 ]) as described previously [ 36 ]. Summarized, the literature-based gene list included a total number of 65 orthologous canine genes for M1-macrophages and 58 orthologous canine genes for M2-macrophages [ 36 ]. Venn diagrams ( http://bioinfogp.cnb.csic.es/tools/venny/index.html ) were used to reveal the intersections between established literature-based genes and the gene sets identified by the present analysis. Blood cell isolation Blood samples were collected from a total number of 12 healthy Beagle dogs, which were kept in regulatory approved animal housing facilities of the Departments of Small Animal Medicine and Surgery and the Institute for Parasitology, University of Veterinary Medicine, Hannover, Germany. Blood collection was done by professional veterinarians from non-anesthetized dogs following the regulations of the German Animal Welfare Law and with permission provided by the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany (permission number: AZ33.9-42502-05-13A303). Following a clinical examination of each animal, which included auscultation of the lung and heart, and rectal measurement of the body temperature, the puncture spot was disinfected. A volume of 20 ml blood was collected from the cephalic vein of each dog. Following blood collection, local manual pressure was used to avoid bleeding. No animals were euthanized for the present study. Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation using a routine protocol as described previously [ 22 ]. Briefly, blood samples were diluted 1:3 in phosphate buffered saline (PBS) containing 1% penicillin/streptomycin (Biochrom GmbH, Berlin, Germany) and added onto a gradient with equal volumes of histopaque 1.077 g/ml and 1.119 g/ml (Sigma Aldrich, Taufkirchen, Germany). After 30 minutes of centrifugation, cells were harvested using a Pasteur pipette, washed with PBS and 1% penicillin/streptomycin followed by depletion of erythrocyte contamination by hypotone lysis with distilled water. Subsequently, cells were resuspended in Roswell Park Memorial Institute (RPMI)-medium 1640 (Biochrom GmbH, Berlin, Germany) containing 10% fetal calf serum (PAA, Cölbe, Germany) and 1% penicillin/streptomycin and seeded onto 96½-well plates (ThermoScientific, Waltham, MA, USA) at a density of 0.3 x 10^6 cells/well. After 24 hours of cultivation under standard conditions (5% CO 2 , 37°C), medium was completely removed and cells were washed twice with PBS and 1% penicillin/streptomycin. Cells attached to the base of wells with strong plastic adherence were referred to as monocytes [ 23 – 26 ]. Cell culture and polarization To culture canine macrophages in vitro from blood-derived monocytes, a protocol according to Durafourt et al. (2012) [ 27 ] was used with slight modifications. Briefly, canine monocytes were cultured over 7 days towards M1-/M2-macrophages by stimulation with hematopoietic growth factors over 5 days followed by activation over 2 days with distinct cytokines, purchased from companies that guarantee an endotoxin level below 0.1 EU/μg of protein. In particular, to obtain M1-macrophages, monocytes were treated with 5 ng/ml canine recombinant granulocyte macrophage colony-stimulating factor (GM-CSF; R&D Systems, Minneapolis, MN, USA), 100 ng/ml LPS (Sigma Aldrich, Taufkirchen, Germany), and 20 ng/ml recombinant canine IFNɣ (Kingfisher Biotech Inc., Saint Paul, Minnesota, USA). M2-macrophages were developed by treatment with 25 ng/ml human macrophage colony-stimulating factor (M-CSF, PeproTech Inc., Rocky Hill, NJ, USA) and 20 ng/ml canine recombinant IL-4 (R&D Systems, Minneapolis, MN, USA). Serving as a control, unstimulated monocytes were cultured under the same conditions with medium change every second day (M0-macrophages). After 7 days of cultivation, morphology, phenotype, and transcriptomic changes of M0-, M1-, and M2-macrophages were assessed. Pictures were taken with an inverted fluorescence microscope (Olympus IX-70, Olympus, Optical Co GmbH, Hamburg, Germany). The overall number of cells for each polarization was counted on at least 4 microscopic pictures (200x magnification) of three individual dogs on day 7 in culture and the mean numbers of cells per 200x field were compared using one-factorial ANOVA and post-hoc t tests applying SPSS version 21 for windows (IBM Inc., Chicago, USA). For quantification of cellular morphology, microphotographs were taken at 200x magnification and the number of cells with a distinct morphologic appearance out of at least 100 randomly selected cells was counted for each polarization (M0, M1, M2). Here, 4 distinct morphologic types were defined. In particular, cells with a cellular diameter ≤ 10 μm and absent cytoplasmic projections on the cellular surface were defined as small/roundish cells (morphology 1); cells with a cellular diameter ≥ 10 μm and numerous cytoplasmic processes were defined as amoeboid (morphology 2); cells obtaining an elongated bipolar morphology were classified as spindeloid (morphology 3); and cells possessing a large, round, and flattened morphology with a cellular diameter ≥ 30 μm and > 1 nuclei were classified as multinucleated giant cells (MNGs; morphology 4). The percentage of each of the four different morphologies was calculated in M0-, M1-, and M2-macrophages from 3 animals. A mixed ANOVA with post-hoc alpha adjustment (Tukey-Kramer) and significance level at p≤0.05 was performed using the statistical software programme SPSS version 21 for windows (IBM Inc., Chicago, USA) in order to compare the percentages of each of the morphologies between M0-, M1-, and M2-macrophages. Graphical compilation was done with GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, USA). Scanning electron microscopy M0-, M1-, and M2-macrophages were prepared for representative ultrastructural characterization using scanning electron microscopy as described previously [ 28 ]. Briefly, cells were fixed in 2.5% glutaraldehyde/cacodylate buffer, post-fixed in 1% osmium tetroxide, and dehydrated in a graded series of alcohol. Subsequently, cells were dried under a Critical Point Dryer (E3000, Polaron, London, UK), and sputter-coated with gold, and examined under a digital scanning electron microscope (DSM 940, Zeiss, Oberkochen, Germany). At least 50 cells were photographed for each morphology and evaluated with regard to ultrastructural morphology. Immunocytochemistry Immunolabelling of canine M0-, M1-, and M2-macrophages from 5 individuals was performed using a routine protocol described previously [ 29 , 30 ]. A panel of different literature-based prototype marker antibodies, reported to distinguish between M1- and M2-macrophages was applied ( Table 1 ; [ 4 , 31 ]). Briefly, cells were fixed with paraformaldehyde (PFA, 4%) for 20 minutes at room temperature (RT) and permeabilized with Triton X (0.25%) diluted in PBS (PBST). Non-specific binding was blocked by treatment of cells with bovine serum albumin (BSA, 3%; Sigma Aldrich, Taufkirchen, Germany) and normal goat serum (5%) diluted in PBST for 15 minutes at RT, except for cells intended to be stained with antibodies directed against Fc-receptors, i . e . CD16 and CD32. Subsequently, primary antibodies ( Table 1 ) were added and incubated for 2 hours at RT. All unconjugated primary antibodies were labeled with secondary goat-anti-mouse antibodies coupled to cyanine 3 (Cy3), goat-anti-rat antibodies coupled to Cy2, and goat-anti-rabbit antibodies coupled to Cy3 (all received from Jackson ImmunoResearch Laboratories, Dianova, Hamburg, Germany; dilution 1:200 in PBS), respectively, and incubated for 2 hours at RT. Following appropriate washing steps with PBS and distilled water, nuclei were counterstained with bisbenzimide (H33258, 0.01% in distilled water) for 5 minutes at RT. Species-specific immunoglobulins from mouse, rat, goat, and rabbit diluted according to the immunoglobulin concentration of the primary antibodies served as appropriate negative controls. M0-, M1-, and M2-macrophages were investigated using an inverted fluorescence microscope (Olympus IX-70, Olympus, Optical Co GmbH, Hamburg, Germany). For quantification of immunopositivity, microphotographs were taken at 200x magnification and at least 100 randomly selected cells were counted for each treatment (M0, M1, M2) with respect to the number of immunopositive cells and the total number of cells. Statistical comparison of the percentage of immunopositive cells between M0-, M1-, and M2-macrophages was done using a Kruskal-Wallis-Test and pairwise Mann-Whitney-U-Tests with significance level at p≤0.05 employing the statistical software programme SPSS version 21 for windows, and box plots were depicted using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, USA). 10.1371/journal.pone.0183572.t001 Table 1 List of antibodies used for immunofluorescence. Polarity Antigen Clone Clonality Source Dilution M1 CD16 LNK16 Monoclonal mouse Abcam ‡ 1:20 CD32 AT10 Monoclonal mouse Abcam ‡ 1:10 MHC class II Dog 26 Monoclonal rat Helmholtz Zentrum †1:10 iNOS n.a. Polyclonal rabbit Merck Milipore ᛭ 1:50 LXN n.a. Polyclonal goat Biologo * 1:10 M2 MS4A2 n.a. Polyclonal rabbit Biologo * 1:10 CD163 AM-3K Monoclonal mouse TransGenic Inc. ‖ 1:20 CD206 3.29B1.10 Monoclonal mouse BeckmanCoulter Inc. ¶ 1:20 Arginase-1 n.a. Polyclonal rabbit Sigma Aldrich § 1:125 CD, cluster of differentiation; MHC, major histocompatibility complex; iNOS, inducible nitric oxide synthase, LXN, latexin; n.a. = not applicable ‡Cambridge, UK †kindly provided by Dr. E Kremmer, Institute of Molecular Immunology, Helmholtz Zentrum, München, German Research Center for Environmental Health (GmbH), Munich, Germany ᛭Darmstadt, Germany ‖Kobe, Japan ¶Krefeld, Germany § Taufkirchen, Germany * Kronshagen, Germany Based on the results of the transcriptome investigations (see below), two additional antibodies targeting LXN and MS4A2 ( Table 1 ) were applied on isolated and polarized cells of 3 dogs. Immunofluorescence was performed analogously to the above mentioned methods. For these antibodies, blocking was done with normal horse serum. Donkey-anti-goat and donkey-anti-rabbit antibodies were used as secondary antibodies. Due to the lower n (3 dogs), statistical comparison of the percentage of immunopositive cells between M0-, M1-, and M2-macrophages was done using parametrical tests (one-factorial ANOVA and pairwise t tests) for these both antibodies. RNA isolation, microarray hybridization, and low level analysis For RNA-isolation a separate and analogous in vitro experiment was performed using blood-derived cells from 6 healthy Beagles. Total RNA was isolated from 6 biological replicates of M0-, M1- and M2- macrophages using the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). Quality and integrity of isolated RNA were controlled using the Agilent 6000 RNA Nano Kit and an Agilent Bioanalyzer 2100 (Agilent, Böblingen, Germany). 200 ng RNA of each sample was amplified and biotin-labeled employing the Ovation RNA Amplification Kit V2 and the Encore Biotin Module (NuGen, San Carlos, USA), and hybridized to GeneChip Canine Genome 2.0 arrays (Affymetrix, Santa Clara, USA) in a rotating oven (45°C, 16 hours). Subsequently, arrays were washed and stained with R-phycoerythrin-streptavidin employing Affymetrix GeneChip Fluidics Station 450 (Affymetrix, Santa Clara, USA). For scanning, an Affymetrix GeneChip Scanner 3000 (Affymetrix, Santa Clara, USA) was employed for signal detection. Background adjustment, quantile normalization, and probe-set summarization were performed using the Gene Chip Robust Multichip Average (GC-RMA) algorithm (Bioconductor gcrma for R package, Version 2.3) as previously described [ 32 ]. Raw and processed data sets of the present study are deposited and publically available in the ArrayExpress database (accession number: E-MTAB-5458; http://www.ebi.ac.uk/arrayexpress ). Differentially expressed probe sets Differentially expressed probe sets (DEPs) were detected using the Linear Models for Microarray Data (LIMMA) algorithm, implemented in Babelomics 4.3 ( http://babelomics.bioinfo.cipf.es ; [ 33 ]), with a maximal false discovery rate (FDR) of 5% (q≤0.05) according to Benjamini and Hochberg, followed by post-hoc pairwise comparison of the expression levels of M0-, M1-, and M2-macrophages [ 32 , 34 ]. The fold change (FC) was calculated as the ratio of the inverse-transformed arithmetic means of the log 2 -transformed expression values. Down-regulations are shown as negative reciprocal values [ 34 , 35 ]. Probe sets were annotated with canine gene symbols and gene names according to the Affymetrix annotation file (release 35; 06. October 2014). A statistical significance filter (LIMMA q≤0.05) and a fold change filter (FC≥2.0 or ≤2.0) were employed to identify differentially expressed probe sets (DEPs) [ 32 ]. Differentially expressed genes (DEGs) were defined as probe sets with a unique canine gene symbol annotation [ 36 ]. Functional annotation and hierarchical clustering analysis Functional enrichment analysis of the DEPs for overexpressed Gene Ontology (GO) terms of the biological process category and the Kyoto encyclopedia of genes and genomes (KEGG) was performed using Web-based Gene Set Analysis Toolkit (WebGestalt; http://bioinfo.vanderbilt.edu/webgestalt/ ; [ 37 – 39 ]). In order to detect the genes in the pairwise comparison of M0-, M1-, and M2-macrophages, whose expression was most severely affected, genes with a FC≥50.0 or ≤-50.0 were selected and grouped into biological categories [ 36 ]. Moreover, unsupervised hierarchical clustering analysis of the DEPs was performed on log 2 -transformed data using TM4 MultiExperimentViewer (MeV) with default settings (Euclidean distance and complete linkage) [ 40 ]. The list of DEPs for each of the resulting clusters was functionally analyzed for significant enrichment applying WebGestalt as described above. Correlation-based marker gene selection Based on the transcriptional profile, a subset of probe sets for polarization prediction was selected using Prophet [ 41 ], provided by Babelomics 4.3 [ 33 , 42 ]. Correlation-based feature selection was used to pre-select the informative subset of probe sets and K-nearest-neighbors (KNN) algorithm was used for class prediction with leave-one out error validation [ 42 ]. The informative subset of 369 probe sets derived from Prophet was further analyzed for the polarization prediction strength of each individual probe set in three independent "M0 versus M1 and M2", "M1 versus M0 and M2", and "M2 versus M0 and M1" tests using Signature evaluation tool (SET; [ 42 , 43 ]). The log 2 -transformed expression values of three biomarkers, suggested by Prophet, were additionally statistically compared between M0-, M1-, and M2-macrophages, employing Kruskal-Wallis-Test and subsequent pairwise Mann-Whitney-U-Tests with significance value p≤0.05. Comparative evaluation of canine M1-/M2-genes with established literature-based human and murine orthologous genes All unique gene symbols, which were up-regulated (FC≥2; q≤0.05) in the comparison of M1 vs . M0 and simultaneously up-regulated in the comparison of M1 vs . M2 were defined as M1-exclusive-genes (n = 404). Likewise, all unique gene symbols, which were up-regulated (FC≥ 2; q≤0.05) in the comparison of M2 vs . M0 and simultaneously up-regulated in the comparison of M2 vs . M1 were defined as M2-exclusive-genes (n = 700). The gene sets of these exclusive M1- or M2-associated canine genes were compared with a list of established human and murine genes specifically associated with M1- or M2-macrophages. This list was generated based on peer-reviewed publications [ 4 , 27 , 31 , 44 ] as previously described [ 8 , 45 ]. This list was translated into canine orthologous gene symbols by employing MADgene ( http://cardioserve.nantes.inserm.fr/madtools/madgene/ ; [ 46 ]) and missing orthologous canine official gene symbols were manually added with the help of the web-based "information hyperlinked over proteins" (ihop; http://www.ihop-net.org/UniPub/iHOP/ ; [ 47 ]) as described previously [ 36 ]. Summarized, the literature-based gene list included a total number of 65 orthologous canine genes for M1-macrophages and 58 orthologous canine genes for M2-macrophages [ 36 ]. Venn diagrams ( http://bioinfogp.cnb.csic.es/tools/venny/index.html ) were used to reveal the intersections between established literature-based genes and the gene sets identified by the present analysis. Results Morphological characterization of polarized canine macrophages On day 7 in culture, the overall number of cells differed between the three polarities (p = 0.02; S1 Fig ). The number of cells was highest in M2-polarized cells (mean = 135 cells per field), while M0-macrophages were lowest in number (mean = 30 cells per field). M1-macrophages had a mean number of 52 cells per field. T tests revealed that the number of M2-macrophages was significantly higher than M0-macrophages (p = 0.02), while comparisons between M0- and M1-macrophages and M1-and M2-macrophages failed to reach the level of significance (p = 0.30 and p = 0.06, respectively). Morphologically, striking differences between M0-, M1-, and M2-polarized cells were observed at day 7 in culture by scanning electron microscopy and phase contrast microscopy ( Fig 1 ). Canine M0-macrophages predominantly appeared as small and roundish cells with an average size of 8 μm in diameter and no or little cytoplasmic extensions that measured up to 1 μm in length ( Fig 1 ). However, a noteworthy proportion of M0-macrophages (about 25%) also obtained an amoeboid morphology, which however was predominantly observed in M1 macrophages ( Fig 1 ). The majority of M1-macrophages was amoeboid and had a mean size of 15 μm and numerous fibrillary cytoplasmic processes on the cellular surface that had a length of up to 5 μm ( Fig 1 ). M2-macrophages appeared as a heterogeneous cell population with a mixture of 4 different morphologies. Besides roundish and amoeboid macrophages, large bipolar spindeloid macrophages were present measuring up to 35 μm that were characterized by an elongated cell body with cytoplasmic extensions with an average length of 7 μm at both poles ( Fig 1 ). Moreover, large MNGs appeared that had a mean size of 40 μm with an extensive cytoplasm and numerous evenly distributed processes that measured up to 7 μm ( Fig 1 ). Allover, the mean percentage of small/roundish macrophages (morphology 1) was significantly higher in untreated cultures (M0) compared to polarity 2 at days 5 and 7 (p≤0.05; Fig 1E ). For amoeboid macrophages (morphology 2), there was a trend towards a higher mean percentage in polarity 1 compared to polarity 0 at day 5 (p = 0.07) and day 7 (p = 0.06; Fig 1F ). The mean percentage of spindeloid macrophages (morphology 3) was significantly higher in polarity 2 at day 5 compared to polarity 1 at day 5 (p≤0.05; Fig 1G ). MNGs (morphology 4) were exclusively present in polarity 2, as compared to the polarities 0 and 1 at day 7 (p≤0.05; Fig 1H ). 10.1371/journal.pone.0183572.g001 Fig 1 Polarization-dependent morphological differences in canine M0-, M1-, and M2-macrophage cultures. A) In scanning electron microscopy, unstimulated macrophages (M0; day 7) obtain a small and roundish morphology, lacking cytoplasmic extensions. B) M1-treated macrophages (day 7) are characterized by an enlarged amoeboid cell shape with roundish cell bodies and numerous delicate cytoplasmic extensions on the cellular surface. C; D) M2-treated macrophage cultures (day 7) demonstrate a marked heterogeneity with two dominating cell types. Large "spindeloid" macrophages with an elongated cell body and cytoplasmic extensions on the apical ends of the cell bodies (C) . Second, in M2-cultures, numerous multinucleated giant cells (MNGs) with abundant cytoplasmic projections on the cellular surface are present (D) . E-H) Dot plot diagrams depicting the morphological changes of macrophage cultures (n = 3) following stimulation (M0, M1, M2) over the time course as calculated with a mixed ANOVA with post-hoc alpha adjustment (Tukey-Kramer) and significance level at p≤0.05 (asterisks). E) Small/roundish macrophages dominate in untreated M0 cell cultures and their relative percentage is significantly higher in M0 when compared to M2 at days 5 and 7. F) Amoeboid macrophages display a statistical trend of predominance in M1 compared to M0 at day 5 and 7. G) The relative percentage of spindeloid macrophages is significantly increased in M2 at day 5 compared to M1. H) MNGs are almost exclusively observed in M2-macrophages at the end of the culturing period and their percentage is significantly higher at day 7 in M2 compared to both M0 and M1. Phenotypical characterization of polarized canine macrophages For the phenotypical characterization of canine macrophages, the percentage of immunopositive cells for selected literature-based M1-/M2-antigens was evaluated in 5 animals and related to the polarization of cells (polarity 0, 1, 2). Interestingly, except for MHC class II and CD206, none of the remaining tested antigens (CD16, CD32, iNOS, CD163, and arginase-1) were differently expressed between canine M0-, M1-, and M2-macrophages (p-values ranging from 0.101 to 0.691; Kruskal-Wallis-Test; Fig 2 ). 10.1371/journal.pone.0183572.g002 Fig 2 Immunofluorescence staining of in vitro cultured canine M0-, M1-, and M2-macrophages labeled with prototypic literature-based antibodies for the M1- (CD16, CD32, MHC class II, and iNOS) and M2-phenotype (CD163, CD206, and arginase 1), respectively. A-C) Low to moderate membranous staining of M0-, M1-, and M2-macrophages for CD16. D-F) Likewise, CD32 shows a low to moderate staining in all three treatment conditions. G-I) M0-, M1-, and M2-macrophages demonstrate a moderate to high membranous staining with CD163. J-L) Intense membranous staining of M0-, M1-, and M2-macrophages with an anti-MHC class II antibody. M-O) Strong intracytoplasmic labeling of macrophages in all treatments (polarity 0, 1, 2) for inducible nitric oxide synthase (iNOS). P-R) High intracytoplasmic expression of arginase 1 in small/roundish, amoeboid, and spindeloid macrophages as well as in multinucleated giant cells (MNGs). a-f) Statistical evaluation of the mean expression percentages of prototypic M1-/M2-markers evaluated in 5 dogs and related to the polarization state of the macrophages (polarity 0, 1, 2). Note that, except for MHC class II, none of the remaining tested antigens (CD16, CD32, iNOS, CD163, and arginase-1) were differently expressed between canine M0-, M1-, and M2-macrophages (* = p≤0.05; Kruskal-Wallis-Test with pairwise Mann-Whitney-U-Tests). Scale bars = 100 μm. Nuclear counterstaining with bisbenzimide. The mean percentage of immunopositive cells expressing MHC class II was significantly higher in M2-macrophages (mean positive cells: 93.64%) when compared to untreated M0-cells (mean positive cells: 72.22%; p = 0.008). However, there was no statistical difference in the percentage of positive cells for MHC class II, when M1-macophages (mean positive cells: 89.92%) were compared to M2-macrophages and M0-macrophages, respectively ( Fig 2 ). For CD206, the mean percentage of immunopositive cells was highest in M2-macrophages (mean: 66.54%) as compared to both M0-macrophages (mean: 33.33%; p = 0.008) and M1-macrophages (mean: 28.67%; p = 0.032, Fig 3 ). No statistical difference was observed between M1- and M0-macrophages ( Fig 3 ). The results were validated in the microarray data set, which similarly revealed significantly higher expression levels of CD206 in canine M2-macrophages as compared to both M1- (p = 0.002) and M0-macrophages (p = 0.004; Fig 3 ). 10.1371/journal.pone.0183572.g003 Fig 3 Phenotypical characterization of canine M0-, M1-, and M2-macrophages. A) Low membranous expression of CD206 antigen by small/roundish M0-macrophages. B) Moderate membranous staining of amoeboid M1-macrophages for CD206. C) Intense membranous expression of CD206 antigen by M2-macrophages. Scale bars = 100 μm. Nuclear counterstaining with bisbenzimide. D) The mean percentage of CD206-immunopositive cells is significantly higher in M2-macrophages as compared to both M1- and M0-macrophages (* = p≤0.05; Kruskal-Wallis-Test with pair-wise Mann-Whitney-U-Tests). E) The log 2 -transformed expression values of the probe set encoding for the gene CD206 is similarly significantly higher in M2-macrophages compared to both M1- and M0-macrophages (* = p≤0.05; Kruskal-Wallis-Test with pair-wise Mann-Whitney-U-Tests), thus confirming the results of the immunofluorescence investigation. Differentially expressed genes between M0-, M1-, and M2-polarized macrophage cultures One-factorial multigroup analysis of microarray data and fold change criteria identified 6358 probe sets that were differentially expressed in at least one of the three post-hoc pairwise comparisons. The total number of DEPs was 3555 in M1 vs . M0, 4831 in M2 vs . M0, and 3141 in M2 vs . M1, respectively ( Table 2 ). In all pairwise comparisons, the number of up- and down-regulated probe sets was nearly equally distributed (M1 vs . M0: 1699 up, 1856 down; M2 vs . M0: 2467 up, 2364 down; M2 vs . M1: 1572 up, 1569 down). Functional annotation of enriched biological processes in DEPs, up–regulated in the comparison M1 vs . M0, revealed terms for "organonitrogen compound metabolic process", "carbohydrate metabolic process", "carboxylic acid metabolic process", and "tricarboxylic acid cycle" ( Table 2 ). In contrast, biological terms in down-regulated DEPs in this comparison reflected terms for "positive regulation of immune response", "immune response-regulating signaling pathway", and "regulation of innate immune response". In both comparisons M2 vs . M0 and M2 vs . M1, up-regulated DEPs displayed significantly enriched gene ontology terms for "M phase of mitotic cell cycle" and "mitotic spindle organization". Additionally, in M2 vs . M0 comparison, up-regulated DEPs were associated with "oxidation-reduction process", "carboxylic acid metabolic process", and "organonitrogen compound metabolic process", whereas down-regulated DEPs were related to the biological term "immune response-activating signal transduction". In the comparisons M2 vs . M1, down-regulated DEPs were functionally associated to biological terms such as "response to other organism", "defense response", "regulation of lymphocyte activation", and "regulation of immune response" ( Table 2 ). 10.1371/journal.pone.0183572.t002 Table 2 Summarized results of the functional annotation of the pairwise comparisons of differentially expressed probe sets (DEPs) in canine M0-, M1-, and M2-macrophages. Pairwise comparison Differentially expressed probe sets Up-/down-regulated probe sets Enriched biological process categories * Enriched KEGG pathways * M1 vs . M0 3555 Up: 1699 • Organonitrogen compound metabolic process • Carbohydrate metabolic process • Tricarboxylic acid cycle • Metabolic pathways • Glutathione metabolism • Steroid biosynthesis • Glycolysis/Gluconeogenesis Down: 1856 • Positive regulation of immune response • Immune response-regulating signaling pathway • Regulation of innate immune response • Hematopoietic cell lineage • RIG-I-like receptor signaling pathway • T cell receptor signaling pathway • Cell adhesion molecules M2 vs . M0 4831 Up: 2467 • Oxidation-reduction process • Carboxylic acid metabolic process • Organonitrogen compound metabolic process • M-phase of mitotic cell cycle • Metabolic pathways • Glycolysis/Gluconeogenesis • Propanoate and pyruvate metabolism • Steroid biosynthesis • Cell cycle Down: 2364 • Immune response-activating signal transduction • T cell receptor signaling pathway • Natural killer cell mediated cytotoxicity • Hematopoietic cell lineage M2 vs . M1 3141 Up: 1572 • Mitotic spindle organization • Cell cycle • Metabolic pathways • Ribosome biogenesis in eukaryotes • PPAR signaling pathway Down: 1569 • Response to other organism • Defense response • Regulation of lymphocyte activation • Regulation of immune response • NOD-like receptor signaling pathway • Osteoclast differentiation • Toll-like receptor signaling pathway • B and T cell receptor signaling pathway * employing Web-based Gene Set Analysis Toolkit (WebGestalt; http://bioinfo.vanderbilt.edu/webgestalt/ ) with default settings adjusted p-value ≤0.05 Enriched KEGG-pathways, significantly associated with up-regulated canine DEPs in M1 vs . M0 (1699 DEPs), were functionally related to "metabolic pathways", "glutathione metabolism", "steroid biosynthesis", and "glycolysis/gluconeogenesis". The comparison M2 vs . M0 (2467 up-regulated DEPs) contained enriched KEGG-pathways for "metabolic pathways", "glycolysis/gluconeogenesis", "propanoate and pyruvate metabolism", "steroid biosynthesis", and "cell cycle". In the comparison M2 vs . M1 (1572 up-regulated DEPs), enriched KEGG-pathways included terms for "cell cycle", "metabolic pathways", "ribosome biogenesis in eukaryotes", and "PPAR signaling pathway". In contrast, down-regulated DEPs in the three comparisons were associated with "hematopoietic cell lineage", "RIG-I-like receptor signaling pathway", "T cell receptor signaling pathway", and "cell adhesion molecules" (M1 vs . M0), "T cell receptor signaling pathway", "natural killer cell mediated cytotoxicity", and "hematopoietic cell lineage" (M2 vs . M0), and "NOD-like receptor signaling pathway", "osteoclast differentiation", "Toll-like receptor signaling pathway", and "B and T cell receptor signaling pathway" (M2 vs . M1; Table 2 ). Genes whose expression was most severely affected (FC ≥50.0 or ≤-50.0) in the pairwise comparison are depicted in Tables 3 , 4 and 5 . Fortynine genes fulfilled these filtering criteria in the M1 vs . M0 contrast (25 up, 24 down), whereas 99 genes were retrieved in the comparison of M2 vs . M0 (41 up; 58 down). Comparing M2 with M1, 66 genes fulfilled the criteria (32 up; 34 down). Focusing on potentially promising cell surface markers, the pairwise comparison of three genes encoding for such surface molecules were up-regulated in M1 vs . M0, namely SUCNR1 , SDC4 , and CHRNA9 , whereas CD209 , CD180 , KLRG1 , COLEC12 , and C3AR1 were down-regulated ( Table 3 ). Comparing M2 vs . M0, nine cell surface makers were up-regulated, i . e . LYVE1 , SUCNR1 , CD1e , MRC1 , TSPAN7 , JAM3 , ANTXR1 , MS4A2 , and CLEC4G ( Table 4 ). In contrast, a total amount of 18 cell surface marker genes was down-regulated, namely ITGB8 , LY6E , TRDC , NCR3 , SELL , SIGLEC1 , KLRD1 , KLRB1 , MPP6 , P2RY14 , TRBC2 , TARP , FCRLA , KCNK5 , P2RX5 , NKG7 , CD69 , and CD7 ( Table 4 ). In the comparison M2 vs . M1, the up-regulated cell surface marker genes contained 10 terms for FCER1A , CD209 , LYVE1 , CLEC4G , COLEC12 , JAM3 , MS4A2 , STAB1 , MRC1 , and SLC15A1 ( Table 5 ). Down-regulated cell surface marker genes were P2RY14 , SLC39A14 , ITGB8 , TMEM150C , TMEM176A , and SLC22A15 ( Table 5 ). 10.1371/journal.pone.0183572.t003 Table 3 List and subgrouping of the top hits of highly differentially expressed genes (fold change ≥ 50 or ≤ -50) in canine M1- vs . M0-macrophages. Gene name Gene symbol Fold change Up-regulated genes Cell surface markers Succinate receptor 1 SUCNR1 212.94 Syndecan 4 SDC4 58.61 Cholinergic receptor, nicotinic, alpha 9 (neuronal) CHRNA9 53.74 Enzymes ADP-ribosylhydrolase like 2 ADPRHL2 535.59 Ceruloplasmin (ferroxidase) CP 161.88 Epoxide hydrolase 2, cytoplasmic EPHX2 119.72 Ectonucleotide pyrophosphatase/phosphodiesterase 2 ENPP2 117.43 Interstitial collagenase-like LOC489428 87.83 E3 ubiquitin-protein ligase NEURL3-like LOC102152163 86.20 NOP2/Sun domain family, member 7 NSUN7 66.00 Nucleoredoxin NXN 64.78 WNK lysine deficient protein kinase 2 WNK2 52.35 Cytokines , chemokines , and their Receptors Interleukin 6 (interferon, beta 2) IL6 252.32 Chemokine (C-C motif) ligand 22 CCL22 187.96 Chemokine (C-X-C motif) receptor 7 ACKR3 178.18 Interleukin 22 receptor, alpha 2 IL22RA2 116.00 Chemokine (C-X-C motif) ligand 14 CXCL14 108.55 Chemokine (C-C motif) ligand 17 CCL17 95.26 Chemokine (C-C motif) ligand 20 CCL20 67.90 Soluble factors Chitinase 3-like 1 (cartilage glycoprotein-39) CHI3L1 83.25 Clusterin CLU 66.23 Miscellaneous Ras homolog family member U RHOU 446.23 Interferon, alpha-inducible protein 6 IFI6 269.13 CXADR-like membrane protein CLMP 213.81 Retinoic acid induced 14 RAI14 118.86 Down-regulated genes Cell surface markers CD209 molecule CD209 -345.12 CD180 molecule CD180 -75.00 Killer cell lectin-like receptor subfamily G, member 1 KLRG1 -66.25 Collectin sub-family member 12 COLEC12 -61.81 Complement component 3a receptor 1 C3AR1 -55.16 Enzymes Carboxypeptidase M CPM -134.50 N-acetylneuraminate pyruvate lyase (dihydrodipicolinate synthase) NPL -78.98 Cathepsin E CTSE -76.30 Cytokines , chemokines , and their receptors Chemokine (C-X-C motif) ligand 12 CXCL12 -401.92 Pro-platelet basic protein (chemokine (C-X-C motif) ligand 7) PPBP -100.42 Interleukin 2 IL2 -95.84 Interleukin 1 receptor, type II IL1R2 -79.71 Chemokine (C-C motif) receptor 3 CCR3 -51.05 Soluble factors Lipocalin 2 LCN2 -180.73 CD5 molecule-like CD5L -153.48 Secreted phosphoprotein 2, 24kDa SPP2 -51.92 Miscellaneous Coagulation factor XIII, A1 polypeptide F13A1 -3305.03 Fatty acid binding protein 4, adipocyte FABP4 -370.34 G protein-coupled receptor 116 GPR116 -130.89 Interferon-induced transmembrane protein 3-like LOC606890 -114.16 Plexin domain containing 2 PLXDC2 -67.07 ADP-ribosylation factor-like 4C ARL4C -56.52 Cyclin J-like CCNJL -54.76 Thrombospondin 1 THBS1 -53.38 10.1371/journal.pone.0183572.t004 Table 4 List and subgrouping of the top hits of highly differentially expressed genes (fold change ≥ 50 or ≤ -50) in canine M2- vs . M0-macrophages. Gene name Gene symbol Fold change Up-regulated genes Cell surface markers Lymphatic vessel endothelial hyaluronan receptor 1 LYVE1 322.32 Succinate receptor 1 SUCNR1 151.91 CD1e molecule CD1E 147.40 Mannose receptor, C type 1 MRC1 104.41 Tetraspanin 7 TSPAN7 83.25 Junctional adhesion molecule 3 JAM3 78.46 Anthrax toxin receptor 1 ANTXR1 72.95 Membrane-spanning 4-domains, subfamily A, member 2 MS4A2 54.98 C-type lectin domain family 4, member G CLEC4G 54.14 Enzymes ADP-ribosylhydrolase like 2 ADPRHL2 614.52 Guanine deaminase GDA 174.44 Ubiquitin-conjugating enzyme E2 C-like LOC481325 151.23 Ribonucleotide reductase M2 RRM2 131.03 Fructose-1,6-bisphosphatase 1 FBP1 57.95 Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase) MMP9 56.03 Lipoprotein lipase LPL 52.03 Trimethyllysine hydroxylase, epsilon TMLHE 51.05 3-hydroxybutyrate dehydrogenase, type 2 BDH2 50.68 Cytokines , chemokines , and their receptors Chemokine (C-C motif) ligand 24 CCL24 1060.82 Chemokine (C-X-C motif) receptor 7 ACKR3 390.67 Chemokine (C-C motif) ligand 17 CCL17 209.36 Chemokine (C-C motif) ligand 13 CCL13 172.58 Interleukin 13 receptor, alpha 2 IL13RA2 170.59 Transforming growth factor, beta 2 TGFB2 61.84 Soluble factors Nephronectin NPNT 444.07 Norrie disease (pseudoglioma) NDP 213.72 Endothelin 1 EDN1 115.81 Cystatin 9 (testatin) CST9 58.21 Miscellaneous Sodium channel, voltage-gated, type II, beta subunit SCN2B 1061.28 Caldesmon 1 CALD1 224.66 CXADR-like membrane protein CLMP 169.27 SHC SH2-domain binding protein 1 SHCBP1 117.77 Dynamin 1 DNM1 112.78 NACC family member 2, BEN and BTB (POZ) domain containing NACC2 110.49 Retinoic acid induced 14 RAI14 84.35 Scinderin SCIN 71.66 Kinesin family member 23 KIF23 66.78 Kinesin family member 11 KIF11 64.20 Cfa-mir-125b-2 cfa-mir-125b-2 58.78 AHNAK nucleoprotein AHNAK 57.19 NUF2, NDC80 kinetochore complex component NUF2 54.62 Down-regulated genes Cell surface markers Integrin, beta 8 ITGB8 -199.37 Lymphocyte antigen 6 complex, locus E LY6E -116.70 T cell receptor delta constant TRDC -112.73 Natural cytotoxicity triggering receptor 3 NCR3 -105.99 Selectin L SELL -95.45 Sialic acid binding Ig-like lectin 1, sialoadhesin SIGLEC1 -91.39 Killer cell lectin-like receptor subfamily D, member 1 KLRD1 -87.12 Killer cell lectin-like receptor subfamily B, member 1 KLRB1 -74.56 Membrane protein, palmitoylated 6 (MAGUK p55 subfamily member 6) MPP6 -72.60 Purinergic receptor P2Y, G-protein coupled, 14 P2RY14 -71.69 T cell receptor beta constant 2 TRBC2 -64.26 TCR gamma alternate reading frame protein TARP -62.39 Fc receptor-like A FCRLA -60.06 Potassium channel, subfamily K, member 5 KCNK5 -58.16 Purinergic receptor P2X, ligand-gated ion channel, 5 P2RX5 -58.16 Natural killer cell group 7 sequence NKG7 -57.26 CD69 molecule CD69 -54.53 CD7 molecule CD7 -52.57 Enzymes Interferon stimulated exonuclease gene 20kDa ISG20 -514.04 Prostaglandin E synthase PTGES -382.20 Cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial CMPK2 -266.50 Granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) GZMA -131.27 Granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1) GZMB -130.63 Hexokinase 3 (white cell) HK3 -127.10 Ubiquitin specific peptidase 18 USP18 -81.72 Phospholipase A1 member A PLA1A -72.65 Cathepsin E CTSE -72.13 GTP cyclohydrolase 1 GCH1 -66.29 Chymase 1, mast cell CMA1 -61.45 Phospholipid scramblase 1-like LOC611500 -61.09 Cytokines , chemokines , and their receptors Chemokine (C-X-C motif) ligand 12 CXCL12 -417.05 Interleukin 2 IL2 -195.40 Interleukin 7 receptor IL7R -66.71 Transforming growth factor, beta receptor III TGFBR3 -50.94 Soluble factors Lipocalin 2 LCN2 -217.74 Adrenomedullin ADM -100.37 Miscellaneous Radical S-adenosyl methionine domain containing 2 RSAD2 -559.34 Interferon-induced transmembrane protein 3-like LOC606890 -435.57 ISG15 ubiquitin-like modifier ISG15 -254.32 Apolipoprotein L, 5 APOL5 -200.23 Fatty acid binding protein 4, adipocyte FABP4 -182.17 Interferon-induced protein with tetratricopeptide repeats 1 IFIT1 -142.72 Interferon regulatory factor 4-like LOC609817 -118.68 Carcinoembryonic antigen-related cell adhesion molecule 25 CAECAM1 -113.27 Interferon regulatory factor 7 IRF7 -102.93 OCIA domain containing 2 OCIAD2 -100.84 Piwi-like RNA-mediated gene silencing 4 PIWIL4 -97.75 Synaptotagmin-like 3 SYTL3 -94.94 Testis expressed 14 TEX14 -92.58 Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) MX1 -91.32 Structural maintenance of chromosomes flexible hinge domain containing 1 SMCHD1 -84.97 Interferon-induced transmembrane protein 1-like LOC475935 -83.91 eukaryotic peptide chain release factor GTP-binding subunit ERF3B-like LOC480921 -74.06 Lactotransferrin LTF -72.08 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 DDX58 -69.54 TNFAIP3 interacting protein 3 TNIP3 -58.03 Syntrophin, beta 1 (dystrophin-associated protein A1, 59kDa, basic component 1) SNTB1 -55.46 Src kinase associated phosphoprotein 1 SKAP1 -50.58 10.1371/journal.pone.0183572.t005 Table 5 List and subgrouping of the top hits of highly differentially expressed genes (fold change ≥ 50 or ≤ -50) in M2- vs . M1-macrophages. Gene name Gene symbol Fold change Up-regulated genes Cell surface markers Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide FCER1A 376.89 CD209 molecule CD209 335.12 Lymphatic vessel endothelial hyaluronan receptor 1 LYVE1 321.11 C-type lectin domain family 4, member G CLEC4G 183.13 Collectin sub-family member 12 COLEC12 181.04 Junctional adhesion molecule 3 JAM3 145.24 Membrane-spanning 4-domains, subfamily A, member 2 MS4A2 137.95 Stabilin 1 STAB1 127.60 Mannose receptor, C type 1 MRC1 52.68 Solute carrier family 15 (oligopeptide transporter), member 1 SLC15A1 52.03 Enzymes Fructose-1,6-bisphosphatase 1 FBP1 149.73 Uronyl-2-sulfotransferase UST 85.20 Alanyl (membrane) aminopeptidase ANPEP 82.13 Lipoprotein lipase LPL 57.39 Cytokines , chemokines , and their receptors Chemokine (C-C motif) ligand 24 CCL24 1050.11 Transforming growth factor, beta 2 TGFB2 66.97 Soluble factors CD5 molecule-like CD5L 209.01 Secreted phosphoprotein 2, 24kDa SPP2 121.14 Nephronectin NPNT 105.07 Secretogranin V (7B2 protein) SCG5 82.69 Complement component 3 C3 67.78 Cystatin 9 (testatin) CST9 58.53 Miscellaneous Coagulation factor XIII, A1 polypeptide F13A1 1309.86 Sodium channel, voltage-gated, type II, beta subunit SCN2B 1120.45 Dynamin 1 DNM1 247.10 Caldesmon 1 CALD1 239.74 Rho GTPase activating protein 6 ARHGAP6 147.58 Fibronectin 1 FN1 141.81 Cfa-mir-125b-2 cfa-mir-125b-2 103.61 Plexin domain containing 2 PLXDC2 71.70 Transforming growth factor, beta-induced, 68kDa TGFBI 59.20 G protein-coupled receptor 116 GPR116 51.83 Down-regulated genes Cell surface markers Purinergic receptor P2Y, G-protein coupled, 14 P2RY14 -344.22 Solute carrier family 39 (zinc transporter), member 14 SLC39A14 -200.01 Integrin, beta 8 ITGB8 -134.58 Transmembrane protein 150C TMEM150C -103.02 Transmembrane protein 176A TMEM176A -78.17 Solute carrier family 22, member 15 SLC22A15 -58.69 Enzymes Prostaglandin E synthase PTGES -1274.66 Epoxide hydrolase 2, cytoplasmic EPHX2 -222.01 Interferon stimulated exonuclease gene 20kDa ISG20 -187.95 Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) PTGS2 -183.48 E3 ubiquitin-protein ligase NEURL3-like LOC102152163 -156.11 Indoleamine 2,3-dioxygenase 2 IDO2 -127.88 STEAP family member 4 STEAP4 -92.42 Ceruloplasmin (ferroxidase) CP -90.30 Granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1) GZMB -69.42 Phospholipase A2, group XVI-like LOC476045 -57.39 cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial CMPK2 -57.00 WNK lysine deficient protein kinase 2 WNK2 -52.30 Interleukin-1 receptor-associated kinase 3 IRAK3 -50.40 Cytokines , chemokines , and their receptors Chemokine (C-X-C motif) receptor 3 CXCR3 -140.97 Chemokine (C-C motif) ligand 20 CCL20 -119.64 Interleukin 6 (interferon, beta 2) IL6 -69.50 Tumor necrosis factor TNF -55.63 Soluble factors Complement component 2 C2 -78.67 Miscellaneous Peptidase inhibitor 3, skin-derived PI3 -235.52 TNFAIP3 interacting protein 3 TNIP3 -178.37 Ras homolog family member U RHOU -168.17 Radical S-adenosyl methionine domain containing 2 RSAD2 -82.43 Cochlin COCH -70.23 Interferon, alpha-inducible protein 6 IFI6 -68.46 multiple C2 domains, transmembrane 2 MCTP2 -66.09 Fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus) FSCN1 -58.64 Interferon regulatory factor 4-like LOC609817 -50.92 ISG15 ubiquitin-like modifier ISG15 -50.28 Interestingly, gene expression of CD209 markedly decreased in the pairwise comparison of M1 vs . M0 (FC = -345.12) whereas it was highly up-regulated in the comparison M2 vs . M1 (FC = 335.12). In both comparisons, M2 vs . M0 and M2 vs . M1, genes for LYVE1 , MRC1 , MS4A2 , JAM3 , and CLEC4G were up-regulated, whereas genes for ITGB8 and P2RY14 were down-regulated. SUCNR1 was up-regulated in both M1 vs . M0 and M2 vs . M0. Hierarchical cluster analysis Unsupervised hierarchical clustering analysis formed 9 different clusters based on similarities and differences in the expression profile of DEPs ( Fig 4 ). Two out of these 9 clusters had an expression profile that was visually clearly associated with either the M1- or M2-phenotype ( Fig 4 ). Functional annotation of these clusters identified that the M1-polarization cluster (cluster 8) was significantly associated with the biological process "respiratory burst involved in defense response" ( Fig 4 ). The M2-cluster (cluster 6) was significantly associated with multiple biological processes of mitosis such as "M phase of mitotic cell cycle" and "mitotic spindle organization" ( Fig 4 ). The genes associated with these M1- and M2-specific clusters are listed with their particular fold changes in S1 and S2 Tables. The remaining 7 clusters were neither specific for M1- nor for M2-macrophages and were associated with biological terms like "peptidyl-lysine mono- and dimethylation" (cluster 1), "immune response-activating signal transduction" (cluster 3), "monosaccharide metabolic process", "organic substance catabolic process", "cellular catabolic process" (cluster 4), "response to other organism", "regulation of lymphocyte proliferation" (cluster 5), "tRNA aminoacylation for protein translation" (cluster 7), "cytokinesis" and "antigen receptor-mediated signaling pathway" (cluster 9, S3 Table ). No significantly enriched biological terms were identified for cluster 2. 10.1371/journal.pone.0183572.g004 Fig 4 Hierarchical clustering analysis. Unsupervised hierarchical clustering analysis of the median centered, log 2 -transformed expression values of 6358 differentially expressed probe sets in canine polarized macrophages as obtained by TM4 MultiExperimentViewer with default settings (Euclidean distance; complete linkage), depicted on a color scale from red (relatively high expression) to green (relatively low expression). A) The analysis identified 9 distinct clusters (I-IX) based on similarities as well as differences in the expression intensity of canine polarized macrophages. Two out of these 9 clusters (VIII and VI) visually displayed an expression profile that was clearly associated with either the M1- or M2-phenotype. B) Functional annotation of the M1-cluster (magnified from A) using Web-based Gene Set Analysis Toolkit (WebGestalt) identified the enriched biological process category "respiratory burst" (adjusted p-value≤0.05). C) The M2-cluster (magnified from A) is associated with enriched biological GO terms such as "M phase of mitotic cell cycle" and "mitotic spindle organization" (adjusted p-value≤0.05). Literature-based gene signatures and marker genes Intersections of genes exclusively up-regulated in M1 vs . M0 and down regulated in M2 vs . M1 (M1-macrophage genes), as well as genes up-regulated in the comparisons M2 vs . M0 and M2 vs . M1 (M2-macrophage genes) were selected and compared to literature-based markers that are known to distinguish between human and murine M1- and M2-macrophages ( Fig 5A ). Notably, many markers identified in the recent study to be specific for canine M1-polarization (404 unique genes in total corresponding to 565 probesets, S5 Table ) did not match with the literature-based M1-markers (65 genes). Similarly, predominating numbers of canine M2-markers (700 unique genes in total corresponding to 1029 probesets, S5 Table ) did not match with the literature-based M2-markers (58 genes; Fig 5A ). However, overlapping expression of genes reflecting M1-polarization state was present for 8 genes, i . e . BIRC3 , CCR7 , CD80 , IL15RA , IL23A , INHBA , NAMPT , and SLC2A6 . For canine M2-polarization, 11 genes matched reported expression in human and murine M2-macrophages, i . e . CCL24 , CCL13 , FCER1A , FN1 , EGR2 , CA2 , LIPA , SLC4A7 , CD163 , ADK , and FGL2 ( Fig 5A ). Conflicting results were present for the genes P2RY14 , TGFBR2 , and TPST2 , which were expected as M2-markers based on the literature but were differentially up-regulated in canine M1-macrophages. Furthermore, PIK3CB was up-regulated by both canine M1- and M2-macrophages in the present study. The differentially expressed genes of the intersections, which are not namely mentioned in Fig 5A are listed in S4 Table . The top 50 candidate canine M1- and M2-macrophage genes, as defined above, are given in Tables 6 and 7 . The full lists of canine probesets, corresponding to the 404 unique M1 genes and 700 unique M2 genes are shown in S5 Table . 10.1371/journal.pone.0183572.g005 Fig 5 Comparative evaluation of canine M1- and M2-associated differentially expressed genes (M1 = 404; M2 = 700) with established literature-based human and murine orthologous genes (A) and detection of polarization-specific biomarkers employing supervised clustering with a K-nearest-neighbors algorithm and correlation-based feature selection using Prophet (B, C). A) The Venn diagram depicts the numbers and intersections of differential and common canine M1- and M2-genes with literature-based human and murine genes. The majority of literature-based M1- (65 genes = 57+8) and M2-markers (58 genes = 44+11+3) did not comply with the present microarray data upon canine macrophages. However, overlapping expression of 8 genes for the M1-phenotype and 11 genes for the M2-phenotype was identified. The genes of the intersections, not specifically mentioned in the figure, are listed in S4 Table B) Biomarker selection detected 4 highly specific probe sets for annotated by the mammalian carboxypeptidase inhibitor latexin ( LXN ) to be highly correlated to the M1-phenotype. The boxplots depict the significantly enhanced log 2 -transformed expression values for LXN in M1-macrophages as compared to both M0- and M2-macrophages (p = 0.002), as well as between M0- and M2-macrophages (p = 0.002). C) For the M2-phenotype, the most significant predicted biomarker was high affinity receptor for IgE, i . e . membrane-spanning 4-domains, subfamily A, member 2 ( MS4A2 ). The expression data of M2-macrophages show significantly higher log 2 -transformed expression values as compared to both M0- and M1-macrophages (p = 0.002) as well as between M0- and M1-macrophages (p = 0.002). Asterisks indicate significance (Kruskal-Wallis-Test with subsequent pairwise Mann-Whitney-U-Tests). 10.1371/journal.pone.0183572.t006 Table 6 Top 50 candidate M1-macrophage associated probesets, which were upregulated in M1- vs. M0-macrophages and simultaneously downregulated in M2- vs. M1-macrophages. Probe Set ID Gene Symbol Fold change M1 vs. M0 Fold change M2 vs. M1 Cfa.12190.1.A1_at PTGES 3.34 -1274.66 CfaAffx.30585.1.S1_s_at PTGES 4.99 -1136.51 CfaAffx.30585.1.S1_at PTGES 4.94 -517.22 CfaAffx.927.1.S1_at P2RY14 4.80 -344.22 Cfa.20456.1.S1_at IFI6 269.13 -284.48 CfaAffx.15155.1.S1_s_at PI3 27.22 -235.52 Cfa.574.1.A1_at EPHX2 119.72 -222.01 Cfa.18083.1.S1_s_at SLC39A14 15.89 -212.19 CfaAffx.14855.1.S1_s_at SLC39A14 13.48 -200.01 Cfa.3449.1.S1_s_at PTGS2 27.78 -183.48 Cfa.15554.1.A1_at TNIP3 3.07 -178.37 Cfa.9253.1.A1_at RHOU 446.23 -168.17 Cfa.12477.1.A1_at LOC102152163 86.20 -156.11 Cfa.16339.1.S1_at CXCR3 5.70 -140.97 Cfa.10779.1.A1_at IDO2 28.65 -127.88 CfaAffx.13394.1.S1_s_at EPHX2 110.24 -122.12 Cfa.15812.1.S1_at CCL20 67.90 -119.64 Cfa.1856.1.S1_at TMEM150C 40.36 -103.02 Cfa.2878.1.A1_s_at CP 425.52 -96.02 CfaAffx.3697.1.S1_at STEAP4 11.45 -92.42 CfaAffx.13209.1.S1_s_at CP 161.88 -90.30 Cfa.8846.1.A1_s_at C2 /// CFB 4.39 -78.67 CfaAffx.7919.1.S1_at TMEM176A 19.58 -78.17 CfaAffx.1718.1.S1_at TNF 47.17 -71.34 Cfa.3528.1.S1_s_at IL6 252.32 -69.50 Cfa.20456.1.S1_s_at IFI6 68.64 -68.46 CfaAffx.17110.1.S1_s_at MCTP2 2.69 -66.09 CfaAffx.15348.1.S1_at SLC22A15 15.67 -58.69 CfaAffx.24565.1.S1_at FSCN1 35.83 -58.64 Cfa.12164.1.A1_at LOC476045 5.65 -57.39 Cfa.54.1.S1_s_at TNF 47.20 -55.63 Cfa.11870.1.A1_at PI3 17.25 -52.33 Cfa.4359.1.S1_at WNK2 52.35 -52.30 CfaAffx.1510.1.S1_s_at IRAK3 10.70 -50.40 CfaAffx.15202.1.S1_s_at SDC4 58.61 -49.56 CfaAffx.1352.1.S1_s_at IL22RA2 116.00 -48.48 CfaAffx.26233.1.S1_s_at CXCR3 32.55 -47.23 CfaAffx.17136.1.S1_s_at MCTP2 6.25 -46.94 CfaAffx.24086.1.S1_at KMO 23.66 -46.48 CfaAffx.15086.1.S1_s_at BMP1 43.14 -43.31 CfaAffx.261.1.S1_at HCAR3 13.88 -42.20 Cfa.18962.1.S1_s_at SGK1 20.11 -39.12 Cfa.3719.1.S1_s_at QPCT 22.78 -38.99 Cfa.4926.1.A1_s_at LOC476045 5.41 -37.14 CfaAffx.6260.1.S1_at C18H7orf10 43.19 -36.13 Cfa.6458.1.A1_s_at C2 /// CFB 5.05 -36.01 Cfa.8282.1.A1_s_at KBTBD7 3.64 -35.38 CfaAffx.8143.1.S1_at KBTBD7 2.59 -35.14 Cfa.21252.1.S1_s_at ENPP2 188.74 -34.85 CfaAffx.16422.1.S1_s_at CCL20 33.43 -34.83 10.1371/journal.pone.0183572.t007 Table 7 Top 50 candidate M2-macrophage associated probesets, which were upregulated in M2- vs. M0-macrophages and simultaneously upregulated in M2- vs. M1-macrophages. Probe Set ID Gene Symbol Fold change M2 vs. M0 Fold change M2 vs. M1 Cfa.11125.1.A1_at SCN2B 1061.28 1120.45 Cfa.15823.1.S1_at CCL24 1060.82 1050.11 CfaAffx.18273.1.S1_at FCER1A 35.63 922.72 CfaAffx.20721.1.S1_s_at CCL24 819.06 819.06 Cfa.17541.1.S1_s_at FBP1 160.02 748.69 CfaAffx.2850.1.S1_s_at FBP1 57.95 422.21 Cfa.3663.1.A1_s_at FCER1A 21.07 376.89 Cfa.3707.1.A1_at FN1 12.68 360.42 CfaAffx.12229.1.S1_at LYVE1 322.32 321.11 Cfa.10966.1.A1_at DNM1 112.78 247.10 Cfa.6272.1.S1_at CALD1 224.66 239.74 CfaAffx.1504.1.S1_s_at UST 7.71 226.47 Cfa.2693.1.A1_at LOC102152647 194.28 194.28 Cfa.3707.1.A1_s_at FN1 7.26 188.32 CfaAffx.28024.1.S1_at CLEC4G 54.14 183.13 CfaAffx.16206.1.S1_at MS4A2 110.05 167.77 Cfa.6369.1.A1_at FBP1 26.98 149.73 Cfa.20346.1.S1_at ARHGAP6 37.99 147.58 Cfa.1705.1.A1_at JAM3 78.46 145.24 Cfa.3662.1.S1_at MS4A2 54.98 137.95 Cfa.19567.1.S1_at UST 8.05 120.81 CfaAffx.16206.1.S1_s_at MS4A2 76.56 108.61 Cfa.3774.1.A1_s_at ANPEP 25.55 105.73 Cfa.14465.1.S1_at NPNT 444.07 105.07 Cfa.688.1.S1_at cfa-mir-125b-2 58.78 103.61 CfaAffx.7700.1.S1_s_at MRC1 136.99 89.14 Cfa.2468.1.A1_at UST 6.54 85.20 Cfa.10794.1.A1_at SCG5 6.75 82.69 Cfa.20798.1.S1_at ANPEP 14.09 82.13 CfaAffx.14701.1.S1_s_at STAB1 4.00 81.41 CfaAffx.28463.1.S1_at C3 36.53 76.45 Cfa.12240.1.A1_at C3 44.14 67.78 CfaAffx.16904.1.S1_s_at TGFB2 61.84 66.97 CfaAffx.7365.1.S1_at CST9 58.21 58.53 CfaAffx.15901.1.S1_s_at LPL 52.03 57.39 CfaAffx.7698.1.S1_at MRC1 104.41 52.68 Cfa.78.1.S1_s_at SLC15A1 14.65 52.03 CfaAffx.27914.1.S1_s_at IL13RA2 170.59 46.70 Cfa.19638.1.S1_s_at TMLHE 51.05 45.55 CfaAffx.22229.1.S1_at GPR34 6.30 42.39 CfaAffx.21392.1.S1_s_at FKBP7 25.81 40.60 CfaAffx.19151.1.S1_s_at ADCY4 14.07 39.02 Cfa.11222.1.A1_s_at FAM185A 23.07 38.03 Cfa.10053.1.A1_at RAB40B 48.86 37.88 CfaAffx.24797.1.S1_at EPHX1 3.75 36.82 Cfa.1930.1.S1_at MRC1 25.70 36.76 Cfa.3367.1.A1_at TGFB2 36.49 36.58 Cfa.15824.1.S1_at CCL13 172.58 34.71 Cfa.14297.1.A1_s_at MAOB 33.75 33.75 Cfa.12621.1.A1_at LRP4 7.01 33.01 Biomarker selection In a hypothesis-driven approach, polarization specific prediction markers were detected with Prophet using the microarray data of unstimulated macrophages (M0), M1-, and M2-macrophages [ 41 , 42 ]. 369 probe sets were identified by the correlation-based feature selection algorithm of Prophet that discriminated between M0-, M1-, and M2-polarity, using the KNN algorithm with 100% correct predictions. Subsequent "one versus all" analyses using SET to create ranked lists of genes, based on their potential to serve as biomarker for M1- or M2-macrophages were performed ( S6 Table ; [ 43 ]). The highest scoring probe set differentiating M1- from M0- and M2-macrophages was CfaAffx.14358.1.S1_at, annotated as latexin ( LXN ), which was up-regulated in M1-macrophages, exhibiting a prediction-accuracy for the M1-phenotype of 100%. Additionally, the probe set ID's occupying the ranks 2 to 4 (Cfa.5195.1.A1_s_at, Cfa.5195.1.A1_at, Cfa.14007.1.A1_x_at) were similarly annotated to LXN ( S6 Table ). Analysis of the expression data of CfaAffx.14358.1.S1_at revealed a relatively high expression in all three polarities with a significantly higher expression in M1-macrophages as compared to M0- and M2-macrophages (pairwise Mann-Whitney-U-Tests; p = 0.02, Fig 5B ). For the comparison of M2 versus M0 and M1, the highest scoring probe set was Cfa.3662.1.S1_at, annotated as membrane-spanning 4-domains , subfamily A , member 2 ( MS4A2 , S6 Table ). This probe set was up-regulated in M2-macrophages and displayed a prediction accuracy of 100% for this phenotype. Statistical evaluation with pairwise Mann-Whitney-U-Tests underlined a low expression of MS4A2 in M0- and M1-macrophages, but a significantly higher expression in M2-macrophages (p = 0.02, Fig 5C ). Testing of antibodies for the detection of predicted biomarkers In order to test, whether the biomarkers predicted to distinguish between canine M0-, M1-, and M2-macrophages during the transcriptome investigations are also mirrored by altered protein expression, an additional experiment using blood from 3 dogs was performed. The cells were isolated and polarized as described and labeled with antibodies targeting MS4A2 and LXN ( Table 1 ). There was protein expression in canine macrophages in vitro for both molecules ( Fig 6 ). However, the percentage of cells labeled with an antibody against MS4A2 showed no differences between M0-, M1-, and M2-macrophages (p = 0.117; Fig 6 ). The percentage of positive cells for LXN was significantly higher in both M1-and M2-macrophages as compared to M0-macrophages (p = 0.02 and p = 0.01, respectively); however, there was no difference in the percentage of LXN-immunopositive cells between M1- and M2-macrophages (p = 0.06; Fig 6 ). 10.1371/journal.pone.0183572.g006 Fig 6 Protein expression of the predicted biomarkers latexin and MS4A2 in canine M0-, M1-, and M2-macrophages. A) Expression of latexin in non-stimulated canine M0-macrophages. B) Expression of latexin in canine M1-macrophages. C) Expression of latexin in canine M2-macrophages. D) Expression of MS4A2 in canine M0-macrophages. E) Expression of MS4A2 in canine M1-macrophages. F) Expression of MS4A2 in canine M2-macrophages. A-F) Scale bars = 100 μm. Nuclear counterstaining with bisbenzimide. G) Dot plots illustrating a significantly higher percentage of immunopositive cells in canine M1- and M2-macrophages as compared to M0-macrophages (One-factorial ANOVA with group-wise t-tests, astrerisk = p<0.05). H) Dot plot showing lack of statistical differences in the percentage of immunopositive cells for MS4A2 in canine M0-, M1-, and M2-macrophages. Morphological characterization of polarized canine macrophages On day 7 in culture, the overall number of cells differed between the three polarities (p = 0.02; S1 Fig ). The number of cells was highest in M2-polarized cells (mean = 135 cells per field), while M0-macrophages were lowest in number (mean = 30 cells per field). M1-macrophages had a mean number of 52 cells per field. T tests revealed that the number of M2-macrophages was significantly higher than M0-macrophages (p = 0.02), while comparisons between M0- and M1-macrophages and M1-and M2-macrophages failed to reach the level of significance (p = 0.30 and p = 0.06, respectively). Morphologically, striking differences between M0-, M1-, and M2-polarized cells were observed at day 7 in culture by scanning electron microscopy and phase contrast microscopy ( Fig 1 ). Canine M0-macrophages predominantly appeared as small and roundish cells with an average size of 8 μm in diameter and no or little cytoplasmic extensions that measured up to 1 μm in length ( Fig 1 ). However, a noteworthy proportion of M0-macrophages (about 25%) also obtained an amoeboid morphology, which however was predominantly observed in M1 macrophages ( Fig 1 ). The majority of M1-macrophages was amoeboid and had a mean size of 15 μm and numerous fibrillary cytoplasmic processes on the cellular surface that had a length of up to 5 μm ( Fig 1 ). M2-macrophages appeared as a heterogeneous cell population with a mixture of 4 different morphologies. Besides roundish and amoeboid macrophages, large bipolar spindeloid macrophages were present measuring up to 35 μm that were characterized by an elongated cell body with cytoplasmic extensions with an average length of 7 μm at both poles ( Fig 1 ). Moreover, large MNGs appeared that had a mean size of 40 μm with an extensive cytoplasm and numerous evenly distributed processes that measured up to 7 μm ( Fig 1 ). Allover, the mean percentage of small/roundish macrophages (morphology 1) was significantly higher in untreated cultures (M0) compared to polarity 2 at days 5 and 7 (p≤0.05; Fig 1E ). For amoeboid macrophages (morphology 2), there was a trend towards a higher mean percentage in polarity 1 compared to polarity 0 at day 5 (p = 0.07) and day 7 (p = 0.06; Fig 1F ). The mean percentage of spindeloid macrophages (morphology 3) was significantly higher in polarity 2 at day 5 compared to polarity 1 at day 5 (p≤0.05; Fig 1G ). MNGs (morphology 4) were exclusively present in polarity 2, as compared to the polarities 0 and 1 at day 7 (p≤0.05; Fig 1H ). 10.1371/journal.pone.0183572.g001 Fig 1 Polarization-dependent morphological differences in canine M0-, M1-, and M2-macrophage cultures. A) In scanning electron microscopy, unstimulated macrophages (M0; day 7) obtain a small and roundish morphology, lacking cytoplasmic extensions. B) M1-treated macrophages (day 7) are characterized by an enlarged amoeboid cell shape with roundish cell bodies and numerous delicate cytoplasmic extensions on the cellular surface. C; D) M2-treated macrophage cultures (day 7) demonstrate a marked heterogeneity with two dominating cell types. Large "spindeloid" macrophages with an elongated cell body and cytoplasmic extensions on the apical ends of the cell bodies (C) . Second, in M2-cultures, numerous multinucleated giant cells (MNGs) with abundant cytoplasmic projections on the cellular surface are present (D) . E-H) Dot plot diagrams depicting the morphological changes of macrophage cultures (n = 3) following stimulation (M0, M1, M2) over the time course as calculated with a mixed ANOVA with post-hoc alpha adjustment (Tukey-Kramer) and significance level at p≤0.05 (asterisks). E) Small/roundish macrophages dominate in untreated M0 cell cultures and their relative percentage is significantly higher in M0 when compared to M2 at days 5 and 7. F) Amoeboid macrophages display a statistical trend of predominance in M1 compared to M0 at day 5 and 7. G) The relative percentage of spindeloid macrophages is significantly increased in M2 at day 5 compared to M1. H) MNGs are almost exclusively observed in M2-macrophages at the end of the culturing period and their percentage is significantly higher at day 7 in M2 compared to both M0 and M1. Phenotypical characterization of polarized canine macrophages For the phenotypical characterization of canine macrophages, the percentage of immunopositive cells for selected literature-based M1-/M2-antigens was evaluated in 5 animals and related to the polarization of cells (polarity 0, 1, 2). Interestingly, except for MHC class II and CD206, none of the remaining tested antigens (CD16, CD32, iNOS, CD163, and arginase-1) were differently expressed between canine M0-, M1-, and M2-macrophages (p-values ranging from 0.101 to 0.691; Kruskal-Wallis-Test; Fig 2 ). 10.1371/journal.pone.0183572.g002 Fig 2 Immunofluorescence staining of in vitro cultured canine M0-, M1-, and M2-macrophages labeled with prototypic literature-based antibodies for the M1- (CD16, CD32, MHC class II, and iNOS) and M2-phenotype (CD163, CD206, and arginase 1), respectively. A-C) Low to moderate membranous staining of M0-, M1-, and M2-macrophages for CD16. D-F) Likewise, CD32 shows a low to moderate staining in all three treatment conditions. G-I) M0-, M1-, and M2-macrophages demonstrate a moderate to high membranous staining with CD163. J-L) Intense membranous staining of M0-, M1-, and M2-macrophages with an anti-MHC class II antibody. M-O) Strong intracytoplasmic labeling of macrophages in all treatments (polarity 0, 1, 2) for inducible nitric oxide synthase (iNOS). P-R) High intracytoplasmic expression of arginase 1 in small/roundish, amoeboid, and spindeloid macrophages as well as in multinucleated giant cells (MNGs). a-f) Statistical evaluation of the mean expression percentages of prototypic M1-/M2-markers evaluated in 5 dogs and related to the polarization state of the macrophages (polarity 0, 1, 2). Note that, except for MHC class II, none of the remaining tested antigens (CD16, CD32, iNOS, CD163, and arginase-1) were differently expressed between canine M0-, M1-, and M2-macrophages (* = p≤0.05; Kruskal-Wallis-Test with pairwise Mann-Whitney-U-Tests). Scale bars = 100 μm. Nuclear counterstaining with bisbenzimide. The mean percentage of immunopositive cells expressing MHC class II was significantly higher in M2-macrophages (mean positive cells: 93.64%) when compared to untreated M0-cells (mean positive cells: 72.22%; p = 0.008). However, there was no statistical difference in the percentage of positive cells for MHC class II, when M1-macophages (mean positive cells: 89.92%) were compared to M2-macrophages and M0-macrophages, respectively ( Fig 2 ). For CD206, the mean percentage of immunopositive cells was highest in M2-macrophages (mean: 66.54%) as compared to both M0-macrophages (mean: 33.33%; p = 0.008) and M1-macrophages (mean: 28.67%; p = 0.032, Fig 3 ). No statistical difference was observed between M1- and M0-macrophages ( Fig 3 ). The results were validated in the microarray data set, which similarly revealed significantly higher expression levels of CD206 in canine M2-macrophages as compared to both M1- (p = 0.002) and M0-macrophages (p = 0.004; Fig 3 ). 10.1371/journal.pone.0183572.g003 Fig 3 Phenotypical characterization of canine M0-, M1-, and M2-macrophages. A) Low membranous expression of CD206 antigen by small/roundish M0-macrophages. B) Moderate membranous staining of amoeboid M1-macrophages for CD206. C) Intense membranous expression of CD206 antigen by M2-macrophages. Scale bars = 100 μm. Nuclear counterstaining with bisbenzimide. D) The mean percentage of CD206-immunopositive cells is significantly higher in M2-macrophages as compared to both M1- and M0-macrophages (* = p≤0.05; Kruskal-Wallis-Test with pair-wise Mann-Whitney-U-Tests). E) The log 2 -transformed expression values of the probe set encoding for the gene CD206 is similarly significantly higher in M2-macrophages compared to both M1- and M0-macrophages (* = p≤0.05; Kruskal-Wallis-Test with pair-wise Mann-Whitney-U-Tests), thus confirming the results of the immunofluorescence investigation. Differentially expressed genes between M0-, M1-, and M2-polarized macrophage cultures One-factorial multigroup analysis of microarray data and fold change criteria identified 6358 probe sets that were differentially expressed in at least one of the three post-hoc pairwise comparisons. The total number of DEPs was 3555 in M1 vs . M0, 4831 in M2 vs . M0, and 3141 in M2 vs . M1, respectively ( Table 2 ). In all pairwise comparisons, the number of up- and down-regulated probe sets was nearly equally distributed (M1 vs . M0: 1699 up, 1856 down; M2 vs . M0: 2467 up, 2364 down; M2 vs . M1: 1572 up, 1569 down). Functional annotation of enriched biological processes in DEPs, up–regulated in the comparison M1 vs . M0, revealed terms for "organonitrogen compound metabolic process", "carbohydrate metabolic process", "carboxylic acid metabolic process", and "tricarboxylic acid cycle" ( Table 2 ). In contrast, biological terms in down-regulated DEPs in this comparison reflected terms for "positive regulation of immune response", "immune response-regulating signaling pathway", and "regulation of innate immune response". In both comparisons M2 vs . M0 and M2 vs . M1, up-regulated DEPs displayed significantly enriched gene ontology terms for "M phase of mitotic cell cycle" and "mitotic spindle organization". Additionally, in M2 vs . M0 comparison, up-regulated DEPs were associated with "oxidation-reduction process", "carboxylic acid metabolic process", and "organonitrogen compound metabolic process", whereas down-regulated DEPs were related to the biological term "immune response-activating signal transduction". In the comparisons M2 vs . M1, down-regulated DEPs were functionally associated to biological terms such as "response to other organism", "defense response", "regulation of lymphocyte activation", and "regulation of immune response" ( Table 2 ). 10.1371/journal.pone.0183572.t002 Table 2 Summarized results of the functional annotation of the pairwise comparisons of differentially expressed probe sets (DEPs) in canine M0-, M1-, and M2-macrophages. Pairwise comparison Differentially expressed probe sets Up-/down-regulated probe sets Enriched biological process categories * Enriched KEGG pathways * M1 vs . M0 3555 Up: 1699 • Organonitrogen compound metabolic process • Carbohydrate metabolic process • Tricarboxylic acid cycle • Metabolic pathways • Glutathione metabolism • Steroid biosynthesis • Glycolysis/Gluconeogenesis Down: 1856 • Positive regulation of immune response • Immune response-regulating signaling pathway • Regulation of innate immune response • Hematopoietic cell lineage • RIG-I-like receptor signaling pathway • T cell receptor signaling pathway • Cell adhesion molecules M2 vs . M0 4831 Up: 2467 • Oxidation-reduction process • Carboxylic acid metabolic process • Organonitrogen compound metabolic process • M-phase of mitotic cell cycle • Metabolic pathways • Glycolysis/Gluconeogenesis • Propanoate and pyruvate metabolism • Steroid biosynthesis • Cell cycle Down: 2364 • Immune response-activating signal transduction • T cell receptor signaling pathway • Natural killer cell mediated cytotoxicity • Hematopoietic cell lineage M2 vs . M1 3141 Up: 1572 • Mitotic spindle organization • Cell cycle • Metabolic pathways • Ribosome biogenesis in eukaryotes • PPAR signaling pathway Down: 1569 • Response to other organism • Defense response • Regulation of lymphocyte activation • Regulation of immune response • NOD-like receptor signaling pathway • Osteoclast differentiation • Toll-like receptor signaling pathway • B and T cell receptor signaling pathway * employing Web-based Gene Set Analysis Toolkit (WebGestalt; http://bioinfo.vanderbilt.edu/webgestalt/ ) with default settings adjusted p-value ≤0.05 Enriched KEGG-pathways, significantly associated with up-regulated canine DEPs in M1 vs . M0 (1699 DEPs), were functionally related to "metabolic pathways", "glutathione metabolism", "steroid biosynthesis", and "glycolysis/gluconeogenesis". The comparison M2 vs . M0 (2467 up-regulated DEPs) contained enriched KEGG-pathways for "metabolic pathways", "glycolysis/gluconeogenesis", "propanoate and pyruvate metabolism", "steroid biosynthesis", and "cell cycle". In the comparison M2 vs . M1 (1572 up-regulated DEPs), enriched KEGG-pathways included terms for "cell cycle", "metabolic pathways", "ribosome biogenesis in eukaryotes", and "PPAR signaling pathway". In contrast, down-regulated DEPs in the three comparisons were associated with "hematopoietic cell lineage", "RIG-I-like receptor signaling pathway", "T cell receptor signaling pathway", and "cell adhesion molecules" (M1 vs . M0), "T cell receptor signaling pathway", "natural killer cell mediated cytotoxicity", and "hematopoietic cell lineage" (M2 vs . M0), and "NOD-like receptor signaling pathway", "osteoclast differentiation", "Toll-like receptor signaling pathway", and "B and T cell receptor signaling pathway" (M2 vs . M1; Table 2 ). Genes whose expression was most severely affected (FC ≥50.0 or ≤-50.0) in the pairwise comparison are depicted in Tables 3 , 4 and 5 . Fortynine genes fulfilled these filtering criteria in the M1 vs . M0 contrast (25 up, 24 down), whereas 99 genes were retrieved in the comparison of M2 vs . M0 (41 up; 58 down). Comparing M2 with M1, 66 genes fulfilled the criteria (32 up; 34 down). Focusing on potentially promising cell surface markers, the pairwise comparison of three genes encoding for such surface molecules were up-regulated in M1 vs . M0, namely SUCNR1 , SDC4 , and CHRNA9 , whereas CD209 , CD180 , KLRG1 , COLEC12 , and C3AR1 were down-regulated ( Table 3 ). Comparing M2 vs . M0, nine cell surface makers were up-regulated, i . e . LYVE1 , SUCNR1 , CD1e , MRC1 , TSPAN7 , JAM3 , ANTXR1 , MS4A2 , and CLEC4G ( Table 4 ). In contrast, a total amount of 18 cell surface marker genes was down-regulated, namely ITGB8 , LY6E , TRDC , NCR3 , SELL , SIGLEC1 , KLRD1 , KLRB1 , MPP6 , P2RY14 , TRBC2 , TARP , FCRLA , KCNK5 , P2RX5 , NKG7 , CD69 , and CD7 ( Table 4 ). In the comparison M2 vs . M1, the up-regulated cell surface marker genes contained 10 terms for FCER1A , CD209 , LYVE1 , CLEC4G , COLEC12 , JAM3 , MS4A2 , STAB1 , MRC1 , and SLC15A1 ( Table 5 ). Down-regulated cell surface marker genes were P2RY14 , SLC39A14 , ITGB8 , TMEM150C , TMEM176A , and SLC22A15 ( Table 5 ). 10.1371/journal.pone.0183572.t003 Table 3 List and subgrouping of the top hits of highly differentially expressed genes (fold change ≥ 50 or ≤ -50) in canine M1- vs . M0-macrophages. Gene name Gene symbol Fold change Up-regulated genes Cell surface markers Succinate receptor 1 SUCNR1 212.94 Syndecan 4 SDC4 58.61 Cholinergic receptor, nicotinic, alpha 9 (neuronal) CHRNA9 53.74 Enzymes ADP-ribosylhydrolase like 2 ADPRHL2 535.59 Ceruloplasmin (ferroxidase) CP 161.88 Epoxide hydrolase 2, cytoplasmic EPHX2 119.72 Ectonucleotide pyrophosphatase/phosphodiesterase 2 ENPP2 117.43 Interstitial collagenase-like LOC489428 87.83 E3 ubiquitin-protein ligase NEURL3-like LOC102152163 86.20 NOP2/Sun domain family, member 7 NSUN7 66.00 Nucleoredoxin NXN 64.78 WNK lysine deficient protein kinase 2 WNK2 52.35 Cytokines , chemokines , and their Receptors Interleukin 6 (interferon, beta 2) IL6 252.32 Chemokine (C-C motif) ligand 22 CCL22 187.96 Chemokine (C-X-C motif) receptor 7 ACKR3 178.18 Interleukin 22 receptor, alpha 2 IL22RA2 116.00 Chemokine (C-X-C motif) ligand 14 CXCL14 108.55 Chemokine (C-C motif) ligand 17 CCL17 95.26 Chemokine (C-C motif) ligand 20 CCL20 67.90 Soluble factors Chitinase 3-like 1 (cartilage glycoprotein-39) CHI3L1 83.25 Clusterin CLU 66.23 Miscellaneous Ras homolog family member U RHOU 446.23 Interferon, alpha-inducible protein 6 IFI6 269.13 CXADR-like membrane protein CLMP 213.81 Retinoic acid induced 14 RAI14 118.86 Down-regulated genes Cell surface markers CD209 molecule CD209 -345.12 CD180 molecule CD180 -75.00 Killer cell lectin-like receptor subfamily G, member 1 KLRG1 -66.25 Collectin sub-family member 12 COLEC12 -61.81 Complement component 3a receptor 1 C3AR1 -55.16 Enzymes Carboxypeptidase M CPM -134.50 N-acetylneuraminate pyruvate lyase (dihydrodipicolinate synthase) NPL -78.98 Cathepsin E CTSE -76.30 Cytokines , chemokines , and their receptors Chemokine (C-X-C motif) ligand 12 CXCL12 -401.92 Pro-platelet basic protein (chemokine (C-X-C motif) ligand 7) PPBP -100.42 Interleukin 2 IL2 -95.84 Interleukin 1 receptor, type II IL1R2 -79.71 Chemokine (C-C motif) receptor 3 CCR3 -51.05 Soluble factors Lipocalin 2 LCN2 -180.73 CD5 molecule-like CD5L -153.48 Secreted phosphoprotein 2, 24kDa SPP2 -51.92 Miscellaneous Coagulation factor XIII, A1 polypeptide F13A1 -3305.03 Fatty acid binding protein 4, adipocyte FABP4 -370.34 G protein-coupled receptor 116 GPR116 -130.89 Interferon-induced transmembrane protein 3-like LOC606890 -114.16 Plexin domain containing 2 PLXDC2 -67.07 ADP-ribosylation factor-like 4C ARL4C -56.52 Cyclin J-like CCNJL -54.76 Thrombospondin 1 THBS1 -53.38 10.1371/journal.pone.0183572.t004 Table 4 List and subgrouping of the top hits of highly differentially expressed genes (fold change ≥ 50 or ≤ -50) in canine M2- vs . M0-macrophages. Gene name Gene symbol Fold change Up-regulated genes Cell surface markers Lymphatic vessel endothelial hyaluronan receptor 1 LYVE1 322.32 Succinate receptor 1 SUCNR1 151.91 CD1e molecule CD1E 147.40 Mannose receptor, C type 1 MRC1 104.41 Tetraspanin 7 TSPAN7 83.25 Junctional adhesion molecule 3 JAM3 78.46 Anthrax toxin receptor 1 ANTXR1 72.95 Membrane-spanning 4-domains, subfamily A, member 2 MS4A2 54.98 C-type lectin domain family 4, member G CLEC4G 54.14 Enzymes ADP-ribosylhydrolase like 2 ADPRHL2 614.52 Guanine deaminase GDA 174.44 Ubiquitin-conjugating enzyme E2 C-like LOC481325 151.23 Ribonucleotide reductase M2 RRM2 131.03 Fructose-1,6-bisphosphatase 1 FBP1 57.95 Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase) MMP9 56.03 Lipoprotein lipase LPL 52.03 Trimethyllysine hydroxylase, epsilon TMLHE 51.05 3-hydroxybutyrate dehydrogenase, type 2 BDH2 50.68 Cytokines , chemokines , and their receptors Chemokine (C-C motif) ligand 24 CCL24 1060.82 Chemokine (C-X-C motif) receptor 7 ACKR3 390.67 Chemokine (C-C motif) ligand 17 CCL17 209.36 Chemokine (C-C motif) ligand 13 CCL13 172.58 Interleukin 13 receptor, alpha 2 IL13RA2 170.59 Transforming growth factor, beta 2 TGFB2 61.84 Soluble factors Nephronectin NPNT 444.07 Norrie disease (pseudoglioma) NDP 213.72 Endothelin 1 EDN1 115.81 Cystatin 9 (testatin) CST9 58.21 Miscellaneous Sodium channel, voltage-gated, type II, beta subunit SCN2B 1061.28 Caldesmon 1 CALD1 224.66 CXADR-like membrane protein CLMP 169.27 SHC SH2-domain binding protein 1 SHCBP1 117.77 Dynamin 1 DNM1 112.78 NACC family member 2, BEN and BTB (POZ) domain containing NACC2 110.49 Retinoic acid induced 14 RAI14 84.35 Scinderin SCIN 71.66 Kinesin family member 23 KIF23 66.78 Kinesin family member 11 KIF11 64.20 Cfa-mir-125b-2 cfa-mir-125b-2 58.78 AHNAK nucleoprotein AHNAK 57.19 NUF2, NDC80 kinetochore complex component NUF2 54.62 Down-regulated genes Cell surface markers Integrin, beta 8 ITGB8 -199.37 Lymphocyte antigen 6 complex, locus E LY6E -116.70 T cell receptor delta constant TRDC -112.73 Natural cytotoxicity triggering receptor 3 NCR3 -105.99 Selectin L SELL -95.45 Sialic acid binding Ig-like lectin 1, sialoadhesin SIGLEC1 -91.39 Killer cell lectin-like receptor subfamily D, member 1 KLRD1 -87.12 Killer cell lectin-like receptor subfamily B, member 1 KLRB1 -74.56 Membrane protein, palmitoylated 6 (MAGUK p55 subfamily member 6) MPP6 -72.60 Purinergic receptor P2Y, G-protein coupled, 14 P2RY14 -71.69 T cell receptor beta constant 2 TRBC2 -64.26 TCR gamma alternate reading frame protein TARP -62.39 Fc receptor-like A FCRLA -60.06 Potassium channel, subfamily K, member 5 KCNK5 -58.16 Purinergic receptor P2X, ligand-gated ion channel, 5 P2RX5 -58.16 Natural killer cell group 7 sequence NKG7 -57.26 CD69 molecule CD69 -54.53 CD7 molecule CD7 -52.57 Enzymes Interferon stimulated exonuclease gene 20kDa ISG20 -514.04 Prostaglandin E synthase PTGES -382.20 Cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial CMPK2 -266.50 Granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) GZMA -131.27 Granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1) GZMB -130.63 Hexokinase 3 (white cell) HK3 -127.10 Ubiquitin specific peptidase 18 USP18 -81.72 Phospholipase A1 member A PLA1A -72.65 Cathepsin E CTSE -72.13 GTP cyclohydrolase 1 GCH1 -66.29 Chymase 1, mast cell CMA1 -61.45 Phospholipid scramblase 1-like LOC611500 -61.09 Cytokines , chemokines , and their receptors Chemokine (C-X-C motif) ligand 12 CXCL12 -417.05 Interleukin 2 IL2 -195.40 Interleukin 7 receptor IL7R -66.71 Transforming growth factor, beta receptor III TGFBR3 -50.94 Soluble factors Lipocalin 2 LCN2 -217.74 Adrenomedullin ADM -100.37 Miscellaneous Radical S-adenosyl methionine domain containing 2 RSAD2 -559.34 Interferon-induced transmembrane protein 3-like LOC606890 -435.57 ISG15 ubiquitin-like modifier ISG15 -254.32 Apolipoprotein L, 5 APOL5 -200.23 Fatty acid binding protein 4, adipocyte FABP4 -182.17 Interferon-induced protein with tetratricopeptide repeats 1 IFIT1 -142.72 Interferon regulatory factor 4-like LOC609817 -118.68 Carcinoembryonic antigen-related cell adhesion molecule 25 CAECAM1 -113.27 Interferon regulatory factor 7 IRF7 -102.93 OCIA domain containing 2 OCIAD2 -100.84 Piwi-like RNA-mediated gene silencing 4 PIWIL4 -97.75 Synaptotagmin-like 3 SYTL3 -94.94 Testis expressed 14 TEX14 -92.58 Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) MX1 -91.32 Structural maintenance of chromosomes flexible hinge domain containing 1 SMCHD1 -84.97 Interferon-induced transmembrane protein 1-like LOC475935 -83.91 eukaryotic peptide chain release factor GTP-binding subunit ERF3B-like LOC480921 -74.06 Lactotransferrin LTF -72.08 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 DDX58 -69.54 TNFAIP3 interacting protein 3 TNIP3 -58.03 Syntrophin, beta 1 (dystrophin-associated protein A1, 59kDa, basic component 1) SNTB1 -55.46 Src kinase associated phosphoprotein 1 SKAP1 -50.58 10.1371/journal.pone.0183572.t005 Table 5 List and subgrouping of the top hits of highly differentially expressed genes (fold change ≥ 50 or ≤ -50) in M2- vs . M1-macrophages. Gene name Gene symbol Fold change Up-regulated genes Cell surface markers Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide FCER1A 376.89 CD209 molecule CD209 335.12 Lymphatic vessel endothelial hyaluronan receptor 1 LYVE1 321.11 C-type lectin domain family 4, member G CLEC4G 183.13 Collectin sub-family member 12 COLEC12 181.04 Junctional adhesion molecule 3 JAM3 145.24 Membrane-spanning 4-domains, subfamily A, member 2 MS4A2 137.95 Stabilin 1 STAB1 127.60 Mannose receptor, C type 1 MRC1 52.68 Solute carrier family 15 (oligopeptide transporter), member 1 SLC15A1 52.03 Enzymes Fructose-1,6-bisphosphatase 1 FBP1 149.73 Uronyl-2-sulfotransferase UST 85.20 Alanyl (membrane) aminopeptidase ANPEP 82.13 Lipoprotein lipase LPL 57.39 Cytokines , chemokines , and their receptors Chemokine (C-C motif) ligand 24 CCL24 1050.11 Transforming growth factor, beta 2 TGFB2 66.97 Soluble factors CD5 molecule-like CD5L 209.01 Secreted phosphoprotein 2, 24kDa SPP2 121.14 Nephronectin NPNT 105.07 Secretogranin V (7B2 protein) SCG5 82.69 Complement component 3 C3 67.78 Cystatin 9 (testatin) CST9 58.53 Miscellaneous Coagulation factor XIII, A1 polypeptide F13A1 1309.86 Sodium channel, voltage-gated, type II, beta subunit SCN2B 1120.45 Dynamin 1 DNM1 247.10 Caldesmon 1 CALD1 239.74 Rho GTPase activating protein 6 ARHGAP6 147.58 Fibronectin 1 FN1 141.81 Cfa-mir-125b-2 cfa-mir-125b-2 103.61 Plexin domain containing 2 PLXDC2 71.70 Transforming growth factor, beta-induced, 68kDa TGFBI 59.20 G protein-coupled receptor 116 GPR116 51.83 Down-regulated genes Cell surface markers Purinergic receptor P2Y, G-protein coupled, 14 P2RY14 -344.22 Solute carrier family 39 (zinc transporter), member 14 SLC39A14 -200.01 Integrin, beta 8 ITGB8 -134.58 Transmembrane protein 150C TMEM150C -103.02 Transmembrane protein 176A TMEM176A -78.17 Solute carrier family 22, member 15 SLC22A15 -58.69 Enzymes Prostaglandin E synthase PTGES -1274.66 Epoxide hydrolase 2, cytoplasmic EPHX2 -222.01 Interferon stimulated exonuclease gene 20kDa ISG20 -187.95 Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) PTGS2 -183.48 E3 ubiquitin-protein ligase NEURL3-like LOC102152163 -156.11 Indoleamine 2,3-dioxygenase 2 IDO2 -127.88 STEAP family member 4 STEAP4 -92.42 Ceruloplasmin (ferroxidase) CP -90.30 Granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1) GZMB -69.42 Phospholipase A2, group XVI-like LOC476045 -57.39 cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial CMPK2 -57.00 WNK lysine deficient protein kinase 2 WNK2 -52.30 Interleukin-1 receptor-associated kinase 3 IRAK3 -50.40 Cytokines , chemokines , and their receptors Chemokine (C-X-C motif) receptor 3 CXCR3 -140.97 Chemokine (C-C motif) ligand 20 CCL20 -119.64 Interleukin 6 (interferon, beta 2) IL6 -69.50 Tumor necrosis factor TNF -55.63 Soluble factors Complement component 2 C2 -78.67 Miscellaneous Peptidase inhibitor 3, skin-derived PI3 -235.52 TNFAIP3 interacting protein 3 TNIP3 -178.37 Ras homolog family member U RHOU -168.17 Radical S-adenosyl methionine domain containing 2 RSAD2 -82.43 Cochlin COCH -70.23 Interferon, alpha-inducible protein 6 IFI6 -68.46 multiple C2 domains, transmembrane 2 MCTP2 -66.09 Fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus) FSCN1 -58.64 Interferon regulatory factor 4-like LOC609817 -50.92 ISG15 ubiquitin-like modifier ISG15 -50.28 Interestingly, gene expression of CD209 markedly decreased in the pairwise comparison of M1 vs . M0 (FC = -345.12) whereas it was highly up-regulated in the comparison M2 vs . M1 (FC = 335.12). In both comparisons, M2 vs . M0 and M2 vs . M1, genes for LYVE1 , MRC1 , MS4A2 , JAM3 , and CLEC4G were up-regulated, whereas genes for ITGB8 and P2RY14 were down-regulated. SUCNR1 was up-regulated in both M1 vs . M0 and M2 vs . M0. Hierarchical cluster analysis Unsupervised hierarchical clustering analysis formed 9 different clusters based on similarities and differences in the expression profile of DEPs ( Fig 4 ). Two out of these 9 clusters had an expression profile that was visually clearly associated with either the M1- or M2-phenotype ( Fig 4 ). Functional annotation of these clusters identified that the M1-polarization cluster (cluster 8) was significantly associated with the biological process "respiratory burst involved in defense response" ( Fig 4 ). The M2-cluster (cluster 6) was significantly associated with multiple biological processes of mitosis such as "M phase of mitotic cell cycle" and "mitotic spindle organization" ( Fig 4 ). The genes associated with these M1- and M2-specific clusters are listed with their particular fold changes in S1 and S2 Tables. The remaining 7 clusters were neither specific for M1- nor for M2-macrophages and were associated with biological terms like "peptidyl-lysine mono- and dimethylation" (cluster 1), "immune response-activating signal transduction" (cluster 3), "monosaccharide metabolic process", "organic substance catabolic process", "cellular catabolic process" (cluster 4), "response to other organism", "regulation of lymphocyte proliferation" (cluster 5), "tRNA aminoacylation for protein translation" (cluster 7), "cytokinesis" and "antigen receptor-mediated signaling pathway" (cluster 9, S3 Table ). No significantly enriched biological terms were identified for cluster 2. 10.1371/journal.pone.0183572.g004 Fig 4 Hierarchical clustering analysis. Unsupervised hierarchical clustering analysis of the median centered, log 2 -transformed expression values of 6358 differentially expressed probe sets in canine polarized macrophages as obtained by TM4 MultiExperimentViewer with default settings (Euclidean distance; complete linkage), depicted on a color scale from red (relatively high expression) to green (relatively low expression). A) The analysis identified 9 distinct clusters (I-IX) based on similarities as well as differences in the expression intensity of canine polarized macrophages. Two out of these 9 clusters (VIII and VI) visually displayed an expression profile that was clearly associated with either the M1- or M2-phenotype. B) Functional annotation of the M1-cluster (magnified from A) using Web-based Gene Set Analysis Toolkit (WebGestalt) identified the enriched biological process category "respiratory burst" (adjusted p-value≤0.05). C) The M2-cluster (magnified from A) is associated with enriched biological GO terms such as "M phase of mitotic cell cycle" and "mitotic spindle organization" (adjusted p-value≤0.05). Literature-based gene signatures and marker genes Intersections of genes exclusively up-regulated in M1 vs . M0 and down regulated in M2 vs . M1 (M1-macrophage genes), as well as genes up-regulated in the comparisons M2 vs . M0 and M2 vs . M1 (M2-macrophage genes) were selected and compared to literature-based markers that are known to distinguish between human and murine M1- and M2-macrophages ( Fig 5A ). Notably, many markers identified in the recent study to be specific for canine M1-polarization (404 unique genes in total corresponding to 565 probesets, S5 Table ) did not match with the literature-based M1-markers (65 genes). Similarly, predominating numbers of canine M2-markers (700 unique genes in total corresponding to 1029 probesets, S5 Table ) did not match with the literature-based M2-markers (58 genes; Fig 5A ). However, overlapping expression of genes reflecting M1-polarization state was present for 8 genes, i . e . BIRC3 , CCR7 , CD80 , IL15RA , IL23A , INHBA , NAMPT , and SLC2A6 . For canine M2-polarization, 11 genes matched reported expression in human and murine M2-macrophages, i . e . CCL24 , CCL13 , FCER1A , FN1 , EGR2 , CA2 , LIPA , SLC4A7 , CD163 , ADK , and FGL2 ( Fig 5A ). Conflicting results were present for the genes P2RY14 , TGFBR2 , and TPST2 , which were expected as M2-markers based on the literature but were differentially up-regulated in canine M1-macrophages. Furthermore, PIK3CB was up-regulated by both canine M1- and M2-macrophages in the present study. The differentially expressed genes of the intersections, which are not namely mentioned in Fig 5A are listed in S4 Table . The top 50 candidate canine M1- and M2-macrophage genes, as defined above, are given in Tables 6 and 7 . The full lists of canine probesets, corresponding to the 404 unique M1 genes and 700 unique M2 genes are shown in S5 Table . 10.1371/journal.pone.0183572.g005 Fig 5 Comparative evaluation of canine M1- and M2-associated differentially expressed genes (M1 = 404; M2 = 700) with established literature-based human and murine orthologous genes (A) and detection of polarization-specific biomarkers employing supervised clustering with a K-nearest-neighbors algorithm and correlation-based feature selection using Prophet (B, C). A) The Venn diagram depicts the numbers and intersections of differential and common canine M1- and M2-genes with literature-based human and murine genes. The majority of literature-based M1- (65 genes = 57+8) and M2-markers (58 genes = 44+11+3) did not comply with the present microarray data upon canine macrophages. However, overlapping expression of 8 genes for the M1-phenotype and 11 genes for the M2-phenotype was identified. The genes of the intersections, not specifically mentioned in the figure, are listed in S4 Table B) Biomarker selection detected 4 highly specific probe sets for annotated by the mammalian carboxypeptidase inhibitor latexin ( LXN ) to be highly correlated to the M1-phenotype. The boxplots depict the significantly enhanced log 2 -transformed expression values for LXN in M1-macrophages as compared to both M0- and M2-macrophages (p = 0.002), as well as between M0- and M2-macrophages (p = 0.002). C) For the M2-phenotype, the most significant predicted biomarker was high affinity receptor for IgE, i . e . membrane-spanning 4-domains, subfamily A, member 2 ( MS4A2 ). The expression data of M2-macrophages show significantly higher log 2 -transformed expression values as compared to both M0- and M1-macrophages (p = 0.002) as well as between M0- and M1-macrophages (p = 0.002). Asterisks indicate significance (Kruskal-Wallis-Test with subsequent pairwise Mann-Whitney-U-Tests). 10.1371/journal.pone.0183572.t006 Table 6 Top 50 candidate M1-macrophage associated probesets, which were upregulated in M1- vs. M0-macrophages and simultaneously downregulated in M2- vs. M1-macrophages. Probe Set ID Gene Symbol Fold change M1 vs. M0 Fold change M2 vs. M1 Cfa.12190.1.A1_at PTGES 3.34 -1274.66 CfaAffx.30585.1.S1_s_at PTGES 4.99 -1136.51 CfaAffx.30585.1.S1_at PTGES 4.94 -517.22 CfaAffx.927.1.S1_at P2RY14 4.80 -344.22 Cfa.20456.1.S1_at IFI6 269.13 -284.48 CfaAffx.15155.1.S1_s_at PI3 27.22 -235.52 Cfa.574.1.A1_at EPHX2 119.72 -222.01 Cfa.18083.1.S1_s_at SLC39A14 15.89 -212.19 CfaAffx.14855.1.S1_s_at SLC39A14 13.48 -200.01 Cfa.3449.1.S1_s_at PTGS2 27.78 -183.48 Cfa.15554.1.A1_at TNIP3 3.07 -178.37 Cfa.9253.1.A1_at RHOU 446.23 -168.17 Cfa.12477.1.A1_at LOC102152163 86.20 -156.11 Cfa.16339.1.S1_at CXCR3 5.70 -140.97 Cfa.10779.1.A1_at IDO2 28.65 -127.88 CfaAffx.13394.1.S1_s_at EPHX2 110.24 -122.12 Cfa.15812.1.S1_at CCL20 67.90 -119.64 Cfa.1856.1.S1_at TMEM150C 40.36 -103.02 Cfa.2878.1.A1_s_at CP 425.52 -96.02 CfaAffx.3697.1.S1_at STEAP4 11.45 -92.42 CfaAffx.13209.1.S1_s_at CP 161.88 -90.30 Cfa.8846.1.A1_s_at C2 /// CFB 4.39 -78.67 CfaAffx.7919.1.S1_at TMEM176A 19.58 -78.17 CfaAffx.1718.1.S1_at TNF 47.17 -71.34 Cfa.3528.1.S1_s_at IL6 252.32 -69.50 Cfa.20456.1.S1_s_at IFI6 68.64 -68.46 CfaAffx.17110.1.S1_s_at MCTP2 2.69 -66.09 CfaAffx.15348.1.S1_at SLC22A15 15.67 -58.69 CfaAffx.24565.1.S1_at FSCN1 35.83 -58.64 Cfa.12164.1.A1_at LOC476045 5.65 -57.39 Cfa.54.1.S1_s_at TNF 47.20 -55.63 Cfa.11870.1.A1_at PI3 17.25 -52.33 Cfa.4359.1.S1_at WNK2 52.35 -52.30 CfaAffx.1510.1.S1_s_at IRAK3 10.70 -50.40 CfaAffx.15202.1.S1_s_at SDC4 58.61 -49.56 CfaAffx.1352.1.S1_s_at IL22RA2 116.00 -48.48 CfaAffx.26233.1.S1_s_at CXCR3 32.55 -47.23 CfaAffx.17136.1.S1_s_at MCTP2 6.25 -46.94 CfaAffx.24086.1.S1_at KMO 23.66 -46.48 CfaAffx.15086.1.S1_s_at BMP1 43.14 -43.31 CfaAffx.261.1.S1_at HCAR3 13.88 -42.20 Cfa.18962.1.S1_s_at SGK1 20.11 -39.12 Cfa.3719.1.S1_s_at QPCT 22.78 -38.99 Cfa.4926.1.A1_s_at LOC476045 5.41 -37.14 CfaAffx.6260.1.S1_at C18H7orf10 43.19 -36.13 Cfa.6458.1.A1_s_at C2 /// CFB 5.05 -36.01 Cfa.8282.1.A1_s_at KBTBD7 3.64 -35.38 CfaAffx.8143.1.S1_at KBTBD7 2.59 -35.14 Cfa.21252.1.S1_s_at ENPP2 188.74 -34.85 CfaAffx.16422.1.S1_s_at CCL20 33.43 -34.83 10.1371/journal.pone.0183572.t007 Table 7 Top 50 candidate M2-macrophage associated probesets, which were upregulated in M2- vs. M0-macrophages and simultaneously upregulated in M2- vs. M1-macrophages. Probe Set ID Gene Symbol Fold change M2 vs. M0 Fold change M2 vs. M1 Cfa.11125.1.A1_at SCN2B 1061.28 1120.45 Cfa.15823.1.S1_at CCL24 1060.82 1050.11 CfaAffx.18273.1.S1_at FCER1A 35.63 922.72 CfaAffx.20721.1.S1_s_at CCL24 819.06 819.06 Cfa.17541.1.S1_s_at FBP1 160.02 748.69 CfaAffx.2850.1.S1_s_at FBP1 57.95 422.21 Cfa.3663.1.A1_s_at FCER1A 21.07 376.89 Cfa.3707.1.A1_at FN1 12.68 360.42 CfaAffx.12229.1.S1_at LYVE1 322.32 321.11 Cfa.10966.1.A1_at DNM1 112.78 247.10 Cfa.6272.1.S1_at CALD1 224.66 239.74 CfaAffx.1504.1.S1_s_at UST 7.71 226.47 Cfa.2693.1.A1_at LOC102152647 194.28 194.28 Cfa.3707.1.A1_s_at FN1 7.26 188.32 CfaAffx.28024.1.S1_at CLEC4G 54.14 183.13 CfaAffx.16206.1.S1_at MS4A2 110.05 167.77 Cfa.6369.1.A1_at FBP1 26.98 149.73 Cfa.20346.1.S1_at ARHGAP6 37.99 147.58 Cfa.1705.1.A1_at JAM3 78.46 145.24 Cfa.3662.1.S1_at MS4A2 54.98 137.95 Cfa.19567.1.S1_at UST 8.05 120.81 CfaAffx.16206.1.S1_s_at MS4A2 76.56 108.61 Cfa.3774.1.A1_s_at ANPEP 25.55 105.73 Cfa.14465.1.S1_at NPNT 444.07 105.07 Cfa.688.1.S1_at cfa-mir-125b-2 58.78 103.61 CfaAffx.7700.1.S1_s_at MRC1 136.99 89.14 Cfa.2468.1.A1_at UST 6.54 85.20 Cfa.10794.1.A1_at SCG5 6.75 82.69 Cfa.20798.1.S1_at ANPEP 14.09 82.13 CfaAffx.14701.1.S1_s_at STAB1 4.00 81.41 CfaAffx.28463.1.S1_at C3 36.53 76.45 Cfa.12240.1.A1_at C3 44.14 67.78 CfaAffx.16904.1.S1_s_at TGFB2 61.84 66.97 CfaAffx.7365.1.S1_at CST9 58.21 58.53 CfaAffx.15901.1.S1_s_at LPL 52.03 57.39 CfaAffx.7698.1.S1_at MRC1 104.41 52.68 Cfa.78.1.S1_s_at SLC15A1 14.65 52.03 CfaAffx.27914.1.S1_s_at IL13RA2 170.59 46.70 Cfa.19638.1.S1_s_at TMLHE 51.05 45.55 CfaAffx.22229.1.S1_at GPR34 6.30 42.39 CfaAffx.21392.1.S1_s_at FKBP7 25.81 40.60 CfaAffx.19151.1.S1_s_at ADCY4 14.07 39.02 Cfa.11222.1.A1_s_at FAM185A 23.07 38.03 Cfa.10053.1.A1_at RAB40B 48.86 37.88 CfaAffx.24797.1.S1_at EPHX1 3.75 36.82 Cfa.1930.1.S1_at MRC1 25.70 36.76 Cfa.3367.1.A1_at TGFB2 36.49 36.58 Cfa.15824.1.S1_at CCL13 172.58 34.71 Cfa.14297.1.A1_s_at MAOB 33.75 33.75 Cfa.12621.1.A1_at LRP4 7.01 33.01 Biomarker selection In a hypothesis-driven approach, polarization specific prediction markers were detected with Prophet using the microarray data of unstimulated macrophages (M0), M1-, and M2-macrophages [ 41 , 42 ]. 369 probe sets were identified by the correlation-based feature selection algorithm of Prophet that discriminated between M0-, M1-, and M2-polarity, using the KNN algorithm with 100% correct predictions. Subsequent "one versus all" analyses using SET to create ranked lists of genes, based on their potential to serve as biomarker for M1- or M2-macrophages were performed ( S6 Table ; [ 43 ]). The highest scoring probe set differentiating M1- from M0- and M2-macrophages was CfaAffx.14358.1.S1_at, annotated as latexin ( LXN ), which was up-regulated in M1-macrophages, exhibiting a prediction-accuracy for the M1-phenotype of 100%. Additionally, the probe set ID's occupying the ranks 2 to 4 (Cfa.5195.1.A1_s_at, Cfa.5195.1.A1_at, Cfa.14007.1.A1_x_at) were similarly annotated to LXN ( S6 Table ). Analysis of the expression data of CfaAffx.14358.1.S1_at revealed a relatively high expression in all three polarities with a significantly higher expression in M1-macrophages as compared to M0- and M2-macrophages (pairwise Mann-Whitney-U-Tests; p = 0.02, Fig 5B ). For the comparison of M2 versus M0 and M1, the highest scoring probe set was Cfa.3662.1.S1_at, annotated as membrane-spanning 4-domains , subfamily A , member 2 ( MS4A2 , S6 Table ). This probe set was up-regulated in M2-macrophages and displayed a prediction accuracy of 100% for this phenotype. Statistical evaluation with pairwise Mann-Whitney-U-Tests underlined a low expression of MS4A2 in M0- and M1-macrophages, but a significantly higher expression in M2-macrophages (p = 0.02, Fig 5C ). Testing of antibodies for the detection of predicted biomarkers In order to test, whether the biomarkers predicted to distinguish between canine M0-, M1-, and M2-macrophages during the transcriptome investigations are also mirrored by altered protein expression, an additional experiment using blood from 3 dogs was performed. The cells were isolated and polarized as described and labeled with antibodies targeting MS4A2 and LXN ( Table 1 ). There was protein expression in canine macrophages in vitro for both molecules ( Fig 6 ). However, the percentage of cells labeled with an antibody against MS4A2 showed no differences between M0-, M1-, and M2-macrophages (p = 0.117; Fig 6 ). The percentage of positive cells for LXN was significantly higher in both M1-and M2-macrophages as compared to M0-macrophages (p = 0.02 and p = 0.01, respectively); however, there was no difference in the percentage of LXN-immunopositive cells between M1- and M2-macrophages (p = 0.06; Fig 6 ). 10.1371/journal.pone.0183572.g006 Fig 6 Protein expression of the predicted biomarkers latexin and MS4A2 in canine M0-, M1-, and M2-macrophages. A) Expression of latexin in non-stimulated canine M0-macrophages. B) Expression of latexin in canine M1-macrophages. C) Expression of latexin in canine M2-macrophages. D) Expression of MS4A2 in canine M0-macrophages. E) Expression of MS4A2 in canine M1-macrophages. F) Expression of MS4A2 in canine M2-macrophages. A-F) Scale bars = 100 μm. Nuclear counterstaining with bisbenzimide. G) Dot plots illustrating a significantly higher percentage of immunopositive cells in canine M1- and M2-macrophages as compared to M0-macrophages (One-factorial ANOVA with group-wise t-tests, astrerisk = p<0.05). H) Dot plot showing lack of statistical differences in the percentage of immunopositive cells for MS4A2 in canine M0-, M1-, and M2-macrophages. Discussion The current investigation is the first report upon the properties of canine polarized macrophages in vitro with a special emphasis on the establishment of unique distinctive gene signatures. Even though in vitro data on macrophage polarization doubtlessly cannot be simply extrapolated to in vivo situations and the paradigm of M1-/M2-polarization represents a simplified approach, which depicts only two extremes of macrophage heterogeneity [ 8 , 48 – 50 ], this initial reductionistic in vitro approach will provide a basis for future investigations on the role of macrophage polarization in both healthy and diseased dogs. Striking morphological differences were observed between canine M0-, M1-, and M2-macrophages, which are most probably attributed to direct effects of cytokine stimulation. Similar to observations in other species [ 51 – 54 ], M1-macrophage cultures appeared to dominantly adapt an amoeboid morphology, although a significant proportion of M0-macrophages also obtained such an amoeboid morphology. Notably, M2-cultures were rather heterogeneous including spindeloid cells and MNGs, which is in line with previous observations in human and murine bone marrow- and blood-derived M2-macrophages [ 51 – 54 ]. Interestingly, stimulation of macrophages with IL-4 and IL-13 together with colony stimulation factors can lead to the formation of MNGs [ 55 – 58 ]. The recent observation of MNG formation, exclusively in canine M2-cultures, is consistent with reports from other species [ 44 , 57 , 59 ]. A variety of different phenotypic markers has been proposed for the differentiation of human and murine M1- and M2-macrophages [ 4 , 31 ]. Even though a multitude of antibodies targeting immune cells including macrophages is known to cross-react with canine antigens [ 60 – 63 ], commercially available antibodies explicitly designed for the use on canine cells and tissues are frequently not available. The prototypical literature-based M1-markers CD16, CD32, and iNOS and the M2-markers CD163 and arginase-1 were demonstrated to be inappropriate for the immunophenotypic discrimination of M0-, M1-, and M2-polarization states in canine macrophages in the present study. This may in part be explained by interspecies differences [ 64 , 65 ]. For instance, following classical stimulation with pro-inflammatory cytokines, murine macrophages produce NO, whereas human macrophages nearly lack synthesis of NO in response to classically activating stimuli [ 66 , 67 ]. Moreover, arginase-1 has been reported as a prototype marker for murine M2-macrophages, which is however inappropriate for the detection of human M2-macrophages [ 13 , 44 , 68 , 69 ]. Based on the present observations, canine macrophages may thus share closer similarities with human than murine macrophages, thus underlining the role of dogs in translational research. Similar to the immunofluorescence data, and supporting the results of the immunophenotyping, expression values for all probesets annotated with the canine genes NOS2 (2 probesets) and ARG1 (3 probesets), which encode iNOS and arginase-1, respectively, also lacked differences in their expression values between M0-, M1-, and M2-macrophages (data available under accession number: E-MTAB-5458; http://www.ebi.ac.uk/arrayexpress ). MHC class II is sometimes regarded as a marker for classically activated M1 macrophages [ 70 ]; however, a substantial fundus of literature also indicates MHC class II as a pan-macrophage marker, which is expressed on both M1 and M2 macrophages in mice and humans without discriminating between both phenotypes [ 4 , 7 , 68 ]. Concordantly, the immunofluorescence data demonstrated that MHC class II was found to distinguish between canine M2- and M0-macrophages with a lower expression in the latter ( Fig 3 ). In fact, it is well known that IL-4 acts as a potent activator of macrophages and induces an up-regulation of MHC class II [ 23 , 71 – 75 ]. However, it must be critically considered that MHC class II appeared inappropriate for the differentiation of canine M0- and M1-macrophages on the protein level, which contradicts reported up-regulation of MHC class II on M1 macrophages [ 7 ]. Whether these differences are attributed to true species effects or whether the low number of investigated animals and the quantification method (immunofluorescence) are responsible for not reaching the level of significance remains to be determined. Conclusively, it is unlikely that a single antibody will be sufficient to specifically detect canine M1- and M2-macrophages in vivo . Due to the paucity of commercially available antibodies detecting canine antigens this will probably also involve the generation of antibodies targeting canine epitopes. The present study thus aimed to set up a transcriptomic basis that should encourage future attempts to establish a broader antibody panel against antigens, which are predicted to potentially represent discriminating markers for canine macrophage phenotypes. Interestingly, in the present study, CD206 was among the top regulated genes of canine M2 macrophages on the transcriptome level, and the results of the immunophenotyping also validated CD206 as a promising marker for canine M2 polarized macrophages. This implies that CD206 might constitute a conserved marker, which is appropriate for labeling M2-macrophages in various species including the dog [ 4 , 27 , 31 , 72 , 76 ]. However, a limitation of the present study is that the suitability of markers identified in the transcriptome analyses either still needs to be verified on the protein level or produced partially conflicting results in the immunofluorescence investigations ( i . e . CD163, LXN, and MS4A2). For M1-associated up-regulated genes, multiple metabolic pathways such as "steroid biosynthesis", and "glycolysis/gluconeogenesis" demonstrated to be enriched in the functional annotation of microarray data. Human and murine monocyte to macrophage differentiation has previously been shown to go along with profound changes in the lipid metabolism as a prerequisite for phagocytosis [ 77 ]. Moreover, in the hierarchical clustering analysis, the M1-specific cluster was functionally annotated to biological processes involved in "respiratory burst". M1 macrophages produce a variety of pro-inflammatory mediators including ROS, whereas in contrast, IL-4 inhibits the respiratory burst [ 78 ]. Enriched terms related to the peroxisome proliferator activated receptor (PPAR) signaling pathway were retrieved for the comparison of M2 vs . M1 up-regulated genes. PPARγ is a member of the nuclear receptor superfamily with potent anti-inflammatory properties and regulatory functions in fatty acid metabolism [ 79 – 82 ]. Interestingly, PPARγ agonists such as rosiglitazone induce an alternative M2a-activation state in murine macrophages and have been used as neuroprotective agents [ 83 , 84 ]. Pharmacological approaches, designed to enhance M2-dominated immune responses may thus similarly represent a promising tool in canine diseases. Interestingly, multiple biological processes related to an enhanced cell cycle were enriched in canine M2-macrophages as compared to both M0- and M1-macrophages. IL-4 has previously been reported to induce local macrophage proliferation in the context of chronic inflammation [ 59 ]. Moreover, human monocyte to macrophage differentiation in the presence of M-CSF is associated with a dramatic regulation of multiple cell-cycle genes [ 44 ]. The transcriptomic link to enhanced cell cycle and proliferation is probably also reflected by the higher cell number of M2-macrophages in the present study as compared to M0-macrophages ( S1 Fig ). Comparison of the canine transcriptome data with published murine and human prototype markers demonstrated a relatively low overlap. This confirms recent reports on marked interspecies differences between polarized macrophages [ 13 ] and suggests that some properties of polarized macrophages are unique to the dog, demonstrating that reported literature-based markers cannot simply be transferred to another species. Based on this observation we sought to predict novel transcriptomic markers using a hypothesis-driven approach. Using a correlation-based algorithm, the carboxypeptidase inhibitor latexin ( LXN ) [ 85 – 88 ] was retrieved for canine M1-macrophages. Though LXN -expression by murine macrophages upon pro-inflammatory stimulation has been demonstrated [ 85 ], LXN has so far not been proposed as a marker for M1-macrophages in the literature for any species. For the canine M2-phenotype, the high-affinity receptor for IgE membrane-spanning 4-domains , subfamily A , member 2 ( MS4A2 ) was predicted to represent the most powerful biomarker. Interestingly, other members of the molecule family, i . e . MS4A4A and MS4A6A , have been previously shown to be associated with M2-polarization [ 4 , 44 , 89 ]. In an attempt to validate LXN and MS4A2 as markers for canine macrophages, we demonstrated that MS4A2 showed no differences in the percentage of immunopositive cells between the three conditions. The percentage of immunopositive cells for LXN was higher in both M1- and M2-macrophages as compared to M0-macrophages. However, similar to MS4A2, LXN failed to distinguish between canine M1- and M2-macrophages. Thus, similar to CD163, the protein data did not accurately mirror the transcriptomic prediction. This could be attributed to conflicting differences between RNA- and protein level for these molecules. However, the low number of dogs tested and the fact that low level differences in protein expression may not be detected by immunofluorescence have certainly influenced the statistical power and sensitivity, and thus future validating experiments both in canine tissues and in vitro remain to be performed. Supporting information S1 Fig Dot plot diagrams depicting differences in the absolute number of cells per field (200x) in canine M0-, M1-, and M2-macrophages derived from 3 dogs on day 7 in culture. One-factorial ANOVA with group-wise t tests reveals a significantly higher number of cells in M2-polarized macrophages as compared to non-stimulated (M0)-macrophages (p≤0.05; asterisk). (TIF) Click here for additional data file. S1 Table List of the genes included in the M1-associated cluster of the hierarchical clustering analysis (refer to Fig 3 ). (DOCX) Click here for additional data file. S2 Table List of the genes included in the M2-associated cluster of the hierarchical clustering analysis (refer to Fig 3 ). (DOCX) Click here for additional data file. S3 Table Overview on retrieved enriched gene ontology biological processes and KEGG pathways of the clusters, resulting from the hierarchical clustering analysis (refer to Fig 3 ). (DOCX) Click here for additional data file. S4 Table List for the intersections of differentially expressed genes in the comparison of literature-based human and murine markers with canine M1- and M2-associated genes as retrieved by the present study (refer to Fig 4 ). Excel table. (XLSX) Click here for additional data file. S5 Table Lists of differentially expressed M1- and M2-macrophage associated probesets with fold change. Sheet 1 depicts all genes, which were upregulated in M1- vs. M0-macrophages and simultaneously downregulated in M2- vs M1-macrophages (i.e. canine M1-macrophage genes). Sheet 2 shows all genes, which were upregulated in M2- vs. M0-macrophages and simultaneously upregulated in M2- vs M1-macrophages (i.e. canine M2-macrophage genes). (XLSX) Click here for additional data file. S6 Table Selected biomarkers predicted to discriminate between canine M1- and M2- macrophages as retrieved and ranked by Prophet. (DOCX) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147598/

Bacillus cereus G9241 Makes Anthrax Toxin and Capsule like Highly Virulent B. anthracis Ames but Behaves like Attenuated Toxigenic Nonencapsulated B. anthracis Sterne in Rabbits and Mice ▿

Bacillus cereus G9241 was isolated from a welder with a pulmonary anthrax-like illness. The organism contains two megaplasmids, pBCXO1 and pBC218. These plasmids are analogous to the Bacillus anthracis Ames plasmids pXO1 and pXO2 that encode anthrax toxins and capsule, respectively. Here we evaluated the virulence of B. cereus G9241 as well as the contributions of pBCXO1 and pBC218 to virulence. B. cereus G9241 was avirulent in New Zealand rabbits after subcutaneous inoculation and attenuated 100-fold compared to the published 50% lethal dose (LD 50 ) values for B. anthracis Ames after aerosol inoculation. A/J and C57BL/6J mice were comparably susceptible to B. cereus G9241 by both subcutaneous and intranasal routes of infection. However, the LD 50 s for B. cereus G9241 in both mouse strains were markedly higher than those reported for B. anthracis Ames and more like those of the toxigenic but nonencapsulated B. anthracis Sterne. Furthermore, B. cereus G9241 spores could germinate and disseminate after intranasal inoculation into A/J mice, as indicated by the presence of vegetative cells in the spleen and blood of animals 48 h after infection. Lastly, B. cereus G9241 derivatives cured of one or both megaplasmids were highly attenuated in A/J mice. We conclude that the presence of the toxin- and capsule-encoding plasmids pBCXO1 and pBC218 in B. cereus G9241 alone is insufficient to render the strain as virulent as B. anthracis Ames. However, like B. anthracis , full virulence of B. cereus G9241 for mice requires the presence of both plasmids.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2542375/

Early indicators of exposure to biological threat agents using host gene profiles in peripheral blood mononuclear cells

Background Effective prophylaxis and treatment for infections caused by biological threat agents (BTA) rely upon early diagnosis and rapid initiation of therapy. Most methods for identifying pathogens in body fluids and tissues require that the pathogen proliferate to detectable and dangerous levels, thereby delaying diagnosis and treatment, especially during the prelatent stages when symptoms for most BTA are indistinguishable flu-like signs. Methods To detect exposures to the various pathogens more rapidly, especially during these early stages, we evaluated a suite of host responses to biological threat agents using global gene expression profiling on complementary DNA arrays. Results We found that certain gene expression patterns were unique to each pathogen and that other gene changes occurred in response to multiple agents, perhaps relating to the eventual course of illness. Nonhuman primates were exposed to some pathogens and the in vitro and in vivo findings were compared. We found major gene expression changes at the earliest times tested post exposure to aerosolized B. anthracis spores and 30 min post exposure to a bacterial toxin. Conclusion Host gene expression patterns have the potential to serve as diagnostic markers or predict the course of impending illness and may lead to new stage-appropriate therapeutic strategies to ameliorate the devastating effects of exposure to biothreat agents. Background Effective prophylaxis and treatment for infections caused by biological threat agents (BTA) rely upon early diagnosis and rapid initiation of therapy. Most methods for identifying pathogens in body fluids and tissues require that the pathogen proliferate to detectable and dangerous levels, thereby delaying diagnosis and treatment, especially during the prelatent stages when symptoms for most BTA are indistinguishable flu-like signs. Methods To detect exposures to the various pathogens more rapidly, especially during these early stages, we evaluated a suite of host responses to biological threat agents using global gene expression profiling on complementary DNA arrays. Results We found that certain gene expression patterns were unique to each pathogen and that other gene changes occurred in response to multiple agents, perhaps relating to the eventual course of illness. Nonhuman primates were exposed to some pathogens and the in vitro and in vivo findings were compared. We found major gene expression changes at the earliest times tested post exposure to aerosolized B. anthracis spores and 30 min post exposure to a bacterial toxin. Conclusion Host gene expression patterns have the potential to serve as diagnostic markers or predict the course of impending illness and may lead to new stage-appropriate therapeutic strategies to ameliorate the devastating effects of exposure to biothreat agents. Background Recent events have demonstrated that the capability to assess exposure and infection of individuals by biological threat agent (BTA) well in advance of onset of illness or at various stages post-exposure could offer important diagnostic and therapeutic benefits. Direct pathogen identification can be elusive since many pathogens sequester in tissues initially. Direct pathogen methods include the classical culture methods, immunoassay, and gene amplification of the pathogen. Although these methods are being improved for incredibly greater sensitivity [ 1 - 3 ], the efficiency of any diagnostic approach for direct pathogen assessment depends upon the presence of agent in a small specimen matrix. By the time detectable levels of pathogen are reached, it is frequently too late to halt the progression of the intractable illness [ 4 , 5 ]. Host responses that occur rapidly after exposure to specific pathogenic agents could provide the needed information for defense against the biothreat agents at time periods when clinical signs might include general malaise or flu-like symptoms that would not differentiate among diverse pathogenic agents (Additional file 1 ). Correlation of the course of the infection and the disease progression with molecular responses provides opportunities to understand pathogenesis resulting from biological agent exposure. Symptoms of exposure to biological toxins such as Vibrio cholerae toxin (CT), Clostridium botulinum toxin A (BoNT-A), and Staphylococcal enterotoxin B (SEB) include violent reactions within hours of exposure and may lead to death in a few days. In contrast, Brucella infection (B. melitensis 16 M) produces late-onset of mild symptoms that persist over a long period of time. Other bacterial pathogens, such as those causing anthrax ( B. anthracis ) or plague ( Yersinia pestis ), induce systemic, flu-like symptoms initially and progress to lethal shock and death days later. In the case of viral threat agents, Venezuelan equine encephalitis (VEE) initially causes serious illness, associated with severe malaise and an extended recovery time. Dengue (DEN-2) patients usually recover fully within days (< 1% mortality), but some individuals, especially those with prior dengue exposure, may develop dengue hemorrhagic fever (DHF). Therefore, the time when an individual is exposed, the incubation period, and the time of manifestation of the illness are crucial to designing diagnostic and therapeutic strategies. In the post-genomic period, it is no longer unrealistic to hope that the examination of host responses, by interrogating large numbers of genes, could reveal unique responses to the various BTA. However, there are a number of obstacles yet to be overcome. Various pathogens may affect different tissues or cells that may not be available for diagnostic purposes. For example, botulinum toxins target neuromuscular synapses and cholera toxin aims for intestinal epithelial cells. As a first step toward the goal, we focused on gene expression changes in human peripheral blood mononuclear cells (PBMC) since they could be readily obtained from an exposed individual, thus taking advantage of their "reconnaissance" role. We carried out in vitro exposure to each of 8 biological threat or infectious agents. We confirmed the in vitro results by using peripheral blood mononuclear cells (PBMC) from nonhuman primates (NHP) exposed to a bacterial pathogen ( Bacillus anthracis ) or, separately, to a toxin (SEB) at various time points post-exposure to compare findings in vitro to those seen in vivo . We have identified host gene expression patterns that can discriminate exposure to various BTA, even at early time periods when flu-like symptoms occur. Methods For the in vitro studies, the bacterial pathogens used were Bacillus anthracis , Yersinia pestis , Brucella melitensis ; toxins: SEB, CT, BoNT-A; viruses: VEE and DEN-2. We isolated RNA from human lymphoid cells after exposure and analyzed the gene expression patterns induced by these agents using cDNA arrays. We have not amplified our message for gene array analysis in order to avoid PCR-mediated bias. Bacterial strain and growth conditions Y. pestis Y. pestis KIM5 Pgm negative mutant bacteria (Laboratory stock) were grown on Brain Heart Infusion (BHI, Invitrogen, Rockville, MD) agar plates 30°C for 48 h. Pre-culture of Y. pestis bacteria were grown in BHI broth at 26°C overnight in shaker incubator at 180 rpm and were diluted 1/25 in fresh BHI medium. The organism was then grown at 26°C for 5 h (OD 600 ~0.5) and used for infection. To determine the MOI we counted serial dilutions of the bacterial culture by plating on (BHI) agar plates followed by incubation at 30°C for 48 h. B. anthracis Spores were prepared from B. anthracis Ames strain (pXO1+, pXO2+). Briefly, 5% sheep blood agar (SBA) plates were inoculated with B. anthracis Ames spores and incubated overnight at 35°C. Several isolated colonies were transferred to a sterile screw capped tube containing 5 ml of sterile PBS. NSM plates (New sporulation medium: per liter added Tryptone; 3 grams. Yeast extract; 3 grams. Agar; 2 grams. Lab Lemco agar; 23 grams. 1% MnCl 2 ·4H 2 O; 1 ml) (150 × 25 mm Petri plate) was inoculated with 200 μl of prepared cell suspension. These plates were incubated for 48 hrs at 35°C, then checked for sporulation progress by microscopic examination. Continued incubation at room temperature was performed until free refractive spores constituted 90–99% of total suspension. Spores were then harvested from plates using 5 ml of sterile water. Spores were then washed 4 times in sterile water. Spores were checked for purity by plating 10 μl in triplicate onto 5% SBA plates and incubating overnight @ 35°C. Enumerations of spores were calculated via CFU/ml (determination of viable spores) and also for actual spores/ml using Petroff Hauser chamber. B. melitensis B. melitensis 16 M strain was grown in shaker flasks overnight in Brucella broth at 37°C, plated on Brucella agar for 48 h at 37°C to obtain confluent growth. Bacteria were scraped from plates in pyrogen-free, sterile 0.9% NaCl for irrigation (saline), washed twice, concentration adjusted in saline to the appropriate OD 600 . Venezuelan Equine Encephalitis Virus (VEE) The Trinidad (Trd) strain used in these studies is a virulent virus of the epizootic IA/B variety of VEEV and was originally isolated from the brain of a donkey. Virus was diluted to an appropriate concentration in Hank's buffered saline solution containing 1% fetal bovine serum. Dengue 2 Virus DV-2 was grown and propagated in mycoplasma-free Vero cell lines. The viral titer was determined by limiting dilution plaque assays on Vero cells. All virus stocks and culture supernatants used in the present study were free from LPS and mycoplasma [ 6 ]. Isolation of cells from Human PBMC using elutriation methods Leukopheresis units were obtained from volunteer donors using the procedures outlined in our approved human use protocol, reviewed by the established Institutional Review Board at WRAIR. The written informed consent document was provided to the volunteers in advance of the procedure. We obtained PBMC (74 blood draws over a period of ~2.5 years, and collected from ~8–10 AM to minimize variability) from healthy human volunteers who had been screened to be HIV and Hepatitis B negative, were from 19–61 years of age and both male and female. Human monocytes and lymphocytes of peripheral blood mononuclear cells were purified from leukopacks of healthy donors by centrifugation over lymphocyte separation medium (Organaon Tecknika, NC). Monocytes and lymphocytes were then further purified by counter flow centrifugation-elutriation with pyrogen-free, Ca 2+ - and Mg 2+ -free phosphate-buffered saline as the eluant. The resulting monocytes and lymphocyte preparations had greater than 95% viability. Monocytes and lymphocytes were mixed in the ratio 1:4 and were used immediately. Cell cultures were maintained in RPMI media at 37°C. Exposure of monocytes and lymphocytes to the various pathogenic agents Lymphoid cells were then exposed to a pathogenic agent under conditions (dose, exposure time) deemed optimal for biological activity for each agent by the pathogen specialist. The multiplicity of infection (MOI) of the bacteria or viruses to lymphoid cells was as follows: anthrax spores (1 or 3), VEE (1 or 3), DEN-2, (0.2 or 1), Brucella (2), and plague (20). Following a 30-min infection period cells were washed once with Hanks Balance Salt Solution (HBSS, Invitrogen, Rockville, MD). Both uninfected and infected cells were maintained in RPMI Medium 1640 with 10% human AB serum at 37°C 5% CO2 for the different time periods post exposure. Cells were harvested and total RNA isolated using Trizol reagent (Invitrogen, Carlsbad, CA). The bacterial toxins, CT (3 nM), SEB (100 ng/mL), and BoNT-A (1 nM), were added to newly plated cells in flasks for the time period specified. Cells, incubated in the absence and presence of the toxins, were collected by centrifugation. Trizol™ (Invitrogen, Carlsbad, CA,) was added to the cells for RNA isolation and the cells were frozen at -70°C until use. RNA isolation and cDNA arrays RNA was isolated according to the Trizol method (Invitrogen, Carlsbad, California) followed with DNAse digestion [ 7 ]. The custom cDNA slides contained ~10,000 genes (Additional file 2 ). Stratagene reference RNA was labeled with Cy3 and used to compare with RNA (Cy5) from either control or exposed samples. RNA was labeled using Micromax-TSA labeling kits (Perkin Elmer, Boston, MA), hybridized and scanned in an Axon scanner. GenePix 3.0 (Axon) was used to analyze the scanned image. For studies using Human cDNA membranes (Clontech Laboratories, Palo Alto, California), RNA samples were labeled with radioactive 33 P. After washing, the blots were exposed to Kodak screen and scanned in a BIORAD multifluor scanner. Atlas Image software (BD Biosciences Clontech) was used for spot alignment and normalization of the scanned arrays. Statistical analyses and data scrutiny We have adhered to "MIAME" (minimum information about microarray experiments) for all our studies. For each pathogen, 3–6 successive time periods were studied and for each time period, data from 2–4 separate experiments were obtained and, using the data from these multiple experiments, 2-way ANOVA analysis were carried out. GeneSpring version 5.0 (Silicon Genetics, San Carlos, CA) and Partek Pro 5.0 (St. Charles, MO) were used to visualize and analyze the data. Welch's ANOVA (p < 0.05) was performed followed by Benjamini Correction [ 8 ] for various sets of data, to find genes that varied significantly across samples and to identify patterns of gene regulation in PBMC exposed to various pathogens. For custom microarrays, we used the scatter plot smoother, Lowess algorithm [ 9 ], to normalize for dye bias among samples. We filtered the array data at 2 steps. In the first step, data filtration allowed only elements for which intensities in both channels were above twice background intensity. In the second step, elements that had intensities below twice the background intensity in one channel only were set at twice background levels. Last, to identify patterns of gene expression among different pathogens, k -means and self-organizing map clustering analyses were performed. Complete linkage hierarchical clustering of an uncentered Pearson correlation similarity matrix was also applied using the Eisen Cluster software [ 10 ], and the results were visualized with the program TreeView. We have used the major dataset (data from Figure 1b ) as a training set to apply a class prediction method (GeneSpring 6.1) that uses the k-nearest neighborhood algorithm to classify blinded samples used as test sets. Figure 1 (a) B. anthracis exposure to PBMC from 3 different donors. Data shown are from exposures at 2, 4, 8 and 24 h. Data from each exposure time period were separately evaluated in order to identify common trends among the three donors (males, ages 61, 41, and 27 years old (respectively) with diverse ethnicity). (b). Comparisons of gene profiles for 8 pathogenic agents. Human PBMC were exposed to each of these pathogenic agents for at least 3 appropriate time periods. RNA was isolated, and the reverse transcript hybridized to cDNA arrays. Unique gene patterns were induced by BTAs. Cluster diagram of gene expression patterns use Gene Spring analysis to illustrate groups of genes that show discriminatory patterns for various threat agents. These genes were compared for their expression patterns across all agents and time points. Red is up regulated, blue is down regulated and black is no change compared to the control sample. The expression patterns illustrate how one can differentiate pathogenic agents by selection of sets of gene expression patterns for examination. (Gene accession ID numbers, rather than gene names, are all provided legibly in the graphs of Additional files). Feature selection, computation and classification Extracting discrete data: We applied the Greedy algorithm approach (46). For each of the 8 pathogens at each time point evaluated (29) (where a condition is the combination of a pathogen and a time interval), we compute for each gene a regulation type which is a nonempty subset of {U, D, S}. For a given condition and gene, the regulation type contains U (respectively D, S) if for at least one array for that condition, using either sum or median normalization techniques and either difference or ratio criteria, the gene appears to be up regulated (respectively down regulated, stay the same). Thus we model both variation between donors and experimental error. The regulation type {U, D} is treated as if it were {U, D, S}; i.e. we consider it to provide no information. Ordering the genes: For each value of n, we would like to select the n genes which best distinguish between the 29 conditions. Since this is an intractable task, we compute an approximation by ordering the genes according to a heuristic, and for each n, choose the first n genes from the list. There are two straightforward greedy approaches to generating such a list. In the grow approach we start with an empty list and at each step add the gene that gives the new list with the best discrimination power. In the shrink approach we generate the list in reverse order by starting with the full set of genes and removing the one that that leaves the remaining set with the best discrimination power. We estimate the discrimination power of a set of genes by computing an integer vector of length 812, containing an entry for each of the 29 × 28 ordered pairs of distinct conditions, which itself is a sum computed over all genes in the set. The values summed are the number of elements in the regulation type for the first condition of the pair that do not occur in the regulation type for the second condition. This vector is then sorted least element first. To compare the discrimination power of two gene sets, their vectors are computed and compared lexicographically, with larger vector considered to correspond to the gene set with better discrimination power. The first 50 genes to be ranked by this method are listed in the Additional file 3 . In vivo anthrax exposure Animal work was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition. Nine anthrax-naïve rhesus macaque NHPs were exposed to approximately 8 LD 50 of B. anthracis spores (Ames strain) by a head-only aerosol exposure system. Blood was drawn before exposure to determine baseline values. After exposure, 3 animals were euthanized at each 24, 48 and 72 h and blood taken for various analyses, including gene response patterns. A full necropsy was performed to collect biological samples for use in B. anthracis diagnostic assay development. Primer set design Primer sets were designed for selected genes for expression profile confirmation. Additional file 4 lists the accession numbers and sequences for both sense and antisense primers. Real-time PCR Total RNA from the all the pathogenic agent studies was reverse-transcribed simultaneously using the same master mix. The cDNA was then used to perform real-time PCR using BIORAD I cycler and the light cycler DNA master SYBR green I kit (Roche Diagnostics, Indianapolis, Indiana). The 18S gene was used as an endogenous control to normalize the HIF-1, GBP, and C5AR genes. Serial 10-fold dilutions of lymphoid cDNA were used to determine the PCR efficiency of each primer set. The slope value was applied to the formula E = 10 -1/m - 1 where m = slope value. The Ct (threshold cycle) values for all the genes were converted to fold change using the formula (1 + E) ΔCt , where E denotes the efficiency of the primer set for a gene. ΔCt denotes the difference between the Ct values of control and treated samples of a given gene. (Personal Communication, C. Baker, National Institutes of Health) Bacterial strain and growth conditions Y. pestis Y. pestis KIM5 Pgm negative mutant bacteria (Laboratory stock) were grown on Brain Heart Infusion (BHI, Invitrogen, Rockville, MD) agar plates 30°C for 48 h. Pre-culture of Y. pestis bacteria were grown in BHI broth at 26°C overnight in shaker incubator at 180 rpm and were diluted 1/25 in fresh BHI medium. The organism was then grown at 26°C for 5 h (OD 600 ~0.5) and used for infection. To determine the MOI we counted serial dilutions of the bacterial culture by plating on (BHI) agar plates followed by incubation at 30°C for 48 h. B. anthracis Spores were prepared from B. anthracis Ames strain (pXO1+, pXO2+). Briefly, 5% sheep blood agar (SBA) plates were inoculated with B. anthracis Ames spores and incubated overnight at 35°C. Several isolated colonies were transferred to a sterile screw capped tube containing 5 ml of sterile PBS. NSM plates (New sporulation medium: per liter added Tryptone; 3 grams. Yeast extract; 3 grams. Agar; 2 grams. Lab Lemco agar; 23 grams. 1% MnCl 2 ·4H 2 O; 1 ml) (150 × 25 mm Petri plate) was inoculated with 200 μl of prepared cell suspension. These plates were incubated for 48 hrs at 35°C, then checked for sporulation progress by microscopic examination. Continued incubation at room temperature was performed until free refractive spores constituted 90–99% of total suspension. Spores were then harvested from plates using 5 ml of sterile water. Spores were then washed 4 times in sterile water. Spores were checked for purity by plating 10 μl in triplicate onto 5% SBA plates and incubating overnight @ 35°C. Enumerations of spores were calculated via CFU/ml (determination of viable spores) and also for actual spores/ml using Petroff Hauser chamber. B. melitensis B. melitensis 16 M strain was grown in shaker flasks overnight in Brucella broth at 37°C, plated on Brucella agar for 48 h at 37°C to obtain confluent growth. Bacteria were scraped from plates in pyrogen-free, sterile 0.9% NaCl for irrigation (saline), washed twice, concentration adjusted in saline to the appropriate OD 600 . Venezuelan Equine Encephalitis Virus (VEE) The Trinidad (Trd) strain used in these studies is a virulent virus of the epizootic IA/B variety of VEEV and was originally isolated from the brain of a donkey. Virus was diluted to an appropriate concentration in Hank's buffered saline solution containing 1% fetal bovine serum. Dengue 2 Virus DV-2 was grown and propagated in mycoplasma-free Vero cell lines. The viral titer was determined by limiting dilution plaque assays on Vero cells. All virus stocks and culture supernatants used in the present study were free from LPS and mycoplasma [ 6 ]. Y. pestis Y. pestis KIM5 Pgm negative mutant bacteria (Laboratory stock) were grown on Brain Heart Infusion (BHI, Invitrogen, Rockville, MD) agar plates 30°C for 48 h. Pre-culture of Y. pestis bacteria were grown in BHI broth at 26°C overnight in shaker incubator at 180 rpm and were diluted 1/25 in fresh BHI medium. The organism was then grown at 26°C for 5 h (OD 600 ~0.5) and used for infection. To determine the MOI we counted serial dilutions of the bacterial culture by plating on (BHI) agar plates followed by incubation at 30°C for 48 h. B. anthracis Spores were prepared from B. anthracis Ames strain (pXO1+, pXO2+). Briefly, 5% sheep blood agar (SBA) plates were inoculated with B. anthracis Ames spores and incubated overnight at 35°C. Several isolated colonies were transferred to a sterile screw capped tube containing 5 ml of sterile PBS. NSM plates (New sporulation medium: per liter added Tryptone; 3 grams. Yeast extract; 3 grams. Agar; 2 grams. Lab Lemco agar; 23 grams. 1% MnCl 2 ·4H 2 O; 1 ml) (150 × 25 mm Petri plate) was inoculated with 200 μl of prepared cell suspension. These plates were incubated for 48 hrs at 35°C, then checked for sporulation progress by microscopic examination. Continued incubation at room temperature was performed until free refractive spores constituted 90–99% of total suspension. Spores were then harvested from plates using 5 ml of sterile water. Spores were then washed 4 times in sterile water. Spores were checked for purity by plating 10 μl in triplicate onto 5% SBA plates and incubating overnight @ 35°C. Enumerations of spores were calculated via CFU/ml (determination of viable spores) and also for actual spores/ml using Petroff Hauser chamber. B. melitensis B. melitensis 16 M strain was grown in shaker flasks overnight in Brucella broth at 37°C, plated on Brucella agar for 48 h at 37°C to obtain confluent growth. Bacteria were scraped from plates in pyrogen-free, sterile 0.9% NaCl for irrigation (saline), washed twice, concentration adjusted in saline to the appropriate OD 600 . Venezuelan Equine Encephalitis Virus (VEE) The Trinidad (Trd) strain used in these studies is a virulent virus of the epizootic IA/B variety of VEEV and was originally isolated from the brain of a donkey. Virus was diluted to an appropriate concentration in Hank's buffered saline solution containing 1% fetal bovine serum. Dengue 2 Virus DV-2 was grown and propagated in mycoplasma-free Vero cell lines. The viral titer was determined by limiting dilution plaque assays on Vero cells. All virus stocks and culture supernatants used in the present study were free from LPS and mycoplasma [ 6 ]. Isolation of cells from Human PBMC using elutriation methods Leukopheresis units were obtained from volunteer donors using the procedures outlined in our approved human use protocol, reviewed by the established Institutional Review Board at WRAIR. The written informed consent document was provided to the volunteers in advance of the procedure. We obtained PBMC (74 blood draws over a period of ~2.5 years, and collected from ~8–10 AM to minimize variability) from healthy human volunteers who had been screened to be HIV and Hepatitis B negative, were from 19–61 years of age and both male and female. Human monocytes and lymphocytes of peripheral blood mononuclear cells were purified from leukopacks of healthy donors by centrifugation over lymphocyte separation medium (Organaon Tecknika, NC). Monocytes and lymphocytes were then further purified by counter flow centrifugation-elutriation with pyrogen-free, Ca 2+ - and Mg 2+ -free phosphate-buffered saline as the eluant. The resulting monocytes and lymphocyte preparations had greater than 95% viability. Monocytes and lymphocytes were mixed in the ratio 1:4 and were used immediately. Cell cultures were maintained in RPMI media at 37°C. Exposure of monocytes and lymphocytes to the various pathogenic agents Lymphoid cells were then exposed to a pathogenic agent under conditions (dose, exposure time) deemed optimal for biological activity for each agent by the pathogen specialist. The multiplicity of infection (MOI) of the bacteria or viruses to lymphoid cells was as follows: anthrax spores (1 or 3), VEE (1 or 3), DEN-2, (0.2 or 1), Brucella (2), and plague (20). Following a 30-min infection period cells were washed once with Hanks Balance Salt Solution (HBSS, Invitrogen, Rockville, MD). Both uninfected and infected cells were maintained in RPMI Medium 1640 with 10% human AB serum at 37°C 5% CO2 for the different time periods post exposure. Cells were harvested and total RNA isolated using Trizol reagent (Invitrogen, Carlsbad, CA). The bacterial toxins, CT (3 nM), SEB (100 ng/mL), and BoNT-A (1 nM), were added to newly plated cells in flasks for the time period specified. Cells, incubated in the absence and presence of the toxins, were collected by centrifugation. Trizol™ (Invitrogen, Carlsbad, CA,) was added to the cells for RNA isolation and the cells were frozen at -70°C until use. RNA isolation and cDNA arrays RNA was isolated according to the Trizol method (Invitrogen, Carlsbad, California) followed with DNAse digestion [ 7 ]. The custom cDNA slides contained ~10,000 genes (Additional file 2 ). Stratagene reference RNA was labeled with Cy3 and used to compare with RNA (Cy5) from either control or exposed samples. RNA was labeled using Micromax-TSA labeling kits (Perkin Elmer, Boston, MA), hybridized and scanned in an Axon scanner. GenePix 3.0 (Axon) was used to analyze the scanned image. For studies using Human cDNA membranes (Clontech Laboratories, Palo Alto, California), RNA samples were labeled with radioactive 33 P. After washing, the blots were exposed to Kodak screen and scanned in a BIORAD multifluor scanner. Atlas Image software (BD Biosciences Clontech) was used for spot alignment and normalization of the scanned arrays. Statistical analyses and data scrutiny We have adhered to "MIAME" (minimum information about microarray experiments) for all our studies. For each pathogen, 3–6 successive time periods were studied and for each time period, data from 2–4 separate experiments were obtained and, using the data from these multiple experiments, 2-way ANOVA analysis were carried out. GeneSpring version 5.0 (Silicon Genetics, San Carlos, CA) and Partek Pro 5.0 (St. Charles, MO) were used to visualize and analyze the data. Welch's ANOVA (p < 0.05) was performed followed by Benjamini Correction [ 8 ] for various sets of data, to find genes that varied significantly across samples and to identify patterns of gene regulation in PBMC exposed to various pathogens. For custom microarrays, we used the scatter plot smoother, Lowess algorithm [ 9 ], to normalize for dye bias among samples. We filtered the array data at 2 steps. In the first step, data filtration allowed only elements for which intensities in both channels were above twice background intensity. In the second step, elements that had intensities below twice the background intensity in one channel only were set at twice background levels. Last, to identify patterns of gene expression among different pathogens, k -means and self-organizing map clustering analyses were performed. Complete linkage hierarchical clustering of an uncentered Pearson correlation similarity matrix was also applied using the Eisen Cluster software [ 10 ], and the results were visualized with the program TreeView. We have used the major dataset (data from Figure 1b ) as a training set to apply a class prediction method (GeneSpring 6.1) that uses the k-nearest neighborhood algorithm to classify blinded samples used as test sets. Figure 1 (a) B. anthracis exposure to PBMC from 3 different donors. Data shown are from exposures at 2, 4, 8 and 24 h. Data from each exposure time period were separately evaluated in order to identify common trends among the three donors (males, ages 61, 41, and 27 years old (respectively) with diverse ethnicity). (b). Comparisons of gene profiles for 8 pathogenic agents. Human PBMC were exposed to each of these pathogenic agents for at least 3 appropriate time periods. RNA was isolated, and the reverse transcript hybridized to cDNA arrays. Unique gene patterns were induced by BTAs. Cluster diagram of gene expression patterns use Gene Spring analysis to illustrate groups of genes that show discriminatory patterns for various threat agents. These genes were compared for their expression patterns across all agents and time points. Red is up regulated, blue is down regulated and black is no change compared to the control sample. The expression patterns illustrate how one can differentiate pathogenic agents by selection of sets of gene expression patterns for examination. (Gene accession ID numbers, rather than gene names, are all provided legibly in the graphs of Additional files). Feature selection, computation and classification Extracting discrete data: We applied the Greedy algorithm approach (46). For each of the 8 pathogens at each time point evaluated (29) (where a condition is the combination of a pathogen and a time interval), we compute for each gene a regulation type which is a nonempty subset of {U, D, S}. For a given condition and gene, the regulation type contains U (respectively D, S) if for at least one array for that condition, using either sum or median normalization techniques and either difference or ratio criteria, the gene appears to be up regulated (respectively down regulated, stay the same). Thus we model both variation between donors and experimental error. The regulation type {U, D} is treated as if it were {U, D, S}; i.e. we consider it to provide no information. Ordering the genes: For each value of n, we would like to select the n genes which best distinguish between the 29 conditions. Since this is an intractable task, we compute an approximation by ordering the genes according to a heuristic, and for each n, choose the first n genes from the list. There are two straightforward greedy approaches to generating such a list. In the grow approach we start with an empty list and at each step add the gene that gives the new list with the best discrimination power. In the shrink approach we generate the list in reverse order by starting with the full set of genes and removing the one that that leaves the remaining set with the best discrimination power. We estimate the discrimination power of a set of genes by computing an integer vector of length 812, containing an entry for each of the 29 × 28 ordered pairs of distinct conditions, which itself is a sum computed over all genes in the set. The values summed are the number of elements in the regulation type for the first condition of the pair that do not occur in the regulation type for the second condition. This vector is then sorted least element first. To compare the discrimination power of two gene sets, their vectors are computed and compared lexicographically, with larger vector considered to correspond to the gene set with better discrimination power. The first 50 genes to be ranked by this method are listed in the Additional file 3 . In vivo anthrax exposure Animal work was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition. Nine anthrax-naïve rhesus macaque NHPs were exposed to approximately 8 LD 50 of B. anthracis spores (Ames strain) by a head-only aerosol exposure system. Blood was drawn before exposure to determine baseline values. After exposure, 3 animals were euthanized at each 24, 48 and 72 h and blood taken for various analyses, including gene response patterns. A full necropsy was performed to collect biological samples for use in B. anthracis diagnostic assay development. Primer set design Primer sets were designed for selected genes for expression profile confirmation. Additional file 4 lists the accession numbers and sequences for both sense and antisense primers. Real-time PCR Total RNA from the all the pathogenic agent studies was reverse-transcribed simultaneously using the same master mix. The cDNA was then used to perform real-time PCR using BIORAD I cycler and the light cycler DNA master SYBR green I kit (Roche Diagnostics, Indianapolis, Indiana). The 18S gene was used as an endogenous control to normalize the HIF-1, GBP, and C5AR genes. Serial 10-fold dilutions of lymphoid cDNA were used to determine the PCR efficiency of each primer set. The slope value was applied to the formula E = 10 -1/m - 1 where m = slope value. The Ct (threshold cycle) values for all the genes were converted to fold change using the formula (1 + E) ΔCt , where E denotes the efficiency of the primer set for a gene. ΔCt denotes the difference between the Ct values of control and treated samples of a given gene. (Personal Communication, C. Baker, National Institutes of Health) Exposure of monocytes and lymphocytes to the various pathogenic agents Lymphoid cells were then exposed to a pathogenic agent under conditions (dose, exposure time) deemed optimal for biological activity for each agent by the pathogen specialist. The multiplicity of infection (MOI) of the bacteria or viruses to lymphoid cells was as follows: anthrax spores (1 or 3), VEE (1 or 3), DEN-2, (0.2 or 1), Brucella (2), and plague (20). Following a 30-min infection period cells were washed once with Hanks Balance Salt Solution (HBSS, Invitrogen, Rockville, MD). Both uninfected and infected cells were maintained in RPMI Medium 1640 with 10% human AB serum at 37°C 5% CO2 for the different time periods post exposure. Cells were harvested and total RNA isolated using Trizol reagent (Invitrogen, Carlsbad, CA). The bacterial toxins, CT (3 nM), SEB (100 ng/mL), and BoNT-A (1 nM), were added to newly plated cells in flasks for the time period specified. Cells, incubated in the absence and presence of the toxins, were collected by centrifugation. Trizol™ (Invitrogen, Carlsbad, CA,) was added to the cells for RNA isolation and the cells were frozen at -70°C until use. RNA isolation and cDNA arrays RNA was isolated according to the Trizol method (Invitrogen, Carlsbad, California) followed with DNAse digestion [ 7 ]. The custom cDNA slides contained ~10,000 genes (Additional file 2 ). Stratagene reference RNA was labeled with Cy3 and used to compare with RNA (Cy5) from either control or exposed samples. RNA was labeled using Micromax-TSA labeling kits (Perkin Elmer, Boston, MA), hybridized and scanned in an Axon scanner. GenePix 3.0 (Axon) was used to analyze the scanned image. For studies using Human cDNA membranes (Clontech Laboratories, Palo Alto, California), RNA samples were labeled with radioactive 33 P. After washing, the blots were exposed to Kodak screen and scanned in a BIORAD multifluor scanner. Atlas Image software (BD Biosciences Clontech) was used for spot alignment and normalization of the scanned arrays. Statistical analyses and data scrutiny We have adhered to "MIAME" (minimum information about microarray experiments) for all our studies. For each pathogen, 3–6 successive time periods were studied and for each time period, data from 2–4 separate experiments were obtained and, using the data from these multiple experiments, 2-way ANOVA analysis were carried out. GeneSpring version 5.0 (Silicon Genetics, San Carlos, CA) and Partek Pro 5.0 (St. Charles, MO) were used to visualize and analyze the data. Welch's ANOVA (p < 0.05) was performed followed by Benjamini Correction [ 8 ] for various sets of data, to find genes that varied significantly across samples and to identify patterns of gene regulation in PBMC exposed to various pathogens. For custom microarrays, we used the scatter plot smoother, Lowess algorithm [ 9 ], to normalize for dye bias among samples. We filtered the array data at 2 steps. In the first step, data filtration allowed only elements for which intensities in both channels were above twice background intensity. In the second step, elements that had intensities below twice the background intensity in one channel only were set at twice background levels. Last, to identify patterns of gene expression among different pathogens, k -means and self-organizing map clustering analyses were performed. Complete linkage hierarchical clustering of an uncentered Pearson correlation similarity matrix was also applied using the Eisen Cluster software [ 10 ], and the results were visualized with the program TreeView. We have used the major dataset (data from Figure 1b ) as a training set to apply a class prediction method (GeneSpring 6.1) that uses the k-nearest neighborhood algorithm to classify blinded samples used as test sets. Figure 1 (a) B. anthracis exposure to PBMC from 3 different donors. Data shown are from exposures at 2, 4, 8 and 24 h. Data from each exposure time period were separately evaluated in order to identify common trends among the three donors (males, ages 61, 41, and 27 years old (respectively) with diverse ethnicity). (b). Comparisons of gene profiles for 8 pathogenic agents. Human PBMC were exposed to each of these pathogenic agents for at least 3 appropriate time periods. RNA was isolated, and the reverse transcript hybridized to cDNA arrays. Unique gene patterns were induced by BTAs. Cluster diagram of gene expression patterns use Gene Spring analysis to illustrate groups of genes that show discriminatory patterns for various threat agents. These genes were compared for their expression patterns across all agents and time points. Red is up regulated, blue is down regulated and black is no change compared to the control sample. The expression patterns illustrate how one can differentiate pathogenic agents by selection of sets of gene expression patterns for examination. (Gene accession ID numbers, rather than gene names, are all provided legibly in the graphs of Additional files). Feature selection, computation and classification Extracting discrete data: We applied the Greedy algorithm approach (46). For each of the 8 pathogens at each time point evaluated (29) (where a condition is the combination of a pathogen and a time interval), we compute for each gene a regulation type which is a nonempty subset of {U, D, S}. For a given condition and gene, the regulation type contains U (respectively D, S) if for at least one array for that condition, using either sum or median normalization techniques and either difference or ratio criteria, the gene appears to be up regulated (respectively down regulated, stay the same). Thus we model both variation between donors and experimental error. The regulation type {U, D} is treated as if it were {U, D, S}; i.e. we consider it to provide no information. Ordering the genes: For each value of n, we would like to select the n genes which best distinguish between the 29 conditions. Since this is an intractable task, we compute an approximation by ordering the genes according to a heuristic, and for each n, choose the first n genes from the list. There are two straightforward greedy approaches to generating such a list. In the grow approach we start with an empty list and at each step add the gene that gives the new list with the best discrimination power. In the shrink approach we generate the list in reverse order by starting with the full set of genes and removing the one that that leaves the remaining set with the best discrimination power. We estimate the discrimination power of a set of genes by computing an integer vector of length 812, containing an entry for each of the 29 × 28 ordered pairs of distinct conditions, which itself is a sum computed over all genes in the set. The values summed are the number of elements in the regulation type for the first condition of the pair that do not occur in the regulation type for the second condition. This vector is then sorted least element first. To compare the discrimination power of two gene sets, their vectors are computed and compared lexicographically, with larger vector considered to correspond to the gene set with better discrimination power. The first 50 genes to be ranked by this method are listed in the Additional file 3 . In vivo anthrax exposure Animal work was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition. Nine anthrax-naïve rhesus macaque NHPs were exposed to approximately 8 LD 50 of B. anthracis spores (Ames strain) by a head-only aerosol exposure system. Blood was drawn before exposure to determine baseline values. After exposure, 3 animals were euthanized at each 24, 48 and 72 h and blood taken for various analyses, including gene response patterns. A full necropsy was performed to collect biological samples for use in B. anthracis diagnostic assay development. Primer set design Primer sets were designed for selected genes for expression profile confirmation. Additional file 4 lists the accession numbers and sequences for both sense and antisense primers. Real-time PCR Total RNA from the all the pathogenic agent studies was reverse-transcribed simultaneously using the same master mix. The cDNA was then used to perform real-time PCR using BIORAD I cycler and the light cycler DNA master SYBR green I kit (Roche Diagnostics, Indianapolis, Indiana). The 18S gene was used as an endogenous control to normalize the HIF-1, GBP, and C5AR genes. Serial 10-fold dilutions of lymphoid cDNA were used to determine the PCR efficiency of each primer set. The slope value was applied to the formula E = 10 -1/m - 1 where m = slope value. The Ct (threshold cycle) values for all the genes were converted to fold change using the formula (1 + E) ΔCt , where E denotes the efficiency of the primer set for a gene. ΔCt denotes the difference between the Ct values of control and treated samples of a given gene. (Personal Communication, C. Baker, National Institutes of Health) Results Host gene expression in vitro Microarray analysis was carried out at 3–6 time periods post exposure of PBMC to each pathogen or vehicle. Prior studies [ 11 ] showed specific gene sets related to sex, age and other parameters, therefore it was important to first identify genes that are normally variant among healthy humans. Data from only the control samples of these healthy donors were subjected to ANOVA (p =< 0.05) and 6% of the genes varied widely among the individuals who were healthy human donors. These genes that showed inconsistent expression profiles were excluded from further comparisons among the data sets from both control and exposed samples. This provided a baseline to confidently identify transcriptional responses induced by bacteria (anthrax, plague, Brucella ), toxins (CT, SEB, BoNTA), or viruses (Dengue, VEE). Expression ratios of 10,000 genes on the custom array (accession numbers of which are listed in Additional file 2 ) and genes (Additional file 5 ) in Human Atlas 1.2 were determined by comparing the levels of mRNA in control and pathogen-treated PBMC paired for each exposure time frame. Each measurement was carried out at least 3 times. Consistency of responses We used PBMC from at least 3 different donors, exposing cells to pathogen or vehicle for specified periods of time. Figure 1a is a cluster analysis of exposures to B. anthracis for 2, 4, 8 and 24 h exposures. The result from the 3 different donors (male, ages 61, 27, 41) is closely replicated among the donors. Unique gene patterns induced by BTAs The gene responses were dissected to identify sets of genes that will differentiate one agent from another based on the patterns of host gene induction. The GeneSpring (Silicon Genetics, California) clustering diagram illustrates gene expression patterns that can discriminate among the various pathogenic agents (Fig. 1b ) by identification of sets of genes where up regulation (red) and down regulation (blue) is seen for specific pathogens. The combination of these selected genes can be the foundation for designing specific diagnostic assays for exposure to one or more agents. For example, gene sets A and B (column labels at the bottom of the dendogram), differentiate B. anthracis from all other pathogenic agents except Dengue; gene sets F and G readily illustrate expression patterns that differentiate gene responses to these 2 pathogenic agent. Similarly, gene sets D and E and H show host responses to SEB that are distinguishing; BoNTA and VEE show similar patterns with gene sets A and B, but are readily separated by gene set C. Principal component analysis (PCA) (Fig. 2a ) illustrates clustering relationships that show marked differences in overall gene patterns among the 3 toxins used in this study. Additionally, gene patterns for the earliest exposure for SEB or CT clustered less closely with the later exposure times (Fig. 2b ), but when observed relative to all pathogens, the four exposure time periods for SEB were relatively closely clustered. A striking observation (Fig. 2b ) is that for all pathogens except SEB, the longest exposure times differ markedly from the clusters of the early time periods. For B. anthracis, Y. pestis, B. melitensis , and CT, those late exposure times cluster together for these various pathogens. This loss of pathogen-specific responses in vitro after lengthy exposure was not seen for the in vivo studies. Figure 2 PCA relational analysis to show how the gene profiles (various exposure times) cluster for each toxin (a) and the relationship among the various pathogens (b). Human PBMC were exposed to each of these pathogenic agents for at least 3 appropriate time periods. RNA was isolated, and the reverse transcript hybridized to cDNA arrays. Use of training and test data sets for classifying test exposures To determine whether the microarray data obtained in this study can be used to predict the exposure type of an uncharacterized sample or condition, we applied a supervised learning method for class prediction (GeneSpring) that uses the k-nearest neighbor algorithm. When algorithm was applied on the data set (training set) to predict the exposure type of a data set obtained from an exposure to Y. pestis (test set), we were able to correctly predict the type of exposure with a p < 0.02. We previously reported that a set of predictor genes was identified when samples from exposures of piglets to SEB were used as test sets [ 12 , 13 ]. Functional classification of genes differentially regulated Gene ontological analysis was carried out for the genes that were differentially expressed. Comparison of gene responses, based on functional similarities, not surprisingly, showed many up regulated genes coding for inflammatory mediators (Fig. 3 ). We clustered and sorted the differentially expressed genes by their functional classification. These functional classifications are depicted in Figure 3 . For gene group ( i ) "Growth Factor, Cytokines & Chemokines," anthrax, Brucella and SEB showed major up regulation of most genes coding for inflammatory mediators; the other 5 agents had mixed or modest effects. Similarly, categories ( iii ) "Interleukins and Interferon Receptors" and ( iv ) "Interleukins" showed up regulation by most pathogens, notable exceptions being the viruses. Down regulated genes, though seen extensively throughout the study, displayed functional clustering for each pathogenic agent such as ( ii ) "Homeostasis & detoxification," ( v ) "Ligand-gated ion channels," and so forth. Plague induced high levels of interleukin-6, macrophage inflammatory protein-1 beta, tumor necrosis factor-alpha (TNF-α), and granulocyte macrophage colony stimulating factor (GM-CSF) when compared with Brucella and anthrax. Not surprisingly, the superantigen SEB displayed kinetic patterns for over expression of interferon-γ, IL-2, IL-6, MIP-1α, and GM-CSF (Fig. 3 ). There are major differences in expression of death receptors, homeostasis, and caspases, examples of which include defensins and certain oxidases (homeostasis) that are down regulated by plague and SEB (Fig. 3 , bullet ii ). A large number of transcription factors are down regulated by anthrax, Brucella , and SEB, but plague consistently down regulated the widest range of these genes. Figure 3 Functional categories of genes that show similarities and differences between these pathogens. Accession numbers are shown associated with Figure 2, Additional files. Human PBMC were exposed to each of these pathogenic agents for at least 3 appropriate time periods. RNA was isolated, and the reverse transcript hybridized to cDNA arrays. Gene responses induced by BTAs in vivo ; comparison with in vitro changes To determine gene changes induced by BTAs in an animal model, NHP were exposed to B. anthracis spores by aerosol challenge. This model has been characterized previously to mimic inhalation anthrax in humans. Blood samples were collected 24 h, 48 h, and 72 h post exposure (by 72 h the NHP were beginning to show signs of the illness, which progresses very rapidly to lethality). The gene expression profiles for in vitro exposure of PBMC to anthrax spores were compared with those found in isolated PBMC at various time periods from NHP. Even by 24 h, a robust response was observed (Fig. 4a ), showing up regulation of genes coding for proteases; proteosome components c2, c3, c5; various cytokines; pro-apoptotic genes; cyclic adenosine monophosphate (cAMP)-related kinases, cAMP regulated transcription factors; and hypoxia inducible factor-1 (HIF-1). Down regulated genes included tyrosine kinases, cytokine receptors, growth factors, and adenosine diphosphate (ADP) ribosylation factors. Comparison of the in vivo results with the in vitro changes induced by anthrax (Fig. 4b ), showed remarkable similarities in gene patterns. Clearly many more changes were observed in vivo than in vitro . Certain surface antigens showed significant alteration that was unique to anthrax exposure. Diagrams were constructed to identify sets of genes that were up regulated (Fig. 5 ) at either 24 (blue) or 72 h (red); other gene sets showed up regulation at both time periods (center of graph, genes in both blue and red). Similarly, certain gene sets showed unique and common down-regulation patterns (far right, Fig. 5 ). Figure 4 Comparison of host gene responses in vivo and in vitro exposures to anthrax. Gene expression profiles in PBMC from healthy human donors exposed to anthrax spores in vitro were compared with gene expression patterns obtained in PBMC taken at 24, 48 and 72 hr after exposure of NHP to anthrax spores by aerosol challenge. (a) Gene cluster analysis of significantly altered genes in vivo. (b) comparison of gene expression profile between in vivo and in vitro exposures. Figure 5 Clustered sets of genes to illustrate stage-specific vs. commonly expressed genes for in vivo exposures of NHP at 24, 72 h or at both time periods. A few genes were selected that showed changes induced by B. anthracis exposure were confirmed by RT-PCR, and the level of expression was compared both in vitro and in vivo after anthrax exposure. The in vivo / in vitro trends were very similar for many genes including IL-6 (Fig. 6a ) and Transducin beta-1 subunit (GNB1) (Fig. 6b ). Altered regulation of that G-protein was not seen with the other pathogenic agents. In an experiment of SEB exposure to NHP (Fig. 7 ), IL-6 and guanylate binding protein GBP-2 were up regulated (6- and 65-fold, respectively) by 30 min post-exposure and the increased expression persisted through 24 h (the last time point tested, data not shown). Among all the pathogens studied so far, SEB was found to be the only pathogen to dramatically alter GBP-2. Figure 6 Confirmation of selected gene changes by RT-PCR with in vitro and in vivo samples for IL-6 (a) and Transducin beta-1 subunit, GNB1, (b). Figure 7 Expression of GBP-2 and IL-6 genes after in vivo SEB exposure of NHP for 30 min. Gene expression profiles in PBMC from NHP exposed to SEB for 30 min. RNA samples were isolated and used in the PCR assays using primers specific for IL-6 and GBP-2. Evaluating the gene selections In order to evaluate the quality of the set of genes selected by the two methods above, a set of 10 simulated samples was generated for each condition by randomly choosing a set of values consistent with the conditions regulation types. Sets of samples in which values had a 10%, 20%, 30%, 40% and 50% chance of being chosen at random were also generated. Each test sample was then classified using the chosen set of genes by scoring it against each of the 29 conditions. A condition scored 1 for every gene on which its regulation type was consistent with the sample. The test sample was considered to be classified correctly if the condition from which it was originally generated had the unique highest score. The results using the list of genes obtained by the "grow" (respectively "shrink") method are shown in Figure 8 . Figure 8 Ordered genes and resulting percentage correct classifications. For a given number of genes, a set of genes most able to discriminate between disease states is selected. Simulations of noisy readings of patient gene expression levels are performed for varying levels of noise. Each colored line plots the percentage of correct classifications versus the number of genes used to make the classification for one particular percentage of random values in the simulated readings. With no noise, very few genes are required to discriminate perfectly. With high noise levels (say, 50%), even 1000 genes cannot reliably discriminate well. Comparison of gene selection approaches: Because there is overlap between the gene types for some pairs of conditions, there must exist a sample consistent with some condition that cannot be classified correctly as that condition. Nevertheless for the generated data, both approaches obtained perfect classification at the 20% noise level for selections of between 150 and 775 genes. Even at the 30% noise level the grow method achieved perfect classification with 297–305 genes and the shrink method achieved perfect classification with 340–342 genes. Interestingly, in the added noise cases, gene selections above a certain size start to perform worse. This is because removing genes that play little role in distinguishing between conditions removes added noise without removing much discrimination power. We have used simulated random noise perturbing the input data samples, since we had no prior basis to assume any specific bias in noise (direction or subset of genes). If noise levels on a given sample were for some reason biased toward the identified patterns of some other disease for that subject, our technique will not perform as well as it does against random noise. Since at this point possible bias or coloration of the noise is unknown, we experimented with a range of noise levels, including levels well higher than the total noise experienced in modern gene expression platforms. Gene profiles to discriminate control from infected lymphoid cells To identify common gene profiles that existed among all these individual donors, we used GeneSpring to identify shared baseline expression levels of genes examined for 75 control samples. As these control datasets were subjected to various analyses, after excluding normally varying genes, we noticed genes that were expressed at low levels in the control samples but were significantly overexpressed in response to one or more pathogens. We selected genes that showed a dramatic change in expression level and could be used to discriminate among the pathogens. Gene profiles from two pathogens are shown with BoNT-A (Fig. 9a ) or B. melitensis (Fig. 9b ) in which the indicated genes readily differentiated it from the other 7 BTAs. Although some of these genes were slightly up regulated by one or more of the other pathogens, even those few genes illustrate the possibilities of distinguishing BoNT-A or B. melitensis from each of the other 7 pathogens. Figure 9 Expression patterns of genes that were at baseline levels in all controls and showed unique expression patterns in (a) BoNT-A exposures or (b) B. melitensis exposures compared to all 8 pathogens. Confirmation of gene changes by real-time PCR To further confirm the expression levels of a few selected genes, we performed semi-quantitative real-time PCR using the same RNA samples isolated from lymphoid cells that had been exposed to the various pathogenic agents/vehicle and used to carry out the microarray experiments. Gene expression for 3 selected genes are compared based on real-time PCR along with gene array results. Expression of GBP-2, which was significantly up regulated by exposure of lymphoid cells to SEB (~10-fold by this technique vs. 6-fold from the microarray analysis), was not altered by any other pathogenic agents studied. Also up regulation was observed for HIF-1 and C5AR by BoNTA and CT and there was good agreement with data from the microarray studies (Fig. 10 ). Figure 10 Real-time PCR determination of gene expression in response to each of 8 pathogenic agents. Primers were designed for these 3 genes and 18S, which was used as a reference gene for comparison of these 3 test genes. GBP-2 was a gene that was identified as being massively up regulated by SEB using differential display PCR (Mendis, et al) and was of particular interest to us. Host gene expression in vitro Microarray analysis was carried out at 3–6 time periods post exposure of PBMC to each pathogen or vehicle. Prior studies [ 11 ] showed specific gene sets related to sex, age and other parameters, therefore it was important to first identify genes that are normally variant among healthy humans. Data from only the control samples of these healthy donors were subjected to ANOVA (p =< 0.05) and 6% of the genes varied widely among the individuals who were healthy human donors. These genes that showed inconsistent expression profiles were excluded from further comparisons among the data sets from both control and exposed samples. This provided a baseline to confidently identify transcriptional responses induced by bacteria (anthrax, plague, Brucella ), toxins (CT, SEB, BoNTA), or viruses (Dengue, VEE). Expression ratios of 10,000 genes on the custom array (accession numbers of which are listed in Additional file 2 ) and genes (Additional file 5 ) in Human Atlas 1.2 were determined by comparing the levels of mRNA in control and pathogen-treated PBMC paired for each exposure time frame. Each measurement was carried out at least 3 times. Consistency of responses We used PBMC from at least 3 different donors, exposing cells to pathogen or vehicle for specified periods of time. Figure 1a is a cluster analysis of exposures to B. anthracis for 2, 4, 8 and 24 h exposures. The result from the 3 different donors (male, ages 61, 27, 41) is closely replicated among the donors. Unique gene patterns induced by BTAs The gene responses were dissected to identify sets of genes that will differentiate one agent from another based on the patterns of host gene induction. The GeneSpring (Silicon Genetics, California) clustering diagram illustrates gene expression patterns that can discriminate among the various pathogenic agents (Fig. 1b ) by identification of sets of genes where up regulation (red) and down regulation (blue) is seen for specific pathogens. The combination of these selected genes can be the foundation for designing specific diagnostic assays for exposure to one or more agents. For example, gene sets A and B (column labels at the bottom of the dendogram), differentiate B. anthracis from all other pathogenic agents except Dengue; gene sets F and G readily illustrate expression patterns that differentiate gene responses to these 2 pathogenic agent. Similarly, gene sets D and E and H show host responses to SEB that are distinguishing; BoNTA and VEE show similar patterns with gene sets A and B, but are readily separated by gene set C. Principal component analysis (PCA) (Fig. 2a ) illustrates clustering relationships that show marked differences in overall gene patterns among the 3 toxins used in this study. Additionally, gene patterns for the earliest exposure for SEB or CT clustered less closely with the later exposure times (Fig. 2b ), but when observed relative to all pathogens, the four exposure time periods for SEB were relatively closely clustered. A striking observation (Fig. 2b ) is that for all pathogens except SEB, the longest exposure times differ markedly from the clusters of the early time periods. For B. anthracis, Y. pestis, B. melitensis , and CT, those late exposure times cluster together for these various pathogens. This loss of pathogen-specific responses in vitro after lengthy exposure was not seen for the in vivo studies. Figure 2 PCA relational analysis to show how the gene profiles (various exposure times) cluster for each toxin (a) and the relationship among the various pathogens (b). Human PBMC were exposed to each of these pathogenic agents for at least 3 appropriate time periods. RNA was isolated, and the reverse transcript hybridized to cDNA arrays. Use of training and test data sets for classifying test exposures To determine whether the microarray data obtained in this study can be used to predict the exposure type of an uncharacterized sample or condition, we applied a supervised learning method for class prediction (GeneSpring) that uses the k-nearest neighbor algorithm. When algorithm was applied on the data set (training set) to predict the exposure type of a data set obtained from an exposure to Y. pestis (test set), we were able to correctly predict the type of exposure with a p < 0.02. We previously reported that a set of predictor genes was identified when samples from exposures of piglets to SEB were used as test sets [ 12 , 13 ]. Functional classification of genes differentially regulated Gene ontological analysis was carried out for the genes that were differentially expressed. Comparison of gene responses, based on functional similarities, not surprisingly, showed many up regulated genes coding for inflammatory mediators (Fig. 3 ). We clustered and sorted the differentially expressed genes by their functional classification. These functional classifications are depicted in Figure 3 . For gene group ( i ) "Growth Factor, Cytokines & Chemokines," anthrax, Brucella and SEB showed major up regulation of most genes coding for inflammatory mediators; the other 5 agents had mixed or modest effects. Similarly, categories ( iii ) "Interleukins and Interferon Receptors" and ( iv ) "Interleukins" showed up regulation by most pathogens, notable exceptions being the viruses. Down regulated genes, though seen extensively throughout the study, displayed functional clustering for each pathogenic agent such as ( ii ) "Homeostasis & detoxification," ( v ) "Ligand-gated ion channels," and so forth. Plague induced high levels of interleukin-6, macrophage inflammatory protein-1 beta, tumor necrosis factor-alpha (TNF-α), and granulocyte macrophage colony stimulating factor (GM-CSF) when compared with Brucella and anthrax. Not surprisingly, the superantigen SEB displayed kinetic patterns for over expression of interferon-γ, IL-2, IL-6, MIP-1α, and GM-CSF (Fig. 3 ). There are major differences in expression of death receptors, homeostasis, and caspases, examples of which include defensins and certain oxidases (homeostasis) that are down regulated by plague and SEB (Fig. 3 , bullet ii ). A large number of transcription factors are down regulated by anthrax, Brucella , and SEB, but plague consistently down regulated the widest range of these genes. Figure 3 Functional categories of genes that show similarities and differences between these pathogens. Accession numbers are shown associated with Figure 2, Additional files. Human PBMC were exposed to each of these pathogenic agents for at least 3 appropriate time periods. RNA was isolated, and the reverse transcript hybridized to cDNA arrays. Gene responses induced by BTAs in vivo ; comparison with in vitro changes To determine gene changes induced by BTAs in an animal model, NHP were exposed to B. anthracis spores by aerosol challenge. This model has been characterized previously to mimic inhalation anthrax in humans. Blood samples were collected 24 h, 48 h, and 72 h post exposure (by 72 h the NHP were beginning to show signs of the illness, which progresses very rapidly to lethality). The gene expression profiles for in vitro exposure of PBMC to anthrax spores were compared with those found in isolated PBMC at various time periods from NHP. Even by 24 h, a robust response was observed (Fig. 4a ), showing up regulation of genes coding for proteases; proteosome components c2, c3, c5; various cytokines; pro-apoptotic genes; cyclic adenosine monophosphate (cAMP)-related kinases, cAMP regulated transcription factors; and hypoxia inducible factor-1 (HIF-1). Down regulated genes included tyrosine kinases, cytokine receptors, growth factors, and adenosine diphosphate (ADP) ribosylation factors. Comparison of the in vivo results with the in vitro changes induced by anthrax (Fig. 4b ), showed remarkable similarities in gene patterns. Clearly many more changes were observed in vivo than in vitro . Certain surface antigens showed significant alteration that was unique to anthrax exposure. Diagrams were constructed to identify sets of genes that were up regulated (Fig. 5 ) at either 24 (blue) or 72 h (red); other gene sets showed up regulation at both time periods (center of graph, genes in both blue and red). Similarly, certain gene sets showed unique and common down-regulation patterns (far right, Fig. 5 ). Figure 4 Comparison of host gene responses in vivo and in vitro exposures to anthrax. Gene expression profiles in PBMC from healthy human donors exposed to anthrax spores in vitro were compared with gene expression patterns obtained in PBMC taken at 24, 48 and 72 hr after exposure of NHP to anthrax spores by aerosol challenge. (a) Gene cluster analysis of significantly altered genes in vivo. (b) comparison of gene expression profile between in vivo and in vitro exposures. Figure 5 Clustered sets of genes to illustrate stage-specific vs. commonly expressed genes for in vivo exposures of NHP at 24, 72 h or at both time periods. A few genes were selected that showed changes induced by B. anthracis exposure were confirmed by RT-PCR, and the level of expression was compared both in vitro and in vivo after anthrax exposure. The in vivo / in vitro trends were very similar for many genes including IL-6 (Fig. 6a ) and Transducin beta-1 subunit (GNB1) (Fig. 6b ). Altered regulation of that G-protein was not seen with the other pathogenic agents. In an experiment of SEB exposure to NHP (Fig. 7 ), IL-6 and guanylate binding protein GBP-2 were up regulated (6- and 65-fold, respectively) by 30 min post-exposure and the increased expression persisted through 24 h (the last time point tested, data not shown). Among all the pathogens studied so far, SEB was found to be the only pathogen to dramatically alter GBP-2. Figure 6 Confirmation of selected gene changes by RT-PCR with in vitro and in vivo samples for IL-6 (a) and Transducin beta-1 subunit, GNB1, (b). Figure 7 Expression of GBP-2 and IL-6 genes after in vivo SEB exposure of NHP for 30 min. Gene expression profiles in PBMC from NHP exposed to SEB for 30 min. RNA samples were isolated and used in the PCR assays using primers specific for IL-6 and GBP-2. Evaluating the gene selections In order to evaluate the quality of the set of genes selected by the two methods above, a set of 10 simulated samples was generated for each condition by randomly choosing a set of values consistent with the conditions regulation types. Sets of samples in which values had a 10%, 20%, 30%, 40% and 50% chance of being chosen at random were also generated. Each test sample was then classified using the chosen set of genes by scoring it against each of the 29 conditions. A condition scored 1 for every gene on which its regulation type was consistent with the sample. The test sample was considered to be classified correctly if the condition from which it was originally generated had the unique highest score. The results using the list of genes obtained by the "grow" (respectively "shrink") method are shown in Figure 8 . Figure 8 Ordered genes and resulting percentage correct classifications. For a given number of genes, a set of genes most able to discriminate between disease states is selected. Simulations of noisy readings of patient gene expression levels are performed for varying levels of noise. Each colored line plots the percentage of correct classifications versus the number of genes used to make the classification for one particular percentage of random values in the simulated readings. With no noise, very few genes are required to discriminate perfectly. With high noise levels (say, 50%), even 1000 genes cannot reliably discriminate well. Comparison of gene selection approaches: Because there is overlap between the gene types for some pairs of conditions, there must exist a sample consistent with some condition that cannot be classified correctly as that condition. Nevertheless for the generated data, both approaches obtained perfect classification at the 20% noise level for selections of between 150 and 775 genes. Even at the 30% noise level the grow method achieved perfect classification with 297–305 genes and the shrink method achieved perfect classification with 340–342 genes. Interestingly, in the added noise cases, gene selections above a certain size start to perform worse. This is because removing genes that play little role in distinguishing between conditions removes added noise without removing much discrimination power. We have used simulated random noise perturbing the input data samples, since we had no prior basis to assume any specific bias in noise (direction or subset of genes). If noise levels on a given sample were for some reason biased toward the identified patterns of some other disease for that subject, our technique will not perform as well as it does against random noise. Since at this point possible bias or coloration of the noise is unknown, we experimented with a range of noise levels, including levels well higher than the total noise experienced in modern gene expression platforms. Gene profiles to discriminate control from infected lymphoid cells To identify common gene profiles that existed among all these individual donors, we used GeneSpring to identify shared baseline expression levels of genes examined for 75 control samples. As these control datasets were subjected to various analyses, after excluding normally varying genes, we noticed genes that were expressed at low levels in the control samples but were significantly overexpressed in response to one or more pathogens. We selected genes that showed a dramatic change in expression level and could be used to discriminate among the pathogens. Gene profiles from two pathogens are shown with BoNT-A (Fig. 9a ) or B. melitensis (Fig. 9b ) in which the indicated genes readily differentiated it from the other 7 BTAs. Although some of these genes were slightly up regulated by one or more of the other pathogens, even those few genes illustrate the possibilities of distinguishing BoNT-A or B. melitensis from each of the other 7 pathogens. Figure 9 Expression patterns of genes that were at baseline levels in all controls and showed unique expression patterns in (a) BoNT-A exposures or (b) B. melitensis exposures compared to all 8 pathogens. Confirmation of gene changes by real-time PCR To further confirm the expression levels of a few selected genes, we performed semi-quantitative real-time PCR using the same RNA samples isolated from lymphoid cells that had been exposed to the various pathogenic agents/vehicle and used to carry out the microarray experiments. Gene expression for 3 selected genes are compared based on real-time PCR along with gene array results. Expression of GBP-2, which was significantly up regulated by exposure of lymphoid cells to SEB (~10-fold by this technique vs. 6-fold from the microarray analysis), was not altered by any other pathogenic agents studied. Also up regulation was observed for HIF-1 and C5AR by BoNTA and CT and there was good agreement with data from the microarray studies (Fig. 10 ). Figure 10 Real-time PCR determination of gene expression in response to each of 8 pathogenic agents. Primers were designed for these 3 genes and 18S, which was used as a reference gene for comparison of these 3 test genes. GBP-2 was a gene that was identified as being massively up regulated by SEB using differential display PCR (Mendis, et al) and was of particular interest to us. Discussion The objective of this study was to use host gene expression responses to aid in detection of exposure to biological threat agents, we aimed to a) discriminate among various pathogenic agents that start with similar flu-like symptoms [ 14 ] yet lead to severe illness by various routes (Additional file 1 ) select sets of genes that can convincingly be used to differentiate normal from exposed samples c) identify sets of genes that could be used for very early detection of exposure or estimating stage of illness post-exposure and d) continue to characterize similarities and differences in host responses for in vitro exposures to show some predictability for host gene profiles in NHP models that replicate the illness induced in humans. In another study, we found sets of genes from SEB exposures in vitro also were predictive of in vivo responses in a piglet model of SEB-induced lethal shock [ 12 ]. The approach depends on circulating lymphoid cell mRNA responses reflecting a historical record (by their mRNA responses) of encounters with pathogenic agents. Use of unique gene patterns for diagnosis has been shown for certain cancers [ 15 - 17 ]; another study showed that various pathogens induced unique gene responses in lymphocytes [ 18 ] and in dendritic cells [ 19 ]. To minimize variability as reported [ 11 ] in baseline gene expression among donors, in our study blood was collected at the same time of the day for all donors. Of the 8 pathogenic agents in this study, most have been characterized as having rapid effects on lymphoid cells [ 14 , 20 ]. For BoNT-A and VEE, the primary target tissues are inaccessible, although VEE has been shown to interact with lymphoid cells [ 21 ]. The gene expression data suggest that pathogen binding to specific receptors on the human PBMC initiates a series of events that contribute to the ultimate illness, producing host responses indicative of a particular pathogenic agent (Additional file 6 ) or showing common responses typical of a severe inflammatory reaction (Fig. 3 ). The kinetics of the course of the infection and the disease process is essential in the study of gene changes induced in the host by these pathogens (Additional file 6 ). Upon inhalation of Y. pestis by NHP, infected alveolar macrophages migrate to the liver and spleen [ 22 , 23 ] where they proliferate rapidly (~24 h). It is thought that LPS from the bacterial cell wall of Y. pestis (Gram-negative) induces circulatory collapse and widespread organ failure, leading to death within days [ 24 ]. SEB, CT, and LPS can induce rapid onset of illness (even less than 1 h), involving loss of regulation of vascular tone, vascular leakage and end-stage organ failure, in 1 to 3 days [ 14 , 25 , 26 ]. B. anthracis (Gram-positive) is transmitted as spores, which, upon inhalation, become engulfed by macrophages and transported to lymph nodes [ 20 , 27 - 31 ]. Upon production of a sufficient bacterial load, flu-like symptoms occur followed by sudden onset of respiratory distress, progressive shock, and death. As recent events have demonstrated, with cases of inhalation anthrax, treatments initiated after patients became seriously symptomatic can be marginally successful [ 27 ]. In this study, host gene expression responses in NHP to B. anthracis exposure were seen at the earliest time point examined, 24 h (Fig. 4b ). In contrast, in these same NHP, a sufficient pathogen load to be identified by culture techniques did not occur until day 3 and clinical diagnosis would not be possible until ~ day 4–5, much too late to initiate reliably effective treatments. Studies of pathogenesis show up regulated cytokine genes as a common response for many pathogens [ 14 , 32 ] and that would not, necessarily, distinguish among them (Fig 3 ). Therefore, we focused on host responses that may potentially identify stage-specific targets and can also serve as early diagnostic markers. The regulation of certain early genes is transient and may relate to factors that participate in recruitment of monocytes to the sites of infection. The genes that are expressed late relate to DNA damage-inducing proteins, hypoxia-inducible proteins, and proteases. Characterization of apoptosis as a result of exposure was reported for SEB, DEN-2 [ 33 ], plague [ 34 ], and anthrax [ 20 , 27 , 35 ] in numerous cell types, including those of lymphoid origin. We observed induction of apoptotic genes by these agents in our studies (Additional file 6 ). In contrast, Brucellae is known to inhibit apoptosis in their mononuclear phagocytic host cells [ 36 ] and we also observed pro-apoptotic genes to be down regulated by Brucella. In regard to the findings with plague exposure, it is important to note that these infections occurred under conditions that limit the ability of Yersinia outer proteins (YOP) to alter host cell physiology by down regulation of cytokines [ 34 ] and oxygen radicals [ 37 ] in calcium-free cultures of macrophages. We observed up regulation of certain cytokines after 1–2 hr of exposure to Y. pestis and a pattern of gene expression that can explain reduced synthesis of oxygen radicals. We suspect that different biochemical mechanisms contribute to pathogenesis when YOPs are produced [ 38 , 39 ]. In contrast to common infectious diseases, human cases of exposure to some of the biological threat agents are rare. Indeed, for certain of these pathogens, there is no appropriate animal model that replicates the illness as it appears in humans. Furthermore, dose effects and other variations suggest the need to investigate in vitro approaches that show some correlations to in vivo findings. For in vivo B. anthracis studies, PBMC from the spore-exposed animals was collected 24, 48, and 72 h. The in vitro study utilized PBMC (from healthy donors) exposed to spores for various time periods. We found many more gene expression changes in vivo than in vitro , perhaps because in vivo changes include both primary and secondary responses. Comparisons of in vitro / in vivo results showed similarities in genes that code for lymphoid receptors/signaling pathways that, when taken as a group, showed a pattern specific for B. anthracis and include a G-protein Transducin beta subunit (GNB1), cAMP related genes, Calmodulin regulated genes, cytokines and MAPKK (mitogen activated protein kinase kinase), some of which had previously been reported in response to anthrax exposures [ 40 ]. Not unexpectedly, genes coding for cytokines showed similarities in vitro vs in vivo (Fig 5 and 6 ). Our microarray data were in accordance with recent reports by Pickering et al. that showed up regulation of some of the cytokines in response to infection by B. anthracis spores including TNF-α, IL-8, IL-1β, GM-CSF, IFN-γ and IL-6 [ 41 ]. However, these genes, alone, would not necessarily distinguish anthrax from other pathogens. In general, the genes expressed by 4 h in vitro and 72 h in vivo were similar and correlated to the symptoms that appear after progression of inhalation anthrax in NHP. Aerosol challenge of SEB in NHPs up regulated (65-fold) the gene coding for interferon-regulated GBP-2 (Fig. 7 ) but in vitro (Figure 10 ) it was up-regulated 10-fold. In vivo , GBP-2 upregulation occurred by 15 min post exposure. Other pathogens showed minor or no effects on the expression of that particular G-protein (Figure 10 ). This may not be surprising in light of seminal studies showing that CT and pertussis toxin work through different guanine triphosphate (GTP)-binding proteins to regulate intracellular cAMP levels [ 42 , 43 ]. This study confirmed gene expression responses induced by CT, anthrax, and Brucella that are known participants in regulation of adenylyl cyclase as well as those relating to ADP ribosylation factor [ 44 - 46 ] (Additional file 6 ). We observed a down regulation of the host adenylyl cyclase but an up regulation of cAMP-related genes upon anthrax exposure in NHP samples (Figure 4a ). Because B. anthracis has its own adenylyl cyclase, it may be playing a role in affecting cAMP-related genes of the host [ 29 , 47 ]. Since most biothreat pathogen exposures start with flu-like symptoms, discriminating them from common pathogenic illnesses for early diagnosis at a treatable stage is one of the critical issue. Even though these 8 pathogens initially cause similar symptoms, such as malaise, fever, headache, and cough, unique sets of genes are induced by each and can be related to the course of illness [ 14 ] (Additional files 1 and 6 ). Using these signature gene profiles to assess possible exposure to pathogenic agents or to differentiate them from non-lethal illnesses, when the classical identification of a pathogen is not conclusive, has the potential to fill a gap in the arsenal of diagnostic tools. Conclusion Rapid detection, before the symptoms appear or even at various stages of illness offers the opportunity to initiate stage-specific therapeutic approaches to ameliorate the devastating results of these pathogenic agents. The use of host genomic markers offers an option to differentiate classes of pathogen exposure, gauge severity of impending illness and apply appropriate therapeutic strategies. 1 Genes were selected and their expression profiles compared with gene array and real-time PCR. 18S was used as a reference gene for comparison of these 3 test genes. 2 GBP-2 was a gene that was identified as being massively up regulated by SEB using differential display PCR (C. Mendis, et al). nd = not determined Competing interests The authors declare that they have no competing interests. Authors' contributions RD and RH drafted the manuscript, performed the genomic analysis, data mining and the apoptosis studies. GVL; BK; XH; CP; MJ; LS; NK; DH and LL carried out the exposures to the various pathogens. SE and PL participated in the statistical analysis of the microarray data. PR; AD; AM; CM; CC; AR; SP and RN participated in the microarray studies for the different pathogens. MJ conceived of the study, and participated in its design and coordination. GVL; DY and EH participated in the design and coordination on the study. All authors read and approved the final manuscript. Disclaimer Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 Comparison of time course of progression of illness for selected BTAs and pathogens: Graph courtesy of COL George Korch, USAMRIID, Ft. Detrick, Maryland. Hatched marks indicate onset of flu-like symptoms (fever, headache, chills) for each agent, X indicates time frame in which death usually occurs (case fatality rate, CFR), and the number of X s suggest the degree of lethality if untreated early in the course of illness. (i) The toxins (yellow bars) cause onset of acute illness within a few hours, but side effects can persist for many weeks. (ii) Bacterial BTAs (pink bars) follow different time course after infection ranging from days to weeks. Many bacterial infections begin with flu-like symptoms, but proceed to respiratory distress and lethal shock within ~ a week. In contrast, a prolonged illness is common in brucellosis, which is caused by Gram-negative bacteria ( B. melitensis. B. suis , and B. abortus ) that are highly infectious via aerosol route. (iii) Viruses (green bars) VEE and DEN infections each progress differently because VEE can proceed to the meninges of the brain, developing into encephalitis. In the case of dengue, the incubation period is 3 to 15 days, with the acute febrile illness lasting 3–5 days. The period of mortality is associated with cessation of the febrile illness or with secondary complications in DHF. Click here for file Additional file 2 A list of genes on the custom array. This table shows a list of the genes that are present on the microarrays used in this study. Click here for file Additional file 3 List of the first 50 genes that were selected using the Grow/Shrink method. This table lists the genes that passed the statistical analysis using the Grow/Shrink method. Click here for file Additional file 4 List of the accession numbers and sequences of the primer sets used in this study. Click here for file Additional file 5 Lists the genes of the Clontech 1.2 human array. Click here for file Additional file 6 Comparisons of gene profiles for 8 pathogenic agents. Cluster analysis of gene expression profiles of PBMC exposed to the 8 pathogens. Human PBMC were exposed to each of these pathogenic agents for appropriate time periods, and the results are sorted based on functional responses, rather than clustering of similar gene expression patterns. RNA was isolated, reverse transcribed and hybridized to cDNA arrays. Red bars indicate up regulation, green bars show down regulation of the genes, and selection of genes for significance is defined in methods. The numbered bullets (right margin of the figure) indicate families of genes showing similarities or unique properties. Gene accession numbers are shown to the left of the figure. Click here for file Acknowledgements This work was supported by the Defense Advanced Research Projects Agency (DARPA) and by the Defense Threat Reduction Agency, Project Number: G0020_04_WR_B.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9697310/

Loop-Mediated Isothermal Amplification (LAMP) for the Rapid and Sensitive Detection of Alternaria alternata (Fr.) Keissl in Apple Alternaria Blotch Disease with Aapg-1 Encoding the Endopolygalacturonase

Apple Alternaria blotch disease, caused by Alternaria alternata (Fr.) Keissl, is one of the most famous leaf diseases. When the disease is prevalent, it causes leaf abscission and influences the formation of flower buds and photosynthesis. Therefore, a simple, rapid, high-specificity and sensitivity method for monitoring infected leaves at early developmental stages is urgently needed, so that the occurrence and expansion of A. alternata can be controlled in time. In our research, a rapid, specific and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect A. alternata within 60 min. Six primers of LAMP detection can only specifically amplify the aapg-1 gene in A. alternata but not in four other important fungi in apples. The aapg-1 gene encodes endopolygalacturonase in A. alternata , and there are significant differences among different species. Thus, it was applied as the target for LAMP primers. Compared to conventional PCR detection, our LAMP method had the same sensitivity as that of detecting as little as 1 fg of pure genomic DNA of A. alternata . When leaves were inoculated with A. alternata conidia, LAMP detected 1 × 10 2 conidia/mL as the minimum concentration. However, the traditional tissue isolation and identification method only isolated A. alternata from leaves inoculated with 1 × 10 5 and 1 × 10 6 conidia/mL, indicating that the LAMP method was more sensitive than the traditional tissue isolation and identification method for A. alternata before symptoms. Further tests also indicated that LAMP detection was more accurate and sensitive than the traditional tissue isolation and identification method for A. alternata in leaves with the Alternaria blotch symptom collected from the field. Our results showed that the LAMP-targeting the aapg-1 gene has the advantages of high sensitivity, specificity and simplicity and can be used for rapid detection and early monitoring of A. alternata in the field. LAMP is instructive for us to effectively prevent and control apple Alternaria blotch disease. 1. Introduction Apples ( Malus domestica ) are widely consumed all over the world. As the largest apple cultivation area in the world, China's total output accounts for more than half of the world's total apple fruit production [ 1 ]. Apple Alternaria blotch disease, also known as brown streak disease, is caused by A. alternata [ 2 ]. It was first discovered in the United States [ 3 ] and is one of the most serious diseases in Asia [ 4 , 5 ]. Apple Alternaria blotch disease causes a loss of up to 50% of apple production [ 6 ]. Especially in recent years, the disease has spread rapidly in the main apple growing areas in north China and has become one of the three major leaf diseases in the main apple producing areas in China [ 7 ]. When the disease is prevalent, it will cause leaf abscission, resulting in worse growth of the tree, which influences the formation of flower buds and photosynthesis [ 8 ]. A. alternata can also infect fruits, causing fruit spots after removing the bag and influencing fruit quality [ 9 ]. There are significant differences in resistance to apple Alternaria blotch disease among different apple varieties [ 10 ]. As the most widely planted variety in China, the 'Fuji' apple is infected by Alternaria alternata and greatly threatened in terms of the quality and output of apples. Those influences pose significant threats to apple production in China. Thus, in order to stabilize the healthy development of apple cultivation, it is urgent to prevent the occurrence and spread of A. alternata . Apple Alternaria blotch disease is an airborne disease, and mainly infects the leaves, especially the 20-day-old leaves. Sometimes it also infects fruits and shoots. By producing AM toxin, A. alternata acts on apple leaves, affecting the normal growth of leaves and then making them susceptible to the disease [ 11 , 12 , 13 , 14 ]. Brown or black lesions with a diameter of 2~5 mm will appear after infection, which causes leaves to dry and fall over, seriously affecting yield [ 15 ]. The infection sources of A. alternata are very extensive, mainly including mycelia in deciduous leaves and dead branches. A. alternata produces spores in May of the following year and spreads with the wind and rain. With the growth of shoots, the disease peaks in July and August [ 16 , 17 ]. A. alternata can also infect the fruit after taking off the outer bags in October, which causes red spots on the peel and affects the quality of the fruit [ 9 ]. However, the prevention and control of apple Alternaria blotch disease is mainly based on chemical control [ 18 ]. Many kinds of chemical agents are used, but there is a lack of evidence and related studies to determine the accurate application time. The continuous use of fungicides will lead to the emergence of many resistant strains and increase the difficulty of disease prevention and control. In order to reduce the use of fungicide and improve the chemical control effect, it is necessary to monitor A. alternata before the symptoms appear. Several methods, including A. alternata isolation, hyphae and spore morphology scanning [ 19 ], and polymerase chain reaction (PCR) detection [ 20 ], are common and practical for the diagnosis of apple Alternaria blotch disease in the laboratory. However, these traditional identification methods are unsuitable for application in the field, as they require specialized technology and equipment, such as microscopes and PCR thermocycle instruments. Moreover, isolating and culturing fungi is time-consuming. Although PCR detection is shorter than traditional isolation methods, it requires expensive equipment, such as PCR instruments and gel imagers. Based on these shortcomings, loop-mediated isothermal amplification (LAMP) was established in our research for the detection of A. alternata . LAMP was first invented and applied in 2000, which is a rapid, specific and efficient method for the amplification of DNA sequences at a stable temperature [ 21 ]. LAMP detection is less sensitive to some inhibitors, such as metal ions and protease, than traditional PCR and has been applied for detection of some plant fungi, including Didymella bryoniae from cucurbit seeds [ 22 ] and Colletotrichum truncatum from soybeans [ 23 ]. Further development of LAMP involves the combination of this technology with other molecular methods, such as reverse transcription and multiplex amplification, for detection of infectious diseases caused by microorganisms in humans, livestock and plants [ 24 ]. LAMP detection applies a set of four to six primers and Bst DNA polymerase with strand displacement activity to amplify target DNA sequences with high specificity and no DNA denaturation stage at a stable temperature [ 25 ]. The product and by-product (magnesium pyrophosphate) of the LAMP reaction can be detected by visual assessment of turbidity or a color change with the addition of color-changing reagents, such as SYBR-Green I and HNB [ 26 ]. LAMP products can also be visualized on agarose gel as a banding pattern [ 27 ]. In general, without specialized technology and equipment, LAMP assays can amplify DNA with high specificity and efficiency. Moreover, testing with a water bath or heating block and the color change of products makes LAMP detection suitable in the field or in limited-resource settings [ 28 ]. In this research, we established a rapid, specific and efficient method for the detection of A. alternata in apple leaves based on the aapg-1 gene, which is unique to A. alternata and encodes endopolygalacturonase, playing an important role in plant cell wall degradation when fungi infect plants [ 29 ]. Finally, the early and rapid LAMP detection can be used to monitor A. alternata and control its development over time. 2. Results 2.1. LAMP Primers The LAMP primers were designed with the target sequence (250 bp) of the aapg-1 gene encoding endopolygalacturonase (AB047682.1) in A. alternate ( Figure 1 , Table 1 ). The target sequence was selected from a region of high homology by comparison with several sequences belonging to Alternaria . The primers exhibiting high specificity and sensitivity did not show similarities to any other fungi sequences. For LAMP primers, ΔG values of 3′ ends of F3/B3 primer, F2/B2 primer and LF/LB primer, and 5′ ends of F1c and B1c primer were determined, and the values were −7.36, −7.42, −4.51, −6.14, −4.90, −6.57, −6.59 and −4.71 Kcal/mole, respectively. All ΔG values were less than −4 Kcal/mol. 2.2. Specificity and Sensitivity of LAMP Detection The specificity of LAMP primers was tested with mycelial DNA of A. alternata and four important pathogenic fungi of apples, i.e., Botryosphaeria dothidea , Glomerella cingulata , Diplocarpon mali , and Trichothecium roseum . In order to verify the results of LAMP detection, we amplified A. alternata and the other four non-target pathogenic fungi with PCR primers ( Table 1 ). The results of LAMP detection can be visualized via color change from orange to green by adding SYBR Green I. The detection of A. alternata was positive in each repeat, and the color of the reaction solution changed markedly from orange to green, while the other fungi remained an orange color. The nuclease-free water templates showed no color change in any validation test ( Figure 2 A). Moreover, a 440 bp product in gel electrophoresis of the PCR amplification indicated the same results as the color change of LAMP detection ( Figure 2 B). Consequently, the newly established LAMP detection using six primers ( Table 1 ) showed high specificity in the detection of A. alternata . After it was determined that the primers were specific for A. alternata , the lowest detection limit was carried out using 10-fold serial dilutions of pure A. alternata mycelial DNA (1 ng to 1 ag). The lowest detection limit for A. alternata was 1 fg of pure A. alternata mycelial DNA as a template within 60 min, along with color change by adding SYBR Green I ( Figure 3 A) and diffuse type bands on gel electrophoresis ( Figure 3 B). As a comparison, conventional PCR detection using primers aapg-1-F/aapg-1-R ( Table 1 ) exhibited the same lowest detection limit as the LAMP assay ( Figure 3 C). 2.3. LAMP Detection of the Minimum Pathogenic Concentration of A. alternata Conidia in Apple Leaves Necrotic spot symptoms were observed after 3 days of incubation in the leaves incubated with 1 × 10 3 conidia/mL to 1 × 10 6 conidia/mL inoculum suspension. Negative controls incubated with tebuconazole [ 30 ] and nuclease-free water were still healthy ( Figure 4 A). Then, we succeeded in isolating A. alternata only from leaves incubated with 1 × 10 3 conidia/mL to 1 × 10 6 conidia/mL inoculum suspension by the traditional isolation method with a frequency of 60.9%, 62.8%, 69.5% and 75.3%. However, the LAMP detection of leaves incubated with inoculum suspension of A. alternata at a concentration of 1 × 10 2 conidia/mL to 1 × 10 6 conidia/mL and the positive control of A. alternata DNA were all positive with a frequency of 100% ( Figure 4 B). Those results suggested that LAMP detection was more accurate and showed higher sensitivity than the traditional isolation method. 2.4. LAMP, PCR and Traditional Isolation Method to Detect A. alternata in Leaf Samples Collected from the Field We collected 20 suspected apple Alternaria blotch samples from the field. LAMP, PCR and the traditional isolation method were applied to detect A. alternata in those leaves. As shown in Figure 5 A, 20 DNA templates numbered 2–21 from suspected apple Alternaria blotch samples with necrotic spot symptoms and the positive control DNA templates numbered 1 from mycelium of A. alternata were all detected with 440 bp products by PCR and displayed a green color in the LAMP assay. For the traditional tissue isolation and identification method, A. alternata was isolated from 16 suspected apple Alternaria blotch samples by observing colony and spore morphology. Colony photographs of some A. alternata strains identified by the traditional isolation method were shown in Figure 5 C. Those results showed that there is the same detection rate between LAMP and PCR detection for suspected apple Alternaria blotch samples, both of which are higher than the traditional isolation method. Furthermore, in order to carry out the early prevention and control of A. alternata before symptoms appear, we collected 20 healthy leaf samples from the field. LAMP, PCR and the traditional isolation method were applied to detect A. alternata in those healthy leaves. As shown in Figure 5 B, DNA templates from healthy leaves numbered 26, 27, 33, 36, 37 and 42, and the positive control DNA templates numbered 23 from A. alternata were all detected with 440 bp products by PCR and displayed green color by LAMP assay. For the traditional tissue isolation and identification method, A. alternata was isolated only from the leaves numbered 26 and 27. Although the leaves did not have any symptoms, Alternaria alternata was still detected, which also confirmed the presence of Alternaria alternata in the leaves. According to Koch's rule, we further cultured all strains of A. alternata isolated by the traditional isolation method and re-inoculated apple leaves. The results showed that all strains obtained by the traditional method successfully caused leaf spot symptoms like in Apple Alternaria blotch disease ( Figure 6 ). Those results showed that, compared with the traditional tissue isolation and identification method, our LAMP detection was more accurate and sensitive when used for early prevention and control of A. alternata in the field. 2.1. LAMP Primers The LAMP primers were designed with the target sequence (250 bp) of the aapg-1 gene encoding endopolygalacturonase (AB047682.1) in A. alternate ( Figure 1 , Table 1 ). The target sequence was selected from a region of high homology by comparison with several sequences belonging to Alternaria . The primers exhibiting high specificity and sensitivity did not show similarities to any other fungi sequences. For LAMP primers, ΔG values of 3′ ends of F3/B3 primer, F2/B2 primer and LF/LB primer, and 5′ ends of F1c and B1c primer were determined, and the values were −7.36, −7.42, −4.51, −6.14, −4.90, −6.57, −6.59 and −4.71 Kcal/mole, respectively. All ΔG values were less than −4 Kcal/mol. 2.2. Specificity and Sensitivity of LAMP Detection The specificity of LAMP primers was tested with mycelial DNA of A. alternata and four important pathogenic fungi of apples, i.e., Botryosphaeria dothidea , Glomerella cingulata , Diplocarpon mali , and Trichothecium roseum . In order to verify the results of LAMP detection, we amplified A. alternata and the other four non-target pathogenic fungi with PCR primers ( Table 1 ). The results of LAMP detection can be visualized via color change from orange to green by adding SYBR Green I. The detection of A. alternata was positive in each repeat, and the color of the reaction solution changed markedly from orange to green, while the other fungi remained an orange color. The nuclease-free water templates showed no color change in any validation test ( Figure 2 A). Moreover, a 440 bp product in gel electrophoresis of the PCR amplification indicated the same results as the color change of LAMP detection ( Figure 2 B). Consequently, the newly established LAMP detection using six primers ( Table 1 ) showed high specificity in the detection of A. alternata . After it was determined that the primers were specific for A. alternata , the lowest detection limit was carried out using 10-fold serial dilutions of pure A. alternata mycelial DNA (1 ng to 1 ag). The lowest detection limit for A. alternata was 1 fg of pure A. alternata mycelial DNA as a template within 60 min, along with color change by adding SYBR Green I ( Figure 3 A) and diffuse type bands on gel electrophoresis ( Figure 3 B). As a comparison, conventional PCR detection using primers aapg-1-F/aapg-1-R ( Table 1 ) exhibited the same lowest detection limit as the LAMP assay ( Figure 3 C). 2.3. LAMP Detection of the Minimum Pathogenic Concentration of A. alternata Conidia in Apple Leaves Necrotic spot symptoms were observed after 3 days of incubation in the leaves incubated with 1 × 10 3 conidia/mL to 1 × 10 6 conidia/mL inoculum suspension. Negative controls incubated with tebuconazole [ 30 ] and nuclease-free water were still healthy ( Figure 4 A). Then, we succeeded in isolating A. alternata only from leaves incubated with 1 × 10 3 conidia/mL to 1 × 10 6 conidia/mL inoculum suspension by the traditional isolation method with a frequency of 60.9%, 62.8%, 69.5% and 75.3%. However, the LAMP detection of leaves incubated with inoculum suspension of A. alternata at a concentration of 1 × 10 2 conidia/mL to 1 × 10 6 conidia/mL and the positive control of A. alternata DNA were all positive with a frequency of 100% ( Figure 4 B). Those results suggested that LAMP detection was more accurate and showed higher sensitivity than the traditional isolation method. 2.4. LAMP, PCR and Traditional Isolation Method to Detect A. alternata in Leaf Samples Collected from the Field We collected 20 suspected apple Alternaria blotch samples from the field. LAMP, PCR and the traditional isolation method were applied to detect A. alternata in those leaves. As shown in Figure 5 A, 20 DNA templates numbered 2–21 from suspected apple Alternaria blotch samples with necrotic spot symptoms and the positive control DNA templates numbered 1 from mycelium of A. alternata were all detected with 440 bp products by PCR and displayed a green color in the LAMP assay. For the traditional tissue isolation and identification method, A. alternata was isolated from 16 suspected apple Alternaria blotch samples by observing colony and spore morphology. Colony photographs of some A. alternata strains identified by the traditional isolation method were shown in Figure 5 C. Those results showed that there is the same detection rate between LAMP and PCR detection for suspected apple Alternaria blotch samples, both of which are higher than the traditional isolation method. Furthermore, in order to carry out the early prevention and control of A. alternata before symptoms appear, we collected 20 healthy leaf samples from the field. LAMP, PCR and the traditional isolation method were applied to detect A. alternata in those healthy leaves. As shown in Figure 5 B, DNA templates from healthy leaves numbered 26, 27, 33, 36, 37 and 42, and the positive control DNA templates numbered 23 from A. alternata were all detected with 440 bp products by PCR and displayed green color by LAMP assay. For the traditional tissue isolation and identification method, A. alternata was isolated only from the leaves numbered 26 and 27. Although the leaves did not have any symptoms, Alternaria alternata was still detected, which also confirmed the presence of Alternaria alternata in the leaves. According to Koch's rule, we further cultured all strains of A. alternata isolated by the traditional isolation method and re-inoculated apple leaves. The results showed that all strains obtained by the traditional method successfully caused leaf spot symptoms like in Apple Alternaria blotch disease ( Figure 6 ). Those results showed that, compared with the traditional tissue isolation and identification method, our LAMP detection was more accurate and sensitive when used for early prevention and control of A. alternata in the field. 3. Discussion Apple Alternaria blotch disease is one of the most important early deciduous diseases in apples, which can cause leaf shedding and influence flower bud formation [ 31 ]. It has become one of the main leaf diseases affecting apple production [ 32 ]. Apple Alternaria blotch disease and other early leaf litter diseases such as brown spot and anthrax leaf blight have similar early symptoms, so it is difficult to distinguish them by sight [ 31 ]. However, the operation of the traditional tissue separation and identification methods is complex and time-consuming. At the same time, due to the different control agents for these early deciduous diseases, the wrong diagnosis will lead to the failure of disease control [ 33 ]. Therefore, it is very important to establish a rapid, sensitive and simple detection method for the early detection of A. alternata and control of apple Alternaria blotch disease. Most LAMP detections use the ITS gene, which is highly conserved in different fungi, to design primers [ 34 , 35 ]. ITS genes have less intraspecific variability and might hinder the development of specific primers for different species. The aapg-1 gene encodes endopolygalacturonase, which plays an important role in the process of fungal infection in plants [ 36 ]. We designed six specific LAMP primers according to the conserved sequence of the aapg-1 gene and successfully performed LAMP detection for A. alternata . These results indicate that the aapg-1 gene is a highly-specific target gene suitable for its LAMP detection. LAMP detection can be used to specifically detect A. alternata from several important pathogens of apples ( Figure 2 ). Although the detection limit of 1 fg of A. alternata DNA of LAMP detection was consistent with that of conventional PCR, LMAP detection is simpler and does not require expensive and complex instruments for reaction and product detection [ 37 ]. In addition, the detection limit of LAMP in this study is close to that of Talaromyces favus [ 38 ]. This result is higher than in other reports, such as the LAMP assay that detected Soybean mosaic virus with the lowest limit of 10 −4 ng/μL [ 39 ]. Since LAMP has such high sensitivity to mycelial DNA, we further carried out detection of its minimum pathogenic concentration of spores. The results confirmed that the lowest concentration of spores detected by LAMP was 10 2 conidia/mL ( Figure 4 B). The lowest detected concentration of A. alternata spores in our study is higher than that of Colletotrichum gloeosporioides spores, which is 100 conidia/μL [ 40 ]. More importantly, we detected A. alternata by LAMP before symptom appearance, so as to monitor A. alternata and prevent the disease spread by fungicide in time. This inference is also consistent with the results of successfully detecting Soybean mosaic virus before the symptoms [ 39 ]. This conclusion was consistent with no disease symptoms on leaves sprayed with tebuconazole and A. alternata spores. Since the early symptoms of A. alternata are mild, it is difficult to determine the species of the spots by sight [ 31 ]. Moreover, the traditional tissue isolation and identification method is time-consuming and has low accuracy, which leads to the difficulty of early identification of A. alternata in the field. The detection rates of LAMP and PCR in this study were higher than those of traditional tissue isolation and identification methods ( Figure 5 ), which was consistent with the detection of pathogenic fungi in most reports, such as the LAMP assay that detected Didymella bryoniae in cucurbit seeds with higher accuracy than that of real-time PCR [ 22 ]. Sixty-one diseased soybean samples of Colletotrichum truncatum were successfully detected from 154 suspected samples using the LAMP assay, but only 29 samples were identified by traditional isolation and culture [ 23 ]. The detection rates for Phytophthora capsici by LAMP, PCR and the traditional isolation method were 55.4%, 57.8% and 25.3% [ 41 ]. The detection rate of Botryosphaeria dothidea by the LAMP method was 68%, while the rate of the traditional isolation method was only 24% [ 42 ]. Although LAMP detection possesses the above advantages, it is susceptible to atmospheric aerosol contamination and has the risk of false positions [ 43 ]. Therefore, this study adopted the method of non-opening detection; that is, SYBR Green I dye was added to the PCR tube cap before the reaction, and the color change was displayed by mixing SYBR Green I dye and the reaction product [ 44 ]. However, before LAMP detection, it is still necessary to strictly divide the districts of DNA extraction and LAMP detection. The sterilization of reaction utensils should be strictly sterilized to prevent contamination. Moreover, to avoid the influence of subjective observation, we placed the tubes on a black table or white paper so that the color change would be more obvious. In conclusion, LAMP targeting the aapg-1 gene has the advantages of high sensitivity, specificity and simplicity, which can be used for rapid detection and early monitoring of A. alternata in the field. 4. Materials and Methods 4.1. Fungal Isolates, Culture Conditions and DNA Extraction We separated A. alternata and four important apple pathogenic fungi, including Botryosphaeria dothidea , Glomerella cingulata , Diplocarpon mali and Trichothecium roseumisolates , from diseased apple samples in Yantai, China. Those fungi were identified using morphological and molecular methods by sequencing ITS sequences. Before all the experiments, all the fungi were transferred to PDA plates and were cultured for 5 d at 25 °C in darkness. Genomic DNA was extracted from each sample using a rapid fungal genomic DNA isolation kit (Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. The quality of the DNA was checked in agarose gels (1.2%) and the quantity was determined in a spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The results of quality and quantity detection of the genomic DNA are shown in Figure S1 . 4.2. LAMP Primers Design and Screen The aapg-1 gene is conserved in A. alternata , encodes endopolygalacturonase, and plays an important role in plant cell wall degradation during fungal infection. Therefore, the aapg-1 gene, as an important pathogenic factor, was chosen to be the specific target for the design of LAMP primers for the detection of A. alternata . The LAMP primers, comprising two outer (F3 and B3), two inner (FIP and BIP) primers and two loop primers (LF and LB), were designed using the Primer Explorer V5 software program ( http://primerexplorer.jp/lampv5e/index.html , accessed on 5 March 2021) based on the A. alternata aapg-1 sequence (AB047682.1). The selection of best primers was based on ΔG values of less than or equal to −4 Kcal/mol at the 3′ end of F3/B3, F2/B2, LF/LB and 5′ end of F1c and B1c, which were all synthesized by Sangon Biotech (Shanghai China) Co., Ltd. 4.3. LAMP and PCR Reaction Mixtures and Conditions LAMP detection was performed using the above primers shown in Figure 1 and Table 1 . Each reaction contained the target DNA sample 1 μL, 2 × LAMP PCR Master Mix 10 μL, 8 U/μL Bst DNA Polymerase 0.5 μL, 10 mM FIP/BIP 2 μL, 10 mM F3/B3 0.5 μL, 10 mM LF/LB 1 μL, adding ddH 2 O to 20 μL. Then 1 μL SYBR Green I was added to the cover of the tube. The reaction mixtures were incubated in a heated block at 65 °C for 60 min followed by incubation at 80 °C for 10 min to terminate the reactions. After the reaction, the results were examined via visual color changes of SYBR Green I (from orange to green) and confirmed by 1.2% agarose gel electrophoresis. PCR reactions were performed using the above primers shown in Table 1 . Each reaction contained the target DNA sample 1 μL, 8 U/μL r Taq DNA Polymerase 0.2 μL, 10 mM aapg-1-F/aapg-1-R 0.5 μL, 2.5 mM dNTPs 2 µL, 25 mM MgSO 4 0.2 µL, 10 × PCR buffer 2 μL, and water to make up the rest to 25 µL adding ddH 2 O to 25 μL. The PCR program was set as: 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s/cycle, 55 °C for 30 s, and 72 °C for 10 min. The reaction results were examined via 1.2% agarose gel electrophoresis. 4.4. Assay of Specificity and Sensitivity of LAMP and PCR Detection The specificity was determined by the LAMP and PCR reaction mixtures with the conditions mentioned above with DNA extracted from A. alternata and four common and important pathogenic fungi of apple. Genomic DNA of A. alternata (AB047682.1) was used to determine the sensitivity of the LAMP and PCR detection. The LAMP and PCR detection limits were defined by the smallest amount of DNA detected in each replicate. Ten-fold serial dilutions of genomic DNA ranging from 1 ng/μL to 1 ag/μL were used as templates for sensitivity detection. Dilution series were prepared with ddH 2 O. LAMP and PCR detections were performed using the same conditions mentioned above. In order to obtain consistent results, each LAMP and PCR reaction was repeated in triplicate. Negative controls contained nuclease-free water in place of genomic DNA. All reactions were performed three times. 4.5. Inoculation of Apple Leaves with A. alternata Conidia Conidia of A. alternata were collected from 7-day-old cultures on PDA medium containing Bengal red and suspended in sterile distilled water. The conidial suspensions were determined using a hemocytometer and adjusted to concentrations of 1 × 10, 1 × 10 2 , 1 × 10 3 , 1 × 10 4 , 1 × 10 5 and 1 × 10 6 conidial/mL. Inoculation of apple leaves with conidial suspensions refers to previously reported methods [ 10 , 15 , 45 ] with some modifications. Leaves, which were 20 to 25 days old, were rinsed with running water and the leaf surface was sterilized with 70% alcohol. Then, the leaves were rinsed with sterile water for 3 times and dried the surface water with sterile filter paper. The leaves were immersed in conidial suspensions of different concentrations for 5 min. Negative controls were ddH 2 O in place of conidial suspension. The petiole was covered with absorbent cotton soaked in water and the inoculated leaves were placed in a light incubator with a 12-h photoperiod and a daytime temperature of 28 °C and 25 °C at night (70–80% RH). Each replicate contained 20 leaves [ 46 ]. 4.6. Detection of A. alternata from Artificially Infested Leaves To assess the detection result by LAMP, the traditional isolation method and the PCR methods, infected and control leaves were both harvested at the same time after inoculation. At 3 days after inoculation, four pieces about 0.5 cm × 0.5 cm were taken from each leaf, immersed in 4% sodium hypochlorite for 4 min, 70% alcohol for 10 s, and rinsed three times in sterile water. Then, those pieces were transferred to PDA medium containing Bengal red and were incubated for 5 d at 25 °C in darkness for traditional isolation and morphological identification. For LAMP detection, four pieces of about 0.5 cm × 0.5 cm were taken from each leaf for DNA extraction with the method mentioned above. Purified DNA from the mycelium of A. alternata was used as a positive control, while DNA from non-inoculated leaves was used as a negative control. 4.7. Detection of A. alternata from Leaves Collected in Fields To further confirm the efficiency of LAMP detection for A. alternata from leaves, 20 naturally infected leaves with necrotic spot symptoms and 20 healthy leaves without necrotic spot symptoms were collected from the fields in Yantai, Shandong Province. Twelve pieces of each leaf were cut for testing; four pieces for LAMP detection, four pieces for PCR detection, and four pieces for traditionally isolated detection as described above. 4.8. Verification of A. alternata Isolated from Leaf Samples by Koch's Rule Healthy leaves that were 25 days old and of the same size were picked and washed with sterile water and quickly air dried. Wounds were made by needling the back of leaves with sterilized insect needles. A. alternata cultured for 5 days was inoculated and the side with hypha was attached to the wound. Five leaves were inoculated with each strain and a blank cake. The inoculated leaves were placed in a light incubator with a 12-h photoperiod at 28 °C during the daytime and 25 °C at night (70–80% RH). 4.1. Fungal Isolates, Culture Conditions and DNA Extraction We separated A. alternata and four important apple pathogenic fungi, including Botryosphaeria dothidea , Glomerella cingulata , Diplocarpon mali and Trichothecium roseumisolates , from diseased apple samples in Yantai, China. Those fungi were identified using morphological and molecular methods by sequencing ITS sequences. Before all the experiments, all the fungi were transferred to PDA plates and were cultured for 5 d at 25 °C in darkness. Genomic DNA was extracted from each sample using a rapid fungal genomic DNA isolation kit (Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. The quality of the DNA was checked in agarose gels (1.2%) and the quantity was determined in a spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The results of quality and quantity detection of the genomic DNA are shown in Figure S1 . 4.2. LAMP Primers Design and Screen The aapg-1 gene is conserved in A. alternata , encodes endopolygalacturonase, and plays an important role in plant cell wall degradation during fungal infection. Therefore, the aapg-1 gene, as an important pathogenic factor, was chosen to be the specific target for the design of LAMP primers for the detection of A. alternata . The LAMP primers, comprising two outer (F3 and B3), two inner (FIP and BIP) primers and two loop primers (LF and LB), were designed using the Primer Explorer V5 software program ( http://primerexplorer.jp/lampv5e/index.html , accessed on 5 March 2021) based on the A. alternata aapg-1 sequence (AB047682.1). The selection of best primers was based on ΔG values of less than or equal to −4 Kcal/mol at the 3′ end of F3/B3, F2/B2, LF/LB and 5′ end of F1c and B1c, which were all synthesized by Sangon Biotech (Shanghai China) Co., Ltd. 4.3. LAMP and PCR Reaction Mixtures and Conditions LAMP detection was performed using the above primers shown in Figure 1 and Table 1 . Each reaction contained the target DNA sample 1 μL, 2 × LAMP PCR Master Mix 10 μL, 8 U/μL Bst DNA Polymerase 0.5 μL, 10 mM FIP/BIP 2 μL, 10 mM F3/B3 0.5 μL, 10 mM LF/LB 1 μL, adding ddH 2 O to 20 μL. Then 1 μL SYBR Green I was added to the cover of the tube. The reaction mixtures were incubated in a heated block at 65 °C for 60 min followed by incubation at 80 °C for 10 min to terminate the reactions. After the reaction, the results were examined via visual color changes of SYBR Green I (from orange to green) and confirmed by 1.2% agarose gel electrophoresis. PCR reactions were performed using the above primers shown in Table 1 . Each reaction contained the target DNA sample 1 μL, 8 U/μL r Taq DNA Polymerase 0.2 μL, 10 mM aapg-1-F/aapg-1-R 0.5 μL, 2.5 mM dNTPs 2 µL, 25 mM MgSO 4 0.2 µL, 10 × PCR buffer 2 μL, and water to make up the rest to 25 µL adding ddH 2 O to 25 μL. The PCR program was set as: 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s/cycle, 55 °C for 30 s, and 72 °C for 10 min. The reaction results were examined via 1.2% agarose gel electrophoresis. 4.4. Assay of Specificity and Sensitivity of LAMP and PCR Detection The specificity was determined by the LAMP and PCR reaction mixtures with the conditions mentioned above with DNA extracted from A. alternata and four common and important pathogenic fungi of apple. Genomic DNA of A. alternata (AB047682.1) was used to determine the sensitivity of the LAMP and PCR detection. The LAMP and PCR detection limits were defined by the smallest amount of DNA detected in each replicate. Ten-fold serial dilutions of genomic DNA ranging from 1 ng/μL to 1 ag/μL were used as templates for sensitivity detection. Dilution series were prepared with ddH 2 O. LAMP and PCR detections were performed using the same conditions mentioned above. In order to obtain consistent results, each LAMP and PCR reaction was repeated in triplicate. Negative controls contained nuclease-free water in place of genomic DNA. All reactions were performed three times. 4.5. Inoculation of Apple Leaves with A. alternata Conidia Conidia of A. alternata were collected from 7-day-old cultures on PDA medium containing Bengal red and suspended in sterile distilled water. The conidial suspensions were determined using a hemocytometer and adjusted to concentrations of 1 × 10, 1 × 10 2 , 1 × 10 3 , 1 × 10 4 , 1 × 10 5 and 1 × 10 6 conidial/mL. Inoculation of apple leaves with conidial suspensions refers to previously reported methods [ 10 , 15 , 45 ] with some modifications. Leaves, which were 20 to 25 days old, were rinsed with running water and the leaf surface was sterilized with 70% alcohol. Then, the leaves were rinsed with sterile water for 3 times and dried the surface water with sterile filter paper. The leaves were immersed in conidial suspensions of different concentrations for 5 min. Negative controls were ddH 2 O in place of conidial suspension. The petiole was covered with absorbent cotton soaked in water and the inoculated leaves were placed in a light incubator with a 12-h photoperiod and a daytime temperature of 28 °C and 25 °C at night (70–80% RH). Each replicate contained 20 leaves [ 46 ]. 4.6. Detection of A. alternata from Artificially Infested Leaves To assess the detection result by LAMP, the traditional isolation method and the PCR methods, infected and control leaves were both harvested at the same time after inoculation. At 3 days after inoculation, four pieces about 0.5 cm × 0.5 cm were taken from each leaf, immersed in 4% sodium hypochlorite for 4 min, 70% alcohol for 10 s, and rinsed three times in sterile water. Then, those pieces were transferred to PDA medium containing Bengal red and were incubated for 5 d at 25 °C in darkness for traditional isolation and morphological identification. For LAMP detection, four pieces of about 0.5 cm × 0.5 cm were taken from each leaf for DNA extraction with the method mentioned above. Purified DNA from the mycelium of A. alternata was used as a positive control, while DNA from non-inoculated leaves was used as a negative control. 4.7. Detection of A. alternata from Leaves Collected in Fields To further confirm the efficiency of LAMP detection for A. alternata from leaves, 20 naturally infected leaves with necrotic spot symptoms and 20 healthy leaves without necrotic spot symptoms were collected from the fields in Yantai, Shandong Province. Twelve pieces of each leaf were cut for testing; four pieces for LAMP detection, four pieces for PCR detection, and four pieces for traditionally isolated detection as described above. 4.8. Verification of A. alternata Isolated from Leaf Samples by Koch's Rule Healthy leaves that were 25 days old and of the same size were picked and washed with sterile water and quickly air dried. Wounds were made by needling the back of leaves with sterilized insect needles. A. alternata cultured for 5 days was inoculated and the side with hypha was attached to the wound. Five leaves were inoculated with each strain and a blank cake. The inoculated leaves were placed in a light incubator with a 12-h photoperiod at 28 °C during the daytime and 25 °C at night (70–80% RH).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7120094/

Ebola in West Africa: Biosocial and Biomedical Reflections

The West Africa ebola epidemic, which killed more than 11,000 people, is fading from urgency and memory. Technical biomedical lessons-learned are being applied in drug and vaccine development, for example in response to Zika. But biosocial issues that arose may not have been directly confronted, or sometimes even noticed, despite their overall importance in the eventual control and ending of the epidemic. The Case This essay is about the scientific and medical response to ebola; and more particularly the actual and potential relationship between biotechnical and biosocial actions. It has been nearly entirely stripped of exposition; nevertheless, the ghost of the critical analysis is, I hope, alive in the narrative choices. This allows great compression of ideas and information within a narrative flow. So it can be read – I hope usefully – as a story. In it, single paragraphs – sometimes single sentences – could have been elaborated into their own chapter in a more technical presentation. In fact, they would seem to require this. Worse yet, a single paranthetic phrase "(very carefully)" represents an entire book that could be written on the production of pharmaceuticals in plants, and the necessary "humanizing" of protein structures such as immunoglobulins. The same compression applies to themes from Agassi 's work. Most of the relationships to his work are in the structure of choices for a general narrative and in the specific direction taken at the end of the paper. There are some direct clues, partly in the section titles. Let me use three examples to illustrate this. One of the skeleton key elements goes back to the original draft of The very idea of modern science which contained much more of Agassi's original PhD thesis from the 1950s than the final manuscript. In it, he displayed an amazing critical empathy for an obscure scientific literature written mainly in the language and references of Greek mythology. I don't think the critical analysis was possible without empathy being the key to unlock the door of their language, because one has to enter into their style of thinking that was embedded in their use of mythological themes. It was a stunning interpretive translation, fully recognizing the way in which the mode of thought was not simply an Aesopian code but influenced how they thought; one could say became integral to how they thought. So: cross-cultural thinking of a kind. Within a culture but cross-cultural to us, now. Agassi moved from one temporally bounded subculture to a different temporally bounded sub-culture; let's say modern philosophy and history of science. One of the themes of my paper is the notion of cross-cultural science – from a broader perspective but perhaps with the same approach. The easiest place to see a parallel at work is how I talk, all too briefly, about Amilcar Cabral . A second hint comes from a section title, in which I use a three word gloss to co-integrate some aspects of Paul Feyerabend's and Joseph Agassi's work: Anything critical goes . This is – among other things – both sloganeering and a reference to Popper and his popular critics . There is more to say about this in terms of Feyerabend and Agassi, but for this essay I was thinking of the immense distances between conceptions and lives of groups, and the possibility of critical evaluation or science as a meeting ground, outside the boundaries of either. Actually, the behavior of the international medical responders fit perfectly with the conceptual actions of witches: their masking in personal protective equipment, their obsession with blood and tissue samples, their restrictions on normal social intercourse and so on. It would be like someone in the USA taking their child to a hospital emergency room, only to be met by giant bats in lab coats. It is a kind of tribute to cross-cultural rationality that the entire rural population of all three countries did not, in fact, revolt. I use the term "convergence" in the paper, instead of discussing "cross-cultural consilience", The great ebola anthropologist Alain Epelboin , who worked mainly in Guinea (Conakry) during this epidemic, seems to maintain a sense of humor about the variety and diversity of human follies. He could probably describe poly-cultural consilience. I think Agassi actually discussed research programs before Imre Lakatos. I do not discuss this directly, but I do mention that the choice of GP (ebola glycoprotein) for target epitopes was common to most of the vaccine and immunomimetic drugs. At least in some of the drug cases, I thought I could see the determining influence of early rodent trials, even though rodents were not a great model for ebola as a human disease. It seemed to me that research programs could be determined by nearly random events, early in their life-cycles, which then became a fixed part of a program or a field-wide dogma. A third cryptic Agassi-related remark, is towards the end: "[…] a kind of Prague Spring of science facing a lethal crisis." This was a complex phenomenon that relates to Agassi's interest in the self-organization of science. It went beyond research to the institutional infrastructure of science and technology, including publications, funding agencies, pharmaceutical companies and governmental bureaucracies. Sometimes, as in the case of the World Bank, it seemed that lower level staff were making up for leadership deficiencies in planning, and actually acting. In other cases, such as Merck, it was the corporate leadership that turned ebola response into a company-wide effort. In may be surprising to many, but the FDA pushed all of its boundaries to facilitate rapid development and deployment of experimental therapeutics and diagnostics. My experience was that people individually responded with great acuity and often forced their agencies and bureaucracies to also respond and actually fulfill their missions. Here, I will just mention some of the changes that were made for communication and publication. At least some journals, such as The New England Journal of Medicine, made all their ebola-related articles open access. Some open access PLoS journals shifted fast publication to their associated (but refereed) blogs, but by Spring 2015 were able to publish new reports fairly quickly. In the meantime many researchers shared data without regard to priority of publication. The social scientists set up something like a temporary fast-publication journal, the Ebola Anthropology Platform in the UK, which played a major role in providing a place for well-researched reports that needed to be put out quickly. Some of these work-arounds became codified by the time the Zika epidemic hit, with a consortium of major journals specifically exempting priority of publication and confidentiality of data in order to encourage, not inhibit, rapid data sharing. Unfortunately, this did not apply to journals in Brazil, apparently; which was the country hardest hit by Zika in 2016. Then a new phenomenon became serious enough to be widely recognized: the rise of the false journal and predatory publishers. The problem of access to publication, which Agassi discussed, became a new version of the problem of demarcation instead. Perhaps this is enough of an indication of the work that lies behind this somewhat unusual structure for a paper. The references can provide access to many of the topics that fly by. Finally, I should say that this was the only way I found I could write about this topic without being lit on fire with fury. Ebola That's Enough! Let's begin at the end. On December 23, 2016, The Lancet published on-line the final report of the "Ebola Ça Suffit!" ring-vaccination trial which took place mainly in Guinea and partly in Sierra Leone (Henao-Restrepo et al. 2016 ). It showed the successful deployment of a vaccine against ebola and confirmed an interim assessment published in September, 2015 (Henao-Restrepo et al. 2015 ). The trial took place late on the downward side of the ebola epicurve. Later, I will discuss issues from the other side of the epicurve, when cases kept doubling faster and faster and case numbers appeared to be going asymptotic to a vertical: the circumstances from late 2013 to the end of 2014. As an investigation and intervention Ebola Ça Suffit! got many things right, under still difficult conditions. Ebola Ça Suffit! got trial design right, for the circumstances. A major issue was designing a trial that would be able to capture actual ebola cases. By the time major resources were made available, in 2015, the ebola epidemic was ending and cases were vanishing. Social scientists I knew working on a proposed multi-vaccine RCT (randomized controlled trial) for Liberia in early 2015 kept hearing the numbers changed: 5000 then 50,000 then 500,000 prospective participants in what was supposed to be a phase II trial and then "a phase II/III trial" trying to capture enough probable cases to have a contrast. There often seems to be a sub-discipline that has more power and authority in a crisis. During the ebola crisis, mathematical epidemiological modelers seemed to have such a position. They played an ambiguous role. The models encouraged a massive response in the fall of 2014 by their projected unthinkable caseloads, if nothing were done. They also used a random population-exposure assumption that was wrong. A natural question would be: why weren't RCT vaccine trials (placebo controlled, double-blind, randomly-assigned treatment trials) used before the ebola case numbers had turned so far down? Part of the answer, lies in the time it took to have adequate non human primate (NHP) data on safety and efficacy and human preliminary trials. Another factor was that objections to the deployment of experimental drugs in RCTs for clinical patient treatment, which had some strong contextual and ethical validity (Adebamowo et al. 2014 ), spilled over into views of vaccine RCTs, even where the same contexts could be seen as absent. Vaccine trials were prospective, when participants could make informed choices. Increased care could be planned for patients who developed ebola or other medical conditions as a result of vaccine trial participation. Trials could have been designed for and with volunteering health care workers at high risk, with advanced medical back-up relief options for all participants, for example in the early Fall of 2014. NIAID, the National Institute of Allergy and Infectious Disease, National Institutes of Health, Department of Health and Human Services in the United States, was one of the strong advocates of RCTs for both drug and vaccine trials, which ultimately they started in Liberia. However, the director of one of the leading vaccine candidates told me in the early fall of 2014, that even mentioning RCT vaccine trials could lead to his vaccine never being trialed in Sierra Leone. There was a clear difference in approaches within the international medical communities. Guinea (Conakry) fell geopolitically and biopolitically on the other side of the divide from the NIAID approach. This is reflected in the composition of the trial funders, from WHO, Norway, UK, MSF (Médicins Sans Frontières) and Canada. Ebola Ça Suffit! could be described as a control measure fused with a trial in a skillful combination. This type of design may have broader future use. One of the only ways novel control measures could legally be used was as trials. Capturing cases was done by defining the participants as contacts and contacts of contacts of remaining ebola cases. These were people at true higher risk of ebola. The trial populations were clusters (actually two rings of risk) of contacts around known cases. The design was based on vaccination procedures used for the final stages of eradicating smallpox under natural (nonmilitary, non-biowarfare) conditions. Vaccination could occur either immediately or following a two-week delay, and the clusters themselves were randomly assigned to either treatment in a double blind manner. By the time they reported their interim results (Henao-Restrepo et al. 2015 ), a separate evaluation team called for the elimination of the delayed vaccination arm of the trial, because sufficient evidence of efficacy of immediate vaccination made it unethical to continue delayed vaccination. The authors describe multiple teams, including for informed consent, separate from the trial medical team. Informed consent, crucially, included community acceptance as well as individual choice (and the right to withdraw). The trial personnel were predominantly from Guinea itself and other African countries. They harmonized the international regulations, country regulations and local participation. Perhaps this is too rosy a picture? There were still many issues in Guinea. Planners could have engaged communities on the trial design itself; or allowed choices between pre-designed approaches that already had regulatory approval, and had sufficient statistical power. The resources brought to bear in this trial would have been unimaginable in August 2014, when basic medical supplies were unavailable and even the supply of the most basic sanitation tool, chlorine bleach was out of date and nearly useless in, for example, Sierra Leone. Remarkably, Ebola Ça Suffit! even had electric power, water, and systems for maintaining a cold chain for vaccine deliveries. In August 2014 Sierra Leone asked the UK military to bring as many helicopters as possible for distribution of supplies to remote areas, and for access to much more of the countryside during the coming rainy season. Roads were often poor at all times. Before ebola, when the critically important Lassa Fever lab was re-equipped in Kenema, Sierra Leone, the only way key components could be brought in was by U.N. troops from Pakistan re-purposing one of the military helicopters (Wilkinson 2016 ). Helicopters could also deliver vaccine on dry ice in coolers; which was cold enough, a vaccine head told me. Nothing so jury-rigged needed to be contemplated by mid-2015. In this ebola epidemic there were over 20,000 infected and over 11,000 dead, due to ebola itself. The secondary effects due to the impact on the overall health system may have been equal or worse. In Sierra Leone with a typical extraordinarily low ratio of doctors or health care workers to population, before the epidemic began, at least 14 senior doctors died in the 7 months from July to December 2014; as well as large numbers of other health care workers. The randomised ring-cluster design probably is statistically under-powered relative to an equal-sized RCT trial, if an RCT could have been finished. Ebola Ça Suffit! is the only phase III vaccine trial to successfully report from the West African ebola epidemic. From Agassi to Ebola By July of 2014 I had been engaged in a multi-year dialog with Joseph Agassi on various aspects of philosophy and history of science. He asked me to read and critique the drafts of two of his books: The Very Idea of Modern Science (Agassi 2013 ) and Popper and His Popular Critics (Agassi 2014 ). Another critical reader was Ian Jarvie. My professional interests came mainly from plant breeding and genetics in agricultural; the intersection with cross-cultural science; the temporal development and intersecting changes of biology, medicine, nutrition and agriculture in North America; and how these interacted with other cultures and countries. These were actually practical matters, but how one thought about them could be crucial. I was also interested in two very different people who specifically credited some of their ideas and methodology to Quine. One was Bob Moses, of the Student Non-Violent Coordinating Committee (SNCC) when he described his later work on math and science education, starting with the Algebra Project (Moses and Cobb 2002 ). Another was Barry Hallen who, with his colleague John Olubi Sodipo, started a long project on cross-cultural understanding of knowledge (itself) and medicine (as we might say), working as colleagues with Yoruba onisegun (Hallen and Sodipo 1997 ). Their self-described pragmatic and experimental uses of concepts and methodologies from Quine in these two different contexts were suggestive and intriguing. Their work is reflected in my discussion at the end of this paper. In early August, 2014, I received a request for assistance on ebola from a colleague who was a senior rice breeder and geneticist, and in fact a World Food Prize laureate. He had returned to Sierra Leone, previously, to accept a position as a Special Advisor to the President and Ambassador-at-Large. I knew Monty Jones from his work at WARDA (the West African Rice Development Association) developing rice varieties ( Oryza sativa ) that were partly based on the indigenous domesticated rice species of West Africa ( Oryza glaberrima ). Now he had the mandate as Presidential liaison for ebola. We had been talking about the experimental drug ZMapp, and he was looking for how to have it be tested in the (then) two ebola epicenters in Sierra Leone: Kenema and Kailahun. They also were looking for anyone who could help provide badly needed equipment, protective gear, vehicles, ambulances, etc. The next day I sat in on the CDC conference call to prepare U.S. medical personnel and institutions for the arrival of two ebola patients to Emory University in Atlanta. Attuned to look for anomalies, I noticed the experts who were presenting briefly mentioned the possibility of some evidence for sexual transmission of ebola. We were off. On Immunochemistry and International Research Policy Ebola is considered by the CDC to be a category A bioterrorism agent; meant to be studied only in level IV biosecure facilities. Because of this designation, and perhaps spurred on by recurring episodes of ebola outbreaks in Central Africa, there had been advanced work done on a number of possible interventions. Unfortunately, none of them were actually ready to be used. It may be that a certain lassitude and comfort level had set in, over a decade after the anthrax attacks in 2001. There is a kind of public biowarfare defense community, so to speak, with their own conferences and journals and associated laboratories and companies. Federal research funding in the United States, in general, includes various small business requirements, and these also apply to funds for biowarfare defense. Such funding can be a useful steady source of income for smaller (or boutique) bioresearch companies. Canada, under the Harper governments, had reached a peak of Thatcher-influenced research policy that required commercial investment, or buy-in, for many areas of public research in the Canadian government laboratories, or their projects would be ended. My guess is that this policy helped lead to the rights to one of the two leading ebola vaccine candidates going from the Public Health Agency of Canada (PHAC) in WInnipeg to NewLink Genetics of the United States for what I recall as two hundred thousand dollars, Canadian. This was rVSV-EBOV, the vaccine that ultimately was used in Ebola Ça Suffit! (by then produced by Merck). A common approach in vaccine development is to use recombinant technology (the small "r") in a live but innocuous virus (to the human population of interest); in this case a vesicular stomatitis virus (the "VSV") to express antigens of interest from the ebola virus ("EBOV"). One alternative approach is to first silence key genes in a dangerous virus, so it can replicate but not cause damage, and then use it as a vehicle for recombinant antigen expression. In both strategies, the immune system can then react to the antigens of a disease without being exposed to the disease itself, or be harmed by the live virus which is vectoring the antigens. Heinz Feldmann originally modified VSV in order to study one of the ebola genes for glycoprotein, GP, in mice without needing level IV containment. Gary Kobinger , who also worked on ZMapp, headed the Winnipeg lab after Feldmann moved to the CDC. ZMapp, in contrast, delivers three monoclonal antibodies against GP (glycoprotein) antigens that have been (very carefully) expressed in plants. Like receiving venom anti-toxin or tetanus antitoxin, the monoclonal antibodies should neutralize a key viral function, in this case glycoprotein fusion into a patient's cell membranes, allowing viral entry into the cell. It is passive immunotherapy. This also gives the patient's immune system time to respond to viral antigens. In general there were three main classes of intervention tools: various means of expressing antigens, leading to an immune system response and defense; various means of providing antibodies to ebola or providing analogs of antibodies for (temporary) passive immunity; and various means of interfering with (RNA) viral specific replication. Some of these were drugs, some were vaccines. There also were immunology based rapid diagnostics for point of care screening under development. Using transfusions or fractions of ebola survivors' blood, which had been done ad hoc in emergencies, was another example of passive immunotherapy. None of them had safety and efficacy data for people. Having rodent data, such as for mice or hamsters, was an advanced stage. Some of them had preliminary small trials of safety or efficacy in nonhuman primates, or these were rapidly undertaken. Proposed repurposed drugs such as favipiravir (Avigan) had extensive human safety data for its intended uses but not in the presence of ebola. There were rodent ebola studies with favipiravir but no human or nonhuman primate efficacy data. Some of the drug classes were so new that I could not find any registered human use in the entire class. I had been developing a network of people who could help evaluate scientific issues of ebola intervention as well as working with institutions that could or did provide direct aid. By mid-August, with the encouragement of Lawrence Gostin at Georgetown University, I had developed a list of the roughly 19 (then) currently known agents. I also included on the list the notion of social mobilization as an intervention, with some possible examples. These could cut the rates of transmission and give the medical response time to catch up. Gostin was also the Director of the WHO Collaborating Center on Public Health Law and Human Rights. He emphasized that informed individualized consent was an ethical obligation. Now, many of the interventions would be hard to explain to anyone not actively working in that particular focused biomedical research subfield. It would be harder to explain to anyone else. It would be harder still to have information translated and explained adequately in a completely different cultural and social context; in the middle of an epidemic. One of the lead tropical disease specialists in the United States commented to me, in early September, that the three most promising interventions on the list were: (A) Immunoglobin purified from ebola survivors (passive immunity). (B) Favipiravir from Fuji/Toyoma (inhibitor of RNA virus replication). (C) The GSK investigational ChAd3-EBOV vaccine. This vaccine was from GlaxoSmithKline: ebola GP antigen carried on chimpanzee adenovirus type 3. It was originally developed by NIAID and Okairos, which later became part of GSK. Two forms were initially proposed and tested: monovalent, carrying GP antigen from the Zaire outbreak strain of ebola; and bivalent, carrying GP antigen from both the Zaire and Sudan ebola outbreaks. Many of the eventual vaccines and immunomimetic drug candidates could be presented in multiple versions depending on these kinds of choices of antigen sources, vaccination schedule, or combinations with other vaccines or related drugs. Almost all of the immunological drug and vaccine interventions were targeted at epitopes (antigenic areas) from forms of the GP ebola protein and not any of the other six ebola proteins. ZMapp was not on his list of high priority interventions to try. This was partly based on his own experience with plant-derived vaccines and their problems. In particular, there was the difficulty in production scale-up during an escalating epidemic even if ZMapp should prove to be clinically successful. When asked what I would choose personally, I said either the GSK or Canadian vaccines, and ZMapp as part of clinical treatment. However, the international interest in ZMapp, starting in August 2014, was based on an anomaly: a single dose, with a long backstory, given to Dr. Kent Brantly in Monrovia. Looking back, it was probably late in his disease course to have expected much, but ZMapp is associated with an immediate (transient) reduction in viral load followed by an increased level of a patient's own IgG response (Zeitlin et al. 2016 ). Brantly had contracted ebola while working for the missionary charity Samaritan's Purse. Later both he and a volunteer colleague, Nancy Writebol were flown to Emory University for advanced medical care under high biocontainment. Nancy Writebol had turned down the ZMapp treatment because Kent Brantly was sicker. Their clinical case history and treatment at Emory are in the New England Journal of Medicine , where they appear thinly disguised as Patient 1 and Patient 2 (Lyon et al. 2014 ). Looking back, the observation of greatest importance to the most patients in West Africa was buried in the case histories and may appear trivial. In Monrovia, Patient 1 tried to maintain fluids and electrolytes by drinking Tang and Gatorade. This was commonly done by Westerners as self-treatment for a number of similarly presenting tropical fevers. It was inadequate for clinical care of ebola. Nutritional support, correct oral hydration therapy (ORT) and (eventually) maintaining intravenous electrolytes (etc) were key to patient survival of ebola in a wide variety of circumstances and facilities (Lamontagne et al. 2014 ). A curious international pattern developed. International medical workers who developed ebola were flown to Europe or the United States, where they received the highest level of monitored intensive care. And they also received one, two or even three of the experimental drugs, with no discussion of enrollment in trials. West African ebola patients, including doctors and medical workers received none of this. This was the context in which MSF, which led the on-the-ground battle against ebola, as well as others, rejected using patients in West Africa as experimental subjects in RCT drug trials as medically unethical. RCT vaccine trials were collateral damage. Well-informed volunteer trials of medical workers were rejected. RCT vaccine trial co-designed with participants, with acceptable risk and conditions from their own perspectives never came up. I was told an RCT of as few as 200 people could give up to 90% confidence of a drug being effective, in the context of projected hundreds of thousands of cases without effective control measures in September, 2014. I wondered about the politics and ethics of the ethical debate itself. Trial designs themselves are imposed as fiats rather than co-designed with the people most at risk. People at risk, including medical workers, were not participants in the ethical debate; nor in the assessment of tolerable risks versus possible benefits of particular trial designs. They were not participants in the decisions to not have RCT trials, even of vaccines. In vaccine trials participants are not under the threat of imminent death; they are healthy and at risk of infection. Drug trials require a constant background of uniformly adequate palliative care to even determine if a treatment is effective This would have been difficult under chaotic medical conditions. Vaccine trials do not. It was perplexing. My initial reason for contacting social scientists, mainly anthropologists, was motivated by the technical concerns about informed individual consent for any of the experimental interventions, however they were deployed. There was an added issue of the need for social or community consent in some contexts. Individual consent could be required by the international community, and that looked hard enough; while some level of social or group consent was a cultural and social requirement for the potential participants. This quickly became subsidiary to much broader issues of social and cultural aspects of disease transmission, prevention, and deliverable clinical care. The Social Epidemiology of Being Dead An ebola disease course starts with fever and mostly non-specific symptoms, which could indicate other diseases, about 8–12 days after effective exposure. The second phase, with the highest classic risk of transmission, starts about 5 days later and includes, notably, massive diarrhea and vomiting. Viral concentration needed for infection is very low (Vetter et al. 2016 ) while blood and other fluids have very high viral concentration (CDC 2016 ). Unfortunately, so do corpses. The handling of ebola corpses can be extremely difficult even under United States' conditions (CDC 2015 ). Consider the impact of just one example of cultural difference: when is a marriage complete? In some Sierra Leone societies a marriage takes years to be complete, involving obligations to both the spouse and her family. When a married woman dies of ebola, where is she buried? It may require near or long distance transport of the body depending on her family of origin and the status of the marriage. Transmission can occur everywhere along the journey. The social scientists I was working with often had decades of experience in West Africa. Many were integrated into the societies they worked with; some because it was their society. Many had medical, biological, ecological or agricultural backgrounds. They had lived through the violent convulsions of West Africa (Richards 1998 ; Suluku et al. 2012 ), the AIDS crisis (Nguyen 2010 ; Benton 2015 ), the crises of governance, and the complex social communication (Ferme 2001 ) elaborated under permanent structural violence (Bardosh et al. 2016 ). They had critical information about the tremendous social impacts of illness care, dying, death, mourning, preparation for burial, burial and post-burial ceremonies. All of these involved high ebola risk to different types of contacts and associates of the dead. My closest anthropology colleague, Professor Paul Richards , prepared a summary of key information which I sent to retired senior CDC staff as a reality check. We then had a formal conference call with active CDC ebola-response staff, in Atlanta, who were joined by multiple other agencies. A later summary was published in 2015 (Richards et al. 2015 ). This is an example of one kind of important but passive engagement. Social scientists from Sierra Leone had useful epidemic-related information needed for planning effective epidemic interventions. Active integration of social scientists with medical intervention was more encouraged by the UK. Several key incidents of social scientist intervention between communities and the medical response were fairly well known, after violence by governments or violence by communities. Active integration of social scientists with communities on epidemic response also occurred but is less well known. Social and cultural issues of burial ranged from the ritually covert and secret to the social, legal and ritually overt and public. Both had to be accommodated. With information on ebola transmission, the physical covert ritual actions could be adjusted to block transmission, for example by women in sodalities such as the sande . The more overt and socially public aspects of burial could also be modified to accommodate both epidemic control and the social requirements of death. This included proposing the use of trained local burial teams. The modification of both social burial and "medical" burial is an area where mutual accommodation eventually succeeded in many areas. The social scientists described people in rural areas who would never have timely access to effective ebola treatment. They still needed assistance. Professor Marianne Ferme , Department of Anthropology, University of California, Berkeley described one of her own journeys to health care (Ferme 2014 ): "When I first did fieldwork in Sierra Leone I came down suddenly with a combination of illnesses that made me completely delirious and incontinent within a few hours (malaria, amoebic dysentery, a staff infection). I was a total mess. I couldn't believe what the two women who took care of me had to do to keep me clean and care for me. They were not only sponging and changing me, they also were constantly laundering all the bed sheets and clothes I was soiling, until I was 'med-evacuated' the indigenous way: the Paramount Chief sent his ceremonial hammock with 4 hammock bearers (who worked in pairs, running at a regular jog up and down hills to keep the momentum, and relieving each other at regular intervals), and I was out of there in no time at all, and on a main road where they flagged down a passing vehicle". This was a walk-in village with no regular transportation, but I was completely in awe at the ingenuity of people in figuring out how to make do with what they had: from fashioning a little "porto-let" on a bucket for me, to figuring out when I was ill enough to need immediate hospitalization. Even with my ready cash to pay for transportation it took me and the man delegated by the village elders to accompany me about 12 h to get to the Serabu hospital emergency ward, which gives you some sense of how unrealistic it may be to expect isolated rural folks who may not have the means to pay for double transportation all the way there to get to hospitals (in my case, 4 different vehicles/legs to the journey, in addition to the bit with the hammock)." We will return to the hammock bearers later. Some of the social scientists saw very early that people were condemned for not bringing ebola patients to medical isolation, even when transportation was impossible and medical centers were nonfunctional. I read medical team reports from all three countries that also recognized the failures in each stage of transportation, facilities, adequate material supplies and successful clinical care that were supposed to make clinical-isolation work. Medical messaging castigated the most basic human need to care for loved ones, rather than providing assistance with material and guides for home or local health care of ebola. It was isolation that was needed, whether the setting was a medical facility, community, social group or at home; not just medical isolation, even when it was unobtainable. In order to break transmission chains in situ , roughly "at home", this meant a patient would have to be isolated, and a single care-giver had to be semi-isolated, physically but not socially from the surrounding community. Many farms had isolated field shelters used at harvest which could have been used. This was similar to social practices used for containing smallpox in Zaire (now DRC) that were repurposed during the first ebola outbreak in 1976. Similar practices had been used historically for other epidemics in West Africa as well. Basic palliative care at home had to be done in a clinical manner, to give caregivers a chance at survival. But these were homes without running water, power, and in significant numbers not even two plastic buckets for water or bleach solution. People could contain the epidemic and break chains of transmission (epidemic intervention) by isolation (patient) and semi-isolation (caregiver) without outside aid. But without that aid, both caregiver and patient would probably have died. With improved communication about burial, at least they could have been buried safely (burial teams or local burial teams) with socially adjusted medical burial. In late August 2014 the U.S. military was going to provide several hundred thousand home health care kits for Liberia. This never happened. The decision making was opaque. No one ever developed a manual for home health care for ebola. The closest I could ever find was an older CDC and WHO manual Infection Control for Viral Haemorrhagic Fever in the African Healthcare Setting (Lloyd and Perry 1998 ) which discusses the layout of care, innovation in personal protective equipment (PPE) and making maximum use of minimal access to supplies in a low resource setting. It took about half a year but eventually the CDC put out a poster giving home caregiving advice while waiting for transportation (CDC 2014 ). I remember similar advice from one MSF facility for people who were waiting for a space to open. At the end of 2015 MSF's internal research unit published a review, in very French-inflected cautious English: noting that home care isolation had been used in other filovirus outbreaks, that they had older guidances for home-based support and risk reduction, that the pragmatic limits of using facility isolation had been reached in 2014, and that perhaps facility isolation was not the only option (Calain and Poncin, December 31, 2015 ). This is in section four: "Facility isolation: an onerous public health measure". Closer to the People: Technologies of Bodies Broadly speaking, social engagement strategies can be monologues and directed from the top down, or dialogues which require mutual recognition. Social science methodology can also reflect these two orientations. In surveys based on questionnaires people were adept at repeating back the ebola messages that came from governments and international medical groups. Don't eat bushmeat along with other more useful messages. Often the surveys were seen as part of the government. When engaged by longer-form more open-ended investigations and asked, for example what they would actually do in response to ebola the answers could be quite different. Medical work in foreign-supported centralized hospitals could involve gruesome triage decisions and 70% death rates in 2014. There were two waves of the international response trying to bring isolation and health-care closer to people: first in what were called Ebola Treatment Units (ETUs) which were relatively more advanced, and then in what were called Community Care Centers (CCCs). These may have suffered from the sociological consequences of top-down implementation. In Liberia, many of the facilities were still being built in 2015, even as case numbers dissolved to nothing. They were burned to the ground for ebola containment reasons and could not be re-purposed for general medical care, following protocol. When the UK teams asked for an ETU plan review, I was able to point out that it might be more appropriate if the patient flow-chart schematics did not have the only outcome, or end-product, be disposing of the patient's body by incineration. They could, for example, show the patient cured and returning home. Incinerating the body, in any case, may have been biologically safer but socially disastrous. What does it mean to be cured of ebola, or ebola-free? Originally it meant that a patient had survived and no longer had detectable virus when blood was tested. This was the diagnostic sign of being ebola-free. It was presumed that this sign derived from blood testing applied to the patient's entire body. However, sexual transmission of ebola developed harder and more convincing evidence. Ebola survival in semen turned out to vary widely and could go on for an indeterminate time in some patients. Gradually it became clear that there were multiple kinds of immune-protected tissues where the virus could remain, be detected, and sometimes be culturable (Vetter et al. 2016 ). Although sex, usually semen, was the main cause of secondary infections, infection of other tissues could cause long-term problems, for example in eyes. Sex was a real but relatively rare means of transmission compared to, say, Zika. Nor was being ebola-free (by blood testing) the end of long-term physical and mental consequences for patients (Etard et al. 2017 ). On the other hand, physical objects from patient-care settings (fomites) did not seem strongly linked to transmission despite the great attention paid to them in all patient-care facilities and the detection of ebola RNA. Ebola was notable for its very high death rate, but people had different risk levels, for example by age. There also were cases of high and intimate exposure followed by minimal symptoms. We received one of these rarer atypical case description from a colleague, Lina Moses working in Kenema, Sierra Leone, at the height of the epidemic. She is Research Assistant Professor in the Department of Global Community Health and Behavioral Sciences, Tulane University (Moses 2014 ): "[…] In the three and a half months doing this Ebola response, I was very surprised to see several cases of what I thought was mild disease. One case in particular, the contact of a case that died with severe hemorrhaging, vomiting, diarrhea, had a fever one day, came in for testing and was positive with low viral load. By the time we got the results he was feeling better, but we had to admit him because he had detectable virus. He was not too happy about this as he never developed any further symptoms. In fact, he occupied his time with push-ups, sit-ups, and pull-ups and was considerably bigger when he was discharged four days after admission. We tested him several times just to be sure it wasn't a lab error." There is a history of some intimate caregivers of ebola patients surviving with only mild ebola symptoms, for example during an earlier outbreak in Gabon. I use the neutral terminology of an atypical disease course, which does not imply any particular mechanism. When blood samples from the Gabon caregivers were tested later they showed anti-ebola antibodies but, interestingly, not to the GP protein. We recommended special attention be paid to the low percentage of West African ebola epidemic survivors who had been identified with an atypical disease course. Some new vaccine and antibody-like drug development strategies are being modeled on a particular survivor's antibodies from prior outbreaks. Others have the goal of broad-spectrum vaccination, seeking exactly those epitopes that could control a range of filoviruses at once. There may be an analog in the natural history and ecology of filovirus exposure that patients with mild symptoms exemplify. Prior exposure to some other disease, which must be rare, may have cross-protected them from most ebola symptoms. A mild disease reaction in enough people could create similar dynamics to immunization in dampening the rate of transmission. Personal protective equipment was critical for medical workers but it was never designed for tropical conditions. In a sense they were being cooked sous vide while they tried to work. Typically a two-hour shift was the longest people could work competently, which meant the effective staffing rate needed was at least four times normal. The large, relatively well-trained Cuban medical teams were reported to have kept to shifts of from 40 minutes to an hour (Kirk and Walker 2016 ). The most dangerous time was disrobing, when liquid contaminating the impermeable surfaces of the PPE could transfer from PPE to exposed skin. It would have made sense for materials science and PPE design teams to have worked with West Africans on better combinations of protection, particularly affordable protection, to reduce the risks and to reduce the heat stress. Cheap single use, absorbent disposable aprons could help when disrobing, for example. PPE designs also made patient interaction difficult. Even medical workers who came down with ebola and were treated by their own friends and peers reported feeling humanly cut off. The very welcome relief offered by the international community at the end of 2014 was always additive in nature. If the epidemic had kept growing exponentially, this too would have been rapidly overtaken and swamped. Tropical adapted PPE would at least have had a clinical-personnel multiplier effect, with better odds of holding the line until vaccines or other effective biotechnical tools were available. On the other hand, something about the pace and conditions of work was quite different for members of the burial teams. Despite the high risk from corpses, and often less adequate PPE, I do not know of any burial team member who contracted or died from ebola. Nevertheless, the team members often faced tremendous social stigma for their work. The clinical-isolation model of epidemic control had two internal contradictions at all levels. First, medical transportation and facilities, if available, were themselves major sites of transmission. Careful design and training could reduce within-facility transmission but peri-nosocomial transmission from transportation, waiting to be admitted, and screening at admission remained. Very effective point-of-care rapid screening diagnostics were needed, with near-zero false negative results. These were under development but not available during the epidemic. Second, access to effective medical treatment, indicated by survival rates, was always unequal; and the more effective the medical care, the more unequal the access. Dr. Olivet Buck was the fourth senior doctor to die in Sierra Leone. When she was diagnosed with ebola, a Level 4 hospital in Germany offered to treat her if she could get there. President Ernest Bai Koroma personally called WHO in Geneva to ask for airplane medical evacuation to Germany. They refused. This was remarkably covered by Joseph Harker in an article in The Guardian newspaper (Harker 2014 ) headlined: "Why are western health workers with ebola flown out, but locals left to die?" In response, we spent quite some time on how to access the two U.S. SARS-ready emergency planes, but the systemic issue remained. The Turning Point There was a nodal point between two very different phases of the epidemic and response; between 2014 and 2015. It was the first questionable possibility of the beginning of the end. Reports that the epicurve was leveling off and perhaps turning down came first from Liberia. This was still uncertain, over the late winter and New Year's holidays, 2014/2015. I could not find anyone who knew whether to believe the reported Liberian numbers, or not. The major aid had only begun to arrive so it seemed too early. On the other hand, the methodology of reporting cases in Liberia seemed to have some strong disincentives, so perhaps it was a methodological artifact. I went back and read Camus ' The Plague , this time as a kind of field handbook to an epidemic. So did others: an article in Emerging Infectious Diseases from April 2015 on ebola in Liberia, mid-year 2014, introduces each section with a quote from The Plague . (Arwady et al. 2015 ). As it happened, the downturn was true, and the same decline in the rate of increase and then the number of new cases would also occur in Guinea (Conakry) and Sierra Leone. One of the under-examined questions of the epidemic is what actually caused the epidemic to slow down, ending the growth phase. I separate social epidemic control measure from classic clinical control measures (medical isolation and contact tracing). They interact: positively when there is adequate medical care for the scale of the outbreak, but negatively when the scale of the epidemic over-runs medical capacity, or medical capacity is limited from the beginning. The outbreak could, possibly, have been controlled early-on by a clinical isolation-contact tracing model, if there had been a timely response to MSF's (Medecins Sans Frontières) first calls for assistance in March, 2014. Later, with comparatively ample resources and many adaptations, the model worked as the epidemic was ending; on the downside of the epicurve controlling re-emerging cases, when single cases or small clusters could have reignited an outbreak. The containment of ebola as an epidemic to Liberia, Sierra Leone and Guinea (Conakry) was a major success due to classical medical isolation and contact tracing. There were only small, brief, outbreaks in Nigeria, Senegal and Mali, for example. Sometimes this was by a combination of preparation, skill and good fortune. Urban Nigeria could have been the beginning of a world-wide epidemic (Shuaib et al. 2014 ). Sometimes, as in Guinea-Bissau, which seemed to have escaped transmission, success may have been due mainly to luck. The international response teams of the Centers for DIsease Control, coordinating with national health systems, had a major role. Jeremy Farrar and Peter Piot described the vastly expanded scope of engagement needed for an ebola epidemic, when classical outbreak control is no longer sufficient (Farrar and Piot 2014 ). As the epidemic expanded exponentially, throughout 2014, clinical isolation was still the main model for epidemic control; ignoring conditions of unsafe or non-existent transportation to inadequately staffed and supplied facilities, with high mortality rates. MSF, whose capacity had been over-run, for first time in its history called for military intervention and assistance, but with the same model for epidemic control. There has been a recurrent amnesia about key social lessons learned about both clinical and epidemiological engagement during ebola outbreaks. The lessons forgotten include the social design of care facilities and community isolation and treatment, among many others (Bremen and Johnson 2014 ; Bremen et al. 2016 ; Hewlett and Hewlett 2007 ; Hewlett 2016 ). The lessons are learned and then forgotten, repeatedly since 1976. This is a bureaucratic and institutional mystery (Abramowitz et al. 2015 ). On the other hand, some key lessons learned, or imagined to have been learned, from the much smaller ebola outbreaks in central Africa were remembered all too well, where they did not apply. For example, messaging on bats, in particular, and bushmeat in general (Frieden et al. 2014 ) were contradicted by what people saw. Hunters and their families were not dying in West Africa; clinic and hospital staff were. In a factually human-human transmission epidemic, this damaged the credibility of international actors and of health program messaging. One hypothesis for the turning of the epicurve is that social epidemic control measures had increasing effectiveness. Examples include social distancing; personal sanitation (chlorine solutions everywhere); mourning and burial changes adapted to local cultural and social requirements; and community-organized isolation control. Temporary shelter-in-place programs, such as in Sierra Leone are another example, although controversial. A variation on the hypothesis stresses community and individual innovation. When there was no effective medical response, I argued that for social epidemic control measures to work: communities needed to think like epidemiologists, and epidemiologists needed to think like villagers and communities. There was, in fact, a process of convergence that could have been much more supported and emphasized. Issues of convergence included evaluation of clinical risk and benefits: which procedures were critical to use in ebola care and worth the risk to health care workers (Ansumana et al. 2015 ) That's part of a longer story. The failure to provide basic supplies to people who would never be able to access any functional or timely clinical care is close to the heart of the matter. Anything Critical Goes! Paul Richards ' book on the ebola social response is titled Ebola: how a people's science helped end an epidemic (Richards 2016 ). This was very kindly and favorably reviewed by Peter Piot , for some time now the Director of the London School of Hygiene and Tropical Medicine (Piot 2016 ). Richards describes the case for how rapid community learning and social adaptation on ebola was a major cause for the turning of the epicurve and for control of the epidemic. These changes appear to be mainly what I call epidemic measures, that limit or end transmission, as opposed to clinical care. His own specific examples come from villages studied in depth in 2014 and 2015 by the team he works with at Njala University in Sierra Leone, including Esther Yei Mokuwa . A large number were in Jawei chiefdom, the hardest hit chiefdom of Kailahun District, the epicenter of the epidemic. In general, he thinks that across Sierra Leone, areas that were hit first and hardest put the greatest adaptive measures in place. How rapid learning was adapted in urban and peri-urban areas, will have to be set aside. Interaction with the epidemic control teams, in these examples, is shown as more of a dialog. Epidemiologists learned from the community and the community learned from medical and epidemic experts. The team set up workshops at Njala specifically for these interactions. Richards' view is that the fact that this was the first epidemic of ebola, as opposed to the smaller prior outbreaks, meant everyone had to learn how to fight and control the epidemic together. Peter Piot agreed (in his review and elsewhere) that there were similar experiences to what he and his team faced with the first ebola outbreak in Yambuku, in what is now the DRC. This included supporting villages using prior social models for fighting small pox. For context, it is worth recalling the nosocomial origins of the first ebola outbreak, where the virus was spread by the use of unsterilized needles at Yambuku Mission Hospital. Eleven of the staff, then the medical director and three Belgian missionaries died. The hospital closed and patients and their contacts fled to their homes, bringing ebola with them (Bremen et al. 1976 ; Breman and Johnson 2014 ). The government and NGO burial teams in Kailahun made some adjustments to the social need of burial but much more could have been done. It was very difficult for the local communities to get support and training for their own burial teams, which they wanted. Where dialog slowed down or disappeared, across the epidemic countries, was over clinical care: supporting the basic palliative care patients need to survive, in a rural community hut as well as in a place designated as a medical facility, or an advanced medical hospital. At a minimum this means, nutritional support (food, broths), oral rehydration therapy (ORT), disinfectants (usually chlorine) and how to prepare them, some PPE, and guidelines on the geometry and procedures of safe patient care to avoid contact with or spread of ebola virus. Facility layout and movement in good ebola patient care has analogies to safe handling of radioisotopes, requiring a notion of the correct structure for the physical environment and how to move through it in sequence. One could say it is a kind of dance. Although ebola patient-care as dance would have made perfect sense for cross-cultural medical training I did not see a discussion in Richards of this kind of interaction. Dance, drumming, and masquerade all have specific cognitive content and communicate structured information in this culture; they are languages as well as other things. Talking drums may be more familiar, and Richards gives examples from fieldwork when planting rice. The Paramount Chief Mussa Kallon and two sande (women) elders went to the Njala meetings, but there were disagreements about what should happen next. When Paul asked Chief Mussa Kallon how this was resolved he replied that the elders went into the bush and danced a solution. Epidemiological concepts became integral to how people approached ebola. The communities Richards worked with were aware of, and developing procedures to prevent, the possibility of ebola transmission when carried by stretcher-bearers. No foreign or domestic medical group ever discussed this. After the epidemic receded, survivors were discussing using ebola, and the history and experience of ebola in their communities, as an important way of teaching science in their children's educational curriculum. When thinking about strengthening a people's science in the context of the West African ebola epidemic, Richards defines the fundamental idea of science, in passing (!), as: prior judgements must be abandoned in the face of compelling empirical evidence. My interpretation of his work is that convergence of understanding and action could happen, even though the knowledge of the social meaning of death, which had to be understood, started more on one side (in communities) and understanding of the nature of the ebola virus and its transmission, which also had to be understood, was more on the other side (the international medical teams). Dialog can lead to cross-cultural convergence. People have a remarkable capacity to maintain prior judgements in the face of compelling empirical evidence. Here, I have been more interested in showing how this warped the international medical response. More broadly applied, cross-cultural convergence can lead to remarkably interesting and enriched kinds of science. Most of the examples I know of come from cross-cultural agriculture, so to speak. Twenty years ago, two of the social scientists we later worked with on ebola, James Fairhead and Melissa Leach, published Misreading the African landscape: society and ecology in a forest-savannah mosaic. A time sequence of aerial photographic surveys of Guinea, which began after the First World War, documented that the integrated cultural use of slash-and-burn annual cropping was part of increasing forest cover and not desertification. Kat Anderson 's Tending the wild (Anderson 2005 ) describes the complex indigenous non-farming cultivation of plants and animals in California. The epistemology, ontology and categories used are different. This is part of a longer story. Medical, epidemiological, ecological and agricultural fields come together in the disease control strategies called one-health (Cohen 2013 ; Rubin et al. 2013 ), which may be a particularly useful area for investigating cross-cultural science. For Amilcar Cabral , Who Would Have Been 76 in the Year 2000 In the Spring of 2015 a fisherman who was a primary contact of an ebola patient disappeared from Guinea (Conakry) into Guinea-Bissau. The CDC social sciences coordinator asked for contacts in Guinea-Bissau; and we had a whole second round of work on the same issues. The medical and governmental conditions were worse. The social scientists now were Portuguese-speaking or from Portuguese language countries. There also were a high proportion of scientists from Nordic countries. Most of this has to be set aside. The history of Guinea-Bissau made it, if anything, even more socially, culturally and ethnically complex than the three primary ebola epidemic countries. Amilcar Cabral had carried out the first agricultural census of Guinea-Bissau, after 500 years of Portuguese rule, published in 1956. Some of this was now available in English translation. Almost all of the population were farmers so an agricultural survey had to deal with most of the complexity of the country. It was still very useful background information 60 years later. Cabral was literally one in a million, one of the very few Africans from then Guinea-Bissau and Cape Verde Islands who received a college education. One can imagine if the United States had only 300 people with bachelor degrees. I read as much of his agricultural work as I could find and re-read the political writing and commentary. Cabral had a very unusual and sophisticated view of critical cross-cultural science, technology, ecology and knowledge. It is framed first within the context of farming, and then in his approach to liberation from Portugal and international support. I could see this in his critical evaluation of rice farming between ethnic and cultural groups, as well as the evaluation of the impact of colonial cash crops. It is nearly completely overlooked in the political literature, except for two cases that could not be ignored but were never analyzed or, I think, understood. He had interesting methods of using theater for education and practice in cross-cultural work. He died before he could use this approach in post-colonial national development. This needs to be followed up. Ebola was not reported found in Guinea-Bissau. They were not so fortunate with (Pacific-Brazilian) Zika virus, which went from Cape Verde to Bissau in 2016. Zika may be harder than ebola to control. Ebola had a striking effect on international biomedical research and development capacity for combatting neglected tropical diseases. It also showed that, in many cases, individuals could wrestle control of scientific, development, government and corporate institutions in a kind of Prague Spring of science facing a lethal crisis. Even advanced biomedical tools have to be used in context. Ebola went undiagnosed in Guinea for over three months. Dr. Michael Van Herp, MSF Brussels, suspected ebola after receiving case reports that included rapid death, probably too rapid to be Lassa fever, and hiccups (Stern 2014 ). Hiccups were toward the bottom of signs and symptoms presented by ebola patients; 17th on the WHO Ebola Response Team table of West African patients, reported in October, 2014 (Team WE 2014 ). But hiccups were associated with ebola among the hemorrhagic fevers. And so it all began.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831098/

LytR-CpsA-Psr Glycopolymer Transferases: Essential Bricks in Gram-Positive Bacterial Cell Wall Assembly

The cell walls of Gram-positive bacteria contain a variety of glycopolymers (CWGPs), a significant proportion of which are covalently linked to the peptidoglycan (PGN) scaffolding structure. Prominent CWGPs include wall teichoic acids of Staphylococcus aureus , streptococcal capsules, mycobacterial arabinogalactan, and rhamnose-containing polysaccharides of lactic acid bacteria. CWGPs serve important roles in bacterial cellular functions, morphology, and virulence. Despite evident differences in composition, structure and underlaying biosynthesis pathways, the final ligation step of CWGPs to the PGN backbone involves a conserved class of enzymes—the LytR-CpsA-Psr (LCP) transferases. Typically, the enzymes are present in multiple copies displaying partly functional redundancy and/or preference for a distinct CWGP type. LCP enzymes require a lipid-phosphate-linked glycan precursor substrate and catalyse, with a certain degree of promiscuity, CWGP transfer to PGN of different maturation stages, according to in vitro evidence. The prototype attachment mode is that to the C6-OH of N -acetylmuramic acid residues via installation of a phosphodiester bond. In some cases, attachment proceeds to N -acetylglucosamine residues of PGN—in the case of the Streptococcus agalactiae capsule, even without involvement of a phosphate bond. A novel aspect of LCP enzymes concerns a predicted role in protein glycosylation in Actinomyces oris . Available crystal structures provide further insight into the catalytic mechanism of this biologically important class of enzymes, which are gaining attention as new targets for antibacterial drug discovery to counteract the emergence of multidrug resistant bacteria. 1. Introduction to the Review This review covers what is currently known about the functions and structures of the LytR-CpsA-Psr (LCP) class of enzymes, which commonly transfer the reducing end of cell wall glycopolymers (CWGPs) of Gram-positive bacteria from a lipid carrier-bound CWGP intermediate to the peptidoglycan (PGN) backbone, usually via a phosphodiester linkage. The review starts with a brief description of the different classes of CWGPs and the LCP enzymes themselves before going on to a species-by-species discussion. Both molecular and structural biology of the systems that have so far been studied are presented as well as the questions that remain to be addressed for the future evaluation of LCP enzymes as novel antibacterial targets. 2. Cell Wall Glycopolymers—Brief Insight into Composition and Biosynthesis 2.1. Peptidoglycan (PGN)—A General Perspective In Gram-positive bacteria, the cytoplasmic membrane is surrounded by a remarkably dynamic and constantly remodelled thick layer of the bacterial cell wall material peptidoglycan (PGN) [ 1 , 2 ]. PGN is a complex macromolecule ("PGN sacculus") that is essential for bacterial survival. It plays an important role against environmental challenges, serves as a protection from rupture due to high internal osmotic pressure, and specifies cell shape [ 3 , 4 ]. PGN is composed of linear glycan backbone strands of alternatingly β-1,4-linked N -acetylmuramic acid (MurNAc) and N -acetylglucosamine (GlcNAc) residues coupled via stem peptides that are attached to the MurNAc residues, resulting in a crosslinked, mesh-like framework. These cross-linkages via different stem peptides together with possible interpeptide bridges, such as the well-known pentaglycine bridge in the PGN of Staphylococcus aureus , form the basis for the classification of different PGN-types [ 5 ]. Importantly, PGN scaffolds numerous proteins and glycopolymers [ 4 , 6 ]. The role of PGN in bacterial survival has made PGN biosynthesis a target for important classes of antibiotics, including the glycopeptides (e.g., vancomycin) and the β-lactams (e.g., the penicillins, cephalosporins, and carbapenems) [ 7 ]. Both of these antibiotics inhibit PGN crosslinking, however, in different ways. While vancomycin binds to D-alanyl-D-alanyl groups at the end of the stem peptides of opposing PGN strands, the β-lactams irreversibly bind transpeptidases (penicillin-binding proteins) [ 8 , 9 , 10 ]. As a mode of induced resistance, bacteria-produced β-lactamases frequently render this group of antibiotics ineffective [ 11 ]. Due to the worldwide increase of antibiotic resistances and the lack of novel drugs in the development pipeline, there is a renewed interest in (unexplored) cell wall components and underlaying biosynthesis pathways to find alternate options to treat infections caused by multidrug-resistant bacteria [ 12 ]. Furthermore, antibiotic inhibition of bacterial cell wall biosynthesis induces both common and compound-specific transcriptional responses, which both can be exploited to increase antibiotic susceptibility [ 13 ]. 2.2. Types of Glycopolymers Attached to PGN 2.2.1. General Considerations The covalent attachment of various glycopolymers to PGN (cell wall glycopolymers—CWGP) is an enigmatic feature of the cell wall assembly of Gram-positive bacteria. CWGPs are mostly found to be anionic, typically composed of individual repeating units (RU) of varying degrees of polymerization, and can make up to 50% of the dry weight of the cell wall [ 14 ]. Frequently, they are tethered to the C6-OH group of MurNAc residues of the PGN glycan backbone strands via a phosphodiester bond, but also the GlcNAc residues can be modified [ 4 , 15 , 16 ]. CWGPs are involved in a myriad of crucial cellular functions and serve as a rich source for both validated and unexploited pathways that are essential for bacterial virulence and survival [ 17 , 18 , 19 ]. On the basis of their structural characteristics, PGN-bound CWGPs of Gram-positive bacteria can be classified into two major groups: (i) "classical" CWGPs, comprising wall teichoic acids (WTAs) [ 20 , 21 ] and teichuronic acids [ 14 , 22 ], and (ii) "non-classical" CWGPs, which is the more diverse group comprising all other anionic and neutral CWGPs [ 23 , 24 ]. To these CWGPs, initially only secondary roles in cell wall function have been attributed; hence, they have been named "secondary" cell wall polymers. However, given the rich body of knowledge that has accumulated over the past 40 years, it is evident that these compounds play pivotal roles in bacterial cell function, physiology and virulence; thus, nowadays, the term "CWGPs" is more appropriate [ 16 ]. Based on detailed investigations of the lipopolysacchaide (LPS) biosynthesis in Gram-negative bacteria [ 25 ], two major principal assembly routes are known for bacterial CWGPs—the ABC-transporter dependent pathway and the Wzy-dependent pathway [ 26 ]. In many bacteria, the genetic information for CWGP biosynthesis is encoded in genomic biosynthesis gene loci or clusters. However, given the interchangeability of modules between the routes, the prediction of a distinct route based on the bacterial genome content is impossible. Generally, the interplay between different CWGP assembly routes—including also that for the PGN scaffold itself—concerns common pools of activated monosaccharides, biosynthetic intermediates, of the C 55 undecaprenylphosphat (undp- P ) lipid carrier and, importantly, enzymatic machineries [ 27 ]. This becomes evident upon inhibition of individual biosynthetic steps. However, it is still largely unknown how the different CWGP biosynthesis pathways, which share building blocks and membrane carriers, function in a coordinated and integrated fashion [ 28 ]. While the coupling of different CWGPs to the PGN has long been discovered [ 29 , 30 ], until the recently exposed LytR-CpsA-Psr (LCP) enzyme family, there were not any plausible candidates for this coupling reaction [ 29 ]. In this review, we are discussing different CWGPs from various bacterial sources, for which information on their ligation to PGN is available in the literature. However, studying LCP enzymes is challenging considering that frequently different types of CWGPs are simultaneously present in a bacterium and that the presence of multiple copies of LCP proteins is the rule rather than the exception. 2.2.2. Wall Teichoic Acids Wall teichoic acids (WTAs) are the most abundant and best-investigated PGN-linked CWGPs in many Gram-positive organisms [ 14 ]. They are composed of repeating units of alditol phosphates linked by phosphodiester bonds and can be further substituted by amino acids (e.g., D-alanine) and/or carbohydrate residues. Typically, Bacillus subtilis produces glycerol phosphate (Gro- P ) or ribitol phosphate (Rbo- P ) WTAs, while in S. aureus , commonly Rbo- P WTAs are present [ 16 , 31 ] ( Figure 1 ). In either case, a conserved β-D-Man p NAc-(1→4)-α-D-Glc p NAc-(1→P murein-linkage unit at the reducing end of the glycopolymer mediates WTA attachment to the PGN. In fact, this "conventional" murein linkage unit is also found in several other CWGPs, as is explained below. The assembly route of WTAs follows the ABC-transporter dependent route [ 18 , 19 ]. This implicates CWGP assembly on a C 55 undp- P carrier in the cytoplasm, involving the formation of the "conventional" β-D-Man p NAc-(1→4)-α-D-Glc p NAc-(1→ P murein linkage unit and polymer elongation using individual nucleoside-diphosphate (NDP)-bound substrates in concert with dedicated glycosyltransferases, followed by glycopolymer export to the outer leaflet of the membrane by an ABC-transporter. Upon reaching the exterior of the cell, WTAs are further functionalized and, finally, transferred to PGN in a ligation reaction involving LCP enzymes [ 19 ]. Conserved enzymes for the biosynthesis of the "conventional" murein linkage unit are TagO and TagA [ 18 , 33 , 34 ]. The first enzyme in this pathway, TagO, is an integral membrane protein that transfers GlcNAc-phosphate from UDP-GlcNAc to the undp- P lipid embedded in the cytoplasmic membrane [ 35 , 36 ]. The lipid-linked monosaccharide is then elongated to a disaccharide by the UDP-ManNAc transferase TagA [ 37 , 38 , 39 ]. This lipid-linked disaccharide constitutes the platform for the subsequent steps of WTA biosynthesis [ 29 , 40 , 41 , 42 , 43 ] and other CWGPs [ 44 , 45 ]. WTAs are intimately involved in many aspects of cell division and essential for maintaining cell shape in rod-shaped organisms [ 41 ]. Importantly, WTAs are key determinants of virulence and antibiotic resistance—e.g., in methicillin-resistant staphylococci (MRSA) or in streptococci—and have, thus, been the target of numerous screening campaigns to find inhibitors [ 46 , 47 ]. 2.2.3. Pyruvylated CWGPs Pyruvylated CWGPs (PyrCWGP) are a less investigated class of PGN-attached CWGPs which are of interest in the context of protein cell surface display in Gram-positive bacteria [ 48 ]. These SCWPs are 5–20 kDa in size, composed of species-specific repeats [ 6 , 49 ], but lack repetitive alditol phosphates and phosphodiester bonds of WTAs [ 15 , 50 , 51 ], hence, categorized as "non-classical" CWGPs. Importantly, they contain 4,6-pyruvateketal-modified-β-D- N -acetylmannosamine (Pyr-β-D-Man p NAc) imparting a negative charge, which serves as a specific cell wall ligand for S-layer homology (SLH) domains that are usually present in triplicates at the termini of cell surface proteins [ 48 , 52 , 53 ]. Prominent examples of SLH-domain proteins are surface (S-) layer proteins which self-assemble into two-dimensional crystalline arrays on the bacterial cell surface [ 54 , 55 ]. S-layer proteins are important for many biological functions such as maintenance of cell integrity, enzyme display, protection to phagocytosis, and interactions with the host and its immune system [ 56 ]. Best investigated pyruvylated CWGPs are those from Paenibacillus alvei CCM 2051 T and Bacillus anthracis . The B. anthracis CWGP is composed of →4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→ trisaccharide RUs with strain-dependent galactosylation occurring at the GlcNAc residues [ 57 , 58 ]; Pyr-β-D-Man p NAc is exclusively contained in the terminal repeat [ 51 , 57 , 58 , 59 ] ( Figure 1 ). In contrast, the CWGP of P. alvei is multiply pyruvylated and consists of →3)-Pyr-β-D-Man p NAc-(1→4)-β-D-Glc p NAc-(1→ RUs [ 60 , 61 ]. Notably, a common structural feature of WTAs and pyruvylated CWGPs is the presence of the "conventional" β-D-Man p NAc-(1→4)-α-D-Glc p NAc-(1→ P murein linkage unit. 2.2.4. Capsules Capsules are long-chain polysaccharides (CPS), which are produced by many bacteria of both Gram-classes [ 62 ]. They can either maintain a strong association with the enveloping bacterial cell and/or be secreted into the immediate environment in the form of exopolysaccharides [ 63 ]. Capsules afford the producing bacteria protection from a wide range of physical, chemical, and biological stresses, support biofilms, and play critical roles in interactions between bacteria and their immediate environments [ 63 ]. The Wzy pathway constitutes a prototypical mechanism to produce these structures. Briefly, the basic CPS RUs are synthesized at the cytosolic side of the membrane by the sequential action of glycosyltransferases using NDP-sugar substrates. The RU is anchored to a undp- P membrane lipid, and it is transferred to the outer side of the membrane via a Wzx flippase, where it is polymerized into the full-length CPS by the addition of new repeat units to the reducing end of the glycopolymer in a reaction requiring the Wzy polymerase and the chain-length regulator Wzz. In some Gram-positive bacteria, the lipid-bound CPS precursors serve as substrates for LCP enzyme-catalysed coupling of CPS to PGN, thus creating the mucoid capsule layer covering the bacterial surface [ 28 , 63 , 64 ]. 2.2.5. Arabinogalactan Arabinogalactan (AG) is a heteropolysaccharide found in covalent attachment to PGN via a phosphoryl- N -acetylglucosaminyl-rhamnosyl linkage unit with the structure →4)-α-L-Rha p -(1→3)-α-D-Glc p NAc-(1→ P to MurNAc residues of the mycobacterial A1γ-type PGN ( Figure 1 ). AG comprises a galactofuran domain bound to the linkage unit which is extended by the arabinofuran domain, which is, in turn, esterified at its non-reducing ends to long-chain (C 70 –C 90 ) mycolic acids forming the inner leaflet of the mycomembrane [ 65 , 66 ]. Notably, mycobacteria stain Gram-indifferently and the compositional and architectural complexity of the mycobacterial cell envelope distinguishes species of the Mycobacterium genus from other prokaryotes. It is the basis of many of the physiological and pathogenic features of mycobacteria and the site of susceptibility and resistance to many anti-tuberculosis drugs [ 67 , 68 ]. The synthesis of AG is initiated in the cytoplasm on a C 50 -undp- P carrier lipid with formation of the murein linkage unit [ 69 , 70 ] followed by the addition of Gal f and Ara f residues [ 71 , 72 , 73 ] and AG export, potentially involving an ABC-transporter [ 74 ]. It is thought that the GlcNAc residue of the linkage unit of the mature AG next forms a 1- O -phosphoryl linkage with the C6-OH position of a MurNAc residue of PGN [ 71 ] and that this transfer reaction is catalysed by an LCP family protein [ 65 ], which requires newly synthesized PGN undergoing concomitant cross-linking [ 75 ]. Despite the fundamental structural differences that exist between AG and WTAs, the structure of the AG-PGN linker shares similarity with the "conventional" murein linkage unit of WTAs with regard to the reducing-end GlcNAc residue and, evidently, LCP proteins are involved in the final ligation of AG to PGN [ 66 ]. 2.2.6. Rhamnose-Containing Cell Wall Glycopolymers Several members of the order Lactobacillales (mainly streptococcal species and Lactococcus lactis ) can generate a specific class of abundant CWGPs, which are composed of individual RUs, where rhamnose is the major constituent, along with variable combinations and linkages of Glc, GlcNAc, Gal, GalNAc, and phosphate [ 76 ]. This heterogeneous group of rhamnose-containing CWGPs is named RhaCWGPs [ 76 ]. For the Group B Streptococcus carbohydrate (GBC) and Group G carbohydrate (GGC), rhamnose is the major antigenic determinant. Species carrying these structures are serologically discriminated based on the presence of either a single rhamnose in GGC versus triterminal α-L-(1→2)-Rha p in GBC in the RhaCWGP RU (for review, see [ 76 ]). Among the streptococcal group antigens, the GBC is unique since it forms a multiantenna branching structure and is negatively charged, due to the presence of phosphodiester bonds that link different GBC repeat units. Similarly, the L. lactis RhaCWGP contains phosphodiester bonds that link branched →6)-β-GlcNAc-(1→3)-Rha-(1→3)[α-Glc–(1→6)]-β-GlcNAc-(1→2)-β-Gal f -(1→6)-α-Glc- P -(1→ hexasaccharide RUs [ 77 ] ( Figure 1 ). RhaCWGPs comprise about 40–60% of the bacterial cell wall by weight, and they are localized on the outermost surface of the cell wall but are likely also intercalated within the PGN layer, since antibodies directed against these structures bind to both sides of isolated cell walls [ 78 ]. According to recent evidence obtained for the glucose-containing RhaCWGP of Streptococcus mutans (also referred to as rhamnose-glucose polysaccharide–RGP) ( Figure 1 ), RhaCWGPs are assembled on a lipid carrier in the cytoplasm and exported via an ABC-transporter followed by covalent attachment to the PGN via the likely activity of an LCP family protein [ 79 ]. Of note, L-rhamnose as integral constituent of RhaCWGPs is often essential for bacterial virulence or even viability [ 80 ], making its biosynthesis pathway an attractive therapeutic target. 2.1. Peptidoglycan (PGN)—A General Perspective In Gram-positive bacteria, the cytoplasmic membrane is surrounded by a remarkably dynamic and constantly remodelled thick layer of the bacterial cell wall material peptidoglycan (PGN) [ 1 , 2 ]. PGN is a complex macromolecule ("PGN sacculus") that is essential for bacterial survival. It plays an important role against environmental challenges, serves as a protection from rupture due to high internal osmotic pressure, and specifies cell shape [ 3 , 4 ]. PGN is composed of linear glycan backbone strands of alternatingly β-1,4-linked N -acetylmuramic acid (MurNAc) and N -acetylglucosamine (GlcNAc) residues coupled via stem peptides that are attached to the MurNAc residues, resulting in a crosslinked, mesh-like framework. These cross-linkages via different stem peptides together with possible interpeptide bridges, such as the well-known pentaglycine bridge in the PGN of Staphylococcus aureus , form the basis for the classification of different PGN-types [ 5 ]. Importantly, PGN scaffolds numerous proteins and glycopolymers [ 4 , 6 ]. The role of PGN in bacterial survival has made PGN biosynthesis a target for important classes of antibiotics, including the glycopeptides (e.g., vancomycin) and the β-lactams (e.g., the penicillins, cephalosporins, and carbapenems) [ 7 ]. Both of these antibiotics inhibit PGN crosslinking, however, in different ways. While vancomycin binds to D-alanyl-D-alanyl groups at the end of the stem peptides of opposing PGN strands, the β-lactams irreversibly bind transpeptidases (penicillin-binding proteins) [ 8 , 9 , 10 ]. As a mode of induced resistance, bacteria-produced β-lactamases frequently render this group of antibiotics ineffective [ 11 ]. Due to the worldwide increase of antibiotic resistances and the lack of novel drugs in the development pipeline, there is a renewed interest in (unexplored) cell wall components and underlaying biosynthesis pathways to find alternate options to treat infections caused by multidrug-resistant bacteria [ 12 ]. Furthermore, antibiotic inhibition of bacterial cell wall biosynthesis induces both common and compound-specific transcriptional responses, which both can be exploited to increase antibiotic susceptibility [ 13 ]. 2.2. Types of Glycopolymers Attached to PGN 2.2.1. General Considerations The covalent attachment of various glycopolymers to PGN (cell wall glycopolymers—CWGP) is an enigmatic feature of the cell wall assembly of Gram-positive bacteria. CWGPs are mostly found to be anionic, typically composed of individual repeating units (RU) of varying degrees of polymerization, and can make up to 50% of the dry weight of the cell wall [ 14 ]. Frequently, they are tethered to the C6-OH group of MurNAc residues of the PGN glycan backbone strands via a phosphodiester bond, but also the GlcNAc residues can be modified [ 4 , 15 , 16 ]. CWGPs are involved in a myriad of crucial cellular functions and serve as a rich source for both validated and unexploited pathways that are essential for bacterial virulence and survival [ 17 , 18 , 19 ]. On the basis of their structural characteristics, PGN-bound CWGPs of Gram-positive bacteria can be classified into two major groups: (i) "classical" CWGPs, comprising wall teichoic acids (WTAs) [ 20 , 21 ] and teichuronic acids [ 14 , 22 ], and (ii) "non-classical" CWGPs, which is the more diverse group comprising all other anionic and neutral CWGPs [ 23 , 24 ]. To these CWGPs, initially only secondary roles in cell wall function have been attributed; hence, they have been named "secondary" cell wall polymers. However, given the rich body of knowledge that has accumulated over the past 40 years, it is evident that these compounds play pivotal roles in bacterial cell function, physiology and virulence; thus, nowadays, the term "CWGPs" is more appropriate [ 16 ]. Based on detailed investigations of the lipopolysacchaide (LPS) biosynthesis in Gram-negative bacteria [ 25 ], two major principal assembly routes are known for bacterial CWGPs—the ABC-transporter dependent pathway and the Wzy-dependent pathway [ 26 ]. In many bacteria, the genetic information for CWGP biosynthesis is encoded in genomic biosynthesis gene loci or clusters. However, given the interchangeability of modules between the routes, the prediction of a distinct route based on the bacterial genome content is impossible. Generally, the interplay between different CWGP assembly routes—including also that for the PGN scaffold itself—concerns common pools of activated monosaccharides, biosynthetic intermediates, of the C 55 undecaprenylphosphat (undp- P ) lipid carrier and, importantly, enzymatic machineries [ 27 ]. This becomes evident upon inhibition of individual biosynthetic steps. However, it is still largely unknown how the different CWGP biosynthesis pathways, which share building blocks and membrane carriers, function in a coordinated and integrated fashion [ 28 ]. While the coupling of different CWGPs to the PGN has long been discovered [ 29 , 30 ], until the recently exposed LytR-CpsA-Psr (LCP) enzyme family, there were not any plausible candidates for this coupling reaction [ 29 ]. In this review, we are discussing different CWGPs from various bacterial sources, for which information on their ligation to PGN is available in the literature. However, studying LCP enzymes is challenging considering that frequently different types of CWGPs are simultaneously present in a bacterium and that the presence of multiple copies of LCP proteins is the rule rather than the exception. 2.2.2. Wall Teichoic Acids Wall teichoic acids (WTAs) are the most abundant and best-investigated PGN-linked CWGPs in many Gram-positive organisms [ 14 ]. They are composed of repeating units of alditol phosphates linked by phosphodiester bonds and can be further substituted by amino acids (e.g., D-alanine) and/or carbohydrate residues. Typically, Bacillus subtilis produces glycerol phosphate (Gro- P ) or ribitol phosphate (Rbo- P ) WTAs, while in S. aureus , commonly Rbo- P WTAs are present [ 16 , 31 ] ( Figure 1 ). In either case, a conserved β-D-Man p NAc-(1→4)-α-D-Glc p NAc-(1→P murein-linkage unit at the reducing end of the glycopolymer mediates WTA attachment to the PGN. In fact, this "conventional" murein linkage unit is also found in several other CWGPs, as is explained below. The assembly route of WTAs follows the ABC-transporter dependent route [ 18 , 19 ]. This implicates CWGP assembly on a C 55 undp- P carrier in the cytoplasm, involving the formation of the "conventional" β-D-Man p NAc-(1→4)-α-D-Glc p NAc-(1→ P murein linkage unit and polymer elongation using individual nucleoside-diphosphate (NDP)-bound substrates in concert with dedicated glycosyltransferases, followed by glycopolymer export to the outer leaflet of the membrane by an ABC-transporter. Upon reaching the exterior of the cell, WTAs are further functionalized and, finally, transferred to PGN in a ligation reaction involving LCP enzymes [ 19 ]. Conserved enzymes for the biosynthesis of the "conventional" murein linkage unit are TagO and TagA [ 18 , 33 , 34 ]. The first enzyme in this pathway, TagO, is an integral membrane protein that transfers GlcNAc-phosphate from UDP-GlcNAc to the undp- P lipid embedded in the cytoplasmic membrane [ 35 , 36 ]. The lipid-linked monosaccharide is then elongated to a disaccharide by the UDP-ManNAc transferase TagA [ 37 , 38 , 39 ]. This lipid-linked disaccharide constitutes the platform for the subsequent steps of WTA biosynthesis [ 29 , 40 , 41 , 42 , 43 ] and other CWGPs [ 44 , 45 ]. WTAs are intimately involved in many aspects of cell division and essential for maintaining cell shape in rod-shaped organisms [ 41 ]. Importantly, WTAs are key determinants of virulence and antibiotic resistance—e.g., in methicillin-resistant staphylococci (MRSA) or in streptococci—and have, thus, been the target of numerous screening campaigns to find inhibitors [ 46 , 47 ]. 2.2.3. Pyruvylated CWGPs Pyruvylated CWGPs (PyrCWGP) are a less investigated class of PGN-attached CWGPs which are of interest in the context of protein cell surface display in Gram-positive bacteria [ 48 ]. These SCWPs are 5–20 kDa in size, composed of species-specific repeats [ 6 , 49 ], but lack repetitive alditol phosphates and phosphodiester bonds of WTAs [ 15 , 50 , 51 ], hence, categorized as "non-classical" CWGPs. Importantly, they contain 4,6-pyruvateketal-modified-β-D- N -acetylmannosamine (Pyr-β-D-Man p NAc) imparting a negative charge, which serves as a specific cell wall ligand for S-layer homology (SLH) domains that are usually present in triplicates at the termini of cell surface proteins [ 48 , 52 , 53 ]. Prominent examples of SLH-domain proteins are surface (S-) layer proteins which self-assemble into two-dimensional crystalline arrays on the bacterial cell surface [ 54 , 55 ]. S-layer proteins are important for many biological functions such as maintenance of cell integrity, enzyme display, protection to phagocytosis, and interactions with the host and its immune system [ 56 ]. Best investigated pyruvylated CWGPs are those from Paenibacillus alvei CCM 2051 T and Bacillus anthracis . The B. anthracis CWGP is composed of →4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→ trisaccharide RUs with strain-dependent galactosylation occurring at the GlcNAc residues [ 57 , 58 ]; Pyr-β-D-Man p NAc is exclusively contained in the terminal repeat [ 51 , 57 , 58 , 59 ] ( Figure 1 ). In contrast, the CWGP of P. alvei is multiply pyruvylated and consists of →3)-Pyr-β-D-Man p NAc-(1→4)-β-D-Glc p NAc-(1→ RUs [ 60 , 61 ]. Notably, a common structural feature of WTAs and pyruvylated CWGPs is the presence of the "conventional" β-D-Man p NAc-(1→4)-α-D-Glc p NAc-(1→ P murein linkage unit. 2.2.4. Capsules Capsules are long-chain polysaccharides (CPS), which are produced by many bacteria of both Gram-classes [ 62 ]. They can either maintain a strong association with the enveloping bacterial cell and/or be secreted into the immediate environment in the form of exopolysaccharides [ 63 ]. Capsules afford the producing bacteria protection from a wide range of physical, chemical, and biological stresses, support biofilms, and play critical roles in interactions between bacteria and their immediate environments [ 63 ]. The Wzy pathway constitutes a prototypical mechanism to produce these structures. Briefly, the basic CPS RUs are synthesized at the cytosolic side of the membrane by the sequential action of glycosyltransferases using NDP-sugar substrates. The RU is anchored to a undp- P membrane lipid, and it is transferred to the outer side of the membrane via a Wzx flippase, where it is polymerized into the full-length CPS by the addition of new repeat units to the reducing end of the glycopolymer in a reaction requiring the Wzy polymerase and the chain-length regulator Wzz. In some Gram-positive bacteria, the lipid-bound CPS precursors serve as substrates for LCP enzyme-catalysed coupling of CPS to PGN, thus creating the mucoid capsule layer covering the bacterial surface [ 28 , 63 , 64 ]. 2.2.5. Arabinogalactan Arabinogalactan (AG) is a heteropolysaccharide found in covalent attachment to PGN via a phosphoryl- N -acetylglucosaminyl-rhamnosyl linkage unit with the structure →4)-α-L-Rha p -(1→3)-α-D-Glc p NAc-(1→ P to MurNAc residues of the mycobacterial A1γ-type PGN ( Figure 1 ). AG comprises a galactofuran domain bound to the linkage unit which is extended by the arabinofuran domain, which is, in turn, esterified at its non-reducing ends to long-chain (C 70 –C 90 ) mycolic acids forming the inner leaflet of the mycomembrane [ 65 , 66 ]. Notably, mycobacteria stain Gram-indifferently and the compositional and architectural complexity of the mycobacterial cell envelope distinguishes species of the Mycobacterium genus from other prokaryotes. It is the basis of many of the physiological and pathogenic features of mycobacteria and the site of susceptibility and resistance to many anti-tuberculosis drugs [ 67 , 68 ]. The synthesis of AG is initiated in the cytoplasm on a C 50 -undp- P carrier lipid with formation of the murein linkage unit [ 69 , 70 ] followed by the addition of Gal f and Ara f residues [ 71 , 72 , 73 ] and AG export, potentially involving an ABC-transporter [ 74 ]. It is thought that the GlcNAc residue of the linkage unit of the mature AG next forms a 1- O -phosphoryl linkage with the C6-OH position of a MurNAc residue of PGN [ 71 ] and that this transfer reaction is catalysed by an LCP family protein [ 65 ], which requires newly synthesized PGN undergoing concomitant cross-linking [ 75 ]. Despite the fundamental structural differences that exist between AG and WTAs, the structure of the AG-PGN linker shares similarity with the "conventional" murein linkage unit of WTAs with regard to the reducing-end GlcNAc residue and, evidently, LCP proteins are involved in the final ligation of AG to PGN [ 66 ]. 2.2.6. Rhamnose-Containing Cell Wall Glycopolymers Several members of the order Lactobacillales (mainly streptococcal species and Lactococcus lactis ) can generate a specific class of abundant CWGPs, which are composed of individual RUs, where rhamnose is the major constituent, along with variable combinations and linkages of Glc, GlcNAc, Gal, GalNAc, and phosphate [ 76 ]. This heterogeneous group of rhamnose-containing CWGPs is named RhaCWGPs [ 76 ]. For the Group B Streptococcus carbohydrate (GBC) and Group G carbohydrate (GGC), rhamnose is the major antigenic determinant. Species carrying these structures are serologically discriminated based on the presence of either a single rhamnose in GGC versus triterminal α-L-(1→2)-Rha p in GBC in the RhaCWGP RU (for review, see [ 76 ]). Among the streptococcal group antigens, the GBC is unique since it forms a multiantenna branching structure and is negatively charged, due to the presence of phosphodiester bonds that link different GBC repeat units. Similarly, the L. lactis RhaCWGP contains phosphodiester bonds that link branched →6)-β-GlcNAc-(1→3)-Rha-(1→3)[α-Glc–(1→6)]-β-GlcNAc-(1→2)-β-Gal f -(1→6)-α-Glc- P -(1→ hexasaccharide RUs [ 77 ] ( Figure 1 ). RhaCWGPs comprise about 40–60% of the bacterial cell wall by weight, and they are localized on the outermost surface of the cell wall but are likely also intercalated within the PGN layer, since antibodies directed against these structures bind to both sides of isolated cell walls [ 78 ]. According to recent evidence obtained for the glucose-containing RhaCWGP of Streptococcus mutans (also referred to as rhamnose-glucose polysaccharide–RGP) ( Figure 1 ), RhaCWGPs are assembled on a lipid carrier in the cytoplasm and exported via an ABC-transporter followed by covalent attachment to the PGN via the likely activity of an LCP family protein [ 79 ]. Of note, L-rhamnose as integral constituent of RhaCWGPs is often essential for bacterial virulence or even viability [ 80 ], making its biosynthesis pathway an attractive therapeutic target. 2.2.1. General Considerations The covalent attachment of various glycopolymers to PGN (cell wall glycopolymers—CWGP) is an enigmatic feature of the cell wall assembly of Gram-positive bacteria. CWGPs are mostly found to be anionic, typically composed of individual repeating units (RU) of varying degrees of polymerization, and can make up to 50% of the dry weight of the cell wall [ 14 ]. Frequently, they are tethered to the C6-OH group of MurNAc residues of the PGN glycan backbone strands via a phosphodiester bond, but also the GlcNAc residues can be modified [ 4 , 15 , 16 ]. CWGPs are involved in a myriad of crucial cellular functions and serve as a rich source for both validated and unexploited pathways that are essential for bacterial virulence and survival [ 17 , 18 , 19 ]. On the basis of their structural characteristics, PGN-bound CWGPs of Gram-positive bacteria can be classified into two major groups: (i) "classical" CWGPs, comprising wall teichoic acids (WTAs) [ 20 , 21 ] and teichuronic acids [ 14 , 22 ], and (ii) "non-classical" CWGPs, which is the more diverse group comprising all other anionic and neutral CWGPs [ 23 , 24 ]. To these CWGPs, initially only secondary roles in cell wall function have been attributed; hence, they have been named "secondary" cell wall polymers. However, given the rich body of knowledge that has accumulated over the past 40 years, it is evident that these compounds play pivotal roles in bacterial cell function, physiology and virulence; thus, nowadays, the term "CWGPs" is more appropriate [ 16 ]. Based on detailed investigations of the lipopolysacchaide (LPS) biosynthesis in Gram-negative bacteria [ 25 ], two major principal assembly routes are known for bacterial CWGPs—the ABC-transporter dependent pathway and the Wzy-dependent pathway [ 26 ]. In many bacteria, the genetic information for CWGP biosynthesis is encoded in genomic biosynthesis gene loci or clusters. However, given the interchangeability of modules between the routes, the prediction of a distinct route based on the bacterial genome content is impossible. Generally, the interplay between different CWGP assembly routes—including also that for the PGN scaffold itself—concerns common pools of activated monosaccharides, biosynthetic intermediates, of the C 55 undecaprenylphosphat (undp- P ) lipid carrier and, importantly, enzymatic machineries [ 27 ]. This becomes evident upon inhibition of individual biosynthetic steps. However, it is still largely unknown how the different CWGP biosynthesis pathways, which share building blocks and membrane carriers, function in a coordinated and integrated fashion [ 28 ]. While the coupling of different CWGPs to the PGN has long been discovered [ 29 , 30 ], until the recently exposed LytR-CpsA-Psr (LCP) enzyme family, there were not any plausible candidates for this coupling reaction [ 29 ]. In this review, we are discussing different CWGPs from various bacterial sources, for which information on their ligation to PGN is available in the literature. However, studying LCP enzymes is challenging considering that frequently different types of CWGPs are simultaneously present in a bacterium and that the presence of multiple copies of LCP proteins is the rule rather than the exception. 2.2.2. Wall Teichoic Acids Wall teichoic acids (WTAs) are the most abundant and best-investigated PGN-linked CWGPs in many Gram-positive organisms [ 14 ]. They are composed of repeating units of alditol phosphates linked by phosphodiester bonds and can be further substituted by amino acids (e.g., D-alanine) and/or carbohydrate residues. Typically, Bacillus subtilis produces glycerol phosphate (Gro- P ) or ribitol phosphate (Rbo- P ) WTAs, while in S. aureus , commonly Rbo- P WTAs are present [ 16 , 31 ] ( Figure 1 ). In either case, a conserved β-D-Man p NAc-(1→4)-α-D-Glc p NAc-(1→P murein-linkage unit at the reducing end of the glycopolymer mediates WTA attachment to the PGN. In fact, this "conventional" murein linkage unit is also found in several other CWGPs, as is explained below. The assembly route of WTAs follows the ABC-transporter dependent route [ 18 , 19 ]. This implicates CWGP assembly on a C 55 undp- P carrier in the cytoplasm, involving the formation of the "conventional" β-D-Man p NAc-(1→4)-α-D-Glc p NAc-(1→ P murein linkage unit and polymer elongation using individual nucleoside-diphosphate (NDP)-bound substrates in concert with dedicated glycosyltransferases, followed by glycopolymer export to the outer leaflet of the membrane by an ABC-transporter. Upon reaching the exterior of the cell, WTAs are further functionalized and, finally, transferred to PGN in a ligation reaction involving LCP enzymes [ 19 ]. Conserved enzymes for the biosynthesis of the "conventional" murein linkage unit are TagO and TagA [ 18 , 33 , 34 ]. The first enzyme in this pathway, TagO, is an integral membrane protein that transfers GlcNAc-phosphate from UDP-GlcNAc to the undp- P lipid embedded in the cytoplasmic membrane [ 35 , 36 ]. The lipid-linked monosaccharide is then elongated to a disaccharide by the UDP-ManNAc transferase TagA [ 37 , 38 , 39 ]. This lipid-linked disaccharide constitutes the platform for the subsequent steps of WTA biosynthesis [ 29 , 40 , 41 , 42 , 43 ] and other CWGPs [ 44 , 45 ]. WTAs are intimately involved in many aspects of cell division and essential for maintaining cell shape in rod-shaped organisms [ 41 ]. Importantly, WTAs are key determinants of virulence and antibiotic resistance—e.g., in methicillin-resistant staphylococci (MRSA) or in streptococci—and have, thus, been the target of numerous screening campaigns to find inhibitors [ 46 , 47 ]. 2.2.3. Pyruvylated CWGPs Pyruvylated CWGPs (PyrCWGP) are a less investigated class of PGN-attached CWGPs which are of interest in the context of protein cell surface display in Gram-positive bacteria [ 48 ]. These SCWPs are 5–20 kDa in size, composed of species-specific repeats [ 6 , 49 ], but lack repetitive alditol phosphates and phosphodiester bonds of WTAs [ 15 , 50 , 51 ], hence, categorized as "non-classical" CWGPs. Importantly, they contain 4,6-pyruvateketal-modified-β-D- N -acetylmannosamine (Pyr-β-D-Man p NAc) imparting a negative charge, which serves as a specific cell wall ligand for S-layer homology (SLH) domains that are usually present in triplicates at the termini of cell surface proteins [ 48 , 52 , 53 ]. Prominent examples of SLH-domain proteins are surface (S-) layer proteins which self-assemble into two-dimensional crystalline arrays on the bacterial cell surface [ 54 , 55 ]. S-layer proteins are important for many biological functions such as maintenance of cell integrity, enzyme display, protection to phagocytosis, and interactions with the host and its immune system [ 56 ]. Best investigated pyruvylated CWGPs are those from Paenibacillus alvei CCM 2051 T and Bacillus anthracis . The B. anthracis CWGP is composed of →4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→ trisaccharide RUs with strain-dependent galactosylation occurring at the GlcNAc residues [ 57 , 58 ]; Pyr-β-D-Man p NAc is exclusively contained in the terminal repeat [ 51 , 57 , 58 , 59 ] ( Figure 1 ). In contrast, the CWGP of P. alvei is multiply pyruvylated and consists of →3)-Pyr-β-D-Man p NAc-(1→4)-β-D-Glc p NAc-(1→ RUs [ 60 , 61 ]. Notably, a common structural feature of WTAs and pyruvylated CWGPs is the presence of the "conventional" β-D-Man p NAc-(1→4)-α-D-Glc p NAc-(1→ P murein linkage unit. 2.2.4. Capsules Capsules are long-chain polysaccharides (CPS), which are produced by many bacteria of both Gram-classes [ 62 ]. They can either maintain a strong association with the enveloping bacterial cell and/or be secreted into the immediate environment in the form of exopolysaccharides [ 63 ]. Capsules afford the producing bacteria protection from a wide range of physical, chemical, and biological stresses, support biofilms, and play critical roles in interactions between bacteria and their immediate environments [ 63 ]. The Wzy pathway constitutes a prototypical mechanism to produce these structures. Briefly, the basic CPS RUs are synthesized at the cytosolic side of the membrane by the sequential action of glycosyltransferases using NDP-sugar substrates. The RU is anchored to a undp- P membrane lipid, and it is transferred to the outer side of the membrane via a Wzx flippase, where it is polymerized into the full-length CPS by the addition of new repeat units to the reducing end of the glycopolymer in a reaction requiring the Wzy polymerase and the chain-length regulator Wzz. In some Gram-positive bacteria, the lipid-bound CPS precursors serve as substrates for LCP enzyme-catalysed coupling of CPS to PGN, thus creating the mucoid capsule layer covering the bacterial surface [ 28 , 63 , 64 ]. 2.2.5. Arabinogalactan Arabinogalactan (AG) is a heteropolysaccharide found in covalent attachment to PGN via a phosphoryl- N -acetylglucosaminyl-rhamnosyl linkage unit with the structure →4)-α-L-Rha p -(1→3)-α-D-Glc p NAc-(1→ P to MurNAc residues of the mycobacterial A1γ-type PGN ( Figure 1 ). AG comprises a galactofuran domain bound to the linkage unit which is extended by the arabinofuran domain, which is, in turn, esterified at its non-reducing ends to long-chain (C 70 –C 90 ) mycolic acids forming the inner leaflet of the mycomembrane [ 65 , 66 ]. Notably, mycobacteria stain Gram-indifferently and the compositional and architectural complexity of the mycobacterial cell envelope distinguishes species of the Mycobacterium genus from other prokaryotes. It is the basis of many of the physiological and pathogenic features of mycobacteria and the site of susceptibility and resistance to many anti-tuberculosis drugs [ 67 , 68 ]. The synthesis of AG is initiated in the cytoplasm on a C 50 -undp- P carrier lipid with formation of the murein linkage unit [ 69 , 70 ] followed by the addition of Gal f and Ara f residues [ 71 , 72 , 73 ] and AG export, potentially involving an ABC-transporter [ 74 ]. It is thought that the GlcNAc residue of the linkage unit of the mature AG next forms a 1- O -phosphoryl linkage with the C6-OH position of a MurNAc residue of PGN [ 71 ] and that this transfer reaction is catalysed by an LCP family protein [ 65 ], which requires newly synthesized PGN undergoing concomitant cross-linking [ 75 ]. Despite the fundamental structural differences that exist between AG and WTAs, the structure of the AG-PGN linker shares similarity with the "conventional" murein linkage unit of WTAs with regard to the reducing-end GlcNAc residue and, evidently, LCP proteins are involved in the final ligation of AG to PGN [ 66 ]. 2.2.6. Rhamnose-Containing Cell Wall Glycopolymers Several members of the order Lactobacillales (mainly streptococcal species and Lactococcus lactis ) can generate a specific class of abundant CWGPs, which are composed of individual RUs, where rhamnose is the major constituent, along with variable combinations and linkages of Glc, GlcNAc, Gal, GalNAc, and phosphate [ 76 ]. This heterogeneous group of rhamnose-containing CWGPs is named RhaCWGPs [ 76 ]. For the Group B Streptococcus carbohydrate (GBC) and Group G carbohydrate (GGC), rhamnose is the major antigenic determinant. Species carrying these structures are serologically discriminated based on the presence of either a single rhamnose in GGC versus triterminal α-L-(1→2)-Rha p in GBC in the RhaCWGP RU (for review, see [ 76 ]). Among the streptococcal group antigens, the GBC is unique since it forms a multiantenna branching structure and is negatively charged, due to the presence of phosphodiester bonds that link different GBC repeat units. Similarly, the L. lactis RhaCWGP contains phosphodiester bonds that link branched →6)-β-GlcNAc-(1→3)-Rha-(1→3)[α-Glc–(1→6)]-β-GlcNAc-(1→2)-β-Gal f -(1→6)-α-Glc- P -(1→ hexasaccharide RUs [ 77 ] ( Figure 1 ). RhaCWGPs comprise about 40–60% of the bacterial cell wall by weight, and they are localized on the outermost surface of the cell wall but are likely also intercalated within the PGN layer, since antibodies directed against these structures bind to both sides of isolated cell walls [ 78 ]. According to recent evidence obtained for the glucose-containing RhaCWGP of Streptococcus mutans (also referred to as rhamnose-glucose polysaccharide–RGP) ( Figure 1 ), RhaCWGPs are assembled on a lipid carrier in the cytoplasm and exported via an ABC-transporter followed by covalent attachment to the PGN via the likely activity of an LCP family protein [ 79 ]. Of note, L-rhamnose as integral constituent of RhaCWGPs is often essential for bacterial virulence or even viability [ 80 ], making its biosynthesis pathway an attractive therapeutic target. 3. Lytr-CpsA-Psr (LCP) Enzymes—A General Perspective The LCP family of proteins is a conserved family of phosphotransferases catalysing the formation of a phosphodiester bond to link CWGPs onto the MurNAc or GlcNAc residues of PGN. These enzymes have aroused great interest because of their role in Gram-positive bacterial cell envelope maintenance and influence on various virulence factors as well as antibiotic resistance of human pathogens [ 40 , 49 , 81 ]. Members of this enzyme family were discovered in eight bacterial phyla— Actinobacteria, Bacteroidetes , Chloroflexi , Cyanobacteria , Deinococcus-Thermus , Firmicutes , Spirochaetes , and Thermotogae , and their Lytr-CpsA-Psr (LCP) domain is unique to the bacterial kingdom [ 49 ]. The "LCP" acronym derives from three proteins initially identified to contain a LytR domain—LytR (lytic repressor, now TagU5), CpsA (capsular polysaccharide expression regulator), and Psr (PBP 5 synthesis repressor). Typically, LCP proteins have a common structural organization made up of an N-terminal transmembrane (TM) domain required for anchoring, an optional, non-conserved accessory domain (CATH 3tflA01), a core catalytic domain that is predicted to be extracellular, and, sometimes, a C-terminal domain of unknown structure. Furthermore, the core LCP domain is a magnesium-dependent enzyme [ 82 ]. Despite their great abundance in Gram-positive bacteria, the precise role of LCP enzymes in cell wall assembly and their catalytic function(s) are only beginning to be discovered. There is evidence that LCP enzymes utilize C 55 lipid-phosphate bound CWGP precursor substrates and that the ligation process likely releases the lipid carrier, which enters new synthesis cycles. Several cardinal points of LCP enzyme activity are remaining to be clarified in future studies. From a catalytic perspective, these concern i) enzyme stringency versus promiscuity for the glycopolymer's PGN linkage unit, ii) relevance of the polyisoprenoid portion of the CWGP precursor, and iii) role of the maturation stage of the PGN acceptor. From a biological perspective, points to be clarified concern iv) the role of multiple LCP proteins in a given bacterium, i.e., if there is (partly) functional redundancy or preference for the transfer of a distinct of coexisting CWGPs, and v) the intracellular control for LCP enzyme expression ( Figure 2 ). 4. LCP Enzymes According to Bacterial Species Experimental evidence of LCP enzyme function is available for bacteria affiliated to the phyla Firmicutes , Actinobacteria , and Cyanobacteria . 4.1. Firmicutes— Order : Bacillales 4.1.1. Staphylococcus aureus S. aureus is a Gram-positive opportunistic pathogen of which certain strains have become resistant to most antibiotic classes [ 83 , 84 , 85 ]. The bacterium can lead to systemic failures, such as infective endocarditis or bacteraemia via nosocomial acquisition [ 83 , 86 ]. The S. aureus WTA is a polymer of 30 to 50 Rbo- P subunits connected via 1,5-phosphodiester bonds [ 16 ], which is tethered to PGN via the "conventional" murein linkage unit [ 87 ] ( Figure 1 ). Furthermore, S. aureus expresses a CPS, which substantially contributes to the bacterium's ability to cause invasive disease [ 88 ]. Specifically, capsular polysaccharide type 5 (CP5) is composed of →4)-β-D-Man p NAcA-(1→3)-α-L-Fuc p NAc-(1→4)-β-D-Fuc p NAc-(1→ repeats with O -acetylation on all L-Fuc p NAc residues except for the one in the reducing-end RU [ 28 ] ( Figure 1 ). Of note, CP5 biosynthesis is TagO-independent resulting in the absence of a "conventional" murein linkage unit in the capsule. S. aureus LcpA, LcpB, LcpC at a Glance The S. aureus genome harbours three LCP proteins, encoded by lcpA (previous name, msrR ), lcpB ( sa0908 ), and lcpC ( sa2103 ) [ 49 , 89 ], which are in part functionally redundant regarding cellular functions [ 29 , 90 ]. S. aureus variants carrying defective alleles of lcp genes resulted in enhanced [ 91 , 92 ] susceptibility to β-lactam antibiotics, deviant septum formation [ 91 , 92 ], autolysis [ 91 ], activation of a cell wall stress response [ 93 ], reduced phosphate content of staphylococcal cell walls [ 93 ], and aberrant biofilm formation [ 92 ]. In strains lacking WTA due to the inactivation of LCP function, major cell division defects were shown, the PGN synthesis machinery was not localized properly, and these strains were unable of nasal epithelial cell colonization [ 94 , 95 ]. Furthermore, methicillin-resistant S. aureus (MRSA) strains were found to become sensitive to β-lactam antibiotics when WTA synthesis was abrogated [ 95 , 96 ]. S. aureus LcpA, LcpB, LcpC in CWGP Biosynthesis Due to their demonstrated biological importance, the biochemical activity of S. aureus LCP proteins is under intense investigation [ 89 ]. Deletion of all three LCP genes resulted in complete WTA loss in the staphylococcal cell wall and deletion of any of individual LCP genes disturbed the attachment of WTA in different degrees [ 89 ]. This partial functional redundancy was also seen for different phenotypes including β-lactam resistance, biofilm formation, and growth defects [ 91 ]. Of note, LcpA was shown to be the most important protein related to WTA-functions [ 84 ]. In a reconstitution approach, cognate Rbo- P WTA precursors including the murein-linkage unit ( Figure 1 ) could be transferred to PGN by truncated versions of either of the three staphylococcal LCP proteins devoid of the TM-segment (ΔTM) [ 29 ], without the requirement of any other proteins, as had been initially suggested [ 82 ]. For the ligation, the WTA substrate needs to be comprised of only two sugar residues (i.e., the "conventional" murein linkage unit) and a hexaprenyl chain, mimicking the truncated native C 55 undp lipid. This is consistent with the length of the hydrophobic channel length observed in the crystal structure of Cps2A—the LCP protein transferring CPS in Sc. pneumoniae [ 82 , 97 ]. Importantly, the S. aureus WTA precursor could be transferred to "nascent" (i.e., un-crosslinked) PGN polymers only [ 98 ], not to lipid II ( i.e ., murein pentapeptides) [ 29 ], indicating that modification of PGN with WTA occurs prior to final PGN cross-linking [ 98 ]. Deletion of lcpC had no effect on the level of WTA that was ligated to PGN, whereas a Δ lcpA or a Δ lcpB mutant showed reduced levels of WTA content [ 89 , 93 ]. This is corroborated by the finding that the Δ lcpC mutant showed no phosphate release compared to single Δ lcpA and Δ lcpB mutants [ 89 ]. Differential localization and regulation of the enzymes might be important factors regarding the greater impact of LcpA and LcpB on WTA synthesis [ 93 ]. Genes involved in S. aureus CPS synthesis, exemplified with CP5 ( Figure 1 ), are clustered presenting conserved genes employing a Wzy-dependent biosynthesis mechanism [ 64 , 90 ], and there is evidence that specifically LcpC plays a key role in catalysis of S. aureus capsule attachment to the PGN [ 28 , 29 , 90 ]. This is corroborated by the lack of a cpsA homologue encoding a CPS ligase, typically involved in streptococcal CPS ligation, in the S. aureus genome [ 49 , 99 ]. Initial evidence of the requirement of LcpC for ligation of S. aureus CP5 to PGN was derived from a mutant approach [ 90 ]. The Δ lcpC mutant accumulated the capsule in the supernatant fraction, while the Δ lcpAB variants did not display any defect in CP5 synthesis [ 90 ]. A triple mutant showed the same levels of CP5 reduction as the variant lacking lcpC. Surprisingly, plasmid-based expression of any of the three lcp genes could restore the CP5 content, indicating that all three LCP proteins are to some extent involved in the attachment [ 90 ]. To investigate the proposed role of LcpC in vitro, different [ 14 C] CP5 lipid intermediates were synthesized, including lipid I cap (i.e., C 55 undp- PP -D-FucNAc), lipid II cap (i.e., C 55 undp- PP -D-FucNAc-L-FucNAc), and lipid III cap (i.e., C 55 undp- PP -D-FucNAc-L-FucNAc-D-ManNAcA). After purification, these were used together with the ultimate PGN precursor lipid II (lipid II PGN ), i.e., C 55 undp- PP -D-MurNAc-D-GlcNAc including a pentapeptide, as a potential acceptor substrate [ 28 ]. In this setup, LcpC was able to catalyse cleavage of the donor substrate lipid I cap and catalyse attachment of the phosphoryl-sugar moiety to the ultimate PGN precursor lipid II. Strikingly, in the presence of CapA1, an activator/phosphodiesterase protein that cleaves lipid-PP-linked CP5 precursors [ 28 ], the transfer rate was increased, possibly by forming an interaction complex between CapA1 and LcpC [ 28 , 100 ]. Surprisingly, in the case of lipid II cap , no transfer to the PGN acceptor could be overserved in the LcpC in vitro assay, which would likely be deleterious in vivo. On the other hand, all CP5 lipid intermediates were effectively processed by LcpC, although the proximal undp- PP -linked FucNAc residue appeared to be sufficient for CP5 precursor recognition. Of note, the natural PGN acceptor substrate of LcpC remains elusive; possible acceptor structures include lipid II PGN , as well as "nascent" and cross-linked PGN. Summarizing, there is evidence that LcpC preferentially recognizes CP5 intermediates rather than WTA intermediates where deletion of lcpC caused only minor reduction levels [ 89 , 90 ]. Whether this is due to the different reducing-end sugars (i.e., D-FucNAc in the CP5 repeat versus D-Glc p NAc in the "conventional" WTA murein linkage unit remains to be investigated. Notably, in LPS O-antigen RU biosynthesis, the first C 55 lipid- PP -linked sugar unit of the O-antigen RU contains all necessary recognition information for the catalytic activity of the O-antigen ligase WaaL [ 101 ]. S. aureus lcpA, lcpB, lcpC Genes and Physiological Effects Deletion of individual S. aureus LCP protein encoding genes showed only minor effects on S. aureus cell wall physiology and growth, whereas a Δ lcp triple mutant was barely viable, showing temperature sensitivity and enlarged cells [ 91 ]. Complementation with LcpA resulted in restoration of growth and cell size almost to wild-type size [ 91 ]. Interestingly, Δ lcp and Δ lcp Δ tagO variants abolished cell division planes by generating aberrant cells with irregular envelopes lacking WTA [ 89 ]. Of note, deletion of tagO alone did not abolish staphylococcal growth [ 35 , 39 , 102 ]. The same phenotype was observed for isolated tagO and lcpA mutants; however, deletion of lcpA did not interrupt WTA synthesis and thus suggests that WTA synthesis as well as assembly are crucial for normal cell division [ 89 , 95 ]. Furthermore, inhibition of WTA synthesis by tunicamycin treatment did not relieve deviant cell separation of the mutant cells and, therefore, did not suppress the cell division defects of Δ lcp variants [ 89 ]. Autolysis was induced by deletion of all three LCP proteins, resulting in an increased resistance of the Δ lcp triple mutant to autolysis compared to the wild-type [ 89 ]. Complementation by LcpA increased autolysis levels the most [ 89 ]. These pleiotropic phenotypes in the Δ lcp mutant are likely owed to WTA cell wall deposition defects and WTA synthesis, as staphylococcal cells without WTA resulted in deviant cell size and septum formation as well as susceptibility to antibiotics and biofilm formation defects [ 49 , 91 , 92 , 93 ]. A comparison of the surface proteomes of methicillin-resistant, laboratory-adapted S. aureus COL strain (COL) and a COL strain in vitro-adapted to high levels of oxacillin (APT) was used to characterize virulence factors showing that LcpC was found uniquely on the APT surface, suggesting a role in adaption to high oxacillin levels [ 84 ]. Deletion of lcpA decreased oxacillin resistance and upregulated lcpA expression was observed by triggered antibiotic stress [ 103 ] and, furthermore, increased cell size and elevated cell wall remodelling was revealed [ 84 , 92 ]. However, overexpression of LcpC did not generate the APT phenotype in COL, suggesting that aggregation and changes in cell morphology are multifactorial [ 84 ]. The adaption of LcpC to high levels of oxacillin in the APT strain prompted the question about the contribution of this enzyme to antimicrobial resistance and pathogenicity [ 104 ]. The finding that deletion of lcpC decreased the resistance to β-lactams in methicillin-resistant S. aureus (MRSA) and in methicillin-susceptible S. aureus (MSSA) and, consequently, the pathogenicity in the host, suggested that the deviant cell shape might allow for an easier access of the antibiotics to the cell [ 104 ]. Thus, LcpC could be an effective target for drug development [ 104 ]. In MSSA and MRSA strains, reduced oxacillin resistance levels were also observed upon lcpA deletion [ 91 , 92 , 103 ]. The Δ lcp triple mutant was hypersusceptible to oxacillin and growth could be restored by any of the three LCP proteins; LcpA had the greatest impact, followed by LcpB and LcpC [ 91 ]. Interestingly, the Δ lcpA mutant produced more biofilm, and in the Δ lcp triple mutant complementation with LcpC revealed the strongest biofilm, LcpA the weakest [ 91 ]. A mutation in lcpA (E146K) was shown to have an impact on β-lactam and vancomycin resistances and led to a reduced resistance to oxacillin, whereby the cells showed abnormal septal placement [ 105 ]. The mutation further led to a decreased autolytic activity, as was also evident form highest autolysis levels obtained after complementation of the Δ lcp triple mutant with LcpA [ 91 , 105 ]. Crystal Structure of S. aureus LcpA The crystal structure of LcpA devoid of its TM (residues 80–327; ΔTM-LcpA) complexed to C 40 - PP -GlcNAc was solved to 1.9 à and provides a relevant target for inhibitor design studies [ 106 ] ( Figure 3 ). The extracellular domain consists of six-stranded β-sheets overlaid amongst several α-helices and double-stranded β-sheets, where a hydrophobic binding pocket narrow opening and a wide base is formed from its center [ 106 ]. The active site of LcpA is enclosed by region A (residues 92–100), B (residues 188–201), C (residues 217–224) and D (residues 296–312) and shows structural variability. The electropositive region in the active site, as seen in other LCP enzymes, consists of conserved arginine residues. R218 located in the loop of the highly flexible region C is suggested to aid in product expulsion and is held away from the active site by a salt bridge. Furthermore, three potential PGN saccharide binding sites were identified in close proximity to the conserved regions of R99, K135, N137, and D224. As D123 is conserved in many LCP enzymes, it is likely that this basic amino acid plays an important role in PGN binding [ 106 ]. 4.1.2. Bacillus subtilis WTAs and lipoteichoic acid constitute up to 60% of the dry weight of the cell wall in B. subtilis providing an overall negative charge to the cell wall [ 14 , 107 ]. Both WTA and LTA are important, as cells that cannot produce either of these compounds show morphological aberrations and can only be grown under certain conditions, whereas the absence of both CWGPs is lethal [ 41 ]. WTA is covalently attached to the MurNAc residues in the B. subtilis cell wall via a "conventional" murein linkage unit; coupled to it is poly(Gro- P ) that can have either D-alanine or glucose bound to the C2, with chain lengths varying from 45 to 60 residues [ 14 ] ( Figure 1 ). WTA biosynthesis in B. subtilis has traditionally been intensely investigated [ 14 , 108 ]. B. subtilis TagT, TagU, TagV, Genes and Physiological Effects In the B. subtilis genome, three LCP genes are encoded— tagT (previously named ywtF ), tagU ( lytR ), and tagV ( yvhJ ) [ 82 ]. As in S. aureus , also in B. subtilis , lcp gene deletion mutants revealed defects in WTA, accompanied by reduced virulence, enhanced antibiotics susceptibility, and deviant cell wall structures [ 82 , 91 , 92 ]. Single mutation variants of the tagTUV genes showed no significant effect on either cell morphology or growth, but a Δ tagTV mutant displayed noticeably slower growth and aberrant cell shape [ 82 ]. In their search for interaction partners for the MreB protein that is involved in later wall PGN synthesis in B. subtilis , Kawai et al. were among the first to describe the LCP enzyme family and identified these enzymes as key players in the attachment of anionic CWGPs such as WTA to the cell wall [ 82 ]. The expression of at least one out of the three lcp gene was found to be required for complete WTA biosynthesis and growth of B. subtilis [ 82 ]. B. subtilis TagT, TagU, TagV in WTA Biosynthesis It was demonstrated in in vitro assays that all LCP enzymes, produced recombinantly without the TM domain, were capable of transferring WTA intermediates to PGN [ 109 ]. Particularly, lipid β (i.e., Man p NAc-β-(1→4)-Glc p NAc-1- PP -undp) was attached to mature PGN in vitro [ 109 ], which is consistent with several crystal structures of LCP enzymes where these proteins showed to bind polyisoprenoid phosphate lipids [ 82 , 109 ]. Of note, the use of a short aliphatic chain (C 13 ) in place of the authentic C 55 undp moiety led to a 95% reduction of LCP activity [ 109 ]. This contradicts the observations made by others for S. aureus LCP proteins, where the lipid portion was assumed to play only a minor role in the ligation reaction [ 82 ]. Currently, it is suggested that key interactions between LCP enzymes and donor CWGP substrates occur in the region of the polyisoprenoid lipid that is proximal to the pyrophosphate moiety. Interestingly, the individual B. subtilis LCP enzymes display significant differences in their in vitro activities [ 109 ]. TagU showed an approximately three-fold higher activity compared to TagT and TagV, which contradicts the proposed functional redundancy of the proteins [ 82 , 109 ]. Thus, it is still possible that each Tag enzyme transfers preferentially a distinct type of CWGP to PGN, representing a scenario comparable to S. aureus , where LcpA [ 98 ] and LcpC [ 90 ] can recognize different substrates [ 109 ]. Conclusively, catalysis of WTA transfer by B. subtilis LCP enzymes requires magnesium ions and a polyisoprenoid moiety of the donor CWGP [ 109 ], and this finding is in accordance with the identity of the first three isoprene units of a WTA substrate used by Schaefer et al. [ 29 , 109 ]. Notably, WTA transfer by TagT, TagU and TagV was successful to higher-order structures of PGN, but it still needs to be investigated how the oligomeric repeats of PGN are recognized. Crystal Structure of B. subtilis TagT, TagU, TagV A ΔTM-TagT (residues 44-322) construct was crystallized, and, consistent with its known pyrophosphatase activity towards polyprenoid-pyrophosphate lipid substrates [ 82 ], an octaprenyl-pyrophosphate fitted well in the hydrophobic tunnel in the electron density map [ 97 ]. Interestingly, longer lipids, such as undp- P fitted worse, and presumably in a full-length protein, the hydrophobic tail of the lipid extends out of the tunnel to interact with the membrane. The ΔTM-TagT structure supports the enzymatic role of TagT in the activity of transferring phosphorylated anionic CWGPs from an undp- P -linked precursor to PGN [ 97 ]. Many interactions between the pyrophosphate head group and charged residues are similar to the Sc. pneumoniae Cps2A enzyme (see below). Asp82, corresponding to Asp234 in Cps2A, is more distant from the phosphate, whereas Asp234 coordinates a magnesium ion interacting with the lipid head group. However, this loop region showed disorder and could not be modelled and possibly is the reason why the magnesium ion did not bind to the pyrophosphate group [ 97 ]. In a more recent study to better understand the substrate preferences of LCP proteins, the ΔTM-TagT protein was crystallized with two lipid-linked WTA precursors, namely LI WTA (i.e., [ 14 C] lipid- PP -GlcNAc) and LII WTA , containing the "conventional" disaccharide murein linkage unit [ 98 ]. The resulting structures differed regarding the orientation of the pyrophosphate and saccharide moieties, where the disaccharide-containing structure exposed a divalent cation in the active site. Changes in the glycosidic linkage and pyrophosphate bonds led to a different orientation of the GlcNAc residue of LII WTA , resulting in a conformation of the pyrophosphate where a divalent cation can bind, as would be necessary for catalysis. Evidently, the PGN substrate binds closely to the anomeric phosphate of LII WTA in a narrow groove, which, similar to the WTA binding pocket, displays three conserved arginine residues. R219 is suggested to play a role in the nucleophilic attack by the PGN substrate, whereas another arginine residue, R118, facilitates deprotonation of the C6-OH of MurNAc [ 98 ]. Li and colleagues provided crystal structures of all three B. subtilis LCP proteins, with additional electron density for the disordered loop region in the TagT apo structure. All enzymes were expressed without their TM segment (TagT, residues 46–322; TagU, residues 62–306; TagV, residues 72–332). All secondary structures consist of four regions, consistent with the structure of LcpA from S. aureus (compare with Figure 3 ), with significant differences in region B, where TagT shows an α-helix, and TagU and TagV a double-stranded β-sheet. On one side of the central β-sheet in TagU, helices 3–7 collapse the lipid binding site, whereas other LCP enzymes, including TagT, show a disorder of helix 6. The exposure of this hydrophobic core is, again, suggested to be necessary for surface interaction with the membrane [ 106 ]. 4.1.3. Bacillus anthracis B. anthracis is a spore-forming Gram-positive pathogen which replicates within vertebrates as chains of vegetative cells by regulating the separation of septal PGN [ 110 ]. The pathogen is the causative agent of anthrax and displays a unique growth pattern by tethering at septal PGN, which results in protection of the bacterium from engulfment by host phagocytes [ 111 ]. Comparing B. anthracis with S. aureus and B. subtilis with regard to the presence of CWGPs, it is important to note that B. anthracis does not harbour a WTA, but expresses a PyrCWGP [ 48 ] ( Figure 1 ) and a poly-D-γ-glutamic acid capsule that is bound to PGN via amide bonds [ 112 , 113 ]. The B. anthracis S-layer proteins and S-layer-associated proteins (BSLs) [ 114 ] function as chain length and cell size determinants and are assembled in the envelope by binding to the bacterium's PyrCWGP [ 48 ]. The biosynthesis of that specific CWGP involves the B. anthracis LCP proteins [ 43 , 115 , 116 ]. B. anthracis LcpB1, LcpB2, LcpB3, LcpB4, LcpC, LcpD, Genes and Physiological Effects B. anthracis encodes on its genome six LCP homologues—BAS1830 (LcpB1), BAS0572 (LcpB2), BAS0746 (LcpB3), BAS3381 (LcpB4), BAS5115 (LcpC) and BAS5047 (LcpD). Mutations in B. anthracis lcpB3 and lcpD caused aberrations in cell size and chain length that could be explained as discrete defects in PyrCWGP assembly. By deleting combinations of lcp genes from the B. anthracis genome, variants with single lcp genes were generated [ 43 ]. B. anthracis expressing lcpB3 alone displayed physiological cell size, vegetative growth, spore formation, and S-layer assembly, which might implicate a direct contribution of this LCP protein to the bacterial cell cycle [ 116 ]. Strains expressing lcpB1 or lcpB4 displayed defects in cell size and shape, S-layer assembly, and spore formation, yet sustained vegetative growth. In contrast, the lcpB2 strain was unable to grow unless the gene was expressed from a multicopy plasmid, and variants expressing lcpC or lcpD displayed severe defects in growth and cell shape. The lcpB2 , lcpC or lcpD strains supported neither S-layer assembly nor spore formation. Conclusively, it is conceivable that B. anthracis LCP enzymes fulfil partially overlapping functions in transferring CWGP to discrete sites within the bacterial envelope. B. anthracis LcpB1, LcpB2, LcpB3, LcpB4, LcpC, LcpD in S. aureus WTA Biosynthesis All six B. anthracis lcp genes were tested for their restoration capability of WTA synthesis in a S. aureus Δ lcp mutant lacking all three lcp genes, revealing that, with lcpB2 , lcpC and lcpD plasmids, full complementation could be achieved. lcpB1 was not able to restore S. aureus WTA synthesis at all and lcpB3 and lcpB4 achieved partial complementation. Thus, evidence has been obtained that LcpB2, LcpC and LcpD could transfer a WTA providing a "conventional" murein linkage unit to PGN implicating that these enzymes would display ligation activity also upon the B. anthracis PyrCWGP, which provides an identical murein linkage unit [ 115 ]. The open question, why B. anthracis employs six LCP enzymes while e.g., B. subtilis has evolved only three (i.e., tagTUV ) [ 39 ] might be, at least in part, explained by the genomic organization of the CWGP biosynthesis machinery in these bacteria. In B. subtilis , tagO is part of a 50-kb genomic region that harbours virtually all genes required for WTA synthesis, including tagTUV [ 82 , 117 ]. In contrast, according to the current understanding, four different genomic loci of B. anthracis are linked to PyrCWGP biosynthesis, of which two encode one LCP protein each. In the scwp1 locus, lcpD is encoded in close proximity to the essential tagO gene as well as the gneY gene required for PyrCWGP synthesis [ 118 , 119 ]. The sps locus ("surface polysaccharide") encodes lcpC and the essential gneZ gene, encoding a UDP-GlcNAc-2-epimerase. The genes for the remaining four LCP enzymes—LcpB1, LcpB2, LcpB3, LcpB4—are encoded elsewhere on the genome and are not linked to genes that are known to contribute to the synthesis of PyrCWGP or a "conventional" murein linkage unit. Thus, the expanded repertoire of B. anthracis lcp genes could implicate that these LCP enzymes are able to attach different, not yet identified, types of B. anthracis CWGPs to PGN [ 43 ]. 4.2. Firmicutes— Order : Lactobacillales 4.2.1. Streptococcus pneumoniae Sc. pneumoniae or pneumococcus is a major human pathogen, which typically resides in the nasopharyngeal cavity. Bacterial colonization requires interaction with host cells, for which the amount of capsule is crucial [ 120 ]. Thus, its CPS is a key virulence factor shielding Sc. pneumoniae from the host immune system and, thus, an important target for protective immune responses. Ninety-three capsular types have been identified serologically, and the RU unit structure of each has been defined [ 64 ]. Sc. pneumoniae is especially dangerous for immunocompromised people where it can cause severe diseases, including meningitis, pneumoniae or sepsis. In asymptomatic humans, Sc. pneumoniae resides in the nasal cavity or the sinuses, where it may cause otitis media and acute sinusitis [ 121 ]. The capsular polysaccharide serotype 2 of Sc. pneumoniae strain D39 (CP2) is composed of branched hexasaccharide RUs with the structure →3)[α-Glc p A-(1→6)-α-Glc-(1→2)]-α-L-Rha-(1→3)-α-L-Rha-(1→3)-β-L-Rha-(1→4)-β-D-Glc- ( Figure 1 ). CP2 is directly glycosidically linked via the reducing end glucose of the RU to β-D-GlcNAc residues of PGN, without involvement of a murein linkage unit and a phosphodiester bond [ 122 ], which contrasts the usual attachment mode of CWGPs to PGN backbone sugars. The cell wall of Sc. pneumoniae further contains an unusually complex WTA, which has identical RUs as the membrane-anchored lipoteichoic acid. Both show pseudo-pentasaccharide RUs containing the rare amino sugar 2-acetamido-4-amino-2,4,6-trideoxygalactose (AATGal) in addition to Glc, Rib- P , and two GalNAc residues that are each modified with phosphorylcholine ( Figure 1 ) [ 123 , 124 , 125 ]. The reducing-end AATGal is proposed to be linked via a phosphodiester bond to MurNAc residues of PGN, based on the in silico identification of LCP family proteins in the Sc. pneumoniae genome, however, without provision of any biochemical evidence [ 124 ]. Sc. pneumoniae Cps2A, LytR, Psr, Genes and Physiological Effects On the Sc. pneumoniae genome, the capsular region of CP 2 begins with the cps2A–D genes; all 17 capsular genes in this region are under control of the promoter upstream of cps2A .The first gene in the region, cps2A , encodes a member of the LCP protein family. Within the biosynthesis pathway of the CP2 of Sc. pneumoniae strain D39, the three paralogous LCP proteins Cps2A, LytR and Psr, with the latter two not localizing to the cps region, have been investigated. Evidence was provided that Cps2A, LytR and Psr contribute to the maintenance of normal capsule levels and to the retention of the CP2 in the Sc. pneumoniae cell wall. Cps2A, LytR and Psr were found to localize at the cell membrane and accumulate at septal sites supportive of their function in cell wall maintenance. Single Δ cps2A and Δ psr mutants produced a reduced amount of capsule, while a Δ cps2AlytR double mutant showed greatly impaired growth and cell morphology and lost approximately half of the total capsule material into the culture supernatant [ 97 ]. Notably, inactivation of lytR proved to be difficult in the background of the encapsulated D39 strain and during exponential growth, LytR expression was continuously high which suggests a housekeeping function of this gene during cell division that is essential for proper septum placement [ 126 ]. According to a current data-based model, CpsA2 is responsible for the covalent attachment of CP2 to the pneumococcal cell wall, and LytR can take over this function in the absence of Cps2A. Crystal Structure of Sc. pneumoniae Cps2A Structural and functional studies of the Cps2A enzyme from Sc. pneumoniae provided the first insight into the catalytic mechanism of an enzyme from the LCP protein family. Cps2A contains a large hydrophobic tunnel that is capped with surface-exposed arginine residues that are important for catalysis [ 82 ]; serendipitously, Cps2A co-crystallizes with octaprenyl-pyrophosphate, where the isoprenyl-tail is nestled within the hydrophobic pocket with the pyrophosphate head group interacting with highly conserved arginine residues within the active site. A ΔTM-Cps2A version of the protein comprising the accessory domain (amino acid residues 111–213) and the LCP domain (amino acid residues 214–481) has been solved at 1.69 à -resolution. Within the LCP domain, a core containing a five-stranded β-sheet surrounded by α-helices on both faces is comprised of an overall α-β-α architecture. Between the two domains, two pairs of β-strands extend from the core site. A hydrophobic pocket is formed between the central β-sheet and α-helices 3–7, where a polyisoprenoid phosphate lipid was found. As it has been seen across the LCP superfamily, these hydrophobic side chains are conserved and arginine residues R267, R362 and R374 play a key role in the interaction between the phosphate oxygens and the formation of the pocket and are stabilized by D371 and Q378. Furthermore, magnesium ions are suggested to contribute to catalysis by implicating neutralization of the developing negative charge and are coordinated by two aspartate residues, D234 and D246 [ 82 ]. Based on the crystal structure of the soluble part of Cps2A, it was inferred that all three homologs in Sc. pneumoniae (Cps2A, LytR, and Psr) might attach polyprenol pyrophosphoryl-linked polymers to PGN without any further specification of the CWGP structure [ 97 ], which leaves open the possibility that these enzymes might additionally be involved in WTA attachment. 4.2.2. Streptococcus agalactiae Sc. agalactiae belongs to the GroupB Streptococcus (GBS); it is a common commensal organism which occurs on vaginal and rectal mucosal surfaces but is also associated with invasive infections, especially in elderly or immunocompromised patients [ 127 ]. CPS represents the main virulence factor of Sc. agalactiae and is a prime target in current vaccine development [ 128 ]. GBS isolates associated with human infection produce one of nine antigenically distinct CPSs [ 127 ]. The Sc. agalactiae CPS serotype III (CPIII) has a branched pentasaccharide structure composed of →6)-[α-NeuAc-(2→3)-Gal p -(1→4)]-β-Glc p NAc-(1→3)-β-Gal-(1→4)-α-Glc-(1→ RUs. CPIII is bound via a phosphodiester bond and an oligosaccharide linker of unknown structure composed of glucose, galactose and arabinose to GlcNAc residues of PGN [ 129 , 130 ] ( Figure 1 ), representing a further variation of the PGN linkage mode. Sc. agalactiae CpsA, Gene and Physiological Effects The investigation of the conserved proteins CpsABCD encoded in the Sc. agalactiae cps operon revealed that cpsA , the first gene in the operon, has a regulatory function and is required for the transcription of the operon and that CpsBCD composes a phosphoregulatory system [ 100 ]. Although having no impact on cps transcription or the synthesis of the CPIII RU, it was suggested that these proteins are required for fine-tuning of the last steps of CPIII biosynthesis, which is balancing repeating unit polymerization and CPIII attachment to the cell wall. As a member of the LCP protein family, the 485-amino acid membrane protein CpsA is unique due to its extracellular accessory domain. It equips CpsA to specifically bind to two promoters in the cps locus [ 131 ]. However, a protein consisting of the accessory domain alone could not complement a Δ cpsA deletion strain for CPIII biosynthesis. Interestingly, even if the truncated form co-existed with the native cpsA , the capsule production was impaired (dominant-negative effect), suggesting involvement of CpsA in cell wall maintenance in addition to capsule expression [ 99 ]. Essential for the dominant-negative effect is a region between amino acids 210 and 245 and probably between amino acids 132 and 153 of the accessory domain of CpsA. Experiments using a fluorescent peptide showed that this effect was not due to a direct interaction of truncated CpsA with wild-type CpsA. Probably, it disturbs the mechanism associated with normal cell wall integrity and CPS synthesis [ 131 ]. Interestingly, zebrafish experiments revealed that expression of a truncated CpsA representing the accessory domain only decreases virulence stronger than the complete absence of CpsA [ 131 ]. Sc. agalactiae CpsA in CPIII Biosynthesis In the final steps of CPIII biosynthesis, the newly synthesized pentasaccharide RU anchored to a polyisoprenoid phosphate lipid is flipped to the outer side of the bacterial membrane, where CpsH acts as the repeating unit polymerase. By analogy with other Wzy-dependent systems, polymerization occurs bottom-up. The nascent CPIII is removed from the lipid through a phosphotransferase reaction and subsequently linked to a single membrane-anchored RU. The final product is a CPS that is removed from the membrane lipid and covalently attached to GlcNAc of the PGN backbone by CpsA activity. This linkage effectively renders further polymerization impossible [ 100 ]. 4.2.3. Streptococcus mutans Sc. mutans is a prototypical member of the lactic acid bacteria group. It inhabits the dental plaque biofilm community of the oral cavity and represents the most tenacious causative agent of the enamel-destructive disease dental caries [ 79 ]. As within several streptococcal species, the major cell wall antigen of Sc. mutans is its RhaCWGP which consists of linear rhamnose-polymers with glucose side chains (also referred to as RGP) [ 132 , 133 ] ( Figure 1 ) of the RU structure →2)-[α-Glc-(1→2)]-α-Rha-(1→3)-α-Rha-(1→2)-[α-Glc-(1→2)]-α-Rha-(1→3)-α-Rha-(1→, making up approxima-tely half of the total weight of the bacterium's cell wall [ 76 , 134 ]. The mature RGP contributes to acid and oxidative stress tolerance of Sc. mutans in the oral habitat [ 135 ] and is required for proper localization of cell division complexes in Sc. mutans translating in a morphogenic role for the bacterium [ 79 ]. Specifically, the serotype-specific glucose branches of the RhaCWGP from strain Sc. mutans serotype c were shown to act as a receptor for phage M102 [ 132 ]. Sc. mutans BrpA and Psr, Genes and Physiological Effects Sc. mutans harbours two paralogues of LCP family proteins, named BrpA and Psr. Deletion of either of these did not show any impact on bacterial growth, but the mutants had major defects in acid and oxidative stress tolerance responses, defects in cell division, alterations in cell envelope morphology, and reduction in biofilm formation, probably due to missing RhaCWGP [ 136 , 137 , 138 ]. BrpA or Psr deficiency was also found to alter the expression of a number of genes, including those known to play a critical role in cell envelope biogenesis and cell division and biofilm formation, although differences exist between the two LCP proteins in the scope and effect of their gene regulation [ 136 , 137 ]. Specifically, in the Δ psr mutant, a decreased expression level of glycosyltransferase C, which is common-ly involved in biofilm formation, was found. Importantly, one of the two LCP homologues is necessary for viability of Sc. mutans cells [ 136 ], which might reveal BrpA and Psr as new potential targets to develop anticaries therapeutics. Sc. mutans BrpA and Psr in RhaCWGP Biosynthesis Six genes ( rgpA through rgpF ) involved in the biosynthesis of the RhaCWGPs have been characterized by heterologous gene expression experiments in E. coli [ 139 , 140 ]. From restoration of plasmid-encoded RhaCWGP biosynthesis in an E. coli Δ wecA mutant by provision of Sc. mutans RgpG, it was concluded that RgpG encodes the initiation enzyme of the RhaCWGP biosynthesis in Sc. mutans , transferring a GlcNAc residue from UDP-GlcNAc to lipid-phosphate. According to that study, it was postulated that RgpA, RgpB, and RgpF, function in rhamnan polymerization, while RgpC and RgpD constitute an ABC transporter. In a more recent study, allelic exchange of RgpG led to impaired cell division, reduced biofilm formation, and altered cell morphology of Sc. mutans . The RgpG deficient strain was used for deletion of brpA and psr . While the double mutants grew comparably to the wild-type, a rgpG brpA psr triple mutant showed swollen giant cells and was totally devoid of the RhaCWGP [ 133 ]. Based on this observation, the authors suggested an involvement of BrpA and Pst in attaching the RhaCWGP to the cell wall PGN, without provision of further details about the linkage. Notably, the involvement of a WecA homologue, which is a well-known UDP-GlcNAc::lipid- P transferase from the LPS biosynthesis pathways [ 25 ], as an initiating glycosyltransferase in RhaCWGP biosynthesis in Sc. mutans , would implicate the presence of a GlcNAc residue at the reducing end of the glycopolymer. This, however, is not consistent with the current knowledge of the Sc. mutans RhaCWGP RU structure, leaving the option of this sugar serving as a potential linker of the capsule to the PGN. 4.2.4. Lactococcus lactis Lc. lactis is a Gram-positive bacterium widely used in dairy fermentations where it metabolizes sugars and converts these to lactic acid. In humans, lactic acid bacteria are naturally present in the gut, and, due to their GRAS (generally regarded as safe), they are considered as a convenient delivery vector for biological molecules for antiinfective and anti-allergic therapies in the gastro-intestinal tract [ 141 , 142 ]. Lc. lactis strains are covered by a RhaCWGP or sugar-phosphate polysaccharide pellicle (PSP), which is likely linked to the cell wall PGN via a "conventional" murein linkage unit as evident from the involvement of the TagO enzyme in its biosynthesis. The PSP protects the bacteria from phagocytosis in vitro and acts as the receptor for members of various lactococcal phage groups, allowing their adsorption through specific recognition events [ 77 , 143 ]. In strain MG1363, the anionic PSP is composed of hexasaccharide-phosphate repeats containing Glc, Gal f , GlcNAc, Rha and a Glc- P residue at the reducing-end of each repeat [ 77 , 144 ] ( Figure 1 ). In addition, the bacterium produces a neutral polyrhamnan, which is composed of linear →2)-α-L-Rha-(1→2)-α-L-Rha-(1→3)-α-L-Rha-(1→ trisaccharide repeating units [ 145 ] ( Figure 1 ). The polyrhamnan is located underneath the surface-exposed PSP and is trapped inside the PGN as evident from a high-resolution magic angle spinning (HR-MAS) NMR analysis of an PSP-deficient Lc. lactis strain. Lc. lactis LcpA and LcpB, Genes and Physiological Implications Lc. lactis strains possess a large chromosomal cwps gene cluster, comprising a highly conserved and a variable region, with the former region involved in polyrhamnan and the latter involved in PSP biosynthesis. Thus, except for the gene tagO encoding the initiating glycosyltransferase, the genetic determinants of polyrhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery. Lc. lactis MG1363 harbours two functional lcp paralogs— lcpA and lcpB –which are located outside the cwps gene cluster and have a monocistronic organization. Of these, only the lcpB gene could be successfully deleted, suggesting an essential role for LcpA in the growth and/or survival of Lc. lactis . An lcpA mutant leading to reduced expression of lcpA was shown to severely affect the cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, polyrhamnan was detected by HR-MAS NMR and was, unlike the situation in the wild-type, flexibly located at the surface. This suggested that LcpA participates in the attachment of polyrhamnan to PGN, but, possibly also in attachment of the PSP, since only its absence would allow the detection of the underlaying polyrhamnan. The current model of polyrhamnan biosynthesis follows an ABC transporter-dependent pathway, involving the production of the lipid-pyrophosphate-linked murein linkage unit, subsequently acting individual rhamnosyltransferases, and addition of a terminal sugar terminating the synthesis of the chain followed by export and ligation to PGN involving LcpA [ 145 ]. 4.2.5. Lactobacillus plantarum The human gut microbiota contains an abundance of symbiotic lactobacilli, amongst which Lb. plantarum is one of the most predominant species. The organism is a key player among probiotic microorganisms and known for its high metabolic versatility [ 146 , 147 ]. Lb. plantarum strains possess 3,4-α-D-diglucosyl-2-Rbo- P and 3,4-α-D-diglucosyl-1-Rbo- P WTAs, respectively ( Figure 1 ). The unique structure of WTA in Lb. plantarum results from the modification of the Rbo- P main chain with multiple glucose residues [ 148 ]. Of note, this WTA structure contains two monomers differing in the position of the phosphoric acid residue. Lb. plantarum FlmA, FlmB and FlmC, Genes and Physiological Effects In the Lb. plantarum genome recently, three genes– flmA , flmB and flmC —encoding proteins to which regulatory and cell wall-related transcriptional attenuator functions were attributed, were identified. The FlmA, FlmB and FlmC proteins all contain highly conserved C-terminal regions which appear closely related to the LCP domain [ 147 , 149 ] and an N-terminal TM anchor domain. By generating Δ flm deletion mutants, it has been shown that FlmC contributes to biofilm development and that lack of this protein results in increased autolytic activity phenotypes, whereas deletion of either of the remaining genes did not result in any significant defects [ 147 , 149 ]. Structural Model of Lb. plantarum FlmC A ΔTM-FlmC (amino acid residues 81-335) structural model could be obtained based on the crystal structure of ΔTM-Cps2A from Sc. pneumoniae , exhibiting a typical topology of the LCP domain where conserved hydrophobic amino acid residues are centred in the inside and polar residues on the outside [ 147 ]. A hydrophobic pocket consistent with the Sc. pneumoniae Cps2A protein between the central β-sheet and helices 3–7 was presented in the model. The central sheet harbours six-strands surrounded on both faces by 5 α-helices [ 147 ]. The modelled structure strongly suggests that FlmC acts as a phosphotransferase, like Cps2A, as evident from the presence of similar conserved arginine residues that stabilize the binding of a lipid molecule in concert with a magnesium ion, which is fundamental for the phosphatase activity of the class of LCP enzymes [ 82 , 147 ]. Overall, the present data suggest that FlmC is involved in cell envelope biogenesis of Lb. plantarum ; whether it directly affects the ligation of the bacterium's WTA to PGN remains to be investigated. 4.2.6. Enterococcus hirae Commensal enterococci such as E. hirae are found in the normal human faecal flora; they are of interest due to their emerging pathogenicity in hospital infections which relates to biofilm formation and antibiotic resistances. Enterococci are intrinsically resistant to β-lactams due to the expression of a penicillin binding protein (PBP) displaying a low affinity for these antibiotics [ 150 ]. As with most bacteria, enterococci are dependent on their cell envelope for growth. Enterococcus species show WTA structures of different complexity which have not been elucidated up to now [ 151 ]. E. hirae LcpA, LcpB and LcpC The E. hirae genome codes for three predicted LCP enzymes named LcpA (formerly Psr protein EHR_11445), LcpB (formerly EHR_11995) and LcpC (formerly EHR_14365), respectively. The three LCP proteins from E. hirae have a common topology according to in silico analysis using the TMHMM program, which shows an N-terminal cytoplasmic tail of different length depending on the protein, (six amino acids for LcpA, 103 amino acids for LcpB and six amino acids for LcpC), followed by a TM α-helix of approximately 20 amino acids (18, 23 and 20 amino acids respectively), and an LCP C-terminal domain located in the extracellular space. E. hirae LcpA Function Of the three E. hirae LCP proteins, only LcpA has been investigated. LcpA is a 293-amino acid Psr orthologue possessing a conserved 150 amino-acid LCP domain. The lcpA gene is located in an operon between the ftsW gene, coding for a SEDS (Shape, Elongation, Division and Sporulation) protein involved in lipid II export, and the pbp5 gene, coding for a low-affinity PBP involved in PGN synthesis [ 20 ]. However, the operon is not flanked by any cluster of known CWGP biosynthetic genes. Initially, LcpA was related to β-lactam resistance of E. hirae and proposed to be a repressor of penicillin-binding protein 5 (PBP5) synthesis because of a deletion found in the lcpA gene of the E. hirae strain R40, which overproduced PBP5 [ 152 ]. In subsequent studies, LcpA was found to be a membrane protein which binds E. hirae lysine-type PGN and localizes at the septation sites together with the low-affinity PBP5, which is involved in the late steps of PGN biosynthesis [ 151 ]. The interaction of recombinant E. hirae LcpA with E. hirae cell walls was investigated by pull-down experiments [ 151 ]. Incubation of purified E. hirae PGN with or without CWGP with LcpA indicated that LcpA binds enterococcal PGN regardless of the presence of WTA. Thus, it seems plausible that this LCP protein plays a role related to the cell wall metabolism, probably acting as a phosphotransferase catalysing the attachment of RhaCWGPs to the PGN of E. hirae [ 151 ]. This assumption is consistent with a previous finding in the E. hirae R40 mutant possessing a truncated lcpA gene which showed a decrease of the rhamnose content in its cell walls by 50%, which was not related to the overproduction of PBP5 nor to other changes in the PGN structure [ 153 ]. 4.3. Actinobacteria— Order : Actinomycetales 4.3.1. Mycobacterium tuberculosis M. tuberculosis is the causative agent of tuberculosis infecting the lungs and causing about 1.5 million deaths per year [ 154 ]. What makes this organism so strong is its unique, low permeable AG-containing cell wall ( Figure 1 ) that provides a high resistance towards antibiotics [ 155 ]. M. tuberculosis Rv0822c, CpsA1, CpsA2 and Rv3840 Function Four genes encoding LCP proteins are annotated in the M. tuberculosis H37Rv genome, namely Rv0822c, Rv3267 ( cpsA1/lcp1 ), Rv3484 ( cpsA/cpsA2 ), and Rv3840 [ 156 ]. To investigate an association of the M. tuberculosis LCP proteins with the ligation of AG to PGN, the predicted enzymes devoid of the TM domain were produced recombinantly in E. coli and assayed in vitro. Functional proof was obtained for CpsA1 and CpsA2, which, under the chosen experimental conditions, showed pyrophosphatase activity on the generic substrate geranyl pyrophosphate in dependence on magnesium ions consistent with other LCP enzymes [ 65 ]. Notably, while individual cpsA1 and cpsA2 knock-outs of M. tuberculosis were readily obtainable, the combined inactivation of both genes appeared to be lethal. M. tuberculosis CpsA1 CpsA1 maps to the AG biosynthetic gene cluster where it is located immediately upstream of two genes involved in linker biosynthesis. M. tuberculosis CpsA1 was suggested to be the predominant enzyme responsible for the covalent attachment of AG to PGN. However, in a Δ cpsA1 deletion mutant, no major effects were seen, probably due to functional compensation of the paralogs [ 65 , 155 ]. CpsA1/Lcp1 was further described to be essential for M. tuberculosis and its activity was verified in a cell-free radiolabelling assay with 14 C-radiolabeled AG and nascent PGN [ 155 ]. To further evaluate the specificity of M. tuberculosis CpsA1, three potential AG binding substrates—L-Rha-α(1¡3)-D-GlcNAc- O -C 8 (compound 1), Gal f 2 -Rha-GlcNAc- O -C 8 (compound 2) and Gal f 3 -Rha-GlcNAc- O -C 8 (compound 3)—were tested using intrinsic tryptophan fluorescence of CpsA1, with compound 2 showing the highest affinity with 5.13 µM [ 155 ]. Although the envelope composition was not drastically changed in the single ligase mutants, the Δ cpsA1 mutant showed increased susceptibility to a range of antibiotics such as penicillins, vancomycin, and CPZEN-45, suggesting changes in cell wall permeability [ 65 ]. M. tuberculosis CpsA2 Although no analytically detectable difference was observed, the Δ cpsA2 deletion mutant in the M. tuberculosis strain H37Rv showed a changed phenotype in an in vivo mouse model, where the mutant was not able to grow, survive and infect. Furthermore, this strain displayed increased resistance to meropenem/clavulanate and rifampicin, which could not be compensated by cpsA1 [ 157 ]. Meropenem belongs to the carbapenem class of β-lactam antibiotics being poor substrates for BlaC, a protein encoded in the genome of M. tuberculosis which hydrolyses β-lactam antibiotics [ 158 ]. Rifampicin did not target the cell wall but the authors argued that the permeability had changed and, therefore, the drug did not efficiently get into the cytoplasm as supported by measuring ethidium bromide uptake and efflux [ 66 , 157 ]. However, Grzegorzewicz et al. could not determine increased resistance against rifampicin. According to Malm et al., this could be due to differences in the experimental set-up or resulting from a secondary effect and not from cpsA2 deletion [ 65 , 157 ]. Additional M. tuberculosis Proteins Related to LCP Enzyme Function Recently, mutants of the "cell envelope integrity" gene ( cei ; Rv2700) of M. tuberculosis and its structural homolog VirR (Rv0431), showed a decreased growth rate at low densities, increased susceptibility towards antibiotics (vancomycin, meropenem and rifampicin), increased sensitivity to nitric oxide (NO), and increased cell envelope permeability. In addition, a Δ cei mutant did not lethally infect mice, where one factor that influences virulence is the growth control by NO after infection [ 66 , 159 ]. Importantly, these newly detected gene products have one predicted TM domain and a LytR_C domain, which are features found in LCP enzymes commonly referred to as LytR_C-only proteins. Due to very similar phenotypes of the knockout mutants of cei and virR to common LCP knockout strains, a relation to and common participation in the same pathway, namely AG ligation to PGN, was hypothesized [ 66 ]. 4.3.2. Mycobacterium marinum M. marinum is a slow-growing, acid-fast bacterium in the category of non-tuberculous mycobacteria which most commonly cause skin and soft tissue infections in patients, particularly those with aquatic exposure [ 160 ]. The bacterium possesses an AG as is characteristic of mycobacteria. M. marinum MMAR_4858, MMAR_1274, MMAR_4966 (CpsA), and MMAR_5392 M. marinum (MMAR) harbours orthologues of all LCP proteins found in M. tuberculosis complex (Mtbc) strains—namely MMAR_4858, MMAR_1274, MMAR_4966 (CpsA), and MMAR_5392 in MM for Rv0822c, Rv3267, Rv3484, and Rv3840 for Mtbc [ 65 , 157 ]. A strain defective in the CpsA2 (Rv3484) orthologue in M. marinum (CpsA) showed impaired growth in vitro in contrast to CpsA deficient strains of M. tuberculosis [ 65 , 157 ]. Furthermore, the M. marinum Δ cpsA mutant revealed alterations in colony morphology and cell surface properties, increased susceptibility to antibiotics (erythromycin, vancomycin and penicillin), and a change in cell wall permeability for hydrophobic components. Traditional analytics of the cell wall composition indicated an imbalance in the AG/PGN ratio indicative of a role of the MMAR_4966 enzyme in AG transfer to PGN. Finally, the transposon mutant was severely attenuated in the zebrafish model and growth impaired in the murine macrophage cell line RAW 264.7 [ 161 ]. 4.3.3. Corynebacterium glutamicum C. glutamicum is a well-established model species for cell wall-related studies in the Corynebacteriales because it shares the complex cell envelope organization with its pathogenic relatives, such as M. tuberculosis [ 162 ]. It contains a mycolyl-AG-PGN complex (compare with Figure 1 ) in addition to lipo(arabino)mannan in its cell wall. Furthermore, C. glutamicum is one of the main species used in the biotechnological industry, especially for the production of amino acids [ 163 ]. C. glutamicum LcpA and LcpB As described for Sc. pneumoniae and E. hirae , C. glutamicum 's two LCP proteins, LcpA and LcpB, localize where nascent cell wall biosynthesis happens. Interestingly, of the two C. glutamicum LCP proteins, a deletion was only feasible for LcpB, but did not lead to any detectable changes in the cell wall as compared to the wild-type strain. LcpA could be conditionally silenced, which influenced bacterial growth, the ratio of cell wall components, and morphology. Compared to the wild-type cell wall, the cell wall of the C. glutamicum Δ lcpA mutant contained significantly less mycolic acids and a reduced amount of AG. In particular, rhamnose, a specific sugar component of the linker that connects AG and PGN was decreased (compare with Figure 1 ). Characteristic of LcpA is the presence of an LCP domain and a LytR_C domain, which is frequently found in actinobacteria. In complementation studies, the importance of the conserved arginine and aspartate residues in the LCP domain as well as the general importance of the LytR_C domain was shown [ 164 ]. LcpA was shown to oligomerize into dimers or tetramers, wherefore the LytR_C domain might be responsible supportive of the LytR_C domain catalysing its own reaction [ 164 ]. 4.3.4. Streptomyces coelicolor Sm. coelicolor is the genetically best-known representative among the soil colonized, filamentous Gram-positive bacteria of the Streptomycetes genus [ 165 ], which play an important role in producing natural antibiotics. In contrast to most other bacteria, which divide by binary fission Sm. coelicolor A3(2) develops a mycelial lifestyle by apical tip extension, which requires a dedicated mode of PGN incorporation [ 166 ]. Sm. coelicolor A3(2) encodes several homologues of Tag proteins [ 167 ] directing WTA synthesis in B. subtilis , although the major glycopolymer of Sm. coelicolor is teichulosonic acid. This teichulosonic acid is a phosphate-free polymer of up to seven RUs composed of galactose and the neuraminic acid-related 2-keto-3-deoxy-D- glycero -D- galacto -nononic acid (Kdn), often substituted with GlcNAc or a methyl group. As a minor component, a polydiglycosylphosphate CWGP (referred to as PDP) consisting of →6)-α-Gal p -(1→6)-α-Glc p NAc- P -(1→- RUs is present in Sm. coelicolor cell walls [ 168 ] ( Figure 1 ). Sm. coelicolor PdtA and Ten Other LCP Proteins The genome of Sm. coelicolor harbours 11 LCP proteins, with seven genes (SCO3042-SCO3048) clustered like the B. subtilis TagT, TagU, or TagV-like phosphotransferae genes [ 169 ]. PdtA (SCO2578), a TagV-like phosphotransferase, is suggested to be co-transcribed with a nicotinate-nucleotide adenylytransferase gene SCO2579 and is in close proximity to other presumed CWGP-linked genes [ 167 , 169 ]. It was identified in a screen of interaction partners of several Streptomyces spore wall-synthesizing complex (SSSC) proteins possibly involved in sporulation [ 170 ]. PdtA inactivation resulted in irregular spore chains; more precisely, the placement of sporulation septa was affected and heterogeneity in spore sizes was displayed [ 169 ]. The other 10 LCP homologs did not show any severe phenotype effects and were not able to substitute for PdtA, upon whose deletion, a 48% reduction of spore wall glycopolymer content was determined, comparable to the situation in other LCP-containing organisms [ 82 , 169 ]. Interestingly, of the two types of Sm. coelicolor CWGPs, only PDP was affected in the spore envelope by the lack of pdtA and resulted in a severe phenotype, including imprecise sporulation septa placement and spore viability decrease by one-third. The remaining spores had increased sensitivity to osmotic stress and lysozyme. Furthermore, the Δ pdtA mutant showed impaired vegetative tip growth and, interestingly, also high sensitivity to rifampicin, which is known to cross the bacterial cell wall targeting the RNA polymerase and to have no effect on cell envelope synthesis [ 169 , 171 ]. Possibly, PDP acts as a barrier to block large-sized antibiotics, such as rifampicin [ 169 ]. It was speculated that PDP is anchored to the hyphal tip by PdtA resulting in apical tip growth, which aids as a framework for PGN synthesis, particularly under stress conditions [ 169 ]. The crucial role of PdtA under stress conditions becomes even clearer, as under high-salt conditions, only 4% of normal biomass was produced and hyphae showed aberrant morphology. Conclusively, of the 11 LCP protein homologs found in Sm. coelicolor , PdtA is the only protein involved in PDP synthesis and plays a key role for the life cycle of the organism [ 169 ]. 4.3.5. Actinomyces oris The actinobacterium A. oris is a colonizer of the oral cavity where it plays a specific role in the formation of supragingival plaque [ 172 ]. A . oris is dependent on the activity of its SrtA enzyme, which is conditionally dependent on glycosylation of the GspA surface protein by the activity of an LCP enzyme [ 173 ]. A. oris LcpA Participates in Protein Glycosylation The LCP homolog LcpA of A. oris provide a so far unique example of an LCP enzyme that is involved in a protein glycosylation process, i.e., the transfer of a saccharide moiety to an amino acid acceptor sequence [ 174 ] instead of a PGN backbone sugar. LcpA is genetically linked to GspA, a glycoprotein that is attached to A. oris PGN by the house-keeping sortase SrtA, which, in turn, recognizes the cell wall sorting signal of the glycoprotein [ 175 ]. Deletion of either the gspA or lcpA gene resulted in rescue effects of srtA depletion, leading to the suggestion that excessive aggregation of GspA proteins causes stress leading to cell death, whereas the absence of GspA in the presence of SrtA and LcpA is not lethal [ 176 ]. The neighbouring genes gspA and lcpA in A. oris implicate that their glycoprotein products are linked to each other and that the high glycosylation level of GspA involves LcpA [ 174 ]. LcpA glycosylates GspA along an unknown pathway, prior translocation across the cytoplasmic membrane and final cell wall anchoring by the sortase SrtA [ 176 ]. However, the exact nature and composition of the GspA glycans remain to be determined. A. oris LcpB, LcpC and LcpD Interestingly, A. oris MG1 encodes three other LCP domain-containing proteins, LcpB (ana_0299), a homolog of the TagF glycosyl/glycerophosphate transferase from Staphylococcus epidermidis [ 177 ], LcpC (ana_1577) and LcpD (ana_1578), which are found in the same transcriptional unit [ 174 ]. Mutant strains were generated by deletion of lcpB and lcpD as well as a triple mutant lcp Δ 3 , devoid of lcpA , lcpB and lcpD , to analyse LcpA-mediated glycosylation. The single mutants did not show any negative effect regarding the formation of high-molecular-mass GspA species with attached glycans, whereas the triple mutant did, supporting that LcpA is necessary and sufficient for the production of glycosylated GspA. Together with the obtained crystal structure (see below), this is the first experimental evidence of the glycosylation capability of LcpA which occurs prior to GspA glycoprotein transfer to PGN [ 174 ]. The data suggest that LcpA is the only enzyme involved in GspA glycosylation in A. oris , but it has to be mentioned that a Δ lcpC deletion mutant could not be generated and possibly may also modify GspA [ 174 ]. Crystal Structure or A. oris LcpA A notable structural variant of LCP enzymes is that of A. oris LcpA, which possesses unique structural features around the active site presumably associated with binding target proteins rather than PGN for glycosylation. The molecular structure of the extracellular LcpA domain has been resolved at 2.5-à (eLcpA, residues 78–360). The core of the protein is formed by seven-stranded antiparallel β-sheets flanked with eight α-helices on both sites, forming ~23-à hydrophobic tunnel [ 174 ]. Its presence is consistent with other members of the LCP protein family, leading into the active sites of LcpA that possibly bind a lipid-linked glycan substrate [ 82 , 174 ]. Conserved arginine residues R128, R149 and R266 were identified clustering within a pocket exposed on the surface, which indicates mediated phosphotransfer of glycopolymers as known in the LCP family [ 82 , 97 , 147 , 174 ]. By generating alanine substitution mutants of these arginine residues, it could be determined that the R149 and R266 residues are essential for LcpA glycosylation activity on GspA. Interestingly, LcpA links the C-terminus to α-helices between C179 and C365 formed by a presumably stabilizing disulphide bond that is also found in other actinobacterial LCP proteins [ 174 ]. Alanine substitution mutants of either one or both Cys residues in LcpA suggested that the disulphide bond is essential for protein stability as evident from a defect of mutant LcpA membrane expression as well as for full enzymatic activity [ 174 ]. Based on the demonstrated in vitro pyrophosphate activity of TagT [ 82 ], an in vitro assay of eLcpA over quantification of released inorganic phosphate in concert with a diphosphate mimic substrate showed that LcpA displays pyrophosphate activity and corroborated the necessity of the disulfide bond for catalysis [ 174 ]. This distinct feature seems to be common in actinobacterial LCP enzymes and has not been found in other LCP enzymes studied to date [ 174 ]. Overall, the structure of eLcpA seems closely related to the TagT enzyme from B. subtilis [ 97 , 174 ]. However, unlike TagT, LcpA is not capable of attaching CWGPs to PGN but to GspA instead [ 174 ]. 4.4. Cyanobacteria— Order: Nostocales 4.4.1. Anabena sp. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is a commonly used model organism to study cyanobacterial nitrogen fixation and cell differentiation [ 178 ]. It is capable of fixing carbon dioxide by oxygenic photosynthesis or of fixing molecular nitrogen when a combined nitrogen source such as ammonium or nitrate is not available; the bacterium segregates these two incompatible processes by multicellular development by differentiating 5–10% of vegetative cells into so-called heterocysts [ 179 ]. A polysaccharide layer is placed over the Anabena sp. proheterocyst during maturation followed by a glycolipid layer between the polysaccharide layer and the outer membrane aimed at diminishing oxygen entry into the cell [ 179 , 180 , 181 ]. Anabena sp. ConR The gene all0817 named conR (constriction regulator) is predicted to contain an LCP domain. While conR was initially predicted to be a transcriptional regulator [ 182 ], its deletion caused diazotrophic growth and heterocyst differentiation defects [ 179 ]. Although the polysaccharide and glycolipid envelope layers were present in the mutant, the polar junctions connecting heterocysts to vegetative cells were incomplete or widely open, which was hypothesized to allow oxygen to enter the heterocysts and inactivate nitrogenase [ 182 ]. Furthermore, the expression of conR was upregulated after nitrogen step-down in differentiating heterocysts and vegetative cells. In nitrate-containing media, filaments of the Δ conR mutant strain also showed aberrant septum formation of vegetative cells and defects in cell separation. However, after nitrogen step-down, the defective vegetative cells seemed less severe compared to filaments in nitrate-containing media [ 179 ]. It was suggested that these phenotypic growth defects do not simply evolve from defective nitrogen fixation but rather from a disrupted delivery of fixed nitrogen from heterocysts to their neighbouring vegetative cells via non-specific intracellular channels. The defective septum formation in the mutant could possibly result in deformation of these channels at the junction between vegetative cells and heterocysts, leading to aberrant metabolite exchange [ 179 ]. Conclusively, the putative LCP protein ConR in Anabena sp. is developmentally regulated and is essential for diazotrophic growth and heterocyst morphogenesis; specifically, it was found to be associated with septum formation and cell wall maintenance. 4.1. Firmicutes— Order : Bacillales 4.1.1. Staphylococcus aureus S. aureus is a Gram-positive opportunistic pathogen of which certain strains have become resistant to most antibiotic classes [ 83 , 84 , 85 ]. The bacterium can lead to systemic failures, such as infective endocarditis or bacteraemia via nosocomial acquisition [ 83 , 86 ]. The S. aureus WTA is a polymer of 30 to 50 Rbo- P subunits connected via 1,5-phosphodiester bonds [ 16 ], which is tethered to PGN via the "conventional" murein linkage unit [ 87 ] ( Figure 1 ). Furthermore, S. aureus expresses a CPS, which substantially contributes to the bacterium's ability to cause invasive disease [ 88 ]. Specifically, capsular polysaccharide type 5 (CP5) is composed of →4)-β-D-Man p NAcA-(1→3)-α-L-Fuc p NAc-(1→4)-β-D-Fuc p NAc-(1→ repeats with O -acetylation on all L-Fuc p NAc residues except for the one in the reducing-end RU [ 28 ] ( Figure 1 ). Of note, CP5 biosynthesis is TagO-independent resulting in the absence of a "conventional" murein linkage unit in the capsule. S. aureus LcpA, LcpB, LcpC at a Glance The S. aureus genome harbours three LCP proteins, encoded by lcpA (previous name, msrR ), lcpB ( sa0908 ), and lcpC ( sa2103 ) [ 49 , 89 ], which are in part functionally redundant regarding cellular functions [ 29 , 90 ]. S. aureus variants carrying defective alleles of lcp genes resulted in enhanced [ 91 , 92 ] susceptibility to β-lactam antibiotics, deviant septum formation [ 91 , 92 ], autolysis [ 91 ], activation of a cell wall stress response [ 93 ], reduced phosphate content of staphylococcal cell walls [ 93 ], and aberrant biofilm formation [ 92 ]. In strains lacking WTA due to the inactivation of LCP function, major cell division defects were shown, the PGN synthesis machinery was not localized properly, and these strains were unable of nasal epithelial cell colonization [ 94 , 95 ]. Furthermore, methicillin-resistant S. aureus (MRSA) strains were found to become sensitive to β-lactam antibiotics when WTA synthesis was abrogated [ 95 , 96 ]. S. aureus LcpA, LcpB, LcpC in CWGP Biosynthesis Due to their demonstrated biological importance, the biochemical activity of S. aureus LCP proteins is under intense investigation [ 89 ]. Deletion of all three LCP genes resulted in complete WTA loss in the staphylococcal cell wall and deletion of any of individual LCP genes disturbed the attachment of WTA in different degrees [ 89 ]. This partial functional redundancy was also seen for different phenotypes including β-lactam resistance, biofilm formation, and growth defects [ 91 ]. Of note, LcpA was shown to be the most important protein related to WTA-functions [ 84 ]. In a reconstitution approach, cognate Rbo- P WTA precursors including the murein-linkage unit ( Figure 1 ) could be transferred to PGN by truncated versions of either of the three staphylococcal LCP proteins devoid of the TM-segment (ΔTM) [ 29 ], without the requirement of any other proteins, as had been initially suggested [ 82 ]. For the ligation, the WTA substrate needs to be comprised of only two sugar residues (i.e., the "conventional" murein linkage unit) and a hexaprenyl chain, mimicking the truncated native C 55 undp lipid. This is consistent with the length of the hydrophobic channel length observed in the crystal structure of Cps2A—the LCP protein transferring CPS in Sc. pneumoniae [ 82 , 97 ]. Importantly, the S. aureus WTA precursor could be transferred to "nascent" (i.e., un-crosslinked) PGN polymers only [ 98 ], not to lipid II ( i.e ., murein pentapeptides) [ 29 ], indicating that modification of PGN with WTA occurs prior to final PGN cross-linking [ 98 ]. Deletion of lcpC had no effect on the level of WTA that was ligated to PGN, whereas a Δ lcpA or a Δ lcpB mutant showed reduced levels of WTA content [ 89 , 93 ]. This is corroborated by the finding that the Δ lcpC mutant showed no phosphate release compared to single Δ lcpA and Δ lcpB mutants [ 89 ]. Differential localization and regulation of the enzymes might be important factors regarding the greater impact of LcpA and LcpB on WTA synthesis [ 93 ]. Genes involved in S. aureus CPS synthesis, exemplified with CP5 ( Figure 1 ), are clustered presenting conserved genes employing a Wzy-dependent biosynthesis mechanism [ 64 , 90 ], and there is evidence that specifically LcpC plays a key role in catalysis of S. aureus capsule attachment to the PGN [ 28 , 29 , 90 ]. This is corroborated by the lack of a cpsA homologue encoding a CPS ligase, typically involved in streptococcal CPS ligation, in the S. aureus genome [ 49 , 99 ]. Initial evidence of the requirement of LcpC for ligation of S. aureus CP5 to PGN was derived from a mutant approach [ 90 ]. The Δ lcpC mutant accumulated the capsule in the supernatant fraction, while the Δ lcpAB variants did not display any defect in CP5 synthesis [ 90 ]. A triple mutant showed the same levels of CP5 reduction as the variant lacking lcpC. Surprisingly, plasmid-based expression of any of the three lcp genes could restore the CP5 content, indicating that all three LCP proteins are to some extent involved in the attachment [ 90 ]. To investigate the proposed role of LcpC in vitro, different [ 14 C] CP5 lipid intermediates were synthesized, including lipid I cap (i.e., C 55 undp- PP -D-FucNAc), lipid II cap (i.e., C 55 undp- PP -D-FucNAc-L-FucNAc), and lipid III cap (i.e., C 55 undp- PP -D-FucNAc-L-FucNAc-D-ManNAcA). After purification, these were used together with the ultimate PGN precursor lipid II (lipid II PGN ), i.e., C 55 undp- PP -D-MurNAc-D-GlcNAc including a pentapeptide, as a potential acceptor substrate [ 28 ]. In this setup, LcpC was able to catalyse cleavage of the donor substrate lipid I cap and catalyse attachment of the phosphoryl-sugar moiety to the ultimate PGN precursor lipid II. Strikingly, in the presence of CapA1, an activator/phosphodiesterase protein that cleaves lipid-PP-linked CP5 precursors [ 28 ], the transfer rate was increased, possibly by forming an interaction complex between CapA1 and LcpC [ 28 , 100 ]. Surprisingly, in the case of lipid II cap , no transfer to the PGN acceptor could be overserved in the LcpC in vitro assay, which would likely be deleterious in vivo. On the other hand, all CP5 lipid intermediates were effectively processed by LcpC, although the proximal undp- PP -linked FucNAc residue appeared to be sufficient for CP5 precursor recognition. Of note, the natural PGN acceptor substrate of LcpC remains elusive; possible acceptor structures include lipid II PGN , as well as "nascent" and cross-linked PGN. Summarizing, there is evidence that LcpC preferentially recognizes CP5 intermediates rather than WTA intermediates where deletion of lcpC caused only minor reduction levels [ 89 , 90 ]. Whether this is due to the different reducing-end sugars (i.e., D-FucNAc in the CP5 repeat versus D-Glc p NAc in the "conventional" WTA murein linkage unit remains to be investigated. Notably, in LPS O-antigen RU biosynthesis, the first C 55 lipid- PP -linked sugar unit of the O-antigen RU contains all necessary recognition information for the catalytic activity of the O-antigen ligase WaaL [ 101 ]. S. aureus lcpA, lcpB, lcpC Genes and Physiological Effects Deletion of individual S. aureus LCP protein encoding genes showed only minor effects on S. aureus cell wall physiology and growth, whereas a Δ lcp triple mutant was barely viable, showing temperature sensitivity and enlarged cells [ 91 ]. Complementation with LcpA resulted in restoration of growth and cell size almost to wild-type size [ 91 ]. Interestingly, Δ lcp and Δ lcp Δ tagO variants abolished cell division planes by generating aberrant cells with irregular envelopes lacking WTA [ 89 ]. Of note, deletion of tagO alone did not abolish staphylococcal growth [ 35 , 39 , 102 ]. The same phenotype was observed for isolated tagO and lcpA mutants; however, deletion of lcpA did not interrupt WTA synthesis and thus suggests that WTA synthesis as well as assembly are crucial for normal cell division [ 89 , 95 ]. Furthermore, inhibition of WTA synthesis by tunicamycin treatment did not relieve deviant cell separation of the mutant cells and, therefore, did not suppress the cell division defects of Δ lcp variants [ 89 ]. Autolysis was induced by deletion of all three LCP proteins, resulting in an increased resistance of the Δ lcp triple mutant to autolysis compared to the wild-type [ 89 ]. Complementation by LcpA increased autolysis levels the most [ 89 ]. These pleiotropic phenotypes in the Δ lcp mutant are likely owed to WTA cell wall deposition defects and WTA synthesis, as staphylococcal cells without WTA resulted in deviant cell size and septum formation as well as susceptibility to antibiotics and biofilm formation defects [ 49 , 91 , 92 , 93 ]. A comparison of the surface proteomes of methicillin-resistant, laboratory-adapted S. aureus COL strain (COL) and a COL strain in vitro-adapted to high levels of oxacillin (APT) was used to characterize virulence factors showing that LcpC was found uniquely on the APT surface, suggesting a role in adaption to high oxacillin levels [ 84 ]. Deletion of lcpA decreased oxacillin resistance and upregulated lcpA expression was observed by triggered antibiotic stress [ 103 ] and, furthermore, increased cell size and elevated cell wall remodelling was revealed [ 84 , 92 ]. However, overexpression of LcpC did not generate the APT phenotype in COL, suggesting that aggregation and changes in cell morphology are multifactorial [ 84 ]. The adaption of LcpC to high levels of oxacillin in the APT strain prompted the question about the contribution of this enzyme to antimicrobial resistance and pathogenicity [ 104 ]. The finding that deletion of lcpC decreased the resistance to β-lactams in methicillin-resistant S. aureus (MRSA) and in methicillin-susceptible S. aureus (MSSA) and, consequently, the pathogenicity in the host, suggested that the deviant cell shape might allow for an easier access of the antibiotics to the cell [ 104 ]. Thus, LcpC could be an effective target for drug development [ 104 ]. In MSSA and MRSA strains, reduced oxacillin resistance levels were also observed upon lcpA deletion [ 91 , 92 , 103 ]. The Δ lcp triple mutant was hypersusceptible to oxacillin and growth could be restored by any of the three LCP proteins; LcpA had the greatest impact, followed by LcpB and LcpC [ 91 ]. Interestingly, the Δ lcpA mutant produced more biofilm, and in the Δ lcp triple mutant complementation with LcpC revealed the strongest biofilm, LcpA the weakest [ 91 ]. A mutation in lcpA (E146K) was shown to have an impact on β-lactam and vancomycin resistances and led to a reduced resistance to oxacillin, whereby the cells showed abnormal septal placement [ 105 ]. The mutation further led to a decreased autolytic activity, as was also evident form highest autolysis levels obtained after complementation of the Δ lcp triple mutant with LcpA [ 91 , 105 ]. Crystal Structure of S. aureus LcpA The crystal structure of LcpA devoid of its TM (residues 80–327; ΔTM-LcpA) complexed to C 40 - PP -GlcNAc was solved to 1.9 à and provides a relevant target for inhibitor design studies [ 106 ] ( Figure 3 ). The extracellular domain consists of six-stranded β-sheets overlaid amongst several α-helices and double-stranded β-sheets, where a hydrophobic binding pocket narrow opening and a wide base is formed from its center [ 106 ]. The active site of LcpA is enclosed by region A (residues 92–100), B (residues 188–201), C (residues 217–224) and D (residues 296–312) and shows structural variability. The electropositive region in the active site, as seen in other LCP enzymes, consists of conserved arginine residues. R218 located in the loop of the highly flexible region C is suggested to aid in product expulsion and is held away from the active site by a salt bridge. Furthermore, three potential PGN saccharide binding sites were identified in close proximity to the conserved regions of R99, K135, N137, and D224. As D123 is conserved in many LCP enzymes, it is likely that this basic amino acid plays an important role in PGN binding [ 106 ]. 4.1.2. Bacillus subtilis WTAs and lipoteichoic acid constitute up to 60% of the dry weight of the cell wall in B. subtilis providing an overall negative charge to the cell wall [ 14 , 107 ]. Both WTA and LTA are important, as cells that cannot produce either of these compounds show morphological aberrations and can only be grown under certain conditions, whereas the absence of both CWGPs is lethal [ 41 ]. WTA is covalently attached to the MurNAc residues in the B. subtilis cell wall via a "conventional" murein linkage unit; coupled to it is poly(Gro- P ) that can have either D-alanine or glucose bound to the C2, with chain lengths varying from 45 to 60 residues [ 14 ] ( Figure 1 ). WTA biosynthesis in B. subtilis has traditionally been intensely investigated [ 14 , 108 ]. B. subtilis TagT, TagU, TagV, Genes and Physiological Effects In the B. subtilis genome, three LCP genes are encoded— tagT (previously named ywtF ), tagU ( lytR ), and tagV ( yvhJ ) [ 82 ]. As in S. aureus , also in B. subtilis , lcp gene deletion mutants revealed defects in WTA, accompanied by reduced virulence, enhanced antibiotics susceptibility, and deviant cell wall structures [ 82 , 91 , 92 ]. Single mutation variants of the tagTUV genes showed no significant effect on either cell morphology or growth, but a Δ tagTV mutant displayed noticeably slower growth and aberrant cell shape [ 82 ]. In their search for interaction partners for the MreB protein that is involved in later wall PGN synthesis in B. subtilis , Kawai et al. were among the first to describe the LCP enzyme family and identified these enzymes as key players in the attachment of anionic CWGPs such as WTA to the cell wall [ 82 ]. The expression of at least one out of the three lcp gene was found to be required for complete WTA biosynthesis and growth of B. subtilis [ 82 ]. B. subtilis TagT, TagU, TagV in WTA Biosynthesis It was demonstrated in in vitro assays that all LCP enzymes, produced recombinantly without the TM domain, were capable of transferring WTA intermediates to PGN [ 109 ]. Particularly, lipid β (i.e., Man p NAc-β-(1→4)-Glc p NAc-1- PP -undp) was attached to mature PGN in vitro [ 109 ], which is consistent with several crystal structures of LCP enzymes where these proteins showed to bind polyisoprenoid phosphate lipids [ 82 , 109 ]. Of note, the use of a short aliphatic chain (C 13 ) in place of the authentic C 55 undp moiety led to a 95% reduction of LCP activity [ 109 ]. This contradicts the observations made by others for S. aureus LCP proteins, where the lipid portion was assumed to play only a minor role in the ligation reaction [ 82 ]. Currently, it is suggested that key interactions between LCP enzymes and donor CWGP substrates occur in the region of the polyisoprenoid lipid that is proximal to the pyrophosphate moiety. Interestingly, the individual B. subtilis LCP enzymes display significant differences in their in vitro activities [ 109 ]. TagU showed an approximately three-fold higher activity compared to TagT and TagV, which contradicts the proposed functional redundancy of the proteins [ 82 , 109 ]. Thus, it is still possible that each Tag enzyme transfers preferentially a distinct type of CWGP to PGN, representing a scenario comparable to S. aureus , where LcpA [ 98 ] and LcpC [ 90 ] can recognize different substrates [ 109 ]. Conclusively, catalysis of WTA transfer by B. subtilis LCP enzymes requires magnesium ions and a polyisoprenoid moiety of the donor CWGP [ 109 ], and this finding is in accordance with the identity of the first three isoprene units of a WTA substrate used by Schaefer et al. [ 29 , 109 ]. Notably, WTA transfer by TagT, TagU and TagV was successful to higher-order structures of PGN, but it still needs to be investigated how the oligomeric repeats of PGN are recognized. Crystal Structure of B. subtilis TagT, TagU, TagV A ΔTM-TagT (residues 44-322) construct was crystallized, and, consistent with its known pyrophosphatase activity towards polyprenoid-pyrophosphate lipid substrates [ 82 ], an octaprenyl-pyrophosphate fitted well in the hydrophobic tunnel in the electron density map [ 97 ]. Interestingly, longer lipids, such as undp- P fitted worse, and presumably in a full-length protein, the hydrophobic tail of the lipid extends out of the tunnel to interact with the membrane. The ΔTM-TagT structure supports the enzymatic role of TagT in the activity of transferring phosphorylated anionic CWGPs from an undp- P -linked precursor to PGN [ 97 ]. Many interactions between the pyrophosphate head group and charged residues are similar to the Sc. pneumoniae Cps2A enzyme (see below). Asp82, corresponding to Asp234 in Cps2A, is more distant from the phosphate, whereas Asp234 coordinates a magnesium ion interacting with the lipid head group. However, this loop region showed disorder and could not be modelled and possibly is the reason why the magnesium ion did not bind to the pyrophosphate group [ 97 ]. In a more recent study to better understand the substrate preferences of LCP proteins, the ΔTM-TagT protein was crystallized with two lipid-linked WTA precursors, namely LI WTA (i.e., [ 14 C] lipid- PP -GlcNAc) and LII WTA , containing the "conventional" disaccharide murein linkage unit [ 98 ]. The resulting structures differed regarding the orientation of the pyrophosphate and saccharide moieties, where the disaccharide-containing structure exposed a divalent cation in the active site. Changes in the glycosidic linkage and pyrophosphate bonds led to a different orientation of the GlcNAc residue of LII WTA , resulting in a conformation of the pyrophosphate where a divalent cation can bind, as would be necessary for catalysis. Evidently, the PGN substrate binds closely to the anomeric phosphate of LII WTA in a narrow groove, which, similar to the WTA binding pocket, displays three conserved arginine residues. R219 is suggested to play a role in the nucleophilic attack by the PGN substrate, whereas another arginine residue, R118, facilitates deprotonation of the C6-OH of MurNAc [ 98 ]. Li and colleagues provided crystal structures of all three B. subtilis LCP proteins, with additional electron density for the disordered loop region in the TagT apo structure. All enzymes were expressed without their TM segment (TagT, residues 46–322; TagU, residues 62–306; TagV, residues 72–332). All secondary structures consist of four regions, consistent with the structure of LcpA from S. aureus (compare with Figure 3 ), with significant differences in region B, where TagT shows an α-helix, and TagU and TagV a double-stranded β-sheet. On one side of the central β-sheet in TagU, helices 3–7 collapse the lipid binding site, whereas other LCP enzymes, including TagT, show a disorder of helix 6. The exposure of this hydrophobic core is, again, suggested to be necessary for surface interaction with the membrane [ 106 ]. 4.1.3. Bacillus anthracis B. anthracis is a spore-forming Gram-positive pathogen which replicates within vertebrates as chains of vegetative cells by regulating the separation of septal PGN [ 110 ]. The pathogen is the causative agent of anthrax and displays a unique growth pattern by tethering at septal PGN, which results in protection of the bacterium from engulfment by host phagocytes [ 111 ]. Comparing B. anthracis with S. aureus and B. subtilis with regard to the presence of CWGPs, it is important to note that B. anthracis does not harbour a WTA, but expresses a PyrCWGP [ 48 ] ( Figure 1 ) and a poly-D-γ-glutamic acid capsule that is bound to PGN via amide bonds [ 112 , 113 ]. The B. anthracis S-layer proteins and S-layer-associated proteins (BSLs) [ 114 ] function as chain length and cell size determinants and are assembled in the envelope by binding to the bacterium's PyrCWGP [ 48 ]. The biosynthesis of that specific CWGP involves the B. anthracis LCP proteins [ 43 , 115 , 116 ]. B. anthracis LcpB1, LcpB2, LcpB3, LcpB4, LcpC, LcpD, Genes and Physiological Effects B. anthracis encodes on its genome six LCP homologues—BAS1830 (LcpB1), BAS0572 (LcpB2), BAS0746 (LcpB3), BAS3381 (LcpB4), BAS5115 (LcpC) and BAS5047 (LcpD). Mutations in B. anthracis lcpB3 and lcpD caused aberrations in cell size and chain length that could be explained as discrete defects in PyrCWGP assembly. By deleting combinations of lcp genes from the B. anthracis genome, variants with single lcp genes were generated [ 43 ]. B. anthracis expressing lcpB3 alone displayed physiological cell size, vegetative growth, spore formation, and S-layer assembly, which might implicate a direct contribution of this LCP protein to the bacterial cell cycle [ 116 ]. Strains expressing lcpB1 or lcpB4 displayed defects in cell size and shape, S-layer assembly, and spore formation, yet sustained vegetative growth. In contrast, the lcpB2 strain was unable to grow unless the gene was expressed from a multicopy plasmid, and variants expressing lcpC or lcpD displayed severe defects in growth and cell shape. The lcpB2 , lcpC or lcpD strains supported neither S-layer assembly nor spore formation. Conclusively, it is conceivable that B. anthracis LCP enzymes fulfil partially overlapping functions in transferring CWGP to discrete sites within the bacterial envelope. B. anthracis LcpB1, LcpB2, LcpB3, LcpB4, LcpC, LcpD in S. aureus WTA Biosynthesis All six B. anthracis lcp genes were tested for their restoration capability of WTA synthesis in a S. aureus Δ lcp mutant lacking all three lcp genes, revealing that, with lcpB2 , lcpC and lcpD plasmids, full complementation could be achieved. lcpB1 was not able to restore S. aureus WTA synthesis at all and lcpB3 and lcpB4 achieved partial complementation. Thus, evidence has been obtained that LcpB2, LcpC and LcpD could transfer a WTA providing a "conventional" murein linkage unit to PGN implicating that these enzymes would display ligation activity also upon the B. anthracis PyrCWGP, which provides an identical murein linkage unit [ 115 ]. The open question, why B. anthracis employs six LCP enzymes while e.g., B. subtilis has evolved only three (i.e., tagTUV ) [ 39 ] might be, at least in part, explained by the genomic organization of the CWGP biosynthesis machinery in these bacteria. In B. subtilis , tagO is part of a 50-kb genomic region that harbours virtually all genes required for WTA synthesis, including tagTUV [ 82 , 117 ]. In contrast, according to the current understanding, four different genomic loci of B. anthracis are linked to PyrCWGP biosynthesis, of which two encode one LCP protein each. In the scwp1 locus, lcpD is encoded in close proximity to the essential tagO gene as well as the gneY gene required for PyrCWGP synthesis [ 118 , 119 ]. The sps locus ("surface polysaccharide") encodes lcpC and the essential gneZ gene, encoding a UDP-GlcNAc-2-epimerase. The genes for the remaining four LCP enzymes—LcpB1, LcpB2, LcpB3, LcpB4—are encoded elsewhere on the genome and are not linked to genes that are known to contribute to the synthesis of PyrCWGP or a "conventional" murein linkage unit. Thus, the expanded repertoire of B. anthracis lcp genes could implicate that these LCP enzymes are able to attach different, not yet identified, types of B. anthracis CWGPs to PGN [ 43 ]. 4.1.1. Staphylococcus aureus S. aureus is a Gram-positive opportunistic pathogen of which certain strains have become resistant to most antibiotic classes [ 83 , 84 , 85 ]. The bacterium can lead to systemic failures, such as infective endocarditis or bacteraemia via nosocomial acquisition [ 83 , 86 ]. The S. aureus WTA is a polymer of 30 to 50 Rbo- P subunits connected via 1,5-phosphodiester bonds [ 16 ], which is tethered to PGN via the "conventional" murein linkage unit [ 87 ] ( Figure 1 ). Furthermore, S. aureus expresses a CPS, which substantially contributes to the bacterium's ability to cause invasive disease [ 88 ]. Specifically, capsular polysaccharide type 5 (CP5) is composed of →4)-β-D-Man p NAcA-(1→3)-α-L-Fuc p NAc-(1→4)-β-D-Fuc p NAc-(1→ repeats with O -acetylation on all L-Fuc p NAc residues except for the one in the reducing-end RU [ 28 ] ( Figure 1 ). Of note, CP5 biosynthesis is TagO-independent resulting in the absence of a "conventional" murein linkage unit in the capsule. S. aureus LcpA, LcpB, LcpC at a Glance The S. aureus genome harbours three LCP proteins, encoded by lcpA (previous name, msrR ), lcpB ( sa0908 ), and lcpC ( sa2103 ) [ 49 , 89 ], which are in part functionally redundant regarding cellular functions [ 29 , 90 ]. S. aureus variants carrying defective alleles of lcp genes resulted in enhanced [ 91 , 92 ] susceptibility to β-lactam antibiotics, deviant septum formation [ 91 , 92 ], autolysis [ 91 ], activation of a cell wall stress response [ 93 ], reduced phosphate content of staphylococcal cell walls [ 93 ], and aberrant biofilm formation [ 92 ]. In strains lacking WTA due to the inactivation of LCP function, major cell division defects were shown, the PGN synthesis machinery was not localized properly, and these strains were unable of nasal epithelial cell colonization [ 94 , 95 ]. Furthermore, methicillin-resistant S. aureus (MRSA) strains were found to become sensitive to β-lactam antibiotics when WTA synthesis was abrogated [ 95 , 96 ]. S. aureus LcpA, LcpB, LcpC in CWGP Biosynthesis Due to their demonstrated biological importance, the biochemical activity of S. aureus LCP proteins is under intense investigation [ 89 ]. Deletion of all three LCP genes resulted in complete WTA loss in the staphylococcal cell wall and deletion of any of individual LCP genes disturbed the attachment of WTA in different degrees [ 89 ]. This partial functional redundancy was also seen for different phenotypes including β-lactam resistance, biofilm formation, and growth defects [ 91 ]. Of note, LcpA was shown to be the most important protein related to WTA-functions [ 84 ]. In a reconstitution approach, cognate Rbo- P WTA precursors including the murein-linkage unit ( Figure 1 ) could be transferred to PGN by truncated versions of either of the three staphylococcal LCP proteins devoid of the TM-segment (ΔTM) [ 29 ], without the requirement of any other proteins, as had been initially suggested [ 82 ]. For the ligation, the WTA substrate needs to be comprised of only two sugar residues (i.e., the "conventional" murein linkage unit) and a hexaprenyl chain, mimicking the truncated native C 55 undp lipid. This is consistent with the length of the hydrophobic channel length observed in the crystal structure of Cps2A—the LCP protein transferring CPS in Sc. pneumoniae [ 82 , 97 ]. Importantly, the S. aureus WTA precursor could be transferred to "nascent" (i.e., un-crosslinked) PGN polymers only [ 98 ], not to lipid II ( i.e ., murein pentapeptides) [ 29 ], indicating that modification of PGN with WTA occurs prior to final PGN cross-linking [ 98 ]. Deletion of lcpC had no effect on the level of WTA that was ligated to PGN, whereas a Δ lcpA or a Δ lcpB mutant showed reduced levels of WTA content [ 89 , 93 ]. This is corroborated by the finding that the Δ lcpC mutant showed no phosphate release compared to single Δ lcpA and Δ lcpB mutants [ 89 ]. Differential localization and regulation of the enzymes might be important factors regarding the greater impact of LcpA and LcpB on WTA synthesis [ 93 ]. Genes involved in S. aureus CPS synthesis, exemplified with CP5 ( Figure 1 ), are clustered presenting conserved genes employing a Wzy-dependent biosynthesis mechanism [ 64 , 90 ], and there is evidence that specifically LcpC plays a key role in catalysis of S. aureus capsule attachment to the PGN [ 28 , 29 , 90 ]. This is corroborated by the lack of a cpsA homologue encoding a CPS ligase, typically involved in streptococcal CPS ligation, in the S. aureus genome [ 49 , 99 ]. Initial evidence of the requirement of LcpC for ligation of S. aureus CP5 to PGN was derived from a mutant approach [ 90 ]. The Δ lcpC mutant accumulated the capsule in the supernatant fraction, while the Δ lcpAB variants did not display any defect in CP5 synthesis [ 90 ]. A triple mutant showed the same levels of CP5 reduction as the variant lacking lcpC. Surprisingly, plasmid-based expression of any of the three lcp genes could restore the CP5 content, indicating that all three LCP proteins are to some extent involved in the attachment [ 90 ]. To investigate the proposed role of LcpC in vitro, different [ 14 C] CP5 lipid intermediates were synthesized, including lipid I cap (i.e., C 55 undp- PP -D-FucNAc), lipid II cap (i.e., C 55 undp- PP -D-FucNAc-L-FucNAc), and lipid III cap (i.e., C 55 undp- PP -D-FucNAc-L-FucNAc-D-ManNAcA). After purification, these were used together with the ultimate PGN precursor lipid II (lipid II PGN ), i.e., C 55 undp- PP -D-MurNAc-D-GlcNAc including a pentapeptide, as a potential acceptor substrate [ 28 ]. In this setup, LcpC was able to catalyse cleavage of the donor substrate lipid I cap and catalyse attachment of the phosphoryl-sugar moiety to the ultimate PGN precursor lipid II. Strikingly, in the presence of CapA1, an activator/phosphodiesterase protein that cleaves lipid-PP-linked CP5 precursors [ 28 ], the transfer rate was increased, possibly by forming an interaction complex between CapA1 and LcpC [ 28 , 100 ]. Surprisingly, in the case of lipid II cap , no transfer to the PGN acceptor could be overserved in the LcpC in vitro assay, which would likely be deleterious in vivo. On the other hand, all CP5 lipid intermediates were effectively processed by LcpC, although the proximal undp- PP -linked FucNAc residue appeared to be sufficient for CP5 precursor recognition. Of note, the natural PGN acceptor substrate of LcpC remains elusive; possible acceptor structures include lipid II PGN , as well as "nascent" and cross-linked PGN. Summarizing, there is evidence that LcpC preferentially recognizes CP5 intermediates rather than WTA intermediates where deletion of lcpC caused only minor reduction levels [ 89 , 90 ]. Whether this is due to the different reducing-end sugars (i.e., D-FucNAc in the CP5 repeat versus D-Glc p NAc in the "conventional" WTA murein linkage unit remains to be investigated. Notably, in LPS O-antigen RU biosynthesis, the first C 55 lipid- PP -linked sugar unit of the O-antigen RU contains all necessary recognition information for the catalytic activity of the O-antigen ligase WaaL [ 101 ]. S. aureus lcpA, lcpB, lcpC Genes and Physiological Effects Deletion of individual S. aureus LCP protein encoding genes showed only minor effects on S. aureus cell wall physiology and growth, whereas a Δ lcp triple mutant was barely viable, showing temperature sensitivity and enlarged cells [ 91 ]. Complementation with LcpA resulted in restoration of growth and cell size almost to wild-type size [ 91 ]. Interestingly, Δ lcp and Δ lcp Δ tagO variants abolished cell division planes by generating aberrant cells with irregular envelopes lacking WTA [ 89 ]. Of note, deletion of tagO alone did not abolish staphylococcal growth [ 35 , 39 , 102 ]. The same phenotype was observed for isolated tagO and lcpA mutants; however, deletion of lcpA did not interrupt WTA synthesis and thus suggests that WTA synthesis as well as assembly are crucial for normal cell division [ 89 , 95 ]. Furthermore, inhibition of WTA synthesis by tunicamycin treatment did not relieve deviant cell separation of the mutant cells and, therefore, did not suppress the cell division defects of Δ lcp variants [ 89 ]. Autolysis was induced by deletion of all three LCP proteins, resulting in an increased resistance of the Δ lcp triple mutant to autolysis compared to the wild-type [ 89 ]. Complementation by LcpA increased autolysis levels the most [ 89 ]. These pleiotropic phenotypes in the Δ lcp mutant are likely owed to WTA cell wall deposition defects and WTA synthesis, as staphylococcal cells without WTA resulted in deviant cell size and septum formation as well as susceptibility to antibiotics and biofilm formation defects [ 49 , 91 , 92 , 93 ]. A comparison of the surface proteomes of methicillin-resistant, laboratory-adapted S. aureus COL strain (COL) and a COL strain in vitro-adapted to high levels of oxacillin (APT) was used to characterize virulence factors showing that LcpC was found uniquely on the APT surface, suggesting a role in adaption to high oxacillin levels [ 84 ]. Deletion of lcpA decreased oxacillin resistance and upregulated lcpA expression was observed by triggered antibiotic stress [ 103 ] and, furthermore, increased cell size and elevated cell wall remodelling was revealed [ 84 , 92 ]. However, overexpression of LcpC did not generate the APT phenotype in COL, suggesting that aggregation and changes in cell morphology are multifactorial [ 84 ]. The adaption of LcpC to high levels of oxacillin in the APT strain prompted the question about the contribution of this enzyme to antimicrobial resistance and pathogenicity [ 104 ]. The finding that deletion of lcpC decreased the resistance to β-lactams in methicillin-resistant S. aureus (MRSA) and in methicillin-susceptible S. aureus (MSSA) and, consequently, the pathogenicity in the host, suggested that the deviant cell shape might allow for an easier access of the antibiotics to the cell [ 104 ]. Thus, LcpC could be an effective target for drug development [ 104 ]. In MSSA and MRSA strains, reduced oxacillin resistance levels were also observed upon lcpA deletion [ 91 , 92 , 103 ]. The Δ lcp triple mutant was hypersusceptible to oxacillin and growth could be restored by any of the three LCP proteins; LcpA had the greatest impact, followed by LcpB and LcpC [ 91 ]. Interestingly, the Δ lcpA mutant produced more biofilm, and in the Δ lcp triple mutant complementation with LcpC revealed the strongest biofilm, LcpA the weakest [ 91 ]. A mutation in lcpA (E146K) was shown to have an impact on β-lactam and vancomycin resistances and led to a reduced resistance to oxacillin, whereby the cells showed abnormal septal placement [ 105 ]. The mutation further led to a decreased autolytic activity, as was also evident form highest autolysis levels obtained after complementation of the Δ lcp triple mutant with LcpA [ 91 , 105 ]. Crystal Structure of S. aureus LcpA The crystal structure of LcpA devoid of its TM (residues 80–327; ΔTM-LcpA) complexed to C 40 - PP -GlcNAc was solved to 1.9 à and provides a relevant target for inhibitor design studies [ 106 ] ( Figure 3 ). The extracellular domain consists of six-stranded β-sheets overlaid amongst several α-helices and double-stranded β-sheets, where a hydrophobic binding pocket narrow opening and a wide base is formed from its center [ 106 ]. The active site of LcpA is enclosed by region A (residues 92–100), B (residues 188–201), C (residues 217–224) and D (residues 296–312) and shows structural variability. The electropositive region in the active site, as seen in other LCP enzymes, consists of conserved arginine residues. R218 located in the loop of the highly flexible region C is suggested to aid in product expulsion and is held away from the active site by a salt bridge. Furthermore, three potential PGN saccharide binding sites were identified in close proximity to the conserved regions of R99, K135, N137, and D224. As D123 is conserved in many LCP enzymes, it is likely that this basic amino acid plays an important role in PGN binding [ 106 ]. S. aureus LcpA, LcpB, LcpC at a Glance The S. aureus genome harbours three LCP proteins, encoded by lcpA (previous name, msrR ), lcpB ( sa0908 ), and lcpC ( sa2103 ) [ 49 , 89 ], which are in part functionally redundant regarding cellular functions [ 29 , 90 ]. S. aureus variants carrying defective alleles of lcp genes resulted in enhanced [ 91 , 92 ] susceptibility to β-lactam antibiotics, deviant septum formation [ 91 , 92 ], autolysis [ 91 ], activation of a cell wall stress response [ 93 ], reduced phosphate content of staphylococcal cell walls [ 93 ], and aberrant biofilm formation [ 92 ]. In strains lacking WTA due to the inactivation of LCP function, major cell division defects were shown, the PGN synthesis machinery was not localized properly, and these strains were unable of nasal epithelial cell colonization [ 94 , 95 ]. Furthermore, methicillin-resistant S. aureus (MRSA) strains were found to become sensitive to β-lactam antibiotics when WTA synthesis was abrogated [ 95 , 96 ]. S. aureus LcpA, LcpB, LcpC in CWGP Biosynthesis Due to their demonstrated biological importance, the biochemical activity of S. aureus LCP proteins is under intense investigation [ 89 ]. Deletion of all three LCP genes resulted in complete WTA loss in the staphylococcal cell wall and deletion of any of individual LCP genes disturbed the attachment of WTA in different degrees [ 89 ]. This partial functional redundancy was also seen for different phenotypes including β-lactam resistance, biofilm formation, and growth defects [ 91 ]. Of note, LcpA was shown to be the most important protein related to WTA-functions [ 84 ]. In a reconstitution approach, cognate Rbo- P WTA precursors including the murein-linkage unit ( Figure 1 ) could be transferred to PGN by truncated versions of either of the three staphylococcal LCP proteins devoid of the TM-segment (ΔTM) [ 29 ], without the requirement of any other proteins, as had been initially suggested [ 82 ]. For the ligation, the WTA substrate needs to be comprised of only two sugar residues (i.e., the "conventional" murein linkage unit) and a hexaprenyl chain, mimicking the truncated native C 55 undp lipid. This is consistent with the length of the hydrophobic channel length observed in the crystal structure of Cps2A—the LCP protein transferring CPS in Sc. pneumoniae [ 82 , 97 ]. Importantly, the S. aureus WTA precursor could be transferred to "nascent" (i.e., un-crosslinked) PGN polymers only [ 98 ], not to lipid II ( i.e ., murein pentapeptides) [ 29 ], indicating that modification of PGN with WTA occurs prior to final PGN cross-linking [ 98 ]. Deletion of lcpC had no effect on the level of WTA that was ligated to PGN, whereas a Δ lcpA or a Δ lcpB mutant showed reduced levels of WTA content [ 89 , 93 ]. This is corroborated by the finding that the Δ lcpC mutant showed no phosphate release compared to single Δ lcpA and Δ lcpB mutants [ 89 ]. Differential localization and regulation of the enzymes might be important factors regarding the greater impact of LcpA and LcpB on WTA synthesis [ 93 ]. Genes involved in S. aureus CPS synthesis, exemplified with CP5 ( Figure 1 ), are clustered presenting conserved genes employing a Wzy-dependent biosynthesis mechanism [ 64 , 90 ], and there is evidence that specifically LcpC plays a key role in catalysis of S. aureus capsule attachment to the PGN [ 28 , 29 , 90 ]. This is corroborated by the lack of a cpsA homologue encoding a CPS ligase, typically involved in streptococcal CPS ligation, in the S. aureus genome [ 49 , 99 ]. Initial evidence of the requirement of LcpC for ligation of S. aureus CP5 to PGN was derived from a mutant approach [ 90 ]. The Δ lcpC mutant accumulated the capsule in the supernatant fraction, while the Δ lcpAB variants did not display any defect in CP5 synthesis [ 90 ]. A triple mutant showed the same levels of CP5 reduction as the variant lacking lcpC. Surprisingly, plasmid-based expression of any of the three lcp genes could restore the CP5 content, indicating that all three LCP proteins are to some extent involved in the attachment [ 90 ]. To investigate the proposed role of LcpC in vitro, different [ 14 C] CP5 lipid intermediates were synthesized, including lipid I cap (i.e., C 55 undp- PP -D-FucNAc), lipid II cap (i.e., C 55 undp- PP -D-FucNAc-L-FucNAc), and lipid III cap (i.e., C 55 undp- PP -D-FucNAc-L-FucNAc-D-ManNAcA). After purification, these were used together with the ultimate PGN precursor lipid II (lipid II PGN ), i.e., C 55 undp- PP -D-MurNAc-D-GlcNAc including a pentapeptide, as a potential acceptor substrate [ 28 ]. In this setup, LcpC was able to catalyse cleavage of the donor substrate lipid I cap and catalyse attachment of the phosphoryl-sugar moiety to the ultimate PGN precursor lipid II. Strikingly, in the presence of CapA1, an activator/phosphodiesterase protein that cleaves lipid-PP-linked CP5 precursors [ 28 ], the transfer rate was increased, possibly by forming an interaction complex between CapA1 and LcpC [ 28 , 100 ]. Surprisingly, in the case of lipid II cap , no transfer to the PGN acceptor could be overserved in the LcpC in vitro assay, which would likely be deleterious in vivo. On the other hand, all CP5 lipid intermediates were effectively processed by LcpC, although the proximal undp- PP -linked FucNAc residue appeared to be sufficient for CP5 precursor recognition. Of note, the natural PGN acceptor substrate of LcpC remains elusive; possible acceptor structures include lipid II PGN , as well as "nascent" and cross-linked PGN. Summarizing, there is evidence that LcpC preferentially recognizes CP5 intermediates rather than WTA intermediates where deletion of lcpC caused only minor reduction levels [ 89 , 90 ]. Whether this is due to the different reducing-end sugars (i.e., D-FucNAc in the CP5 repeat versus D-Glc p NAc in the "conventional" WTA murein linkage unit remains to be investigated. Notably, in LPS O-antigen RU biosynthesis, the first C 55 lipid- PP -linked sugar unit of the O-antigen RU contains all necessary recognition information for the catalytic activity of the O-antigen ligase WaaL [ 101 ]. S. aureus lcpA, lcpB, lcpC Genes and Physiological Effects Deletion of individual S. aureus LCP protein encoding genes showed only minor effects on S. aureus cell wall physiology and growth, whereas a Δ lcp triple mutant was barely viable, showing temperature sensitivity and enlarged cells [ 91 ]. Complementation with LcpA resulted in restoration of growth and cell size almost to wild-type size [ 91 ]. Interestingly, Δ lcp and Δ lcp Δ tagO variants abolished cell division planes by generating aberrant cells with irregular envelopes lacking WTA [ 89 ]. Of note, deletion of tagO alone did not abolish staphylococcal growth [ 35 , 39 , 102 ]. The same phenotype was observed for isolated tagO and lcpA mutants; however, deletion of lcpA did not interrupt WTA synthesis and thus suggests that WTA synthesis as well as assembly are crucial for normal cell division [ 89 , 95 ]. Furthermore, inhibition of WTA synthesis by tunicamycin treatment did not relieve deviant cell separation of the mutant cells and, therefore, did not suppress the cell division defects of Δ lcp variants [ 89 ]. Autolysis was induced by deletion of all three LCP proteins, resulting in an increased resistance of the Δ lcp triple mutant to autolysis compared to the wild-type [ 89 ]. Complementation by LcpA increased autolysis levels the most [ 89 ]. These pleiotropic phenotypes in the Δ lcp mutant are likely owed to WTA cell wall deposition defects and WTA synthesis, as staphylococcal cells without WTA resulted in deviant cell size and septum formation as well as susceptibility to antibiotics and biofilm formation defects [ 49 , 91 , 92 , 93 ]. A comparison of the surface proteomes of methicillin-resistant, laboratory-adapted S. aureus COL strain (COL) and a COL strain in vitro-adapted to high levels of oxacillin (APT) was used to characterize virulence factors showing that LcpC was found uniquely on the APT surface, suggesting a role in adaption to high oxacillin levels [ 84 ]. Deletion of lcpA decreased oxacillin resistance and upregulated lcpA expression was observed by triggered antibiotic stress [ 103 ] and, furthermore, increased cell size and elevated cell wall remodelling was revealed [ 84 , 92 ]. However, overexpression of LcpC did not generate the APT phenotype in COL, suggesting that aggregation and changes in cell morphology are multifactorial [ 84 ]. The adaption of LcpC to high levels of oxacillin in the APT strain prompted the question about the contribution of this enzyme to antimicrobial resistance and pathogenicity [ 104 ]. The finding that deletion of lcpC decreased the resistance to β-lactams in methicillin-resistant S. aureus (MRSA) and in methicillin-susceptible S. aureus (MSSA) and, consequently, the pathogenicity in the host, suggested that the deviant cell shape might allow for an easier access of the antibiotics to the cell [ 104 ]. Thus, LcpC could be an effective target for drug development [ 104 ]. In MSSA and MRSA strains, reduced oxacillin resistance levels were also observed upon lcpA deletion [ 91 , 92 , 103 ]. The Δ lcp triple mutant was hypersusceptible to oxacillin and growth could be restored by any of the three LCP proteins; LcpA had the greatest impact, followed by LcpB and LcpC [ 91 ]. Interestingly, the Δ lcpA mutant produced more biofilm, and in the Δ lcp triple mutant complementation with LcpC revealed the strongest biofilm, LcpA the weakest [ 91 ]. A mutation in lcpA (E146K) was shown to have an impact on β-lactam and vancomycin resistances and led to a reduced resistance to oxacillin, whereby the cells showed abnormal septal placement [ 105 ]. The mutation further led to a decreased autolytic activity, as was also evident form highest autolysis levels obtained after complementation of the Δ lcp triple mutant with LcpA [ 91 , 105 ]. Crystal Structure of S. aureus LcpA The crystal structure of LcpA devoid of its TM (residues 80–327; ΔTM-LcpA) complexed to C 40 - PP -GlcNAc was solved to 1.9 à and provides a relevant target for inhibitor design studies [ 106 ] ( Figure 3 ). The extracellular domain consists of six-stranded β-sheets overlaid amongst several α-helices and double-stranded β-sheets, where a hydrophobic binding pocket narrow opening and a wide base is formed from its center [ 106 ]. The active site of LcpA is enclosed by region A (residues 92–100), B (residues 188–201), C (residues 217–224) and D (residues 296–312) and shows structural variability. The electropositive region in the active site, as seen in other LCP enzymes, consists of conserved arginine residues. R218 located in the loop of the highly flexible region C is suggested to aid in product expulsion and is held away from the active site by a salt bridge. Furthermore, three potential PGN saccharide binding sites were identified in close proximity to the conserved regions of R99, K135, N137, and D224. As D123 is conserved in many LCP enzymes, it is likely that this basic amino acid plays an important role in PGN binding [ 106 ]. 4.1.2. Bacillus subtilis WTAs and lipoteichoic acid constitute up to 60% of the dry weight of the cell wall in B. subtilis providing an overall negative charge to the cell wall [ 14 , 107 ]. Both WTA and LTA are important, as cells that cannot produce either of these compounds show morphological aberrations and can only be grown under certain conditions, whereas the absence of both CWGPs is lethal [ 41 ]. WTA is covalently attached to the MurNAc residues in the B. subtilis cell wall via a "conventional" murein linkage unit; coupled to it is poly(Gro- P ) that can have either D-alanine or glucose bound to the C2, with chain lengths varying from 45 to 60 residues [ 14 ] ( Figure 1 ). WTA biosynthesis in B. subtilis has traditionally been intensely investigated [ 14 , 108 ]. B. subtilis TagT, TagU, TagV, Genes and Physiological Effects In the B. subtilis genome, three LCP genes are encoded— tagT (previously named ywtF ), tagU ( lytR ), and tagV ( yvhJ ) [ 82 ]. As in S. aureus , also in B. subtilis , lcp gene deletion mutants revealed defects in WTA, accompanied by reduced virulence, enhanced antibiotics susceptibility, and deviant cell wall structures [ 82 , 91 , 92 ]. Single mutation variants of the tagTUV genes showed no significant effect on either cell morphology or growth, but a Δ tagTV mutant displayed noticeably slower growth and aberrant cell shape [ 82 ]. In their search for interaction partners for the MreB protein that is involved in later wall PGN synthesis in B. subtilis , Kawai et al. were among the first to describe the LCP enzyme family and identified these enzymes as key players in the attachment of anionic CWGPs such as WTA to the cell wall [ 82 ]. The expression of at least one out of the three lcp gene was found to be required for complete WTA biosynthesis and growth of B. subtilis [ 82 ]. B. subtilis TagT, TagU, TagV in WTA Biosynthesis It was demonstrated in in vitro assays that all LCP enzymes, produced recombinantly without the TM domain, were capable of transferring WTA intermediates to PGN [ 109 ]. Particularly, lipid β (i.e., Man p NAc-β-(1→4)-Glc p NAc-1- PP -undp) was attached to mature PGN in vitro [ 109 ], which is consistent with several crystal structures of LCP enzymes where these proteins showed to bind polyisoprenoid phosphate lipids [ 82 , 109 ]. Of note, the use of a short aliphatic chain (C 13 ) in place of the authentic C 55 undp moiety led to a 95% reduction of LCP activity [ 109 ]. This contradicts the observations made by others for S. aureus LCP proteins, where the lipid portion was assumed to play only a minor role in the ligation reaction [ 82 ]. Currently, it is suggested that key interactions between LCP enzymes and donor CWGP substrates occur in the region of the polyisoprenoid lipid that is proximal to the pyrophosphate moiety. Interestingly, the individual B. subtilis LCP enzymes display significant differences in their in vitro activities [ 109 ]. TagU showed an approximately three-fold higher activity compared to TagT and TagV, which contradicts the proposed functional redundancy of the proteins [ 82 , 109 ]. Thus, it is still possible that each Tag enzyme transfers preferentially a distinct type of CWGP to PGN, representing a scenario comparable to S. aureus , where LcpA [ 98 ] and LcpC [ 90 ] can recognize different substrates [ 109 ]. Conclusively, catalysis of WTA transfer by B. subtilis LCP enzymes requires magnesium ions and a polyisoprenoid moiety of the donor CWGP [ 109 ], and this finding is in accordance with the identity of the first three isoprene units of a WTA substrate used by Schaefer et al. [ 29 , 109 ]. Notably, WTA transfer by TagT, TagU and TagV was successful to higher-order structures of PGN, but it still needs to be investigated how the oligomeric repeats of PGN are recognized. Crystal Structure of B. subtilis TagT, TagU, TagV A ΔTM-TagT (residues 44-322) construct was crystallized, and, consistent with its known pyrophosphatase activity towards polyprenoid-pyrophosphate lipid substrates [ 82 ], an octaprenyl-pyrophosphate fitted well in the hydrophobic tunnel in the electron density map [ 97 ]. Interestingly, longer lipids, such as undp- P fitted worse, and presumably in a full-length protein, the hydrophobic tail of the lipid extends out of the tunnel to interact with the membrane. The ΔTM-TagT structure supports the enzymatic role of TagT in the activity of transferring phosphorylated anionic CWGPs from an undp- P -linked precursor to PGN [ 97 ]. Many interactions between the pyrophosphate head group and charged residues are similar to the Sc. pneumoniae Cps2A enzyme (see below). Asp82, corresponding to Asp234 in Cps2A, is more distant from the phosphate, whereas Asp234 coordinates a magnesium ion interacting with the lipid head group. However, this loop region showed disorder and could not be modelled and possibly is the reason why the magnesium ion did not bind to the pyrophosphate group [ 97 ]. In a more recent study to better understand the substrate preferences of LCP proteins, the ΔTM-TagT protein was crystallized with two lipid-linked WTA precursors, namely LI WTA (i.e., [ 14 C] lipid- PP -GlcNAc) and LII WTA , containing the "conventional" disaccharide murein linkage unit [ 98 ]. The resulting structures differed regarding the orientation of the pyrophosphate and saccharide moieties, where the disaccharide-containing structure exposed a divalent cation in the active site. Changes in the glycosidic linkage and pyrophosphate bonds led to a different orientation of the GlcNAc residue of LII WTA , resulting in a conformation of the pyrophosphate where a divalent cation can bind, as would be necessary for catalysis. Evidently, the PGN substrate binds closely to the anomeric phosphate of LII WTA in a narrow groove, which, similar to the WTA binding pocket, displays three conserved arginine residues. R219 is suggested to play a role in the nucleophilic attack by the PGN substrate, whereas another arginine residue, R118, facilitates deprotonation of the C6-OH of MurNAc [ 98 ]. Li and colleagues provided crystal structures of all three B. subtilis LCP proteins, with additional electron density for the disordered loop region in the TagT apo structure. All enzymes were expressed without their TM segment (TagT, residues 46–322; TagU, residues 62–306; TagV, residues 72–332). All secondary structures consist of four regions, consistent with the structure of LcpA from S. aureus (compare with Figure 3 ), with significant differences in region B, where TagT shows an α-helix, and TagU and TagV a double-stranded β-sheet. On one side of the central β-sheet in TagU, helices 3–7 collapse the lipid binding site, whereas other LCP enzymes, including TagT, show a disorder of helix 6. The exposure of this hydrophobic core is, again, suggested to be necessary for surface interaction with the membrane [ 106 ]. B. subtilis TagT, TagU, TagV, Genes and Physiological Effects In the B. subtilis genome, three LCP genes are encoded— tagT (previously named ywtF ), tagU ( lytR ), and tagV ( yvhJ ) [ 82 ]. As in S. aureus , also in B. subtilis , lcp gene deletion mutants revealed defects in WTA, accompanied by reduced virulence, enhanced antibiotics susceptibility, and deviant cell wall structures [ 82 , 91 , 92 ]. Single mutation variants of the tagTUV genes showed no significant effect on either cell morphology or growth, but a Δ tagTV mutant displayed noticeably slower growth and aberrant cell shape [ 82 ]. In their search for interaction partners for the MreB protein that is involved in later wall PGN synthesis in B. subtilis , Kawai et al. were among the first to describe the LCP enzyme family and identified these enzymes as key players in the attachment of anionic CWGPs such as WTA to the cell wall [ 82 ]. The expression of at least one out of the three lcp gene was found to be required for complete WTA biosynthesis and growth of B. subtilis [ 82 ]. B. subtilis TagT, TagU, TagV in WTA Biosynthesis It was demonstrated in in vitro assays that all LCP enzymes, produced recombinantly without the TM domain, were capable of transferring WTA intermediates to PGN [ 109 ]. Particularly, lipid β (i.e., Man p NAc-β-(1→4)-Glc p NAc-1- PP -undp) was attached to mature PGN in vitro [ 109 ], which is consistent with several crystal structures of LCP enzymes where these proteins showed to bind polyisoprenoid phosphate lipids [ 82 , 109 ]. Of note, the use of a short aliphatic chain (C 13 ) in place of the authentic C 55 undp moiety led to a 95% reduction of LCP activity [ 109 ]. This contradicts the observations made by others for S. aureus LCP proteins, where the lipid portion was assumed to play only a minor role in the ligation reaction [ 82 ]. Currently, it is suggested that key interactions between LCP enzymes and donor CWGP substrates occur in the region of the polyisoprenoid lipid that is proximal to the pyrophosphate moiety. Interestingly, the individual B. subtilis LCP enzymes display significant differences in their in vitro activities [ 109 ]. TagU showed an approximately three-fold higher activity compared to TagT and TagV, which contradicts the proposed functional redundancy of the proteins [ 82 , 109 ]. Thus, it is still possible that each Tag enzyme transfers preferentially a distinct type of CWGP to PGN, representing a scenario comparable to S. aureus , where LcpA [ 98 ] and LcpC [ 90 ] can recognize different substrates [ 109 ]. Conclusively, catalysis of WTA transfer by B. subtilis LCP enzymes requires magnesium ions and a polyisoprenoid moiety of the donor CWGP [ 109 ], and this finding is in accordance with the identity of the first three isoprene units of a WTA substrate used by Schaefer et al. [ 29 , 109 ]. Notably, WTA transfer by TagT, TagU and TagV was successful to higher-order structures of PGN, but it still needs to be investigated how the oligomeric repeats of PGN are recognized. Crystal Structure of B. subtilis TagT, TagU, TagV A ΔTM-TagT (residues 44-322) construct was crystallized, and, consistent with its known pyrophosphatase activity towards polyprenoid-pyrophosphate lipid substrates [ 82 ], an octaprenyl-pyrophosphate fitted well in the hydrophobic tunnel in the electron density map [ 97 ]. Interestingly, longer lipids, such as undp- P fitted worse, and presumably in a full-length protein, the hydrophobic tail of the lipid extends out of the tunnel to interact with the membrane. The ΔTM-TagT structure supports the enzymatic role of TagT in the activity of transferring phosphorylated anionic CWGPs from an undp- P -linked precursor to PGN [ 97 ]. Many interactions between the pyrophosphate head group and charged residues are similar to the Sc. pneumoniae Cps2A enzyme (see below). Asp82, corresponding to Asp234 in Cps2A, is more distant from the phosphate, whereas Asp234 coordinates a magnesium ion interacting with the lipid head group. However, this loop region showed disorder and could not be modelled and possibly is the reason why the magnesium ion did not bind to the pyrophosphate group [ 97 ]. In a more recent study to better understand the substrate preferences of LCP proteins, the ΔTM-TagT protein was crystallized with two lipid-linked WTA precursors, namely LI WTA (i.e., [ 14 C] lipid- PP -GlcNAc) and LII WTA , containing the "conventional" disaccharide murein linkage unit [ 98 ]. The resulting structures differed regarding the orientation of the pyrophosphate and saccharide moieties, where the disaccharide-containing structure exposed a divalent cation in the active site. Changes in the glycosidic linkage and pyrophosphate bonds led to a different orientation of the GlcNAc residue of LII WTA , resulting in a conformation of the pyrophosphate where a divalent cation can bind, as would be necessary for catalysis. Evidently, the PGN substrate binds closely to the anomeric phosphate of LII WTA in a narrow groove, which, similar to the WTA binding pocket, displays three conserved arginine residues. R219 is suggested to play a role in the nucleophilic attack by the PGN substrate, whereas another arginine residue, R118, facilitates deprotonation of the C6-OH of MurNAc [ 98 ]. Li and colleagues provided crystal structures of all three B. subtilis LCP proteins, with additional electron density for the disordered loop region in the TagT apo structure. All enzymes were expressed without their TM segment (TagT, residues 46–322; TagU, residues 62–306; TagV, residues 72–332). All secondary structures consist of four regions, consistent with the structure of LcpA from S. aureus (compare with Figure 3 ), with significant differences in region B, where TagT shows an α-helix, and TagU and TagV a double-stranded β-sheet. On one side of the central β-sheet in TagU, helices 3–7 collapse the lipid binding site, whereas other LCP enzymes, including TagT, show a disorder of helix 6. The exposure of this hydrophobic core is, again, suggested to be necessary for surface interaction with the membrane [ 106 ]. 4.1.3. Bacillus anthracis B. anthracis is a spore-forming Gram-positive pathogen which replicates within vertebrates as chains of vegetative cells by regulating the separation of septal PGN [ 110 ]. The pathogen is the causative agent of anthrax and displays a unique growth pattern by tethering at septal PGN, which results in protection of the bacterium from engulfment by host phagocytes [ 111 ]. Comparing B. anthracis with S. aureus and B. subtilis with regard to the presence of CWGPs, it is important to note that B. anthracis does not harbour a WTA, but expresses a PyrCWGP [ 48 ] ( Figure 1 ) and a poly-D-γ-glutamic acid capsule that is bound to PGN via amide bonds [ 112 , 113 ]. The B. anthracis S-layer proteins and S-layer-associated proteins (BSLs) [ 114 ] function as chain length and cell size determinants and are assembled in the envelope by binding to the bacterium's PyrCWGP [ 48 ]. The biosynthesis of that specific CWGP involves the B. anthracis LCP proteins [ 43 , 115 , 116 ]. B. anthracis LcpB1, LcpB2, LcpB3, LcpB4, LcpC, LcpD, Genes and Physiological Effects B. anthracis encodes on its genome six LCP homologues—BAS1830 (LcpB1), BAS0572 (LcpB2), BAS0746 (LcpB3), BAS3381 (LcpB4), BAS5115 (LcpC) and BAS5047 (LcpD). Mutations in B. anthracis lcpB3 and lcpD caused aberrations in cell size and chain length that could be explained as discrete defects in PyrCWGP assembly. By deleting combinations of lcp genes from the B. anthracis genome, variants with single lcp genes were generated [ 43 ]. B. anthracis expressing lcpB3 alone displayed physiological cell size, vegetative growth, spore formation, and S-layer assembly, which might implicate a direct contribution of this LCP protein to the bacterial cell cycle [ 116 ]. Strains expressing lcpB1 or lcpB4 displayed defects in cell size and shape, S-layer assembly, and spore formation, yet sustained vegetative growth. In contrast, the lcpB2 strain was unable to grow unless the gene was expressed from a multicopy plasmid, and variants expressing lcpC or lcpD displayed severe defects in growth and cell shape. The lcpB2 , lcpC or lcpD strains supported neither S-layer assembly nor spore formation. Conclusively, it is conceivable that B. anthracis LCP enzymes fulfil partially overlapping functions in transferring CWGP to discrete sites within the bacterial envelope. B. anthracis LcpB1, LcpB2, LcpB3, LcpB4, LcpC, LcpD in S. aureus WTA Biosynthesis All six B. anthracis lcp genes were tested for their restoration capability of WTA synthesis in a S. aureus Δ lcp mutant lacking all three lcp genes, revealing that, with lcpB2 , lcpC and lcpD plasmids, full complementation could be achieved. lcpB1 was not able to restore S. aureus WTA synthesis at all and lcpB3 and lcpB4 achieved partial complementation. Thus, evidence has been obtained that LcpB2, LcpC and LcpD could transfer a WTA providing a "conventional" murein linkage unit to PGN implicating that these enzymes would display ligation activity also upon the B. anthracis PyrCWGP, which provides an identical murein linkage unit [ 115 ]. The open question, why B. anthracis employs six LCP enzymes while e.g., B. subtilis has evolved only three (i.e., tagTUV ) [ 39 ] might be, at least in part, explained by the genomic organization of the CWGP biosynthesis machinery in these bacteria. In B. subtilis , tagO is part of a 50-kb genomic region that harbours virtually all genes required for WTA synthesis, including tagTUV [ 82 , 117 ]. In contrast, according to the current understanding, four different genomic loci of B. anthracis are linked to PyrCWGP biosynthesis, of which two encode one LCP protein each. In the scwp1 locus, lcpD is encoded in close proximity to the essential tagO gene as well as the gneY gene required for PyrCWGP synthesis [ 118 , 119 ]. The sps locus ("surface polysaccharide") encodes lcpC and the essential gneZ gene, encoding a UDP-GlcNAc-2-epimerase. The genes for the remaining four LCP enzymes—LcpB1, LcpB2, LcpB3, LcpB4—are encoded elsewhere on the genome and are not linked to genes that are known to contribute to the synthesis of PyrCWGP or a "conventional" murein linkage unit. Thus, the expanded repertoire of B. anthracis lcp genes could implicate that these LCP enzymes are able to attach different, not yet identified, types of B. anthracis CWGPs to PGN [ 43 ]. B. anthracis LcpB1, LcpB2, LcpB3, LcpB4, LcpC, LcpD, Genes and Physiological Effects B. anthracis encodes on its genome six LCP homologues—BAS1830 (LcpB1), BAS0572 (LcpB2), BAS0746 (LcpB3), BAS3381 (LcpB4), BAS5115 (LcpC) and BAS5047 (LcpD). Mutations in B. anthracis lcpB3 and lcpD caused aberrations in cell size and chain length that could be explained as discrete defects in PyrCWGP assembly. By deleting combinations of lcp genes from the B. anthracis genome, variants with single lcp genes were generated [ 43 ]. B. anthracis expressing lcpB3 alone displayed physiological cell size, vegetative growth, spore formation, and S-layer assembly, which might implicate a direct contribution of this LCP protein to the bacterial cell cycle [ 116 ]. Strains expressing lcpB1 or lcpB4 displayed defects in cell size and shape, S-layer assembly, and spore formation, yet sustained vegetative growth. In contrast, the lcpB2 strain was unable to grow unless the gene was expressed from a multicopy plasmid, and variants expressing lcpC or lcpD displayed severe defects in growth and cell shape. The lcpB2 , lcpC or lcpD strains supported neither S-layer assembly nor spore formation. Conclusively, it is conceivable that B. anthracis LCP enzymes fulfil partially overlapping functions in transferring CWGP to discrete sites within the bacterial envelope. B. anthracis LcpB1, LcpB2, LcpB3, LcpB4, LcpC, LcpD in S. aureus WTA Biosynthesis All six B. anthracis lcp genes were tested for their restoration capability of WTA synthesis in a S. aureus Δ lcp mutant lacking all three lcp genes, revealing that, with lcpB2 , lcpC and lcpD plasmids, full complementation could be achieved. lcpB1 was not able to restore S. aureus WTA synthesis at all and lcpB3 and lcpB4 achieved partial complementation. Thus, evidence has been obtained that LcpB2, LcpC and LcpD could transfer a WTA providing a "conventional" murein linkage unit to PGN implicating that these enzymes would display ligation activity also upon the B. anthracis PyrCWGP, which provides an identical murein linkage unit [ 115 ]. The open question, why B. anthracis employs six LCP enzymes while e.g., B. subtilis has evolved only three (i.e., tagTUV ) [ 39 ] might be, at least in part, explained by the genomic organization of the CWGP biosynthesis machinery in these bacteria. In B. subtilis , tagO is part of a 50-kb genomic region that harbours virtually all genes required for WTA synthesis, including tagTUV [ 82 , 117 ]. In contrast, according to the current understanding, four different genomic loci of B. anthracis are linked to PyrCWGP biosynthesis, of which two encode one LCP protein each. In the scwp1 locus, lcpD is encoded in close proximity to the essential tagO gene as well as the gneY gene required for PyrCWGP synthesis [ 118 , 119 ]. The sps locus ("surface polysaccharide") encodes lcpC and the essential gneZ gene, encoding a UDP-GlcNAc-2-epimerase. The genes for the remaining four LCP enzymes—LcpB1, LcpB2, LcpB3, LcpB4—are encoded elsewhere on the genome and are not linked to genes that are known to contribute to the synthesis of PyrCWGP or a "conventional" murein linkage unit. Thus, the expanded repertoire of B. anthracis lcp genes could implicate that these LCP enzymes are able to attach different, not yet identified, types of B. anthracis CWGPs to PGN [ 43 ]. 4.2. Firmicutes— Order : Lactobacillales 4.2.1. Streptococcus pneumoniae Sc. pneumoniae or pneumococcus is a major human pathogen, which typically resides in the nasopharyngeal cavity. Bacterial colonization requires interaction with host cells, for which the amount of capsule is crucial [ 120 ]. Thus, its CPS is a key virulence factor shielding Sc. pneumoniae from the host immune system and, thus, an important target for protective immune responses. Ninety-three capsular types have been identified serologically, and the RU unit structure of each has been defined [ 64 ]. Sc. pneumoniae is especially dangerous for immunocompromised people where it can cause severe diseases, including meningitis, pneumoniae or sepsis. In asymptomatic humans, Sc. pneumoniae resides in the nasal cavity or the sinuses, where it may cause otitis media and acute sinusitis [ 121 ]. The capsular polysaccharide serotype 2 of Sc. pneumoniae strain D39 (CP2) is composed of branched hexasaccharide RUs with the structure →3)[α-Glc p A-(1→6)-α-Glc-(1→2)]-α-L-Rha-(1→3)-α-L-Rha-(1→3)-β-L-Rha-(1→4)-β-D-Glc- ( Figure 1 ). CP2 is directly glycosidically linked via the reducing end glucose of the RU to β-D-GlcNAc residues of PGN, without involvement of a murein linkage unit and a phosphodiester bond [ 122 ], which contrasts the usual attachment mode of CWGPs to PGN backbone sugars. The cell wall of Sc. pneumoniae further contains an unusually complex WTA, which has identical RUs as the membrane-anchored lipoteichoic acid. Both show pseudo-pentasaccharide RUs containing the rare amino sugar 2-acetamido-4-amino-2,4,6-trideoxygalactose (AATGal) in addition to Glc, Rib- P , and two GalNAc residues that are each modified with phosphorylcholine ( Figure 1 ) [ 123 , 124 , 125 ]. The reducing-end AATGal is proposed to be linked via a phosphodiester bond to MurNAc residues of PGN, based on the in silico identification of LCP family proteins in the Sc. pneumoniae genome, however, without provision of any biochemical evidence [ 124 ]. Sc. pneumoniae Cps2A, LytR, Psr, Genes and Physiological Effects On the Sc. pneumoniae genome, the capsular region of CP 2 begins with the cps2A–D genes; all 17 capsular genes in this region are under control of the promoter upstream of cps2A .The first gene in the region, cps2A , encodes a member of the LCP protein family. Within the biosynthesis pathway of the CP2 of Sc. pneumoniae strain D39, the three paralogous LCP proteins Cps2A, LytR and Psr, with the latter two not localizing to the cps region, have been investigated. Evidence was provided that Cps2A, LytR and Psr contribute to the maintenance of normal capsule levels and to the retention of the CP2 in the Sc. pneumoniae cell wall. Cps2A, LytR and Psr were found to localize at the cell membrane and accumulate at septal sites supportive of their function in cell wall maintenance. Single Δ cps2A and Δ psr mutants produced a reduced amount of capsule, while a Δ cps2AlytR double mutant showed greatly impaired growth and cell morphology and lost approximately half of the total capsule material into the culture supernatant [ 97 ]. Notably, inactivation of lytR proved to be difficult in the background of the encapsulated D39 strain and during exponential growth, LytR expression was continuously high which suggests a housekeeping function of this gene during cell division that is essential for proper septum placement [ 126 ]. According to a current data-based model, CpsA2 is responsible for the covalent attachment of CP2 to the pneumococcal cell wall, and LytR can take over this function in the absence of Cps2A. Crystal Structure of Sc. pneumoniae Cps2A Structural and functional studies of the Cps2A enzyme from Sc. pneumoniae provided the first insight into the catalytic mechanism of an enzyme from the LCP protein family. Cps2A contains a large hydrophobic tunnel that is capped with surface-exposed arginine residues that are important for catalysis [ 82 ]; serendipitously, Cps2A co-crystallizes with octaprenyl-pyrophosphate, where the isoprenyl-tail is nestled within the hydrophobic pocket with the pyrophosphate head group interacting with highly conserved arginine residues within the active site. A ΔTM-Cps2A version of the protein comprising the accessory domain (amino acid residues 111–213) and the LCP domain (amino acid residues 214–481) has been solved at 1.69 à -resolution. Within the LCP domain, a core containing a five-stranded β-sheet surrounded by α-helices on both faces is comprised of an overall α-β-α architecture. Between the two domains, two pairs of β-strands extend from the core site. A hydrophobic pocket is formed between the central β-sheet and α-helices 3–7, where a polyisoprenoid phosphate lipid was found. As it has been seen across the LCP superfamily, these hydrophobic side chains are conserved and arginine residues R267, R362 and R374 play a key role in the interaction between the phosphate oxygens and the formation of the pocket and are stabilized by D371 and Q378. Furthermore, magnesium ions are suggested to contribute to catalysis by implicating neutralization of the developing negative charge and are coordinated by two aspartate residues, D234 and D246 [ 82 ]. Based on the crystal structure of the soluble part of Cps2A, it was inferred that all three homologs in Sc. pneumoniae (Cps2A, LytR, and Psr) might attach polyprenol pyrophosphoryl-linked polymers to PGN without any further specification of the CWGP structure [ 97 ], which leaves open the possibility that these enzymes might additionally be involved in WTA attachment. 4.2.2. Streptococcus agalactiae Sc. agalactiae belongs to the GroupB Streptococcus (GBS); it is a common commensal organism which occurs on vaginal and rectal mucosal surfaces but is also associated with invasive infections, especially in elderly or immunocompromised patients [ 127 ]. CPS represents the main virulence factor of Sc. agalactiae and is a prime target in current vaccine development [ 128 ]. GBS isolates associated with human infection produce one of nine antigenically distinct CPSs [ 127 ]. The Sc. agalactiae CPS serotype III (CPIII) has a branched pentasaccharide structure composed of →6)-[α-NeuAc-(2→3)-Gal p -(1→4)]-β-Glc p NAc-(1→3)-β-Gal-(1→4)-α-Glc-(1→ RUs. CPIII is bound via a phosphodiester bond and an oligosaccharide linker of unknown structure composed of glucose, galactose and arabinose to GlcNAc residues of PGN [ 129 , 130 ] ( Figure 1 ), representing a further variation of the PGN linkage mode. Sc. agalactiae CpsA, Gene and Physiological Effects The investigation of the conserved proteins CpsABCD encoded in the Sc. agalactiae cps operon revealed that cpsA , the first gene in the operon, has a regulatory function and is required for the transcription of the operon and that CpsBCD composes a phosphoregulatory system [ 100 ]. Although having no impact on cps transcription or the synthesis of the CPIII RU, it was suggested that these proteins are required for fine-tuning of the last steps of CPIII biosynthesis, which is balancing repeating unit polymerization and CPIII attachment to the cell wall. As a member of the LCP protein family, the 485-amino acid membrane protein CpsA is unique due to its extracellular accessory domain. It equips CpsA to specifically bind to two promoters in the cps locus [ 131 ]. However, a protein consisting of the accessory domain alone could not complement a Δ cpsA deletion strain for CPIII biosynthesis. Interestingly, even if the truncated form co-existed with the native cpsA , the capsule production was impaired (dominant-negative effect), suggesting involvement of CpsA in cell wall maintenance in addition to capsule expression [ 99 ]. Essential for the dominant-negative effect is a region between amino acids 210 and 245 and probably between amino acids 132 and 153 of the accessory domain of CpsA. Experiments using a fluorescent peptide showed that this effect was not due to a direct interaction of truncated CpsA with wild-type CpsA. Probably, it disturbs the mechanism associated with normal cell wall integrity and CPS synthesis [ 131 ]. Interestingly, zebrafish experiments revealed that expression of a truncated CpsA representing the accessory domain only decreases virulence stronger than the complete absence of CpsA [ 131 ]. Sc. agalactiae CpsA in CPIII Biosynthesis In the final steps of CPIII biosynthesis, the newly synthesized pentasaccharide RU anchored to a polyisoprenoid phosphate lipid is flipped to the outer side of the bacterial membrane, where CpsH acts as the repeating unit polymerase. By analogy with other Wzy-dependent systems, polymerization occurs bottom-up. The nascent CPIII is removed from the lipid through a phosphotransferase reaction and subsequently linked to a single membrane-anchored RU. The final product is a CPS that is removed from the membrane lipid and covalently attached to GlcNAc of the PGN backbone by CpsA activity. This linkage effectively renders further polymerization impossible [ 100 ]. 4.2.3. Streptococcus mutans Sc. mutans is a prototypical member of the lactic acid bacteria group. It inhabits the dental plaque biofilm community of the oral cavity and represents the most tenacious causative agent of the enamel-destructive disease dental caries [ 79 ]. As within several streptococcal species, the major cell wall antigen of Sc. mutans is its RhaCWGP which consists of linear rhamnose-polymers with glucose side chains (also referred to as RGP) [ 132 , 133 ] ( Figure 1 ) of the RU structure →2)-[α-Glc-(1→2)]-α-Rha-(1→3)-α-Rha-(1→2)-[α-Glc-(1→2)]-α-Rha-(1→3)-α-Rha-(1→, making up approxima-tely half of the total weight of the bacterium's cell wall [ 76 , 134 ]. The mature RGP contributes to acid and oxidative stress tolerance of Sc. mutans in the oral habitat [ 135 ] and is required for proper localization of cell division complexes in Sc. mutans translating in a morphogenic role for the bacterium [ 79 ]. Specifically, the serotype-specific glucose branches of the RhaCWGP from strain Sc. mutans serotype c were shown to act as a receptor for phage M102 [ 132 ]. Sc. mutans BrpA and Psr, Genes and Physiological Effects Sc. mutans harbours two paralogues of LCP family proteins, named BrpA and Psr. Deletion of either of these did not show any impact on bacterial growth, but the mutants had major defects in acid and oxidative stress tolerance responses, defects in cell division, alterations in cell envelope morphology, and reduction in biofilm formation, probably due to missing RhaCWGP [ 136 , 137 , 138 ]. BrpA or Psr deficiency was also found to alter the expression of a number of genes, including those known to play a critical role in cell envelope biogenesis and cell division and biofilm formation, although differences exist between the two LCP proteins in the scope and effect of their gene regulation [ 136 , 137 ]. Specifically, in the Δ psr mutant, a decreased expression level of glycosyltransferase C, which is common-ly involved in biofilm formation, was found. Importantly, one of the two LCP homologues is necessary for viability of Sc. mutans cells [ 136 ], which might reveal BrpA and Psr as new potential targets to develop anticaries therapeutics. Sc. mutans BrpA and Psr in RhaCWGP Biosynthesis Six genes ( rgpA through rgpF ) involved in the biosynthesis of the RhaCWGPs have been characterized by heterologous gene expression experiments in E. coli [ 139 , 140 ]. From restoration of plasmid-encoded RhaCWGP biosynthesis in an E. coli Δ wecA mutant by provision of Sc. mutans RgpG, it was concluded that RgpG encodes the initiation enzyme of the RhaCWGP biosynthesis in Sc. mutans , transferring a GlcNAc residue from UDP-GlcNAc to lipid-phosphate. According to that study, it was postulated that RgpA, RgpB, and RgpF, function in rhamnan polymerization, while RgpC and RgpD constitute an ABC transporter. In a more recent study, allelic exchange of RgpG led to impaired cell division, reduced biofilm formation, and altered cell morphology of Sc. mutans . The RgpG deficient strain was used for deletion of brpA and psr . While the double mutants grew comparably to the wild-type, a rgpG brpA psr triple mutant showed swollen giant cells and was totally devoid of the RhaCWGP [ 133 ]. Based on this observation, the authors suggested an involvement of BrpA and Pst in attaching the RhaCWGP to the cell wall PGN, without provision of further details about the linkage. Notably, the involvement of a WecA homologue, which is a well-known UDP-GlcNAc::lipid- P transferase from the LPS biosynthesis pathways [ 25 ], as an initiating glycosyltransferase in RhaCWGP biosynthesis in Sc. mutans , would implicate the presence of a GlcNAc residue at the reducing end of the glycopolymer. This, however, is not consistent with the current knowledge of the Sc. mutans RhaCWGP RU structure, leaving the option of this sugar serving as a potential linker of the capsule to the PGN. 4.2.4. Lactococcus lactis Lc. lactis is a Gram-positive bacterium widely used in dairy fermentations where it metabolizes sugars and converts these to lactic acid. In humans, lactic acid bacteria are naturally present in the gut, and, due to their GRAS (generally regarded as safe), they are considered as a convenient delivery vector for biological molecules for antiinfective and anti-allergic therapies in the gastro-intestinal tract [ 141 , 142 ]. Lc. lactis strains are covered by a RhaCWGP or sugar-phosphate polysaccharide pellicle (PSP), which is likely linked to the cell wall PGN via a "conventional" murein linkage unit as evident from the involvement of the TagO enzyme in its biosynthesis. The PSP protects the bacteria from phagocytosis in vitro and acts as the receptor for members of various lactococcal phage groups, allowing their adsorption through specific recognition events [ 77 , 143 ]. In strain MG1363, the anionic PSP is composed of hexasaccharide-phosphate repeats containing Glc, Gal f , GlcNAc, Rha and a Glc- P residue at the reducing-end of each repeat [ 77 , 144 ] ( Figure 1 ). In addition, the bacterium produces a neutral polyrhamnan, which is composed of linear →2)-α-L-Rha-(1→2)-α-L-Rha-(1→3)-α-L-Rha-(1→ trisaccharide repeating units [ 145 ] ( Figure 1 ). The polyrhamnan is located underneath the surface-exposed PSP and is trapped inside the PGN as evident from a high-resolution magic angle spinning (HR-MAS) NMR analysis of an PSP-deficient Lc. lactis strain. Lc. lactis LcpA and LcpB, Genes and Physiological Implications Lc. lactis strains possess a large chromosomal cwps gene cluster, comprising a highly conserved and a variable region, with the former region involved in polyrhamnan and the latter involved in PSP biosynthesis. Thus, except for the gene tagO encoding the initiating glycosyltransferase, the genetic determinants of polyrhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery. Lc. lactis MG1363 harbours two functional lcp paralogs— lcpA and lcpB –which are located outside the cwps gene cluster and have a monocistronic organization. Of these, only the lcpB gene could be successfully deleted, suggesting an essential role for LcpA in the growth and/or survival of Lc. lactis . An lcpA mutant leading to reduced expression of lcpA was shown to severely affect the cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, polyrhamnan was detected by HR-MAS NMR and was, unlike the situation in the wild-type, flexibly located at the surface. This suggested that LcpA participates in the attachment of polyrhamnan to PGN, but, possibly also in attachment of the PSP, since only its absence would allow the detection of the underlaying polyrhamnan. The current model of polyrhamnan biosynthesis follows an ABC transporter-dependent pathway, involving the production of the lipid-pyrophosphate-linked murein linkage unit, subsequently acting individual rhamnosyltransferases, and addition of a terminal sugar terminating the synthesis of the chain followed by export and ligation to PGN involving LcpA [ 145 ]. 4.2.5. Lactobacillus plantarum The human gut microbiota contains an abundance of symbiotic lactobacilli, amongst which Lb. plantarum is one of the most predominant species. The organism is a key player among probiotic microorganisms and known for its high metabolic versatility [ 146 , 147 ]. Lb. plantarum strains possess 3,4-α-D-diglucosyl-2-Rbo- P and 3,4-α-D-diglucosyl-1-Rbo- P WTAs, respectively ( Figure 1 ). The unique structure of WTA in Lb. plantarum results from the modification of the Rbo- P main chain with multiple glucose residues [ 148 ]. Of note, this WTA structure contains two monomers differing in the position of the phosphoric acid residue. Lb. plantarum FlmA, FlmB and FlmC, Genes and Physiological Effects In the Lb. plantarum genome recently, three genes– flmA , flmB and flmC —encoding proteins to which regulatory and cell wall-related transcriptional attenuator functions were attributed, were identified. The FlmA, FlmB and FlmC proteins all contain highly conserved C-terminal regions which appear closely related to the LCP domain [ 147 , 149 ] and an N-terminal TM anchor domain. By generating Δ flm deletion mutants, it has been shown that FlmC contributes to biofilm development and that lack of this protein results in increased autolytic activity phenotypes, whereas deletion of either of the remaining genes did not result in any significant defects [ 147 , 149 ]. Structural Model of Lb. plantarum FlmC A ΔTM-FlmC (amino acid residues 81-335) structural model could be obtained based on the crystal structure of ΔTM-Cps2A from Sc. pneumoniae , exhibiting a typical topology of the LCP domain where conserved hydrophobic amino acid residues are centred in the inside and polar residues on the outside [ 147 ]. A hydrophobic pocket consistent with the Sc. pneumoniae Cps2A protein between the central β-sheet and helices 3–7 was presented in the model. The central sheet harbours six-strands surrounded on both faces by 5 α-helices [ 147 ]. The modelled structure strongly suggests that FlmC acts as a phosphotransferase, like Cps2A, as evident from the presence of similar conserved arginine residues that stabilize the binding of a lipid molecule in concert with a magnesium ion, which is fundamental for the phosphatase activity of the class of LCP enzymes [ 82 , 147 ]. Overall, the present data suggest that FlmC is involved in cell envelope biogenesis of Lb. plantarum ; whether it directly affects the ligation of the bacterium's WTA to PGN remains to be investigated. 4.2.6. Enterococcus hirae Commensal enterococci such as E. hirae are found in the normal human faecal flora; they are of interest due to their emerging pathogenicity in hospital infections which relates to biofilm formation and antibiotic resistances. Enterococci are intrinsically resistant to β-lactams due to the expression of a penicillin binding protein (PBP) displaying a low affinity for these antibiotics [ 150 ]. As with most bacteria, enterococci are dependent on their cell envelope for growth. Enterococcus species show WTA structures of different complexity which have not been elucidated up to now [ 151 ]. E. hirae LcpA, LcpB and LcpC The E. hirae genome codes for three predicted LCP enzymes named LcpA (formerly Psr protein EHR_11445), LcpB (formerly EHR_11995) and LcpC (formerly EHR_14365), respectively. The three LCP proteins from E. hirae have a common topology according to in silico analysis using the TMHMM program, which shows an N-terminal cytoplasmic tail of different length depending on the protein, (six amino acids for LcpA, 103 amino acids for LcpB and six amino acids for LcpC), followed by a TM α-helix of approximately 20 amino acids (18, 23 and 20 amino acids respectively), and an LCP C-terminal domain located in the extracellular space. E. hirae LcpA Function Of the three E. hirae LCP proteins, only LcpA has been investigated. LcpA is a 293-amino acid Psr orthologue possessing a conserved 150 amino-acid LCP domain. The lcpA gene is located in an operon between the ftsW gene, coding for a SEDS (Shape, Elongation, Division and Sporulation) protein involved in lipid II export, and the pbp5 gene, coding for a low-affinity PBP involved in PGN synthesis [ 20 ]. However, the operon is not flanked by any cluster of known CWGP biosynthetic genes. Initially, LcpA was related to β-lactam resistance of E. hirae and proposed to be a repressor of penicillin-binding protein 5 (PBP5) synthesis because of a deletion found in the lcpA gene of the E. hirae strain R40, which overproduced PBP5 [ 152 ]. In subsequent studies, LcpA was found to be a membrane protein which binds E. hirae lysine-type PGN and localizes at the septation sites together with the low-affinity PBP5, which is involved in the late steps of PGN biosynthesis [ 151 ]. The interaction of recombinant E. hirae LcpA with E. hirae cell walls was investigated by pull-down experiments [ 151 ]. Incubation of purified E. hirae PGN with or without CWGP with LcpA indicated that LcpA binds enterococcal PGN regardless of the presence of WTA. Thus, it seems plausible that this LCP protein plays a role related to the cell wall metabolism, probably acting as a phosphotransferase catalysing the attachment of RhaCWGPs to the PGN of E. hirae [ 151 ]. This assumption is consistent with a previous finding in the E. hirae R40 mutant possessing a truncated lcpA gene which showed a decrease of the rhamnose content in its cell walls by 50%, which was not related to the overproduction of PBP5 nor to other changes in the PGN structure [ 153 ]. 4.2.1. Streptococcus pneumoniae Sc. pneumoniae or pneumococcus is a major human pathogen, which typically resides in the nasopharyngeal cavity. Bacterial colonization requires interaction with host cells, for which the amount of capsule is crucial [ 120 ]. Thus, its CPS is a key virulence factor shielding Sc. pneumoniae from the host immune system and, thus, an important target for protective immune responses. Ninety-three capsular types have been identified serologically, and the RU unit structure of each has been defined [ 64 ]. Sc. pneumoniae is especially dangerous for immunocompromised people where it can cause severe diseases, including meningitis, pneumoniae or sepsis. In asymptomatic humans, Sc. pneumoniae resides in the nasal cavity or the sinuses, where it may cause otitis media and acute sinusitis [ 121 ]. The capsular polysaccharide serotype 2 of Sc. pneumoniae strain D39 (CP2) is composed of branched hexasaccharide RUs with the structure →3)[α-Glc p A-(1→6)-α-Glc-(1→2)]-α-L-Rha-(1→3)-α-L-Rha-(1→3)-β-L-Rha-(1→4)-β-D-Glc- ( Figure 1 ). CP2 is directly glycosidically linked via the reducing end glucose of the RU to β-D-GlcNAc residues of PGN, without involvement of a murein linkage unit and a phosphodiester bond [ 122 ], which contrasts the usual attachment mode of CWGPs to PGN backbone sugars. The cell wall of Sc. pneumoniae further contains an unusually complex WTA, which has identical RUs as the membrane-anchored lipoteichoic acid. Both show pseudo-pentasaccharide RUs containing the rare amino sugar 2-acetamido-4-amino-2,4,6-trideoxygalactose (AATGal) in addition to Glc, Rib- P , and two GalNAc residues that are each modified with phosphorylcholine ( Figure 1 ) [ 123 , 124 , 125 ]. The reducing-end AATGal is proposed to be linked via a phosphodiester bond to MurNAc residues of PGN, based on the in silico identification of LCP family proteins in the Sc. pneumoniae genome, however, without provision of any biochemical evidence [ 124 ]. Sc. pneumoniae Cps2A, LytR, Psr, Genes and Physiological Effects On the Sc. pneumoniae genome, the capsular region of CP 2 begins with the cps2A–D genes; all 17 capsular genes in this region are under control of the promoter upstream of cps2A .The first gene in the region, cps2A , encodes a member of the LCP protein family. Within the biosynthesis pathway of the CP2 of Sc. pneumoniae strain D39, the three paralogous LCP proteins Cps2A, LytR and Psr, with the latter two not localizing to the cps region, have been investigated. Evidence was provided that Cps2A, LytR and Psr contribute to the maintenance of normal capsule levels and to the retention of the CP2 in the Sc. pneumoniae cell wall. Cps2A, LytR and Psr were found to localize at the cell membrane and accumulate at septal sites supportive of their function in cell wall maintenance. Single Δ cps2A and Δ psr mutants produced a reduced amount of capsule, while a Δ cps2AlytR double mutant showed greatly impaired growth and cell morphology and lost approximately half of the total capsule material into the culture supernatant [ 97 ]. Notably, inactivation of lytR proved to be difficult in the background of the encapsulated D39 strain and during exponential growth, LytR expression was continuously high which suggests a housekeeping function of this gene during cell division that is essential for proper septum placement [ 126 ]. According to a current data-based model, CpsA2 is responsible for the covalent attachment of CP2 to the pneumococcal cell wall, and LytR can take over this function in the absence of Cps2A. Crystal Structure of Sc. pneumoniae Cps2A Structural and functional studies of the Cps2A enzyme from Sc. pneumoniae provided the first insight into the catalytic mechanism of an enzyme from the LCP protein family. Cps2A contains a large hydrophobic tunnel that is capped with surface-exposed arginine residues that are important for catalysis [ 82 ]; serendipitously, Cps2A co-crystallizes with octaprenyl-pyrophosphate, where the isoprenyl-tail is nestled within the hydrophobic pocket with the pyrophosphate head group interacting with highly conserved arginine residues within the active site. A ΔTM-Cps2A version of the protein comprising the accessory domain (amino acid residues 111–213) and the LCP domain (amino acid residues 214–481) has been solved at 1.69 à -resolution. Within the LCP domain, a core containing a five-stranded β-sheet surrounded by α-helices on both faces is comprised of an overall α-β-α architecture. Between the two domains, two pairs of β-strands extend from the core site. A hydrophobic pocket is formed between the central β-sheet and α-helices 3–7, where a polyisoprenoid phosphate lipid was found. As it has been seen across the LCP superfamily, these hydrophobic side chains are conserved and arginine residues R267, R362 and R374 play a key role in the interaction between the phosphate oxygens and the formation of the pocket and are stabilized by D371 and Q378. Furthermore, magnesium ions are suggested to contribute to catalysis by implicating neutralization of the developing negative charge and are coordinated by two aspartate residues, D234 and D246 [ 82 ]. Based on the crystal structure of the soluble part of Cps2A, it was inferred that all three homologs in Sc. pneumoniae (Cps2A, LytR, and Psr) might attach polyprenol pyrophosphoryl-linked polymers to PGN without any further specification of the CWGP structure [ 97 ], which leaves open the possibility that these enzymes might additionally be involved in WTA attachment. Sc. pneumoniae Cps2A, LytR, Psr, Genes and Physiological Effects On the Sc. pneumoniae genome, the capsular region of CP 2 begins with the cps2A–D genes; all 17 capsular genes in this region are under control of the promoter upstream of cps2A .The first gene in the region, cps2A , encodes a member of the LCP protein family. Within the biosynthesis pathway of the CP2 of Sc. pneumoniae strain D39, the three paralogous LCP proteins Cps2A, LytR and Psr, with the latter two not localizing to the cps region, have been investigated. Evidence was provided that Cps2A, LytR and Psr contribute to the maintenance of normal capsule levels and to the retention of the CP2 in the Sc. pneumoniae cell wall. Cps2A, LytR and Psr were found to localize at the cell membrane and accumulate at septal sites supportive of their function in cell wall maintenance. Single Δ cps2A and Δ psr mutants produced a reduced amount of capsule, while a Δ cps2AlytR double mutant showed greatly impaired growth and cell morphology and lost approximately half of the total capsule material into the culture supernatant [ 97 ]. Notably, inactivation of lytR proved to be difficult in the background of the encapsulated D39 strain and during exponential growth, LytR expression was continuously high which suggests a housekeeping function of this gene during cell division that is essential for proper septum placement [ 126 ]. According to a current data-based model, CpsA2 is responsible for the covalent attachment of CP2 to the pneumococcal cell wall, and LytR can take over this function in the absence of Cps2A. Crystal Structure of Sc. pneumoniae Cps2A Structural and functional studies of the Cps2A enzyme from Sc. pneumoniae provided the first insight into the catalytic mechanism of an enzyme from the LCP protein family. Cps2A contains a large hydrophobic tunnel that is capped with surface-exposed arginine residues that are important for catalysis [ 82 ]; serendipitously, Cps2A co-crystallizes with octaprenyl-pyrophosphate, where the isoprenyl-tail is nestled within the hydrophobic pocket with the pyrophosphate head group interacting with highly conserved arginine residues within the active site. A ΔTM-Cps2A version of the protein comprising the accessory domain (amino acid residues 111–213) and the LCP domain (amino acid residues 214–481) has been solved at 1.69 à -resolution. Within the LCP domain, a core containing a five-stranded β-sheet surrounded by α-helices on both faces is comprised of an overall α-β-α architecture. Between the two domains, two pairs of β-strands extend from the core site. A hydrophobic pocket is formed between the central β-sheet and α-helices 3–7, where a polyisoprenoid phosphate lipid was found. As it has been seen across the LCP superfamily, these hydrophobic side chains are conserved and arginine residues R267, R362 and R374 play a key role in the interaction between the phosphate oxygens and the formation of the pocket and are stabilized by D371 and Q378. Furthermore, magnesium ions are suggested to contribute to catalysis by implicating neutralization of the developing negative charge and are coordinated by two aspartate residues, D234 and D246 [ 82 ]. Based on the crystal structure of the soluble part of Cps2A, it was inferred that all three homologs in Sc. pneumoniae (Cps2A, LytR, and Psr) might attach polyprenol pyrophosphoryl-linked polymers to PGN without any further specification of the CWGP structure [ 97 ], which leaves open the possibility that these enzymes might additionally be involved in WTA attachment. 4.2.2. Streptococcus agalactiae Sc. agalactiae belongs to the GroupB Streptococcus (GBS); it is a common commensal organism which occurs on vaginal and rectal mucosal surfaces but is also associated with invasive infections, especially in elderly or immunocompromised patients [ 127 ]. CPS represents the main virulence factor of Sc. agalactiae and is a prime target in current vaccine development [ 128 ]. GBS isolates associated with human infection produce one of nine antigenically distinct CPSs [ 127 ]. The Sc. agalactiae CPS serotype III (CPIII) has a branched pentasaccharide structure composed of →6)-[α-NeuAc-(2→3)-Gal p -(1→4)]-β-Glc p NAc-(1→3)-β-Gal-(1→4)-α-Glc-(1→ RUs. CPIII is bound via a phosphodiester bond and an oligosaccharide linker of unknown structure composed of glucose, galactose and arabinose to GlcNAc residues of PGN [ 129 , 130 ] ( Figure 1 ), representing a further variation of the PGN linkage mode. Sc. agalactiae CpsA, Gene and Physiological Effects The investigation of the conserved proteins CpsABCD encoded in the Sc. agalactiae cps operon revealed that cpsA , the first gene in the operon, has a regulatory function and is required for the transcription of the operon and that CpsBCD composes a phosphoregulatory system [ 100 ]. Although having no impact on cps transcription or the synthesis of the CPIII RU, it was suggested that these proteins are required for fine-tuning of the last steps of CPIII biosynthesis, which is balancing repeating unit polymerization and CPIII attachment to the cell wall. As a member of the LCP protein family, the 485-amino acid membrane protein CpsA is unique due to its extracellular accessory domain. It equips CpsA to specifically bind to two promoters in the cps locus [ 131 ]. However, a protein consisting of the accessory domain alone could not complement a Δ cpsA deletion strain for CPIII biosynthesis. Interestingly, even if the truncated form co-existed with the native cpsA , the capsule production was impaired (dominant-negative effect), suggesting involvement of CpsA in cell wall maintenance in addition to capsule expression [ 99 ]. Essential for the dominant-negative effect is a region between amino acids 210 and 245 and probably between amino acids 132 and 153 of the accessory domain of CpsA. Experiments using a fluorescent peptide showed that this effect was not due to a direct interaction of truncated CpsA with wild-type CpsA. Probably, it disturbs the mechanism associated with normal cell wall integrity and CPS synthesis [ 131 ]. Interestingly, zebrafish experiments revealed that expression of a truncated CpsA representing the accessory domain only decreases virulence stronger than the complete absence of CpsA [ 131 ]. Sc. agalactiae CpsA in CPIII Biosynthesis In the final steps of CPIII biosynthesis, the newly synthesized pentasaccharide RU anchored to a polyisoprenoid phosphate lipid is flipped to the outer side of the bacterial membrane, where CpsH acts as the repeating unit polymerase. By analogy with other Wzy-dependent systems, polymerization occurs bottom-up. The nascent CPIII is removed from the lipid through a phosphotransferase reaction and subsequently linked to a single membrane-anchored RU. The final product is a CPS that is removed from the membrane lipid and covalently attached to GlcNAc of the PGN backbone by CpsA activity. This linkage effectively renders further polymerization impossible [ 100 ]. Sc. agalactiae CpsA, Gene and Physiological Effects The investigation of the conserved proteins CpsABCD encoded in the Sc. agalactiae cps operon revealed that cpsA , the first gene in the operon, has a regulatory function and is required for the transcription of the operon and that CpsBCD composes a phosphoregulatory system [ 100 ]. Although having no impact on cps transcription or the synthesis of the CPIII RU, it was suggested that these proteins are required for fine-tuning of the last steps of CPIII biosynthesis, which is balancing repeating unit polymerization and CPIII attachment to the cell wall. As a member of the LCP protein family, the 485-amino acid membrane protein CpsA is unique due to its extracellular accessory domain. It equips CpsA to specifically bind to two promoters in the cps locus [ 131 ]. However, a protein consisting of the accessory domain alone could not complement a Δ cpsA deletion strain for CPIII biosynthesis. Interestingly, even if the truncated form co-existed with the native cpsA , the capsule production was impaired (dominant-negative effect), suggesting involvement of CpsA in cell wall maintenance in addition to capsule expression [ 99 ]. Essential for the dominant-negative effect is a region between amino acids 210 and 245 and probably between amino acids 132 and 153 of the accessory domain of CpsA. Experiments using a fluorescent peptide showed that this effect was not due to a direct interaction of truncated CpsA with wild-type CpsA. Probably, it disturbs the mechanism associated with normal cell wall integrity and CPS synthesis [ 131 ]. Interestingly, zebrafish experiments revealed that expression of a truncated CpsA representing the accessory domain only decreases virulence stronger than the complete absence of CpsA [ 131 ]. Sc. agalactiae CpsA in CPIII Biosynthesis In the final steps of CPIII biosynthesis, the newly synthesized pentasaccharide RU anchored to a polyisoprenoid phosphate lipid is flipped to the outer side of the bacterial membrane, where CpsH acts as the repeating unit polymerase. By analogy with other Wzy-dependent systems, polymerization occurs bottom-up. The nascent CPIII is removed from the lipid through a phosphotransferase reaction and subsequently linked to a single membrane-anchored RU. The final product is a CPS that is removed from the membrane lipid and covalently attached to GlcNAc of the PGN backbone by CpsA activity. This linkage effectively renders further polymerization impossible [ 100 ]. 4.2.3. Streptococcus mutans Sc. mutans is a prototypical member of the lactic acid bacteria group. It inhabits the dental plaque biofilm community of the oral cavity and represents the most tenacious causative agent of the enamel-destructive disease dental caries [ 79 ]. As within several streptococcal species, the major cell wall antigen of Sc. mutans is its RhaCWGP which consists of linear rhamnose-polymers with glucose side chains (also referred to as RGP) [ 132 , 133 ] ( Figure 1 ) of the RU structure →2)-[α-Glc-(1→2)]-α-Rha-(1→3)-α-Rha-(1→2)-[α-Glc-(1→2)]-α-Rha-(1→3)-α-Rha-(1→, making up approxima-tely half of the total weight of the bacterium's cell wall [ 76 , 134 ]. The mature RGP contributes to acid and oxidative stress tolerance of Sc. mutans in the oral habitat [ 135 ] and is required for proper localization of cell division complexes in Sc. mutans translating in a morphogenic role for the bacterium [ 79 ]. Specifically, the serotype-specific glucose branches of the RhaCWGP from strain Sc. mutans serotype c were shown to act as a receptor for phage M102 [ 132 ]. Sc. mutans BrpA and Psr, Genes and Physiological Effects Sc. mutans harbours two paralogues of LCP family proteins, named BrpA and Psr. Deletion of either of these did not show any impact on bacterial growth, but the mutants had major defects in acid and oxidative stress tolerance responses, defects in cell division, alterations in cell envelope morphology, and reduction in biofilm formation, probably due to missing RhaCWGP [ 136 , 137 , 138 ]. BrpA or Psr deficiency was also found to alter the expression of a number of genes, including those known to play a critical role in cell envelope biogenesis and cell division and biofilm formation, although differences exist between the two LCP proteins in the scope and effect of their gene regulation [ 136 , 137 ]. Specifically, in the Δ psr mutant, a decreased expression level of glycosyltransferase C, which is common-ly involved in biofilm formation, was found. Importantly, one of the two LCP homologues is necessary for viability of Sc. mutans cells [ 136 ], which might reveal BrpA and Psr as new potential targets to develop anticaries therapeutics. Sc. mutans BrpA and Psr in RhaCWGP Biosynthesis Six genes ( rgpA through rgpF ) involved in the biosynthesis of the RhaCWGPs have been characterized by heterologous gene expression experiments in E. coli [ 139 , 140 ]. From restoration of plasmid-encoded RhaCWGP biosynthesis in an E. coli Δ wecA mutant by provision of Sc. mutans RgpG, it was concluded that RgpG encodes the initiation enzyme of the RhaCWGP biosynthesis in Sc. mutans , transferring a GlcNAc residue from UDP-GlcNAc to lipid-phosphate. According to that study, it was postulated that RgpA, RgpB, and RgpF, function in rhamnan polymerization, while RgpC and RgpD constitute an ABC transporter. In a more recent study, allelic exchange of RgpG led to impaired cell division, reduced biofilm formation, and altered cell morphology of Sc. mutans . The RgpG deficient strain was used for deletion of brpA and psr . While the double mutants grew comparably to the wild-type, a rgpG brpA psr triple mutant showed swollen giant cells and was totally devoid of the RhaCWGP [ 133 ]. Based on this observation, the authors suggested an involvement of BrpA and Pst in attaching the RhaCWGP to the cell wall PGN, without provision of further details about the linkage. Notably, the involvement of a WecA homologue, which is a well-known UDP-GlcNAc::lipid- P transferase from the LPS biosynthesis pathways [ 25 ], as an initiating glycosyltransferase in RhaCWGP biosynthesis in Sc. mutans , would implicate the presence of a GlcNAc residue at the reducing end of the glycopolymer. This, however, is not consistent with the current knowledge of the Sc. mutans RhaCWGP RU structure, leaving the option of this sugar serving as a potential linker of the capsule to the PGN. Sc. mutans BrpA and Psr, Genes and Physiological Effects Sc. mutans harbours two paralogues of LCP family proteins, named BrpA and Psr. Deletion of either of these did not show any impact on bacterial growth, but the mutants had major defects in acid and oxidative stress tolerance responses, defects in cell division, alterations in cell envelope morphology, and reduction in biofilm formation, probably due to missing RhaCWGP [ 136 , 137 , 138 ]. BrpA or Psr deficiency was also found to alter the expression of a number of genes, including those known to play a critical role in cell envelope biogenesis and cell division and biofilm formation, although differences exist between the two LCP proteins in the scope and effect of their gene regulation [ 136 , 137 ]. Specifically, in the Δ psr mutant, a decreased expression level of glycosyltransferase C, which is common-ly involved in biofilm formation, was found. Importantly, one of the two LCP homologues is necessary for viability of Sc. mutans cells [ 136 ], which might reveal BrpA and Psr as new potential targets to develop anticaries therapeutics. Sc. mutans BrpA and Psr in RhaCWGP Biosynthesis Six genes ( rgpA through rgpF ) involved in the biosynthesis of the RhaCWGPs have been characterized by heterologous gene expression experiments in E. coli [ 139 , 140 ]. From restoration of plasmid-encoded RhaCWGP biosynthesis in an E. coli Δ wecA mutant by provision of Sc. mutans RgpG, it was concluded that RgpG encodes the initiation enzyme of the RhaCWGP biosynthesis in Sc. mutans , transferring a GlcNAc residue from UDP-GlcNAc to lipid-phosphate. According to that study, it was postulated that RgpA, RgpB, and RgpF, function in rhamnan polymerization, while RgpC and RgpD constitute an ABC transporter. In a more recent study, allelic exchange of RgpG led to impaired cell division, reduced biofilm formation, and altered cell morphology of Sc. mutans . The RgpG deficient strain was used for deletion of brpA and psr . While the double mutants grew comparably to the wild-type, a rgpG brpA psr triple mutant showed swollen giant cells and was totally devoid of the RhaCWGP [ 133 ]. Based on this observation, the authors suggested an involvement of BrpA and Pst in attaching the RhaCWGP to the cell wall PGN, without provision of further details about the linkage. Notably, the involvement of a WecA homologue, which is a well-known UDP-GlcNAc::lipid- P transferase from the LPS biosynthesis pathways [ 25 ], as an initiating glycosyltransferase in RhaCWGP biosynthesis in Sc. mutans , would implicate the presence of a GlcNAc residue at the reducing end of the glycopolymer. This, however, is not consistent with the current knowledge of the Sc. mutans RhaCWGP RU structure, leaving the option of this sugar serving as a potential linker of the capsule to the PGN. 4.2.4. Lactococcus lactis Lc. lactis is a Gram-positive bacterium widely used in dairy fermentations where it metabolizes sugars and converts these to lactic acid. In humans, lactic acid bacteria are naturally present in the gut, and, due to their GRAS (generally regarded as safe), they are considered as a convenient delivery vector for biological molecules for antiinfective and anti-allergic therapies in the gastro-intestinal tract [ 141 , 142 ]. Lc. lactis strains are covered by a RhaCWGP or sugar-phosphate polysaccharide pellicle (PSP), which is likely linked to the cell wall PGN via a "conventional" murein linkage unit as evident from the involvement of the TagO enzyme in its biosynthesis. The PSP protects the bacteria from phagocytosis in vitro and acts as the receptor for members of various lactococcal phage groups, allowing their adsorption through specific recognition events [ 77 , 143 ]. In strain MG1363, the anionic PSP is composed of hexasaccharide-phosphate repeats containing Glc, Gal f , GlcNAc, Rha and a Glc- P residue at the reducing-end of each repeat [ 77 , 144 ] ( Figure 1 ). In addition, the bacterium produces a neutral polyrhamnan, which is composed of linear →2)-α-L-Rha-(1→2)-α-L-Rha-(1→3)-α-L-Rha-(1→ trisaccharide repeating units [ 145 ] ( Figure 1 ). The polyrhamnan is located underneath the surface-exposed PSP and is trapped inside the PGN as evident from a high-resolution magic angle spinning (HR-MAS) NMR analysis of an PSP-deficient Lc. lactis strain. Lc. lactis LcpA and LcpB, Genes and Physiological Implications Lc. lactis strains possess a large chromosomal cwps gene cluster, comprising a highly conserved and a variable region, with the former region involved in polyrhamnan and the latter involved in PSP biosynthesis. Thus, except for the gene tagO encoding the initiating glycosyltransferase, the genetic determinants of polyrhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery. Lc. lactis MG1363 harbours two functional lcp paralogs— lcpA and lcpB –which are located outside the cwps gene cluster and have a monocistronic organization. Of these, only the lcpB gene could be successfully deleted, suggesting an essential role for LcpA in the growth and/or survival of Lc. lactis . An lcpA mutant leading to reduced expression of lcpA was shown to severely affect the cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, polyrhamnan was detected by HR-MAS NMR and was, unlike the situation in the wild-type, flexibly located at the surface. This suggested that LcpA participates in the attachment of polyrhamnan to PGN, but, possibly also in attachment of the PSP, since only its absence would allow the detection of the underlaying polyrhamnan. The current model of polyrhamnan biosynthesis follows an ABC transporter-dependent pathway, involving the production of the lipid-pyrophosphate-linked murein linkage unit, subsequently acting individual rhamnosyltransferases, and addition of a terminal sugar terminating the synthesis of the chain followed by export and ligation to PGN involving LcpA [ 145 ]. Lc. lactis LcpA and LcpB, Genes and Physiological Implications Lc. lactis strains possess a large chromosomal cwps gene cluster, comprising a highly conserved and a variable region, with the former region involved in polyrhamnan and the latter involved in PSP biosynthesis. Thus, except for the gene tagO encoding the initiating glycosyltransferase, the genetic determinants of polyrhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery. Lc. lactis MG1363 harbours two functional lcp paralogs— lcpA and lcpB –which are located outside the cwps gene cluster and have a monocistronic organization. Of these, only the lcpB gene could be successfully deleted, suggesting an essential role for LcpA in the growth and/or survival of Lc. lactis . An lcpA mutant leading to reduced expression of lcpA was shown to severely affect the cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, polyrhamnan was detected by HR-MAS NMR and was, unlike the situation in the wild-type, flexibly located at the surface. This suggested that LcpA participates in the attachment of polyrhamnan to PGN, but, possibly also in attachment of the PSP, since only its absence would allow the detection of the underlaying polyrhamnan. The current model of polyrhamnan biosynthesis follows an ABC transporter-dependent pathway, involving the production of the lipid-pyrophosphate-linked murein linkage unit, subsequently acting individual rhamnosyltransferases, and addition of a terminal sugar terminating the synthesis of the chain followed by export and ligation to PGN involving LcpA [ 145 ]. 4.2.5. Lactobacillus plantarum The human gut microbiota contains an abundance of symbiotic lactobacilli, amongst which Lb. plantarum is one of the most predominant species. The organism is a key player among probiotic microorganisms and known for its high metabolic versatility [ 146 , 147 ]. Lb. plantarum strains possess 3,4-α-D-diglucosyl-2-Rbo- P and 3,4-α-D-diglucosyl-1-Rbo- P WTAs, respectively ( Figure 1 ). The unique structure of WTA in Lb. plantarum results from the modification of the Rbo- P main chain with multiple glucose residues [ 148 ]. Of note, this WTA structure contains two monomers differing in the position of the phosphoric acid residue. Lb. plantarum FlmA, FlmB and FlmC, Genes and Physiological Effects In the Lb. plantarum genome recently, three genes– flmA , flmB and flmC —encoding proteins to which regulatory and cell wall-related transcriptional attenuator functions were attributed, were identified. The FlmA, FlmB and FlmC proteins all contain highly conserved C-terminal regions which appear closely related to the LCP domain [ 147 , 149 ] and an N-terminal TM anchor domain. By generating Δ flm deletion mutants, it has been shown that FlmC contributes to biofilm development and that lack of this protein results in increased autolytic activity phenotypes, whereas deletion of either of the remaining genes did not result in any significant defects [ 147 , 149 ]. Structural Model of Lb. plantarum FlmC A ΔTM-FlmC (amino acid residues 81-335) structural model could be obtained based on the crystal structure of ΔTM-Cps2A from Sc. pneumoniae , exhibiting a typical topology of the LCP domain where conserved hydrophobic amino acid residues are centred in the inside and polar residues on the outside [ 147 ]. A hydrophobic pocket consistent with the Sc. pneumoniae Cps2A protein between the central β-sheet and helices 3–7 was presented in the model. The central sheet harbours six-strands surrounded on both faces by 5 α-helices [ 147 ]. The modelled structure strongly suggests that FlmC acts as a phosphotransferase, like Cps2A, as evident from the presence of similar conserved arginine residues that stabilize the binding of a lipid molecule in concert with a magnesium ion, which is fundamental for the phosphatase activity of the class of LCP enzymes [ 82 , 147 ]. Overall, the present data suggest that FlmC is involved in cell envelope biogenesis of Lb. plantarum ; whether it directly affects the ligation of the bacterium's WTA to PGN remains to be investigated. Lb. plantarum FlmA, FlmB and FlmC, Genes and Physiological Effects In the Lb. plantarum genome recently, three genes– flmA , flmB and flmC —encoding proteins to which regulatory and cell wall-related transcriptional attenuator functions were attributed, were identified. The FlmA, FlmB and FlmC proteins all contain highly conserved C-terminal regions which appear closely related to the LCP domain [ 147 , 149 ] and an N-terminal TM anchor domain. By generating Δ flm deletion mutants, it has been shown that FlmC contributes to biofilm development and that lack of this protein results in increased autolytic activity phenotypes, whereas deletion of either of the remaining genes did not result in any significant defects [ 147 , 149 ]. Structural Model of Lb. plantarum FlmC A ΔTM-FlmC (amino acid residues 81-335) structural model could be obtained based on the crystal structure of ΔTM-Cps2A from Sc. pneumoniae , exhibiting a typical topology of the LCP domain where conserved hydrophobic amino acid residues are centred in the inside and polar residues on the outside [ 147 ]. A hydrophobic pocket consistent with the Sc. pneumoniae Cps2A protein between the central β-sheet and helices 3–7 was presented in the model. The central sheet harbours six-strands surrounded on both faces by 5 α-helices [ 147 ]. The modelled structure strongly suggests that FlmC acts as a phosphotransferase, like Cps2A, as evident from the presence of similar conserved arginine residues that stabilize the binding of a lipid molecule in concert with a magnesium ion, which is fundamental for the phosphatase activity of the class of LCP enzymes [ 82 , 147 ]. Overall, the present data suggest that FlmC is involved in cell envelope biogenesis of Lb. plantarum ; whether it directly affects the ligation of the bacterium's WTA to PGN remains to be investigated. 4.2.6. Enterococcus hirae Commensal enterococci such as E. hirae are found in the normal human faecal flora; they are of interest due to their emerging pathogenicity in hospital infections which relates to biofilm formation and antibiotic resistances. Enterococci are intrinsically resistant to β-lactams due to the expression of a penicillin binding protein (PBP) displaying a low affinity for these antibiotics [ 150 ]. As with most bacteria, enterococci are dependent on their cell envelope for growth. Enterococcus species show WTA structures of different complexity which have not been elucidated up to now [ 151 ]. E. hirae LcpA, LcpB and LcpC The E. hirae genome codes for three predicted LCP enzymes named LcpA (formerly Psr protein EHR_11445), LcpB (formerly EHR_11995) and LcpC (formerly EHR_14365), respectively. The three LCP proteins from E. hirae have a common topology according to in silico analysis using the TMHMM program, which shows an N-terminal cytoplasmic tail of different length depending on the protein, (six amino acids for LcpA, 103 amino acids for LcpB and six amino acids for LcpC), followed by a TM α-helix of approximately 20 amino acids (18, 23 and 20 amino acids respectively), and an LCP C-terminal domain located in the extracellular space. E. hirae LcpA Function Of the three E. hirae LCP proteins, only LcpA has been investigated. LcpA is a 293-amino acid Psr orthologue possessing a conserved 150 amino-acid LCP domain. The lcpA gene is located in an operon between the ftsW gene, coding for a SEDS (Shape, Elongation, Division and Sporulation) protein involved in lipid II export, and the pbp5 gene, coding for a low-affinity PBP involved in PGN synthesis [ 20 ]. However, the operon is not flanked by any cluster of known CWGP biosynthetic genes. Initially, LcpA was related to β-lactam resistance of E. hirae and proposed to be a repressor of penicillin-binding protein 5 (PBP5) synthesis because of a deletion found in the lcpA gene of the E. hirae strain R40, which overproduced PBP5 [ 152 ]. In subsequent studies, LcpA was found to be a membrane protein which binds E. hirae lysine-type PGN and localizes at the septation sites together with the low-affinity PBP5, which is involved in the late steps of PGN biosynthesis [ 151 ]. The interaction of recombinant E. hirae LcpA with E. hirae cell walls was investigated by pull-down experiments [ 151 ]. Incubation of purified E. hirae PGN with or without CWGP with LcpA indicated that LcpA binds enterococcal PGN regardless of the presence of WTA. Thus, it seems plausible that this LCP protein plays a role related to the cell wall metabolism, probably acting as a phosphotransferase catalysing the attachment of RhaCWGPs to the PGN of E. hirae [ 151 ]. This assumption is consistent with a previous finding in the E. hirae R40 mutant possessing a truncated lcpA gene which showed a decrease of the rhamnose content in its cell walls by 50%, which was not related to the overproduction of PBP5 nor to other changes in the PGN structure [ 153 ]. E. hirae LcpA, LcpB and LcpC The E. hirae genome codes for three predicted LCP enzymes named LcpA (formerly Psr protein EHR_11445), LcpB (formerly EHR_11995) and LcpC (formerly EHR_14365), respectively. The three LCP proteins from E. hirae have a common topology according to in silico analysis using the TMHMM program, which shows an N-terminal cytoplasmic tail of different length depending on the protein, (six amino acids for LcpA, 103 amino acids for LcpB and six amino acids for LcpC), followed by a TM α-helix of approximately 20 amino acids (18, 23 and 20 amino acids respectively), and an LCP C-terminal domain located in the extracellular space. E. hirae LcpA Function Of the three E. hirae LCP proteins, only LcpA has been investigated. LcpA is a 293-amino acid Psr orthologue possessing a conserved 150 amino-acid LCP domain. The lcpA gene is located in an operon between the ftsW gene, coding for a SEDS (Shape, Elongation, Division and Sporulation) protein involved in lipid II export, and the pbp5 gene, coding for a low-affinity PBP involved in PGN synthesis [ 20 ]. However, the operon is not flanked by any cluster of known CWGP biosynthetic genes. Initially, LcpA was related to β-lactam resistance of E. hirae and proposed to be a repressor of penicillin-binding protein 5 (PBP5) synthesis because of a deletion found in the lcpA gene of the E. hirae strain R40, which overproduced PBP5 [ 152 ]. In subsequent studies, LcpA was found to be a membrane protein which binds E. hirae lysine-type PGN and localizes at the septation sites together with the low-affinity PBP5, which is involved in the late steps of PGN biosynthesis [ 151 ]. The interaction of recombinant E. hirae LcpA with E. hirae cell walls was investigated by pull-down experiments [ 151 ]. Incubation of purified E. hirae PGN with or without CWGP with LcpA indicated that LcpA binds enterococcal PGN regardless of the presence of WTA. Thus, it seems plausible that this LCP protein plays a role related to the cell wall metabolism, probably acting as a phosphotransferase catalysing the attachment of RhaCWGPs to the PGN of E. hirae [ 151 ]. This assumption is consistent with a previous finding in the E. hirae R40 mutant possessing a truncated lcpA gene which showed a decrease of the rhamnose content in its cell walls by 50%, which was not related to the overproduction of PBP5 nor to other changes in the PGN structure [ 153 ]. 4.3. Actinobacteria— Order : Actinomycetales 4.3.1. Mycobacterium tuberculosis M. tuberculosis is the causative agent of tuberculosis infecting the lungs and causing about 1.5 million deaths per year [ 154 ]. What makes this organism so strong is its unique, low permeable AG-containing cell wall ( Figure 1 ) that provides a high resistance towards antibiotics [ 155 ]. M. tuberculosis Rv0822c, CpsA1, CpsA2 and Rv3840 Function Four genes encoding LCP proteins are annotated in the M. tuberculosis H37Rv genome, namely Rv0822c, Rv3267 ( cpsA1/lcp1 ), Rv3484 ( cpsA/cpsA2 ), and Rv3840 [ 156 ]. To investigate an association of the M. tuberculosis LCP proteins with the ligation of AG to PGN, the predicted enzymes devoid of the TM domain were produced recombinantly in E. coli and assayed in vitro. Functional proof was obtained for CpsA1 and CpsA2, which, under the chosen experimental conditions, showed pyrophosphatase activity on the generic substrate geranyl pyrophosphate in dependence on magnesium ions consistent with other LCP enzymes [ 65 ]. Notably, while individual cpsA1 and cpsA2 knock-outs of M. tuberculosis were readily obtainable, the combined inactivation of both genes appeared to be lethal. M. tuberculosis CpsA1 CpsA1 maps to the AG biosynthetic gene cluster where it is located immediately upstream of two genes involved in linker biosynthesis. M. tuberculosis CpsA1 was suggested to be the predominant enzyme responsible for the covalent attachment of AG to PGN. However, in a Δ cpsA1 deletion mutant, no major effects were seen, probably due to functional compensation of the paralogs [ 65 , 155 ]. CpsA1/Lcp1 was further described to be essential for M. tuberculosis and its activity was verified in a cell-free radiolabelling assay with 14 C-radiolabeled AG and nascent PGN [ 155 ]. To further evaluate the specificity of M. tuberculosis CpsA1, three potential AG binding substrates—L-Rha-α(1¡3)-D-GlcNAc- O -C 8 (compound 1), Gal f 2 -Rha-GlcNAc- O -C 8 (compound 2) and Gal f 3 -Rha-GlcNAc- O -C 8 (compound 3)—were tested using intrinsic tryptophan fluorescence of CpsA1, with compound 2 showing the highest affinity with 5.13 µM [ 155 ]. Although the envelope composition was not drastically changed in the single ligase mutants, the Δ cpsA1 mutant showed increased susceptibility to a range of antibiotics such as penicillins, vancomycin, and CPZEN-45, suggesting changes in cell wall permeability [ 65 ]. M. tuberculosis CpsA2 Although no analytically detectable difference was observed, the Δ cpsA2 deletion mutant in the M. tuberculosis strain H37Rv showed a changed phenotype in an in vivo mouse model, where the mutant was not able to grow, survive and infect. Furthermore, this strain displayed increased resistance to meropenem/clavulanate and rifampicin, which could not be compensated by cpsA1 [ 157 ]. Meropenem belongs to the carbapenem class of β-lactam antibiotics being poor substrates for BlaC, a protein encoded in the genome of M. tuberculosis which hydrolyses β-lactam antibiotics [ 158 ]. Rifampicin did not target the cell wall but the authors argued that the permeability had changed and, therefore, the drug did not efficiently get into the cytoplasm as supported by measuring ethidium bromide uptake and efflux [ 66 , 157 ]. However, Grzegorzewicz et al. could not determine increased resistance against rifampicin. According to Malm et al., this could be due to differences in the experimental set-up or resulting from a secondary effect and not from cpsA2 deletion [ 65 , 157 ]. Additional M. tuberculosis Proteins Related to LCP Enzyme Function Recently, mutants of the "cell envelope integrity" gene ( cei ; Rv2700) of M. tuberculosis and its structural homolog VirR (Rv0431), showed a decreased growth rate at low densities, increased susceptibility towards antibiotics (vancomycin, meropenem and rifampicin), increased sensitivity to nitric oxide (NO), and increased cell envelope permeability. In addition, a Δ cei mutant did not lethally infect mice, where one factor that influences virulence is the growth control by NO after infection [ 66 , 159 ]. Importantly, these newly detected gene products have one predicted TM domain and a LytR_C domain, which are features found in LCP enzymes commonly referred to as LytR_C-only proteins. Due to very similar phenotypes of the knockout mutants of cei and virR to common LCP knockout strains, a relation to and common participation in the same pathway, namely AG ligation to PGN, was hypothesized [ 66 ]. 4.3.2. Mycobacterium marinum M. marinum is a slow-growing, acid-fast bacterium in the category of non-tuberculous mycobacteria which most commonly cause skin and soft tissue infections in patients, particularly those with aquatic exposure [ 160 ]. The bacterium possesses an AG as is characteristic of mycobacteria. M. marinum MMAR_4858, MMAR_1274, MMAR_4966 (CpsA), and MMAR_5392 M. marinum (MMAR) harbours orthologues of all LCP proteins found in M. tuberculosis complex (Mtbc) strains—namely MMAR_4858, MMAR_1274, MMAR_4966 (CpsA), and MMAR_5392 in MM for Rv0822c, Rv3267, Rv3484, and Rv3840 for Mtbc [ 65 , 157 ]. A strain defective in the CpsA2 (Rv3484) orthologue in M. marinum (CpsA) showed impaired growth in vitro in contrast to CpsA deficient strains of M. tuberculosis [ 65 , 157 ]. Furthermore, the M. marinum Δ cpsA mutant revealed alterations in colony morphology and cell surface properties, increased susceptibility to antibiotics (erythromycin, vancomycin and penicillin), and a change in cell wall permeability for hydrophobic components. Traditional analytics of the cell wall composition indicated an imbalance in the AG/PGN ratio indicative of a role of the MMAR_4966 enzyme in AG transfer to PGN. Finally, the transposon mutant was severely attenuated in the zebrafish model and growth impaired in the murine macrophage cell line RAW 264.7 [ 161 ]. 4.3.3. Corynebacterium glutamicum C. glutamicum is a well-established model species for cell wall-related studies in the Corynebacteriales because it shares the complex cell envelope organization with its pathogenic relatives, such as M. tuberculosis [ 162 ]. It contains a mycolyl-AG-PGN complex (compare with Figure 1 ) in addition to lipo(arabino)mannan in its cell wall. Furthermore, C. glutamicum is one of the main species used in the biotechnological industry, especially for the production of amino acids [ 163 ]. C. glutamicum LcpA and LcpB As described for Sc. pneumoniae and E. hirae , C. glutamicum 's two LCP proteins, LcpA and LcpB, localize where nascent cell wall biosynthesis happens. Interestingly, of the two C. glutamicum LCP proteins, a deletion was only feasible for LcpB, but did not lead to any detectable changes in the cell wall as compared to the wild-type strain. LcpA could be conditionally silenced, which influenced bacterial growth, the ratio of cell wall components, and morphology. Compared to the wild-type cell wall, the cell wall of the C. glutamicum Δ lcpA mutant contained significantly less mycolic acids and a reduced amount of AG. In particular, rhamnose, a specific sugar component of the linker that connects AG and PGN was decreased (compare with Figure 1 ). Characteristic of LcpA is the presence of an LCP domain and a LytR_C domain, which is frequently found in actinobacteria. In complementation studies, the importance of the conserved arginine and aspartate residues in the LCP domain as well as the general importance of the LytR_C domain was shown [ 164 ]. LcpA was shown to oligomerize into dimers or tetramers, wherefore the LytR_C domain might be responsible supportive of the LytR_C domain catalysing its own reaction [ 164 ]. 4.3.4. Streptomyces coelicolor Sm. coelicolor is the genetically best-known representative among the soil colonized, filamentous Gram-positive bacteria of the Streptomycetes genus [ 165 ], which play an important role in producing natural antibiotics. In contrast to most other bacteria, which divide by binary fission Sm. coelicolor A3(2) develops a mycelial lifestyle by apical tip extension, which requires a dedicated mode of PGN incorporation [ 166 ]. Sm. coelicolor A3(2) encodes several homologues of Tag proteins [ 167 ] directing WTA synthesis in B. subtilis , although the major glycopolymer of Sm. coelicolor is teichulosonic acid. This teichulosonic acid is a phosphate-free polymer of up to seven RUs composed of galactose and the neuraminic acid-related 2-keto-3-deoxy-D- glycero -D- galacto -nononic acid (Kdn), often substituted with GlcNAc or a methyl group. As a minor component, a polydiglycosylphosphate CWGP (referred to as PDP) consisting of →6)-α-Gal p -(1→6)-α-Glc p NAc- P -(1→- RUs is present in Sm. coelicolor cell walls [ 168 ] ( Figure 1 ). Sm. coelicolor PdtA and Ten Other LCP Proteins The genome of Sm. coelicolor harbours 11 LCP proteins, with seven genes (SCO3042-SCO3048) clustered like the B. subtilis TagT, TagU, or TagV-like phosphotransferae genes [ 169 ]. PdtA (SCO2578), a TagV-like phosphotransferase, is suggested to be co-transcribed with a nicotinate-nucleotide adenylytransferase gene SCO2579 and is in close proximity to other presumed CWGP-linked genes [ 167 , 169 ]. It was identified in a screen of interaction partners of several Streptomyces spore wall-synthesizing complex (SSSC) proteins possibly involved in sporulation [ 170 ]. PdtA inactivation resulted in irregular spore chains; more precisely, the placement of sporulation septa was affected and heterogeneity in spore sizes was displayed [ 169 ]. The other 10 LCP homologs did not show any severe phenotype effects and were not able to substitute for PdtA, upon whose deletion, a 48% reduction of spore wall glycopolymer content was determined, comparable to the situation in other LCP-containing organisms [ 82 , 169 ]. Interestingly, of the two types of Sm. coelicolor CWGPs, only PDP was affected in the spore envelope by the lack of pdtA and resulted in a severe phenotype, including imprecise sporulation septa placement and spore viability decrease by one-third. The remaining spores had increased sensitivity to osmotic stress and lysozyme. Furthermore, the Δ pdtA mutant showed impaired vegetative tip growth and, interestingly, also high sensitivity to rifampicin, which is known to cross the bacterial cell wall targeting the RNA polymerase and to have no effect on cell envelope synthesis [ 169 , 171 ]. Possibly, PDP acts as a barrier to block large-sized antibiotics, such as rifampicin [ 169 ]. It was speculated that PDP is anchored to the hyphal tip by PdtA resulting in apical tip growth, which aids as a framework for PGN synthesis, particularly under stress conditions [ 169 ]. The crucial role of PdtA under stress conditions becomes even clearer, as under high-salt conditions, only 4% of normal biomass was produced and hyphae showed aberrant morphology. Conclusively, of the 11 LCP protein homologs found in Sm. coelicolor , PdtA is the only protein involved in PDP synthesis and plays a key role for the life cycle of the organism [ 169 ]. 4.3.5. Actinomyces oris The actinobacterium A. oris is a colonizer of the oral cavity where it plays a specific role in the formation of supragingival plaque [ 172 ]. A . oris is dependent on the activity of its SrtA enzyme, which is conditionally dependent on glycosylation of the GspA surface protein by the activity of an LCP enzyme [ 173 ]. A. oris LcpA Participates in Protein Glycosylation The LCP homolog LcpA of A. oris provide a so far unique example of an LCP enzyme that is involved in a protein glycosylation process, i.e., the transfer of a saccharide moiety to an amino acid acceptor sequence [ 174 ] instead of a PGN backbone sugar. LcpA is genetically linked to GspA, a glycoprotein that is attached to A. oris PGN by the house-keeping sortase SrtA, which, in turn, recognizes the cell wall sorting signal of the glycoprotein [ 175 ]. Deletion of either the gspA or lcpA gene resulted in rescue effects of srtA depletion, leading to the suggestion that excessive aggregation of GspA proteins causes stress leading to cell death, whereas the absence of GspA in the presence of SrtA and LcpA is not lethal [ 176 ]. The neighbouring genes gspA and lcpA in A. oris implicate that their glycoprotein products are linked to each other and that the high glycosylation level of GspA involves LcpA [ 174 ]. LcpA glycosylates GspA along an unknown pathway, prior translocation across the cytoplasmic membrane and final cell wall anchoring by the sortase SrtA [ 176 ]. However, the exact nature and composition of the GspA glycans remain to be determined. A. oris LcpB, LcpC and LcpD Interestingly, A. oris MG1 encodes three other LCP domain-containing proteins, LcpB (ana_0299), a homolog of the TagF glycosyl/glycerophosphate transferase from Staphylococcus epidermidis [ 177 ], LcpC (ana_1577) and LcpD (ana_1578), which are found in the same transcriptional unit [ 174 ]. Mutant strains were generated by deletion of lcpB and lcpD as well as a triple mutant lcp Δ 3 , devoid of lcpA , lcpB and lcpD , to analyse LcpA-mediated glycosylation. The single mutants did not show any negative effect regarding the formation of high-molecular-mass GspA species with attached glycans, whereas the triple mutant did, supporting that LcpA is necessary and sufficient for the production of glycosylated GspA. Together with the obtained crystal structure (see below), this is the first experimental evidence of the glycosylation capability of LcpA which occurs prior to GspA glycoprotein transfer to PGN [ 174 ]. The data suggest that LcpA is the only enzyme involved in GspA glycosylation in A. oris , but it has to be mentioned that a Δ lcpC deletion mutant could not be generated and possibly may also modify GspA [ 174 ]. Crystal Structure or A. oris LcpA A notable structural variant of LCP enzymes is that of A. oris LcpA, which possesses unique structural features around the active site presumably associated with binding target proteins rather than PGN for glycosylation. The molecular structure of the extracellular LcpA domain has been resolved at 2.5-à (eLcpA, residues 78–360). The core of the protein is formed by seven-stranded antiparallel β-sheets flanked with eight α-helices on both sites, forming ~23-à hydrophobic tunnel [ 174 ]. Its presence is consistent with other members of the LCP protein family, leading into the active sites of LcpA that possibly bind a lipid-linked glycan substrate [ 82 , 174 ]. Conserved arginine residues R128, R149 and R266 were identified clustering within a pocket exposed on the surface, which indicates mediated phosphotransfer of glycopolymers as known in the LCP family [ 82 , 97 , 147 , 174 ]. By generating alanine substitution mutants of these arginine residues, it could be determined that the R149 and R266 residues are essential for LcpA glycosylation activity on GspA. Interestingly, LcpA links the C-terminus to α-helices between C179 and C365 formed by a presumably stabilizing disulphide bond that is also found in other actinobacterial LCP proteins [ 174 ]. Alanine substitution mutants of either one or both Cys residues in LcpA suggested that the disulphide bond is essential for protein stability as evident from a defect of mutant LcpA membrane expression as well as for full enzymatic activity [ 174 ]. Based on the demonstrated in vitro pyrophosphate activity of TagT [ 82 ], an in vitro assay of eLcpA over quantification of released inorganic phosphate in concert with a diphosphate mimic substrate showed that LcpA displays pyrophosphate activity and corroborated the necessity of the disulfide bond for catalysis [ 174 ]. This distinct feature seems to be common in actinobacterial LCP enzymes and has not been found in other LCP enzymes studied to date [ 174 ]. Overall, the structure of eLcpA seems closely related to the TagT enzyme from B. subtilis [ 97 , 174 ]. However, unlike TagT, LcpA is not capable of attaching CWGPs to PGN but to GspA instead [ 174 ]. 4.3.1. Mycobacterium tuberculosis M. tuberculosis is the causative agent of tuberculosis infecting the lungs and causing about 1.5 million deaths per year [ 154 ]. What makes this organism so strong is its unique, low permeable AG-containing cell wall ( Figure 1 ) that provides a high resistance towards antibiotics [ 155 ]. M. tuberculosis Rv0822c, CpsA1, CpsA2 and Rv3840 Function Four genes encoding LCP proteins are annotated in the M. tuberculosis H37Rv genome, namely Rv0822c, Rv3267 ( cpsA1/lcp1 ), Rv3484 ( cpsA/cpsA2 ), and Rv3840 [ 156 ]. To investigate an association of the M. tuberculosis LCP proteins with the ligation of AG to PGN, the predicted enzymes devoid of the TM domain were produced recombinantly in E. coli and assayed in vitro. Functional proof was obtained for CpsA1 and CpsA2, which, under the chosen experimental conditions, showed pyrophosphatase activity on the generic substrate geranyl pyrophosphate in dependence on magnesium ions consistent with other LCP enzymes [ 65 ]. Notably, while individual cpsA1 and cpsA2 knock-outs of M. tuberculosis were readily obtainable, the combined inactivation of both genes appeared to be lethal. M. tuberculosis CpsA1 CpsA1 maps to the AG biosynthetic gene cluster where it is located immediately upstream of two genes involved in linker biosynthesis. M. tuberculosis CpsA1 was suggested to be the predominant enzyme responsible for the covalent attachment of AG to PGN. However, in a Δ cpsA1 deletion mutant, no major effects were seen, probably due to functional compensation of the paralogs [ 65 , 155 ]. CpsA1/Lcp1 was further described to be essential for M. tuberculosis and its activity was verified in a cell-free radiolabelling assay with 14 C-radiolabeled AG and nascent PGN [ 155 ]. To further evaluate the specificity of M. tuberculosis CpsA1, three potential AG binding substrates—L-Rha-α(1¡3)-D-GlcNAc- O -C 8 (compound 1), Gal f 2 -Rha-GlcNAc- O -C 8 (compound 2) and Gal f 3 -Rha-GlcNAc- O -C 8 (compound 3)—were tested using intrinsic tryptophan fluorescence of CpsA1, with compound 2 showing the highest affinity with 5.13 µM [ 155 ]. Although the envelope composition was not drastically changed in the single ligase mutants, the Δ cpsA1 mutant showed increased susceptibility to a range of antibiotics such as penicillins, vancomycin, and CPZEN-45, suggesting changes in cell wall permeability [ 65 ]. M. tuberculosis CpsA2 Although no analytically detectable difference was observed, the Δ cpsA2 deletion mutant in the M. tuberculosis strain H37Rv showed a changed phenotype in an in vivo mouse model, where the mutant was not able to grow, survive and infect. Furthermore, this strain displayed increased resistance to meropenem/clavulanate and rifampicin, which could not be compensated by cpsA1 [ 157 ]. Meropenem belongs to the carbapenem class of β-lactam antibiotics being poor substrates for BlaC, a protein encoded in the genome of M. tuberculosis which hydrolyses β-lactam antibiotics [ 158 ]. Rifampicin did not target the cell wall but the authors argued that the permeability had changed and, therefore, the drug did not efficiently get into the cytoplasm as supported by measuring ethidium bromide uptake and efflux [ 66 , 157 ]. However, Grzegorzewicz et al. could not determine increased resistance against rifampicin. According to Malm et al., this could be due to differences in the experimental set-up or resulting from a secondary effect and not from cpsA2 deletion [ 65 , 157 ]. Additional M. tuberculosis Proteins Related to LCP Enzyme Function Recently, mutants of the "cell envelope integrity" gene ( cei ; Rv2700) of M. tuberculosis and its structural homolog VirR (Rv0431), showed a decreased growth rate at low densities, increased susceptibility towards antibiotics (vancomycin, meropenem and rifampicin), increased sensitivity to nitric oxide (NO), and increased cell envelope permeability. In addition, a Δ cei mutant did not lethally infect mice, where one factor that influences virulence is the growth control by NO after infection [ 66 , 159 ]. Importantly, these newly detected gene products have one predicted TM domain and a LytR_C domain, which are features found in LCP enzymes commonly referred to as LytR_C-only proteins. Due to very similar phenotypes of the knockout mutants of cei and virR to common LCP knockout strains, a relation to and common participation in the same pathway, namely AG ligation to PGN, was hypothesized [ 66 ]. M. tuberculosis Rv0822c, CpsA1, CpsA2 and Rv3840 Function Four genes encoding LCP proteins are annotated in the M. tuberculosis H37Rv genome, namely Rv0822c, Rv3267 ( cpsA1/lcp1 ), Rv3484 ( cpsA/cpsA2 ), and Rv3840 [ 156 ]. To investigate an association of the M. tuberculosis LCP proteins with the ligation of AG to PGN, the predicted enzymes devoid of the TM domain were produced recombinantly in E. coli and assayed in vitro. Functional proof was obtained for CpsA1 and CpsA2, which, under the chosen experimental conditions, showed pyrophosphatase activity on the generic substrate geranyl pyrophosphate in dependence on magnesium ions consistent with other LCP enzymes [ 65 ]. Notably, while individual cpsA1 and cpsA2 knock-outs of M. tuberculosis were readily obtainable, the combined inactivation of both genes appeared to be lethal. M. tuberculosis CpsA1 CpsA1 maps to the AG biosynthetic gene cluster where it is located immediately upstream of two genes involved in linker biosynthesis. M. tuberculosis CpsA1 was suggested to be the predominant enzyme responsible for the covalent attachment of AG to PGN. However, in a Δ cpsA1 deletion mutant, no major effects were seen, probably due to functional compensation of the paralogs [ 65 , 155 ]. CpsA1/Lcp1 was further described to be essential for M. tuberculosis and its activity was verified in a cell-free radiolabelling assay with 14 C-radiolabeled AG and nascent PGN [ 155 ]. To further evaluate the specificity of M. tuberculosis CpsA1, three potential AG binding substrates—L-Rha-α(1¡3)-D-GlcNAc- O -C 8 (compound 1), Gal f 2 -Rha-GlcNAc- O -C 8 (compound 2) and Gal f 3 -Rha-GlcNAc- O -C 8 (compound 3)—were tested using intrinsic tryptophan fluorescence of CpsA1, with compound 2 showing the highest affinity with 5.13 µM [ 155 ]. Although the envelope composition was not drastically changed in the single ligase mutants, the Δ cpsA1 mutant showed increased susceptibility to a range of antibiotics such as penicillins, vancomycin, and CPZEN-45, suggesting changes in cell wall permeability [ 65 ]. M. tuberculosis CpsA2 Although no analytically detectable difference was observed, the Δ cpsA2 deletion mutant in the M. tuberculosis strain H37Rv showed a changed phenotype in an in vivo mouse model, where the mutant was not able to grow, survive and infect. Furthermore, this strain displayed increased resistance to meropenem/clavulanate and rifampicin, which could not be compensated by cpsA1 [ 157 ]. Meropenem belongs to the carbapenem class of β-lactam antibiotics being poor substrates for BlaC, a protein encoded in the genome of M. tuberculosis which hydrolyses β-lactam antibiotics [ 158 ]. Rifampicin did not target the cell wall but the authors argued that the permeability had changed and, therefore, the drug did not efficiently get into the cytoplasm as supported by measuring ethidium bromide uptake and efflux [ 66 , 157 ]. However, Grzegorzewicz et al. could not determine increased resistance against rifampicin. According to Malm et al., this could be due to differences in the experimental set-up or resulting from a secondary effect and not from cpsA2 deletion [ 65 , 157 ]. Additional M. tuberculosis Proteins Related to LCP Enzyme Function Recently, mutants of the "cell envelope integrity" gene ( cei ; Rv2700) of M. tuberculosis and its structural homolog VirR (Rv0431), showed a decreased growth rate at low densities, increased susceptibility towards antibiotics (vancomycin, meropenem and rifampicin), increased sensitivity to nitric oxide (NO), and increased cell envelope permeability. In addition, a Δ cei mutant did not lethally infect mice, where one factor that influences virulence is the growth control by NO after infection [ 66 , 159 ]. Importantly, these newly detected gene products have one predicted TM domain and a LytR_C domain, which are features found in LCP enzymes commonly referred to as LytR_C-only proteins. Due to very similar phenotypes of the knockout mutants of cei and virR to common LCP knockout strains, a relation to and common participation in the same pathway, namely AG ligation to PGN, was hypothesized [ 66 ]. 4.3.2. Mycobacterium marinum M. marinum is a slow-growing, acid-fast bacterium in the category of non-tuberculous mycobacteria which most commonly cause skin and soft tissue infections in patients, particularly those with aquatic exposure [ 160 ]. The bacterium possesses an AG as is characteristic of mycobacteria. M. marinum MMAR_4858, MMAR_1274, MMAR_4966 (CpsA), and MMAR_5392 M. marinum (MMAR) harbours orthologues of all LCP proteins found in M. tuberculosis complex (Mtbc) strains—namely MMAR_4858, MMAR_1274, MMAR_4966 (CpsA), and MMAR_5392 in MM for Rv0822c, Rv3267, Rv3484, and Rv3840 for Mtbc [ 65 , 157 ]. A strain defective in the CpsA2 (Rv3484) orthologue in M. marinum (CpsA) showed impaired growth in vitro in contrast to CpsA deficient strains of M. tuberculosis [ 65 , 157 ]. Furthermore, the M. marinum Δ cpsA mutant revealed alterations in colony morphology and cell surface properties, increased susceptibility to antibiotics (erythromycin, vancomycin and penicillin), and a change in cell wall permeability for hydrophobic components. Traditional analytics of the cell wall composition indicated an imbalance in the AG/PGN ratio indicative of a role of the MMAR_4966 enzyme in AG transfer to PGN. Finally, the transposon mutant was severely attenuated in the zebrafish model and growth impaired in the murine macrophage cell line RAW 264.7 [ 161 ]. M. marinum MMAR_4858, MMAR_1274, MMAR_4966 (CpsA), and MMAR_5392 M. marinum (MMAR) harbours orthologues of all LCP proteins found in M. tuberculosis complex (Mtbc) strains—namely MMAR_4858, MMAR_1274, MMAR_4966 (CpsA), and MMAR_5392 in MM for Rv0822c, Rv3267, Rv3484, and Rv3840 for Mtbc [ 65 , 157 ]. A strain defective in the CpsA2 (Rv3484) orthologue in M. marinum (CpsA) showed impaired growth in vitro in contrast to CpsA deficient strains of M. tuberculosis [ 65 , 157 ]. Furthermore, the M. marinum Δ cpsA mutant revealed alterations in colony morphology and cell surface properties, increased susceptibility to antibiotics (erythromycin, vancomycin and penicillin), and a change in cell wall permeability for hydrophobic components. Traditional analytics of the cell wall composition indicated an imbalance in the AG/PGN ratio indicative of a role of the MMAR_4966 enzyme in AG transfer to PGN. Finally, the transposon mutant was severely attenuated in the zebrafish model and growth impaired in the murine macrophage cell line RAW 264.7 [ 161 ]. 4.3.3. Corynebacterium glutamicum C. glutamicum is a well-established model species for cell wall-related studies in the Corynebacteriales because it shares the complex cell envelope organization with its pathogenic relatives, such as M. tuberculosis [ 162 ]. It contains a mycolyl-AG-PGN complex (compare with Figure 1 ) in addition to lipo(arabino)mannan in its cell wall. Furthermore, C. glutamicum is one of the main species used in the biotechnological industry, especially for the production of amino acids [ 163 ]. C. glutamicum LcpA and LcpB As described for Sc. pneumoniae and E. hirae , C. glutamicum 's two LCP proteins, LcpA and LcpB, localize where nascent cell wall biosynthesis happens. Interestingly, of the two C. glutamicum LCP proteins, a deletion was only feasible for LcpB, but did not lead to any detectable changes in the cell wall as compared to the wild-type strain. LcpA could be conditionally silenced, which influenced bacterial growth, the ratio of cell wall components, and morphology. Compared to the wild-type cell wall, the cell wall of the C. glutamicum Δ lcpA mutant contained significantly less mycolic acids and a reduced amount of AG. In particular, rhamnose, a specific sugar component of the linker that connects AG and PGN was decreased (compare with Figure 1 ). Characteristic of LcpA is the presence of an LCP domain and a LytR_C domain, which is frequently found in actinobacteria. In complementation studies, the importance of the conserved arginine and aspartate residues in the LCP domain as well as the general importance of the LytR_C domain was shown [ 164 ]. LcpA was shown to oligomerize into dimers or tetramers, wherefore the LytR_C domain might be responsible supportive of the LytR_C domain catalysing its own reaction [ 164 ]. C. glutamicum LcpA and LcpB As described for Sc. pneumoniae and E. hirae , C. glutamicum 's two LCP proteins, LcpA and LcpB, localize where nascent cell wall biosynthesis happens. Interestingly, of the two C. glutamicum LCP proteins, a deletion was only feasible for LcpB, but did not lead to any detectable changes in the cell wall as compared to the wild-type strain. LcpA could be conditionally silenced, which influenced bacterial growth, the ratio of cell wall components, and morphology. Compared to the wild-type cell wall, the cell wall of the C. glutamicum Δ lcpA mutant contained significantly less mycolic acids and a reduced amount of AG. In particular, rhamnose, a specific sugar component of the linker that connects AG and PGN was decreased (compare with Figure 1 ). Characteristic of LcpA is the presence of an LCP domain and a LytR_C domain, which is frequently found in actinobacteria. In complementation studies, the importance of the conserved arginine and aspartate residues in the LCP domain as well as the general importance of the LytR_C domain was shown [ 164 ]. LcpA was shown to oligomerize into dimers or tetramers, wherefore the LytR_C domain might be responsible supportive of the LytR_C domain catalysing its own reaction [ 164 ]. 4.3.4. Streptomyces coelicolor Sm. coelicolor is the genetically best-known representative among the soil colonized, filamentous Gram-positive bacteria of the Streptomycetes genus [ 165 ], which play an important role in producing natural antibiotics. In contrast to most other bacteria, which divide by binary fission Sm. coelicolor A3(2) develops a mycelial lifestyle by apical tip extension, which requires a dedicated mode of PGN incorporation [ 166 ]. Sm. coelicolor A3(2) encodes several homologues of Tag proteins [ 167 ] directing WTA synthesis in B. subtilis , although the major glycopolymer of Sm. coelicolor is teichulosonic acid. This teichulosonic acid is a phosphate-free polymer of up to seven RUs composed of galactose and the neuraminic acid-related 2-keto-3-deoxy-D- glycero -D- galacto -nononic acid (Kdn), often substituted with GlcNAc or a methyl group. As a minor component, a polydiglycosylphosphate CWGP (referred to as PDP) consisting of →6)-α-Gal p -(1→6)-α-Glc p NAc- P -(1→- RUs is present in Sm. coelicolor cell walls [ 168 ] ( Figure 1 ). Sm. coelicolor PdtA and Ten Other LCP Proteins The genome of Sm. coelicolor harbours 11 LCP proteins, with seven genes (SCO3042-SCO3048) clustered like the B. subtilis TagT, TagU, or TagV-like phosphotransferae genes [ 169 ]. PdtA (SCO2578), a TagV-like phosphotransferase, is suggested to be co-transcribed with a nicotinate-nucleotide adenylytransferase gene SCO2579 and is in close proximity to other presumed CWGP-linked genes [ 167 , 169 ]. It was identified in a screen of interaction partners of several Streptomyces spore wall-synthesizing complex (SSSC) proteins possibly involved in sporulation [ 170 ]. PdtA inactivation resulted in irregular spore chains; more precisely, the placement of sporulation septa was affected and heterogeneity in spore sizes was displayed [ 169 ]. The other 10 LCP homologs did not show any severe phenotype effects and were not able to substitute for PdtA, upon whose deletion, a 48% reduction of spore wall glycopolymer content was determined, comparable to the situation in other LCP-containing organisms [ 82 , 169 ]. Interestingly, of the two types of Sm. coelicolor CWGPs, only PDP was affected in the spore envelope by the lack of pdtA and resulted in a severe phenotype, including imprecise sporulation septa placement and spore viability decrease by one-third. The remaining spores had increased sensitivity to osmotic stress and lysozyme. Furthermore, the Δ pdtA mutant showed impaired vegetative tip growth and, interestingly, also high sensitivity to rifampicin, which is known to cross the bacterial cell wall targeting the RNA polymerase and to have no effect on cell envelope synthesis [ 169 , 171 ]. Possibly, PDP acts as a barrier to block large-sized antibiotics, such as rifampicin [ 169 ]. It was speculated that PDP is anchored to the hyphal tip by PdtA resulting in apical tip growth, which aids as a framework for PGN synthesis, particularly under stress conditions [ 169 ]. The crucial role of PdtA under stress conditions becomes even clearer, as under high-salt conditions, only 4% of normal biomass was produced and hyphae showed aberrant morphology. Conclusively, of the 11 LCP protein homologs found in Sm. coelicolor , PdtA is the only protein involved in PDP synthesis and plays a key role for the life cycle of the organism [ 169 ]. Sm. coelicolor PdtA and Ten Other LCP Proteins The genome of Sm. coelicolor harbours 11 LCP proteins, with seven genes (SCO3042-SCO3048) clustered like the B. subtilis TagT, TagU, or TagV-like phosphotransferae genes [ 169 ]. PdtA (SCO2578), a TagV-like phosphotransferase, is suggested to be co-transcribed with a nicotinate-nucleotide adenylytransferase gene SCO2579 and is in close proximity to other presumed CWGP-linked genes [ 167 , 169 ]. It was identified in a screen of interaction partners of several Streptomyces spore wall-synthesizing complex (SSSC) proteins possibly involved in sporulation [ 170 ]. PdtA inactivation resulted in irregular spore chains; more precisely, the placement of sporulation septa was affected and heterogeneity in spore sizes was displayed [ 169 ]. The other 10 LCP homologs did not show any severe phenotype effects and were not able to substitute for PdtA, upon whose deletion, a 48% reduction of spore wall glycopolymer content was determined, comparable to the situation in other LCP-containing organisms [ 82 , 169 ]. Interestingly, of the two types of Sm. coelicolor CWGPs, only PDP was affected in the spore envelope by the lack of pdtA and resulted in a severe phenotype, including imprecise sporulation septa placement and spore viability decrease by one-third. The remaining spores had increased sensitivity to osmotic stress and lysozyme. Furthermore, the Δ pdtA mutant showed impaired vegetative tip growth and, interestingly, also high sensitivity to rifampicin, which is known to cross the bacterial cell wall targeting the RNA polymerase and to have no effect on cell envelope synthesis [ 169 , 171 ]. Possibly, PDP acts as a barrier to block large-sized antibiotics, such as rifampicin [ 169 ]. It was speculated that PDP is anchored to the hyphal tip by PdtA resulting in apical tip growth, which aids as a framework for PGN synthesis, particularly under stress conditions [ 169 ]. The crucial role of PdtA under stress conditions becomes even clearer, as under high-salt conditions, only 4% of normal biomass was produced and hyphae showed aberrant morphology. Conclusively, of the 11 LCP protein homologs found in Sm. coelicolor , PdtA is the only protein involved in PDP synthesis and plays a key role for the life cycle of the organism [ 169 ]. 4.3.5. Actinomyces oris The actinobacterium A. oris is a colonizer of the oral cavity where it plays a specific role in the formation of supragingival plaque [ 172 ]. A . oris is dependent on the activity of its SrtA enzyme, which is conditionally dependent on glycosylation of the GspA surface protein by the activity of an LCP enzyme [ 173 ]. A. oris LcpA Participates in Protein Glycosylation The LCP homolog LcpA of A. oris provide a so far unique example of an LCP enzyme that is involved in a protein glycosylation process, i.e., the transfer of a saccharide moiety to an amino acid acceptor sequence [ 174 ] instead of a PGN backbone sugar. LcpA is genetically linked to GspA, a glycoprotein that is attached to A. oris PGN by the house-keeping sortase SrtA, which, in turn, recognizes the cell wall sorting signal of the glycoprotein [ 175 ]. Deletion of either the gspA or lcpA gene resulted in rescue effects of srtA depletion, leading to the suggestion that excessive aggregation of GspA proteins causes stress leading to cell death, whereas the absence of GspA in the presence of SrtA and LcpA is not lethal [ 176 ]. The neighbouring genes gspA and lcpA in A. oris implicate that their glycoprotein products are linked to each other and that the high glycosylation level of GspA involves LcpA [ 174 ]. LcpA glycosylates GspA along an unknown pathway, prior translocation across the cytoplasmic membrane and final cell wall anchoring by the sortase SrtA [ 176 ]. However, the exact nature and composition of the GspA glycans remain to be determined. A. oris LcpB, LcpC and LcpD Interestingly, A. oris MG1 encodes three other LCP domain-containing proteins, LcpB (ana_0299), a homolog of the TagF glycosyl/glycerophosphate transferase from Staphylococcus epidermidis [ 177 ], LcpC (ana_1577) and LcpD (ana_1578), which are found in the same transcriptional unit [ 174 ]. Mutant strains were generated by deletion of lcpB and lcpD as well as a triple mutant lcp Δ 3 , devoid of lcpA , lcpB and lcpD , to analyse LcpA-mediated glycosylation. The single mutants did not show any negative effect regarding the formation of high-molecular-mass GspA species with attached glycans, whereas the triple mutant did, supporting that LcpA is necessary and sufficient for the production of glycosylated GspA. Together with the obtained crystal structure (see below), this is the first experimental evidence of the glycosylation capability of LcpA which occurs prior to GspA glycoprotein transfer to PGN [ 174 ]. The data suggest that LcpA is the only enzyme involved in GspA glycosylation in A. oris , but it has to be mentioned that a Δ lcpC deletion mutant could not be generated and possibly may also modify GspA [ 174 ]. Crystal Structure or A. oris LcpA A notable structural variant of LCP enzymes is that of A. oris LcpA, which possesses unique structural features around the active site presumably associated with binding target proteins rather than PGN for glycosylation. The molecular structure of the extracellular LcpA domain has been resolved at 2.5-à (eLcpA, residues 78–360). The core of the protein is formed by seven-stranded antiparallel β-sheets flanked with eight α-helices on both sites, forming ~23-à hydrophobic tunnel [ 174 ]. Its presence is consistent with other members of the LCP protein family, leading into the active sites of LcpA that possibly bind a lipid-linked glycan substrate [ 82 , 174 ]. Conserved arginine residues R128, R149 and R266 were identified clustering within a pocket exposed on the surface, which indicates mediated phosphotransfer of glycopolymers as known in the LCP family [ 82 , 97 , 147 , 174 ]. By generating alanine substitution mutants of these arginine residues, it could be determined that the R149 and R266 residues are essential for LcpA glycosylation activity on GspA. Interestingly, LcpA links the C-terminus to α-helices between C179 and C365 formed by a presumably stabilizing disulphide bond that is also found in other actinobacterial LCP proteins [ 174 ]. Alanine substitution mutants of either one or both Cys residues in LcpA suggested that the disulphide bond is essential for protein stability as evident from a defect of mutant LcpA membrane expression as well as for full enzymatic activity [ 174 ]. Based on the demonstrated in vitro pyrophosphate activity of TagT [ 82 ], an in vitro assay of eLcpA over quantification of released inorganic phosphate in concert with a diphosphate mimic substrate showed that LcpA displays pyrophosphate activity and corroborated the necessity of the disulfide bond for catalysis [ 174 ]. This distinct feature seems to be common in actinobacterial LCP enzymes and has not been found in other LCP enzymes studied to date [ 174 ]. Overall, the structure of eLcpA seems closely related to the TagT enzyme from B. subtilis [ 97 , 174 ]. However, unlike TagT, LcpA is not capable of attaching CWGPs to PGN but to GspA instead [ 174 ]. A. oris LcpA Participates in Protein Glycosylation The LCP homolog LcpA of A. oris provide a so far unique example of an LCP enzyme that is involved in a protein glycosylation process, i.e., the transfer of a saccharide moiety to an amino acid acceptor sequence [ 174 ] instead of a PGN backbone sugar. LcpA is genetically linked to GspA, a glycoprotein that is attached to A. oris PGN by the house-keeping sortase SrtA, which, in turn, recognizes the cell wall sorting signal of the glycoprotein [ 175 ]. Deletion of either the gspA or lcpA gene resulted in rescue effects of srtA depletion, leading to the suggestion that excessive aggregation of GspA proteins causes stress leading to cell death, whereas the absence of GspA in the presence of SrtA and LcpA is not lethal [ 176 ]. The neighbouring genes gspA and lcpA in A. oris implicate that their glycoprotein products are linked to each other and that the high glycosylation level of GspA involves LcpA [ 174 ]. LcpA glycosylates GspA along an unknown pathway, prior translocation across the cytoplasmic membrane and final cell wall anchoring by the sortase SrtA [ 176 ]. However, the exact nature and composition of the GspA glycans remain to be determined. A. oris LcpB, LcpC and LcpD Interestingly, A. oris MG1 encodes three other LCP domain-containing proteins, LcpB (ana_0299), a homolog of the TagF glycosyl/glycerophosphate transferase from Staphylococcus epidermidis [ 177 ], LcpC (ana_1577) and LcpD (ana_1578), which are found in the same transcriptional unit [ 174 ]. Mutant strains were generated by deletion of lcpB and lcpD as well as a triple mutant lcp Δ 3 , devoid of lcpA , lcpB and lcpD , to analyse LcpA-mediated glycosylation. The single mutants did not show any negative effect regarding the formation of high-molecular-mass GspA species with attached glycans, whereas the triple mutant did, supporting that LcpA is necessary and sufficient for the production of glycosylated GspA. Together with the obtained crystal structure (see below), this is the first experimental evidence of the glycosylation capability of LcpA which occurs prior to GspA glycoprotein transfer to PGN [ 174 ]. The data suggest that LcpA is the only enzyme involved in GspA glycosylation in A. oris , but it has to be mentioned that a Δ lcpC deletion mutant could not be generated and possibly may also modify GspA [ 174 ]. Crystal Structure or A. oris LcpA A notable structural variant of LCP enzymes is that of A. oris LcpA, which possesses unique structural features around the active site presumably associated with binding target proteins rather than PGN for glycosylation. The molecular structure of the extracellular LcpA domain has been resolved at 2.5-à (eLcpA, residues 78–360). The core of the protein is formed by seven-stranded antiparallel β-sheets flanked with eight α-helices on both sites, forming ~23-à hydrophobic tunnel [ 174 ]. Its presence is consistent with other members of the LCP protein family, leading into the active sites of LcpA that possibly bind a lipid-linked glycan substrate [ 82 , 174 ]. Conserved arginine residues R128, R149 and R266 were identified clustering within a pocket exposed on the surface, which indicates mediated phosphotransfer of glycopolymers as known in the LCP family [ 82 , 97 , 147 , 174 ]. By generating alanine substitution mutants of these arginine residues, it could be determined that the R149 and R266 residues are essential for LcpA glycosylation activity on GspA. Interestingly, LcpA links the C-terminus to α-helices between C179 and C365 formed by a presumably stabilizing disulphide bond that is also found in other actinobacterial LCP proteins [ 174 ]. Alanine substitution mutants of either one or both Cys residues in LcpA suggested that the disulphide bond is essential for protein stability as evident from a defect of mutant LcpA membrane expression as well as for full enzymatic activity [ 174 ]. Based on the demonstrated in vitro pyrophosphate activity of TagT [ 82 ], an in vitro assay of eLcpA over quantification of released inorganic phosphate in concert with a diphosphate mimic substrate showed that LcpA displays pyrophosphate activity and corroborated the necessity of the disulfide bond for catalysis [ 174 ]. This distinct feature seems to be common in actinobacterial LCP enzymes and has not been found in other LCP enzymes studied to date [ 174 ]. Overall, the structure of eLcpA seems closely related to the TagT enzyme from B. subtilis [ 97 , 174 ]. However, unlike TagT, LcpA is not capable of attaching CWGPs to PGN but to GspA instead [ 174 ]. 4.4. Cyanobacteria— Order: Nostocales 4.4.1. Anabena sp. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is a commonly used model organism to study cyanobacterial nitrogen fixation and cell differentiation [ 178 ]. It is capable of fixing carbon dioxide by oxygenic photosynthesis or of fixing molecular nitrogen when a combined nitrogen source such as ammonium or nitrate is not available; the bacterium segregates these two incompatible processes by multicellular development by differentiating 5–10% of vegetative cells into so-called heterocysts [ 179 ]. A polysaccharide layer is placed over the Anabena sp. proheterocyst during maturation followed by a glycolipid layer between the polysaccharide layer and the outer membrane aimed at diminishing oxygen entry into the cell [ 179 , 180 , 181 ]. Anabena sp. ConR The gene all0817 named conR (constriction regulator) is predicted to contain an LCP domain. While conR was initially predicted to be a transcriptional regulator [ 182 ], its deletion caused diazotrophic growth and heterocyst differentiation defects [ 179 ]. Although the polysaccharide and glycolipid envelope layers were present in the mutant, the polar junctions connecting heterocysts to vegetative cells were incomplete or widely open, which was hypothesized to allow oxygen to enter the heterocysts and inactivate nitrogenase [ 182 ]. Furthermore, the expression of conR was upregulated after nitrogen step-down in differentiating heterocysts and vegetative cells. In nitrate-containing media, filaments of the Δ conR mutant strain also showed aberrant septum formation of vegetative cells and defects in cell separation. However, after nitrogen step-down, the defective vegetative cells seemed less severe compared to filaments in nitrate-containing media [ 179 ]. It was suggested that these phenotypic growth defects do not simply evolve from defective nitrogen fixation but rather from a disrupted delivery of fixed nitrogen from heterocysts to their neighbouring vegetative cells via non-specific intracellular channels. The defective septum formation in the mutant could possibly result in deformation of these channels at the junction between vegetative cells and heterocysts, leading to aberrant metabolite exchange [ 179 ]. Conclusively, the putative LCP protein ConR in Anabena sp. is developmentally regulated and is essential for diazotrophic growth and heterocyst morphogenesis; specifically, it was found to be associated with septum formation and cell wall maintenance. 4.4.1. Anabena sp. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is a commonly used model organism to study cyanobacterial nitrogen fixation and cell differentiation [ 178 ]. It is capable of fixing carbon dioxide by oxygenic photosynthesis or of fixing molecular nitrogen when a combined nitrogen source such as ammonium or nitrate is not available; the bacterium segregates these two incompatible processes by multicellular development by differentiating 5–10% of vegetative cells into so-called heterocysts [ 179 ]. A polysaccharide layer is placed over the Anabena sp. proheterocyst during maturation followed by a glycolipid layer between the polysaccharide layer and the outer membrane aimed at diminishing oxygen entry into the cell [ 179 , 180 , 181 ]. Anabena sp. ConR The gene all0817 named conR (constriction regulator) is predicted to contain an LCP domain. While conR was initially predicted to be a transcriptional regulator [ 182 ], its deletion caused diazotrophic growth and heterocyst differentiation defects [ 179 ]. Although the polysaccharide and glycolipid envelope layers were present in the mutant, the polar junctions connecting heterocysts to vegetative cells were incomplete or widely open, which was hypothesized to allow oxygen to enter the heterocysts and inactivate nitrogenase [ 182 ]. Furthermore, the expression of conR was upregulated after nitrogen step-down in differentiating heterocysts and vegetative cells. In nitrate-containing media, filaments of the Δ conR mutant strain also showed aberrant septum formation of vegetative cells and defects in cell separation. However, after nitrogen step-down, the defective vegetative cells seemed less severe compared to filaments in nitrate-containing media [ 179 ]. It was suggested that these phenotypic growth defects do not simply evolve from defective nitrogen fixation but rather from a disrupted delivery of fixed nitrogen from heterocysts to their neighbouring vegetative cells via non-specific intracellular channels. The defective septum formation in the mutant could possibly result in deformation of these channels at the junction between vegetative cells and heterocysts, leading to aberrant metabolite exchange [ 179 ]. Conclusively, the putative LCP protein ConR in Anabena sp. is developmentally regulated and is essential for diazotrophic growth and heterocyst morphogenesis; specifically, it was found to be associated with septum formation and cell wall maintenance. Anabena sp. ConR The gene all0817 named conR (constriction regulator) is predicted to contain an LCP domain. While conR was initially predicted to be a transcriptional regulator [ 182 ], its deletion caused diazotrophic growth and heterocyst differentiation defects [ 179 ]. Although the polysaccharide and glycolipid envelope layers were present in the mutant, the polar junctions connecting heterocysts to vegetative cells were incomplete or widely open, which was hypothesized to allow oxygen to enter the heterocysts and inactivate nitrogenase [ 182 ]. Furthermore, the expression of conR was upregulated after nitrogen step-down in differentiating heterocysts and vegetative cells. In nitrate-containing media, filaments of the Δ conR mutant strain also showed aberrant septum formation of vegetative cells and defects in cell separation. However, after nitrogen step-down, the defective vegetative cells seemed less severe compared to filaments in nitrate-containing media [ 179 ]. It was suggested that these phenotypic growth defects do not simply evolve from defective nitrogen fixation but rather from a disrupted delivery of fixed nitrogen from heterocysts to their neighbouring vegetative cells via non-specific intracellular channels. The defective septum formation in the mutant could possibly result in deformation of these channels at the junction between vegetative cells and heterocysts, leading to aberrant metabolite exchange [ 179 ]. Conclusively, the putative LCP protein ConR in Anabena sp. is developmentally regulated and is essential for diazotrophic growth and heterocyst morphogenesis; specifically, it was found to be associated with septum formation and cell wall maintenance. 5. Conclusions The LytR-CpsA-Psr (LCP) phosphotransferases are present in virtually all Gram-positive bacteria, which characteristically contain a high proportion of PGN-attached CWGPs ( Table 1 ). Accumulated structural and biochemical data on several LCP proteins from various bacterial species provide strong evidence that this family of proteins carries out the key step of attaching CWGPs to the cell wall PGN. Thus, in-depth investigations of LCP proteins may not only aid our understanding of Gram-positive cell wall assembly but may also reveal important aspects of a novel antibiotic target. It is noteworthy that LCP-encoding genes often but not necessarily localize in close proximity to the gene cluster encoding the CWGP for which they exert their catalytic effects [ 89 ] and that, in most bacteria, LCP proteins are present in multiple copies expressing in part functional redundancy. Mechanistically, LCP proteins typically hydrolyse the pyrophosphate linkage between the lipid-carrier and the reducing-end sugar of the CWGP (i.e., they hydrolyse the linkage created by an NDP-sugar::lipid phosphate transferase)—in most but not all cases, this sugar is part of a dedicated murein linkage unit—and attach the CWGP to either MurNAc or GlcNAc residues of the PGN backbone via a phosphate ester linkage [ 82 , 89 , 97 ]. Recent exceptions from this picture of LCP proteins come from Sc. pneumoniae , where CP2 is directly glycosidically attached to GlcNAc residues of PGN by LCP activity of CpsA2 [ 122 ] and from A. oris , where the LCP domain-containing LcpA protein is involved in protein glycosylation [ 174 ]. The extracellular, soluble domains of several homologous LCP proteins in, e.g., Sc. pneumoniae and B. subtilis , have been crystallized and were proposed to be responsible for hydrolysis of the pyrophosphate linkage between the CWGPs and the membrane lipid anchor and subsequent attachment of CPS to the PGN. Further structural characterization of LCP enzymes is required to investigate PGN binding and to clarify the structural relationship between the donor lipid headgroup and the enzyme [ 106 ].

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335645/

Recent progress concerning CpG DNA and its use as a vaccine adjuvant

CpG Oligonucleotides (ODN) are immunomodulatory synthetic oligonucleotides designed to specifically agonize Toll-like receptor 9. Here, we review recent progress in understanding the mechanism of action of CpG ODN and provide an overview of human clinical trial results using CpG ODN to improve the vaccines for cancer, allergy and infectious disease.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10796458/

Regulation and functions of the NLRP3 inflammasome in RNA virus infection

Virus infection is one of the greatest threats to human life and health. In response to viral infection, the host's innate immune system triggers an antiviral immune response mostly mediated by inflammatory processes. Among the many pathways involved, the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome has received wide attention in the context of viral infection. The NLRP3 inflammasome is an intracellular sensor composed of three components, including the innate immune receptor NLRP3, adaptor apoptosis-associated speck-like protein containing CARD (ASC), and the cysteine protease caspase-1. After being assembled, the NLRP3 inflammasome can trigger caspase-1 to induce gasdermin D (GSDMD)-dependent pyroptosis, promoting the maturation and secretion of proinflammatory cytokines such as interleukin-1 (IL-1β) and interleukin-18 (IL-18). Recent studies have revealed that a variety of viruses activate or inhibit the NLRP3 inflammasome via viral particles, proteins, and nucleic acids. In this review, we present a variety of regulatory mechanisms and functions of the NLRP3 inflammasome upon RNA viral infection and demonstrate multiple therapeutic strategies that target the NLRP3 inflammasome for anti-inflammatory effects in viral infection. 1 Introduction The innate immune response is a natural immune defense mechanism that was gradually formed during evolution. It is considered the first line of defense against pathogen invasion. Innate immune responses are vital for eliminating external pathogens as well as for generating a powerful adaptive immune response ( Medzhitov and Janeway, 1997 ; Medzhitov, 2007 ; Koyama et al., 2008 ). Pattern recognition receptors (PRRs) are highly conserved sensors that quickly detect viral infection and launch antiviral immune responses ( Tan et al., 2018 ). Inflammasome is an important member of PRR. At present, there are four main types of inflammasome, mainly including: pyrin domain containing 1 (NLRP1), absent in melanoma 2 (AIM2), pyrin domain containing 3 (NLRP3) and caspase activation recruitment domain containing 4 (NLRC4) ( Broz and Dixit, 2016 ; Guo et al., 2018 ). Many different types of PAMPs, DAMPs, and environmental irritants might trigger their activation ( Schroder and Tschopp, 2010 ). The NLRP1 can be activated by lethal toxin (LeTx) ( Boyden and Dietrich, 2006 ); AIM2 can be activated by dsRNA ( Bürckstümmer et al., 2009 ; Hornung et al., 2009 ); activation of the NLRP3 inflammasome is triggered by a wide range of stimuli, including microbial infections, cellular stress, tissue damage, and some metabolic disturbances ( Kelley et al., 2019 ; Wang and Hauenstein, 2020 ). NLRC4 can be activated by Salmonella typhimurium, Legionella pneumophila (Legionella), etc. ( Mariathasan et al., 2004 ; Schroder and Tschopp, 2010 ). The nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome plays a vital role in innate immune responses and has garnered considerable attention in the context of virus infection ( Wang et al., 2014 ; He et al., 2016 ). The NLRP3 inflammasome is composed of three components: the innate immune receptor NLRP3, the effector cysteine protease caspase-1, and the adaptor apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (CARD) (ASC). To initiate inflammatory responses and activate the inflammasome, NLRP3 identifies pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) ( Jin and Flavell, 2010 ; Kelley et al., 2019 ). It has three different domains: a central nucleotide-binding and oligomerization domain (NOD), an amino-terminal pyrin (PYD) domain, and a carboxy-terminal leucine-rich repeat (LRR) domain. These three domains play vital roles in NLRP3 activation. The PYD domain recruits the pyrin domain of ASC to initiate the assembly of the inflammasome; on the other hand, the NOD domain, which exhibits ATPase activity, is vital for NLRP3 oligomerization after activation; the LRR domain is the region that recognizes ligands ( Schroder and Tschopp, 2010 ). The activation of the NLRP3 inflammasome results in the maturation of proinflammatory cytokines and secretion of active interleukin (IL)-1β and IL-18; these cytokines subsequently trigger downstream immune responses and inflammation ( Sagulenko et al., 2013 ). NLRP3 inflammasome activation occurs via two steps: signal 1 priming and signal 2 activation ( Figure 1 ) ( Sutterwala et al., 2014 ; He et al., 2016 ; Abad and Danthi, 2020 ). In the first step, NLRP3 and active IL-1β expression is upregulated via the Toll-like receptor (TLR) and nuclear factor-kappaB (NF-κB) signaling pathways. This is regulated at the transcriptional and translational levels ( Bauernfeind et al., 2009 ). Under the action of microbial components or endogenous cytokines such as tumor necrosis factor (TNF) and lipopolysaccharide, the transcription factor NF-κB is activated via receptors such as TLR4 or TNF receptor superfamily (TNFR), resulting in the transcription of NLRP3, pro-caspase-1, pro-IL-1β, and pro-IL-18 ( Sutterwala et al., 2014 ). In the second step, the NLRP3 inflammasome is activated via PAMPs and DAMPs, where NLRP3, caspase-1, and ASC combine to form the NLRP3 inflammasome, subsequently activating caspase-1 to drive the maturation and secretion of IL-1β. This encompasses the regulation of NLRP3 inflammasome activation at the post-translational level ( Bauernfeind et al., 2009 ; Cai et al., 2014 ). Tissue injury, metabolic imbalance, or different stress signals such as ATP, particulate matter, bacteria, and viruses can activate the NLRP3 inflammasome. Furthermore, the stimulation can be activated via three modes: potassium efflux (K + efflux), lysosomal destabilization, and reactive oxygen species (ROS) production ( Pétrilli et al., 2007 ; Dostert et al., 2008 ; Halle et al., 2008 ; Tschopp and Schroder, 2010 ; Lamkanfi and Dixit, 2014 ; Swanson et al., 2019 ). Once activated, NLRP3 can be further linked to the ASC via its PYD domain. ASC binds to pro-caspase-1 via its CARD domain to form a complete structure; thereafter, it forms apoptosis-related spot-like proteins to form inflammasome complexes. Active caspase-1 is produced by autocatalytic cleavage, and subsequently activated caspase-1 catalyzes the transcription of IL-1β precursor proteins to produce active IL-1β ( Fernandes-Alnemri et al., 2007 ; Franchi et al., 2009 ; Latz, 2010 ). This suggests that activating the NLRP3 inflammasome is a complicated process involving multiple cellular events. Consequently, further elucidations are needed to understand the regulatory processes of NLRP3 inflammasome activation. Figure 1 Schematic diagram of the two signaling pathways warranted for activating the NLRP3 inflammasome. Two signals are needed for activating the NLRP3 inflammasome. The first is the priming signal: tumor necrosis factor (TNF) and lipopolysaccharide act on the cell surface receptors TNFR and Toll-like receptor (TLR) 4, which activate the nuclear factor- kappaB (NF-κB) pathway and induce gene expression to produce large amounts of pro-IL-1β, pro-IL-18, and NLRP3. The second is the activation signal: pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) induce the assembly and activation of the NLRP3 inflammasome. There are three main DAMP models: (1) ionic flux model, (2) reactive oxygen species (ROS) production, and (3) lysosomal damage. The activated NLRP3 inflammasome activates caspase-1, pro-IL-1β, and pro-IL-18 to produce active IL-1β and IL-18 under the action of active caspase-1, finally releasing them outside the cell. In addition, activated caspase-1 can also cleave GSDMD to N-GSDMD and liberate them to insert into membrane and form the pyroptotic pores. The mature IL-1β and IL-18 release out of the cell together with cell content through GSDMD-pores to induce pyroptosis. When the NLRP3 inflammasome is activated by virus infection, it disrupts the replication niche within the pathogen cell, thereby promoting the release of proinflammatory factors; this, in turn, leads to the activation of a highly inflammatory form of cell death, namely pyroptosis; subsequently, it plays a barrier role in innate immunity ( Miao et al., 2010 ; Shi et al., 2017 ). Pyroptosis is an innate immune effector mechanism in the host antiviral defense system and a type of lytic cell death that further triggers the inflammatory cascade and activates immune surveillance systems to promote virus clearance ( Vande Walle and Lamkanfi, 2016 ; de Vasconcelos et al., 2019 ; Imre, 2020 ). Activated caspase-1 cleaves gasdermin D (GSDMD) to generate independent N- and C-terminal fragments. The N-terminal fragment of GSDMD induces osmotic pressure imbalance and membrane rupture by forming plasma membrane pores, thereby facilitating cell leakage and dissolution and extracellular active IL-1β secretion ( Fink and Cookson, 2006 ; Sborgi et al., 2016 ; Evavold et al., 2018 ; Huang et al., 2021 ). RNA viruses and emerging human pathogens are public health concerns. Various RNA viruses have developed various strategies to evade innate immune responses; they can induce the production of inflammatory factors that can lead to fatal diseases ( Nelemans and Kikkert, 2019 ). However, whether the virus can activate or inhibit the NLRP3 inflammasome remains controversial. Therefore, we elucidate and discuss the regulatory relationship between the NLRP3 inflammasome and RNA virus infection. Herein, we classified RNA viruses according to positive-sense single-stranded, negative-sense single-stranded and double-stranded RNA viruses. The various mechanisms by which RNA viruses regulate the activation or inhibition of the NLRP3 inflammasome were summarized ( Table 1 ). We briefly discuss the clinical significance of NLRP3 inflammasome activation in viral disease. We also discuss several therapeutic strategies targeting the NLRP3 inflammasome for anti-inflammatory effects in virus infection. The hope is to provide valuable experience for the clinical treatment of NLRP3-related diseases caused by RNA viruses. Table 1 Regulatory mechanism of the NLRP3 inflammasome in RNA virus infection. Viruses Regulatory mechanism Reference Severe acute respiratory syndrome coronavirus (SARS-CoV) 1. Signal 1-Priming: ORF3a, E 2. Signal 2-Activation: E, ORF3a, ORF8b ( DeDiego et al., 2014 ; Nieto-Torres et al., 2015 ; Chen et al., 2019 ; Shi et al., 2019 ; Siu et al., 2019 ). Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) 1. Signal 1-Priming: S, ORF3a 2. Signal 2-Activation: ORF3a, N, C3a, MAC ( Asgari et al., 2013 ; Sluyter, 2017 ; Magro et al., 2020 ; Ratajczak and Kucia, 2020 ; Olajide et al., 2021 ; Pan et al., 2021 ; Zhao et al., 2021 ; Xu et al., 2022 ; Triantafilou et al., 2013a ) Dengue virus (DENV) 1. Signal 1-Priming: DENV viral RNA, E(EDIII) 2. Signal 2-Activation: E(EDIII), NS2A, NS2B, M, ( Khan et al., 2019 ; Pan et al., 2019a ; Shrivastava et al., 2020a ; Shrivastava et al., 2020b ) Zika virus (ZIKV) Enterovirus 71 (EV71) 1. Signal 2-Activation: NS5, 2. Inhibition mechanism: NS3 ( He et al., 2018 ; Wang et al., 2018 ; Gim et al., 2019 ) Enterovirus 71 (EV71) 1. Signal 1-Priming: VIM-ERK-NF-κB 2. Signal 2-Activation: 3D, TXNIP 3. Inhibition mechanism: 2A, 3C ( Wang et al., 2015 ; Wang et al., 2017 ; Xiao et al., 2019 ; You et al., 2020 ; Qayyum et al., 2021 ; Gong et al., 2022 ; Hu et al., 2023 ) Human Immunodeficiency Virus (HIV) 1. Signal 1-Priming: HIV-1, Vpr, Tat 2. Signal 2-Activation: Tat, HIV-1 RNA, E ( Demarchi et al., 1996 ; Benelli et al., 2000 ; Séror et al., 2011 ; Lee et al., 2012 ; Hernandez et al., 2014 ; Chivero et al., 2017 ; Paoletti et al., 2019 ; He et al., 2020 ; Li et al., 2020 ; Stunnenberg et al., 2021 ) Influenza virus (FLU) 1. Signal 1-Priming: NP, RNA 2. Signal 2-Activation: M2, PB1-F2 protein, RNA 3. Inhibition mechanism: NS1 ( Allen et al., 2009 ; Ichinohe et al., 2009 ; Ichinohe et al., 2010 ; Pang and Iwasaki, 2011 ; McAuley et al., 2013 ; Yoshizumi et al., 2014 ; Chung et al., 2015 ; Moriyama et al., 2016 ; Kuriakose and Kanneganti, 2017 ; Moriyama et al., 2019 ; Wang et al., 2021 ; Kim et al., 2022 ) Respiratory syncytial virus (RSV) 1. Signal 2-Activation : SH, ORMDL3 ( Triantafilou et al., 2013b ; Cantero-Recasens et al., 2010 ) 2 Regulatory mechanisms and functions of the NLRP3 inflammasome during RNA virus infection 2.1 Positive-sense single-stranded RNA virus 2.1.1 Human coronaviruses Coronaviruses belong to the family Coronaviridae and comprise a positive-sense single-stranded RNA that is 27–32 kb long ( Van Der Hoek et al., 2004 ; Lu et al., 2020 ). Both severe acute respiratory syndrome coronavirus (SARS-CoV) and novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are types of human coronaviruses ( Hasöksüz et al., 2020 ). The genomes of SARS-CoV and SARS-CoV-2 encode four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The RNA genome is encapsulated by the N proteins. The M and E proteins ensure that the N proteins can be incorporated into the virus particles during assembly, whereas the S proteins provide specificity for cellular entry receptors ( Alanagreh et al., 2020 ; Kim et al., 2020 ; Liu et al., 2020a ). After binding to specific receptors such as angiotensin-converting enzyme 2, coronaviruses can either directly fuse with the cell surface or be engulfed by the endosomes, thereby entering the cell. Then, the viral N protein enters the cytoplasm, releasing the RNA genome of the virus into the cell. It synthesizes new virus RNA and proteins and assembles them into virus particles for release ( Fehr and Perlman, 2015 ; Hartenian et al., 2020 ; Prydz and Saraste, 2022 ). 2.1.1.1 SARS-CoV virus infection and NLRP3 inflammasome SARS-CoV-encoded proteins can activate the NLRP3 inflammasome via the NF-κB pathway. For example, ORF3a and E proteins play vital roles in activating the NLRP3 inflammasome. ORF3a and E proteins of SARS-CoV can activate NF-κB, resulting in the transcription of pro-IL-1β ( DeDiego et al., 2014 ; Siu et al., 2019 ). Furthermore, ORF3a promotes the ubiquitination of p105 and ASC via TNFR-associated factors, promoting the activation of NF-κB and the NLRP3 inflammasome ( Siu et al., 2019 ). ORF3a and E proteins of SARS-CoV play roles in the second activation step. ORF3a can lead to lysosomal dysfunction in the host cells and initiate caspase-1 activation either directly or via increased K + efflux. The ion channel function of ORF3a is vital for activating the NLRP3 inflammasome, mitochondrial ROS production, and K + efflux during active IL-1β secretion ( Chen et al., 2019 ). On the other hand, E proteins can alter the permeability of Ca 2+ in the plasma membrane to activate the inflammasome. To be specific, Ca 2+ leakage via the E protein ion channels and increased cytoplasmic Ca 2+ levels may activate the NLRP3 inflammasome in the endoplasmic reticulum–Golgi intermediate compartment/Golgi membranes ( Nieto-Torres et al., 2015 ). In addition, the activation of the inflammasome involves other proteins. In macrophages, ORF8b directly interacts with the LRR domain of NLRP3 and co-localizes with NLRP3 and ASC in the cytosol for activation ( Shi et al., 2019 ). 2.1.1.2 SARS-CoV-2 virus infection and NLRP3 inflammasome Some patients with severe COVID-19 experience a cytokine storm and exhibit inflammasome activation; this increases the amount of active IL-1β and IL-18 in the lungs, cerebrospinal fluid, and serum ( Conti et al., 2020 ; Guasp et al., 2022 ). The mechanism of activation of the NLRP3 inflammasome by SARS-CoV-2 infection is depicted in Figure 2 . Figure 2 Schematic diagram of the mechanism by which SARS-CoV-2 activates the NLRP3 inflammasome. SARS-CoV-2 infection may activate the NLRP3 inflammasome in the following ways: (1) one of the subunits of the spike glycoprotein S1 can release proinflammatory cytokines via mechanisms involving the activation of the NF-κB pathway. (2) The E protein of SARS-CoV-2 can cause lysosomal damage to release a large amount of Ca 2+ , thereby activating the NLRP3 inflammasome. (3) The N viral protein interact with NLRP3 to promote inflammasome assembly; the complement cascade induced by the N protein–MBL-MASP2 axis may lead to the activation of the NLRP3 inflammasome via the different functions of C3a, C5a, and membrane attack complex (MAC). (4) ORF3a primes the inflammasome via NF-κB-mediated transcriptional activation of pro-IL-1β. Furthermore, ORF3a activates the NLRP3 inflammasome via K + efflux and NEK7. The activated NLRP3 inflammasome can activate transcription of pro-IL-1β and pro-IL-18 and produce mature IL-1β and IL-18. When infected with SARS-CoV-2, the NF-κB pathway is activated, leading to the increased expression and production of NLRP3 and active IL-1β. For instance, the SARS-CoV-2 spike glycoprotein S1 activates NF-κB, p38 MAPK, and the NLRP3 inflammasome, leading to the release of proinflammatory cytokines in peripheral blood mononuclear cells ( Olajide et al., 2021 ). Furthermore, ORF3a of SARS-CoV-2 can activate the NLRP3 inflammasome by triggering pro-IL-1β transcription via the NF-κB pathway ( Xu et al., 2022 ). SARS-CoV-2 can also regulate NLRP3 through the second activation phase. Furthermore, ORF3a can use NEK7 and K + efflux to trigger the NLRP3 inflammasome activation, resulting in the conversion of IL-1β precursors into mature IL-1β ( Xu et al., 2022 ). Studies have revealed that the N proteins of SARS-CoV-2 interact with the inflammasome pathway to induce the transcription of NLRP3, pro-IL-1β and pro-IL-18 and to promote inflammasome assembly leading to active IL-18 and IL-1β release and accelerating inflammation ( Figure 2 ) ( Pan et al., 2021 ). Furthermore, some studies have revealed that the N protein of SARS-CoV-2 may activate NLRP3 by initiating the complement cascade pathway by forming the Mannan-binding lectin (MBL; alias: mannose-binding lectin) and MBL-associated serine protease 2 (MBL-MASP-2) complex. The complement cascade induced by N protein-MBL-MASP-2 may activate the NLRP3 inflammasome via the different functions of C3a, C5a, and membrane attack complex (MAC) ( Asgari et al., 2013 ; Sluyter, 2017 ; Magro et al., 2020 ; Ratajczak and Kucia, 2020 ; Zhao et al., 2021 ). The insertion of MAC triggers Ca 2+ influx and increases Ca 2+ levels in the cytoplasm, thereby inducing mitochondrial damage and activating the NLRP3 inflammasome ( Triantafilou et al., 2013a ). 2.1.2 Flavivirus Flaviviruses remain a major public health concern worldwide. Dengue virus (DENV) and Zika virus (ZIKV) are two important pathogens in the genus Flavivirus , which comprise enveloped, positive, and approximately 11 kb single-stranded RNA ( Xie et al., 2021 ). The encoded protein comprises three structural proteins: capsid (C), pre-membrane (prM), and envelope (E) in the N-terminal region and seven nonstructural proteins (i.e., NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) in the C-terminal region ( Chambers et al., 1990 ; Javed et al., 2018 ; Ngono and Shresta, 2018 ; Xie et al., 2021 ). The viral glycoprotein E binds to cell receptors and initiates endocytosis-mediated internalization. After the process of virus uncoating in the cytoplasm, the genomic RNA undergoes subsequent replication and transcription to produce new viral RNA and proteins. Finally, they are assembled into new virus particles and released ( Mukhopadhyay et al., 2005 ; Barrows et al., 2018 ; Diosa-Toro et al., 2020 ; van Leur et al., 2021 ). There are four primary dengue virus serotypes of DENV: DENV-1 to 4; they cause mild fever to severe dengue hemorrhagic fever or dengue shock syndrome, which is potentially life-threatening ( Diamond and Pierson, 2015 ; Mustafa et al., 2015 ; Harapan et al., 2020 ). On the other hand, ZIKV infection can result in considerable neurological damage, including congenital Zika virus syndrome ( Cao-Lormeau et al., 2016 ; Polonio and Peron, 2021 ). 2.1.2.1 DENV virus infection and NLRP3 inflammasome Dengue fever may occur because of a cytokine storm ( de-Oliveira-Pinto et al., 2012 ). Patients with severe dengue have higher active IL-1β levels in their blood and gene expression profiles; this indicates its role in disease severity in clinical settings ( Bozza et al., 2008 ; Pan et al., 2019b ). Active IL-1β potentially recruits neutrophils to the inflammation site to help to clear DENV infection ( Niu et al., 2019 ). During host cell infection with DENV, first, signal 1 is activated: cytoplasmic pattern recognition receptors sense DENV viral RNA and trigger the NF-κB signaling pathway, thereby upregulating the transcription of pro-caspase-1, the NLRP3 inflammasome, pro-IL-1β, and pro-IL-18 ( Shrivastava et al., 2020a ). Many DENV viral proteins exert a strong effect on NLRP3 inflammasome activation and induce host immune responses. For example, in THP-1 cells (human monocyte leukemia cell line), the EDIII domain of E protein activates the NLRP3 inflammasome via the NF-κB pathway, resulting in the transcription of pro-IL-1β and TNF-α ( Khan et al., 2019 ). DENV can also regulate NLRP3 via the second activation phase. For example, EDIII can promote IL-1β secretion by inducing ROS generation and potassium efflux in the second step leading to the activation of the NLRP3 inflammasome and caspase-1-mediated maturation of pro-IL-1β ( Khan et al., 2019 ). Further understanding of the role of EDIII in regulating inflammatory responses will help to understand the pathogenesis of DENV infections. Furthermore, the nonstructural proteins NS2A and NS2B of DENV can trigger the activation of the NLRP3 inflammasome via ROS and Ca 2+ influx ( Shrivastava et al., 2020b ). The M protein of DENV is important in the innate immune response of the host. The interaction of M protein with NLRP3 suggests its crucial role in inflammasome activation, active IL-1β release, and subsequent pathogenic responses ( Pan et al., 2019a ). Therefore, DENV viral proteins are crucial in regulating the innate inflammatory responses of the host. 2.1.2.2 ZIKV virus infection and NLRP3 inflammasome A study has reported that the levels of proinflammatory cytokines such as IL-1β, interferon-γ, and IL-8 are increased in patients with ZIKV infection ( Tappe et al., 2016 ). ZIKV infection can increase the transcription of proinflammatory cytokines such as IL-1β and IL-6 by activating the NF-κB signaling pathway ( Gim et al., 2019 ). Furthermore, ZIKV can regulate NLRP3 via the second activation phase. The NS5 protein plays a vital role in NLRP3 inflammasome activation ( Figure 3 ). Wang et al. have suggested that the NS5 protein of ZIKV directly binds to the NLRP3 inflammasome for activation. NS5 promotes the assembly of the NLRP3–ASC inflammasome complex, resulting in pro-caspase-1 cleavage and secretion of active IL-1β ( Wang et al., 2018 ). Furthermore, He et al. have discovered a novel role of the NS5 protein of ZIKV in regulating NLRP3 activation. NS5 induces ROS production and directly binds to NLRP3 to stimulate the assembly of the NLRP3 inflammasome complex triggering the maturation of pro-IL-1β ( He et al., 2018 ; Wang et al., 2018 ). In addition, ZIKV results in oxidative stress in glial cells, resulting in NLRP3 inflammasome activation and mature IL-1β release for cell death ( Tricarico et al., 2017 ). Figure 3 Schematic diagram of the mechanism by which Zika virus activates the NLRP3 inflammasome. After the Zika virus invades and infects the cells, it activates the NF-κB pathway, inducing gene expression to produce pro-IL-1β and NLRP3. (1) The NS5 protein interacts with NLRP3 to promote the assembly of the NLRP3 inflammasome. The induced mitochondrial ROS leads to pro-caspase-1 cleavage and IL-1β release (2): The NS3 protein inhibits the activation of the NLRP3 inflammasome by cleaving NLRP3, decreasing caspase-1 activation and mature IL-1β secretion. In contrast, ZIKV NS3 protein can prevent the activation of NLRP3 inflammasome. ZIKV NS3 protein expression can decrease the extent of caspase-1 activation and active IL-1β secretion in macrophages, whereas its overexpression in 293T cells can lead to NLRP3 degradation ( Gim et al., 2019 ). 2.1.3 Enterovirus Enterovirus 71 (EV71) belongs to the family Picornaviridae and is a positive-sense single-stranded RNA virus with a genomic length of 7.4 kb ( Solomon et al., 2010 ; Wen et al., 2020 ). EV71 has four structural proteins, namely, VP1, VP2, VP3, and VP4, that are primarily involved in virus particle formation and virus binding to host cells. On the other hand, the nonstructural proteins 2A, 2B, 2C, 3A, 3B, 3C, and 3D are vital for virus infection, replication, and innate immune responses ( Solomon et al., 2010 ; Wen et al., 2020 ). EV71 infection begins with receptor interaction. Then, it enters the cells via endocytosis and undergoes early uncoating. The viral RNA undergoes replication and transcription to generate new viral RNA and proteins. The resulting positive-sense viral RNA is loaded into the procapsid, eventually maturing into infectious virus particles ( Solomon et al., 2010 ; Tan et al., 2014 ). EV71 has caused several epidemics worldwide. It can induce hand-foot-mouth disease, neonatal sepsis, meningoencephalitis, and pulmonary edema in children as well as other severe systemic disorders ( McMinn, 2002 ; Ooi et al., 2010 ). 2.1.3.1 EV71 virus infection and NLRP3 inflammasome The infection of cells by EV71 can activate the NLRP3 inflammasome, which then causes the creation and release of proinflammatory cytokines including active IL-1β. This activation of the NLRP3 inflammasome may be involved in the antiviral defense mechanism of the host against EV71 infection ( Wang et al., 2015 ). EV71 can activate the NLRP3 inflammasome in the first step. EV71 uses cellular vimentin (VIM) to activate extracellular regulatory protein kinase (ERK) and translocate NF-κB. Ultimately, ERK phosphorylation triggers the activation of the NF-κB signaling pathway, which leads to the activation of the NLRP3 inflammasome and the transcription of IL-1β precursors. Therefore, Gong et al. concluded that EV71 activates the NLRP3 inflammasome via the VIM-ERK-NF-κB pathway ( Gong et al., 2022 ). EV71 can also regulate NLRP3 via a second activation phase. The nonstructural protein 3D of the virus plays a role in activating NLRP3 inflammasome-mediated transcription of pro-IL-1β. The 3D protein functions as an RNA-dependent RNA polymerase in EV71 and directly interact with the LRR and NACHT domains of NLRP3 to trigger the formation of the 3D–NLRP3–ASC inflammasome complex ( Wang et al., 2017 ; You et al., 2020 ). Recent studies have reported that methylation of adenosine in RNA molecules generating N6-methyladenosine (m6A RNA) plays an important role in regulating the activation of the NLRP3 inflammasome during EV71 infection. The depletion of fat mass and obesity-associated protein can enhance the mRNA stability and expression of thioredoxin-interacting protein (TXNIP), thereby enhancing ROS production and NLRP3 inflammasome activation ( Qayyum et al., 2021 ; Hu et al., 2023 ). On the other hand, the EV71 viral proteins has a role in inhibition of NLRP3 activation. The nonstructural proteins 2A and 3C of EV71 inhibit NLRP3 inflammasome activation by lysing it ( Wang et al., 2017 ). 2A and 3C can cleave NLRP3 at the G493–L494 and Q225–G226 junction, respectively, to prevent inflammasome activation. Furthermore, the 3C protein of EV71 interacts with NLRP3 and inhibits the secretion of active IL-1β in mammalian cells ( Wang et al., 2015 ; Xiao et al., 2019 ). In summary, EV71 virus proteins exhibit dual roles in NLRP3 interaction. 2.1.4 Retrovirus Retroviruses comprise positive-sense single-stranded RNA genomes that infect cells via reverse transcription and genomic integration ( Hu and Hughes, 2012 ; Zhang et al., 2018 ). The characteristic of retroviruses is that the virus-encoded reverse transcriptase reversely transcribes the viral RNA into DNA and integrates it into the chromosomes of host cells. Subsequently, all structural and regulatory proteins are transcribed and translated ( Zhang et al., 2018 ). After viral protein and genome assembly, new virions bud out ( Krebs et al., 2021 ). HIV, a well-known retrovirus, has considerably affected humanity since the discovery of acquired immunodeficiency syndrome in the early 1980s ( Lucas and Nelson, 2015 ). HIV infection severely damages the immune system, and the virus targets critical CD4 T lymphocytes, resulting in severe cell damage and impairing immune function ( Fanales-Belasio et al., 2010 ; Doitsh et al., 2014 ). HIV-1 and HIV-2 are two types of HIV. HIV-1 accounts for most infections. On the other hand, HIV-2, originating from West Africa, is less prevalent and less virulent than HIV-1 ( Sharp and Hahn, 2011 ; Visseaux et al., 2019 ). 2.1.4.1 HIV virus infection and NLRP3 inflammasome HIV can interact with the NLRP3 inflammasome. Furthermore, HIV infection can trigger NLRP3 inflammasome activation, leading to the production and release of proinflammatory cytokines, including IL-1β ( Reis et al., 2019 ; Zhang et al., 2019 ). A previous study has suggested that monocytes from HIV-1-infected individuals produce more IL-1β than those from noninfected individuals, indicating that HIV-1 triggers NLRP3 inflammasome activation ( Jalbert et al., 2013 ). HIV-1 can activate the NLRP3 inflammasome via the first activation step. HIV-1 activates the NLRP3 inflammasome in primary human monocyte-derived macrophages via the NF-κB pathway, promoting IL-1β secretion by increasing the amount of its precursor in the cell ( Hernandez et al., 2014 ). Furthermore, HIV viral proteins can activate the NLRP3 inflammasome. For example, when it comes to HIV-1 replication and infection, viral protein R (Vpr) plays a crucial role as an accessory protein. Vpr can induce the activation of proinflammatory markers such as TNF-α and the NF-κB signaling pathway ( Li et al., 2020 ). In addition, the HIV-1 transactivator of transcription (Tat) protein can prime the NLRP3 inflammasome by activating NF-κB. Subsequently, the interaction the interaction of the Tat protein with the inflammasome leads to caspase-1 maturation and IL-1β release ( Demarchi et al., 1996 ; Chivero et al., 2017 ). HIV can also regulate NLRP3 via a second activation phase. Tat can induce inflammasome activation by regulating Ca 2+ flux ( Benelli et al., 2000 ; Lee et al., 2012 ). HIV-1 RNA can be recognized by the pattern recognition receptor protein kinase RNA-activated (PKR), triggering the activation of the NLRP3 inflammasome by inducing ROS generation and activating the MAP kinases ERK1/2, JNK, and p38 ( Stunnenberg et al., 2021 ). Furthermore, during the virus attachment step, the HIV-1 envelope glycoprotein causes extracellular ATP and K + efflux by associating with PNX1, leading to NLRP3 activation ( Séror et al., 2011 ; Paoletti et al., 2019 ; He et al., 2020 ). 2.2 Negative-sense single-stranded RNA virus 2.2.1 Influenza virus At present, influenza is an infectious disease worldwide and a serious global health concern; approximately 3 million to 5 million severe cases and 290,000–650,000 deaths are reported each year ( Kim et al., 2022 ). Influenza viruses belong to the family Orthomyxoviridae and contain a negative-sense single-stranded RNA genome ( Bouvier and Palese, 2008 ; Te Velthuis and Fodor, 2016 ). Human influenza viruses come in three different types: Influenza A, B, and C viruses (IAV, IBV, and ICV); they are the pathogens of the human respiratory disease influenza ( Blut and Hemotherapy, 2009 ; Hutchinson, 2018 ). IAV and IBV have caused significant morbidity and mortality worldwide as well as an economic burden; in contrast, ICV causes a mild respiratory disease that rarely generates epidemics, mostly in children ( Te Velthuis and Fodor, 2016 ). The virus binds to the cell surface receptors containing sialic acid and enters the cell via endocytosis. The viral core is released into the nucleus, where the viral genome is transcribed into mRNA and subsequently translated into viral proteins. Thereafter, the viral proteins and newly synthesized RNA assemble into new virus particles. After the maturation of the virus particles, they are released via membrane fusion or penetration ( Watanabe et al., 2010 ; Te Velthuis and Fodor, 2016 ). 2.2.1.1 Influenza virus infection and NLRP3 inflammasome Influenza often triggers inflammatory responses in the immune system of humans ( Kuriakose and Kanneganti, 2017 ). During influenza, host immune cells release cytokines such as IL-1β and IL-6 in response to virus invasion. Among these cytokines, IL-1β is a vital proinflammatory cytokine that regulates immune and inflammatory responses. Virus components may function as signals that activate the NLRP3 inflammasome, the maturation of pro-IL-1β into their active forms ( Allen et al., 2009 ; Tate and Mansell, 2018 ). Influenza can regulate NLRP3 via the first step. The viral nucleoprotein (NP) can stimulate neighboring cells via TLR2 and TLR4 activating the NF-κB pathway and thereby inducing the production of pro-IL-1β and IL-6; this subsequently leads to the production of trypsin which can increase the infectivity of influenza virus ( Kim et al., 2022 ). For the priming associated with NLRP3 inflammasome activation, TLRs and retinoic acid-inducible gene I (RIG-I) are critical. Influenza viral RNA can indirectly promote inflammasome assembly and the release of inflammasome-dependent cytokines by interacting with known RNA sensors such as TLR-7 and RIG-I ( Ichinohe et al., 2009 ; Kuriakose and Kanneganti, 2017 ). After influenza virus invasion, viral ribonucleoproteins are released into the nucleus for transcription and replication. SsRNA is recognized by TLR7 and triggers the expression of NF-κB, pro-IL-1β, pro-IL-18, and other pro-inflammatory cytokines ( Niu and Meng, 2023 ). Studies have shown a lack of IL-1β release by infecting influenza virus into TLR7-deficient bone marrow-derived DCs ( Ichinohe et al., 2010 ). In addition, when the viral genome reaches the cytoplasm, the 5α-triphosphate dsRNA of influenza A virus can activate RIG-I, which is involved in NF-κB activation and pro-IL-18 and pro-IL-1β transcription ( Cui et al., 2008 ). Influenza can also regulate NLRP3 via a second activation phase. The M2 protein of the influenza virus is a proton-selective ion channel that is important in NLRP3 inflammasome activation. The M2 protein can activate the NLRP3 inflammasome in macrophages and dendritic cells and plays a role in viral pathogenesis ( Ichinohe et al., 2010 ). The M2 protein promotes inflammasome activation by locating the acidified Golgi apparatus, promoting proton outflow, causing ion imbalance, causing K efflux binding with Na efflux, and producing ROS, thereby activating the NLRP3 inflammasome. Furthermore, it triggers the inflammasome by modulating ion flux or stimulating mitochondrial DNA release into the cytoplasm ( Ichinohe et al., 2010 ; Pang and Iwasaki, 2011 ; Moriyama et al., 2019 ). Moreover, a study has reported that NLRP3 can be recruited into dispersed Trans-Golgi-Network (TGN), where it oligomerizes, alters the conformation, and recruits ASC to activate the inflammasome ( Pandey and Zhou, 2022 ). The PB1-F2 protein of IAV activates NLRP3 via various mechanisms. The PB1-F2 protein is translocated into the mitochondrial intima through Tom40 channel; its accumulation decreases the potential of the mitochondrial intima (Δφm), accelerates mitochondrial fragmentation, and activates the NLRP3 inflammasome ( Yoshizumi et al., 2014 ). The aggregated form of the C-terminal region of PB1-F2 protein can activate NLRP3 via mitochondrial autophagy, promoting mitochondrial ROS production and mitochondrial DNA release ( McAuley et al., 2013 ; Wang et al., 2021 ). Furthermore, IAV RNA activates the NLRP3 inflammasome. The activation of the NLRP3 inflammasome by influenza virus RNA depends on lysosomal maturation and ROS production ( Allen et al., 2009 ). However, the NLRP3 inflammasome's activation can also be inhibited by influenza viruses. For example, IAVs can inhibit NLRP3 inflammasome activation via viral proteins. The NS1 protein of IAV interacts with NLRP3 to inhibit single-speck formation required for NLRP3 and ASC-induced inflammasome activation; this results in decreased secretion of active IL-1β ( Moriyama et al., 2016 ). Chung et al. have reported that NS1 overexpression can significantly disrupt the transcription of proinflammatory cytokines by inhibiting the activation of NF-κB. Furthermore, inflammasome NLRP3 activation is inhibited in NS1-expressing THP-1 cells ( Chung et al., 2015 ). 2.2.2 Ebola virus Ebola virus (EBOV) is one of the deadly zoonotic epidemic viruses that can cause fatal systemic disease ( Marcinkiewicz et al., 2014 ; Malvy et al., 2019 ). The Ebola virus genome is composed of single negative strand RNA; the genome size is about 19kb; and it belongs to the filoviridae family ( Baseler et al., 2017 ). The main characteristics of patients are high fever, fatigue, body aches, gastrointestinal symptoms, abnormal inflammatory response, immune suppression, large fluid and electrolyte loss, and high mortality ( De Clercq, 2015 ; Malvy et al., 2019 ). When a virus infects, the virus first binds to the host cell membrane, triggering endocytosis of the virus particles. The particle envelope fuses with the endosomal membrane, thereby releasing the ribonucleoprotein complex into the cytoplasm. In the cytoplasm the viral genome is replicated and transcribed into mRNA. The viral proteins are then translated into the cytoplasm and into the endoplasmic reticulum. Mature daughter ribonucleoprotein complexes and viral proteins are transported to the plasma membrane. Finally, mature virions are released in the form of budding ( Sakurai, 2015 ; Baseler et al., 2017 ; Jacob et al., 2020 ). 2.2.2.1 Ebola virus infection and NLRP3 inflammasome The immune response and inflammatory cascade of the innate immune system are key factors in the pathogenesis and mortality of EBOV ( Leroy et al., 2001 ). The release of proinflammatory cytokines IL-1β, TNF-α, and IL-5 was found in asymptomatic individuals ( Leroy et al., 2001 ). In addition, when EBOV infects monocytes, it also leads to the maturation and secretion of the pro-inflammatory cytokine IL-1β ( Ströher et al., 2001 ). It has been reported that Ebola virus-like particles stimulate the expression of type I interferon and proinflammatory cytokines through Toll-like receptors and interferon signaling pathways ( Ayithan et al., 2014 ). Halfmann et al. found that EBOV stimulates the secretion of proinflammatory cytokines IL-1β and IL-18 by activating the NLRP3 inflammasome in a caspase 1-dependent manner ( Halfmann et al., 2018 ). However, more research is still needed to study how EBOV activates the NLRP3 inflammasome to trigger the release of pro-inflammatory cytokines like IL-1β and the downstream role of IL-1β signal transduction. 2.2.3 Respiratory syncytial virus Respiratory syncytial virus (RSV) infection is a constant public health problem, and the impact on children, the elderly, and immunocompromised patients can be very significant ( Yang et al., 2023 ). RSV is an enveloped, negatively-sense single-stranded RNA virus belonging to the Paramyxoviridae family ( Collins et al., 2013 ). RSV has eight known structural proteins. Fusion protein (F), attached glycoprotein (G), small hydrophobic protein (SH), matrix protein (M), nucleocapsid protein (N), large protein (L), phosphoprotein (P), and M2 gene product M2-1, as well as two non-structural proteins (NS1 and NS1) ( Borchers et al., 2013 ; Griffiths et al., 2017 ; Agac et al., 2023 ). The virus is mainly transmitted through close contact with saliva or mucus droplets. Common symptoms include fever, runny nose, cough, and chest tightness ( Linder and Malani, 2017 ). RSV first binds to receptors on the surface of the host cell through endocytosis, or the viral F protein on its surface, and then the virus fuses with the cell membrane to enter the host cell. Viral RNA is released into the cytoplasm of the host cell. In host cells, viral RNA acts as a transcription template, producing mRNA. The mRNA is then translated into viral proteins. The newly synthesized viral protein and the replicating RNA are assembled into new viral particles, which bud out from the infected cell to complete the viral life cycle ( Shang et al., 2021 ). 2.2.3.1 Respiratory syncytial virus infection and NLRP3 inflammasome Activation of the inflammasome pathway also plays an important role in RSV infection. Excessive inflammatory responses can lead to the release of pro-inflammatory cytokines, including IL-1β and IL-18, leading to lung inflammation and damage ( Shen et al., 2018 ). Studies have shown that NLRP3, ASC, and caspase-1 are critical for IL-1β production during RSV infection ( Segovia et al., 2012 ; Shen et al., 2018 ). RSV can regulate NLRP3 via the first step. RSV activates NF-κB during infection ( Sabbah et al., 2009 ). NF-κB signaling has been shown to be critical for NLRP3 expression during RSV infection ( Shen et al., 2019 ). TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection ( Segovia et al., 2012 ). After the administration of NF-κB inhibitors in RSV-infected cells, the production of IL-1β decreased significantly ( Segovia et al., 2012 ). Respiratory syncytial virus can also regulate NLRP3 via a second activation phase. During RSV infection, activation of the NLRP3 inflammasome is dependent on K efflux and ROS production, followed by caspase-1-mediated maturation and secretion of IL-1β ( Segovia et al., 2012 ; Yang et al., 2023 ). It has been shown that RSV SH viroporin induces membrane permeability to ions or small molecules that are essential for triggering the NLRP3 inflammasome. After RSV infection, RSV SH virus channel proteins accumulate in the Golgi within the lipid raft structure, possibly forming ion channels that trigger the translocation of NLRP3 from cytoplasm to Golgi apparatus. This leads to NLRP3 inflammasome activation as well as pro-IL1β transcriptional activation ( Triantafilou et al., 2013b ). It has also been reported that orosomucoid 1-like protein 3 (ORMDL3) can inhibit calcium pump function, resulting in increased calcium levels in the cytoplasm and decreased ER levels, thereby inducing ER stress ( Cantero-Recasens et al., 2010 ). RSV may induce NLRP3 inflammasome expression by activating ORMDL3 overexpression ( Cheng et al., 2023 ). 2.2.4 Rift Valley Fever virus Rift Valley Fever virus (RVFV) is a negative sense segmented single-stranded RNA virus with a size of about 12kb that belongs to the family Phenuiviridae and the genus Phlebovirus ( Ahsan et al., 2016 ; Gaudreault et al., 2019 ). In humans, RVFV can cause a variety of disease manifestations, ranging from febrile illness to hemorrhagic fever and death ( Ermler et al., 2014 ). The viral genome has three segments: the negative sense L (large) and negative sense M (medium) segments, and the double sense S (small) segment. These three segments encode multifunctional proteins. The S segment encodes nuclear protein (N) and non-structural protein S (NSs), the M segment encodes viral glycoprotein (Gn and Gc) and non-structural protein (NSm and a 78-kDa protein), and the L segment encodes viral RNA-dependent RNA polymerase ( Baer et al., 2016 ; Wright et al., 2019 ; Tercero et al., 2021 ; Lean and Johnson, 2022 ; Ganaie et al., 2023 ). The virus first attaches to the host membrane and enters the host cell through endocytosis. The viral genome is released into the cytoplasm. Negative RNA is transcribed into positive mRNA encoding viral proteins, and positive RNA serves as a template for the synthesis of new negative RNA. The newly synthesized viral protein and the replicated genome are combined in the cytoplasm to form new viral particles, which are released when they mature ( Wright et al., 2019 ). 2.2.4.1 Rift Valley Fever virus infection and NLRP3 inflammasome RVFV infection induces strong cytokines, which are essential for the recruitment of innate immune cells to the site of infection ( Nair et al., 2023 ). RVFV infected-cells secrete IL-1β, which is involved in the NLRP3 inflammasome, ASC oligomerization, and caspase-1 maturation. It has been reported that RVFV activates the NLRP3 inflammasome by inducing the formation of an inflammasome complex containing NLRP3 and MAVS, where MAVS are localized to NLRP3 during RVFV infection, leading to the maturation and secretion of IL-1β ( Ermler et al., 2014 ). 2.2.5 Hantavirus Hantavirus (HTNV) is a coated single-stranded negative sense RNA virus belonging to the Bunyaviridae family ( Stock, 2008 ). The HTNV genome consists of three single-stranded negative sense RNA fragments: small (S), medium (M), and large (L) genome fragments encode four structural proteins (nuclear proteins N, glycoproteins Gn and Gc, and L proteins) ( Muyangwa et al., 2015 ; Meier et al., 2021 ). HTNV can cause acute febrile illness in humans. Hemorrhagic Fever with Renal Syndrome (HFRS) caused in Asia and Europe, and Hantavirus Pulmonary Syndrome (HPS) caused in the Americas ( Sargianou et al., 2012 ). Once Hantavirus enters the body, the virions bind to cell surface membrane receptors and enter the cell through endocytosis. The specific mechanism involves the transcription of the viral genome and the synthesis of viral RNA and viral proteins. The newly synthesized vRNA is coated with N protein to form ribonucleoprotein, which is then sent to the perinuclear membrane system. The synthesized viral proteins and genome are assembled into new viral particles inside the host cell. When the virion matures, it is released ( Muyangwa et al., 2015 ; Meier et al., 2021 ). 2.2.5.1 Hantavirus infection and NLRP3 inflammasome HTNV infection causes cells to enter a stress condition and induces the production of inflammatory cytokines ( Zhang et al., 2021 ). Studies have found that IL-1β is significantly elevated during HFRS. Induction of the human monocyte line THP-1 by HTNV revealed the secretion of IL-1β. The specific mechanism found that the induction of IL-1β by HTNV depended on the activation of caspase-1. Hantavirus thus induces the formation of the NLRP3 inflammasome in THP-1 cells, which may be an important factor in IL-1β levels in patients with HFRS ( Ye et al., 2015 ). 2.3 Double-stranded RNA virus 2.3.1 Reovirus DsRNA viruses have complementary dsRNA. This virus family displays two distinctive features: (1) their virus genome typically comprises 10-12 double-stranded RNA segments, and (2) the virus contains a double capsid structure but lacks an envelope ( Danthi et al., 2013 ). dsRNA viruses form a large group of RNA disease viruses, including reoviruses. Reoviruses comprise two concentric protein shells (the outer capsid and the core) that contain 10 segments of the dsRNA genome ( Ahlquist, 2006 ; Abad and Danthi, 2020 ). The infectious life cycle of reovirus starts with the attachment of the viral protein σ1 to sialic acid residues on the target cell surface. Alternatively, proteolysis by extracellular proteases leads to the formation of infectious subvirion particles; these particles directly enter the cell via membrane penetration. Thereafter, transcriptionally active virus core particles are released into the cytoplasm. After virus replication and assembly, mature virus particles are released ( Comins et al., 2008 ; Gong and Mita, 2014 ; Lemay, 2018 ; Abad and Danthi, 2020 ). 2.3.1.1 Reovirus virus infection and NLRP3 inflammasome Reoviruses use the host protein EphA2 to counteract the activation of the NLRP3 inflammasome ( Zhang et al., 2020 ). Zhang et al. have reported that reovirus infection of airway epithelial cells increases EPHA2-dependent NLRP3 phosphorylation; this inhibits the activation of the inflammasome by inhibiting the recruitment of other inflammasome components. Upon virus infection, EphA2 −/− mice exhibited increased inflammatory infiltration, resulting in the secretion of active IL-1β and IL-18 ( Zhang and Ricci, 2020 ; Zhang et al., 2020 ). 2.1 Positive-sense single-stranded RNA virus 2.1.1 Human coronaviruses Coronaviruses belong to the family Coronaviridae and comprise a positive-sense single-stranded RNA that is 27–32 kb long ( Van Der Hoek et al., 2004 ; Lu et al., 2020 ). Both severe acute respiratory syndrome coronavirus (SARS-CoV) and novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are types of human coronaviruses ( Hasöksüz et al., 2020 ). The genomes of SARS-CoV and SARS-CoV-2 encode four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The RNA genome is encapsulated by the N proteins. The M and E proteins ensure that the N proteins can be incorporated into the virus particles during assembly, whereas the S proteins provide specificity for cellular entry receptors ( Alanagreh et al., 2020 ; Kim et al., 2020 ; Liu et al., 2020a ). After binding to specific receptors such as angiotensin-converting enzyme 2, coronaviruses can either directly fuse with the cell surface or be engulfed by the endosomes, thereby entering the cell. Then, the viral N protein enters the cytoplasm, releasing the RNA genome of the virus into the cell. It synthesizes new virus RNA and proteins and assembles them into virus particles for release ( Fehr and Perlman, 2015 ; Hartenian et al., 2020 ; Prydz and Saraste, 2022 ). 2.1.1.1 SARS-CoV virus infection and NLRP3 inflammasome SARS-CoV-encoded proteins can activate the NLRP3 inflammasome via the NF-κB pathway. For example, ORF3a and E proteins play vital roles in activating the NLRP3 inflammasome. ORF3a and E proteins of SARS-CoV can activate NF-κB, resulting in the transcription of pro-IL-1β ( DeDiego et al., 2014 ; Siu et al., 2019 ). Furthermore, ORF3a promotes the ubiquitination of p105 and ASC via TNFR-associated factors, promoting the activation of NF-κB and the NLRP3 inflammasome ( Siu et al., 2019 ). ORF3a and E proteins of SARS-CoV play roles in the second activation step. ORF3a can lead to lysosomal dysfunction in the host cells and initiate caspase-1 activation either directly or via increased K + efflux. The ion channel function of ORF3a is vital for activating the NLRP3 inflammasome, mitochondrial ROS production, and K + efflux during active IL-1β secretion ( Chen et al., 2019 ). On the other hand, E proteins can alter the permeability of Ca 2+ in the plasma membrane to activate the inflammasome. To be specific, Ca 2+ leakage via the E protein ion channels and increased cytoplasmic Ca 2+ levels may activate the NLRP3 inflammasome in the endoplasmic reticulum–Golgi intermediate compartment/Golgi membranes ( Nieto-Torres et al., 2015 ). In addition, the activation of the inflammasome involves other proteins. In macrophages, ORF8b directly interacts with the LRR domain of NLRP3 and co-localizes with NLRP3 and ASC in the cytosol for activation ( Shi et al., 2019 ). 2.1.1.2 SARS-CoV-2 virus infection and NLRP3 inflammasome Some patients with severe COVID-19 experience a cytokine storm and exhibit inflammasome activation; this increases the amount of active IL-1β and IL-18 in the lungs, cerebrospinal fluid, and serum ( Conti et al., 2020 ; Guasp et al., 2022 ). The mechanism of activation of the NLRP3 inflammasome by SARS-CoV-2 infection is depicted in Figure 2 . Figure 2 Schematic diagram of the mechanism by which SARS-CoV-2 activates the NLRP3 inflammasome. SARS-CoV-2 infection may activate the NLRP3 inflammasome in the following ways: (1) one of the subunits of the spike glycoprotein S1 can release proinflammatory cytokines via mechanisms involving the activation of the NF-κB pathway. (2) The E protein of SARS-CoV-2 can cause lysosomal damage to release a large amount of Ca 2+ , thereby activating the NLRP3 inflammasome. (3) The N viral protein interact with NLRP3 to promote inflammasome assembly; the complement cascade induced by the N protein–MBL-MASP2 axis may lead to the activation of the NLRP3 inflammasome via the different functions of C3a, C5a, and membrane attack complex (MAC). (4) ORF3a primes the inflammasome via NF-κB-mediated transcriptional activation of pro-IL-1β. Furthermore, ORF3a activates the NLRP3 inflammasome via K + efflux and NEK7. The activated NLRP3 inflammasome can activate transcription of pro-IL-1β and pro-IL-18 and produce mature IL-1β and IL-18. When infected with SARS-CoV-2, the NF-κB pathway is activated, leading to the increased expression and production of NLRP3 and active IL-1β. For instance, the SARS-CoV-2 spike glycoprotein S1 activates NF-κB, p38 MAPK, and the NLRP3 inflammasome, leading to the release of proinflammatory cytokines in peripheral blood mononuclear cells ( Olajide et al., 2021 ). Furthermore, ORF3a of SARS-CoV-2 can activate the NLRP3 inflammasome by triggering pro-IL-1β transcription via the NF-κB pathway ( Xu et al., 2022 ). SARS-CoV-2 can also regulate NLRP3 through the second activation phase. Furthermore, ORF3a can use NEK7 and K + efflux to trigger the NLRP3 inflammasome activation, resulting in the conversion of IL-1β precursors into mature IL-1β ( Xu et al., 2022 ). Studies have revealed that the N proteins of SARS-CoV-2 interact with the inflammasome pathway to induce the transcription of NLRP3, pro-IL-1β and pro-IL-18 and to promote inflammasome assembly leading to active IL-18 and IL-1β release and accelerating inflammation ( Figure 2 ) ( Pan et al., 2021 ). Furthermore, some studies have revealed that the N protein of SARS-CoV-2 may activate NLRP3 by initiating the complement cascade pathway by forming the Mannan-binding lectin (MBL; alias: mannose-binding lectin) and MBL-associated serine protease 2 (MBL-MASP-2) complex. The complement cascade induced by N protein-MBL-MASP-2 may activate the NLRP3 inflammasome via the different functions of C3a, C5a, and membrane attack complex (MAC) ( Asgari et al., 2013 ; Sluyter, 2017 ; Magro et al., 2020 ; Ratajczak and Kucia, 2020 ; Zhao et al., 2021 ). The insertion of MAC triggers Ca 2+ influx and increases Ca 2+ levels in the cytoplasm, thereby inducing mitochondrial damage and activating the NLRP3 inflammasome ( Triantafilou et al., 2013a ). 2.1.2 Flavivirus Flaviviruses remain a major public health concern worldwide. Dengue virus (DENV) and Zika virus (ZIKV) are two important pathogens in the genus Flavivirus , which comprise enveloped, positive, and approximately 11 kb single-stranded RNA ( Xie et al., 2021 ). The encoded protein comprises three structural proteins: capsid (C), pre-membrane (prM), and envelope (E) in the N-terminal region and seven nonstructural proteins (i.e., NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) in the C-terminal region ( Chambers et al., 1990 ; Javed et al., 2018 ; Ngono and Shresta, 2018 ; Xie et al., 2021 ). The viral glycoprotein E binds to cell receptors and initiates endocytosis-mediated internalization. After the process of virus uncoating in the cytoplasm, the genomic RNA undergoes subsequent replication and transcription to produce new viral RNA and proteins. Finally, they are assembled into new virus particles and released ( Mukhopadhyay et al., 2005 ; Barrows et al., 2018 ; Diosa-Toro et al., 2020 ; van Leur et al., 2021 ). There are four primary dengue virus serotypes of DENV: DENV-1 to 4; they cause mild fever to severe dengue hemorrhagic fever or dengue shock syndrome, which is potentially life-threatening ( Diamond and Pierson, 2015 ; Mustafa et al., 2015 ; Harapan et al., 2020 ). On the other hand, ZIKV infection can result in considerable neurological damage, including congenital Zika virus syndrome ( Cao-Lormeau et al., 2016 ; Polonio and Peron, 2021 ). 2.1.2.1 DENV virus infection and NLRP3 inflammasome Dengue fever may occur because of a cytokine storm ( de-Oliveira-Pinto et al., 2012 ). Patients with severe dengue have higher active IL-1β levels in their blood and gene expression profiles; this indicates its role in disease severity in clinical settings ( Bozza et al., 2008 ; Pan et al., 2019b ). Active IL-1β potentially recruits neutrophils to the inflammation site to help to clear DENV infection ( Niu et al., 2019 ). During host cell infection with DENV, first, signal 1 is activated: cytoplasmic pattern recognition receptors sense DENV viral RNA and trigger the NF-κB signaling pathway, thereby upregulating the transcription of pro-caspase-1, the NLRP3 inflammasome, pro-IL-1β, and pro-IL-18 ( Shrivastava et al., 2020a ). Many DENV viral proteins exert a strong effect on NLRP3 inflammasome activation and induce host immune responses. For example, in THP-1 cells (human monocyte leukemia cell line), the EDIII domain of E protein activates the NLRP3 inflammasome via the NF-κB pathway, resulting in the transcription of pro-IL-1β and TNF-α ( Khan et al., 2019 ). DENV can also regulate NLRP3 via the second activation phase. For example, EDIII can promote IL-1β secretion by inducing ROS generation and potassium efflux in the second step leading to the activation of the NLRP3 inflammasome and caspase-1-mediated maturation of pro-IL-1β ( Khan et al., 2019 ). Further understanding of the role of EDIII in regulating inflammatory responses will help to understand the pathogenesis of DENV infections. Furthermore, the nonstructural proteins NS2A and NS2B of DENV can trigger the activation of the NLRP3 inflammasome via ROS and Ca 2+ influx ( Shrivastava et al., 2020b ). The M protein of DENV is important in the innate immune response of the host. The interaction of M protein with NLRP3 suggests its crucial role in inflammasome activation, active IL-1β release, and subsequent pathogenic responses ( Pan et al., 2019a ). Therefore, DENV viral proteins are crucial in regulating the innate inflammatory responses of the host. 2.1.2.2 ZIKV virus infection and NLRP3 inflammasome A study has reported that the levels of proinflammatory cytokines such as IL-1β, interferon-γ, and IL-8 are increased in patients with ZIKV infection ( Tappe et al., 2016 ). ZIKV infection can increase the transcription of proinflammatory cytokines such as IL-1β and IL-6 by activating the NF-κB signaling pathway ( Gim et al., 2019 ). Furthermore, ZIKV can regulate NLRP3 via the second activation phase. The NS5 protein plays a vital role in NLRP3 inflammasome activation ( Figure 3 ). Wang et al. have suggested that the NS5 protein of ZIKV directly binds to the NLRP3 inflammasome for activation. NS5 promotes the assembly of the NLRP3–ASC inflammasome complex, resulting in pro-caspase-1 cleavage and secretion of active IL-1β ( Wang et al., 2018 ). Furthermore, He et al. have discovered a novel role of the NS5 protein of ZIKV in regulating NLRP3 activation. NS5 induces ROS production and directly binds to NLRP3 to stimulate the assembly of the NLRP3 inflammasome complex triggering the maturation of pro-IL-1β ( He et al., 2018 ; Wang et al., 2018 ). In addition, ZIKV results in oxidative stress in glial cells, resulting in NLRP3 inflammasome activation and mature IL-1β release for cell death ( Tricarico et al., 2017 ). Figure 3 Schematic diagram of the mechanism by which Zika virus activates the NLRP3 inflammasome. After the Zika virus invades and infects the cells, it activates the NF-κB pathway, inducing gene expression to produce pro-IL-1β and NLRP3. (1) The NS5 protein interacts with NLRP3 to promote the assembly of the NLRP3 inflammasome. The induced mitochondrial ROS leads to pro-caspase-1 cleavage and IL-1β release (2): The NS3 protein inhibits the activation of the NLRP3 inflammasome by cleaving NLRP3, decreasing caspase-1 activation and mature IL-1β secretion. In contrast, ZIKV NS3 protein can prevent the activation of NLRP3 inflammasome. ZIKV NS3 protein expression can decrease the extent of caspase-1 activation and active IL-1β secretion in macrophages, whereas its overexpression in 293T cells can lead to NLRP3 degradation ( Gim et al., 2019 ). 2.1.3 Enterovirus Enterovirus 71 (EV71) belongs to the family Picornaviridae and is a positive-sense single-stranded RNA virus with a genomic length of 7.4 kb ( Solomon et al., 2010 ; Wen et al., 2020 ). EV71 has four structural proteins, namely, VP1, VP2, VP3, and VP4, that are primarily involved in virus particle formation and virus binding to host cells. On the other hand, the nonstructural proteins 2A, 2B, 2C, 3A, 3B, 3C, and 3D are vital for virus infection, replication, and innate immune responses ( Solomon et al., 2010 ; Wen et al., 2020 ). EV71 infection begins with receptor interaction. Then, it enters the cells via endocytosis and undergoes early uncoating. The viral RNA undergoes replication and transcription to generate new viral RNA and proteins. The resulting positive-sense viral RNA is loaded into the procapsid, eventually maturing into infectious virus particles ( Solomon et al., 2010 ; Tan et al., 2014 ). EV71 has caused several epidemics worldwide. It can induce hand-foot-mouth disease, neonatal sepsis, meningoencephalitis, and pulmonary edema in children as well as other severe systemic disorders ( McMinn, 2002 ; Ooi et al., 2010 ). 2.1.3.1 EV71 virus infection and NLRP3 inflammasome The infection of cells by EV71 can activate the NLRP3 inflammasome, which then causes the creation and release of proinflammatory cytokines including active IL-1β. This activation of the NLRP3 inflammasome may be involved in the antiviral defense mechanism of the host against EV71 infection ( Wang et al., 2015 ). EV71 can activate the NLRP3 inflammasome in the first step. EV71 uses cellular vimentin (VIM) to activate extracellular regulatory protein kinase (ERK) and translocate NF-κB. Ultimately, ERK phosphorylation triggers the activation of the NF-κB signaling pathway, which leads to the activation of the NLRP3 inflammasome and the transcription of IL-1β precursors. Therefore, Gong et al. concluded that EV71 activates the NLRP3 inflammasome via the VIM-ERK-NF-κB pathway ( Gong et al., 2022 ). EV71 can also regulate NLRP3 via a second activation phase. The nonstructural protein 3D of the virus plays a role in activating NLRP3 inflammasome-mediated transcription of pro-IL-1β. The 3D protein functions as an RNA-dependent RNA polymerase in EV71 and directly interact with the LRR and NACHT domains of NLRP3 to trigger the formation of the 3D–NLRP3–ASC inflammasome complex ( Wang et al., 2017 ; You et al., 2020 ). Recent studies have reported that methylation of adenosine in RNA molecules generating N6-methyladenosine (m6A RNA) plays an important role in regulating the activation of the NLRP3 inflammasome during EV71 infection. The depletion of fat mass and obesity-associated protein can enhance the mRNA stability and expression of thioredoxin-interacting protein (TXNIP), thereby enhancing ROS production and NLRP3 inflammasome activation ( Qayyum et al., 2021 ; Hu et al., 2023 ). On the other hand, the EV71 viral proteins has a role in inhibition of NLRP3 activation. The nonstructural proteins 2A and 3C of EV71 inhibit NLRP3 inflammasome activation by lysing it ( Wang et al., 2017 ). 2A and 3C can cleave NLRP3 at the G493–L494 and Q225–G226 junction, respectively, to prevent inflammasome activation. Furthermore, the 3C protein of EV71 interacts with NLRP3 and inhibits the secretion of active IL-1β in mammalian cells ( Wang et al., 2015 ; Xiao et al., 2019 ). In summary, EV71 virus proteins exhibit dual roles in NLRP3 interaction. 2.1.4 Retrovirus Retroviruses comprise positive-sense single-stranded RNA genomes that infect cells via reverse transcription and genomic integration ( Hu and Hughes, 2012 ; Zhang et al., 2018 ). The characteristic of retroviruses is that the virus-encoded reverse transcriptase reversely transcribes the viral RNA into DNA and integrates it into the chromosomes of host cells. Subsequently, all structural and regulatory proteins are transcribed and translated ( Zhang et al., 2018 ). After viral protein and genome assembly, new virions bud out ( Krebs et al., 2021 ). HIV, a well-known retrovirus, has considerably affected humanity since the discovery of acquired immunodeficiency syndrome in the early 1980s ( Lucas and Nelson, 2015 ). HIV infection severely damages the immune system, and the virus targets critical CD4 T lymphocytes, resulting in severe cell damage and impairing immune function ( Fanales-Belasio et al., 2010 ; Doitsh et al., 2014 ). HIV-1 and HIV-2 are two types of HIV. HIV-1 accounts for most infections. On the other hand, HIV-2, originating from West Africa, is less prevalent and less virulent than HIV-1 ( Sharp and Hahn, 2011 ; Visseaux et al., 2019 ). 2.1.4.1 HIV virus infection and NLRP3 inflammasome HIV can interact with the NLRP3 inflammasome. Furthermore, HIV infection can trigger NLRP3 inflammasome activation, leading to the production and release of proinflammatory cytokines, including IL-1β ( Reis et al., 2019 ; Zhang et al., 2019 ). A previous study has suggested that monocytes from HIV-1-infected individuals produce more IL-1β than those from noninfected individuals, indicating that HIV-1 triggers NLRP3 inflammasome activation ( Jalbert et al., 2013 ). HIV-1 can activate the NLRP3 inflammasome via the first activation step. HIV-1 activates the NLRP3 inflammasome in primary human monocyte-derived macrophages via the NF-κB pathway, promoting IL-1β secretion by increasing the amount of its precursor in the cell ( Hernandez et al., 2014 ). Furthermore, HIV viral proteins can activate the NLRP3 inflammasome. For example, when it comes to HIV-1 replication and infection, viral protein R (Vpr) plays a crucial role as an accessory protein. Vpr can induce the activation of proinflammatory markers such as TNF-α and the NF-κB signaling pathway ( Li et al., 2020 ). In addition, the HIV-1 transactivator of transcription (Tat) protein can prime the NLRP3 inflammasome by activating NF-κB. Subsequently, the interaction the interaction of the Tat protein with the inflammasome leads to caspase-1 maturation and IL-1β release ( Demarchi et al., 1996 ; Chivero et al., 2017 ). HIV can also regulate NLRP3 via a second activation phase. Tat can induce inflammasome activation by regulating Ca 2+ flux ( Benelli et al., 2000 ; Lee et al., 2012 ). HIV-1 RNA can be recognized by the pattern recognition receptor protein kinase RNA-activated (PKR), triggering the activation of the NLRP3 inflammasome by inducing ROS generation and activating the MAP kinases ERK1/2, JNK, and p38 ( Stunnenberg et al., 2021 ). Furthermore, during the virus attachment step, the HIV-1 envelope glycoprotein causes extracellular ATP and K + efflux by associating with PNX1, leading to NLRP3 activation ( Séror et al., 2011 ; Paoletti et al., 2019 ; He et al., 2020 ). 2.1.1 Human coronaviruses Coronaviruses belong to the family Coronaviridae and comprise a positive-sense single-stranded RNA that is 27–32 kb long ( Van Der Hoek et al., 2004 ; Lu et al., 2020 ). Both severe acute respiratory syndrome coronavirus (SARS-CoV) and novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are types of human coronaviruses ( Hasöksüz et al., 2020 ). The genomes of SARS-CoV and SARS-CoV-2 encode four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The RNA genome is encapsulated by the N proteins. The M and E proteins ensure that the N proteins can be incorporated into the virus particles during assembly, whereas the S proteins provide specificity for cellular entry receptors ( Alanagreh et al., 2020 ; Kim et al., 2020 ; Liu et al., 2020a ). After binding to specific receptors such as angiotensin-converting enzyme 2, coronaviruses can either directly fuse with the cell surface or be engulfed by the endosomes, thereby entering the cell. Then, the viral N protein enters the cytoplasm, releasing the RNA genome of the virus into the cell. It synthesizes new virus RNA and proteins and assembles them into virus particles for release ( Fehr and Perlman, 2015 ; Hartenian et al., 2020 ; Prydz and Saraste, 2022 ). 2.1.1.1 SARS-CoV virus infection and NLRP3 inflammasome SARS-CoV-encoded proteins can activate the NLRP3 inflammasome via the NF-κB pathway. For example, ORF3a and E proteins play vital roles in activating the NLRP3 inflammasome. ORF3a and E proteins of SARS-CoV can activate NF-κB, resulting in the transcription of pro-IL-1β ( DeDiego et al., 2014 ; Siu et al., 2019 ). Furthermore, ORF3a promotes the ubiquitination of p105 and ASC via TNFR-associated factors, promoting the activation of NF-κB and the NLRP3 inflammasome ( Siu et al., 2019 ). ORF3a and E proteins of SARS-CoV play roles in the second activation step. ORF3a can lead to lysosomal dysfunction in the host cells and initiate caspase-1 activation either directly or via increased K + efflux. The ion channel function of ORF3a is vital for activating the NLRP3 inflammasome, mitochondrial ROS production, and K + efflux during active IL-1β secretion ( Chen et al., 2019 ). On the other hand, E proteins can alter the permeability of Ca 2+ in the plasma membrane to activate the inflammasome. To be specific, Ca 2+ leakage via the E protein ion channels and increased cytoplasmic Ca 2+ levels may activate the NLRP3 inflammasome in the endoplasmic reticulum–Golgi intermediate compartment/Golgi membranes ( Nieto-Torres et al., 2015 ). In addition, the activation of the inflammasome involves other proteins. In macrophages, ORF8b directly interacts with the LRR domain of NLRP3 and co-localizes with NLRP3 and ASC in the cytosol for activation ( Shi et al., 2019 ). 2.1.1.2 SARS-CoV-2 virus infection and NLRP3 inflammasome Some patients with severe COVID-19 experience a cytokine storm and exhibit inflammasome activation; this increases the amount of active IL-1β and IL-18 in the lungs, cerebrospinal fluid, and serum ( Conti et al., 2020 ; Guasp et al., 2022 ). The mechanism of activation of the NLRP3 inflammasome by SARS-CoV-2 infection is depicted in Figure 2 . Figure 2 Schematic diagram of the mechanism by which SARS-CoV-2 activates the NLRP3 inflammasome. SARS-CoV-2 infection may activate the NLRP3 inflammasome in the following ways: (1) one of the subunits of the spike glycoprotein S1 can release proinflammatory cytokines via mechanisms involving the activation of the NF-κB pathway. (2) The E protein of SARS-CoV-2 can cause lysosomal damage to release a large amount of Ca 2+ , thereby activating the NLRP3 inflammasome. (3) The N viral protein interact with NLRP3 to promote inflammasome assembly; the complement cascade induced by the N protein–MBL-MASP2 axis may lead to the activation of the NLRP3 inflammasome via the different functions of C3a, C5a, and membrane attack complex (MAC). (4) ORF3a primes the inflammasome via NF-κB-mediated transcriptional activation of pro-IL-1β. Furthermore, ORF3a activates the NLRP3 inflammasome via K + efflux and NEK7. The activated NLRP3 inflammasome can activate transcription of pro-IL-1β and pro-IL-18 and produce mature IL-1β and IL-18. When infected with SARS-CoV-2, the NF-κB pathway is activated, leading to the increased expression and production of NLRP3 and active IL-1β. For instance, the SARS-CoV-2 spike glycoprotein S1 activates NF-κB, p38 MAPK, and the NLRP3 inflammasome, leading to the release of proinflammatory cytokines in peripheral blood mononuclear cells ( Olajide et al., 2021 ). Furthermore, ORF3a of SARS-CoV-2 can activate the NLRP3 inflammasome by triggering pro-IL-1β transcription via the NF-κB pathway ( Xu et al., 2022 ). SARS-CoV-2 can also regulate NLRP3 through the second activation phase. Furthermore, ORF3a can use NEK7 and K + efflux to trigger the NLRP3 inflammasome activation, resulting in the conversion of IL-1β precursors into mature IL-1β ( Xu et al., 2022 ). Studies have revealed that the N proteins of SARS-CoV-2 interact with the inflammasome pathway to induce the transcription of NLRP3, pro-IL-1β and pro-IL-18 and to promote inflammasome assembly leading to active IL-18 and IL-1β release and accelerating inflammation ( Figure 2 ) ( Pan et al., 2021 ). Furthermore, some studies have revealed that the N protein of SARS-CoV-2 may activate NLRP3 by initiating the complement cascade pathway by forming the Mannan-binding lectin (MBL; alias: mannose-binding lectin) and MBL-associated serine protease 2 (MBL-MASP-2) complex. The complement cascade induced by N protein-MBL-MASP-2 may activate the NLRP3 inflammasome via the different functions of C3a, C5a, and membrane attack complex (MAC) ( Asgari et al., 2013 ; Sluyter, 2017 ; Magro et al., 2020 ; Ratajczak and Kucia, 2020 ; Zhao et al., 2021 ). The insertion of MAC triggers Ca 2+ influx and increases Ca 2+ levels in the cytoplasm, thereby inducing mitochondrial damage and activating the NLRP3 inflammasome ( Triantafilou et al., 2013a ). 2.1.1.1 SARS-CoV virus infection and NLRP3 inflammasome SARS-CoV-encoded proteins can activate the NLRP3 inflammasome via the NF-κB pathway. For example, ORF3a and E proteins play vital roles in activating the NLRP3 inflammasome. ORF3a and E proteins of SARS-CoV can activate NF-κB, resulting in the transcription of pro-IL-1β ( DeDiego et al., 2014 ; Siu et al., 2019 ). Furthermore, ORF3a promotes the ubiquitination of p105 and ASC via TNFR-associated factors, promoting the activation of NF-κB and the NLRP3 inflammasome ( Siu et al., 2019 ). ORF3a and E proteins of SARS-CoV play roles in the second activation step. ORF3a can lead to lysosomal dysfunction in the host cells and initiate caspase-1 activation either directly or via increased K + efflux. The ion channel function of ORF3a is vital for activating the NLRP3 inflammasome, mitochondrial ROS production, and K + efflux during active IL-1β secretion ( Chen et al., 2019 ). On the other hand, E proteins can alter the permeability of Ca 2+ in the plasma membrane to activate the inflammasome. To be specific, Ca 2+ leakage via the E protein ion channels and increased cytoplasmic Ca 2+ levels may activate the NLRP3 inflammasome in the endoplasmic reticulum–Golgi intermediate compartment/Golgi membranes ( Nieto-Torres et al., 2015 ). In addition, the activation of the inflammasome involves other proteins. In macrophages, ORF8b directly interacts with the LRR domain of NLRP3 and co-localizes with NLRP3 and ASC in the cytosol for activation ( Shi et al., 2019 ). 2.1.1.2 SARS-CoV-2 virus infection and NLRP3 inflammasome Some patients with severe COVID-19 experience a cytokine storm and exhibit inflammasome activation; this increases the amount of active IL-1β and IL-18 in the lungs, cerebrospinal fluid, and serum ( Conti et al., 2020 ; Guasp et al., 2022 ). The mechanism of activation of the NLRP3 inflammasome by SARS-CoV-2 infection is depicted in Figure 2 . Figure 2 Schematic diagram of the mechanism by which SARS-CoV-2 activates the NLRP3 inflammasome. SARS-CoV-2 infection may activate the NLRP3 inflammasome in the following ways: (1) one of the subunits of the spike glycoprotein S1 can release proinflammatory cytokines via mechanisms involving the activation of the NF-κB pathway. (2) The E protein of SARS-CoV-2 can cause lysosomal damage to release a large amount of Ca 2+ , thereby activating the NLRP3 inflammasome. (3) The N viral protein interact with NLRP3 to promote inflammasome assembly; the complement cascade induced by the N protein–MBL-MASP2 axis may lead to the activation of the NLRP3 inflammasome via the different functions of C3a, C5a, and membrane attack complex (MAC). (4) ORF3a primes the inflammasome via NF-κB-mediated transcriptional activation of pro-IL-1β. Furthermore, ORF3a activates the NLRP3 inflammasome via K + efflux and NEK7. The activated NLRP3 inflammasome can activate transcription of pro-IL-1β and pro-IL-18 and produce mature IL-1β and IL-18. When infected with SARS-CoV-2, the NF-κB pathway is activated, leading to the increased expression and production of NLRP3 and active IL-1β. For instance, the SARS-CoV-2 spike glycoprotein S1 activates NF-κB, p38 MAPK, and the NLRP3 inflammasome, leading to the release of proinflammatory cytokines in peripheral blood mononuclear cells ( Olajide et al., 2021 ). Furthermore, ORF3a of SARS-CoV-2 can activate the NLRP3 inflammasome by triggering pro-IL-1β transcription via the NF-κB pathway ( Xu et al., 2022 ). SARS-CoV-2 can also regulate NLRP3 through the second activation phase. Furthermore, ORF3a can use NEK7 and K + efflux to trigger the NLRP3 inflammasome activation, resulting in the conversion of IL-1β precursors into mature IL-1β ( Xu et al., 2022 ). Studies have revealed that the N proteins of SARS-CoV-2 interact with the inflammasome pathway to induce the transcription of NLRP3, pro-IL-1β and pro-IL-18 and to promote inflammasome assembly leading to active IL-18 and IL-1β release and accelerating inflammation ( Figure 2 ) ( Pan et al., 2021 ). Furthermore, some studies have revealed that the N protein of SARS-CoV-2 may activate NLRP3 by initiating the complement cascade pathway by forming the Mannan-binding lectin (MBL; alias: mannose-binding lectin) and MBL-associated serine protease 2 (MBL-MASP-2) complex. The complement cascade induced by N protein-MBL-MASP-2 may activate the NLRP3 inflammasome via the different functions of C3a, C5a, and membrane attack complex (MAC) ( Asgari et al., 2013 ; Sluyter, 2017 ; Magro et al., 2020 ; Ratajczak and Kucia, 2020 ; Zhao et al., 2021 ). The insertion of MAC triggers Ca 2+ influx and increases Ca 2+ levels in the cytoplasm, thereby inducing mitochondrial damage and activating the NLRP3 inflammasome ( Triantafilou et al., 2013a ). 2.1.2 Flavivirus Flaviviruses remain a major public health concern worldwide. Dengue virus (DENV) and Zika virus (ZIKV) are two important pathogens in the genus Flavivirus , which comprise enveloped, positive, and approximately 11 kb single-stranded RNA ( Xie et al., 2021 ). The encoded protein comprises three structural proteins: capsid (C), pre-membrane (prM), and envelope (E) in the N-terminal region and seven nonstructural proteins (i.e., NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) in the C-terminal region ( Chambers et al., 1990 ; Javed et al., 2018 ; Ngono and Shresta, 2018 ; Xie et al., 2021 ). The viral glycoprotein E binds to cell receptors and initiates endocytosis-mediated internalization. After the process of virus uncoating in the cytoplasm, the genomic RNA undergoes subsequent replication and transcription to produce new viral RNA and proteins. Finally, they are assembled into new virus particles and released ( Mukhopadhyay et al., 2005 ; Barrows et al., 2018 ; Diosa-Toro et al., 2020 ; van Leur et al., 2021 ). There are four primary dengue virus serotypes of DENV: DENV-1 to 4; they cause mild fever to severe dengue hemorrhagic fever or dengue shock syndrome, which is potentially life-threatening ( Diamond and Pierson, 2015 ; Mustafa et al., 2015 ; Harapan et al., 2020 ). On the other hand, ZIKV infection can result in considerable neurological damage, including congenital Zika virus syndrome ( Cao-Lormeau et al., 2016 ; Polonio and Peron, 2021 ). 2.1.2.1 DENV virus infection and NLRP3 inflammasome Dengue fever may occur because of a cytokine storm ( de-Oliveira-Pinto et al., 2012 ). Patients with severe dengue have higher active IL-1β levels in their blood and gene expression profiles; this indicates its role in disease severity in clinical settings ( Bozza et al., 2008 ; Pan et al., 2019b ). Active IL-1β potentially recruits neutrophils to the inflammation site to help to clear DENV infection ( Niu et al., 2019 ). During host cell infection with DENV, first, signal 1 is activated: cytoplasmic pattern recognition receptors sense DENV viral RNA and trigger the NF-κB signaling pathway, thereby upregulating the transcription of pro-caspase-1, the NLRP3 inflammasome, pro-IL-1β, and pro-IL-18 ( Shrivastava et al., 2020a ). Many DENV viral proteins exert a strong effect on NLRP3 inflammasome activation and induce host immune responses. For example, in THP-1 cells (human monocyte leukemia cell line), the EDIII domain of E protein activates the NLRP3 inflammasome via the NF-κB pathway, resulting in the transcription of pro-IL-1β and TNF-α ( Khan et al., 2019 ). DENV can also regulate NLRP3 via the second activation phase. For example, EDIII can promote IL-1β secretion by inducing ROS generation and potassium efflux in the second step leading to the activation of the NLRP3 inflammasome and caspase-1-mediated maturation of pro-IL-1β ( Khan et al., 2019 ). Further understanding of the role of EDIII in regulating inflammatory responses will help to understand the pathogenesis of DENV infections. Furthermore, the nonstructural proteins NS2A and NS2B of DENV can trigger the activation of the NLRP3 inflammasome via ROS and Ca 2+ influx ( Shrivastava et al., 2020b ). The M protein of DENV is important in the innate immune response of the host. The interaction of M protein with NLRP3 suggests its crucial role in inflammasome activation, active IL-1β release, and subsequent pathogenic responses ( Pan et al., 2019a ). Therefore, DENV viral proteins are crucial in regulating the innate inflammatory responses of the host. 2.1.2.2 ZIKV virus infection and NLRP3 inflammasome A study has reported that the levels of proinflammatory cytokines such as IL-1β, interferon-γ, and IL-8 are increased in patients with ZIKV infection ( Tappe et al., 2016 ). ZIKV infection can increase the transcription of proinflammatory cytokines such as IL-1β and IL-6 by activating the NF-κB signaling pathway ( Gim et al., 2019 ). Furthermore, ZIKV can regulate NLRP3 via the second activation phase. The NS5 protein plays a vital role in NLRP3 inflammasome activation ( Figure 3 ). Wang et al. have suggested that the NS5 protein of ZIKV directly binds to the NLRP3 inflammasome for activation. NS5 promotes the assembly of the NLRP3–ASC inflammasome complex, resulting in pro-caspase-1 cleavage and secretion of active IL-1β ( Wang et al., 2018 ). Furthermore, He et al. have discovered a novel role of the NS5 protein of ZIKV in regulating NLRP3 activation. NS5 induces ROS production and directly binds to NLRP3 to stimulate the assembly of the NLRP3 inflammasome complex triggering the maturation of pro-IL-1β ( He et al., 2018 ; Wang et al., 2018 ). In addition, ZIKV results in oxidative stress in glial cells, resulting in NLRP3 inflammasome activation and mature IL-1β release for cell death ( Tricarico et al., 2017 ). Figure 3 Schematic diagram of the mechanism by which Zika virus activates the NLRP3 inflammasome. After the Zika virus invades and infects the cells, it activates the NF-κB pathway, inducing gene expression to produce pro-IL-1β and NLRP3. (1) The NS5 protein interacts with NLRP3 to promote the assembly of the NLRP3 inflammasome. The induced mitochondrial ROS leads to pro-caspase-1 cleavage and IL-1β release (2): The NS3 protein inhibits the activation of the NLRP3 inflammasome by cleaving NLRP3, decreasing caspase-1 activation and mature IL-1β secretion. In contrast, ZIKV NS3 protein can prevent the activation of NLRP3 inflammasome. ZIKV NS3 protein expression can decrease the extent of caspase-1 activation and active IL-1β secretion in macrophages, whereas its overexpression in 293T cells can lead to NLRP3 degradation ( Gim et al., 2019 ). 2.1.2.1 DENV virus infection and NLRP3 inflammasome Dengue fever may occur because of a cytokine storm ( de-Oliveira-Pinto et al., 2012 ). Patients with severe dengue have higher active IL-1β levels in their blood and gene expression profiles; this indicates its role in disease severity in clinical settings ( Bozza et al., 2008 ; Pan et al., 2019b ). Active IL-1β potentially recruits neutrophils to the inflammation site to help to clear DENV infection ( Niu et al., 2019 ). During host cell infection with DENV, first, signal 1 is activated: cytoplasmic pattern recognition receptors sense DENV viral RNA and trigger the NF-κB signaling pathway, thereby upregulating the transcription of pro-caspase-1, the NLRP3 inflammasome, pro-IL-1β, and pro-IL-18 ( Shrivastava et al., 2020a ). Many DENV viral proteins exert a strong effect on NLRP3 inflammasome activation and induce host immune responses. For example, in THP-1 cells (human monocyte leukemia cell line), the EDIII domain of E protein activates the NLRP3 inflammasome via the NF-κB pathway, resulting in the transcription of pro-IL-1β and TNF-α ( Khan et al., 2019 ). DENV can also regulate NLRP3 via the second activation phase. For example, EDIII can promote IL-1β secretion by inducing ROS generation and potassium efflux in the second step leading to the activation of the NLRP3 inflammasome and caspase-1-mediated maturation of pro-IL-1β ( Khan et al., 2019 ). Further understanding of the role of EDIII in regulating inflammatory responses will help to understand the pathogenesis of DENV infections. Furthermore, the nonstructural proteins NS2A and NS2B of DENV can trigger the activation of the NLRP3 inflammasome via ROS and Ca 2+ influx ( Shrivastava et al., 2020b ). The M protein of DENV is important in the innate immune response of the host. The interaction of M protein with NLRP3 suggests its crucial role in inflammasome activation, active IL-1β release, and subsequent pathogenic responses ( Pan et al., 2019a ). Therefore, DENV viral proteins are crucial in regulating the innate inflammatory responses of the host. 2.1.2.2 ZIKV virus infection and NLRP3 inflammasome A study has reported that the levels of proinflammatory cytokines such as IL-1β, interferon-γ, and IL-8 are increased in patients with ZIKV infection ( Tappe et al., 2016 ). ZIKV infection can increase the transcription of proinflammatory cytokines such as IL-1β and IL-6 by activating the NF-κB signaling pathway ( Gim et al., 2019 ). Furthermore, ZIKV can regulate NLRP3 via the second activation phase. The NS5 protein plays a vital role in NLRP3 inflammasome activation ( Figure 3 ). Wang et al. have suggested that the NS5 protein of ZIKV directly binds to the NLRP3 inflammasome for activation. NS5 promotes the assembly of the NLRP3–ASC inflammasome complex, resulting in pro-caspase-1 cleavage and secretion of active IL-1β ( Wang et al., 2018 ). Furthermore, He et al. have discovered a novel role of the NS5 protein of ZIKV in regulating NLRP3 activation. NS5 induces ROS production and directly binds to NLRP3 to stimulate the assembly of the NLRP3 inflammasome complex triggering the maturation of pro-IL-1β ( He et al., 2018 ; Wang et al., 2018 ). In addition, ZIKV results in oxidative stress in glial cells, resulting in NLRP3 inflammasome activation and mature IL-1β release for cell death ( Tricarico et al., 2017 ). Figure 3 Schematic diagram of the mechanism by which Zika virus activates the NLRP3 inflammasome. After the Zika virus invades and infects the cells, it activates the NF-κB pathway, inducing gene expression to produce pro-IL-1β and NLRP3. (1) The NS5 protein interacts with NLRP3 to promote the assembly of the NLRP3 inflammasome. The induced mitochondrial ROS leads to pro-caspase-1 cleavage and IL-1β release (2): The NS3 protein inhibits the activation of the NLRP3 inflammasome by cleaving NLRP3, decreasing caspase-1 activation and mature IL-1β secretion. In contrast, ZIKV NS3 protein can prevent the activation of NLRP3 inflammasome. ZIKV NS3 protein expression can decrease the extent of caspase-1 activation and active IL-1β secretion in macrophages, whereas its overexpression in 293T cells can lead to NLRP3 degradation ( Gim et al., 2019 ). 2.1.3 Enterovirus Enterovirus 71 (EV71) belongs to the family Picornaviridae and is a positive-sense single-stranded RNA virus with a genomic length of 7.4 kb ( Solomon et al., 2010 ; Wen et al., 2020 ). EV71 has four structural proteins, namely, VP1, VP2, VP3, and VP4, that are primarily involved in virus particle formation and virus binding to host cells. On the other hand, the nonstructural proteins 2A, 2B, 2C, 3A, 3B, 3C, and 3D are vital for virus infection, replication, and innate immune responses ( Solomon et al., 2010 ; Wen et al., 2020 ). EV71 infection begins with receptor interaction. Then, it enters the cells via endocytosis and undergoes early uncoating. The viral RNA undergoes replication and transcription to generate new viral RNA and proteins. The resulting positive-sense viral RNA is loaded into the procapsid, eventually maturing into infectious virus particles ( Solomon et al., 2010 ; Tan et al., 2014 ). EV71 has caused several epidemics worldwide. It can induce hand-foot-mouth disease, neonatal sepsis, meningoencephalitis, and pulmonary edema in children as well as other severe systemic disorders ( McMinn, 2002 ; Ooi et al., 2010 ). 2.1.3.1 EV71 virus infection and NLRP3 inflammasome The infection of cells by EV71 can activate the NLRP3 inflammasome, which then causes the creation and release of proinflammatory cytokines including active IL-1β. This activation of the NLRP3 inflammasome may be involved in the antiviral defense mechanism of the host against EV71 infection ( Wang et al., 2015 ). EV71 can activate the NLRP3 inflammasome in the first step. EV71 uses cellular vimentin (VIM) to activate extracellular regulatory protein kinase (ERK) and translocate NF-κB. Ultimately, ERK phosphorylation triggers the activation of the NF-κB signaling pathway, which leads to the activation of the NLRP3 inflammasome and the transcription of IL-1β precursors. Therefore, Gong et al. concluded that EV71 activates the NLRP3 inflammasome via the VIM-ERK-NF-κB pathway ( Gong et al., 2022 ). EV71 can also regulate NLRP3 via a second activation phase. The nonstructural protein 3D of the virus plays a role in activating NLRP3 inflammasome-mediated transcription of pro-IL-1β. The 3D protein functions as an RNA-dependent RNA polymerase in EV71 and directly interact with the LRR and NACHT domains of NLRP3 to trigger the formation of the 3D–NLRP3–ASC inflammasome complex ( Wang et al., 2017 ; You et al., 2020 ). Recent studies have reported that methylation of adenosine in RNA molecules generating N6-methyladenosine (m6A RNA) plays an important role in regulating the activation of the NLRP3 inflammasome during EV71 infection. The depletion of fat mass and obesity-associated protein can enhance the mRNA stability and expression of thioredoxin-interacting protein (TXNIP), thereby enhancing ROS production and NLRP3 inflammasome activation ( Qayyum et al., 2021 ; Hu et al., 2023 ). On the other hand, the EV71 viral proteins has a role in inhibition of NLRP3 activation. The nonstructural proteins 2A and 3C of EV71 inhibit NLRP3 inflammasome activation by lysing it ( Wang et al., 2017 ). 2A and 3C can cleave NLRP3 at the G493–L494 and Q225–G226 junction, respectively, to prevent inflammasome activation. Furthermore, the 3C protein of EV71 interacts with NLRP3 and inhibits the secretion of active IL-1β in mammalian cells ( Wang et al., 2015 ; Xiao et al., 2019 ). In summary, EV71 virus proteins exhibit dual roles in NLRP3 interaction. 2.1.3.1 EV71 virus infection and NLRP3 inflammasome The infection of cells by EV71 can activate the NLRP3 inflammasome, which then causes the creation and release of proinflammatory cytokines including active IL-1β. This activation of the NLRP3 inflammasome may be involved in the antiviral defense mechanism of the host against EV71 infection ( Wang et al., 2015 ). EV71 can activate the NLRP3 inflammasome in the first step. EV71 uses cellular vimentin (VIM) to activate extracellular regulatory protein kinase (ERK) and translocate NF-κB. Ultimately, ERK phosphorylation triggers the activation of the NF-κB signaling pathway, which leads to the activation of the NLRP3 inflammasome and the transcription of IL-1β precursors. Therefore, Gong et al. concluded that EV71 activates the NLRP3 inflammasome via the VIM-ERK-NF-κB pathway ( Gong et al., 2022 ). EV71 can also regulate NLRP3 via a second activation phase. The nonstructural protein 3D of the virus plays a role in activating NLRP3 inflammasome-mediated transcription of pro-IL-1β. The 3D protein functions as an RNA-dependent RNA polymerase in EV71 and directly interact with the LRR and NACHT domains of NLRP3 to trigger the formation of the 3D–NLRP3–ASC inflammasome complex ( Wang et al., 2017 ; You et al., 2020 ). Recent studies have reported that methylation of adenosine in RNA molecules generating N6-methyladenosine (m6A RNA) plays an important role in regulating the activation of the NLRP3 inflammasome during EV71 infection. The depletion of fat mass and obesity-associated protein can enhance the mRNA stability and expression of thioredoxin-interacting protein (TXNIP), thereby enhancing ROS production and NLRP3 inflammasome activation ( Qayyum et al., 2021 ; Hu et al., 2023 ). On the other hand, the EV71 viral proteins has a role in inhibition of NLRP3 activation. The nonstructural proteins 2A and 3C of EV71 inhibit NLRP3 inflammasome activation by lysing it ( Wang et al., 2017 ). 2A and 3C can cleave NLRP3 at the G493–L494 and Q225–G226 junction, respectively, to prevent inflammasome activation. Furthermore, the 3C protein of EV71 interacts with NLRP3 and inhibits the secretion of active IL-1β in mammalian cells ( Wang et al., 2015 ; Xiao et al., 2019 ). In summary, EV71 virus proteins exhibit dual roles in NLRP3 interaction. 2.1.4 Retrovirus Retroviruses comprise positive-sense single-stranded RNA genomes that infect cells via reverse transcription and genomic integration ( Hu and Hughes, 2012 ; Zhang et al., 2018 ). The characteristic of retroviruses is that the virus-encoded reverse transcriptase reversely transcribes the viral RNA into DNA and integrates it into the chromosomes of host cells. Subsequently, all structural and regulatory proteins are transcribed and translated ( Zhang et al., 2018 ). After viral protein and genome assembly, new virions bud out ( Krebs et al., 2021 ). HIV, a well-known retrovirus, has considerably affected humanity since the discovery of acquired immunodeficiency syndrome in the early 1980s ( Lucas and Nelson, 2015 ). HIV infection severely damages the immune system, and the virus targets critical CD4 T lymphocytes, resulting in severe cell damage and impairing immune function ( Fanales-Belasio et al., 2010 ; Doitsh et al., 2014 ). HIV-1 and HIV-2 are two types of HIV. HIV-1 accounts for most infections. On the other hand, HIV-2, originating from West Africa, is less prevalent and less virulent than HIV-1 ( Sharp and Hahn, 2011 ; Visseaux et al., 2019 ). 2.1.4.1 HIV virus infection and NLRP3 inflammasome HIV can interact with the NLRP3 inflammasome. Furthermore, HIV infection can trigger NLRP3 inflammasome activation, leading to the production and release of proinflammatory cytokines, including IL-1β ( Reis et al., 2019 ; Zhang et al., 2019 ). A previous study has suggested that monocytes from HIV-1-infected individuals produce more IL-1β than those from noninfected individuals, indicating that HIV-1 triggers NLRP3 inflammasome activation ( Jalbert et al., 2013 ). HIV-1 can activate the NLRP3 inflammasome via the first activation step. HIV-1 activates the NLRP3 inflammasome in primary human monocyte-derived macrophages via the NF-κB pathway, promoting IL-1β secretion by increasing the amount of its precursor in the cell ( Hernandez et al., 2014 ). Furthermore, HIV viral proteins can activate the NLRP3 inflammasome. For example, when it comes to HIV-1 replication and infection, viral protein R (Vpr) plays a crucial role as an accessory protein. Vpr can induce the activation of proinflammatory markers such as TNF-α and the NF-κB signaling pathway ( Li et al., 2020 ). In addition, the HIV-1 transactivator of transcription (Tat) protein can prime the NLRP3 inflammasome by activating NF-κB. Subsequently, the interaction the interaction of the Tat protein with the inflammasome leads to caspase-1 maturation and IL-1β release ( Demarchi et al., 1996 ; Chivero et al., 2017 ). HIV can also regulate NLRP3 via a second activation phase. Tat can induce inflammasome activation by regulating Ca 2+ flux ( Benelli et al., 2000 ; Lee et al., 2012 ). HIV-1 RNA can be recognized by the pattern recognition receptor protein kinase RNA-activated (PKR), triggering the activation of the NLRP3 inflammasome by inducing ROS generation and activating the MAP kinases ERK1/2, JNK, and p38 ( Stunnenberg et al., 2021 ). Furthermore, during the virus attachment step, the HIV-1 envelope glycoprotein causes extracellular ATP and K + efflux by associating with PNX1, leading to NLRP3 activation ( Séror et al., 2011 ; Paoletti et al., 2019 ; He et al., 2020 ). 2.1.4.1 HIV virus infection and NLRP3 inflammasome HIV can interact with the NLRP3 inflammasome. Furthermore, HIV infection can trigger NLRP3 inflammasome activation, leading to the production and release of proinflammatory cytokines, including IL-1β ( Reis et al., 2019 ; Zhang et al., 2019 ). A previous study has suggested that monocytes from HIV-1-infected individuals produce more IL-1β than those from noninfected individuals, indicating that HIV-1 triggers NLRP3 inflammasome activation ( Jalbert et al., 2013 ). HIV-1 can activate the NLRP3 inflammasome via the first activation step. HIV-1 activates the NLRP3 inflammasome in primary human monocyte-derived macrophages via the NF-κB pathway, promoting IL-1β secretion by increasing the amount of its precursor in the cell ( Hernandez et al., 2014 ). Furthermore, HIV viral proteins can activate the NLRP3 inflammasome. For example, when it comes to HIV-1 replication and infection, viral protein R (Vpr) plays a crucial role as an accessory protein. Vpr can induce the activation of proinflammatory markers such as TNF-α and the NF-κB signaling pathway ( Li et al., 2020 ). In addition, the HIV-1 transactivator of transcription (Tat) protein can prime the NLRP3 inflammasome by activating NF-κB. Subsequently, the interaction the interaction of the Tat protein with the inflammasome leads to caspase-1 maturation and IL-1β release ( Demarchi et al., 1996 ; Chivero et al., 2017 ). HIV can also regulate NLRP3 via a second activation phase. Tat can induce inflammasome activation by regulating Ca 2+ flux ( Benelli et al., 2000 ; Lee et al., 2012 ). HIV-1 RNA can be recognized by the pattern recognition receptor protein kinase RNA-activated (PKR), triggering the activation of the NLRP3 inflammasome by inducing ROS generation and activating the MAP kinases ERK1/2, JNK, and p38 ( Stunnenberg et al., 2021 ). Furthermore, during the virus attachment step, the HIV-1 envelope glycoprotein causes extracellular ATP and K + efflux by associating with PNX1, leading to NLRP3 activation ( Séror et al., 2011 ; Paoletti et al., 2019 ; He et al., 2020 ). 2.2 Negative-sense single-stranded RNA virus 2.2.1 Influenza virus At present, influenza is an infectious disease worldwide and a serious global health concern; approximately 3 million to 5 million severe cases and 290,000–650,000 deaths are reported each year ( Kim et al., 2022 ). Influenza viruses belong to the family Orthomyxoviridae and contain a negative-sense single-stranded RNA genome ( Bouvier and Palese, 2008 ; Te Velthuis and Fodor, 2016 ). Human influenza viruses come in three different types: Influenza A, B, and C viruses (IAV, IBV, and ICV); they are the pathogens of the human respiratory disease influenza ( Blut and Hemotherapy, 2009 ; Hutchinson, 2018 ). IAV and IBV have caused significant morbidity and mortality worldwide as well as an economic burden; in contrast, ICV causes a mild respiratory disease that rarely generates epidemics, mostly in children ( Te Velthuis and Fodor, 2016 ). The virus binds to the cell surface receptors containing sialic acid and enters the cell via endocytosis. The viral core is released into the nucleus, where the viral genome is transcribed into mRNA and subsequently translated into viral proteins. Thereafter, the viral proteins and newly synthesized RNA assemble into new virus particles. After the maturation of the virus particles, they are released via membrane fusion or penetration ( Watanabe et al., 2010 ; Te Velthuis and Fodor, 2016 ). 2.2.1.1 Influenza virus infection and NLRP3 inflammasome Influenza often triggers inflammatory responses in the immune system of humans ( Kuriakose and Kanneganti, 2017 ). During influenza, host immune cells release cytokines such as IL-1β and IL-6 in response to virus invasion. Among these cytokines, IL-1β is a vital proinflammatory cytokine that regulates immune and inflammatory responses. Virus components may function as signals that activate the NLRP3 inflammasome, the maturation of pro-IL-1β into their active forms ( Allen et al., 2009 ; Tate and Mansell, 2018 ). Influenza can regulate NLRP3 via the first step. The viral nucleoprotein (NP) can stimulate neighboring cells via TLR2 and TLR4 activating the NF-κB pathway and thereby inducing the production of pro-IL-1β and IL-6; this subsequently leads to the production of trypsin which can increase the infectivity of influenza virus ( Kim et al., 2022 ). For the priming associated with NLRP3 inflammasome activation, TLRs and retinoic acid-inducible gene I (RIG-I) are critical. Influenza viral RNA can indirectly promote inflammasome assembly and the release of inflammasome-dependent cytokines by interacting with known RNA sensors such as TLR-7 and RIG-I ( Ichinohe et al., 2009 ; Kuriakose and Kanneganti, 2017 ). After influenza virus invasion, viral ribonucleoproteins are released into the nucleus for transcription and replication. SsRNA is recognized by TLR7 and triggers the expression of NF-κB, pro-IL-1β, pro-IL-18, and other pro-inflammatory cytokines ( Niu and Meng, 2023 ). Studies have shown a lack of IL-1β release by infecting influenza virus into TLR7-deficient bone marrow-derived DCs ( Ichinohe et al., 2010 ). In addition, when the viral genome reaches the cytoplasm, the 5α-triphosphate dsRNA of influenza A virus can activate RIG-I, which is involved in NF-κB activation and pro-IL-18 and pro-IL-1β transcription ( Cui et al., 2008 ). Influenza can also regulate NLRP3 via a second activation phase. The M2 protein of the influenza virus is a proton-selective ion channel that is important in NLRP3 inflammasome activation. The M2 protein can activate the NLRP3 inflammasome in macrophages and dendritic cells and plays a role in viral pathogenesis ( Ichinohe et al., 2010 ). The M2 protein promotes inflammasome activation by locating the acidified Golgi apparatus, promoting proton outflow, causing ion imbalance, causing K efflux binding with Na efflux, and producing ROS, thereby activating the NLRP3 inflammasome. Furthermore, it triggers the inflammasome by modulating ion flux or stimulating mitochondrial DNA release into the cytoplasm ( Ichinohe et al., 2010 ; Pang and Iwasaki, 2011 ; Moriyama et al., 2019 ). Moreover, a study has reported that NLRP3 can be recruited into dispersed Trans-Golgi-Network (TGN), where it oligomerizes, alters the conformation, and recruits ASC to activate the inflammasome ( Pandey and Zhou, 2022 ). The PB1-F2 protein of IAV activates NLRP3 via various mechanisms. The PB1-F2 protein is translocated into the mitochondrial intima through Tom40 channel; its accumulation decreases the potential of the mitochondrial intima (Δφm), accelerates mitochondrial fragmentation, and activates the NLRP3 inflammasome ( Yoshizumi et al., 2014 ). The aggregated form of the C-terminal region of PB1-F2 protein can activate NLRP3 via mitochondrial autophagy, promoting mitochondrial ROS production and mitochondrial DNA release ( McAuley et al., 2013 ; Wang et al., 2021 ). Furthermore, IAV RNA activates the NLRP3 inflammasome. The activation of the NLRP3 inflammasome by influenza virus RNA depends on lysosomal maturation and ROS production ( Allen et al., 2009 ). However, the NLRP3 inflammasome's activation can also be inhibited by influenza viruses. For example, IAVs can inhibit NLRP3 inflammasome activation via viral proteins. The NS1 protein of IAV interacts with NLRP3 to inhibit single-speck formation required for NLRP3 and ASC-induced inflammasome activation; this results in decreased secretion of active IL-1β ( Moriyama et al., 2016 ). Chung et al. have reported that NS1 overexpression can significantly disrupt the transcription of proinflammatory cytokines by inhibiting the activation of NF-κB. Furthermore, inflammasome NLRP3 activation is inhibited in NS1-expressing THP-1 cells ( Chung et al., 2015 ). 2.2.2 Ebola virus Ebola virus (EBOV) is one of the deadly zoonotic epidemic viruses that can cause fatal systemic disease ( Marcinkiewicz et al., 2014 ; Malvy et al., 2019 ). The Ebola virus genome is composed of single negative strand RNA; the genome size is about 19kb; and it belongs to the filoviridae family ( Baseler et al., 2017 ). The main characteristics of patients are high fever, fatigue, body aches, gastrointestinal symptoms, abnormal inflammatory response, immune suppression, large fluid and electrolyte loss, and high mortality ( De Clercq, 2015 ; Malvy et al., 2019 ). When a virus infects, the virus first binds to the host cell membrane, triggering endocytosis of the virus particles. The particle envelope fuses with the endosomal membrane, thereby releasing the ribonucleoprotein complex into the cytoplasm. In the cytoplasm the viral genome is replicated and transcribed into mRNA. The viral proteins are then translated into the cytoplasm and into the endoplasmic reticulum. Mature daughter ribonucleoprotein complexes and viral proteins are transported to the plasma membrane. Finally, mature virions are released in the form of budding ( Sakurai, 2015 ; Baseler et al., 2017 ; Jacob et al., 2020 ). 2.2.2.1 Ebola virus infection and NLRP3 inflammasome The immune response and inflammatory cascade of the innate immune system are key factors in the pathogenesis and mortality of EBOV ( Leroy et al., 2001 ). The release of proinflammatory cytokines IL-1β, TNF-α, and IL-5 was found in asymptomatic individuals ( Leroy et al., 2001 ). In addition, when EBOV infects monocytes, it also leads to the maturation and secretion of the pro-inflammatory cytokine IL-1β ( Ströher et al., 2001 ). It has been reported that Ebola virus-like particles stimulate the expression of type I interferon and proinflammatory cytokines through Toll-like receptors and interferon signaling pathways ( Ayithan et al., 2014 ). Halfmann et al. found that EBOV stimulates the secretion of proinflammatory cytokines IL-1β and IL-18 by activating the NLRP3 inflammasome in a caspase 1-dependent manner ( Halfmann et al., 2018 ). However, more research is still needed to study how EBOV activates the NLRP3 inflammasome to trigger the release of pro-inflammatory cytokines like IL-1β and the downstream role of IL-1β signal transduction. 2.2.3 Respiratory syncytial virus Respiratory syncytial virus (RSV) infection is a constant public health problem, and the impact on children, the elderly, and immunocompromised patients can be very significant ( Yang et al., 2023 ). RSV is an enveloped, negatively-sense single-stranded RNA virus belonging to the Paramyxoviridae family ( Collins et al., 2013 ). RSV has eight known structural proteins. Fusion protein (F), attached glycoprotein (G), small hydrophobic protein (SH), matrix protein (M), nucleocapsid protein (N), large protein (L), phosphoprotein (P), and M2 gene product M2-1, as well as two non-structural proteins (NS1 and NS1) ( Borchers et al., 2013 ; Griffiths et al., 2017 ; Agac et al., 2023 ). The virus is mainly transmitted through close contact with saliva or mucus droplets. Common symptoms include fever, runny nose, cough, and chest tightness ( Linder and Malani, 2017 ). RSV first binds to receptors on the surface of the host cell through endocytosis, or the viral F protein on its surface, and then the virus fuses with the cell membrane to enter the host cell. Viral RNA is released into the cytoplasm of the host cell. In host cells, viral RNA acts as a transcription template, producing mRNA. The mRNA is then translated into viral proteins. The newly synthesized viral protein and the replicating RNA are assembled into new viral particles, which bud out from the infected cell to complete the viral life cycle ( Shang et al., 2021 ). 2.2.3.1 Respiratory syncytial virus infection and NLRP3 inflammasome Activation of the inflammasome pathway also plays an important role in RSV infection. Excessive inflammatory responses can lead to the release of pro-inflammatory cytokines, including IL-1β and IL-18, leading to lung inflammation and damage ( Shen et al., 2018 ). Studies have shown that NLRP3, ASC, and caspase-1 are critical for IL-1β production during RSV infection ( Segovia et al., 2012 ; Shen et al., 2018 ). RSV can regulate NLRP3 via the first step. RSV activates NF-κB during infection ( Sabbah et al., 2009 ). NF-κB signaling has been shown to be critical for NLRP3 expression during RSV infection ( Shen et al., 2019 ). TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection ( Segovia et al., 2012 ). After the administration of NF-κB inhibitors in RSV-infected cells, the production of IL-1β decreased significantly ( Segovia et al., 2012 ). Respiratory syncytial virus can also regulate NLRP3 via a second activation phase. During RSV infection, activation of the NLRP3 inflammasome is dependent on K efflux and ROS production, followed by caspase-1-mediated maturation and secretion of IL-1β ( Segovia et al., 2012 ; Yang et al., 2023 ). It has been shown that RSV SH viroporin induces membrane permeability to ions or small molecules that are essential for triggering the NLRP3 inflammasome. After RSV infection, RSV SH virus channel proteins accumulate in the Golgi within the lipid raft structure, possibly forming ion channels that trigger the translocation of NLRP3 from cytoplasm to Golgi apparatus. This leads to NLRP3 inflammasome activation as well as pro-IL1β transcriptional activation ( Triantafilou et al., 2013b ). It has also been reported that orosomucoid 1-like protein 3 (ORMDL3) can inhibit calcium pump function, resulting in increased calcium levels in the cytoplasm and decreased ER levels, thereby inducing ER stress ( Cantero-Recasens et al., 2010 ). RSV may induce NLRP3 inflammasome expression by activating ORMDL3 overexpression ( Cheng et al., 2023 ). 2.2.4 Rift Valley Fever virus Rift Valley Fever virus (RVFV) is a negative sense segmented single-stranded RNA virus with a size of about 12kb that belongs to the family Phenuiviridae and the genus Phlebovirus ( Ahsan et al., 2016 ; Gaudreault et al., 2019 ). In humans, RVFV can cause a variety of disease manifestations, ranging from febrile illness to hemorrhagic fever and death ( Ermler et al., 2014 ). The viral genome has three segments: the negative sense L (large) and negative sense M (medium) segments, and the double sense S (small) segment. These three segments encode multifunctional proteins. The S segment encodes nuclear protein (N) and non-structural protein S (NSs), the M segment encodes viral glycoprotein (Gn and Gc) and non-structural protein (NSm and a 78-kDa protein), and the L segment encodes viral RNA-dependent RNA polymerase ( Baer et al., 2016 ; Wright et al., 2019 ; Tercero et al., 2021 ; Lean and Johnson, 2022 ; Ganaie et al., 2023 ). The virus first attaches to the host membrane and enters the host cell through endocytosis. The viral genome is released into the cytoplasm. Negative RNA is transcribed into positive mRNA encoding viral proteins, and positive RNA serves as a template for the synthesis of new negative RNA. The newly synthesized viral protein and the replicated genome are combined in the cytoplasm to form new viral particles, which are released when they mature ( Wright et al., 2019 ). 2.2.4.1 Rift Valley Fever virus infection and NLRP3 inflammasome RVFV infection induces strong cytokines, which are essential for the recruitment of innate immune cells to the site of infection ( Nair et al., 2023 ). RVFV infected-cells secrete IL-1β, which is involved in the NLRP3 inflammasome, ASC oligomerization, and caspase-1 maturation. It has been reported that RVFV activates the NLRP3 inflammasome by inducing the formation of an inflammasome complex containing NLRP3 and MAVS, where MAVS are localized to NLRP3 during RVFV infection, leading to the maturation and secretion of IL-1β ( Ermler et al., 2014 ). 2.2.5 Hantavirus Hantavirus (HTNV) is a coated single-stranded negative sense RNA virus belonging to the Bunyaviridae family ( Stock, 2008 ). The HTNV genome consists of three single-stranded negative sense RNA fragments: small (S), medium (M), and large (L) genome fragments encode four structural proteins (nuclear proteins N, glycoproteins Gn and Gc, and L proteins) ( Muyangwa et al., 2015 ; Meier et al., 2021 ). HTNV can cause acute febrile illness in humans. Hemorrhagic Fever with Renal Syndrome (HFRS) caused in Asia and Europe, and Hantavirus Pulmonary Syndrome (HPS) caused in the Americas ( Sargianou et al., 2012 ). Once Hantavirus enters the body, the virions bind to cell surface membrane receptors and enter the cell through endocytosis. The specific mechanism involves the transcription of the viral genome and the synthesis of viral RNA and viral proteins. The newly synthesized vRNA is coated with N protein to form ribonucleoprotein, which is then sent to the perinuclear membrane system. The synthesized viral proteins and genome are assembled into new viral particles inside the host cell. When the virion matures, it is released ( Muyangwa et al., 2015 ; Meier et al., 2021 ). 2.2.5.1 Hantavirus infection and NLRP3 inflammasome HTNV infection causes cells to enter a stress condition and induces the production of inflammatory cytokines ( Zhang et al., 2021 ). Studies have found that IL-1β is significantly elevated during HFRS. Induction of the human monocyte line THP-1 by HTNV revealed the secretion of IL-1β. The specific mechanism found that the induction of IL-1β by HTNV depended on the activation of caspase-1. Hantavirus thus induces the formation of the NLRP3 inflammasome in THP-1 cells, which may be an important factor in IL-1β levels in patients with HFRS ( Ye et al., 2015 ). 2.2.1 Influenza virus At present, influenza is an infectious disease worldwide and a serious global health concern; approximately 3 million to 5 million severe cases and 290,000–650,000 deaths are reported each year ( Kim et al., 2022 ). Influenza viruses belong to the family Orthomyxoviridae and contain a negative-sense single-stranded RNA genome ( Bouvier and Palese, 2008 ; Te Velthuis and Fodor, 2016 ). Human influenza viruses come in three different types: Influenza A, B, and C viruses (IAV, IBV, and ICV); they are the pathogens of the human respiratory disease influenza ( Blut and Hemotherapy, 2009 ; Hutchinson, 2018 ). IAV and IBV have caused significant morbidity and mortality worldwide as well as an economic burden; in contrast, ICV causes a mild respiratory disease that rarely generates epidemics, mostly in children ( Te Velthuis and Fodor, 2016 ). The virus binds to the cell surface receptors containing sialic acid and enters the cell via endocytosis. The viral core is released into the nucleus, where the viral genome is transcribed into mRNA and subsequently translated into viral proteins. Thereafter, the viral proteins and newly synthesized RNA assemble into new virus particles. After the maturation of the virus particles, they are released via membrane fusion or penetration ( Watanabe et al., 2010 ; Te Velthuis and Fodor, 2016 ). 2.2.1.1 Influenza virus infection and NLRP3 inflammasome Influenza often triggers inflammatory responses in the immune system of humans ( Kuriakose and Kanneganti, 2017 ). During influenza, host immune cells release cytokines such as IL-1β and IL-6 in response to virus invasion. Among these cytokines, IL-1β is a vital proinflammatory cytokine that regulates immune and inflammatory responses. Virus components may function as signals that activate the NLRP3 inflammasome, the maturation of pro-IL-1β into their active forms ( Allen et al., 2009 ; Tate and Mansell, 2018 ). Influenza can regulate NLRP3 via the first step. The viral nucleoprotein (NP) can stimulate neighboring cells via TLR2 and TLR4 activating the NF-κB pathway and thereby inducing the production of pro-IL-1β and IL-6; this subsequently leads to the production of trypsin which can increase the infectivity of influenza virus ( Kim et al., 2022 ). For the priming associated with NLRP3 inflammasome activation, TLRs and retinoic acid-inducible gene I (RIG-I) are critical. Influenza viral RNA can indirectly promote inflammasome assembly and the release of inflammasome-dependent cytokines by interacting with known RNA sensors such as TLR-7 and RIG-I ( Ichinohe et al., 2009 ; Kuriakose and Kanneganti, 2017 ). After influenza virus invasion, viral ribonucleoproteins are released into the nucleus for transcription and replication. SsRNA is recognized by TLR7 and triggers the expression of NF-κB, pro-IL-1β, pro-IL-18, and other pro-inflammatory cytokines ( Niu and Meng, 2023 ). Studies have shown a lack of IL-1β release by infecting influenza virus into TLR7-deficient bone marrow-derived DCs ( Ichinohe et al., 2010 ). In addition, when the viral genome reaches the cytoplasm, the 5α-triphosphate dsRNA of influenza A virus can activate RIG-I, which is involved in NF-κB activation and pro-IL-18 and pro-IL-1β transcription ( Cui et al., 2008 ). Influenza can also regulate NLRP3 via a second activation phase. The M2 protein of the influenza virus is a proton-selective ion channel that is important in NLRP3 inflammasome activation. The M2 protein can activate the NLRP3 inflammasome in macrophages and dendritic cells and plays a role in viral pathogenesis ( Ichinohe et al., 2010 ). The M2 protein promotes inflammasome activation by locating the acidified Golgi apparatus, promoting proton outflow, causing ion imbalance, causing K efflux binding with Na efflux, and producing ROS, thereby activating the NLRP3 inflammasome. Furthermore, it triggers the inflammasome by modulating ion flux or stimulating mitochondrial DNA release into the cytoplasm ( Ichinohe et al., 2010 ; Pang and Iwasaki, 2011 ; Moriyama et al., 2019 ). Moreover, a study has reported that NLRP3 can be recruited into dispersed Trans-Golgi-Network (TGN), where it oligomerizes, alters the conformation, and recruits ASC to activate the inflammasome ( Pandey and Zhou, 2022 ). The PB1-F2 protein of IAV activates NLRP3 via various mechanisms. The PB1-F2 protein is translocated into the mitochondrial intima through Tom40 channel; its accumulation decreases the potential of the mitochondrial intima (Δφm), accelerates mitochondrial fragmentation, and activates the NLRP3 inflammasome ( Yoshizumi et al., 2014 ). The aggregated form of the C-terminal region of PB1-F2 protein can activate NLRP3 via mitochondrial autophagy, promoting mitochondrial ROS production and mitochondrial DNA release ( McAuley et al., 2013 ; Wang et al., 2021 ). Furthermore, IAV RNA activates the NLRP3 inflammasome. The activation of the NLRP3 inflammasome by influenza virus RNA depends on lysosomal maturation and ROS production ( Allen et al., 2009 ). However, the NLRP3 inflammasome's activation can also be inhibited by influenza viruses. For example, IAVs can inhibit NLRP3 inflammasome activation via viral proteins. The NS1 protein of IAV interacts with NLRP3 to inhibit single-speck formation required for NLRP3 and ASC-induced inflammasome activation; this results in decreased secretion of active IL-1β ( Moriyama et al., 2016 ). Chung et al. have reported that NS1 overexpression can significantly disrupt the transcription of proinflammatory cytokines by inhibiting the activation of NF-κB. Furthermore, inflammasome NLRP3 activation is inhibited in NS1-expressing THP-1 cells ( Chung et al., 2015 ). 2.2.1.1 Influenza virus infection and NLRP3 inflammasome Influenza often triggers inflammatory responses in the immune system of humans ( Kuriakose and Kanneganti, 2017 ). During influenza, host immune cells release cytokines such as IL-1β and IL-6 in response to virus invasion. Among these cytokines, IL-1β is a vital proinflammatory cytokine that regulates immune and inflammatory responses. Virus components may function as signals that activate the NLRP3 inflammasome, the maturation of pro-IL-1β into their active forms ( Allen et al., 2009 ; Tate and Mansell, 2018 ). Influenza can regulate NLRP3 via the first step. The viral nucleoprotein (NP) can stimulate neighboring cells via TLR2 and TLR4 activating the NF-κB pathway and thereby inducing the production of pro-IL-1β and IL-6; this subsequently leads to the production of trypsin which can increase the infectivity of influenza virus ( Kim et al., 2022 ). For the priming associated with NLRP3 inflammasome activation, TLRs and retinoic acid-inducible gene I (RIG-I) are critical. Influenza viral RNA can indirectly promote inflammasome assembly and the release of inflammasome-dependent cytokines by interacting with known RNA sensors such as TLR-7 and RIG-I ( Ichinohe et al., 2009 ; Kuriakose and Kanneganti, 2017 ). After influenza virus invasion, viral ribonucleoproteins are released into the nucleus for transcription and replication. SsRNA is recognized by TLR7 and triggers the expression of NF-κB, pro-IL-1β, pro-IL-18, and other pro-inflammatory cytokines ( Niu and Meng, 2023 ). Studies have shown a lack of IL-1β release by infecting influenza virus into TLR7-deficient bone marrow-derived DCs ( Ichinohe et al., 2010 ). In addition, when the viral genome reaches the cytoplasm, the 5α-triphosphate dsRNA of influenza A virus can activate RIG-I, which is involved in NF-κB activation and pro-IL-18 and pro-IL-1β transcription ( Cui et al., 2008 ). Influenza can also regulate NLRP3 via a second activation phase. The M2 protein of the influenza virus is a proton-selective ion channel that is important in NLRP3 inflammasome activation. The M2 protein can activate the NLRP3 inflammasome in macrophages and dendritic cells and plays a role in viral pathogenesis ( Ichinohe et al., 2010 ). The M2 protein promotes inflammasome activation by locating the acidified Golgi apparatus, promoting proton outflow, causing ion imbalance, causing K efflux binding with Na efflux, and producing ROS, thereby activating the NLRP3 inflammasome. Furthermore, it triggers the inflammasome by modulating ion flux or stimulating mitochondrial DNA release into the cytoplasm ( Ichinohe et al., 2010 ; Pang and Iwasaki, 2011 ; Moriyama et al., 2019 ). Moreover, a study has reported that NLRP3 can be recruited into dispersed Trans-Golgi-Network (TGN), where it oligomerizes, alters the conformation, and recruits ASC to activate the inflammasome ( Pandey and Zhou, 2022 ). The PB1-F2 protein of IAV activates NLRP3 via various mechanisms. The PB1-F2 protein is translocated into the mitochondrial intima through Tom40 channel; its accumulation decreases the potential of the mitochondrial intima (Δφm), accelerates mitochondrial fragmentation, and activates the NLRP3 inflammasome ( Yoshizumi et al., 2014 ). The aggregated form of the C-terminal region of PB1-F2 protein can activate NLRP3 via mitochondrial autophagy, promoting mitochondrial ROS production and mitochondrial DNA release ( McAuley et al., 2013 ; Wang et al., 2021 ). Furthermore, IAV RNA activates the NLRP3 inflammasome. The activation of the NLRP3 inflammasome by influenza virus RNA depends on lysosomal maturation and ROS production ( Allen et al., 2009 ). However, the NLRP3 inflammasome's activation can also be inhibited by influenza viruses. For example, IAVs can inhibit NLRP3 inflammasome activation via viral proteins. The NS1 protein of IAV interacts with NLRP3 to inhibit single-speck formation required for NLRP3 and ASC-induced inflammasome activation; this results in decreased secretion of active IL-1β ( Moriyama et al., 2016 ). Chung et al. have reported that NS1 overexpression can significantly disrupt the transcription of proinflammatory cytokines by inhibiting the activation of NF-κB. Furthermore, inflammasome NLRP3 activation is inhibited in NS1-expressing THP-1 cells ( Chung et al., 2015 ). 2.2.2 Ebola virus Ebola virus (EBOV) is one of the deadly zoonotic epidemic viruses that can cause fatal systemic disease ( Marcinkiewicz et al., 2014 ; Malvy et al., 2019 ). The Ebola virus genome is composed of single negative strand RNA; the genome size is about 19kb; and it belongs to the filoviridae family ( Baseler et al., 2017 ). The main characteristics of patients are high fever, fatigue, body aches, gastrointestinal symptoms, abnormal inflammatory response, immune suppression, large fluid and electrolyte loss, and high mortality ( De Clercq, 2015 ; Malvy et al., 2019 ). When a virus infects, the virus first binds to the host cell membrane, triggering endocytosis of the virus particles. The particle envelope fuses with the endosomal membrane, thereby releasing the ribonucleoprotein complex into the cytoplasm. In the cytoplasm the viral genome is replicated and transcribed into mRNA. The viral proteins are then translated into the cytoplasm and into the endoplasmic reticulum. Mature daughter ribonucleoprotein complexes and viral proteins are transported to the plasma membrane. Finally, mature virions are released in the form of budding ( Sakurai, 2015 ; Baseler et al., 2017 ; Jacob et al., 2020 ). 2.2.2.1 Ebola virus infection and NLRP3 inflammasome The immune response and inflammatory cascade of the innate immune system are key factors in the pathogenesis and mortality of EBOV ( Leroy et al., 2001 ). The release of proinflammatory cytokines IL-1β, TNF-α, and IL-5 was found in asymptomatic individuals ( Leroy et al., 2001 ). In addition, when EBOV infects monocytes, it also leads to the maturation and secretion of the pro-inflammatory cytokine IL-1β ( Ströher et al., 2001 ). It has been reported that Ebola virus-like particles stimulate the expression of type I interferon and proinflammatory cytokines through Toll-like receptors and interferon signaling pathways ( Ayithan et al., 2014 ). Halfmann et al. found that EBOV stimulates the secretion of proinflammatory cytokines IL-1β and IL-18 by activating the NLRP3 inflammasome in a caspase 1-dependent manner ( Halfmann et al., 2018 ). However, more research is still needed to study how EBOV activates the NLRP3 inflammasome to trigger the release of pro-inflammatory cytokines like IL-1β and the downstream role of IL-1β signal transduction. 2.2.2.1 Ebola virus infection and NLRP3 inflammasome The immune response and inflammatory cascade of the innate immune system are key factors in the pathogenesis and mortality of EBOV ( Leroy et al., 2001 ). The release of proinflammatory cytokines IL-1β, TNF-α, and IL-5 was found in asymptomatic individuals ( Leroy et al., 2001 ). In addition, when EBOV infects monocytes, it also leads to the maturation and secretion of the pro-inflammatory cytokine IL-1β ( Ströher et al., 2001 ). It has been reported that Ebola virus-like particles stimulate the expression of type I interferon and proinflammatory cytokines through Toll-like receptors and interferon signaling pathways ( Ayithan et al., 2014 ). Halfmann et al. found that EBOV stimulates the secretion of proinflammatory cytokines IL-1β and IL-18 by activating the NLRP3 inflammasome in a caspase 1-dependent manner ( Halfmann et al., 2018 ). However, more research is still needed to study how EBOV activates the NLRP3 inflammasome to trigger the release of pro-inflammatory cytokines like IL-1β and the downstream role of IL-1β signal transduction. 2.2.3 Respiratory syncytial virus Respiratory syncytial virus (RSV) infection is a constant public health problem, and the impact on children, the elderly, and immunocompromised patients can be very significant ( Yang et al., 2023 ). RSV is an enveloped, negatively-sense single-stranded RNA virus belonging to the Paramyxoviridae family ( Collins et al., 2013 ). RSV has eight known structural proteins. Fusion protein (F), attached glycoprotein (G), small hydrophobic protein (SH), matrix protein (M), nucleocapsid protein (N), large protein (L), phosphoprotein (P), and M2 gene product M2-1, as well as two non-structural proteins (NS1 and NS1) ( Borchers et al., 2013 ; Griffiths et al., 2017 ; Agac et al., 2023 ). The virus is mainly transmitted through close contact with saliva or mucus droplets. Common symptoms include fever, runny nose, cough, and chest tightness ( Linder and Malani, 2017 ). RSV first binds to receptors on the surface of the host cell through endocytosis, or the viral F protein on its surface, and then the virus fuses with the cell membrane to enter the host cell. Viral RNA is released into the cytoplasm of the host cell. In host cells, viral RNA acts as a transcription template, producing mRNA. The mRNA is then translated into viral proteins. The newly synthesized viral protein and the replicating RNA are assembled into new viral particles, which bud out from the infected cell to complete the viral life cycle ( Shang et al., 2021 ). 2.2.3.1 Respiratory syncytial virus infection and NLRP3 inflammasome Activation of the inflammasome pathway also plays an important role in RSV infection. Excessive inflammatory responses can lead to the release of pro-inflammatory cytokines, including IL-1β and IL-18, leading to lung inflammation and damage ( Shen et al., 2018 ). Studies have shown that NLRP3, ASC, and caspase-1 are critical for IL-1β production during RSV infection ( Segovia et al., 2012 ; Shen et al., 2018 ). RSV can regulate NLRP3 via the first step. RSV activates NF-κB during infection ( Sabbah et al., 2009 ). NF-κB signaling has been shown to be critical for NLRP3 expression during RSV infection ( Shen et al., 2019 ). TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection ( Segovia et al., 2012 ). After the administration of NF-κB inhibitors in RSV-infected cells, the production of IL-1β decreased significantly ( Segovia et al., 2012 ). Respiratory syncytial virus can also regulate NLRP3 via a second activation phase. During RSV infection, activation of the NLRP3 inflammasome is dependent on K efflux and ROS production, followed by caspase-1-mediated maturation and secretion of IL-1β ( Segovia et al., 2012 ; Yang et al., 2023 ). It has been shown that RSV SH viroporin induces membrane permeability to ions or small molecules that are essential for triggering the NLRP3 inflammasome. After RSV infection, RSV SH virus channel proteins accumulate in the Golgi within the lipid raft structure, possibly forming ion channels that trigger the translocation of NLRP3 from cytoplasm to Golgi apparatus. This leads to NLRP3 inflammasome activation as well as pro-IL1β transcriptional activation ( Triantafilou et al., 2013b ). It has also been reported that orosomucoid 1-like protein 3 (ORMDL3) can inhibit calcium pump function, resulting in increased calcium levels in the cytoplasm and decreased ER levels, thereby inducing ER stress ( Cantero-Recasens et al., 2010 ). RSV may induce NLRP3 inflammasome expression by activating ORMDL3 overexpression ( Cheng et al., 2023 ). 2.2.3.1 Respiratory syncytial virus infection and NLRP3 inflammasome Activation of the inflammasome pathway also plays an important role in RSV infection. Excessive inflammatory responses can lead to the release of pro-inflammatory cytokines, including IL-1β and IL-18, leading to lung inflammation and damage ( Shen et al., 2018 ). Studies have shown that NLRP3, ASC, and caspase-1 are critical for IL-1β production during RSV infection ( Segovia et al., 2012 ; Shen et al., 2018 ). RSV can regulate NLRP3 via the first step. RSV activates NF-κB during infection ( Sabbah et al., 2009 ). NF-κB signaling has been shown to be critical for NLRP3 expression during RSV infection ( Shen et al., 2019 ). TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection ( Segovia et al., 2012 ). After the administration of NF-κB inhibitors in RSV-infected cells, the production of IL-1β decreased significantly ( Segovia et al., 2012 ). Respiratory syncytial virus can also regulate NLRP3 via a second activation phase. During RSV infection, activation of the NLRP3 inflammasome is dependent on K efflux and ROS production, followed by caspase-1-mediated maturation and secretion of IL-1β ( Segovia et al., 2012 ; Yang et al., 2023 ). It has been shown that RSV SH viroporin induces membrane permeability to ions or small molecules that are essential for triggering the NLRP3 inflammasome. After RSV infection, RSV SH virus channel proteins accumulate in the Golgi within the lipid raft structure, possibly forming ion channels that trigger the translocation of NLRP3 from cytoplasm to Golgi apparatus. This leads to NLRP3 inflammasome activation as well as pro-IL1β transcriptional activation ( Triantafilou et al., 2013b ). It has also been reported that orosomucoid 1-like protein 3 (ORMDL3) can inhibit calcium pump function, resulting in increased calcium levels in the cytoplasm and decreased ER levels, thereby inducing ER stress ( Cantero-Recasens et al., 2010 ). RSV may induce NLRP3 inflammasome expression by activating ORMDL3 overexpression ( Cheng et al., 2023 ). 2.2.4 Rift Valley Fever virus Rift Valley Fever virus (RVFV) is a negative sense segmented single-stranded RNA virus with a size of about 12kb that belongs to the family Phenuiviridae and the genus Phlebovirus ( Ahsan et al., 2016 ; Gaudreault et al., 2019 ). In humans, RVFV can cause a variety of disease manifestations, ranging from febrile illness to hemorrhagic fever and death ( Ermler et al., 2014 ). The viral genome has three segments: the negative sense L (large) and negative sense M (medium) segments, and the double sense S (small) segment. These three segments encode multifunctional proteins. The S segment encodes nuclear protein (N) and non-structural protein S (NSs), the M segment encodes viral glycoprotein (Gn and Gc) and non-structural protein (NSm and a 78-kDa protein), and the L segment encodes viral RNA-dependent RNA polymerase ( Baer et al., 2016 ; Wright et al., 2019 ; Tercero et al., 2021 ; Lean and Johnson, 2022 ; Ganaie et al., 2023 ). The virus first attaches to the host membrane and enters the host cell through endocytosis. The viral genome is released into the cytoplasm. Negative RNA is transcribed into positive mRNA encoding viral proteins, and positive RNA serves as a template for the synthesis of new negative RNA. The newly synthesized viral protein and the replicated genome are combined in the cytoplasm to form new viral particles, which are released when they mature ( Wright et al., 2019 ). 2.2.4.1 Rift Valley Fever virus infection and NLRP3 inflammasome RVFV infection induces strong cytokines, which are essential for the recruitment of innate immune cells to the site of infection ( Nair et al., 2023 ). RVFV infected-cells secrete IL-1β, which is involved in the NLRP3 inflammasome, ASC oligomerization, and caspase-1 maturation. It has been reported that RVFV activates the NLRP3 inflammasome by inducing the formation of an inflammasome complex containing NLRP3 and MAVS, where MAVS are localized to NLRP3 during RVFV infection, leading to the maturation and secretion of IL-1β ( Ermler et al., 2014 ). 2.2.4.1 Rift Valley Fever virus infection and NLRP3 inflammasome RVFV infection induces strong cytokines, which are essential for the recruitment of innate immune cells to the site of infection ( Nair et al., 2023 ). RVFV infected-cells secrete IL-1β, which is involved in the NLRP3 inflammasome, ASC oligomerization, and caspase-1 maturation. It has been reported that RVFV activates the NLRP3 inflammasome by inducing the formation of an inflammasome complex containing NLRP3 and MAVS, where MAVS are localized to NLRP3 during RVFV infection, leading to the maturation and secretion of IL-1β ( Ermler et al., 2014 ). 2.2.5 Hantavirus Hantavirus (HTNV) is a coated single-stranded negative sense RNA virus belonging to the Bunyaviridae family ( Stock, 2008 ). The HTNV genome consists of three single-stranded negative sense RNA fragments: small (S), medium (M), and large (L) genome fragments encode four structural proteins (nuclear proteins N, glycoproteins Gn and Gc, and L proteins) ( Muyangwa et al., 2015 ; Meier et al., 2021 ). HTNV can cause acute febrile illness in humans. Hemorrhagic Fever with Renal Syndrome (HFRS) caused in Asia and Europe, and Hantavirus Pulmonary Syndrome (HPS) caused in the Americas ( Sargianou et al., 2012 ). Once Hantavirus enters the body, the virions bind to cell surface membrane receptors and enter the cell through endocytosis. The specific mechanism involves the transcription of the viral genome and the synthesis of viral RNA and viral proteins. The newly synthesized vRNA is coated with N protein to form ribonucleoprotein, which is then sent to the perinuclear membrane system. The synthesized viral proteins and genome are assembled into new viral particles inside the host cell. When the virion matures, it is released ( Muyangwa et al., 2015 ; Meier et al., 2021 ). 2.2.5.1 Hantavirus infection and NLRP3 inflammasome HTNV infection causes cells to enter a stress condition and induces the production of inflammatory cytokines ( Zhang et al., 2021 ). Studies have found that IL-1β is significantly elevated during HFRS. Induction of the human monocyte line THP-1 by HTNV revealed the secretion of IL-1β. The specific mechanism found that the induction of IL-1β by HTNV depended on the activation of caspase-1. Hantavirus thus induces the formation of the NLRP3 inflammasome in THP-1 cells, which may be an important factor in IL-1β levels in patients with HFRS ( Ye et al., 2015 ). 2.2.5.1 Hantavirus infection and NLRP3 inflammasome HTNV infection causes cells to enter a stress condition and induces the production of inflammatory cytokines ( Zhang et al., 2021 ). Studies have found that IL-1β is significantly elevated during HFRS. Induction of the human monocyte line THP-1 by HTNV revealed the secretion of IL-1β. The specific mechanism found that the induction of IL-1β by HTNV depended on the activation of caspase-1. Hantavirus thus induces the formation of the NLRP3 inflammasome in THP-1 cells, which may be an important factor in IL-1β levels in patients with HFRS ( Ye et al., 2015 ). 2.3 Double-stranded RNA virus 2.3.1 Reovirus DsRNA viruses have complementary dsRNA. This virus family displays two distinctive features: (1) their virus genome typically comprises 10-12 double-stranded RNA segments, and (2) the virus contains a double capsid structure but lacks an envelope ( Danthi et al., 2013 ). dsRNA viruses form a large group of RNA disease viruses, including reoviruses. Reoviruses comprise two concentric protein shells (the outer capsid and the core) that contain 10 segments of the dsRNA genome ( Ahlquist, 2006 ; Abad and Danthi, 2020 ). The infectious life cycle of reovirus starts with the attachment of the viral protein σ1 to sialic acid residues on the target cell surface. Alternatively, proteolysis by extracellular proteases leads to the formation of infectious subvirion particles; these particles directly enter the cell via membrane penetration. Thereafter, transcriptionally active virus core particles are released into the cytoplasm. After virus replication and assembly, mature virus particles are released ( Comins et al., 2008 ; Gong and Mita, 2014 ; Lemay, 2018 ; Abad and Danthi, 2020 ). 2.3.1.1 Reovirus virus infection and NLRP3 inflammasome Reoviruses use the host protein EphA2 to counteract the activation of the NLRP3 inflammasome ( Zhang et al., 2020 ). Zhang et al. have reported that reovirus infection of airway epithelial cells increases EPHA2-dependent NLRP3 phosphorylation; this inhibits the activation of the inflammasome by inhibiting the recruitment of other inflammasome components. Upon virus infection, EphA2 −/− mice exhibited increased inflammatory infiltration, resulting in the secretion of active IL-1β and IL-18 ( Zhang and Ricci, 2020 ; Zhang et al., 2020 ). 2.3.1 Reovirus DsRNA viruses have complementary dsRNA. This virus family displays two distinctive features: (1) their virus genome typically comprises 10-12 double-stranded RNA segments, and (2) the virus contains a double capsid structure but lacks an envelope ( Danthi et al., 2013 ). dsRNA viruses form a large group of RNA disease viruses, including reoviruses. Reoviruses comprise two concentric protein shells (the outer capsid and the core) that contain 10 segments of the dsRNA genome ( Ahlquist, 2006 ; Abad and Danthi, 2020 ). The infectious life cycle of reovirus starts with the attachment of the viral protein σ1 to sialic acid residues on the target cell surface. Alternatively, proteolysis by extracellular proteases leads to the formation of infectious subvirion particles; these particles directly enter the cell via membrane penetration. Thereafter, transcriptionally active virus core particles are released into the cytoplasm. After virus replication and assembly, mature virus particles are released ( Comins et al., 2008 ; Gong and Mita, 2014 ; Lemay, 2018 ; Abad and Danthi, 2020 ). 2.3.1.1 Reovirus virus infection and NLRP3 inflammasome Reoviruses use the host protein EphA2 to counteract the activation of the NLRP3 inflammasome ( Zhang et al., 2020 ). Zhang et al. have reported that reovirus infection of airway epithelial cells increases EPHA2-dependent NLRP3 phosphorylation; this inhibits the activation of the inflammasome by inhibiting the recruitment of other inflammasome components. Upon virus infection, EphA2 −/− mice exhibited increased inflammatory infiltration, resulting in the secretion of active IL-1β and IL-18 ( Zhang and Ricci, 2020 ; Zhang et al., 2020 ). 2.3.1.1 Reovirus virus infection and NLRP3 inflammasome Reoviruses use the host protein EphA2 to counteract the activation of the NLRP3 inflammasome ( Zhang et al., 2020 ). Zhang et al. have reported that reovirus infection of airway epithelial cells increases EPHA2-dependent NLRP3 phosphorylation; this inhibits the activation of the inflammasome by inhibiting the recruitment of other inflammasome components. Upon virus infection, EphA2 −/− mice exhibited increased inflammatory infiltration, resulting in the secretion of active IL-1β and IL-18 ( Zhang and Ricci, 2020 ; Zhang et al., 2020 ). 3 The clinical significance of NLRP3 inflammasome activation in the viral diseases The inflammasome plays a crucial role in sensing viral infections and related pathologies. The main physiological function of inflammasomes is to initiate immune responses and help maintain tissue homeostasis and repair ( Spel and Martinon, 2021 ). Infection with RNA viruses can induce the production of inflammatory factors that can lead to fatal diseases ( Choudhury et al., 2021 ). Overstimulation of innate immunity is the direct cause of persistent morbidity and death from pathogenic virus infection ( Deng et al., 2023 ). In some cases, it may also be associated with the development and exacerbation of diseases, such as chronic viral infections or autoimmune conditions ( Choudhury et al., 2021 ; Bonaventura et al., 2022 ). Targeting inflammasome or inflammasome-dependent cytokines such as IL-1β can be used as a therapeutic strategy ( Iannitti et al., 2016 ). Therefore, clinically targeting the activation of the inflammasome as a therapeutic approach to control viral infections requires further in-depth research. This will aid in the development of effective treatment strategies for managing viral infections. 4 Therapeutic strategies targeting the NLRP3 inflammasome for anti-inflammatory effects in virus infection In virus infections, NLRP3-mediated inflammation and cytokine storms are associated with disease severity; therefore, determining ways to regulate the inflammasome during virus infection is vital for decreasing inflammation and disease severity. As a result, studying and developing small-molecule drugs targeting the regulation of the NLRP3 inflammasome are significant. Cannabidiol exerts anti-inflammatory and immunomodulatory activities in the lungs and can inhibit the cytotoxicity and inflammation induced by SARS-CoV-2 spike proteins in Caco-2 cell lines via the peroxisome proliferator-activated receptor gamma-dependent TLR4/NLRP3/caspase-1 signaling pathway ( Corpetti et al., 2021 ). Statins also exert anti-inflammatory effects, and studies have discussed their potential beneficial effects in patients with COVID-19. Statins improve cytokine storms by inhibiting many molecular mechanisms, including the NF-κB pathway and NLRP3 inflammasome ( Rodrigues-Diez et al., 2020 ). Berberine is an isoquinoline alkaloid extracted from Chinese herbs. Liu et al. have reported that berberine inhibits the activation of the NLRP3 inflammasome induced by influenza virus in macrophages by inducing mitophagy and decreasing mitochondrial ROS ( Liu et al., 2020b ). In addition, probenecid and AZ11645373 can target the P2X7 receptor signaling pathway and inhibit the responses of the NLRP3 inflammasome during IAV infection, subsequently limiting excessive inflammation and illness during influenza ( Rosli et al., 2019 ). ERK and NF-κB inhibitors also play vital roles in regulating the NLRP3 inflammasome during virus infection ( Wei et al., 2016 ; Gong et al., 2022 ). The EV71-mediated NLRP3 inflammasome can be activated via the VIM–ERK–NF-κB pathway. As previously described, EV71 induces the activation of the NF-κB signaling pathway, which leads to the activation of the NLRP3 inflammasome and the transcription of IL-1β and IL-18 precursors. Therefore, VIM plays an important role in EV71-induced ERK phosphorylation, which triggers the activation of the NF-κB signaling pathway ( Gong et al., 2022 ). PD098059 is a p-ERK inhibitor that significantly inhibits VIM-mediated ERK1/2 phosphorylation in EV71-infected cells, thereby preventing inflammasome activation ( Gong et al., 2022 ). On the other hand, caffeic acid phenethyl ester is an NF-κB inhibitor that regulates the NLRP3 inflammasome after virus infection ( Lee et al., 2016 ; Gong et al., 2022 ). Disulfiram (DSF) plays an essential role in NLRP3 inflammasome-associated diseases ( Deng et al., 2020 ; Huang et al., 2021 ). Deng et al. have reported that DSF can effectively inhibit NLRP3 inflammasome activation and active IL-1β release. Mechanistically, DSF prevents lysosomal rupture and subsequent cathepsin B release into the cytoplasm. Furthermore, it decreases mitochondrial-independent ROS production. Cathepsin B and ROS are important upstream signals for activating the NLRP3 inflammasome ( Deng et al., 2020 ). MCC950, a potent and selective NLRP3 inhibitor, is active in mice and human cells in vitro . It can specifically act on the NRLP3 inflammasome and does not inhibit the NLRP1, AIM2, or NLRC4 inflammasome. MCC950 targets the NLRP3 inflammasome by blocking caspase-1-dependent IL-1β processing by inhibiting NLRP3-induced ASC oligomerization. Therefore, future clinical development of MCC950 or its derivatives may lead to new anti-inflammatory therapies ( Coll et al., 2015 ). For example, MCC950 has been used to inhibit NLRP3 inflammasome activity after RSV infection ( Malinczak et al., 2021 ). CY-09 specifically inhibits the activation of the NLRP3 inflammasome. CY-09 directly binds to the ATP-binding motif of the NACHT domain of NLRP3 and inhibits its ATPase activity, thereby inhibiting the assembly and activation of the NLRP3 inflammasome. Therefore, CY-09 provides a direct and selective small-molecule NLRP3 inhibitor for the targeted treatment of NLRP3-associated diseases ( Jiang et al., 2017 ). In summary, we can use various approaches to inhibit the activation of the NLRP3 inflammasome during virus infections. For example, the NLRP3 inflammasome can be inhibited by inhibiting upstream signaling molecules, inflammasome assembly, caspase-1 activation, GSDMD cleavage, etc. Furthermore, the use of inhibitors targeting the P2X7 receptor, K + efflux, ROS, or ATPase activity of NRLP3 is an efficient approach ( Lamkanfi et al., 2009 ; Hu et al., 2014 ; Jiang et al., 2017 ). All the abovementioned methods may contribute to antiviral immune defense strategies that inhibit inflammasome activation in virus-infected hosts. 5 Conclusions Virus infections threaten public health and economic growth worldwide. During virus invasion, the excessive activation of the NLRP3 inflammasome can lead to a cytokine storm that increases virus infection and damages the tissues. However, insufficient activation of the NLRP3 inflammasome prevents the organism from responding to the virus invasion, facilitating the survival of harmful microorganisms and resulting in infections and diseases ( Abdin et al., 2020 ; Sharma and Kanneganti, 2021 ). Therefore, the NLRP3 inflammasome is a double-edged sword in host defense against virus infection. The regulation of the NLRP3 inflammasome plays a crucial role in controlling inflammation. Summarizing studies on NLRP3 inflammasome activation during RNA virus infections, understanding virus-induced pyroptosis, and exploring approaches to inhibit virus-triggered activation can help to understand the pathogenesis of inflammatory diseases caused by RNA virus infections and discover new therapeutic targets. This will, in turn, help control virus infection and develop therapies based on the severity of the illness. Author contributions ZY: Writing – original draft, Writing – review & editing. XZ: Writing – original draft, Software. YG: Writing – original draft. YL: Writing – original draft. L-ML: Writing – original draft. YLL: Writing – original draft. YKL: Writing – original draft, Supervision. GY: Supervision, Writing – original draft, Writing – review & editing. PW: Supervision, Writing – original draft, Writing – review & editing. XC: Conceptualization, Funding acquisition, Supervision, Validation, Writing – original draft, Writing – review & editing. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404811/

Syndecan-1 Displays a Protective Role in Aortic Aneurysm Formation by Modulating T Cell-Mediated Responses

Objective Chronic inflammation drives progressive and pathological remodeling inherent to formation of abdominal aortic aneurysm (AAA). Syndecan-1 (Sdc-1) is a cell surface heparan sulfate (HS) proteoglycan that displays the capacity to modulate inflammatory processes within the vascular wall. In the current investigation, the role of Sdc-1 in AAA formation was examined using two models of experimental aneurysm induction, angiotensin II infusion and elastase perfusion. Methods and Results Sdc-1 deficiency exacerbated AAA formation in both experimental models and was associated with increased degradation of elastin, greater protease activity, and enhanced inflammatory cell recruitment into the aortic wall. Bone marrow transplantation studies indicated that deficiency of Sdc-1 in marrow-derived cells significantly contributed to AAA severity. Immunostaining revealed augmented Sdc-1 expression in a subset of AAA localized macrophages. We specifically characterized a higher percentage of CD4 + T cells in Sdc-1 deficient AAA and antibody depletion studies established the active role of T cells in aneurysmal dilatation. Finally, we confirmed the ability of Sdc-1 macrophage to modulate the inflammatory chemokine environment. Conclusions These investigations identify crosstalk between Sdc-1 expressing macrophages and AAA-localized CD4 + T cells, with Sdc-1 providing an important counterbalance to T cell driven inflammation in the vascular wall. Objective Chronic inflammation drives progressive and pathological remodeling inherent to formation of abdominal aortic aneurysm (AAA). Syndecan-1 (Sdc-1) is a cell surface heparan sulfate (HS) proteoglycan that displays the capacity to modulate inflammatory processes within the vascular wall. In the current investigation, the role of Sdc-1 in AAA formation was examined using two models of experimental aneurysm induction, angiotensin II infusion and elastase perfusion. Methods and Results Sdc-1 deficiency exacerbated AAA formation in both experimental models and was associated with increased degradation of elastin, greater protease activity, and enhanced inflammatory cell recruitment into the aortic wall. Bone marrow transplantation studies indicated that deficiency of Sdc-1 in marrow-derived cells significantly contributed to AAA severity. Immunostaining revealed augmented Sdc-1 expression in a subset of AAA localized macrophages. We specifically characterized a higher percentage of CD4 + T cells in Sdc-1 deficient AAA and antibody depletion studies established the active role of T cells in aneurysmal dilatation. Finally, we confirmed the ability of Sdc-1 macrophage to modulate the inflammatory chemokine environment. Conclusions These investigations identify crosstalk between Sdc-1 expressing macrophages and AAA-localized CD4 + T cells, with Sdc-1 providing an important counterbalance to T cell driven inflammation in the vascular wall.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679849/

Ethno-medicinal study of plants used for treatment of human and livestock ailments by traditional healers in South Omo, Southern Ethiopia

Background Plants have traditionally been used for treatment of human and livestock ailments in Ethiopia by different ethnic and social groups. However, this valuable source of knowledge is not adequately documented, which impedes their widespread use, evaluation and validation. Here, we recorded indigenous knowledge and standard practices for human and livestock disease control, of three ethnic groups (Aari, Maale and Bena-Tsemay) in South Omo Zone of Southern Nations, Nationalities and Peoples Regional State, Ethiopia. Methods A cross-sectional study was carried out using a semi-structured questionnaire to document knowledge of 50 traditional healers (40 male and 10 female) in medicinal plant use for treatment of human and livestock ailments. Descriptive statistics were used to analyze and summarize the ethno-botanical data. Results Ninety-one plants, with claimed medicinal properties against a total of 34 human and livestock ailments, were reported and botanically identified as belonging to 57 genera and 33 plant families. Most of the plant species reported belonged to one of seven major families: Lamiaceae , Solanaceae , Menispermiaceae, Fabaceae , Asteraceae , Plumbaginaceae and Geraniaceae . Woody plants (shrubs 21% and trees 29%) were the major growth form used, whilst roots (40%) and leaves (35%) were the major plant parts used in the study areas. Healers mostly practice oral administration of plant preparations (65%). Multiple medicinal plants were cited against particular ailments, and mixing of two or more different medicinal plants (14.3%) against a single ailment was also commonly reported. Conclusion This study showed that traditional medicine, mainly involving the use of medicinal plants, is playing a significant role in meeting the primary healthcare needs of the three ethnic groups. Acceptance of traditional medicine and limited access to modern healthcare facilities could be considered as the main factors for the continuation of the practice. Documented knowledge of the traditional healers can be used to support the country's human and livestock health care system and improve lives and livelihoods. Information generated will be used in future studies to validate bioactivity of selected medicinal plants used by traditional healers, so to increase their acceptability in health care systems both nationally and internationally. Background Plants have traditionally been used for treatment of human and livestock ailments in Ethiopia by different ethnic and social groups. However, this valuable source of knowledge is not adequately documented, which impedes their widespread use, evaluation and validation. Here, we recorded indigenous knowledge and standard practices for human and livestock disease control, of three ethnic groups (Aari, Maale and Bena-Tsemay) in South Omo Zone of Southern Nations, Nationalities and Peoples Regional State, Ethiopia. Methods A cross-sectional study was carried out using a semi-structured questionnaire to document knowledge of 50 traditional healers (40 male and 10 female) in medicinal plant use for treatment of human and livestock ailments. Descriptive statistics were used to analyze and summarize the ethno-botanical data. Results Ninety-one plants, with claimed medicinal properties against a total of 34 human and livestock ailments, were reported and botanically identified as belonging to 57 genera and 33 plant families. Most of the plant species reported belonged to one of seven major families: Lamiaceae , Solanaceae , Menispermiaceae, Fabaceae , Asteraceae , Plumbaginaceae and Geraniaceae . Woody plants (shrubs 21% and trees 29%) were the major growth form used, whilst roots (40%) and leaves (35%) were the major plant parts used in the study areas. Healers mostly practice oral administration of plant preparations (65%). Multiple medicinal plants were cited against particular ailments, and mixing of two or more different medicinal plants (14.3%) against a single ailment was also commonly reported. Conclusion This study showed that traditional medicine, mainly involving the use of medicinal plants, is playing a significant role in meeting the primary healthcare needs of the three ethnic groups. Acceptance of traditional medicine and limited access to modern healthcare facilities could be considered as the main factors for the continuation of the practice. Documented knowledge of the traditional healers can be used to support the country's human and livestock health care system and improve lives and livelihoods. Information generated will be used in future studies to validate bioactivity of selected medicinal plants used by traditional healers, so to increase their acceptability in health care systems both nationally and internationally. Background Knowledge can arise from scientific or traditional sources [ 1 ]. Traditional knowledge has been described as a cumulative body of knowledge, practice and belief, evolving through adaptive processes and handed over through generations by cultural transmission [ 2 ]. Traditional medicine is used throughout the world as it is heavily dependent on locally available plant species and plant-based products and capitalizes on traditional wisdom-repository of knowledge [ 3 ]. The wide spread use of traditional medicine could be attributed to cultural acceptability, economic affordability and efficacy against certain type of diseases as compared to modern medicines. Thus, different local communities in countries across the world have indigenous experience in various medicinal plants where they use their perceptions and experience to categorize plants and plant parts to be used when dealing with different ailments [ 4 ]. Plants have played a central part in combating many ailments in human and livestock in many indigenous communities, including Africa [ 5 ]. Traditional healers, and particularly medicinal plant herbalists, in Africa have a detailed knowledge-base of traditional medicine [ 6 , 7 ], which is transferred orally from one generation to the next through professional healers, knowledgeable elders and/or ordinary people [ 8 ]. In Ethiopia, traditional medicine has played a significant role in treating health problems in both livestock and humans [ 9 - 12 ]. Knowledge of medicinal plants of Ethiopia and of their uses provides vital contribution to human and livestock health care needs throughout the country [ 13 ]. The plant- based human and livestock health care persists and remains as the main alternative treatment for different ailments in Ethiopia, largely due to shortage of pharmaceutical products, prohibitive distance of the health service stations, unaffordable prices by small holder farmers and pastoralists for conventional drugs, emergence and re-emergence of certain diseases and appearance of drug resistant microbes and/or helminthes [ 14 ]. Much of the Ethiopian South Omo population is made up of nomadic pastoralists who depend upon livestock as their main source of livelihood [ 15 ]. The traditional medicinal plant lore and potentials of the three ethnic groups have not been investigated to a conspicuous level. Similar to many other rural communities in Ethiopia, the use of traditional medicinal plants plays a vital role in human and livestock health care systems in the pastoral and agro-pastoral communities of the study areas. The three ethnic groups (Aari, Maale and Bena-Tsemay) in South Omo are expected to be custodians of valuable indigenous knowledge on the use of their traditional medicinal plants, which they use for treating of human and livestock ailments. Currently, access to modern health services for both human and livestock is very limited and/or non-existent for some community members of the study areas. This study is basically focusing on a remote and pastoralist areas where the accessibility, affordability and cultural acceptability of the use of medicinal plants for treatment of human and livestock ailments is very significant. The dependence of the plant-based health care system could partly be attributed to underdeveloped infrastructures and modern medical health care system in the general area. Unless the plants are conserved and the ethno-medicinal knowledge is documented, there is a danger that both the valuable medicinal plants and the associated indigenous knowledge of the ethnic groups could vanish forever due to lack of documentation [ 6 ] and loss of valuable medicinal plants due to population pressure, agricultural expansion and deforestation [ 16 ], as well as due to drought, urbanization and acculturation [ 17 ]. Furthermore, pastoral and agro-pastoral communities of these ethnic groups have remained ethno-medicinally unexplored and there is no comprehensive account of the medicinal plant-based practices. Therefore, the objective of this study was to document the indigenous knowledge and practices of the healers in the study areas (the three ethnic societies in the South Omo zone of the Southern Nations, Nationalities and Peoples Region (SNNPR) of Ethiopia) on medicinal plants for human and livestock disease control. Below, we describe the study area, how informants were selected, the type of information we have gathered, and the use of the informant consensus factor to synthesize the information obtained, followed by presenting our findings and discussing them in context of existing literature. Materials and methods Description of the study area and the people The study was conducted in selected areas of the South Omo zone, inhabited by three ethnic groups (Aari, Maale and Bena-Tsemay). The administrative zone is bordered on the south by Kenya, on the west by Bench Maji, on the northwest by Kefa-Sheka, on the north by North Omo, on the northeast by the Derashe and Konso Special Woredas, and on the east by the Oromia Regional State (Figure 1 ). Figure 1 Map of South Omo zone indicated by the shaded region. The annual average temperature of the area is 28°C with an annual average rainfall of 1190 mm [ 18 ]. The 2007 census data revealed that the zone has a total population of 573,435 people (286,607 male and 286,828 female) of which 4.45% are pastoralists and semi-pastoralists. Their culture places highest value on cattle, with relatively less on mixed farming (Central Statistics Agency, 2007). Among the six largest ethnic groups living in the area, Aari, Maale and Hamer-Bena (Bena-Tsemay) comprise 42.9%, 13.5% and 12.9%, respectively. Aari, Maale and Hamer are the major languages spoken by 43.3%, 13.7% and 13% of the people in the area, respectively [ 19 ]. Selection of informants A total of 50 traditional healers, i.e. 24 from the Aari (20 males and 4 females), 16 from the Maale (13 males and 3 females) and 10 from Bena-Tsemay (7 males and 3 females) ethnic groups of different ages (32–81 years) were selected with the help of local elders, agricultural and health extension workers and administrative personnel, and interviewed as key informants. The selected healers were well-known in the community due to their long practice in providing services related to traditional health care to the community. Prior to the interview process, discussion was held with the informants through assistance of local elders to elaborate the objective of the study. This was done to clarify the purpose and build confidence of the respondents to provide reliable information without suspicion. After the discussion, 6 potential informants (2 from Aari, 1 from Maale and 3 from Bena-Tsemay) showed unwillingness to share their medicinal plant knowledge and withdrew from the study. The 50 healers that participated in the study were asked to provide information on plant(s) use against any kind of illness in humans and livestock, and in particular the type of plant (e.g. trees, shrubs, herbs, climbers or others) and the parts used (e.g. roots, leaves, seeds, flowers, stems or others), the methods of remedy preparation (e.g. concoction, filtrate, paste on, smoke bath, pounded or others), the routes of administration (e.g. oral, topical, smoke bath, nasal or others) and the dosage. Specimen of the reported medicinal plants were collected during the interview from the field, coded and sent to the National Herbarium of Addis Ababa University for botanical identification and archiving. Data collection and analysis Descriptive statistics were used to analyze and summarize the ethno-botanical data. Based on the information obtained from the informants, the ailments reported were grouped into a total of 12 categories (Table 1 ). To estimate medicinal plant use variability and to assist prioritizing medicinal plants for further studies, the informant consensus factor (ICF) was calculated [ 20 ]. The ICF is calculated using the number of use citations (Nuc) in each category and the number of plant species (Ns) cited through the following formula: ICF = Nuc − Ns Nuc − 1 Thus, ICF values range from 0 to 1, with high values (i.e. close to 1) indicating that relatively few plants are used by a large proportion of informants, while low values (<0.5) indicate that informants do not agree on the plant species to be used to treat a category of ailments. Table 1 Informant consensus factor (ICF) values of use category of multiple plants claimed as having medicinal values by informants of the three ethnic groups from South Omo zone, southern Ethiopia Use category Plant species Number of use citation * % of all citations ICF value Anthrax Dombeya spp. (7), Achyrospremum schimperi (6), Tragia doryodes (5), Geranium aculeolatum (8) 26 6.5 0.88 Poisonous plants Solanum spp. (5), Oxalis corniculata (7) 12 2.9 0.91 Skin infections and external parasites Solanum incanum (6), Desmodium dichotomum ( 7), Laggera tomentosa (8), Geranium arabicum (6), Nicotiana tabacum (6), Premna schimperi (9), Calpurnia aurea (8) 50 12.4 0.88 Pain related illnesses Senna spp. (4), Tagetes spp . (9), Monsonia parvifolia (8), Plumbago caerulea (4), Desmodium delotum (3) 28 6.9 0.85 Malaria and anemia like syndrome with jaundice Carphalea glaucescens (5), Cissampeolos mucronata (6), Indigofera arrecta (6), Cissampelos spp. (2), Lantana trifolia (4), Luecas stachydiformis (2), Premna oligotricha (5), Droguetia iners (6), Zornia glochidiata (3), Galinsoga parviflora (3) 40 9.9 0.77 Abdominal/stomach disorders and internal parasites Pycnostachys meyeri (5), Zornia apiculata (4), Achyranthes aspera (4), Stereospermum kunthianum (7), Cissampelos pariera (6), Cissampelos capensis (3), Lagenaria siceraria (3), Momordica charantia (2), Tagetes minuta (6), Premna spp. (5), Salvia acuminata (4), Echinacea spp. (2), Pelargonium alchemilloides (5), Orthosiphon surmentosus (6), Mucuna melanocarpa (8), Sida spp. (5), Chasmanthera welwitschii (2), Rytigynia spp. (5), Zornia latifolia (3), Centella asiatica (3), Ipomoea eriocarpa (7), Chlaenandra ovata (2) 97 24.1 0.78 Snake bite/poisoning Alonsoa acutifolia (5), Plumbago pulchella (6), Opericulicarya gummifera (6), Plectranthus globosus (6), Barthlottia madagascariensis (3), Ludwigia abyssinica (6), Claoxylopsis andapensis (8), Carissa carandas (5), Hyparrhenia hirta (4), Verbena officinalis (5) 54 13.4 0.83 Mich and Megagna ** Dobera spp. (6), Droguetia debilis (5), Justica dianthera (4) 15 3.7 0.86 Coughing in equines and ruminants Justicia diffusa (5), Solanum bellum (5), Datura stramonium (4), Vaccaria hispanica (6), Ozoroa insignis (6) 26 6.5 0.84 Removal of retained placenta Solanum acaule (2), Solanum acuminatum (3), Dovyalis spp. (5), Galinsoga quadriradiata (6), Colocasia esculenta (4), Plumbago zeylanica (6), Momordica spp . (2) 28 6.9 0.78 Evil eye Drymaria spp. (4), Plectranthus glabriflorus (3), Cissus quadrangularis (4), C ryptocarpus spp . (3), Colignonia ovalifolia (4), Achyrospermum africanum (5), Drymaria cordata ( 3), Plumbago auriculata (4), Chelonopsis moschata (2), Withania somnifera (3), Plumbago spp. (2) 37 9.2 0.72 Black leg Momordica foetida (7), Pentas suswaensis (8), Chasmanthera dependens (3) 18 4.5 0.88 Rabies Caylusea abyssinica (3) 3 0.7 1.00 Improve milk production in cows Indigofera trita (6) 6 1.4 1.00 * Numbers in parenthesis indicate the number of citation of that plant by informants (traditional healers and community members) against a particular ailment category. ** Ailment characterized with fever, head ache and sweating. Description of the study area and the people The study was conducted in selected areas of the South Omo zone, inhabited by three ethnic groups (Aari, Maale and Bena-Tsemay). The administrative zone is bordered on the south by Kenya, on the west by Bench Maji, on the northwest by Kefa-Sheka, on the north by North Omo, on the northeast by the Derashe and Konso Special Woredas, and on the east by the Oromia Regional State (Figure 1 ). Figure 1 Map of South Omo zone indicated by the shaded region. The annual average temperature of the area is 28°C with an annual average rainfall of 1190 mm [ 18 ]. The 2007 census data revealed that the zone has a total population of 573,435 people (286,607 male and 286,828 female) of which 4.45% are pastoralists and semi-pastoralists. Their culture places highest value on cattle, with relatively less on mixed farming (Central Statistics Agency, 2007). Among the six largest ethnic groups living in the area, Aari, Maale and Hamer-Bena (Bena-Tsemay) comprise 42.9%, 13.5% and 12.9%, respectively. Aari, Maale and Hamer are the major languages spoken by 43.3%, 13.7% and 13% of the people in the area, respectively [ 19 ]. Selection of informants A total of 50 traditional healers, i.e. 24 from the Aari (20 males and 4 females), 16 from the Maale (13 males and 3 females) and 10 from Bena-Tsemay (7 males and 3 females) ethnic groups of different ages (32–81 years) were selected with the help of local elders, agricultural and health extension workers and administrative personnel, and interviewed as key informants. The selected healers were well-known in the community due to their long practice in providing services related to traditional health care to the community. Prior to the interview process, discussion was held with the informants through assistance of local elders to elaborate the objective of the study. This was done to clarify the purpose and build confidence of the respondents to provide reliable information without suspicion. After the discussion, 6 potential informants (2 from Aari, 1 from Maale and 3 from Bena-Tsemay) showed unwillingness to share their medicinal plant knowledge and withdrew from the study. The 50 healers that participated in the study were asked to provide information on plant(s) use against any kind of illness in humans and livestock, and in particular the type of plant (e.g. trees, shrubs, herbs, climbers or others) and the parts used (e.g. roots, leaves, seeds, flowers, stems or others), the methods of remedy preparation (e.g. concoction, filtrate, paste on, smoke bath, pounded or others), the routes of administration (e.g. oral, topical, smoke bath, nasal or others) and the dosage. Specimen of the reported medicinal plants were collected during the interview from the field, coded and sent to the National Herbarium of Addis Ababa University for botanical identification and archiving. Data collection and analysis Descriptive statistics were used to analyze and summarize the ethno-botanical data. Based on the information obtained from the informants, the ailments reported were grouped into a total of 12 categories (Table 1 ). To estimate medicinal plant use variability and to assist prioritizing medicinal plants for further studies, the informant consensus factor (ICF) was calculated [ 20 ]. The ICF is calculated using the number of use citations (Nuc) in each category and the number of plant species (Ns) cited through the following formula: ICF = Nuc − Ns Nuc − 1 Thus, ICF values range from 0 to 1, with high values (i.e. close to 1) indicating that relatively few plants are used by a large proportion of informants, while low values (<0.5) indicate that informants do not agree on the plant species to be used to treat a category of ailments. Table 1 Informant consensus factor (ICF) values of use category of multiple plants claimed as having medicinal values by informants of the three ethnic groups from South Omo zone, southern Ethiopia Use category Plant species Number of use citation * % of all citations ICF value Anthrax Dombeya spp. (7), Achyrospremum schimperi (6), Tragia doryodes (5), Geranium aculeolatum (8) 26 6.5 0.88 Poisonous plants Solanum spp. (5), Oxalis corniculata (7) 12 2.9 0.91 Skin infections and external parasites Solanum incanum (6), Desmodium dichotomum ( 7), Laggera tomentosa (8), Geranium arabicum (6), Nicotiana tabacum (6), Premna schimperi (9), Calpurnia aurea (8) 50 12.4 0.88 Pain related illnesses Senna spp. (4), Tagetes spp . (9), Monsonia parvifolia (8), Plumbago caerulea (4), Desmodium delotum (3) 28 6.9 0.85 Malaria and anemia like syndrome with jaundice Carphalea glaucescens (5), Cissampeolos mucronata (6), Indigofera arrecta (6), Cissampelos spp. (2), Lantana trifolia (4), Luecas stachydiformis (2), Premna oligotricha (5), Droguetia iners (6), Zornia glochidiata (3), Galinsoga parviflora (3) 40 9.9 0.77 Abdominal/stomach disorders and internal parasites Pycnostachys meyeri (5), Zornia apiculata (4), Achyranthes aspera (4), Stereospermum kunthianum (7), Cissampelos pariera (6), Cissampelos capensis (3), Lagenaria siceraria (3), Momordica charantia (2), Tagetes minuta (6), Premna spp. (5), Salvia acuminata (4), Echinacea spp. (2), Pelargonium alchemilloides (5), Orthosiphon surmentosus (6), Mucuna melanocarpa (8), Sida spp. (5), Chasmanthera welwitschii (2), Rytigynia spp. (5), Zornia latifolia (3), Centella asiatica (3), Ipomoea eriocarpa (7), Chlaenandra ovata (2) 97 24.1 0.78 Snake bite/poisoning Alonsoa acutifolia (5), Plumbago pulchella (6), Opericulicarya gummifera (6), Plectranthus globosus (6), Barthlottia madagascariensis (3), Ludwigia abyssinica (6), Claoxylopsis andapensis (8), Carissa carandas (5), Hyparrhenia hirta (4), Verbena officinalis (5) 54 13.4 0.83 Mich and Megagna ** Dobera spp. (6), Droguetia debilis (5), Justica dianthera (4) 15 3.7 0.86 Coughing in equines and ruminants Justicia diffusa (5), Solanum bellum (5), Datura stramonium (4), Vaccaria hispanica (6), Ozoroa insignis (6) 26 6.5 0.84 Removal of retained placenta Solanum acaule (2), Solanum acuminatum (3), Dovyalis spp. (5), Galinsoga quadriradiata (6), Colocasia esculenta (4), Plumbago zeylanica (6), Momordica spp . (2) 28 6.9 0.78 Evil eye Drymaria spp. (4), Plectranthus glabriflorus (3), Cissus quadrangularis (4), C ryptocarpus spp . (3), Colignonia ovalifolia (4), Achyrospermum africanum (5), Drymaria cordata ( 3), Plumbago auriculata (4), Chelonopsis moschata (2), Withania somnifera (3), Plumbago spp. (2) 37 9.2 0.72 Black leg Momordica foetida (7), Pentas suswaensis (8), Chasmanthera dependens (3) 18 4.5 0.88 Rabies Caylusea abyssinica (3) 3 0.7 1.00 Improve milk production in cows Indigofera trita (6) 6 1.4 1.00 * Numbers in parenthesis indicate the number of citation of that plant by informants (traditional healers and community members) against a particular ailment category. ** Ailment characterized with fever, head ache and sweating. Results and discussion Knowledge of informants on medicinal plants Indigenous people of different localities have their own specific knowledge on plant use, management and conservation [ 21 ]. Medicinal plants represent a significant contribution to human and livestock health and it has been suggested that their use is one of the most significant ways in which humans directly reap the benefits provided from biodiversity [ 22 , 23 ]. During the field survey in our study areas, informants reported ethno-medicinal data of 91 species of plants distributed across 33 families and 57 genera as having medicinal properties against 34 ailments (12 in humans, 11 in livestock and 11 in both human and livestock). The 91 plant species that are used by traditional healers among the three ethnic groups interviewed were identified and documented. Among the medicinal plants identified most of them belong to the seven families as shown in Figure 2 . The plant family Lamiaceae was most frequently represented amongst the documented useful species, with a total of 12 species out of the 91 plants identified, followed by Solanaceae with a total of 8 species and Menispermiaceae , and Fabaceae with total of 7 species each, and others constitute one up to six plant species per family. Figure 2 The percentage of plant species distributed over seven major families. The informants cited 32 (35.2%), 35 (38.5%) and 24 (26.4%) plants as having medicinal properties against ailments of livestock, humans or both livestock and humans, respectively (Table 2 ). The informants also reported multiple plant remedies against certain ailments, such as snake bite/poisoning for both humans and livestock. Depending upon the type of illness, the use of two or more parts of medicinal plants was also reported by some healers as common practice. For example, of the total 32 medicinal plants purely claimed for livestock illnesses, eight were used in two-plant combination preparations, and these target three different ailments, i.e. to treat epizootic lymphangitis, removal of fetal membrane, and anemia with jaundice (Table 2 ). Table 2 Medicinal plants, with family, scientific and local name, for selected ailments of human and/or veterinary importance, with parts used and preparations, as claimed by informants of the three ethnic groups from South Omo zone, Southern Ethiopia Family Scientific name Local name * Voucher number Use(s) Parts used and preparation Importance Amaranthaceae Achyranthes aspera Linn. Busino (M) KTG28 Abdominal pain and tonsillitis Root chopped and mixed with water and taken orally. Leaf chewed and the extract kept near the inflammation area Human Lamiaceae Achyrospermum africanum Hook.f. ex Baker Kebit buda (A) KTG54 Evil eye Leaf and root chopped and soaked with water Human Achyrospermum schimperi (Hochst. ex Briq.) Perkins Abasanga medihanit (A,Amh) KTG65 Anthrax Leaf and flower chopped and soaked with hot water and drenched Veterinary Scrophulariaceae Alonsoa acutifolia Ruiz & Pav. Shosha tesha (M) KTG66 Snake bite/poison Root chopped and mixed with Plectranthus glandulosus in water and the filtrate drenched Human and Veterinary Barthlottia madagascariensis E.Fisch. Unkown (A) KTG18 Snake bite/poison Concoction Human and Veterinary Fabaceae Calpurnia aurea (Aiton)Benth. Kaino(M) KTG91 Flea and louse infestation Freshly chopped or dried and ground leaf mixed with water and applied to the flea and louse infested areas Veterinary Apocynaceae Carissa carandas L. Goiti(B),ebab medihanit (Amh) KTG45 Snake bite/poison The leaf chopped, mixed with water taken orally Human and Veterinary Rubiaceae Carphalea glaucescens (Hiern) Verdc Wariamo (M) KTG31 Anaemia (known as Airo) Leaf powdered mixed with Ipomoea kiwuensis smoked for three days Human Recedaceae Caylusea abyssinica (fresen.) Fisch. & Mey Giesilla (M) KTG64 Rabies (effective even when clinical signs are present) Root chopped and mixed with cold water and drenched Human and Veterinary Apiaceae Centella asiatica - (L.)Urb. Busino (M) KTG87 Abdominal ache Root dried, ground and mixed with cold water when needed (on cup or glass full) Human Menispermaceae Chasmanthera dependens (Hochst) Moshito (M) KTG33 Black leg Root bark and leaf dried and ground and given to emaciate calf as much as possible Veterinary Chasmanthera welwitschii Troupin Heilho (M) KTG27 Antiamoeba Root bark and leaf dried and ground and given to emaciate calf as much as possible Veterinary Lamiaceae Chelonopsis moschata Miq. Kebit buda (A) KTG47 Evil eye Leaf and root chopped and soaked with water Human Menispermaceae Chlaenandra ovata . Miq. Eincht (A) KTG73 Abdominal ache Root chopped and mixed with water and drenched Human Cissampelos capensis L.f . Wontin kanna (A) KTG76 Abdominal cramp Root chopped, powdered, soaked with water, filtered and drenched Human Cissampelos mucronata A.Rich. Kawuro (M) KTG37 Anaemia with jaundice Leaf collected, dried, ground and mixed with hot water and two spoonful taken at once Human Cissampelos pareira L . Shelindo (M) KTG70 Broad spectrum anti- helminthiasis Root ground mixed with large amount of water and drenched. It causes fever diarrhea then animals are cured Veterinary Cissampelos spp. Kawto (M) KTG32 Anaemia like syndrome with jaundice Leaf collected, dried, ground with local mill and mixed with hot water and two spoonful taken at once (bitter) Human Vitaceae Cissus quadrangularis L. Bararo (M) KTG20 Evil eye Tied under belly Veterinary Euphorbiaceae Claoxylopsis andapensis Radcl.-Sm. Dorba (A) KTG43 Snake bite/poison Bark and leaf chopped, soaked in water and drenched Human Nyctaginaceae Colignonia ovalifolia Heimerl Afesha (A) KTG41 Evil eye Leaf squeezed and inhaled Human Araceae Colocasia esculenta (L.) Schott Haleko (A,M,BT) KTG77 To detach retained fetal membrane Root dried, ground and mixed with powdered root of Momordica spp. and all soaked in warm water and one cupful drenched Veterinary Nyctaginaceae Cryptocarpus spp . Afei tesha (M) KTG23 Evil eye Root chopped and mixed with cold/hot water Human Solanaceae Datura stramonium L. Onidod (A) KTG05 Coughing (for horses, mules and donkeys) Leaf chopped and mixed with cold water and drenched via nose Veterinary Papilionaceae Desmodium dichotomum (Willd.) DC. Muasii (A) KTG07 Epizootic lymphangitis (tushita) Root chopped, mixed with cold water and drenched via nose Veterinary Desmodium delotum J.F. Macbr. Not known (A) KTG42 Eye illness Leaf apex chopped, soaked in water, applied to sick eye Human and Veterinary Salvadoraceae Dobera spp. Mitch medihanit (A, Am) KTG89 Mitch Leaf boiled with water and inhaled Human Sterculiaceae Dombeya spp. Bata (A) KTG15 Anthrax The leaf is chopped and mixed with Tragia doryodes and the filtrate taken orally Human and Veterinary Flacortiaceae Dovyalis spp. Mukale (M) KTG85 To detach retained placenta Leaf chopped and mixed with hot water and given as ad libtum Veterinary Urticaceae Droguetia debilis Rendle Megagna medanit (Amh) KTG52 Megagna Leaf apex chopped and pasted on the pain area Human Droguetia iners (Forssk.) Schweinf. Yewoba medihanit (A) KTG61 Malaria Leaf chopped and mixed with Premna oligotricha and boiled together one glassful drenched Human Caryophyllaceae Drymaria cordata (L.) Willd. ex Schult. Yebuda medihanit (A, Amh) KTG59 Evil eye in animals Leaf and root chopped mixed in water and the filtrate is sprayed on animal body and the sediments are drenched Human and Veterinary Drymaria spp . Unknown (A) KTG17 Evil eye The leaf is chopped and mixed with water and the filtrate taken orally Human Asteraceae Echinacea spp. Unkown KTG53 Diarrhoea alone Root chopped and soaked in warm water taken orally Human and Veterinary Galinsoga parviflora Cav. Midirberbere, (Amh) KTG14 Anaemia with jaundice The flower is chopped and mixed with Monosonia longipes and warmed on and applied of gum of achy tooth Human Galinsoga quadriradiata Cav. Mukala (M) KTG74 To detach retained fetal membrane and/or placenta Leaf chopped and mixed with Plumbango zeylanica and then drenched Veterinary Geraniaceae Geranium aculeolatum Oliv. Abasanga medihanit (A, AM) KTG72 Anthrax Leaf chopped and rubbed on wounded part Veterinary Geranium arabicum Forssk. Tushita (A) KTG26 Epizootic lymphangitis Root chopped and mixed with chopped root of Laggera tomentosa and one bear bottle drenched through left nose of horse. Veterinary Poaceae Hyparrhenia hirta (L.) Stapf Goiti ebab" medihanit (B) KTG46 Snake bite/poison Plant material chopped and soaked in hot water and the filtrate drenched Human and Veterinary Fabaceae Indigofera arrecta A.Rich. Wareami (A) KTG80 Anaemia with jaundice Leaf dried and smoked to patients Human Indigofera trita L.f. Wusis (A) KTG01 To improve milk production of cows Root chopped, mixed with water and drenched Veterinary Convolvulaceae Ipomoea eriocarpa R. Br. Choko (M) KTG40 Endoparasite Root chopped and mixed with water, then the filtrate is drenched and rest sediments are poured on the wound part Veterinary Acanthaceae Justica dianthera Vell. Mitch(A, Amh) KTG51 Mitch The leaf apex boiled with water and the vapor inhaled and/or the filtrate drenched Human Justicia diffusa Willd. Makaiso (A) KTG04 For coughing of equines Leaf chopped and mixed with cold water and drenched via nose Veterinary Cucurbitaceae Lagenaria siceraria (Molina) Standl. Busino (M) KTG35 Diarrhoea and vomiting Leaf chopped and ground and the drench the filtrate Human Asteraceae Laggera tomentosa Schultz Bip. Tushita (A) KTG71 Epizootic lymphangitis Root chopped and mixed with chopped root of Geranium arabicum and one bear bottle drenched through horses nose Veterinary Verbenaceae Lantana Trifolia L. Yewoba medihanit (A) KTG55 Malaria (shivering type, vivax) Root chopped and soaked with water and mixed with local alcoholic drink (Areke) Human Lamiaceae Leucas stachydiformis (Benth.) Hochst. ex Briq. Businae (M) KTG19 Anaemia with jaundice Leaf and bark chopped and drench the filtrate or inhalation Human Onagraceae Ludwigia abyssinica A.Rich. Yechira ebab medihanit (Amh) KTG44 Snake bite/poison Stem and root mixed with other plants and applied orally Human Cucurbitaceae Momordica foetida Schumach. Chekko (M,B) KTG03 Black leg Root chopped, soaked in water for half a day and a filtrate is drenched Veterinary Momordica charantia L. Unknown (A) KTG90 Diarrhea and vomiting Leaf and root ground well and mixed with milk and taken orally Human Momordica spp. Kill (M) KTG79 To detach fetal membrane Root dried, ground and mixed with Colocasia esculenta , all soaked in warm water and one cupful filtrate drenched Veterinary Geraniaceae Monsonia parvifolia Schinz Not known (A) KTG58 Tooth ache Seed and leaf crushed and mixed with salt and Galinsoga parvifolia ; made hot on fire with "enset leaf" and applied on gum. Human Papilionaceae Mucuna melanocarpa Hochst Salabano (M) KTG86 For calf Ascariasis Leaf ground and mixed with water and drenched that induces diarrhea Veterinary Solanaceae Nicotiana tabacum L. Bangiso(M) KTG78 Tick infestation Root chopped and mixed with water and dressed to the tick infested area on cow and calf Veterinary Anacardiaceae Operculicarya gummifera (Sprague) Capuron Dorba (M) KTG38 Snake bite/poison Orally taken butt its preparation is not specified due to unwillingness of the respondent Human and Veterinary Lamiaceae Orthosiphon sarmentosus A.J. Paton & Hedge Zititu (A) KTG67 Ascariasis Leaf chopped, soaked in water and a glass full filtrate drunken Human Oxalidaceae Oxalis corniculata L . Dani (M) KTG02 Toxic Root chopped, cooked for two days and more and the paste rubbed on arrow tip to hunt wild animals Human and veterinary Anacardiaceae Ozoroa insignis Delile Bussa (M) KTG39 For coughing of equines Bark dried, powdered and mixed with cold water and the filtrate drenched Veterinary Geraniaceae Pelargonium alchemilloides (L.) Aiton Unkown KTG13 Constipation Root chopped and soaked in warm water taken orally Human Rubiaceae Pentas suswaensis Verdc. Haromato (M) KTG63 Aba gorba "Black leg" Leaf chopped and mixed with boiled water and the filtrate is drenched Veterinary Lamiaceae Plectranthus glabriflorus P.I.Forst. Gullo/Karika (A) KTG09 Evil eye Leaf soaked in hot water and drunken Human Plectranthus globosus Ryding Chambuase (M) KTG60 Snake bite/poison Leaf chopped mixed with Alectra sessiliflora mixed in cold water and taken orally Human Plumbaginaceae Plumbago auriculata Lam. Masilok (M) KTG81 For animal evil spirit Leaf chopped and soaked in water and the filtrated drenched and the remaining sediments pasted on the body Human and Veterinary Plumbago caerulea Kunth Wugat medihanit (Amh) KTG68 Back and side pain Root and seed are chopped and mixed with hot water and onion Human Plumbago pulchella Boiss. Not known (A) KTG12 Snake bite/poison Fresh leaf chopped and mixed with cold water Human and Veterinary Plumbago spp. Misirich (M) KTG49 Evil eye in animals Leaf chopped and soaked in water and the filtrated drenched and the remaining sediments pasted on the body Veterinary Plumbago zeylanica L . Telba (M) KTG75 To detach retained fetal membrane Seed ground by traditional mortar mixed with Galinsoga Parviflora and boiled with water and drenched Veterinary Lamiaceae Premna oligotricha Baker Yewoba medihanit (A) KTG34 Malaria (non-shivering type, falciparium) Leaf collected and ground and mixed with water Human Premna schimperi Engl. Bangizo(M) KTG21 Dermatophilous and mite infestation Root chopped and soaked in warm water over night and filtrate applied topically to treat dermatophytes and tick mite infestations Veterinary Premna spp. Anchiphi (M) KTG57 Diarrhea in calf Leaf powdered and mixed with water and the filtrate drenched Veterinary Pycnostachys meyeri Gürke ex Engl. Unkown (A) KTG83 Abdominal pain (children) Fresh root chopped and mixed with cold water and drenched Human Rubiaceae Rytigynia spp. Golodo (M) KTG30 Typhoid (Micho) Leaf chopped and mixed with water and taken orally Human Lamiaceae Salvia acuminata Ruiz & Pav. Anchino (M) KTG48 Diarrhoea alone Chewing the leaf Human Fabaceae Senna spp. Diko (M) KTG84 Joint ache and breakage of bones Leaf rubbed on affected parts and some leaf chopped and soaked in warm water and drenched Human and Veterinary Malvaceae Sida spp. Moishita (M) KTG25 Anti-parasitic/ to fatten calf Leaf chopped and soaked in water and the filtrate is drenched repeatedly Veterinary Solanaceae Solanum bellum S. Knapp Ondod (M) KTG22 Coughing of equines(Busa) Root chopped and mixed with cold water and the filtrate drenched either by nose or mouth Veterinary Solanum acaule Bitter Mushta (A) KTG62 Removes retained placenta Root chopped and mixed with cold water and the filtrate is applied nasally Veterinary Solanum acuminatum Ruiz & Pav. raki (A) KTG82 To detach retained placenta Root chopped, mixed with cold water and drenched orally Human and Veterinary Solanum incanum L. Garint (A) KTG06 Epizootic lymphangitis(tushita) Root chopped and mixed with cold water and drenched via nose Veterinary Solanum spp. Danni (M) KTG50 Poisonous to animals Root chopped and concoction with water until paste is formed, rubbed on arrow tip & used for hunting Human and Veterinary Bignoniaceae Stereospermum kunthianum Cham. Addi (M) KTG29 Abdominal pain Root chopped and mixed with water/coffee and taken in ad libitum Human Asteraceae Tagetes spp. Businae (A) KTG88 Muscle cramp and joint pain Leaf rubbed well and oils from the leaf are swabbed on areas where pain felt. Fumigation is also possible. Boiled filtrate is drenched Human Tagetes minuta L. Kawato (M) KTG36 Diarrhea and vomiting Leaf chopped and ground and the drench the filtrate Human Euphorbiaceae Tragia doryodes M.G.Gilbert Anderta (A) KTG16 Anthrax The leaf is chopped and mixed with Dombya spp. and the filtrate taken orally Veterinary Caryophyllaceae Vaccaria hispanica (Mill.) Rauschert Sanba tesha (M) KTG69 For contagious bovine pleuropneumonia and contagious caprine pleuropneumonia Root chopped and mixed with large amount of water. It gets bloody color (the bloody color indicates appropriate concentration), then drenched Veterinary Verbenaceae Verbena officinalis L. Guni tesha (A) KTG56 Snake bite/poison Leaf squeezed by hand and mixed with water and drenched by water Human and Veterinary Solanaceae Withania somnifera (L.) Dunal Buto/wogare (M) KTG24 Night mare Roots powdered and children smoked until they cough Human Fabaceae Zornia apiculata Milne-Redh. Medhanit (A) KTG10 Abdomen ache and vomiting in children Fresh root chopped and mixed with cold water and drenched Human Zornia glochidiata DC. Halimi (A) KTG11 Malaria Root bark is chopped and boiled/concoction with local drinks and boiled coffee leaf Human Zornia latifolia Sm . Medihanit (A) KTG08 Abdominal pain, vomiting Fresh leaf chopped and mixed to form filtrate Human * A=Aari; M=Maale; B=Benigna; Amh = Amharic. This is the first study that documented plants used for disease control by the three ethnic groups in South Ethiopia. Previous studies have documented indigenous knowledge of medicinal plants and medicinal plant practices used in other parts of the country and by other ethnic groups including those in southern Ethiopia [ 24 , 25 ], northern and northwestern Ethiopia [ 8 , 26 - 28 ], and southwestern Ethiopia [ 29 - 31 ]. Our study thus complements existing studies but also extends them to pastoral areas where the ecology, practices, biodiversity, accessibility and cultural acceptability of medicinal plants are very different from the highlands. The aforementioned reports and our study taken together capture a wide range of different ethnic and social groups, which is a reflection of the richness of knowledge in use of plants for medicinal purposes, and the significance and cultural acceptability of plant based medicinal practice in large parts of Ethiopia. At the same time, this indicates that plant diversity and use of plant based remedies remain decisive for managing human and livestock health in countries like Ethiopia, as is the case for many other countries [ 7 , 32 - 46 ]. Ailments treated and ICF Plants were clustered into 12 different groups based on the use citations by the informants and other end users (Table 1 ) in order to calculate the ICF. In our study, the ICF values range from 0.72 for evil eye and to 1.00 for rabies. Thus, all clusters had an ICF value greater than 0.5 and hence all of them could be considered for validation of bioactivity and isolation and characterization of the active principles by interested and potential researchers in each cluster. The highest number of plant species were reported to be used for treatment of abdominal/stomach disorders and internal parasites (22 species, 24.2%), followed by evil eye spirit (11 species, 12.1%), malaria and anemia like syndrome with jaundice, and snake bite/poisoning (10 species, 10.9% each), skin conditions (skin infections and ecto-parasites) and removal of retained placenta (7 species, 7.7% each), coughing in equines and ruminants and pain related illness (5 species, 5.5% each), anthrax (4 species, 4.4%), mich and megagna (an ailment characterized with fever, headache and sweating) and black leg (3 species, 3.3% each) and rabies (1 species, 1.1%) as shown in Table 1 . Animal diseases are one of the major reasons for poor livestock performance in Ethiopia [ 33 ], and the use of conventional medicine by smallholder livestock owners is constrained by their high prices and inaccessibility. On the other hand, Ethiopia is characterized by having diverse ecology and diverse mix of socio-cultural and linguistic groups, which might have contributed to the existence of rich knowledge in managing and using large numbers of different medicinal plants against both human and livestock ailments [ 32 ]. Therefore, in the absence of use of modern medicine to treat livestock diseases in smallholder livestock production systems, the use of traditional medicinal plants will remain a vital component of Ethiopian livestock production for some years to come. For instance, ethnoveterinary uses of the plant species Caylusea abyssinica, Cissampelos mucronata, Cissampelos pariera, Desmodium dichotomum, Ipomoea eriocarpa, Justicia diffusa, Premna schimperi, and Zornia glochidiata are reported by these ethnic groups to be effective against selected ecto- and endo-parasites of livestock. Validation of the later through in vitro and in vivo assessment of their anti-parasitic properties is required to better inform their use by pastoralists and smallholder farmers. Furthermore, bioactivity evaluation of these plant species also help to isolate and purify the active principles by bio-assay guided fractionation for new drug development. The ICF results could be useful in prioritizing medicinal plants for further scientific validation of plants and plant products [ 7 , 8 , 27 , 47 - 49 ], as pharmacologically effective remedies are expected from plants with higher ICF values [ 50 , 51 ]. Indeed, documentation of inherently rich traditional ethno-medicinal knowledge based on ICF values have provided valuable information on new pharmacological dimensions for better health care of livestock and humans regarding many ailments [ 50 ], and also assist conservation and management of rare, gradually vanishing important ethno-medicinal plant species. If validated, the claim for medicinal plants used in traditional medicine for a number of ailments of humans and livestock could provide new applications in supporting health care systems that are urgently needed. In our study, medicinal plant species claimed for anthrax, skin infection and external parasites, pain related illnesses and black leg were cited with the highest ICF values followed by those used to treat coughing in equines and ruminants, malaria and anemia like syndrome with jaundice, abdominal/stomach disorders and internal parasite and retained placenta. The lowest ICF value was recorded for the medicinal plant used to treat evil eye spirit. However, none were below 0.5, which would typically result from plant use to treat rare diseases [ 27 , 49 ], suggesting that our survey addressed medicinal plant species commonly used to treat common human and veterinary ailments in the study areas. Moreover, the highest numbers of plant species were reported to be used for treatment of abdominal/stomach disorders and internal parasites whereas the lowest number of medicinal plant species were reported for the treatment of rabies (Table 1 ). This implies that stomach disorders and endoparasite infections are likely the more common health problems of human and livestock in the three ethnic groups. Parasite-based health problems in human may be due to domestic hygiene, shared use of water from the same source for themselves and for their livestock, and zoonotic parasite infection. The parasitic health problem in livestock in the study areas could be associated with the ectoparasites particularly ticks and mange mites, increasing the risk for vector born diseases. The internal parasitic health problem in livestock in the study areas are a serious threat during humid season as the condition favors the infection, multiplication and transmission of endoparasites. Habits of growth Figure 3 a shows that woody plants made up 50% of the growth form of the plants claimed by the healers for having medicinal properties (29% trees and 21% shrubs), followed by herbs (36%) and climbers (14%). The high proportion of woody plants in our survey is likely associated to the ability of trees and shrubs to withstand long dry seasons, thus resulting in their abundance and year round availability in arid and semi-arid areas. This finding is contrary to the general patterns seen in most medicinal plant inventories where herbs are the largest plant growth forms [ 23 , 25 , 27 , 53 ]. A high usage of herbs in some studies could be an indication of their abundance, especially in areas receiving year round rainfall. Thus, the variation in parts of medicinal plants used may be related to differences in seasonality though also arise from differences in socio-cultural beliefs, and practices of the healers of different regions or countries. Figure 3 Proportions of growth form (a) and form of use (b) of medicinal plants identified in South Omo for treatment of different human and livestock ailments in South Omo zone, southern Ethiopia. Mode of preparation (form of use) Concoction, filtrate (a liquid from which insoluble impurities have been removed), paste on (topical), pounded and smoke bath are common use forms or modes of preparations reported in our study, with concoction (71%) and filtrate (11%) as the major use forms of the plants cited (Figure 3 b). The remedies are prepared using water (hot or warm), local drinks, boiled coffee or milk as a carrier and taken either orally or through inhalation of the vapor after boiling (smoke bath treatment). Within the total number of claimed medicinal plants, healers used 14 plants (15.4%) by mixing of two plants to treat selected ailments. For instance, Geranium arabicum mixed with Laggera tomentosa is used for the treatment of epizootic lymphangitis in animals; Droguetia iners mixed with Premna oligotricha is used for the treatment of malaria in humans, and Dombeya spp. mixed with Tragia doryodes is used for the treatment of anthrax. The frequent use of concoction and the mixing of two or more plants by healers could be associated with healer's belief of synergistic effects of certain plant components for healing the illnesses. This finding is consistent with earlier reports [ 26 , 54 , 55 ] but disagrees with other studies where crushing and squeezing [ 27 , 28 ] and homogenizing and crushing [ 24 ] were the main use forms. It is likely that these differences are associated with the differences in culture and knowledge in different socio-cultural groups. Parts of plant used Almost all plant parts, including roots, leaves, stem, bark, fruits, young shoots and flowers, were cited for use in preparing the different remedies. However, roots followed by leaves represented the most common parts used (Figure 4 a) for treating ailments in humans and livestock, respectively. Roots appeared to be the main plant part commonly used by the healers in the current study area. This could be associated with the fact that roots remain in the soil and are easily available, even during the long dry seasons in arid and semi-arid areas. In addition, the use of plants root could also be associated with early African beliefs in their powerful therapeutic effects. For example, early African diasporas in the Americas and those migrants to Caribbean countries during the colonial period used plant roots to protect against malaria and venereal diseases and to induce abortions, but also to prepare favorite household alcoholic drinks, as roots contributed to alcohol fermentation, color, flavor, and foam formation [ 20 , 56 , 57 ]. However, the use of medicinal plant roots, either for immediate use of treating ailments or for commercialization purpose to generate income, could also negatively contribute to local biological diversity and conservation because of complete plant removal from its natural habitat. The common use of leaf in the preparation of remedies could partly be due to the relative ease of finding this plant part. In agreement with our study, similar studies in other parts of Ethiopia reported that roots and leaves are indeed the most commonly used medicinal plant parts [ 27 , 28 ]. Figure 4 Proportion of plant parts used for medicinal purposes (a) and route of administration of plant preparations (b) for treatment of human and livestock ailments in South Omo zone, southern Ethiopia. Routes of administration and dosage used Both internal and external applications were reported by the informants in the treatment of various human and livestock ailments in our study. The commonly reported routes of administration are oral (65%), followed by topical (15%), nasal (10%) and smoke bath treatment (10%; Figure 4 b). The choice of oral administration may be related to the use of some solvents or additives (milk, butter, alcoholic drinks, boiled coffee, and food) that are commonly believed to serve as a vehicle to transport the remedies. The additives are also important to minimize discomfort, improve the taste and reduce adverse effects such as vomiting and diarrhoea, and enhance the efficacy and healing conditions [ 31 ]. Similar findings were reported by many other researchers, indicating the oral route as the most preferred mode of administration [ 25 , 28 , 58 - 64 ]. However, there is no consensus on the dosage used and frequency of the medication among healers. For example, the dosage varied according to the type of illness ranging from two spoonfuls (e.g. for treatment of anemia like syndrome with jaundice using concoct prepared from Cissampelos spp . ) to a cup or glass full (e.g. for treating "busino" or abdominal pain using decoct from Centella asiatica). Knowledge of informants on medicinal plants Indigenous people of different localities have their own specific knowledge on plant use, management and conservation [ 21 ]. Medicinal plants represent a significant contribution to human and livestock health and it has been suggested that their use is one of the most significant ways in which humans directly reap the benefits provided from biodiversity [ 22 , 23 ]. During the field survey in our study areas, informants reported ethno-medicinal data of 91 species of plants distributed across 33 families and 57 genera as having medicinal properties against 34 ailments (12 in humans, 11 in livestock and 11 in both human and livestock). The 91 plant species that are used by traditional healers among the three ethnic groups interviewed were identified and documented. Among the medicinal plants identified most of them belong to the seven families as shown in Figure 2 . The plant family Lamiaceae was most frequently represented amongst the documented useful species, with a total of 12 species out of the 91 plants identified, followed by Solanaceae with a total of 8 species and Menispermiaceae , and Fabaceae with total of 7 species each, and others constitute one up to six plant species per family. Figure 2 The percentage of plant species distributed over seven major families. The informants cited 32 (35.2%), 35 (38.5%) and 24 (26.4%) plants as having medicinal properties against ailments of livestock, humans or both livestock and humans, respectively (Table 2 ). The informants also reported multiple plant remedies against certain ailments, such as snake bite/poisoning for both humans and livestock. Depending upon the type of illness, the use of two or more parts of medicinal plants was also reported by some healers as common practice. For example, of the total 32 medicinal plants purely claimed for livestock illnesses, eight were used in two-plant combination preparations, and these target three different ailments, i.e. to treat epizootic lymphangitis, removal of fetal membrane, and anemia with jaundice (Table 2 ). Table 2 Medicinal plants, with family, scientific and local name, for selected ailments of human and/or veterinary importance, with parts used and preparations, as claimed by informants of the three ethnic groups from South Omo zone, Southern Ethiopia Family Scientific name Local name * Voucher number Use(s) Parts used and preparation Importance Amaranthaceae Achyranthes aspera Linn. Busino (M) KTG28 Abdominal pain and tonsillitis Root chopped and mixed with water and taken orally. Leaf chewed and the extract kept near the inflammation area Human Lamiaceae Achyrospermum africanum Hook.f. ex Baker Kebit buda (A) KTG54 Evil eye Leaf and root chopped and soaked with water Human Achyrospermum schimperi (Hochst. ex Briq.) Perkins Abasanga medihanit (A,Amh) KTG65 Anthrax Leaf and flower chopped and soaked with hot water and drenched Veterinary Scrophulariaceae Alonsoa acutifolia Ruiz & Pav. Shosha tesha (M) KTG66 Snake bite/poison Root chopped and mixed with Plectranthus glandulosus in water and the filtrate drenched Human and Veterinary Barthlottia madagascariensis E.Fisch. Unkown (A) KTG18 Snake bite/poison Concoction Human and Veterinary Fabaceae Calpurnia aurea (Aiton)Benth. Kaino(M) KTG91 Flea and louse infestation Freshly chopped or dried and ground leaf mixed with water and applied to the flea and louse infested areas Veterinary Apocynaceae Carissa carandas L. Goiti(B),ebab medihanit (Amh) KTG45 Snake bite/poison The leaf chopped, mixed with water taken orally Human and Veterinary Rubiaceae Carphalea glaucescens (Hiern) Verdc Wariamo (M) KTG31 Anaemia (known as Airo) Leaf powdered mixed with Ipomoea kiwuensis smoked for three days Human Recedaceae Caylusea abyssinica (fresen.) Fisch. & Mey Giesilla (M) KTG64 Rabies (effective even when clinical signs are present) Root chopped and mixed with cold water and drenched Human and Veterinary Apiaceae Centella asiatica - (L.)Urb. Busino (M) KTG87 Abdominal ache Root dried, ground and mixed with cold water when needed (on cup or glass full) Human Menispermaceae Chasmanthera dependens (Hochst) Moshito (M) KTG33 Black leg Root bark and leaf dried and ground and given to emaciate calf as much as possible Veterinary Chasmanthera welwitschii Troupin Heilho (M) KTG27 Antiamoeba Root bark and leaf dried and ground and given to emaciate calf as much as possible Veterinary Lamiaceae Chelonopsis moschata Miq. Kebit buda (A) KTG47 Evil eye Leaf and root chopped and soaked with water Human Menispermaceae Chlaenandra ovata . Miq. Eincht (A) KTG73 Abdominal ache Root chopped and mixed with water and drenched Human Cissampelos capensis L.f . Wontin kanna (A) KTG76 Abdominal cramp Root chopped, powdered, soaked with water, filtered and drenched Human Cissampelos mucronata A.Rich. Kawuro (M) KTG37 Anaemia with jaundice Leaf collected, dried, ground and mixed with hot water and two spoonful taken at once Human Cissampelos pareira L . Shelindo (M) KTG70 Broad spectrum anti- helminthiasis Root ground mixed with large amount of water and drenched. It causes fever diarrhea then animals are cured Veterinary Cissampelos spp. Kawto (M) KTG32 Anaemia like syndrome with jaundice Leaf collected, dried, ground with local mill and mixed with hot water and two spoonful taken at once (bitter) Human Vitaceae Cissus quadrangularis L. Bararo (M) KTG20 Evil eye Tied under belly Veterinary Euphorbiaceae Claoxylopsis andapensis Radcl.-Sm. Dorba (A) KTG43 Snake bite/poison Bark and leaf chopped, soaked in water and drenched Human Nyctaginaceae Colignonia ovalifolia Heimerl Afesha (A) KTG41 Evil eye Leaf squeezed and inhaled Human Araceae Colocasia esculenta (L.) Schott Haleko (A,M,BT) KTG77 To detach retained fetal membrane Root dried, ground and mixed with powdered root of Momordica spp. and all soaked in warm water and one cupful drenched Veterinary Nyctaginaceae Cryptocarpus spp . Afei tesha (M) KTG23 Evil eye Root chopped and mixed with cold/hot water Human Solanaceae Datura stramonium L. Onidod (A) KTG05 Coughing (for horses, mules and donkeys) Leaf chopped and mixed with cold water and drenched via nose Veterinary Papilionaceae Desmodium dichotomum (Willd.) DC. Muasii (A) KTG07 Epizootic lymphangitis (tushita) Root chopped, mixed with cold water and drenched via nose Veterinary Desmodium delotum J.F. Macbr. Not known (A) KTG42 Eye illness Leaf apex chopped, soaked in water, applied to sick eye Human and Veterinary Salvadoraceae Dobera spp. Mitch medihanit (A, Am) KTG89 Mitch Leaf boiled with water and inhaled Human Sterculiaceae Dombeya spp. Bata (A) KTG15 Anthrax The leaf is chopped and mixed with Tragia doryodes and the filtrate taken orally Human and Veterinary Flacortiaceae Dovyalis spp. Mukale (M) KTG85 To detach retained placenta Leaf chopped and mixed with hot water and given as ad libtum Veterinary Urticaceae Droguetia debilis Rendle Megagna medanit (Amh) KTG52 Megagna Leaf apex chopped and pasted on the pain area Human Droguetia iners (Forssk.) Schweinf. Yewoba medihanit (A) KTG61 Malaria Leaf chopped and mixed with Premna oligotricha and boiled together one glassful drenched Human Caryophyllaceae Drymaria cordata (L.) Willd. ex Schult. Yebuda medihanit (A, Amh) KTG59 Evil eye in animals Leaf and root chopped mixed in water and the filtrate is sprayed on animal body and the sediments are drenched Human and Veterinary Drymaria spp . Unknown (A) KTG17 Evil eye The leaf is chopped and mixed with water and the filtrate taken orally Human Asteraceae Echinacea spp. Unkown KTG53 Diarrhoea alone Root chopped and soaked in warm water taken orally Human and Veterinary Galinsoga parviflora Cav. Midirberbere, (Amh) KTG14 Anaemia with jaundice The flower is chopped and mixed with Monosonia longipes and warmed on and applied of gum of achy tooth Human Galinsoga quadriradiata Cav. Mukala (M) KTG74 To detach retained fetal membrane and/or placenta Leaf chopped and mixed with Plumbango zeylanica and then drenched Veterinary Geraniaceae Geranium aculeolatum Oliv. Abasanga medihanit (A, AM) KTG72 Anthrax Leaf chopped and rubbed on wounded part Veterinary Geranium arabicum Forssk. Tushita (A) KTG26 Epizootic lymphangitis Root chopped and mixed with chopped root of Laggera tomentosa and one bear bottle drenched through left nose of horse. Veterinary Poaceae Hyparrhenia hirta (L.) Stapf Goiti ebab" medihanit (B) KTG46 Snake bite/poison Plant material chopped and soaked in hot water and the filtrate drenched Human and Veterinary Fabaceae Indigofera arrecta A.Rich. Wareami (A) KTG80 Anaemia with jaundice Leaf dried and smoked to patients Human Indigofera trita L.f. Wusis (A) KTG01 To improve milk production of cows Root chopped, mixed with water and drenched Veterinary Convolvulaceae Ipomoea eriocarpa R. Br. Choko (M) KTG40 Endoparasite Root chopped and mixed with water, then the filtrate is drenched and rest sediments are poured on the wound part Veterinary Acanthaceae Justica dianthera Vell. Mitch(A, Amh) KTG51 Mitch The leaf apex boiled with water and the vapor inhaled and/or the filtrate drenched Human Justicia diffusa Willd. Makaiso (A) KTG04 For coughing of equines Leaf chopped and mixed with cold water and drenched via nose Veterinary Cucurbitaceae Lagenaria siceraria (Molina) Standl. Busino (M) KTG35 Diarrhoea and vomiting Leaf chopped and ground and the drench the filtrate Human Asteraceae Laggera tomentosa Schultz Bip. Tushita (A) KTG71 Epizootic lymphangitis Root chopped and mixed with chopped root of Geranium arabicum and one bear bottle drenched through horses nose Veterinary Verbenaceae Lantana Trifolia L. Yewoba medihanit (A) KTG55 Malaria (shivering type, vivax) Root chopped and soaked with water and mixed with local alcoholic drink (Areke) Human Lamiaceae Leucas stachydiformis (Benth.) Hochst. ex Briq. Businae (M) KTG19 Anaemia with jaundice Leaf and bark chopped and drench the filtrate or inhalation Human Onagraceae Ludwigia abyssinica A.Rich. Yechira ebab medihanit (Amh) KTG44 Snake bite/poison Stem and root mixed with other plants and applied orally Human Cucurbitaceae Momordica foetida Schumach. Chekko (M,B) KTG03 Black leg Root chopped, soaked in water for half a day and a filtrate is drenched Veterinary Momordica charantia L. Unknown (A) KTG90 Diarrhea and vomiting Leaf and root ground well and mixed with milk and taken orally Human Momordica spp. Kill (M) KTG79 To detach fetal membrane Root dried, ground and mixed with Colocasia esculenta , all soaked in warm water and one cupful filtrate drenched Veterinary Geraniaceae Monsonia parvifolia Schinz Not known (A) KTG58 Tooth ache Seed and leaf crushed and mixed with salt and Galinsoga parvifolia ; made hot on fire with "enset leaf" and applied on gum. Human Papilionaceae Mucuna melanocarpa Hochst Salabano (M) KTG86 For calf Ascariasis Leaf ground and mixed with water and drenched that induces diarrhea Veterinary Solanaceae Nicotiana tabacum L. Bangiso(M) KTG78 Tick infestation Root chopped and mixed with water and dressed to the tick infested area on cow and calf Veterinary Anacardiaceae Operculicarya gummifera (Sprague) Capuron Dorba (M) KTG38 Snake bite/poison Orally taken butt its preparation is not specified due to unwillingness of the respondent Human and Veterinary Lamiaceae Orthosiphon sarmentosus A.J. Paton & Hedge Zititu (A) KTG67 Ascariasis Leaf chopped, soaked in water and a glass full filtrate drunken Human Oxalidaceae Oxalis corniculata L . Dani (M) KTG02 Toxic Root chopped, cooked for two days and more and the paste rubbed on arrow tip to hunt wild animals Human and veterinary Anacardiaceae Ozoroa insignis Delile Bussa (M) KTG39 For coughing of equines Bark dried, powdered and mixed with cold water and the filtrate drenched Veterinary Geraniaceae Pelargonium alchemilloides (L.) Aiton Unkown KTG13 Constipation Root chopped and soaked in warm water taken orally Human Rubiaceae Pentas suswaensis Verdc. Haromato (M) KTG63 Aba gorba "Black leg" Leaf chopped and mixed with boiled water and the filtrate is drenched Veterinary Lamiaceae Plectranthus glabriflorus P.I.Forst. Gullo/Karika (A) KTG09 Evil eye Leaf soaked in hot water and drunken Human Plectranthus globosus Ryding Chambuase (M) KTG60 Snake bite/poison Leaf chopped mixed with Alectra sessiliflora mixed in cold water and taken orally Human Plumbaginaceae Plumbago auriculata Lam. Masilok (M) KTG81 For animal evil spirit Leaf chopped and soaked in water and the filtrated drenched and the remaining sediments pasted on the body Human and Veterinary Plumbago caerulea Kunth Wugat medihanit (Amh) KTG68 Back and side pain Root and seed are chopped and mixed with hot water and onion Human Plumbago pulchella Boiss. Not known (A) KTG12 Snake bite/poison Fresh leaf chopped and mixed with cold water Human and Veterinary Plumbago spp. Misirich (M) KTG49 Evil eye in animals Leaf chopped and soaked in water and the filtrated drenched and the remaining sediments pasted on the body Veterinary Plumbago zeylanica L . Telba (M) KTG75 To detach retained fetal membrane Seed ground by traditional mortar mixed with Galinsoga Parviflora and boiled with water and drenched Veterinary Lamiaceae Premna oligotricha Baker Yewoba medihanit (A) KTG34 Malaria (non-shivering type, falciparium) Leaf collected and ground and mixed with water Human Premna schimperi Engl. Bangizo(M) KTG21 Dermatophilous and mite infestation Root chopped and soaked in warm water over night and filtrate applied topically to treat dermatophytes and tick mite infestations Veterinary Premna spp. Anchiphi (M) KTG57 Diarrhea in calf Leaf powdered and mixed with water and the filtrate drenched Veterinary Pycnostachys meyeri Gürke ex Engl. Unkown (A) KTG83 Abdominal pain (children) Fresh root chopped and mixed with cold water and drenched Human Rubiaceae Rytigynia spp. Golodo (M) KTG30 Typhoid (Micho) Leaf chopped and mixed with water and taken orally Human Lamiaceae Salvia acuminata Ruiz & Pav. Anchino (M) KTG48 Diarrhoea alone Chewing the leaf Human Fabaceae Senna spp. Diko (M) KTG84 Joint ache and breakage of bones Leaf rubbed on affected parts and some leaf chopped and soaked in warm water and drenched Human and Veterinary Malvaceae Sida spp. Moishita (M) KTG25 Anti-parasitic/ to fatten calf Leaf chopped and soaked in water and the filtrate is drenched repeatedly Veterinary Solanaceae Solanum bellum S. Knapp Ondod (M) KTG22 Coughing of equines(Busa) Root chopped and mixed with cold water and the filtrate drenched either by nose or mouth Veterinary Solanum acaule Bitter Mushta (A) KTG62 Removes retained placenta Root chopped and mixed with cold water and the filtrate is applied nasally Veterinary Solanum acuminatum Ruiz & Pav. raki (A) KTG82 To detach retained placenta Root chopped, mixed with cold water and drenched orally Human and Veterinary Solanum incanum L. Garint (A) KTG06 Epizootic lymphangitis(tushita) Root chopped and mixed with cold water and drenched via nose Veterinary Solanum spp. Danni (M) KTG50 Poisonous to animals Root chopped and concoction with water until paste is formed, rubbed on arrow tip & used for hunting Human and Veterinary Bignoniaceae Stereospermum kunthianum Cham. Addi (M) KTG29 Abdominal pain Root chopped and mixed with water/coffee and taken in ad libitum Human Asteraceae Tagetes spp. Businae (A) KTG88 Muscle cramp and joint pain Leaf rubbed well and oils from the leaf are swabbed on areas where pain felt. Fumigation is also possible. Boiled filtrate is drenched Human Tagetes minuta L. Kawato (M) KTG36 Diarrhea and vomiting Leaf chopped and ground and the drench the filtrate Human Euphorbiaceae Tragia doryodes M.G.Gilbert Anderta (A) KTG16 Anthrax The leaf is chopped and mixed with Dombya spp. and the filtrate taken orally Veterinary Caryophyllaceae Vaccaria hispanica (Mill.) Rauschert Sanba tesha (M) KTG69 For contagious bovine pleuropneumonia and contagious caprine pleuropneumonia Root chopped and mixed with large amount of water. It gets bloody color (the bloody color indicates appropriate concentration), then drenched Veterinary Verbenaceae Verbena officinalis L. Guni tesha (A) KTG56 Snake bite/poison Leaf squeezed by hand and mixed with water and drenched by water Human and Veterinary Solanaceae Withania somnifera (L.) Dunal Buto/wogare (M) KTG24 Night mare Roots powdered and children smoked until they cough Human Fabaceae Zornia apiculata Milne-Redh. Medhanit (A) KTG10 Abdomen ache and vomiting in children Fresh root chopped and mixed with cold water and drenched Human Zornia glochidiata DC. Halimi (A) KTG11 Malaria Root bark is chopped and boiled/concoction with local drinks and boiled coffee leaf Human Zornia latifolia Sm . Medihanit (A) KTG08 Abdominal pain, vomiting Fresh leaf chopped and mixed to form filtrate Human * A=Aari; M=Maale; B=Benigna; Amh = Amharic. This is the first study that documented plants used for disease control by the three ethnic groups in South Ethiopia. Previous studies have documented indigenous knowledge of medicinal plants and medicinal plant practices used in other parts of the country and by other ethnic groups including those in southern Ethiopia [ 24 , 25 ], northern and northwestern Ethiopia [ 8 , 26 - 28 ], and southwestern Ethiopia [ 29 - 31 ]. Our study thus complements existing studies but also extends them to pastoral areas where the ecology, practices, biodiversity, accessibility and cultural acceptability of medicinal plants are very different from the highlands. The aforementioned reports and our study taken together capture a wide range of different ethnic and social groups, which is a reflection of the richness of knowledge in use of plants for medicinal purposes, and the significance and cultural acceptability of plant based medicinal practice in large parts of Ethiopia. At the same time, this indicates that plant diversity and use of plant based remedies remain decisive for managing human and livestock health in countries like Ethiopia, as is the case for many other countries [ 7 , 32 - 46 ]. Ailments treated and ICF Plants were clustered into 12 different groups based on the use citations by the informants and other end users (Table 1 ) in order to calculate the ICF. In our study, the ICF values range from 0.72 for evil eye and to 1.00 for rabies. Thus, all clusters had an ICF value greater than 0.5 and hence all of them could be considered for validation of bioactivity and isolation and characterization of the active principles by interested and potential researchers in each cluster. The highest number of plant species were reported to be used for treatment of abdominal/stomach disorders and internal parasites (22 species, 24.2%), followed by evil eye spirit (11 species, 12.1%), malaria and anemia like syndrome with jaundice, and snake bite/poisoning (10 species, 10.9% each), skin conditions (skin infections and ecto-parasites) and removal of retained placenta (7 species, 7.7% each), coughing in equines and ruminants and pain related illness (5 species, 5.5% each), anthrax (4 species, 4.4%), mich and megagna (an ailment characterized with fever, headache and sweating) and black leg (3 species, 3.3% each) and rabies (1 species, 1.1%) as shown in Table 1 . Animal diseases are one of the major reasons for poor livestock performance in Ethiopia [ 33 ], and the use of conventional medicine by smallholder livestock owners is constrained by their high prices and inaccessibility. On the other hand, Ethiopia is characterized by having diverse ecology and diverse mix of socio-cultural and linguistic groups, which might have contributed to the existence of rich knowledge in managing and using large numbers of different medicinal plants against both human and livestock ailments [ 32 ]. Therefore, in the absence of use of modern medicine to treat livestock diseases in smallholder livestock production systems, the use of traditional medicinal plants will remain a vital component of Ethiopian livestock production for some years to come. For instance, ethnoveterinary uses of the plant species Caylusea abyssinica, Cissampelos mucronata, Cissampelos pariera, Desmodium dichotomum, Ipomoea eriocarpa, Justicia diffusa, Premna schimperi, and Zornia glochidiata are reported by these ethnic groups to be effective against selected ecto- and endo-parasites of livestock. Validation of the later through in vitro and in vivo assessment of their anti-parasitic properties is required to better inform their use by pastoralists and smallholder farmers. Furthermore, bioactivity evaluation of these plant species also help to isolate and purify the active principles by bio-assay guided fractionation for new drug development. The ICF results could be useful in prioritizing medicinal plants for further scientific validation of plants and plant products [ 7 , 8 , 27 , 47 - 49 ], as pharmacologically effective remedies are expected from plants with higher ICF values [ 50 , 51 ]. Indeed, documentation of inherently rich traditional ethno-medicinal knowledge based on ICF values have provided valuable information on new pharmacological dimensions for better health care of livestock and humans regarding many ailments [ 50 ], and also assist conservation and management of rare, gradually vanishing important ethno-medicinal plant species. If validated, the claim for medicinal plants used in traditional medicine for a number of ailments of humans and livestock could provide new applications in supporting health care systems that are urgently needed. In our study, medicinal plant species claimed for anthrax, skin infection and external parasites, pain related illnesses and black leg were cited with the highest ICF values followed by those used to treat coughing in equines and ruminants, malaria and anemia like syndrome with jaundice, abdominal/stomach disorders and internal parasite and retained placenta. The lowest ICF value was recorded for the medicinal plant used to treat evil eye spirit. However, none were below 0.5, which would typically result from plant use to treat rare diseases [ 27 , 49 ], suggesting that our survey addressed medicinal plant species commonly used to treat common human and veterinary ailments in the study areas. Moreover, the highest numbers of plant species were reported to be used for treatment of abdominal/stomach disorders and internal parasites whereas the lowest number of medicinal plant species were reported for the treatment of rabies (Table 1 ). This implies that stomach disorders and endoparasite infections are likely the more common health problems of human and livestock in the three ethnic groups. Parasite-based health problems in human may be due to domestic hygiene, shared use of water from the same source for themselves and for their livestock, and zoonotic parasite infection. The parasitic health problem in livestock in the study areas could be associated with the ectoparasites particularly ticks and mange mites, increasing the risk for vector born diseases. The internal parasitic health problem in livestock in the study areas are a serious threat during humid season as the condition favors the infection, multiplication and transmission of endoparasites. Habits of growth Figure 3 a shows that woody plants made up 50% of the growth form of the plants claimed by the healers for having medicinal properties (29% trees and 21% shrubs), followed by herbs (36%) and climbers (14%). The high proportion of woody plants in our survey is likely associated to the ability of trees and shrubs to withstand long dry seasons, thus resulting in their abundance and year round availability in arid and semi-arid areas. This finding is contrary to the general patterns seen in most medicinal plant inventories where herbs are the largest plant growth forms [ 23 , 25 , 27 , 53 ]. A high usage of herbs in some studies could be an indication of their abundance, especially in areas receiving year round rainfall. Thus, the variation in parts of medicinal plants used may be related to differences in seasonality though also arise from differences in socio-cultural beliefs, and practices of the healers of different regions or countries. Figure 3 Proportions of growth form (a) and form of use (b) of medicinal plants identified in South Omo for treatment of different human and livestock ailments in South Omo zone, southern Ethiopia. Mode of preparation (form of use) Concoction, filtrate (a liquid from which insoluble impurities have been removed), paste on (topical), pounded and smoke bath are common use forms or modes of preparations reported in our study, with concoction (71%) and filtrate (11%) as the major use forms of the plants cited (Figure 3 b). The remedies are prepared using water (hot or warm), local drinks, boiled coffee or milk as a carrier and taken either orally or through inhalation of the vapor after boiling (smoke bath treatment). Within the total number of claimed medicinal plants, healers used 14 plants (15.4%) by mixing of two plants to treat selected ailments. For instance, Geranium arabicum mixed with Laggera tomentosa is used for the treatment of epizootic lymphangitis in animals; Droguetia iners mixed with Premna oligotricha is used for the treatment of malaria in humans, and Dombeya spp. mixed with Tragia doryodes is used for the treatment of anthrax. The frequent use of concoction and the mixing of two or more plants by healers could be associated with healer's belief of synergistic effects of certain plant components for healing the illnesses. This finding is consistent with earlier reports [ 26 , 54 , 55 ] but disagrees with other studies where crushing and squeezing [ 27 , 28 ] and homogenizing and crushing [ 24 ] were the main use forms. It is likely that these differences are associated with the differences in culture and knowledge in different socio-cultural groups. Parts of plant used Almost all plant parts, including roots, leaves, stem, bark, fruits, young shoots and flowers, were cited for use in preparing the different remedies. However, roots followed by leaves represented the most common parts used (Figure 4 a) for treating ailments in humans and livestock, respectively. Roots appeared to be the main plant part commonly used by the healers in the current study area. This could be associated with the fact that roots remain in the soil and are easily available, even during the long dry seasons in arid and semi-arid areas. In addition, the use of plants root could also be associated with early African beliefs in their powerful therapeutic effects. For example, early African diasporas in the Americas and those migrants to Caribbean countries during the colonial period used plant roots to protect against malaria and venereal diseases and to induce abortions, but also to prepare favorite household alcoholic drinks, as roots contributed to alcohol fermentation, color, flavor, and foam formation [ 20 , 56 , 57 ]. However, the use of medicinal plant roots, either for immediate use of treating ailments or for commercialization purpose to generate income, could also negatively contribute to local biological diversity and conservation because of complete plant removal from its natural habitat. The common use of leaf in the preparation of remedies could partly be due to the relative ease of finding this plant part. In agreement with our study, similar studies in other parts of Ethiopia reported that roots and leaves are indeed the most commonly used medicinal plant parts [ 27 , 28 ]. Figure 4 Proportion of plant parts used for medicinal purposes (a) and route of administration of plant preparations (b) for treatment of human and livestock ailments in South Omo zone, southern Ethiopia. Routes of administration and dosage used Both internal and external applications were reported by the informants in the treatment of various human and livestock ailments in our study. The commonly reported routes of administration are oral (65%), followed by topical (15%), nasal (10%) and smoke bath treatment (10%; Figure 4 b). The choice of oral administration may be related to the use of some solvents or additives (milk, butter, alcoholic drinks, boiled coffee, and food) that are commonly believed to serve as a vehicle to transport the remedies. The additives are also important to minimize discomfort, improve the taste and reduce adverse effects such as vomiting and diarrhoea, and enhance the efficacy and healing conditions [ 31 ]. Similar findings were reported by many other researchers, indicating the oral route as the most preferred mode of administration [ 25 , 28 , 58 - 64 ]. However, there is no consensus on the dosage used and frequency of the medication among healers. For example, the dosage varied according to the type of illness ranging from two spoonfuls (e.g. for treatment of anemia like syndrome with jaundice using concoct prepared from Cissampelos spp . ) to a cup or glass full (e.g. for treating "busino" or abdominal pain using decoct from Centella asiatica). Conclusion This study showed that traditional medicine, mainly involving the use of medicinal plants, is playing a significant role in meeting the primary healthcare needs of the three ethnic groups. Acceptance of traditional medicine and limited access to modern healthcare facilities could be considered as the main factors for the continuation of the practice. This field survey has documented 91 plant species distributed across 33 families and 57 genera as having medicinal properties against 34 human and livestock ailments as reported by healers from Aari, Maale and Bena-Tsemay ethnic groups, complementing previous studies from other ethnic groups in Ethiopia. The highest number of plant species was reported to be used for treatment of abdominal/stomach disorders and internal parasites. Woody plants (trees and shrubs) were the main form used, likely related to the long dry seasons typically occurring in the residential area of the ethnic groups studied. Concoction appeared to be the most popular use form in the current study. The most commonly used route of administration is oral. This study contributes to the enormous indigenous knowledge on medicinal plants and plant-based remedies practiced among ethnic groups, and it assists knowledge and practice preservation, which remain mostly with elderly traditional practitioners. Furthermore, the information generated will also inform future validation studies, so as to increase the acceptability of plant-based remedies in human and animal health care systems both nationally and internationally. Competing interests The authors declare that they have no competing interests. Authors' contributions KT, ED and AT carried out field survey and data analysis, ED and KT prepared the initial structure of the draft manuscript and KT, ED and AT revised the manuscript critically to the present form. GG introduced us to the people in the study and was involved in a preliminary survey. SA and JH secured funding for the project, assisted data interpretation, manuscript structuring and provided input to previous drafts resulting in the present form. All authors read the final manuscript and agreed on its submission. Acknowledgements This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBRSC), Department for International Development (DFID) and Scottish Government (SG) under the umbrella of their CIDLID initiative (BB/H009299/1). RCBP-NARF provided support for preliminary survey work. We are also thankful to the informants from the three ethnic groups who without reservation shared their medicinal plant knowledge with us, the elders and the local administration for their support in facilitating the interview process. We also thank the staff of the National herbarium of Addis Ababa University for plant identification.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8495834/

Rolling pits of Hartmann’s mountain zebra ( Zebra equus hartmannae ) increase vegetation diversity and landscape heterogeneity in the Pre‐Namib

Abstract Microsites created by soil‐disturbing animals are important landscape elements in arid environments. In the Pre‐Namib, dust‐bathing behavior of the near‐endemic Hartmann's mountain zebra creates unique rolling pits that persist in the landscape. However, the ecohydrological characteristics and the effects of those microsites on the vegetation and on organisms of higher trophic levels are still unknown. In our study, we characterized the soil grain size composition and infiltration properties of rolling pits and reference sites and recorded vegetation and arthropod assemblages during the rainy season of five consecutive years with different amounts of seasonal rainfall. We further used the excess green vegetation index derived from drone imagery to demonstrate the different green up and wilting of pits and references after a rainfall event. In contrast to the surrounding grassland, rolling pits had finer soil with higher nutrient content, collected runoff, showed a higher infiltration, and kept soil moisture longer. Vegetation in the rolling pits was denser, dominated by annual forbs and remained green for longer periods. The denser vegetation resulted in a slightly higher activity density of herbivorous arthropods, which in turn increased the activity density of omnivorous and predatory arthropods. In times of drought, the rolling pits could act as safe sites and refuges for forbs and arthropods. With their rolling pits, Hartmann's mountain zebras act as ecosystem engineers, contributing to the diversity of forb communities and heterogeneity of the landscape in the Pre‐Namib. 1 INTRODUCTION Biopedturbation, the disturbance of soils by animals, is an important and often essential functional component of many ecosystems worldwide (Coggan et al., 2018 ). It determines the spatiotemporal characteristics of soil patches and thereby contributes to the ecosystems' diversity and heterogeneity (Mallen‐Cooper et al., 2019 ). Examples of the ecological importance of biopedturbation can be found in all climatic zones, but the focus is on arid regions (Coggan et al., 2018 ; Mallen‐Cooper et al., 2019 ). The spectrum of animals for these disturbances spans almost all animal groups (Coggan et al., 2018 ; Whitford & Kay, 1999 ) and even includes marine species such as the Californian Gray Whale ( Eschrichtius robustus ; Johnson et al., 1983 ) or the Antillean Manatee ( Trichechus manatus manatus ; Bacchus et al., 2009 ). The soil disturbances by animals affect both physical and chemical soil properties. These disturbances create vegetation‐free areas, shape soil topography, alter soil density and structure, change infiltration properties and soil moisture, influence the nutrient situation, and contribute to carbon cycling and nutrient turnover (for a detailed account of species and how they affect soil properties, see Coggan et al., 2018 , Whitford & Kay, 1999 and Mallen‐Cooper et al., 2019 ). The large majority of documented soil disturbances by animals are due to burrows, mounds, or foraging digs, whereas reports about ground disturbances related to other behavior are relatively rare (Coggan et al., 2018 ; Whitford & Kay, 1999 ). Such described behavior‐related soil disturbances are all due to larger mammals and consequently result in relatively large structures; predominantly, depressions in the ground that serve as resting places are used for self‐grooming or even serve a social function. Examples of resting sites are the beds of the Nubian Ibex ( Capra nubiana ; Gutterman, 1997 , 2001 ), the shelter sites of Gemsbok ( Oryx gazella ; Dean & Milton, 1991a , 1991b ), or the hip holes of Kangaroos ( Macropus spp.; Eldridge & Rath, 2002 ). As an example of resting depressions of a marine species, the resting holes of the Antillean Manatee (Bacchus et al., 2009 ) are known. Extensively studied and well‐documented are the wallows of the American Bison ( Bison bison ; McMillan et al., 2011 , Nickell et al., 2018 ), which, in addition to rest and self‐grooming, also serve as a meeting place to promote social cohesion (Reinhardt, 1985 ). Also known are soil disturbances caused by the dust‐bathing behavior of many equids such as horses, donkeys (Moehlman, 1998 ), and zebras (Joubert, 1972 ; Skinner & Chimimba, 2005 ) that serves mainly for self‐grooming and the removal of ectoparasites. The altered soil properties caused by the soil disturbance first directly affect the vegetation and via the trophic cascade eventually organisms of higher trophic levels (Coggan et al., 2018 ). The importance of such disturbances is especially high in arid environments. Here, with nutrient‐poor soils and the scarcity of water, the patterns and composition of vegetation are often linked to the geomorphological heterogeneity of the landscape (Bestelmeyer et al., 2006 ; Tongway & Ludwig, 2005 ). Small‐scale topographical features such as depressions and changes in soil properties form favorable microsites with higher nutrient and water availability (Bestelmeyer et al., 2006 ) that affect density and composition of the vegetation. The depressions created by animals collect runoff after rain and have better infiltration properties, increased soil moisture, and a higher nutrient content due to the accumulation of dung and urine (Mallen‐Cooper et al., 2019 ). The removal of existing vegetation further changes the competitive balance of plant communities (Mallen‐Cooper et al., 2019 ; Romero et al., 2015 ). Hence, such microsites offer more favorable conditions for plant recruitment and establishment than the surrounding matrix (Dean & Milton, 1991a , 1991b ; Mallen‐Cooper et al., 2019 ). The vegetation of such microsites is therefore often characterized by higher annual plant species richness, higher biomass production, and different plant community composition (Mallen‐Cooper et al., 2019 ). The more favorable conditions provided by these depressions are particularly important for annual forbs (Mallen‐Cooper et al., 2019 ; Whitford & Kay, 1999 ), which account for the largest share of species richness in arid grasslands, contribute to soil organic matter, and provide shelter and forage for arthropods, as well as larger animals and mammals (Siebert & Dreber, 2019 ). This in turn increases the activity density and diversity of arthropods (Nickell et al., 2018 ; Ruttan et al., 2016 ) or mammals (Ewacha et al., 2016 ). Such ecological effects can persist over many years or even decades (Coggan et al., 2018 ; Hastings et al., 2007 ; McMillan et al., 2011 ). Consequently, many soil‐disturbing animals act as ecosystem engineers and play an essential role in maintaining the diversity and productivity of the respective ecosystem (Mallen‐Cooper et al., 2019 ). Furthermore, such ecosystem engineers are often of particular concern for nature conservation (Hastings et al., 2007 ; Mullan Crain & Bertness, 2006 ), as their loss can lead to a deterioration in ecosystem function (Eldridge & James, 2009 ). Coggan et al. ( 2018 ) list 121 species in their ecosystem engineering database, of which many occur in arid or dry regions. A species not yet included is the Hartmann's mountain zebra ( Equus zebra hartmannae ), a species that is endemic in the semidesert of Namibia's great escarpment, the Pre‐Namib, and a small part of Southern Angola and South Africa (Gosling et al., 2019 ). With their unique dust‐bathing behavior, the Hartmann's mountain zebras create characteristic small depressions, which even when abandoned remain in the landscape for many years (Skinner & Chimimba, 2005 ). None of the co‐occurring larger mammal species in the Namibian escarpment, Oryx ( Oryx gazella ), Greater Kudu ( Strepsiceros zambesiensis ), or Springbok ( Antidorcas marsuialis ), exhibits a similar behavior (Barandongo et al., 2018 ). The exact effects of such rolling pits on the vegetation of arid savanna ecosystems and within the trophic cascade are largely unknown and have not been reported yet. With our study, we aim to elucidate the role of Hartmann's mountain zebras as ecosystem engineers in arid savanna ecosystems. Therefore, we investigated the effect of the rolling pits of Hartmann's mountain zebras over a period of five consecutive vegetation growing seasons with different amounts of rainfall on soil properties and density and composition of the vegetation. Further, we tested whether and how these soil and vegetation differences also affect ground‐dwelling arthropods. 2 METHODS 2.1 Study site and study species Our study was conducted between 2014 and 2018 on the farm Rooiklip, Khomas, Namibia (33 K 612,448 7,411,832), situated at 1,000 m.a.s.l. in the center of the escarpment halfway between Windhoek and Walvis Bay (Figure A1 ). Namibia's great escarpment is an approximately 100‐km wide strip of rugged semidesert reaching from the border with South Africa in the South to Angola in the North. The climate is hot‐arid, mean annual precipitation ranges from ~50 mm in the West to ~200 mm at the upper edge in the East (Mendelson et al., 2009 ), but rainfall is highly variable and often erratic. At our study site, the mean annual precipitation is 120 mm, but ranged from 60 mm to 180 mm during the study period. Rainfall was measured on a daily basis with an on‐site standard rain gauge. The main vegetation growth period occurs from February to March, where two‐thirds of the annual precipitation occurs (Wagner et al., 2016 ). The vegetation is arid grassland dominated by perennial grasses of the genus Stipagrostis that is sparsely scattered with trees and occasional dwarf shrubs. Depending on the amount of rainfall, various annual forbs and annual grasses join these perennials. Total vegetation cover seldom reaches more than 15% (Wagner et al., 2016 ). Soils are leptosols and consist of a shallow layer of differently sized weathered particles of the underlying schist bedrock. Rooiklip and its neighboring farms are largely unfenced and allow wildlife to roam freely between the farms and from the edge of the escarpment to the Namib Desert. The escarpment and the Pre‐Namib are home to the near‐endemic Hartmann's mountain zebra (Figure A1 ; Environmental Information Service, 2020 ; Gosling et al., 2019 ). The habitat of Hartmann's mountain zebra is mountainous areas and flats. Hartmann's mountain zebras further exhibit a marked migration pattern, following the rainfall and forage availability and moving between dry season and rainy season home ranges (Muntifering et al., 2019 ). Hartmann's mountain zebras usually live as family groups that consist of a stallion and his harem of mares and their foals. However, stallion groups or solitary stallions are frequent (Skinner & Chimimba, 2005 ), and occasionally, several family groups can join into herds with up to 30 individuals (Skinner & Chimimba, 2005 ). Similar to many other equines, the Hartmann's mountain zebras show a regular dust‐bathing behavior (Joubert, 1972a ), that in contrast to many other equines is carried out quite frequently several times during the day: The animal first moves into an incomplete lateral position and then turns alternately around its longitudinal axis to the left and right and then gives itself a push and stretches itself (McGreevy, 2012 ; Panzera et al., 2020 ). The exact reason for this behavior is unknown, but it is assumed that it is mainly for self‐grooming and removal of ectoparasites but partially also for resting (Joubert, 1972a ). Typically, a family of Hartmann's mountain zebras prefers a very specific area for this procedure and often the individual spots are used by the group members, so that soon pronounced depressions in the soil are created that have on roughly 2.5 m diameter and can become up to 30 cm deep (Joubert, 1972b ). When the zebras are migrating, the pits of the previous home range become abandoned but are sometimes reactivated during the next season, where they are used by the same or other families. 2.2 Microsite characteristics In 2014, we mapped all detectable zebra pits within a 1 × 1 km area unused for livestock‐keeping since 2001. We searched the pits for signs of current use (tracks, fresh dung, lack of vegetation), noted the presence of zebra feces, and classified the pits as either active or abandoned. From all pits mapped in 2014, we randomly selected 16 abandoned pits, marked them, and paired them with a 2 m × 2 m reference site 5 m south of the center of the respective pit (Figure A4 ). We chose this particular size to match the average size of the rolling pits. The distance of 5 m was chosen to ensure similar topography and ground conditions. We measured diameter and depth of the depression and determined the depth of the loose soil layer of the pit and reference sites using a simple penetrometer (Sutherland, 2006 ). We further characterized soil grain size composition of pits and reference sites by taking soil samples of the upper ten‐cm soil layer of a 40 × 40‐cm‐sized area. The samples were sieved into the different grain size classes: cobble and boulders, very coarse gravel, coarse gravel, fine‐to‐medium gravel, and sand and smaller particles, according to ISO 14688 (International Organization for Standardization, 2017 ), and the respective percentage by weight was calculated. Water‐soluble soil nutrients were determined by eluting an aliquot of 10 g the soil fraction sand and finer particles (oven‐dried at 105°C for 24 hr) with 100 ml of ultrapure water for 3 hr at room temperature. The eluate was left to sediment for 20 min, the supernatant was centrifuged for 15 min at 3,500 g , and 5 ml of the supernatant was used to analyze nitrogen and phosphate contents using standard ion chromatography (Michalski et al., 2019 ). A simulated precipitation event equivalent to 20 mm rainfall (applied within 1 hr) was used to measure the infiltration depth and determine the time course of soil moisture within the upper 10 cm of soil. Soil infiltration depth was determined 30 min after the precipitation event by digging and measuring the depth of the visually identified seepage front. The time course of soil moisture was determined using a TDR probe (Delta‐T Devices, ML3; Rajkai & Ryden, 1992 ) in two separate runs over 6 days each. The probe was initially calibrated once with an aliquot of soils taken from the pits and reference sites, respectively (Delta‐T Devices Ltd, 2013 ). 2.3 Vegetation assessment From 2014 until 2018, we consecutively mapped the vegetation of pits and reference sites in two rounds at an interval of about 4 weeks during the respective vegetation period (Table A1 ). We determined the plant species present and their respective abundance and visually estimated the percentage of cover. Cover data were aggregated to the percentage of total plant cover, cover of annual forbs, and cover of annual grasses. To show the development of vegetation over time and the differences in greening and wilting of pits and reference sites, we used high‐resolution RGB images acquired with a commercial DJI Phantom 4pro UAV. During the rainy season 2017, between 27 February and 6 April 2017, in total, 15 flyovers, 2 to 3 days between each other, were carried out over a transect covering the study area. The last relevant rain event occurred 14 days before the start of the flyovers and was 8 mm. During the flyovers, four distinct rainfall events occurred with 5 mm on 28 February, 22 mm on 2 March, 9 mm on 8 March, and 4 mm on 13 March. Flight altitude was 40 m.a.g.l., and image overlap was 75% to allow proper image alignment. All images of each flyover were processed and stitched as described in Woellner and Wagner ( 2019 ), resulting in a georeferenced (UTM, 33S) orthomosaic with a resolution of ~2 px/cm and a digital elevation model (DEM) of the study area. 2.4 Arthropod sampling Coincident in time with each vegetation mapping, the occurrence, and abundance of ground‐dwelling arthropods were determined using pitfall traps. As the numbers of trapped arthropods depend not only on their abundance but also on their activity levels (e.g., Topping & Sunderland, 1992 ), the obtained numbers of arthropods are commonly referred to as "activity density" rather than abundance (Thiele, 1977 ). The resulting data were used to test for differences among arthropod assemblages between pits and references and to study bottom‐up effects along the trophic cascade. Pitfall traps had a volume of 650 ml and a diameter of 9 cm and were filled with 250 ml of a 50% ethylene glycol—water mixture and a drop of detergent to reduce surface tension. Traps were installed on a level with the soil surface in the center of each pit or reference site (Lange et al., 2011 ) and kept open for 7 days after the vegetation was mapped. After each sampling round, arthropods were transferred into 70% ethanol and identified up to order and classified according to feeding guilds into herbivores, omnivores, and predators (Table A2 ; Picker et al., 2004 ; Scholtz & Holm, 2008 ) following the rapid assessment of biodiversity proposed by Oliver and Beattie ( 1996 ) used in comparable studies, for example, by Nickell et al. 2018 on Bison wallows. Though this somewhat limits deductions in the community structure, this was necessary as the majority of invertebrates have not been described (Samways & Samways, 1994 ). However, the method has been successfully applied to demonstrate habitat‐related changes in arthropod assemblages (see Blaum et al., 2009 ; Fabricius et al., 2003 ). Beetles ( Coleoptera ) and true bugs ( Hemiptera ) that included both species with herbivorous and predatory feeding preference were assigned to omnivores. Larvae that were not further determinable were excluded from analysis. 2.5 Statistics and data analysis Statistical analysis was performed with R, version 3.6.3 (R Core Team, 2020 ). Comparisons of soil grain sizes and nutrients between pits and reference sites were done by permutational paired t tests ( perm.t.test , library RVAideMemoire , Hervé, 2020 ). Time course of soil moisture was modeled as linear mixed effect model ( lme ; library nlme version 3.1‐120; Pinheiro et al., 2020 ). Soil moisture was log(x+1) transformed to obtain normality of variances. Day (since simulated rain event) and Type (pit/reference site) were used as explanatory variable. Plant diversity was calculated as "effective number of species" (ENS), the equally common species that result in the respective Shannon–Wiener index and reflect the true diversity of the communities considered (Jost, 2006 ) using the function diversity from the vegan library (Oksanen et al., 2019 ). Indicator species, that is, plant species predominantly associated with either pits or reference sites, were identified using the multipatt function with IndVal.g as association function (library indicspecies ; De Caceres & Legendre, 2009 ). The function provides estimates for the probability of a species occurring in the respective site group ( pO ) and a probability with which a site belongs to the respective group when the species is found (pT). It further calculates for each species an indicator value ( IndVal ) based on the product of its relative frequency and its relative average abundance in either pits or reference site. Analysis of the drone data was done using the raster package (library raster , version 3.1‐5; Hijmans, 2020 ). Based on the orthomosaic, the "greenness" of each pixel was determined by using the calc function to calculate the excess green minus excess red vegetation index (ExGR; Meyer & Neto, 2008 ) that is suitable to differentiate vegetation from ground. The ExGR vegetation index is defined as 2*g‐r‐b – (1.4*r‐b), with r = R/(R+G+B), g = G/(R+G+B), and b = B/(R+G+B) and R, G, and B being the red, green, and blue values of the respective pixel. All zebra pits and the respective reference sites were digitized as polygons. The ExGR values within each polygon were sampled, and the mean was calculated with the extract function. A baseline value was established using the total mean of the ExGR values of all pits and reference sites at the beginning and end of the flyovers (days 0 and 38) when the vegetation was dry and wilted. In order to determine the slope at each zebra pit position, we used the terrain function to determine the slope of each cell of the DEM and aggregated the results to 5 m × 5 m cells applying the aggregate function with mean . Differences between greenness of pits and reference sites were tested for each day using permutational t tests with holm adjustment for paired samples; differences between greenness of pits or reference sites to baseline were tested using standard permutational t tests ( perm.t.test , library RVAideMemoire , Hervé, 2020 ) with holm adjustment. To characterize the vegetation composition with regard to the prevalence of a certain functional group (annual grass, annual forb), we calculated the annual grass‐to‐forb ratio for each pit and reference site as the difference between the cover of annual grasses (annual grass cover/total cover) and proportional forbs cover (annual forb cover/total cover). This resulted in an index ranging between −1 for forbs only and +1 for grasses only. The relation between this grass‐to‐forb ratio and rainfall received within the preceding 30 days was modeled as linear mixed effect models ( lme ; library nlme version 3.1‐120; Pinheiro et al., 2020 ) using maximum likelihood with amount of rainfall ( Rain30d ), Type , and the two‐way interaction as explanatory variables. For testing bottom‐up effects on arthropods, the activity density of the respective feeding group was modeled as linear mixed effect model ( lme ; library nlme version 3.1‐120; Pinheiro et al., 2020 ) using maximum likelihood. Models for herbivores included total vegetation cover and Type (pit/reference site) as explanatory variables, models for omnivores additionally contained the activity density of herbivores, and models for predators additionally contained the activity density of potential prey (herbivores and omnivores), as well as two‐way interactions of all explanatory variables. To obtain normality of errors, the response variables were log(x+1) transformed. For all lmes, we used maximum likelihood and random intercept with pit/reference id as random factor to ensure independence of errors with respect to spatial and temporal autocorrelations (Pinheiro & Bates, 2000 ). Model simplifications were done in a backward stepwise model selection procedure by stepAIC (library MASS; Venables & Ripley, 2002 ) until the respective minimal adequate model was reached. 2.1 Study site and study species Our study was conducted between 2014 and 2018 on the farm Rooiklip, Khomas, Namibia (33 K 612,448 7,411,832), situated at 1,000 m.a.s.l. in the center of the escarpment halfway between Windhoek and Walvis Bay (Figure A1 ). Namibia's great escarpment is an approximately 100‐km wide strip of rugged semidesert reaching from the border with South Africa in the South to Angola in the North. The climate is hot‐arid, mean annual precipitation ranges from ~50 mm in the West to ~200 mm at the upper edge in the East (Mendelson et al., 2009 ), but rainfall is highly variable and often erratic. At our study site, the mean annual precipitation is 120 mm, but ranged from 60 mm to 180 mm during the study period. Rainfall was measured on a daily basis with an on‐site standard rain gauge. The main vegetation growth period occurs from February to March, where two‐thirds of the annual precipitation occurs (Wagner et al., 2016 ). The vegetation is arid grassland dominated by perennial grasses of the genus Stipagrostis that is sparsely scattered with trees and occasional dwarf shrubs. Depending on the amount of rainfall, various annual forbs and annual grasses join these perennials. Total vegetation cover seldom reaches more than 15% (Wagner et al., 2016 ). Soils are leptosols and consist of a shallow layer of differently sized weathered particles of the underlying schist bedrock. Rooiklip and its neighboring farms are largely unfenced and allow wildlife to roam freely between the farms and from the edge of the escarpment to the Namib Desert. The escarpment and the Pre‐Namib are home to the near‐endemic Hartmann's mountain zebra (Figure A1 ; Environmental Information Service, 2020 ; Gosling et al., 2019 ). The habitat of Hartmann's mountain zebra is mountainous areas and flats. Hartmann's mountain zebras further exhibit a marked migration pattern, following the rainfall and forage availability and moving between dry season and rainy season home ranges (Muntifering et al., 2019 ). Hartmann's mountain zebras usually live as family groups that consist of a stallion and his harem of mares and their foals. However, stallion groups or solitary stallions are frequent (Skinner & Chimimba, 2005 ), and occasionally, several family groups can join into herds with up to 30 individuals (Skinner & Chimimba, 2005 ). Similar to many other equines, the Hartmann's mountain zebras show a regular dust‐bathing behavior (Joubert, 1972a ), that in contrast to many other equines is carried out quite frequently several times during the day: The animal first moves into an incomplete lateral position and then turns alternately around its longitudinal axis to the left and right and then gives itself a push and stretches itself (McGreevy, 2012 ; Panzera et al., 2020 ). The exact reason for this behavior is unknown, but it is assumed that it is mainly for self‐grooming and removal of ectoparasites but partially also for resting (Joubert, 1972a ). Typically, a family of Hartmann's mountain zebras prefers a very specific area for this procedure and often the individual spots are used by the group members, so that soon pronounced depressions in the soil are created that have on roughly 2.5 m diameter and can become up to 30 cm deep (Joubert, 1972b ). When the zebras are migrating, the pits of the previous home range become abandoned but are sometimes reactivated during the next season, where they are used by the same or other families. 2.2 Microsite characteristics In 2014, we mapped all detectable zebra pits within a 1 × 1 km area unused for livestock‐keeping since 2001. We searched the pits for signs of current use (tracks, fresh dung, lack of vegetation), noted the presence of zebra feces, and classified the pits as either active or abandoned. From all pits mapped in 2014, we randomly selected 16 abandoned pits, marked them, and paired them with a 2 m × 2 m reference site 5 m south of the center of the respective pit (Figure A4 ). We chose this particular size to match the average size of the rolling pits. The distance of 5 m was chosen to ensure similar topography and ground conditions. We measured diameter and depth of the depression and determined the depth of the loose soil layer of the pit and reference sites using a simple penetrometer (Sutherland, 2006 ). We further characterized soil grain size composition of pits and reference sites by taking soil samples of the upper ten‐cm soil layer of a 40 × 40‐cm‐sized area. The samples were sieved into the different grain size classes: cobble and boulders, very coarse gravel, coarse gravel, fine‐to‐medium gravel, and sand and smaller particles, according to ISO 14688 (International Organization for Standardization, 2017 ), and the respective percentage by weight was calculated. Water‐soluble soil nutrients were determined by eluting an aliquot of 10 g the soil fraction sand and finer particles (oven‐dried at 105°C for 24 hr) with 100 ml of ultrapure water for 3 hr at room temperature. The eluate was left to sediment for 20 min, the supernatant was centrifuged for 15 min at 3,500 g , and 5 ml of the supernatant was used to analyze nitrogen and phosphate contents using standard ion chromatography (Michalski et al., 2019 ). A simulated precipitation event equivalent to 20 mm rainfall (applied within 1 hr) was used to measure the infiltration depth and determine the time course of soil moisture within the upper 10 cm of soil. Soil infiltration depth was determined 30 min after the precipitation event by digging and measuring the depth of the visually identified seepage front. The time course of soil moisture was determined using a TDR probe (Delta‐T Devices, ML3; Rajkai & Ryden, 1992 ) in two separate runs over 6 days each. The probe was initially calibrated once with an aliquot of soils taken from the pits and reference sites, respectively (Delta‐T Devices Ltd, 2013 ). 2.3 Vegetation assessment From 2014 until 2018, we consecutively mapped the vegetation of pits and reference sites in two rounds at an interval of about 4 weeks during the respective vegetation period (Table A1 ). We determined the plant species present and their respective abundance and visually estimated the percentage of cover. Cover data were aggregated to the percentage of total plant cover, cover of annual forbs, and cover of annual grasses. To show the development of vegetation over time and the differences in greening and wilting of pits and reference sites, we used high‐resolution RGB images acquired with a commercial DJI Phantom 4pro UAV. During the rainy season 2017, between 27 February and 6 April 2017, in total, 15 flyovers, 2 to 3 days between each other, were carried out over a transect covering the study area. The last relevant rain event occurred 14 days before the start of the flyovers and was 8 mm. During the flyovers, four distinct rainfall events occurred with 5 mm on 28 February, 22 mm on 2 March, 9 mm on 8 March, and 4 mm on 13 March. Flight altitude was 40 m.a.g.l., and image overlap was 75% to allow proper image alignment. All images of each flyover were processed and stitched as described in Woellner and Wagner ( 2019 ), resulting in a georeferenced (UTM, 33S) orthomosaic with a resolution of ~2 px/cm and a digital elevation model (DEM) of the study area. 2.4 Arthropod sampling Coincident in time with each vegetation mapping, the occurrence, and abundance of ground‐dwelling arthropods were determined using pitfall traps. As the numbers of trapped arthropods depend not only on their abundance but also on their activity levels (e.g., Topping & Sunderland, 1992 ), the obtained numbers of arthropods are commonly referred to as "activity density" rather than abundance (Thiele, 1977 ). The resulting data were used to test for differences among arthropod assemblages between pits and references and to study bottom‐up effects along the trophic cascade. Pitfall traps had a volume of 650 ml and a diameter of 9 cm and were filled with 250 ml of a 50% ethylene glycol—water mixture and a drop of detergent to reduce surface tension. Traps were installed on a level with the soil surface in the center of each pit or reference site (Lange et al., 2011 ) and kept open for 7 days after the vegetation was mapped. After each sampling round, arthropods were transferred into 70% ethanol and identified up to order and classified according to feeding guilds into herbivores, omnivores, and predators (Table A2 ; Picker et al., 2004 ; Scholtz & Holm, 2008 ) following the rapid assessment of biodiversity proposed by Oliver and Beattie ( 1996 ) used in comparable studies, for example, by Nickell et al. 2018 on Bison wallows. Though this somewhat limits deductions in the community structure, this was necessary as the majority of invertebrates have not been described (Samways & Samways, 1994 ). However, the method has been successfully applied to demonstrate habitat‐related changes in arthropod assemblages (see Blaum et al., 2009 ; Fabricius et al., 2003 ). Beetles ( Coleoptera ) and true bugs ( Hemiptera ) that included both species with herbivorous and predatory feeding preference were assigned to omnivores. Larvae that were not further determinable were excluded from analysis. 2.5 Statistics and data analysis Statistical analysis was performed with R, version 3.6.3 (R Core Team, 2020 ). Comparisons of soil grain sizes and nutrients between pits and reference sites were done by permutational paired t tests ( perm.t.test , library RVAideMemoire , Hervé, 2020 ). Time course of soil moisture was modeled as linear mixed effect model ( lme ; library nlme version 3.1‐120; Pinheiro et al., 2020 ). Soil moisture was log(x+1) transformed to obtain normality of variances. Day (since simulated rain event) and Type (pit/reference site) were used as explanatory variable. Plant diversity was calculated as "effective number of species" (ENS), the equally common species that result in the respective Shannon–Wiener index and reflect the true diversity of the communities considered (Jost, 2006 ) using the function diversity from the vegan library (Oksanen et al., 2019 ). Indicator species, that is, plant species predominantly associated with either pits or reference sites, were identified using the multipatt function with IndVal.g as association function (library indicspecies ; De Caceres & Legendre, 2009 ). The function provides estimates for the probability of a species occurring in the respective site group ( pO ) and a probability with which a site belongs to the respective group when the species is found (pT). It further calculates for each species an indicator value ( IndVal ) based on the product of its relative frequency and its relative average abundance in either pits or reference site. Analysis of the drone data was done using the raster package (library raster , version 3.1‐5; Hijmans, 2020 ). Based on the orthomosaic, the "greenness" of each pixel was determined by using the calc function to calculate the excess green minus excess red vegetation index (ExGR; Meyer & Neto, 2008 ) that is suitable to differentiate vegetation from ground. The ExGR vegetation index is defined as 2*g‐r‐b – (1.4*r‐b), with r = R/(R+G+B), g = G/(R+G+B), and b = B/(R+G+B) and R, G, and B being the red, green, and blue values of the respective pixel. All zebra pits and the respective reference sites were digitized as polygons. The ExGR values within each polygon were sampled, and the mean was calculated with the extract function. A baseline value was established using the total mean of the ExGR values of all pits and reference sites at the beginning and end of the flyovers (days 0 and 38) when the vegetation was dry and wilted. In order to determine the slope at each zebra pit position, we used the terrain function to determine the slope of each cell of the DEM and aggregated the results to 5 m × 5 m cells applying the aggregate function with mean . Differences between greenness of pits and reference sites were tested for each day using permutational t tests with holm adjustment for paired samples; differences between greenness of pits or reference sites to baseline were tested using standard permutational t tests ( perm.t.test , library RVAideMemoire , Hervé, 2020 ) with holm adjustment. To characterize the vegetation composition with regard to the prevalence of a certain functional group (annual grass, annual forb), we calculated the annual grass‐to‐forb ratio for each pit and reference site as the difference between the cover of annual grasses (annual grass cover/total cover) and proportional forbs cover (annual forb cover/total cover). This resulted in an index ranging between −1 for forbs only and +1 for grasses only. The relation between this grass‐to‐forb ratio and rainfall received within the preceding 30 days was modeled as linear mixed effect models ( lme ; library nlme version 3.1‐120; Pinheiro et al., 2020 ) using maximum likelihood with amount of rainfall ( Rain30d ), Type , and the two‐way interaction as explanatory variables. For testing bottom‐up effects on arthropods, the activity density of the respective feeding group was modeled as linear mixed effect model ( lme ; library nlme version 3.1‐120; Pinheiro et al., 2020 ) using maximum likelihood. Models for herbivores included total vegetation cover and Type (pit/reference site) as explanatory variables, models for omnivores additionally contained the activity density of herbivores, and models for predators additionally contained the activity density of potential prey (herbivores and omnivores), as well as two‐way interactions of all explanatory variables. To obtain normality of errors, the response variables were log(x+1) transformed. For all lmes, we used maximum likelihood and random intercept with pit/reference id as random factor to ensure independence of errors with respect to spatial and temporal autocorrelations (Pinheiro & Bates, 2000 ). Model simplifications were done in a backward stepwise model selection procedure by stepAIC (library MASS; Venables & Ripley, 2002 ) until the respective minimal adequate model was reached. 3 RESULTS Between 2014 and 2018, in total, 656 pits were mapped in the study area, all restricted to elevated, but level and flat grounds, free of rocks, with an average inclination of <5° (Figure A5 ). From these 656 pits, 341 (~52%) were created in new places, where no pit has been mapped before, the rest was either re‐used or abandoned during the study period. Fresh or dried dung was found in 89% of the mapped pits. After heavy rainfall events, many rolling pits collected runoff and rainfall and it took up to three days for the water to evaporate (Figure A3b ). Pit diameter ranged from 105 cm to 350 cm with a mean of 229 ± 55 cm (mean ± SD ), resulting in an average area of 4.1 ± 0.3 m² per pit. The average depth of the pits (distance from the surface of the matrix to the soil layer of the pit) was 9.5 ± 3.6 cm. The substrate of pits contained considerably more fine‐grained material than the reference sites. In pits, sand and finer grained particles (84%) were the highest share, followed by gravel (11%), whereas reference sites contained 60% gravel and only 36% sand and finer material (Table A3 ). Water‐soluble soil nutrients were low (Table A4 ), with nitrogen and phosphate contents being significantly higher in pits (water‐soluble N: 4.8 mg/kg; PO 4 : 1.3 mg/kg) than on reference sites (water‐soluble N: 1.2 mg/kg; PO 4 : 0.5 mg/kg). The depth of the loose soil layer of pits was 29.3 ± 8.1 cm and thereby significantly higher than the average loose soil depth 5.5 ± 2.1 cm of the reference sites ( p < .002). After our simulated rain event, the soil infiltration depth in pits reached 9.9 ± 1.9 cm and only 4.7 ± 1.7 cm on reference sites, which corresponded with the depth of loose soil. During the first 24 hr after the event, the average soil moisture in pits reached 6.31 ± 0.24% and remained above the permanent wilting point for coarse sand (~2%) for over four days. In contrast, soil moisture never exceeded the permanent wilting point on reference sites (Figure 1 ). FIGURE 1 Trend of soil moisture in pits and reference sites after a simulated rainfall event with 20 mm applied within 1 h. Dashed horizontal line indicates permanent wilting point of sandy "soils" The temporal course of greening and drying out of the vegetation following natural rain events showed clear differences between pits and reference sites. The ExGR vegetation index and hence greenness of the zebra pits increased with each rainfall event until day 14 and then decreased steadily over the next 14 days until it reached the baseline at day 28 when the vegetation was wilted. From day six on, it was always above the greenness of the reference sites. In contrast, the greenness of the reference sites increased only after the first rain and then remained constant, apart from a slight decrease on days six and eight, until day 18. After that, it decreases and reaches the baseline around day 26, about two days earlier than the pits (Figure 2 ; Table A5 ). FIGURE 2 Greenness of pits and reference sites expressed by the ExGR vegetation index, derived from RGB UAV imagery taken on March 2017. Dashed line represents baseline of soil without green vegetation. Gray bars indicate rainfall events with 5, 22, 9, and 5 mm Total vegetation cover did not exceed 23% in pits and 13% on reference sites and correlated with rainfall received within the preceding 30 days. Both total vegetation cover and cover of annual forbs were higher (total cover on average 1.4‐fold, cover of annuals 1.8‐fold) in pits compared to reference sites throughout all years (Table A6 ). The proportion of forbs and the total cover of annual plants was significantly higher in pits and increased significantly with the amount of rainfall received during the preceding 30 days (Figure 3 ; Table A6 ). FIGURE 3 Relative proportion of annual forbs and grasses in relation to rainfall in the preceding 30 days in pits and reference sites. Dashed line indicates equal grass‐to‐forb ratio In total, we found 26 annual plant species, from which six were annual grasses and 20 annual forbs, but all native to the area. General plant diversity of pits and reference sites was similar with 4.8–8.4 species in pits and 4.5–7.1 species in reference sites (Table A6 ) but varied throughout the years as a response to different amounts of rainfall. Pits were mainly characterized by the presence of annual forbs (seven indicator species), whereas annual grasses mainly characterized the reference sites (three indicator species; Table 1 ). TABLE 1 Species associated predominantly with pits or reference sites as identified by indicator analysis Growth form pT pO IndVal p ‐value Rolling pits Heliotropium ciliatum Annual forb 0.89 0.26 0.527 .001 Hirpicum gazanoides Annual forb 0.84 0.27 0.511 .001 Sesbania pachycarpa Annual forb 0.97 0.22 0.443 .001 Cucumis sagittatus Annual forb 1.00 0.16 0.432 .001 Hermannia modesta Annual forb 0.83 0.06 0.248 .025 Cleome suffruticosa Annual forb 0.80 0.05 0.204 .052 Citrullus lanatus Annual forb 1.00 0.01 0.122 .513 Reference sites Enneapogon desvauxii Annual grass 0.84 0.79 0.843 .001 Eragrostis nindensis Annual grass 0.96 0.15 0.377 .001 Entoplocamia aristulata Annual grass 0.75 0.15 0.367 .007 Note IndVal, indicator value, product of the relative frequency and relative average abundance in pits or reference sites (De Caceres & Legendre, 2009 ); pO, probability of finding the species within group; pT, estimate of probability that site belongs to target group when species has been found. John Wiley & Sons, Ltd Herbivorous arthropods showed a significant positive correlation with total vegetation cover with slightly higher activity density in pits compared to reference sites. Omnivores were positively related to the activity density of herbivores, but without differences between pits and reference sites, whereas predators were showing a significant positive correlation with the activity density of prey (omnivores and herbivores) but no significant difference between pits and reference sites (Table 2 ). TABLE 2 Coefficients of the relationship between the activity density of the different arthropod groups and the respective explanatory variables determined with the mixed effect models Explanatory variable Activity density herbivores Activity density omnivores Activity density predators Vegetation cover 1.02*** – – Herbivores n.t. 1.04*** 1.03*** Omnivores n.t. n.t. 1.02*** Type a 0.84* – – Note All interactions were excluded during stepwise simplification. Back‐transformed, exponentiated coefficients, and levels of significance are given. "n.t.": not tested. "Herbivores" include the orders Caelifera , Cicadina , and Stenorrhyncha ; "omnivores" include Blattodea , Coleoptera , Ensifera , Heteroptera , and Psocoptera ; "predators" include Acarina , Araneae , Chilopoda , Pseudoscorpiones , and Scorpiones . * p < .05, ** p < .01, *** p < .001. a "pit" versus "reference site"; reference level was "reference site." John Wiley & Sons, Ltd 4 DISCUSSION The characteristic rolling pits of the Hartmann's mountain zebra in the Pre‐Namib, created by their dust‐bathing behavior, produce favorable microsites that clearly differ from their surroundings. Their special ecohydrological properties promote a denser and different vegetation composition, characterized by annual forbs, and, to a lesser extent, increase the activity density of herbivorous arthropods. In the escarpment region of Namibia, no other larger mammal species exhibits a comparable rolling or digging behavior leading to characteristic depressions similar to the rolling pits of the Hartmann's mountain zebra (Barandongo et al., 2018 ). With an average diameter of 230 cm and a total depth of up to over 40 cm, the rolling pits of the Hartmann's mountain zebra are within the larger discrete structures created by soil‐disturbing animals, although they did not reach the size of bison wallows (Miller et al., 2013 ) that cover in average almost twice the area. The rolling pits are filled up to ¾ with a 30‐cm thick layer of predominantly loose sand. By their dust‐bathing behavior, the mountain zebras remove the extant vegetation dominated by perennial grasses of the genus Stipagrostis and coarser soil components such as gravel and cobble are moved outside of the pit, leaving the finer material in the pits themselves. The characteristic of the rolling pits generally corresponds to those known from depressions and resting places of larger antelopes, equines, or kangaroos. Similar to the bedding sites of the Nubian ibex (Gutterman, 1997 ) or the wallows of American bison (McMillan et al., 2011 ), the rolling pits of Hartmann's mountain zebra collect runoff after rainfall. The loose soil filling of the pits allows a better infiltration, and once infiltrated, this moist soil acts as a storage for the runoff water that keeps the soil moist for a few days longer than the surrounding soil. In addition, the loose soil of rolling pits has a higher water‐soluble nitrogen and phosphate contents. Nine of ten pits we mapped had considerable remains of dry zebra dung. Deposits of feces and higher urine concentrations in the soil are a typical feature known from other resting places of larger animals such as Gemsbok (Dean & Milton, 1991a , 1991b ) or Kangaroo (Eldridge & Rath, 2002 ). In addition, such depressions frequently accumulate litter (Mallen‐Cooper et al., 2019 ), which is then together with the dung quickly converted into nutrients by the stronger mineralization due to a higher soil moisture (Eldridge & Rath, 2002 ; Veldhuis et al., 2018 ). As a result, these resting places and so the rolling pits of the Hartmann's mountain zebra form relatively moist and nutrient‐rich patches in the otherwise nutrient‐poor savanna (Augustine & McNaughton, 2006 ; McNaughton et al., 1988 ). The pits are mainly used for only one season but remain as depressions for several years. When the vegetation dries up, the Hartmann's mountain zebras move to their dry season home ranges (Muntifering et al., 2019 ) and the pits become abandoned. In the following rainy season, when the zebras return, new pits are created. From 656 abandoned pits, between 2014 and 2018, only 71 (11%) were reactivated, while at the same time, 341 were newly created. When abandoned, the higher nutrient availability and an increased and longer lasting soil moisture allow the establishment of vegetation that is clearly denser and differs in terms of vegetation composition from the surrounding grassland similar to the effects of the abandoned wallows of the American bison or the bedding sites of the Nubian ibex (Gutterman, 1997 ; Nickell et al., 2018 ). Pits and reference sites show a simultaneous green flush, but the greenness of pits soon exceeds that of the reference sites, a clear sign of denser vegetation within the pits. The greenness of pits remains higher until it reaches the baseline of dry ground about two to four days later than the reference sites. This delayed wilting corresponds with the finding of our infiltration experiments, where soil moisture in pits remained over the permanent wilting point for two days longer. Ground truthing and our assessment of the vegetation in the field confirms a higher cover and a different structure of the vegetation of the pits. Though the general plant diversity of pits and reference sites is similar, the vegetation of pits and reference sites differs in terms of species composition and dominant functional types. The typical tufted perennial grasses of the genus Stipagrostis that make up the dominant vegetation of the surrounding grassland are practically absent in the pits. Instead, the pits are mainly vegetated with annuals, with a clear dominance of forbs. This dominance of forbs becomes even more pronounced with higher rainfall, when sufficient runoff can be collected and stored by the soil in the pits. The general importance of animal‐created depressions in arid environments for annual forbs has been documented for a number of animal species (Coggan et al., 2018 ; Gutterman, 2001 ). The higher and longer lasting soil moisture of the rolling pits also plays a crucial role in the occurrence of annual forbs. Together with higher nutrient availability, it is a decisive factor for the establishment of forbs in arid environments (Siebert & Dreber, 2019 ), which need longer lasting favorable conditions for the successful establishment of their seedlings (Hillel & Kozlovsky, 2012 ). The lack of perennial grasses in the pits further ensures that freshly germinated ephemerals are subject to less competition (Dean & Milton, 1991a , 1991b ). Many of the annual forbs found in the pits such as H. ciliatum , H. modesta , or S. pachycarpa are otherwise mainly found at sites with better water supply, such as dry riverbeds or topographic depressions (personal observation, Strohbach, 2017 ). Annual forbs represent the main component of species diversity in savanna ecosystems. Annual forbs contribute substantially to various ecosystem functions and provide forage and shelter for numerous arthropod species (Siebert & Dreber, 2019 ). Given the generally sparse vegetation and the particularly low density of forbs in the study region, zebra pits are likely to be an important factor shaping the local forb communities and providing safe sites for forbs particularly under dry conditions. Therefore, pits may act as stepping stones, linking the vegetation of the intermittent streams and allowing forb species to spread not only along these rivers but also via the surrounding landscape. Similar to Nickell et al. ( 2018 ) or Ewacha et al. ( 2016 ), we found that the effects of pits were not restricted to vegetation but also affected higher trophic levels and slightly increased the activity density of arthropods. Independent of vegetation cover, herbivorous arthropods were generally more abundant in the pits. The different and denser vegetation of the abandoned pits provides a good source of nutrition for herbivores (Dalerum et al., 2017 ) and the larvae of the other feeding guilds. The structural diversity and conditions of the herbaceous vegetation further offer a variety of niches and provide habitats and refuges for arthropod species and their larvae (Dalerum et al., 2017 ). However, the size of these effects on arthropod activity density is not very pronounced, which is probably due to the limited size of the rolling pits and arthropod mobility, as well as the low resolution of the determination of the different arthropod taxa. Even though our study was only carried out on a single farm and an area comprising only 1 km 2 , our results can be transferred to the entire geographic region. Flyovers with UAV and observations on other farms in the region confirm the more or less dense occurrence of rolling pits within larger areas. Climate, precipitation, and vegetation in the distribution range of Hartmann's mountain zebra are largely similar (see Mendelson et al., 2009 ). The rolling pits persist for many years after they have been abandoned and thus most likely have a long‐lasting influence on the ecosystem. The current number of mountain zebras is estimated to be 32.000 individuals (Gosling et al., 2019 ). Their seasonally shifting home ranges (Muntifering et al., 2019 ), the long persistence, and limited reuse of abandoned pits result in an occurrence of mountain zebra pits throughout the Namibian escarpment in a varying density (Wagner TC & Fischer C, unpublished data). In conclusion, with their rolling pits, the Hartmann's mountain zebras clearly contribute to a higher biodiversity and structural heterogeneity of the arid savanna landscape in the Pre‐Namib and can therefore be, like other soil‐disturbing animals (Mallen‐Cooper et al., 2019 ; Romero et al., 2015 ), considered as an ecosystem engineer. In the future, the importance of the rolling pits for the local forb and arthropod communities might even increase, due to prolonged severe drought periods, which are expected in the region as a result of climate change (Archer et al., 2019 ; Maúre et al., 2018 ). CONFLICT OF INTEREST The corresponding author confirms on behalf of all authors that there have been no involvements that might raise the question of bias in the work reported or in the conclusions, implications, or opinions stated. AUTHOR CONTRIBUTIONS Thomas C Wagner: Conceptualization (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Supervision (equal); Writing‐original draft (equal). Kenneth Uiseb: Formal analysis (equal); Investigation (equal); Writing‐original draft (equal); Writing‐review & editing (equal). Christina Fischer: Conceptualization (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Supervision (equal); Writing‐original draft (equal); Writing‐review & editing (equal). DATA AVAILABILITY STATEMENT Data are available in the OSF Data repository under https://doi.org/10.17605/OSF.IO/QWX6K . 1 FIGURE A1 Distribution area of the Hartmann's mountain zebra ( Zebra equus hartmannae ) and location of the study site in Namibia. Position of the study site Rooiklip (S 23°24′23.29″, E 016°03′37.35″) and current distribution range of Hartmann's mountain zebra (transparent white area; www.iucnredlist.org , updated with data from the Environmental Information Service Namibia, Atlas of Mammals). The Hartmann's mountain zebra occurs in two disjunct areas within the Namibian escarpment, a smaller one in the southern part of Namibia, bordering South Africa, and a larger one, stretching from the Maltahöhe District north into the Iona National Park, southern Angola (Gosling et al., 2019 ) FIGURE A2 Distribution of zebra pits. Distribution of active (green) and abandoned (orange) zebra pits within a 500 m × 500 m section of the study site in 2017. The pits are clustered on level, open areas between intermittent rivers (blue lines) and ravines and along tracks (brown lines) FIGURE A3 Zebra pits as microsites. Typical pits of Hartmann's mountain zebra. (a) active, with a pronounced ring of vegetation. (b) filled with runoff after rainfall. (c) abandoned and vegetated with various forbs FIGURE A4 Aerial photograph with densely vegetated rolling pits. Details from an aerial photograph of the study site with zebra tracks (dotted lines) and six abandoned rolling pits (dashed circles) of mountain zebra 14 days after a rainfall event with ~30 mm of rainfall within 1 day. The dense annual vegetation clearly stands out against the less dense cover of the perennial Stipagrostis grasses of the matrix. The configuration of a research plot consisting of pit and reference site and the position of the pitfall traps (red dot) in the center are schematically illustrated FIGURE A5 Position of the rolling pits within a section of the study site. Rolling pits only occur on level ground and often elevated grounds that make up about 1/3rd of the study site. Ground with an inclination of less than 5° is highlighted with magenta, red dots mark the respective pits TABLE A1 Vegetation mapping and arthropod sampling dates Year Run Date of vegetation sampling and opening of pitfall traps Rainfall within the preceding 30 days [mm] Remark 2014 1 10.03.2014 0 No vegetation 2 22.03.2014 54 2015 1 07.03.2015 4 2 08.04.2015 18 2016 1 12.03.2016 48 2 23.03.2016 48 2017 1 11.03.2017 36 2 30.03.2017 41 2018 1 20.03.2018 0 No vegetation 2 24.04.2018 42 Note Dates when vegetation sampling and pitfall trapping took place and the total amount of rainfall received in the preceding 30 days. The remark "no vegetation" indicates that no vegetation was found. John Wiley & Sons, Ltd TABLE A2 Arthropod groups Group Included taxonomic units Herbivores Caelifera (Short‐horned grasshoppers) Cicadina (Cicadas) Stenorrhyncha (Aphids and scale insects) Omnivores Blattodea (Cockroaches) Coleoptera (Beetles) Ensifera (Long‐horned grasshoppers) Heteroptera (True bugs) Psocoptera (Psocids and book lice) Predators Acarina (Ticks and mites) Araneae (Spiders) Chilopoda (Centipedes) Pseudoscorpiones (False scorpions) Scorpiones (Scorpions) Solifugae (Solifugae) Note Classification of arthropod orders according to their feeding preference. Arthropods were determined to order and grouped according to their feeding behavior using Picker et al., 2004 ; Scholtz & Holm, 2008 ; http://biodiversity.org.na/taxondisplay.php?nr=8 , downloaded: 04.07.2017. Orders with both herbivorous and predatory representatives were classified as omnivores. John Wiley & Sons, Ltd TABLE A3 Soil grain size composition and chemistry Grain size according to EN/ISO 14688‐1:2002 Reference (mean ± SD ) [%] Pit (mean ± SD ) [%] p ‐value Sand and finer 35.5 ± 15.5 84.3 ± 10.7 .001 ** Fine to medium gravel 30.1 ± 11.6 11.7 ± 4.7 .004 * Coarse gravel 30.3 ± 8.4 1.1 ± 0.8 .001 ** Very coarse gravel 3.4 ± 3.4 2.3 ± 3.4 .400 n.s. Cobble 0.6 ± 1.3 0.6 ± 0.7 .944 n.s. Boulders 0.1 ± 0.2 0.0 ± 0.1 .000 n.s. Note Soil sample from the upper 10 cm of a 40 × 40‐cm‐sized plot of pits and references, taken after the end of the study. Percentage by weight of the different grain size classes (ISO 14,688). Significance of difference determined with paired permutational t tests ( perm.t.test , RVAideMemoire ; n = 999); * p < .05, ** p < .01, *** p < .001; n.s: not significant. John Wiley & Sons, Ltd TABLE A4 Water‐soluble soil nutrients Parameter Reference (mean ± SD ) [mg/kg] Pit (mean ± SD ) [mg/kg] Significance of difference pH 7.41 ± 0.27 7.52 ± 0.28 0.180 n.s. N 1.22 ± 0.07 4.80 ± 3.46 0.012 ** PO4 0.48 ± 0.28 1.33 ± 0.78 0.012 ** Na 0.22 ± 0.08 0.45 ± 0.15 0.631 n.s. K 6.76 ± 5.00 6.63 ± 5.31 0.924 n.s. Mg 3.02 ± 2.27 5.78 ± 4.78 0.250 n.s. Ca 6.84 ± 3.91 8.77 ± 1.82 0.344 n.s. Note Aliquot of 10 g oven‐dried substrate (sand and finer) eluted with 100ml of ultrapure water for 3h at room temperature; eluate left to sediment for 20min, supernatant centrifuged for 15min at 3,500 g; 5m of supernatant analyzed using standard ion chromatography. Significance of difference between pits and reference determined with paired permutational t tests ( perm.t.test , RVAideMemoire ; n = 999); * p < .05, ** p < .01, *** p < .001; n.s: not significant. John Wiley & Sons, Ltd TABLE A5 Greenness Day ExGR Significance Pit Reference Pit–Reference Pit‐Baseline Reference‐Baseline 0 −0.208 ± 0.030 −0.221 ± 0.016 *** *** * 4 −0.170 ± 0.028 −0.172 ± 0.029 n.s. *** *** 7 −0.173 ± 0.023 −0.193 ± 0.019 *** *** *** 9 −0.161 ± 0.025 −0.186 ± 0.019 *** *** *** 11 −0.153 ± 0.028 −0.186 ± 0.024 *** *** *** 15 −0.129 ± 0.031 −0.170 ± 0.024 *** *** *** 18 −0.140 ± 0.027 −0.172 ± 0.021 *** *** *** 20 −0.145 ± 0.031 −0.182 ± 0.020 *** *** *** 22 −0.141 ± 0.043 −0.198 ± 0.018 *** *** *** 24 −0.184 ± 0.026 −0.215 ± 0.016 *** *** * 26 −0.196 ± 0.050 −0.223 ± 0.021 *** *** n.s. 28 −0.224 ± 0.021 −0.230 ± 0.017 n.s. n.s. n.s. 31 −0.214 ± 0.026 −0.214 ± 0.031 n.s. * n.s. 34 −0.219 ± 0.027 −0.223 ± 0.026 * n.s. n.s. 38 −0.223 ± 0.024 −0.224 ± 0.021 n.s. n.s. n.s. Note n.s: not significant, *** p < .001; ** p < .01, * p < .05. Average ExGR values and standard deviation of pits and references during the flyovers and the significance of difference between pits and references and the respective difference to baseline. John Wiley & Sons, Ltd TABLE A6 Total plant cover, cover of annual forbs and grasses and effective number of species (mean ± SD ) in pits and on references Param. 2014 2015 2016 2017 2018 Rain 30 d [mm] 52 18 48 41 36 Pit Ref. Pit Ref. Pit Ref. Pit Ref. Pit Ref. Total plant cover [%] 6.7 ± 3.9 6.6 ± 5.5 5.0 ± 4.9 3.7 ± 2.0 18.3 ± 13.9 13.1 ± 8.3 22.9 ± 14.0 11.7 ± 6.7 0.3 ± 0.3 0.2 ± 0.2 Annual forb cover [%] 6.5 ± 3.9 3.9 ± 2.6 3.9 ± 0.4 2.9 ± 2.0 16.7 ± 12.5 10.1 ± 8.1 22.8 ± 14.0 8.1 ± 6.4 0.3 ± 0.3 0.2 ± 0.2 Annual grass cover [%] 0.19 ± 0.28 0.96 ± 0.60 0.65 ± 0.53 1.19 ± 0.84 1.67 ± 1.29 2.42 ± 1.69 0.03 ± 0.09 0.10 ± 0.15 0.07 ± 0.14 0.19 ± 0.26 Species 8.4 ± 2.7 6.7 ± 1.7 6.2 ± 2.2 7.1 ± 2.2 5.3 ± 1.5 5.6 ± 1.7 6.8 ± 1.7 7.1 ± 1.7 4.9 ± 1.3 4.5 ± 2.1 ENS 5.8 ± 2.3 3.8 ± 1.4 3.1 ± 1.9 3.5 ± 1.5 3.0 ± 1.3 2.8 ± 1.3 4.6 ± 1.5 5.3 ± 1.5 3.0 ± 1.2 2.2 ± 1.0 Note Plant diversity of pits and references as average number of species and effective number of species (ENS, Jost, 2006 ; Tuomisto, 2010 ). No relevant vegetation was found in 2019. John Wiley & Sons, Ltd

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794041/

Potential Risk of Regional Disease Spread in West Africa through Cross-Border Cattle Trade

Background Transboundary animal movements facilitate the spread of pathogens across large distances. Cross-border cattle trade is of economic and cultural importance in West Africa. This study explores the potential disease risk resulting from large-scale, cross-border cattle trade between Togo, Burkina Faso, Ghana, Benin, and Nigeria for the first time. Methods and Principal Findings A questionnaire-based survey of livestock movements of 226 cattle traders was conducted in the 9 biggest cattle markets of northern Togo in February-March 2012. More than half of the traders (53.5%) operated in at least one other country. Animal flows were stochastically simulated based on reported movements and the risk of regional disease spread assessed. More than three quarters (79.2%, range: 78.1–80.0%) of cattle flowing into the market system originated from other countries. Through the cattle market system of northern Togo, non-neighbouring countries were connected via potential routes for disease spread. Even for diseases with low transmissibility and low prevalence in a given country, there was a high risk of disease introduction into other countries. Conclusions By stochastically simulating data collected by interviewing cattle traders in northern Togo, this study identifies potential risks for regional disease spread in West Africa through cross-border cattle trade. The findings highlight that surveillance for emerging infectious diseases as well as control activities targeting endemic diseases in West Africa are likely to be ineffective if only conducted at a national level. A regional approach to disease surveillance, prevention and control is essential. Background Transboundary animal movements facilitate the spread of pathogens across large distances. Cross-border cattle trade is of economic and cultural importance in West Africa. This study explores the potential disease risk resulting from large-scale, cross-border cattle trade between Togo, Burkina Faso, Ghana, Benin, and Nigeria for the first time. Methods and Principal Findings A questionnaire-based survey of livestock movements of 226 cattle traders was conducted in the 9 biggest cattle markets of northern Togo in February-March 2012. More than half of the traders (53.5%) operated in at least one other country. Animal flows were stochastically simulated based on reported movements and the risk of regional disease spread assessed. More than three quarters (79.2%, range: 78.1–80.0%) of cattle flowing into the market system originated from other countries. Through the cattle market system of northern Togo, non-neighbouring countries were connected via potential routes for disease spread. Even for diseases with low transmissibility and low prevalence in a given country, there was a high risk of disease introduction into other countries. Conclusions By stochastically simulating data collected by interviewing cattle traders in northern Togo, this study identifies potential risks for regional disease spread in West Africa through cross-border cattle trade. The findings highlight that surveillance for emerging infectious diseases as well as control activities targeting endemic diseases in West Africa are likely to be ineffective if only conducted at a national level. A regional approach to disease surveillance, prevention and control is essential. Introduction Animal movements within countries and across borders can facilitate the rapid spread of pathogens across large distances [1] . Recent examples include the spread of highly pathogenic avian influenza H5N1 globally [2] and of Trypanosoma brucei rhodesiense , one of the agents of Human African Trypanosomiasis, through cattle trade in Uganda [3] . In industrialised countries, this risk can be mitigated through strict importation controls. However, in developing countries, cross-border trade of live animals is often an important component of livestock production systems from both an economic and cultural perspective. In West Africa, long-distance, cross-border cattle trade was estimated to be worth US$150 million in 2000 [4] . Traditionally, livestock have been raised in the semi-arid Sahel zone and traded with countries in the southerly forested zones [4] , [5] . These cross-border movements continue to occur frequently and often outside of official veterinary control. Similar trends exist in East Africa, where cross-border trade involving Somalia, Ethiopia, Sudan, Kenya, and Tanzania was estimated to be worth US$61 million per annum in 2009, with only 10% of trade occurring through official channels [6] . In South-East Asia, interviews with cattle traders uncovered trade routes involving Thailand, Laos, Cambodia, and Vietnam. Almost half (45%) of the 60 Cambodian traders interviewed admitted to trading animals which they suspected to be infected with Foot-and-Mouth Disease (FMD) virus [7] . Understanding the potential pathways and risk for regional spread of infectious diseases is essential for tailoring appropriate control interventions in West Africa. In this context, a questionnaire-based survey was undertaken with cattle traders operating in cattle markets in northern Togo in 2012, and the potential risk of regional disease spread through trade routes was assessed through stochastic simulations. Methods Ethics Statement This research was a component of a larger study of zoonotic disease epidemiology in Togo [8] , and was approved by the Ethics Committee for Health Research (Comité de Bioéthique pour la Recherche en Santé) of the Ministry of Health of Togo. In Switzerland, approval was given by the Ethics Commission of the Cantons of Basel-Stadt and Basel-Land and the Research Commission of the Swiss Tropical and Public Health Institute of Basel, Switzerland. The information to be communicated to participants was provided as a written document to the interviewers and they received training regarding the consent process. Prior to interviewing, the study objectives, procedures and questionnaire content were explained to participants in their local language and they were assured that the questionnaire data would be treated anonymously. As the interviews were conducted in the cattle traders' busy, outdoor workplaces, obtaining written consent was determined to be impractical in this setting. Similar to previous cross-sectional surveys conducted with traders in marketplaces [9] , [10] , all participants provided informed verbal consent before the interview, as approved by the aforementioned ethics and research commissions. The informed consent of each participant was recorded by the interviewer on the questionnaire form at the time of interview, and refusals to participate were recorded on a separate sheet. Study Site The study was conducted in the northernmost region of Togo, the Savannah Region, which is bordered by Burkina Faso, Ghana, and Benin. The Savannah Region is a pastoral zone important for livestock raising, with approximately half of Togo's cattle population found in this region, estimated to be 138,000 in 2011 (Direction de l'Elevage - Togo, personal communication). The area also receives a large number of transhumant (i.e. semi-nomadic) herds each dry season, the official period of transhumance being from January-May. These herds are mainly from Burkina Faso, as well as from Benin and Niger. Questionnaire Survey Through discussion with regional veterinary services and livestock traders, the nine biggest cattle markets in the region were identified. Markets were open 1–2 days per week. The target population was traders of live cattle operating in markets in northern Togo. The survey was conducted in February-March 2012 and, in order to capture as many traders as possible, larger markets were visited up to 5 times. Structured questionnaire-based interviews were conducted by two trained interviewers. Although the questionnaire was in French, the official language of Togo, interviews were also conducted in four local languages. Traders were asked to name all of the sites that they visited to purchase or sell cattle during the current dry season and the previous wet season. The information recorded by the interviewers included the nature of the site (market or informal trading place), its full location (village, district, province and country), and the type of the stakeholders with whom they were trading (such as traders, farmers, or butchers). If the traders reported visiting markets, the frequency of their visits was recorded. For each location, traders were asked to specify the minimum and maximum number of cattle sold or purchased per month, if they visited that location every month in a given season. If a location was visited only sporadically, they instead specified a minimum and maximum per season. Additionally, traders were asked whether they sold animals from, or bought animals for, herds that they personally owned. The locations of these herds, the minimum and maximum number of cattle sold/purchased and the frequencies of sales/purchases were recorded. As the definition of dry and wet seasons may vary between individuals, participants were asked to define the months corresponding to these periods. Additionally, the manager of each market was asked to estimate the number of traders operating in the market each open day in both seasons. In order to minimise data entry errors, all data were entered twice into a pre-designed Microsoft Access 2003 database and cross-checked for discrepancies using EpiInfo 3.5.3 (Centers for Disease Control and Prevention, USA). Market Catchment Area Locations where cattle were bought or sold by traders operating in the Savannah market system were visualised using MapInfo Professional Version 7.0. The centroid of each province was plotted in order to show the geographic distribution. Cattle Flows Through the Market System The analysis was conducted using R statistical software Version 2.12.2 [11] . Characterisation of the flows of cattle into and out of the market system of the Savannah Region could not be directly deduced from the empirical data, due to two constraints. Firstly, it was not possible to sample every trader operating in the market system during the course of the survey. Secondly, although the number of cattle purchased and sold in each location was known for each trader, information about the actual origin or destination of these animals was missing. In other words, the number of animals purchased by a trader from locations A and B was known, as was the number sold to locations C and D, but the proportions of cattle purchased in A or B that were then sold to C or D were unknown. Consequently, it was necessary to estimate the flows of cattle into and out of the Savannah market system through stochastic simulations. The simulation algorithm is described below, which was repeated over 10,000 simulations. In order to assess the proportion of the total trader population in the Savannah market system that was captured by the survey, the number of trader-days per season per market was first calculated from the market visit frequencies reported by the traders. This was compared with the estimate of the number of traders provided by the market manager. If the trader interview data gave a lower estimation of the number of trader-days per season than the market manager's estimation for a given market, it was assumed that the trader sample did not capture the full trader population of the market. In these markets, the trader data were completed by randomly re-sampling from the group of traders visiting this market until the number of trader-days estimated by the manager was reached. This modified dataset was then used to reconstruct the flow of cattle between locations. Cattle movements were simulated for each individual trader as follows. The minimum and maximum numbers of cattle reported to be sold or purchased at each site by a given trader were summed over one season (dry or wet). These minimum and maximum values accounted for the monthly variability reported by traders as well as the uncertainty associated with the recall of the number of cattle traded. As traders were often livestock owners who bought and sold cattle for their own herds, the location of their herd was considered as the sale location for cattle which they purchased for themselves. For those traders selling cattle from their own herds, the location of the herd was considered as the purchase location. For each trader t , the number of cattle sold in location j (with ) and the number of cattle purchased in location i (with ) were then randomly drawn from their respective ranges extending from the minimum to maximum value. In order to ensure that a given simulated trader t sold as many cattle as he purchased over an entire season, the total number of cattle traded was randomly drawn from the range extending from the total of number sold, and the total number purchased, The simulated number of cattle sold and purchased in each location was then defined as and respectively. The sold animals were attributed to an origin by exploring two different scenarios, Location Scenarios 1 and 2. In the first scenario, Location Scenario 1, the probability of an animal being sold in any of the sale locations did not depend on its purchase location. For each animal sold by a trader, an origin was allocated by randomly sampling from the locations given for the animals purchased by this trader, without replacement. In other words, each animal sold by a given trader was randomly matched with a unique animal purchased by this trader. The flows of cattle resulting from each individual trader were then summed. However, given the possibility that cattle may have been more likely to be sold at a site closer to the purchase location, an assumption of dependence of purchase and sale sites was also explored. In this second cattle flow scenario, Location Scenario 2, cattle were preferentially sold in the same country as where they were purchased; or, for purchases in Togo, they were preferentially sold in the same administrative region. For each simulation, the number of cattle entering the market system did not necessarily equal the number leaving. The number of cattle flowing through the Savannah market system in each simulation was, therefore, assumed to be the greater of these two values. After running the algorithm over 10,000 simulations, the mean, minimum and maximum numbers of cattle flowing between locations was assessed. Market Network A network of contacts between markets in the Savannah Region was simulated using R package sna [12] , with markets as nodes and animal movements as edges, which were simulated according to the algorithm described above. The network was directed, meaning that the direction of animal movements was accounted for. Each directed edge connecting two markets was given a weight, equal to the number of cattle traded between these two locations. Network connectivity was assessed via the giant strongly connected component (GSCC) and giant weakly connected component (GWCC). The GSCC refers to the part of the network within which all nodes can reach one another through directed paths, whereas the GWCC includes nodes that can reach one another through undirected paths. The in- and out-degree distributions of the binary and weighted networks were assessed. For the binary network, the in- and out-degree referred to the number of Savannah markets sending cattle to and receiving cattle from a given Savannah market, respectively. For the weighted network, the in-degree referred to the number of cattle being moved to a given Savannah market from other Savannah markets, with the out-degree being the converse. Cattle in the network were moved directly from their place of purchase to their place of sale based on the results of the cattle flow simulations described above. It is possible, however, that a trader visiting several markets may conduct his visits in a particular order. For example, a trader may purchase all of his cattle in market A, move these cattle to market B where some would be sold, and then finally visit market C to sell those remaining. Information about the order of market visits was not available, but could influence the distribution of links between markets. In order to explore this, an algorithm which stochastically ordered the market visits of each trader was run over 1,000 simulations. Further information is given in a supplementary file, Text S1 . Risk of Disease Spread Through Market System The risk of cross-border disease spread through the simulated cross-border livestock flows was assessed for Location Scenario 1. The risk was defined as the probability of a disease invading an area j through importation of cattle from a disease-endemic area i within a one year period. The pathway was divided into two steps: firstly, the introduction of an infectious animal into area j from area i ; secondly, the spread of disease within the cattle population of area j due to the introduction of this infectious animal. Given that border crossings rarely involve veterinary assessment, it is assumed that infectious animals would be able to cross into neighbouring countries without detection and quarantine. Therefore, the probability p i of exporting an infectious animal from an area i can be approximated by the prevalence of the disease in area i . The number of animals moved from area i to j within the one year period was taken from the results of the simulations previously described. When an infectious animal has been introduced into an area j , the disease may either fade out or spread within the cattle population of area j . The basic reproduction number of a disease ( R 0 ) is the number of secondary cases resulting from the introduction of one infectious case into a fully susceptible population. It refers to a pathogen's potential to spread in a given population. The cattle population in area j was assumed to be fully susceptible and to mix homogeneously. With being the reproduction number in area j , the likelihood of disease extinction soon after the introduction of one infected animal into this population was equal to [13] , and the probability of a sustained outbreak was The risk P of disease invasion was therefore: By varying and the risk of an exotic disease invading the cattle population of the Savannah Region in Togo through cattle trade from Burkina Faso was explored. Here, refers to the disease prevalence in Burkina Faso, and refers to the potential of a disease to invade the Savannah cattle population following the introduction of an infectious animal into one of its herds through the Savannah market system. The Savannah cattle population was assumed to mix homogeneously. Additionally, the probability of a disease invading at least three other countries through cattle trade from Savannah herds was investigated. Here, refers to the disease prevalence in the cattle population in the Savannah region, and refers to the potential of a disease to invade a cattle population in a given country after the introduction of an infectious animal from the Savannah Region into the market system of this country. was assumed to be the same in all countries trading with the Savannah Region and, in each given country, the local cattle population and cattle traded through the local market system were assumed to mix homogeneously. This assumption was necessary because data relating to flow of cattle from the market systems into herds in these countries were not available. Ethics Statement This research was a component of a larger study of zoonotic disease epidemiology in Togo [8] , and was approved by the Ethics Committee for Health Research (Comité de Bioéthique pour la Recherche en Santé) of the Ministry of Health of Togo. In Switzerland, approval was given by the Ethics Commission of the Cantons of Basel-Stadt and Basel-Land and the Research Commission of the Swiss Tropical and Public Health Institute of Basel, Switzerland. The information to be communicated to participants was provided as a written document to the interviewers and they received training regarding the consent process. Prior to interviewing, the study objectives, procedures and questionnaire content were explained to participants in their local language and they were assured that the questionnaire data would be treated anonymously. As the interviews were conducted in the cattle traders' busy, outdoor workplaces, obtaining written consent was determined to be impractical in this setting. Similar to previous cross-sectional surveys conducted with traders in marketplaces [9] , [10] , all participants provided informed verbal consent before the interview, as approved by the aforementioned ethics and research commissions. The informed consent of each participant was recorded by the interviewer on the questionnaire form at the time of interview, and refusals to participate were recorded on a separate sheet. Study Site The study was conducted in the northernmost region of Togo, the Savannah Region, which is bordered by Burkina Faso, Ghana, and Benin. The Savannah Region is a pastoral zone important for livestock raising, with approximately half of Togo's cattle population found in this region, estimated to be 138,000 in 2011 (Direction de l'Elevage - Togo, personal communication). The area also receives a large number of transhumant (i.e. semi-nomadic) herds each dry season, the official period of transhumance being from January-May. These herds are mainly from Burkina Faso, as well as from Benin and Niger. Questionnaire Survey Through discussion with regional veterinary services and livestock traders, the nine biggest cattle markets in the region were identified. Markets were open 1–2 days per week. The target population was traders of live cattle operating in markets in northern Togo. The survey was conducted in February-March 2012 and, in order to capture as many traders as possible, larger markets were visited up to 5 times. Structured questionnaire-based interviews were conducted by two trained interviewers. Although the questionnaire was in French, the official language of Togo, interviews were also conducted in four local languages. Traders were asked to name all of the sites that they visited to purchase or sell cattle during the current dry season and the previous wet season. The information recorded by the interviewers included the nature of the site (market or informal trading place), its full location (village, district, province and country), and the type of the stakeholders with whom they were trading (such as traders, farmers, or butchers). If the traders reported visiting markets, the frequency of their visits was recorded. For each location, traders were asked to specify the minimum and maximum number of cattle sold or purchased per month, if they visited that location every month in a given season. If a location was visited only sporadically, they instead specified a minimum and maximum per season. Additionally, traders were asked whether they sold animals from, or bought animals for, herds that they personally owned. The locations of these herds, the minimum and maximum number of cattle sold/purchased and the frequencies of sales/purchases were recorded. As the definition of dry and wet seasons may vary between individuals, participants were asked to define the months corresponding to these periods. Additionally, the manager of each market was asked to estimate the number of traders operating in the market each open day in both seasons. In order to minimise data entry errors, all data were entered twice into a pre-designed Microsoft Access 2003 database and cross-checked for discrepancies using EpiInfo 3.5.3 (Centers for Disease Control and Prevention, USA). Market Catchment Area Locations where cattle were bought or sold by traders operating in the Savannah market system were visualised using MapInfo Professional Version 7.0. The centroid of each province was plotted in order to show the geographic distribution. Cattle Flows Through the Market System The analysis was conducted using R statistical software Version 2.12.2 [11] . Characterisation of the flows of cattle into and out of the market system of the Savannah Region could not be directly deduced from the empirical data, due to two constraints. Firstly, it was not possible to sample every trader operating in the market system during the course of the survey. Secondly, although the number of cattle purchased and sold in each location was known for each trader, information about the actual origin or destination of these animals was missing. In other words, the number of animals purchased by a trader from locations A and B was known, as was the number sold to locations C and D, but the proportions of cattle purchased in A or B that were then sold to C or D were unknown. Consequently, it was necessary to estimate the flows of cattle into and out of the Savannah market system through stochastic simulations. The simulation algorithm is described below, which was repeated over 10,000 simulations. In order to assess the proportion of the total trader population in the Savannah market system that was captured by the survey, the number of trader-days per season per market was first calculated from the market visit frequencies reported by the traders. This was compared with the estimate of the number of traders provided by the market manager. If the trader interview data gave a lower estimation of the number of trader-days per season than the market manager's estimation for a given market, it was assumed that the trader sample did not capture the full trader population of the market. In these markets, the trader data were completed by randomly re-sampling from the group of traders visiting this market until the number of trader-days estimated by the manager was reached. This modified dataset was then used to reconstruct the flow of cattle between locations. Cattle movements were simulated for each individual trader as follows. The minimum and maximum numbers of cattle reported to be sold or purchased at each site by a given trader were summed over one season (dry or wet). These minimum and maximum values accounted for the monthly variability reported by traders as well as the uncertainty associated with the recall of the number of cattle traded. As traders were often livestock owners who bought and sold cattle for their own herds, the location of their herd was considered as the sale location for cattle which they purchased for themselves. For those traders selling cattle from their own herds, the location of the herd was considered as the purchase location. For each trader t , the number of cattle sold in location j (with ) and the number of cattle purchased in location i (with ) were then randomly drawn from their respective ranges extending from the minimum to maximum value. In order to ensure that a given simulated trader t sold as many cattle as he purchased over an entire season, the total number of cattle traded was randomly drawn from the range extending from the total of number sold, and the total number purchased, The simulated number of cattle sold and purchased in each location was then defined as and respectively. The sold animals were attributed to an origin by exploring two different scenarios, Location Scenarios 1 and 2. In the first scenario, Location Scenario 1, the probability of an animal being sold in any of the sale locations did not depend on its purchase location. For each animal sold by a trader, an origin was allocated by randomly sampling from the locations given for the animals purchased by this trader, without replacement. In other words, each animal sold by a given trader was randomly matched with a unique animal purchased by this trader. The flows of cattle resulting from each individual trader were then summed. However, given the possibility that cattle may have been more likely to be sold at a site closer to the purchase location, an assumption of dependence of purchase and sale sites was also explored. In this second cattle flow scenario, Location Scenario 2, cattle were preferentially sold in the same country as where they were purchased; or, for purchases in Togo, they were preferentially sold in the same administrative region. For each simulation, the number of cattle entering the market system did not necessarily equal the number leaving. The number of cattle flowing through the Savannah market system in each simulation was, therefore, assumed to be the greater of these two values. After running the algorithm over 10,000 simulations, the mean, minimum and maximum numbers of cattle flowing between locations was assessed. Market Network A network of contacts between markets in the Savannah Region was simulated using R package sna [12] , with markets as nodes and animal movements as edges, which were simulated according to the algorithm described above. The network was directed, meaning that the direction of animal movements was accounted for. Each directed edge connecting two markets was given a weight, equal to the number of cattle traded between these two locations. Network connectivity was assessed via the giant strongly connected component (GSCC) and giant weakly connected component (GWCC). The GSCC refers to the part of the network within which all nodes can reach one another through directed paths, whereas the GWCC includes nodes that can reach one another through undirected paths. The in- and out-degree distributions of the binary and weighted networks were assessed. For the binary network, the in- and out-degree referred to the number of Savannah markets sending cattle to and receiving cattle from a given Savannah market, respectively. For the weighted network, the in-degree referred to the number of cattle being moved to a given Savannah market from other Savannah markets, with the out-degree being the converse. Cattle in the network were moved directly from their place of purchase to their place of sale based on the results of the cattle flow simulations described above. It is possible, however, that a trader visiting several markets may conduct his visits in a particular order. For example, a trader may purchase all of his cattle in market A, move these cattle to market B where some would be sold, and then finally visit market C to sell those remaining. Information about the order of market visits was not available, but could influence the distribution of links between markets. In order to explore this, an algorithm which stochastically ordered the market visits of each trader was run over 1,000 simulations. Further information is given in a supplementary file, Text S1 . Risk of Disease Spread Through Market System The risk of cross-border disease spread through the simulated cross-border livestock flows was assessed for Location Scenario 1. The risk was defined as the probability of a disease invading an area j through importation of cattle from a disease-endemic area i within a one year period. The pathway was divided into two steps: firstly, the introduction of an infectious animal into area j from area i ; secondly, the spread of disease within the cattle population of area j due to the introduction of this infectious animal. Given that border crossings rarely involve veterinary assessment, it is assumed that infectious animals would be able to cross into neighbouring countries without detection and quarantine. Therefore, the probability p i of exporting an infectious animal from an area i can be approximated by the prevalence of the disease in area i . The number of animals moved from area i to j within the one year period was taken from the results of the simulations previously described. When an infectious animal has been introduced into an area j , the disease may either fade out or spread within the cattle population of area j . The basic reproduction number of a disease ( R 0 ) is the number of secondary cases resulting from the introduction of one infectious case into a fully susceptible population. It refers to a pathogen's potential to spread in a given population. The cattle population in area j was assumed to be fully susceptible and to mix homogeneously. With being the reproduction number in area j , the likelihood of disease extinction soon after the introduction of one infected animal into this population was equal to [13] , and the probability of a sustained outbreak was The risk P of disease invasion was therefore: By varying and the risk of an exotic disease invading the cattle population of the Savannah Region in Togo through cattle trade from Burkina Faso was explored. Here, refers to the disease prevalence in Burkina Faso, and refers to the potential of a disease to invade the Savannah cattle population following the introduction of an infectious animal into one of its herds through the Savannah market system. The Savannah cattle population was assumed to mix homogeneously. Additionally, the probability of a disease invading at least three other countries through cattle trade from Savannah herds was investigated. Here, refers to the disease prevalence in the cattle population in the Savannah region, and refers to the potential of a disease to invade a cattle population in a given country after the introduction of an infectious animal from the Savannah Region into the market system of this country. was assumed to be the same in all countries trading with the Savannah Region and, in each given country, the local cattle population and cattle traded through the local market system were assumed to mix homogeneously. This assumption was necessary because data relating to flow of cattle from the market systems into herds in these countries were not available. Results Descriptive Analysis of Empirical Data Two hundred and twenty-six traders were interviewed, with a refusal rate of 12%, mainly due to lack of time to participate. In each of the nine markets, 9–55 traders were interviewed (median 20, IQR: 11–36). For three markets, the number of trader-days calculated from the trader interview data accounted for only 24–77% of the trader-days estimated by the market manager, requiring modification (inflation) of their datasets. For the other six markets, the market manager estimated less trader-days than the trader interview data and modification of the dataset was not required. Only 8.4% of traders (19 of 226) transported their animals between locations solely by vehicle, with the remainder herding their animals by foot, either exclusively or in combination with road transport. The majority of traders defined the dry season as extending from October – April, and the wet season from May – September. Given the higher number of cattle traded at a greater number of locations during the dry season compared to the wet season, these data will be presented in detail below. The corresponding information for the wet season is provided in a supplementary file, Data S1 . Most interviewees (193/226) not only acted as traders, but also bought and sold cattle for their own private herds. However, the proportions of their purchases and sales that involved their own herds were small. In the dry season, the median proportion of their purchases that represented cattle taken from their own herds was only 1.1% (IQR: 0.7–1.8%), and the proportion of their sales corresponding to cattle being added to their own herds was 1.5% (IQR: 1.0–2.3%). In the dry season, cattle were sold from 179 herds. Most of these herds (146 of 179) were located in the Savannah Region, as well as other areas of Togo (5 of 179), Burkina Faso (23 of 179) and Ghana (5 of 179). Similarly, most of the 175 herds which received purchased cattle during the dry season were in the Savannah Region (141 of 175), as well as other areas of Togo (5 of 175), Burkina Faso (23 of 175), Ghana (4 of 175), Benin (1 of 175) and Niger (1 of 175). The distribution of the average number of cattle traded per trader during the dry season was right-skewed. While the median was 500 (IQR: 173–639), as many as 2,916 cattle were reported to be traded by a single trader during the season. The median numbers of purchase and sale locations of the traders were 4 (IQR:3–5) and 3 (IQR:2–4), respectively, regardless of season, with a maximum of 8. Most of these sites were cattle markets within the Savannah Region. The median number of Savannah markets in which traders operated for purchase or sale was 3 (IQR: 2–4) in the dry season. Nearly three quarters of traders (166 of 226, 73.5%) in the dry season bought cattle in at least one Savannah market and sold cattle in at least one other Savannah market. Around half of the cattle purchases and sales reported by traders (55.2% and 51.4%, respectively) took place in Savannah markets, whilst 35.8% and 21.6% of purchases and sales, respectively, took place in another country. Of all the cattle purchases and sales taking place in the 28 Savannah markets, 83% and 82% respectively took place in only 4 markets. Outside of Togo, most of the foreign cattle purchases (85.3%) were conducted in Burkina Faso, and 38.4% of foreign cattle sales took place in Nigeria. The number of traders operating at different purchase and sale locations as well as the numbers of cattle traded at these sites are summarised for the dry season in Table 1 . 10.1371/journal.pone.0075570.t001 Table 1 Empirical data from interviews with 226 cattle traders – dry season. Purchase and sale locations Savannah markets Savannah herds Savannah butchers Other Togo markets Other Togo herds Benin Burkina Faso Ghana Niger Nigeria No. of traders purchasing or selling * 226 (100%) 172 (76.1%) 39 (17.3%) 91 (40.3%) 7 (3.1%) 37 (16.4%) 81 (35.8%) 22 (9.7%) 2 (0.09%) 31 (13.7%) No. of traders purchasing * 202 (89.4%) 163 (72.1%) 0 16 (7.1%) 6 (2.7%) 10 (4.4%) 79 (35.0%) 11 (4.9%) 0 0 No. of traders selling * 190 (84.1%) 152 (67.3%) 39 (17.3%) 80 (35.4%) 5 (2.2%) 34 (15.5%) 32 (14.2%) 18 (8.0%) 2 (0.09%) 31 (13.7%) No. of cattle purchased, ranging from min. to max.° 58334–66285 (55.2%) 6605–7370 (6.2%) 0 2761–3094 (2.6%) 299–327 (0.3%) 2480–2908 (2.4%) 32776–36067 (30.5%) 3120–3391 (2.9%) 0 0 Median proportion (%) of total cattle purchased per trader + (IQR) 77.0 (53.0–99.0) 2 (1.0–18.5) 0 28.5 (19.0–34.5) 2.5 (1.3–13.5) 33.5 (27.0–38.0) 55 (29.0–95.0) 29 (18.5–49.5) 0 0 No. of cattle sold, ranging from min. to max.° 54675–61312 (51.4%) 2519–2806 (2.4%) 2961–3388 (2.8%) 23177–26084 (21.8%) 67–72 (0.1%) 6422–6997 (5.9%) 2933–3533 (2.9%) 4796–5282 (4.5%) 36–36 (<0.1%) 8779–9914 (8.3%) Median proportion (%) of total cattle sold per trader + (IQR) 83.5 (50.0–99.0) 2.0 (1.0–4.0) 15.0 (10.0–22.0) 45.0 (30.8–61.3) 2.0 (1.0–2.0) 26.5 (21.0–32.8) 5.0 (1.0–27.0) 26.0 (20.3–39.3) 6.5 (4.3–8.8) 28.0 (19.0–35.0) * The numbers of traders purchasing from, and selling to, different locations over the dry season are shown. The proportion of the total number of traders interviewed is given in brackets as a percentage. Markets and herds located outside of the study zone, the Savannah Region, are referred to as "Other Togo markets" and "Other Togo herds", respectively. No butchers were located outside of the Savannah Region. °The minimum and maximum numbers of cattle purchased and sold in these locations are presented for the dry season, expressed in brackets as percentages of the average number of cattle purchased or sold. + This is the median value of the proportion of each trader's purchases or sales taking place in the given locations, expressed as a percentage. The interquartile range (IQR) is given in brackets. Traders that did not purchase in a given location were excluded. The market catchment area is shown in Figure 1 , with traders operating in the Savannah market system also operating in Burkina Faso, Ghana, Benin, Nigeria, and Niger, as far as 500 km from the Savannah Region. Overall, more than half of the traders (121 of 226, 53.5%) operated in at least one other country outside of Togo. Among those traders operating in multiple countries, only one quarter (32 of 121, 26.4%) conducted both purchase and sale activities in at least two countries. In the dry season, almost two thirds of the traders operating in Burkina Faso (49 of 81, 60.5%) conducted only purchases in Burkina Faso, without any sales. Half (11 of 22, 50.0%) of traders operating in the dry season in Ghana, three quarters (27 of 37, 73.0%) of traders operating in Benin and all (31 of 31, 100%) of the traders operating in Nigeria only sold cattle in these countries, without purchasing. 10.1371/journal.pone.0075570.g001 Figure 1 Study zone in West Africa. The study zone, the Savannah Region of northern Togo, is shaded red. The centroids of the other districts where the interviewed traders also bought or sold cattle are shown as red dots. Simulated Livestock Flows The 10,000 model simulations gave means of 32,370 cattle (range: 31,070–34,180) flowing into the Savannah market system during the dry season and 28,860 (range: 27,940–29,970) during the wet season. Location Scenario 2 with non-independence of buying and selling locations did not have a notable impact on the results, with a mean cattle flow into the Savannah market system during the dry season of 28,260 (range: 26,810–29,500) and 24,900 (range: 23,670–25,930) in the wet season. Overall trends of animal flows were the same regardless of season. Given the greater cattle flows during the dry season, these data will be presented in detail below. Results from the wet season are provided in a supplementary file, Data S1 . A summary of results for Location Scenarios 1 and 2 for both the dry and wet seasons is provided as a table in a supplementary file, Data S2 . Figure 2 shows the mean proportions of animals entering into and leaving the Savannah market system obtained by the simulations for the Location Scenario 1 for the dry season. More than three quarters (79.2%, range: 78.1–80.0%) of cattle flowing into the Savannah market system during the dry season originated from other countries, with 68.0% (range: 65.2–70.2%) of inflow coming from Burkina Faso, 7.6% (range: 7.2–8.0%) from Ghana and 3.6% (range: 3.1–4.7%) from Benin. Half of the cattle leaving the Savannah market system in the dry season (49.3%, range: 47.0–51.7%) were sent to Togolese markets outside of the Savannah Region. The majority (93.7%, range: 92.7%–94.5%) of these were sent to one large market near the coastal capital city of Lomé, where animals are generally slaughtered for meat consumption. One third of the cattle (38.8%, range: 36.7–40.7%) leaving the Savannah market system moved into other countries, with 7.8% (range: 7.3–8.4%) of outflow into Ghana, 12.3% (range: 11.1–13.8%) into Benin and 14.7% (range: 13.5–15.9%) into Nigeria. The results of Location Scenario 2 did not demonstrate any major differences in flow, as detailed in Data S2 . 10.1371/journal.pone.0075570.g002 Figure 2 Simulated flows of cattle into and out of the market system of Savannah Region, Togo. These simulated mean flows are shown for the six month dry season period, as a proportion of total flow into or out of the Savannah market system. The range of simulated values from minimum to maximum are shown as black bars. In the dry season, 2,992 (range: 2775–3284) cattle flowed into herds in the Savannah Region, equating to 2.2% (range: 2.0–2.4%) of the estimated total cattle population size in the Savannah Region. Given that most herds likely breed their own replacement animals, many more animals flowed in the reverse direction from Savannah herds into the market system. There was a mean of 13,633 cattle (range: 9,096–24,506) leaving herds in the dry season, equating to 9.9% (range: 6.6–17.8%) of the estimated total cattle population size. Location Scenario 2 produced similar results with 2,989 cattle (range: 2,733–3,305) flowing into herds and 13,730 (range: 9,408–23,940) leaving herds. Flows into and out of the Savannah herds followed the same trends as the aforementioned market system flows. Market Network during Dry Season In the dry season, the market system consisted of 28 markets. They formed a well connected network incorporating all but one of the markets. The GWCC, estimating the upper bound of the maximum epidemic size, was 27. Nearly half of these markets (13) formed the GSCC, estimating the lower bound of the maximum epidemic size. When using the alternative algorithm for reconstructing the order of market visits, the GSCC was even higher, with a median of 20 markets (range: 14–23). Further details of this algorithm are provided in a supplementary file, Text S1 . Figure 3 shows the distribution of markets during the dry season as a function of their binary and weighted in- and out-degrees. The majority of markets (17 of 28) received cattle from at least two other markets, with a maximum of 13 other markets. Approximately half of the markets (15 of 28) sent cattle to at least two other markets, with a maximum of 14 other markets. However, most cattle movements within the Savannah market network were mediated by a small number of markets: 4 markets accounted for 73.7% and 78.6% of the total weighted in- and out-degrees, respectively. The markets with the highest degrees were those included in the survey, shown as blue circles in Figure 3 . The Savannah market network for the wet season is presented in a supplementary file, Data S1 . 10.1371/journal.pone.0075570.g003 Figure 3 Market network of the Savannah Region of Togo and degree distributions. The graph on the left shows the binary in-degree as a function of the binary out-degree, and the graph on the right shows the weighted in-degree as a function of the weighted out-degree, during the dry season. The 9 markets where the survey was conducted are coloured blue and the other 19 are red. Disease Risk Figure 4 shows the average probability over the course of one year that a disease present in Burkina Faso will be introduced through the Savannah market system into Togolese herds and result in an outbreak. For a hypothetical disease, even at a low prevalence of less than 1% in Burkina Faso and low transmissibility with an R 0 of around 1.25, there was a high probability (80%) of an outbreak in Togo. When disease prevalence is higher, between 1–10%, this probability reaches 100%. Similarly, if a hypothetical disease with an R 0 of around 1.25 is present in Savannah herds at a prevalence of less than 1%, the probability of disease being introduced into at least 3 other countries in the region through the Savannah market system is also 80% ( Figure 5 ). 10.1371/journal.pone.0075570.g004 Figure 4 Probability of a disease invading the cattle population of the Savannah Region in Togo through cattle trade from Burkina Faso. The probability of a disease invading the cattle population of the Savannah Region is shown as a function of the disease prevalence in Burkina Faso and the basic reproduction number of the disease, R 0 . Here, R 0 relates to the potential of a disease to invade the Savannah cattle population following the introduction of an infectious animal into one of its herds. 10.1371/journal.pone.0075570.g005 Figure 5 Probability of a disease invading at least three other countries through cattle trade from Savannah herds. The probability of disease invasion is shown as a function of the disease prevalence in the cattle population in the Savannah region and the basic reproduction number of the disease, R 0 . Here, R 0 relates to the potential of a disease to invade a cattle population in a given country, after the introduction of an infectious animal into the market system of this country. Descriptive Analysis of Empirical Data Two hundred and twenty-six traders were interviewed, with a refusal rate of 12%, mainly due to lack of time to participate. In each of the nine markets, 9–55 traders were interviewed (median 20, IQR: 11–36). For three markets, the number of trader-days calculated from the trader interview data accounted for only 24–77% of the trader-days estimated by the market manager, requiring modification (inflation) of their datasets. For the other six markets, the market manager estimated less trader-days than the trader interview data and modification of the dataset was not required. Only 8.4% of traders (19 of 226) transported their animals between locations solely by vehicle, with the remainder herding their animals by foot, either exclusively or in combination with road transport. The majority of traders defined the dry season as extending from October – April, and the wet season from May – September. Given the higher number of cattle traded at a greater number of locations during the dry season compared to the wet season, these data will be presented in detail below. The corresponding information for the wet season is provided in a supplementary file, Data S1 . Most interviewees (193/226) not only acted as traders, but also bought and sold cattle for their own private herds. However, the proportions of their purchases and sales that involved their own herds were small. In the dry season, the median proportion of their purchases that represented cattle taken from their own herds was only 1.1% (IQR: 0.7–1.8%), and the proportion of their sales corresponding to cattle being added to their own herds was 1.5% (IQR: 1.0–2.3%). In the dry season, cattle were sold from 179 herds. Most of these herds (146 of 179) were located in the Savannah Region, as well as other areas of Togo (5 of 179), Burkina Faso (23 of 179) and Ghana (5 of 179). Similarly, most of the 175 herds which received purchased cattle during the dry season were in the Savannah Region (141 of 175), as well as other areas of Togo (5 of 175), Burkina Faso (23 of 175), Ghana (4 of 175), Benin (1 of 175) and Niger (1 of 175). The distribution of the average number of cattle traded per trader during the dry season was right-skewed. While the median was 500 (IQR: 173–639), as many as 2,916 cattle were reported to be traded by a single trader during the season. The median numbers of purchase and sale locations of the traders were 4 (IQR:3–5) and 3 (IQR:2–4), respectively, regardless of season, with a maximum of 8. Most of these sites were cattle markets within the Savannah Region. The median number of Savannah markets in which traders operated for purchase or sale was 3 (IQR: 2–4) in the dry season. Nearly three quarters of traders (166 of 226, 73.5%) in the dry season bought cattle in at least one Savannah market and sold cattle in at least one other Savannah market. Around half of the cattle purchases and sales reported by traders (55.2% and 51.4%, respectively) took place in Savannah markets, whilst 35.8% and 21.6% of purchases and sales, respectively, took place in another country. Of all the cattle purchases and sales taking place in the 28 Savannah markets, 83% and 82% respectively took place in only 4 markets. Outside of Togo, most of the foreign cattle purchases (85.3%) were conducted in Burkina Faso, and 38.4% of foreign cattle sales took place in Nigeria. The number of traders operating at different purchase and sale locations as well as the numbers of cattle traded at these sites are summarised for the dry season in Table 1 . 10.1371/journal.pone.0075570.t001 Table 1 Empirical data from interviews with 226 cattle traders – dry season. Purchase and sale locations Savannah markets Savannah herds Savannah butchers Other Togo markets Other Togo herds Benin Burkina Faso Ghana Niger Nigeria No. of traders purchasing or selling * 226 (100%) 172 (76.1%) 39 (17.3%) 91 (40.3%) 7 (3.1%) 37 (16.4%) 81 (35.8%) 22 (9.7%) 2 (0.09%) 31 (13.7%) No. of traders purchasing * 202 (89.4%) 163 (72.1%) 0 16 (7.1%) 6 (2.7%) 10 (4.4%) 79 (35.0%) 11 (4.9%) 0 0 No. of traders selling * 190 (84.1%) 152 (67.3%) 39 (17.3%) 80 (35.4%) 5 (2.2%) 34 (15.5%) 32 (14.2%) 18 (8.0%) 2 (0.09%) 31 (13.7%) No. of cattle purchased, ranging from min. to max.° 58334–66285 (55.2%) 6605–7370 (6.2%) 0 2761–3094 (2.6%) 299–327 (0.3%) 2480–2908 (2.4%) 32776–36067 (30.5%) 3120–3391 (2.9%) 0 0 Median proportion (%) of total cattle purchased per trader + (IQR) 77.0 (53.0–99.0) 2 (1.0–18.5) 0 28.5 (19.0–34.5) 2.5 (1.3–13.5) 33.5 (27.0–38.0) 55 (29.0–95.0) 29 (18.5–49.5) 0 0 No. of cattle sold, ranging from min. to max.° 54675–61312 (51.4%) 2519–2806 (2.4%) 2961–3388 (2.8%) 23177–26084 (21.8%) 67–72 (0.1%) 6422–6997 (5.9%) 2933–3533 (2.9%) 4796–5282 (4.5%) 36–36 (<0.1%) 8779–9914 (8.3%) Median proportion (%) of total cattle sold per trader + (IQR) 83.5 (50.0–99.0) 2.0 (1.0–4.0) 15.0 (10.0–22.0) 45.0 (30.8–61.3) 2.0 (1.0–2.0) 26.5 (21.0–32.8) 5.0 (1.0–27.0) 26.0 (20.3–39.3) 6.5 (4.3–8.8) 28.0 (19.0–35.0) * The numbers of traders purchasing from, and selling to, different locations over the dry season are shown. The proportion of the total number of traders interviewed is given in brackets as a percentage. Markets and herds located outside of the study zone, the Savannah Region, are referred to as "Other Togo markets" and "Other Togo herds", respectively. No butchers were located outside of the Savannah Region. °The minimum and maximum numbers of cattle purchased and sold in these locations are presented for the dry season, expressed in brackets as percentages of the average number of cattle purchased or sold. + This is the median value of the proportion of each trader's purchases or sales taking place in the given locations, expressed as a percentage. The interquartile range (IQR) is given in brackets. Traders that did not purchase in a given location were excluded. The market catchment area is shown in Figure 1 , with traders operating in the Savannah market system also operating in Burkina Faso, Ghana, Benin, Nigeria, and Niger, as far as 500 km from the Savannah Region. Overall, more than half of the traders (121 of 226, 53.5%) operated in at least one other country outside of Togo. Among those traders operating in multiple countries, only one quarter (32 of 121, 26.4%) conducted both purchase and sale activities in at least two countries. In the dry season, almost two thirds of the traders operating in Burkina Faso (49 of 81, 60.5%) conducted only purchases in Burkina Faso, without any sales. Half (11 of 22, 50.0%) of traders operating in the dry season in Ghana, three quarters (27 of 37, 73.0%) of traders operating in Benin and all (31 of 31, 100%) of the traders operating in Nigeria only sold cattle in these countries, without purchasing. 10.1371/journal.pone.0075570.g001 Figure 1 Study zone in West Africa. The study zone, the Savannah Region of northern Togo, is shaded red. The centroids of the other districts where the interviewed traders also bought or sold cattle are shown as red dots. Simulated Livestock Flows The 10,000 model simulations gave means of 32,370 cattle (range: 31,070–34,180) flowing into the Savannah market system during the dry season and 28,860 (range: 27,940–29,970) during the wet season. Location Scenario 2 with non-independence of buying and selling locations did not have a notable impact on the results, with a mean cattle flow into the Savannah market system during the dry season of 28,260 (range: 26,810–29,500) and 24,900 (range: 23,670–25,930) in the wet season. Overall trends of animal flows were the same regardless of season. Given the greater cattle flows during the dry season, these data will be presented in detail below. Results from the wet season are provided in a supplementary file, Data S1 . A summary of results for Location Scenarios 1 and 2 for both the dry and wet seasons is provided as a table in a supplementary file, Data S2 . Figure 2 shows the mean proportions of animals entering into and leaving the Savannah market system obtained by the simulations for the Location Scenario 1 for the dry season. More than three quarters (79.2%, range: 78.1–80.0%) of cattle flowing into the Savannah market system during the dry season originated from other countries, with 68.0% (range: 65.2–70.2%) of inflow coming from Burkina Faso, 7.6% (range: 7.2–8.0%) from Ghana and 3.6% (range: 3.1–4.7%) from Benin. Half of the cattle leaving the Savannah market system in the dry season (49.3%, range: 47.0–51.7%) were sent to Togolese markets outside of the Savannah Region. The majority (93.7%, range: 92.7%–94.5%) of these were sent to one large market near the coastal capital city of Lomé, where animals are generally slaughtered for meat consumption. One third of the cattle (38.8%, range: 36.7–40.7%) leaving the Savannah market system moved into other countries, with 7.8% (range: 7.3–8.4%) of outflow into Ghana, 12.3% (range: 11.1–13.8%) into Benin and 14.7% (range: 13.5–15.9%) into Nigeria. The results of Location Scenario 2 did not demonstrate any major differences in flow, as detailed in Data S2 . 10.1371/journal.pone.0075570.g002 Figure 2 Simulated flows of cattle into and out of the market system of Savannah Region, Togo. These simulated mean flows are shown for the six month dry season period, as a proportion of total flow into or out of the Savannah market system. The range of simulated values from minimum to maximum are shown as black bars. In the dry season, 2,992 (range: 2775–3284) cattle flowed into herds in the Savannah Region, equating to 2.2% (range: 2.0–2.4%) of the estimated total cattle population size in the Savannah Region. Given that most herds likely breed their own replacement animals, many more animals flowed in the reverse direction from Savannah herds into the market system. There was a mean of 13,633 cattle (range: 9,096–24,506) leaving herds in the dry season, equating to 9.9% (range: 6.6–17.8%) of the estimated total cattle population size. Location Scenario 2 produced similar results with 2,989 cattle (range: 2,733–3,305) flowing into herds and 13,730 (range: 9,408–23,940) leaving herds. Flows into and out of the Savannah herds followed the same trends as the aforementioned market system flows. Market Network during Dry Season In the dry season, the market system consisted of 28 markets. They formed a well connected network incorporating all but one of the markets. The GWCC, estimating the upper bound of the maximum epidemic size, was 27. Nearly half of these markets (13) formed the GSCC, estimating the lower bound of the maximum epidemic size. When using the alternative algorithm for reconstructing the order of market visits, the GSCC was even higher, with a median of 20 markets (range: 14–23). Further details of this algorithm are provided in a supplementary file, Text S1 . Figure 3 shows the distribution of markets during the dry season as a function of their binary and weighted in- and out-degrees. The majority of markets (17 of 28) received cattle from at least two other markets, with a maximum of 13 other markets. Approximately half of the markets (15 of 28) sent cattle to at least two other markets, with a maximum of 14 other markets. However, most cattle movements within the Savannah market network were mediated by a small number of markets: 4 markets accounted for 73.7% and 78.6% of the total weighted in- and out-degrees, respectively. The markets with the highest degrees were those included in the survey, shown as blue circles in Figure 3 . The Savannah market network for the wet season is presented in a supplementary file, Data S1 . 10.1371/journal.pone.0075570.g003 Figure 3 Market network of the Savannah Region of Togo and degree distributions. The graph on the left shows the binary in-degree as a function of the binary out-degree, and the graph on the right shows the weighted in-degree as a function of the weighted out-degree, during the dry season. The 9 markets where the survey was conducted are coloured blue and the other 19 are red. Disease Risk Figure 4 shows the average probability over the course of one year that a disease present in Burkina Faso will be introduced through the Savannah market system into Togolese herds and result in an outbreak. For a hypothetical disease, even at a low prevalence of less than 1% in Burkina Faso and low transmissibility with an R 0 of around 1.25, there was a high probability (80%) of an outbreak in Togo. When disease prevalence is higher, between 1–10%, this probability reaches 100%. Similarly, if a hypothetical disease with an R 0 of around 1.25 is present in Savannah herds at a prevalence of less than 1%, the probability of disease being introduced into at least 3 other countries in the region through the Savannah market system is also 80% ( Figure 5 ). 10.1371/journal.pone.0075570.g004 Figure 4 Probability of a disease invading the cattle population of the Savannah Region in Togo through cattle trade from Burkina Faso. The probability of a disease invading the cattle population of the Savannah Region is shown as a function of the disease prevalence in Burkina Faso and the basic reproduction number of the disease, R 0 . Here, R 0 relates to the potential of a disease to invade the Savannah cattle population following the introduction of an infectious animal into one of its herds. 10.1371/journal.pone.0075570.g005 Figure 5 Probability of a disease invading at least three other countries through cattle trade from Savannah herds. The probability of disease invasion is shown as a function of the disease prevalence in the cattle population in the Savannah region and the basic reproduction number of the disease, R 0 . Here, R 0 relates to the potential of a disease to invade a cattle population in a given country, after the introduction of an infectious animal into the market system of this country. Discussion Although livestock market networks and implications for disease spread have been described in detail in developed countries [14] , [15] , few data are available from developing countries and are not captured by official datasets such as FAOSTAT from the Food and Agricultural Organization of the United Nations (FAO) [16] . This study quantitatively captures the potential disease risk resulting from large-scale, cross-border cattle trade between Togo, Burkina Faso, Ghana, Benin, and Nigeria for the first time. The findings will serve as the basis for further research hypotheses and should lead to strengthened collaboration at the regional level in the planning of disease control measures. The number of animals flowing into the northern Togolese Savannah market system during the dry season equals nearly one quarter of the resident cattle population. Although more than half of these animals were reported by sellers as having been purchased in Burkina Faso, it is possible that some animals originated from further afield, such as Mali or Niger. Through the cattle market system of northern Togo, non-neighbouring countries are potentially epidemiologically connected via trade routes. The GSCC of a network is an estimate of the lower bound of the maximum epidemic size for a given disease, whilst the GWCC estimates the upper bound [17] . The Savannah market network displayed high connectivity, with nearly half of the markets being incorporated into the GSCC and all but one forming the GWCC. In the alternative algorithm for reconstructing the sequence of market visits ( Text S1 ), the GSCC was higher, incorporating the majority of markets. This suggests that a disease introduced into one market could rapidly spread to other markets. The market network of northern Togo is, therefore, a potential conduit for disease spread between West African countries. Although no data are available, it is likely that the scale of cross-border trade through the Togolese market network is not a unique scenario, but is rather the norm for West Africa. These findings are relevant not only to the surveillance and control of newly emerging diseases, but also endemic diseases. Cross-border cattle trade and transhumance may have contributed to the genetic diversity of Brucella abortus strains circulating in the study zone [8] . These cross-border movements could also potentially explain the overlapping distribution of FMD serotypes O, A, SAT1 and SAT2 across West African countries [18] – [20] , particularly given that wildlife play a less important role in disease transmission than in East Africa. The results of this study are of direct relevance to the effective implementation of the Global FMD Control Strategy of the FAO and the World Animal Organisation for Animal Health (OIE) announced in 2012, a 15 year program which seeks to reduce the global impact of this devastating livestock disease [21] . Estimates of the R 0 of FMD in sub-Saharan Africa are not available. However, the R 0 of Rinderpest, a viral disease of cattle officially eradicated in 2011, has been estimated to range between 1.2–4.4 in Somalian and Sudanese cattle populations [22] , [23] . The R 0 of Contagious Bovine Pleuropneumonia, a severe respiratory disease of cattle in Africa, is estimated to fall between 3.2–4.6 in pastoral herds of southern Sudan [24] . Therefore, the R 0 range of 1–4 used in this study to assess the risk of disease spread through regional cattle trade in West Africa is appropriate. In addition to animal movements through trade, the Savannah Region of northern Togo also receives a large number of transhumant herds from the Sahel zone in search of grazing pasture and water sources during the dry season. Members of the Economic Community of West African States (ECOWAS) are legally bound to permit seasonal cross-border movements of herds [25] . In 2011, 47,000 transhumant cattle entered the Savannah Region through official Togolese government check points (Direction de l'Elevage - Togo, personal communication), although it is likely that an even greater number entered the country unofficially. The risk of regional disease spread in West Africa through trade is, therefore, further compounded by transhumance. In northern Togo, there is evidence that anthrax outbreaks occur along the routes followed by transhumant herds. As many herds do not follow the official routes designated by the Togolese authorities, conducting disease surveillance in this zone is particularly challenging [26] . According to the market managers, the higher cattle flow through markets during the dry season reflected contributions from transhumant herds temporarily visiting Togo. This study highlights the importance of a regional approach to disease control activities in West Africa. Prior to the annual childhood polio vaccination campaign implemented in 20 countries in Central and West Africa in March 2012, health care providers of border districts in the Savannah Region met and discussed with their counterparts in neighbouring countries. The animal health sector should also invest in cross-border disease prevention activities, such as synchronised vaccination campaigns or formal systems for the communication of unusual animal health events. Limitations The questionnaire was written in French, the official language of Togo, Burkina Faso, and Benin. However, interviews were predominantly conducted orally in local languages by two trained multilingual interviewers. Occasionally, interpreters were also required. Due to the linguistic complexity of the study zone, data errors due to incorrect interpretation are possible. As the survey results are based on estimations of cattle transactions by traders, rather than witnessed transactions, there is a risk of recall bias. The flow of cattle through the Savannah market system was reconstructed using simulations. While it is possible that the complexities of the market system have not been fully captured, these are unlikely to have a major impact on the results: estimates obtained when assuming independence or dependence of purchase and sale locations (Location Scenarios 1 and 2) were similar. This is likely due to the fact that when operating in locations outside of the Savannah region, most traders either purchased or sold cattle, not both. However, the data only capture cattle trade through the formal market system. Given that informal trade also occurs, the scale of cross-border cattle trade is likely to have been underestimated. Furthermore, small ruminant cross-border trade has not been considered in this study. Although further data collection would improve the accuracy and applicability of the study findings, substantial investment of resources would be required. The order in which traders visited markets in the Savannah Region was not known and the network of cattle movements between these markets had to be reconstructed. While the GSCC size varied, it was always greater than 45% of all markets, meaning that all simulated networks displayed high connectivity. Moreover, most of the purchases and sales were always mediated by only a few markets. These dominant markets were those where the interviews were conducted. This may suggest that the sampling approach may have potentially introduced bias. However, given that these markets were identified by the local veterinary services as the largest markets in the Savannah Region, the constructed network may indeed reflect the true structure. The estimations of the risk of disease spread through the market system assumed a homogenously mixing population, simplifying animal contact patterns and the underlying disease dynamics. Collecting further information on cattle population dynamics in this region would be useful for refining this estimate. Moreover, pathogen amplification within herds and within markets was not accounted for. If such amplification did occur, this would only serve to further increase the risk of disease spread. Conclusions By stochastically simulating data collected by interviewing cattle traders in northern Togo, this study identifies potential risks for regional disease spread in West Africa through cross-border cattle trade. The findings highlight that surveillance for emerging infectious diseases as well as control activities targeting endemic diseases in West Africa, such as FMD, are likely to be ineffective if only conducted at a national level. A regional approach to animal disease surveillance, prevention and control is essential. Limitations The questionnaire was written in French, the official language of Togo, Burkina Faso, and Benin. However, interviews were predominantly conducted orally in local languages by two trained multilingual interviewers. Occasionally, interpreters were also required. Due to the linguistic complexity of the study zone, data errors due to incorrect interpretation are possible. As the survey results are based on estimations of cattle transactions by traders, rather than witnessed transactions, there is a risk of recall bias. The flow of cattle through the Savannah market system was reconstructed using simulations. While it is possible that the complexities of the market system have not been fully captured, these are unlikely to have a major impact on the results: estimates obtained when assuming independence or dependence of purchase and sale locations (Location Scenarios 1 and 2) were similar. This is likely due to the fact that when operating in locations outside of the Savannah region, most traders either purchased or sold cattle, not both. However, the data only capture cattle trade through the formal market system. Given that informal trade also occurs, the scale of cross-border cattle trade is likely to have been underestimated. Furthermore, small ruminant cross-border trade has not been considered in this study. Although further data collection would improve the accuracy and applicability of the study findings, substantial investment of resources would be required. The order in which traders visited markets in the Savannah Region was not known and the network of cattle movements between these markets had to be reconstructed. While the GSCC size varied, it was always greater than 45% of all markets, meaning that all simulated networks displayed high connectivity. Moreover, most of the purchases and sales were always mediated by only a few markets. These dominant markets were those where the interviews were conducted. This may suggest that the sampling approach may have potentially introduced bias. However, given that these markets were identified by the local veterinary services as the largest markets in the Savannah Region, the constructed network may indeed reflect the true structure. The estimations of the risk of disease spread through the market system assumed a homogenously mixing population, simplifying animal contact patterns and the underlying disease dynamics. Collecting further information on cattle population dynamics in this region would be useful for refining this estimate. Moreover, pathogen amplification within herds and within markets was not accounted for. If such amplification did occur, this would only serve to further increase the risk of disease spread. Conclusions By stochastically simulating data collected by interviewing cattle traders in northern Togo, this study identifies potential risks for regional disease spread in West Africa through cross-border cattle trade. The findings highlight that surveillance for emerging infectious diseases as well as control activities targeting endemic diseases in West Africa, such as FMD, are likely to be ineffective if only conducted at a national level. A regional approach to animal disease surveillance, prevention and control is essential. Supporting Information Data S1 Detailed results for wet season. Empirical data as well as results of the livestock flow simulations and network analysis are presented for the wet season, complementing the information provided in the manuscript for the dry season. (DOC) Click here for additional data file. Data S2 Summarised simulation results for wet and dry seasons. The results of the 10,000 model simulations of livestock flows through the Savannah market system in the dry and wet seasons are provided in a table. This includes results for the independent and non-independent purchase and sale location scenarios, Location Scenario 1 and Location Scenario 2, respectively. (DOC) Click here for additional data file. Text S1 Accounting for the order of market visits in the analysis of the Savannah market network. The process of stochastically ordering the market visits undertaken by traders in the Savannah market network is described for both dry and wet seasons. (DOC) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2870367/

Can syndromic surveillance data detect local outbreaks of communicable disease? A model using a historical cryptosporidiosis outbreak

SUMMARY A national UK surveillance system currently uses data from a health helpline (NHS Direct) in an attempt to provide early warning of a bio-terrorist attack, or an outbreak caused by a more common infection. To test this syndromic surveillance system we superimposed data from a historical outbreak of cryptosporidiosis onto a statistical model of NHS Direct call data. We modelled whether calls about diarrhoea (a proxy for cryptosporidiosis) exceeded a statistical threshold, thus alerting the surveillance team to the outbreak. On the date that the public health team were first notified of the outbreak our model predicted a 4% chance of detection when we assumed that one-twentieth of cryptosporidiosis cases telephoned the helpline. This rose to a 72% chance when we assumed nine-tenths of cases telephoned. The NHS Direct surveillance system is currently unlikely to detect an event similar to the cryptosporidiosis outbreak used here and may be most suited to detecting more widespread rises in syndromes in the community, as previously demonstrated. However, the expected rise in NHS Direct call rates, should improve early warning of outbreaks using call data.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7110601/

Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A

Mucosal epithelia constitute the first barriers to be overcome by pathogens during infection. The induction of protective IgA in this location is important for the prevention of infection and can be achieved through different mucosal immunization strategies. Lactic acid bacteria have been tested in the last few years as live vectors for the delivery of antigens at mucosal sites, with promising results. In this work, Streptococcus pneumoniae PsaA antigen was expressed in different species of lactic acid bacteria, such as Lactococcus lactis , Lactobacillus casei , Lactobacillus plantarum , and Lactobacillus helveticus . After nasal inoculation of C57Bl/6 mice, their ability to induce both systemic (IgG in serum) and mucosal (IgA in saliva, nasal and bronchial washes) anti-PsaA antibodies was determined. Immunization with L. lactis MG1363 induced very low levels of IgA and IgG, possibly by the low amount of PsaA expressed in this strain and its short persistence in the nasal mucosa. All three lactobacilli persisted in the nasal mucosa for 3 days and produced a similar amount of PsaA protein (150–250 ng per 10 9 CFU). However, L. plantarum NCDO1193 and L. helveticus ATCC15009 elicited the highest antibody response (IgA and IgG). Vaccination with recombinant lactobacilli but not with recombinant L. lactis led to a decrease in S. pneumoniae recovery from nasal mucosa upon a colonization challenge. Our results confirm that certain Lactobacillus strains have intrinsic properties that make them suitable candidates for mucosal vaccination experiments. 1 Introduction Streptococcus pneumoniae is the major agent of pneumonia around the world, causing up to one million deaths per year, mainly in developing countries [1] . The high costs of medical care and the appearance of new clinical isolates with multidrug resistance led to the search for efficient new vaccines to prevent pneumococcal infection. Since the pathogen enters the host through the respiratory mucosa, a vaccine inducing the production of protective secretory IgA at this site, as well as systemic IgG antibodies, would be desirable. Pneumococcal surface antigen A (PsaA) is a membrane-anchored virulence factor, possibly involved in Mn 2+ and Zn 2+ transport as predicted by its crystal structure [2] . PsaA deletion mutants display low ability to adhere to mucosal cells and therefore are less pathogenic [3] . This characteristic may be due to differences in the modulation of pneumococcal adhesins caused by the absence of Mn 2+ or Zn 2+ in the cell [4] . PsaA is conserved among the 90 described S. pneumoniae serotypes and is also immunogenic, which makes it a good candidate for vaccine formulations. In fact, antibodies produced against PsaA by nasal immunization, using cholera toxin B subunit as adjuvant, were shown to protect mice against nasopharyngeal colonization by S. pneumoniae . This protection can be further increased by the co-administration of PsaA with the pneumococcal surface protein A (PspA), another pneumococcal virulence factor [5] , [6] . In another approach, oral immunization of mice with PsaA encapsulated in microspheres induced the production of IgG and IgA and resulted in protection against lung colonization and septicemia with five different S. pneumoniae strains [7] . Live bacterial vaccine vectors are being extensively studied for mucosal immunization in the prevention of different infectious diseases [8] , [9] . Among them, lactic acid bacteria (LAB) are especially attractive since they are microorganisms present in the gastrointestinal mucosa of healthy individuals, are widely used in dietary products and possess a GRAS (generally recognized as safe) status. This characteristic is not shared by attenuated pathogen derived live vectors, due to the possibility of reversion of the attenuated phenotype, which could be dangerous mainly for immunocompromised individuals. Interaction of LAB with the immune system and their potential as antigen carriers are the subjects of a number of recently published studies [10] , [11] , [12] , [13] , [14] , [15] . Different strains and routes of inoculation were evaluated using the fragment C of the tetanus toxin (TTFC), which is so far the best characterized antigen expressed in LAB [9] , [16] , [17] , [18] . Most of these approaches resulted in protection against tetanus toxin lethal challenge [16] , [19] . Other antigens like the protective antigen from Bacillus anthracis [20] , the E7 protein from human papilloma virus 16 [21] , the L7/L12 antigen from Brucella abortus [22] , the Env protein from HIV [23] , the M protein from Streptococcus pyogenes [24] and the spike glycoprotein from gastroenteritis coronavirus [25] were expressed in LAB and their potential as vaccines against the associated diseases are currently being evaluated. In some cases, such as the immunization with L. lactis expressing the HIV Env protein or the M protein from Streptococcus pyogenes , protection was observed using adequate animal models [23] , [24] . In other cases, in vitro neutralizing effects of the antibodies were observed [25] . Using an inducible expression system based on the lactose operon from Lactobacillus casei [26] we expressed the PsaA and the PspA antigens from S. pneumoniae [27] either intracellularly or secreted to the culture media. These recombinant L. casei are being tested as potential anti-pneumococcal vaccines through nasal immunization of mice, but so far we could not detect significant levels of anti-PsaA IgA or IgG (unpublished data). The failure in stimulating the production of antibodies may be a result of the lack of PsaA or PspA expression in the recombinant L. casei after nasal immunization, due to the absence of the inducer in the host mucosa. For this reason, we decided to use a system that allows the constitutive expression of PsaA in different LAB strains. In this work, recombinant Lactococcus lactis , Lactobacillus casei , Lactobacillus plantarum and Lactobacillus helveticus expressing PsaA were evaluated for their ability to induce systemic and mucosal immune responses in nasally immunized C57Bl/6 mice. Nasal colonization of Streptococcus pneumoniae in these mice was also analyzed. 2 Materials and methods 2.1 Bacterial strains and growth conditions L. casei CECT5275 (formerly ATCC 393 [pLZ15] − ), L. plantarum NCDO1193 (kindly provided by Dr K. Thompsom from the Food and Agricultural Microbiology Research Division, Department of Agriculture, Northern Ireland, UK) and L. helveticus ATCC 15009 were routinely grown in MRS medium (Difco), at 37 °C, without shaking. L. lactis MG1363 was grown in M17 medium (Difco) containing 0.5% glucose (GM17) at 30 °C without shaking. Plating of bacteria was performed on the respective media with 1.8% agar. For the selection of transformants, 5 μg/ml of erythromycin was used in the media. 2.2 Plasmids and recombinant DNA procedures The pT1NX vector, kindly provided by Dr Lothar Steidler (Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Belgium) contains the lactococcal P1 constitutive promoter and the usp-45 signal sequence [28] . The 867 bp gene encoding PsaA from S. pneumoniae serotype 6B (strain St 472/96 from the Instituto Adolpho Lutz, São Paulo, SP, Brazil) was amplified from pCI- psaA plasmid [29] by PCR using the following oligonucleotides: PsaA-I-forw: 5′ATG CAT CGA TAT CAG CTA GCG GAA AAA AAG AT3′ and PsaA-I-rev: 5′CCA AGC TTT TAT TTT GCC AAT CCT TCA GC3′ PCR was performed using Taq DNA polymerase (Invitrogen), 200 mM of each deoxynucleoside triphosphate and 20 pmol of each primer. PCR amplification conditions were as follows: 94 °C, 4 min; 30 cycles of 94 °C, 45 s; 45 °C, 45 s; 72 °C 1.5 min; 72 °C, 7 min for final extension. PCR products were cloned into the pGEM-T vector (Promega) for sequence confirmation. One clone was chosen for preparation of the PsaA fragment as follows. The pGEM-T-psaA plasmid was digested with Hin dIII and treated with Klenow polymerase (Invitrogen) for generation of a blunt end and was further digested with Nsi I. The resulting fragment was cloned into pT1NX previously digested with Bam HI and treated with Klenow DNA polymerase (Invitrogen) for generation of a blunt end and then digested with Pst I, providing a protrude end compatible with the Nsi I end from the insert. Nucleic acid manipulation and general cloning procedures were performed according to laboratory manuals [30] . For the preparation of competent L. lactis , an overnight culture was diluted 1:50 in GM17 containing 0.5% glycine and incubated at 30 °C until OD 600 reached 0.6. Bacteria were collected by centrifugation at 10,000 × g , for 10 min at 4 °C and the pellet was washed two times with 0.5 M saccharose, 10% glycerol (v/v). Bacteria were resuspended in 1:100 of the same solution and electroporated immediately or kept at −80 °C for further use. L. lactis was electroporated with ligation mixtures at 2.5 kV, 200 Ω, 25 mF in 0.2 cm cuvettes using a BioRad GenePulser (BioRad, Life Science Research Products, CA, USA). Plasmids isolated from L. lactis were used for electroporation of all Lactobacillus strains. Electroporation of L. casei was carried out as previously described [31] . The same protocol was used for the other lactobacillus strains except that the electroporation buffer was three-fold concentrated. Antibiotic resistant L. lactis and Lactobacillus clones were screened for the presence of the insert of interest by PCR, using specific primers as described above. Positive clones were frozen in GM17 or MRS containing 15% glycerol, at −80 °C. 2.3 Protein expression and Western blot analysis Isolated L. lactis and Lactobacillus clones were grown overnight in GM17 or MRS, respectively. Cultures (10 ml) were collected by centrifugation at 4000 × g for 10 min and bacterial pellet was suspended in 1 ml of 100 mM Tris–HCl, pH 8.0. Cell suspensions were transferred to 2 ml tubes containing the same volume of glass beads and lysates were prepared by vigorous shaking in a Bead-Beater (Biospec, Bartlesville, OK, USA) (four cycles of 30 s at maximal speed). Lysates were centrifuged at 15,000 × g for 5 min and supernatants were maintained at −20 °C for further analysis. For quantification of PsaA, cultures were grown until OD 600 reached 2. The CFU values of each culture were determined and extracts corresponding to 10 9 CFU were analyzed by Western blot. A concentration curve was loaded on the same gel using recombinant PsaA expressed and purified from E. coli [32] . For the analysis of PsaA in the cell wall, bacteria were incubated with 20 mg/ml of lysozyme in 50 mM glucose, 25 mM Tris–HCl pH 8.0 and 10 mM EDTA, at 37 °C for 30 min. The suspension was centrifuged at 15,000 × g for 5 min and the supernatants were collected. Protein extracts or supernatants were fractionated by SDS-PAGE and electrotransferred to nitrocellulose membranes using the Mini Protean II equipment (BioRad, Life Science Research Products, CA, USA). Mouse polyclonal anti-PsaA antiserum was developed against recombinant S. pneumoniae PsaA (from strain St 472/96) expressed in E. coli . Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma Chemical, St. Louis, MO, USA) was used according to the manufacturer's instructions. Detection was performed using the chemiluminescent ECL kit (GE Healthcare). 2.4 Immunization and analysis of immune responses Six- to eight-week-old female C57Bl/6 mice (five per group) received either the recombinant bacteria expressing PsaA or the respective control bacteria harboring the empty vector. An additional control group received saline. LAB strains were grown until cultures reached an OD 600 of 2.0; bacteria were collected by centrifugation (4000 × g , 20 min at room temperature), washed with saline and then suspended at 10 9 cells in 10 μl. For nasal immunization, mice were anesthetized with a mixture of 0.5% xilazine and 0.2% ketamine and 10 μl of a saline suspension containing 10 9 cells were inoculated into the nostrils with the help of a micropipette on days 0, 1, 14, 15, 28 and 29. Ten days after the last booster mice were bled by the retrorbital plexus. For the collection of saliva, a 0.01% pilocarpine solution was injected intraperitoneally. Nasal and bronchial washes were performed as described elsewhere, using 200 μl and 300 μl of saline, respectively [33] . Anti-PsaA antibodies were detected from sera, saliva, nasal and bronchial washes by enzyme-linked immunoabsorbent assay (ELISA) as previously described [29] , using rPsaA purified from E. coli as coating and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or anti-mouse IgA (Sigma Chemical, St. Louis, MO, USA). Titers were defined as the last dilution in which absorbances at 492 nm reached 0.1. The maintenance of the LAB strains harboring the pT1NX or the pT1NX-PsaA plasmids in respiratory mucosa was analyzed up to 7 days after a nasal inoculation of 10 9 bacteria. For this, we performed nasal and bronchial washes in groups of four animals on days 1, 3, 5 and 7 after inoculation (day 0). Dilutions of these washes were plated in MRS or GM17 containing 5 μg/ml of erythromycin. Colonies were counted after 48 h incubation at 37 °C. 2.5 Streptococcus pneumoniae nasal colonization Six- to eight-week-old C57Bl/6 mice (12 per group) were immunized as described above. Individual serum and pooled saliva were collected for analysis of anti-PsaA IgG and IgA, respectively. Fifteen days after the last inoculation, animals were anesthetized and 10 μl of a suspension containing 5 × 10 6 CFU of Streptococcus pneumoniae strain 0603 serotype 6B [34] were inoculated nasally. After 5 days, nasal washes were performed using 200 μl of saline. Serial dilutions of the samples were plated in blood agar containing 8 μg/ml gentamicin. Alpha-hemolytic colonies were counted after incubation of the plates for 24 h at 37 °C, considering the volume recovered. For representation in the graphic and statistical analysis log 10 was applied to the values and recovery of 0 CFU was considered 1 CFU. 2.6 Statistical analysis Differences in antibody titers were analyzed by the Mann–Whitney U test. ( P ≤ 0.05 was considered significantly different). The same test was used for the analysis of S. pneumoniae colonization. ( P ≤ 0.02 was considered significantly different). 2.1 Bacterial strains and growth conditions L. casei CECT5275 (formerly ATCC 393 [pLZ15] − ), L. plantarum NCDO1193 (kindly provided by Dr K. Thompsom from the Food and Agricultural Microbiology Research Division, Department of Agriculture, Northern Ireland, UK) and L. helveticus ATCC 15009 were routinely grown in MRS medium (Difco), at 37 °C, without shaking. L. lactis MG1363 was grown in M17 medium (Difco) containing 0.5% glucose (GM17) at 30 °C without shaking. Plating of bacteria was performed on the respective media with 1.8% agar. For the selection of transformants, 5 μg/ml of erythromycin was used in the media. 2.2 Plasmids and recombinant DNA procedures The pT1NX vector, kindly provided by Dr Lothar Steidler (Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Belgium) contains the lactococcal P1 constitutive promoter and the usp-45 signal sequence [28] . The 867 bp gene encoding PsaA from S. pneumoniae serotype 6B (strain St 472/96 from the Instituto Adolpho Lutz, São Paulo, SP, Brazil) was amplified from pCI- psaA plasmid [29] by PCR using the following oligonucleotides: PsaA-I-forw: 5′ATG CAT CGA TAT CAG CTA GCG GAA AAA AAG AT3′ and PsaA-I-rev: 5′CCA AGC TTT TAT TTT GCC AAT CCT TCA GC3′ PCR was performed using Taq DNA polymerase (Invitrogen), 200 mM of each deoxynucleoside triphosphate and 20 pmol of each primer. PCR amplification conditions were as follows: 94 °C, 4 min; 30 cycles of 94 °C, 45 s; 45 °C, 45 s; 72 °C 1.5 min; 72 °C, 7 min for final extension. PCR products were cloned into the pGEM-T vector (Promega) for sequence confirmation. One clone was chosen for preparation of the PsaA fragment as follows. The pGEM-T-psaA plasmid was digested with Hin dIII and treated with Klenow polymerase (Invitrogen) for generation of a blunt end and was further digested with Nsi I. The resulting fragment was cloned into pT1NX previously digested with Bam HI and treated with Klenow DNA polymerase (Invitrogen) for generation of a blunt end and then digested with Pst I, providing a protrude end compatible with the Nsi I end from the insert. Nucleic acid manipulation and general cloning procedures were performed according to laboratory manuals [30] . For the preparation of competent L. lactis , an overnight culture was diluted 1:50 in GM17 containing 0.5% glycine and incubated at 30 °C until OD 600 reached 0.6. Bacteria were collected by centrifugation at 10,000 × g , for 10 min at 4 °C and the pellet was washed two times with 0.5 M saccharose, 10% glycerol (v/v). Bacteria were resuspended in 1:100 of the same solution and electroporated immediately or kept at −80 °C for further use. L. lactis was electroporated with ligation mixtures at 2.5 kV, 200 Ω, 25 mF in 0.2 cm cuvettes using a BioRad GenePulser (BioRad, Life Science Research Products, CA, USA). Plasmids isolated from L. lactis were used for electroporation of all Lactobacillus strains. Electroporation of L. casei was carried out as previously described [31] . The same protocol was used for the other lactobacillus strains except that the electroporation buffer was three-fold concentrated. Antibiotic resistant L. lactis and Lactobacillus clones were screened for the presence of the insert of interest by PCR, using specific primers as described above. Positive clones were frozen in GM17 or MRS containing 15% glycerol, at −80 °C. 2.3 Protein expression and Western blot analysis Isolated L. lactis and Lactobacillus clones were grown overnight in GM17 or MRS, respectively. Cultures (10 ml) were collected by centrifugation at 4000 × g for 10 min and bacterial pellet was suspended in 1 ml of 100 mM Tris–HCl, pH 8.0. Cell suspensions were transferred to 2 ml tubes containing the same volume of glass beads and lysates were prepared by vigorous shaking in a Bead-Beater (Biospec, Bartlesville, OK, USA) (four cycles of 30 s at maximal speed). Lysates were centrifuged at 15,000 × g for 5 min and supernatants were maintained at −20 °C for further analysis. For quantification of PsaA, cultures were grown until OD 600 reached 2. The CFU values of each culture were determined and extracts corresponding to 10 9 CFU were analyzed by Western blot. A concentration curve was loaded on the same gel using recombinant PsaA expressed and purified from E. coli [32] . For the analysis of PsaA in the cell wall, bacteria were incubated with 20 mg/ml of lysozyme in 50 mM glucose, 25 mM Tris–HCl pH 8.0 and 10 mM EDTA, at 37 °C for 30 min. The suspension was centrifuged at 15,000 × g for 5 min and the supernatants were collected. Protein extracts or supernatants were fractionated by SDS-PAGE and electrotransferred to nitrocellulose membranes using the Mini Protean II equipment (BioRad, Life Science Research Products, CA, USA). Mouse polyclonal anti-PsaA antiserum was developed against recombinant S. pneumoniae PsaA (from strain St 472/96) expressed in E. coli . Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma Chemical, St. Louis, MO, USA) was used according to the manufacturer's instructions. Detection was performed using the chemiluminescent ECL kit (GE Healthcare). 2.4 Immunization and analysis of immune responses Six- to eight-week-old female C57Bl/6 mice (five per group) received either the recombinant bacteria expressing PsaA or the respective control bacteria harboring the empty vector. An additional control group received saline. LAB strains were grown until cultures reached an OD 600 of 2.0; bacteria were collected by centrifugation (4000 × g , 20 min at room temperature), washed with saline and then suspended at 10 9 cells in 10 μl. For nasal immunization, mice were anesthetized with a mixture of 0.5% xilazine and 0.2% ketamine and 10 μl of a saline suspension containing 10 9 cells were inoculated into the nostrils with the help of a micropipette on days 0, 1, 14, 15, 28 and 29. Ten days after the last booster mice were bled by the retrorbital plexus. For the collection of saliva, a 0.01% pilocarpine solution was injected intraperitoneally. Nasal and bronchial washes were performed as described elsewhere, using 200 μl and 300 μl of saline, respectively [33] . Anti-PsaA antibodies were detected from sera, saliva, nasal and bronchial washes by enzyme-linked immunoabsorbent assay (ELISA) as previously described [29] , using rPsaA purified from E. coli as coating and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or anti-mouse IgA (Sigma Chemical, St. Louis, MO, USA). Titers were defined as the last dilution in which absorbances at 492 nm reached 0.1. The maintenance of the LAB strains harboring the pT1NX or the pT1NX-PsaA plasmids in respiratory mucosa was analyzed up to 7 days after a nasal inoculation of 10 9 bacteria. For this, we performed nasal and bronchial washes in groups of four animals on days 1, 3, 5 and 7 after inoculation (day 0). Dilutions of these washes were plated in MRS or GM17 containing 5 μg/ml of erythromycin. Colonies were counted after 48 h incubation at 37 °C. 2.5 Streptococcus pneumoniae nasal colonization Six- to eight-week-old C57Bl/6 mice (12 per group) were immunized as described above. Individual serum and pooled saliva were collected for analysis of anti-PsaA IgG and IgA, respectively. Fifteen days after the last inoculation, animals were anesthetized and 10 μl of a suspension containing 5 × 10 6 CFU of Streptococcus pneumoniae strain 0603 serotype 6B [34] were inoculated nasally. After 5 days, nasal washes were performed using 200 μl of saline. Serial dilutions of the samples were plated in blood agar containing 8 μg/ml gentamicin. Alpha-hemolytic colonies were counted after incubation of the plates for 24 h at 37 °C, considering the volume recovered. For representation in the graphic and statistical analysis log 10 was applied to the values and recovery of 0 CFU was considered 1 CFU. 2.6 Statistical analysis Differences in antibody titers were analyzed by the Mann–Whitney U test. ( P ≤ 0.05 was considered significantly different). The same test was used for the analysis of S. pneumoniae colonization. ( P ≤ 0.02 was considered significantly different). 3 Results 3.1 Expression of PsaA in different LAB The psaA gene was cloned into the pT1NX vector Pst I site, which produced a fusion of a truncated usp45 signal peptide carried by the vector to the PsaA sequence ( Fig. 1 ), under the control of a constitutive promoter. Ligation products were used to transform L. lactis and expression of PsaA was analyzed by Western blot of cell lysates. L. lactis carrying the constructed vector showed the expression of a protein around 37 kDa reacting against anti-PsaA antibodies, which was not present in extracts from cells carrying the empty vector ( Fig. 2 A). Recombinant plasmid isolated from L. lactis was used to transform different Lactobacillus strains. Western blot analysis of protein extracts from erythromycin resistant colonies showed the expression of PsaA in L. casei , L. plantarum and L. helveticus . The PsaA band was not observed in the respective strains carrying the empty vector ( Fig. 2 A). Using recombinant PsaA as reference, it could be calculated that 10 9 L. lactis CFU expressed approximately 20 ng of PsaA, while the amounts synthesized by the different Lactobacillus strains were about 150 ng ( L. plantarum ), 200 ng ( L. casei ) and 250 ng ( L. helveticus ) of PsaA ( Fig. 2 B). PsaA was not detected in 10-fold concentrated culture media from these clones, indicating that secretion of the protein was not occurring (data not shown). However, analysis of the supernatant obtained after incubation of the cells with lysozyme, showed that at least part of the protein is being directed to the cell wall ( Fig. 2 C). Moreover, when we incubated whole cells with mouse polyclonal anti-PsaA followed by incubation with anti-mouse IgG-peroxidase conjugate and further revealed with OPD and H 2 O 2 , positive reactions were observed in all LAB expressing PsaA, indicating that at least part of the protein is exposed outside of the cell, in contrast to negative reactions observed for the LAB strains carrying the empty vector (data not shown). Fig. 1 Schematic representation of the PsaA-expressing plasmid and amino acid sequence resulting from the genetic fusion. P1 represents the constitutive promoter; SD, Shine-Dalgarno sequence; SS, first codons of the Usp45 signal peptide fused to the psaA gene. Fig. 2 Expression of PsaA in different LAB strains. (A) Western blot analysis from protein extracts show the constitutive expression of PsaA in L. lactis ( L. l. PsaA), L. casei , ( L. c. PsaA), L. plantarum ( L. p. PsaA) and L. helveticus ( L. h. PsaA). PsaA bands are pointed by an arrow. Lysates from respective control strains carrying the pT1NX empty vector are also shown in the figure. (B) Lysates from 10 9 cells of each recombinant LAB were loaded onto SDS-PAGE and transferred to a nitrocellulose membrane. Concentrations from 40 to 640 ng of rPsaA were used as reference. The estimated amounts of PsaA are shown below the panel (20 ng for L. lactis ; 200 ng for L. casei , 150 ng for L. plantarum and 250 ng for L. helveticus ). (C) Supernatants recovered after treatment of the different strains with lyzosyme. Arrows point to the PsaA band. 3.2 Induction of anti-PsaA antibodies by nasal immunization with recombinant LAB Recombinant LAB were used for nasal immunization of C57Bl/6 mice. As can be observed in Fig. 3 A, nasal inoculation with L. casei , L. plantarum or L. helveticus expressing PsaA induced specific anti-PsaA IgA in pooled saliva. No detectable levels of anti-PsaA IgA antibodies were observed in saliva collected from animals that received L. lactis expressing PsaA, the LAB strains carrying the pT1NX vector or saline. The animals were then subjected to nasal and bronchial washes for the analysis of the presence of IgA in the respiratory tract. In these samples, the highest IgA titers could be observed in animal groups that received either L. plantarum or L. helveticus expressing PsaA ( Fig. 3 A and B) with means significantly different from groups that received saline ( P  0.05). The group that received L. lactis expressing PsaA displayed low levels of anti-PsaA IgA in nasal washes or even no detectable levels in bronchial washes ( Fig. 3 A and B), with the mean values being not different from the saline group or the L. lactis pT1NX group ( P > 0.05). Analysis of the sera collected from the same animals revealed that the animals that displayed the highest levels of anti-PsaA IgA also displayed the highest levels of anti-PsaA IgG, showing correlation in the production of these two classes of antibodies (data not shown). Fig. 3 Analysis of the induction of IgA in saliva, nasal and bronchial washes. Saliva and nasal washes (A) and bronchial washes (B) from individual mice were analyzed by ELISA for anti-PsaA antibodies. Log 10 of reciprocal antibody titers are shown. Animals that received the respective LAB strains carrying the pT1NX empty vector ( L. l. ; L. c. ; L. p. and L. h. ) and saline were used as controls. Results are representative of two independent experiments. The individual sera that displayed non-detectable anti-PsaA IgA titers were represented as  0.05). Comparisons with their control strains carrying the empty vector show that no differences were found for L. lactis ( P > 0.05), whereas L. casei PsaA induced higher levels of anti-PsaA IgG when compared with L. casei pT1NX ( P = 0.01). Fig. 4 Induction of IgG by nasal immunization of mice with different LAB strains. Sera from individual mice were analyzed by ELISA for anti-PsaA antibodies. Log 10 of reciprocal antibody titers are shown. Animals that received the respective LAB strains carrying the pT1NX empty vector ( L. l. ; L. c. ; L. p. and L. h. ) or saline were used as controls. Results are representative of two independent experiments. The individual sera that displayed non-detectable anti-PsaA IgG titers were represented as  0.02), but administration of L. helveticus PsaA led to a significant reduction in S. pneumoniae colonization in relation to L. helveticus -pT1NX ( P = 0.02). Administration of L. lactis PsaA did not induce significant reduction of S. pneumoniae colonization either when compared with the saline group or the L. lactis pT1NX group ( P > 0.02). Interestingly, administration of L. casei pT1NX led to an inhibition in S. pneumoniae colonization when compared with the saline group ( P  0.02). No other LAB strain carrying the empty vector induced significant reduction in S. pneumoniae colonization when compared with the saline group. Fig. 5 Nasal colonization by S. pneumoniae . Dilutions of individual nasal washes were plated on blood agar and α-hemolytic colonies were counted after 24 h incubation. Log 10 of total CFU is shown. Animals that received the respective LAB strains carrying the pT1NX empty vector ( L. l. ; L. c. ; L. p. and L. h. ) or saline were used as controls. Absence of colonies in individual nasal washes is represented as 1. 3.4 Recovery of recombinant LAB strains from nasal mucosa The differences observed in the ability to induce anti-PsaA antibodies as well as the differences in S. pneumoniae colonization led us to analyze possible correlations with the permanency of each recombinant strain in mice respiratory mucosa. All LAB strains could be recovered from nasal washes on day 1 in almost the totality of animals analyzed (3 of 4 animals for L. lactis PsaA, L. casei PsaA and L. helveticus PsaA, 4 of 4 animals for L. plantarum PsaA). On the other hand, erythromycin resistant L. lactis were already absent on day 3, although all Lactobacillus strains were recovered in practically all of the animals analyzed on that day (3 of 4 animals for L. casei and L. plantarum and 4 of 4 animals for L. helveticus ). None of the LAB was recovered from nasal washes on days 5 or 7 or from bronchial washes on any of the tested days. Nasal inoculation of the respective strains carrying the empty vector produced similar results, showing that PsaA expression does not exert any effect in bacteria permanency on mice respiratory mucosa (data not shown). Protein extracts from L. lactis recovered on day 1 and Lactobacillus strains recovered on day 3 were tested for the expression of PsaA. For this, three colonies of each strain were grown overnight and protein extracts were analyzed by Western blot. All recovered colonies tested were still able to express PsaA in vitro (data not shown). 3.1 Expression of PsaA in different LAB The psaA gene was cloned into the pT1NX vector Pst I site, which produced a fusion of a truncated usp45 signal peptide carried by the vector to the PsaA sequence ( Fig. 1 ), under the control of a constitutive promoter. Ligation products were used to transform L. lactis and expression of PsaA was analyzed by Western blot of cell lysates. L. lactis carrying the constructed vector showed the expression of a protein around 37 kDa reacting against anti-PsaA antibodies, which was not present in extracts from cells carrying the empty vector ( Fig. 2 A). Recombinant plasmid isolated from L. lactis was used to transform different Lactobacillus strains. Western blot analysis of protein extracts from erythromycin resistant colonies showed the expression of PsaA in L. casei , L. plantarum and L. helveticus . The PsaA band was not observed in the respective strains carrying the empty vector ( Fig. 2 A). Using recombinant PsaA as reference, it could be calculated that 10 9 L. lactis CFU expressed approximately 20 ng of PsaA, while the amounts synthesized by the different Lactobacillus strains were about 150 ng ( L. plantarum ), 200 ng ( L. casei ) and 250 ng ( L. helveticus ) of PsaA ( Fig. 2 B). PsaA was not detected in 10-fold concentrated culture media from these clones, indicating that secretion of the protein was not occurring (data not shown). However, analysis of the supernatant obtained after incubation of the cells with lysozyme, showed that at least part of the protein is being directed to the cell wall ( Fig. 2 C). Moreover, when we incubated whole cells with mouse polyclonal anti-PsaA followed by incubation with anti-mouse IgG-peroxidase conjugate and further revealed with OPD and H 2 O 2 , positive reactions were observed in all LAB expressing PsaA, indicating that at least part of the protein is exposed outside of the cell, in contrast to negative reactions observed for the LAB strains carrying the empty vector (data not shown). Fig. 1 Schematic representation of the PsaA-expressing plasmid and amino acid sequence resulting from the genetic fusion. P1 represents the constitutive promoter; SD, Shine-Dalgarno sequence; SS, first codons of the Usp45 signal peptide fused to the psaA gene. Fig. 2 Expression of PsaA in different LAB strains. (A) Western blot analysis from protein extracts show the constitutive expression of PsaA in L. lactis ( L. l. PsaA), L. casei , ( L. c. PsaA), L. plantarum ( L. p. PsaA) and L. helveticus ( L. h. PsaA). PsaA bands are pointed by an arrow. Lysates from respective control strains carrying the pT1NX empty vector are also shown in the figure. (B) Lysates from 10 9 cells of each recombinant LAB were loaded onto SDS-PAGE and transferred to a nitrocellulose membrane. Concentrations from 40 to 640 ng of rPsaA were used as reference. The estimated amounts of PsaA are shown below the panel (20 ng for L. lactis ; 200 ng for L. casei , 150 ng for L. plantarum and 250 ng for L. helveticus ). (C) Supernatants recovered after treatment of the different strains with lyzosyme. Arrows point to the PsaA band. 3.2 Induction of anti-PsaA antibodies by nasal immunization with recombinant LAB Recombinant LAB were used for nasal immunization of C57Bl/6 mice. As can be observed in Fig. 3 A, nasal inoculation with L. casei , L. plantarum or L. helveticus expressing PsaA induced specific anti-PsaA IgA in pooled saliva. No detectable levels of anti-PsaA IgA antibodies were observed in saliva collected from animals that received L. lactis expressing PsaA, the LAB strains carrying the pT1NX vector or saline. The animals were then subjected to nasal and bronchial washes for the analysis of the presence of IgA in the respiratory tract. In these samples, the highest IgA titers could be observed in animal groups that received either L. plantarum or L. helveticus expressing PsaA ( Fig. 3 A and B) with means significantly different from groups that received saline ( P  0.05). The group that received L. lactis expressing PsaA displayed low levels of anti-PsaA IgA in nasal washes or even no detectable levels in bronchial washes ( Fig. 3 A and B), with the mean values being not different from the saline group or the L. lactis pT1NX group ( P > 0.05). Analysis of the sera collected from the same animals revealed that the animals that displayed the highest levels of anti-PsaA IgA also displayed the highest levels of anti-PsaA IgG, showing correlation in the production of these two classes of antibodies (data not shown). Fig. 3 Analysis of the induction of IgA in saliva, nasal and bronchial washes. Saliva and nasal washes (A) and bronchial washes (B) from individual mice were analyzed by ELISA for anti-PsaA antibodies. Log 10 of reciprocal antibody titers are shown. Animals that received the respective LAB strains carrying the pT1NX empty vector ( L. l. ; L. c. ; L. p. and L. h. ) and saline were used as controls. Results are representative of two independent experiments. The individual sera that displayed non-detectable anti-PsaA IgA titers were represented as  0.05). Comparisons with their control strains carrying the empty vector show that no differences were found for L. lactis ( P > 0.05), whereas L. casei PsaA induced higher levels of anti-PsaA IgG when compared with L. casei pT1NX ( P = 0.01). Fig. 4 Induction of IgG by nasal immunization of mice with different LAB strains. Sera from individual mice were analyzed by ELISA for anti-PsaA antibodies. Log 10 of reciprocal antibody titers are shown. Animals that received the respective LAB strains carrying the pT1NX empty vector ( L. l. ; L. c. ; L. p. and L. h. ) or saline were used as controls. Results are representative of two independent experiments. The individual sera that displayed non-detectable anti-PsaA IgG titers were represented as  0.02), but administration of L. helveticus PsaA led to a significant reduction in S. pneumoniae colonization in relation to L. helveticus -pT1NX ( P = 0.02). Administration of L. lactis PsaA did not induce significant reduction of S. pneumoniae colonization either when compared with the saline group or the L. lactis pT1NX group ( P > 0.02). Interestingly, administration of L. casei pT1NX led to an inhibition in S. pneumoniae colonization when compared with the saline group ( P  0.02). No other LAB strain carrying the empty vector induced significant reduction in S. pneumoniae colonization when compared with the saline group. Fig. 5 Nasal colonization by S. pneumoniae . Dilutions of individual nasal washes were plated on blood agar and α-hemolytic colonies were counted after 24 h incubation. Log 10 of total CFU is shown. Animals that received the respective LAB strains carrying the pT1NX empty vector ( L. l. ; L. c. ; L. p. and L. h. ) or saline were used as controls. Absence of colonies in individual nasal washes is represented as 1. 3.4 Recovery of recombinant LAB strains from nasal mucosa The differences observed in the ability to induce anti-PsaA antibodies as well as the differences in S. pneumoniae colonization led us to analyze possible correlations with the permanency of each recombinant strain in mice respiratory mucosa. All LAB strains could be recovered from nasal washes on day 1 in almost the totality of animals analyzed (3 of 4 animals for L. lactis PsaA, L. casei PsaA and L. helveticus PsaA, 4 of 4 animals for L. plantarum PsaA). On the other hand, erythromycin resistant L. lactis were already absent on day 3, although all Lactobacillus strains were recovered in practically all of the animals analyzed on that day (3 of 4 animals for L. casei and L. plantarum and 4 of 4 animals for L. helveticus ). None of the LAB was recovered from nasal washes on days 5 or 7 or from bronchial washes on any of the tested days. Nasal inoculation of the respective strains carrying the empty vector produced similar results, showing that PsaA expression does not exert any effect in bacteria permanency on mice respiratory mucosa (data not shown). Protein extracts from L. lactis recovered on day 1 and Lactobacillus strains recovered on day 3 were tested for the expression of PsaA. For this, three colonies of each strain were grown overnight and protein extracts were analyzed by Western blot. All recovered colonies tested were still able to express PsaA in vitro (data not shown). 4 Discussion Several strategies to induce mucosal immune responses against S. pneumoniae antigens are currently being tested [6] , [32] . One strategy can be the use of LAB as live vectors for the delivery of specific antigens to mucosal surfaces due to their adhesion to the epithelium and to their claimed adjuvant properties [17] , [35] , [36] . In order to circumvent a possible bacterial host dependent effect that could compromise the success when designing live vectors for mucosal vaccination, we have expressed the PsaA protein from S. pneumoniae in L. casei CECT 5275, L. plantarum NCDO1193 and L. helveticus ATCC 15009 as well as in the model LAB L. lactis MG1363 strain. After the fusion to the Usp45 signal sequence, the recombinant PsaA protein was directed to the cell wall in the four LAB assayed, with no protein detected in culture supernatants. In a previous report, expression of PsaA in fusion with the leader sequence from the L. casei cell wall proteinase PrtP also resulted in the localization of the protein in the cell wall and no secretion to the culture media [27] . Since the sequence which specifies PsaA covalent anchoring to the cell surface in its natural streptococcal host is absent from our construct, it seems that this protein has the particularity to be retained in the cell wall in different bacteria. The recombinant LAB inoculated by the nasal route were able to induce different levels of specific anti-PsaA IgA and IgG in C57Bl/6 mice ( Fig. 3 , Fig. 4 ). L. plantarum and L. helveticus turned out to be better in the development of mucosal and systemic anti-PsaA immune response than L. casei and L. lactis . Pooled saliva from the L. casei PsaA group displayed an IgA induction similar to those found in the L. plantarum PsaA or the L. helveticus PsaA groups ( Fig. 3 A). However, when nasal or bronchial washes of individual animals in the L. casei PsaA group were analyzed, most of them displayed low or even no detectable levels of IgA, with only one animal producing a high titer of anti-PsaA IgA ( Fig. 3 A and B). The high titer in this individual may be the cause of the anti-PsaA IgA observed in pooled saliva from the L. casei PsaA group ( Fig. 3 A). Since L. casei expressed about the same amount of PsaA as L. plantarum and L. helveticus and was also recovered from mice nasal mucosa in the same period, the differences observed among L. casei and the other lactobacilli may reflect differences in their intrinsic adjuvant potential. In another study, Shaw and collaborators have shown that L. plantarum NCIMB 8826 expressing TTFC produced better results in oral immunization when compared to L. casei 393 [37] . In this case, L. plantarum persisted for up to 12 days in mice gastrointestinal tract whereas L. casei remained for about 72 h [37] . The inability of L. lactis expressing PsaA to significantly induce serum IgG or secreted IgA after nasal inoculation could be explained by the low level of expression in this strain compared to lactobacilli. In addition, it was observed that lactococci carrying the expression vector, remained in mice nasal mucosa only 1 day after the inoculation, while lactobacilli could be detected up to 3 days later. Nasal inoculation of mice with L. lactis PsaA did not exert any effect on S. pneumoniae colonization, in comparison with inoculation of saline or L. lactis carrying the empty vector ( Fig. 5 ). On the other hand, colonization of S. pneumoniae was significantly reduced in mice immunized with L. helveticus PsaA, when compared with the animals that received saline or L. helveticus pT1NX. A significant decrease in S. pneumoniae colonization is also observed in the group that received L. plantarum PsaA in relation to the group that received saline, but not in relation to the group that received L. plantarum pT1NX. In this case, a possible inhibition effect related to the strain may be taking place. However, it is important to notice that simply inoculation of the L. plantarum strain carrying the empty vector is not sufficient to cause a decrease in S. pneumoniae colonization. Interestingly the group that received L. casei carrying the empty vector displayed a significant reduction in S. pneumoniae colonization and this reduction was more accentuated in the group that received L. casei PsaA. Since the administration of this strain induced low levels of anti-PsaA antibodies, other mechanisms may be causing this reduction. In addition, although a correlation between antibody induction and inhibition of S. pneumoniae colonization was observed when we compared the groups that received L. lactis PsaA, L. plantarum PsaA and L. helveticus PsaA, this correlation is not always maintained when we analyzed the animals individually. Thus, the induction of anti-PsaA antibodies may be only one of the factors contributing to the reduction of S. pneumoniae colonization. Nasal immunization with recombinant PsaA in combination with other pneumococcal proteins and CTB as adjuvant [6] as well as oral immunization with microencapsulated PsaA and CTB [38] were able to induce mucosal and systemic antibodies and protect mice against S. pneumoniae colonization. These studies suggest a correlation between antibody production and inhibition of colonization. In another study using a colonization model in mice, nasal inoculation of S. pneumoniae resulted in mucosal antibody induction that was associated with clearance of the bacteria. However, no correlation was observed between the amount of antibodies detected in the sera or mucosa and the density of colonization of individual animals. In addition, animals with impaired humoral immunity displayed similar densities and durations of S. pneumoniae colonization than their normal counterparts. The authors discuss that other components of the adaptative immune response as well as innate immune response may be the main contributors for pneumococcal clearance in their model [39] . The probiotic effects of certain Lactobacillus strains have been extensively studied [40] . Among these studies, the L. casei strain Shirota has been shown to induce cellular immunity and to reduce influenza virus titers in mice respiratory tract [41] . The administration of Lactobacillus fermentum prior to a S. pneumoniae challenge led to an increase in anti- S. pneumoniae antibodies as well as in activated macrophages in mice lung and a decrease in S. pneumoniae colonization [42] . Although the regimen of administration in these type of studies differ from that used in this work, a possible probiotic effect of the Lactobacillus strains used here cannot be ruled out and may explain the reduction in S. pneumoniae colonization in animals immunized with L. casei . Our results support the use of lactobacilli for vaccination purposes and experimentally proved the importance of two key issues in the design of live vectors for oral vaccination which include the amount of antigen delivered in relation to the expression system and the strain chosen. The relevance of the immunization protocols in these experiments should be stressed and they could still be improved, perhaps to achieve stimulation of the immune system with fewer doses and more efficiently. As an attempt, co-administration with lactobacilli strains expressing other pneumococcal antigens, such as the PspA, is currently being evaluated.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5710159/

Using Telemetry Data to Refine Endpoints for New Zealand White Rabbits Challenged with Bacillus anthracis

We used a continuous-monitoring digital telemetry system to investigate temperature response in New Zealand White rabbits after inhalation or subcutaneous challenge with Bacillus anthracis . Two spore preparations of B. anthracis Ames A2084 were evaluated by using a nose-only inhalation model, and 2 strains, B. anthracis Ames A2084 and B. anthracis UT500, were evaluated in a subcutaneous model. Animal body temperature greater than 3 SD above the mean baseline temperature was considered a significant increase in body temperature (SIBT). All rabbits that exhibited SIBT after challenge by either route of infection or bacterial strain eventually died or were euthanized due to infection, and all rabbits that died or were euthanized due to infection exhibited SIBT during the course of disease. The time at onset of SIBT preceded clinical signs of disease in 94% of the rabbits tested by as long as 2 days. In addition, continuous temperature monitoring facilitated discrimination between the 2 B. anthracis strains with regard to the time interval between SIBT and death. These data suggest that for the New Zealand White rabbit anthrax model, SIBT is a reliable indicator of infection, is predictive of experimental outcome in the absence of treatment, and is measurable prior to the appearance of more severe signs of disease. The use of digital telemetry to monitor infectious disease course in animal models of anthrax can potentially be used in conjunction with other clinical score metrics to refine endpoint euthanasia criteria.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957342/

Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018

In Italy anthrax is an endemic disease, with a few outbreaks occurring almost every year. We surveyed 234 B . anthracis strains from animals (n = 196), humans (n = 3) and the environment (n = 35) isolated during Italian outbreaks in the years 1972–2018. Despite the considerable genetic homogeneity of B . anthracis , the strains were effectively differentiated using canonical single nucleotide polymorphisms (CanSNPs) assay and multiple-locus variable-number tandem repeat analysis (MLVA). The phylogenetic identity was determined through the characterization of 14 CanSNPs. In addition, a subsequent 31-loci MLVA assay was also used to further discriminate B . anthracis genotypes into subgroups. The analysis of 14 CanSNPs allowed for the identification of four main lineages: A.Br.011/009, A.Br.008/011 (respectively belonging to A.Br.008/009 sublineage, also known Trans-Eurasian or TEA group), A.Br.005/006 and B.Br.CNEVA. A.Br.011/009, the most common subgroup of lineage A, is the major genotype of B . anthracis in Italy. The MLVA analysis revealed the presence of 55 different genotypes in Italy. Most of the genotypes are genetically very similar, supporting the hypothesis that all strains evolved from a local common ancestral strain, except for two genotypes representing the branch A.Br.005/006 and B.Br.CNEVA. The genotyping analysis applied in this study remains a very valuable tool for studying the diversity, evolution, and molecular epidemiology of B . anthracis . Introduction Anthrax is a non-contagious zoonotic disease affecting a broad range of animal species including humans. Bacillus anthracis , the etiological agent of anthrax, forms highly resistant spores that can to persist in the environment for several decades [ 1 ]. Domestic and wild ruminants are species most susceptible to anthrax [ 2 ]. Animals are infected during grazing in areas contaminated with anthrax spores, while humans can contract the disease by contact with anthrax-infected animals or anthrax-contaminated animal products. Most frequently this involves employment in specific high risk occupation; such a farmer, butcher, tanner, wool carder, shearer and veterinarian. Exposure most commonly occurs during the skinning and butchering of cattle that are sick or dead because of anthrax [ 3 ]. Three forms of anthrax occur in humans, depending on exposure type: cutaneous (which is usually non-fatal), gastrointestinal, and inhalational, both of which can be potentially fatal [ 4 ]. Recently, a fourth form of the disease was reported in drug users who inject drugs contaminated with anthrax spores [ 5 ]. Further, since it is relatively easy and inexpensive to obtain B . anthracis , the bacterium is one of the preferred pathogenic agents for use as a bacteriological weapon in bio-terrorist attacks [ 6 ]. In Italy, anthrax is typically a sporadic disease, particularly occurring during the summer (with a few exceptions) in the central and southern regions, and in the major islands, where it almost exclusively affects animals at pasture [ 7 ]. Between 1972 and 2018, approximately 200 outbreaks of animal anthrax were recorded (unpublished data). Very rarely, anthrax infection takes the form of an epidemic-like disease, characterized by outbreaks in limited areas involving a great number of animals. In Italy, two major epidemic-like anthrax outbreaks have been reported: one during the summer of 2004 in Basilicata, and one during the summer of 2011, in an area between Basilicata and Campania [ 8 , 9 ]. Molecular tools, such as the canonical SNPs assay (CanSNPs) and multiple-locus variable-number tandem repeat analysis (MLVA) are highly effective for differentiating B . anthracis strains. The overall goal of this study was to utilize SNP analysis to establish the phylogenetic relationship between the B . anthracis strains examined, and further discriminate them in the context of the MLVA assay, in order to examine correlations among the B . anthracis isolates associated with the Italian anthrax outbreaks and to assess genetic diversity at regional and broader scales. Materials and methods Ethics statement The animal and environmental strains used in the current study were isolated at the Anthrax Reference Institute of Italy (Ce.R.N.A.), a public laboratory mandated by the Italian Ministry of Health to confirm diagnosis of all animal anthrax cases in Italy. During outbreaks, samples were collected by the veterinary services of the Ministry of Health. Specific permission for soil sampling was not required. B . anthracis DNAs from anthrax human cutaneous cases were also included in the current study; two came from the "San Carlo" Hospital, Department of Infectious Disease, Potenza, Italy, and one from the "L. Spallanzani" National Institute for Infectious Disease, Rome, Italy [ 10 ]. Bacterial strains A collection of 234 B . anthracis strains, including 196 strains isolated from animal and 35 from the environment, isolated during Italian anthrax outbreaks in the years 1972–2018, were analyzed in the current study ( Table 1 ). Furthermore, 3 B . anthracis DNAs from anthrax human cutaneous cases were also analyzed. 10.1371/journal.pone.0227875.t001 Table 1 Overview of Bacillus anthracis isolates from the years 1972–2018 analyzed in the current study. Sample type Source No. of isolates Regions Environmental samples Water 3 Tuscany Soil 32 Basilicata, Tuscany Animal samples Bovine 101 Basilicata, Campania, Lazio, Apulia, Sardinia, Sicily, Tuscany, Umbria, Veneto, Lombardy Caprine 20 Abruzzo, Basilicata, Calabria, Campania, Apulia, Sardinia, Trentino Deer 7 Basilicata Equine 12 Basilicata, Campania, Apulia Ovine 53 Basilicata, Campania, Lazio, Apulia, Sicily Swine 3 Basilicata Human samples (DNAs) Human 3 Basilicata, Lazio DNA extraction B . anthracis strains were seeded on 5% sheep blood agar plates and then incubated at +37°C for 24 h. Bacterial DNA was extracted using the DNAeasy Blood and Tissue kit (Qiagen, Hilden, Germany), following the protocol for Gram-positive bacteria. All manipulations of B . anthracis strains were performed in a biosafety level 3 laboratory at the Experimental Zooprophylactic Institute of Apulia and Basilicata Regions in a class II type A 2 biosafety cabinet. Real-time polymerase chain reaction (PCR) assay Molecular identification of B . anthracis was performed using qualitative real-time PCR. The method is based on the amplification of specific DNA sequences using three pairs of specific primers [ 11 ] as follows: R1/R2 primers, specific for the BA813 gene located on the B . anthracis chromosome; PAG 23/24 primers, specific for the protective antigen (PA) gene located on the virulence plasmid pXO1; and CAP 57/58 primers, specific for the capsule (CAP) gene located on the virulence plasmid pXO2. Each 20 μl reaction mixture contained 1x Sso Advanced TM SYBR® Green Supermix (BIORAD), 300 nM each forward and reverse primer, and approximately 10 ng DNA template. The amplification was performed using the CFX Connect Real Time PCR Detection System (BIORAD). A melting curve was generated at 0.5°C increments between 65°C and 95°C, and was analyzed by CFX Manager TM Software, Version 3.0 (BIORAD). CanSNP analysis CanSNP profiles were obtained using 13 allelic discrimination assays involving specific oligonucleotides and probes, as described by Van Ert et al. [ 12 ]. Each 10 μl reaction mixture contained 1x TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, CA, USA), 250 nM probe, 600 nM each of forward and reverse primer, and approximately 10 ng DNA template. For all assays, the thermal cycling parameters used were as follows: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Endpoint fluorescent data were acquired by using the ABI 7900HT instrument. The CanSNPs data were compared with the data for 12 recognized sublineage or subgroups. The 14th SNP was detected using a High Resolution Melting (HRM) assay for a specific A.Br.011 CanSNP [ 13 , 14 ]. Position 2,552,486, based on the Ames Ancestor genome (NC_007530.2), was analyzed. Amplification was performed using the CFX Connect Real Time PCR Detection System (BIORAD) and Precision Melt Supermix (BIORAD). The reaction mixture contained 0.2 μM of each primer and 1x Precision Melt Supermix (BIORAD) in a 20 μl final volume. The following cycling parameters were used: 2 min at 95°C, were followed by 35 cycles of 10 s at 95°C and 30 s at 60°C. The samples were then heated to 95°C for 30 s, cooled down to 60°C over 1 min, and then heated from 65°C to 95°C at a rate of 0.5°C/s. High-resolution melting data were analyzed using Precision Melt Analysis Software (BIORAD). 31-loci MLVA analysis For the 31-marker MLVA, 5' fluorescently labeled oligonucleotides (6-FAM, VIC, NED and PET), specifically selected for variable number tandem repeats (VNTR) analysis were used. Twenty-seven chromosomal VNTR loci (vrrA, vrrB1, cg3, vrrB2, vntr19, vrrC1, vrrC2, vntr32, vntr12, vntr35, vntr23, bams03, bams05, bams13, bams15, bams21, bams22, bams23, bams24, bams25, bams28, bams30, bams31, bams34, bams44, bams51 and bams53) and four plasmid loci (vntr16, vntr17, pxO1 and pxO2) [ 12 , 15 – 18 ] were analyzed. The MLVA assay involved preparation of two singleplex and nine multiplex reactions, in a final volume of 15 μl. Each reaction mixture contained the following: 1x PCR reaction buffer (Qiagen, Hilden, Germany); 3 mM MgCl 2 , 0.2 mM for each dNTP; 1 U Hot Star Plus Taq DNA polymerase (Qiagen, Hilden, Germany), the appropriate concentration of each primer (as described in Table 2 ); and approximately 10 ng DNA template. 10.1371/journal.pone.0227875.t002 Table 2 Primer concentration for each set of marker in PCR reactions of MLVA analysis. PCR Reactions Primers conc. [μM] Singleplex 1 vrrC1 [0.2 μM] Singleplex 2 vrrC2 [0.2 μM] Multiplex 1 vrrA, vrrB1 [0.2 μM]; CG3 [0.4 μM] Multiplex 2 vrrB2 [0.25 μM]; pXO1 [0.3 μM]; pXO2 [0.1 μM] Multiplex 3 vntr12 [0.25 μM]; vntr19 [0.2 μμM]; vntr35 [0.2 μM] Multiplex 4 vntr16 [0.25 μM]; vntr23 [0.2 μM] Multiplex 5 vntr17 [0.1 μM]; vntr32 [0.4 μM] Multiplex 6 bams03 [0.8 μM]; bams05 [0.2 μM]; bams15, bams44 [0.5 μM] Multiplex 7 bams21 [0.4 μM]; bams24, bams25 [0.3 μM]; bams34 [0.2 μM] Multiplex 8 bams13 [0.3 μM]; bams28 [0.15 μM]; bams31, bams53 [0.6 μM] Multiplex 9 bams22, bams51 [0.3 μM]; bams23 [0.2 μM]; bams30 [0.6 μM] The following PCR cycling program was used for the two singleplex reactions and for multiplex reactions 1 and 2: 5 min at 95°C; followed by 35 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C, with a final step of 5 min at 72°C. The following amplification program was used for multiplex reactions 3: 5 min at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 54°C, 45 s at 72°C, and 5 min at 72°C. The following amplification program was used for multiplex reaction 4: 5 min at 95°C, followed by 35 cycles of 30 s at 94°C, 45 s at 56°C, 1 min at 72°C, and 5 min at 72°. The following amplification program was used for multiplex reaction 5: 5 min at 95°C, followed by 35 cycles of 30 s at 94°C, 45 s at 59°, 1 min at 72°C, and 5 min at 72°C. The following amplification program was used for multiplex reactions 6 to 9: 5 min at 94°C, followed by 35 cycles of 1 min at 94°C, 90 s at 60°, 90 s at 72°C, and 15 min at 72°C. Automated genotype analysis The MLVA PCR products were diluted 1:80 and analyzed by capillary electrophoresis using the ABI Prism 3130 genetic analyzer (Applied Biosystems) and 0.25 μl GeneScan 1200, and were sized by using Gene Mapper 4.0 (Applied Biosystems Inc.). Assignment of the sizes and corresponding repeating unit numbers for each locus was performed using the following strains as references: Ames Ancestor (NCBI Reference Sequence: NC_007530.2, chromosome), pXO1 (NCBI Reference Sequence: NC_007322.2, plasmid), and pXO2 (NCBI Reference Sequence: NC_007323.2, plasmid). Data were analyzed using conventional values proposed in the updated version of the 2016 Bacillus anthracis MLVA database, available at MLVAbank ( http://mlva.u-psud.fr/ ). A phylogram was derived by clustering with the unweighted pair group method with arithmetic means (UPGMA), using 'categorical' character table values. All markers were given equal weight, irrespective of the number of repeats. The discriminatory ability of the MLVA technique was determined by calculating the discriminatory index (D) for 234 typed strains. The discriminatory power of each VNTR was estimated by the number of alleles detected and the allele diversity using BioNumerics 7.6 software (Applied Maths, Belgium) [ 19 ]. Ethics statement The animal and environmental strains used in the current study were isolated at the Anthrax Reference Institute of Italy (Ce.R.N.A.), a public laboratory mandated by the Italian Ministry of Health to confirm diagnosis of all animal anthrax cases in Italy. During outbreaks, samples were collected by the veterinary services of the Ministry of Health. Specific permission for soil sampling was not required. B . anthracis DNAs from anthrax human cutaneous cases were also included in the current study; two came from the "San Carlo" Hospital, Department of Infectious Disease, Potenza, Italy, and one from the "L. Spallanzani" National Institute for Infectious Disease, Rome, Italy [ 10 ]. Bacterial strains A collection of 234 B . anthracis strains, including 196 strains isolated from animal and 35 from the environment, isolated during Italian anthrax outbreaks in the years 1972–2018, were analyzed in the current study ( Table 1 ). Furthermore, 3 B . anthracis DNAs from anthrax human cutaneous cases were also analyzed. 10.1371/journal.pone.0227875.t001 Table 1 Overview of Bacillus anthracis isolates from the years 1972–2018 analyzed in the current study. Sample type Source No. of isolates Regions Environmental samples Water 3 Tuscany Soil 32 Basilicata, Tuscany Animal samples Bovine 101 Basilicata, Campania, Lazio, Apulia, Sardinia, Sicily, Tuscany, Umbria, Veneto, Lombardy Caprine 20 Abruzzo, Basilicata, Calabria, Campania, Apulia, Sardinia, Trentino Deer 7 Basilicata Equine 12 Basilicata, Campania, Apulia Ovine 53 Basilicata, Campania, Lazio, Apulia, Sicily Swine 3 Basilicata Human samples (DNAs) Human 3 Basilicata, Lazio DNA extraction B . anthracis strains were seeded on 5% sheep blood agar plates and then incubated at +37°C for 24 h. Bacterial DNA was extracted using the DNAeasy Blood and Tissue kit (Qiagen, Hilden, Germany), following the protocol for Gram-positive bacteria. All manipulations of B . anthracis strains were performed in a biosafety level 3 laboratory at the Experimental Zooprophylactic Institute of Apulia and Basilicata Regions in a class II type A 2 biosafety cabinet. Real-time polymerase chain reaction (PCR) assay Molecular identification of B . anthracis was performed using qualitative real-time PCR. The method is based on the amplification of specific DNA sequences using three pairs of specific primers [ 11 ] as follows: R1/R2 primers, specific for the BA813 gene located on the B . anthracis chromosome; PAG 23/24 primers, specific for the protective antigen (PA) gene located on the virulence plasmid pXO1; and CAP 57/58 primers, specific for the capsule (CAP) gene located on the virulence plasmid pXO2. Each 20 μl reaction mixture contained 1x Sso Advanced TM SYBR® Green Supermix (BIORAD), 300 nM each forward and reverse primer, and approximately 10 ng DNA template. The amplification was performed using the CFX Connect Real Time PCR Detection System (BIORAD). A melting curve was generated at 0.5°C increments between 65°C and 95°C, and was analyzed by CFX Manager TM Software, Version 3.0 (BIORAD). CanSNP analysis CanSNP profiles were obtained using 13 allelic discrimination assays involving specific oligonucleotides and probes, as described by Van Ert et al. [ 12 ]. Each 10 μl reaction mixture contained 1x TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, CA, USA), 250 nM probe, 600 nM each of forward and reverse primer, and approximately 10 ng DNA template. For all assays, the thermal cycling parameters used were as follows: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Endpoint fluorescent data were acquired by using the ABI 7900HT instrument. The CanSNPs data were compared with the data for 12 recognized sublineage or subgroups. The 14th SNP was detected using a High Resolution Melting (HRM) assay for a specific A.Br.011 CanSNP [ 13 , 14 ]. Position 2,552,486, based on the Ames Ancestor genome (NC_007530.2), was analyzed. Amplification was performed using the CFX Connect Real Time PCR Detection System (BIORAD) and Precision Melt Supermix (BIORAD). The reaction mixture contained 0.2 μM of each primer and 1x Precision Melt Supermix (BIORAD) in a 20 μl final volume. The following cycling parameters were used: 2 min at 95°C, were followed by 35 cycles of 10 s at 95°C and 30 s at 60°C. The samples were then heated to 95°C for 30 s, cooled down to 60°C over 1 min, and then heated from 65°C to 95°C at a rate of 0.5°C/s. High-resolution melting data were analyzed using Precision Melt Analysis Software (BIORAD). 31-loci MLVA analysis For the 31-marker MLVA, 5' fluorescently labeled oligonucleotides (6-FAM, VIC, NED and PET), specifically selected for variable number tandem repeats (VNTR) analysis were used. Twenty-seven chromosomal VNTR loci (vrrA, vrrB1, cg3, vrrB2, vntr19, vrrC1, vrrC2, vntr32, vntr12, vntr35, vntr23, bams03, bams05, bams13, bams15, bams21, bams22, bams23, bams24, bams25, bams28, bams30, bams31, bams34, bams44, bams51 and bams53) and four plasmid loci (vntr16, vntr17, pxO1 and pxO2) [ 12 , 15 – 18 ] were analyzed. The MLVA assay involved preparation of two singleplex and nine multiplex reactions, in a final volume of 15 μl. Each reaction mixture contained the following: 1x PCR reaction buffer (Qiagen, Hilden, Germany); 3 mM MgCl 2 , 0.2 mM for each dNTP; 1 U Hot Star Plus Taq DNA polymerase (Qiagen, Hilden, Germany), the appropriate concentration of each primer (as described in Table 2 ); and approximately 10 ng DNA template. 10.1371/journal.pone.0227875.t002 Table 2 Primer concentration for each set of marker in PCR reactions of MLVA analysis. PCR Reactions Primers conc. [μM] Singleplex 1 vrrC1 [0.2 μM] Singleplex 2 vrrC2 [0.2 μM] Multiplex 1 vrrA, vrrB1 [0.2 μM]; CG3 [0.4 μM] Multiplex 2 vrrB2 [0.25 μM]; pXO1 [0.3 μM]; pXO2 [0.1 μM] Multiplex 3 vntr12 [0.25 μM]; vntr19 [0.2 μμM]; vntr35 [0.2 μM] Multiplex 4 vntr16 [0.25 μM]; vntr23 [0.2 μM] Multiplex 5 vntr17 [0.1 μM]; vntr32 [0.4 μM] Multiplex 6 bams03 [0.8 μM]; bams05 [0.2 μM]; bams15, bams44 [0.5 μM] Multiplex 7 bams21 [0.4 μM]; bams24, bams25 [0.3 μM]; bams34 [0.2 μM] Multiplex 8 bams13 [0.3 μM]; bams28 [0.15 μM]; bams31, bams53 [0.6 μM] Multiplex 9 bams22, bams51 [0.3 μM]; bams23 [0.2 μM]; bams30 [0.6 μM] The following PCR cycling program was used for the two singleplex reactions and for multiplex reactions 1 and 2: 5 min at 95°C; followed by 35 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C, with a final step of 5 min at 72°C. The following amplification program was used for multiplex reactions 3: 5 min at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 54°C, 45 s at 72°C, and 5 min at 72°C. The following amplification program was used for multiplex reaction 4: 5 min at 95°C, followed by 35 cycles of 30 s at 94°C, 45 s at 56°C, 1 min at 72°C, and 5 min at 72°. The following amplification program was used for multiplex reaction 5: 5 min at 95°C, followed by 35 cycles of 30 s at 94°C, 45 s at 59°, 1 min at 72°C, and 5 min at 72°C. The following amplification program was used for multiplex reactions 6 to 9: 5 min at 94°C, followed by 35 cycles of 1 min at 94°C, 90 s at 60°, 90 s at 72°C, and 15 min at 72°C. Automated genotype analysis The MLVA PCR products were diluted 1:80 and analyzed by capillary electrophoresis using the ABI Prism 3130 genetic analyzer (Applied Biosystems) and 0.25 μl GeneScan 1200, and were sized by using Gene Mapper 4.0 (Applied Biosystems Inc.). Assignment of the sizes and corresponding repeating unit numbers for each locus was performed using the following strains as references: Ames Ancestor (NCBI Reference Sequence: NC_007530.2, chromosome), pXO1 (NCBI Reference Sequence: NC_007322.2, plasmid), and pXO2 (NCBI Reference Sequence: NC_007323.2, plasmid). Data were analyzed using conventional values proposed in the updated version of the 2016 Bacillus anthracis MLVA database, available at MLVAbank ( http://mlva.u-psud.fr/ ). A phylogram was derived by clustering with the unweighted pair group method with arithmetic means (UPGMA), using 'categorical' character table values. All markers were given equal weight, irrespective of the number of repeats. The discriminatory ability of the MLVA technique was determined by calculating the discriminatory index (D) for 234 typed strains. The discriminatory power of each VNTR was estimated by the number of alleles detected and the allele diversity using BioNumerics 7.6 software (Applied Maths, Belgium) [ 19 ]. Results Real Time PCR, CanSNPs and MLVA analysis of anthrax strains All the analyzed strains tested positive after the PCR amplification of chromosomal, plasmid pXO1 (toxins coding) and pXO2 (capsule formation) targets. The analysis of 13 classical CanSNPs revealed that 231 analyzed strains belonged to the sublineage A.Br. 008/009, also known as Trans-Eurasian (TEA) group. The TEA group was established in southern and eastern Europe and represents the dominant subgroup in Italy, Bulgaria, Hungary and Albania [ 7 , 12 , 20 – 22 ]. The analysis of an additional 14th CanSNP (A.Br.011), recently allowed for the differentiation of the A.Br.008/009 group into 2 subgroups. Accordingly, the analysis of the 14th CanSNP in the current study revealed that 207 of the 231 strains belonged to the main sub-lineage A.Br.011/009, while 24 strains belonged to the sublineage A.Br.008/011. All but one strain belonging to the latter sublineage were isolated in Sicily; one strain was isolated in Umbria. Further, one strain isolated in Veneto belonged to the main lineage A, sublineage A.Br.005/006, while two other strains, one from Veneto and one from Trentino, belonged to the main lineage B, sublineage B.Br.CNEVA. MLVA based on the analysis of 31 VNTRs revealed 55 different genotypes, as shown in S1 Table , distributed in the Italian regions named GT-1 to GT-55, accordingly ( Fig 1 ). The GT-14 genotype was the most common and was represented by 34 strains, mostly from Basilicata, Apulia, and Calabria. The GT-31 genotype was represented by 19 isolates: 16 from Tuscany, two from Apulia and one from Sardinia. The GT-26 and GT-27 genotypes were only isolated in the Basilicata and Campania regions. Other genotypes were characteristic for single regions, as showed in Table 3 . 10.1371/journal.pone.0227875.g001 Fig 1 The geographical distribution of 55 Bacillus anthracis genotypes in Italy. Image modified from the "Map of Italy"; "World of Maps" Public Domain ( https : //www . worldofmaps . net/europa/landkarten-und-stadtplaene-von-italien/landkarte-italien-administrative-bezirke-regioni . htm ) . 10.1371/journal.pone.0227875.t003 Table 3 Distribution of Bacillus anthracis CanSNPs and genotypes isolated in Italy in the years 1972–2018. Number of isolates Regions CanSNPs sublineage Genotype 1 Apulia A.Br. 011/009 MLVA31-1 1 Apulia A.Br. 011/009 MLVA31-2 1 Apulia A.Br. 011/009 MLVA31-3 3 Campania A.Br. 011/009 MLVA31-4 1 Sardinia A.Br. 011/009 MLVA31-5 3 Sardinia A.Br. 011/009 MLVA31-6 2 Apulia A.Br. 011/009 MLVA31-7 1 Umbria A.Br. 008/011 MLVA31-8 14 Tuscany A.Br. 011/009 MLVA31-9 3 Sicily A.Br. 011/009 MLVA31-10 1 Tuscany A.Br. 011/009 MLVA31-11 3 Sicily A.Br. 011/009 MLVA31-12 1 Lombardy A.Br. 011/009 MLVA31-13 34 Basilicata/Apulia/Calabria A.Br. 011/009 MLVA31-14 1 Apulia A.Br. 011/009 MLVA31-15 2 Apulia A.Br. 011/009 MLVA31-16 1 Apulia A.Br. 011/009 MLVA31-17 1 Basilicata A.Br. 011/009 MLVA31-18 1 Apulia A.Br. 011/009 MLVA31-19 1 Apulia A.Br. 011/009 MLVA31-20 1 Apulia A.Br. 011/009 MLVA31-21 1 Apulia A.Br. 011/009 MLVA31-22 1 Apulia A.Br. 011/009 MLVA31-23 57 Basilicata A.Br. 011/009 MLVA31-24 3 Basilicata A.Br. 011/009 MLVA31-25 3 Campania/Basilicata A.Br. 011/009 MLVA31-26 9 Campania/Basilicata A.Br. 011/009 MLVA31-27 5 Basilicata A.Br. 011/009 MLVA31-28 1 Apulia A.Br. 011/009 MLVA31-29 1 Sardinia A.Br. 011/009 MLVA31-30 19 Tuscany/Apulia/Sardinia A.Br. 011/009 MLVA31-31 1 Apulia A.Br. 011/009 MLVA31-32 1 Apulia A.Br. 011/009 MLVA31-33 5 Apulia A.Br. 011/009 MLVA31-34 6 Apulia A.Br. 011/009 MLVA31-35 2 Apulia A.Br. 011/009 MLVA31-36 1 Apulia A.Br. 011/009 MLVA31-37 1 Lazio A.Br. 011/009 MLVA31-38 1 Lazio A.Br. 011/009 MLVA31-39 1 Tuscany A.Br. 011/009 MLVA31-40 1 Apulia A.Br. 011/009 MLVA31-41 1 Apulia A.Br. 011/009 MLVA31-42 1 Campania A.Br. 011/009 MLVA31-43 1 Abruzzo A.Br. 011/009 MLVA31-44 2 Lazio A.Br. 011/009 MLVA31-45 1 Lazio A.Br. 011/009 MLVA31-46 5 Lazio A.Br. 011/009 MLVA31-47 3 Sicily A.Br. 008/011 MLVA31-48 1 Sicily A.Br. 008/011 MLVA31-49 2 Sicily A.Br. 008/011 MLVA31-50 9 Sicily A.Br. 008/011 MLVA31-51 7 Sicily A.Br. 008/011 MLVA31-52 1 Sicily A.Br. 008/011 MLVA31-53 1 Veneto A.Br. 005/006 MLVA31-54 2 Trentino/Veneto B.Br. CNEVA MLVA31-55 The number of different alleles ranged from 1 for bams21 and bams25 to 10 for bams15. Highest allelic diversities measured by Shannon Diversity Index (0.40632) was observed for the locus bams15 ( Table 4 ). The relationship among the strains based on MLVA results is represented in Fig 2 . 10.1371/journal.pone.0227875.g002 Fig 2 A UPGMA phylogram of MLVA profiles. The phylogram was built using BioNumerics 7.6 software (Applied Maths, Belgium). The visualization and the annotation of the genetic distances were performed using the web-based tool Interactive Tree of Life (iTOL). Circling the phylogram from the external to internal region are: genotype number, sublineage, species, year, regions (differently colored) of isolation and identification number of each analyzed strain. 10.1371/journal.pone.0227875.t004 Table 4 Shannon Diversity Index and allele numbers of MLVA markers with respect to the collection investigated. Locus No. alleles Diversity Index (Shannon) vrrA 4 0.172297 vrrB1 2 0.021373 vrrB2 3 0.073064 vrrC1 2 0.021373 vrrC2 2 0.082347 CG3 2 0.02979 pXO1aat 4 0.344872 pXO2at 4 0.118086 vntr32 3 0.033334 bams03 2 0.021373 bams05 5 0.08735 bams13 5 0.1482 bams15 10 0.40632 bams21 1 0 bams22 3 0.09788 bams23 4 0.06145 bams24 4 0.208345 bams25 1 0 bams28 2 0.23682 bams30 6 0.11232 bams31 7 0.224167 bams34 3 0.030103 bams44 2 0.147596 bams51 5 0.183046 bams53 3 0.021602 vntr12 4 0.08852 vntr16 5 0.219688 vntr17 4 0.215683 vntr19 2 0.234608 vntr23 2 0.0708 vntr35 2 0.159057 Real Time PCR, CanSNPs and MLVA analysis of anthrax strains All the analyzed strains tested positive after the PCR amplification of chromosomal, plasmid pXO1 (toxins coding) and pXO2 (capsule formation) targets. The analysis of 13 classical CanSNPs revealed that 231 analyzed strains belonged to the sublineage A.Br. 008/009, also known as Trans-Eurasian (TEA) group. The TEA group was established in southern and eastern Europe and represents the dominant subgroup in Italy, Bulgaria, Hungary and Albania [ 7 , 12 , 20 – 22 ]. The analysis of an additional 14th CanSNP (A.Br.011), recently allowed for the differentiation of the A.Br.008/009 group into 2 subgroups. Accordingly, the analysis of the 14th CanSNP in the current study revealed that 207 of the 231 strains belonged to the main sub-lineage A.Br.011/009, while 24 strains belonged to the sublineage A.Br.008/011. All but one strain belonging to the latter sublineage were isolated in Sicily; one strain was isolated in Umbria. Further, one strain isolated in Veneto belonged to the main lineage A, sublineage A.Br.005/006, while two other strains, one from Veneto and one from Trentino, belonged to the main lineage B, sublineage B.Br.CNEVA. MLVA based on the analysis of 31 VNTRs revealed 55 different genotypes, as shown in S1 Table , distributed in the Italian regions named GT-1 to GT-55, accordingly ( Fig 1 ). The GT-14 genotype was the most common and was represented by 34 strains, mostly from Basilicata, Apulia, and Calabria. The GT-31 genotype was represented by 19 isolates: 16 from Tuscany, two from Apulia and one from Sardinia. The GT-26 and GT-27 genotypes were only isolated in the Basilicata and Campania regions. Other genotypes were characteristic for single regions, as showed in Table 3 . 10.1371/journal.pone.0227875.g001 Fig 1 The geographical distribution of 55 Bacillus anthracis genotypes in Italy. Image modified from the "Map of Italy"; "World of Maps" Public Domain ( https : //www . worldofmaps . net/europa/landkarten-und-stadtplaene-von-italien/landkarte-italien-administrative-bezirke-regioni . htm ) . 10.1371/journal.pone.0227875.t003 Table 3 Distribution of Bacillus anthracis CanSNPs and genotypes isolated in Italy in the years 1972–2018. Number of isolates Regions CanSNPs sublineage Genotype 1 Apulia A.Br. 011/009 MLVA31-1 1 Apulia A.Br. 011/009 MLVA31-2 1 Apulia A.Br. 011/009 MLVA31-3 3 Campania A.Br. 011/009 MLVA31-4 1 Sardinia A.Br. 011/009 MLVA31-5 3 Sardinia A.Br. 011/009 MLVA31-6 2 Apulia A.Br. 011/009 MLVA31-7 1 Umbria A.Br. 008/011 MLVA31-8 14 Tuscany A.Br. 011/009 MLVA31-9 3 Sicily A.Br. 011/009 MLVA31-10 1 Tuscany A.Br. 011/009 MLVA31-11 3 Sicily A.Br. 011/009 MLVA31-12 1 Lombardy A.Br. 011/009 MLVA31-13 34 Basilicata/Apulia/Calabria A.Br. 011/009 MLVA31-14 1 Apulia A.Br. 011/009 MLVA31-15 2 Apulia A.Br. 011/009 MLVA31-16 1 Apulia A.Br. 011/009 MLVA31-17 1 Basilicata A.Br. 011/009 MLVA31-18 1 Apulia A.Br. 011/009 MLVA31-19 1 Apulia A.Br. 011/009 MLVA31-20 1 Apulia A.Br. 011/009 MLVA31-21 1 Apulia A.Br. 011/009 MLVA31-22 1 Apulia A.Br. 011/009 MLVA31-23 57 Basilicata A.Br. 011/009 MLVA31-24 3 Basilicata A.Br. 011/009 MLVA31-25 3 Campania/Basilicata A.Br. 011/009 MLVA31-26 9 Campania/Basilicata A.Br. 011/009 MLVA31-27 5 Basilicata A.Br. 011/009 MLVA31-28 1 Apulia A.Br. 011/009 MLVA31-29 1 Sardinia A.Br. 011/009 MLVA31-30 19 Tuscany/Apulia/Sardinia A.Br. 011/009 MLVA31-31 1 Apulia A.Br. 011/009 MLVA31-32 1 Apulia A.Br. 011/009 MLVA31-33 5 Apulia A.Br. 011/009 MLVA31-34 6 Apulia A.Br. 011/009 MLVA31-35 2 Apulia A.Br. 011/009 MLVA31-36 1 Apulia A.Br. 011/009 MLVA31-37 1 Lazio A.Br. 011/009 MLVA31-38 1 Lazio A.Br. 011/009 MLVA31-39 1 Tuscany A.Br. 011/009 MLVA31-40 1 Apulia A.Br. 011/009 MLVA31-41 1 Apulia A.Br. 011/009 MLVA31-42 1 Campania A.Br. 011/009 MLVA31-43 1 Abruzzo A.Br. 011/009 MLVA31-44 2 Lazio A.Br. 011/009 MLVA31-45 1 Lazio A.Br. 011/009 MLVA31-46 5 Lazio A.Br. 011/009 MLVA31-47 3 Sicily A.Br. 008/011 MLVA31-48 1 Sicily A.Br. 008/011 MLVA31-49 2 Sicily A.Br. 008/011 MLVA31-50 9 Sicily A.Br. 008/011 MLVA31-51 7 Sicily A.Br. 008/011 MLVA31-52 1 Sicily A.Br. 008/011 MLVA31-53 1 Veneto A.Br. 005/006 MLVA31-54 2 Trentino/Veneto B.Br. CNEVA MLVA31-55 The number of different alleles ranged from 1 for bams21 and bams25 to 10 for bams15. Highest allelic diversities measured by Shannon Diversity Index (0.40632) was observed for the locus bams15 ( Table 4 ). The relationship among the strains based on MLVA results is represented in Fig 2 . 10.1371/journal.pone.0227875.g002 Fig 2 A UPGMA phylogram of MLVA profiles. The phylogram was built using BioNumerics 7.6 software (Applied Maths, Belgium). The visualization and the annotation of the genetic distances were performed using the web-based tool Interactive Tree of Life (iTOL). Circling the phylogram from the external to internal region are: genotype number, sublineage, species, year, regions (differently colored) of isolation and identification number of each analyzed strain. 10.1371/journal.pone.0227875.t004 Table 4 Shannon Diversity Index and allele numbers of MLVA markers with respect to the collection investigated. Locus No. alleles Diversity Index (Shannon) vrrA 4 0.172297 vrrB1 2 0.021373 vrrB2 3 0.073064 vrrC1 2 0.021373 vrrC2 2 0.082347 CG3 2 0.02979 pXO1aat 4 0.344872 pXO2at 4 0.118086 vntr32 3 0.033334 bams03 2 0.021373 bams05 5 0.08735 bams13 5 0.1482 bams15 10 0.40632 bams21 1 0 bams22 3 0.09788 bams23 4 0.06145 bams24 4 0.208345 bams25 1 0 bams28 2 0.23682 bams30 6 0.11232 bams31 7 0.224167 bams34 3 0.030103 bams44 2 0.147596 bams51 5 0.183046 bams53 3 0.021602 vntr12 4 0.08852 vntr16 5 0.219688 vntr17 4 0.215683 vntr19 2 0.234608 vntr23 2 0.0708 vntr35 2 0.159057 Discussion Bacillus anthracis is clonal in nature and often exhibits a high degree of genetic homogeneity due to the fact that is has a single stranded chromosome and reproduces asexually. This characteristic has traditionally made the discrimination of isolates for epidemiological purposes difficult. Furthermore the high survivability of spores in the soils, allowed B . anthracis to reproduce a relatively limited number of times during its evolution [ 23 ]. The 31-loci MLVA analysis carried out on 234 B . anthracis strains, isolated in Italy during the years 1972–2018, revealed the circulation of 55 B . anthracis genotypes. The performed CanSNPs analysis placed 53 of the 55 identified genotypes in a common cluster (TEA). The analysis of the classical 13 CanSNPs revealed that most of the analyzed strains (98%) belonged to the sublineage A.Br.008/009 (the TEA group), which is the most common group in Europe and Asia [ 15 ]. However, except for the genotypes of strains isolated in Umbria and some others isolated in Sicily belonging to sublineage A.Br.008/011, all strains belonged to the sublineage A.Br.011/009. Interestingly, genotype GT-54 isolated in Veneto was very different from the other characteristic Italian strains. CanSNPs analysis confirmed this observation placing this genotype in the branch A.Br.005/006. This branch is generally present in the central-southern Africa, although it was also identified in Europe [ 12 , 24 ].Furthermore, genotype GT-55; B.Br.CNEVA, isolated in Veneto and Trentino is highly differentiated from most other Italian strains examined here. This genotype is widespread in Europe and found in France, Switzerland and Germany [ 12 , 25 , 26 ]. In Italy, the population of B . anthracis is mainly divided into two sublineages: A.Br.011/009, definitely the most common and A.Br.008/011 present only in Umbria and Sicily. Both these sublineages belong to the large TEA group ( Fig 2 ). The TEA group A.Br.008/009 contains a B . anthracis subpopulation that is well adapted to the northern hemisphere and predominant in Europe, Russia, Kazakhstan, Caucasus and western China [ 12 , 27 ]. It has also been detected in Africa [ 18 , 28 ]. This group is thought to have given rise to the western north American sublineage (A.Br.WNA), which is dominant in central Canada and much of the western USA. The presence of strains belonging to sublineages A.Br.008/011 and A.Br.011/009 might represent an effect of evolution on a common ancestral strain at the territorial level. In particular, A.Br.008/011 represents a rare and deep branching sublineage, also observed in Bulgaria, France and Turkey [ 29 ]. The spread of the TEA group to Europe and Asia is postulated to be linked to animal handling along the ancient East-West commercial routes of the Silk Road [ 30 ]. In the current study, strains belonging to the B.Br.CNEVA lineage were isolated in a relatively small area of north-eastern Italy. The relatively low diversity between the two strains demonstrated in the current study is consistent with a single introduction event of the B.Br.CNEVA lineage into the country, followed by ecological establishment and progressive in situ differentiation around the Italian Alps area [ 21 ]. Consistent with this hypothesis, the Italian strains form a cluster that is distinct from the other European B.Br.CNEVA strains. Identification of one A.Br.005/006 strain in Italy could be associated with the trade exchanges dating back when city states competed for trade and commerce throughout the Mediterranean [ 7 ]. This subgroup is well represented in Africa, but rare in Europe [ 12 ]. It is therefore quite surprising that past importations of ill or dying animals or spore-infected items from Africa, the Middle East, or even Asia, did not have a greater impact on the genetic structure of B . anthracis in the region. The higher variety of B . anthracis genotypes identified in southern Italy relative to genotypes from other Italian regions may be explained by the differences in the breeding systems between northern and southern Italy. In southern Italy, many livestock farmers use extensive farming methods, which increases the chances of grazer exposure to historical spore sites and deposits. The possibility of exposure is lower in northern Italy because most livestock farmers use intensive breeding systems. Another observation from the current study was that the neighboring regions share just a few genotypes. In particular, the GT-24 genotype was present in Apulia, Basilicata and Calabria; the GT-26 and GT-27 genotypes were identified in Basilicata and Campania; and the GT-55 genotype was identified in Veneto and Trentino. Noteworthy and difficult to explain is the dislocation of genotype GT-31, identified in Apulia, Tuscany and Sardinia. These are not neighboring regions; on the contrary, they are quite far from one another. Also in this national scenario one of the explanations could be the trade of animals or animal products within the country over the years. Nevertheless, since most genotypes are exclusive to each region, it appears that Italian B . anthracis strains may be autochthonous for a single territory. Interestingly, genotypes exclusive to specific regions were detected especially in Sicily and Sardinia, probably because of low animal movements between these islands and the rest of Italy. The analysis of chromosomal and plasmid hypervariable regions using such methods as MLVA constitutes a valuable approach for studying the diversity, evolution and molecular epidemiology of B . anthracis . Therefore, MLVA is a valid method that enables the understanding of the distribution of B . anthracis within a country. Supporting information S1 Table Allele distribution of the 55 genotypes identified using 31 VNTR analysis. (XLSX) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924283/

Anthrax Lethal Toxin Inhibits Translation of Hypoxia-inducible Factor 1α and Causes Decreased Tolerance to Hypoxic Stress *

Background: Hypoxia is proposed as a mediator of anthrax lethal factor (LT)-induced pathology. Results: LT inhibits hypoxia-inducible factor 1α (HIF-1α) translation and causes increased cellular toxicity in response to hypoxic stress. Conclusion: LT reduces HIF-1α translation, dysregulating host responses to hypoxia. Significance: Inhibition of HIF-1α translation is a novel mechanism underlying LT-induced pathology.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208855/

The Vibrio cholerae VctPDGC System Transports Catechol Siderophores and a Siderophore-Free Iron Ligand

Vibrio cholerae , the causative agent of cholera, has an absolute requirement for iron. It transports the catechol siderophores vibriobactin, which it synthesizes and secretes, and enterobactin. These siderophores are transported across the inner membrane by one of two periplasmic binding protein-dependent ABC transporters, VctPDGC or ViuPDGC. We show here that one of these inner membrane transport systems, VctPDGC, also promotes iron acquisition in the absence of siderophores. Plasmids carrying the vctPDGC genes stimulated growth in both rich and minimal media of a Shigella flexneri mutant that produces no siderophores. vctPDGC also stimulated the growth of an E. coli enterobactin biosynthetic mutant in low iron medium, and this effect did not require feoB , tonB or aroB . A tyrosine to phenylalanine substitution in the periplasmic binding protein VctP did not alter enterobactin transport, but eliminated growth stimulation in the absence of a siderophore. These data suggest that the VctPDGC system has the capacity to transport both catechol siderophores and a siderophore-free iron ligand. We also show that VctPDGC is the previously unidentified siderophore-independent iron transporter in V. cholerae , and this appears to complete the list of iron transport systems in V. cholerae .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10722900/

Nanopore Single-molecule Analysis of Biomarkers: Providing Possible Clues to Disease Diagnosis

Biomarker detection has attracted increasing interest in recent years due to the minimally or non-invasive sampling process. Single entity analysis of biomarkers is expected to provide real-time and accurate biological information for early disease diagnosis and prognosis, which is critical to the effective disease treatment and is also important in personalized medicine. As an innovative single entity analysis method, nanopore sensing is a pioneering single-molecule detection technique that is widely used in analytical bioanalytical fields. In this review, we overview the recent progress of nanopore biomarker detection as new approaches to disease diagnosis. In highlighted studies, nanopore was focusing on detecting biomarkers of different categories of communicable and noncommunicable diseases, such as pandemic Covid-19, AIDS, cancers, neurologic diseases, etc. Various sensitive and selective nanopore detecting strategies for different types of biomarkers are summarized. In addition, the challenges, opportunities, and direction for future development of nanopore-based biomarker sensors are also discussed.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9827366/

Monkeypox virus detection in rodents using real-time 3′-minor groove binder TaqMan® assays on the Roche LightCycler

During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan -orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan -orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism. Main Monkeypox virus (MPXV) is a zoonotic virus in the family Poxviridae , genus Orthopoxvirus . It was first isolated from lesions seen among captive monkeys in Copenhagen, Denmark. 1 Human monkeypox was subsequently identified in 1970 in the Democratic Republic of the Congo (DRC). 2 The virus causes a disease in humans that is similar to smallpox, but results in a lower case-fatality rate. The disease is endemic in the rainforests of central and western Africa, where squirrels and monkeys have been suggested to play a role in the life cycle of the virus. 3 , 4 , 5 With the eradication of smallpox and widespread discontinuation of smallpox vaccination, human monkeypox has re-emerged as a human health threat with major outbreaks occurring in 1996–1997 and 2001 in the DRC. 6 , 7 More recently, MPXV was found to be the cause of a cluster of cases of disease in the Midwest region of the US. 8 , 9 , 10 Epidemiologic investigations confirm that the human cases of monkeypox resulted from contact with infected prairie dogs that had been housed or transported with African rodents imported from Ghana. 11 This represents the first time that a monkeypox outbreak has occurred in the Western Hemisphere and highlights the need for rapid and accurate diagnostics to detect these emerging viral pathogens. Several molecular-based techniques have been developed to detect and discriminate orthopoxviruses; 12 , 13 , 14 , 15 however, they require conventional polymerase chain reaction (PCR) followed by restriction endonuclease digestion and subsequent gel electrophoresis. Recently, rapid real-time PCR methods were developed for detecting Orthopoxvirus DNA. 16 , 17 , 18 The goal of this study was to develop real-time LightCycler PCR assays based on the conserved F3L and N3R genes for specifically detecting MPXV and validating these assays by using a panel of DNAs from orthopox viral isolates that vary both temporally and geographically and tissues from various MPXV-infected rodents from the recent US outbreak. 8 Two previously developed pan -orthopox real-time PCR assays based on the hemagglutinin ( HA ) and E9L genes 19 and a pan -orthopox electrochemiluminescence (ECL) immunoassay were also used to evaluate potentially monkeypox-infected tissues. Materials and methods PCR Primers, Target Sequences, and Fluorogenic Probe The real-time PCR assay primers and TaqMan®-MGB probe sequences are listed in Table 1 . The Vaccinia F3L gene (also called the D14L gene) (GenBank Accession #'s: AF380138 for MPXV-ZRE (bp 48 048–48 509); M35027 for VAC-COP (bp 50 911–51 483); Y16780 for VMN-GAR (bp 39 619–40 197); L22579 for VAR-BSH (bp 39 258–39 836); AF482758 for CPXV-BR (bp 64 465–65 037); NC_001611 for VAR-IND (bp 38 632–39 204); AF095689 for VAC-TAN (bp 47 348–47 921); AF438165 for CMPV-M96 (bp 50 654–51 226)) and the N3R gene (GenBank Accession #'s: AF380138 for MPXV-ZRE (bp 190 003–190 733); X94355 for CPXV-BR (bp 19 910–20 625 reverse)) sequences were selected as potential monkeypox-specific targets. All sequence alignments were carried out using the EMBL-EBI ClustalW (1.82) Multiple Sequence Alignment Tool ( http://www.ebi.ac.uk/clustalw/index.html ). Regions of nonhomology were used as target sequences for each potential MPXV-specific assay. The specific primer and TaqMan®-MGB probe sequences were designed using Primer Express Version 2.0 for Windows (Applied Biosystems). All primers were synthesized using standard phosphoramidite chemistry with an ABI 394 DNA/RNA synthesizer. The TaqMan®-MGB probes were also synthesized by ABI and contained 6-carboxyfluorescein (6FAM) at the 5′ end. An NFQ and the MGB were added to the 3′ end. Table 1 Primer/probe sequence of the monkeypox-specific assays Organism Gene target Amplicon size (bp) Primers/probe Sequence Final conc ( μ M) MgCl 2 (mM) Monkeypox F3L 107 F3L-F290 5′-CTC ATT GAT TTT TCG CGG GAT A-3′ 0.5 5 F3L-R396 5′-GAC GAT ACT CCT CCT CGT TGG T-3′ 0.5 F3Lp333S-MGB 5′-6FAM-CAT CAG AAT CTG TAG GCC GT-MGBNFQ-3′ 0.05 Monkeypox N3R 139 N3R-F319 5′-AAC AAC CGT CCT ACA ATT AAA CAA CA-3′ 0.5 5 N3R-R457 5′-CGC TAT CGA ACC ATT TTT GTA GTC T-3′ 0.5 N3Rp352S-MGB 5′-6FAM-TAT AAC GGC GAA GAA TAT ACT-MGBNFQ-3′ 0.05 5′ Nuclease PCR (TaqMan®-MGB) Assays Using the Primer Express 2.0. software to design potential MPXV-specific TaqMan®-MGB assays ( F3L -MGB and N3R -MGB), we optimized each assay according to a standard protocol instituted by the Diagnostic Systems Division at US Army Medical Research Institute of Infectious Diseases (USAMRIID). Briefly, potential primer pairs were initially tested in the LightCycler by the fluorescent dye SYBR Green I (Roche Biochemicals). The optimum primer pair was selected based on specificity (a single, appropriately sized amplicon) and efficiency of amplification (lowest C t value which is defined as the real-time PCR cycle at which the LightCycler software determines the reaction to be positive). The selected primer pair was then optimized to the final concentration (0.1–1.0 μ M) with the lowest C t value and the highest fluorescent signal. Next, several potential TaqMan®-MGB probes were tested with the optimized primer pair by varying the probe and MgCl 2 concentrations. The final assay consisted of the primer/probe pair concentrations and reaction conditions that combined the lowest level of detection (LOD—the gene copy number that was detected by the assay at least 58/60 times), lowest C t value, and highest fluorescent signal-to-noise ratio. The LODs of the assays were determined from serial dilutions of genomic DNA purified from monkeypox virions (Zaire 79-I-05). All assays were carried out in 20 μ l volumes for the LightCycler with each reaction made up in PCR buffer (50 mM Tris, pH 8.3); 25 μ g/ml of bovine serum albumin (BSA) and 0.2 mM dinucleotide triphosphate (dNTP) mix (Idaho Technology, Salt Lake City, UT, USA). Eight-tenths (0.8) unit of Platinum Taq DNA polymerase (Invitrogen) was added to each reaction. The final MgCl 2 , primer and probe concentrations for each assay are listed in Table 1 . Thermal cycling for the LightCycler was performed as follows: one cycle at 95°C for 2 min, followed by 45 cycles of 95°C for 1 s and 60°C for 20 s. A fluorescence reading was taken at the end of each 60°C step. Each reaction capillary tube was read in Channel 1 (F1) at a gain setting of 16 with data being analyzed by the LightCycler Data Analysis software (version 3.5.3). Sample curves were analyzed using the 'second derivative maximum' with the baseline adjustment set to Arithmetic . Extended Assay Evaluation Both assays were extensively evaluated, first against various genomic orthopox and nonorthopox virus nearest-neighbor DNAs ( Table 2 ). The panel consisted of 17 monkeypox viral isolate DNAs (10–100 pg/ μ l), 25 Variola isolate DNAs (100 pg/ μ l), and DNAs from Vaccinia , camelpox, cowpox, Herpes simplex , Varicella zoster viruses, and human genomic DNA. Utrecht is a nonhuman primate monkeypox isolate. The other MPXV DNAs are human monkeypox isolates from Zaire/DRC; MPX-Sierra Leone is an isolate from 1970, 12003KI is an isolate from 1999 (DRC), and V96-I-16 is a strain of MPX-monkeypox Zaire. Within the MPXV panel are 11 monkeypox-human isolates obtained from clinical samples during the 1996 outbreak in the Congo (unpublished data). All testing with Variola virus genomic DNA was conducted at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA. Both assays were also tested against various bacterial isolates and strains from the USAMRIID bacterial DNA cross-reactivity panel (100 pg of each DNA) (data not shown). In all, 5 μ l of each sample was added to the appropriate MPXV assay LightCycler Master Mix (15 μ l) and cycled as described above. All test runs included at least one positive control that contained 2.3 × 10 4 copies (5 pg total) of purified monkeypox genomic DNA (Zaire 79-I-05) and two no-template controls (NTC): reagent NTC and sample NTC. Table 2 USAMRIID/CDC DNA panel DNA species (Genomic) Concentration (pg/ μ l) F3L-MGB N3R-MGB Human genomic DNA 1000 − − MPV Zaire 79-I-05 10 + + MPXR Zaire 79-I-05 (Cidofovir-resistant) 10 + + MPV Utrecht 10 + + MPV Sierra Leone 10 + + MPV 12003k 10 + + MPV DCR (Zaire) 96-I-16 10 + + MPX V97-I-04 100 + + MPX V97-I-16 100 + + MPX V97-I-03 100 + + MPX V98-I-0035 100 + + MPX V97-I-07 100 + + MPX V97-I-05 100 + + MPX V97-I-12 100 + + MPX V97-I-17 100 + + MPX V97-I-11 100 + + MPX V97-I-06 100 + + MPX V97-I-08 100 + + Variola Horn 100 − − V77-I-1605 100 − − V70-I-46 100 − − Afgan Variolator 4 100 − − Nepal V73-I-175 100 − − India 7124 100 − − Bangladesh 1975 (V75-I-550) 100 − − Kali Muthu 50 100 − − Iran 2602 100 − − K1629 100 − − Higgins 100 − − Hinden 100 − − Juba 100 − − Lee 100 − − Rafig-Lahore 100 − − Harper 100 − − ETH-17R14-X-72 100 − − 102 100 − − Yamada 100 − − V70-I-222 100 − − Butler 100 − − V72-I-119 100 − − V72-I-143 100 − − V73-I-225 100 − − V77-I-1252 100 − − IHD— Vaccinia strain IHD 100 − − MVA—modified Vaccinia Ankara 100 − − MDV—Maerek's Disease Vaccine virus 100 − − VAC WR— Vaccinia Western Reserve 100 − − CML—Camelpox Somalia 100 − − CMLR—Cidofovir-resistant Camelpox Somalia 100 − − CPX—cowpox Brighton Red (BR) 100 − − CPXR—Cidofovir-resistant cowpox Brighton Red 100 − − VAC— Vaccinia Copenhagen 100 − − VACR—Cidofovir-resistant Vaccinia Copenhagen 100 − − HSV-1—Herpes Simplex virus type 1 100 − − HSV-2—Herpes Simplex virus type 2 100 − − Varicella OKA (purified virus) 4000 − − Varicella WEB (purified virus) 18 000 − − Varicella V2V 123j (purified virus) 48 000 − − Study Animals All animals used in this study were collected from either an exotic pet dealer or private homes in the Chicago, Illinois metropolitan area. The animals were voluntarily released to public health and veterinary health officials as a result of either cohabitation of these animals with known positive animals or those that exhibited signs of orthopox infection. All animals were euthanized and frozen for transport to USAMRIID. Animal species tested during the course of this study included: prairie dog ( Cynomys sp.), rat ( Rattus sp.), hamster ( Cricetus spp.), dwarf hamster ( Allocricetulus sp.), gerbil ( Gerbillus sp.), Jerboa ( Jaculus sp.), mole rat ( Heterocephalus sp.), and chinchilla ( Chinchilla sp.). Postmortem Examination and Tissue Collection The 52 frozen rodent carcasses (see Table 3 ) collected in Illinois that were linked to possible MPXV transmission were thawed overnight in a Class II laminar hood. Complete necropsies were performed on each animal by or under the direct supervision of a board-certified veterinary pathologist. Tissue samples including brain, lung, liver, spleen, kidney, and ovary/testis of each rodent, and dermal lesions with the draining lymph nodes of affected rodents were harvested for PCR, immunoassay, and viral isolation. However, not all tissues were taken from each animal. Table 3 Animal real-time PCR Animal Total # of animals pan HA-MGB pan E9L-MGB MPXV F3L-MGB MPXV N3R-MGB Total a Prarie dog 8 b 3/8 2/8 3/8 3/8 3/8 Rat 2 0/2 0/2 0/2 0/2 0/2 Mole rat 1 0/1 0/1 0/1 0/1 0/1 Jerboas 2 0/2 0/2 0/2 0/2 0/2 Hamster 12 2/12 0/12 1/12 1/12 2/12 Gerbil 13 4/13 5/13 6/13 5/13 6/13 Dwarf Hamster 12 0/12 0/12 0/12 0/12 0/12 Chinchilla 2 1/2 1/2 1/2 1/2 1/2 52 12/52 a Total positive animals for at least one pan -orthopox/MPXV PCR assay in one tissue=12/52 (23% positive animals for orthopox). b Prairie dog #5 was pan -OP (PCR) and MPXV positive for 8/8 tissues (although liver was only positive for 2/4 assays: panE9L -MGB and MPXV- F3L -MGB; all other tissues were positive for 4/4 assays). Tissue Preparation and DNA Extraction A 10% homogenate of each tissue specimen was made in Dulbecco's high-glucose medium using a PCR tissue homogenizer (Omni International, Inc., Warrenton, VA, USA). The homogenate was centrifuged for 10 min at 2000 rpm at 4°C. DNA was extracted from 100 μ l of the supernatant using the QIAamp DNA Mini kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions with the following minor modification: during the initial lysis step, the incubation time was increased to overnight at 56°C to ensure complete lysis of any virus present in the sample. The remaining supernatant was stored at −70°C for immunoassay and virus isolation. Inhibition Testing of Animal DNA Inhibition testing of DNA isolated from animals was performed incorporating an internal positive control (IPC) testing system developed at USAMRIID (manuscript submitted). Briefly, both forward and reverse primer sites and a TaqMan® probe site of the Bacillus anthracis protective antigen gene was mutated to create a novel sequence with no known homology to published sequences with a resulting amplicon of 153 bp. The IPC was shown to be sensitive to a variety of inhibitors including hemoglobin, heparin, ethylenediamine tetraacetic acid (EDTA), humic acids, and fulvic acid. All extracted animal samples were tested for inhibition by adding either 5 μ l of the sample to 15 μ l of the same IPC LightCycler Master Mix and compared to an IPC control (5 μ l of H 2 O instead of sample). Cycling conditions on the LightCycler were as described above. All sample curves were analyzed using the LightCycler Data Analysis software (LCDA version 3.5.3) with 'second derivative maximum' and baseline adjustment set to arithmetic. Testing for inhibition with the IPC assay resulted in three possible outcomes: (a) complete inhibition; (b) partial inhibition (the cycle threshold ( C t ) was delayed and/or end point fluorescence (EPF) was reduced); or (c) no inhibition ( C t and EPF were comparable to the H 2 O blank). A C t shift of three cycles was quantitatively equivalent to an approximately one log decrease in copy number (detection limit). Therefore, a C t shift of greater than three cycles beyond the water blank was considered inhibitory. Inhibition was indicated when a sample had a C t shift of greater than three cycles OR a reduction in EPF of greater than or equal to 50% when compared to water controls. If inhibition was observed in a sample, two-fold serial dilutions were performed until inhibition was completely eliminated. C t shifts less than three cycles or less than 50% loss of EPF were not considered significant, and dilution of these samples would unnecessarily dilute out the target DNA. PCR Primers, Target Sequences, and Fluorogenic Probe The real-time PCR assay primers and TaqMan®-MGB probe sequences are listed in Table 1 . The Vaccinia F3L gene (also called the D14L gene) (GenBank Accession #'s: AF380138 for MPXV-ZRE (bp 48 048–48 509); M35027 for VAC-COP (bp 50 911–51 483); Y16780 for VMN-GAR (bp 39 619–40 197); L22579 for VAR-BSH (bp 39 258–39 836); AF482758 for CPXV-BR (bp 64 465–65 037); NC_001611 for VAR-IND (bp 38 632–39 204); AF095689 for VAC-TAN (bp 47 348–47 921); AF438165 for CMPV-M96 (bp 50 654–51 226)) and the N3R gene (GenBank Accession #'s: AF380138 for MPXV-ZRE (bp 190 003–190 733); X94355 for CPXV-BR (bp 19 910–20 625 reverse)) sequences were selected as potential monkeypox-specific targets. All sequence alignments were carried out using the EMBL-EBI ClustalW (1.82) Multiple Sequence Alignment Tool ( http://www.ebi.ac.uk/clustalw/index.html ). Regions of nonhomology were used as target sequences for each potential MPXV-specific assay. The specific primer and TaqMan®-MGB probe sequences were designed using Primer Express Version 2.0 for Windows (Applied Biosystems). All primers were synthesized using standard phosphoramidite chemistry with an ABI 394 DNA/RNA synthesizer. The TaqMan®-MGB probes were also synthesized by ABI and contained 6-carboxyfluorescein (6FAM) at the 5′ end. An NFQ and the MGB were added to the 3′ end. Table 1 Primer/probe sequence of the monkeypox-specific assays Organism Gene target Amplicon size (bp) Primers/probe Sequence Final conc ( μ M) MgCl 2 (mM) Monkeypox F3L 107 F3L-F290 5′-CTC ATT GAT TTT TCG CGG GAT A-3′ 0.5 5 F3L-R396 5′-GAC GAT ACT CCT CCT CGT TGG T-3′ 0.5 F3Lp333S-MGB 5′-6FAM-CAT CAG AAT CTG TAG GCC GT-MGBNFQ-3′ 0.05 Monkeypox N3R 139 N3R-F319 5′-AAC AAC CGT CCT ACA ATT AAA CAA CA-3′ 0.5 5 N3R-R457 5′-CGC TAT CGA ACC ATT TTT GTA GTC T-3′ 0.5 N3Rp352S-MGB 5′-6FAM-TAT AAC GGC GAA GAA TAT ACT-MGBNFQ-3′ 0.05 5′ Nuclease PCR (TaqMan®-MGB) Assays Using the Primer Express 2.0. software to design potential MPXV-specific TaqMan®-MGB assays ( F3L -MGB and N3R -MGB), we optimized each assay according to a standard protocol instituted by the Diagnostic Systems Division at US Army Medical Research Institute of Infectious Diseases (USAMRIID). Briefly, potential primer pairs were initially tested in the LightCycler by the fluorescent dye SYBR Green I (Roche Biochemicals). The optimum primer pair was selected based on specificity (a single, appropriately sized amplicon) and efficiency of amplification (lowest C t value which is defined as the real-time PCR cycle at which the LightCycler software determines the reaction to be positive). The selected primer pair was then optimized to the final concentration (0.1–1.0 μ M) with the lowest C t value and the highest fluorescent signal. Next, several potential TaqMan®-MGB probes were tested with the optimized primer pair by varying the probe and MgCl 2 concentrations. The final assay consisted of the primer/probe pair concentrations and reaction conditions that combined the lowest level of detection (LOD—the gene copy number that was detected by the assay at least 58/60 times), lowest C t value, and highest fluorescent signal-to-noise ratio. The LODs of the assays were determined from serial dilutions of genomic DNA purified from monkeypox virions (Zaire 79-I-05). All assays were carried out in 20 μ l volumes for the LightCycler with each reaction made up in PCR buffer (50 mM Tris, pH 8.3); 25 μ g/ml of bovine serum albumin (BSA) and 0.2 mM dinucleotide triphosphate (dNTP) mix (Idaho Technology, Salt Lake City, UT, USA). Eight-tenths (0.8) unit of Platinum Taq DNA polymerase (Invitrogen) was added to each reaction. The final MgCl 2 , primer and probe concentrations for each assay are listed in Table 1 . Thermal cycling for the LightCycler was performed as follows: one cycle at 95°C for 2 min, followed by 45 cycles of 95°C for 1 s and 60°C for 20 s. A fluorescence reading was taken at the end of each 60°C step. Each reaction capillary tube was read in Channel 1 (F1) at a gain setting of 16 with data being analyzed by the LightCycler Data Analysis software (version 3.5.3). Sample curves were analyzed using the 'second derivative maximum' with the baseline adjustment set to Arithmetic . Extended Assay Evaluation Both assays were extensively evaluated, first against various genomic orthopox and nonorthopox virus nearest-neighbor DNAs ( Table 2 ). The panel consisted of 17 monkeypox viral isolate DNAs (10–100 pg/ μ l), 25 Variola isolate DNAs (100 pg/ μ l), and DNAs from Vaccinia , camelpox, cowpox, Herpes simplex , Varicella zoster viruses, and human genomic DNA. Utrecht is a nonhuman primate monkeypox isolate. The other MPXV DNAs are human monkeypox isolates from Zaire/DRC; MPX-Sierra Leone is an isolate from 1970, 12003KI is an isolate from 1999 (DRC), and V96-I-16 is a strain of MPX-monkeypox Zaire. Within the MPXV panel are 11 monkeypox-human isolates obtained from clinical samples during the 1996 outbreak in the Congo (unpublished data). All testing with Variola virus genomic DNA was conducted at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA. Both assays were also tested against various bacterial isolates and strains from the USAMRIID bacterial DNA cross-reactivity panel (100 pg of each DNA) (data not shown). In all, 5 μ l of each sample was added to the appropriate MPXV assay LightCycler Master Mix (15 μ l) and cycled as described above. All test runs included at least one positive control that contained 2.3 × 10 4 copies (5 pg total) of purified monkeypox genomic DNA (Zaire 79-I-05) and two no-template controls (NTC): reagent NTC and sample NTC. Table 2 USAMRIID/CDC DNA panel DNA species (Genomic) Concentration (pg/ μ l) F3L-MGB N3R-MGB Human genomic DNA 1000 − − MPV Zaire 79-I-05 10 + + MPXR Zaire 79-I-05 (Cidofovir-resistant) 10 + + MPV Utrecht 10 + + MPV Sierra Leone 10 + + MPV 12003k 10 + + MPV DCR (Zaire) 96-I-16 10 + + MPX V97-I-04 100 + + MPX V97-I-16 100 + + MPX V97-I-03 100 + + MPX V98-I-0035 100 + + MPX V97-I-07 100 + + MPX V97-I-05 100 + + MPX V97-I-12 100 + + MPX V97-I-17 100 + + MPX V97-I-11 100 + + MPX V97-I-06 100 + + MPX V97-I-08 100 + + Variola Horn 100 − − V77-I-1605 100 − − V70-I-46 100 − − Afgan Variolator 4 100 − − Nepal V73-I-175 100 − − India 7124 100 − − Bangladesh 1975 (V75-I-550) 100 − − Kali Muthu 50 100 − − Iran 2602 100 − − K1629 100 − − Higgins 100 − − Hinden 100 − − Juba 100 − − Lee 100 − − Rafig-Lahore 100 − − Harper 100 − − ETH-17R14-X-72 100 − − 102 100 − − Yamada 100 − − V70-I-222 100 − − Butler 100 − − V72-I-119 100 − − V72-I-143 100 − − V73-I-225 100 − − V77-I-1252 100 − − IHD— Vaccinia strain IHD 100 − − MVA—modified Vaccinia Ankara 100 − − MDV—Maerek's Disease Vaccine virus 100 − − VAC WR— Vaccinia Western Reserve 100 − − CML—Camelpox Somalia 100 − − CMLR—Cidofovir-resistant Camelpox Somalia 100 − − CPX—cowpox Brighton Red (BR) 100 − − CPXR—Cidofovir-resistant cowpox Brighton Red 100 − − VAC— Vaccinia Copenhagen 100 − − VACR—Cidofovir-resistant Vaccinia Copenhagen 100 − − HSV-1—Herpes Simplex virus type 1 100 − − HSV-2—Herpes Simplex virus type 2 100 − − Varicella OKA (purified virus) 4000 − − Varicella WEB (purified virus) 18 000 − − Varicella V2V 123j (purified virus) 48 000 − − Study Animals All animals used in this study were collected from either an exotic pet dealer or private homes in the Chicago, Illinois metropolitan area. The animals were voluntarily released to public health and veterinary health officials as a result of either cohabitation of these animals with known positive animals or those that exhibited signs of orthopox infection. All animals were euthanized and frozen for transport to USAMRIID. Animal species tested during the course of this study included: prairie dog ( Cynomys sp.), rat ( Rattus sp.), hamster ( Cricetus spp.), dwarf hamster ( Allocricetulus sp.), gerbil ( Gerbillus sp.), Jerboa ( Jaculus sp.), mole rat ( Heterocephalus sp.), and chinchilla ( Chinchilla sp.). Postmortem Examination and Tissue Collection The 52 frozen rodent carcasses (see Table 3 ) collected in Illinois that were linked to possible MPXV transmission were thawed overnight in a Class II laminar hood. Complete necropsies were performed on each animal by or under the direct supervision of a board-certified veterinary pathologist. Tissue samples including brain, lung, liver, spleen, kidney, and ovary/testis of each rodent, and dermal lesions with the draining lymph nodes of affected rodents were harvested for PCR, immunoassay, and viral isolation. However, not all tissues were taken from each animal. Table 3 Animal real-time PCR Animal Total # of animals pan HA-MGB pan E9L-MGB MPXV F3L-MGB MPXV N3R-MGB Total a Prarie dog 8 b 3/8 2/8 3/8 3/8 3/8 Rat 2 0/2 0/2 0/2 0/2 0/2 Mole rat 1 0/1 0/1 0/1 0/1 0/1 Jerboas 2 0/2 0/2 0/2 0/2 0/2 Hamster 12 2/12 0/12 1/12 1/12 2/12 Gerbil 13 4/13 5/13 6/13 5/13 6/13 Dwarf Hamster 12 0/12 0/12 0/12 0/12 0/12 Chinchilla 2 1/2 1/2 1/2 1/2 1/2 52 12/52 a Total positive animals for at least one pan -orthopox/MPXV PCR assay in one tissue=12/52 (23% positive animals for orthopox). b Prairie dog #5 was pan -OP (PCR) and MPXV positive for 8/8 tissues (although liver was only positive for 2/4 assays: panE9L -MGB and MPXV- F3L -MGB; all other tissues were positive for 4/4 assays). Tissue Preparation and DNA Extraction A 10% homogenate of each tissue specimen was made in Dulbecco's high-glucose medium using a PCR tissue homogenizer (Omni International, Inc., Warrenton, VA, USA). The homogenate was centrifuged for 10 min at 2000 rpm at 4°C. DNA was extracted from 100 μ l of the supernatant using the QIAamp DNA Mini kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions with the following minor modification: during the initial lysis step, the incubation time was increased to overnight at 56°C to ensure complete lysis of any virus present in the sample. The remaining supernatant was stored at −70°C for immunoassay and virus isolation. Inhibition Testing of Animal DNA Inhibition testing of DNA isolated from animals was performed incorporating an internal positive control (IPC) testing system developed at USAMRIID (manuscript submitted). Briefly, both forward and reverse primer sites and a TaqMan® probe site of the Bacillus anthracis protective antigen gene was mutated to create a novel sequence with no known homology to published sequences with a resulting amplicon of 153 bp. The IPC was shown to be sensitive to a variety of inhibitors including hemoglobin, heparin, ethylenediamine tetraacetic acid (EDTA), humic acids, and fulvic acid. All extracted animal samples were tested for inhibition by adding either 5 μ l of the sample to 15 μ l of the same IPC LightCycler Master Mix and compared to an IPC control (5 μ l of H 2 O instead of sample). Cycling conditions on the LightCycler were as described above. All sample curves were analyzed using the LightCycler Data Analysis software (LCDA version 3.5.3) with 'second derivative maximum' and baseline adjustment set to arithmetic. Testing for inhibition with the IPC assay resulted in three possible outcomes: (a) complete inhibition; (b) partial inhibition (the cycle threshold ( C t ) was delayed and/or end point fluorescence (EPF) was reduced); or (c) no inhibition ( C t and EPF were comparable to the H 2 O blank). A C t shift of three cycles was quantitatively equivalent to an approximately one log decrease in copy number (detection limit). Therefore, a C t shift of greater than three cycles beyond the water blank was considered inhibitory. Inhibition was indicated when a sample had a C t shift of greater than three cycles OR a reduction in EPF of greater than or equal to 50% when compared to water controls. If inhibition was observed in a sample, two-fold serial dilutions were performed until inhibition was completely eliminated. C t shifts less than three cycles or less than 50% loss of EPF were not considered significant, and dilution of these samples would unnecessarily dilute out the target DNA. Assaying of animal tissues Real-Time PCR ( panHA -MGB, panE9L -MGB, F3L -MGB, and N3R -MGB) Each animal tissue-extracted DNA (appropriately diluted based on the IPC assay results) was tested with four real-time PCR assays. The first two assays, panHA -MGB and panE9L -MGB, were described previously 19 and detect the presence of orthopoxvirus DNAs. Each tissue DNA was then tested in singlet with the MPXV-specific assays, F3L -MGB and N3R -MGB. All resulting positive samples were then retested in triplicate. A 3/4 or 4/4 positive result on any given assay indicated that the sample was positive for either/both Orthopoxvirus DNA and/or MPXV DNA. If a sample was only 1/4 positive, the sample was called negative. If the sample was 2/4 positive, the sample was re-extracted and retested by both the F3L -MGB and N3R -MGB PCR assays. If any sample remained only '50% positive', the sample result would have been labeled 'indeterminate.' pan -Orthopox ECL A mixture of four monoclonal antibodies (Mab) was used as capture antibodies. An additional two Mabs were added as detector antibodies (total of six Mabs). All antibodies were originally developed against Vaccinia virus and were selected for this assay based on their ability to recognize four different Vaccinia proteins and their cross-reactivity with multiple members of the Orthopoxvirus genus including Vaccinia , monkeypox, cowpox, camel pox, Variola major , and Variola minor . These antibodies were produced in bioreactors, purified using protein G chromatography medium, and labeled with biotin or ruthenium (II) tris-bipyridal chelate (Ru) using standard coupling methods. 20 Biotinylated antibodies were prebound to streptavidin-coated 2.8 μ m diameter paramagnetic beads. Concentrations of labeled antibodies were then optimized as previously described. 20 Finally, the optimized capture antibody-bound beads and Ru-labeled detector antibodies were mixed and lyophilized into single-use assay tubes. The ECL Orthopoxvirus -genus detection assay was conducted as follows: 50 μ l of an unknown sample (liquid) or controls were added in duplicate to tubes containing the lyophilized capture and detector antibodies. The tubes were then incubated for 15 min on the vortexing Origen® Analyzer 1.5 carousel (Igen International Inc., Gaithersburg, MD, USA) after which 300 μ l of 10 mM phosphate-buffered saline with 0.3% Tween-20 (PBS-T) was added to each tube. The analyzer then drew the processed sample from the carousel to a flow cell where it captured and washed the magnetic beads and measured the ECL signal. Tissue preparations from uninfected mice and Dulbecco's high glucose medium containing 1% fetal calf serum served as matrix controls and were used to determine assay cutoffs. The data were analyzed using custom prepared Microsoft® Excel 2000 spreadsheets. Samples were considered positive if the ECL signal was greater than the average plus three times the standard deviation or 1.2 times the average ECL signal of the negative matrix controls, whichever was higher. Differences in ECL signals of the various machines was negated by determining the S / N ratio of each sample, calculated by dividing the sample ECL value by the average ECL value of the negative matrix controls. Positive controls near the sensitivity level of the assay were included on every run. The assay could detect as little as 5 × 10 5 plaque-forming units (PFU)/ml or 2.5 × 10 4 PFU per assay of Vaccinia virus and was quantitative within a dynamic range of at least 2.5 logs of virus. The size of the dynamic range and sensitivity level varied slightly depending on the Orthopoxvirus strain tested. The assay was not used in a quantitative manner for this work. The total assay time is 15 min plus up to 1 min per sample reading time. Real-Time PCR ( panHA -MGB, panE9L -MGB, F3L -MGB, and N3R -MGB) Each animal tissue-extracted DNA (appropriately diluted based on the IPC assay results) was tested with four real-time PCR assays. The first two assays, panHA -MGB and panE9L -MGB, were described previously 19 and detect the presence of orthopoxvirus DNAs. Each tissue DNA was then tested in singlet with the MPXV-specific assays, F3L -MGB and N3R -MGB. All resulting positive samples were then retested in triplicate. A 3/4 or 4/4 positive result on any given assay indicated that the sample was positive for either/both Orthopoxvirus DNA and/or MPXV DNA. If a sample was only 1/4 positive, the sample was called negative. If the sample was 2/4 positive, the sample was re-extracted and retested by both the F3L -MGB and N3R -MGB PCR assays. If any sample remained only '50% positive', the sample result would have been labeled 'indeterminate.' pan -Orthopox ECL A mixture of four monoclonal antibodies (Mab) was used as capture antibodies. An additional two Mabs were added as detector antibodies (total of six Mabs). All antibodies were originally developed against Vaccinia virus and were selected for this assay based on their ability to recognize four different Vaccinia proteins and their cross-reactivity with multiple members of the Orthopoxvirus genus including Vaccinia , monkeypox, cowpox, camel pox, Variola major , and Variola minor . These antibodies were produced in bioreactors, purified using protein G chromatography medium, and labeled with biotin or ruthenium (II) tris-bipyridal chelate (Ru) using standard coupling methods. 20 Biotinylated antibodies were prebound to streptavidin-coated 2.8 μ m diameter paramagnetic beads. Concentrations of labeled antibodies were then optimized as previously described. 20 Finally, the optimized capture antibody-bound beads and Ru-labeled detector antibodies were mixed and lyophilized into single-use assay tubes. The ECL Orthopoxvirus -genus detection assay was conducted as follows: 50 μ l of an unknown sample (liquid) or controls were added in duplicate to tubes containing the lyophilized capture and detector antibodies. The tubes were then incubated for 15 min on the vortexing Origen® Analyzer 1.5 carousel (Igen International Inc., Gaithersburg, MD, USA) after which 300 μ l of 10 mM phosphate-buffered saline with 0.3% Tween-20 (PBS-T) was added to each tube. The analyzer then drew the processed sample from the carousel to a flow cell where it captured and washed the magnetic beads and measured the ECL signal. Tissue preparations from uninfected mice and Dulbecco's high glucose medium containing 1% fetal calf serum served as matrix controls and were used to determine assay cutoffs. The data were analyzed using custom prepared Microsoft® Excel 2000 spreadsheets. Samples were considered positive if the ECL signal was greater than the average plus three times the standard deviation or 1.2 times the average ECL signal of the negative matrix controls, whichever was higher. Differences in ECL signals of the various machines was negated by determining the S / N ratio of each sample, calculated by dividing the sample ECL value by the average ECL value of the negative matrix controls. Positive controls near the sensitivity level of the assay were included on every run. The assay could detect as little as 5 × 10 5 plaque-forming units (PFU)/ml or 2.5 × 10 4 PFU per assay of Vaccinia virus and was quantitative within a dynamic range of at least 2.5 logs of virus. The size of the dynamic range and sensitivity level varied slightly depending on the Orthopoxvirus strain tested. The assay was not used in a quantitative manner for this work. The total assay time is 15 min plus up to 1 min per sample reading time. Results Development of Assays The final primer/TaqMan®-MGB probe assay sequences and reaction conditions for each MPXV-specific assay are shown in Table 1 . The pan -orthopox assays ( panHA -MGB and panE9L -MGB) were previously published. 19 The MPXV assays reproducibly detected 11–55 fg of monkeypox genomic DNA, which represented approximately 50–250 copies of each gene. Results from the genomic DNA LOD experiments also correlated linearly with a dynamic range of six orders of magnitude representing approximately 25 to 2 500 000 copies (data not shown). USAMRIID/CDC DNA Panel Evaluation Both MPXV-specific assays were tested against two DNA panels: (1) a USAMRIID/CDC orthopox DNA panel ( Table 2 ) and the USAMRIID DNA cross-reactivity panel (99 DNAs—data not shown). The results in Table 2 indicate that both the F3L -MGB and N3R -MGB assays were capable of detecting all of the MPXV genomic DNA species available in the orthopox DNA panel. Non-MPXV DNAs (to include human DNA, 25 strains of Variola virus DNA, five strains of Vaccinia , camelpox, cowpox, fowlpox viruses, two strains of Herpes simplex virus, and three strains of Varicella virus) were not detected by either assay. There were no false positives among the non-MPXV DNA samples for either of the assays on the LightCycler. Data from the USAMRIID bacterial cross-reactivity panel demonstrated that both assays were also negative (not detected) for 99 different isolates and strains of bacterial DNA. Animal Results A total of 52 animals were screened for the presence of orthopox DNA using the two previously tested orthopox spp-MGB assays: panHA -MGB and panE9L -MGB. 19 The sensitivities of the panHA -MGB and panE9L -MGB were retested with monkeypox DNA. The LODs for both assays was 2.5 fg (12 copies). The animals were also tested for the presence of monkeypox DNA using the F3L -MGB and N3R -MGB assays. Results ( Table 3 ) indicate that a total of 12 out of the 52 animals had at least one tissue PCR positive for MPXV DNA. Table 4 shows the results of testing 318 total tissue samples from the 52 animals. Note that some tissues (particularly from the prairie dogs) exhibited some level of PCR inhibition. The inhibition was overcome by diluting the sample 1:2, 1:4, or 1:8 with the dilution selected, which restored the C t values when compared to the noninhibited control. The total positives for one or more PCR assays were 25 tissues from 12 different animals ( Table 5 ). The ECL data are also presented in Table 5 . In all, 12.6% of all tissues exhibited some level of PCR inhibition. However, the vast majority (75%) of PCR-inhibited tissue samples were from prairie dogs. We attribute this fact to the possible way the prairie dogs were handled both before and during shipment to USAMRIID. Also noted during virus culture was the fact that the prairie dog tissues were highly contaminated with bacteria that may have contributed to the PCR inhibition. Table 5 shows the breakdown of each positive animal, which tissue was positive, and the results of the individual PCR and ECL tests. All other tissue samples (a total of 292 with the exception of the spleen of gerbil #32, which was PCR-negative but ECL-positive) were negative for all four PCR tests. An additional 17 tissues from randomly chosen PCR-negative animals were also negative for ECL (data not shown). Animals testing positive for all four PCR assays (in at least one tissue) were 7/12 (58%) of positive animals or 7/52 (15% of all animals tested). Animals testing positive for at least three PCR assays (in at least one tissue) were 10/12 (83%) of positive animals or 10/52 (19% of all animals tested). Animals testing positive for at least two PCR assays (in at least one tissue) were 10/12 (83%) of positive animals or 10/52 (19% of all animals tested). Animals testing positive for only one PCR assay (in at least one tissue) were 2/12 (17%) of positive animals or 2/52 (3.8% of all animals tested). Interestingly, there was only one case where a tissue (liver of hamster #21) was positive for panHA -MGB but not positive in either MPXV assay. This tissue was also ECL positive. We, therefore conclude that all virus detected by PCR was MPXV and that the liver tissue from hamster #21 contained MPXV virus DNA very near the detection limit of our four PCR assays. It is also possible that the tissue may have been infected by some other orthopox virus (ie, ectromelia). Table 4 Animal tissue real-time PCR assays Tissue Total # of tissues a # Tissues w/inhibition b rtPCR Total pan HA-MGB pan E9L-MGB MPXV F3L-MGB MPXV N3R-MGB Brain 52 0 4/52 3/52 3/52 4/52 4/52 Kidney 52 11 3/52 2/52 3/52 3/52 3/52 Liver 51 7 2/51 3/51 3/51 2/51 4/52 c Lung 51 4 2/51 2/51 4/51 2/51 4/51 Lymph node 10 2 2/10 2/10 2/10 2/10 2/10 Skin 3 0 2/3 1/3 2/3 2/3 2/3 Spleen 49 9 3/49 3/49 3/49 3/49 3/49 Testis/ovary 49 6 3/49 3/49 3/49 2/49 3/49 Scab d 1 1 0/1 0/1 0/1 0/1 0/1 Subtotal 318 40 21/318 19/318 23/318 20/318 25 a Not all tissues were taken from all animals. b In all, 12.6% of all tissue exhibited some level of inhibition; 75% (20/30) of tissues showing a level of inhibition were from prairie dogs. c One liver sample was only positive for panHA -MGB. d Scab was from prairie dog #6 (Department of Agriculture #5587). Table 5 Positive animal tissue assays Animal # Animal type Tissue type Inhibition assay Dilution pan HA-MGB pan E9L-MGB MPXV F3L-MGB MPXV N3R-MGB pan- OP ECL 5 Prairie dog Brain No 0 + + + + + 5 Prairie dog Kidney Yes 1:2 + + + + + 5 Prairie dog Liver Yes 1:2 − + + − + 5 Prairie dog Lung No 0 + + + + − 5 Prairie dog Lymph node No 0 + + + + − 5 Prairie dog Skin No 0 + + + + − 5 Prairie dog Spleen Yes 1:8 + + + + − 5 Prairie dog Testis/ovary No 0 + + + + − 7 Prairie dog Lung No 0 + + + + − 7 Prairie dog Lymph node No 0 + + + + − 2 Prairie dog Skin No 0 + − + + − 19 Hamster Brain No 0 + − − + + 19 Hamster Kidney No 0 + − + + + 21 Hamster Liver No 0 + − − − + 24 Gerbil Brain No 0 + + + + + 24 Gerbil Liver No 0 + + + + + 25 Gerbil Brain No 0 + + + + − 27 Gerbil Lung No 0 − − + − − 28 Gerbil Liver No 0 − + + + + 30 Gerbil Spleen No 0 + + + + + 49 Gerbil Kidney No 0 + + + + + 49 Gerbil Spleen No 0 + + + + + 49 Gerbil Testis/ovary No 0 + + + − − 45 Chinchilla Lung No 0 − − + − − 45 Chinchilla Testis/ovary Yes 1:2 + + + + − 32 Gerbil Spleen No 0 − − − − + All eight tested tissues from prairie dog #8 tested positive by PCR. Three of these tissues were also ECL positive. This animal was therefore chosen for culture attempts of the virus (specifically the skin and lung tissue) because they had the lowest PCR C t values indicating that they potentially contained the highest concentration of viable virus. DNA Sequencing The 139 and 107-bp amplicons produced from the MPXV-specific N3R and F3L assays, respectively, were sequenced and compared to related orthopoxviruses in GenBank. When aligned to other MPXV sequences, using BLASTN, both amplicons exhibited 100% identity to the Zaire-96-I-16 strain of MPXV (data not shown). Development of Assays The final primer/TaqMan®-MGB probe assay sequences and reaction conditions for each MPXV-specific assay are shown in Table 1 . The pan -orthopox assays ( panHA -MGB and panE9L -MGB) were previously published. 19 The MPXV assays reproducibly detected 11–55 fg of monkeypox genomic DNA, which represented approximately 50–250 copies of each gene. Results from the genomic DNA LOD experiments also correlated linearly with a dynamic range of six orders of magnitude representing approximately 25 to 2 500 000 copies (data not shown). USAMRIID/CDC DNA Panel Evaluation Both MPXV-specific assays were tested against two DNA panels: (1) a USAMRIID/CDC orthopox DNA panel ( Table 2 ) and the USAMRIID DNA cross-reactivity panel (99 DNAs—data not shown). The results in Table 2 indicate that both the F3L -MGB and N3R -MGB assays were capable of detecting all of the MPXV genomic DNA species available in the orthopox DNA panel. Non-MPXV DNAs (to include human DNA, 25 strains of Variola virus DNA, five strains of Vaccinia , camelpox, cowpox, fowlpox viruses, two strains of Herpes simplex virus, and three strains of Varicella virus) were not detected by either assay. There were no false positives among the non-MPXV DNA samples for either of the assays on the LightCycler. Data from the USAMRIID bacterial cross-reactivity panel demonstrated that both assays were also negative (not detected) for 99 different isolates and strains of bacterial DNA. Animal Results A total of 52 animals were screened for the presence of orthopox DNA using the two previously tested orthopox spp-MGB assays: panHA -MGB and panE9L -MGB. 19 The sensitivities of the panHA -MGB and panE9L -MGB were retested with monkeypox DNA. The LODs for both assays was 2.5 fg (12 copies). The animals were also tested for the presence of monkeypox DNA using the F3L -MGB and N3R -MGB assays. Results ( Table 3 ) indicate that a total of 12 out of the 52 animals had at least one tissue PCR positive for MPXV DNA. Table 4 shows the results of testing 318 total tissue samples from the 52 animals. Note that some tissues (particularly from the prairie dogs) exhibited some level of PCR inhibition. The inhibition was overcome by diluting the sample 1:2, 1:4, or 1:8 with the dilution selected, which restored the C t values when compared to the noninhibited control. The total positives for one or more PCR assays were 25 tissues from 12 different animals ( Table 5 ). The ECL data are also presented in Table 5 . In all, 12.6% of all tissues exhibited some level of PCR inhibition. However, the vast majority (75%) of PCR-inhibited tissue samples were from prairie dogs. We attribute this fact to the possible way the prairie dogs were handled both before and during shipment to USAMRIID. Also noted during virus culture was the fact that the prairie dog tissues were highly contaminated with bacteria that may have contributed to the PCR inhibition. Table 5 shows the breakdown of each positive animal, which tissue was positive, and the results of the individual PCR and ECL tests. All other tissue samples (a total of 292 with the exception of the spleen of gerbil #32, which was PCR-negative but ECL-positive) were negative for all four PCR tests. An additional 17 tissues from randomly chosen PCR-negative animals were also negative for ECL (data not shown). Animals testing positive for all four PCR assays (in at least one tissue) were 7/12 (58%) of positive animals or 7/52 (15% of all animals tested). Animals testing positive for at least three PCR assays (in at least one tissue) were 10/12 (83%) of positive animals or 10/52 (19% of all animals tested). Animals testing positive for at least two PCR assays (in at least one tissue) were 10/12 (83%) of positive animals or 10/52 (19% of all animals tested). Animals testing positive for only one PCR assay (in at least one tissue) were 2/12 (17%) of positive animals or 2/52 (3.8% of all animals tested). Interestingly, there was only one case where a tissue (liver of hamster #21) was positive for panHA -MGB but not positive in either MPXV assay. This tissue was also ECL positive. We, therefore conclude that all virus detected by PCR was MPXV and that the liver tissue from hamster #21 contained MPXV virus DNA very near the detection limit of our four PCR assays. It is also possible that the tissue may have been infected by some other orthopox virus (ie, ectromelia). Table 4 Animal tissue real-time PCR assays Tissue Total # of tissues a # Tissues w/inhibition b rtPCR Total pan HA-MGB pan E9L-MGB MPXV F3L-MGB MPXV N3R-MGB Brain 52 0 4/52 3/52 3/52 4/52 4/52 Kidney 52 11 3/52 2/52 3/52 3/52 3/52 Liver 51 7 2/51 3/51 3/51 2/51 4/52 c Lung 51 4 2/51 2/51 4/51 2/51 4/51 Lymph node 10 2 2/10 2/10 2/10 2/10 2/10 Skin 3 0 2/3 1/3 2/3 2/3 2/3 Spleen 49 9 3/49 3/49 3/49 3/49 3/49 Testis/ovary 49 6 3/49 3/49 3/49 2/49 3/49 Scab d 1 1 0/1 0/1 0/1 0/1 0/1 Subtotal 318 40 21/318 19/318 23/318 20/318 25 a Not all tissues were taken from all animals. b In all, 12.6% of all tissue exhibited some level of inhibition; 75% (20/30) of tissues showing a level of inhibition were from prairie dogs. c One liver sample was only positive for panHA -MGB. d Scab was from prairie dog #6 (Department of Agriculture #5587). Table 5 Positive animal tissue assays Animal # Animal type Tissue type Inhibition assay Dilution pan HA-MGB pan E9L-MGB MPXV F3L-MGB MPXV N3R-MGB pan- OP ECL 5 Prairie dog Brain No 0 + + + + + 5 Prairie dog Kidney Yes 1:2 + + + + + 5 Prairie dog Liver Yes 1:2 − + + − + 5 Prairie dog Lung No 0 + + + + − 5 Prairie dog Lymph node No 0 + + + + − 5 Prairie dog Skin No 0 + + + + − 5 Prairie dog Spleen Yes 1:8 + + + + − 5 Prairie dog Testis/ovary No 0 + + + + − 7 Prairie dog Lung No 0 + + + + − 7 Prairie dog Lymph node No 0 + + + + − 2 Prairie dog Skin No 0 + − + + − 19 Hamster Brain No 0 + − − + + 19 Hamster Kidney No 0 + − + + + 21 Hamster Liver No 0 + − − − + 24 Gerbil Brain No 0 + + + + + 24 Gerbil Liver No 0 + + + + + 25 Gerbil Brain No 0 + + + + − 27 Gerbil Lung No 0 − − + − − 28 Gerbil Liver No 0 − + + + + 30 Gerbil Spleen No 0 + + + + + 49 Gerbil Kidney No 0 + + + + + 49 Gerbil Spleen No 0 + + + + + 49 Gerbil Testis/ovary No 0 + + + − − 45 Chinchilla Lung No 0 − − + − − 45 Chinchilla Testis/ovary Yes 1:2 + + + + − 32 Gerbil Spleen No 0 − − − − + All eight tested tissues from prairie dog #8 tested positive by PCR. Three of these tissues were also ECL positive. This animal was therefore chosen for culture attempts of the virus (specifically the skin and lung tissue) because they had the lowest PCR C t values indicating that they potentially contained the highest concentration of viable virus. DNA Sequencing The 139 and 107-bp amplicons produced from the MPXV-specific N3R and F3L assays, respectively, were sequenced and compared to related orthopoxviruses in GenBank. When aligned to other MPXV sequences, using BLASTN, both amplicons exhibited 100% identity to the Zaire-96-I-16 strain of MPXV (data not shown). Discussion In the United States, smallpox vaccinations were discontinued after the eradication of the disease in 1979. However, recent world events have prompted the US military to begin vaccinating troops against smallpox. The anthrax attacks during the fall of 2001 demonstrate that there is a high potential for additional bioterrorism attacks. It is conceivable that an individual or possibly a rogue government could use their technical expertise to mass-produce an infectious Orthopoxvirus such as smallpox or MPXVs (or a genetically engineered variant of either). It is, therefore, crucial that fast, reliable yet simple molecular diagnostic tests be developed with the latest detection and identification technology available. We present the development and extended evaluation of two real-time PCR assays to confirm the presence of monkeypox DNA (MPXV-specific assays) on the LightCycler. Each assay is an entirely new assay ( F3L -MGB or N3R -MGB) incorporating MPXV-specific target sequences. The primers were specifically designed to generate amplicons less than 150 bp because these assays were to be used in a rapid cycling machine (one cycle/15–20 s). We chose primer/TaqMan®-MGB probe pairs that exhibited the maximum efficiency of amplicon synthesis, the lowest C t value, and the maximum LOD. We opted to use TaqMan®-MGB probe technology because it possesses significantly improved hybridization properties. 21 TaqMan®-MGB probes are more stable, display increased mismatch discrimination, and have an improved S / N ratio due to the use of an NFQ instead of the fluorescent quencher dye TAMRA. 22 In addition, the MGB stabilizes A/T rich duplexes, resulting in increased probe T m (that temperature at which 50% of an oligonucleotide is annealed to its complement strand). Therefore, the MGB probes simplified assay design for the MPXVs, which have a high A/T ratio (∼66%). 23 In this study, the LOD for each TaqMan®-MGB assay was evaluated on the LightCycler using purified monkeypox (Zaire 79-I-05) virus genomic DNA. The LOD with genomic DNA was 11–55 fg (50–250 copies). Both assays were also highly quantitative over a range of 1 ng (5 × 10 6 copies) to 11 fg (50 copies) when tested in the LightCycler. Initial testing against genomic DNAs available at USAMRIID and from the CDC increased our confidence in the overall specificity of each assay. While each assay detected only the appropriate DNA samples in the USAMRIID/CDC orthopox DNA panel, none of the assays detected any DNAs in the cross-reactivity panel. These data showed that both MPXV-specific TaqMan®-MGB assays were highly specific and exceptionally sensitive. The MPXV-infected animal results presented in this study confirm that both previously reported pan -orthopox assays 19 along with the two new MPXV-specific assays ( F3L -MGB and N3R -MGB) achieve the same level of sensitivity and specificity with clinical (animal) samples as they do with pure cultures. Of the 52 animals ultimately tested, 12 were shown to have at least one tissue positive for MPXV DNA with one animal positive for all eight of its tissues tested. ECL testing of this same animal's tissues showed that only three of the PCR-positive tissues were ECL-positive. This result is not surprising for two reasons: ECL for orthopox viruses has been shown to be less sensitive than PCR (ECL limit of detection for the orthopox viruses is approximately 5 × 10 5 PFU/ml or 2.5 × 10 4 PFU/assay); secondly, we were not able to detect monkeypox virions by electron microscopy and no viable virus was isolated from the PCR-positive skin and lung tissue of prairie dog #5. These observations suggest that viral loads were either present at concentrations below the LOD of these techniques or samples were degraded such that antigen was not available in a form that could be recognized by the antibodies used in the ECL assay. Spleen from one animal was ECL-positive but PCR-negative. This result is at present not understood but could represent a false-positive ECL result or a false-negative PCR result. Alternatively, it is possible that viral antigen could have been present in the absence of agent-specific nucleic acid. Sequencing of the PCR products obtained from the MPXV-infected tissues revealed a 100% identity with the Zaire 96-I-16 strain of MPXV. This strain was originally isolated from an infected human during the 1996 outbreak in the Democratic Republic of Congo (formerly Zaire). 24 , 25 It is known that two clades of MPXV exist in Africa, those isolated from Zaire and those from West Africa (H Meyer, personal communication). As the source of this outbreak was from rodents collected from Ghana in West Africa, we presumed that they carried viruses of the West African clade. It is possible, although highly unlikely, that our samples and/or PCRs were contaminated with MPXV strain Zaire 96-I-16. We do not believe this to be the case for several reasons. First, testing of additional samples from this outbreak, which were processed independently by the Poxvirus Section at the CDC, yielded PCR products that also exhibited 100% identity to the Zaire 96-I-16 strain. Second, although our laboratory possesses the Zaire 96-I-16 strain of MPXV, it had not been worked with or grown in the laboratory in over 2 years, and it was not used as positive control DNA for any of the PCR assays used in this study. Another, more likely possibility, is that either the size of the amplicons sequenced, or the specific gene targets themselves (ie, F3L and N3R ) do not allow for differentiation of the two clades. In support of this idea, Reed et al 10 recently showed that the sequence of a PCR amplicon, using the HA gene as a target, from a patient and a prairie dog from the 2003 outbreak grouped within the West African clade. Complete genome sequencing of isolated virus will be needed to resolve these issues. In addition to using these assays for diagnostics during an outbreak, they have potential uses in monitoring viral-load in real-time in animal models of Orthopoxvirus disease. For example, Jahrling and colleagues recently developed a monkey model for studying the progression of smallpox infections. 26 In these studies, our quantitative pan- orthopox HA -MGB assay was used to monitor viral load in monkey blood and tissues after infection with smallpox virus. Smallpox infections in monkeys are an important surrogate model for human smallpox infections. 27 Likewise, the two MPXV-specific assays reported here could be used to monitor, in real time, the progression of monkeypox in nonhuman primates as has been done in smallpox animal models (unpublished data). In conclusion, this study demonstrates the reliable and specific identification of MPXV virus DNA from clinical (animal) samples by TaqMan®-MGB real-time PCR on the LightCycler. By using these assays as part of a battery of PCR and ECL tests, one can first establish the presence of orthopox viral DNA and/proteins in a sample. Then MPVX can be quickly and accurately distinguished from smallpox virus by the use of the MPXV-specific PCR confirmatory assays in conjunction with three previously published smallpox-specific assays. 19 Accession codes Accessions GenBank/EMBL/DDBJ • AF095689 • AF380138 • AF438165 • AF482758 • L22579 • M35027 • NC_001611 • X94355 • Y16780 Accessions GenBank/EMBL/DDBJ • AF095689 • AF380138 • AF438165 • AF482758 • L22579 • M35027 • NC_001611 • X94355 • Y16780

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393728/

Transcriptome Changes Affecting Hedgehog and Cytokine Signalling in the Umbilical Cord: Implications for Disease Risk

Background Babies born at lower gestational ages or smaller birthweights have a greater risk of poorer health in later life. Both the causes of these sub-optimal birth outcomes and the mechanism by which the effects are transmitted over decades are the subject of extensive study. We investigated whether a transcriptomic signature of either birthweight or gestational age could be detected in umbilical cord RNA. Methods The gene expression patterns of 32 umbilical cords from Singaporean babies of Chinese ethnicity across a range of birthweights (1698–4151 g) and gestational ages (35–41 weeks) were determined. We confirmed the differential expression pattern by gestational age for 12 genes in a series of 127 umbilical cords of Chinese, Malay and Indian ethnicity. Results We found that the transcriptome is substantially influenced by gestational age; but less so by birthweight. We show that some of the expression changes dependent on gestational age are enriched in signal transduction pathways, such as Hedgehog and in genes with roles in cytokine signalling and angiogenesis. We show that some of the gene expression changes we report are reflected in the epigenome. Conclusions We studied the umbilical cord which is peripheral to disease susceptible tissues. The results suggest that soma-wide transcriptome changes, preserved at the epigenetic level, may be a mechanism whereby birth outcomes are linked to the risk of adult metabolic and arthritic disease and suggest that greater attention be given to the association between premature birth and later disease risk. Background Babies born at lower gestational ages or smaller birthweights have a greater risk of poorer health in later life. Both the causes of these sub-optimal birth outcomes and the mechanism by which the effects are transmitted over decades are the subject of extensive study. We investigated whether a transcriptomic signature of either birthweight or gestational age could be detected in umbilical cord RNA. Methods The gene expression patterns of 32 umbilical cords from Singaporean babies of Chinese ethnicity across a range of birthweights (1698–4151 g) and gestational ages (35–41 weeks) were determined. We confirmed the differential expression pattern by gestational age for 12 genes in a series of 127 umbilical cords of Chinese, Malay and Indian ethnicity. Results We found that the transcriptome is substantially influenced by gestational age; but less so by birthweight. We show that some of the expression changes dependent on gestational age are enriched in signal transduction pathways, such as Hedgehog and in genes with roles in cytokine signalling and angiogenesis. We show that some of the gene expression changes we report are reflected in the epigenome. Conclusions We studied the umbilical cord which is peripheral to disease susceptible tissues. The results suggest that soma-wide transcriptome changes, preserved at the epigenetic level, may be a mechanism whereby birth outcomes are linked to the risk of adult metabolic and arthritic disease and suggest that greater attention be given to the association between premature birth and later disease risk. Introduction Birth outcomes defined by gestational age and birth weight have far reaching consequences across the life-course. For instance young adults born prematurely or at very low birthweights have significantly lower bone density than do their larger and term-born peers [1] . The prenatal environment has been linked to the risk of disease in later life [2] , [3] , [4] , [5] , [6] , [7] , [8] . Children born either prematurely or small for gestational age have reduced insulin sensitivity and are at higher risk for type 2 diabetes mellitus [7] , [8] . Little is known of the molecular mechanisms by which these longer-term consequences for the offspring are transmitted, although epigenetic mechanisms appear to be implicated [9] . Umbilical cord tissue is a readily available tissue and offers a source of RNA and DNA for an assessment of the genomic and epigenomic state of the neonate. Molecular biomarkers that reveal early life experience and predict later disease risk would be valuable as a means to identify high-risk patients, and could be a starting point to designing therapeutic interventions [9] , [10] , [11] . While several groups have found transcriptomic [12] , [13] and epigenetic marks [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] in the umbilical cord associated with extreme intrauterine experience or birth outcomes, of particular interest to us and relevant to global health concerns are the molecular mechanisms of inter-individual variability operating within the normal range [22] , as long-term effects of the prenatal state can be readily demonstrated within children of normal birthweight and gestation [9] , [23] , [24] . GUSTO (Growing Up in Singapore Towards healthy Outcomes) is a Singapore-based birth cohort study [25] . From this deeply phenotyped cohort we profiled the transcriptome of 32 umbilical cords collected at birth from ethnic Chinese babies in Singapore hospitals. Transcriptomic change significantly related to gestational age were discovered and then validated by q-PCR analysis across an expanded set of 127 multiethnic samples. Pathway analysis revealed enrichment of differential transcription by gestational age in hedgehog signalling genes: GLI2, GLI3, and SMO, and others downstream of the pathway with a role in skeletal development, defects of which are associated with early-onset osteoarthritis. In addition, inflammatory mediators such as CXCL14 and Il1RL1 were differentially expressed, as well as HSD11B1, a gene with known implications in development [26] . Furthermore, a subset of the differentially expressed genes was found to also differ in DNA methylation levels in a manner associated with gestational age. We suggest that lower birth weight and particularly earlier (within the near term range) gestational ages, leaves an epigenetic echo that affects the risk for diseases such as type 2 diabetes mellitus and osteoarthritis in later life. Results To ascertain the independent roles of both gestational age and birth weight, which are positively correlated, we selected normal birth weight samples to match the gestational ages of the extreme birth weight samples [ Figure 1 and Table 1 ], and subjected them to expression microarray analysis. After probe quality control and inter-sample quantile normalisation of the 32 RNA samples interrogated on the array, the data for two samples failed quality control (MAD scores 37 w_NBW groups and are highlighted in Figure 1 . Three series of technical replicates were included in the experiment, they clustered together in 10 cases out of 12; when the full dataset was subjected to unsupervised hierarchical clustering demonstrating that the intra-sample variation is lower than the inter-sample variation [ Figure S1 ]. Therefore the data were declared of acceptable quality and the replicates were combined. 10.1371/journal.pone.0039744.g001 Figure 1 RNA Expression Microarray Study Design. Gestational age in weeks (y-axis) and birth-weight in grams (x-axis) of the samples analysed by expression microarrays are symmetrical to allow somewhat independent comparisons for birth-weight and gestational age. Samples are classified into high birth weight group (>3700 g) in orange; low birthweight group (37 weeks 18 (56%) 39 (1) 111 (93%) 39 (1) 10 (50%) 40 (1) BW (g) LBW (3700) 8 (25%) 4076 (62) 29 (24%) 4073 (319) 5 (25%) 4086 (51) Ethnic Group Chinese 32 (100%) 58 (48%) 20 (100%) Malay 37 (31%) Indian 25 (21%) Parity 0.6 (1) 0.9 (1) 0.6 (1) Maternal age 34 (4) 34 (4) 31 (5) 35 (3) To examine the pattern of relatedness between individual transcriptomes and to identify potential underlying associations with birth outcomes, principal component analysis was performed on the dataset. We found that within the normal birth weight samples, PC1 separated the samples by gestational age ( p 3700 g) in orange; low birthweight group (37 w_NBW), 64 probes passed an FDR correction for multiplicity of q3700 g) in orange; low birthweight group (37 w_NBW group. No probes passed an FDR correction for multiplicity (adjusted p1.5 in both ANOVA tests [ Table S4 and Figure S3 ]. We selected some of the strongest expression changes along with some of the more interesting genes from a disease-related perspective and assayed their expression levels in 127 additional samples. Twenty-two genes in all were tested with a range of significance in the array data ( Table S5 ). Generally there was a concordance between the magnitude of the fold change in the ANOVA and repetition in the expansion set (average absolute fold change replicating group (shown in figure 4 and table S5)  = 2.52, average absolute fold change non-replicating group (shown in table S5)  = 1.47, t -test p = 0.026). The replicating group comprises genes whose mRNA levels had a significant relationship with gestational age in the qPCR expanded study. The non-replicating group are those genes whose mRNA levels did not achieve significance against gestational age in the expanded qPCR study. Of this set, 12 genes showed p37 w) groups, they are illustrated in Figure 4 . 10.1371/journal.pone.0039744.g004 Figure 4 Twelve transcripts have differential expression levels in gestational age groups across the 120 sample replication set. Fold change with regard to the median sample of the more than 37 weeks gestation group, is shown on the y-axis. Gene names are shown above each panel. P-values from the 2 group tests are shown within each panel. Data is represented as a box plot where the 2–3 quartile range is within the box, the median is denoted by a horizontal line within the box, the min and max are denoted by horizontal lines outside of the box and single outliers are represented by crosses. Pathway enrichment analysis was performed on the 45 genes mapping to the 64 probes which had FDR 37 weeks gestation neonates is in line with published observations [26] . It is tempting to speculate whether enzymatic activities of the HSD11 isoforms are changed in a gestational age dependent manner, as this has been demonstrated in children born small for gestational age with lack of catch-up growth who were found to have lower activity of HSD11B1 [77] . We suggest that differential expression observed in the umbilical cord transcriptome corresponding to gestational age is partly driven by specific DNA methylation changes. Schroeder et al (2011) [28] found that gestational age within the normal range significantly drives DNA methylation. Interestingly one of the CpGs which reached experiment-wide significance in their study was located in GLI3 , a gene with transcript levels significantly varying with gestational age in our study. We have also measured the methylation state of the umbilical cords in our study and report concordant relationships with DNA methylation for 8 of the 16 genes replicated in our qPCR study as having significant concordance with gestational age at the expression level, including hedgehog transcription factor GLI3 . DNA methylation is an epigenetic mechanism that could explain the persistence of birth outcomes on disease susceptibility in later life [78] . Relton et al (2012) [79] recently reported DNA methylation variance in nine genes at birth, persisting to gene expression differences at 11–13 years of age and associating with body composition at nine years of age. Specific methylation differences correlating to gestational age could drive both the transcriptome of the umbilical cord at birth and have consequences in specific tissues at later ages. We chose here to assay the genomics of umbilical cord tissue as the only available somatic tissue. It is unknown if the genomic state of the umbilical cord correlates with that of the other tissues of the fetus, especially in those that give rise to diseases that have developmental origins. However, the available data suggests that DNA methylation patterns are generally conserved across tissues and there are examples of individually-variable epigenetic marks being soma-wide [80] . Godfrey et al (2011) [9] have shown that methylation of a site in the RXRα gene promoter in the umbilical cord correlates with body composition in later life, suggesting widespread influences across multiple tissues. It is a limitation of our study that results on whole umbilical cord tissue cannot be attributed to specific cell lineages. However, by using this heterogeneous tissue we anticipate that we have enriched for soma-wide changes. In this study, we have made use of frozen umbilical cord tissue derived from an Asian population. By designing the study to independently examine both birth weight and gestational age, we were able to find expression changes significant for gestational age. Furthermore, by combining gene expression microarray data with corresponding data from the latest Infinium 450 K human methylation bead array chip (Illumina), our results provide insights into the epigenetic profile of babies with differing birth outcomes. This may allow the development of novel predictive epigenetic markers for non-communicable diseases prevalent in Asia. Materials and Methods Clinical Populations and Sample Collection All specimens were from babies born at the KK Women's and Children's Hospital (KKH) and the National University Hospital (NUH), in Singapore. These hospitals are part of the GUSTO birth cohort study [25] . Written parental consent to participate in the study was given and hard copies are stored by the GUSTO data team. Ethical approval for the study and the consent forms and contents was granted, by the ethics boards of both KKH and NUH, which are centralised Institute Review Board (CIRB) and Domain Specific Review Board (DSRB), respectively. Gestational age was defined from a dating ultrasound (10–12 weeks) followed by an additional scan at 18–22 weeks. The discovery microarray analysis sample set consisted of a total of 32 umbilical cords from babies of Chinese ethnicity. Maternal ages were restricted to between 20–40 years. It was designed to include eight low birth weight samples (defined as 3700 g) samples and sixteen normal birth weight babies with gestational ages matching the extreme birthweight samples. The average birth weight in the GUSTO cohort was 3081 g, which is comparable to the average across a larger Singaporean sample of 3183 g. for a term infant (unpublished data). All children had a gestational age in the range of 35–37 (shorter gestational age) or 38–41 weeks (longer gestational age), to allow comparison across the range of normal gestational age. This resulted in four groups, each comparable for birthweight and/or gestational age and matched for gender and maternal age. The expanded replication sample set of 127 umbilical cords for qPCR analysis did not include any of those from the discovery set, and were from babies of Chinese, Indian and Malay ethnicities. The 20 samples analysed for DNA methylation were a subset of the discovery microarray analysis set of 32. [See Table 1 for sample characteristics]. Umbilical cord tissue samples were collected at the time of delivery, flushed with saline to remove fetal blood and flash-frozen in liquid nitrogen within 30 min of collection. RNA Extraction Umbilical cord tissue (300 mg) was first placed in a sterile Dispomix tube and homogenized for 55 s for 3 cycles in 3 ml of Trizol using the Dispomix (Medic Tools, AG, Zug, Switzerland). After spinning down the debris, the supernatants were divided equally into three 2 ml tubes. 200 ul of chloroform were added to each tube, vortexed vigorously and centrifuged for 15 min at 4°C. The aqueous phase was carefully transferred to a new tube containing 1 ul of linear acrylamide. An equal amount of isopropanol was added and mixed by inversion. After incubating at −20°C overnight to precipitate the RNA, the pellet was obtained by centrifuging at 13,200 rpm for 10 min at 4°C. The RNA pellet was washed twice in 70% (v/v) ethanol, air-dried and resuspended in RNase-free water. The isolated RNA was then purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany). On-column DNase digestion was carried out before the first wash step according to the manufacturer's instructions. The purified RNA was then eluted in 30 µl of RNase-free water and stored at −80°C. RNA concentration and purity were measured using a nanodrop ND-8000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), and RNA integrity was determined using the Agilent 2100 Bioanalyzer and RNA 6000 Nano Labchips (Agilent Technologies, Santa Clara, CA, USA). Expression Microarray Gene expression analysis was carried out with 3 sets of duplicate technical replicates from the 32 study subjects. All subsequent experimental steps followed the manufactures instructions. Briefly, Cy3-labelled cRNA was generated from 100 ng of total RNA using the Quick Amp Labelling Kit (One-Color) (Agilent Technologies, Santa Clara, CA, USA). Hybridization performance was assessed by means of 10 proprietary spike-in controls incorporated into the cRNA synthesis procedure. The labelled cRNA was then purified and hybridized onto Agilent SurePrint G3 Human Gene Expression (8×60 K) microarrays in a rotating (10 rpm) hybridization oven for 17 h at 65°C, after which they were washed and processed with proprietary buffers and solutions. The microarrays were then scanned at a resolution of 3 µm on an Agilent scanner using an extended dynamic range (PMT 10/100). The image data were processed using default values in feature extraction version 10.7.1.1 (Agilent Technologies, Santa Clara, CA, USA). Agilent ".txt" files were outputted from the scanner and loaded into Arraystudio (Omicsoft). Signal extraction was performed from the gProcessedSignal value incorporating background subtraction. All expression values were log transformed. All probes with expression levels less than two standard deviations above background were removed. Values across replicate probes were averaged. Data were normalised amongst samples using quantile normalisation. Two samples with MAD scores 37 weeks gestation group was then selected as the reference and then the of all samples were normalized by this reference using . Finally, the fold changes of all samples were calculated by . All samples were grouped as either 37 weeks gestation. The differences between the two groups were examined by one way ANOVA. Pathway and Network Analysis Pathway enrichment and de novo network discovery were performed in GeneGO metacore [82] . Pathway enrichment was calculated using a hypergeometric distribution against both Gene Ontologies and GeneGo pathway maps. Results were corrected for FDR using Benjamini-Hochberg. Network discovery was performed using the "shortest path" and "direct interactions" module. Low confidence, indirect and "influence by expression" interactions were pre-filtered and canonical pathway interactions were retained. Illumina® Infinium® HD Genome-wide Methylation Assay Genomic DNA methylation analysis was carried out with 3 sets of duplicate technical replicates from 20 study subjects. All subsequent experimental steps followed the manufacturers' instructions. After extraction of genomic DNA from frozen umbilical cord specimens according to standard procedures, 1 mg was bisulfite converted using EZ-96 DNA Methylation™ Gold Kit (Zymo Research, Irvine, CA, USA). Successful conversion was confirmed via methylation-specific PCR prior to proceeding with subsequent steps of the Infinium assay protocol. The bisulfite converted genomic DNA was isothermally amplified at 37°C for 22 hrs, enzymatically fragmented, purified and hybridized on an Infinium® HumanMethlyation 450 BeadChip (Illumina Inc., San Diego, CA, USA) at 48°C for 18 hrs. After which, the BeadChip was then washed to remove any un-hybridized or non-specific hybridized DNA. Labelled single-base extension was performed on primers hybridized with DNA, and the hybridized DNA was removed. The extended primers were stained with multiple layers of fluorescence, the BeadChip was then coated using a proprietary solution and scanned using the Illumina® iScan system. The image data were processed with the Genome Studio™ Methylation Module software. The intensity files (.idat) produced by the Illumina iSCAN system were loaded into GenomeStudio's methylation module for signal extraction. Background subtraction was performed by averaging the signals from the internal negative control beads. CpGs with less than three beads for either probe for any sample (18,603), or with signal detection p- values (calculated from the individual bead intensities) less than 0.05 (2,949) for any sample, were discarded for all samples. This step removed 4.4% of the 485,577 CpGs assayed. Data were normalized to the internal controls, which were designed to be housekeeping genes with no CpGs in the probe (samples the variation inherent in the array). β-values were then calculated, which are the ratio of the methylated probe intensity and the overall intensity. The β-value for an i th interrogated CpG site was calculated by: Where and are the intensities measured by the i th methylated and unmethylated probes respectively, averaged over the replicate beads, and " " is a constant offset(by default 100). Therefore β -values range between 0–1, with 0 representing no methylation and 1 representing 100% methylation. Tables of CpG β-values across samples were exported from GenomeStudio and loaded into Arraystudio for downstream analysis. As a further QC step MAD scores were calculated for the sample sets. MAD is a robust measure of statistical dispersion and is defined as the median of the absolute deviations from the data's median: Samples with a MAD score of less than −5 were discarded. Principal component analysis and hierarchical clustering were performed to observe the clustering of technical replicates and discernible batch effects. No batch effects were observed. The intra-sample deviation was lower than the inter-sample deviation. Regression analysis was performed against gestational age to identify CpGs whose methylation levels co-varied. CpGs with a regression p3700 g) samples and sixteen normal birth weight babies with gestational ages matching the extreme birthweight samples. The average birth weight in the GUSTO cohort was 3081 g, which is comparable to the average across a larger Singaporean sample of 3183 g. for a term infant (unpublished data). All children had a gestational age in the range of 35–37 (shorter gestational age) or 38–41 weeks (longer gestational age), to allow comparison across the range of normal gestational age. This resulted in four groups, each comparable for birthweight and/or gestational age and matched for gender and maternal age. The expanded replication sample set of 127 umbilical cords for qPCR analysis did not include any of those from the discovery set, and were from babies of Chinese, Indian and Malay ethnicities. The 20 samples analysed for DNA methylation were a subset of the discovery microarray analysis set of 32. [See Table 1 for sample characteristics]. Umbilical cord tissue samples were collected at the time of delivery, flushed with saline to remove fetal blood and flash-frozen in liquid nitrogen within 30 min of collection. RNA Extraction Umbilical cord tissue (300 mg) was first placed in a sterile Dispomix tube and homogenized for 55 s for 3 cycles in 3 ml of Trizol using the Dispomix (Medic Tools, AG, Zug, Switzerland). After spinning down the debris, the supernatants were divided equally into three 2 ml tubes. 200 ul of chloroform were added to each tube, vortexed vigorously and centrifuged for 15 min at 4°C. The aqueous phase was carefully transferred to a new tube containing 1 ul of linear acrylamide. An equal amount of isopropanol was added and mixed by inversion. After incubating at −20°C overnight to precipitate the RNA, the pellet was obtained by centrifuging at 13,200 rpm for 10 min at 4°C. The RNA pellet was washed twice in 70% (v/v) ethanol, air-dried and resuspended in RNase-free water. The isolated RNA was then purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany). On-column DNase digestion was carried out before the first wash step according to the manufacturer's instructions. The purified RNA was then eluted in 30 µl of RNase-free water and stored at −80°C. RNA concentration and purity were measured using a nanodrop ND-8000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), and RNA integrity was determined using the Agilent 2100 Bioanalyzer and RNA 6000 Nano Labchips (Agilent Technologies, Santa Clara, CA, USA). Expression Microarray Gene expression analysis was carried out with 3 sets of duplicate technical replicates from the 32 study subjects. All subsequent experimental steps followed the manufactures instructions. Briefly, Cy3-labelled cRNA was generated from 100 ng of total RNA using the Quick Amp Labelling Kit (One-Color) (Agilent Technologies, Santa Clara, CA, USA). Hybridization performance was assessed by means of 10 proprietary spike-in controls incorporated into the cRNA synthesis procedure. The labelled cRNA was then purified and hybridized onto Agilent SurePrint G3 Human Gene Expression (8×60 K) microarrays in a rotating (10 rpm) hybridization oven for 17 h at 65°C, after which they were washed and processed with proprietary buffers and solutions. The microarrays were then scanned at a resolution of 3 µm on an Agilent scanner using an extended dynamic range (PMT 10/100). The image data were processed using default values in feature extraction version 10.7.1.1 (Agilent Technologies, Santa Clara, CA, USA). Agilent ".txt" files were outputted from the scanner and loaded into Arraystudio (Omicsoft). Signal extraction was performed from the gProcessedSignal value incorporating background subtraction. All expression values were log transformed. All probes with expression levels less than two standard deviations above background were removed. Values across replicate probes were averaged. Data were normalised amongst samples using quantile normalisation. Two samples with MAD scores 37 weeks gestation group was then selected as the reference and then the of all samples were normalized by this reference using . Finally, the fold changes of all samples were calculated by . All samples were grouped as either 37 weeks gestation. The differences between the two groups were examined by one way ANOVA. Pathway and Network Analysis Pathway enrichment and de novo network discovery were performed in GeneGO metacore [82] . Pathway enrichment was calculated using a hypergeometric distribution against both Gene Ontologies and GeneGo pathway maps. Results were corrected for FDR using Benjamini-Hochberg. Network discovery was performed using the "shortest path" and "direct interactions" module. Low confidence, indirect and "influence by expression" interactions were pre-filtered and canonical pathway interactions were retained. Illumina® Infinium® HD Genome-wide Methylation Assay Genomic DNA methylation analysis was carried out with 3 sets of duplicate technical replicates from 20 study subjects. All subsequent experimental steps followed the manufacturers' instructions. After extraction of genomic DNA from frozen umbilical cord specimens according to standard procedures, 1 mg was bisulfite converted using EZ-96 DNA Methylation™ Gold Kit (Zymo Research, Irvine, CA, USA). Successful conversion was confirmed via methylation-specific PCR prior to proceeding with subsequent steps of the Infinium assay protocol. The bisulfite converted genomic DNA was isothermally amplified at 37°C for 22 hrs, enzymatically fragmented, purified and hybridized on an Infinium® HumanMethlyation 450 BeadChip (Illumina Inc., San Diego, CA, USA) at 48°C for 18 hrs. After which, the BeadChip was then washed to remove any un-hybridized or non-specific hybridized DNA. Labelled single-base extension was performed on primers hybridized with DNA, and the hybridized DNA was removed. The extended primers were stained with multiple layers of fluorescence, the BeadChip was then coated using a proprietary solution and scanned using the Illumina® iScan system. The image data were processed with the Genome Studio™ Methylation Module software. The intensity files (.idat) produced by the Illumina iSCAN system were loaded into GenomeStudio's methylation module for signal extraction. Background subtraction was performed by averaging the signals from the internal negative control beads. CpGs with less than three beads for either probe for any sample (18,603), or with signal detection p- values (calculated from the individual bead intensities) less than 0.05 (2,949) for any sample, were discarded for all samples. This step removed 4.4% of the 485,577 CpGs assayed. Data were normalized to the internal controls, which were designed to be housekeeping genes with no CpGs in the probe (samples the variation inherent in the array). β-values were then calculated, which are the ratio of the methylated probe intensity and the overall intensity. The β-value for an i th interrogated CpG site was calculated by: Where and are the intensities measured by the i th methylated and unmethylated probes respectively, averaged over the replicate beads, and " " is a constant offset(by default 100). Therefore β -values range between 0–1, with 0 representing no methylation and 1 representing 100% methylation. Tables of CpG β-values across samples were exported from GenomeStudio and loaded into Arraystudio for downstream analysis. As a further QC step MAD scores were calculated for the sample sets. MAD is a robust measure of statistical dispersion and is defined as the median of the absolute deviations from the data's median: Samples with a MAD score of less than −5 were discarded. Principal component analysis and hierarchical clustering were performed to observe the clustering of technical replicates and discernible batch effects. No batch effects were observed. The intra-sample deviation was lower than the inter-sample deviation. Regression analysis was performed against gestational age to identify CpGs whose methylation levels co-varied. CpGs with a regression p37w_NBW = red) and gestational age (green  =  low, white = intermediate, red  = high). (TIF) Click here for additional data file. Figure S2 Tests for differential expression by gestational age return transcript levels co-varying with gestational age and significantly different between gestational age groups. A and B, Examples of significantly co-varying transcript level by gestational age: probe A_23_P209246, mapping to the GLI2 (A) and probe A_23_P70818, mapping to the SMO (B) log2 expression levels across samples are represented on the y-axis and gestational ages of those samples are represented on the x-axis. Samples with gestational age ≤37 weeks are denoted in blue, >37 weeks in red. C and D , Results from 1-way ANOVA tests for transcripts whose expression levels are significantly different between samples with gestational age ≤37 weeks and samples with gestational age >37 weeks. Average differences in expression levels between the two gestational age groups are represented on the x-axis, −log10 pvalues from the ANOVA tests are represented on the y-axis. Probes above the horizontal red line have nominal pvalues 37w_NBW. D, includes all samples i.e. LBW and ≤37w_NBW vs. >37w_NBW and HBW. (TIF) Click here for additional data file. Figure S3 Example of significantly co-varying transcript level by birthweight: probe A_33_P331451 (mapping to the TGFBR1 ) log2 expression levels across samples are represented on the y-axis and birth weights of those samples are represented on the x-axis. LBW samples are denoted in green, NBW samples in purple and HBW samples in blue. (TIF) Click here for additional data file. Table S1 Probes whose expression levels co-varied with gestational age with a p1.5. (DOCX) Click here for additional data file. Table S5 20 genes studied in expanded sample set of 120 by qPCR. The replicating group comprises genes whose mRNA levels had a significant relationship with gestational age in the qPCR expanded study. The non-replicating group are those genes whose mRNA levels did not achieve significance against gestational age in the expanded qPCR study. (DOCX) Click here for additional data file. Table S6 Genes containing probes whose transcript levels were significantly different between birthweight groups in the microarray analysis and also containing CpGs whose methylation levels correlated with birthweight in the Infinium analysis. (DOCX) Click here for additional data file. Text S1 Detailed description analysis of correlation between mRNA level and DNA methylation state across samples paired fro gestational age. Four CpGs from table 2 in CHRDL2, GLI2 and HSD11B1 had significant (p<0.05) correlation of GA ratios between RNA expression and DNA methylation, when all the samples were used to create 17 non-unique pairs. (DOCX) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494345/

CpG Oligonucleotides as Cancer Vaccine Adjuvants

Adjuvants improve host responsiveness to co-delivered vaccines through a variety of mechanisms. Agents that trigger cells expressing Toll-like receptors (TLR) activate an innate immune response that enhances the induction of vaccine-specific immunity. When administered in combination with vaccines designed to prevent or slow tumor growth, TLR agonists have significantly improved the generation of cytotoxic T lymphocytes. Unfortunately, vaccines containing TLR agonists have rarely been able to eliminate large established tumors when administered systemically. To improve efficacy, attention has focused on delivering TLR agonists intra-tumorally with the intent of altering the tumor microenvironment. Agonists targeting TLRs 7/8 or 9 can reduce the frequency of Tregs while causing immunosuppressive MDSC in the tumor bed to differentiate into tumoricidal macrophages thereby enhancing tumor elimination. This work reviews pre-clinical and clinical studies concerning the utility of TLR 7/8/9 agonists as adjuvants for tumor vaccines. 1. Introduction Adjuvants are immunological agents that function to enhance the magnitude, breadth, quality and/or longevity of specific immune responses generated against co-administered antigens (Ag). Adjuvants are also used to reduce the dose and frequency of immunizations required to achieve protective immunity. Historically, vaccines were produced from live attenuated or heated inactivated organisms. While not appreciated at the time, those original vaccines contained bacterial contaminants that served as adjuvants [ 1 ]. There are several ways in which an adjuvant can promote immunity including: (1) Stabilizing or entrapping the Ag to extend release and thus prolong immune stimulation; (2) Promoting an inflammatory response at the site of Ag deposition thereby attracting activated macrophages and dendritic cells to improve Ag uptake and presentation; (3) Presenting co-stimulatory signals to T and B cells to enhance induction of Ag-specific immunity. There is considerable interest in identifying safer and more effective adjuvants to enhance the utility of novel vaccines targeting infectious pathogens, allergy and cancer. In support of these goals, immunologists and microbiologists have sought to elucidate the mechanism(s) of action of adjuvants. Notable success was achieved in the discovery of Toll like receptors (TLRs) and their role is promoting innate and adaptive immune responses, leading to a Nobel prize for Drs. Hoffmann and Beutler in 2011 [ 2 , 3 ]. 2. Background Information Concerning TLRs TLRs are an important component of the host's pathogen sensing mechanism [ 4 , 5 ]. TLRs are typically classified into two families based on their localization: TLRs 1, 2, and 4–6 are expressed on the cell surface and sense bacterial cell wall components whereas TLRs 3 and 7–9 are expressed in endosomes and sense viral or bacterial nucleic acids [ 6 ]. The molecular structures recognized by TLRs have been evolutionarily conserved, are expressed by a wide variety of infectious microorganisms, and are termed pathogen-associated molecular patterns (PAMPs) [ 4 , 5 ]. The innate immune response elicited by TLR activation is characterized by the production of pro-inflammatory cytokines, chemokines, type I interferons and anti-microbial peptides. This innate response promotes and modulates the adaptive immune system. A common result is the expansion of Ag specific B cells that produce high affinity antibodies and of cytotoxic T cells including long-lasting memory cells that protect against subsequent infection through enhanced cytotoxic function targeting the effector phase [ 7 , 8 ]. Although several TLRs utilize similar signaling pathways, there are reproducible differences in the cytokine profile and adaptive immune response each elicits. Understanding which elements of the immune response are best supported by which TLR ligands should enable the development of adjuvants specifically tailored to enhance desired vaccination outcomes. 3. CpG ODN and TLR9 TLR9 recognizes and is activated by CpG motifs (consisting of a central unmethylated CG dinucleotide embedded within specific flanking regions) present at high frequency in bacterial DNA [ 9 , 10 ]. TLR9 molecules differ between species, with the structure of human versus mouse TLR9 varying by 24% [ 9 ]. There is also variation between species in terms of which cell types express TLR9. For example, the TLR9 receptor is present in rodent but not in primate macrophages and myeloid dendritic cells (DC). In humans, TLR9 is expressed primarily by plasmacytoid DC and B cells [ 11 , 12 , 13 , 14 ]. Reflecting their utility as vaccine adjuvants, B lymphocytes exposed to TLR9 agonists become more susceptible to activation by Ag [ 15 , 16 , 17 ] while TLR9 stimulated pDC produce type I interferons and more efficiently present Ag to T cells [ 18 , 19 , 20 ]. The signaling pathway triggered when CpG interacts with TLR9 proceeds through the recruitment of myeloid differentiation factor 88 (MyD88), IL-1R-associated kinase (IRAK), and tumor necrosis factor receptor-associated factor 6 (TRAF6) [ 5 ]. This signaling cascade subsequently leads to the activation of several mitogen-activated kinases (MAPK) and transcription factors (such as NF-kB and AP-1), culminating in the transcription of pro-inflammatory chemokines and cytokines [ 5 ]. In humans, four distinct classes of CpG ODN have been identified based on differences in structure and the nature of the immune response they induce. Although each class contains at least one motif composed of a central unmethylated CG dinucleotide plus flanking regions, they differ in structure and immunological activity. "K"-type ODNs (also referred to as "B" type) contain from one to five CpG motifs typically on a phosphorothioate backbone. This backbone enhances resistance to nuclease digestion and substantially prolongs in vivo half-life (30–60 min compared with 5–10 min for phosphodiester) [ 21 ]. K-type ODNs trigger pDC to differentiate and produce TNFα and stimulate B cells to proliferate and secrete IgM [ 22 , 23 ]. Unless otherwise mentioned, the studies described below reflect the activity of K ODN as these have been studied most extensively in pre-clinical and clinical trials. "D"-type ODNs (also referred to as "A" type) have a phosphodiester core flanked by phosphorothioate terminal nucleotides. They carry a single CpG motif flanked by palindromic sequences that enables the formation of a stem-loop structure. D ODN also have poly G motifs at the 3' and 5' ends that facilitate concatamer formation. D-type ODNs trigger pDC to mature and secrete IFNα but have no effect on B cells [ 22 , 24 ]. C-type ODNs resemble K-type in being composed entirely of phosphorothioate nucleotides but resemble D-type in containing palindromic CpG motifs that can form stem loop structures or dimers. This class of ODN stimulates B cells to secrete IL-6 and pDC to produce IFNα [ 25 , 26 ]. P-Class CpG ODN contains double palindromes that can form hairpins at their GC-rich 3' ends as well as concatamerize due to the presence of the 5' palindromes. These highly ordered structures are credited with inducing the strongest type I IFN production of any class of CpG ODN [ 27 , 28 ]. 4. Effect of CpG ODN on Human pDC and B Cells In humans, TLR9 is expressed primarily by B cells and plasmacytoid DC (pDC) [ 29 ]. By comparison, multiple cells of the myeloid lineage including conventional DCs, monocytes and macrophages express TLR9 and respond to CpG ODN in mice [ 30 ]. pDC contribute to the initiation of many immune responses: they promote the generation of protective immunity to viral infection via their rapid and massive production of type I IFNs that support the generation of strong CTL responses [ 31 , 32 , 33 ]. Human pDC express TLRs 7 and 9 whereas myeloid DC (mDC) recognize TLRs 2, 3, 4, 5, 6 and 8 [ 29 ]. These divergent patterns of TLR expression support the hypothesis that distinct DC subsets generate unique/tailored responses optimized for the elimination of different pathogens [ 34 , 35 ]. Thus, CpG ODN should be particularly useful as adjuvants for vaccines targeting viral infections and cancer, both of which require the type of strong CTL response elicited by pDC activation [ 36 , 37 ]. TLR9 activation also induces human memory B cells to proliferate, undergo class switching to IgG2a and secrete antibodies in a T cell independent manner [ 38 ]. By comparison, naive human B cells express low levels of TLR9 and do not respond directly to CpG ODN [ 14 ]. Ag stimulation via the B cell receptor induces naive B cells to up-regulate TLR9 expression and acquire responsiveness to CpG DNA. The requirement that naive B cells interact with cognate Ag before acquiring responsiveness to CpG prevents polyclonal B cell activation and reduces the risk of autoimmunity [ 39 ]. This synergy between BCR ligation and CpG ODN stimulation was verified in studies using CpG-Ag complexes to enhance Ag-specific class switching in vivo and supports the use of CpG ODN as adjuvants for vaccines designed to induce strong humoral responses [ 39 ]. 5. CpG ODN as Vaccine Adjuvants: Importance of CpG-Ag Co-Delivery A number of preclinical (murine) studies examined the immunogenicity of CpG-adjuvanted vaccines. Most reported that CpG ODN enhanced both the humoral and cellular (Th1 cells and CTL) immune response elicited by vaccines against pathogens, allergens and/or tumors [ 40 ]. To optimize the efficiency of Ag presentation by DCs requires that they encounter CpG ODN in the presence of vaccine Ag. Co-delivery of ODN plus Ag to the same APC accelerates the induction, increases the maximal level and extends the duration of the induced immune response [ 41 ]. It also supports modulation of Ab isotype and increases the immunogenicity of weak Ags [ 42 ]. Examples include studies in which ovalbumin or the hepatitis B surface antigen vaccine were administered with CpG ODN in which co-delivery to the same site significantly enhanced humoral protective immunity [ 21 , 43 , 44 ]. Based on such findings, a number of delivery strategies were examined to optimize the co-delivery of CpG ODN plus Ag to the same APCs. These approaches included the preparation of CpG-Ag conjugates, co-encapsulation in liposomes or on biodegradable microparticles, and the use of multicomponent nanorods [ 40 , 45 , 46 ]. Murine studies show that conjugating CpG ODN directly to Ag can boost immunity by up to 100-fold over that induced by simply mixing CpG ODN with immunogen [ 47 , 48 ]. The mechanisms by which CpG ODN-Ag conjugates enhance immunogenicity include insuring that both Ag and TLR agonist are taken up by the same APC and improving such uptake via DNA-binding receptors on the APCs (the latter effect is independent of the nature of the ODN but requires physical conjugation of DNA to target antigen). While early murine studies focused on administering CpG ODN with defined vaccine Ags, their ability to support immunity when combined with complex vaccines expressing multiple tumor Ags has also been examined. One such endeavor examined the effect of conjugating CpG ODN to apoptotic tumor cells [ 49 ]. Whole mouse tumor cells were used because they expressed all possible tumor-associated Ags, allowing the host's immune system to select the most immunogenic determinant based on presentation in the context of self MHC. Apoptotic tumor cells alone lack a TLR signaling moiety and thus fail to trigger the innate immune system in support of tumor specific immunity. To overcome this limitation, CpG was conjugated directly to tumor cells. The resultant adjuvant/vaccine combination triggered the expansion of tumor-specific CTL in the periphery that reduced the growth of small tumors and prevented their metastatic spread in murine experiments [ 49 ]. There is concern that inclusion of CpG ODN may increase the risk of vaccine-induced autoimmunity. CpG ODN and immune complexes that contain nucleic acids interact with TLR9 to increase the production of type I IFNs. While IFNa and IFNb can induce/exacerbate autoimmune disease [ 50 , 51 , 52 , 53 ], whether CpG based adjuvants have such an effect remains controversial, having not been observed in clinical vaccine trials [ 40 , 54 ]. We conclude that when a vaccine against cancer capable of overcoming tolerance to tumor Ags is required, the benefit of including a strong adjuvant (such as CpG ODN) outweighs the potential risk. 6. Effect of CpG DNA on MDSC and Macrophages in the Tumor Microenvironment The anti-tumor activity of CpG-adjuvanted tumor vaccines was initially examined by delivering the vaccines systemically (by i.m. or s.c. routes) [ 55 ]. In murine studies, CpG-adjuvanted vaccines were effective against small tumors (<300 mm 3 ) but were unable to eliminate large established tumors (of the size typical present in humans when first diagnosed). Whether delivered to mice with large or small tumors, the CpG-adjuvanted vaccines continued to induce tumor-specific CTL that were readily detected in the peripheral circulation. The problem was that immunosuppressive leukocytes present in the microenvironment of large tumors down-regulated the activity of these CTL. To overcome this limitation, CpG ODN were injected directly into the tumor bed with the goal of activating intratumoral DCs and facilitating tumor Ag presentation in situ . Unexpectedly, local delivery interfered with the function of tolerogenic cells in the tumor milieu. Intra-tumoral injection of CpG ODN reduced the number and suppressive activity of tumor infiltrating monocyte-derived suppressor cells (MDSC) [ 56 ]. This was true of both free and vaccine associated CpG ODN, and led to a re-interpretation of data from clinical trials in which CpG ODN were delivered intratumorally to treat skin tumors or lymphoma. For example, Hofmann et al. induced complete or partial tumor remission in half of all patients with basal cell carcinoma or melanoma by intra-tumoral CpG injection [ 57 ]. Molenkamp et al. showed that intratumoral CpG increased the frequency of tumor-specific CD8 T cells in half of patients with melanoma [ 58 ]. Brody et al. showed that intratumoral CpG administration combined with radiation therapy induced systemic tumor regression (including at untreated sites) and tumor-reactive CD8 T cells in patients with low-grade B-cell lymphoma [ 59 ]. Kim et al. showed intratumoral injection of CpG ODN combined with radiation induced the regression of distal tumors, significantly decreased the frequency of FoxP3+ regulatory T cells (Tregs) and increased the frequency of CD123+ pDC at the site of CpG administration in patients with lymphoma [ 60 ]. These strategies are referred to as " in situ tumor vaccination" as they do not require the use of a customized vaccine. These findings are consistent with animal studies showing that local CpG ODN treatment increased the number of tumor infiltrating T and NK cells while decreasing the frequency and inhibitory activity of tumor resident MDSC. The monocytic MDSC studied in that work expressed TLR9 and exposure to CpG ODN (i) triggered their rapid production of Th1-type cytokines (including IL-6, IL-12 and TNFa); (ii) impaired their ability to secrete arginase 1 and nitric oxide (factors critical to their suppression of T cell activity) and (iii) induced them to differentiation into tumoricidal macrophages [ 56 ]. These results suggest the existence of additional mechanisms through which CpG ODN could promote tumor regression. Unfortunately, human mMDSC do not express TLR9 or respond to CpG ODN, limiting the clinical applicability of the murine findings. However, we find that the suppressive activity of mMDSC isolated from cancer patients can be reversed by treatment with TLR 7/8 agonists which induce them to differentiate into tumoricidal M1-like macrophages in a manner very similar to CpG ODN in mice [ 61 ]. 7. TLR 7 and TLR8 Agonists as Cancer Vaccine Adjuvants TLR7 and TLR8 are closely related receptors. They have similar structures and trigger a similar signaling cascade but differ in their pattern of cellular expression and thus the array of cytokines they elicit. In humans, TLR7 receptors are present on B cells and pDC which when activated secrete IFNa. TLR8 receptors are prevalent on neutrophils, monocytes and mDC which when triggered secrete TNFa, IL-12 and MIP1a [ 62 , 63 ]. Most ligands that interact with TLR7 also bind to TLR8. These include synthetic imidzaquinolines (such as resiquimod/R-848), and the natural ligand ssRNA containing GU-rich sequences. Imiquimod and the guanosine analogue loxoribine are considered selective for TLR7 [ 64 , 65 , 66 ]) and a new generation of TLR8 selective agents has been described [ 63 ]. 8. Trials Utilizing TLR 7/8 Agonists The utility of TLR 7/8 ligands as vaccine adjuvants was evaluated in pre-clinical studies. Smorlesi et al. used transgenic mice expressing the HER2/neu oncogene that spontaneously develop mammary tumors [ 67 ]. When immunized with a DNA vaccine plus imiquimod, the incidence and growth rate of breast tumors was reduced when compared to DNA vaccination alone. Ab titers in mice receiving the imiquimod adjuvanted vaccine were higher and biased towards IgG2a and the number of CD8 T cells producing IFNg was also increased [ 68 ]. Narusawa et al. evaluated imiquimod as an adjuvant when used in combination with GM-CSF plus a gene-transduced tumor vaccine (GVAX). This vaccine combination significantly reduced the rate of tumor growth while increasing the number of pDC [ 69 ]. It should be noted that neither of these studies examined the effect of TLR 7/8 adjuvanted vaccines on large established tumors, limiting the ability to draw conclusions concerning their utility under conditions similar to those found in patients with cancer. The TLR7/8 agonists currently approved by the FDA are designed for topical administration and are used primarily to treat HPV-induced warts, lentigo maligna, actinic keratoses, and basal or squamous cell carcinoma [ 70 , 71 , 72 , 73 ]. Clinical studies therefore routinely relied on topical administration to evaluate TLR 7/8 ligand activity. In a trial of patients with prostate cancer characterized by rising PSA titers (indicative of tumor growth), a prostate-specific peptide vaccine was combined with one of several adjuvants or immunomodulatory treatments. Patients receiving topical imiquimod over the vaccine injection site had the best clinical outcome with the slowest rise in PSA when compared to other modalities (including GM-CSF, hyperthermia, and mucin-1-mRNA/protamine complex) [ 74 ]. The ability of imiquimod to act as a topical adjuvant was also evaluated in patients with melanoma. When administered in conjunction with a variety of melanoma specific peptides plus Flt3 ligand, imiquimod stimulated an increase in the frequency of peptide-specific CD8 T cells [ 75 ]. When used in combination with a vaccine containing the NY-ESO-1 cancer Ag, four out of nine patients with melanoma developed specific Abs while seven out of nine developed CD4 T cell responses. CD8 T cell responses were not enhanced in these subjects nor did disease progression correlate with the induction of the types of immunity observed [ 76 ]. In an effort to improve outcome, resiquimod was substituted for imiquimod based on animal studies showing that this TLR 7/8 agonist was better at generating Ag specific CD8 T cells [ 77 , 78 ]. A clinical study of NY-ESO-1 immunized melanoma patients found that the addition of resiquimod improved CD8 T cell responses in 25% of patients. No change in Ab or CD4 T cells was observed nor was time to progression improved (interestingly CD8 T cell responders also expressed the TLR7 SNP rs179008) [ 79 ]. 9. Trials Utilizing CpG ODN Agonists targeting TLR9 have been studied more extensively than those against TLR7/8. CpG ODN showed activity in murine models as monotherapy, in combination with cancer vaccines, and when paired with other modalities including radiotherapy, cryotherapy and chemotherapy. Numerous preclinical studies showed that including CpG ODN increased CTL frequency and that this effect correlated with slower tumor progression. For example, the immunogenicity of DC-based tumor vaccines was improved by the addition of CpG ODN as characterized by a marked improvement in CD8 T cell activity [ 80 ]. Most such studies delivered the CpG adjuvanted vaccines before or shortly after challenge and thus targeted tumors that were relatively small [ 81 , 82 , 83 , 84 , 85 , 86 ]. However, recent studies indicate that CpG ODN adjuvanted vaccines can eradicate even large established tumors. In one report, combining CpG ODN with a peptide vaccine targeting HPV16 E7 resulted in the elimination of tumors up to 250 mm 3 in size. The growth of even larger tumors was significantly delayed although eradication was not achieved [ 87 ]. Eradication of very large tumors (1.2 cm in diameter) was observed in 50% of mice using a fusion protein vaccine targeting the E7 epitope in combination with CpG ODN plus a chemotherapeutic agent [ 88 ]. Several clinical trials examined the use of CpG ODN combined with peptide-based vaccines targeting tumor antigens. These studies commonly evaluated additional immunostimulatory agents such as Montanide ISA-51, GM-CSF and IFA. Phase I trials of the MART-1 peptide vaccine in patients with melanoma reported that the inclusion of CpG ODN increased the number of Ag-specific CD8 T cells by 10-fold [ 89 , 90 ]. Higher levels of IFNg, TNFa, and IL-2 were also detected [ 91 ]. In another trial using a multi-epitope peptide vaccine that included MART-1, gp100, and tyrosinase 40%–50% of patients developed IFNg secreting CD8 T cells and two-thirds of these had stable disease or partial regression. Unfortunately, these benefits lasted only 2–7 months and did not result in a significant difference in outcome when compared to other therapies in patients with stage IV or recurrent melanoma [ 92 ]. Expansion of CD8 T cells was also observed in studies utilizing the NY-ESO-1 peptide. In two trials, three out of three and nine out of 18 patients responded [ 93 , 94 ]. Elevated CD8 T cell responses were also observed against cancers expressing the NY-ESO-1 or LAGE-1 tumor Ag, and such responses were associated with improved clinical outcomes [ 95 ]. CpG ODN were also evaluated in combination with a vaccine targeting the Wilms' Tumor-1 Ag (WT-1). Among patients receiving the CpG adjuvanted vaccine, 60% had stable disease compared to 15%–20% of those lacking the CpG component [ 96 ]. A study of patients with metastatic esophageal squamous cell carcinoma used a vaccine targeting the cancer-testis Ag peptides LY6K and TTK. Inclusion of CpG ODN led to an increase in CD8 T cells and secretion of IFNa. More patients receiving the CpG vaccine had stable disease compared to patients who did not (33% vs. 66%) although no complete or partial remissions were induced [ 97 ]. A reasonable conclusion from these human trials is that the addition of TLR adjuvants modestly boosts vaccine induced immunity but rarely results in tumor eradication. One explanation for this limited success is the ability of established tumors to evade immune elimination. The microenvironment in which tumors reside is rich in factors that support growth and contains Tregs and MDSCs that down-regulate tumor-specific immunity [ 98 , 99 ]. Tregs aid the host by suppressing autoreactive T cells and thus prevent autoimmunity [ 100 , 101 , 102 ]. However, when present in the tumor milieu they disrupt the host's ability to destroy cancer cells [ 100 , 103 , 104 ]. Many tumors actively secrete factors such as CCL2 that recruit Tregs or that induce naive T cells to differentiate into Tregs [ 105 , 106 , 107 ]. Myeloid-derived suppressor cells are also present at high frequency in established tumors. They inhibit the tumoricidal activity of T and NK cells by interfering with l-arginine metabolism through the production of Arg-1 and iNOS or ROS [ 108 , 109 ]. Tregs and MDSC within the tumor microenvironment thus block the effector function of immune cells generated by TLR adjuvanted vaccines [ 110 , 111 ]. One way to limit the activity of these immunosuppressive cells is to induce their differentiation. For example, TGFb, IL-10 and other factors can induce Tregs to differentiate while IL-6, IL-10 and TNFa can drive MDSC to differentiate into macrophages [ 100 , 112 , 113 , 114 ]. Intratumoral delivery of CpG ODN has been shown to slow tumor growth by altering the balance between suppression and immunity. While TLR9 stimulation increases systemic production of NK and CD8 T cells [ 55 , 115 , 116 , 117 ], local delivery improves tumor infiltration by such cells. Moreover, murine studies show that local delivery reduces the frequency of immunosuppressive Tregs and monocytic MDSCs in the tumor microenvironment [ 55 , 115 , 116 , 117 ]. In vitro studies demonstrate that MDSCs lose their immunosuppressive activity when treated with CpG DNA in association with reduced expression of NO and Arg-1 [ 56 , 118 , 119 ]. These effects were driven by the differentiation of mMDSC into tumoricidal M1 macrophages (as characterized by decreased expression of Ly6c and Gr-1 and increased expression of F4/80). Transferring these differentiated cells into tumor-bearing animals significantly slowed tumor growth, indicating that intra-tumoral delivery of CpG ODN (alone or in conjunction with vaccine) might profoundly alter the balance between tumoricidal and immunosuppressive cells [ 118 , 119 ]. Recent reports suggest that TLR 7/8 agonists also trigger MDSC maturation. Resquimod induces murine MDSC to differentiate into F4/80+ macrophages and CD11c+/I-Ad+ dendritic cells that support the expansion of CD4 and CD8 T cells [ 120 ]. Our group found that mMDSCs isolated from the peripheral blood of normal volunteers and cancer patients differentiated into M1-like macrophages when exposed to several TLR7 and TLR8 agonists. Interestingly, this was not a universal effect of all TLR agonists. PAM3, a ligand for TLR1/2, causes mMDSC to differentiate into M2-like macrophages that support tumor growth [ 61 ]. TLR7 agonists have additional effects on immune cells. For example, loxorubin can inhibit tumor growth by promoting CD4 T cell proliferation and modulating the suppressive activity of Tregs [ 121 ]. 10. TLR Agonist Combinations Many strategies have been identified that enable the immune system to eliminate small tumors in animal models (including the intra-tumoral delivery of TLR agonists). Such strategies become increasingly less effective as larger cancers are targeted, in part because large tumors are infiltrated by immunosuppressive cells that inhibit the activity of tumoricidal CLT and NK cells [ 113 , 122 , 123 ]. Our group examined the effect of combining a novel TLR7/8 agonist (3M-052) with CpG ODN. While each TLR agonist alone slowed tumor growth, neither prevented the eventual outgrowth of established CT26 cancers. In contrast, the combination of both agonists cleared large established tumors in 87% of mice [ 119 ]. Mechanistically, this combination of TLR agonists reduced the number of mMDSCs and increased the number of CD8 T cells much more effectively than either agent alone. Intra-tumoral delivery of this TLR agonist combination up-regulated the expression of IL12, IFNg and granzyme B while lowering levels of Arg-1, Nos 2, CTLA-4 and TGFb [ 119 ]. The effect of combining TLR7 plus TLR9 agonists was evaluated in a single clinical trial. A virus-like nanoparticle containing CpG ODN plus the melanoma protein MelQbG10 was used in conjunction with imiquimod. The MelQbG10/CpG vaccine elicited tumor specific CD8 T cell responses. Inclusion of the TLR7 agonists significantly increased the magnitude of that response and improved the generation of memory T cells [ 124 ]. TLRs 7, 8 and 9 are all endosomal receptors, are expressed on overlapping cell types, and utilize similar signaling pathways [ 62 , 63 , 125 ]. What then accounts for their synergistic anti-tumor activity (particularly since TLR7 triggering may inhibit cytokine secretion induced by TLR9 agonists) [ 126 , 127 ]? A recent report shows that the expression of receptors by individual cells is stochastic and that cells with high levels of one receptor can have much lower levels of another [ 128 ]. We postulate that a greater fraction of APCs are activated by the combination of TLR7 plus TLR9 agonists than by either alone. Consistent with such a conclusion, the fraction of MDSC activated to differentiate and secrete cytokines was significantly increased when these cells were stimulated with both agonists vs. either one singly [ 119 ]. First generation TLR7/8 agonists were short acting agents designed for topical use. A new generation designed for in vivo administration and use as vaccine adjuvants is now available. Preliminary studies indicate that they are safe and persist at the injection site [ 63 ]. Delivering these agents in combination with CpG ODN into the tumor microenvironment as adjuvants for tumor vaccines thus represents a promising approach to the immunotherapy of large established cancers. 11. Conclusions TLR agonists have complex and pleiotropic effects on the immune system. When used as adjuvants, TLR agonists boost Ag-specific cellular and humoral immunity. Stimulating endosomal TLRs is particularly effective at promoting the generation of CTL capable of eliminating viral pathogens and cancer. Simultaneous activation of multiple TLRs further improves the breadth and efficacy of such responses. When targeting cancer, intra-tumoral delivery of agonists against TLRs 7, 8 and 9 provides the added benefit of altering the tumor microenvironment. Such treatment reduces the frequency of immunosuppressive Tregs, MDSC and M2 macrophages while increasing the frequency of tumoricidal M1 macrophage. We believe that intra-tumoral delivery of vaccines that include TLR agonists should be of considerable benefit in the immunotherapy of cancer.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5076642/

Evaluation of Two Loop-mediated Isothermal Amplification Methods for the Detection of Salmonella Enteritidis and Listeria Monocytogenes in Artificially Contaminated Ready-to-Eat Fresh Produce

In the present study, the effectiveness of two loop-mediated isothermal amplification (LAMP) assays was evaluated. Samples of romaine lettuce, strawberries, cherry tomatoes, green onions and sour berries were inoculated with known dilutions (10 0 -10 8 CFU/g of produce) of S. Enteritidis and L. monocytogenes. With LAMP, assay pathogens can be detected in less than 60 min. The limits of detection of S. Enteritidis and L. monocytogenes depended on the food sample tested and on the presence of enrichment step. After enrichment steps, all food samples were found positive even at low initial pathogen levels. The developed LAMP, assays, are expected to become a valuable, robust, innovative, powerful, cheap and fast monitoring tool, which can be extensively used for routine analysis, and screening of contaminated foods by the food industry and the Public Food Health Authorities. Introduction The consumption of ready-to-eat (RTE) produce, such as salads, fresh fruits and raw vegetables has been increased significantly. RTE fresh produce can become a vehicle for the transmission of pathogens capable of causing human illness (Abadias et al ., 2008 ). Numerous outbreaks in RTE fresh produce have been reported due to extensive human handling during their preparation or by cross-contamination (Escalona et al ., 2010 ). L. monocytogenes and Salmonella spp. are important pathogens detected in vegetables and fruits (Beuchat, 1999 ; Bracket, 1999 ). Salmonella has been shown to survive or grow in a wide variety of products (Hanning et al ., 2009 ). An increasing number of product-associated foodborne outbreaks in the United States associated with bacterial contamination are primarily from Salmonella (Tauxe et al ., 1997 ; Harris et al ., 2003 ). L. monocytogenes has been isolated from 15% of environmental samples in New York, emphasizing the prevalence of this pathogen on product fields (Strawn et al ., 2013 ). Prevalence estimates reported in the literature for products grown in the United States range from 0% to more than 35% (Hoelzer et al ., 2014 ). Salmonella remains the most frequently detected causative agent in the food-borne outbreaks reported (22.5 % of total outbreaks). For Listeria foodborne outbreaks a total of 13 outbreaks have been reported in 2013, which was slightly higher than in the previous years (EFSA and ECDC, 2015 ). Their control and prevention is essential throughout the food chain, as they can be related with many foodborne outbreaks registered in the European Union (European Food Safety Authority, 2012 ). Moreover, in case of foodborne outbreaks they cause high mortality rates within high-risk population groups (Kotzekidou, 2013 ). The fatality rate of L. monocytogenes is relatively rare but can reach 30% (Ponniah et al ., 2010 ). Salmonellosis constitutes a major foodborne infectious disease worldwide and is caused by 2500 serovars. It is most often attributed to the consumption of contaminated foods such as poultry, beef, pork, eggs, milk, seafood, nut products, and fresh products (Techathuvanan et al ., 2010 ; Wang et al ., 2008 ). S. enterica serovar Enteritidis is often associated with salmonellosis outbreaks (Techathuvanan and D'Souza, 2012 ). Traditionally, Salmonella detection includes pre-enrichment, then selective enrichment, culture plating, biochemical and serological tests, which in total require approximately 5 days for completion (Kokkinos et al ., 2014 ). Therefore, it is time consuming, cost and labor intensive to meet the criteria for food safety control in routine analysis in food companies (Techathuvanan et al ., 2010 ). Listeriosis is another major foodborne infectious disease that has the potential to cause serious life-threatening diseases, like septicemia, meningitis, meningoencephalitis and abortion (Dogany, 2003 ). Listeriosis can lead to a severe illness for individuals with compromised immune systems as the elderly, pregnant women, and young children (Ahmed et al ., 2015 ). In USA approximately 1600 illnesses and 260 deaths have been caused each year due to listeriosis (Scallan et al ., 2011 ). It is well known that a zero tolerance policy for L. monocytogenes in RTE fresh product exists (Gombas et al ., 2003 ). However, for RTE foods which are able to support the growth of L. monocytogenes , EU Regulation 2073 specifies that the 100 CFU/g criterion applies if the manufacturer is able to demonstrate, to the satisfaction of the competent authority, that the product will not exceed the limit of 100 CFU/g throughout the shelf-life and the absence in 25 g criterion applies only when the manufacturer is not able to demonstrate, to the satisfaction of the competent authority, that the product will not exceed the limit of 100 CFU/mL throughout the shelf-life (Koutsoumanis and Angelidis, 2007 ; EFSA, 2007 ). Traditionally, food companies test the existence of L. monocytogenes with routine culture methods, which demand at least 5 days for completion. The lack of any heating step prior to consumption of the RTE produce emphasizes the need for taking appropriate sanitary measures for the prevention and reduction of the microbial load, but simultaneously without altering the fresh status and the organoleptic and quality properties of the products, intended to be consumed raw (Baert et al ., 2009 ). Increased public awareness related to health and economic impacts of foodborne diseases has resulted in greater efforts in order to develop sensitive, rapid and inexpensive screening methods of pathogens detection (Wang et al ., 2008 ). To satisfy the expected rapidity, molecular methods have been developed. However, they still require time and expensive, sophisticated equipment, as well as expertise (Techathuvanan et al ., 2011 ). Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification assay that is rapid, specific, and simple, and can be selected as a new alternative for molecular diagnosis (Notomi et al ., 2000 ). The advantages of LAMP are high specificity and sensitivity, simple operation, and low cost, which constitutes a potentially valuable tool for rapid diagnosis of food-borne pathogens (Shao et al ., 2011 ). LAMP works under isothermal conditions between 60 and 65°C resulting in 10 9 copies of target DNA within an hour (Parida et al ., 2008 ). The aim of the present study was the evaluation of two specific LAMP assays which are simple, cost-effective, one-step, single-tube, for the detection of S. Enteritidis and L. monocytogenes seperately, on a series of artificially inoculated fresh ready-to-eat (RTE) produce samples. The main objective of our study was the application and further evaluation of two diagnostic LAMP-based assays, and the simultaneous-complementary testing with classic ISO methods for the detection of different concentrations of S. Enteritidis and L. monocytogenes in RTE produce, and their potential use in routine food analysis by the food industry and the Public Food Authorities. Materials and Methods Bacterial strains Bacterial strains used were S. Enteritidis NCTC 6676 and L. monocytogenes NCTC 11994 [Health Protection Agency (HPA), Colingdale, UK]. Lenticules with the microorganisms were rehydrated in 9 mL of peptone saline (0.1%) (Oxoid, Basingstoke, UK), and after 20 min, working cultures were streaked onto Tryptic Soy Agar (TSA; Oxoid), incubated at 37°C for 24 h, and stored at 4°C. Culture preparation Each bacterial type was cultured in 20 mL Tryptone Soya Broth (TSB; Merck KGaA, Darmstadt, Germany) at 37°C for 17 h, harvested by centrifugation at 4000 x g for 20 min at 4°C and washed three times with buffered peptone water (BPW; Oxoid). The final pellets were resuspended in BPW, corresponding to approximately 10 8 -10 9 CFU/mL. Food samples collection and processing Five different RTE fresh produce, were purchased from a local supermarket (Patras, Greece) the day of the experiment, and stored under refrigerated conditions (4°C) until the time of the experiment. Fresh RTE produce, such as romaine lettuce ( Lactuca sativa L. var. longifolia ), strawberries ( Fragaria x ananassa ), cherry tomatoes ( Solanum lycopersicum var. cerasiforme ), green onions ( Allium spp ), and sour berries ( Prunus cerasus ), were used for the specificity and evaluation tests of the developed LAMP assay. Sample inoculation All RTE fresh produce samples were rinsed with sterile water to remove some of the natural microbiota or any other matter ( e.g. soil) before treatment (Bermúdez-Aguirre and Barbosa-Cánovas, 2013 ). For the inoculation of the samples, a spot-inoculation method was applied to inoculate the bacteria on their surface. Briefly, 100 µL of S. Enteritidis and L. monocytogenes corresponding to concentrations of 10 8 CFU/g to 10 0 CFU/g (which were determined by culture method) were spotted separately with a micropipette on 10 different areas of the surface of each RTE fresh produce sample weighing 10g, in order to simulate real conditions. After spiking, the samples were dried, for 1 hour at 22±2°C, to allow bacterial attachment. All processes were performed in a class II biosafety cabinet (Cytair 155; FluFrance, Wissous, France). Culture-based confirmation of Salmonella spp. detection The method used was based on protocol ISO 6579: 2002 (ISO, 2002), with slight modifications. Artificially inoculated RTE fresh produce (10 g) were added to 90 mL Buffered Peptone Water (BPW; Oxoid). Secondary enrichment was performed using only Rappaport-Vassiliadis medium (RVS broth; Oxoid). Finally, plating out was performed using xylose lysine desoxycholate agar (XLD; Oxoid). At the same time a LAMP-friendly secondary enrichment with YPCE broth (Knuttson et al ., 2002) was assessed, with incubation for 24h at 37°C ( Figure 1 ). Culture-based confirmation of Listeria spp. detection The method used was based on protocol ISO 11290-1:1996 (ISO, 1996 ). Artificially inoculated RTE fresh produce (10 g) were added to 90 mL Half Fraser Broth (HFB) (Oxoid) and enrichment for 22-24h at 30°C was followed. Secondary enrichment was performed with Fraser Broth (FB) (Oxoid) for 24h at 37°C. Finally, plating out was performed using Oxford Listeria selective agar (Oxford Listeria; Oxoid). Samples from different stages (samples without enrichment, samples from first enrichment, and samples from secondary enrichment) were taken for culture and LAMP assays ( Figure 2 ). L. monocytogenes nucleic acids were extracted using the Genomic DNA from tissue (Nucleospin tissue; Macherey-Nagel, Düren, Germany), according to the manufacturer's instructions. Sample preparation for loop-mediated isothermal amplification methods Buffered Peptone Water (90 mL) and Half Fraser Broth (90 mL) were added to 10 g of each RTE fresh produce sample prior inoculation with S. Enteritidis and L. monocytogenes , respectively. The samples were homogenized for Salmonella and Listeria testing, respectively. Sample was taken at day 0 (fresh produce sample with PBW or HFB). Then, 100µL were added to 100ml of selective enrichment broth YPCE and enrichment at 37°C for 24-48h (day 1 and day 2 respectively) followed for Salmonella detection. The LAMP friendly enrichment broth was prepared according to D'Agostino et al . ( 2015 ). For L. monocytogenes detection, enrichment of samples with HFB at 37°C for 24h (day 1) followed and finally 0.1 µL of HFB was added to 10ml of FB and the samples were enriched at 37°C for 24h (day 2). Loop-mediated isothermal amplification assay for Salmonella Enteritidis detection In this study, six primers (two inner primers, two outer primers and two loop primers), targeting Salmonella enterica invasion protein (invA) gene were used for the LAMP reactions (Hara-Kudo et al ., 2005 ; Ziros et al ., 2012 ). The reaction was carried out in a total of 25µL and contained 16µL of Tin Isothermal Mastermix, 25 µM invASalm FIP, 25 µM invASalm BIP, 5 µM invASalmF3, 5 µM invASalmB3, 12,5 µM invASalmF-Loop, 12,5 µM invASalmB-Loop and 3µL of template DNA (D'Agostino et al ., 2015 ). The primers were high-performance liquid chromatography-purified. The thermal profile of the reaction was the following: 95°C for 2 min (cell lysis), followed by 65°C for 50 min (nucleic acid amplification). Amplicon annealing profiling was performed by heating to 98°C then cooling to 80°C at a rate of 0.05°C sec-1. The samples were analyzed using a LightCycler Nano Instrument (Roche, Basel, Switzerland). Positive and negative controls were included in each run. Aliquots of 10 µL of LAMP products were electrophoresed on 2% agarose gels and were visualized by ethidium bromide (Sigma, St. Louis, MO, USA) staining. The amplified products were also detected by adding 1 µL of 1000 X SYBR green dye to each reaction tube. After incubation for 15 min in the dark at room temperature, a yellowish green colour indicated a positive reaction, while a reddish orange (the colour of the unbound dye) indicated a negative reaction. Loop-mediated isothermal amplification assay for Listeria monocytogenes detection This study used six primers (two inner primers, two outer primers and two loop primers) targeting hly A gene of L. monocytogenes . The primers were high-performance liquid chromatography-purified. Positive and negative controls were included in each run. The LAMP reaction was carried out in a total volume of 25 µL. The optimal conditions as well as the thermal profile were based on the assay of Wang et al . ( 2012 ). The samples were analyzed using a LightCycler Nano Instrument (Roche). Aliquots of 10 µL of LAMP products were electrophoresed on 2% agarose gels and were visualized by ethidium bromide (Sigma) staining with UV light transillumination. The amplified products were also detected by adding 1 µL of 1000 X SYBR green dye to each reaction tube. After incubation for 15 min in the dark at room temperature, a yellowish green colour indicated a positive reaction, while a reddish orange (the colour of the unbound dye) indicated a negative reaction. Prevention of polymerase chain reaction carryover contamination To avoid any LAMP carryover contamination strict laboratory practices were followed throughout the experimental procedure. The manipulations before the LAMP assay (DNA isolation and LAMP set-up) were performed in a clean UV room that was separated from the LAMP PCR machine and the post-LAMP processing area. Negative controls were run with all assays, and no indications of contamination were detected. Attention was paid when the caps of the used reaction tubes were opened for the addition of SYBR green dye or subsequent electrophoresis. Specificity and sensitivity of the loop-mediated isothermal amplification assays The specificity of the developed LAMP assays in real-world situations were evaluated in the context of the present study by analyzing different fresh produce matrices, after inoculation of non- Salmonella and non- Listeria DNA. Strains other than Salmonella and Listeria (human adenovirus-35, hAdV35; human adenovirus 40/41, hAdV40/41; E. coli , NCTC 9001) were selected in order to check if cross-reaction was observed in LAMP assays. The negative controls which have been used in the study were the following: i) for every batch of analyzed food samples, a control sample (non-inoculated with the target microorganism) was tested to investigate any potential initial contamination or contamination during the enrichment procedures; ii) for every LAMP assay, two negative controls with the aforementioned non-target microorganisms, and one additional negative control consisting of water (ddH2O) (instead of the target microorganism), were used. In addition, ten-fold dilutions of Salmonella and Listeria , which were previously quantified by culture methods, were inoculated in RTE fresh produce samples to determine the sensitivity of the assay for food analysis. Finally, for measuring the limit of detection (LOD) of the LAMP assay, RTE fresh produce samples were tested in triplicate and the lowest concentration of colony forming units (CFUs) was taken as the limit when all of the triplicate samples (for each dilution) were positive. Statistical analysis All experiments were carried out in triplicates. During each experiment two samples were run in agarose gels (2%) or tested after the addition of SYBR Green. The data for the detection of two pathogens with LAMP assays were analyzed for statistical significance using SPSS 21.0 (SPSS Inc., Chicago, IL, USA). Results were compared by an analysis of variance (ANOVA) followed by Tukey's method with a significance level of P0.05) for both pathogens tested and for all enrichment steps. This imposes the implementation of enrichment steps, which for Salmonella , one day enrichment was found to be sufficient, whereas for Listeria , two enrichment days were important for the detection of lower levels of pathogen ( Tables 1 and 2 ). Different researchers have studied LAMP assays for Salmonella detection in RTE fresh produce like lettuce, tomatoes and milk (Zhang et al ., 2011 ; Yang et al ., 2015 ; Wu et al ., 2015 ). More precisely Zhang et al. ( 2011 ) have found the detection limit of L. monocytogenes in lettuce (2 CFU/25g) and tomato (19 CFU/25g). Whereas, the detection limit of L. monocytogenes using the ISO 11290-1:1996 method (Amd. 2004) was found to be 5-100 CFU/25g (Zunabovic et al ., 2011 ). Other researchers have studied the detection limit of L. monocytogenes in food samples like chicken and milk (Wu et al ., 2014 ; Wang et al ., 2011 ; Tang et al ., 2011). The present study did not aim at proposing LAMP as an alternative to ISO methods, but aimed to highlight the potential of LAMP assays to complement ISO methods in order to enhance their potential use in routine food analysis by the food industry and the Public Food Authorities. LAMP-based assays may be used as sensitive, specific and cheap tools for a fast initial screening of food samples for the target microorganisms of interest. Negative samples may be rapidly released to the market, while positive samples may be further analyzed and confirmed by ISO methods. This is to the best of our knowledge the first report on the evaluation of -one Gram negative ( Salmonella ) and one Gram positive ( Listeria ) bacterium-specific LAMP assays on different food samples. This is the first time that LAMP assays are used for pathogen detection in five RTE fresh produce. Conclusions The results showed that Salmonella spp. and L. monocytogenes were detected successfully on the five fresh RTE fresh produce samples tested (romaine lettuce, strawberries, cherry tomatoes, green onions, sour berries) by both LAMP assays and conventional culture assays. The greatest advantage of the proposed LAMP assays is the reduction of the required analysis time depending on the initial contamination of the products, compared to the classical culture methods. The developed LAMP assays are expected to become a valuable tool and be used extensively for routine analysis and screening of contaminated foods by the food industry and the Public Health Authorities.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9558170/

Single Domain Antibody application in bacterial infection diagnosis and neutralization

Increasing antibiotic resistance to bacterial infections causes a serious threat to human health. Efficient detection and treatment strategies are the keys to preventing and reducing bacterial infections. Due to the high affinity and antigen specificity, antibodies have become an important tool for diagnosis and treatment of various human diseases. In addition to conventional antibodies, a unique class of "heavy-chain-only" antibodies (HCAbs) were found in the serum of camelids and sharks. HCAbs binds to the antigen through only one variable domain Referred to as VHH (variable domain of the heavy chain of HCAbs). The recombinant format of the VHH is also called single domain antibody (sdAb) or nanobody (Nb). Sharks might also have an ancestor HCAb from where SdAbs or V-NAR might be engineered. Compared with traditional Abs, Nbs have several outstanding properties such as small size, high stability, strong antigen-binding affinity, high solubility and low immunogenicity. Furthermore, they are expressed at low cost in microorganisms and amenable to engineering. These superior properties make Nbs a highly desired alternative to conventional antibodies, which are extensively employed in structural biology, unravelling biochemical mechanisms, molecular imaging, diagnosis and treatment of diseases. In this review, we summarized recent progress of nanobody-based approaches in diagnosis and neutralization of bacterial infection and further discussed the challenges of Nbs in these fields. Introduction With the increasing antibiotic resistance, bacterial infection constitutes a serious threat to human health. It can lead to tremendous morbidity and mortality, emphasizing the need for rapid and effective identification and treatment of bacteria pathogens ( 1 ). At present, clinical bacterial diagnosis mainly involves bacterial culture, molecular diagnostics and colony formation methods which are time-consuming, labor intensive and requiring expensive equipment, all of which limit the utility, especially in resource limited settings ( 2 – 4 ). Oral and intravenous antibiotics are the most common treatments against bacterial infections; however, they are usually administered against ill-defined pathogens. This abuse of antibiotics plays an important role in the increase of antibiotic resistance ( 5 – 8 ). Therefore, it is important to develop fast, cost-effective, and accurate methods for the detection, identification and treatment of bacterial infections. Antibodies became promising molecules for bacterial detection and treatment due to their high sensitivity and specificity. Antibodies are essential components of adaptive immunity. Antibody-based diagnosis and therapeutics are the fastest growing classes of drugs on the market. The US FDA has approved over 100 antibodies mainly for treating cancer (45%) and immune-mediated disorders (27%) while only 8% against infectious diseases ( 9 ). The high production cost, low stability and large size may be the main obstacles to develop the antibodies for treating infectious diseases ( 10 ). Therefore, single domain antibodies (sdAbs), which have low production costs, high stability and small size become a promising alternative to canonical antibodies ( 11 ). In 1990s, scientists found a unique class of "heavy-chain-only" antibodies (HCAbs) in the serum of camelids and sharks. Owing to the absence of light chains, HCAbs binds to the antigen through only one variable region, referred to as VHH or also sdAb or nanobody (Nbs). The antigen-binding domain of shark HCAbs are known as VNAR ( 12 , 13 ). Their special structure endowed sdAbs with superior properties and enabled them to be extensively employed in structural biology ( 14 – 16 ), unravelling biochemical mechanisms ( 17 ), molecular imaging ( 18 – 20 ), diagnosis and treatment of tumors ( 21 , 22 ) and infection diseases ( 23 – 27 ). As for infectious diseases, sdAb have been widely used in the diagnosis and treatment of a variety of viral infections ( 28 ). It is noteworthy that a lot of nanobodies have been generated targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19), and there have been excellent reviews to summarize the current research progress ( 29 , 30 ). This review will focus on the current progress and perspectives of diagnostic and neutralizing sdAbs against bacterial infection ( Figure 1 ). Figure 1 The nanobodies take effects in several ways against bacterial infection. In the early stage of infection, pathogenic microorganisms are confined to the lesion. At this time, the PAMPs and the toxins are released into the bloodstream. The nanobodies binding onto the receptors prevent PAMPs recognition by PRRs, such as Toll-like receptors, Nod-like receptors and C-type lectins such as Clec4f, leading to a series of bodily reactions. The toxins, such as CDT, Tcd and BoNT are the major part in bacterial pathogenicity, which serve in different ways to cause damage in hosts and assist in enlargement of infection foci. The neutralizing nanobodies protect the host through specifically binding the toxins. At late stage of infection, pathogens are released into blood causing bacteremia. The nanobodies recognizing surface antigens, such as pilus and flagellum, bind onto the pathogen surface, preventing bacterial attachment. Structural and physicochemical features of Single Domain Antibodies SdAbs are the smallest known natural antigen-specific binding functional fragment, with dimensions of 2.5 nm in diameter and 4 nm in length. They consist of approximately 120 amino acids and merely 12~17 kD in weight, which is only one-tenth of canonical antibodies (150kD) ( 31 ). Similar to the VH domain of canonical antibodies, sdAbs consist of three hypervariable antigen-binding loops (complementarity determining regions, CDR1-CDR3) and four conserved framework regions (FR1-FR4) ( 32 – 34 ). There are mainly two differences between sdAbs and VHs of canonical antibodies. SdAbs have elongated CDR1 and CDR3, which to some extent compensate the loss of antigen-binding surface contributed by the light chain CDRs. In addition, the elongated CDR3 can adopt larger variety of structures and has a preference to interact with concave shaped antigen surfaces ( 35 ). Another notable difference is the conservative hydrophobic amino acids (Val47, Gly49, Leu50, and Trp52) in canonical antibody FR2 substitution by hydrophilic amino acids (Phe42, Glu49, Arg50, and Gly52), increasing solubility and stability of sdAbs ( 33 , 36 ). Due to the specific structure, sdAbs possess several outstanding characteristics compared to the traditional antibodies. 1) SdAbs represent the smallest naturally derived antigen-binding functional fragments (~15 kD). The small size allows sdAbs to penetrate deeper in dense tissues and might cross the blood-brain barrier(BBB) ( 37 , 38 ), and to be quickly eliminated via the kidney ( 39 ). Besides, the higher isoelectric point makes SdAbs positively charged and easier to penetrate the BBB. Therefore, sdAbs are more suitable for targeting solid tumors ( 40 , 41 ) and brain diseases ( 37 , 42 , 43 ). 2) Compared with traditional antibodies, the sdAbs have only one domain with disulfide bonds, which folds into a relatively stable structure. Amazingly, sdAbs maintained their antigenic binding ability after being incubated for one week at 37°C. They even can tolerate higher temperatures of 60-80°C ( 36 ). In some cases, they can regain antigen-binding activity after thermal denaturation by exposure at high temperatures (90°C) ( 44 ).When exposed to chemical denaturing agents and proteases, as well as non-physiological pH (pH range 3.0-9.0), sdAbs can also retain most antigen-binding capabilities ( 45 ). These properties permit the use of more demanding chemical and physical conditions during treatments or modifications of sdAbs than other types of antibodies. 3) The longer amino acid sequences of the CDR3 enlarges the antigen binding surface of the sdAbs, increases their structural repertoire, and further expands the binding ability to some hidden antigenic epitopes by creating new finger-like structures. Thus, they are enhancing the recognition ability of concave epitopes as well as binding to such epitope architectures with high affinity ( 31 , 46 – 51 ). 4) The hydrophilic amino acids on the side of VHH, corresponding to the VL interface of VH domains, improve the solubility in aqueous solutions and lower the tendency to aggregate ( 52 ). 5) The high degree of sequence identity with human VH domains of family-3 of VHHs, their small size, resistance to form aggregates and rapid blood clearance favor a low immunogenicity. VNARs may have a higher immunogenicity due to low sequence identity between VNARs and human VH or VL domains (~ 30% overall) ( 53 ). Overall, the immunogenicity risks with VHHs are low ( 54 ). In addition, the humanization of sdAbs provides a safe option for long-term treatment ( 55 , 56 ). 6) SdAbs can be efficiently, easily and economically produced recombinantly in bacteria, mammalian cell lines, yeast and plants at an affordable cost ( 11 , 57 ), while the production of canonical monoclonal antibodies requires mammalian expression system, which is complex in technology and expensive to maintain. Apart from these outstanding characteristics, sdAbs also have some limitations. The biggest drawback of sdAbs is their inadequate pharmacokinetics. Compared with conventional antibodies, sdAbs have a faster serum clearance rate, which limits their application in the field of therapy. In addition, sdAbs may have some adverse effects and a humanized tetravalent Nb have been reported with hepatotoxicity. The characteristics compared between nanobodies and conventional antibodies are listed in Table 1 . Table 1 Characteristics compared between nanobodies and conventional antibodies. Characteristics Nanobodies Conventional antibodies The molecular weight Low (~15 kDa) High (~150 kDa) Stability High Low Affinity High Low Solubility High Low Immunogenicity Low High Cost Economic Expensive Serum clearance rate Fast Slow Single Domain Antibody use to diagnose and neutralize infections by Gram- negative bacteria Enterotoxigenic E. coli Enterotoxigenic E. coli (ETEC) is one of the most common causes of diarrhea in toddlers, adults in the developing world and in travelers to endemic areas. According to WHO reports, ETEC related diarrhea is one of the leading causes of death in the children under the age of 5 in developing countries ( 58 ). In addition, ETEC strains causing severe, watery diarrhea are responsible for significant death and morbidity in neonatal and post-weaned piglets, leading to worldwide tremendous economic losses in pork industry ( 59 ). ETEC is a non-invasive pathogen that mediates small intestine adherence through bacterial surface structures, known as colonization factors (CFs). Once bound to the small intestine, the bacteria produce toxins causing a net flow of water from enterocytes, leading to watery diarrhea ( 60 ). ETEC strains can also produce many types of fimbriae that are involved in bacterial attachment. F4 fimbriae are commonly found on ETEC from diarrheic piglets ( 61 , 62 ). In 2005, Harmsen et al. immunized a llama with F4ac fimbriae from the F4-positive (F4+) ETEC strain CVI-1000 and obtained a few monoclonal VHHs. However, the best monovalent VHH, K609, could not significantly reduce diarrhea and reduced piglet mortality was poor ( 63 ). In contrast, orally administration of the linker connected bivalent VHH could enhance the clearance of F4+ ETEC and decrease the number of infected piglets ( 64 ). The different in vivo activity of mono- and bivalent VHH suggested that the ability to agglutinate bacteria may have a higher impact on infection, consistent with other studies where only bivalent antibodies showed in vivo protection ( 65 – 67 ). In another study, Moonens et al. ( 59 ) fused four different variable domains of llama heavy chain-only antibodies (V1-4), raised against F4ac, to the Fc domain of a porcine immunoglobulin IgA. These four different VHH targeted conserved epitopes of FaeG, a major adhesive subunit of F4. The four VHHs were fused to porcine IgA-Fc and subsequently expressed in Arabidopsis thaliana seeds to feed piglets. The oral feed-based passive immunization strategy protected piglets as demonstrated by (i) the progressive decline in shedding of F4 positive ETEC bacteria, (ii) the significantly lower immune responses of the piglets to F4 fimbriae, which suggest a reduced exposure to the ETEC pathogen, and (iii) a significantly higher body weight in comparison with control piglets ( 63 , 68 ). The structural study of V1-4 in complex with FaeG indicated that they sterically hindered FaeG associating with the F4 receptor but they did not directly interfere with the carbohydrate binding site ( 59 ). Besides F4+ ETEC, four VHHs targeting F18 fimbriae FedF domain were generated by llama immunization and selection as well. They could inhibit F18+ ETEC attaching to piglet enterocytes in vitro , and either sterically hinder or induce conformational changes of the binding surface of FedF ( 69 ). In a recent study, Amcheslavsky ( 60 ) and his colleagues immunized two male llamas with N-terminal fragments of eight class-5 ETEC adhesins to generate nanobodies with broad cross-reactivity against ETEC adhesins. They identified single nanobodies that show cross protective potency against eleven major pathogenic ETEC strains in vitro and inhibited ETEC colonization in vivo . Molecular docking and mutagenesis analysis revealed that nanobodies recognized a highly conserved epitope within the putative receptor binding region of ETEC adhesins ( 60 ). Shiga toxin-producing Escherichia coli Shiga toxin-producing Escherichia coli (STEC) are a subset of E. coli pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome (HUS) and even death. Shiga toxins, the main virulence factors are divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans and newly weaned pigs ( 70 ). Stx2e consists of an enzymatically active A subunit and five B subunits that bind to globotriaosylceramide (Gb3) on host cells ( 71 ). Lo et al. reported the discovery and characterization of a VHH, NbStx2e1, isolated from a llama phage display library that confers potent neutralizing capacity against Stx2e toxin. Structural analysis revealed that for each B subunit of Stx2e, one NbStx2e1 is interacting in a head-to-head orientation and directly competing with the glycolipid receptor binding site on the surface of the B subunit. The neutralizing NbStx2e1 can be used to prevent or treat edema disease in the future. Tremblay et al. immunized llama with Stx1 and 2 together and identified a panel of neutralizing VHHs, two of which demonstrated cross activity to Stx1 and 2 ( 72 ). A VHH heterodimer consisting of one Stx1-specific VHH/Stx2-specific VHH, and one Stx1/Stx2 cross-specific VHH, significantly improved the survival and reduced the kidney damage of mice challenged with Stx1 or 2. In addition, co-administration of the heterodimeric VHH with an effector Ab that binds to the VHH heterodimer, was effective in preventing all symptoms of intoxication from Stx1 and Stx2. In 2016, Mejías and his colleagues reported the generation of a family of Stx2B-binding VHHs that neutralize Stx2 in vitro at a nanomolar to subnanomolar range. The anti-Stx2B VHH, 2vb27, was selected and two copies were fused to an anti-human serum albumin VHH. This engineered antibody showed increased retention in circulation and was able to neutralize Stx2 in three different mouse models. This novel and simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome ( 73 ). In another study, Navarro et al. described the identification and characterization of a nanobody (Nb113) with the potential to neutralize the Stx2a and Stx2c toxins that are associated with human clinical infections. The crystal structural study revealed that each B subunit in the pentameric B5 ring is associated with a single Nb113 molecule. A detailed analysis of the epitope targeted by Nb113 suggests that this Nb prevents the formation of the Stx2a–Gb3 complex, thereby impeding the subsequent steps of the internalization and enzymatic activity of the Stx2a holotoxin ( 70 ). Besides Stx2-neutralizing VHHs, two VHHs were identified from immunized llama for detection of Stx2 using ELISA, which was even more sensitive than commercial ELISA kits ( 74 ). The ELISA was best for the major subtype Stx2a and less sensitive for Stx2f. VHH based ELISA is expected to be more cost effective than IgG ELISA. Other Gram- negative bacteria Pseudomonas aeruginosa is one of the leading causes of hospital-acquired infections. It is difficult to treat the infections due to the high intrinsic antibiotic resistance and the organism's capability to occur in biofilms in the host. Adams et al. immunized a llama with P. aeruginosa antigens and identified monoclonal anti-flagellin VHHs. In an in vitro assay, they showed that the anti-flagellin VHHs are capable of inhibiting P. aeruginosa from swimming and that they prevented biofilm formation ( 75 ). Helicobacter pylori infection is associated with gastritis, gastric and duodenal ulcers, and even gastric adenocarcinoma. It is important to seek alternative therapeutic strategies due to the increasing occurrence of antibiotic resistance. Some studies reported the isolation and purification of nanobodies with high affinity against UreC subunit of urease enzyme from H. pylori . These nanobodies could be a novel class of treatments against H. pylori infection ( 76 , 77 ). The sdAbs employed for diagnosis and neutralization of Gram- negative bacterial infection are listed in Table 2 . Table 2 SdAb reports to diagnose and/or neutralizing infections by Gram-negative bacteria. Nanobody Source Target Structure (IC50)/KD Function Diagnosis/Neutralizing Ref. K609 Immune library ETEC F4 fimbriae – – prevented F4+ ETEC attachment Neutralizing ( 63 ) V1 V2 V3 – ETEC F4 FaeG 4WEM 4WEN 4WEU 0.1 to 7.7 µM prevent F4+ ETEC attachment Neutralizing ( 59 ) NbFedF6 NbFedF7 NbFedF9 Immune library ETEC F18 FedF 4W6W 4W6X 4W6Y – inhibit F18+ ETEC attachment Neutralizing ( 69 ) 2R215 2R23 naive library ETEC CfaE – 0.4125 to 13.3 µM(IC100) broad cross-protection against 11 major disease causing ETEC strains and prevented colonization in vivo Neutralizing ( 60 ) 1D7 1H4 Immune library ETEC CfaE – – prevented bacterial colonization in animals. Neutralizing ( 60 ) NbStx2e1 Immune library STEC Stx2e 4P2C 8 nM direct interaction with the Stx2e B subunit binding site for glycolipid, thereby impeding toxin-host cell receptor contacts Neutralizing ( 71 ) 2VB27 Immune library STEC Stx2B neutralized Stx2 in vitro at subnanomolar concentrations Neutralizing ( 73 ) Nb113 Immune library STEC rStx2aB 6FE4 9.6 nM neutralized Stx2a by competing for the Gb3 receptor Neutralizing ( 70 ) Stx-A4 Stx-A5 Immune library STEC Stx1/Stx2 – 7.2-12.5 nM neutralized Stx1 and Stx2 and prevented all symptoms of intoxication from Stx1 and Stx2 Neutralizing ( 72 ) 1vb1- 2vb10 2vb21-2vb10 Immune library STEC Stx2 – – early detection of STEC infections Diagnosis ( 74 ) 7G 9D Immune library P. aeruginosa flagellum – 2.5 nM 4.7 nM inhibit P. aeruginosa from swimming and prevent biofilm formation in vitro Neutralizing ( 75 ) nanobody against UreC Immune library UreC – 0.05nM bind to UreC and inhibit urease activity Neutralizing ( 76 ) HMR23 Immune library UreC – 0.0263nM bind to UreC and inhibit urease activity Neutralizing ( 77 ) Enterotoxigenic E. coli Enterotoxigenic E. coli (ETEC) is one of the most common causes of diarrhea in toddlers, adults in the developing world and in travelers to endemic areas. According to WHO reports, ETEC related diarrhea is one of the leading causes of death in the children under the age of 5 in developing countries ( 58 ). In addition, ETEC strains causing severe, watery diarrhea are responsible for significant death and morbidity in neonatal and post-weaned piglets, leading to worldwide tremendous economic losses in pork industry ( 59 ). ETEC is a non-invasive pathogen that mediates small intestine adherence through bacterial surface structures, known as colonization factors (CFs). Once bound to the small intestine, the bacteria produce toxins causing a net flow of water from enterocytes, leading to watery diarrhea ( 60 ). ETEC strains can also produce many types of fimbriae that are involved in bacterial attachment. F4 fimbriae are commonly found on ETEC from diarrheic piglets ( 61 , 62 ). In 2005, Harmsen et al. immunized a llama with F4ac fimbriae from the F4-positive (F4+) ETEC strain CVI-1000 and obtained a few monoclonal VHHs. However, the best monovalent VHH, K609, could not significantly reduce diarrhea and reduced piglet mortality was poor ( 63 ). In contrast, orally administration of the linker connected bivalent VHH could enhance the clearance of F4+ ETEC and decrease the number of infected piglets ( 64 ). The different in vivo activity of mono- and bivalent VHH suggested that the ability to agglutinate bacteria may have a higher impact on infection, consistent with other studies where only bivalent antibodies showed in vivo protection ( 65 – 67 ). In another study, Moonens et al. ( 59 ) fused four different variable domains of llama heavy chain-only antibodies (V1-4), raised against F4ac, to the Fc domain of a porcine immunoglobulin IgA. These four different VHH targeted conserved epitopes of FaeG, a major adhesive subunit of F4. The four VHHs were fused to porcine IgA-Fc and subsequently expressed in Arabidopsis thaliana seeds to feed piglets. The oral feed-based passive immunization strategy protected piglets as demonstrated by (i) the progressive decline in shedding of F4 positive ETEC bacteria, (ii) the significantly lower immune responses of the piglets to F4 fimbriae, which suggest a reduced exposure to the ETEC pathogen, and (iii) a significantly higher body weight in comparison with control piglets ( 63 , 68 ). The structural study of V1-4 in complex with FaeG indicated that they sterically hindered FaeG associating with the F4 receptor but they did not directly interfere with the carbohydrate binding site ( 59 ). Besides F4+ ETEC, four VHHs targeting F18 fimbriae FedF domain were generated by llama immunization and selection as well. They could inhibit F18+ ETEC attaching to piglet enterocytes in vitro , and either sterically hinder or induce conformational changes of the binding surface of FedF ( 69 ). In a recent study, Amcheslavsky ( 60 ) and his colleagues immunized two male llamas with N-terminal fragments of eight class-5 ETEC adhesins to generate nanobodies with broad cross-reactivity against ETEC adhesins. They identified single nanobodies that show cross protective potency against eleven major pathogenic ETEC strains in vitro and inhibited ETEC colonization in vivo . Molecular docking and mutagenesis analysis revealed that nanobodies recognized a highly conserved epitope within the putative receptor binding region of ETEC adhesins ( 60 ). Shiga toxin-producing Escherichia coli Shiga toxin-producing Escherichia coli (STEC) are a subset of E. coli pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome (HUS) and even death. Shiga toxins, the main virulence factors are divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans and newly weaned pigs ( 70 ). Stx2e consists of an enzymatically active A subunit and five B subunits that bind to globotriaosylceramide (Gb3) on host cells ( 71 ). Lo et al. reported the discovery and characterization of a VHH, NbStx2e1, isolated from a llama phage display library that confers potent neutralizing capacity against Stx2e toxin. Structural analysis revealed that for each B subunit of Stx2e, one NbStx2e1 is interacting in a head-to-head orientation and directly competing with the glycolipid receptor binding site on the surface of the B subunit. The neutralizing NbStx2e1 can be used to prevent or treat edema disease in the future. Tremblay et al. immunized llama with Stx1 and 2 together and identified a panel of neutralizing VHHs, two of which demonstrated cross activity to Stx1 and 2 ( 72 ). A VHH heterodimer consisting of one Stx1-specific VHH/Stx2-specific VHH, and one Stx1/Stx2 cross-specific VHH, significantly improved the survival and reduced the kidney damage of mice challenged with Stx1 or 2. In addition, co-administration of the heterodimeric VHH with an effector Ab that binds to the VHH heterodimer, was effective in preventing all symptoms of intoxication from Stx1 and Stx2. In 2016, Mejías and his colleagues reported the generation of a family of Stx2B-binding VHHs that neutralize Stx2 in vitro at a nanomolar to subnanomolar range. The anti-Stx2B VHH, 2vb27, was selected and two copies were fused to an anti-human serum albumin VHH. This engineered antibody showed increased retention in circulation and was able to neutralize Stx2 in three different mouse models. This novel and simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome ( 73 ). In another study, Navarro et al. described the identification and characterization of a nanobody (Nb113) with the potential to neutralize the Stx2a and Stx2c toxins that are associated with human clinical infections. The crystal structural study revealed that each B subunit in the pentameric B5 ring is associated with a single Nb113 molecule. A detailed analysis of the epitope targeted by Nb113 suggests that this Nb prevents the formation of the Stx2a–Gb3 complex, thereby impeding the subsequent steps of the internalization and enzymatic activity of the Stx2a holotoxin ( 70 ). Besides Stx2-neutralizing VHHs, two VHHs were identified from immunized llama for detection of Stx2 using ELISA, which was even more sensitive than commercial ELISA kits ( 74 ). The ELISA was best for the major subtype Stx2a and less sensitive for Stx2f. VHH based ELISA is expected to be more cost effective than IgG ELISA. Other Gram- negative bacteria Pseudomonas aeruginosa is one of the leading causes of hospital-acquired infections. It is difficult to treat the infections due to the high intrinsic antibiotic resistance and the organism's capability to occur in biofilms in the host. Adams et al. immunized a llama with P. aeruginosa antigens and identified monoclonal anti-flagellin VHHs. In an in vitro assay, they showed that the anti-flagellin VHHs are capable of inhibiting P. aeruginosa from swimming and that they prevented biofilm formation ( 75 ). Helicobacter pylori infection is associated with gastritis, gastric and duodenal ulcers, and even gastric adenocarcinoma. It is important to seek alternative therapeutic strategies due to the increasing occurrence of antibiotic resistance. Some studies reported the isolation and purification of nanobodies with high affinity against UreC subunit of urease enzyme from H. pylori . These nanobodies could be a novel class of treatments against H. pylori infection ( 76 , 77 ). The sdAbs employed for diagnosis and neutralization of Gram- negative bacterial infection are listed in Table 2 . Table 2 SdAb reports to diagnose and/or neutralizing infections by Gram-negative bacteria. Nanobody Source Target Structure (IC50)/KD Function Diagnosis/Neutralizing Ref. K609 Immune library ETEC F4 fimbriae – – prevented F4+ ETEC attachment Neutralizing ( 63 ) V1 V2 V3 – ETEC F4 FaeG 4WEM 4WEN 4WEU 0.1 to 7.7 µM prevent F4+ ETEC attachment Neutralizing ( 59 ) NbFedF6 NbFedF7 NbFedF9 Immune library ETEC F18 FedF 4W6W 4W6X 4W6Y – inhibit F18+ ETEC attachment Neutralizing ( 69 ) 2R215 2R23 naive library ETEC CfaE – 0.4125 to 13.3 µM(IC100) broad cross-protection against 11 major disease causing ETEC strains and prevented colonization in vivo Neutralizing ( 60 ) 1D7 1H4 Immune library ETEC CfaE – – prevented bacterial colonization in animals. Neutralizing ( 60 ) NbStx2e1 Immune library STEC Stx2e 4P2C 8 nM direct interaction with the Stx2e B subunit binding site for glycolipid, thereby impeding toxin-host cell receptor contacts Neutralizing ( 71 ) 2VB27 Immune library STEC Stx2B neutralized Stx2 in vitro at subnanomolar concentrations Neutralizing ( 73 ) Nb113 Immune library STEC rStx2aB 6FE4 9.6 nM neutralized Stx2a by competing for the Gb3 receptor Neutralizing ( 70 ) Stx-A4 Stx-A5 Immune library STEC Stx1/Stx2 – 7.2-12.5 nM neutralized Stx1 and Stx2 and prevented all symptoms of intoxication from Stx1 and Stx2 Neutralizing ( 72 ) 1vb1- 2vb10 2vb21-2vb10 Immune library STEC Stx2 – – early detection of STEC infections Diagnosis ( 74 ) 7G 9D Immune library P. aeruginosa flagellum – 2.5 nM 4.7 nM inhibit P. aeruginosa from swimming and prevent biofilm formation in vitro Neutralizing ( 75 ) nanobody against UreC Immune library UreC – 0.05nM bind to UreC and inhibit urease activity Neutralizing ( 76 ) HMR23 Immune library UreC – 0.0263nM bind to UreC and inhibit urease activity Neutralizing ( 77 ) Single Domain Antibody usage for diagnosis and neutralization of Gram-positive bacterial infection Clostridium difficile Clostridium difficile is an opportunistic pathogen residing in the gastrointestinal tract of humans, causing antibiotic-associated diarrhea and pseudomembranous colitis ( 78 ). Antibiotics metronidazole and/or vancomycin are the primary treatment for C. difficile -associated disease (CDI) and surgeries are often required in the case of fulminant CDI ( 79 ). Due to the difficulties of treatment and high rates of recurrence, it's necessary to explore new therapeutic agents ( 80 ). The Gram-positive bacterium produces two large clostridial exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for CDI and are potential targets for CDI therapy ( 81 ). TcdA and TcdB are homologous to each other, having a similar domain organization including glucosyltransferase domain (GTD), cysteine protease domain (CPD), delivery and receptor binding domain (RBD) and combined repetitive oligopeptide domain (CROPs) ( 82 , 83 ). In 2011, Hussack and his colleagues isolated after phage display from an immune sdAb llama library four VHHs specifically targeting partial CROPs of TcdA or TcdB. In vitro assay on fibroblast cells demonstrated potent protection from the cytopathic effects of toxin A by these VHHs. Moreover, the protection efficiency was further enhanced when VHHs were administered in a manner of paired or triplet combinations ( 81 ). In another study, they characterized a panel of VHHs against partial RBD and CROPs of TcdB. Unfortunately, none of these VHHs exhibited inhibitory effects against TcdB cytotoxicity in a cell-based assay, given that several VHHs showed high affinity to toxin. This incapability of neutralization is probably due to TcdB accepting multiple proteins as receptors ( 84 – 86 ) and blockage of a single epitope might not be effective inhibition of TcdB toxicity. Nevertheless, when bivalent VHHs fused to the Fc fragment, their neutralization efficiency reached to the level of the recently approved anti-toxin B monoclonal antibody, bezlotoxumab ( 87 ). Furthermore, VHHs targeting different vulnerable regions on TcdB were also developed. SdAb named E3, 7F and 5D were demonstrated to bind with GTD, the connecting region between GTD and CPD, and RBD, respectively. Among which, E3 showed the best inhibition of TcdB cytotoxicity ( 88 , 89 ). Yang and his colleagues created a tetravalent and bispecific antibody called "ABA" which comprised two VHHs against both, TcdA and TcdB. ABA was capable of binding to both toxins simultaneously and neutralizing toxins from clinical C. difficile isolates . Therefore, ABA showed a significantly enhanced neutralizing activity both in vitro and in vivo ( 90 ). Schimdt and colleagues constructed a heteromultimeric VHH-based neutralizing agent, which potently neutralized both C.difficile toxins in cell assays and protected animals from CDI to different extents ( 88 ). In addition to development of VHHs, strategies to administer VHHs were also explored. For example, adenovirus, engineered Lactobacillus and probiotic Saccharomyces boulardii, expressing different forms of VHHs, were utilized to treat CDI effectively in animal models and proved to be promiscuous for combating the diseases invoked by C. difficile ( 91 – 93 ). Beside TcdA and TcdB, surface layer proteins (SLPs), mediating adherence to host cells, represents an alternative target for CDI treatment. Kandalaft and his colleagues used SLPs isolated from C. difficile hypervirulent strain QCD32g58 (027 ribotype) to immunize a llama and identified a panel of SLP-specific VHHs, which exhibited inhibition of C. difficile QCD32g58 motility in vitro . Therefore, targeting SLPs with VHHs may be a viable therapeutic approach against CDI ( 94 ). Bacillus anthracis Anthrax is a severe and fatal disease caused by the Gram-positive Bacillus anthracis . Anthrax toxin is a mixture of one non-toxic protein, protective antigen (PA) and two toxins, edema factor (EF) and lethal factor (LF). Protective antigen (PA) could bind to anthrax toxin receptors on cell surface forming oligomer pore and translocate the lethal factor (LF) and edema factor (EF) into the cytosol to take effects ( 95 ). In 2015, Moayeri and his colleagues identified two classes VHHs (JIK-B8 and JKH-C7) targeting two epitopes of PA from immunized alpacas. The two VHHs were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA) and displayed improved neutralizing potency in in vitro and in vivo assays compared with monomeric VHH ( 96 ). In another study, they used a gene therapy approach using recombinant replication-incompetent human adenovirus serotype 5 (Ad5) vector to express and secret the VNA (Ad/VNA2-PA) into the serum, and found that it can protect mice against an anthrax toxin challenge and anthrax spore infection ( 97 ). Apart from PA, the same group identified a set of 15 VHHs against EF and/or LF. Six of these VHHs were cross-reactive with both, EF and LF N-terminal domain, which is responsible for association with PA. Unlike the other selected VHHs, one LF specific VHH bound the C-terminal of LF inhibiting its enzymatic activity. Two bispecific heterodimers of the selected neutralizing VHHs demonstrated full protection against lethal anthrax spore infection ( 98 ). The cell surface of B. anthracis is covered by a protective surface layer or S-layer, composed of the highly-conserved S-layer protein (Sap). S-layers are proposed to function (i) as exoskeletons, (ii) as protection against harmful environments, (iii) as scaffolding structures for surface-localized enzymes and adhesins, (iv) as molecular sieves for nutrient uptake and (v) as a contact zone with the extracellular environment, including host cells in case of pathogenic bacteria ( 99 ). Fioravanti et al. generated Sap self-assembly inhibiting nanobodies, which exhibited disruption of the S-layer and attenuated the bacterial growth. Subcutaneous injection of the Sap inhibiting nanobodies cleared anthrax infection and prevented death in a mouse model of anthrax ( 100 ). Clostridium Botulinum Botulinum neurotoxins (BoNTs) are a category of bacterial toxins produced by Clostridium Botulinum and related strains, they are dangerous potential bioterrorism agents (Category A and Tier 1 select agent) ( 101 ). BoNTs cause a life-threatening disease called botulism, which develops flaccid paralysis and autonomic dysfunctions. Once infected, patients have to stay in the intensive care unit (ICU) and rely on mechanical ventilation for weeks to months, which is costly and time consuming ( 102 ). There are seven known serotypes of BoNTs (BoNT/A to BoNT/G), in which serotypes A, B and E are often associated with human botulism ( 103 ). Currently, antitoxins such as equine antitoxin and human botulism immunoglobulin represent the main strategy for treatment. However, adverse reactions, including early anaphylactic shock and late serum sickness, have been reported ( 103 ), which poses the necessity for developing new therapeutics to treat botulism. To this end, nanobodies could play an important role in such tasks. For this purpose, a variety of VHHs against BoNT/A were generated in the past years from phage or yeast display libraries derived from camel, alpaca and llama, respectively. Thanongsaksrikul et al. reported a neutralizing nanobody, VHH17, binding specifically to the catalytic cleft in light chain of BoNT/A via its CDR2 region, which is inaccessible to conventional antibodies due to their large size ( 104 ). In a similar study, Dong et al. identified a VHH Aa1 using yeast display. Rather than binding to the catalytic site of BoNT/A, Aa1 targeted the non-catalytic α-exosite binding region and inhibited enzyme activity of the toxin. Besides, Aa1 exhibited extraordinary thermal and reducing stability, which is optimal for therapeutic purposes ( 105 ). Tremblay and colleagues identified and characterized two VHHs ALc-B8 and ALc-H7 having affinity up to the nanomolar level to the light chain of BoNT/A. They further confirmed that ALc-B8 was able to inhibit SNAP-25 proteolysis in neuronal cells intoxicated by BoNT/A ( 106 ), which demonstrated its potential for therapy. In a recent study, Lam et al. discussed the inhibitory mechanism of VHHs against BoNT/A light chain via structural studies and found that the recognized epitopes of the light chain are quite conserved across different subtypes, laying the foundation for structure-based drug design ( 107 , 108 ). Besides the protease domain, VHHs such as ciA-C2, specifically recognizing the receptor binding domain of BoNT/A were also identified and proven to exert an inhibitory function ( 109 ). Furthermore, various strategies to enhance the efficacy of VHHs neutralization of BoNT/A have been exploited, such as (i) tagging the VHHs for better and faster clearance of bound toxin ( 110 ), (ii) fusing the VHHs with human Fc fragment or Glycophorin A on red blood cell surface to increase their circulation half-life ( 111 , 112 ), or (iii) expressing VHHs in replication-incompetent adenovirus to provide prolonged protection ( 113 ). With similar strategies, several VHHs bound to BoNT/E were also produced and characterized. Bakherad et al. selected a VHH, BMR2, specifically targeting the receptor binding domain of BoNT/E, which completely neutralized 3LD 50 of BoNT/E in mice ( 103 ). Lately, Tremblay et al. identified plenty of BoNT/E-neutralizing VHHs and Lam et al. characterized two of them, JLE-E5 and JLE-E9, targeting the translocation domain of BoNT/E. They confirmed that these two VHHs blocked a structural change of BoNT/E in acidic pH, a process necessary for its biological function, which could hamper toxicity of BoNT/E ( 114 ). The pitfall to treat botulism is that no drugs are able entering into neurons to take effect once the toxins are endocytosed. A hallmark application of VHH for treating botulism was to deliver VHHs into neural cells by coupling them to intoxicated BoNTs. Utilizing this strategy, two independent groups successfully delivered VHHs into neurons and provided animals with full recovery from botulism, which opened new avenues of using VHHs to treat diseases ( 115 , 116 ). Other Gram-positive bacteria In addition to the bacteria mentioned above, nanobodies also play an important role in the diagnosis and therapy of other bacteria. Nanobodies can also be used to establish immuno-assays to uncover bacteria contaminations in foods. Staphylococcus aureus is one of the most common food-borne pathogens. Hu et al. selected a specific nanobody Nb147 to develop an immuno-assay detecting S. aureus in milk ( 117 ). Staphylococcal enterotoxins (SEs) are the major causes of staphylococcal food poisoning (SFP) and various other diseases. Ji et al. developed a double nanobody-based sandwich immunoassay for the detection of staphylococcal enterotoxin C in dairy products ( 118 ) while Zanganeh et al. developed a rapid and sensitive detection of staphylococcal enterotoxin B by recombinant nanobodies ( 119 ). Listeria monocytogenes (LM) causes listeriosis, a potentially fatal food-borne disease especially harmful to pregnant women. Tu and his colleagues developed an ELISA using the VHH clone L5-79 and a monoclonal antibody to detect LM in pasteurized milk ( 120 ). King et al. identified a group of VHHs targeting internalin B (InlB) of LM which were competitive inhibitors preventing bacterial invasion. These results point to the potential of VHH as a novel class of therapeutics for the prevention of listeriosis ( 121 ). The sdAbs applications to diagnose and neutralize Gram- positive bacterial infection are overviewed in Table 3 . Table 3 SdAb reports to diagnosis and neutralization of infection by Gram-positive bacteria. Nanobody Source Target Structure (IC50)/KD Function Diagnosis/Neutralizing Ref. A4.2 A5.1 A20.1 A26.8 Immune library CD TcdA – – neutralized toxin A by binding to sites other than the carbohydrate binding pocket of the toxin Neutralizing ( 81 ) B39 B69 B71 B74 B94 B131 B167 Immune library CD TcdB – – neutralized toxin B when formatted as bivalent VHH-Fc fusions Neutralizing ( 87 ) 5D,E3,7F Immune library CD TcdB 6oQ6 6oQ7 6oQ8 – neutralized toxin B Neutralizing ( 89 ) ABA Immune library CD TcdA TcdB – – bound to both toxins simultaneously and displayed a significantly enhanced neutralizing activity both in vitro and in vivo Neutralizing ( 90 ) SLP-VHH Immune library CD-SLP – – bound SLPs with high affinity bloking the adherence to host cells Neutralizing ( 94 ) VNA2-PA Immune library Bacillus anthracis PA – – displayed improved neutralizing potency in vitro and in vivo than the separate component VHHs Neutralizing ( 96 ) ( 97 ) JMN-D10 JMO-G1 Immune library Bacillus anthracis EF/LF – – block binding of EF/LF to the protective antigen C-terminal binding interface and preventing toxin entry into the cell Neutralizing ( 98 ) Nbs-NbAF684 nbaf694 Immune library Bacillus anthracis SAP – – prevented the assembly of Sap and depolymerized existing Sap S-layers Neutralizing ( 100 ) VHH17 naive library BoNTs BoTxA/ LC – 11.6nm neutralized the SNAP25 hydrolytic activity of BoTxA/LC Neutralizing ( 104 ) BMR2 Immune library BONT/E HC – – neutralized BoNT/E Neutralizing ( 103 ) Aa1 naive library BONT/A-LC 3K3Q 4.7×10 -10 M targeted the non-catalytic α-exosite binding region and inhibited enzyme activity of toxin Neutralizing ( 105 ) ALc-B8 ALc-H7 Immune library BONT/A-LC – – neutralized BoNT/A-LC and inhibit SNAP-25 proteolysis in neuronal cells Neutralizing ( 106 ) JLK-G12 JLO-G11 JLI-G10 JLI-H11 Immune library BONT/B-HC 6UFT 6UL4 6UHT 6UC6 – block BoNT/B1 binding to host receptors Neutralizing ( 108 ) ciA-B5 ciA-H7 ciA-C2 Immune library BONT/A1- HN LC HC 6UL6 6UI1 5L21 – block membrane insertion of boNT/A1 translocation domain, interfere with the unfolding of the protease domain, block host receptor binding Neutralizing ( 108 , 109 ) B11 G3 Immune library BoNT/A – – neutralized BoNT/A Neutralizing ( 111 ) H7/B5/ABP Immune library BoNT/A – <3 nM neutralized BoNT/A Neutralizing ( 122 ) ( 113 ) ( 112 ) JLE-E5 JLE-E9 Immune library BoNT/E1 7K84 7K7Y – block membrane association of BoNT/E1 Neutralizing ( 114 ) A8-J10-ciBoNT/XA Immune library BoNT/A BoNT/B – – neutralize both BoNT/A and BoNT/B Neutralizing ( 115 ) Nb147 Immune library S. aureus – – screen for S. aureus contaminations in foods Diagnosis ( 117 ) C6 C11 Immune library SEC – – detected SEC in dairy products Diagnosis ( 118 ) nanobody against SEB Immune library SEB – – detected SEB in suspicious foods Diagnosis ( 119 ) L5-78 L5-79 naive library LM – – detected foodborne LM in food Diagnosis ( 120 ) R303 R330 R326 naive library LM InlB 6DBA 6DBE 6DBD – bound at the c-Met interaction site on InlB and preventing bacterial invasion Neutralizing ( 121 ) Clostridium difficile Clostridium difficile is an opportunistic pathogen residing in the gastrointestinal tract of humans, causing antibiotic-associated diarrhea and pseudomembranous colitis ( 78 ). Antibiotics metronidazole and/or vancomycin are the primary treatment for C. difficile -associated disease (CDI) and surgeries are often required in the case of fulminant CDI ( 79 ). Due to the difficulties of treatment and high rates of recurrence, it's necessary to explore new therapeutic agents ( 80 ). The Gram-positive bacterium produces two large clostridial exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for CDI and are potential targets for CDI therapy ( 81 ). TcdA and TcdB are homologous to each other, having a similar domain organization including glucosyltransferase domain (GTD), cysteine protease domain (CPD), delivery and receptor binding domain (RBD) and combined repetitive oligopeptide domain (CROPs) ( 82 , 83 ). In 2011, Hussack and his colleagues isolated after phage display from an immune sdAb llama library four VHHs specifically targeting partial CROPs of TcdA or TcdB. In vitro assay on fibroblast cells demonstrated potent protection from the cytopathic effects of toxin A by these VHHs. Moreover, the protection efficiency was further enhanced when VHHs were administered in a manner of paired or triplet combinations ( 81 ). In another study, they characterized a panel of VHHs against partial RBD and CROPs of TcdB. Unfortunately, none of these VHHs exhibited inhibitory effects against TcdB cytotoxicity in a cell-based assay, given that several VHHs showed high affinity to toxin. This incapability of neutralization is probably due to TcdB accepting multiple proteins as receptors ( 84 – 86 ) and blockage of a single epitope might not be effective inhibition of TcdB toxicity. Nevertheless, when bivalent VHHs fused to the Fc fragment, their neutralization efficiency reached to the level of the recently approved anti-toxin B monoclonal antibody, bezlotoxumab ( 87 ). Furthermore, VHHs targeting different vulnerable regions on TcdB were also developed. SdAb named E3, 7F and 5D were demonstrated to bind with GTD, the connecting region between GTD and CPD, and RBD, respectively. Among which, E3 showed the best inhibition of TcdB cytotoxicity ( 88 , 89 ). Yang and his colleagues created a tetravalent and bispecific antibody called "ABA" which comprised two VHHs against both, TcdA and TcdB. ABA was capable of binding to both toxins simultaneously and neutralizing toxins from clinical C. difficile isolates . Therefore, ABA showed a significantly enhanced neutralizing activity both in vitro and in vivo ( 90 ). Schimdt and colleagues constructed a heteromultimeric VHH-based neutralizing agent, which potently neutralized both C.difficile toxins in cell assays and protected animals from CDI to different extents ( 88 ). In addition to development of VHHs, strategies to administer VHHs were also explored. For example, adenovirus, engineered Lactobacillus and probiotic Saccharomyces boulardii, expressing different forms of VHHs, were utilized to treat CDI effectively in animal models and proved to be promiscuous for combating the diseases invoked by C. difficile ( 91 – 93 ). Beside TcdA and TcdB, surface layer proteins (SLPs), mediating adherence to host cells, represents an alternative target for CDI treatment. Kandalaft and his colleagues used SLPs isolated from C. difficile hypervirulent strain QCD32g58 (027 ribotype) to immunize a llama and identified a panel of SLP-specific VHHs, which exhibited inhibition of C. difficile QCD32g58 motility in vitro . Therefore, targeting SLPs with VHHs may be a viable therapeutic approach against CDI ( 94 ). Bacillus anthracis Anthrax is a severe and fatal disease caused by the Gram-positive Bacillus anthracis . Anthrax toxin is a mixture of one non-toxic protein, protective antigen (PA) and two toxins, edema factor (EF) and lethal factor (LF). Protective antigen (PA) could bind to anthrax toxin receptors on cell surface forming oligomer pore and translocate the lethal factor (LF) and edema factor (EF) into the cytosol to take effects ( 95 ). In 2015, Moayeri and his colleagues identified two classes VHHs (JIK-B8 and JKH-C7) targeting two epitopes of PA from immunized alpacas. The two VHHs were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA) and displayed improved neutralizing potency in in vitro and in vivo assays compared with monomeric VHH ( 96 ). In another study, they used a gene therapy approach using recombinant replication-incompetent human adenovirus serotype 5 (Ad5) vector to express and secret the VNA (Ad/VNA2-PA) into the serum, and found that it can protect mice against an anthrax toxin challenge and anthrax spore infection ( 97 ). Apart from PA, the same group identified a set of 15 VHHs against EF and/or LF. Six of these VHHs were cross-reactive with both, EF and LF N-terminal domain, which is responsible for association with PA. Unlike the other selected VHHs, one LF specific VHH bound the C-terminal of LF inhibiting its enzymatic activity. Two bispecific heterodimers of the selected neutralizing VHHs demonstrated full protection against lethal anthrax spore infection ( 98 ). The cell surface of B. anthracis is covered by a protective surface layer or S-layer, composed of the highly-conserved S-layer protein (Sap). S-layers are proposed to function (i) as exoskeletons, (ii) as protection against harmful environments, (iii) as scaffolding structures for surface-localized enzymes and adhesins, (iv) as molecular sieves for nutrient uptake and (v) as a contact zone with the extracellular environment, including host cells in case of pathogenic bacteria ( 99 ). Fioravanti et al. generated Sap self-assembly inhibiting nanobodies, which exhibited disruption of the S-layer and attenuated the bacterial growth. Subcutaneous injection of the Sap inhibiting nanobodies cleared anthrax infection and prevented death in a mouse model of anthrax ( 100 ). Clostridium Botulinum Botulinum neurotoxins (BoNTs) are a category of bacterial toxins produced by Clostridium Botulinum and related strains, they are dangerous potential bioterrorism agents (Category A and Tier 1 select agent) ( 101 ). BoNTs cause a life-threatening disease called botulism, which develops flaccid paralysis and autonomic dysfunctions. Once infected, patients have to stay in the intensive care unit (ICU) and rely on mechanical ventilation for weeks to months, which is costly and time consuming ( 102 ). There are seven known serotypes of BoNTs (BoNT/A to BoNT/G), in which serotypes A, B and E are often associated with human botulism ( 103 ). Currently, antitoxins such as equine antitoxin and human botulism immunoglobulin represent the main strategy for treatment. However, adverse reactions, including early anaphylactic shock and late serum sickness, have been reported ( 103 ), which poses the necessity for developing new therapeutics to treat botulism. To this end, nanobodies could play an important role in such tasks. For this purpose, a variety of VHHs against BoNT/A were generated in the past years from phage or yeast display libraries derived from camel, alpaca and llama, respectively. Thanongsaksrikul et al. reported a neutralizing nanobody, VHH17, binding specifically to the catalytic cleft in light chain of BoNT/A via its CDR2 region, which is inaccessible to conventional antibodies due to their large size ( 104 ). In a similar study, Dong et al. identified a VHH Aa1 using yeast display. Rather than binding to the catalytic site of BoNT/A, Aa1 targeted the non-catalytic α-exosite binding region and inhibited enzyme activity of the toxin. Besides, Aa1 exhibited extraordinary thermal and reducing stability, which is optimal for therapeutic purposes ( 105 ). Tremblay and colleagues identified and characterized two VHHs ALc-B8 and ALc-H7 having affinity up to the nanomolar level to the light chain of BoNT/A. They further confirmed that ALc-B8 was able to inhibit SNAP-25 proteolysis in neuronal cells intoxicated by BoNT/A ( 106 ), which demonstrated its potential for therapy. In a recent study, Lam et al. discussed the inhibitory mechanism of VHHs against BoNT/A light chain via structural studies and found that the recognized epitopes of the light chain are quite conserved across different subtypes, laying the foundation for structure-based drug design ( 107 , 108 ). Besides the protease domain, VHHs such as ciA-C2, specifically recognizing the receptor binding domain of BoNT/A were also identified and proven to exert an inhibitory function ( 109 ). Furthermore, various strategies to enhance the efficacy of VHHs neutralization of BoNT/A have been exploited, such as (i) tagging the VHHs for better and faster clearance of bound toxin ( 110 ), (ii) fusing the VHHs with human Fc fragment or Glycophorin A on red blood cell surface to increase their circulation half-life ( 111 , 112 ), or (iii) expressing VHHs in replication-incompetent adenovirus to provide prolonged protection ( 113 ). With similar strategies, several VHHs bound to BoNT/E were also produced and characterized. Bakherad et al. selected a VHH, BMR2, specifically targeting the receptor binding domain of BoNT/E, which completely neutralized 3LD 50 of BoNT/E in mice ( 103 ). Lately, Tremblay et al. identified plenty of BoNT/E-neutralizing VHHs and Lam et al. characterized two of them, JLE-E5 and JLE-E9, targeting the translocation domain of BoNT/E. They confirmed that these two VHHs blocked a structural change of BoNT/E in acidic pH, a process necessary for its biological function, which could hamper toxicity of BoNT/E ( 114 ). The pitfall to treat botulism is that no drugs are able entering into neurons to take effect once the toxins are endocytosed. A hallmark application of VHH for treating botulism was to deliver VHHs into neural cells by coupling them to intoxicated BoNTs. Utilizing this strategy, two independent groups successfully delivered VHHs into neurons and provided animals with full recovery from botulism, which opened new avenues of using VHHs to treat diseases ( 115 , 116 ). Other Gram-positive bacteria In addition to the bacteria mentioned above, nanobodies also play an important role in the diagnosis and therapy of other bacteria. Nanobodies can also be used to establish immuno-assays to uncover bacteria contaminations in foods. Staphylococcus aureus is one of the most common food-borne pathogens. Hu et al. selected a specific nanobody Nb147 to develop an immuno-assay detecting S. aureus in milk ( 117 ). Staphylococcal enterotoxins (SEs) are the major causes of staphylococcal food poisoning (SFP) and various other diseases. Ji et al. developed a double nanobody-based sandwich immunoassay for the detection of staphylococcal enterotoxin C in dairy products ( 118 ) while Zanganeh et al. developed a rapid and sensitive detection of staphylococcal enterotoxin B by recombinant nanobodies ( 119 ). Listeria monocytogenes (LM) causes listeriosis, a potentially fatal food-borne disease especially harmful to pregnant women. Tu and his colleagues developed an ELISA using the VHH clone L5-79 and a monoclonal antibody to detect LM in pasteurized milk ( 120 ). King et al. identified a group of VHHs targeting internalin B (InlB) of LM which were competitive inhibitors preventing bacterial invasion. These results point to the potential of VHH as a novel class of therapeutics for the prevention of listeriosis ( 121 ). The sdAbs applications to diagnose and neutralize Gram- positive bacterial infection are overviewed in Table 3 . Table 3 SdAb reports to diagnosis and neutralization of infection by Gram-positive bacteria. Nanobody Source Target Structure (IC50)/KD Function Diagnosis/Neutralizing Ref. A4.2 A5.1 A20.1 A26.8 Immune library CD TcdA – – neutralized toxin A by binding to sites other than the carbohydrate binding pocket of the toxin Neutralizing ( 81 ) B39 B69 B71 B74 B94 B131 B167 Immune library CD TcdB – – neutralized toxin B when formatted as bivalent VHH-Fc fusions Neutralizing ( 87 ) 5D,E3,7F Immune library CD TcdB 6oQ6 6oQ7 6oQ8 – neutralized toxin B Neutralizing ( 89 ) ABA Immune library CD TcdA TcdB – – bound to both toxins simultaneously and displayed a significantly enhanced neutralizing activity both in vitro and in vivo Neutralizing ( 90 ) SLP-VHH Immune library CD-SLP – – bound SLPs with high affinity bloking the adherence to host cells Neutralizing ( 94 ) VNA2-PA Immune library Bacillus anthracis PA – – displayed improved neutralizing potency in vitro and in vivo than the separate component VHHs Neutralizing ( 96 ) ( 97 ) JMN-D10 JMO-G1 Immune library Bacillus anthracis EF/LF – – block binding of EF/LF to the protective antigen C-terminal binding interface and preventing toxin entry into the cell Neutralizing ( 98 ) Nbs-NbAF684 nbaf694 Immune library Bacillus anthracis SAP – – prevented the assembly of Sap and depolymerized existing Sap S-layers Neutralizing ( 100 ) VHH17 naive library BoNTs BoTxA/ LC – 11.6nm neutralized the SNAP25 hydrolytic activity of BoTxA/LC Neutralizing ( 104 ) BMR2 Immune library BONT/E HC – – neutralized BoNT/E Neutralizing ( 103 ) Aa1 naive library BONT/A-LC 3K3Q 4.7×10 -10 M targeted the non-catalytic α-exosite binding region and inhibited enzyme activity of toxin Neutralizing ( 105 ) ALc-B8 ALc-H7 Immune library BONT/A-LC – – neutralized BoNT/A-LC and inhibit SNAP-25 proteolysis in neuronal cells Neutralizing ( 106 ) JLK-G12 JLO-G11 JLI-G10 JLI-H11 Immune library BONT/B-HC 6UFT 6UL4 6UHT 6UC6 – block BoNT/B1 binding to host receptors Neutralizing ( 108 ) ciA-B5 ciA-H7 ciA-C2 Immune library BONT/A1- HN LC HC 6UL6 6UI1 5L21 – block membrane insertion of boNT/A1 translocation domain, interfere with the unfolding of the protease domain, block host receptor binding Neutralizing ( 108 , 109 ) B11 G3 Immune library BoNT/A – – neutralized BoNT/A Neutralizing ( 111 ) H7/B5/ABP Immune library BoNT/A – <3 nM neutralized BoNT/A Neutralizing ( 122 ) ( 113 ) ( 112 ) JLE-E5 JLE-E9 Immune library BoNT/E1 7K84 7K7Y – block membrane association of BoNT/E1 Neutralizing ( 114 ) A8-J10-ciBoNT/XA Immune library BoNT/A BoNT/B – – neutralize both BoNT/A and BoNT/B Neutralizing ( 115 ) Nb147 Immune library S. aureus – – screen for S. aureus contaminations in foods Diagnosis ( 117 ) C6 C11 Immune library SEC – – detected SEC in dairy products Diagnosis ( 118 ) nanobody against SEB Immune library SEB – – detected SEB in suspicious foods Diagnosis ( 119 ) L5-78 L5-79 naive library LM – – detected foodborne LM in food Diagnosis ( 120 ) R303 R330 R326 naive library LM InlB 6DBA 6DBE 6DBD – bound at the c-Met interaction site on InlB and preventing bacterial invasion Neutralizing ( 121 ) Single Domain Antibodies against pattern recognition receptor Pattern recognition receptors (PRRs) are a class of receptors that play crucial roles in detecting conserved pathogen associated molecular patterns (PAMPs) shared among many microorganisms or endogenous damage-associated molecular patterns (DAMPs) to initiate downstream signaling ( 123 – 126 ). PRRs have been identified and are notably classified into the following families: Toll-like receptors (TLRs), the Ctype lectin receptors(CLRs), the nucleotide-binding oligomerisation (NOD)-like receptors (NLRs), the RIG-I-like receptors, the absent in melanoma 2 (AIM2)-like receptors and the OAS like receptors ( 127 – 130 ). PRRs connect PAMPs or DAMPs to trigger a variety of signal pathways, eventually activating interferon regulatory factor (IRFs), nuclear factor-kappa B (NF-κ B), mitogen-activated protein kinase (MAPKs) and etc., which promotes the expression of pro-inflammatory cytokines ( 131 – 133 ). The sdAbs against Pattern Recognition Receptor are listed in Table 4 . Table 4 Single Domain Antibody against Pattern Recognition Receptor. Nanobody Source Target Structure (IC50)/KD Function Diagnosis/Neutralizing Ref. nanobody against TLR4 Immune library TLR4 – – reduce the release of inflammatory factors and improve the survival rate of animals Neutralizing ( 134 ) Nb1.46 Nb2.22 Immune library Clec4F 7DJX 7DJY 0.2-2 nM structural and functional investigation and as molecular imaging and therapeutic agents Diagnosis Neutralizing ( 135 ) TLR4 Toll-like receptor 4 (TLR4) is a member of the TLR family, which participates in innate immunity and mediates inflammation by recognizing lipopolysaccharide (LPS) or bacterial endotoxin ( 125 , 136 , 137 ). Overactivation of TLR4 can trigger the production of various inflammatory factors, which are related to the occurrence and development of a series of diseases including sepsis ( 138 ), endotoxemia, pregnancy-related disorders ( 139 , 140 ), cardiovascular disease ( 141 , 142 ), intestinal inflammation ( 143 ), rheumatoid arthritis ( 144 ), acute kidney injury (AKI) ( 145 , 146 ), and acute lung injury ( 147 ). Therefore, the drug design and development for this target have high therapeutic potential and the anti-inflammatory effect of TLR4 inhibitors has been confirmed by several studies ( 148 – 150 ). Liao and his colleagues ( 134 ) identified an anti-TLR4 intermediate and C-terminal domain-recognizing nanobodies using phage display. Then, through in vitro and in vivo experiments, they confirmed that the anti-TLR4 nanobody can effectively reduce the release of inflammatory factors and improve the animal survival rate. The effect is even more pronounced when two different nanobodies are combined. Clec4f C-type lectins can recognize a variety of ligands and play an important role in a variety of physiological functions. Particularly, C-type lectins contribute to innate and adaptive antibacterial immune responses by recognizing surface polysaccharides of specific pathogens ( 151 ). Clec4f is a member of the type II C-type lectin family and is only expressed by Kupffer cells ( 152 – 154 ). In addition, studies have shown that Clec4f is involved in α-galactose ceramide presentation and Listeria monocytogenes infection in mouse liver ( 155 ). Zheng et al. developed a series of nanobodies from an alpaca immunized with recombinant mouse Kupffer cell receptor Clec4F by using a phage display. After bio-panning selections, they obtained 14 different nanobodies against Clec4F with an affinity ranging from 0.2 to 2 nM. Furthermore, they have characterized the structure of two Clec4F nanobodies, Nb1.46 and Nb2.22, with different CDR2 and CDR3 sequence features. These works may contribute to the study of Clec4F structure and function as well as its use as a molecular imaging agent and therapeutic agent ( 135 ). In another study, they indicated that Clec4F nanobodies could be used to track changes in Kupffer cell (KCs) dynamics in mice via non-invasive imaging ( 153 ). TLR4 Toll-like receptor 4 (TLR4) is a member of the TLR family, which participates in innate immunity and mediates inflammation by recognizing lipopolysaccharide (LPS) or bacterial endotoxin ( 125 , 136 , 137 ). Overactivation of TLR4 can trigger the production of various inflammatory factors, which are related to the occurrence and development of a series of diseases including sepsis ( 138 ), endotoxemia, pregnancy-related disorders ( 139 , 140 ), cardiovascular disease ( 141 , 142 ), intestinal inflammation ( 143 ), rheumatoid arthritis ( 144 ), acute kidney injury (AKI) ( 145 , 146 ), and acute lung injury ( 147 ). Therefore, the drug design and development for this target have high therapeutic potential and the anti-inflammatory effect of TLR4 inhibitors has been confirmed by several studies ( 148 – 150 ). Liao and his colleagues ( 134 ) identified an anti-TLR4 intermediate and C-terminal domain-recognizing nanobodies using phage display. Then, through in vitro and in vivo experiments, they confirmed that the anti-TLR4 nanobody can effectively reduce the release of inflammatory factors and improve the animal survival rate. The effect is even more pronounced when two different nanobodies are combined. Clec4f C-type lectins can recognize a variety of ligands and play an important role in a variety of physiological functions. Particularly, C-type lectins contribute to innate and adaptive antibacterial immune responses by recognizing surface polysaccharides of specific pathogens ( 151 ). Clec4f is a member of the type II C-type lectin family and is only expressed by Kupffer cells ( 152 – 154 ). In addition, studies have shown that Clec4f is involved in α-galactose ceramide presentation and Listeria monocytogenes infection in mouse liver ( 155 ). Zheng et al. developed a series of nanobodies from an alpaca immunized with recombinant mouse Kupffer cell receptor Clec4F by using a phage display. After bio-panning selections, they obtained 14 different nanobodies against Clec4F with an affinity ranging from 0.2 to 2 nM. Furthermore, they have characterized the structure of two Clec4F nanobodies, Nb1.46 and Nb2.22, with different CDR2 and CDR3 sequence features. These works may contribute to the study of Clec4F structure and function as well as its use as a molecular imaging agent and therapeutic agent ( 135 ). In another study, they indicated that Clec4F nanobodies could be used to track changes in Kupffer cell (KCs) dynamics in mice via non-invasive imaging ( 153 ). Conclusion and perspectives As bacterial antibiotic resistance is developed at increasing pace, there is a great urgency to develop a non-antibiotic approach to treat bacterial infections. SdAbs are versatile molecules with favorable properties representing an alternative tactic for both therapeutic and diagnostic applications in bacterial infections. SdAbs are characterized by minimal size, high stability, strong affinity, good solubility, and low immunogenicity which open pathways to target antigens that were previously inaccessible during bacterial infection. Therapeutic nanobodies are still in early phase development, however they have a promising future. The first therapeutic nanobody-based drug, Caplicizumab (Cablivi), was approved by EMA in August 2018 and by FDA in March 2019 for the treatment of blood clotting disorder. Since then, Ciltacabtagene autoleucel (Carvykti) a nanobody based Chimeric Antigen Receptor T cell (CAR-T)-based medication against relapsed or refractory multiple myeloma was approved by FDA and EMA (February and May, 2022) and Envafolimab, a subcutaneous injectable sdAb directed against PD-L1 (approved in November 2021) by the Chinese National Medical Products Administration (NMPA) for adult patients with microsatellite instability-high or mismatch repair deficient advanced solid tumors followed soon after. These successes demonstrate the flexibility in engineering and administration of sdAbs as well as the variety of diseases that can be tackled. It will probably not take long before sdAbs with their considerable potential as a diagnostic and therapeutic agent will enter the market for bacterial infectious diseases and will contribute to public health. Author contributions QQ, HL wrote the review under the supervision of YW and SZ. WH, YG, JZ, FZ, JS and SM made the figure, tables and revised the manuscript. All authors contributed to the article and approved the submitted version. Funding This work was supported by the National Natural Science Foundation of China (No. 31870132, No. 82072237), Shaanxi Province Natural Science Funding, and Institutional Foundation of the First Affiliated Hospital of Xi'an Jiaotong University. SZ was supported by Northwest A&F University Star-up Funding. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9010362/

Group 3 innate lymphocytes (ILC3s) upregulate IL-22 in response to elevated intracellular cAMP levels

Group 3 innate lymphocytes (ILC3s) are important immune cells within mucosal tissues and protect against bacterial infections. They can be activated in response to the innate cytokines IL-23 or IL-1β, which rapidly increases their production of effector molecules that regulate barrier functions. Pathogens can subvert these anti-bacterial effects to evade mucosal defenses to infect the host. Bacillus anthracis , the causative agent of anthrax, produces two major toxins that can modulate the immune response. We have previously shown that lethal toxin downmodulates the function of ILC3s. On the other hand, edema toxin has been shown promote T helper 17 (Th17) cell differentiation, adaptive counterparts of ILC3s, via elevation of cyclic AMP (cAMP). We hypothesized that edema toxin may also modulate ILC3 function. In this study, we show that edema toxin has the opposite effect of lethal toxin; edema toxin directly activates ILC3s independently of innate cytokine stimulation. Treatment of a mouse ILC3-like cell line with edema toxin, a potent adenylate cyclase, upregulated production of the cytokine IL-22, a major effector molecule of ILC3s and a critical factor in maintaining mucosal barriers. Forskolin treatment phenocopied the effect observed with edema toxin and led to an increase in CREB phosphorylation in ILC3s. This observation has potential implications for a role for cAMP signaling in the activation of ILC3s.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641822/

Proteomics identifies differences in fibrotic potential of extracellular vesicles from human tendon and muscle fibroblasts

Background Fibroblasts are the powerhouses responsible for the production and assembly of extracellular matrix (ECM). Their activity needs to be tightly controlled especially within the musculoskeletal system, where changes to ECM composition affect force transmission and mechanical loading that are required for effective movement of the body. Extracellular vesicles (EVs) are a mode of cell-cell communication within and between tissues, which has been largely characterised in cancer. However, it is unclear what the role of healthy fibroblast-derived EVs is during tissue homeostasis. Methods Here, we performed proteomic analysis of small EVs derived from primary human muscle and tendon cells to identify the potential functions of healthy fibroblast-derived EVs. Results Mass spectrometry-based proteomics revealed comprehensive profiles for small EVs released from healthy human fibroblasts from different tissues. We found that fibroblast-derived EVs were more similar than EVs from differentiating myoblasts, but there were significant differences between tendon fibroblast and muscle fibroblast EVs. Small EVs from tendon fibroblasts contained higher levels of proteins that support ECM synthesis, including TGFβ1, and muscle fibroblast EVs contained proteins that support myofiber function and components of the skeletal muscle matrix. Conclusions Our data demonstrates a marked heterogeneity among healthy fibroblast-derived EVs, indicating shared tasks between EVs of skeletal muscle myoblasts and fibroblasts, whereas tendon fibroblast EVs could play a fibrotic role in human tendon tissue. These findings suggest an important role for EVs in tissue homeostasis of both tendon and skeletal muscle in humans. Video abstract Supplementary information Supplementary information accompanies this paper at 10.1186/s12964-020-00669-9. Background Fibroblasts are the powerhouses responsible for the production and assembly of extracellular matrix (ECM). Their activity needs to be tightly controlled especially within the musculoskeletal system, where changes to ECM composition affect force transmission and mechanical loading that are required for effective movement of the body. Extracellular vesicles (EVs) are a mode of cell-cell communication within and between tissues, which has been largely characterised in cancer. However, it is unclear what the role of healthy fibroblast-derived EVs is during tissue homeostasis. Methods Here, we performed proteomic analysis of small EVs derived from primary human muscle and tendon cells to identify the potential functions of healthy fibroblast-derived EVs. Results Mass spectrometry-based proteomics revealed comprehensive profiles for small EVs released from healthy human fibroblasts from different tissues. We found that fibroblast-derived EVs were more similar than EVs from differentiating myoblasts, but there were significant differences between tendon fibroblast and muscle fibroblast EVs. Small EVs from tendon fibroblasts contained higher levels of proteins that support ECM synthesis, including TGFβ1, and muscle fibroblast EVs contained proteins that support myofiber function and components of the skeletal muscle matrix. Conclusions Our data demonstrates a marked heterogeneity among healthy fibroblast-derived EVs, indicating shared tasks between EVs of skeletal muscle myoblasts and fibroblasts, whereas tendon fibroblast EVs could play a fibrotic role in human tendon tissue. These findings suggest an important role for EVs in tissue homeostasis of both tendon and skeletal muscle in humans. Video abstract Supplementary information Supplementary information accompanies this paper at 10.1186/s12964-020-00669-9. Background Fibroblasts are cells responsible for producing extracellular matrix (ECM) and their activity is tightly regulated. For examples, too much or too little ECM causes fibrosis or tissue frailty, respectively, and changes to the ECM composition will affect both the mechanical properties and the biochemistry of the tissue. A relatively new mechanism by which fibroblasts can be regulated is via extracellular vesicles (EVs), which are cell-released lipid membrane encapsulated particles. The two most studied subtypes of EVs are exosomes, which are 50–150 nm vesicles that are derived from the endosomal pathway and microvesicles (sometimes referred to as ectosomes), which are from 100 nm up to 1 μm in size and are formed by direct budding of the plasma membrane [ 1 , 2 ]. Although they have different routes to their formation there are no specific protein markers to differentiate them [ 2 , 3 ]; Many marker proteins for exosomes are also present in microvesicles [ 2 ], which include tetraspannins (e.g. CD81), heat shock proteins, components of the endosomal sorting complexes required for transport (ESCRT), integrins and regulators of intracellular trafficking (e.g. Ras-associated binding proteins, annexins and clathrins). The molecular content of exosomes is, however, highly specific to the cell of origin and can be passed on to other cells as part of intra- and inter-tissue communication [ 4 – 7 ]. How exosomes and other EVs from healthy fibroblasts regulate tissue homeostasis is unknown and the lack of biomarkers makes them difficult to study. Multiple studies have demonstrated that exosomes and microvesicles are able to dock and fuse with cells to deliver functional protein cargo, as well as micro RNAs and messenger RNAs [ 2 , 3 , 5 ]. The most studied examples of exosome-mediated crosstalk with fibroblasts are in tumour growth and metastasis [ 8 , 9 ]. Exosomes produced by cancer cells activate stromal fibroblasts to become cancer-associated fibroblasts (CAFs) and exosomes released from CAFs induce metastatic properties in cancer cells lines and have also been demonstrated to reprogram stromal fibroblasts in the pre-metastatic niche [ 6 , 10 , 11 ]. Induction of the pro-tumour progression phenotype in normal stromal fibroblasts is attributed to transforming growth factor β1 (TGFβ1) that is localised inside CAF exosomes [ 6 , 11 ]. We hypothesised that EVs from healthy fibroblasts also regulates the ECM during tissue homeostasis and they do so via their tissue-specific, functional cargo. In the musculoskeletal system tendon fibroblasts make large amounts of type I collagen whereas muscle fibroblasts make more type III than type I collagen, and in smaller amounts than tendon [ 12 , 13 ]. Treatment of tenocytes in a number of in vitro and in vivo experimental models with EVs from various sources, e.g. plasma [ 14 ], adipose stem cells [ 15 ], tendon progenitors [ 16 ], macrophages [ 17 ], have demonstrated that signalling through EVs can modulate the expression of genes that regulate ECM synthesis and remodelling. Exosomes derived from tendon cells have also been reported [ 18 , 19 ]; and one study showed that tendon cell-derived exosomes induce the expression of tenogenic genes in bone marrow-derived mesenchymal stem cells in a TGFβ-dependent manner [ 19 ]. Compared to tendon, muscle is a more complex tissue with more than one cell type. Collagen synthesis by muscle fibroblasts is tightly regulated and exosomes derived from satellite cells play a role in preventing fibrosis in healthy muscle tissues [ 20 ]. Interestingly, tendon rupture in humans and animal models causes an increase of type I collagen in the adjacent muscle tissue, which correlates to increased muscle stiffness and decreased muscle function [ 12 , 21 – 26 ]. Establishing the biochemistry of healthy fibroblast EVs may be a crucial first step in understanding their role when tissues are damaged. Exosomes and other EVs have great potential as a non-invasive source of biomarkers for disease detection and monitoring as they can be isolated from various bodily fluids in addition to blood, including urine, saliva, breast milk and sweat [ 7 , 27 , 28 ]. In order to determine the health status of a specific tissue from a pool of EVs derived from all the tissues in the body, we must be able to isolate tissue-specific exosomes and the identification of membrane-localised proteins can aid the capture of EVs for targeted analyses. Recent studies have demonstrated that EV surface proteins bear characteristics of their tissue of origin and these EVs can be captured from bodily fluids using antibody-based assays targeting these proteins [ 29 , 30 ]. Proteomic profiling of EVs is a novel and sensitive approach to increase understanding of EV function and its use has been successful in unravelling their role in cancer [ 31 ]. A proteomic approach will also reveal potential protein biomarkers for isolating tissue-specific EVs. In this study, we took a proteomics approach to investigate and compare the proteome of small EVs isolated from the major cell populations in human tendon and muscle to elucidate key molecules and understand how homeostasis of the different ECMs in these two tissues is regulated. Methods Ethics Informed consent was obtained from all tissue donors (ethics approval H-3-2010-070 by the Regional Ethical Committee for the Hospital Region of Greater Copenhagen, in accordance with the Declaration of Helsinki II). The study was reported to the Danish register (Datatilsynet) and was performed in accordance with Danish law (Lov om behandling af personoplysninger). Human tissues Human tissue was obtained in connection with anterior cruciate ligament reconstruction surgery. Gracilis and semitendinosus tendon and muscle tissues were harvested under general anaesthetic. After the surgeon had obtained the tendon required for the reconstruction, any waste tissues were transferred to ice-cold PBS containing 50 U/ml penicillin and 50 μg/ml streptomycin (Thermo Fisher Scientific). Tissues from five biological samples (2 females, 3 males) with a mean age 29.6 ± 7.2 (standard deviation (SD)) years old were used (Supplementary Figure 1 a and 1 b). Muscle fibroblasts derived from one of the samples did not proliferate enough and so they were not included in the study. Tendon fibroblasts Tendon tissues that were cleaned of muscle tissue were cut into small pieces and incubated overnight in 400 U/ml collagenase type 2 (Worthington Biochemical Corporation) prepared in DMEM/F-12 (Thermo Fisher Scientific) supplemented with 20% FCS (BioWest) and 50 U/ml penicillin and 50 μg/ml streptomycin at 37 °C in 5% CO 2 . After, cells were strained through a 70 μm filter (BD Biosciences) and centrifuged for 6 min at 600 x g, washed with PBS, pelleted again and then resuspended in medium. Tendon fibroblasts were cultured in DMEM/F-12 medium supplemented with 10% FCS and 50 U/ml penicillin and 50 μg/ml streptomycin at 37 °C in 5% CO 2 . Myoblasts and muscle fibroblasts Muscle tissues were cut into small pieces and incubated for 1 h in 2 mg/ml collagenase B (Roche), 2 mg/ml dispase II (Roche) in Skeletal Muscle Basal Medium (Promocell) at 37 °C in 5% CO 2 , with agitation by pipetting every 15 min. After, cells were strained through a 70 μm filter and centrifuged for 6 min at 600 x g, washed with PBS, pelleted again and then resuspended in medium. Muscle tissue cells were cultured for 1 week in Human Skeletal Growth Medium (Promocell) supplemented with 15% FCS, 2 mM L-glutamine, 20 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in 5% CO 2 . After, CD56+ myogenic cells (myoblasts) were sorted from CD56- non-myogenic cells (muscle fibroblasts) as described previously [ 32 ]. In brief, muscle cells were treated for a maximum of 2 min at 37 °C in 5% CO 2 with trypsin EDTA solution C (Biological Industries) diluted 1:2 in PBS. Detached cells were pelleted, washed in PBS and pelleted again. The cell pellet was resuspended in 170 μl MACS Separation Buffer (Miltenyi Biotec) and 35 μl CD56 magnetic beads (Human CD56 MicroBead Kit; Miltenyi Biotec) and incubated for 15 min at 4 °C. After, cells were centrifuged for 6 min at 600 x g, resuspended in MACS Separation Buffer and passed through a 30 μm pre-separation filter into a large cell column attached to a MiniMACS Separator (all from Miltenyi Biotec) following the manufacturer's protocol. Both CD56- muscle fibroblasts and CD56+ myoblasts were collected and cultured separately in DMEM/F-12 medium supplemented with 10% FCS and 50 U/ml penicillin and 50 μg/ml streptomycin and in Human Skeletal Growth Medium supplemented with 15% FCS, 2 mM L-glutamine, 20 U/ml penicillin and 100 μg/ml streptomycin, respectively, at 37 °C in 5% CO 2 . Before EV isolation, myoblasts were differentiated by culturing for 4 days in Skeletal Muscle Basal Medium supplemented with 2 mM L-glutamine, 20 U/ml penicillin and 100 μg/ml streptomycin, at 37 °C in 5% CO 2 . Small extracellular vesicle isolation For each sample, cells ~ 80% confluency from one T175 flask were used for small EV isolation. Cells were cultured without serum, in DMEM/F-12 supplemented with 0.035% sodium bicarbonate, 10 mM HEPES, 1x GlutaMAX supplement and 50 U/ml penicillin and 50 μg/ml streptomycin at 37 °C in 5% CO 2 . After 16 h, the conditioned medium was collected. The conditioned media was ultra-filtered through 0.22 μm filter (Satorius) to remove any intact cells, contaminating microvesicles and apoptotic bodies, and then centrifuged at 2000 x g for 20 min at 4 °C to remove any remaining cell debris. The supernatant was further centrifuged at 4566 x g for 1 h at 4 °C to remove any remaining microvesicles. The supernatant was ultracentrifuged in 5-ml polypropylene centrifuge tubes (Beckman Coulter) at 100000 x g for 2 h at 4 °C using an SW55Ti rotor and Optima L-80 XP Ultracentrifuge (Beckman Coulter). The pellet containing crude EV extract was resuspended in 40 μl PBS containing protease inhibitor cocktail (Roche). The EV extract was mixed with 4 ml BioUltra PBS (Sigma-Aldrich) and ultracentrifuged in 5-ml polypropylene centrifuge tubes at 100000 x g for 2 h at 4 °C. The pellet of small EVs was then resuspended in 20 μl 6 M guanidine hydrochloride, 10 mM Tris (2-carboxyethyl) phosphine hydrochloride, 40 mM 2-chloroacetamide, 100 mM Tris pH 8.5 for mass spectrometry analysis. Samples were stored at -80 °C. Transmission electron microscopy For TEM analysis of EVs, EV pellets were re-suspended and fixed in 2% glutaraldehyde in 100 mM phosphate buffer. Fixed EVs were mounted on to glow-discharged copper grids (Agar Scientific) coated with a continuous carbon film, then stained with 1% (w/v) uranyl acetate (Sigma Aldrich) in ddH 2 O for 1 min at RT, and washed with ddH 2 O. Grids were examined with a CM 100 TWIN (Philips) fitted with a 2 k × 2 k side-mounted TEM CCD camera (Olympus Veleta). EV diameter measurements were made using FIJI. Median diameter was calculated from at least 300 measurements per sample, the average and SD of median EV diameters were calculated from 3 biological samples. Tryptic digestion of small EV proteins Protein concentrations were determined by Quick Start Bradford Protein Assay (Bio-Rad). In 1.5 ml low-bind eppendorf tubes proteins ( 7 or unknown were excluded. MS performance was verified for consistency by running complex cell lysate quality control standards, and chromatography was monitored to check for reproducibility. Label-free quantitative proteomics analysis The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository ( http://www.ebi.ac.uk/pride/archive/ ) with the data set identifier PXD018233. The mass spectrometry raw files were analysed using Proteome Discoverer 2.4 and can be found in Supplementary Data 1 . Label-free quantitation (LFQ) was enabled in the processing and consensus steps, and spectra were matched against the Homo sapiens database obtained from Uniprot. Dynamic modifications were set as oxidation (M), deamidation (N,Q) and acetyl on protein N-termini. Cysteine carbamidomethyl was set as a static modification. All results were filtered to a 1% false discovery rate (FDR), and protein quantitation done using the built-in Minora Feature Detector. Proteins suggest by the Minimal Information of Studies for EVs 2018 [ 33 ] were used for protein content-based EV characterisation. Statistical analysis of LC-MS data The normalised protein intensities generated by LC-MS were analysed using the R-based integrated web application Differential Expression and Pathway version 0.90 (iDEP) [ 34 ]. In the interest of identifying fibroblast EV-enriched proteins, only those detected in at least 2 biological replicates of small EVs derived from tendon fibroblasts (TenX) and small EVs derived from muscle fibroblasts (FibX) samples were further analysed (612 proteins). We did not find that removing proteins affected the distribution pattern of TenX and FibX samples. Supplementary Data 2 contains the customised R code for the iDEP workflow. Supplementary Data 3 contains the log transformed protein intensities with missing values filled in by imputation of the median intensity for the protein within the sample group. iDEP-generated values for heatmaps can be found in Supplementary Data 4 and 5 . Supplementary Data 6 contains the results from the DESeq2 (an iDEP package), using a threshold of false discovery rate (FDR) p  ± 2. Functional enrichment analysis was performed on high abundance proteins, e.g. by combining proteins identified as high in one type of small EV, when compared to the other two types of small EVs using the online tool DAVID version 6.7 [ 35 ] and the resultant enrichment clusters are contained in Supplementary Data 7 and 8 . Venny version 2.1 (BioinfoGP, Spanish National Biotechnology Centre) was used to identify common and unique proteins between groups and the output can be found in Supplementary Data 9 . Statistical significance indicated in figures (* p  7 or unknown were excluded. MS performance was verified for consistency by running complex cell lysate quality control standards, and chromatography was monitored to check for reproducibility. Label-free quantitative proteomics analysis The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository ( http://www.ebi.ac.uk/pride/archive/ ) with the data set identifier PXD018233. The mass spectrometry raw files were analysed using Proteome Discoverer 2.4 and can be found in Supplementary Data 1 . Label-free quantitation (LFQ) was enabled in the processing and consensus steps, and spectra were matched against the Homo sapiens database obtained from Uniprot. Dynamic modifications were set as oxidation (M), deamidation (N,Q) and acetyl on protein N-termini. Cysteine carbamidomethyl was set as a static modification. All results were filtered to a 1% false discovery rate (FDR), and protein quantitation done using the built-in Minora Feature Detector. Proteins suggest by the Minimal Information of Studies for EVs 2018 [ 33 ] were used for protein content-based EV characterisation. Statistical analysis of LC-MS data The normalised protein intensities generated by LC-MS were analysed using the R-based integrated web application Differential Expression and Pathway version 0.90 (iDEP) [ 34 ]. In the interest of identifying fibroblast EV-enriched proteins, only those detected in at least 2 biological replicates of small EVs derived from tendon fibroblasts (TenX) and small EVs derived from muscle fibroblasts (FibX) samples were further analysed (612 proteins). We did not find that removing proteins affected the distribution pattern of TenX and FibX samples. Supplementary Data 2 contains the customised R code for the iDEP workflow. Supplementary Data 3 contains the log transformed protein intensities with missing values filled in by imputation of the median intensity for the protein within the sample group. iDEP-generated values for heatmaps can be found in Supplementary Data 4 and 5 . Supplementary Data 6 contains the results from the DESeq2 (an iDEP package), using a threshold of false discovery rate (FDR) p  ± 2. Functional enrichment analysis was performed on high abundance proteins, e.g. by combining proteins identified as high in one type of small EV, when compared to the other two types of small EVs using the online tool DAVID version 6.7 [ 35 ] and the resultant enrichment clusters are contained in Supplementary Data 7 and 8 . Venny version 2.1 (BioinfoGP, Spanish National Biotechnology Centre) was used to identify common and unique proteins between groups and the output can be found in Supplementary Data 9 . Statistical significance indicated in figures (* p  2-fold mean differential abundance in TenX when compared to MyoX (90 proteins) and in TenX when compared to FibX (68 proteins), with 10 proteins in common. b Venn diagram showing the number of proteins with > − 2-fold mean differential abundance in TenX when compared to FibX (21 proteins) and in MyoX when compared to FibX (83 proteins), with 9 proteins in common. See Supplementary Data 9 for full list of proteins (FDR  2-fold mean differential abundance in TenX when compared to MyoX (90 proteins) and in TenX when compared to FibX (68 proteins), with 10 proteins in common. b Venn diagram showing the number of proteins with > − 2-fold mean differential abundance in TenX when compared to FibX (21 proteins) and in MyoX when compared to FibX (83 proteins), with 9 proteins in common. See Supplementary Data 9 for full list of proteins (FDR < 0.01). c Plots showing the log transformed intensities of the 10 commonly over abundant TenX proteins. A Membrane-localised proteins: ANTXR2, PMCA4 ( ATP2B4 ), PTGFRN, syntaxin-4 and syntaxin-7. d Plots showing the log transformed intensities of the 9 commonly over abundant FibX proteins. B Membrane-localised protein: CD73 ( NT5E ). *p < 0.05, **p < 0.01, *** p < 0.001 and **** p < 0.0001. See Supplementary Data 6 for all adjusted p-values from DESeq2 analysis Discussion In this study, we identified key functional differences of small EVs produced by healthy fibroblasts isolated from human musculoskeletal tissues. By performing label-free quantitative LC-MS-based proteomics analysis we have, in an unprecedented manner, established three different proteome profiles of small EVs isolated from primary cultures of tendon fibroblasts, muscle fibroblasts and differentiating myoblasts. We found a high abundance of proteins that support substantial ECM synthesis in tendon fibroblasts EVs but not in muscle fibroblast EVs. In differentiating myoblast EVs a high myofibrillar synthesis was indicated, and in skeletal muscle fibroblast EV proteins supporting myoblast differentiation and the skeletal muscle ECM were present (Fig. 6 ). Fig. 6 Summary of proposed functional roles of small EVs from human tendon and muscle cells in tissue homeostasis. In tendon, tendon fibroblasts produce EVs (TenX), which act through autocrine or paracrine signalling to induce type I collagen expression, synthesis and assembly into the ECM. In skeletal muscle, EVs produced by myoblast progenitors (satellite cells, activated satellite cells) and myofibers (MyoX) regulate myoblast differentiation and myofibrillogenesis. There are EVs that are produced by muscle fibroblasts (FibX), which also help regulate myoblast differentiation but they also regulate the homeostasis of the basement membrane enveloping the myofibers and the fine collagen network of the endomysium Proteome studies on EVs have focused on cancer, in which high numbers of EVs containing many more proteins are produced [ 31 , 36 , 37 ]. We identified over 1000 proteins in small EVs purified from conditioned media and confirmed the presence of EV marker proteins. No one has to our knowledge previously performed EV proteomics on human tendon tissue. The only other tendon fibroblast EV proteome data published is of porcine tendon exosomes, where only 199 proteins were identified [ 18 ], making the current TenX proteome profile the most comprehensive to date. MyoX contained significantly more of some of EV marker proteins, which was expected as Hsc70 and Hsp90-β upregulation are required during myoblast differentiation [ 38 – 40 ], and the presence of these chaperones might be a mechanism of autocrine or paracrine signalling. Integrin α6 is a laminin receptor and their enrichment in MyoX is reflective of the basement membrane adhesion profile of differentiating myoblasts [ 41 ], and ESCRT-III molecules are enriched in myoblasts as they are responsible for the shedding of injured membranes [ 42 ]. MyoX were also enriched in proteins required for translation elongation, and this enrichment is consistent with previously published biochemical contents of differentiating human myoblasts-derived exosomes and microvesicles [ 43 ]. Taken together, our data suggest that the main role of MyoX is to regulate myoblast differentiation and/or myofiber homeostasis (Fig. 6 ). We used iDEP, an R-based web application designed to easily analyse transcriptomic and proteomic data [ 34 ]. The analysis revealed that the largest source of variability was between fibroblast- and non-fibroblast-derived EVs and the second largest source of variance was between TenX and FibX proteome profiles. So, despite the fibroblasts having been removed from their native in vivo environments and put into the same culture conditions, these two EV proteome profiles remained distinct. Functional analysis of the TenX proteome identified an enrichment of ECM proteins (collagens), translational proteins (ribosomal proteins) and cytoskeletal proteins (keratins and myosins), which is consistent with the proteome of porcine tendon exosomes [ 18 ]. On the other hand FibX distinctly lacked a high abundance of ribosomal proteins, Golgi transport proteins and ATPase ion-transporters but contained significantly higher levels of the collagenase MMP2 than TenX. FibX were also uniquely enriched in the ECM proteins fibulin-1, nidogen-2 and tenascin X, all of which are components of the skeletal muscle ECM [ 44 ]; TIMP3, a negative regulator of tumour necrosis factor α autocrine signalling in satellite cells during muscle regeneration [ 45 ]; and acetyl-CoA acyl-transferase, a subunit of the mitochondrial trifunctional protein complex and autosomal recessive mutations of this gene causes muscle weakness [ 46 – 48 ]. The presence of these proteins could indicate the mechanisms of interaction between the skeletal muscle fibroblasts and the muscle cell (Fig. 6 ), an idea that is supported by earlier findings that demonstrated an intimate interplay between different cell types in skeletal muscle through EVs [ 20 ]. Further, it has been shown that de-adhesion and adhesion activity is involved in muscle regeneration after heavy mechanical loading [ 49 ], suggesting that the intramuscular connective tissue is prepared to coordinate tasks in adaptation and regeneration with the myonuclei of the muscle cell and the satellite cells. More than half of secreted TGFβ1 are localised to EVs [ 11 ] and it is commonly identified in EVs released by cancer associated fibroblasts [ 8 ]. Here we report that small EVs released by healthy human tendon fibroblasts also contained a significantly high abundance of TGFβ1 compared to MyoX and FibX. A recent study showed that injection of exosomes isolated from cultures of tendon progenitors into an in vivo collagenase-induced tendinopathy model in rat Achilles increased type I collagen synthesis and improved tissue biomechanics [ 16 ]. Another study showed that the ability of tendon exosomes to reprogram mesenchymal stem cells to produce type I collagen could be blocked using a TGFβ inhibitor [ 19 ]. Thus, we hypothesise that it is likely that TGFβ1 is also a functional cargo of tendon EVs that may regulate the tendon ECM. Further studies confirming TGFβ localisation in TenX and its specific function are required and these should be performed carefully to exclude activity associated with co-isolated soluble mediators [ 33 ]. Together these data suggest that tendon EVs have the potential to induce collagen synthesis, transportation of new proteins for post-translational modifications and ECM assembly in recipient cells (Fig. 6 ), whereas EVs derived from muscle fibroblasts may maintain the skeletal muscle ECM as well as the low fibrotic potential of healthy muscle fibroblasts [ 50 ]. Muscle tissue-derived exosomes are released into the circulation upon exercise and are targeted to the liver [ 51 ]. We propose that small EVs produced by tendon cells, however, remain in the tissue under normal conditions and are used to synchronise ECM remodelling via autocrine and paracrine signalling. Rupture of the tissue could lead to the release of tendon EVs into the tissue surroundings, such as the adjacent muscle, potentially being responsible for the initiation of a transient fibrotic response. Isolation of specific types of EVs is challenging with small samples. Although we were able to identify many exosome-enriched proteins in our samples, the protocol we used, without the inclusion of a density-gradient separation step that produces a higher purity of exosomes at the cost of yield, does not separate out other small vesicles [ 52 , 53 ]. We observed a large number of ribosomal proteins that are commonly co-isolated with EVs (Supplementary Figure 3 d). The role of these ribosomal proteins may include forming protein-RNA complexes inside exosomes and other EVs [ 54 , 55 ] but it is possible that they are co-isolated as non-EV protein aggregates rather than promiscuous loading [ 56 , 57 ]. Further investigations of purer exosome isolations in combination with immuno-electron microscopy would establish the location of ribosomal and ECM proteins identified by proteomics. The proteome profiles revealed the potential functions of fibroblast EVs but it is clear that the EV protein contents capture a snap shot of the cell's biochemistry at a given time, e.g. the differentiation of myo-progenitors. Therefore EVs could permit long-term monitoring of tissue health in a non-invasive manner in tissues including tendon [ 58 ] that cannot be sampled repeatedly. A very recent study demonstrated that exosomes from different sources are characterised by specific combinations of their surface proteins that can be quantified using antibody-based barcoding assay [ 59 ]. We identified five membrane proteins that are significantly enriched in tendon EVs (ANTXR2, PMCA4, PTGFRN, syntaxin-4 and syntaxin-7) and one membrane protein that is significantly enriched in muscle fibroblast EVs (CD73) that, hypothetically, could be targeted in combination with other exosome-specific surface proteins in antibody-binding assays to capture themfrom various bodily fluids [ 29 , 30 ] for analysis. The ability to identify tissue-specific EVs expands the potential of exosomes as biomarker carriers in non-cancerous diseases. Conclusions This study reports, for the first time, comprehensive proteome profiles for small EVs released from healthy human fibroblasts and their potential roles in tissue homeostasis. Our results also demonstrate that with LC-MS-based proteome profiling it was possible to reveal a marked heterogeneity among fibroblast-derived small EVs, indicating shared tasks between EVs in skeletal muscle myoblasts and fibroblast, whereas tendon fibroblast extracellular vesicles demonstrated a potential to be pro-fibrotic in human tendon tissue. Supplementary information Additional file 1. Supplementary Data 1. Mass spectrometry data of list of proteins and normalised intensities in all samples. Additional file 2. Supplementary Data 2. Customised R code for iDEP analyses. Additional file 3. Supplementary Data 3. Log transformed intensity list with missing values filled in by imputation. Additional file 4. Supplementary Data 4. Heatmap values for proteins ranked by SD. Additional file 5. Supplementary Data 5. Lists of proteins, fold changes and FDR values from DESeq2 analyses of TenX-MyoX, TenX-FibX and MyoX-FibX comparisons. Additional file 6. Supplementary Data 6. Fold change values of heatmaps from TenX-MyoX, TenX-FibX and MyoX-FibX DESeq2 analyses. Additional file 7. Supplementary Data 7. Functional enrichment analysis results for high abundance proteins in MyoX, TenX and FibX. Additional file 8. Supplementary Data 8. Functional enrichment analysis result for proteins with differential abundance between TenX and FibX. Additional file 9. Supplementary Data 9. Venn diagram outputs. Additional file 10 Supplementary Figure 1. Overview of expansion of muscle tissue and tendon cells for small EV isolation. a Illustrative overview of cell isolation and expansion for small EV isolation from differentiating myoblasts, muscle fibroblasts and tendon fibroblasts ( n = 5 except n = 4 for FibX because muscle fibroblasts from prep 5* did not proliferate enough for small EV isolation). b Table of information for the five samples used for LC-MS. c Transmission electron microscopy images (TEM) of negative-stained EV isolates. Bars, 200 nm. (D) Measured diameter of EVs from TEM images. At least 300 measurements for each sample ( n = 3 biological samples). Supplementary Figure 2. Variances of small EV protein abundance detected by LC/MS. a Summary of protein exclusion for statistical analysis by iDEP. b-c Distribution of log transformed protein abundances in MyoX, TenX and FibX as detected by LC-MS shown in a box plot ( b ) and a density plot ( c ). Supplementary Figure 3. Characterisation of protein content of isolated EVs. a-f Heatmaps showing the levels of protein detected in MyoX, TenX and FibX samples that are proteins enriched in EVs ( a ), cytosolic proteins recovered in EVs via lipid or membrane protein-binding ability ( b ) or promiscuous incorporation ( c ), proteins that have are commonly co-isolated with EVs ( d ), and secreted proteins recovered with EVs ( e ). Additional file 1. Supplementary Data 1. Mass spectrometry data of list of proteins and normalised intensities in all samples. Additional file 2. Supplementary Data 2. Customised R code for iDEP analyses. Additional file 3. Supplementary Data 3. Log transformed intensity list with missing values filled in by imputation. Additional file 4. Supplementary Data 4. Heatmap values for proteins ranked by SD. Additional file 5. Supplementary Data 5. Lists of proteins, fold changes and FDR values from DESeq2 analyses of TenX-MyoX, TenX-FibX and MyoX-FibX comparisons. Additional file 6. Supplementary Data 6. Fold change values of heatmaps from TenX-MyoX, TenX-FibX and MyoX-FibX DESeq2 analyses. Additional file 7. Supplementary Data 7. Functional enrichment analysis results for high abundance proteins in MyoX, TenX and FibX. Additional file 8. Supplementary Data 8. Functional enrichment analysis result for proteins with differential abundance between TenX and FibX. Additional file 9. Supplementary Data 9. Venn diagram outputs. Additional file 10 Supplementary Figure 1. Overview of expansion of muscle tissue and tendon cells for small EV isolation. a Illustrative overview of cell isolation and expansion for small EV isolation from differentiating myoblasts, muscle fibroblasts and tendon fibroblasts ( n = 5 except n = 4 for FibX because muscle fibroblasts from prep 5* did not proliferate enough for small EV isolation). b Table of information for the five samples used for LC-MS. c Transmission electron microscopy images (TEM) of negative-stained EV isolates. Bars, 200 nm. (D) Measured diameter of EVs from TEM images. At least 300 measurements for each sample ( n = 3 biological samples). Supplementary Figure 2. Variances of small EV protein abundance detected by LC/MS. a Summary of protein exclusion for statistical analysis by iDEP. b-c Distribution of log transformed protein abundances in MyoX, TenX and FibX as detected by LC-MS shown in a box plot ( b ) and a density plot ( c ). Supplementary Figure 3. Characterisation of protein content of isolated EVs. a-f Heatmaps showing the levels of protein detected in MyoX, TenX and FibX samples that are proteins enriched in EVs ( a ), cytosolic proteins recovered in EVs via lipid or membrane protein-binding ability ( b ) or promiscuous incorporation ( c ), proteins that have are commonly co-isolated with EVs ( d ), and secreted proteins recovered with EVs ( e ).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8454293/

Innate immune detection of lipid oxidation as a threat assessment strategy

Oxidized phospholipids that result from tissue injury operate as immunomodulatory signals that, depending on the context, lead to proinflammatory or anti-inflammatory responses. In this Perspective, we posit that cells of the innate immune system use the presence of oxidized lipids as a generic indicator of threat to the host. Similarly to how pathogen-associated molecular patterns represent general indicators of microbial encounters, oxidized lipids may be the most common molecular feature of an injured tissue. Therefore, microbial detection in the absence of oxidized lipids may indicate encounters with avirulent microorganisms. By contrast, microbial detection and detection of oxidized lipids would indicate encounters with replicating microorganisms, thereby inducing a heightened inflammatory and defensive response. Here we review recent studies supporting this idea. We focus on the biology of oxidized phosphocholines, which have emerged as context-dependent regulators of immunity. We highlight emerging functions of oxidized phosphocholines in dendritic cells and macrophages that drive unique inflammasome and migratory activities and hypermetabolic states. We describe how these lipids hyperactivate dendritic cells to stimulate antitumour CD8 + T cell immunity and discuss the potential implications of the newly described activities of oxidized phosphocholines in host defence. Introduction The innate and adaptive arms of immunity operate as generalists and specialists, respectively. General molecular patterns that are associated with microbial life are used by pattern recognition receptors (PRRs) to detect potential infections 1 , 2 (Box 1 ). The microbial molecules are referred to as pathogen-associated molecular patterns (PAMPs). However, the specific identity of the potential pathogen is not identified by PRRs 1 . This specificity is provided by lymphocytes that bear antigen receptors, which are precise in their identification of individual pathogens. The link between generalists (PRRs) and specialists (antigen receptors) is causal, as genetic evidence indicates that PRR signalling on dendritic cells (DCs) is critical to stimulate antigen-specific responses by T cells 3 – 6 . While PRRs are not designed to identify specific microorganisms, increasing evidence indicates that innate immune networks operate as threat-assessment stations, which can distinguish between microbial and pathogenic encounters 7 . The most commonly discussed means of distinguishing microorganisms from pathogens is through the coincident detection of PAMPs and virulence factor activities by distinct innate immune receptors 8 , 9 . Microorganisms contain PAMPs but have no virulence factors, whereas pathogens contain both 9 . An example of threat assessment involves the induction of pyroptosis, an inflammatory form of cell death that can be triggered upon coincident detection of PAMPs and virulence factor activities 10 – 13 . One of the challenges of this strategy of threat assessment is the unique repertoire of virulence factors encoded by individual pathogens. A general means of identifying threats to the host, regardless of the virulence factor repertoire, may be useful and more symmetrical to the PAMP-based strategy of microbial detection used by PRRs. Herein we discuss data suggesting a distinct strategy of threat assessment by the innate immune system, which is not mediated by specific virulence factors. This process is rather mediated by one of the most common consequences of pathogen replication — oxidation of phospholipids in the environment following tissue injury. We discuss how oxidized phospholipids (oxPLs) operate as immunomodulatory signals that may provide a generic indication of the context of PAMP detection to the host. PAMP detection in the absence of oxPLs may represent contexts where non-replicating microorganisms have been encountered (that is, low threat encounters). By contrast, PAMP detection in the presence of oxidized products may represent contexts of high threat, where replicating microorganisms (that is, pathogens) have been encountered. The benefit of such a strategy is that tissue injury-induced oxidation may be considered a common (and perhaps unavoidable) outcome of pathogen replication, regardless of the virulence factor repertoire. Thus, akin to the generic use of PAMPs as an indicator of microorganisms, the innate immune system may use PAMPs plus oxPLs as generic indicators of heightened threat. The mechanisms and consequences of oxPL detection by the innate immune system are discussed herein. Box 1 Pattern recognition receptors sensing PAMPs and DAMPs Mammalian cells detect detrimental changes to the host such as potentially pathogenic microorganisms or host-encoded molecules indicative of tissue injury. This threat surveillance is achieved via the actions of a superfamily of pattern recognition receptors (PRRs), which recognize conserved ligands defined by classical terms as pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) 2 , 68 , 69 . Some of the most inflammatory PAMPs include lipopolysaccharides (LPS), outer-membrane components of Gram-negative bacteria. Upon bacterial encounters, LPS-binding protein (LBP) and CD14 operate in tandem to extract individual LPS molecules from bacterial cell walls and deliver these lipids to membrane-associated MD2 and Toll-like receptor 4 (TLR4). LPS-bound CD14 also induces an inflammatory endocytosis pathway that delivers TLR4 to endosomes 22 . This interaction activates inflammatory gene expression through nuclear factor-κB and mitogen-activated protein kinase signalling. Other LPS-interacting proteins have been described in detail in recent reviews 13 , 70 . Recently, it was shown that mammalian PRRs are unable to detect the LPS of most bacteria from a different ecosystem, such as deep sea bacteria, despite retaining most structural features of Escherichia coli LPS 71 . These data suggest that pattern recognition of structurally conserved ligands may be defined locally, not globally. In addition to detecting microbial products, several PRRs recognize DAMPs, such as oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine derivatives (oxPAPCs) 72 . As opposed to our increasing understanding of how PAMPs and virulence factors are recognized, numerous questions remain unanswered regarding DAMP detection and signalling. Box 1 Pattern recognition receptors sensing PAMPs and DAMPs Mammalian cells detect detrimental changes to the host such as potentially pathogenic microorganisms or host-encoded molecules indicative of tissue injury. This threat surveillance is achieved via the actions of a superfamily of pattern recognition receptors (PRRs), which recognize conserved ligands defined by classical terms as pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) 2 , 68 , 69 . Some of the most inflammatory PAMPs include lipopolysaccharides (LPS), outer-membrane components of Gram-negative bacteria. Upon bacterial encounters, LPS-binding protein (LBP) and CD14 operate in tandem to extract individual LPS molecules from bacterial cell walls and deliver these lipids to membrane-associated MD2 and Toll-like receptor 4 (TLR4). LPS-bound CD14 also induces an inflammatory endocytosis pathway that delivers TLR4 to endosomes 22 . This interaction activates inflammatory gene expression through nuclear factor-κB and mitogen-activated protein kinase signalling. Other LPS-interacting proteins have been described in detail in recent reviews 13 , 70 . Recently, it was shown that mammalian PRRs are unable to detect the LPS of most bacteria from a different ecosystem, such as deep sea bacteria, despite retaining most structural features of Escherichia coli LPS 71 . These data suggest that pattern recognition of structurally conserved ligands may be defined locally, not globally. In addition to detecting microbial products, several PRRs recognize DAMPs, such as oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine derivatives (oxPAPCs) 72 . As opposed to our increasing understanding of how PAMPs and virulence factors are recognized, numerous questions remain unanswered regarding DAMP detection and signalling. Phospholipid oxidation and recognition Phospholipids such as phosphocholines are major constituents of mammalian cells. Under conditions of non-inflammatory or inflammatory tissue injury, any cell death that occurs in the local environment will result in the generation of reactive oxygen species (ROS). The source of the ROS may result from the actions of infiltrating neutrophils, dying mitochondria or the air itself. Under any of these conditions, phosphocholines in the plasma membrane can be oxidized. Defined examples of oxPL generation include tissue injury, lung infection in which an initial encounter with a pathogen is followed by tissue damage 14 , and during chronic inflammatory diseases such as atherosclerosis 15 . Mechanisms of oxPL generation are summarized in Box 2 . In recent years, much focus has been on the arachidonic acid-containing phospholipid 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine (PAPC), which is a constituent of membranes in mammalian cells. Unlike their non-oxidized counterparts, oxPLs serve as damage-associated molecular patterns (DAMPs) and can modulate inflammation. Upon exposure to ROS, PAPC is oxidized at various positions to create a heterogeneous mixture of lipids collectively referred to as oxPAPCs. oxPAPCs are composed of a mixture of full-chain and truncated oxidized phosphocholine derivatives, which can be detected on dying cells and approach millimolar concentration in damaged tissues 16 . Growing interest has been focused on two components of oxPAPC — namely, 1-palmitoyl-2-glutaroyl- sn -glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)- sn -glycero-3-phosphocholine (POVPC) 17 . As opposed to PAMPs, which are intrinsically inflammatory, it is impossible to describe oxPAPCs as inducers or inhibitors of inflammation in all contexts. Rather, oxPAPCs display context-dependent activities, which is likely to be an important aspect of threat assessment. Several oxPAPC receptors have been defined, each of which may impact oxPAPC-mediated inflammatory activity in different ways. On macrophages, the scavenger receptor CD36 binds oxPAPCs and induces their uptake. This process promotes foam cell formation during atherosclerosis 18 . The structural basis of oxPAPC binding to CD36 has been defined, with Lys164 and Lys166 being required for the binding with sn -2 acyl chains of oxPAPC 19 . oxPAPC binding to CD36 is blocked by E06, a monoclonal IgM antibody that recognizes the phosphocholine headgroup of oxPAPC (see Box 3 ), demonstrating the importance of the oxPAPC headgroup in binding to CD36 (ref. 20 ). In addition to CD36, oxPAPCs bind the bacterial lipopolysaccharide (LPS) receptor CD14 on myeloid cells, thereby allowing oxPAPC internalization into endosomes and transport to the cell cytosol 21 . In the context of oxPAPC-induced inflammatory activities (described in detail later), CD14 serves as the primary receptor for these oxPLs 21 . CD14 functions as a transporter associated with the execution of inflammation (TAXI) protein 22 , which delivers LPS from the bacterial cell wall to membrane-associated MD2 and Toll-like receptor 4 (TLR4). CD14 then transports LPS and TLR4 to signalling-competent subcellular regions 22 . Similarly to LPS, the interaction between oxPAPCs and CD14 results in CD14 endocytosis and depletion of CD14 from the plasma membrane 21 . Therefore, oxPAPCs compete with LPS for CD14 binding in cell-free experimental systems, an activity that likely explains why the pretreatment of naive cells with oxPAPCs blocks subsequent responses to LPS via TLR4 (ref. 23 ). Upon CD14-dependent endocytosis in macrophages and DCs, oxPAPCs gain access to the cytosol, where they bind the inflammasome regulators caspase 1 and caspase 11 (ref. 24 ). oxPAPC–caspase interactions are thought to stimulate NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome assembly in a potassium-independent manner 24 . Like CD14, caspase 11 interacts with oxPAPCs and LPS. In vitro studies demonstrated that LPS interacts with the caspase activation and recruitment domain (CARD) of caspase 11, whereas oxPAPCs bind the catalytic domain of caspase 11 (ref. 24 ). As a consequence, the functional impacts of oxPAPCs and LPS on caspase 11 are distinct. When LPS binds to caspase 11 in macrophages or DCs, this interaction induces inflammasome-dependent IL-1β release from macrophages, which is associated with pyroptosis. By contrast, oxPAPC binding to caspase 11 induces the release of IL-1β from living DCs, in the absence of pyroptosis 24 . The ability of oxPAPCs to bind the catalytic domain of caspase 11 was not associated with stimulation of its enzymatic activity, as is typically observed when inflammatory LPS structures interact with the caspase 11 CARD 25 , 26 . Rather, oxPAPCs prevent intrinsic and LPS-induced caspase 11 activity 24 . This finding, coupled with the aforementioned competition between oxPAPCs and LPS for CD14, may explain why LPS-induced inflammation can be prevented by pretreatment of cells or mice with oxPAPCs. Many questions remain unanswered with regard to the biochemical interaction of oxPAPCs with CD14 and inflammatory caspases. In addition, how CD14 delivers oxPAPCs to the cytosol remains ambiguous, but it is possible that oxPAPCs alter the endosomal membrane and provoke their own leakage into the cytosol 27 . In summary, oxPAPCs are recognized by multiple receptors on myeloid cells, which may lead to the crosstalk of diverse signalling pathways and the modulation of myeloid cell functions that we further describe in the following sections. Box 2 Mechanisms of oxidized phosphocholine generation and physiological accumulation Phospholipids such as the arachidonic acid-containing phospholipid 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine (PAPC) are essential components of the plasma membrane of every cell type 73 , lung surfactant 74 and circulating lipoproteins 75 . PAPCs are composed of fatty acids bound to a glycerol backbone containing a polar head group. The second position, also known as the sn -2 chain, in the glycerol backbone contains esterified monounsaturated or polyunsaturated fatty acids that are prone to oxidation by free radicals or enzymatically by myeloperoxidase or lipoxygenase. PAPCs react with oxygen to create a mixture of oxidized phospholipids, collectedly referred to as oxPAPCs. Enzymatically oxidized phospholipids are key signalling mediators that regulate various cellular and physiological processes, including thrombosis, metabolism and vascular inflammation 76 . Accordingly, oxidized lipids were shown to modulate responses by innate immune cells, including neutrophils, platelets and peritoneal macrophages 62 . A detailed description of the mechanism of generation of enzymatically oxidized lipids as well as their regulatory role in innate immunity is provided elsewhere 77 . Physiologically, the pool of oxPAPCs can also be generated non-enzymatically from cellular membranes, by reactive oxygen and nitrogen species from endogenous sources (for example, mitochondrial respiratory chain and NADPH oxidase) or exogenous sources (air pollution, smoking and UVB light) 30 . In the absence of pathogenic infections, oxPAPCs accumulate in dying cells, in membrane vesicles released from activated cells 78 , 79 and in oxidized low-density lipoproteins 80 and oxidized pulmonary surfactant 14 . Furthermore, diverse inducers of lung inflammation, such as influenza viruses (H5N1, H1N1 and H3N2), monkeypox virus, Yersinia pestis , Bacillus anthracis and severe acute respiratory syndrome coronavirus, can increase the levels of oxPAPCs 14 . Studies demonstrating evidence of oxPAPC accumulation in different infectious diseases, ranging from mycobacterial infections to sepsis and during various respiratory viral infections, have recently been reviewed 29 . In addition, oxPAPCs are generated in the cerebrospinal fluid of individuals with multiple sclerosis 81 , 82 , suggesting that oxPAPCs may contribute to the pathogenesis of autoimmune demyelination. The level of bioactive 1-palmitoyl-2-glutaroyl- sn -glycero-3-phosphocholine was found to be elevated in a broad range of cardiovascular injuries, such as atherosclerosis and ischaemia–reperfusion injury, during age-related macular degeneration in humans and in patients with alcoholic liver inflammation 30 and non-alcoholic steatohepatitis 83 . There is a growing interest in using oxPAPCs as biomarkers of human diseases, particularly for cardiovascular diseases 84 . Increasing evidence indicates that individual moieties contained in oxPAPCs exert non-redundant proinflammatory effects in macrophages and dendritic cells. Further work is needed to better understand the scope of threats to the host where these bioactive oxidized phospholipids could arise and their influence on immune responses and diseases. Box 3 Natural antibody-mediated recognition of oxidized phosphocholines Oxidized phospholipids (oxPLs) released from dying cells serve as antigens for natural antibodies. Therefore, antibody-based enzyme-linked immunosorbent assays are used as a method to detect and quantify oxPLs. Three monoclonal antibodies are specific for oxPLs (E06, DLH3 and 509) 30 . E06 and DLH3 are specific for phosphatidylcholine moieties, whereas 509 specifically recognizes oxidized phosphatidylethanolamine 85 . E06 is the best characterized oxPL-specific antibody, and was cloned from hybridomas generated from APOE-deficient mice and is commonly used to quantify oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine derivatives (oxPAPCs). However, E06 cannot distinguish between the distinct molecular species of oxPAPCs such as 1-palmitoyl-2-glutaroyl- sn -glycero-3-phosphocholine, 1-palmitoyl-2-(5-oxovaleroyl)- sn -glycero-3-phosphocholine (and 1-palmitoyl-2-(5,6-epoxyisoprostane E 2 )- sn -glycero-3-phosphocholine. Such differentiation is important because available data show that individual molecular species often have different biological activities. Imai et al. used E06 to quantify the levels of oxPAPCs in murine lung injuries caused by H5N1 avian influenza virus and acid instillation 14 . Furthermore, using E06 antibody, this group showed that oxPAPCs accumulate during infections in the lungs of humans and animals infected with anthrax or severe acute respiratory syndrome coronavirus. The accumulation of E06-reactive oxPAPCs was also detected in dermal fibroblasts upon skin exposure to UV radiation 86 . It is speculated that E06 may be useful for therapeutic targeting of inflamed tissues in various inflammatory diseases. As a proof of concept, within mice, E06 activities in mice reduced inflammation and attenuated the progression of atherosclerosis, aortic stenosis and hepatic steatosis 87 . In addition, treatment with the monoclonal IgG1 antibody X19-mu, which exhibits properties similar to those of the endogenous IgM E06 antibody, preserved coronary function and attenuated atherosclerosis in mice 88 . The presence of natural oxPL-reactive antibodies was detected during inflammation using an anti-idiotypic antibody that specifically recognizes the idiotope of E06, and oxPAPC-reactive antibodies were found in plaques of patients with multiple sclerosis and in mouse models 81 . The role of these anti-oxPL antibodies is still unknown and requires further evaluation. Box 2 Mechanisms of oxidized phosphocholine generation and physiological accumulation Phospholipids such as the arachidonic acid-containing phospholipid 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine (PAPC) are essential components of the plasma membrane of every cell type 73 , lung surfactant 74 and circulating lipoproteins 75 . PAPCs are composed of fatty acids bound to a glycerol backbone containing a polar head group. The second position, also known as the sn -2 chain, in the glycerol backbone contains esterified monounsaturated or polyunsaturated fatty acids that are prone to oxidation by free radicals or enzymatically by myeloperoxidase or lipoxygenase. PAPCs react with oxygen to create a mixture of oxidized phospholipids, collectedly referred to as oxPAPCs. Enzymatically oxidized phospholipids are key signalling mediators that regulate various cellular and physiological processes, including thrombosis, metabolism and vascular inflammation 76 . Accordingly, oxidized lipids were shown to modulate responses by innate immune cells, including neutrophils, platelets and peritoneal macrophages 62 . A detailed description of the mechanism of generation of enzymatically oxidized lipids as well as their regulatory role in innate immunity is provided elsewhere 77 . Physiologically, the pool of oxPAPCs can also be generated non-enzymatically from cellular membranes, by reactive oxygen and nitrogen species from endogenous sources (for example, mitochondrial respiratory chain and NADPH oxidase) or exogenous sources (air pollution, smoking and UVB light) 30 . In the absence of pathogenic infections, oxPAPCs accumulate in dying cells, in membrane vesicles released from activated cells 78 , 79 and in oxidized low-density lipoproteins 80 and oxidized pulmonary surfactant 14 . Furthermore, diverse inducers of lung inflammation, such as influenza viruses (H5N1, H1N1 and H3N2), monkeypox virus, Yersinia pestis , Bacillus anthracis and severe acute respiratory syndrome coronavirus, can increase the levels of oxPAPCs 14 . Studies demonstrating evidence of oxPAPC accumulation in different infectious diseases, ranging from mycobacterial infections to sepsis and during various respiratory viral infections, have recently been reviewed 29 . In addition, oxPAPCs are generated in the cerebrospinal fluid of individuals with multiple sclerosis 81 , 82 , suggesting that oxPAPCs may contribute to the pathogenesis of autoimmune demyelination. The level of bioactive 1-palmitoyl-2-glutaroyl- sn -glycero-3-phosphocholine was found to be elevated in a broad range of cardiovascular injuries, such as atherosclerosis and ischaemia–reperfusion injury, during age-related macular degeneration in humans and in patients with alcoholic liver inflammation 30 and non-alcoholic steatohepatitis 83 . There is a growing interest in using oxPAPCs as biomarkers of human diseases, particularly for cardiovascular diseases 84 . Increasing evidence indicates that individual moieties contained in oxPAPCs exert non-redundant proinflammatory effects in macrophages and dendritic cells. Further work is needed to better understand the scope of threats to the host where these bioactive oxidized phospholipids could arise and their influence on immune responses and diseases. Box 3 Natural antibody-mediated recognition of oxidized phosphocholines Oxidized phospholipids (oxPLs) released from dying cells serve as antigens for natural antibodies. Therefore, antibody-based enzyme-linked immunosorbent assays are used as a method to detect and quantify oxPLs. Three monoclonal antibodies are specific for oxPLs (E06, DLH3 and 509) 30 . E06 and DLH3 are specific for phosphatidylcholine moieties, whereas 509 specifically recognizes oxidized phosphatidylethanolamine 85 . E06 is the best characterized oxPL-specific antibody, and was cloned from hybridomas generated from APOE-deficient mice and is commonly used to quantify oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine derivatives (oxPAPCs). However, E06 cannot distinguish between the distinct molecular species of oxPAPCs such as 1-palmitoyl-2-glutaroyl- sn -glycero-3-phosphocholine, 1-palmitoyl-2-(5-oxovaleroyl)- sn -glycero-3-phosphocholine (and 1-palmitoyl-2-(5,6-epoxyisoprostane E 2 )- sn -glycero-3-phosphocholine. Such differentiation is important because available data show that individual molecular species often have different biological activities. Imai et al. used E06 to quantify the levels of oxPAPCs in murine lung injuries caused by H5N1 avian influenza virus and acid instillation 14 . Furthermore, using E06 antibody, this group showed that oxPAPCs accumulate during infections in the lungs of humans and animals infected with anthrax or severe acute respiratory syndrome coronavirus. The accumulation of E06-reactive oxPAPCs was also detected in dermal fibroblasts upon skin exposure to UV radiation 86 . It is speculated that E06 may be useful for therapeutic targeting of inflamed tissues in various inflammatory diseases. As a proof of concept, within mice, E06 activities in mice reduced inflammation and attenuated the progression of atherosclerosis, aortic stenosis and hepatic steatosis 87 . In addition, treatment with the monoclonal IgG1 antibody X19-mu, which exhibits properties similar to those of the endogenous IgM E06 antibody, preserved coronary function and attenuated atherosclerosis in mice 88 . The presence of natural oxPL-reactive antibodies was detected during inflammation using an anti-idiotypic antibody that specifically recognizes the idiotope of E06, and oxPAPC-reactive antibodies were found in plaques of patients with multiple sclerosis and in mouse models 81 . The role of these anti-oxPL antibodies is still unknown and requires further evaluation. Consequences of oxPAPC recognition The detection of oxPAPCs by macrophages or DCs can impact inflammatory activities in a diverse range of diseases, including acute inflammation, atherosclerosis, cancer, lung injury and age-related diseases 16 , 28 , 29 . Depending on the context in which oxPAPCs are encountered, these lipids exert proinflammatory or anti-inflammatory effects 30 . For example, during conditions representative of sterile tissue injury, oxPAPCs exert anti-inflammatory functions, perhaps by preventing efficient detection of other molecules that stimulate CD14, TLR4 or caspase 11. By contrast, at the site of infection where tissue damage is common, oxPAPCs exhibit proinflammatory activities in cells that have also detected PAMPs. In the following subsections we describe how oxPAPCs regulate innate immune functions of myeloid cells and influence the induction of adaptive immunity. Impact of oxPAPCs on phagocytosis and endocytosis Accumulating evidence demonstrates that oxPAPCs modulate phagocytosis by antigen-presenting cells in a cell type-specific and context-specific manner. For example, oxPAPC pretreatment of peritoneal macrophages diminished their ability to internalize Escherichia coli . In mice, pre-exposure to oxPAPCs led to a reduction in host defence, resulting in bacterial outgrowth and systemic dissemination 31 . Similarly, intratracheal instillation of an oxPAPC mixture or pure phosphocholine moieties (for example, POVPC or PGPC) rendered mice unable to eradicate subsequent infections with Pseudomonas aeruginosa 32 . oxPAPCs generated in the bronchoalveolar space of mice exposed to cigarette smoke impaired phagocytosis by alveolar macrophages 32 . The link between these in vivo defects in phagocytosis after oxPAPC exposure was established by means of the monoclonal antibody E06, which binds to and prevents the activities of several oxPAPC component lipids. E06 treatment prevented the inhibition of bacterial phagocytosis and restored bacterial clearance 32 . In contrast to the bacterial exposures described above, oxPLs can stimulate phagocytosis of apoptotic cells by macrophages via CD36 (ref. 33 ). In the context of non-phagocytic cargo, oxPAPCs did not affect dextran or soluble ovalbumin uptake by endocytosis. Moreover, in the presence of oxPAPCs, the ability of LPS-primed DCs to cross-present peptides on MHC class I molecules was enhanced 34 . These data are in accordance with reports indicating that within DCs, antigen leakage from endosomes for cross-presentation may occur upon direct oxidation of endosomal lipids and generation of oxPLs 27 . Thus, depending on the cargo to be internalized, oxPAPCs exhibit contrasting effects on phagocytic and endocytic activities. The molecular basis for these context-dependent behaviours is undefined. Impact of oxPAPCs on inflammasome activities Following priming with various TLR ligands, oxPAPCs induce a long-lived state of hyperactivation in myeloid cells 24 . This activation state is distinct from naive, active or pyroptotic cell states. A description of these cell states is provided in Box 4 and Fig. 1 . A hallmark of oxPAPC-stimulated hyperactive cells is their ability to secrete inflammasome-dependent IL-1β while remaining viable. Extracellular oxPAPC components are detected by CD14 (ref. 21 ). CD14-dependent endocytosis delivers these lipids to intracellular caspase 11. oxPAPC binding with caspase 11 leads to NLRP3-dependent inflammasome assembly and IL-1β release in the absence of cell death 24 . In contrast to the rapid but transient burst of IL-1β release that occurs upon exposure of phagocytes to pyroptotic stimuli, oxPAPCs or PGPC promote IL-1β release from LPS-primed cells for several days 21 . Fig. 1 The T cell stimulatory activities of dendritic cells at distinct activation states. To regulate T cell activity, dendritic cells (DCs) provide T cells with several signals that are important for the establishment of an appropriate T cell response. Naive DCs are quiescent DCs that have the ability to take up antigens. Active DCs take up antigens and have an enhanced ability to present antigen peptides on MHC molecules. In addition, active DCs upregulate co-stimulatory molecules to stimulate T cells. Hyperactive DCs share similar activities with their active counterparts, but also gain the ability to hypermigrate to lymph nodes and to secrete IL-1β. Pyroptotic DCs secrete high levels of IL-1β. However, pyroptotic DCs are dead and lose their T cell stimulatory capacity. IL-1β is a highly inflammatory cytokine and its secretion is tightly regulated 35 . TLR signalling induces production of pro-IL-1β, but this pro-protein form lacks an amino-terminal secretion signal and cannot be secreted via the endoplasmic reticulum–Golgi network 35 . To gain inflammatory activities, pro-IL-1β must be cleaved, most commonly by inflammatory caspases present within inflammasomes 36 . Inflammasomes are supramolecular organizing centres, which assemble in the cytoplasm following their activation 36 . Caspases that are activated within inflammasomes (most commonly caspase 1) cleave pro-IL-1β and gasdermin D (GSDMD). GSDMD cleavage unleashes the latent ability of its amino terminus to form pores in the plasma membrane. Pore formation is facilitated by interactions between GSDMD and acidic phosphoinositides present in the inner leaflet of the plasma membrane, such as phosphatidylinositol 4,5-bisphosphate and phosphatidylserine 37 . GSDMD pores serve as conduits for mature IL-1β secretion in a cell lysis-independent event 38 , 39 . Cargo transport across the negatively charged GSDMD conduit occurs via a size-exclusion mechanism 38 , 39 , as well as by electrostatic filtering of cytosolic proteins, which favours the release of the positively charged mature IL-1β, while repelling pro-IL-1β 40 . If GSDMD pores are not repaired by the cell, pyroptotic cell death may occur, during which IL-1β and other cytosolic contents can be released non-selectively. Of the well-defined inflammasome agonists, including intracellular LPS, ATP, nigericin and alum, all induce pyroptosis. By contrast, within TLR ligand-stimulated cells, oxPAPCs induce inflammasome-dependent release of IL-1β through GSDMD pores in the absence of cell death. The ability of oxPAPCs to add IL-1β to the repertoire of factors that are normally secreted by living phagocytes renders these cells more immunostimulatory than their activated counterparts that have been stimulated only with PAMPs — hence the term 'hyperactive'. Hyperactive cells display evidence of fewer GSDMD pores at their surface than their pyroptotic counterparts 39 . Whether GSDMD pores in hyperactive cells are regulated similarly as in pyroptotic cells downstream of inflammasome activation is unknown. Hyperactive cells may undergo rapid membrane repair events that ensure viability. Indeed, pyroptotic events increase in cells that are defective for membrane repair mediated by the endosomal sorting complex required for transport machinery 41 . Individual lipid moieties such as PGPC and POVPC, which constitute less than 10% of the total lipids present in the parental oxPAPC mixture 21 , induce NLRP3 inflammasome activation and the release of IL-1β from DCs and macrophages 24 . However, while the oxPAPC mixture induces a state of hyperactivation in DCs generated using granulocyte–macrophage colony-stimulating factor (GM-CSF), oxPAPCs were poor inducers of IL-1β secretion from the type 1 conventional DC (cDC1) subset or cDC2 subset of DCs 34 . In addition, oxPAPCs were capable of eliciting these responses only from DCs, not macrophages. Conversely, PGPC was able to induce inflammasome-dependent IL-1β secretion from living cDC1s, cDC2s and macrophages 34 . All these oxPAPC components engage CD14 and promote inflammasome-mediated IL-1β release from living cells (Fig. 2 ). Much work remains to be done to define the molecular events that dictate the specific cell fate commitments induced by oxPAPCs or the component lipids. Additional activities of oxPAPC components have been identified, which are described in the following sections. Fig. 2 Mechanism of myeloid cell hyperactivation by oxidized phosphocholines. Lipopolysaccharide (LPS) is captured by surface CD14, which delivers this lipid to membrane-associated MD2 and Toll-like receptor 4 (TLR4). CD14 then transports LPS and TLR4 into the cell. TLR4 activation unleashes signalling pathways that stimulate an inflammatory response, resulting in the upregulation of many genes, including IL1B and NLRP3 . This signal is recognized as a priming signal. Oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine derivatives (oxPAPCs) are also captured by CD14 and transported into the cell. Somehow oxPAPCs leave the endosomes and bind to caspase 11. This interaction leads to the oligomerization of an NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome that results in a hyperactive state, in which caspase 1 cleaves pro-IL-1β into its mature form and gasdermin D (GSDMD) into its active form. Hyperactive dendritic cells (DCs) induce IL-1β release without the commitment to pyroptosis. As depicted in the cell at the top of the figure, oxPAPC binding to CD14 prevents subsequent interactions with LPS. Box 4 Distinct activation states of dendritic cells and implication for immunotherapy In the absence of infection or damage, dendritic cells (DCs) exist in a non-inflammatory resting state. Upon detection of pathogen-associated molecular patterns or damage-associated molecular patterns, pattern recognition receptor activation unleashes signalling pathways that lead to a shift in DC activities from a naive state to an 'activated' state 89 , 90 . Activated DCs upregulate several factors, including co-stimulatory molecules (such as CD69 and CD80) and chemokine receptors (such as CC-chemokine receptor 7 (CCR7)) that are essential for their migration to adjacent lymphoid tissues to initiate an adaptive immune response 4 , 91 . During this activation process, polarizing cytokines are produced by DCs that determine the progressive differentiation of CD4 + T cells and CD8 + T cells into effector cells and terminally differentiated memory cells. Active DCs have a life expectancy of only a few days, and are viewed as well-equipped antigen-presenting cells to stimulate de novo T cell priming 92 , and for boosting memory T cells. Therefore, several immunotherapies for cancer have been undertaken to use DCs to drive protective immunity 93 , 94 . These strategies often involve the use of microbial products that stimulate Toll-like receptors (TLRs), such as intratumoural administration of the TLR3 ligand poly(I:C) 94 . The clinical efficacy of DC vaccines remains unsubstantiated. A potential reason for this clinical failure is that we lack a consensus on the optimal means to stimulate DC activities that drive protective immunity 95 , 96 . Among the polarizing cytokines that DCs need to produce is IL-1β, a critical regulator of T cell differentiation, memory T cell generation and effector function 53 , 97 , 98 . However, TLR-stimulated DCs do not release this important cytokine, a finding that undermines the value of TLR-alone stimulations for cancer immunotherapy. DCs can be prompted to release IL-1β by treatments with chemical agonists of the inflammasome pathway, with the classic inflammasome agonists being those that stimulate an inflammatory form of cell death known as pyroptosis 10 , 13 , 99 , 100 . Pyroptotic DCs, however, cannot participate in the days-long process needed to prime and differentiate naive T cells in the draining lymph node 101 . Accordingly, stimuli that promote pyroptosis, such as alum (the vaccine adjuvant approved by the US Food and Drug administration) 102 , 103 , are recognized as weak inducers of T cell-mediated protective immunity to infection or cancer 104 . The ideal strategy of stimulating memory T cell immunity is achieved by generating hyperactive DCs, a recently identified activation state in which DCs have the ability to secrete inflammasome-dependent IL-1β while maintaining their viability 24 . When DCs are exposed to TLR ligands and oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine derivatives (oxPAPCs) or pure components of oxPAPCs (such as 1-palmitoyl-2-glutaroyl- sn -glycero-3-phosphocholine or 1-palmitoyl-2-(5-oxovaleroyl)- sn -glycero-3-phosphocholine), DCs achieve a long-lived state of 'hyperactivation' 21 , 24 . Notably, the type 1 conventional DC (cDC1) subset achieves a state of hyperactivation in vivo. Hyperactive cDC1s display common features with activated DCs (such as secretion of tumour necrosis factor), but they also gain the ability to simultaneously provide T cells with IL-1β over the course of several days 21 . Consequently, hyperactive cDC1s were identified as the most potent DCs in generating long-lived memory CD8 + T cells that eradicate tumours 34 . The identification of these distinct DC activation states provides a mandate to further explore the differential impact of hyperactive DCs compared with their naive, active or pyroptotic counterparts during immunopathogenesis. Impact of oxPAPCs on DC migratory activity Independently of their inflammasome-stimulatory activities, oxPAPCs promote the robust migratory capacity of TLR-activated DCs. Therefore, the hyperactivating stimuli LPS plus oxPAPCs (or PGPC) exceeded other activation stimuli (for example, LPS alone) in their ability to stimulate DC migration to adjacent draining lymph nodes 34 . RNA sequencing of the cDC1 and cDC2 subsets revealed that hyperactive DCs (primed with LPS and then stimulated with PGPC) upregulated the expression of several gene clusters involved in DC migration and chemotaxis 34 . Notably, DCs that were stimulated with LPS plus oxPAPCs or PGPC induced the upregulation of CC-chemokine receptor 7 (CCR7). Accordingly, LPS plus oxPL treatment of DCs enhanced their migratory activities to the draining lymph nodes after adoptive transfer into the skin 34 . The ability of oxPAPCs or their components to stimulate DC migration was independent of inflammasome activities, as no defects in DC migration were observed when studies were performed with cells lacking the inflammasome components NLRP3 or caspases 1 and 11. Although these findings expand the scope of immunological activities mediated by oxPAPCs, the mechanism by which oxPAPCs or PGPC modulates DC motility is undefined. oxPAPC-mediated mitochondrial reprogramming in macrophages Within macrophages, oxPAPCs stimulate metabolic reprogramming, termed 'hypermetabolism' 42 . This metabolic state translates tissue oxidation status into heightened inflammatory gene expression. While macrophages activated by LPS downregulate their oxidative phosphorylation activities and rely on glycolysis for energy production, macrophages exposed to LPS and oxPAPCs used both oxidative phosphorylation and glycolysis. Under these conditions, the Krebs cycle is fed with glutamine, and pro-IL-1β production is enhanced by the increased activity of the transcription factor hypoxia-inducible factor 1α (HIF1α). This process is mediated by the accumulation of the metabolite oxaloacetate in the cytoplasm 42 . The significance of the hypermetabolic state induced by oxPAPCs was revealed in atherosclerosis models, such as Ldlr −/− or Apoe −/− mice. When these mice were fed a high-fat diet, which drives oxPAPC accumulation in oxidized low-density lipoproteins, hypermetabolic phagocytes were identified in the blood. Interestingly, drugs that interfered with oxPAPC-driven metabolic changes reduced atherosclerotic plaque formation and protected mice against atherosclerosis development. It is speculated that this metabolic rewiring of macrophages increases mitochondrial fitness, which may prolong the lifespan of macrophages. In the context of infection, this hypermetabolic state may enhance host defence, but the same mechanism may underpin symptoms associated with heart disease and potentially other chronic inflammatory diseases. Pure lipids contained within the oxPAPC mixture elicited distinct contributions to metabolic hyperinflammation. Whereas POVPC and PGPC drove inflammasome activation in macrophages, 1-palmitoyl-2-(5,6-epoxyisoprostane E 2 )- sn -glycero-3-phosphocholine uniquely induced the hypermetabolic activities described above 42 . In another study, macrophages that were exposed to oxPAPCs acquired a unique redox-regulatory phenotype (referred to as Mox cells). Mox macrophages induce glutathione synthesis and depend on glucose to fuel cellular activities. This unusual macrophage phenotype was observed in atherosclerotic plaques of Ldlr −/− mice. Additional details about the metabolic activities of oxPLs in phagocytes are described in a recent review 43 . Impact of oxPAPCs on phagocytosis and endocytosis Accumulating evidence demonstrates that oxPAPCs modulate phagocytosis by antigen-presenting cells in a cell type-specific and context-specific manner. For example, oxPAPC pretreatment of peritoneal macrophages diminished their ability to internalize Escherichia coli . In mice, pre-exposure to oxPAPCs led to a reduction in host defence, resulting in bacterial outgrowth and systemic dissemination 31 . Similarly, intratracheal instillation of an oxPAPC mixture or pure phosphocholine moieties (for example, POVPC or PGPC) rendered mice unable to eradicate subsequent infections with Pseudomonas aeruginosa 32 . oxPAPCs generated in the bronchoalveolar space of mice exposed to cigarette smoke impaired phagocytosis by alveolar macrophages 32 . The link between these in vivo defects in phagocytosis after oxPAPC exposure was established by means of the monoclonal antibody E06, which binds to and prevents the activities of several oxPAPC component lipids. E06 treatment prevented the inhibition of bacterial phagocytosis and restored bacterial clearance 32 . In contrast to the bacterial exposures described above, oxPLs can stimulate phagocytosis of apoptotic cells by macrophages via CD36 (ref. 33 ). In the context of non-phagocytic cargo, oxPAPCs did not affect dextran or soluble ovalbumin uptake by endocytosis. Moreover, in the presence of oxPAPCs, the ability of LPS-primed DCs to cross-present peptides on MHC class I molecules was enhanced 34 . These data are in accordance with reports indicating that within DCs, antigen leakage from endosomes for cross-presentation may occur upon direct oxidation of endosomal lipids and generation of oxPLs 27 . Thus, depending on the cargo to be internalized, oxPAPCs exhibit contrasting effects on phagocytic and endocytic activities. The molecular basis for these context-dependent behaviours is undefined. Impact of oxPAPCs on inflammasome activities Following priming with various TLR ligands, oxPAPCs induce a long-lived state of hyperactivation in myeloid cells 24 . This activation state is distinct from naive, active or pyroptotic cell states. A description of these cell states is provided in Box 4 and Fig. 1 . A hallmark of oxPAPC-stimulated hyperactive cells is their ability to secrete inflammasome-dependent IL-1β while remaining viable. Extracellular oxPAPC components are detected by CD14 (ref. 21 ). CD14-dependent endocytosis delivers these lipids to intracellular caspase 11. oxPAPC binding with caspase 11 leads to NLRP3-dependent inflammasome assembly and IL-1β release in the absence of cell death 24 . In contrast to the rapid but transient burst of IL-1β release that occurs upon exposure of phagocytes to pyroptotic stimuli, oxPAPCs or PGPC promote IL-1β release from LPS-primed cells for several days 21 . Fig. 1 The T cell stimulatory activities of dendritic cells at distinct activation states. To regulate T cell activity, dendritic cells (DCs) provide T cells with several signals that are important for the establishment of an appropriate T cell response. Naive DCs are quiescent DCs that have the ability to take up antigens. Active DCs take up antigens and have an enhanced ability to present antigen peptides on MHC molecules. In addition, active DCs upregulate co-stimulatory molecules to stimulate T cells. Hyperactive DCs share similar activities with their active counterparts, but also gain the ability to hypermigrate to lymph nodes and to secrete IL-1β. Pyroptotic DCs secrete high levels of IL-1β. However, pyroptotic DCs are dead and lose their T cell stimulatory capacity. IL-1β is a highly inflammatory cytokine and its secretion is tightly regulated 35 . TLR signalling induces production of pro-IL-1β, but this pro-protein form lacks an amino-terminal secretion signal and cannot be secreted via the endoplasmic reticulum–Golgi network 35 . To gain inflammatory activities, pro-IL-1β must be cleaved, most commonly by inflammatory caspases present within inflammasomes 36 . Inflammasomes are supramolecular organizing centres, which assemble in the cytoplasm following their activation 36 . Caspases that are activated within inflammasomes (most commonly caspase 1) cleave pro-IL-1β and gasdermin D (GSDMD). GSDMD cleavage unleashes the latent ability of its amino terminus to form pores in the plasma membrane. Pore formation is facilitated by interactions between GSDMD and acidic phosphoinositides present in the inner leaflet of the plasma membrane, such as phosphatidylinositol 4,5-bisphosphate and phosphatidylserine 37 . GSDMD pores serve as conduits for mature IL-1β secretion in a cell lysis-independent event 38 , 39 . Cargo transport across the negatively charged GSDMD conduit occurs via a size-exclusion mechanism 38 , 39 , as well as by electrostatic filtering of cytosolic proteins, which favours the release of the positively charged mature IL-1β, while repelling pro-IL-1β 40 . If GSDMD pores are not repaired by the cell, pyroptotic cell death may occur, during which IL-1β and other cytosolic contents can be released non-selectively. Of the well-defined inflammasome agonists, including intracellular LPS, ATP, nigericin and alum, all induce pyroptosis. By contrast, within TLR ligand-stimulated cells, oxPAPCs induce inflammasome-dependent release of IL-1β through GSDMD pores in the absence of cell death. The ability of oxPAPCs to add IL-1β to the repertoire of factors that are normally secreted by living phagocytes renders these cells more immunostimulatory than their activated counterparts that have been stimulated only with PAMPs — hence the term 'hyperactive'. Hyperactive cells display evidence of fewer GSDMD pores at their surface than their pyroptotic counterparts 39 . Whether GSDMD pores in hyperactive cells are regulated similarly as in pyroptotic cells downstream of inflammasome activation is unknown. Hyperactive cells may undergo rapid membrane repair events that ensure viability. Indeed, pyroptotic events increase in cells that are defective for membrane repair mediated by the endosomal sorting complex required for transport machinery 41 . Individual lipid moieties such as PGPC and POVPC, which constitute less than 10% of the total lipids present in the parental oxPAPC mixture 21 , induce NLRP3 inflammasome activation and the release of IL-1β from DCs and macrophages 24 . However, while the oxPAPC mixture induces a state of hyperactivation in DCs generated using granulocyte–macrophage colony-stimulating factor (GM-CSF), oxPAPCs were poor inducers of IL-1β secretion from the type 1 conventional DC (cDC1) subset or cDC2 subset of DCs 34 . In addition, oxPAPCs were capable of eliciting these responses only from DCs, not macrophages. Conversely, PGPC was able to induce inflammasome-dependent IL-1β secretion from living cDC1s, cDC2s and macrophages 34 . All these oxPAPC components engage CD14 and promote inflammasome-mediated IL-1β release from living cells (Fig. 2 ). Much work remains to be done to define the molecular events that dictate the specific cell fate commitments induced by oxPAPCs or the component lipids. Additional activities of oxPAPC components have been identified, which are described in the following sections. Fig. 2 Mechanism of myeloid cell hyperactivation by oxidized phosphocholines. Lipopolysaccharide (LPS) is captured by surface CD14, which delivers this lipid to membrane-associated MD2 and Toll-like receptor 4 (TLR4). CD14 then transports LPS and TLR4 into the cell. TLR4 activation unleashes signalling pathways that stimulate an inflammatory response, resulting in the upregulation of many genes, including IL1B and NLRP3 . This signal is recognized as a priming signal. Oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine derivatives (oxPAPCs) are also captured by CD14 and transported into the cell. Somehow oxPAPCs leave the endosomes and bind to caspase 11. This interaction leads to the oligomerization of an NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome that results in a hyperactive state, in which caspase 1 cleaves pro-IL-1β into its mature form and gasdermin D (GSDMD) into its active form. Hyperactive dendritic cells (DCs) induce IL-1β release without the commitment to pyroptosis. As depicted in the cell at the top of the figure, oxPAPC binding to CD14 prevents subsequent interactions with LPS. Box 4 Distinct activation states of dendritic cells and implication for immunotherapy In the absence of infection or damage, dendritic cells (DCs) exist in a non-inflammatory resting state. Upon detection of pathogen-associated molecular patterns or damage-associated molecular patterns, pattern recognition receptor activation unleashes signalling pathways that lead to a shift in DC activities from a naive state to an 'activated' state 89 , 90 . Activated DCs upregulate several factors, including co-stimulatory molecules (such as CD69 and CD80) and chemokine receptors (such as CC-chemokine receptor 7 (CCR7)) that are essential for their migration to adjacent lymphoid tissues to initiate an adaptive immune response 4 , 91 . During this activation process, polarizing cytokines are produced by DCs that determine the progressive differentiation of CD4 + T cells and CD8 + T cells into effector cells and terminally differentiated memory cells. Active DCs have a life expectancy of only a few days, and are viewed as well-equipped antigen-presenting cells to stimulate de novo T cell priming 92 , and for boosting memory T cells. Therefore, several immunotherapies for cancer have been undertaken to use DCs to drive protective immunity 93 , 94 . These strategies often involve the use of microbial products that stimulate Toll-like receptors (TLRs), such as intratumoural administration of the TLR3 ligand poly(I:C) 94 . The clinical efficacy of DC vaccines remains unsubstantiated. A potential reason for this clinical failure is that we lack a consensus on the optimal means to stimulate DC activities that drive protective immunity 95 , 96 . Among the polarizing cytokines that DCs need to produce is IL-1β, a critical regulator of T cell differentiation, memory T cell generation and effector function 53 , 97 , 98 . However, TLR-stimulated DCs do not release this important cytokine, a finding that undermines the value of TLR-alone stimulations for cancer immunotherapy. DCs can be prompted to release IL-1β by treatments with chemical agonists of the inflammasome pathway, with the classic inflammasome agonists being those that stimulate an inflammatory form of cell death known as pyroptosis 10 , 13 , 99 , 100 . Pyroptotic DCs, however, cannot participate in the days-long process needed to prime and differentiate naive T cells in the draining lymph node 101 . Accordingly, stimuli that promote pyroptosis, such as alum (the vaccine adjuvant approved by the US Food and Drug administration) 102 , 103 , are recognized as weak inducers of T cell-mediated protective immunity to infection or cancer 104 . The ideal strategy of stimulating memory T cell immunity is achieved by generating hyperactive DCs, a recently identified activation state in which DCs have the ability to secrete inflammasome-dependent IL-1β while maintaining their viability 24 . When DCs are exposed to TLR ligands and oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphocholine derivatives (oxPAPCs) or pure components of oxPAPCs (such as 1-palmitoyl-2-glutaroyl- sn -glycero-3-phosphocholine or 1-palmitoyl-2-(5-oxovaleroyl)- sn -glycero-3-phosphocholine), DCs achieve a long-lived state of 'hyperactivation' 21 , 24 . Notably, the type 1 conventional DC (cDC1) subset achieves a state of hyperactivation in vivo. Hyperactive cDC1s display common features with activated DCs (such as secretion of tumour necrosis factor), but they also gain the ability to simultaneously provide T cells with IL-1β over the course of several days 21 . Consequently, hyperactive cDC1s were identified as the most potent DCs in generating long-lived memory CD8 + T cells that eradicate tumours 34 . The identification of these distinct DC activation states provides a mandate to further explore the differential impact of hyperactive DCs compared with their naive, active or pyroptotic counterparts during immunopathogenesis. Impact of oxPAPCs on DC migratory activity Independently of their inflammasome-stimulatory activities, oxPAPCs promote the robust migratory capacity of TLR-activated DCs. Therefore, the hyperactivating stimuli LPS plus oxPAPCs (or PGPC) exceeded other activation stimuli (for example, LPS alone) in their ability to stimulate DC migration to adjacent draining lymph nodes 34 . RNA sequencing of the cDC1 and cDC2 subsets revealed that hyperactive DCs (primed with LPS and then stimulated with PGPC) upregulated the expression of several gene clusters involved in DC migration and chemotaxis 34 . Notably, DCs that were stimulated with LPS plus oxPAPCs or PGPC induced the upregulation of CC-chemokine receptor 7 (CCR7). Accordingly, LPS plus oxPL treatment of DCs enhanced their migratory activities to the draining lymph nodes after adoptive transfer into the skin 34 . The ability of oxPAPCs or their components to stimulate DC migration was independent of inflammasome activities, as no defects in DC migration were observed when studies were performed with cells lacking the inflammasome components NLRP3 or caspases 1 and 11. Although these findings expand the scope of immunological activities mediated by oxPAPCs, the mechanism by which oxPAPCs or PGPC modulates DC motility is undefined. oxPAPC-mediated mitochondrial reprogramming in macrophages Within macrophages, oxPAPCs stimulate metabolic reprogramming, termed 'hypermetabolism' 42 . This metabolic state translates tissue oxidation status into heightened inflammatory gene expression. While macrophages activated by LPS downregulate their oxidative phosphorylation activities and rely on glycolysis for energy production, macrophages exposed to LPS and oxPAPCs used both oxidative phosphorylation and glycolysis. Under these conditions, the Krebs cycle is fed with glutamine, and pro-IL-1β production is enhanced by the increased activity of the transcription factor hypoxia-inducible factor 1α (HIF1α). This process is mediated by the accumulation of the metabolite oxaloacetate in the cytoplasm 42 . The significance of the hypermetabolic state induced by oxPAPCs was revealed in atherosclerosis models, such as Ldlr −/− or Apoe −/− mice. When these mice were fed a high-fat diet, which drives oxPAPC accumulation in oxidized low-density lipoproteins, hypermetabolic phagocytes were identified in the blood. Interestingly, drugs that interfered with oxPAPC-driven metabolic changes reduced atherosclerotic plaque formation and protected mice against atherosclerosis development. It is speculated that this metabolic rewiring of macrophages increases mitochondrial fitness, which may prolong the lifespan of macrophages. In the context of infection, this hypermetabolic state may enhance host defence, but the same mechanism may underpin symptoms associated with heart disease and potentially other chronic inflammatory diseases. Pure lipids contained within the oxPAPC mixture elicited distinct contributions to metabolic hyperinflammation. Whereas POVPC and PGPC drove inflammasome activation in macrophages, 1-palmitoyl-2-(5,6-epoxyisoprostane E 2 )- sn -glycero-3-phosphocholine uniquely induced the hypermetabolic activities described above 42 . In another study, macrophages that were exposed to oxPAPCs acquired a unique redox-regulatory phenotype (referred to as Mox cells). Mox macrophages induce glutathione synthesis and depend on glucose to fuel cellular activities. This unusual macrophage phenotype was observed in atherosclerotic plaques of Ldlr −/− mice. Additional details about the metabolic activities of oxPLs in phagocytes are described in a recent review 43 . Impact of oxPAPC on immune responses oxPAPC-induced acute inflammatory responses In the absence of PAMPs, oxPAPCs are weak inducers of cytokine production by myeloid cells 24 . In contrast to LPS, oxPAPCs did not induce TLR4 dimerization and endocytosis, myddosome formation or the expression of proinflammatory cytokines in macrophages 21 . However, in the context of animals, in which exposures to microbial products are difficult to control, oxPAPCs exhibit proinflammatory responses via TLR4 (refs 14 , 43 ). Upon lung injury, oxPAPCs promoted IL-6 secretion from alveolar macrophages, and played a critical role in lung disease pathogenesis via a TLR4–TICAM pathway. Neutralization of oxPAPCs by the antibody E06 attenuated IL-6 production in the bronchoalveolar fluid. Furthermore, Shirey et al. described strong inhibition of oxPAPC-induced IL-6 production after treatment with the TLR4 antagonist eritoran 43 . Thus, in airways, oxPAPCs are potent stimulators of inflammation. In contrast to the activities of oxPAPCs in the lung, studies of the peritoneal cavity revealed that oxPAPCs prevented TLR4 signalling 21 and were described as LPS antagonists 44 , 45 . These anti-inflammatory activities of oxPAPCs in the peritoneum can be reproduced in vitro, where oxPAPC-treated phagocytes that were subsequently exposed to LPS exhibited defects in TLR4 dimerization and signalling. As a consequence, oxPAPC pretreatment inhibited LPS-inducible expression of interferon-stimulated genes and secretion of proinflammatory cytokines 21 . In addition, several studies indicated that pretreatment of DCs or macrophages with oxPAPCs blocks nuclear factor-κB responses to subsequent LPS treatments 46 – 49 . Mechanistic studies revealed that this inhibition of TLR signalling occurred by competitive interaction of LPS or oxPAPCs with CD14 (ref. 21 ). Indeed, oxPAPCs and LPS interact with CD14 using the identical amino acids, thus providing a biochemical explanation for their competitive activities 21 . These mechanistic findings likely explain why co-injection of oxPAPCs and LPS into mice protected mice from lethal sepsis 46 , as under these conditions, both of these lipids compete with one another for CD14. Recent studies identified PECPC as the most potent anti-inflammatory oxPAPC component 50 . Additionally, a small synthetic analogue of PGPC named 'VB-201', in which the ester bonds at sn -1 and sn -2 of the glycerol backbone are replaced with ether bonds to enhance the molecule's stability against enzymatic degradation in vivo, was also shown to bind to CD14. VB-201 inhibits TLR2 and TLR4 in human and mouse monocytes and monocyte-derived DCs 51 . In addition, VB-201 administered orally ameliorated the severity of experimental autoimmune encephalomyelitis 51 , and inhibited liver inflammation in a mouse model of non-alcoholic steatohepatosis 52 . These data highlight circumstances in which oxPAPCs are acutely inflammatory or immunosuppressive. These seemingly disparate observations may be explained by the mechanistic studies that have been performed. The proinflammatory activities of oxPAPCs in the lung may be explained by the presence of TLR4 ligands at this mucosal surface, which synergize with oxPAPCs to drive inflammation. By contrast, the anti-inflammatory activities in the peritoneal cavity may be explained by the lack of resident TLR ligands to synergize with oxPAPCs. In this latter context, oxPAPC injection leads to reduced inflammatory responses to subsequent bacterial infections, perhaps because oxPAPC pre-injection saturates cellular pools of CD14 that would normally be stimulated by bacterial products. Support for this idea was provided by recent data demonstrating that pretreatment of the peritoneal cavity of mice with LPS enabled oxPAPCs to induce inflammation 21 . By contrast, oxPAPC injection into the peritoneum in the absence of LPS priming elicits no inflammatory response 21 . The immunomodulatory activities of oxPAPCs described above are associated with rapid innate immune responses that occur within minutes to hours of cellular exposure. In the next section we describe recent assessments of the impact of oxPAPCs on the long-term adaptive immune responses in the context of cancer. oxPAPC-mediated control of adaptive immunity oxPAPCs can regulate adaptive immunity by acting on antigen-presenting cells. Hyperactive DCs stimulated with LPS and PGPC induced antitumour CD8 + T cell responses in mice that resulted from inflammasome activities within living DCs, and their hypermigratory ability that enables tumour antigens to reach lymphoid tissues (Fig. 3 ). These migratory DCs continuously secrete IL-1β to prime de novo T cells and activate memory T cells 34 , 53 . Consequently, LPS plus PGPC was a strong adjuvant to complex tumour antigens (for example, whole tumour lysates) during prophylactic or therapeutic immunizations against a range of murine tumours. Recent studies in humans revealed the need for multiple neoantigens (up to 20 peptides) for effective generation of personalized cancer therapies 54 – 56 . The ability of oxPLs to adjuvant whole tumour lysates may bypass the challenge of neoantigen identification and enable immunotherapies to be developed that are agnostic to the identity of tumour antigens 34 . It is likely that hyperactivating stimuli uniquely elicit the generation of a wide diversity of antigen-specific T cells, perhaps due to their abundance in the draining lymph node. Indeed, oxPAPC-containing or PGPC-containing adjuvants potentiated the generation of antigen-specific T cell responses and increased the kinetics of memory and effector T cell generation 34 . Fig. 3 Hyperactive DCs control antitumour T cell immunity. Upon pathogen encounter, immature dendritic cells (DCs) circulating in the peripheral tissue recognize pathogen-associated molecular patterns (PAMPs) via Toll like-receptors (TLRs) and shift their activities from a naive state to an active state. In the presence of tissue damage, CD14 captures 1-palmitoyl-2-glutaroyl- sn -glycero-3-phosphocholine (PGPC) and delivers the lipids to caspase 11 to induce NLR family pyrin domain-containing protein 3 (NLRP3) activation. These DCs achieve a long-lived state of hyperactivation, leading to IL-1β release from living DCs. Hyperactive DCs upregulate CC-chemokine receptor 7 (CCR7) expression and retain the ability for antigen uptake and processing of antigenic peptide on MHC class I. Hyperactive DCs carrying antigens then migrate to the draining lymph node via CCR7, where they activate CD8 + T cells. IL-1β secreted by hyperactive DCs signal via IL-1 receptor (IL-1R) to stimulate T cell effector function. IFNγ, interferon-γ; TCR, T cell receptor. In considering the underlying logic to the benefit of hyperactivating stimuli on antitumour immunity, we return to the central thesis we have proposed. We propose that the conditions of DC hyperactivation — where cells are exposed to generic indicators of microorganisms (TLR ligands) and tissue injury (oxPAPCs) — mimic the microenvironment of a virulent infection. Under these conditions, DCs receive multiple signals necessary to maximally enhance their migratory activities, metabolic activities and T cell stimulatory activities. The result of this enhancement is a superior adaptive immune response that enhances antitumour immunity. While the genesis of our logic derives from consideration of infectious encounters, these ideas have yet to be tested in this context. Of note, oxPLs, including oxidized low-density lipoprotein, are highly enriched in the tumour microenvironment. It was shown that oxidized low-density lipoprotein induces lipid peroxidation that suppresses CD8 + T cell effector functions in the tumour in a CD36-dependent manner 57 . These data provide the mandate to explore the links between lipid oxidation and cancer immunotherapy. Unrecognized sources of oxPAPCs may underlie diverse immunotherapies Although the role of hyperactive DCs in adaptive immunity needs further exploration, it is worth considering whether the protective activities of current therapies may depend on previously unrecognized oxPAPC sources that drive cell hyperactivation. For example, the chemotherapeutic chemicals oxaliplatin and anthracycline stimulate IL‐1β production via NLRP3 inflammasomes, leading to tumour-specific CD8 + T cell responses 57 . Oxaliplatin is known to stimulate ROS, whose activities may generate PGPC and other oxPAPCs within the tumour. These factors would act with any coexisting PAMPs to hyperactivate DCs and prime antitumour T cell responses. Consistent with this idea is evidence that patients with breast cancer bearing mutations in the inflammasome regulator P2X7R can develop rapid metastatic disease 57 , 58 . Total body irradiation should also induce oxPAPC formation, via the generation of ROS that occurs in these contexts. The impact of radiation-induced (or chemotherapy-induced) oxPAPC production on tumour immunity remains speculative. If these ideas are proven in humans, it is possible that vaccines could be engineered to specifically introduce oxPL moieties as adjuvants to promote more effective antigen-specific T cell responses. oxPAPC-induced acute inflammatory responses In the absence of PAMPs, oxPAPCs are weak inducers of cytokine production by myeloid cells 24 . In contrast to LPS, oxPAPCs did not induce TLR4 dimerization and endocytosis, myddosome formation or the expression of proinflammatory cytokines in macrophages 21 . However, in the context of animals, in which exposures to microbial products are difficult to control, oxPAPCs exhibit proinflammatory responses via TLR4 (refs 14 , 43 ). Upon lung injury, oxPAPCs promoted IL-6 secretion from alveolar macrophages, and played a critical role in lung disease pathogenesis via a TLR4–TICAM pathway. Neutralization of oxPAPCs by the antibody E06 attenuated IL-6 production in the bronchoalveolar fluid. Furthermore, Shirey et al. described strong inhibition of oxPAPC-induced IL-6 production after treatment with the TLR4 antagonist eritoran 43 . Thus, in airways, oxPAPCs are potent stimulators of inflammation. In contrast to the activities of oxPAPCs in the lung, studies of the peritoneal cavity revealed that oxPAPCs prevented TLR4 signalling 21 and were described as LPS antagonists 44 , 45 . These anti-inflammatory activities of oxPAPCs in the peritoneum can be reproduced in vitro, where oxPAPC-treated phagocytes that were subsequently exposed to LPS exhibited defects in TLR4 dimerization and signalling. As a consequence, oxPAPC pretreatment inhibited LPS-inducible expression of interferon-stimulated genes and secretion of proinflammatory cytokines 21 . In addition, several studies indicated that pretreatment of DCs or macrophages with oxPAPCs blocks nuclear factor-κB responses to subsequent LPS treatments 46 – 49 . Mechanistic studies revealed that this inhibition of TLR signalling occurred by competitive interaction of LPS or oxPAPCs with CD14 (ref. 21 ). Indeed, oxPAPCs and LPS interact with CD14 using the identical amino acids, thus providing a biochemical explanation for their competitive activities 21 . These mechanistic findings likely explain why co-injection of oxPAPCs and LPS into mice protected mice from lethal sepsis 46 , as under these conditions, both of these lipids compete with one another for CD14. Recent studies identified PECPC as the most potent anti-inflammatory oxPAPC component 50 . Additionally, a small synthetic analogue of PGPC named 'VB-201', in which the ester bonds at sn -1 and sn -2 of the glycerol backbone are replaced with ether bonds to enhance the molecule's stability against enzymatic degradation in vivo, was also shown to bind to CD14. VB-201 inhibits TLR2 and TLR4 in human and mouse monocytes and monocyte-derived DCs 51 . In addition, VB-201 administered orally ameliorated the severity of experimental autoimmune encephalomyelitis 51 , and inhibited liver inflammation in a mouse model of non-alcoholic steatohepatosis 52 . These data highlight circumstances in which oxPAPCs are acutely inflammatory or immunosuppressive. These seemingly disparate observations may be explained by the mechanistic studies that have been performed. The proinflammatory activities of oxPAPCs in the lung may be explained by the presence of TLR4 ligands at this mucosal surface, which synergize with oxPAPCs to drive inflammation. By contrast, the anti-inflammatory activities in the peritoneal cavity may be explained by the lack of resident TLR ligands to synergize with oxPAPCs. In this latter context, oxPAPC injection leads to reduced inflammatory responses to subsequent bacterial infections, perhaps because oxPAPC pre-injection saturates cellular pools of CD14 that would normally be stimulated by bacterial products. Support for this idea was provided by recent data demonstrating that pretreatment of the peritoneal cavity of mice with LPS enabled oxPAPCs to induce inflammation 21 . By contrast, oxPAPC injection into the peritoneum in the absence of LPS priming elicits no inflammatory response 21 . The immunomodulatory activities of oxPAPCs described above are associated with rapid innate immune responses that occur within minutes to hours of cellular exposure. In the next section we describe recent assessments of the impact of oxPAPCs on the long-term adaptive immune responses in the context of cancer. oxPAPC-mediated control of adaptive immunity oxPAPCs can regulate adaptive immunity by acting on antigen-presenting cells. Hyperactive DCs stimulated with LPS and PGPC induced antitumour CD8 + T cell responses in mice that resulted from inflammasome activities within living DCs, and their hypermigratory ability that enables tumour antigens to reach lymphoid tissues (Fig. 3 ). These migratory DCs continuously secrete IL-1β to prime de novo T cells and activate memory T cells 34 , 53 . Consequently, LPS plus PGPC was a strong adjuvant to complex tumour antigens (for example, whole tumour lysates) during prophylactic or therapeutic immunizations against a range of murine tumours. Recent studies in humans revealed the need for multiple neoantigens (up to 20 peptides) for effective generation of personalized cancer therapies 54 – 56 . The ability of oxPLs to adjuvant whole tumour lysates may bypass the challenge of neoantigen identification and enable immunotherapies to be developed that are agnostic to the identity of tumour antigens 34 . It is likely that hyperactivating stimuli uniquely elicit the generation of a wide diversity of antigen-specific T cells, perhaps due to their abundance in the draining lymph node. Indeed, oxPAPC-containing or PGPC-containing adjuvants potentiated the generation of antigen-specific T cell responses and increased the kinetics of memory and effector T cell generation 34 . Fig. 3 Hyperactive DCs control antitumour T cell immunity. Upon pathogen encounter, immature dendritic cells (DCs) circulating in the peripheral tissue recognize pathogen-associated molecular patterns (PAMPs) via Toll like-receptors (TLRs) and shift their activities from a naive state to an active state. In the presence of tissue damage, CD14 captures 1-palmitoyl-2-glutaroyl- sn -glycero-3-phosphocholine (PGPC) and delivers the lipids to caspase 11 to induce NLR family pyrin domain-containing protein 3 (NLRP3) activation. These DCs achieve a long-lived state of hyperactivation, leading to IL-1β release from living DCs. Hyperactive DCs upregulate CC-chemokine receptor 7 (CCR7) expression and retain the ability for antigen uptake and processing of antigenic peptide on MHC class I. Hyperactive DCs carrying antigens then migrate to the draining lymph node via CCR7, where they activate CD8 + T cells. IL-1β secreted by hyperactive DCs signal via IL-1 receptor (IL-1R) to stimulate T cell effector function. IFNγ, interferon-γ; TCR, T cell receptor. In considering the underlying logic to the benefit of hyperactivating stimuli on antitumour immunity, we return to the central thesis we have proposed. We propose that the conditions of DC hyperactivation — where cells are exposed to generic indicators of microorganisms (TLR ligands) and tissue injury (oxPAPCs) — mimic the microenvironment of a virulent infection. Under these conditions, DCs receive multiple signals necessary to maximally enhance their migratory activities, metabolic activities and T cell stimulatory activities. The result of this enhancement is a superior adaptive immune response that enhances antitumour immunity. While the genesis of our logic derives from consideration of infectious encounters, these ideas have yet to be tested in this context. Of note, oxPLs, including oxidized low-density lipoprotein, are highly enriched in the tumour microenvironment. It was shown that oxidized low-density lipoprotein induces lipid peroxidation that suppresses CD8 + T cell effector functions in the tumour in a CD36-dependent manner 57 . These data provide the mandate to explore the links between lipid oxidation and cancer immunotherapy. Unrecognized sources of oxPAPCs may underlie diverse immunotherapies Although the role of hyperactive DCs in adaptive immunity needs further exploration, it is worth considering whether the protective activities of current therapies may depend on previously unrecognized oxPAPC sources that drive cell hyperactivation. For example, the chemotherapeutic chemicals oxaliplatin and anthracycline stimulate IL‐1β production via NLRP3 inflammasomes, leading to tumour-specific CD8 + T cell responses 57 . Oxaliplatin is known to stimulate ROS, whose activities may generate PGPC and other oxPAPCs within the tumour. These factors would act with any coexisting PAMPs to hyperactivate DCs and prime antitumour T cell responses. Consistent with this idea is evidence that patients with breast cancer bearing mutations in the inflammasome regulator P2X7R can develop rapid metastatic disease 57 , 58 . Total body irradiation should also induce oxPAPC formation, via the generation of ROS that occurs in these contexts. The impact of radiation-induced (or chemotherapy-induced) oxPAPC production on tumour immunity remains speculative. If these ideas are proven in humans, it is possible that vaccines could be engineered to specifically introduce oxPL moieties as adjuvants to promote more effective antigen-specific T cell responses. Conclusions and perspectives We suggest that heterogeneous oxPAPC mixtures or pure oxPAPC components cannot be classified as intrinsically proinflammatory or anti-inflammatory molecules. It is likely that the pleiotropic effects of oxPAPCs work in concert in vivo. In this regard, our knowledge is currently limited, as little is known about the identity and function of specific oxPAPC compounds. Further research is required to better understand the biological role of oxPAPCs during infectious diseases and other threats to the host. Recent evidence indicates that PGPC and POVPC can prevent vesicular stomatitis virus entry, thereby limiting viral replication 59 . Although the mechanism by which oxPAPCs inhibit viral entry is undetermined, these data provide the mandate to explore the interplay of pathogens and oxPLs, which may open new avenues for novel therapeutic targets. Furthermore, a better understanding of how endogenous oxPLs impact phagocyte function is crucial for uncovering the impact of oxPLs on homeostasis and pathogenesis. POVPC abundance is increased in the lungs of aged mice, and these levels are further augmented upon inflammatory stimulation 60 . In addition, the clearance of oxPLs from lungs was delayed in aged mice 60 , and correlated with severe lung injury and delayed resolution. In this regard, several questions remain unexplored. How do these oxPLs regulate phagocyte function in the ageing host? Can these lipids regulate the inflammaging process? This knowledge could support the development of effective vaccination strategies designed for elderly patients. oxPLs enzymatically derived from innate immune cells were shown to promote coagulation factor function which restored thrombin generation. Therefore, it was proposed that bioactive oxPLs may be a target in bleeding and vascular inflammation disorders 61 , 62 . Interestingly, pyroptotic macrophages were shown to release tissue factor (an essential initiator of coagulation cascades) following inflammasome activation, which triggered blood clotting 63 . Whether oxPLs can regulate blood clotting via inflammasome activation is a possibility. Finally, oxPLs are not the only stimuli that can induce a state of DC hyperactivation. Other hyperactivating stimuli besides oxPLs have been reported 64 , including bacterial peptidoglycan 65 , Staphylococcus aureus ΔoatA mutants 66 and the TLR agonist R848 plus muramyl dipeptide 67 . The latter induces human cDC2 hyperactivation that enhances T helper 1 and T helper 17 cell responses. Whether hyperactivating stimuli could be generally classified as boosters of adaptive T cell immunity or context-dependent regulators of immunity requires further investigation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147595/

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on " Intracellular Traffic and Transport of Bacterial Protein Toxins ", reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis , α-hemolysin of Staphylococcus aureus , and binary toxin of Bacillus anthracis , which have found their "second life" in a variety of developing medical and technological applications. 1. Introduction: Channel-Forming Bacterial Toxins During intoxication or internalization, many bacterial exotoxins form ion-conducting channels in membranes of the targeted cells or intracellular organelles. Channel formation is potentiated by a unique ability of many of these molecules to exist in two states: a stable water-soluble conformation and an integral membrane pore [ 1 ]. The resulting channels contribute to the toxin's virulent action either directly or indirectly. Some channel-forming toxins directly kill compromised cells by inducing uncontrollable leaks of ions, water, and/or water-soluble metabolites [ 2 , 3 , 4 ]. The toxin-induced membrane perforation allows virulent cells to combat the host defense systems, to mediate escape of invasive bacterial cells into host cytoplasm from encapsulating phagosomes, to provide nutrients for bacteria, and to eliminate or control the competing bacterial cells [ 4 , 5 , 6 , 7 , 8 ]. Membrane-perforating bacterial toxins are often called pore-forming toxins (PFTs). Distinctively different are the AB-type toxins that employ channel-forming parts for intracellular delivery of specialized catalytic subunits. In contrast to PFTs, AB-type toxins act in the cytosol, targeting specific intracellular substrates. The AB-type toxins are secreted as either single-chain proteins containing at least two functional domains, the receptor binding B domain and the active/enzymatic A domain, or as two (or three in the case of anthrax toxin) individual non-linked binary toxin subunits, an enzymatic/active A component and a binding/translocation B component [ 9 ]. For the AB-type toxin intracellular action, the enzymatic A component has to be delivered across the target cell membrane into the cytosol. Remarkably, the B components of the binary bacterial toxins secreted by several pathogenic species of Bacillus and Clostridium not only provide the binding site for the A components but also mediate the A component intracellular transport [ 9 ]. In particular, following the receptor mediated endocytosis, the B components form oligomeric transmembrane channels, that have been suggested to serve as active translocation pathways for the A component transport. The exact role of the B domain in transport of the single-chain AB-type toxins is debated; in particular, it is not always established whether the uptake of these toxins involves formation of ion channels. However, ion channel activity in planar bilayer membranes has been documented for at least two members of the family, namely diphtheria toxin, DT of Corynebacterium diphtheriae [ 10 , 11 , 12 ] and botulinum neurotoxin, BoNT of Clostridium botulinum [ 13 , 14 ]. Moreover, even though formation of ion channels in the artificial lipid bilayer systems has been reported for a number of binary bacterial toxin B components [ 15 , 16 , 17 ], the traditional pore-facilitated model of anthrax toxin internalization was recently challenged by an alternative "membrane rupturing" scenario [ 18 ]. This review is written to discuss several "classical" channel-forming bacterial toxins, which won their "second life" in a variety of developing biotechnological applications. Because the focus of this "Toxins" issue is on intracellular traffic and transport of bacterial protein toxins, we will mostly discuss the questions related to biosensing properties of the channel-forming bacterial toxins. More specifically, we highlight the applications where bacterial toxin pores were used to probe mechanisms of molecule detection and macromolecule transport. Remarkably, some of these applications, for instance those that develop the targeted toxin therapies to battle cancer, directly employ the unique ability of these proteins to intoxicate cells, whereas many other approaches use the channel-forming bacterial toxins as suitable tools able to respond to electrical, chemical or mechanical stimuli [ 19 ]. Note that the channel-forming proteins play quite distinct roles in the cell intoxication by PFTs and AB-type bacterial toxins. However, for the practical purpose of this review, we will use the term "channel-forming bacterial toxins" when referring to any of these types of toxins. 2. Biosensing and Polymer Translocation with Nanopores The channel-forming toxins, and, more broadly, biological nanopores are molecules of immediate interest for biotechnology [ 19 , 20 , 21 , 22 ]. The ability of these molecules to generate selective and regulated pathways for water, ions, and water-soluble small and macromolecules in biological and artificial membranes offers exciting possibilities for a variety of medical and technological applications. To be directly examined for these applications, the biological nanopores are often incorporated into model lipid membranes where changes in the channel permeability to ions could be detected directly by applying transmembrane voltage and measuring ion current. The reconstitution methods range from the "classical" planar lipid bilayer and liposomes-based approaches to membranes supported on solid substrates and droplet interface bilayers (reviewed in refs. [ 19 , 23 ]). After gaining momentum from the pioneered studies by Kasianowicz and Bezrukov [ 24 , 25 ] where α-hemolysin (αHL) of Staphylococcus aureus was suggested to serve as a "nanoscopic cuvette" to study reaction dynamic some 20 years ago, the field rapidly exploded. Sensing with biological nanopores is based on reversible interruptions of ion current through an assembled pore in the model membrane that are generated by individual analytes, molecular complexes or nanoscale objects. Changes in this current resolved with uttermost precision using low noise feedback-loop operational amplifiers [ 26 ] could be used to determine presence, identity, structure, sequence, charged state, and kinetics of the studied objects or phenomena. In a way, this method is a molecular-scaled version of the resistive-pulse sensing technique suggested by William Coulter to count particles suspended in a fluid [ 27 ]. Within the reconstituted pore, single molecule binding affects the simultaneous concurrent transport of ~10 4 s −1 of the small ions, thus providing great electrical amplification of the studied single-molecule process. Pores formed by the membrane-perforating toxins may act as elementary on/off "switches" activated in response to interaction with specific analyte molecules, and as sensors reporting on their membrane surrounding and providing a specific volume for the studied compounds to pass and/or to interact. 3. GrA, αHL, and PA 63 as Nanopores of Choice in Biotechnology Among a variety of channel-forming bacterial toxins potentially suitable for the biotechnological applications, one can highlight several dominant toxin molecules that, for a number of reasons, are in the lead of the field. Those are small peptide channel-forming gramicidin A of Bacillus brevis , which induces lesions in cell membranes; α-hemolysin of Staphylococcus aureus , a β-barrel heptameric pore-former, which also perforates cellular membranes; and binary toxin of Bacillus anthracis , in which the channel-forming component mediates delivery of the enzymatic subunits into target cell cytoplasm. The small channel-forming peptides, such as gramicidin A, can be chemically modified to serve as ideal on/off switches reacting to different stimuli and sensors of the bilayer membrane physical properties. The large β-barrel channels, such as α-hemolysin, are more suitable for the stochastic resistive-pulse sensing of the entering or passing small and macromolecules. The binary anthrax toxin is a unique tool to investigate the fundamental principles of protein translocation across the membranes and to reengineer the toxin's properties for targeted killing of the cancer cells. In the following sections, we will first briefly review the biological functions of gramicidin A, α-hemolysin, and anthrax toxin and then discuss the main current and potential future applications of these three toxins in the growing field of biotechnology. 3.1. Gramicidin A of Bacillus Brevis Antibiotic gramicidin A (GrA) is secreted by soil bacteria Bacillus brevis in a mixture containing closely related peptide compounds [ 28 ]. GrA, which is a linear pentadecapeptide composed of 15 alternating L-D amino acids [ 29 , 30 , 31 ], is active against many gram-positive and several gram-negative bacteria [ 32 ]. Arguably one of the first ever described pore-formers, GrA generates defined and reproducible transmembrane water-filled channels [ 33 ] ( Figure 1 ). Figure 1 ( a ) Side and end views of the bilayer-spanning gramicidin A (GrA) channel. The energy-minimized structure represents a composite consistent with several NMR structures (PDB 1GRM, 1MAG); ( b ) GrA channels formed by the transbilayer dimerization of two β-helical subunits; ( c ) Single-channel current trace obtained with GrA in diphytanoylphosphatidylcholine bilayer [ 34 ]. Reprinted and modified with permission from reference [ 34 ]. GrA membrane activity has been studied extensively over the last 40 years. Solution and solid-state NMR reveals a dimeric structure of the GrA pore where each molecular monomer adopts β-helical conformation and interacts with another one through hydrogen bonding between terminal formyl groups [ 35 , 36 ]. The luminal diameter of the pore that is formed by a peptide backbone is 4 à , as determined from the channel's permeability to alkali metal cations, H + , and water [ 37 , 38 ] ( Figure 1 a). Despite relatively low molecular weight of ~2 kDa, GrA creates well-defined and reproducible pores in planar lipid membranes ( Figure 1 c). The presented ion current recording, obtained under voltage-clamp experimental conditions, demonstrates sharp step-wise increments of the transmembrane current, where each upward "step" corresponds to formation of a GrA conductive dimer in a membrane, and the downward step indicates the dimer dissociation; maximum of four concurrently open GrA channels are seen in the record (numbered on the right side of Figure 1 c). This type of single-channel recording allows for direct estimation of the major parameters of ion channel activity, current amplitude of the single pore and its average open time. These channels exhibit the structural and functional features typical for complex membrane proteins: defined conductive levels, sharp transitions between open and closed states ( i.e. , spontaneous gating), and low excess electric noise of the pore open state. The gramicidin channels show an ideal selectivity for monovalent inorganic cations [ 38 ], that originates from the side chain dipoles of the peptide backbone facing the channel lumen [ 39 , 40 ]. The cation transport across the narrow GrA pore occurs in a single-file fashion through consecutive ion bindings to two specific sites in the pore [ 41 ]. The gramicidin channels are specifically interesting because they show a unique sensitivity to the properties of the surrounding lipid bilayer. The length of a pore-forming dimer is only ~26 à , which roughly matches the thickness of the hydrophobic part of the lipid bilayer. Since the hydrophobic thickness of the gramicidin dimer is less than the thickness of the membrane [ 42 , 43 ], pore formation is almost always associated with a local membrane deformation [ 44 , 45 ] ( Figure 1 b). The energetic cost of that deformation is therefore a primary factor determining the channel's functions. Because of that, the membrane lipid environment is a strong modulator of the GrA channel activity. Subtle changes in membrane properties such as membrane thickness, lipid composition, and ordered state influence parameters of GrA channels. Due to its relative simplicity, gramicidin has been recognized as unique ion channel for modeling and simulation of the fundamental principles of lipid/protein interactions, and membrane protein structure and function. The GrA channels are good candidates for the molecular sensing applications because of the relative simplicity of the peptide, defined characteristics of the ion channel conductance, and unique sensitivity of conductive parameters channel to the membrane structure [ 46 , 47 ]. In the developed applications, GrA is used to study mechanical properties [ 48 ] and charged state of membranes [ 34 , 49 , 50 ], to detect protein-membrane interactions [ 51 ], to probe enzyme activity [ 52 ] and protein-protein interactions, to create conductive components of bio-inspired diodes [ 53 ], and to fabricate components of drug delivery systems in nanomedicine [ 54 ]. 3.2. α-Hemolysin of Staphylococcus Aureus Alpha-hemolysin (αHL) is a water-soluble toxin secreted by the pathogenic bacterium Staphylococcus aureus [ 55 , 56 ]. Released as a 293-amino acid monomeric polypeptide with a molecular mass of 33 kDa, αHL is believed to bind to membrane protein receptors [ 57 , 58 ] and/or to specific lipids in the susceptible target cells [ 59 , 60 ]. Seven adsorbed monomeric subunits then associate and form a nonlytic prepore complex on the membrane. The subunits further penetrate the membrane to form a lytic pore [ 61 , 62 ]. Recent evidence suggests that the lytic pore formation may be not the only way of αHL toxicity. A specific toxin-induced activation of its putative metalloprotease receptor may trigger activation of intracellular cascades, leading to increased toxicity (for a review see reference [ 63 ]). However, the correct assembly of the αHL pores remains a necessary step for its toxic action [ 64 ]. The first original study on αHL channel reconstitution in the model bilayers membranes was performed by Krasilnikov and co-workers over three decades ago [ 65 ]. Further electrophysiological studies refined our understanding of an αHL pore's conductance, geometric, and gating properties [ 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 ]. The pore is weakly anion-selective at neutral pH [ 66 ] and a stable conductance of ~1 nS is observed in 1 M KCl solution at room temperature ( Figure 2 a). Figure 2 ( a ) Typical recording of a single αHL channel reconstituted into diphytanoyl-phosphatidylcholine membrane. Applied voltage is 100 mV. Channel current corresponds to ~100 pA in 1M KCl, pH 7.4; ( b ) Crystal structure of the αHL heptamer (top and side views are shown) (PDB 7AHL) [ 78 ]. The mushroom-shaped complex is approximately 100 à tall and up to 100 à in diameter, and the stem domain measures about 52 à in height and 26 à in diameter. Crystal structure of the αHL pore was solved in 1996 at 1.9-à resolution [ 78 ] ( Figure 2 b). The pore heptamer has a hollow mushroom-like shape consisting of the "stem", "cap", and "rim" domains. The cap of the mushroom, which together with the rim forms the core of the protein complex, resides outside the target membrane and is formed by the N- and C-terminal ends of the monomers; it is composed of a β-sandwich and has a diameter of 100 à . The stem part is a lytic transmembrane 14-stranded β-barrel assembled from seven β-hairpins, each contributed by an individual monomer. Two apparent constrictions with radii of 0.9 nm and 0.6–0.7 nm are located in the channel lumen, with the larger one being closer to the side of the cap. The interior of the β-barrel is primarily hydrophilic while the exterior has a hydrophobic surface. Molecular dynamic simulations were applied to estimate the diameter of the pore and the permeability of water through its side channels of the mushroom "head" [ 79 ]. By applying an electric potential across the pore, the authors were able to calculate the open-channel conductivity and the electrostatic potential along the pore length. Large pore dimensions and structural robustness of the αHL heptamer permit the wide usage of this bacterial toxin in a variety of developing biotechnological applications. 3.3. PA 63 Component of Anthrax Toxin of Bacillus Anthracis Bacillus anthracis , the bacterium that causes anthrax, is a large Gram-positive, rod-shaped, aerobic, spore-forming bacterial pathogen. The tripartite exotoxin (anthrax toxin) [ 80 , 81 ] and phagocytosis-inhibiting poly-D-glutamic acid capsule are the main virulence factors of B. anthracis [ 82 ]. The deliberate dissemination of B. anthracis spores in 2001 via the "anthrax letters" and their fatal consequences led to almost 13 years of continuing political and scientific efforts to develop medical countermeasures to protect humans from anthrax bioterrorism [ 83 ]. Because anthrax infections are infrequent, bacterial resistance of B. anthracis , even though described [ 84 , 85 ], is not a major focus of ongoing research. The recent efforts to disable the anthrax infection mostly focused on inhibiting the tripartite anthrax toxin (reviewed in [ 86 ]). Anthrax infection, especially in its inhalational form, is extremely difficult to treat because flu-like symptoms appear only after B. anthracis have multiplied inside the host and started to produce the anthrax exotoxin [ 87 , 88 ]. At this stage, the aggressive antibacterial therapy can inhibit the bacterium growth, but the infection can still be lethal because of the accumulation of the toxin [ 89 ]. The recent progress made in understanding of anthrax toxin structure, arrangement, cellular uptake and functions is significant (reviewed in refs. [ 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 ]). Briefly, the binary anthrax toxin consists of two enzymatically active A moieties: lethal factor (LF) and edema factor (EF), and a single shared binding and translocation moiety: protective antigen (PA). The term "protective antigen" originates from the ability of this protein to stimulate production of the protective antibodies when used in anthrax vaccines. Individually, anthrax toxin's PA, LF and EF components are nontoxic; however, PA combinations with lethal toxin (LT = LF + PA) or with edema toxin (ET = EF + PA) are primarily responsible for the anthrax symptoms and lethality. The internalization process of anthrax toxin involves several stages ( Figure 3 a). Figure 3 ( a ) A schematic model of Bacillus anthracis toxins cell entry; ( b ) Side and top views of anthrax toxin PA 63 component (the symmetric model, PDB:1V36) [ 101 ]. The Phe427s are marked because of their importance in the transport properties; ( c ) Conductance of a single PA 63 channel reconstituted into planar lipid membranes demonstrate fast flickering between open and closed states at 1-ms time resolution [ 102 ]. Reprinted with permission from references [ 86 , 101 , 102 ]. Copyright 2011 Wiley, 2012 Elsevier. First, full length PA 83 binds to the CMG2 and TEM8 receptors [ 103 , 104 ] on a host cell surface and is subsequently cleaved by extracellular furin protease to PA 63 [ 105 ]. PA 63 then undergoes oligomerization, which leads to formation of heptameric, (PA 63 ) 7 [ 106 ] and octameric, (PA 63 ) 8 [ 107 ] ring-shaped prepores. The formation of (PA 63 ) 7 and (PA 63 ) 8 generates, correspondingly, three [ 108 ] and four [ 107 ] LF/EF binding sites at the interface of two adjacent PA 63 molecules and causes receptor-mediated endocytosis of the anthrax toxin tripartite complexes [ 109 ]. The oligomeric PA 63 prepore then undergoes significant structural changes promoted by the acidic endosomal environment, which results in the formation of a mushroom-shaped ion channel (125 à in diameter with 70 à long cap, and 100 à long stem [ 110 ]) ( Figure 3 b), preferentially selective to cations [ 15 ]. When incorporated into the bilayer lipid membranes, PA 63 was reported to form ion-permeable cation-selective ion channels [ 15 , 111 , 112 ] prone to voltage-dependent closures typical for the other β-barrel channels. The characteristic property of PA 63 single channels as well as the channel-forming components of binary clostridial C2 and iota toxins reconstituted into bilayer membranes is the non-voltage-dependent flickering of ion current between the open and completely closed states [ 16 , 17 , 113 , 114 , 115 ] ( Figure 3 c). Interestingly, the current noise of the F427A (ϕ-clamp) mutant (see Section 5.2 ) of PA 63 is mostly free of this complex behavior [ 115 ]. A PA oligomer then is believed to act as an effective translocase, which unfolds and translocates LF and EF inside the cell using the proton gradient across the endosomal membrane (pH endosome pH cis ( Figure 10 c) [ 117 , 239 , 240 ]. At the same time, the ΔpH that the authors used to mimic a proton gradient across an endosomal membrane was more efficient in stimulating protein transport, allowing translocation not only LF N but also full-length LF and EF. The conductance rise was S-shaped, and its rate increased systematically both with increasing of Δψ and ΔpH. With the minimum PA 63 -binding translocation component domain of LF, LF N, it was shown that, under small Δψ, the N terminus of bound LF N enters the oligomeric channel and initiates the threading of the substrate across the bilayer membrane [ 116 ]. Figure 10 The interaction of LF N with the PA 63 multichannel membrane [ 239 ]. ( a ) After the macroscopic PA 63 -induced current had reached a steady state at +20 mV, ~3 nM LF N was added to the cis compartment of the chamber, which resulted in a rapid fall in current. The cis compartment was then perfused of unbound LF N . At time zero, the voltage was increased to +50 mV, and LF N translocation kinetics through the PA 63 channels was indicated by the increase in conductance vs. time; ( b ) Kinetic transients for LF N translocation at +60 mV and the indicated symmetrical pH values; ( c ) The rate of LF N translocation is controlled by the magnitude and sign of the transmembrane ΔpH (pH trans > pH cis ); ( d ) A schematic illustration of the tandem Brownian ratchet translocation mechanism shows a model of several partially unfolded intermediates of LF N during translocation [ 239 ]. The scheme demonstrates how the hydrophobic, ϕ-clamp ratchet and the protonation-state ratchet may work together facilitating translocation of LF N . Reprinted with permission from reference [ 239 ]. In order to identify residues in the PA 63 lumen important for the channel-mediated transport of a substrate, the authors used cysteine-scanning mutagenesis coupled to [2-(trimethylammonium ethyl]methanethiosulfonate (MTS-ET) modifications [ 241 ]. The channels were the most significantly affected by Phe427/Cys427 modifications, and Phe427 (named ϕ-clamp is the most hydrophobic residue in the otherwise hydrophilic pore lining [ 118 ]. In particular, when the bulky 427 phenylalanine residue was replaced by the small alanine, the single-channel conductance expectably increased but the rate of LF N translocation reduced by a factor of six [ 239 ]. Based on the fact that the ϕ-clamp mutation blocked LF translocation in cell assays [ 242 ] or significantly slowed it in vitro [ 118 ], a chaperone model was suggested in which the ϕ-clamp's phenyl rings directly interacted with the translocating polypeptide. According to the model [ 118 ], the ϕ-clamp recognizes substrates through the hydrophobic effects, enhanced by aromatic-aromatic, π-π, and cation-π interactions, and stabilizes the fragments it an unfolded state. This study for the first time provided evidence that PA 63 was not a passive tunnel through which proteins electrophorese but rather an active player in facilitating the transport of a substrate molecule. At the same time, the lumen of a 14-strand β-barrel PA 63 is believed to be as narrow as 12–15 à [ 101 , 174 , 243 ], which would allow the pore to accommodate secondary structure only as large as an α-helix [ 244 ]. Therefore, translocation through PA 63 would require the enzymatic substrates to unfold. If so, what are the factors that cause the folded LF and EF with tertiary structures to unfold? The authors show that one critical factor is low endosomal pH. Thus, the acidic conditions in the endosome are sufficient to destabilize the native structure of LF N and EF N proteins [ 244 ]. In vitro experiments have also revealed that the translocation process is driven by cis positive voltages [ 117 ] and that the PA 63 channel is selective to cations [ 175 ]. Paradoxically, LF N bears a net negative charge (about six negative charges) even at pH values as low as 5.5 [ 240 ]. To explain this finding, the authors developed a model describing PA 63 channel as a protein translocase rather than an unselective long tunnel. The easiest way for the substrates to achieve a net positive charge, which is thermodynamically and electrostatically required for the translocation, would be to get their aspartic and glutamic residues neutralized, for instance protonated [ 239 ]. Under ΔpH, the substrate molecule would get protons from the cis compartment solution (or an endosome) and discharge them into the trans compartment solution (or cytosol) following translocation. At the same time, the portion of LF/EF located inside the pore lumen would carry a net positive charge and PA 63 would work as a proton-protein symporter [ 119 ]. To address the fundamental question of the translocation driving force, the authors developed a novel charge state-dependent Brownian-ratchet model for the ΔpH-driven translocation, which is based on the chemical asymmetry created by ΔpH [ 239 ]. The probability of an aspartate or glutamate being in the protonated form is greater in the more acidic cis side compared to the more basic trans side, and, therefore, the rate of their entry is higher from the cis side than that from the trans side [ 240 ]. After the protonated aspartate and glutamate groups reach the cytosolic part of the membrane, they deprotonate, becoming negatively charged again, resulting in the electrostatic repulsion of the negatively charged chain fragment from the channel lined with the negatively charged residues. This electrostatic repulsion drives the translocation per se and enforces its directionality [ 100 , 239 ]. Therefore, the random Brownian motion of the substrate inside PA 63 is biased toward the more basic trans compartment. Based on the experimental evidence, the authors suggest that the PA 63 symporter achieves protein translocation using a tandem of two synergistic Brownian ratchets: the ϕ-clamp ratchet, promoting the substrate unfolding, and the charge-state ratchet, which biases the entry rates of the substrates into the pore ( Figure 10 d). Remarkably, further experiments with planar lipid bilayers have imparted support to this model using essentially non-titratable negatively charged SO 3 − groups introduced at most positions in LF N [ 119 ]. The voltage-driven translocation of the resulting LF N variants was dramatically reduced and the ϕ-clamp was determined as a significant factor in the exclusion of SO 3 − from the channel. In another study, semisynthetic variants of LF N (12-263) in which selected acidic residues were replaced with the unnatural amino acid, cysteic acid, were examined [ 245 ]. The cysteic acid has a negatively charged side chain protonated only at pH values below the physiological range. Depending on the number of acidic residues replaced, transport of these mutants was either significantly suppressed or completely inhibited, whereas their binding and channel-blocking properties were comparable with those of wild type LF N . To determine if the substrate's secondary structure is preserved during the pore-facilitated translocation, a method of trapping the polypeptide chain of the translocating protein within a pore was developed [ 121 ]. In order to determine the minimum number of residues that could traverse the PA 63 oligomeric pore, the authors attached biotin to the N terminus of LF N and used molecular stoppers at the different positions. The trans -side streptavidin addition was used to determine whether the N terminus has reached the trans compartment solution. If the N terminus-stopper distance was long enough to allow for LF N to appear from the pore, streptavidin was able to bind the biotin. Otherwise, no biotin binding was recorded. The authors showed that an LF N polypeptide chain adopts a fully extended conformation, because it is being translocated through the channel's stem. A typical characteristic of LF N translocation is its non-exponential S-shaped kinetics. At the same time, in most of the experiments the substrate translocation was investigated under conditions where two or three LF N molecules were bound to PA 63 channels. To understand whether the S-shaped kinetics is an intrinsic characteristic of translocation kinetics or a consequence of the translocation in a tandem of two or three LF N molecules, a kinetic analysis of protein transport via the macroscopic and single-channel PA 63 channels was performed [ 120 ]. The study showed that even with one LF N bound to PA 63 , the translocation kinetics is S-shaped, being, however, slower, with more than one LF N bound. The authors also propose a simple drift-diffusion model of LF N transport, where LF N is represented as a charged rod that moves subject to both Brownian motion and an applied electric field across the membrane [ 120 ]. A dramatically different model of anthrax toxin transmembrane uptake was recently suggested by Kasianowicz and colleagues [ 18 ]. The new model suggests that instead of LF and EF being threaded through the pore, anthrax toxin complexes ( i.e. , LF or EF bound to the PA 63 channel) rupture membranes ( Figure 11 ). Figure 11 ( a ) A schematic illustration of two proposed mechanisms for PA 63 channel-mediated lethal factor (LF) and edema factor (EF) transport into the cytoplasm. The channel-mediated translocation model suggests that LF and EF pass through the pore [ 118 , 239 ]. The recent membrane rupturing model suggests that the enzymatically active anthrax toxin complexes (namely, LF or EF bound to the PA 63 channel) rupture membranes [ 18 ]. Time course of the PA 63 channel conductance with ( b ) LF removed before or ( c ) maintained at 1 nM during cis -side acidification. ( Top rows ) ~60 PA 63 channels were reconstituted into a planar bilayer membrane at pH cis | trans 7.2|7.2 (black) and, 1 nM LF was added to the cis chamber (blue). ( Middle rows ) The pH cis | trans 5.5|7.2 gradient was formed by perfusing the cis chamber with pH 5.5 buffer that contained either [LF] = 0 (red) or [LF] = 1 nM (green). ( Bottom rows ) The neutral pH condition (pH cis | trans 7.2|7.2) (black) was restored by perfusing the cis chamber with pH 7.2 buffer. If LF was present, the cis chamber was first perfused with pH 5.5 buffer (red) then pH 7.2 buffer (black). Reprinted with permission from reference [ 18 ]. Kasianowicz et al. , question the translocation model, noting that the in vitro pH gradient formation used in the previous translocation studies is precisely opposite to what occurs in vivo [ 18 ]. The translocation, manifested by PA 63 ion current recovery ( Figure 10 ), was observed either in static low pH conditions on both sites of the membrane (mostly with LF N ) or starting with the symmetrical acid conditions and forming a pH gradient by raising the trans solution pH (both with LF N , EF N , LF, and EF). In the real in vivo systems, PA 63 oligomeric prepore complexes would first bind the LF and/or EF subunits on the cell surface, then undergo endocytosis, and, after that, under pH endosome pH cis ( Figure 10 c) [ 117 , 239 , 240 ]. At the same time, the ΔpH that the authors used to mimic a proton gradient across an endosomal membrane was more efficient in stimulating protein transport, allowing translocation not only LF N but also full-length LF and EF. The conductance rise was S-shaped, and its rate increased systematically both with increasing of Δψ and ΔpH. With the minimum PA 63 -binding translocation component domain of LF, LF N, it was shown that, under small Δψ, the N terminus of bound LF N enters the oligomeric channel and initiates the threading of the substrate across the bilayer membrane [ 116 ]. Figure 10 The interaction of LF N with the PA 63 multichannel membrane [ 239 ]. ( a ) After the macroscopic PA 63 -induced current had reached a steady state at +20 mV, ~3 nM LF N was added to the cis compartment of the chamber, which resulted in a rapid fall in current. The cis compartment was then perfused of unbound LF N . At time zero, the voltage was increased to +50 mV, and LF N translocation kinetics through the PA 63 channels was indicated by the increase in conductance vs. time; ( b ) Kinetic transients for LF N translocation at +60 mV and the indicated symmetrical pH values; ( c ) The rate of LF N translocation is controlled by the magnitude and sign of the transmembrane ΔpH (pH trans > pH cis ); ( d ) A schematic illustration of the tandem Brownian ratchet translocation mechanism shows a model of several partially unfolded intermediates of LF N during translocation [ 239 ]. The scheme demonstrates how the hydrophobic, ϕ-clamp ratchet and the protonation-state ratchet may work together facilitating translocation of LF N . Reprinted with permission from reference [ 239 ]. In order to identify residues in the PA 63 lumen important for the channel-mediated transport of a substrate, the authors used cysteine-scanning mutagenesis coupled to [2-(trimethylammonium ethyl]methanethiosulfonate (MTS-ET) modifications [ 241 ]. The channels were the most significantly affected by Phe427/Cys427 modifications, and Phe427 (named ϕ-clamp is the most hydrophobic residue in the otherwise hydrophilic pore lining [ 118 ]. In particular, when the bulky 427 phenylalanine residue was replaced by the small alanine, the single-channel conductance expectably increased but the rate of LF N translocation reduced by a factor of six [ 239 ]. Based on the fact that the ϕ-clamp mutation blocked LF translocation in cell assays [ 242 ] or significantly slowed it in vitro [ 118 ], a chaperone model was suggested in which the ϕ-clamp's phenyl rings directly interacted with the translocating polypeptide. According to the model [ 118 ], the ϕ-clamp recognizes substrates through the hydrophobic effects, enhanced by aromatic-aromatic, π-π, and cation-π interactions, and stabilizes the fragments it an unfolded state. This study for the first time provided evidence that PA 63 was not a passive tunnel through which proteins electrophorese but rather an active player in facilitating the transport of a substrate molecule. At the same time, the lumen of a 14-strand β-barrel PA 63 is believed to be as narrow as 12–15 à [ 101 , 174 , 243 ], which would allow the pore to accommodate secondary structure only as large as an α-helix [ 244 ]. Therefore, translocation through PA 63 would require the enzymatic substrates to unfold. If so, what are the factors that cause the folded LF and EF with tertiary structures to unfold? The authors show that one critical factor is low endosomal pH. Thus, the acidic conditions in the endosome are sufficient to destabilize the native structure of LF N and EF N proteins [ 244 ]. In vitro experiments have also revealed that the translocation process is driven by cis positive voltages [ 117 ] and that the PA 63 channel is selective to cations [ 175 ]. Paradoxically, LF N bears a net negative charge (about six negative charges) even at pH values as low as 5.5 [ 240 ]. To explain this finding, the authors developed a model describing PA 63 channel as a protein translocase rather than an unselective long tunnel. The easiest way for the substrates to achieve a net positive charge, which is thermodynamically and electrostatically required for the translocation, would be to get their aspartic and glutamic residues neutralized, for instance protonated [ 239 ]. Under ΔpH, the substrate molecule would get protons from the cis compartment solution (or an endosome) and discharge them into the trans compartment solution (or cytosol) following translocation. At the same time, the portion of LF/EF located inside the pore lumen would carry a net positive charge and PA 63 would work as a proton-protein symporter [ 119 ]. To address the fundamental question of the translocation driving force, the authors developed a novel charge state-dependent Brownian-ratchet model for the ΔpH-driven translocation, which is based on the chemical asymmetry created by ΔpH [ 239 ]. The probability of an aspartate or glutamate being in the protonated form is greater in the more acidic cis side compared to the more basic trans side, and, therefore, the rate of their entry is higher from the cis side than that from the trans side [ 240 ]. After the protonated aspartate and glutamate groups reach the cytosolic part of the membrane, they deprotonate, becoming negatively charged again, resulting in the electrostatic repulsion of the negatively charged chain fragment from the channel lined with the negatively charged residues. This electrostatic repulsion drives the translocation per se and enforces its directionality [ 100 , 239 ]. Therefore, the random Brownian motion of the substrate inside PA 63 is biased toward the more basic trans compartment. Based on the experimental evidence, the authors suggest that the PA 63 symporter achieves protein translocation using a tandem of two synergistic Brownian ratchets: the ϕ-clamp ratchet, promoting the substrate unfolding, and the charge-state ratchet, which biases the entry rates of the substrates into the pore ( Figure 10 d). Remarkably, further experiments with planar lipid bilayers have imparted support to this model using essentially non-titratable negatively charged SO 3 − groups introduced at most positions in LF N [ 119 ]. The voltage-driven translocation of the resulting LF N variants was dramatically reduced and the ϕ-clamp was determined as a significant factor in the exclusion of SO 3 − from the channel. In another study, semisynthetic variants of LF N (12-263) in which selected acidic residues were replaced with the unnatural amino acid, cysteic acid, were examined [ 245 ]. The cysteic acid has a negatively charged side chain protonated only at pH values below the physiological range. Depending on the number of acidic residues replaced, transport of these mutants was either significantly suppressed or completely inhibited, whereas their binding and channel-blocking properties were comparable with those of wild type LF N . To determine if the substrate's secondary structure is preserved during the pore-facilitated translocation, a method of trapping the polypeptide chain of the translocating protein within a pore was developed [ 121 ]. In order to determine the minimum number of residues that could traverse the PA 63 oligomeric pore, the authors attached biotin to the N terminus of LF N and used molecular stoppers at the different positions. The trans -side streptavidin addition was used to determine whether the N terminus has reached the trans compartment solution. If the N terminus-stopper distance was long enough to allow for LF N to appear from the pore, streptavidin was able to bind the biotin. Otherwise, no biotin binding was recorded. The authors showed that an LF N polypeptide chain adopts a fully extended conformation, because it is being translocated through the channel's stem. A typical characteristic of LF N translocation is its non-exponential S-shaped kinetics. At the same time, in most of the experiments the substrate translocation was investigated under conditions where two or three LF N molecules were bound to PA 63 channels. To understand whether the S-shaped kinetics is an intrinsic characteristic of translocation kinetics or a consequence of the translocation in a tandem of two or three LF N molecules, a kinetic analysis of protein transport via the macroscopic and single-channel PA 63 channels was performed [ 120 ]. The study showed that even with one LF N bound to PA 63 , the translocation kinetics is S-shaped, being, however, slower, with more than one LF N bound. The authors also propose a simple drift-diffusion model of LF N transport, where LF N is represented as a charged rod that moves subject to both Brownian motion and an applied electric field across the membrane [ 120 ]. A dramatically different model of anthrax toxin transmembrane uptake was recently suggested by Kasianowicz and colleagues [ 18 ]. The new model suggests that instead of LF and EF being threaded through the pore, anthrax toxin complexes ( i.e. , LF or EF bound to the PA 63 channel) rupture membranes ( Figure 11 ). Figure 11 ( a ) A schematic illustration of two proposed mechanisms for PA 63 channel-mediated lethal factor (LF) and edema factor (EF) transport into the cytoplasm. The channel-mediated translocation model suggests that LF and EF pass through the pore [ 118 , 239 ]. The recent membrane rupturing model suggests that the enzymatically active anthrax toxin complexes (namely, LF or EF bound to the PA 63 channel) rupture membranes [ 18 ]. Time course of the PA 63 channel conductance with ( b ) LF removed before or ( c ) maintained at 1 nM during cis -side acidification. ( Top rows ) ~60 PA 63 channels were reconstituted into a planar bilayer membrane at pH cis | trans 7.2|7.2 (black) and, 1 nM LF was added to the cis chamber (blue). ( Middle rows ) The pH cis | trans 5.5|7.2 gradient was formed by perfusing the cis chamber with pH 5.5 buffer that contained either [LF] = 0 (red) or [LF] = 1 nM (green). ( Bottom rows ) The neutral pH condition (pH cis | trans 7.2|7.2) (black) was restored by perfusing the cis chamber with pH 7.2 buffer. If LF was present, the cis chamber was first perfused with pH 5.5 buffer (red) then pH 7.2 buffer (black). Reprinted with permission from reference [ 18 ]. Kasianowicz et al. , question the translocation model, noting that the in vitro pH gradient formation used in the previous translocation studies is precisely opposite to what occurs in vivo [ 18 ]. The translocation, manifested by PA 63 ion current recovery ( Figure 10 ), was observed either in static low pH conditions on both sites of the membrane (mostly with LF N ) or starting with the symmetrical acid conditions and forming a pH gradient by raising the trans solution pH (both with LF N , EF N , LF, and EF). In the real in vivo systems, PA 63 oligomeric prepore complexes would first bind the LF and/or EF subunits on the cell surface, then undergo endocytosis, and, after that, under pH endosome < pH cytosol conditions, the prepore/pore transition would occur. To support the alternative anthrax toxin uptake model, the authors show that anthrax toxin complexes can rupture both the planar bilayer and the droplet membranes [ 18 ]. Moreover, the transmembrane pH gradients alter the ion conducting properties of the PA 63 pores and LF/PA 63 interaction. In particular, under conditions that mimic those across the endosomal membrane, the strength of LF/PA 63 interaction in the absence of LF excess in the solution is relatively weak. However the LF binding is irreversible when LF is present in bulk during acidification [ 18 ]. To understand the binding of LF/EF to the channel, the authors designed an experiment starting from essentially different experimental conditions ( Figure 11 b) compared with those used for the translocation experiments. PA 63 channels were incorporated into the planar bilayer membranes under symmetrical pH cis = pH trans = 7.2 conditions ( Figure 11 b, top). Following channel formation, LF was added to the cis compartment solution and the remaining unbound LF was flushed from the chamber. Lowering the pH cis solution to 5.5 led to a slow current increase ( Figure 11 b, middle) explained by dissociation of LF from the channel; the current continued to increase when pH was increased to 7.2 ( Figure 11 b, bottom). About 90% of current recovery was observed after 30 min [ 18 ]. To show that acidification promotes the LF dissociation from the channel, the authors repeated the experiment ( Figure 11 c) maintaining the bulk cis -solution LF concentration at constant during the acidification. For that purpose, the cis solution was perfused with the LF containing pH 5.5 buffer ( Figure 11 c, middle). After that, the solution was additionally perfused with LF-less pH 5.5 buffer ( Figure 11 c, middle). The described procedure led to only minor increase in the current even after pH was return to 7.2 ( Figure 11 c, bottom). Thus, about 90 min after returning to initial pH cis = pH trans = 7.2 conditions, only 17% of the initial PA 63 current was recorded, indicating strong, nearly irreversible LF/PA 63 interaction when [LF] bulk = 1 nM during acidification. The authors also showed that when LF or EF were in the bulk during acidification of the cis side, the formation of the essentially irreversibly bound LF:PA 63 and EF:PA 63 complexes led to membrane rupture. In addition, the article contains interesting suggestions about a possible alternative mechanism of the anthrax toxin uptake, which the authors support with carefully designed experiments and detailed discussion [ 18 ]. We believe these data must be addressed in the future. On the other hand, it is important to emphasize that the earlier studies showed nearly ideal positive correlation between the translocation rate measured for different LF, EF, and PA 63 variants in vitro and cytotoxicity of the complexes in cell assays and in vivo . Moreover, a variety of available and rationally designed small molecule and polyvalent compounds aimed to specifically obstruct the PA 63 's translocation pathway were shown to be very effective in inhibiting anthrax toxin [ 111 , 118 , 163 , 165 , 167 , 176 , 246 ]. The positive correlation between binding activity of these blockers to the PA 63 channel in vitro and their protective action in vivo was reported. The dominant-negative PA mutants that co-assemble with the wild-type PA 63 and block its ability to translocate the LF and EF components have been also described [ 247 , 248 , 249 , 250 ]. 6. Channel-Forming Bacterial Toxins for Cancer Therapy Many traditional anticancer agents, while being highly effective, also show their well-known toxic properties toward normal fast proliferating cells. These drugs were often discovered in cellular screens of extracts from natural sources, or in in vivo screens using a leukemic P388 mouse model, and entered the clinical studies and market before the exact mechanism of their action was understood [ 251 ]. In contrast, emergent next-generation strategies of the anticancer drug discovery focus on targeted therapies, where the agents are designed rationally to target the unique features of malignant cells. Thus, cancer cells overexpress specific tumor antigens, carbohydrate structures, and growth factor receptors on their surface or they express cancer specific proteases [ 252 ]. Targeting these factors is a widespread strategy under development for a selective killing of cancer cell using small molecules, monoclonal antibodies, modified bacterial, plant and fungal toxins, viral nanoparticles, and any other inhibitors, which follow the principles required to selectively destroy cancer cells. The bacterial toxins are naturally cytotoxic: a property that makes them attractive to use in the targeted therapies. Thus, only one molecule of diphtheria toxin fragment A introduced into a cell can kill the cell [ 253 ]. The major task is to direct the cell-binding properties of the bacterial toxins for selective action on cancer cells while minimizing their ability to destroy healthy cells. At first, the targeted toxin research focused on design of so-called "immunotoxins", molecules consisting of a protein toxin fused to a tumor cell-specific antibody or antibody fragments. This approach afterwards expanded to include other target-specific ligands, such as growth factors or cytokines. These proteins are often referred to as "targeted toxins" (TTs). Pseudomonas exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheria , which enzymatically inhibit protein synthesis, are commonly used for the immunotoxin construction. The Clostridium and Bacillus binary toxins are also considered excellent candidates for the targeted toxin design. Being composed of two (or three in the case of anthrax toxin) nontoxic proteins, the binary toxins offer a significant advantage over the single-chain AB-type toxins, because activities of their binding and enzymatic components can be redirected independently towards targeted cancer cells. The membrane-perforating bacterial toxins and pore-forming antimicrobial peptides have also served as excellent tools for the targeted toxin research. For the specific aim of the current review, here we highlight examples of the therapeutic strategies that are focused on a targeted modification of the membrane-perforating bacterial toxins, such as αHL, and on the channel-forming components of the binary toxins, such as PA 63 . For examples of other targeted toxin applications and for a general overview of the field, we direct the reader to a series of recent reviews [ 254 , 255 , 256 , 257 , 258 , 259 , 260 , 261 , 262 , 263 , 264 ]. Anthrax toxin is an excellent choice for targeted toxin research [ 265 ]. The Bacillus anthracis infections are rare and currently only a limited number of people have been immunized against anthrax. According to the CDC, only laboratory workers who work with anthrax, some veterinarians, and some members of the US army are routinely vaccinated. Therefore, in contrast to diphtheria toxin (childhood immunization) and Pseudomonas exotoxin (earlier infections), most people lack pre-existing immunity for anthrax. Besides, the tripartite nature of the anthrax toxin offers a variety of tuning strategies for targeted modifications of the non-linked toxin components. Moreover, substantial recent progress made in the understanding of anthrax toxin protein structures and uptake mechanisms facilitates design of tailored antitumor agents. Targeted anthrax toxin research progresses in at least three different directions, which are often combined. First, because PA binding to the CMG2 and TEM8 cell surface receptors is the first event in the multistep anthrax toxin's intracellular transit, redirecting the protein towards alternate tumor cell specific receptors was investigated. To demonstrate the feasibility of this approach, it was shown that tailor-made PA can be targeted towards the human p62 (c-Myc)-specific hybridoma cell line 9E10 [ 266 , 267 ]. The C-terminus of wild-type PA was fused to the amino acid residues 410–419 of the human p62 c−Myc epitope via a (G 2 S) 2 linker; ac-Myc IgG was proven to act as alternate receptor. The PA-c-Myc fusion protein was shown to kill RAW cells, which do not express the c-Myc receptor. The addition of the c-Myc epitope to the C-terminus of PA did not interfere with the ability of the fusion protein to bind to the PA TEM8 receptor. Therefore, the presence of competitive inhibitor PA SNKEΔFF (a non-toxic receptor-binding mutant) and the fusion protein FP59 (amino acid residues 1–254 of LF and the ADP-ribosylation domain of Pseudomonas exotoxin) was required to fully protect 9E10 cells from a challenge with PA-c-Myc and FP59. Another receptor-redirected approach is to disrupt the native receptor-binding function of the toxin and then to specifically link the mutated protein to a heterologous receptor-binding protein. Recently, the native receptor-binding activity of PA was ablated by introducing N682A and D683A mutations in domain 4 [ 268 ]. The resulting C terminus of the mutated protein was then fused to one of two heterologous receptor-binding proteins: human epidermal growth factor or the receptor-binding domain of diphtheria toxin ( Figure 12 ). The resulting PA variants mediated the cell entry of the active components of the toxin. The developed approach was used to redirect toxin action to cells bearing the HER2 receptor [ 269 , 270 ]. Figure 12 The receptor-based approach to re-engineer PA by disrupting the native receptor-binding function of the toxin and specific linking of the mutated protein to a heterologous receptor-binding protein [ 268 ]. Composite representation of the heptameric prepore formed by PA 63 (PDB: 1TZO) with EGF (PDB: 1JL9) linked to the C-terminus. An axial view of the heptameric prepore is shown, with domains 1, 2, 3, and 4 in a single subunit of PA 63 colored magenta, green, gold, and purple, respectively. EGF is in red. Broken lines represent an 8-amino-acid linker (SPGHKTQP) connecting the N terminus of EGF to the C terminus of PA 63 . Reprinted with permission from reference [ 268 ]. The unique requirement that certain toxins have to be active on the cell surface of the targeted cell provides strategies to tailor these toxins to make them dependent on the cancer cell-specific proteases. Therefore, a second approach to re-engineered PA is to replace the furin cleavage site with cleavage sites for proteases that are present on the surface of cancer cells. For the broad-spectrum TTs, it is practical to focus on the cell surface-associated proteases that are overexpressed in a variety of tumor tissues and tumor cell lines, namely on the matrix metalloproteases (MMPs) and urokinase plasminogen activator (uPA, not to be confused with PA, the binding/translocation subunit of anthrax toxin). To examine the role of MMPs in the design of TTs, two mutated PA proteins were constructed in which the furin protease recognition site RKKR was replaced by the GPLGMLSQ (PA-L1) and GPLGLWAQ (PA-L2) sequences [ 271 ]. These fusion proteins were designed to be susceptible to cleavage by MMP-2, MMP-9 and MT1-MMP and were rapidly and selectively activated on the surface of MMP-overexpressing cancel cells. The resulting PA-L1 and PA-L2 oligomers were used to internalize a recombinant FP59 fusion protein and these combinations were shown to be selectively toxic to MMP-overexpressing tumor cells, which included human fibrosarcoma, breast cancer, melanoma and thyroid carcinoma [ 267 , 271 , 272 , 273 , 274 , 275 ]. To examine the role of uPA in the design of TTs, a set of mutated PA proteins in which the furin activation site was replaced by uPA recognition sequences was also constructed [ 276 ]. The uPA substrate sequences, GSGRSA and GSGKSA were used to replace the furin RKKR sequence in PA, which yielded mutated PA proteins, PA-U2 and PA-U3 that were efficiently activated by uPA. PA-U2/FP59 was later investigated as a wide-range, highly selective, and highly potent chimeric toxin that specifically targets uPA-expressing tumors, independently of their tissue origin [ 277 , 278 , 279 , 280 ]. In an elegant study, Leppla and colleagues reengineered PA-constructing mutants with mutations affecting different LF-binding subsites and containing either uPA or MMP cleavage sites [ 281 ] ( Figure 13 a). Figure 13 ( a ) Scheme for reengineering anthrax toxin protective antigen (PA) to modify its action towards two distinct proteolytic activities overexpressed by the cancer cells [ 281 ]; ( b ) Scheme for discovery of PA mutants that exclusively form octamers [ 282 ]; ( c ) EM images of heptameric and octameric PA species [ 282 ]. Reprinted with permission from references [ 281 , 282 ]. These proteins contained additional mutations so that PA-U2 and PA-L1 monomeric subunits had to be adjacent in an oligomer to form native LF binding sites. As a result, individually the constructed PA variants showed decreased toxicity due to the impaired LF binding; however, when administered together to uPA and MMP overexpressing cancer cells formed functional LF-binding heteroheptamers. The mixture of these two mutants was highly effective in vivo to treat diverse aggressive transplanted tumors. In this study, the authors established proof-of-principle that anthrax toxin can be re-engineered in a way so that its cytotoxicity relies on two distinct proteolytic activities overexpressed by the cancer cell [ 281 ]. This strategy was further examined in a study where the therapeutic window of the anthrax toxin-based TTs was enlarged with PA variants rationally designed to selectively and exclusively form oligomers [ 282 ] ( Figure 13 b). The idea of this design was based on a finding made by Krantz and colleagues that PA, which for a long time was believed to form heptameric complexes, is also able to form octamers, and they are functional [ 107 ]. Moreover, conditions under which octameric oligomerization predominates were determined and heptamer/octamer structural comparison showed that there are two orientations of the receptor-binding PA domain 4, which alternate in the octamer. The molecular determinants that influence the stoichiometry of PA complexes were identified, e.g., the relative proportion of PA heptamers and octamers could be controlled by tethering domain 4 to domain 2 with two different length cross-links [ 283 ]. By screening a highly directed library of PA mutants, Leppla and colleagues identified variants that complemented each other to form octamers exclusively [ 282 ]. The authors started with the PA mutant D512K, which is incapable of forming oligomers ( Figure 13 b, top), and prepared a <50,000-members library of PA variants that have the D512K substitution together with random mutations in several residues on the complementary face of PA 63 within the oligomers ( Figure 13 b, middle). The library was screened for regaining of oligomer-forming ability and then, after the sought variants were identified, D512K and new complementary mutations were placed into two separate PA proteins. As a result, the authors achieved formation of oligomers through the use of the two unique interfaces (wild type and mutated), which resulted in formation of even-numbered oligomers ( Figure 13 b, bottom), predominately octamers ( Figure 13 c). In particular, the authors focused on the transformants containing substitutions at residues 245 and 252. By combining PA-D512K with either PA K245G/R252N (abbreviated as PA-GN) or PA K245N/R252S (abbreviated as PA-NS) generated high toxicity comparable to that of wild type PA both in vitro and in vivo . However these variant were non-toxic when used individually [ 282 ]. The third strategy is based on the fact that the PA 63 -mediated protein uptake machinery is effective enough to deliver multiple engineered variants of the native anthrax toxin LF and EF components. To increase the therapeutic benefit of the TTs, the native LF and EF were replaced with a variety of fusion proteins containing the PA 63 binding N-terminal domain of LF (LF N ) or EF (EF N ) and different toxophores (the so called "cut and paste" approach [ 265 ]). The resulting chimeric variants included LF N fused to Pseudomonas exotoxin A enzymatic domain [ 284 ], Shiga toxin enzymatic domain, diphtheria toxin A chain [ 285 , 286 ], tetanus toxin light chain [ 287 ], Clostridia difficile B toxin glycosylating domain [ 288 ], Haemophilus ducreyi cytolethal distending toxin B subunit [ 289 ], Bcl-XL protein [ 290 ], ricin toxin A chain [ 269 ], flagellin [ 291 ], and beta-lactamase [ 292 , 293 ]. Many of these heterologous proteins have been successfully delivered by PA 63 (native or mutated) into the cytosol. However, some proteins, apparently those that were not able to adopt a partially or fully unfolded state at the acidic endosomal pH, have failed [ 267 , 294 ]. Another factor that can limit the potency of TTs is the stability of the enzymatic domains in the cytosol [ 295 ]. Recently, this issue has been examined with LF N -PEIII variants constructed with insertion of a ubiquitin domain (wild type and mutated) between the targeting domain (LF N ) and the catalytic "payload" (PEIII) [ 295 ]. The variants were designed to address the previously described bias against the presence of lysine residues in enzymatic domains of several AB-type bacterial toxins [ 296 ]. Thus, the catalytic domains of cholera toxin, E. coli heat-labile toxin, Shiga-like toxin, and ricin have strong bias towards Arg relative to Lys. This characteristic is believed to limit the attachment of ubiquitin followed by the proteasomal degradation of the toxins [ 295 , 297 ]. Ubiquitin is a small eukaryotic regulatory protein, which, among its multiple functions, also has a signaling role in protein degradation. The cytosolic stability of LF and LF N -based chimeric proteins was shown to follow the so-called N-end rule [ 294 , 298 ] described by Alexander Varshavsky in 1986 [ 299 ]. In accordance with the N-end rule, the specific destabilizing N-terminal amino acid of a protein controls the effectiveness with which side chain lysine residues are ubiquitinated for the following proteasomal degradation. Ubiquitin, after binding to a target protein, can be in its turn ubiquitinated, forming polyubiquitin chains on any of the seven lysine residues within ubiquitin. This process is controlled by deubiquitinating (DUBs) enzymes that allow for ubiquitin to be released from the protein. Interestingly, LF N -Ub-PEIII fusion proteins, in which all seven lysines of the wild type ubiquitin were retained while the site cleaved by cytosolic DUBs was removed, were nontoxic, which the authors explain by a rapid ubiquitination and proteasomal degradation [ 295 ]. The authors also showed that the fusion protein, in which all seven lysines were substituted by arginine (Ub Knull ), had high potency exceeding that of FP59. In general, the potency of these proteins was highly dependent on the number of lysines retained in the ubiquitin domain and on retention of the C-terminal ubiquitin sequences cleaved by DUBs [ 295 ]. The stability of LF N -PEIII fusion proteins was also significantly improved in a study where N-terminal amino acids of LF N were mutated and all the lysines reductively dimethylated [ 300 ]. Besides the single chain and binary toxins, membrane-perforating bacterial toxins were also considered as potential candidates for the tailored modifications. Among the bacterial pore-forming toxins which were investigated for their cytolytic or cytocidal properties against tumorous cells were α-hemolysin of Staphylococcus aureus [ 26 , 301 , 302 , 303 ], parasporin-4 of Bacillus thuringiensis [ 304 , 305 , 306 , 307 ], listeriolysin O of Listeria monocytogenes [ 308 ], aerolysin of Aeromonas hydrophila [ 309 , 310 ], the S component of Panton-Valentine lekocidin (LukS-PV) of Staphylococcus aureus [ 311 ] and epsilon toxin of Clostridium perfringens [ 312 ]. In particular, Bayley and colleagues explored αHL mutants in which channel-forming activity can be triggered or switched on and off by biochemical, chemical, or physical stimuli [ 26 , 301 , 302 ]. The approach was based on earlier studies, which showed that the subunits of a functional αHL with nicks near the midpoint of a central glycine-rich loop are supported by domain-domain interactions, whereas αHL oligomers containing two truncated subunits that overlap in the central loop had greatly reduced channel-forming activity [ 313 , 314 , 315 ]. Based on these data, the group designed overlap αHL mutants that were activated when redundant amino acids in the loop were removed by proteases that inactivate the wild-type protein [ 301 ]. The authors proposed that this strategy could be used to construct proimmunolysins, variants of αHL that would be preferentially activated by the cancer cell surface proteases [ 301 ]. αHL would then perforate and kill the tumor cell or permeabilize the cell membrane for the cytotoxic drugs that have no or low permeability [ 303 ]. Thus, combinatorial mutagenesis was used to obtain αHL mutants that were rapidly and preferentially activated by cathepsin B [ 303 ]. Cathepsin B, normally an enzymatic protein, is involved in various pathologies and oncogenic processes in humans; its overexpression is correlated with invasive and metastatic phenotypes in cancer [ 316 ]. A series of tailor-made pore-forming bacterial toxins were generated for intracellular delivery of different types of macromolecules [ 317 , 318 ]. One of the main challenges drug designers face is poor bioavailability of the compounds as a result of poor membrane permeability. Loaded pH-sensitive listeriolysin O-containing liposomes were developed [ 319 , 320 , 321 , 322 , 323 , 324 , 325 ] and tested both in vitro and in vivo (see refs. [ 19 , 317 ] for details). Targeted drug delivery to generate protective antiviral immunity was also examined using the anthrax toxin [ 266 , 326 , 327 , 328 ]. Interestingly, gramicidin, the first marketed natural antimicrobial agent (1939) [ 329 ], was also studied for inhibition of human immunodeficiency virus (HIV) infection [ 330 , 331 , 332 , 333 ]. 7. Concluding Remarks Promising new developments in nanopore biotechnology continually emerge [ 334 ]. Because of the overwhelming number of the reports in this popular field, the current review is not exhaustive. In this article, written for a special volume on " Intracellular Traffic and Transport of Bacterial Protein Toxins ", we highlight many interesting applications of channel-forming bacterial toxins in small and macromolecule sensing and polymer transport. For clarity, instead of discussing all pore types previously investigated, we focus on three structurally and functionally different bacterial toxins: (1) peptide gramicidin A (GrA) of Bacillus brevis , which induces lesions in cell membranes forming small exclusively cation-permeable channels; (2) α-hemolysin (αHL) of Staphylococcus aureus , which targets the host cell membranes by forming large β-barrel pores; and (3) protective antigen (PA 63 ), the pore-forming B component of the anthrax toxin, which mediates translocation of the toxin's enzymatic components inside the target cell. There is no doubt that the number of related publications on biotechnological applications of any of these toxins far exceeds all the residual reports in the bionanopore field. At the same time, the reasons why these particular molecules were chosen for development are mani-fold. Gramicidin A can serve as an ideal single molecule on/off switch reacting to different stimuli and as a sensor for lipid membranes properties. The αHL channel is a structurally stable pore, which remains in the electrically low-noise open state over a wide range of experimental conditions, including extreme salt concentrations, pH, and temperatures. The crystal structure of αHL was solved at a 1.9-à resolution more than 15 years ago and the protein could be genetically or chemically modified in a number of ways. αHL was recently reengineered to form a functional bottom-up dimeric pore than spans two adjacent lipid bilayers [ 335 ]. On the other hand, when αHL was so severely truncated that the protein heptamer could not span a bilayer, the channel insertions were still observed, supporting the idea that membrane proteins could stabilize the toroidal lipid pores [ 336 ]. Interestingly, the channel-forming B component of the anthrax toxin, PA 63 is often compared with the membrane pore of αHL. This is mostly because these channels were reported to form functional mushroom-shaped heptamers. However, electrical characteristics of PA 63 differ dramatically from those of αHL. First, despite having comparable diameters at the most narrow constrictions, the αHL conductance in 1 M KCl solution is ~5 times higher, which allows for a significant improvement of the signal-to-noise ratio at the single channel level. Second, since formation of the PA 63 channels are triggered by the low endosomal pH, the available pH range for the molecular sensing experiments is significantly limited for subacidic pH. Finally, two types of gating were reported for the PA 63 channels. The typical voltage gating, intrinsic to many β-barrel channels incorporated into the artificial bilayers [ 337 , 338 ], shows a pronounced voltage asymmetry in the case of PA 63 pore, somewhat limiting the voltage range available for the statistically reliable measurements. More importantly, the current noise through the open PA 63 channel is far from being electrically quiet, showing persistent 1/f fluctuations between open and closed states. Similar to αHL, PA 63 was shown to be able to accommodate βCD molecules. However, the current blockage events were complete, which limits the possibilities to use βCD as PA 63 -modifying adapters. Therefore, it was not the PA 63 channel of being used as a biosensor per se , but rather an elevated interest in the anthrax toxin and several strong research groups working in the field that advanced biotechnological applications for the anthrax toxin. Attempting to understand the anthrax toxin uptake mechanism by investigating protein transport on the one hand, and the unique ability of reengineered PA 63 to deliver enzymatic components inside the target tumor cell on the other hand, made this "noisy" channel a nanopore of choice for different studies. Some of these reports created directions for the rational modifications of PA 63 that could significantly alter the pore properties. For instance, F427A PA 63 mutant was reported to have ~3-times higher conductance than the wild type pore and did not show the wild type PA 63 's 1/f noise current fluctuations [ 102 , 118 ]. A number of interesting dominant negative [ 247 , 248 , 249 , 250 ] and predominantly octameric PA 63 mutants was reported [ 282 ]. The anthrax toxin-neutralizing antibodies reconfiguring PA into supercomplexes, which are yet to be tested in the bilayer membranes, were also described [ 339 ]. GrA, αHL, and anthrax toxin's channel forming component PA 63 are available commercially and, in contrast to other toxins, for instance epsilon toxin and botulinum neurotoxin, there are no firm biosafety restrictions on the usage of small quantities of these proteins under regular laboratory conditions. There is no doubt that the nanopore biotechnology is and will remain an exciting field of research. In the future, with the development of single-channel electrical recording techniques, X-ray crystallography, protein engineering, computational methods, and, importantly, human curiosity, we will see many interesting new pore types being explored. As an example, a group of peptide phytotoxins produced by Pseudomonas syringae , syringomycin E (SRE), and similar compounds were extensively studied by Schagina and colleagues [ 135 , 340 ]. Similarly to gramicidin A, SRE-produced pores can be used as sensitive probes of the membrane physical state, surface charge, orientation of membrane inner dipoles, and the interaction of membrane-active molecules. A cytolytic pore-forming toxin aerolysin of Aeromonas hydrophila represents an interesting alterative to αHL. Recently, X-ray crystallography, cryo-EM, MD and computational modeling approaches have been used to resolve a near-atomistic structure of the aerolysin pore and to study intermediate states leading to the pore formation [ 341 ]. Both biosensing and protein translocation properties of the channel have been reported [ 342 , 343 , 344 , 345 ]. Another "αHL-like" oligomeric pore is formed by cholesterol-dependent Vibrio cholerae cytolysin [ 346 , 347 , 348 ]. Remarkably, the X-ray crystallography [ 349 ] revealed an interesting structural detail of cytolysin—a narrow constriction region formed by an unexpected aromatic tryptophan W318 ring of residues within the pore that is otherwise rich in charged amino acid residues. The authors compare this region with the ϕ-clamp of the B component of the binary bacterial toxins, such as anthrax, where it is believed to be critical for the A component's translocation. At the same time, so far there is no evidence indicating that Vibrio cholerae cytolysin serves as a transmembrane protein translocase. We wonder if this structural property could be employed to mimic the Brownian ratchet mechanism of anthrax toxin translocation [ 239 ] with this significantly different protein in vitro . Currently, one of the greatest problems hampering the study of polymer transport and nucleic acid pore-assisted sequencing is the high translocation rate of these molecules. Several approaches have been developed to slow down the single-molecule transport through a protein nanopore [ 192 , 350 , 351 , 352 ]. Thus, genetically optimized porin MspA of Myobacterium smegmatis is believed [ 19 ] to be a promising pore for biosensing and sequencing applications due to its conical shape providing a very narrow (~10-à ) sensing zone [ 194 ]. Interestingly, epsilon toxin of Clostridium perfringens (reviewed in refs. [ 353 , 354 , 355 ]) was reported to form slightly anion-selective stable low-noise pores with a single-channel conductance in the range of 440–640 pS in 1 M KCl [ 356 , 357 ]. The polymer-partitioning studies to access the epsilon toxin pore's functional shape and size suggested that the channel is highly asymmetric, i.e. , conical with the tentative radii of openings of 0.4 and 1.0 nm on the cis and trans sides, respectively [ 358 ]. However the single channel studies on this channel are limited to three publications [ 356 , 357 , 358 ], which could be partially explained by lack of the epsilon toxin oligomer's crystal structure and by certain CDC regulations placed on use of this category B agent. Besides, the channel-forming B components of the clostridial binary bacterial toxins show remarkable structural and functional similarities with PA 63 [ 9 , 318 ]. Moreover, PA 63 was shown to be effective in transporting the His-tagged enzymatic C2I component of the binary C2 toxin into the cytosol [ 359 , 360 ]. Along with the anthrax toxin, binary clostridial ADP-ribosylating toxins have been examined for their ability to deliver heterologous catalytic domains inside the tumor cells. Many of the reported examples, which mainly consist of the chimeric toxins constructed on the basis of the active component of C2 toxin, C2I are intensively reviewed elsewhere [ 9 , 318 ]. More recent reports describe genetically engineered chimeric C2IN-streptavidin complexes, which were designed to delivered biotin-labeled molecules into the cytosol of diverse eukaryotic cell lines by the binding/translocation subunit of the toxin [ 361 , 362 , 363 ]. The C2-streptavidin delivery system was used to internalize biotin-labeled p53 tumor suppressor into different mammalian cell lines, including human tumor cells [ 364 ]. The direct C2IN-p53 constructs were also investigated [ 365 ]. Chemical conjugation strategies as alternatives to engineering fusion proteins have also recently been explored resulting in the assembly of C2 toxin-based Janus-like supramolecular fusion proteins based on the iminobiotin-avidin linkage responding to external stimuli, such as pH [ 366 ]. Currently, it is unclear to what extent the channel-forming B components of the clostridial binary toxins serve as active translocase of their A components, similarly to what is suggested for PA 63 . On the one hand, the preserved ϕ-clamp in position 428 was shown to be important for pore formation and for cytotoxicity and rounding up of cells by the enzymatic C2I subunits of the C2 toxin [ 367 ]. On the other hand, the host cell chaperones Hsp90 and the peptidyl-prolyl cis/trans isomerase cyclophilin A were reported to be critical for membrane translocation of the active moieties of clostridial C2, iota, and CDT toxins but not for LF of the anthrax toxin [ 368 , 369 , 370 ]. Provided that the corresponding bilayer measurements show successful translocation events similar to those described for the anthrax toxin, we believe these findings would add some useful arguments to the anthrax toxin uptake debate [ 18 ]. For that purpose, not only the native but also the PA 63 /C2I-His cross-combination of components could be tested.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC275416/

Xp38γ/SAPK3 promotes meiotic G 2 /M transition in Xenopus oocytes and activates Cdc25C

We have studied the role of p38 mitogen-activated protein kinases (MAPKs) in the meiotic maturation of Xenopus oocytes. Overexpression of a constitutively active mutant of the p38 activator MKK6 accelerates progesterone-induced maturation. Immunoprecipit ation experiments indicate that p38γ/SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38γ (Xp38γ) and show that co-expression of active MKK6 with Xp38γ induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38γ requires neither protein synthesis nor activation of the p42 MAPK–p90Rsk pathway, but it is blocked by cAMP-dependent protein kinase. A role for the endogenous Xp38γ in progesterone-induced maturation is supported by the inhibitory effect of kinase-dead mutants of MKK6 and Xp38γ. Furthermore, MKK6 can rescue the inhibition of oocyte maturation by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38γ can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38γ. Our results indicate that phosphorylation of XCdc25C by Xp38γ/SAPK3 is important for the meiotic G 2 /M progression of Xenopus oocytes.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3398800/

Progress in filovirus vaccine development: evaluating the potential for clinical use

Marburg and Ebola viruses cause severe hemorrhagic fever in humans and nonhuman primates. Currently, there are no effective treatments and no licensed vaccines; although a number of vaccine platforms have proven successful in animal models. The ideal filovirus vaccine candidate should be able to provide rapid protection following a single immunization, have the potential to work postexposure and be cross-reactive or multivalent against all Marburg virus strains and all relevant Ebola virus species and strains. Currently, there are multiple platforms that have provided prophylactic protection in nonhuman primates, including DNA, recombinant adenovirus serotype 5, recombinant human parainfluenza virus 3 and virus-like particles. In addition, a single platform, recombinant vesicular stomatitis virus, has demonstrated both prophylactic and postexposure protection in nonhuman primates. These results demonstrate that achieving a vaccine that is protective against filoviruses is possible; the challenge now is to prove its safety and efficacy in order to obtain a vaccine that is ready for human use.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3577763/

Adenovirus-Based Vaccine with Epitopes Incorporated in Novel Fiber Sites to Induce Protective Immunity against Pseudomonas aeruginosa

Adenovirus (Ad) vector-based vaccines displaying pathogen-derived epitopes on Ad capsid proteins can elicit anti-pathogen immunity. This approach seems to be particularly efficient with epitopes incorporated into the Ad fiber protein. Here, we explore epitope insertion into various sites of the Ad fiber to elicit epitope-specific immunity. Ad vectors expressing the 14-mer Pseudomonas aeruginosa immune-dominant outer membrane protein F (OprF) epitope 8 (Epi8) in five distinct sites of the Ad5 fiber, loops CD (AdZ.F(CD)Epi8), DE (AdZ.F(DE)Epi8), FG (AdZ.F(FG)Epi8), HI (AdZ.F(HI)Epi8) and C terminus (AdZ.F(CT)Epi8), or the hexon HVR5 loop (AdZ.HxEpi8) were compared in their capacity to elicit anti- P. aeruginosa immunity to AdOprF, an Ad expressing the entire OprF protein. Intramuscular immunization of BALB/c mice with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 elicited higher anti-OprF humoral and cellular CD4 and CD8 responses as well as enhanced protection against respiratory infection with P. aeruginosa compared to immunization with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8. Importantly, repeat administration of the fiber- and hexon-modified Ad vectors boosted the OprF-specific humoral immune response in contrast to immunization with AdOprF. Strikingly, following three doses of AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 anti-OprF immunity surpassed that induced by AdOprF. Furthermore, in the presence of anti-Ad5 immunity, immunization with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8, but not with AdOprF, induced protective immunity against P. aeruginosa . This suggests that incorporation of epitopes into distinct sites of the Ad fiber is a promising vaccine strategy. Introduction Pseudomonas aeruginosa is one of the leading nosocomial bacterial pathogens worldwide and can cause serious infections of the respiratory tract. A vaccine against P. aeruginosa would be useful as treatment is often challenged by antibiotic resistance of the organism. No efficient and marketable vaccine is yet available [1] , [2] . P. aeruginosa outer membrane protein F (OprF) is one of the promising vaccine antigens. OprF is surface exposed, antigenically conserved in wild-type strains of P. aeruginosa and elicits cross-reactive, opsonizing and protective antibodies in various animal models and humans [1] , [3] – [7] . Various immunogenic peptides have been identified in the outer loops of OprF, including the 14-mer peptide Epi8 [8] – [10] . Adenovirus (Ad) vectors are attractive delivery vehicles for genetic vaccines due to their ability to act as immune system adjuvants and to rapidly evoke robust immune responses against the transgene product and viral capsid proteins [11] – [14] . Ad vectors could also serve as a vaccine platform against P. aeruginosa . Human Ad serotype 5 (Ad5) or non-human primate Ad serotype C7 (AdC7) expressing OprF induced robust protective immunity against pulmonary infections with P. aeruginosa in mice [15] , [16] . One of the limitations of Ad as vaccine carrier is that anti-Ad immunity elicited by the initial immunization usually prevents productive infection with subsequent immunizations, critical to achieve boosting of the anti-transgene immunity [13] , [17] , [18] . One of the prime-boost strategies for Ad-based vaccines is to incorporate vaccine epitopes into the Ad capsid [10] , [19] – [22] . Various Ad outer capsid proteins including hexon, fiber knob, penton base and protein IX have been targets for genetic modification [23] . Incorporation of influenza hemagglutinin (HA) epitopes into the fiber HI loop of the Ad5 fiber elicits stronger humoral and cellular immunity compared to incorporation of the same epitope into the more abundant hexon protein [20] . Here we explore different epitope-insertion sites within the Ad fiber protein to enhance the epitope-specific immune response of an Ad-based vaccine. We identify a novel site in the FG loop for epitope insertion to elicit robust epitope-specific immunity that can be boosted and is effective in Ad pre-immune animals. Materials and Methods Ethics statement All animal studies were conducted in accordance to the protocols reviewed and approved by the Weill Cornell Institutional Animal Care and Use Committee (Permit Number 0703-594A). All efforts were made to minimize suffering to the animals. Ad vectors AdEasy™ adenoviral vector system (Agilent Technologies, Santa Clara, CA) was used to construct the replication-defective recombinant human Ad5 vectors. The vectors expressed either β-galactosidase, referred to as "Z" in the vector (AdZ), or no transgene (AdNull) [24] . The plasmid pAdEasy-1 (Agilent Technologies) was modified to insert gene encoding OprF 14-mer epitope Epi8 (NATAEGRAINRRVE) into loops CD (Gly450/Thr451), DE (Asn464/Gly465), FG (Gly509/Lys510), HI (Gly543/Asp544 ) or C terminus (CT) of the Ad5 fiber gene ( Figure 1 ). The resultant plasmids and pAdEasy-1 were recombined with pShuttle-CMV-lacZ (Agilent Technologies) to obtain recombinant plasmids pAdZ.F(CD)Epi8, pAdZ.F(DE)Epi8, pAdZ.F(FG)Epi8, pAdZ.F(HI)Epi8, pAdZ.F(CT)Epi8 and pAdZ that were used for transfection to generate the fiber-modified Ad vectors AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8 and AdZ respectively. Fiber-modified Ad vectors were generated using a previously described strategy [25] . Because of potential inhibitory effects of the modified Ad fibers with the cellular Ad receptors, it is difficult to generate fiber-modified vectors in regular human embryonic kidney (HEK) 293 cells. Therefore, a HEK 293-derived cell line that constitutively expresses the Ad5 fiber protein (293F) was developed and used as packaging cell line. The 293F cell line was generated by transfection of a fiber-expressing plasmid (pcDNA3.1/Hyg-Fiber) and then screening single cell clones under hygromycin selection pressure. In addition to the modified fiber protein derived from the viral DNA, Ad vectors generated in 293F cells also carry the wild-type fiber proteins derived from the cell line and can thus effectively infect the unmodified HEK 293 cells. Fiber-modified Ad vectors generated in 293F cells were subsequently propagated in HEK 293 cells to recover the viruses that only carry the modified fiber proteins. AdZ.HxEpi8 has Epi8 inserted into loop 1 of the hypervariable region 5 (HVR5) [10] and AdOprF expresses entire OprF as a transgene [16] as previously described. The vectors were used with equal physical particle concentrations (pu) and were propagated, purified and quantified as described previously [26] , [27] . 10.1371/journal.pone.0056996.g001 Figure 1 Epitope insertion sites in Ad5 fiber knob. The arrow indicates the location of P. aeruginosa OprF 14-mer epitope Epi8 inserted into loops CD (Green; Gly450/Thr451), DE (Brown; Asn464/Gly465), FG (Red; Gly509/Lys510), HI (Blue; Gly543/Asp544 ) and C terminus (CT) of the Ad5 fiber protein (PDB 1KNB). Mice Female BALB/c mice were obtained from Taconic Farms. The animals were housed under specific pathogen–free conditions and were used at 6–8 weeks of age. Mice were immunized by injection of 50 µl of the Ad vectors AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8, AdOprF, AdZ or AdNull at a dose of 10 10 pu/animal diluted in PBS to the left thigh muscle using a 0.5 ml insulin syringe (Becton, Dickinson and Company, Franklin Lakes, NJ). Western analysis To evaluate the presence of the Epi8 epitope on the Ad fiber protein, purified Ad vectors (10 10 virus particles) were denatured (95°C for 5 min) in NuPAGE sample buffer (Invitrogen, Carlsbad, CA) and separated by 4 to 12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; NuPAGE system; Invitrogen). Following transfer to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA) equal loading was confirmed by GelCode silver staining (Pierce, Rockford, IL). The membrane was exposed to blocking solution (5% fat-free milk; Bio-Rad Laboratories) in PBS for 1 h and then incubated with either polyclonal anti-OprF serum, obtained from C57Bl6 mice immunized with recombinant OprF protein (1∶500) [16] , or anti-Ad fiber antibody (Abcam, Cambridge, MA) (1∶500) for 1 h. Following addition of a peroxidase-conjugated goat anti-mouse antibody (Sigma-Aldrich, St. Louis, MO) (1∶10,000) for 1 h, a chemiluminescent peroxidase substrate (ECL reagent; Amersham Biosciences, Piscataway, NJ) was used for detection. Infection with capsid-modified Ad vectors in vitro To evaluate if incorporation of Epi8 in the different fiber sites interferes with the coxsackie-adenovirus receptor (CAR)-dependent or -independent infection in vitro , infection of A549 cells (high expression of CAR) or dendritic cells (DC; low expression of CAR) was analyzed. A549 cells (CCL185; American Type Culture Collection, Manassas, VA), maintained in complete Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum, 100 U of penicillin/ml, 100 mg of streptomycin/ml (all from GIBCO BRL, Gaithersburg, MD), were infected with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8 or AdZ (1,000 pu/cell) in low-serum medium (2% fetal bovine serum) for 2 h, washed, and then maintained in complete medium for 24 h. Bone marrow-derived DC were generated from bone marrow precursors as described previously [28] . In brief, bone marrow cells harvested from BALB/c mice were grown in complete RPMI 1640 medium [10% fetal bovine serum, 100 U of penicillin/ml, 100 µg of streptomycin/ml supplemented with 10 ng/ml recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and 2 ng/ml recombinant mouse interleukin-4 (IL-4) (both from R&D Systems, Minneapolis, MN) for 6–7 days. The DC were then washed and suspended in PBS, infected with the Ad vectors (50,000 pu/cell) for 4 h, and washed and maintained in complete medium for 36 h. The infected A549 cells and DCs were harvested and β-galactosidase activity evaluated using the β-gal assay kit (Invitrogen) as per manufacturer's instructions. Anti-OprF, anti-Ad and anti-β-galactosidase humoral immune response Mice were immunized intramuscularly with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8, AdOprF or AdZ at a dose of 10 10 pu/animal and boosted twice with the same virus after 2 and 5 wk. Serum was collected after 2, 5 and 8 wk of initial immunization. Lung bronchioalveolar lavage fluid was collected by intratracheal instillation and aspiration of 0.5 ml PBS, pH 7.4., which was centrifuged at 6000 rpm at 4°C for 10 min and the supernatant was stored at −80°C. Anti-OprF, anti-Ad and anti-β-galactosidase IgG were assessed by ELISA using flat bottomed 96-well EIA/RIA plates (Corning, New York, NY) coated with recombinant OprF (0.5 µg/well) [10] , Ad5 (10 9 pu/well) or β-galactosidase (0.1 ug/well) in 0.05 M carbonate buffer, pH 7.4. The plates were blocked with 5% dry milk in PBS for 1 h at 25°C and two-fold serial serum dilutions were added to each well and incubated for 1 h at 25°C. Following three washes with PBS containing 0.05% Tween (PBS-Tween) a peroxidase-conjugated sheep anti-mouse IgG (Sigma-Aldrich), diluted 1∶10,000 in PBS containing 1% dry milk, was added and incubated for 1 h at 25°C. Absorbance at 415 nm was measured with a microplate reader (Bio-Rad Laboratories) and the antibody titers were calculated with a log (OD)–log (dilution) interpolation model and a cutoff value equal to 2-fold the absorbance of the background. To assess surface exposure of Epi8 on the capsid-modified Ad vectors, ELISA plates were coated with intact or disrupted AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8 or AdZ (10 10 pu/well). Disruption of Ad5 vector was carried out in 0.5% sodium dodecyl sulfate (56°C, 45 seconds) [29] . The plates were blocked with 5% dry milk in PBS for 1 h at 25°C and two-fold serial dilutions of anti-OprF or anti-Ad5 were added to each well and incubated for 2 h at 25°C. The plates were further processed and evaluated as described above. OprF-specific cellular immune response To evaluate OprF-specific cellular immune responses induced by the capsid-modified Ad vectors, BALB/c mice were immunized intramuscularly with 10 10 pu of AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8, AdOprF or AdZ and boosted after 2 and 5 wk with the same vector. The frequency of OprF-specific CD4 and CD8 T lymphocytes was determined with a IL-4- and/or interferon-γ (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assay (R&D Systems) 7 days following the last immunization. Splenic CD4 or CD8 T cells were purified by positive selection with CD4 (L3T4) or CD8 (Ly-2) MACS microbeads (Miltenyi Biotec, Auburn, CA). The purity of the T cells was more than 95%. To serve as antigen-presenting cells, splenic DCs were purified from syngeneic naive animals by positive selection with CD11c MACS beads (Miltenyi Biotec) and two consecutive purifications over MACS LS columns (Miltenyi Biotec). The purity of the DC was more than 90%. DC (5×10 6 /ml) were incubated for 2 h with purified recombinant OprF protein (100 µg/ml) in RPMI medium supplemented with 2% fetal bovine serum (HyClone, Logan, UT), 10 mM HEPES (pH 7.5; BioSource International, Camarillo, CA), and 10 5 µM β-mercaptoethanol (Sigma-Aldrich). CD4 or CD8 T cells (2×10 5 ) were incubated with splenic DC with or without recombinant OprF protein at a ratio of 4∶1 in IL-4 and/or IFN-γ plates (R&D Systems) for 48 h. Following washing, biotinylated anti-IFN-γ or anti-IL-4 (both from R&D Systems) antibodies were added and incubated overnight at 4°C. For final spot detection a streptavidin-alkaline phosphatase conjugate followed by 3-amino-9-ethylcarbazole substrate (both R&D Systems) was added. The spots were counted by computer-assisted ELISPOT image analysis (Zellnet Consulting, New York, NY). Protection against pulmonary challenge with P. aeruginosa The P. aeruginosa strain PAO1 was used to assess protective immunity. PAO1-containing agar beads were prepared based on the method of Starke et al. [30] and were used as described previously [31] , [32] . Briefly, a log-phase culture of PAO1 suspended in warm tryptic soy agar (52°C) was added to mineral oil with vigorous stirring and the mixture was cooled on ice. The PAO1 -impregnated beads were washed extensively with PBS, and the density of viable bacteria enmeshed in agar beads was determined by plating of serial dilutions of homogenized beads. To evaluate if immunization with Epi8 capsid-modified Ad vectors resulted in protective immunity against a pulmonary challenge with P. aeruginosa , BALB/c mice were immunized intramuscularly with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8, AdOprF or AdZ (all 10 10 pu/mouse) followed by boost immunizations after 2 and 5 wk. Eight weeks following initial immunization, the mice were challenged intranasally with PAO1 (4×10 6 cfu in 50 ul) encapsulated in agar beads. Mice were sacrificed 24 h post challenge and lung homogenates were plated on McConkey agar plates. The numbers of colonies were quantified after 48 h. Statistical Analysis Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using Two-Way ANOVA and statistical significance was determined at pAd.Hx.Epi8>AdZ.F(FG)Epi8 and (AdZ.F(HI)Epi8>AdZ.F(CD)Epi8, AdZ.F(DE)Epi8 and AdZ.F(CT)Epi8 (p0.05; Figure 4B ). Compared to the disrupted fiber-modified vectors the interaction of disrupted AdZ.HxEpi8 with the OprF serum was stronger (p0.05) that were higher compared to immunization with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8 (pAd.Hx.Epi8>AdZ.F(FG)Epi8 and (AdZ.F(HI)Epi8>AdZ.F(CD)Epi8, AdZ.F(DE)Epi8 and AdZ.F(CT)Epi8 (p0.05; Figure 4B ). Compared to the disrupted fiber-modified vectors the interaction of disrupted AdZ.HxEpi8 with the OprF serum was stronger (p0.05) that were higher compared to immunization with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8 (p<0.05; Figure 5A ). Interestingly, repeat administration of the capsid-modified Ad vectors boosted the OprF-specific humoral immune response in contrast to repeat administration of AdOprF ( Figure 5A ). Anti-OprF IgG was higher following three doses of AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 compared to AdOprF (p<0.05). As expected, anti-Ad IgG titers increased with repeat administration of all vectors ( Figure 5B ). Antibodies against the β-galactosidase transgene were induced by all vectors and were higher with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 compared to AdZ.F(CD)Epi8, AdZ.F(DE)Epi8 or AdZ.F(CT)Epi8 ( Figure 5C ). Repeat administration did not result in increase of anti-β-galactosidase titers. 10.1371/journal.pone.0056996.g005 Figure 5 Immunization with Epi8 fiber-modified Ad vectors induces anti- P. aeruginosa systemic humoral immunity. BALB/c mice were immunized via the intramuscular route with the fiber-modified Ad vectors AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8 and AdZ.F(CT)Epi8, the hexon-modified AdZ.HxEpi8 or AdOprF (all 10 10 pu/mouse). Mice were boosted with the same vectors after 2 and 5 wk, respectively, and anti-OprF, anti-Ad and anti- β-galactosidase antibodies in serum were analyzed at 2, 5 and 8 wks by ELISA. A. Anti-OprF IgG. B. Anti-Ad IgG. C. Anti-β-galactosidase IgG. Data are shown as the mean ± SEM of 5 mice/group. Limit of detection is indicated by the dashed line. * denotes p<0.05. Lung mucosal humoral immune response to Epi8 capsid-modified Ad vectors To assess the lung mucosal anti-OprF humoral immunity, anti-OprF titers were evaluated in bronchioalveolar lavage fluid. Anti-OprF IgG was detected in all mice immunized with the Epi8 capsid-modified or OprF expressing vectors but not in mice immunized with AdZ ( Figure 6 ). Immunization with AdZ.F(FG)Epi8, AdZ.F(HI)Epi8 and AdOprF elicited comparable titers that were higher compared to AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8 (p<0.05). 10.1371/journal.pone.0056996.g006 Figure 6 Immunization with Epi8 fiber-modified Ad vectors induces anti- P. aeruginosa lung mucosal humoral immunity. BALB/c mice were immunized via the intramuscular route with the fiber-modified Ad vectors AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8 and AdZ.F(CT)Epi8, the hexon-modified AdZ.HxEpi8, AdOprF or AdZ (all 10 10 pu/mouse) and boosted with the same vectors at 2 and 5 wk. Anti-OprF IgG in bronchioalveolar lavage fluid was analyzed by ELISA after 8 wk. Data are shown as the mean ± SEM of 5 mice/group. Limit of detection is indicated by the dashed line. * denotes p<0.05, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8 or AdOprF compared to all others. Cellular immune response to Epi8 capsid-modified Ad vectors To evaluate the cellular immune responses, mice were immunized intramuscularly with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8, AdOprF or AdZ and boosted twice with the same vector. The frequencies of OprF-specific splenic CD4 and CD8 T cells stimulated by syngeneic DC pulsed with recombinant OprF protein were analyzed by ELISPOT. Immunization with AdOprF induced the highest OprF-specific IFN-γ CD4 ( Figure 7A ), IL-4 CD4 ( Figure 7B ) and IFN-γ CD8 T cell ( Figure 7C ) responses (p<0.05). Of the capsid-modified vectors, AdZ.F(FG)Epi8 and AdZ.F(HI)Epi8 induced higher OprF-specific IFN-γ CD4 ( Figure 7A ), IL-4 CD4 ( Figure 7B ) and IFN-γ CD8 T cell ( Figure 7C ) compared to AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8 (p<0.05). 10.1371/journal.pone.0056996.g007 Figure 7 Immunization with Epi8 fiber-modified Ad vectors induces anti- P. aeruginosa cellular immunity. BALB/c mice were immunized via the intramuscular route with the fiber-modified Ad vectors AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8 and AdZ.F(CT)Epi8, the hexon-modified AdZ.HxEpi8, AdOprF or AdZ (all 10 10 pu/mouse) and boosted with the same vectors at 2 and 5 wk. Splenic CD4 and CD8 T cells were isolated 7 days following the last administration and incubated in vitro with syngeneic DC pulsed with recombinant OprF or DC alone. IL-4 and IFN-γ were determined by ELISPOT assay. A. CD4 IFN-γ; B. CD4 IL-4; C. CD8 IFN-γ. The data represent the mean of pooled cells from five mice per group from three separate experiments ± SEM. * denotes p<0.05, AdOprF compared to all others. § denotes p<0.05, AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 compared to AdZ, AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8. Protection against pulmonary infection with P. aeruginosa To evaluate if the most immunogenic Epi8 fiber-modified Ad vectors protect against pulmonary infection with P. aeruginosa , mice were immunized with AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdOprF or AdZ, boosted twice with the same vector and challenged by intranasal administration of agar-encapsulated PAO1 three weeks after the last vector administration. Mice immunized with AdZ.F(FG)Epi8, AdZ.F(HI)Epi8 and AdOprF showed reduction in the P. aeruginosa colony count compared to the AdZ group (p<0.05; Figure 8 ). This suggests that the protective immunity generated by AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 is comparable to that induced by AdOprF. 10.1371/journal.pone.0056996.g008 Figure 8 Protective anti- P. aeruginosa immunity induced by immunization with Epi8 fiber-modified Ad vectors. BALB/c mice were immunized via the intramuscular route with AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdOprF and AdZ at a dose of 10 10 pu/mouse followed by boost immunizations after 2 and 5 wk. Eight weeks after immunization, mice were challenged with agar-encapsulated P. aeruginosa (4×10 6 cfu). Bacterial counts in lung homogenate were performed after 24 h. Data are shown as means ± SEM of 5 mice/group. * denotes p<0.05, compared to AdZ-immunized mice. Efficacy of Epi8 fiber-modified Ad vectors in the presence of anti-Ad immunity To evaluate the efficacy of fiber-modified Ad vectors in the presence of pre-existing anti-Ad immunity, Ad-immune mice, induced by repeat administration of AdNull, were immunized with AdZ.F(FG)Epi8 or AdOprF. In the presence of pre-existing anti-Ad immunity, AdOprF inoculated mice showed a marked reduction in anti-OprF titers that were close to basal levels ( Figure 9A ). In contrast, immunization with AdZ.F(FG)Epi8 elicited robust levels of anti-OprF IgG irrespective of pre-existing Ad immunity. Likewise, protection against P. aeruginosa , was similar when AdZ.F(FG)Epi8 was administered in the presence or absence of anti-Ad immunity ( Figure 9B ). This suggests that, in contrast to AdOprF, AdZ.F(FG)Epi8 can elicit protective anti- P. aeruginosa immunity even in the presence of anti-Ad immunity. 10.1371/journal.pone.0056996.g009 Figure 9 AdZ.F(FG)Epi8 induces robust humoral and protective anti- P. aeruginosa in the presence of pre-existing anti-Ad immunity. BALB/c mice were immunized with AdZ.F(FG)Epi8, AdOprF or AdNull (all 10 10 pu)/mouse in the presence or absence of anti-Ad5 immunity. Mice were inoculated with 10 10 pu/mouse of AdNull and boosted twice at 2 weeks interval in order to mimic pre-existing immunity A. Anti-OprF IgG in serum determined by ELISA at 4 wk. Limit of detection is indicated by dotted line. * denotes p<0.05, compared to AdNull-immunized mice. B. Protective immunity against pulmonary challenge with agar-encapsulated P. aeruginosa (4×10 6 cfu) 5 wk after immunization. Bacterial counts in lung homogenate were performed after 24 h. Data are shown as means ± SEM of seven mice per group. * denotes p<0.05, compared to AdNull-immunized mice. Discussion A potent and effective vaccine against P. aeruginosa has long been sought after, but is so far not available. The present study demonstrates that fiber-modified Ad vectors expressing Epi8 induce anti- P. aeruginosa humoral and cellular protective immunity that can be boosted on repeated administration and is effective in presence of anti-Ad5 immunity. Incorporation of epitopes into Ad vector capsid to induce epitope-specific immunity One attractive feature of Ad-based vaccines is the feasibility to modify the Ad capsid to enhance immune responses or change the Ad tropism [23] , [33] . Incorporation of Epi8 into loop 1 of HVR5 of the Ad hexon protein has been shown to induce anti-epitope humoral and cellular immunity to protect against infections with P. aeruginosa in a murine model [16] . Incorporation of influenza HA or ovalbumin epitopes into various Ad capsid proteins demonstrated that incorporation into the fiber HI loop induces the strongest anti-epitope response [20] , [21] . The structure of the Ad5 fiber is known and there are multiple loops and sites that could theoretically be used as insertion sites for peptide sequences without disrupting the overall structure [34] . We developed and compared various Ad vectors that display Epi8 on the CD, DE, FG, HI loops or CT of Ad fiber knob. Immunization with fiber-modified Epi8 Ad vectors induced robust humoral and cellular responses. Following single administration anti-OprF immunity induced by the fiber-modified vectors was less compared to anti-OprF immunity induced by a vector expressing the entire OprF protein as transgene. Among the capsid-modified vectors, the strongest humoral response against the OprF protein was induced by AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8. The AdZ.HxEpi8, AdZ.F(CD)Epi8, AdZ.F(DE)Epi8 and AdZ.F(CT)Epi8 induced only low levels of anti-OprF humoral immunity. The stronger induction of Epi8-specific immune response by fiber-modified Ad vector compared to hexon-modified vector is consistent with our previous observations with HA epitope [20] . The low efficiency of CD, DE fiber loops CT or loop 1 of HVR5 of Ad hexon to generate epitope-specific immune responses can be explained by the interference of the inserted peptide with cellular infectivity, in particular of antigen-presenting cells or the impaired folding and exposure of the epitopes on the capsid. In contrast to our results, higher humoral immunity after single immunization was elicited against an ovalbumin epitope incorporated into the hexon protein compared to insertion of the epitope into the fiber HI loop [21] . It is likely that the nature of the two different epitopes and the location of the epitope within the hexon (insertion within HVR5 in this study versus replacement of HVR5 by Lanzi et al. ) influenced the epitope-specific immune responses. Consistent with our results, upon second administration a stronger humoral response was elicited when ovalbumin epitope was inserted into fiber protein compared to hexon [21] . Protective immunity against extracellular bacteria such as P. aeruginosa is mainly dependent on sufficient levels of humoral immunity induced by the vaccine. T cell-mediated immunity has received less attention in the development of a vaccine against P. aeruginosa but is a part of the response against natural infection with the organism [35] , [36] . Consistent with the humoral response, FG- and HI- modified vectors elicited strong OprF-specific Th1 and Th2 type cellular immunity, which was lower compared to AdOprF. The presence of multiple T-cell epitopes in the full length OprF protein used to pulse DCs, is likely responsible for higher T-cell activation by AdOprF. Importantly, the protective immunity generated by AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 was comparable to AdOprF. Although both humoral and cellular responses were induced after immunization with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 in the present study, their individual contribution to the protection against P. aeruginosa challenge is not clear. The HI loop in the fiber knob has been the usual choice for incorporation of antigenic epitopes or targeting moieties [20] , [21] , [35] , [36] . In the present study we identified the FG loop as a novel location in the fiber knob for peptide insertion. This site is comparable to the HI loop regarding in vitro infectivity and its capacity to elicit anti-peptide immunity. It would be interesting to assess infectivity and immunogenicity of a bispecific Ad vector with peptides at both FG and HI loops. Boosting of anti-OprF immune response by repeat administration of fiber-modified Ad vectors Anti-Ad immune responses impair efficacy of Ad vectors of the same serotype, as pre-existing neutralizing antibodies against Ad prevent the cellular uptake of Ad and expression of the transgene in a previously immunized host [37] , [38] . To circumvent this issue Ad vaccine vectors derived from rare human or nonhuman Ad serotypes that evade anti-Ad5 immunity have been developed [39] – [41] . However, all of these Ad vectors generate potent anti-vector immunity that diminishes the utility of vector re-administration/boosting. Anti-Ad immunity can be boosted by repeated infection with wild-type Ad or Ad vectors, leading to an increase in anti-Ad humoral responses with subsequent infections [37] , [38] , [42] . Consequently, immune response against a foreign epitope placed on an Ad capsid protein can be boosted by repeated vector administration. Immunization with the capsid-modified Ad vectors in this study enabled repeated administration of the same vector resulting in boosting of the anti-epitope and not the anti-transgene humoral response. Strikingly, anti-OprF IgG was higher following three doses of FG- or HI-modified vector compared to AdOprF, thus highlighting the utility of fiber-modified Ad vectors for vaccine delivery. One of the mechanisms that explains the boosting of humoral response is the Fcγ receptor-mediated uptake of Ad vector-antibody immune complexes by antigen-presenting cells and subsequent increased stimulation of specific immune cells [43] . The prevalence of pre-existing vector immunity in humans may limit the utility of human Ad serotype vaccines. Notably, in sharp contrast to immunization with AdOprF, AdZ.F(FG)Epi8 induced protective anti- P. aeruginosa immunity even in the presence of high levels of pre-existing anti-Ad immunity. This suggests that pre-existing Ad vector immunity can be effectively circumvented by the incorporation of antigenic epitopes into the fiber protein. Taken together, incorporation of antigenic peptides on the FG or HI loop of Ad fiber knob is an efficient strategy to generate protective immunity that can be boosted by repeated administration and is effective in the presence of pre-existing anti-Ad immunity. The display of antigenic epitopes on the Ad fiber should also be valuable in the development of Ad-based vaccines against other pathogens. The robust protective immunity induced by AdZ.F(FG)Epi8 and AdZ.F(HI)Epi8 make both of these sites attractive for the insertion of epitopes as a general vaccine strategy. Incorporation of epitopes into Ad vector capsid to induce epitope-specific immunity One attractive feature of Ad-based vaccines is the feasibility to modify the Ad capsid to enhance immune responses or change the Ad tropism [23] , [33] . Incorporation of Epi8 into loop 1 of HVR5 of the Ad hexon protein has been shown to induce anti-epitope humoral and cellular immunity to protect against infections with P. aeruginosa in a murine model [16] . Incorporation of influenza HA or ovalbumin epitopes into various Ad capsid proteins demonstrated that incorporation into the fiber HI loop induces the strongest anti-epitope response [20] , [21] . The structure of the Ad5 fiber is known and there are multiple loops and sites that could theoretically be used as insertion sites for peptide sequences without disrupting the overall structure [34] . We developed and compared various Ad vectors that display Epi8 on the CD, DE, FG, HI loops or CT of Ad fiber knob. Immunization with fiber-modified Epi8 Ad vectors induced robust humoral and cellular responses. Following single administration anti-OprF immunity induced by the fiber-modified vectors was less compared to anti-OprF immunity induced by a vector expressing the entire OprF protein as transgene. Among the capsid-modified vectors, the strongest humoral response against the OprF protein was induced by AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8. The AdZ.HxEpi8, AdZ.F(CD)Epi8, AdZ.F(DE)Epi8 and AdZ.F(CT)Epi8 induced only low levels of anti-OprF humoral immunity. The stronger induction of Epi8-specific immune response by fiber-modified Ad vector compared to hexon-modified vector is consistent with our previous observations with HA epitope [20] . The low efficiency of CD, DE fiber loops CT or loop 1 of HVR5 of Ad hexon to generate epitope-specific immune responses can be explained by the interference of the inserted peptide with cellular infectivity, in particular of antigen-presenting cells or the impaired folding and exposure of the epitopes on the capsid. In contrast to our results, higher humoral immunity after single immunization was elicited against an ovalbumin epitope incorporated into the hexon protein compared to insertion of the epitope into the fiber HI loop [21] . It is likely that the nature of the two different epitopes and the location of the epitope within the hexon (insertion within HVR5 in this study versus replacement of HVR5 by Lanzi et al. ) influenced the epitope-specific immune responses. Consistent with our results, upon second administration a stronger humoral response was elicited when ovalbumin epitope was inserted into fiber protein compared to hexon [21] . Protective immunity against extracellular bacteria such as P. aeruginosa is mainly dependent on sufficient levels of humoral immunity induced by the vaccine. T cell-mediated immunity has received less attention in the development of a vaccine against P. aeruginosa but is a part of the response against natural infection with the organism [35] , [36] . Consistent with the humoral response, FG- and HI- modified vectors elicited strong OprF-specific Th1 and Th2 type cellular immunity, which was lower compared to AdOprF. The presence of multiple T-cell epitopes in the full length OprF protein used to pulse DCs, is likely responsible for higher T-cell activation by AdOprF. Importantly, the protective immunity generated by AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 was comparable to AdOprF. Although both humoral and cellular responses were induced after immunization with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 in the present study, their individual contribution to the protection against P. aeruginosa challenge is not clear. The HI loop in the fiber knob has been the usual choice for incorporation of antigenic epitopes or targeting moieties [20] , [21] , [35] , [36] . In the present study we identified the FG loop as a novel location in the fiber knob for peptide insertion. This site is comparable to the HI loop regarding in vitro infectivity and its capacity to elicit anti-peptide immunity. It would be interesting to assess infectivity and immunogenicity of a bispecific Ad vector with peptides at both FG and HI loops. Boosting of anti-OprF immune response by repeat administration of fiber-modified Ad vectors Anti-Ad immune responses impair efficacy of Ad vectors of the same serotype, as pre-existing neutralizing antibodies against Ad prevent the cellular uptake of Ad and expression of the transgene in a previously immunized host [37] , [38] . To circumvent this issue Ad vaccine vectors derived from rare human or nonhuman Ad serotypes that evade anti-Ad5 immunity have been developed [39] – [41] . However, all of these Ad vectors generate potent anti-vector immunity that diminishes the utility of vector re-administration/boosting. Anti-Ad immunity can be boosted by repeated infection with wild-type Ad or Ad vectors, leading to an increase in anti-Ad humoral responses with subsequent infections [37] , [38] , [42] . Consequently, immune response against a foreign epitope placed on an Ad capsid protein can be boosted by repeated vector administration. Immunization with the capsid-modified Ad vectors in this study enabled repeated administration of the same vector resulting in boosting of the anti-epitope and not the anti-transgene humoral response. Strikingly, anti-OprF IgG was higher following three doses of FG- or HI-modified vector compared to AdOprF, thus highlighting the utility of fiber-modified Ad vectors for vaccine delivery. One of the mechanisms that explains the boosting of humoral response is the Fcγ receptor-mediated uptake of Ad vector-antibody immune complexes by antigen-presenting cells and subsequent increased stimulation of specific immune cells [43] . The prevalence of pre-existing vector immunity in humans may limit the utility of human Ad serotype vaccines. Notably, in sharp contrast to immunization with AdOprF, AdZ.F(FG)Epi8 induced protective anti- P. aeruginosa immunity even in the presence of high levels of pre-existing anti-Ad immunity. This suggests that pre-existing Ad vector immunity can be effectively circumvented by the incorporation of antigenic epitopes into the fiber protein. Taken together, incorporation of antigenic peptides on the FG or HI loop of Ad fiber knob is an efficient strategy to generate protective immunity that can be boosted by repeated administration and is effective in the presence of pre-existing anti-Ad immunity. The display of antigenic epitopes on the Ad fiber should also be valuable in the development of Ad-based vaccines against other pathogens. The robust protective immunity induced by AdZ.F(FG)Epi8 and AdZ.F(HI)Epi8 make both of these sites attractive for the insertion of epitopes as a general vaccine strategy.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5267348/

Inflammasomes in Myeloid Cells: Warriors within

The inflammasome is a large multimeric protein complex comprised of an effector protein that demonstrates a specificity for a variety of activators or ligands, an adaptor molecule and pro-caspase-1 which is converted to caspase-1 upon inflammasome activation. Inflammasomes are expressed primarily by myeloid cells and are located within the cell. The macromolecular inflammasome structure can be visualized by cryo-electron microscopy. This complex has been found to play a role in a variety of disease models in mice and several have been genetically linked to human diseases. In most cases, the effector protein is a member of the NLR (nucleotide-binding domain leucine rich repeat containing), or NOD (nucleotide oligomerization domain)-like receptor protein family. However, other effectors have also been described, with the most notable being AIM2 (absence in melanoma 2), which recognizes DNA to elicit inflammasome function. This chapter will focus on the role of the inflammasome in myeloid cells and its role in health and disease.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544442/

Occupational Disease Registries–Characteristics and Experiences

Introduction: Due to growth of occupational diseases and also increase of public awareness about their consequences, attention to various aspects of diseases and improve occupational health and safety has found great importance. Therefore, there is the need for appropriate information management tools such as registries in order to recognitions of diseases patterns and then making decision about prevention, early detection and treatment of them. These registries have different characteristics in various countries according to their occupational health priorities. Aim: Aim of this study is evaluate dimensions of occupational diseases registries including objectives, data sources, responsible institutions, minimum data set, classification systems and process of registration in different countries. Material and Methods: In this study, the papers were searched using the MEDLINE (PubMed) Google scholar, Scopus, ProQuest and Google. The search was done based on keyword in English for all motor engines including "occupational disease", "work related disease", "surveillance", "reporting", "registration system" and "registry" combined with name of the countries including all subheadings. After categorizing search findings in tables, results were compared with each other. Results: Important aspects of the registries studied in ten countries including Finland, France, United Kingdom, Australia, Czech Republic, Malaysia, United States, Singapore, Russia and Turkey. The results show that surveyed countries have statistical, treatment and prevention objectives. Data sources in almost the rest of registries were physicians and employers. The minimum data sets in most of them consist of information about patient, disease, occupation and employer. Some of countries have special occupational related classification systems for themselves and some of them apply international classification systems such as ICD-10. Finally, the process of registration system was different in countries. Conclusion: Because occupational diseases are often preventable, but not curable, it is necessary to all countries, to consider prevention and early detection of occupational diseases as the objectives of their registry systems. Also it is recommended that all countries reach an agreement about global characteristics of occupational disease registries. This enables country to compare their data at international levels. Introduction: Due to growth of occupational diseases and also increase of public awareness about their consequences, attention to various aspects of diseases and improve occupational health and safety has found great importance. Therefore, there is the need for appropriate information management tools such as registries in order to recognitions of diseases patterns and then making decision about prevention, early detection and treatment of them. These registries have different characteristics in various countries according to their occupational health priorities. Aim: Aim of this study is evaluate dimensions of occupational diseases registries including objectives, data sources, responsible institutions, minimum data set, classification systems and process of registration in different countries. Material and Methods: In this study, the papers were searched using the MEDLINE (PubMed) Google scholar, Scopus, ProQuest and Google. The search was done based on keyword in English for all motor engines including "occupational disease", "work related disease", "surveillance", "reporting", "registration system" and "registry" combined with name of the countries including all subheadings. After categorizing search findings in tables, results were compared with each other. Results: Important aspects of the registries studied in ten countries including Finland, France, United Kingdom, Australia, Czech Republic, Malaysia, United States, Singapore, Russia and Turkey. The results show that surveyed countries have statistical, treatment and prevention objectives. Data sources in almost the rest of registries were physicians and employers. The minimum data sets in most of them consist of information about patient, disease, occupation and employer. Some of countries have special occupational related classification systems for themselves and some of them apply international classification systems such as ICD-10. Finally, the process of registration system was different in countries. Conclusion: Because occupational diseases are often preventable, but not curable, it is necessary to all countries, to consider prevention and early detection of occupational diseases as the objectives of their registry systems. Also it is recommended that all countries reach an agreement about global characteristics of occupational disease registries. This enables country to compare their data at international levels. 1. INTRODUCTION Occupational diseases caused by occupational activities and working conditions ( 1 ). In fact, any disease occurs at early stage as a result of exposure to occupational (physical, chemical or biological) risk factors is an occupational disease ( 1 - 3 ). Occupational diseases impose considerable costs to workers, their family, health care system and society ( 4 ) and reduce productivity and work capacity. According to ILOs' estimates, occupational diseases and injuries causes the loss of 4% of global GDP annually, in other words direct and indirect costs of these diseases and injuries is about 2.8 trillion dollars ( 5 ). In addition, due to social and technological changes, the nature of occupational diseases is changing and new occupational diseases are emerging ( 6 ). In the other hand, occupational diseases are not curable or have long-term and difficult treatment. But most of these diseases are preventable ( 7 , 8 ). Preventing these diseases requires correct information about prevalence of them ( 9 ). Nevertheless, statistical and basic information about some of occupational diseases are not available due to lack of awareness, diagnostic problems and insufficient attention to these diseases. And there are many limitations in reporting and systematic collection of data relating to occupational diseases. To overcome these challenges developing occupational diseases registries, as an effective solution, is very useful. Disease or patient registries are the rich sources of information for any decision making in the field of health ( 10 ). Like other registries, occupational disease registry is a set of information about work related disease and injuries with different levels of complexity. And applies for multiple purposes such as administrative, statistical, preventive, diagnostic, treatment follow up and research ( 11 ). Registry information is crucial to the recognition and then planning for treatment, and prevention of occupational injuries and disease ( 12 ). Using this information to detect patterns of disease, can be taken as an effective action to prevent disease and reduce the health, economic and social costs ( 13 , 14 ). 2. AIM Considering that there is not comprehensive information about the status of occupational diseases registries in other countries. In this paper, we studied the status of occupational diseases in selected countries, and the results are presented in comparative tables; also some information is described narrative. 3. MATERIAL AND METHODS In this study, the papers were searched according to keywords in English using the MEDLINE (PubMed) during 1989 to 2017, Google scholar, Scopus, ProQuest and Google. Keywords included "occupational disease", "work related disease", "surveillance", "reporting", "registration system" and "registry" combined with name of the countries including all subheadings. After completed search, all search results were reviewed separately in databases based on titles and related articles were selected. Then we excluded duplicated documents. Two reviewers reviewed all documents separately. Then unrelated documents were excluded and data collection forms were filled with accepted documents. Then articles have been categorized based on their developer countries. After that the results were presented in the comparative tables. In all stages, the disagreements between reviewers were addressed by group discussion. 4. RESULTS In this review, documents were included from mentioned database. The included documents arranged according to their selected countries. At last the result presented in two narrative and table format ( Table 1 and Table 2 ). Table 1 Name, responsible institution and objectives of registries Table 2 Minimum data set, data sources and classification system of registries In this part other characteristics of mentioned registries are describing according the name of the countries. Finland In Finland the occupational disease registry covers people who are working under a contract for an employer, public services, public administration, or as entrepreneurs like farmers. There is three ways to reports occupational disease cases to the FROD (Finnish register of occupational disease) ( 15 ). In one way, insurance companies send reports from physicians and employers to the FAII (The Federation of Accident Insurance Institution) then FAII send these reports to the FROD. In other way MELA (the farmers' social insurance institution) sends physicians and farmers reports to FROD. In the last way regional state administrative agencies send physicians' reports to the FROD directly. In Finland occupational diseases reportable including diseases caused by asbestos, skin diseases, Allergic respiratory disease, Injuries caused by repeated pressure, hearing loss, and other diseases including infectious diseases, vibration syndrome, conjunctivitis and different types of poisoning ( 16 - 19 ). France RNV 3 is a National Network for Monitoring and Prevention of Occupational Diseases in the France that coordinates Knowledge of the registry for monitoring purposes, recovery and prevention of occupational risks. This network also contributes through Modernet Network (a network of monitoring trends in occupational diseases in the European countries) with other European counterparts ( 20 ). The Occupational Disease Consultation Centers (CCPPs) and occupational health services (SSTs), report new cases of occupational diseases to the RNV3 without any inclusion and exclusion criteria. Registration has been done by occupational physicians, general practitioners and other specialists, nurses, medical secretaries and trained intern. RNV3P includes 110 tables of all occupational diseases in France. Most tables' present diseases are caused by chemical substances but some of them including diseases caused by noise, repetitive movements and working conditions ( 21 , 22 ). United Kingdom In United Kingdom the THOR (the health and occupation research network) program created in 2002 Based on the voluntary participation of more than a thousand specialists, including physicians, consultants and dermatologists transmitted diseases, as well as trained general practitioners to report cases of occupational diseases. Since 2005, occupational data from the Republic of Ireland are collected by THOR. It also contributes with other European countries through Modernet Network ( 23 - 25 ). THOR allows physicians report every item that they believe created by occupational factors addition to the occupational disease list provided by this program ( 26 ). Occupational diseases that are included in THOR are musculoskeletal disorders, stress, depression and anxiety, skin diseases, respiratory diseases and other diseases. THOR covers all people with occupational problems who connect to doctors' offices and clinics ( 24 ). Australia The National Data Set for Compensation-based Statistics (NDS) is a national occupational registration system in Australia. NDS provides information on workers' compensation claims that involve work-related disease in fact every compensation claim request from workers is a new case in the registry system ( 27 , 28 ). Occupational diseases included in the registry are musculoskeletal diseases, mental disorders, cardiovascular diseases, occupational cancers, respiratory diseases, infectious and parasitic diseases, contact dermatitis and noise-induced hearing loss ( 29 ). Range of cases recorded in NDS, are included all new cases (all verified, rejected cases and cases in the decision making process) reported in the current year. Claims that were subsequently withdrawn by the worker and the ones that are outside the scope of application of the program are removed ( 30 ). Czech Republic The NRNP (the Czech National Registry of Occupational Disease) created in 1991 and is maintained by Centre of occupational medicine of the State Institute of public health in Prague as Central register of occupational diseases. NRNP since 2003 is connected with EODS (European Occupational Diseases Statistics) ( 31 - 33 ). Occupational diseases caused by chemical substances, occupational diseases caused by physical factors, occupational diseases relating to the respiratory pathways, lungs, pleura and peritoneum, occupational skin diseases caused by physical, chemical or biological factors, infectious and parasitic occupational diseases, occupational diseases due to other factors or agents are included in the registry system ( 34 ). Malaysia The occupational disease registration system in Malaysia was created and is maintained by DOSH (department of occupational safety and health). According to law, employers and physicians are obliged to report new cases of occupational diseases to DOSH ( 35 - 37 ). Occupational diseases in this registry system including Occupational lung diseases, occupational skin diseases, noise-induced hearing loss, diseases caused by chemical agents (poisoning), diseases caused by biological agents, occupational cancers and other occupational diseases ( 38 ). Registry covers workers in occupations such as manufacturing, mining and quarrying, construction, agriculture, forestry, logging and fishery, utility, transport, storage and communication, wholesale and retail trade, hotel and restaurant, financial, insurance, real estate and business services, public services and statutory bodies ( 38 ). United States In the United States IIF (Injuries, Illnesses, and Fatalities) provide rates and the number of occupational diseases, injuries and the number of fatal cases annually. Two main data sources of this program are SOII (The Survey of Occupational Injuries and Illnesses) and CFOI (Census of Fatal Occupational Injuries). SOII is a federal program in which employer's reports (OSHA 300 form) are collected from private industry and public sector annually. These reports are processed by BLS (Bureau of Labor Statistics). Diseases that are included in the registry contain occupational musculoskeletal diseases, infectious diseases, respiratory diseases, skin diseases and other diseases ( 39 - 41 ). The registry provides data from all full-time and part-time wage and salary workers in nonfarm industries. The excluded items are self-employed, owners and partners in unincorporated firms, household workers, or unpaid family workers ( 42 ). Singapore In Singapore "iReport" was introduced as a national system of electronic reporting for occupational diseases in 2006 under the supervision of MOM (Ministry of Manpower) ( 43 ). Physicians and employers are required to report cases of occupational diseases. Physicians during ten days from the time of diagnosis should register cases in the iReport system. Employers too within ten days of receiving a written diagnosis of the disease should report it ( 44 ). Occupational diseases list in Singapore including anthrax, asbestosis, barotrauma, byssinosis, chrome ulceration, compressed air illness, epitheliomatous ulceration, occupational skin diseases, liver angiosarcoma, mesothelioma, noise-induced deafness, occupational asthma, repetitive strain disorder of the upper limb, silicosis, toxic anemia, toxic hepatitis and poisonings due to Aniline, Arsenical, Beryllium, Cadmium, Carbamate, Carbon bisulphide, chronic benzene, Cyanide, Hydrogen sulphide, Lead, Manganese, Mercurial, Organophosphate, Phosphorous and halogen derivatives of hydrocarbon compounds ( 45 ). Russia Before 2007, Russia was not mandated evaluation of the working environment and working conditions monitoring was done selective. A law was passed in 2007 by the Ministry of Health asked all employers that to measure and quantify workplace hazards every 5 years by standardized methods. After that in 2013 Russia adopted the federal law on the basis of a special assessment of working conditions. This law classified working condition in to the 4 level including optimal, permissible, harmful and dangerous ( 46 ). According to working condition, workers receive different types of compensation fees ( 47 ). The medical commission in suspected cases of occupational diseases reports the results of medical examination to the employers and Rospotrebnadzor (Territorial Department of Federal Service for Oversight of Consumer Protection and Welfare). Then hygienists of Rospotrebnadzor prepare a reports including a description of the sanitary-hygienic characteristics of working conditions, containing the occupational history, description of the working process, information about applied materials and equipment, and levels of occupational exposures during two weeks ( 48 , 49 ). Turkey The SSI (Social Security Institution) is the governing authority of the Turkish social security system and according to law reporting all occupational diseases and injuries to SSI is mandatory. The report arranged and classified by SSI in accordance with the rules of the International Labour Organization. According to statistics released by SSI in Turkey occupational diseases are divided into 5 groups including occupational diseases caused by chemicals, occupational skin disorders, pneumoconiosis and other respiratory occupational diseases, communicable occupational diseases and occupational diseases caused by physical factors. In total 74 cases of occupational disease are defined in 5 groups. In this field the main challenge is underreporting of occupational disease in compare with other counties reports such as Germany, United States and Finland ( 50 , 51 ). Finland In Finland the occupational disease registry covers people who are working under a contract for an employer, public services, public administration, or as entrepreneurs like farmers. There is three ways to reports occupational disease cases to the FROD (Finnish register of occupational disease) ( 15 ). In one way, insurance companies send reports from physicians and employers to the FAII (The Federation of Accident Insurance Institution) then FAII send these reports to the FROD. In other way MELA (the farmers' social insurance institution) sends physicians and farmers reports to FROD. In the last way regional state administrative agencies send physicians' reports to the FROD directly. In Finland occupational diseases reportable including diseases caused by asbestos, skin diseases, Allergic respiratory disease, Injuries caused by repeated pressure, hearing loss, and other diseases including infectious diseases, vibration syndrome, conjunctivitis and different types of poisoning ( 16 - 19 ). France RNV 3 is a National Network for Monitoring and Prevention of Occupational Diseases in the France that coordinates Knowledge of the registry for monitoring purposes, recovery and prevention of occupational risks. This network also contributes through Modernet Network (a network of monitoring trends in occupational diseases in the European countries) with other European counterparts ( 20 ). The Occupational Disease Consultation Centers (CCPPs) and occupational health services (SSTs), report new cases of occupational diseases to the RNV3 without any inclusion and exclusion criteria. Registration has been done by occupational physicians, general practitioners and other specialists, nurses, medical secretaries and trained intern. RNV3P includes 110 tables of all occupational diseases in France. Most tables' present diseases are caused by chemical substances but some of them including diseases caused by noise, repetitive movements and working conditions ( 21 , 22 ). United Kingdom In United Kingdom the THOR (the health and occupation research network) program created in 2002 Based on the voluntary participation of more than a thousand specialists, including physicians, consultants and dermatologists transmitted diseases, as well as trained general practitioners to report cases of occupational diseases. Since 2005, occupational data from the Republic of Ireland are collected by THOR. It also contributes with other European countries through Modernet Network ( 23 - 25 ). THOR allows physicians report every item that they believe created by occupational factors addition to the occupational disease list provided by this program ( 26 ). Occupational diseases that are included in THOR are musculoskeletal disorders, stress, depression and anxiety, skin diseases, respiratory diseases and other diseases. THOR covers all people with occupational problems who connect to doctors' offices and clinics ( 24 ). Australia The National Data Set for Compensation-based Statistics (NDS) is a national occupational registration system in Australia. NDS provides information on workers' compensation claims that involve work-related disease in fact every compensation claim request from workers is a new case in the registry system ( 27 , 28 ). Occupational diseases included in the registry are musculoskeletal diseases, mental disorders, cardiovascular diseases, occupational cancers, respiratory diseases, infectious and parasitic diseases, contact dermatitis and noise-induced hearing loss ( 29 ). Range of cases recorded in NDS, are included all new cases (all verified, rejected cases and cases in the decision making process) reported in the current year. Claims that were subsequently withdrawn by the worker and the ones that are outside the scope of application of the program are removed ( 30 ). Czech Republic The NRNP (the Czech National Registry of Occupational Disease) created in 1991 and is maintained by Centre of occupational medicine of the State Institute of public health in Prague as Central register of occupational diseases. NRNP since 2003 is connected with EODS (European Occupational Diseases Statistics) ( 31 - 33 ). Occupational diseases caused by chemical substances, occupational diseases caused by physical factors, occupational diseases relating to the respiratory pathways, lungs, pleura and peritoneum, occupational skin diseases caused by physical, chemical or biological factors, infectious and parasitic occupational diseases, occupational diseases due to other factors or agents are included in the registry system ( 34 ). Malaysia The occupational disease registration system in Malaysia was created and is maintained by DOSH (department of occupational safety and health). According to law, employers and physicians are obliged to report new cases of occupational diseases to DOSH ( 35 - 37 ). Occupational diseases in this registry system including Occupational lung diseases, occupational skin diseases, noise-induced hearing loss, diseases caused by chemical agents (poisoning), diseases caused by biological agents, occupational cancers and other occupational diseases ( 38 ). Registry covers workers in occupations such as manufacturing, mining and quarrying, construction, agriculture, forestry, logging and fishery, utility, transport, storage and communication, wholesale and retail trade, hotel and restaurant, financial, insurance, real estate and business services, public services and statutory bodies ( 38 ). United States In the United States IIF (Injuries, Illnesses, and Fatalities) provide rates and the number of occupational diseases, injuries and the number of fatal cases annually. Two main data sources of this program are SOII (The Survey of Occupational Injuries and Illnesses) and CFOI (Census of Fatal Occupational Injuries). SOII is a federal program in which employer's reports (OSHA 300 form) are collected from private industry and public sector annually. These reports are processed by BLS (Bureau of Labor Statistics). Diseases that are included in the registry contain occupational musculoskeletal diseases, infectious diseases, respiratory diseases, skin diseases and other diseases ( 39 - 41 ). The registry provides data from all full-time and part-time wage and salary workers in nonfarm industries. The excluded items are self-employed, owners and partners in unincorporated firms, household workers, or unpaid family workers ( 42 ). Singapore In Singapore "iReport" was introduced as a national system of electronic reporting for occupational diseases in 2006 under the supervision of MOM (Ministry of Manpower) ( 43 ). Physicians and employers are required to report cases of occupational diseases. Physicians during ten days from the time of diagnosis should register cases in the iReport system. Employers too within ten days of receiving a written diagnosis of the disease should report it ( 44 ). Occupational diseases list in Singapore including anthrax, asbestosis, barotrauma, byssinosis, chrome ulceration, compressed air illness, epitheliomatous ulceration, occupational skin diseases, liver angiosarcoma, mesothelioma, noise-induced deafness, occupational asthma, repetitive strain disorder of the upper limb, silicosis, toxic anemia, toxic hepatitis and poisonings due to Aniline, Arsenical, Beryllium, Cadmium, Carbamate, Carbon bisulphide, chronic benzene, Cyanide, Hydrogen sulphide, Lead, Manganese, Mercurial, Organophosphate, Phosphorous and halogen derivatives of hydrocarbon compounds ( 45 ). Russia Before 2007, Russia was not mandated evaluation of the working environment and working conditions monitoring was done selective. A law was passed in 2007 by the Ministry of Health asked all employers that to measure and quantify workplace hazards every 5 years by standardized methods. After that in 2013 Russia adopted the federal law on the basis of a special assessment of working conditions. This law classified working condition in to the 4 level including optimal, permissible, harmful and dangerous ( 46 ). According to working condition, workers receive different types of compensation fees ( 47 ). The medical commission in suspected cases of occupational diseases reports the results of medical examination to the employers and Rospotrebnadzor (Territorial Department of Federal Service for Oversight of Consumer Protection and Welfare). Then hygienists of Rospotrebnadzor prepare a reports including a description of the sanitary-hygienic characteristics of working conditions, containing the occupational history, description of the working process, information about applied materials and equipment, and levels of occupational exposures during two weeks ( 48 , 49 ). Turkey The SSI (Social Security Institution) is the governing authority of the Turkish social security system and according to law reporting all occupational diseases and injuries to SSI is mandatory. The report arranged and classified by SSI in accordance with the rules of the International Labour Organization. According to statistics released by SSI in Turkey occupational diseases are divided into 5 groups including occupational diseases caused by chemicals, occupational skin disorders, pneumoconiosis and other respiratory occupational diseases, communicable occupational diseases and occupational diseases caused by physical factors. In total 74 cases of occupational disease are defined in 5 groups. In this field the main challenge is underreporting of occupational disease in compare with other counties reports such as Germany, United States and Finland ( 50 , 51 ). Finland In Finland the occupational disease registry covers people who are working under a contract for an employer, public services, public administration, or as entrepreneurs like farmers. There is three ways to reports occupational disease cases to the FROD (Finnish register of occupational disease) ( 15 ). In one way, insurance companies send reports from physicians and employers to the FAII (The Federation of Accident Insurance Institution) then FAII send these reports to the FROD. In other way MELA (the farmers' social insurance institution) sends physicians and farmers reports to FROD. In the last way regional state administrative agencies send physicians' reports to the FROD directly. In Finland occupational diseases reportable including diseases caused by asbestos, skin diseases, Allergic respiratory disease, Injuries caused by repeated pressure, hearing loss, and other diseases including infectious diseases, vibration syndrome, conjunctivitis and different types of poisoning ( 16 - 19 ). France RNV 3 is a National Network for Monitoring and Prevention of Occupational Diseases in the France that coordinates Knowledge of the registry for monitoring purposes, recovery and prevention of occupational risks. This network also contributes through Modernet Network (a network of monitoring trends in occupational diseases in the European countries) with other European counterparts ( 20 ). The Occupational Disease Consultation Centers (CCPPs) and occupational health services (SSTs), report new cases of occupational diseases to the RNV3 without any inclusion and exclusion criteria. Registration has been done by occupational physicians, general practitioners and other specialists, nurses, medical secretaries and trained intern. RNV3P includes 110 tables of all occupational diseases in France. Most tables' present diseases are caused by chemical substances but some of them including diseases caused by noise, repetitive movements and working conditions ( 21 , 22 ). United Kingdom In United Kingdom the THOR (the health and occupation research network) program created in 2002 Based on the voluntary participation of more than a thousand specialists, including physicians, consultants and dermatologists transmitted diseases, as well as trained general practitioners to report cases of occupational diseases. Since 2005, occupational data from the Republic of Ireland are collected by THOR. It also contributes with other European countries through Modernet Network ( 23 - 25 ). THOR allows physicians report every item that they believe created by occupational factors addition to the occupational disease list provided by this program ( 26 ). Occupational diseases that are included in THOR are musculoskeletal disorders, stress, depression and anxiety, skin diseases, respiratory diseases and other diseases. THOR covers all people with occupational problems who connect to doctors' offices and clinics ( 24 ). Australia The National Data Set for Compensation-based Statistics (NDS) is a national occupational registration system in Australia. NDS provides information on workers' compensation claims that involve work-related disease in fact every compensation claim request from workers is a new case in the registry system ( 27 , 28 ). Occupational diseases included in the registry are musculoskeletal diseases, mental disorders, cardiovascular diseases, occupational cancers, respiratory diseases, infectious and parasitic diseases, contact dermatitis and noise-induced hearing loss ( 29 ). Range of cases recorded in NDS, are included all new cases (all verified, rejected cases and cases in the decision making process) reported in the current year. Claims that were subsequently withdrawn by the worker and the ones that are outside the scope of application of the program are removed ( 30 ). Czech Republic The NRNP (the Czech National Registry of Occupational Disease) created in 1991 and is maintained by Centre of occupational medicine of the State Institute of public health in Prague as Central register of occupational diseases. NRNP since 2003 is connected with EODS (European Occupational Diseases Statistics) ( 31 - 33 ). Occupational diseases caused by chemical substances, occupational diseases caused by physical factors, occupational diseases relating to the respiratory pathways, lungs, pleura and peritoneum, occupational skin diseases caused by physical, chemical or biological factors, infectious and parasitic occupational diseases, occupational diseases due to other factors or agents are included in the registry system ( 34 ). Malaysia The occupational disease registration system in Malaysia was created and is maintained by DOSH (department of occupational safety and health). According to law, employers and physicians are obliged to report new cases of occupational diseases to DOSH ( 35 - 37 ). Occupational diseases in this registry system including Occupational lung diseases, occupational skin diseases, noise-induced hearing loss, diseases caused by chemical agents (poisoning), diseases caused by biological agents, occupational cancers and other occupational diseases ( 38 ). Registry covers workers in occupations such as manufacturing, mining and quarrying, construction, agriculture, forestry, logging and fishery, utility, transport, storage and communication, wholesale and retail trade, hotel and restaurant, financial, insurance, real estate and business services, public services and statutory bodies ( 38 ). United States In the United States IIF (Injuries, Illnesses, and Fatalities) provide rates and the number of occupational diseases, injuries and the number of fatal cases annually. Two main data sources of this program are SOII (The Survey of Occupational Injuries and Illnesses) and CFOI (Census of Fatal Occupational Injuries). SOII is a federal program in which employer's reports (OSHA 300 form) are collected from private industry and public sector annually. These reports are processed by BLS (Bureau of Labor Statistics). Diseases that are included in the registry contain occupational musculoskeletal diseases, infectious diseases, respiratory diseases, skin diseases and other diseases ( 39 - 41 ). The registry provides data from all full-time and part-time wage and salary workers in nonfarm industries. The excluded items are self-employed, owners and partners in unincorporated firms, household workers, or unpaid family workers ( 42 ). Singapore In Singapore "iReport" was introduced as a national system of electronic reporting for occupational diseases in 2006 under the supervision of MOM (Ministry of Manpower) ( 43 ). Physicians and employers are required to report cases of occupational diseases. Physicians during ten days from the time of diagnosis should register cases in the iReport system. Employers too within ten days of receiving a written diagnosis of the disease should report it ( 44 ). Occupational diseases list in Singapore including anthrax, asbestosis, barotrauma, byssinosis, chrome ulceration, compressed air illness, epitheliomatous ulceration, occupational skin diseases, liver angiosarcoma, mesothelioma, noise-induced deafness, occupational asthma, repetitive strain disorder of the upper limb, silicosis, toxic anemia, toxic hepatitis and poisonings due to Aniline, Arsenical, Beryllium, Cadmium, Carbamate, Carbon bisulphide, chronic benzene, Cyanide, Hydrogen sulphide, Lead, Manganese, Mercurial, Organophosphate, Phosphorous and halogen derivatives of hydrocarbon compounds ( 45 ). Russia Before 2007, Russia was not mandated evaluation of the working environment and working conditions monitoring was done selective. A law was passed in 2007 by the Ministry of Health asked all employers that to measure and quantify workplace hazards every 5 years by standardized methods. After that in 2013 Russia adopted the federal law on the basis of a special assessment of working conditions. This law classified working condition in to the 4 level including optimal, permissible, harmful and dangerous ( 46 ). According to working condition, workers receive different types of compensation fees ( 47 ). The medical commission in suspected cases of occupational diseases reports the results of medical examination to the employers and Rospotrebnadzor (Territorial Department of Federal Service for Oversight of Consumer Protection and Welfare). Then hygienists of Rospotrebnadzor prepare a reports including a description of the sanitary-hygienic characteristics of working conditions, containing the occupational history, description of the working process, information about applied materials and equipment, and levels of occupational exposures during two weeks ( 48 , 49 ). Turkey The SSI (Social Security Institution) is the governing authority of the Turkish social security system and according to law reporting all occupational diseases and injuries to SSI is mandatory. The report arranged and classified by SSI in accordance with the rules of the International Labour Organization. According to statistics released by SSI in Turkey occupational diseases are divided into 5 groups including occupational diseases caused by chemicals, occupational skin disorders, pneumoconiosis and other respiratory occupational diseases, communicable occupational diseases and occupational diseases caused by physical factors. In total 74 cases of occupational disease are defined in 5 groups. In this field the main challenge is underreporting of occupational disease in compare with other counties reports such as Germany, United States and Finland ( 50 , 51 ). 5. CONCLUSION Obviously, in order to identify and prevent occupational diseases, the existences of valid and powerful information systems such as occupational diseases registries are essential. However, in most countries still appropriate and comprehensive registry systems, for these purposes, does not exist. On the other hand, despite development and implementation of the occupational diseases registry in some countries, due to lack of international agreements and standards, comparing of data at international level is not possible. Creating such standards will accelerate the development of these systems in other countries.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1448514/

Rural Public Health Service Delivery: Promising New Directions

I describe variations in the structure and in the practice of rural public health and how rural communities meet the challenges of current public health practice, including primary methods of service delivery and partnership development. I present examples of promising models for the creation of rural public health capacity—the ability of local health departments to carry out core public health responsibilities.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629933/

GSK3β and CREB3 Gene Expression Profiling in Benign and Malignant Salivary Gland Tumors

Background: Salivary gland tumors (SGT) are rare lesions with uncertain histopathology. One of the major signaling pathways that participate in the development of several tumors is protein kinase A. In this pathway, glycogen synthase kinase β (GSK3β) and cAMP responsive element binding protein (CREB3) are two genes which are supposed to be down regulated in most human tumors. The expression level of the genes was evaluated in SGT to scrutinize their possible under expression in these tumors. Methods: Forty eight fresh tissue samples were obtained from patients with benign and malignant SGT, including pleomorphic adenoma, warthin's tumor, mucoepidermoid carcinoma (MEC), salivary duct carcinoma and carcinoma ex pleomorphic adenoma. Eight normal samples were used as controls. Quantitative real-time PCR was used to analyze the expression level of interest genes. Results: Data was analyzed by statistical methods. GSK3β was downregulate in all samples and all results were statistically significant ( P <0.05). CREB3 did not show a significant decrease or increase in its mRNA expression, but the results were significant in MEC and salivary duct carcinoma. Conclusion: GSK3β down regulation has been reported in many human tumors. This gene stimulates CREB3, inducing cell proliferation and oncogenesis. Our findings showed GSK3β down regulation; however, CREB3 expression level was close to normal group. No association between CREB3 expression and inactivated GSK3β could be postulated in SGT.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899236/

Decontamination of soil contaminated at the surface with Bacillus anthracis spores using dry thermal treatment

In the event of a large, aerosol release of Bacillus anthracis spores in a major metropolitan area, soils and other outdoor materials may become contaminated with the biological agent. A study was conducted to assess the in-situ remediation of soil using a dry thermal treatment approach to inactivate a B. anthracis spore surrogate inoculated into soil samples. The study was conducted in two phases, using loam, clay and sand-based soils, as well as biological indicators and spore-inoculated stainless-steel coupons. Initial experiments were performed in an environmental test chamber with temperatures controlled between 80 and 110 °C, with and without added humidity, and with contact times ranging from 4 h to 7 weeks. Tests were then scaled up to assess the thermal inactivation of spores in small soil columns, in which a heating plate set to 141 °C was applied to the soil surface. These column tests were conducted to assess time requirements to inactivate spores as a function of soil depth and soil type. Results from the initial phase of testing showed that increasing the temperature and relative humidity reduced the time requirements to achieve samples in which no surrogate spores were detected. For the test at 80 °C with no added humidity, 49 days were required to achieve soil samples with no spores detected in clay and loam. At 110 °C, 24 h were required to achieve samples in which no spores were detected. In the column tests, no spores were detected at the 2.5 cm depth at four days and at the 5.1 cm depth at 21 days, for two of the three soils. The experiments described in the study demonstrate the feasibility of using dry thermal techniques to decontaminate soils that have been surficially contaminated with B. anthracis spores.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4225133/

Cathelicidin Administration Protects Mice from Bacillus anthracis Spore Challenge 1

Cathelicidins are a family of cationic peptides expressed in mammals that possess numerous bactericidal and immunomodulatory properties. In vitro analyses showed that human, mouse, and pig cathelicidins inhibited Bacillus anthracis bacterial growth at micromolar concentrations in the presence or absence of capsule. Combined in vitro analyses of the effects of each peptide on spore germination and vegetative outgrowth by time lapse phase contrast microscopy, transmission electron microscopy, and flow cytometric analysis showed that only the pig cathelicidin was capable of directly arresting vegetative outgrowth and killing the developing bacilli within the confines of the exosporium. C57BL/6 mice were protected from spore-induced death by each cathelicidin in a time- and dose-dependent manner. Protection afforded by the porcine cathelicidin was due to its bactericidal effects, whereas the human and mouse cathelicidins appeared to mediate protection through increased recruitment of neutrophils to the site of infection. These findings suggest that cathelicidins might be utilized to augment the initial innate immune response to B. anthracis spore exposure and prevent the development of anthrax.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881705/

Inducible Colonic M Cells Are Dependent on TNFR2 but Not Ltβr, Identifying Distinct Signalling Requirements for Constitutive Versus Inducible M Cells

Abstract Background and Aims: M cells associated with organised lymphoid tissues such as intestinal Peyer's patches provide surveillance of the intestinal lumen. Inflammation or infection in the colon can induce an M cell population associated with lymphoid infiltrates; paradoxically, induction is dependent on the inflammatory cytokine tumour necrosis factor [TNF]-α. Anti-TNFα blockade is an important therapeutic in inflammatory bowel disease, so understanding the effects of TNFα signalling is important in refining therapeutics. Methods: To dissect pro-inflammatory signals from M cell inductive signals, we used confocal microscopy image analysis to assess requirements for specific cytokine receptor signals using TNF receptor 1 [TNFR1] and 2 [TNFR2] knockouts [ko] back-crossed to the PGRP-S-dsRed transgene; separate groups were treated with soluble lymphotoxin β receptor [sLTβR] to block LTβR signalling. All groups were treated with dextran sodium sulphate [DSS] to induce colitis. Results: Deficiency of TNFR1 or TNFR2 did not prevent DSS-induced inflammation nor induction of stromal cell expression of receptor activator of nuclear factor kappa-B ligand [RANKL], but absence of TNFR2 prevented M cell induction. LTβR blockade had no effect on M cell induction, but it appeared to reduce RANKL induction below adjacent M cells. Conclusions: TNFR2 is required for inflammation-inducible M cells, indicating that constitutive versus inflammation-inducible M cells depend on different triggers. The inducible M cell dependence on TNFR2 suggests that this specific subset is dependent on TNFα in addition to a presumed requirement for RANKL. Since inducible M cell function will influence immune responses, selective blockade of TNFα may affect colonic inflammation. Background and Aims: M cells associated with organised lymphoid tissues such as intestinal Peyer's patches provide surveillance of the intestinal lumen. Inflammation or infection in the colon can induce an M cell population associated with lymphoid infiltrates; paradoxically, induction is dependent on the inflammatory cytokine tumour necrosis factor [TNF]-α. Anti-TNFα blockade is an important therapeutic in inflammatory bowel disease, so understanding the effects of TNFα signalling is important in refining therapeutics. Methods: To dissect pro-inflammatory signals from M cell inductive signals, we used confocal microscopy image analysis to assess requirements for specific cytokine receptor signals using TNF receptor 1 [TNFR1] and 2 [TNFR2] knockouts [ko] back-crossed to the PGRP-S-dsRed transgene; separate groups were treated with soluble lymphotoxin β receptor [sLTβR] to block LTβR signalling. All groups were treated with dextran sodium sulphate [DSS] to induce colitis. Results: Deficiency of TNFR1 or TNFR2 did not prevent DSS-induced inflammation nor induction of stromal cell expression of receptor activator of nuclear factor kappa-B ligand [RANKL], but absence of TNFR2 prevented M cell induction. LTβR blockade had no effect on M cell induction, but it appeared to reduce RANKL induction below adjacent M cells. Conclusions: TNFR2 is required for inflammation-inducible M cells, indicating that constitutive versus inflammation-inducible M cells depend on different triggers. The inducible M cell dependence on TNFR2 suggests that this specific subset is dependent on TNFα in addition to a presumed requirement for RANKL. Since inducible M cell function will influence immune responses, selective blockade of TNFα may affect colonic inflammation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519738/

Loading and Releasing Ciprofloxacin in Photoactivatable Liposomes

We demonstrate that ciprofloxacin can be actively loaded into liposomes that contain small amounts of porphyrin-phospholipid (PoP). PoP renders the liposomes photoactivatable, so that the antibiotic is released from the carrier under red light irradiation (665 nm). The use of 2 molar % PoP in the liposomes accommodated active loading of ciprofloxacin. Further inclusion of 2 molar % of an unsaturated phospholipid accelerated light-triggered drug release, with more than 90 % antibiotic release from the liposomes occurring in less than 30 seconds. With or without laser treatment, ciprofloxacin PoP liposomes inhibited the growth of Bacillus subtilis in liquid media, apparently due to uptake of the liposomes by the bacteria. However, when liposomes were first separated from smaller molecules with centrifugal filtration, only the filtrate from laser-treated liposomes was bactericidal, confirming effective release of active antibiotic. These results establish the feasibility of remote loading antibiotics into photoactivatable liposomes, which could lead to opportunities for enhanced localized antibiotic therapy.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815476/

Axial Ligand Replacement Mechanism in Heme Transfer from Streptococcal Heme-Binding Protein Shp to HtsA of the HtsABC Transporter

The heme-binding protein Shp of Group A Streptococcus rapidly transfers its heme to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. Heme axial residue-to-alanine replacement mutant proteins of Shp and HtsA (Shp M66A , Shp M153A , HtsA M79A , and HtsA H229A ) were used to probe the axial displacement mechanism of this heme transfer reaction. Ferric Shp M66A at high pH and Shp M153A have a pentacoordinate heme iron complex with a methionine axial ligand. ApoHtsA M79A efficiently acquires heme from ferric Shp but alters the reaction mechanism to two kinetic phases from a single phase in the wild-type protein reactions. In contrast, apoHtsA H229A cannot assimilate heme from ferric Shp. The conversion of pentacoordinate holoShp M66A into pentacoordinate holoHtsA H229A involves an intermediate, whereas holoHtsA H229A is directly formed from pentacoordinate holoShp M153A . Conversely, apoHtsA M79A reacts with holoShp M66A and holoShp M153A in the mechanisms with one and two kinetic phases, respectively. These results imply that the Met79 and His229 residues of HtsA displace the Met66 and Met153 residues of Shp, respectively. Structural docking analysis supports this mechanism of the specific axial residue displacement. Furthermore, the rates of the cleavage of the axial bond in Shp in the presence of a replacing HtsA axial residue are greater than that in the absence of a replacing HtsA axial residue. These findings reveal a novel heme transfer mechanism of the specific displacement of the Shp axial residues with the HtsA axial residues and the involvement of the HtsA axial residues in the displacement.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127683/

Biosecurity practices in Belgian veal calf farming: Level of implementation, attitudes, strengths, weaknesses and constraints

Highlights • Biosecurity awareness among veal farmers was very low. • On average, veal farms were filled in 11.4 days. • On average, 124 calves originated from 100 farms of origin. • No difference in biosecurity level could be found between different veal companies. • Fundamental changes are needed to improve biosecurity regarding introduction of animals. 1 Introduction The impact of infectious animal diseases and the measures to control them are of great importance for animal health, public health, food safety, and the economy. In order to implement the European Commission's Animal Health Strategy vision, "prevention is better than cure,' and the European Union Animal Health Law states that biosecurity is one of the key tools in preventing the introduction, development, and spread of transmissible animal diseases to, from, and within an animal population. In the recent past, some studies regarding biosecurity in cattle farms have found that the overall application of biosecurity measures was low ( Sarrazin et al., 2014 ; Renault et al., 2018a ). As far as we are aware, no studies regarding biosecurity in intensive veal-rearing systems have yet been executed. Biosecurity is defined as all measures that aim to prevent pathogens from entering or leaving a herd, referred to as external biosecurity, and all measures aiming to reduce the spread of pathogens within a herd, referred to as internal biosecurity ( Damiaans et al., 2018 ). External biosecurity contains measures concerning animal movements, e.g., purchase and transport of live animals. Biosecurity also includes the entrance of visitors, such as the herd veterinarian, and possible contact with other animals of the same or other species. Internal biosecurity contains measures concerning the health management of the animals, compartmentation of different age groups, and cleaning and disinfection. In Europe and North America, a high number of excess dairy and, to a lesser degree, beef calves are reared in the highly integrated veal industry ( Brown and Claxton, 2011 ). The veal sector is strongly integrated and industrialized and is therefore substantially different from conventional cattle farms ( Pardon et al., 2014 ). Therefore, it cannot be assumed that biosecurity measures and levels of implementation on veal farms are comparable to conventional dairy and beef farms. The veal-rearing system is highly similar throughout the majority of the main veal-producing countries, often with veal companies working across borders ( Sans and Fontguyon, 2009 ). Therefore, biosecurity in Belgian veal farms could, to a certain extent, be considered representative of European veal production. In Europe, before entering the veal sector, calves from dairy or beef farms are collected by salesmen and transported to a sorting center. The age when leaving the farm of origin differs between countries. In the sorting center, calves are sorted by breed, bodyweight, and conformation, and are thereafter transported to the veal farms ( Schoonmaker et al., 2002 ). White veal calves are slaughtered at a maximum age of 8 months. Most veal calf farms in Belgium are part of a veal company ( Pardon et al., 2014 ). Veal companies organize the veal farming process from the top down, with their own sorting centers, feed factories, and slaughterhouses. The companies generally own the calves, distribute feed to the farms, and impose some management requirements, while the farmer gets paid for each calf he raises on his farm. The veal sector might benefit from improved biosecurity since several researchers have suggested that improved disease prevention is possible through increased biosecurity on the farm ( Roca et al., 2015 ). Due to the high degree of commingling calves from different farms of origin, infected calves can lead to a rapid spread of disease on the veal calf farm, causing severe health and welfare issues and economic losses. As biosecurity may (partially) prevent these losses, it is considered a cost-effective method of prevention ( Van Schaik et al., 2001 ). The high level of antimicrobial use in veal-rearing is causing considerable concerns ( Pardon et al., 2012 ) as it facilitates development of antimicrobial resistance ( McEwen and Fedorka-Cray, 2002 ) as has previously been demonstrated ( Catry et al., 2016 ). As shown in other animal species, a possible way to reduce the level of antimicrobial use and its subsequent resistance selection is to improve the level of biosecurity ( Postma et al., 2016 ; Collineau et al., 2017 ). Biosecurity practices are often neglected by cattle farmers who assume that the risk of infection in their animals is low ( Nöremark et al., 2016 ). This assumption is likely not the case for veal farmers since the risk of infection is known to be high ( Pardon et al., 2011 ; Knight et al., 2013 ). Moreover, cattle farmers have indicated a lack of information regarding biosecurity ( Damiaans et al., 2018 ; Higgins et al., 2018 ). This lack can be presumed to be similar among veal farmers because comparable channels of information are available. Thus, in order to improve biosecurity on veal farms, its strengths, weaknesses, and constraints should first be identified. Therefore this study aimed to determine the main biosecurity measures in veal production and the application level of these measures in Belgian veal farms as reported or observed during a visit. 2 Material and methods 2.1 Disease selection First, a list of cattle diseases that are either endemic in Belgium or at risk of (re)emergence was developed according to the methodology previously described by Renault et al. (2018b) . An initial list of diseases was based on a literature review after a search of the PubMed database. In the list, both calf diseases and diseases of high importance in cattle, or with zoonotic potential, were included. Diseases not occurring in, or not at risk of emergence in Belgium (never reported in Europe), were removed from the initial list. Second, three different data sources were accessed to select the most important diseases from this list: 1) a combination of recently described prioritization methods applied to the literature search, including all notifiable diseases ( ANSES, 2010 ; Havelaar et al., 2010 ; Humblet et al., 2012 ; McIntyre et al., 2014 ; Ciliberti et al., 2015 ); 2) data on disease occurrences in the last three years, provided by regional animal health centers; and 3) an online survey among bovine veterinary practitioners ( Renault et al., 2018a ). 2.2 Building the questionnaire Based on the final list of diseases ( Table 1 ), a review of the literature on risk factors and biosecurity measures related to each of the diseases was performed. This review was kept as broad as possible to have a complete overview of all factors concerning biosecurity, and then cross-referenced with previous biosecurity questionnaires and a biosecurity reference work ( Dewulf and Van Immerseel, 2018 ). For this reason, a search of the PubMed database was performed with this combination of terms: "name of disease and/or pathogen," or "cattle," "risk factors" or "epidemiology" or "prevalence" or "biosecurity measures" or "control measures." The list of risk factors and biosecurity measures for each disease was integrated into an exhaustive list with all known (published) risk factors and biosecurity measures relevant for veal calves. If possible, a corresponding biosecurity measure was identified for each risk factor. Risk factors that cannot be controlled, or for which no biosecurity measure is available (e.g., birth weight, weather), as well as risk factors related to parturition or shortly thereafter (e.g., hygiene at parturition and provision of colostrum) were discarded. Though this last category is considered important, these risk factors are outside the control of the veal farmer because the animals arrive at two weeks of age. The total list of biosecurity measures is provided in Annex 1. This table also provides the number of risk factors each measure addresses, and the number of diseases for which it was cited in the literature. In Table 2 , an overview of the 12 most important biosecurity measures, and their relation to the 34 most important calf diseases is provided. Table 1 List of the 34 most important calf diseases with their respective transmission routes. Table 1 Disease Transmission Pathways Selection Criteria Direct contact Transplacental Venereal Indirect/fomite Ingestion Inhalation Vector Prioritization exercises Labresults Veterinary survey Bovine respiratory diseases (including Pasteurella spp., Mannheimia haemolytica, bovine adenovirus, …) 1 1 1 1 1 1 1 Bovine viral diarrhea 1 1 1 1 1 1 1 1 1 Infectious Bovine Rhinotracheitis(IBR) 1 1 1 1 1 1 Mycoplasma bovis 1 1 1 1 1 1 1 Salmonellosis 1 1 1 1 1 1 1 Anaplasmosis 1 1 1 1 1 Babesiosis 1 1 1 Botulism 1 1 1 1 1 1 BRSV 1 1 1 1 1 Campylobacteriosis 1 1 1 1 1 Coccidiosis 1 1 1 1 1 Cryptosporidiosis 1 1 1 1 1 Diarrhea/enteritis, neonatal (Rotavirus, coronavirus, E. coli, adenovirus, …) 1 1 1 1 1 Leptospirosis 1 1 1 1 1 1 1 Lice and ectoparasites 1 1 1 1 Q Fever/Coxiellosis 1 1 1 1 1 1 1 Schmallenberg disease 1 1 1 1 Anthrax 1 1 1 1 1 Aujeszky's Disease 1 1 1 1 1 Bluetongue 1 1 1 1 1 Bovine Spongiform Encephalopathy 1 1 1 Brucellosis 1 1 1 1 1 1 Dermatophytosis/-mycosis 1 1 1 1 E. Coli verotoxic 1 1 1 1 Enterotoxemia ( Clostridium spp) 1 1 1 1 Enzootic bovine leucosis 1 1 1 1 Foot and Mouth Disease 1 1 1 1 1 Giardiasis 1 1 1 1 Infectious Bovine Keratoconjunctivitis 1 1 1 a 1 Listeriosis 1 1 1 1 Necrobacillosis (laryngitis) 1 1 1 Rabies 1 1 1 1 1 Scabies 1 1 1 Tuberculosis (bovine) 1 1 1 a Only mechanical vector. Table 2 Twelve biosecurity measures considered most important, and their presence in literature for the 34 most important calf diseases. Table 2 Disease Biosecurity Measures References Ensure free source of origin on purchase Prevention of transmission by visitors Prevention of direct and indirect contact with animals of neighboring farms Prevention of (in)direct contact with wildlife, pets, rodents, birds and insects Proper carcass disposal Prevention of feed and water contamination Separation of infected animals Elimination of permanently infected animals Working organization and compartmentation All in/all out system of separate stables Cleaning and disinfection of stables and equipment Sanitary vacancy after cleaning Anaplasmosis 2 0 2 0 0 0 0 2 0 0 0 0 1–4 Anthrax 0 0 0 0 2 0 0 0 0 0 0 0 5,6 Aujeszky's Disease 2 2 2 0 0 1 0 0 0 2 0 0 4,7 Babesiosis 0 0 2 0 0 0 0 2 0 0 0 0 4 Bluetongue 2 0 0 0 0 0 0 2 0 2 0 0 4,7,8 Botulism 0 0 0 0 2 2 0 0 0 0 0 0 9–19 Bovine respiratory diseases 2 2 1 2 0 2 2 2 2 2 2 2 20–92 Bovine respiratory syncytial virus 1 2 2 2 0 0 1 0 1 1 0 0 21, 29, 32, 40, 58–62, 93–105 Bovine Spongiform Encephalopathy 1 0 0 0 0 2 0 2 0 0 0 0 4,7,106,107 Bovine viral diarrhea 2 2 2 2 0 0 2 2 2 2 2 2 43,58,108–119 Brucellosis 2 1 2 2 2 2 2 2 1 2 1 0 4,7,120–126 Campylobacteriosis 0 0 0 0 0 0 0 2 0 0 1 1 4,8 Coccidiosis 1 1 1 1 0 0 1 0 2 2 2 1 127-142 Cryptosporidiosis 2 2 1 1 0 0 2 2 1 1 2 2 130,132,143–158 Dermatophytosis/ -mycosis 0 0 0 0 0 0 1 0 2 0 2 2 4,159 Diarrhea/enteritis 2 2 1 2 0 0 2 2 2 2 2 1 51, 54, 59, 60, 143, 147, 149, 152, 158, 160–179 E. Coli (verotoxic) 2 2 2 1 0 1 2 0 1 1 2 1 180–190 Enterotoxemia (Clostridium spp) 0 0 0 0 0 0 0 0 2 0 0 0 191–201 Enzootic bovine leucosis 2 0 0 0 0 0 2 2 0 0 0 0 202–205 Foot and Mouth Disease 2 2 2 0 2 0 1 2 1 2 2 2 4, 206–212 Giardiasis 2 2 1 1 0 0 2 2 1 1 2 2 150,155,213 Infectious Bovine Keratoconjunctivitis 1 0 1 0 1 0 0 2 0 1 0 0 214–224 Infectious Bovine Rhinotracheitis(IBR) 2 2 2 1 0 0 2 2 2 2 2 2 43, 58, 61, 89, 225–248 Leptospirosis 1 0 2 2 0 2 0 2 0 0 1 1 4,249 Lice and ectoparasites 2 2 2 0 0 0 0 0 0 2 2 2 250–265 Listeriosis 0 1 2 2 0 2 1 0 2 0 2 2 266–290 Mycoplasma bovis 2 2 2 0 0 1 2 2 2 2 2 2 21, 26, 29, 32, 63, 66, 95, 291–308 Necrobacillosis (laryngitis) 0 0 0 0 0 0 0 0 1 0 0 0 309–327 Q Fever/Coxiellosis 2 2 0 0 0 0 1 2 2 2 2 2 4,328–330 Rabies 2 0 2 2 0 0 0 0 0 0 0 0 331–346 Salmonellosis 2 2 1 1 0 2 1 2 1 2 2 1 143,347–363 Scabies 1 1 1 0 0 0 0 0 0 1 1 1 364–378 Schmallenberg disease 0 0 0 0 0 0 0 0 0 0 0 0 8,379–383 Tuberculosis 2 1 2 2 0 0 1 2 2 1 1 0 4,384,385 "2" means the measure was mentioned as such in literature for the disease, "1" means the relevance of the measure could be deduced from context, while "0" means that the risk factor was not found in literature for that disease. The numbered references in the last column are provided in Annex 3. Based on the list of biosecurity measures and complemented with content and experience from previous questionnaires concerning biosecurity in pig and broiler production ( www.biocheck.ugent.be ), a questionnaire assessing the implementation of biosecurity on veal farms was created. In addition to questions about the implementation of biosecurity, questions about motivators or hurdles when implementing biosecurity measures were also asked, as well as general attitudes and knowledge regarding disease prevention and biosecurity. A draft questionnaire was tested on two veal farms. The final questionnaire consisted of 40 open-ended questions and a maximum of 114 multiple choice questions (Annex 2) and is available upon request by readers. Part of the multiple choice questions, 57 in total, were arranged into 3 tables to facilitate data collection. 2.3 Visiting farms A random sample of 60 farms from all Belgian veal farms (241 farms in 2016) was obtained from the Flemish Animal Health Service (Diergezondheidszorg Vlaanderen). A computer-generated random number (Excel®, Microsoft) was assigned to each of the 60 farms, and the numbers were sorted from low to high. Selected farmers were contacted, starting with the farm assigned to number 1, and were asked to collaborate until 20 farmers willing to cooperate were selected. The sample size was limited to 20 farms due to limited time and resources, as it was part of a research project to study and quantify biosecurity on different types of cattle farms. A total of 28 farmers were contacted to obtain a sample from 20 veal farms. Of the 8 farmers not willing to participate, 1 was no longer active, 3 cited a lack of time, 3 wished to receive no visitors to keep a closed farm, and 1 farmer did not give a reason. The study farms were visited between November 2016 and February 2017, and face-to-face interviews were conducted by the first author in Dutch, the native tongue of both farmers and interviewer. The visit consisted of a tour of the farm and the interview itself. Participants were informed beforehand of the procedure. Written informed consent was obtained from the participating farmers. 2.4 Data processing After the survey, all data was entered into a Limesurvey-form and exported to the statistical package IBM® SPSS® Statistics 25.0. The results were analyzed using basic descriptive analysis. The frequency of each answer and, when possible, the mean, median, standard deviation (SD), quartiles, minimum, and maximum were calculated. A biosecurity scoring system was created with binary variables, where 1 indicated the presence of a biosecurity measure and 0 indicated the absence. These scores were added up to generate a score on a scale of 0 to 10 for each biosecurity category, with a total of seven categories describing measures concerning animal movements, visitors, contact with other animals, disease management, compartmentation, cleaning and disinfection, and calf management. Next, a categorical principal components analysis (CATPCA; SPSS 25.0) and clustering analysis, as previously described by Van Steenwinkel et al. (2011) and Sarrazin et al. (2014) , were performed to combine the information originating from multiple variables. Based on this information, the researchers assessed whether the veal companies influenced biosecurity levels. To this end, the categories were given an ordinal measurement scale in the CATPCA analysis. The veal companies were included as a supplementary nominal measurement to explore their relationship with the biosecurity levels. For the analysis, 3 major and a group of minor veal companies, as described in Pardon et al. (2014) , were randomly assigned a number from 1 to 4. The object scores, following the CATPCA analysis, were included in a k-means cluster analysis (KMCA; SPSS 25.0) to compare the clusters to the veal companies. 2.1 Disease selection First, a list of cattle diseases that are either endemic in Belgium or at risk of (re)emergence was developed according to the methodology previously described by Renault et al. (2018b) . An initial list of diseases was based on a literature review after a search of the PubMed database. In the list, both calf diseases and diseases of high importance in cattle, or with zoonotic potential, were included. Diseases not occurring in, or not at risk of emergence in Belgium (never reported in Europe), were removed from the initial list. Second, three different data sources were accessed to select the most important diseases from this list: 1) a combination of recently described prioritization methods applied to the literature search, including all notifiable diseases ( ANSES, 2010 ; Havelaar et al., 2010 ; Humblet et al., 2012 ; McIntyre et al., 2014 ; Ciliberti et al., 2015 ); 2) data on disease occurrences in the last three years, provided by regional animal health centers; and 3) an online survey among bovine veterinary practitioners ( Renault et al., 2018a ). 2.2 Building the questionnaire Based on the final list of diseases ( Table 1 ), a review of the literature on risk factors and biosecurity measures related to each of the diseases was performed. This review was kept as broad as possible to have a complete overview of all factors concerning biosecurity, and then cross-referenced with previous biosecurity questionnaires and a biosecurity reference work ( Dewulf and Van Immerseel, 2018 ). For this reason, a search of the PubMed database was performed with this combination of terms: "name of disease and/or pathogen," or "cattle," "risk factors" or "epidemiology" or "prevalence" or "biosecurity measures" or "control measures." The list of risk factors and biosecurity measures for each disease was integrated into an exhaustive list with all known (published) risk factors and biosecurity measures relevant for veal calves. If possible, a corresponding biosecurity measure was identified for each risk factor. Risk factors that cannot be controlled, or for which no biosecurity measure is available (e.g., birth weight, weather), as well as risk factors related to parturition or shortly thereafter (e.g., hygiene at parturition and provision of colostrum) were discarded. Though this last category is considered important, these risk factors are outside the control of the veal farmer because the animals arrive at two weeks of age. The total list of biosecurity measures is provided in Annex 1. This table also provides the number of risk factors each measure addresses, and the number of diseases for which it was cited in the literature. In Table 2 , an overview of the 12 most important biosecurity measures, and their relation to the 34 most important calf diseases is provided. Table 1 List of the 34 most important calf diseases with their respective transmission routes. Table 1 Disease Transmission Pathways Selection Criteria Direct contact Transplacental Venereal Indirect/fomite Ingestion Inhalation Vector Prioritization exercises Labresults Veterinary survey Bovine respiratory diseases (including Pasteurella spp., Mannheimia haemolytica, bovine adenovirus, …) 1 1 1 1 1 1 1 Bovine viral diarrhea 1 1 1 1 1 1 1 1 1 Infectious Bovine Rhinotracheitis(IBR) 1 1 1 1 1 1 Mycoplasma bovis 1 1 1 1 1 1 1 Salmonellosis 1 1 1 1 1 1 1 Anaplasmosis 1 1 1 1 1 Babesiosis 1 1 1 Botulism 1 1 1 1 1 1 BRSV 1 1 1 1 1 Campylobacteriosis 1 1 1 1 1 Coccidiosis 1 1 1 1 1 Cryptosporidiosis 1 1 1 1 1 Diarrhea/enteritis, neonatal (Rotavirus, coronavirus, E. coli, adenovirus, …) 1 1 1 1 1 Leptospirosis 1 1 1 1 1 1 1 Lice and ectoparasites 1 1 1 1 Q Fever/Coxiellosis 1 1 1 1 1 1 1 Schmallenberg disease 1 1 1 1 Anthrax 1 1 1 1 1 Aujeszky's Disease 1 1 1 1 1 Bluetongue 1 1 1 1 1 Bovine Spongiform Encephalopathy 1 1 1 Brucellosis 1 1 1 1 1 1 Dermatophytosis/-mycosis 1 1 1 1 E. Coli verotoxic 1 1 1 1 Enterotoxemia ( Clostridium spp) 1 1 1 1 Enzootic bovine leucosis 1 1 1 1 Foot and Mouth Disease 1 1 1 1 1 Giardiasis 1 1 1 1 Infectious Bovine Keratoconjunctivitis 1 1 1 a 1 Listeriosis 1 1 1 1 Necrobacillosis (laryngitis) 1 1 1 Rabies 1 1 1 1 1 Scabies 1 1 1 Tuberculosis (bovine) 1 1 1 a Only mechanical vector. Table 2 Twelve biosecurity measures considered most important, and their presence in literature for the 34 most important calf diseases. Table 2 Disease Biosecurity Measures References Ensure free source of origin on purchase Prevention of transmission by visitors Prevention of direct and indirect contact with animals of neighboring farms Prevention of (in)direct contact with wildlife, pets, rodents, birds and insects Proper carcass disposal Prevention of feed and water contamination Separation of infected animals Elimination of permanently infected animals Working organization and compartmentation All in/all out system of separate stables Cleaning and disinfection of stables and equipment Sanitary vacancy after cleaning Anaplasmosis 2 0 2 0 0 0 0 2 0 0 0 0 1–4 Anthrax 0 0 0 0 2 0 0 0 0 0 0 0 5,6 Aujeszky's Disease 2 2 2 0 0 1 0 0 0 2 0 0 4,7 Babesiosis 0 0 2 0 0 0 0 2 0 0 0 0 4 Bluetongue 2 0 0 0 0 0 0 2 0 2 0 0 4,7,8 Botulism 0 0 0 0 2 2 0 0 0 0 0 0 9–19 Bovine respiratory diseases 2 2 1 2 0 2 2 2 2 2 2 2 20–92 Bovine respiratory syncytial virus 1 2 2 2 0 0 1 0 1 1 0 0 21, 29, 32, 40, 58–62, 93–105 Bovine Spongiform Encephalopathy 1 0 0 0 0 2 0 2 0 0 0 0 4,7,106,107 Bovine viral diarrhea 2 2 2 2 0 0 2 2 2 2 2 2 43,58,108–119 Brucellosis 2 1 2 2 2 2 2 2 1 2 1 0 4,7,120–126 Campylobacteriosis 0 0 0 0 0 0 0 2 0 0 1 1 4,8 Coccidiosis 1 1 1 1 0 0 1 0 2 2 2 1 127-142 Cryptosporidiosis 2 2 1 1 0 0 2 2 1 1 2 2 130,132,143–158 Dermatophytosis/ -mycosis 0 0 0 0 0 0 1 0 2 0 2 2 4,159 Diarrhea/enteritis 2 2 1 2 0 0 2 2 2 2 2 1 51, 54, 59, 60, 143, 147, 149, 152, 158, 160–179 E. Coli (verotoxic) 2 2 2 1 0 1 2 0 1 1 2 1 180–190 Enterotoxemia (Clostridium spp) 0 0 0 0 0 0 0 0 2 0 0 0 191–201 Enzootic bovine leucosis 2 0 0 0 0 0 2 2 0 0 0 0 202–205 Foot and Mouth Disease 2 2 2 0 2 0 1 2 1 2 2 2 4, 206–212 Giardiasis 2 2 1 1 0 0 2 2 1 1 2 2 150,155,213 Infectious Bovine Keratoconjunctivitis 1 0 1 0 1 0 0 2 0 1 0 0 214–224 Infectious Bovine Rhinotracheitis(IBR) 2 2 2 1 0 0 2 2 2 2 2 2 43, 58, 61, 89, 225–248 Leptospirosis 1 0 2 2 0 2 0 2 0 0 1 1 4,249 Lice and ectoparasites 2 2 2 0 0 0 0 0 0 2 2 2 250–265 Listeriosis 0 1 2 2 0 2 1 0 2 0 2 2 266–290 Mycoplasma bovis 2 2 2 0 0 1 2 2 2 2 2 2 21, 26, 29, 32, 63, 66, 95, 291–308 Necrobacillosis (laryngitis) 0 0 0 0 0 0 0 0 1 0 0 0 309–327 Q Fever/Coxiellosis 2 2 0 0 0 0 1 2 2 2 2 2 4,328–330 Rabies 2 0 2 2 0 0 0 0 0 0 0 0 331–346 Salmonellosis 2 2 1 1 0 2 1 2 1 2 2 1 143,347–363 Scabies 1 1 1 0 0 0 0 0 0 1 1 1 364–378 Schmallenberg disease 0 0 0 0 0 0 0 0 0 0 0 0 8,379–383 Tuberculosis 2 1 2 2 0 0 1 2 2 1 1 0 4,384,385 "2" means the measure was mentioned as such in literature for the disease, "1" means the relevance of the measure could be deduced from context, while "0" means that the risk factor was not found in literature for that disease. The numbered references in the last column are provided in Annex 3. Based on the list of biosecurity measures and complemented with content and experience from previous questionnaires concerning biosecurity in pig and broiler production ( www.biocheck.ugent.be ), a questionnaire assessing the implementation of biosecurity on veal farms was created. In addition to questions about the implementation of biosecurity, questions about motivators or hurdles when implementing biosecurity measures were also asked, as well as general attitudes and knowledge regarding disease prevention and biosecurity. A draft questionnaire was tested on two veal farms. The final questionnaire consisted of 40 open-ended questions and a maximum of 114 multiple choice questions (Annex 2) and is available upon request by readers. Part of the multiple choice questions, 57 in total, were arranged into 3 tables to facilitate data collection. 2.3 Visiting farms A random sample of 60 farms from all Belgian veal farms (241 farms in 2016) was obtained from the Flemish Animal Health Service (Diergezondheidszorg Vlaanderen). A computer-generated random number (Excel®, Microsoft) was assigned to each of the 60 farms, and the numbers were sorted from low to high. Selected farmers were contacted, starting with the farm assigned to number 1, and were asked to collaborate until 20 farmers willing to cooperate were selected. The sample size was limited to 20 farms due to limited time and resources, as it was part of a research project to study and quantify biosecurity on different types of cattle farms. A total of 28 farmers were contacted to obtain a sample from 20 veal farms. Of the 8 farmers not willing to participate, 1 was no longer active, 3 cited a lack of time, 3 wished to receive no visitors to keep a closed farm, and 1 farmer did not give a reason. The study farms were visited between November 2016 and February 2017, and face-to-face interviews were conducted by the first author in Dutch, the native tongue of both farmers and interviewer. The visit consisted of a tour of the farm and the interview itself. Participants were informed beforehand of the procedure. Written informed consent was obtained from the participating farmers. 2.4 Data processing After the survey, all data was entered into a Limesurvey-form and exported to the statistical package IBM® SPSS® Statistics 25.0. The results were analyzed using basic descriptive analysis. The frequency of each answer and, when possible, the mean, median, standard deviation (SD), quartiles, minimum, and maximum were calculated. A biosecurity scoring system was created with binary variables, where 1 indicated the presence of a biosecurity measure and 0 indicated the absence. These scores were added up to generate a score on a scale of 0 to 10 for each biosecurity category, with a total of seven categories describing measures concerning animal movements, visitors, contact with other animals, disease management, compartmentation, cleaning and disinfection, and calf management. Next, a categorical principal components analysis (CATPCA; SPSS 25.0) and clustering analysis, as previously described by Van Steenwinkel et al. (2011) and Sarrazin et al. (2014) , were performed to combine the information originating from multiple variables. Based on this information, the researchers assessed whether the veal companies influenced biosecurity levels. To this end, the categories were given an ordinal measurement scale in the CATPCA analysis. The veal companies were included as a supplementary nominal measurement to explore their relationship with the biosecurity levels. For the analysis, 3 major and a group of minor veal companies, as described in Pardon et al. (2014) , were randomly assigned a number from 1 to 4. The object scores, following the CATPCA analysis, were included in a k-means cluster analysis (KMCA; SPSS 25.0) to compare the clusters to the veal companies. 3 Results 3.1 Literature research After selection, as described in the Material & Methods section, the final list contained the 34 most important calf diseases ( Table 1 ). A total of 385 articles related to these diseases were reviewed to list all risk factors and biosecurity measures as input for the questionnaire. The full list of biosecurity measures can be found in Annex 1. One of the most frequently mentioned risk factors was animal movement. Animal movement includes the purchase of animals and all associated biosecurity measures, such as ensuring that the farm of origin is free from infection, limiting the number of source farms, and collecting information on animal and farm of origin as well as testing the animals after purchase and quarantining new animals. These measures were described as risk factors for multiple diseases and were considered important for the questionnaire, especially since the veal sector has its own system for purchase. Another frequently mentioned group of measures is related to visitors. The use of farm-specific clothing and footwear before entering the stables is often mentioned as well as the use of a disinfection footbath and hand-washing facilities before and between the animals' lodgings. Measures concerning management of diseased animals, such as quick recognition, good assessment and correct treatment of disease, and elimination of disease carriers were also frequently cited. Finally, all measures related to cleaning and disinfection of housing and equipment after each use were considered important, according to the literature. 3.2 Farm characteristics and attitude toward biosecurity The majority of the participating farms ( Fig. 1 ) were located in the province Antwerp (n = 13), which corresponds to the area with the highest density of veal farms in Belgium. The other participants were located in West-Flanders (n = 4), Limburg (n = 2) and East-Flanders (n = 1). The maximum number of calves present on the farm ranged from 212 to 1700 calves. Other farm characteristics can be found in Annex 4. Fig. 1 Map of all Belgian veal farms. Visited farms are marked with a yellow arrow, while non-selected farms are represented by a blue arrow. Fig. 1 Sixteen farms were part of three veal companies coordinating the highest number of Belgian veal farms (veal company 1: 6 farms; veal company 2: 6 farms; veal company 3: 4 farms), and four farms belonged to three smaller veal companies. Of 20 farmers, only 4 (20%) could give a partial definition of biosecurity, mainly focusing on external biosecurity. Other farmers had no idea (n = 4), defined it vaguely as the reduction of antimicrobial usage (n = 6; 30%), improvement of food safety (n = 3; 15%), or organic production (n = 3; 15%). After explaining the term, 19 farmers (95%) considered biosecurity to be important. All of them considered disease prevention to be cheaper than treatment. Only slightly more than half (11/20; 55%) of the farmers could list five or more biosecurity measures they implemented on their farm, and 19 participants (95%) considered the measures as implemented sufficiently to prevent disease transmission. Seven farmers (35%) preferred that the veterinarian provide them with information on biosecurity or disease prevention. Six farmers (30%) considered professional organizations, such as the animal healthcare association or the veal calf producers association, their preferred source of information. Nine farmers (45%) did not consult any information sources because they believed no such information was available. Two farmers (10%) mentioned the role of the veal company. No farmers seemed to gain information from the internet or from magazines for agricultural professionals. 3.3 Implementation of external biosecurity measures 3.3.1 Measures concerning animal movements Inherent to the production system in the veal sector, all farms bought calves every 7.5 to 8 months. There was a large difference in the time required to fill the stables for one cycle, ranging from 2 to 52 days. On average, a stable was filled in 11.4 days (SD: 9.6). During the filling of the stable, all farms received animals on three fixed delivery days per week. On three farms, the age difference between calves was larger than two weeks due to a large spread of calves entering the stables. All calves were collected by cattle salesmen at the farm of origin, moved to a sorting center, and delivered by the veal company to the veal calf farms ( Table 3 ). Table 3 Implementation of external biosecurity measures. Column one contains the biosecurity measure, the second column contains the maximum number of farms that can adhere to the measure, while the third to fifth columns contain the adherence to the measure. Table 3 Biosecurity measure concerning animal movements N Yes Sometimes No Purchasing animals from the same source 20 0 20 Possible contact with other calves before arrival 20 20 0 Check of sanitary status and health management of farms of origin 20 4 2 14 Separation of calves in high/low risk groups based on risk classification 20 12 8 Testing for specific diseases when purchasing 20 0 20 Calves leaving and re-entering the farm 20 0 20 Applying quarantine 20 0 20 Access of transport vehicle to calves' residence prohibited 20 20 0 Transport empty before entering the farm (for loading animals) 20 15 5 Transport clean and disinfected before entering the farm (if empty) 15 14 1 Only the calves on the transport that are supposed to be delivered 20 9 11 Biosecurity measure concerning animal contact N Yes Sometimes No Rodent control program 20 13 7 Use of insect repellants on the animals 20 1 19 Use of insect repellants in the environment 20 14 6 Use of insect traps/nets 20 3 17 Handling of manure to limit insects 20 1 19 No contact possible between calves and other cattle 20 19 1 No contact possible between calves and wild ruminants or boar 20 20 0 No access to the stable by cats 20 9 11 No access to the stable by dogs 20 16 4 No access to the stable by birds 20 12 8 No access to the stable by rodents 20 0 20 No (frequent) contact of employees with animals from other farms 20 14 6 Carcass storage space with solid, easy to clean floors 20 17 3 Carcass storage space protected from pets and vermin 17 14 3 Carcass storage space cleaned after use 17 10 7 Carcass storage cleaned and disinfected after use 17 5 12 Carcass removal possible without entering the farm 20 9 11 Carcass manipulation with gloves (or hands cleaned and disinfected afterwards) 20 20 0 No manure from other farms spread within 500 m of the farm 20 5 15 No access to the food storage by cats 20 8 12 No access to the food storage by dogs 20 15 5 No access to the food storage by birds 20 13 7 No access to the food storage by rodents 20 3 17 Feeding utensils cleaned after use 20 8 12 Feeding utensils cleaned and disinfected after use 20 2 18 No use of feeding utensils for manure 20 17 3 Testing of microbial quality of animals' drinking water at least once a year 20 16 4 No sharing of equipment with other farms 20 20 0 Six farmers indicated that they paid attention to sanitary status and health management, which refers to the presence of specific diseases on the farm of origin ( Table 3 ). This procedure was based on previous experiences with the farm of origin, in consultation with the veal company. The remaining participants argued that the veal company decides which calves are sent to them, and four farmers emphasized their trust in the company to cover this issue. One farmer believed reviewing the health status of all new calves was unfeasible. A shared opinion was that it is virtually impossible to check all farms of origin, since their number is almost equal to the number of calves. This number is confirmed, since the average degree of commingling for the 20 farms was calculated to be 1.24 (SD = 0.16), meaning that, on average, 124 calves originated from 100 farms. As such, a farm with 500 calves will harbor animals from over 400 different origins. Upon arrival, calves were divided into high and low risk groups based on visual appraisals by 12 of the 20 farmers (60%). On these farms, weaker calves were housed together and received more attention. Half of the farmers (50%) felt that taking blood samples from all the animals to test for disease is neither feasible nor affordable. Other reasons for not testing upon arrival included that there is no obligation to the government (n = 3; 15%) or to the veal company (n = 3; 15%), or that it would provide little additional information (n = 4; 20%). As the stables are filled in a short period, the farmers mostly felt quarantine was neither feasible nor necessary (n = 19; 95%). Before animals are transported to slaughter, transport vehicles are generally empty, cleaned and disinfected prior to picking up animals ready for slaughter, according to the majority of the farmers (n = 15; 75%). However, upon delivery of animals to the farm, on 11 farms (55%), not all animals were unloaded, indicating that trucks were not empty and so were not cleaned between farms. 3.3.2 Measures relating to visitors In 13 farms (65%), access to the stables was controlled by a closed gate and a requirement for visitors to announce themselves before entering. The remaining 7 farmers (35%) believed this was not feasible. The same farmers did not require visitors to register, either because it was not considered important (n = 3; 43%), regularly forgotten (n = 2; 29%), unknown (n = 1; 14%), or not mandatory (n = 1; 14%). Measures regarding farm-specific clothing and boots were not well implemented by most visitors ( Table 4 ), despite farm-specific clothing and boots being available in a high number of farms ( Table 5 ). Other measures for visitors were rarely implemented. Disinfection footbaths were generally present but were either dirty, empty, or ignored. Footbaths were not used by most farmers and staff, mainly because they believed it was not important on their own farm. Very few participants always washed their hands or wore gloves before entering the stables. Those not washing their hands assumed it was not important. On the few farms where a hygiene lock (a room to change into farm-specific boots and clothing before a visitor can enter the stables) was present, it was consistently used by farm personnel and visitors. For one-third of the farmers (n = 6; 35%) that did not have a hygiene lock, the practice was unknown. Table 4 Implemented biosecurity measures by different visitors before entering the stables. Column one contains the biosecurity measure, the second column contains the maximum number of farms that can adhere to the measure, while the third to fifth columns contain the number of visitors complying to the biosecurity measure. Table 4 Biosecurity measure related to visitors N Farmer/Employees Veterinarian Advisor Restricted access to the stables 20 / 14 14 Wearing farm specific clothes before entrance 20 16 6 4 Wearing farm specific boots before entrance 20 17 8 6 Using a hygiene lock 20 3 3 2 Washing and disinfecting hands before entrance 20 2 4 4 Wearing gloves before entrance 20 1 3 1 Using disinfection footbath before entrance 20 4 5 5 Table 5 Implementation of internal biosecurity measures. Column one contains the biosecurity measure, the second column contains the maximum number of farms that can adhere to the measure, while the third to fifth columns contain the adherence to the measure. Table 5 Biosecurity measure concerning disease management N Yes Sometimes No Protocols for vaccination 20 1 19 Preventive measures for endoparasites 20 14 2 6 Preventive measures for ectoparasites 20 20 0 Isolation of sick calves 20 0 3 17 Hospital pen placed physically separated from the other calves 3 1 2 Specific equipment available for the hospital pen 3 3 0 Specific equipment for the hospital pen cleaned after use 3 1 2 Feed and water troughs cleaned after use 3 1 2 Handling sick animals in hospital pen last 3 1 2 Registration of animal health data 20 8 12 Elimination of carriers of infection 20 7 5 8 Segregation of carriers of infection (if no elimination) 13 5 5 3 Biosecurity measure concerning compartmentation N Yes Sometimes No Multiple age groups present on farm 20 9 11 Separation of age groups 9 9 0 No contact possible between age groups 9 8 1 Working from young to old 9 8 1 Specific equipment available for each age group/stable 20 4 16 Specific equipment per age group/stable cleaned after use 4 0 4 Specific equipment recognizable per age group/stable 4 0 4 Farm specific clothing available before entering the farm 20 14 6 Farm specific boots available before entering the farm 20 17 3 Hygiene lock before entering the farm 20 3 17 Hygiene lock only entrance to the stable 3 0 3 Clean and dirty area of the hygiene lock designated and physically separated 3 0 3 Gloves available before entering the farm 20 1 19 Disinfection footbath available and ready for use before entering the farm 20 5 15 Hand washing facilities available and ready for use before entering the farm 20 3 17 Biosecurity measure concerning cleaning and disinfection N Yes Sometimes No Sanitary vacancy of the calf stables after removal of animals 20 17 2 1 Calf stables cleaned after removal of animals 17 17 0 Calf stables cleaned and disinfected after removal of animals 17 11 6 Calf stables dry before next use 17 13 4 All-in, all-out system in the calf stables 17 15 2 Hospital pen available on farm 20 7 13 No direct contact possible in the hospital pen 7 3 4 No indirect contact possible in the hospital pen 7 1 6 Sanitary vacancy of the hospital pen after removal of animals 7 1 6 Hospital pen cleaned after removal of animals 7 2 5 Hospital pen cleaned and disinfected after removal of animals 7 2 5 Hospital pen dry before next use 7 2 5 All-in, all-out system in the hospital pen 7 4 3 Biosecurity measure concerning calf management N Yes Sometimes No Age groups <2 weeks age difference 20 17 3 Draught-free hutches 20 17 3 Slatted floors 20 20 0 Regular cleaning of floors during production rounds 20 0 20 Clean and dry bedding 20 8 12 Always the same bucket (for milk) for a calf 20 20 0 Buckets for milk cleaned after each use 20 3 17 Automated climate control system (temperature, humidity) 20 14 6 Air flow in the stable from young to old 20 20 0 3.3.3 Measures concerning direct or indirect contact with other animals or insects A standard rodent control program usually consisted of the implementation of rodenticides. Farmers without a rodent control program deemed it not important or only took measures when visibly infested ( Table 3 ). All farmers that implemented measures for insect control (n = 14; 70%) treated the environment, sometimes combined with additional measures ( Table 3 ). These measures were mostly intended to control fly populations during summer. The use of a well-equipped carcass storage space was often implemented (85%; n = 17), although few (25%; n = 5) regularly cleaned and disinfected the carcass storage area. Removal of carcasses by the rendering company without entering the premises was considered very important, although this was only possible on 11 farms (55%). 3.4 Implementation of internal biosecurity measures 3.4.1 Measures concerning disease management More than half of the farmers (n = 13; 65%) believed vaccination was not important or too expensive because of the short duration of a production cycle and because most vaccines can only be administered at a certain age ( Table 5 ). According to these farmers, most disease outbreaks are observed during the first weeks after introduction, a moment when vaccines are considered not yet effective. Some farmers also mentioned that since the veal companies own the calves, the companies should decide whether to vaccinate. Measures for ectoparasites consisted of preventive treatments, mainly to avoid outbreaks of scabies. Specific treatment for endoparasites was administered only curatively. Seven of twenty (35%) farmers thought it was not feasible to isolate sick animals and five (25%) farmers applied partial isolation, where the animals were not separated from the other animals (direct contact possible) ( Table 5 ). Although a hospital pen was present on seven farms (35%), only three farmers (43%) indicated that they sometimes isolated sick animals when they were lame or unable to function in the group (e.g., unable to eat, drink, or stand up). Only two out of seven hospital pens were cleaned, disinfected and dried before new animals entered, and an "all-in, all-out" system was used in four hospital pens. Only one farmer implemented all these measures and had a fully, physically separated hospital pen. The farmers that did not take these measures declared them infeasible because their hospital pen was located inside the regular stables, making thorough cleaning unfeasible. For five participants (25%), elimination or segregation of a carrier of infection depended on the age or clinical status of the animal. An older animal would often go to slaughter while younger animals would be separated. 3.4.2 Measures concerning compartmentation On the nine farms with multiple age groups, eight farmers performed work from old to young, contrary to established wisdom ( Table 5 ). On 16 farms (80%), equipment, such as wheelbarrows and feeding utensils, were moved between compartments (same age group) without cleaning or disinfection. None of the farms used compartment-specific measures, such as changing clothes and footwear or washing hands between different compartments or age groups. Within the compartment, calves were sorted by drinking speed for economic reasons, since the difference between the animals would impair the growth of slower animals. Between compartments, animals were only moved to segregate carriers of infection. Only one farm (5%) could not prevent direct contact with another age group due to the structure of the stable. In two farms, the "all-in, all-out" system was not always well applied, i.e., young calves entered the stables while (some) older animals were still present, resulting in possible contact between the age groups. The calf stables were empty after each production cycle on the other 17 farms (85%). The duration of the sanitary vacancy, often also referred to as downtime, a period between production cycles where the stable is not used, was on average 9.8 days (SD = 4.1; range 3–15 days). 3.4.3 Measures concerning cleaning and disinfection All farmers who always applied a sanitary vacancy (n = 17; 85%), also cleaned their stables during the vacancy. However, only 11 out of 17 farmers also disinfected them. Pipelines used for milk were cleaned once or twice a week. Water and feed troughs were rinsed with water on a daily (n = 5; 25%), weekly (n = 4; 20%), or monthly (n = 1; 5%) basis, or once per production cycle (n = 8; 40%). Two farmers (10%) never cleaned the feed troughs. All farmers used reusable needles to inject the animals. 3.4.4 Measures concerning calf management In general, calves were housed in individual boxes with both visual and physical contact during the first six weeks. Calves were then sorted by drinking speed within the compartment. Poorly growing calves were isolated in one compartment with a different diet. As one compartment only contained animals of the same age group, air flow within the compartment was considered irrelevant concerning disease spread from younger to older animals ( Table 5 ). 3.5 CATPCA and KMCA The two-dimensional solution of the CATPCA explained 69.7% of the variance of the seven biosecurity categories in the 20 herds ( Fig. 2 ). The percentage accounted for was 41.7% for the first dimension, and 28.0% for the second dimension. The vectorial component loadings represent the contributions of each category to the dimensions, while the different categories of the nominal variable "veal companies" are represented by their centroid coordinates. The vectors appear in the upper and lower right quadrant. The projection of the vector for contact with other animals has the largest contribution to the first dimension (x-axis), followed by the vector for cleaning and disinfection. The vector for compartmentation, which has the lowest contribution for dimension 1, has the largest contribution to the second dimension (y-axis). Veal companies 3 and 4, whose centroids are located in the directions of the vectors, have, on average, the highest biosecurity scores. For veal company 3, this result is mainly related to a higher score for compartmentation and measures for visitors, while these farms score lower on disease management and cleaning and disinfection. In the farms of veal company 4, the opposite applies. On average, the farms of veal company 2 have the lowest biosecurity score. However, the overall differences in biosecurity between veal companies are limited (centroids close to the center). Fig. 2 Triplot of component loadings (the position of the original variables in the two-dimensional space, represented by vectors), multiple nominal category points (veal companies) and objects (individual farms) labeled by the clusters, resulting from the categorical principal component analysis and K-means clustering analysis. The vector of a variable points in the direction of the highest category of the variable, indicating in this case a higher level in biosecurity. The veal companies are located close to the center of the plot, meaning no distinction can be made between the veal companies. The first and second dimension distinguish between the different clusters. The green circles with number 1–4 represent the individual farms part of cluster 1–4. The first cluster has on average the lowest biosecurity, while the second and third cluster tend to have the highest scores. The fourth cluster is located in the center. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 2 Based on the KMCA, four clusters were identified ( Fig. 2 ). The first cluster contains the highest number of farms (13) that scored lowest for biosecurity overall. Clusters 2 and 3 scored high for biosecurity. The farms in cluster 2 scored, on average, higher for disease management and cleaning and disinfection, while farms in cluster 3 score higher for compartmentation and visitors. Though this seems similar to the results of veal companies 3 and 4, the clusters consist of farms of multiple veal companies. Overall no clear veal company effect was observed in the clusters. 3.1 Literature research After selection, as described in the Material & Methods section, the final list contained the 34 most important calf diseases ( Table 1 ). A total of 385 articles related to these diseases were reviewed to list all risk factors and biosecurity measures as input for the questionnaire. The full list of biosecurity measures can be found in Annex 1. One of the most frequently mentioned risk factors was animal movement. Animal movement includes the purchase of animals and all associated biosecurity measures, such as ensuring that the farm of origin is free from infection, limiting the number of source farms, and collecting information on animal and farm of origin as well as testing the animals after purchase and quarantining new animals. These measures were described as risk factors for multiple diseases and were considered important for the questionnaire, especially since the veal sector has its own system for purchase. Another frequently mentioned group of measures is related to visitors. The use of farm-specific clothing and footwear before entering the stables is often mentioned as well as the use of a disinfection footbath and hand-washing facilities before and between the animals' lodgings. Measures concerning management of diseased animals, such as quick recognition, good assessment and correct treatment of disease, and elimination of disease carriers were also frequently cited. Finally, all measures related to cleaning and disinfection of housing and equipment after each use were considered important, according to the literature. 3.2 Farm characteristics and attitude toward biosecurity The majority of the participating farms ( Fig. 1 ) were located in the province Antwerp (n = 13), which corresponds to the area with the highest density of veal farms in Belgium. The other participants were located in West-Flanders (n = 4), Limburg (n = 2) and East-Flanders (n = 1). The maximum number of calves present on the farm ranged from 212 to 1700 calves. Other farm characteristics can be found in Annex 4. Fig. 1 Map of all Belgian veal farms. Visited farms are marked with a yellow arrow, while non-selected farms are represented by a blue arrow. Fig. 1 Sixteen farms were part of three veal companies coordinating the highest number of Belgian veal farms (veal company 1: 6 farms; veal company 2: 6 farms; veal company 3: 4 farms), and four farms belonged to three smaller veal companies. Of 20 farmers, only 4 (20%) could give a partial definition of biosecurity, mainly focusing on external biosecurity. Other farmers had no idea (n = 4), defined it vaguely as the reduction of antimicrobial usage (n = 6; 30%), improvement of food safety (n = 3; 15%), or organic production (n = 3; 15%). After explaining the term, 19 farmers (95%) considered biosecurity to be important. All of them considered disease prevention to be cheaper than treatment. Only slightly more than half (11/20; 55%) of the farmers could list five or more biosecurity measures they implemented on their farm, and 19 participants (95%) considered the measures as implemented sufficiently to prevent disease transmission. Seven farmers (35%) preferred that the veterinarian provide them with information on biosecurity or disease prevention. Six farmers (30%) considered professional organizations, such as the animal healthcare association or the veal calf producers association, their preferred source of information. Nine farmers (45%) did not consult any information sources because they believed no such information was available. Two farmers (10%) mentioned the role of the veal company. No farmers seemed to gain information from the internet or from magazines for agricultural professionals. 3.3 Implementation of external biosecurity measures 3.3.1 Measures concerning animal movements Inherent to the production system in the veal sector, all farms bought calves every 7.5 to 8 months. There was a large difference in the time required to fill the stables for one cycle, ranging from 2 to 52 days. On average, a stable was filled in 11.4 days (SD: 9.6). During the filling of the stable, all farms received animals on three fixed delivery days per week. On three farms, the age difference between calves was larger than two weeks due to a large spread of calves entering the stables. All calves were collected by cattle salesmen at the farm of origin, moved to a sorting center, and delivered by the veal company to the veal calf farms ( Table 3 ). Table 3 Implementation of external biosecurity measures. Column one contains the biosecurity measure, the second column contains the maximum number of farms that can adhere to the measure, while the third to fifth columns contain the adherence to the measure. Table 3 Biosecurity measure concerning animal movements N Yes Sometimes No Purchasing animals from the same source 20 0 20 Possible contact with other calves before arrival 20 20 0 Check of sanitary status and health management of farms of origin 20 4 2 14 Separation of calves in high/low risk groups based on risk classification 20 12 8 Testing for specific diseases when purchasing 20 0 20 Calves leaving and re-entering the farm 20 0 20 Applying quarantine 20 0 20 Access of transport vehicle to calves' residence prohibited 20 20 0 Transport empty before entering the farm (for loading animals) 20 15 5 Transport clean and disinfected before entering the farm (if empty) 15 14 1 Only the calves on the transport that are supposed to be delivered 20 9 11 Biosecurity measure concerning animal contact N Yes Sometimes No Rodent control program 20 13 7 Use of insect repellants on the animals 20 1 19 Use of insect repellants in the environment 20 14 6 Use of insect traps/nets 20 3 17 Handling of manure to limit insects 20 1 19 No contact possible between calves and other cattle 20 19 1 No contact possible between calves and wild ruminants or boar 20 20 0 No access to the stable by cats 20 9 11 No access to the stable by dogs 20 16 4 No access to the stable by birds 20 12 8 No access to the stable by rodents 20 0 20 No (frequent) contact of employees with animals from other farms 20 14 6 Carcass storage space with solid, easy to clean floors 20 17 3 Carcass storage space protected from pets and vermin 17 14 3 Carcass storage space cleaned after use 17 10 7 Carcass storage cleaned and disinfected after use 17 5 12 Carcass removal possible without entering the farm 20 9 11 Carcass manipulation with gloves (or hands cleaned and disinfected afterwards) 20 20 0 No manure from other farms spread within 500 m of the farm 20 5 15 No access to the food storage by cats 20 8 12 No access to the food storage by dogs 20 15 5 No access to the food storage by birds 20 13 7 No access to the food storage by rodents 20 3 17 Feeding utensils cleaned after use 20 8 12 Feeding utensils cleaned and disinfected after use 20 2 18 No use of feeding utensils for manure 20 17 3 Testing of microbial quality of animals' drinking water at least once a year 20 16 4 No sharing of equipment with other farms 20 20 0 Six farmers indicated that they paid attention to sanitary status and health management, which refers to the presence of specific diseases on the farm of origin ( Table 3 ). This procedure was based on previous experiences with the farm of origin, in consultation with the veal company. The remaining participants argued that the veal company decides which calves are sent to them, and four farmers emphasized their trust in the company to cover this issue. One farmer believed reviewing the health status of all new calves was unfeasible. A shared opinion was that it is virtually impossible to check all farms of origin, since their number is almost equal to the number of calves. This number is confirmed, since the average degree of commingling for the 20 farms was calculated to be 1.24 (SD = 0.16), meaning that, on average, 124 calves originated from 100 farms. As such, a farm with 500 calves will harbor animals from over 400 different origins. Upon arrival, calves were divided into high and low risk groups based on visual appraisals by 12 of the 20 farmers (60%). On these farms, weaker calves were housed together and received more attention. Half of the farmers (50%) felt that taking blood samples from all the animals to test for disease is neither feasible nor affordable. Other reasons for not testing upon arrival included that there is no obligation to the government (n = 3; 15%) or to the veal company (n = 3; 15%), or that it would provide little additional information (n = 4; 20%). As the stables are filled in a short period, the farmers mostly felt quarantine was neither feasible nor necessary (n = 19; 95%). Before animals are transported to slaughter, transport vehicles are generally empty, cleaned and disinfected prior to picking up animals ready for slaughter, according to the majority of the farmers (n = 15; 75%). However, upon delivery of animals to the farm, on 11 farms (55%), not all animals were unloaded, indicating that trucks were not empty and so were not cleaned between farms. 3.3.2 Measures relating to visitors In 13 farms (65%), access to the stables was controlled by a closed gate and a requirement for visitors to announce themselves before entering. The remaining 7 farmers (35%) believed this was not feasible. The same farmers did not require visitors to register, either because it was not considered important (n = 3; 43%), regularly forgotten (n = 2; 29%), unknown (n = 1; 14%), or not mandatory (n = 1; 14%). Measures regarding farm-specific clothing and boots were not well implemented by most visitors ( Table 4 ), despite farm-specific clothing and boots being available in a high number of farms ( Table 5 ). Other measures for visitors were rarely implemented. Disinfection footbaths were generally present but were either dirty, empty, or ignored. Footbaths were not used by most farmers and staff, mainly because they believed it was not important on their own farm. Very few participants always washed their hands or wore gloves before entering the stables. Those not washing their hands assumed it was not important. On the few farms where a hygiene lock (a room to change into farm-specific boots and clothing before a visitor can enter the stables) was present, it was consistently used by farm personnel and visitors. For one-third of the farmers (n = 6; 35%) that did not have a hygiene lock, the practice was unknown. Table 4 Implemented biosecurity measures by different visitors before entering the stables. Column one contains the biosecurity measure, the second column contains the maximum number of farms that can adhere to the measure, while the third to fifth columns contain the number of visitors complying to the biosecurity measure. Table 4 Biosecurity measure related to visitors N Farmer/Employees Veterinarian Advisor Restricted access to the stables 20 / 14 14 Wearing farm specific clothes before entrance 20 16 6 4 Wearing farm specific boots before entrance 20 17 8 6 Using a hygiene lock 20 3 3 2 Washing and disinfecting hands before entrance 20 2 4 4 Wearing gloves before entrance 20 1 3 1 Using disinfection footbath before entrance 20 4 5 5 Table 5 Implementation of internal biosecurity measures. Column one contains the biosecurity measure, the second column contains the maximum number of farms that can adhere to the measure, while the third to fifth columns contain the adherence to the measure. Table 5 Biosecurity measure concerning disease management N Yes Sometimes No Protocols for vaccination 20 1 19 Preventive measures for endoparasites 20 14 2 6 Preventive measures for ectoparasites 20 20 0 Isolation of sick calves 20 0 3 17 Hospital pen placed physically separated from the other calves 3 1 2 Specific equipment available for the hospital pen 3 3 0 Specific equipment for the hospital pen cleaned after use 3 1 2 Feed and water troughs cleaned after use 3 1 2 Handling sick animals in hospital pen last 3 1 2 Registration of animal health data 20 8 12 Elimination of carriers of infection 20 7 5 8 Segregation of carriers of infection (if no elimination) 13 5 5 3 Biosecurity measure concerning compartmentation N Yes Sometimes No Multiple age groups present on farm 20 9 11 Separation of age groups 9 9 0 No contact possible between age groups 9 8 1 Working from young to old 9 8 1 Specific equipment available for each age group/stable 20 4 16 Specific equipment per age group/stable cleaned after use 4 0 4 Specific equipment recognizable per age group/stable 4 0 4 Farm specific clothing available before entering the farm 20 14 6 Farm specific boots available before entering the farm 20 17 3 Hygiene lock before entering the farm 20 3 17 Hygiene lock only entrance to the stable 3 0 3 Clean and dirty area of the hygiene lock designated and physically separated 3 0 3 Gloves available before entering the farm 20 1 19 Disinfection footbath available and ready for use before entering the farm 20 5 15 Hand washing facilities available and ready for use before entering the farm 20 3 17 Biosecurity measure concerning cleaning and disinfection N Yes Sometimes No Sanitary vacancy of the calf stables after removal of animals 20 17 2 1 Calf stables cleaned after removal of animals 17 17 0 Calf stables cleaned and disinfected after removal of animals 17 11 6 Calf stables dry before next use 17 13 4 All-in, all-out system in the calf stables 17 15 2 Hospital pen available on farm 20 7 13 No direct contact possible in the hospital pen 7 3 4 No indirect contact possible in the hospital pen 7 1 6 Sanitary vacancy of the hospital pen after removal of animals 7 1 6 Hospital pen cleaned after removal of animals 7 2 5 Hospital pen cleaned and disinfected after removal of animals 7 2 5 Hospital pen dry before next use 7 2 5 All-in, all-out system in the hospital pen 7 4 3 Biosecurity measure concerning calf management N Yes Sometimes No Age groups <2 weeks age difference 20 17 3 Draught-free hutches 20 17 3 Slatted floors 20 20 0 Regular cleaning of floors during production rounds 20 0 20 Clean and dry bedding 20 8 12 Always the same bucket (for milk) for a calf 20 20 0 Buckets for milk cleaned after each use 20 3 17 Automated climate control system (temperature, humidity) 20 14 6 Air flow in the stable from young to old 20 20 0 3.3.3 Measures concerning direct or indirect contact with other animals or insects A standard rodent control program usually consisted of the implementation of rodenticides. Farmers without a rodent control program deemed it not important or only took measures when visibly infested ( Table 3 ). All farmers that implemented measures for insect control (n = 14; 70%) treated the environment, sometimes combined with additional measures ( Table 3 ). These measures were mostly intended to control fly populations during summer. The use of a well-equipped carcass storage space was often implemented (85%; n = 17), although few (25%; n = 5) regularly cleaned and disinfected the carcass storage area. Removal of carcasses by the rendering company without entering the premises was considered very important, although this was only possible on 11 farms (55%). 3.3.1 Measures concerning animal movements Inherent to the production system in the veal sector, all farms bought calves every 7.5 to 8 months. There was a large difference in the time required to fill the stables for one cycle, ranging from 2 to 52 days. On average, a stable was filled in 11.4 days (SD: 9.6). During the filling of the stable, all farms received animals on three fixed delivery days per week. On three farms, the age difference between calves was larger than two weeks due to a large spread of calves entering the stables. All calves were collected by cattle salesmen at the farm of origin, moved to a sorting center, and delivered by the veal company to the veal calf farms ( Table 3 ). Table 3 Implementation of external biosecurity measures. Column one contains the biosecurity measure, the second column contains the maximum number of farms that can adhere to the measure, while the third to fifth columns contain the adherence to the measure. Table 3 Biosecurity measure concerning animal movements N Yes Sometimes No Purchasing animals from the same source 20 0 20 Possible contact with other calves before arrival 20 20 0 Check of sanitary status and health management of farms of origin 20 4 2 14 Separation of calves in high/low risk groups based on risk classification 20 12 8 Testing for specific diseases when purchasing 20 0 20 Calves leaving and re-entering the farm 20 0 20 Applying quarantine 20 0 20 Access of transport vehicle to calves' residence prohibited 20 20 0 Transport empty before entering the farm (for loading animals) 20 15 5 Transport clean and disinfected before entering the farm (if empty) 15 14 1 Only the calves on the transport that are supposed to be delivered 20 9 11 Biosecurity measure concerning animal contact N Yes Sometimes No Rodent control program 20 13 7 Use of insect repellants on the animals 20 1 19 Use of insect repellants in the environment 20 14 6 Use of insect traps/nets 20 3 17 Handling of manure to limit insects 20 1 19 No contact possible between calves and other cattle 20 19 1 No contact possible between calves and wild ruminants or boar 20 20 0 No access to the stable by cats 20 9 11 No access to the stable by dogs 20 16 4 No access to the stable by birds 20 12 8 No access to the stable by rodents 20 0 20 No (frequent) contact of employees with animals from other farms 20 14 6 Carcass storage space with solid, easy to clean floors 20 17 3 Carcass storage space protected from pets and vermin 17 14 3 Carcass storage space cleaned after use 17 10 7 Carcass storage cleaned and disinfected after use 17 5 12 Carcass removal possible without entering the farm 20 9 11 Carcass manipulation with gloves (or hands cleaned and disinfected afterwards) 20 20 0 No manure from other farms spread within 500 m of the farm 20 5 15 No access to the food storage by cats 20 8 12 No access to the food storage by dogs 20 15 5 No access to the food storage by birds 20 13 7 No access to the food storage by rodents 20 3 17 Feeding utensils cleaned after use 20 8 12 Feeding utensils cleaned and disinfected after use 20 2 18 No use of feeding utensils for manure 20 17 3 Testing of microbial quality of animals' drinking water at least once a year 20 16 4 No sharing of equipment with other farms 20 20 0 Six farmers indicated that they paid attention to sanitary status and health management, which refers to the presence of specific diseases on the farm of origin ( Table 3 ). This procedure was based on previous experiences with the farm of origin, in consultation with the veal company. The remaining participants argued that the veal company decides which calves are sent to them, and four farmers emphasized their trust in the company to cover this issue. One farmer believed reviewing the health status of all new calves was unfeasible. A shared opinion was that it is virtually impossible to check all farms of origin, since their number is almost equal to the number of calves. This number is confirmed, since the average degree of commingling for the 20 farms was calculated to be 1.24 (SD = 0.16), meaning that, on average, 124 calves originated from 100 farms. As such, a farm with 500 calves will harbor animals from over 400 different origins. Upon arrival, calves were divided into high and low risk groups based on visual appraisals by 12 of the 20 farmers (60%). On these farms, weaker calves were housed together and received more attention. Half of the farmers (50%) felt that taking blood samples from all the animals to test for disease is neither feasible nor affordable. Other reasons for not testing upon arrival included that there is no obligation to the government (n = 3; 15%) or to the veal company (n = 3; 15%), or that it would provide little additional information (n = 4; 20%). As the stables are filled in a short period, the farmers mostly felt quarantine was neither feasible nor necessary (n = 19; 95%). Before animals are transported to slaughter, transport vehicles are generally empty, cleaned and disinfected prior to picking up animals ready for slaughter, according to the majority of the farmers (n = 15; 75%). However, upon delivery of animals to the farm, on 11 farms (55%), not all animals were unloaded, indicating that trucks were not empty and so were not cleaned between farms. 3.3.2 Measures relating to visitors In 13 farms (65%), access to the stables was controlled by a closed gate and a requirement for visitors to announce themselves before entering. The remaining 7 farmers (35%) believed this was not feasible. The same farmers did not require visitors to register, either because it was not considered important (n = 3; 43%), regularly forgotten (n = 2; 29%), unknown (n = 1; 14%), or not mandatory (n = 1; 14%). Measures regarding farm-specific clothing and boots were not well implemented by most visitors ( Table 4 ), despite farm-specific clothing and boots being available in a high number of farms ( Table 5 ). Other measures for visitors were rarely implemented. Disinfection footbaths were generally present but were either dirty, empty, or ignored. Footbaths were not used by most farmers and staff, mainly because they believed it was not important on their own farm. Very few participants always washed their hands or wore gloves before entering the stables. Those not washing their hands assumed it was not important. On the few farms where a hygiene lock (a room to change into farm-specific boots and clothing before a visitor can enter the stables) was present, it was consistently used by farm personnel and visitors. For one-third of the farmers (n = 6; 35%) that did not have a hygiene lock, the practice was unknown. Table 4 Implemented biosecurity measures by different visitors before entering the stables. Column one contains the biosecurity measure, the second column contains the maximum number of farms that can adhere to the measure, while the third to fifth columns contain the number of visitors complying to the biosecurity measure. Table 4 Biosecurity measure related to visitors N Farmer/Employees Veterinarian Advisor Restricted access to the stables 20 / 14 14 Wearing farm specific clothes before entrance 20 16 6 4 Wearing farm specific boots before entrance 20 17 8 6 Using a hygiene lock 20 3 3 2 Washing and disinfecting hands before entrance 20 2 4 4 Wearing gloves before entrance 20 1 3 1 Using disinfection footbath before entrance 20 4 5 5 Table 5 Implementation of internal biosecurity measures. Column one contains the biosecurity measure, the second column contains the maximum number of farms that can adhere to the measure, while the third to fifth columns contain the adherence to the measure. Table 5 Biosecurity measure concerning disease management N Yes Sometimes No Protocols for vaccination 20 1 19 Preventive measures for endoparasites 20 14 2 6 Preventive measures for ectoparasites 20 20 0 Isolation of sick calves 20 0 3 17 Hospital pen placed physically separated from the other calves 3 1 2 Specific equipment available for the hospital pen 3 3 0 Specific equipment for the hospital pen cleaned after use 3 1 2 Feed and water troughs cleaned after use 3 1 2 Handling sick animals in hospital pen last 3 1 2 Registration of animal health data 20 8 12 Elimination of carriers of infection 20 7 5 8 Segregation of carriers of infection (if no elimination) 13 5 5 3 Biosecurity measure concerning compartmentation N Yes Sometimes No Multiple age groups present on farm 20 9 11 Separation of age groups 9 9 0 No contact possible between age groups 9 8 1 Working from young to old 9 8 1 Specific equipment available for each age group/stable 20 4 16 Specific equipment per age group/stable cleaned after use 4 0 4 Specific equipment recognizable per age group/stable 4 0 4 Farm specific clothing available before entering the farm 20 14 6 Farm specific boots available before entering the farm 20 17 3 Hygiene lock before entering the farm 20 3 17 Hygiene lock only entrance to the stable 3 0 3 Clean and dirty area of the hygiene lock designated and physically separated 3 0 3 Gloves available before entering the farm 20 1 19 Disinfection footbath available and ready for use before entering the farm 20 5 15 Hand washing facilities available and ready for use before entering the farm 20 3 17 Biosecurity measure concerning cleaning and disinfection N Yes Sometimes No Sanitary vacancy of the calf stables after removal of animals 20 17 2 1 Calf stables cleaned after removal of animals 17 17 0 Calf stables cleaned and disinfected after removal of animals 17 11 6 Calf stables dry before next use 17 13 4 All-in, all-out system in the calf stables 17 15 2 Hospital pen available on farm 20 7 13 No direct contact possible in the hospital pen 7 3 4 No indirect contact possible in the hospital pen 7 1 6 Sanitary vacancy of the hospital pen after removal of animals 7 1 6 Hospital pen cleaned after removal of animals 7 2 5 Hospital pen cleaned and disinfected after removal of animals 7 2 5 Hospital pen dry before next use 7 2 5 All-in, all-out system in the hospital pen 7 4 3 Biosecurity measure concerning calf management N Yes Sometimes No Age groups <2 weeks age difference 20 17 3 Draught-free hutches 20 17 3 Slatted floors 20 20 0 Regular cleaning of floors during production rounds 20 0 20 Clean and dry bedding 20 8 12 Always the same bucket (for milk) for a calf 20 20 0 Buckets for milk cleaned after each use 20 3 17 Automated climate control system (temperature, humidity) 20 14 6 Air flow in the stable from young to old 20 20 0 3.3.3 Measures concerning direct or indirect contact with other animals or insects A standard rodent control program usually consisted of the implementation of rodenticides. Farmers without a rodent control program deemed it not important or only took measures when visibly infested ( Table 3 ). All farmers that implemented measures for insect control (n = 14; 70%) treated the environment, sometimes combined with additional measures ( Table 3 ). These measures were mostly intended to control fly populations during summer. The use of a well-equipped carcass storage space was often implemented (85%; n = 17), although few (25%; n = 5) regularly cleaned and disinfected the carcass storage area. Removal of carcasses by the rendering company without entering the premises was considered very important, although this was only possible on 11 farms (55%). 3.4 Implementation of internal biosecurity measures 3.4.1 Measures concerning disease management More than half of the farmers (n = 13; 65%) believed vaccination was not important or too expensive because of the short duration of a production cycle and because most vaccines can only be administered at a certain age ( Table 5 ). According to these farmers, most disease outbreaks are observed during the first weeks after introduction, a moment when vaccines are considered not yet effective. Some farmers also mentioned that since the veal companies own the calves, the companies should decide whether to vaccinate. Measures for ectoparasites consisted of preventive treatments, mainly to avoid outbreaks of scabies. Specific treatment for endoparasites was administered only curatively. Seven of twenty (35%) farmers thought it was not feasible to isolate sick animals and five (25%) farmers applied partial isolation, where the animals were not separated from the other animals (direct contact possible) ( Table 5 ). Although a hospital pen was present on seven farms (35%), only three farmers (43%) indicated that they sometimes isolated sick animals when they were lame or unable to function in the group (e.g., unable to eat, drink, or stand up). Only two out of seven hospital pens were cleaned, disinfected and dried before new animals entered, and an "all-in, all-out" system was used in four hospital pens. Only one farmer implemented all these measures and had a fully, physically separated hospital pen. The farmers that did not take these measures declared them infeasible because their hospital pen was located inside the regular stables, making thorough cleaning unfeasible. For five participants (25%), elimination or segregation of a carrier of infection depended on the age or clinical status of the animal. An older animal would often go to slaughter while younger animals would be separated. 3.4.2 Measures concerning compartmentation On the nine farms with multiple age groups, eight farmers performed work from old to young, contrary to established wisdom ( Table 5 ). On 16 farms (80%), equipment, such as wheelbarrows and feeding utensils, were moved between compartments (same age group) without cleaning or disinfection. None of the farms used compartment-specific measures, such as changing clothes and footwear or washing hands between different compartments or age groups. Within the compartment, calves were sorted by drinking speed for economic reasons, since the difference between the animals would impair the growth of slower animals. Between compartments, animals were only moved to segregate carriers of infection. Only one farm (5%) could not prevent direct contact with another age group due to the structure of the stable. In two farms, the "all-in, all-out" system was not always well applied, i.e., young calves entered the stables while (some) older animals were still present, resulting in possible contact between the age groups. The calf stables were empty after each production cycle on the other 17 farms (85%). The duration of the sanitary vacancy, often also referred to as downtime, a period between production cycles where the stable is not used, was on average 9.8 days (SD = 4.1; range 3–15 days). 3.4.3 Measures concerning cleaning and disinfection All farmers who always applied a sanitary vacancy (n = 17; 85%), also cleaned their stables during the vacancy. However, only 11 out of 17 farmers also disinfected them. Pipelines used for milk were cleaned once or twice a week. Water and feed troughs were rinsed with water on a daily (n = 5; 25%), weekly (n = 4; 20%), or monthly (n = 1; 5%) basis, or once per production cycle (n = 8; 40%). Two farmers (10%) never cleaned the feed troughs. All farmers used reusable needles to inject the animals. 3.4.4 Measures concerning calf management In general, calves were housed in individual boxes with both visual and physical contact during the first six weeks. Calves were then sorted by drinking speed within the compartment. Poorly growing calves were isolated in one compartment with a different diet. As one compartment only contained animals of the same age group, air flow within the compartment was considered irrelevant concerning disease spread from younger to older animals ( Table 5 ). 3.4.1 Measures concerning disease management More than half of the farmers (n = 13; 65%) believed vaccination was not important or too expensive because of the short duration of a production cycle and because most vaccines can only be administered at a certain age ( Table 5 ). According to these farmers, most disease outbreaks are observed during the first weeks after introduction, a moment when vaccines are considered not yet effective. Some farmers also mentioned that since the veal companies own the calves, the companies should decide whether to vaccinate. Measures for ectoparasites consisted of preventive treatments, mainly to avoid outbreaks of scabies. Specific treatment for endoparasites was administered only curatively. Seven of twenty (35%) farmers thought it was not feasible to isolate sick animals and five (25%) farmers applied partial isolation, where the animals were not separated from the other animals (direct contact possible) ( Table 5 ). Although a hospital pen was present on seven farms (35%), only three farmers (43%) indicated that they sometimes isolated sick animals when they were lame or unable to function in the group (e.g., unable to eat, drink, or stand up). Only two out of seven hospital pens were cleaned, disinfected and dried before new animals entered, and an "all-in, all-out" system was used in four hospital pens. Only one farmer implemented all these measures and had a fully, physically separated hospital pen. The farmers that did not take these measures declared them infeasible because their hospital pen was located inside the regular stables, making thorough cleaning unfeasible. For five participants (25%), elimination or segregation of a carrier of infection depended on the age or clinical status of the animal. An older animal would often go to slaughter while younger animals would be separated. 3.4.2 Measures concerning compartmentation On the nine farms with multiple age groups, eight farmers performed work from old to young, contrary to established wisdom ( Table 5 ). On 16 farms (80%), equipment, such as wheelbarrows and feeding utensils, were moved between compartments (same age group) without cleaning or disinfection. None of the farms used compartment-specific measures, such as changing clothes and footwear or washing hands between different compartments or age groups. Within the compartment, calves were sorted by drinking speed for economic reasons, since the difference between the animals would impair the growth of slower animals. Between compartments, animals were only moved to segregate carriers of infection. Only one farm (5%) could not prevent direct contact with another age group due to the structure of the stable. In two farms, the "all-in, all-out" system was not always well applied, i.e., young calves entered the stables while (some) older animals were still present, resulting in possible contact between the age groups. The calf stables were empty after each production cycle on the other 17 farms (85%). The duration of the sanitary vacancy, often also referred to as downtime, a period between production cycles where the stable is not used, was on average 9.8 days (SD = 4.1; range 3–15 days). 3.4.3 Measures concerning cleaning and disinfection All farmers who always applied a sanitary vacancy (n = 17; 85%), also cleaned their stables during the vacancy. However, only 11 out of 17 farmers also disinfected them. Pipelines used for milk were cleaned once or twice a week. Water and feed troughs were rinsed with water on a daily (n = 5; 25%), weekly (n = 4; 20%), or monthly (n = 1; 5%) basis, or once per production cycle (n = 8; 40%). Two farmers (10%) never cleaned the feed troughs. All farmers used reusable needles to inject the animals. 3.4.4 Measures concerning calf management In general, calves were housed in individual boxes with both visual and physical contact during the first six weeks. Calves were then sorted by drinking speed within the compartment. Poorly growing calves were isolated in one compartment with a different diet. As one compartment only contained animals of the same age group, air flow within the compartment was considered irrelevant concerning disease spread from younger to older animals ( Table 5 ). 3.5 CATPCA and KMCA The two-dimensional solution of the CATPCA explained 69.7% of the variance of the seven biosecurity categories in the 20 herds ( Fig. 2 ). The percentage accounted for was 41.7% for the first dimension, and 28.0% for the second dimension. The vectorial component loadings represent the contributions of each category to the dimensions, while the different categories of the nominal variable "veal companies" are represented by their centroid coordinates. The vectors appear in the upper and lower right quadrant. The projection of the vector for contact with other animals has the largest contribution to the first dimension (x-axis), followed by the vector for cleaning and disinfection. The vector for compartmentation, which has the lowest contribution for dimension 1, has the largest contribution to the second dimension (y-axis). Veal companies 3 and 4, whose centroids are located in the directions of the vectors, have, on average, the highest biosecurity scores. For veal company 3, this result is mainly related to a higher score for compartmentation and measures for visitors, while these farms score lower on disease management and cleaning and disinfection. In the farms of veal company 4, the opposite applies. On average, the farms of veal company 2 have the lowest biosecurity score. However, the overall differences in biosecurity between veal companies are limited (centroids close to the center). Fig. 2 Triplot of component loadings (the position of the original variables in the two-dimensional space, represented by vectors), multiple nominal category points (veal companies) and objects (individual farms) labeled by the clusters, resulting from the categorical principal component analysis and K-means clustering analysis. The vector of a variable points in the direction of the highest category of the variable, indicating in this case a higher level in biosecurity. The veal companies are located close to the center of the plot, meaning no distinction can be made between the veal companies. The first and second dimension distinguish between the different clusters. The green circles with number 1–4 represent the individual farms part of cluster 1–4. The first cluster has on average the lowest biosecurity, while the second and third cluster tend to have the highest scores. The fourth cluster is located in the center. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 2 Based on the KMCA, four clusters were identified ( Fig. 2 ). The first cluster contains the highest number of farms (13) that scored lowest for biosecurity overall. Clusters 2 and 3 scored high for biosecurity. The farms in cluster 2 scored, on average, higher for disease management and cleaning and disinfection, while farms in cluster 3 score higher for compartmentation and visitors. Though this seems similar to the results of veal companies 3 and 4, the clusters consist of farms of multiple veal companies. Overall no clear veal company effect was observed in the clusters. 4 Discussion To our knowledge, this paper is the first to describe biosecurity on farms with an intensive veal-rearing system. Because of the strong integration and industrialization in the veal sector, it could be theorized that the implementation of biosecurity on veal farms would differ from that of conventional dairy and beef farms. This study was designed to describe the biosecurity level on veal farms as the first step of a larger research project to develop a risk-based biosecurity scoring system for cattle farms. Though no comparison was made with conventional farms, based on the results of this study, a difference in biosecurity level can be expected due to differences in the purchase policy, contact with other animals, compartmentation and cleaning and disinfection ( Renault et al., 2018a ). The random sample of 20 veal farms in this study may be considered small, yet it represented about 8.3% of all Belgian veal farms since the sector consists of a limited number of farms. The selected farms were distributed among veal companies corresponding to their market share. Furthermore, the size of the selected farms was representative (average veal farms house 200–1200 veal calves) for the population, and different veterinary practices advised the farmers. Therefore, the selected farms were considered to be representative of the veal calf industry. To a certain extent, selection bias cannot be excluded, due to the possibility that better farms might be more willing to participate. The face-to-face interviews, in combination with a herd visit, allowed the investigator to observe the majority of the practices and measures, which limited the amount of interview bias due to the socially desired response rather than the true situation ( Sarrazin et al., 2014 ). However, only a single visit to the farm was made, so the actual compliance for some measures could not be determined. Because the herd visits were performed by a single interviewer, investigator variability was avoided. Therefore, it is believed that the presented results provide an accurate image of the biosecurity situation on Belgian veal farms. Most of the veal farmers considered biosecurity important, though they were not familiar with the term itself and most were not able to list five biosecurity measures, thus indicating that the perceived importance is only sparsely translated into actions. Furthermore, a number of the farmers considered several measures to be unimportant or impossible to implement. This finding confirms previous observations that there is a substantial lack of information on how to improve farm management and how to implement these improvements ( Damiaans et al., 2018 ). The finding also shows that the results of previous studies that suggested measures like risk classification, limitation of the arrival period, and the farms of origin have not been translated into practice ( Pardon, 2012 ). Contrary to, e.g., mortality and antibiotic use on veal farms, biosecurity seems affected by the veal company only in a limited capacity ( Bokma et al., 2019 ). Though biosecurity improvements are partly within the power of the farmers, this limited impact may show the veal companies' lack of policy regarding biosecurity. Some characteristics inherent to veal production, as it is currently organized, largely hamper the implementation of several biosecurity measures. The most important issue is the huge number of farms of origin. As the purchase of animals is often described as one of the most important risks in disease introduction ( Cuttance and Cuttance, 2014 ; Sarrazin et al., 2017 ), this procedure is a significant disadvantage for the veal sector. This disadvantage is even aggravated by the induction of stress through the transport and commingling of the calves, resulting in increased susceptibility to new infections ( Stokka, 2010 ). Solving this calf-sourcing risk will require fundamental changes in the organization of the industry. A first step toward limiting the farms of origin could be grouping births in larger dairy farms to increase the number of calves originating from one farm. Regardless of this fundamental challenge, other measures regarding animal introduction can be taken. For instance, animals with the same disease status could be grouped in the same stable to limit contamination of other calves and the environment. This measure requires more upstream information on the sanitary status of the herds of origin and additional testing, measures that are currently poorly implemented. The national eradication programs for infectious bovine rhinotracheitis (IBR) and bovine viral diarrhea (BVD), currently implemented in Belgium ( Royal Decree KB2017-09-18/09, 2017KB-09-18/09, Royal Decree, 2017 Royal Decree KB2017-09-18/09, 2017; Royal Decree KB2018-04-27/03, 2018KB-04-27/03, Royal Decree, 2018 Royal Decree KB2018-04-27/03, 2018) are expected to decrease the infection pressure caused by these diseases. This decrease is especially important for BVD, as it has been described as one of the major contributors to disease in veal calf-rearing ( Pardon et al., 2011 ). Furthermore, in collaboration with the veal companies, previous experiences with farms of origin could serve as a valuable source of information, provided that this information is recorded and shared ( Hobbs, 2004 ; Pardon, 2012 ). This type of information could improve the risk classification of animals, which is currently performed only through visual inspection. As shown by van Schaik et al. (1998) and Brennan et al. (2008) , a higher number of visitors is a risk factor for disease introduction. In veal farms, only two types of visitors visit the farm frequently: the veterinarian and the representative of the veal company. Conventional farms often have more types of visitors, such as salesmen, feed suppliers, hoof trimmers and drivers of milk trucks ( Renault et al., 2018a ). Nevertheless, the frequency with which some visitors enter influences the risk for introduction of disease. Although only a limited number of visitors enter the farm, the precautionary measures they take upon entrance are insufficient ( Table 4 ). As these professional visitors are, by definition, high-risk visitors since they have frequent contact with animals from different farms, the risk of spreading infection through this route remains high. The implementation of a minimum of preventive measures, such as wearing herd-specific clothing and footwear, by professional visitors is a relatively easy and cheap measure that can be implemented on short notice. Very few farmers considered themselves or their staff as a risk when entering their own farm, forgetting that they may also transmit disease ( Sarrazin et al., 2017 ). This shows that they do not fully understand disease transmission and the risks associated. This lack of knowledge might reflect in the execution of other biosecurity measures. Sick animals are rarely physically isolated, even though keeping sick animals in a group has been described as detrimental to the health of other animals ( Edwards, 2010 ). Furthermore, during the first weeks of the rearing period, farmers believe the calves are sufficiently separated. This lack of isolation is likely linked to the observation that during these first weeks, disease outbreaks usually cannot be limited to one or a few animals in the current rearing system. Moreover, most farmers did not consider investing in a hospital pen, even though the benefit in limiting disease transmission by separating the source of infection has been shown repeatedly ( Edwards, 2010 ). Since the most crucial period for disease prevention is during the first few weeks of the rearing period, farmers consider a number of preventive practices, such as vaccination, unnecessary. However, Lava et al. (2016) concluded that farms where calves were vaccinated had a lower mortality rate. Lava and colleagues also remarked that an ideal vaccination scheme should start at the farm of origin, thus reiterating the importance of information exchange between the origin farms and the veal farm. Admittedly, the calves in the study by Lava et al. (2016) were, on average, one month old upon purchase while, in Belgium, calves are sold at the age of two to four weeks ( Pardon et al., 2015 ). Besides vaccination, maternal immunity is of the utmost importance for the calf's immunity ( Delafosse et al., 2015 ). Measuring serum IgG concentrations of all calves upon arrival, as described by Weaver et al. (2000) , could be a measure to ensure the adequate function of the herd's immune system. Furthermore, a higher serum IgG concentration decreased the risk of mortality, according to Renaud et al. (2018) . A concentration of less than 7.5 g/L IgG was shown to decrease average daily gain ( Pardon et al., 2015 ). Moreover, measuring the blood serum values would likely stimulate the farmers of the origin herds to ensure sufficient colostrum administration. Nonetheless, taking blood samples upon arrival is considered infeasible by the majority of the farmers, even though blood samples to check for iron deficiency are taken regularly, and the value of this measure has been described ( Maunsell and Donovan, 2009 ; Maunsell et al., 2011 ). Most farmers considered it better not to follow conventional working lines from youngest to oldest, as described by Sarrazin et al. (2014) . These farmers prefer to start with the oldest animals, reasoning that a younger group carries and spreads more pathogens from their farm of origin, having only recently arrived. However, the farmers seem to ignore that the older animals have a higher immune status and can be carriers of quickly spreading, high impact diseases, such as Mycoplasma bovis and Salmonella spp. ( Radaelli et al., 2008 ; Pardon et al., 2011 ), and therefore can spread disease to the younger animals. By handling the youngest animals first and the sick and quarantined animals last, farmers can reduce the spread of disease within the farm ( Sarrazin et al., 2013 ). Due to the organization of the veal industry, the application of an "all-in, all-out" system as well as clear compartmentation, which has been described as an adequate biosecurity measure ( Maunsell and Donovan, 2009 ; Maunsell et al., 2011 ), is easily implementable. Besides the advantages of keeping young, susceptible calves separated from the older cohorts ( Sarrazin et al., 2014 ), each compartment can be cleaned, disinfected, and thoroughly dried during the sanitary vacancy. A clean and disinfected environment is recommended in the literature for multiple diseases, such as Cryptosporidium parvum , Salmonella spp., and BVD ( Daugschies and Najdrowski, 2005 ; Fossler et al., 2005 ; Villarroel et al., 2007 ). Next to the frequency of cleaning and disinfection, a well-designed and adhered-to protocol, including the seven steps described in Van Immerseel et al. (2018) , is equally important. These seven steps consist of removal of all organic material, soaking all surfaces, high pressure cleaning, drying, disinfection, drying and testing the efficiency of the procedure. If the stables are not cleaned and disinfected properly, pathogens can survive even after a sufficiently long sanitary vacancy. Research suggests that the length of the sanitary vacancy, which in this study was, on average, ten days, is not as important as a proper cleaning and disinfection procedure ( Luyckx et al., 2016 ). The farmers indicated that they thoroughly cleaned and disinfected their stables more often during recent years due to the distribution of cleaning and disinfection products by the veal company. This example illustrates that the veal company could play a crucial role in the motivation toward the implementation of biosecurity measures. It also illustrates that the farmers are not the sole decision makers and can be influenced regarding their biosecurity policies. Possibly, this understanding explains why several farmers answered that they were not obliged by government or veal company to apply certain measures, but were waiting for guidelines to follow. In the CATPCA analysis, no clear distinction between the levels of biosecurity in the different veal companies was observed. However, these results do not signify that the veal companies cannot guide and motivate their farmers in improving biosecurity. Instead, the analysis suggests that, at this moment, they do not take the opportunity to address biosecurity, leaving room for improvement. Most farmers in this study were willing to invest money and time to solve shortcomings on their farm, which is in agreement with previous findings ( Damiaans et al., 2018 ). However, farmers are often hindered by their beliefs that many biosecurity measures are not feasible or important. Farmers often feel they lack information on both the efficacy and feasibility of disease prevention through biosecurity measures ( Sarrazin et al., 2014 ; Damiaans et al., 2018 ), The data from this study provides a first indication of the biosecurity level of veal farms, starting with the Belgian situation. Given the fact that the industry is organized in a comparable manner to most European veal-producing countries, often with the same veal companies working in different countries, it can be hypothesized that the obtained results are comparable to production in Europe. This study provides insights on current biosecurity measures in veal herds and identifies potential priority areas for short, middle, and long term improvements. Several biosecurity measures of high importance, such as "all-in, all-out" and compartmentation, are implemented relatively well whereas other measures, such as cleaning and disinfection, isolation of sick animals, and measures for visitors can easily be improved. The improvement of some measures regarding the introduction of animals from a huge number of different origins with variable infectious and immunity status will require more fundamental changes in the organization of the industry. In the implementation of these improvements, the collaboration between farmers, veal companies, and veterinarians will be crucial. Funding source This study was supported by the Belgian Federal Public Service for Health, Food Safety and Environment (Contract RT 15/4 BOBIOSEC1). The funding source had no other involvement. Research data All research data are available with the author. Transparency declaration The lead author affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained. Declaration of Competing Interest None. Appendix A Supplementary data The following are Supplementary data to this article:

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077580/

Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae

Background Susceptibility to Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance. Methods We introduced SNPs into the A. gambiae MAPK kinase MEK to alter key residues in the N-terminal docking site (D-site), thus interfering with its ability to interact with the downstream kinase target ERK. ERK phosphorylation levels in vitro and in vivo were evaluated to confirm the effects of MEK D-site mutations. In addition, overexpression of various MEK D-site alleles was used to assess P. berghei infection in A. gambiae. Results The MEK D-site contains conserved lysine residues predicted to mediate protein-protein interaction with ERK. As anticipated, each of the D-site mutations (K3M, K6M) suppressed ERK phosphorylation and this inhibition was significant when both mutations were present. Tissue-targeted overexpression of alleles encoding MEK D-site polymorphisms resulted in reduced ERK phosphorylation in the midgut of A. gambiae . Furthermore, as expected, inhibition of MEK-ERK signaling due to D-site mutations resulted in reduction in P. berghei development relative to infection in the presence of overexpressed catalytically active MEK. Conclusion MEK-ERK signaling in A. gambiae , as in model organisms and humans, depends on the integrity of conserved key residues within the MEK D-site. Disruption of signal transmission via engineered SNPs provides a purposeful proof-of-principle model for the study of naturally occurring mutations that may be associated with mosquito resistance to parasite infection as well as an alternative genetic basis for manipulation of this important immune signaling pathway. Background Susceptibility to Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance. Methods We introduced SNPs into the A. gambiae MAPK kinase MEK to alter key residues in the N-terminal docking site (D-site), thus interfering with its ability to interact with the downstream kinase target ERK. ERK phosphorylation levels in vitro and in vivo were evaluated to confirm the effects of MEK D-site mutations. In addition, overexpression of various MEK D-site alleles was used to assess P. berghei infection in A. gambiae. Results The MEK D-site contains conserved lysine residues predicted to mediate protein-protein interaction with ERK. As anticipated, each of the D-site mutations (K3M, K6M) suppressed ERK phosphorylation and this inhibition was significant when both mutations were present. Tissue-targeted overexpression of alleles encoding MEK D-site polymorphisms resulted in reduced ERK phosphorylation in the midgut of A. gambiae . Furthermore, as expected, inhibition of MEK-ERK signaling due to D-site mutations resulted in reduction in P. berghei development relative to infection in the presence of overexpressed catalytically active MEK. Conclusion MEK-ERK signaling in A. gambiae , as in model organisms and humans, depends on the integrity of conserved key residues within the MEK D-site. Disruption of signal transmission via engineered SNPs provides a purposeful proof-of-principle model for the study of naturally occurring mutations that may be associated with mosquito resistance to parasite infection as well as an alternative genetic basis for manipulation of this important immune signaling pathway. Background Malaria is caused by protozoan parasites of the genus Plasmodium , transmitted by female anopheline mosquitoes to humans during blood feeding. Prevention and treatment of the disease requires extensive efforts and coordination among the general public, health care organizations, and government agencies. Despite increased global efforts, malaria remains a top-ranked vector-borne disease and major global health concern, affecting over half of the world's population every year [ 1 ]. Reports from populations in Sub-Saharan Africa recorded the highest numbers of cases and deaths estimated at 80% and 90% of the global burden, respectively, in 2012 [ 1 ]. In this region, P. falciparum is responsible for the largest number of infections and is the most deadly species, transmitted by Anopheles gambiae , the main mosquito vector. A network of highly conserved cell signaling pathways controls malaria parasite development in and transmission by the anopheline mosquito host. Among these are the mitogen-activated protein kinase (MAPK) pathways, which function in growth, differentiation, and immune processes from nematodes to humans [ 2 - 5 ]. MAPKs function in multi-tiered sequential signaling cascades, in which an activated MAP4K phosphorylates and activates a MAP3K which, in turn, activates a downstream MAP2K, which activates a MAPK that can phosphorylate effector proteins or transcription factors to positively or negatively regulate a wide variety of cellular functions [ 6 - 8 ]. The subgroup involved in cellular proliferation and differentiation includes the extracellular signal-related kinase (ERK) and its upstream dual specificity MAPK/ERK kinase (MEK) [ 8 ]. Efficient propagation of MEK-ERK signaling requires a stable docking interaction between the upstream activating kinase and its downstream target. The N-terminal ERK docking site or D-site of MEK interfaces with the common docking or CD domain of ERK [ 9 - 13 ]. In humans, the first 32 or 36 residues of MEK1 or MEK2, respectively, comprise the D-site that mediates interaction with the common docking or CD domain of ERK [ 12 , 13 ]. The MEK D-site shares a conserved motif found in other MAPK-interacting proteins that includes a basic region, a ØA-X-ØB motif where Ø is leucine, isoleucine, or valine, and a hydrophobic-X-hydrophobic spacer region [ 14 - 17 ]. Deletion and mutational studies have revealed that the D-site is essential for enhancing the rate of MEK phosphorylation of ERK, and that the loss of the domain or substitution of the conserved basic and hydrophobic residues diminished the ability of MEK to bind to ERK [ 12 , 18 ]. In addition to the role of the MEK D-site in facilitating efficient activation, it is thought to tether ERK in the cytosol in resting cells [ 9 ]. The MEK-ERK signaling module plays a central role in the regulation of malaria parasite development in Anopheles stephensi , the Indian malaria mosquito [ 19 - 22 ]. In particular, human transforming growth factor-beta1 (TGF-β1) ingested with a P. falciparum -infected blood meal induces ERK activation in the midgut [ 20 - 22 ]. The provision of small molecule inhibitors of MEK in the blood meal reproducibly reduced ERK activation in the A. stephensi midgut and enhanced nitric oxide synthase ( NOS ) transcription within 24 h after infection, resulting in the production of inflammatory levels of reactive oxygen and nitrogen species in the midgut lumen [ 23 ] that are directly toxic to P. falciparum [ 24 ] and leading to significant reductions in oocyst numbers on the midgut epithelium [ 21 ]. Confirmation that small molecule inhibition of MEK can significantly reduce mosquito infectivity suggests that overexpression of altered MEK alleles could form the basis of a genetic strategy to generate parasite-resistant mosquitoes. Accordingly, we hypothesized that the introduction of non-synonymous single nucleotide polymorphisms (SNPs) into the highly conserved D-site of MEK could reduce ERK phosphorylation and decrease malaria parasite development in the mosquito host in vivo . Herein, we demonstrate that overexpression of a catalytically active MEK allele in A. gambiae cells in vitro resulted in enhanced ERK phosphorylation in these cells, while overexpression of a MEK allele with D-site mutations reduced ERK phosphorylation. Using a transient transformation strategy, midgut-specific overexpression of the same mutated MEK allele in vivo reduced ERK phosphorylation in this tissue and reduced development of naturally acquired Plasmodium berghei in vivo , suggesting for the first time that tissue-specific overexpression of mutated MEK could be used as the basis for a malaria transmission blocking strategy. Methods Cell culture, mosquito rearing and mosquito feeding The immortalized A. gambiae Sua5B cell line [ 25 ] was maintained in Schneider's medium (Invitrogen) with 10% heat-inactivated fetal bovine serum at 28°C. Anopheles gambiae (G3 strain) mosquitoes were reared and maintained at 27°C and 75% humidity. Mosquitoes were maintained under a 12 h light/dark cycle. Mosquito eggs were placed in water and fed 0.2% baker's yeast on the day collected. After hatching, larvae were fed a mixture of liquid food containing 2% w/v powdered fish food (Sera Micron) and baker's yeast in a 2:1 ratio, and Game Fish Chow pellet food (Purina). Adult mosquitoes were maintained on 10% sucrose solution-soaked cotton pads. All mosquito-rearing protocols were approved and in accord with regulatory guidelines and standards set by the Institutional Animal Care and Use Committee of the University of California, Davis. For in vivo studies, 3–5 d old female mosquitoes were allowed to feed for 30 min on artificial blood meals of washed human erythrocytes and heat-inactivated human serum provided through a Hemotek Insect Feeding System (Discovery Workshops). MEK allele plasmid construction and transfection for in vitro studies The complete mRNA sequence of A. gambiae MEK [GenBank: XM_322064] in the pDREAM 2.1 vector (Genscript) (wild type MEK or wtMEK) was used to generate five additional plasmids encoding MEK mRNA with various combinations of SNPs: pMEK1, pMEK2, pMEK3, pMEK4 and pMEK5 (see Table 1 for a summary of these mutations). In brief, SNPs were introduced at codon positions 3 and 6 to convert lysines (K) to methionines (M) and at positions 243 and 247 to convert serines (S) to glutamic acid (E) and aspartic acid (D), respectively (Figure 1 ). Table 1 pMEK plasmid nucleotide changes to D-site lysines and catalytic site serines Domain Codon Position wtMEK pMEK1 pMEK2 pMEK3 pMEK4 pMEK5 Docking (D)-Site 3 AAA (K) AAA (K) AAA (K) ATG (M)* AAA (K) ATG (M)* 6 AAA (K) AAA (K) AAA (K) AAA (K) ATG (M)* ATG (M)* Catalytic 243 TCA (S) GAA (E)* GAA (E)* GAA (E)* GAA (E)* GAA (E)* 247 TCT (S) TCT (S) GAT (D)* GAT (D)* GAT (D)* GAT (D)* Mutations at positions 3 and 6 in the D-site were introduced to disrupt MEK-ERK interaction and corresponding phosphorylation of ERK. Mutations at positions 243 and 247 mimic MEK phosphorylation and, hence, activation. The codons are shown with encoded amino acids in parentheses. wtMEK = wild type MEK; amino acid substitutions are noted with asterisks. Figure 1 Amino acid alignment of human MEK1 and MEK2 with Anopheles gambiae MEK. Human (Hs) MEK1 and MEK2 and A. gambiae (Ag) MEK show significant overall conservation with high amino acid identity and similarity, including conservation in the docking site or D-site (blue box) and the catalytic domain (orange box). Lysine residues at positions 3 and 6 (yellow boxes) in the A. gambiae MEK allele were mutated to methionine (M). The key serine residues within the catalytic domain at positions 243 and 247 (red boxes) were mutated to aspartic acid (D) and glutamic acid (E), respectively. Human MEK1 [Genbank: NP_002746], human MEK2 [Genbank: NP_109587] and A. gambiae MEK1 [Genbank: XP_322064] protein sequences were aligned using the MUSCLE method with default settings in Geneious [ 26 ]. To introduce SNPs into the MEK -encoding sequence, paired synthetic primers that encoded the desired mutations were synthesized (See Table 2 for primer sequences; Sigma-Aldrich) and utilized for mutagenic primer-directed replication of both plasmid strands with high-fidelity PfuUltra DNA polymerase (Agilent). The following conditions were used for plasmid replication: 15–17 cycles of denaturation at 95°C for 30 sec, primer annealing for 1 min at 55°C, followed by extension at 68°C for 1 min per 1 kb amplified. The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA. The nicked synthesized plasmid DNAs with the desired mutations were transformed into E. coli TOP10 chemically competent cells (Invitrogen). Eight to ten transformed colonies for every desired mutation were screened for plasmid DNA using the Qiagen Miniprep Kit and the manufacturer's instructions (Qiagen). Among those, four to five plasmids were sequenced for confirmation of the introduced functional nucleotide changes (CDC Sequencing Facility, Fort Collins, CO). Table 2 Primer sequences for site-directed mutagenesis Plasmid constructs Resulting amino acid mutations Primer sequence 5′ ➔ 3′ Docking domain mutations pMEK3 K3M F: GACGACGACAAGATGAGTA TG ATGACAAAAAACAAACTTAA R: TTAAGTTTGTTTTTTGTCAT CA TACTCATCTTGTCGTCGTC pMEK4 K6M F: CAAGATGAGTAAAATGACAA TG AACAAACTTAATTTGACGTTG R: CAACGTCAAATTAAGTTTGTT CA TTGTCATTTTACTCATCTTG pMEK5 K3M F: GACGACGACAAGATGAGTA TG ATGACAA TG AACAAACTTAATTTGACGTTG K6M R: CAACGTCAAATTAAGTTTGTT CA TTGTCAT CA TACTCATCTTGTCGTCGTC Catalytic domain mutations pMEK1 S243E F: GATTGAT GA AATGGCCAATTCTTTTGTAGGTACTCGAAG R: CTTCGAGTACCTACAAAAGAATTGGCCATT TC ATCAATC pMEK2 S243E F: GATTTCGGCGTTTCCGGTCAGTTGATTGAT GA AATGGCCAAT GA TTTTGTAGGTACTCGAAG pMEK3 pMEK4 S247D R: CTTCGAGTACCTACAAAA TC ATTGGCCATT TC ATCAATCAACTGACCGGAAACGCCGAAATC pMEK5 Paired synthetic primers encoding the engineered SNPs (bold) were used to introduce mutations into the MEK -encoding sequence through mutagenic primer-directed replication of both plasmid strands. MEK -encoding plasmids were transfected into A. gambiae Sua5B cells using Effectene Reagent (Qiagen) and the manufacturer's recommended protocol. In brief, 1×10 6 Sua5B cells in 2 mL medium were plated in 6-well tissue culture plates overnight at 28°C. At 24 h after plating, cells were transfected with 0.6 μg of plasmid DNA and incubated at 28°C. At 36 h post-transfection, medium was removed and cells were washed with ice-cold phosphate buffered saline (PBS) in preparation for immunoblotting. MEK allele plasmid construction for in vivo studies and microinjection of female A. gambiae The plasmid for transgene overexpression in adult female A. gambiae was described previously [ 27 ]. To ensure midgut-specific expression of the transgene post-blood feeding, the A. gambiae carboxypeptidase promoter was engineered into the plasmid [ 28 , 29 ]. The MEK inserts wtMEK, pMEK2 and pMEK5 were cloned into the plasmid using 5′-PstI and 3′-SalI restriction sites. For each experiment, at least twenty laboratory reared 3–5 d old female A. gambiae were allowed to feed for 30 min on artificial blood meals at 16–24 h prior to MEK -encoding plasmid inoculation. For our studies, a mixture of 0.5 μg/μl MEK -encoding plasmid DNA, the in vivo transfection reagent jetPEI TM (Polyplus-transfection Inc.) and glucose at a final concentration of 5% [ 27 ] was injected into the hemocoel of vitellogenic females (0.1 to 0.5 μg DNA/female) using the Nanoject II Auto Nanoliter Injector (Drummond Scientific Company). At 24 h post injection, the mosquitoes were provided small cups of water for oviposition. F0 eggs were collected and reared through to the adult stage. Mosquito cell and tissue preparation and immunoblotting To harvest proteins from A. gambiae Sua5B cells, cells were lysed in 200 μl cell lysis buffer (10 mM Tris–HCl pH 7.4, 1 mM EDTA, 100 mM NaCl, 1 mM NaF, 1 mM EGTA, 2 mM Na 3 VO 4 20 mM Na 4 P 2 O 7 , 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol, 60 mg/mL aprotinin, 10 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mg/ml calyculin A). Cellular debris was removed by centrifugation at 14,000 × g for 10 min at 4°C. The resulting supernatants were mixed with Laemmli sample buffer (125 mM Tris–HCl pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue, 130 mM dithiothreitol) and the proteins were denatured at 95°C for 4 min prior to electrophoresis. Mosquito midguts were dissected into PBS and mixed to release blood, if any, by pipetting up and down. The midguts were washed in a filter column fitted with a fine mesh with a mixture of protease and phosphatase inhibitor cocktails (Sigma) in ice-cold PBS until all of the blood was removed. Fresh PBS mixture was added to loosen the midgut tissue from the filter, transferred to a fresh tube, and then centrifuged and prepared for electrophoresis as for cell culture lysate above. Proteins were separated on 10% SDS-PAGE polyacrylamide gels at 135 V for 1 h, 50 min. Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) for 1 h, 15 min at 7 V. Coomassie blue staining of the polyacrylamide gel was used to visually assess consistency of protein loading. Membranes were blocked in nonfat dry milk (5% w/v) in 1X Tris-buffered saline (TBS; pH 7.0) containing 0.1% Tween (TBS-T) for 1 h at room temperature, and then reacted overnight in primary antibody at 4°C. The membrane was washed 3 times, 5 min each with 1X TBS-T followed by incubation with appropriate secondary antibody 4°C overnight. The membrane was washed again 3 times, 5 min each with 1X TBS-T and then incubated in SuperSignal West Dura Extended Duration Substrate (Pierce). The Kodak Image Station 4000MM Pro Imaging System (Carestream Health, Inc.) was used to capture the image of the membrane and Quantity One (Bio-Rad Laboratories) software was used for densitometry analysis of the antibody-bound proteins. Levels of phosphorylated ERK (pERK) in Sua5B cells for each treatment were normalized to total ERK levels for protein loading and then normalized to pERK levels in the control cells transfected with wtMEK plasmid construct for in vitro experiments. Levels of pERK in the midgut for each group of A. gambiae were normalized to GAPDH levels and then to pERK levels in control mosquitoes transformed with wtMEK plasmid construct for in vivo experiments. Primary antibodies and dilutions included anti-FLAG-M2 (A2220; Sigma-Aldrich) (1:7,500), anti-GAPDH (G9545; Sigma-Aldrich) (1:10,000), anti-diphosphorylated ERK (pERK) (M8159; Sigma-Aldrich) (1:10,000) and anti-ERK1/2 (total ERK) (9102; Cell Signaling Technology) (1:1,250). Anti-rabbit IgG-peroxidase (A0545; Sigma-Aldrich) (pERK 1:20,000; FLAG 1:2,000) and anti-mouse IgG-peroxidase (A9044; Sigma-Aldrich) (GAPDH and total ERK 1:20,000) were used as secondary antibodies for immunoblotting. Real-time quantitative PCR Total RNA was isolated from dissected individual midguts and carcasses (all tissue remaining after dissection) using TRIzol reagent (Invitrogen) and genomic DNA was removed using TURBO DNA- free (Invitrogen). Quantitative RT-PCR was performed on an ABI Prism 7300 Sequence Detection System (Applied Biosystems). Primers and Taqman probes (Applied Biosystems) were designed to distinguish over-expressed alleles from endogenous A. gambiae MEK mRNA: MEK -RT forward, 5′CCGAGCAACATTCTTGTAAATAGCAGTGG3′; MEK -RT reverse, 5′AAGCGCTCGGGCGACATATAAC3′; S7 forward, 3′GAAGGCCTTCCAGAAGGTACAGA3′; S7 reverse 5′CATCGGTTTGGGCAGAATG3′; wtMEK probe, 6FAM-GATTCAATGGCCAATTCTTTTGTAGG-MGBNFQ; MEK2/5 probe, 6FAM-GATGAAATGGCCAATGATTTTGTAGG-MGBNFQ; and S7 probe, VIC-AGAAGTTCTCCGGCAAGCACGTCGT-6-carboxytetramethylrhodamine. Amplification conditions were defined as reverse transcription at 48°C for 30 min, AmpliTaq Gold activation at 95°C for 10 min, and then 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 1 min. Laboratory infection of mice with P. berghei and mosquito blood feeding Female CD1 mice were infected with P. berghei for transmission to A. gambiae . When parasitemia reached 5-10% of peripheral red blood cells (typically at 4 d post-infection), mice were anesthetized and exposed to mosquitoes for feeding. Thirty 3–5 d old F0 female mosquitoes transformed for midgut-specific overexpression of wtMEK, pMEK2 or pMEK5 were aspirated into individual cartons. Non-transformed A. gambiae females in a fourth carton served as an additional control. Mosquitoes were allowed to rest for 24 h and starved 2–4 h prior to blood feeding on anesthetized P. berghei- infected mice for 30 min. All non-blood fed females were removed from the containers using a mechanical aspirator while the remainder were maintained at 19°C and 80% humidity. At 12 d post-blood feeding, mosquito midguts were dissected in PBS and stained with mercurochrome for direct counting of P. berghei oocysts. Protocols involving the culture and handling of P. berghei were approved and in accord with regulatory guidelines and standards set by the Biological Safety Administrative Advisory Committee of the University of California, Davis. Experiments involving the use of animals were reviewed and deemed to be in accord with all relevant institutional policies and federal guidelines by the UC Davis Institutional Animal Care and Use Committee (protocol #17619, expiration 26 June 2014). Statistical analyses Differences in levels of ERK phosphorylation in Sua5B cells in vitro were analyzed by ANOVA (α = 0.05) for overall significance and by Bonferroni's test for pairwise comparisons of means. Differences in exogenous MEK allele expression in vivo were analyzed by ANOVA (α = 0.05) and by Bonferroni's test for pairwise comparisons of means. A Pearson's r test was performed to assess the relationship between docking site mutations and ERK phosphorylation levels in midgut tissue of transformed F0 females (α = 0.05). Significant differences in control (non-transformed mosquitoes) mean oocyst counts among replicates were determined by one-way ANOVA (α = 0.05); no differences were detected, so replicates were combined for analyses. Significant differences in oocyst counts from combined replicates were detected by unpaired t-tests (α = 0.05). All calculations were performed using GraphPad Prism version 5.02 for Windows (GraphPad Software, San Diego, California USA). Cell culture, mosquito rearing and mosquito feeding The immortalized A. gambiae Sua5B cell line [ 25 ] was maintained in Schneider's medium (Invitrogen) with 10% heat-inactivated fetal bovine serum at 28°C. Anopheles gambiae (G3 strain) mosquitoes were reared and maintained at 27°C and 75% humidity. Mosquitoes were maintained under a 12 h light/dark cycle. Mosquito eggs were placed in water and fed 0.2% baker's yeast on the day collected. After hatching, larvae were fed a mixture of liquid food containing 2% w/v powdered fish food (Sera Micron) and baker's yeast in a 2:1 ratio, and Game Fish Chow pellet food (Purina). Adult mosquitoes were maintained on 10% sucrose solution-soaked cotton pads. All mosquito-rearing protocols were approved and in accord with regulatory guidelines and standards set by the Institutional Animal Care and Use Committee of the University of California, Davis. For in vivo studies, 3–5 d old female mosquitoes were allowed to feed for 30 min on artificial blood meals of washed human erythrocytes and heat-inactivated human serum provided through a Hemotek Insect Feeding System (Discovery Workshops). MEK allele plasmid construction and transfection for in vitro studies The complete mRNA sequence of A. gambiae MEK [GenBank: XM_322064] in the pDREAM 2.1 vector (Genscript) (wild type MEK or wtMEK) was used to generate five additional plasmids encoding MEK mRNA with various combinations of SNPs: pMEK1, pMEK2, pMEK3, pMEK4 and pMEK5 (see Table 1 for a summary of these mutations). In brief, SNPs were introduced at codon positions 3 and 6 to convert lysines (K) to methionines (M) and at positions 243 and 247 to convert serines (S) to glutamic acid (E) and aspartic acid (D), respectively (Figure 1 ). Table 1 pMEK plasmid nucleotide changes to D-site lysines and catalytic site serines Domain Codon Position wtMEK pMEK1 pMEK2 pMEK3 pMEK4 pMEK5 Docking (D)-Site 3 AAA (K) AAA (K) AAA (K) ATG (M)* AAA (K) ATG (M)* 6 AAA (K) AAA (K) AAA (K) AAA (K) ATG (M)* ATG (M)* Catalytic 243 TCA (S) GAA (E)* GAA (E)* GAA (E)* GAA (E)* GAA (E)* 247 TCT (S) TCT (S) GAT (D)* GAT (D)* GAT (D)* GAT (D)* Mutations at positions 3 and 6 in the D-site were introduced to disrupt MEK-ERK interaction and corresponding phosphorylation of ERK. Mutations at positions 243 and 247 mimic MEK phosphorylation and, hence, activation. The codons are shown with encoded amino acids in parentheses. wtMEK = wild type MEK; amino acid substitutions are noted with asterisks. Figure 1 Amino acid alignment of human MEK1 and MEK2 with Anopheles gambiae MEK. Human (Hs) MEK1 and MEK2 and A. gambiae (Ag) MEK show significant overall conservation with high amino acid identity and similarity, including conservation in the docking site or D-site (blue box) and the catalytic domain (orange box). Lysine residues at positions 3 and 6 (yellow boxes) in the A. gambiae MEK allele were mutated to methionine (M). The key serine residues within the catalytic domain at positions 243 and 247 (red boxes) were mutated to aspartic acid (D) and glutamic acid (E), respectively. Human MEK1 [Genbank: NP_002746], human MEK2 [Genbank: NP_109587] and A. gambiae MEK1 [Genbank: XP_322064] protein sequences were aligned using the MUSCLE method with default settings in Geneious [ 26 ]. To introduce SNPs into the MEK -encoding sequence, paired synthetic primers that encoded the desired mutations were synthesized (See Table 2 for primer sequences; Sigma-Aldrich) and utilized for mutagenic primer-directed replication of both plasmid strands with high-fidelity PfuUltra DNA polymerase (Agilent). The following conditions were used for plasmid replication: 15–17 cycles of denaturation at 95°C for 30 sec, primer annealing for 1 min at 55°C, followed by extension at 68°C for 1 min per 1 kb amplified. The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA. The nicked synthesized plasmid DNAs with the desired mutations were transformed into E. coli TOP10 chemically competent cells (Invitrogen). Eight to ten transformed colonies for every desired mutation were screened for plasmid DNA using the Qiagen Miniprep Kit and the manufacturer's instructions (Qiagen). Among those, four to five plasmids were sequenced for confirmation of the introduced functional nucleotide changes (CDC Sequencing Facility, Fort Collins, CO). Table 2 Primer sequences for site-directed mutagenesis Plasmid constructs Resulting amino acid mutations Primer sequence 5′ ➔ 3′ Docking domain mutations pMEK3 K3M F: GACGACGACAAGATGAGTA TG ATGACAAAAAACAAACTTAA R: TTAAGTTTGTTTTTTGTCAT CA TACTCATCTTGTCGTCGTC pMEK4 K6M F: CAAGATGAGTAAAATGACAA TG AACAAACTTAATTTGACGTTG R: CAACGTCAAATTAAGTTTGTT CA TTGTCATTTTACTCATCTTG pMEK5 K3M F: GACGACGACAAGATGAGTA TG ATGACAA TG AACAAACTTAATTTGACGTTG K6M R: CAACGTCAAATTAAGTTTGTT CA TTGTCAT CA TACTCATCTTGTCGTCGTC Catalytic domain mutations pMEK1 S243E F: GATTGAT GA AATGGCCAATTCTTTTGTAGGTACTCGAAG R: CTTCGAGTACCTACAAAAGAATTGGCCATT TC ATCAATC pMEK2 S243E F: GATTTCGGCGTTTCCGGTCAGTTGATTGAT GA AATGGCCAAT GA TTTTGTAGGTACTCGAAG pMEK3 pMEK4 S247D R: CTTCGAGTACCTACAAAA TC ATTGGCCATT TC ATCAATCAACTGACCGGAAACGCCGAAATC pMEK5 Paired synthetic primers encoding the engineered SNPs (bold) were used to introduce mutations into the MEK -encoding sequence through mutagenic primer-directed replication of both plasmid strands. MEK -encoding plasmids were transfected into A. gambiae Sua5B cells using Effectene Reagent (Qiagen) and the manufacturer's recommended protocol. In brief, 1×10 6 Sua5B cells in 2 mL medium were plated in 6-well tissue culture plates overnight at 28°C. At 24 h after plating, cells were transfected with 0.6 μg of plasmid DNA and incubated at 28°C. At 36 h post-transfection, medium was removed and cells were washed with ice-cold phosphate buffered saline (PBS) in preparation for immunoblotting. MEK allele plasmid construction for in vivo studies and microinjection of female A. gambiae The plasmid for transgene overexpression in adult female A. gambiae was described previously [ 27 ]. To ensure midgut-specific expression of the transgene post-blood feeding, the A. gambiae carboxypeptidase promoter was engineered into the plasmid [ 28 , 29 ]. The MEK inserts wtMEK, pMEK2 and pMEK5 were cloned into the plasmid using 5′-PstI and 3′-SalI restriction sites. For each experiment, at least twenty laboratory reared 3–5 d old female A. gambiae were allowed to feed for 30 min on artificial blood meals at 16–24 h prior to MEK -encoding plasmid inoculation. For our studies, a mixture of 0.5 μg/μl MEK -encoding plasmid DNA, the in vivo transfection reagent jetPEI TM (Polyplus-transfection Inc.) and glucose at a final concentration of 5% [ 27 ] was injected into the hemocoel of vitellogenic females (0.1 to 0.5 μg DNA/female) using the Nanoject II Auto Nanoliter Injector (Drummond Scientific Company). At 24 h post injection, the mosquitoes were provided small cups of water for oviposition. F0 eggs were collected and reared through to the adult stage. Mosquito cell and tissue preparation and immunoblotting To harvest proteins from A. gambiae Sua5B cells, cells were lysed in 200 μl cell lysis buffer (10 mM Tris–HCl pH 7.4, 1 mM EDTA, 100 mM NaCl, 1 mM NaF, 1 mM EGTA, 2 mM Na 3 VO 4 20 mM Na 4 P 2 O 7 , 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol, 60 mg/mL aprotinin, 10 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mg/ml calyculin A). Cellular debris was removed by centrifugation at 14,000 × g for 10 min at 4°C. The resulting supernatants were mixed with Laemmli sample buffer (125 mM Tris–HCl pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue, 130 mM dithiothreitol) and the proteins were denatured at 95°C for 4 min prior to electrophoresis. Mosquito midguts were dissected into PBS and mixed to release blood, if any, by pipetting up and down. The midguts were washed in a filter column fitted with a fine mesh with a mixture of protease and phosphatase inhibitor cocktails (Sigma) in ice-cold PBS until all of the blood was removed. Fresh PBS mixture was added to loosen the midgut tissue from the filter, transferred to a fresh tube, and then centrifuged and prepared for electrophoresis as for cell culture lysate above. Proteins were separated on 10% SDS-PAGE polyacrylamide gels at 135 V for 1 h, 50 min. Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) for 1 h, 15 min at 7 V. Coomassie blue staining of the polyacrylamide gel was used to visually assess consistency of protein loading. Membranes were blocked in nonfat dry milk (5% w/v) in 1X Tris-buffered saline (TBS; pH 7.0) containing 0.1% Tween (TBS-T) for 1 h at room temperature, and then reacted overnight in primary antibody at 4°C. The membrane was washed 3 times, 5 min each with 1X TBS-T followed by incubation with appropriate secondary antibody 4°C overnight. The membrane was washed again 3 times, 5 min each with 1X TBS-T and then incubated in SuperSignal West Dura Extended Duration Substrate (Pierce). The Kodak Image Station 4000MM Pro Imaging System (Carestream Health, Inc.) was used to capture the image of the membrane and Quantity One (Bio-Rad Laboratories) software was used for densitometry analysis of the antibody-bound proteins. Levels of phosphorylated ERK (pERK) in Sua5B cells for each treatment were normalized to total ERK levels for protein loading and then normalized to pERK levels in the control cells transfected with wtMEK plasmid construct for in vitro experiments. Levels of pERK in the midgut for each group of A. gambiae were normalized to GAPDH levels and then to pERK levels in control mosquitoes transformed with wtMEK plasmid construct for in vivo experiments. Primary antibodies and dilutions included anti-FLAG-M2 (A2220; Sigma-Aldrich) (1:7,500), anti-GAPDH (G9545; Sigma-Aldrich) (1:10,000), anti-diphosphorylated ERK (pERK) (M8159; Sigma-Aldrich) (1:10,000) and anti-ERK1/2 (total ERK) (9102; Cell Signaling Technology) (1:1,250). Anti-rabbit IgG-peroxidase (A0545; Sigma-Aldrich) (pERK 1:20,000; FLAG 1:2,000) and anti-mouse IgG-peroxidase (A9044; Sigma-Aldrich) (GAPDH and total ERK 1:20,000) were used as secondary antibodies for immunoblotting. Real-time quantitative PCR Total RNA was isolated from dissected individual midguts and carcasses (all tissue remaining after dissection) using TRIzol reagent (Invitrogen) and genomic DNA was removed using TURBO DNA- free (Invitrogen). Quantitative RT-PCR was performed on an ABI Prism 7300 Sequence Detection System (Applied Biosystems). Primers and Taqman probes (Applied Biosystems) were designed to distinguish over-expressed alleles from endogenous A. gambiae MEK mRNA: MEK -RT forward, 5′CCGAGCAACATTCTTGTAAATAGCAGTGG3′; MEK -RT reverse, 5′AAGCGCTCGGGCGACATATAAC3′; S7 forward, 3′GAAGGCCTTCCAGAAGGTACAGA3′; S7 reverse 5′CATCGGTTTGGGCAGAATG3′; wtMEK probe, 6FAM-GATTCAATGGCCAATTCTTTTGTAGG-MGBNFQ; MEK2/5 probe, 6FAM-GATGAAATGGCCAATGATTTTGTAGG-MGBNFQ; and S7 probe, VIC-AGAAGTTCTCCGGCAAGCACGTCGT-6-carboxytetramethylrhodamine. Amplification conditions were defined as reverse transcription at 48°C for 30 min, AmpliTaq Gold activation at 95°C for 10 min, and then 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 1 min. Laboratory infection of mice with P. berghei and mosquito blood feeding Female CD1 mice were infected with P. berghei for transmission to A. gambiae . When parasitemia reached 5-10% of peripheral red blood cells (typically at 4 d post-infection), mice were anesthetized and exposed to mosquitoes for feeding. Thirty 3–5 d old F0 female mosquitoes transformed for midgut-specific overexpression of wtMEK, pMEK2 or pMEK5 were aspirated into individual cartons. Non-transformed A. gambiae females in a fourth carton served as an additional control. Mosquitoes were allowed to rest for 24 h and starved 2–4 h prior to blood feeding on anesthetized P. berghei- infected mice for 30 min. All non-blood fed females were removed from the containers using a mechanical aspirator while the remainder were maintained at 19°C and 80% humidity. At 12 d post-blood feeding, mosquito midguts were dissected in PBS and stained with mercurochrome for direct counting of P. berghei oocysts. Protocols involving the culture and handling of P. berghei were approved and in accord with regulatory guidelines and standards set by the Biological Safety Administrative Advisory Committee of the University of California, Davis. Experiments involving the use of animals were reviewed and deemed to be in accord with all relevant institutional policies and federal guidelines by the UC Davis Institutional Animal Care and Use Committee (protocol #17619, expiration 26 June 2014). Statistical analyses Differences in levels of ERK phosphorylation in Sua5B cells in vitro were analyzed by ANOVA (α = 0.05) for overall significance and by Bonferroni's test for pairwise comparisons of means. Differences in exogenous MEK allele expression in vivo were analyzed by ANOVA (α = 0.05) and by Bonferroni's test for pairwise comparisons of means. A Pearson's r test was performed to assess the relationship between docking site mutations and ERK phosphorylation levels in midgut tissue of transformed F0 females (α = 0.05). Significant differences in control (non-transformed mosquitoes) mean oocyst counts among replicates were determined by one-way ANOVA (α = 0.05); no differences were detected, so replicates were combined for analyses. Significant differences in oocyst counts from combined replicates were detected by unpaired t-tests (α = 0.05). All calculations were performed using GraphPad Prism version 5.02 for Windows (GraphPad Software, San Diego, California USA). Results S243E and S247D mutations in the catalytic core of A. gambiae MEK mimicked kinase activation in Sua5B cells in vitro The substitution mutations in A. gambiae MEK (S243E and S247D; Figure 1 , Table 1 ) resulted in negatively charged residues that mimic phosphorylation in the absence of exogenous stimuli and, therefore, mimicked kinase activation as described for the analogous mammalian MEK mutations S218E/S222D [ 30 , 31 ]. Specifically, A. gambiae Sua5B cells that were transfected with pMEK1 (S243E) or with pMEK2 (S243E/S247D) had 50-70% higher levels of ERK phosphorylation relative to cells transfected with wtMEK plasmid, which encoded the unaltered MEK allele (Figure 2 A). ERK phosphorylation levels were approximately 20% higher, albeit not significantly, in cells transfected with pMEK2 relative to cells transfected with pMEK1 (Figure 2 A), suggesting a modest additive effect of the activating mutations [ 30 , 31 ]. These increases in ERK phosphorylation were comparable to human TGF-β1-induced ERK phosphorylation in A. gambiae cells (70% to 2.5-fold above control) [ 3 ], but were substantially lower than those observed following analogous overexpression studies in mammalian cells. In particular, overexpression of human MEK S218E/S222D in human kidney 293 or monkey kidney COS-7 cells increased ERK activity by more than 100-fold above wild type MEK levels [ 30 , 31 ]. However, background ERK phosphorylation in the absence of stimulation in both 293 and COS-7 cells is nearly undetectable [ 30 , 31 ], whereas previously observed basal ERK phosphorylation levels in A. gambiae cells in the absence of treatment [ 3 ] are nearly comparable to levels following transfection with wtMEK (Figure 2 B). Figure 2 SNPs within MEK D-site and catalytic domain alter Anopheles gambiae ERK phosphorylation in vitro . A , ERK phosphorylation (pERK) levels in Sua5B cells transfected with pMEK1-5 were first normalized to total ERK, and then to pERK levels in control cells transfected with the unaltered allele (wtMEK; set here as 1.0). The introduction of S243E and S243E/S247D mutations in pMEK1 and pMEK2, respectively, increased pERK levels, although not significantly, in transfected cells relative to control cells. However, pERK levels in cells transfected with catalytically active MEK K3M/K6M (pMEK5) were significantly lower than levels observed in cells transfected with catalytically active pMEK2 (ANOVA, Bonferroni's test for pairwise comparisons, P < 0.05). Data are represented as means ± SEMs (N = 5). B , Representative western blots showing phosphorylated ERK, total ERK, and FLAG in Sua5B cells transfected with plasmids as in A. Detection of FLAG confirmed MEK overexpression. D-site mutations in catalytically active A. gambiae MEK reduced ERK phosphorylation in Sua5B cells in vitro Based on functional interactions of MEK and ERK in mammalian cells [ 16 ], we predicted that conserved lysine residues K3 and K6 encoded in the A. gambiae MEK D-site should interact directly with two conserved aspartic acids in the CD domain of ERK (Figure 3 ). As such, MEK S243E/S247D is catalytically active, but the addition of K3M and K6M mutations would be expected to block the interaction of activated MEK with ERK and, hence, block ERK activation (Table 1 ). While not significant, overexpression of catalytically active MEK K3M (pMEK3) or catalytically active MEK K6M (pMEK4) in A. gambiae Sua5B cells reduced ERK phosphorylation relative to cells overexpressing catalytically active MEK (pMEK2; Figure 2 A). Furthermore, overexpression of catalytically active MEK K3M/K6M (pMEK5) resulted in a significant reduction in ERK phosphorylation relative to cells that were transfected with pMEK2 (Figure 2 A), suggesting that D-site lysine residues in A. gambiae are essential for functional docking and phosphorylation of ERK by MEK. Figure 3 MEK-ERK signaling transmission is dependent on a docking interaction between key residues. A , The A. gambiae MEK (Ag-MEK) D-site shares similarities with human MEK D-sites (Hs-MEK1 and Hs-MEK2). The key D-site residues (blue boxes) are predicted to interact with aspartic acid residues within the CD-domain of ERK (green boxes). A. gambiae ERK [Genbank: XP_319983] and human ERK2 [Genbank: NP_002736] protein sequences were aligned using the MUSCLE method with default settings in Geneious [ 26 ]. B , A schematic illustration of MEK-ERK protein interaction. The key lysine residues within the MEK D-site interact with the aspartic acids within the CD-domain of ERK. Following the binding of the two proteins, MEK phosphorylates threonine and tyrosine within the activation motif of ERK. Transovarially acquired pMEK2 and pMEK5 resulted in midgut-biased transgene overexpression in F0 mosquitoes Consumption of blood initiate vitellogenesis in the female mosquito during which the fat body produces yolk protein precursors that are absorbed by the developing oocytes [ 32 , 33 ]. Peng et al . [ 27 ] exploited this physiology to develop a "vertical DNA vector delivery method" to transiently manipulate gene expression in F0 offspring of plasmid-injected female mosquitoes. We used this strategy to investigate whether MEK D-site mutations could alter ERK phosphorylation in the A. gambiae midgut. Specifically, pMEK2 and pMEK5 plasmids were microinjected into the hemocoels of vitellogenic female mosquitoes that had fed on blood 16–24 h earlier. Following injection, mosquitoes were allowed to oviposit and eggs (F0) were collected for rearing. To confirm midgut-specific overexpression of the variant MEK alleles, 4 d old F0 adult female offspring of plasmid-injected A. gambiae were allowed to feed on blood for 30 min. Age-matched F0 offspring from mosquitoes from the same cohorts that were not injected with plasmid and, hence, not transformed were fed alongside transformed F0 females as controls to assess specificity of transcript detection. At 2 h post-feeding, midguts from six female A. gambiae in each group were dissected into PBS. TaqMan qRT-PCR with probes specific to exogenous variant MEK alleles revealed no detectable signals from midguts or carcasses of non-transformed F0 females; hence, relative levels of endogenous MEK mRNA in non-transformed females (NT) were used for comparison to variant MEK allele expression in transformed F0 females (pMEK2, pMEK5; Figure 4 ). From replicated F0 cohorts, midgut expression of mRNAs encoding catalytically active MEK (pMEK2) and catalytically active MEK K3M/K6M (pMEK5) were detected at levels four- and three-fold higher ( P < 0.05) than endogenous MEK mRNA levels in non-transformed mosquitoes (Figure 4 ). Surprisingly, expression of exogenous variant MEK alleles in the carcass appeared to be equivalent to endogenous MEK mRNA levels in the same tissue in non-transformed F0 females (compare carcass levels of pMEK2 and pMEK5 to carcass levels of endogenous MEK in non-transformed females; Figure 4 ). While variant MEK allele probes specifically detected transcripts in transformed mosquitoes, we cannot exclude the possibility that some level of endogenous MEK mRNA is also detected by these probes, which would explain signal detection in the carcass of transformed F0 females despite use of a midgut-specific promoter [ 29 , 34 ]. Figure 4 Midgut-specific overexpression of exogenous variant MEK alleles was confirmed by qRT-PCR. Total RNA was isolated from the midguts or carcasses of non-transformed (NT) female mosquitoes and female mosquitoes transformed with either pMEK2 or pMEK5 for qRT-PCR with probes that distinguished between exogenous variant and endogenous MEK alleles. Relative to levels of endogenous MEK mRNA in NT females, expression levels of exogenous variant MEK allele were four- or three-fold higher ( P < 0.05) in midguts of females transformed with plasmids encoding catalytically active MEK (pMEK2) or catalytically active MEK K3M/K6M (pMEK5), respectively. Variant MEK allele expression levels in the carcass of transformed mosquitoes were comparable to endogenous MEK allele expression levels in NT female mosquitoes. Five independent mosquito cohorts were used in this experiment. Midgut-directed overexpression of MEK alleles with D-site polymorphisms decreased ERK phosphorylation Based on midgut-biased variant MEK mRNA expression in transformed F0 mosquitoes (Figure 4 ), we examined this tissue from transformed F0 females for relative levels of ERK phosphorylation. At 2 h post-blood meal, 30–45 midguts from each group were dissected, pooled and processed for immunoblotting (Figure 5 A). As shown in Figure 5 B, the response to pMEK2 transformation was correlated with the response to pMEK5 across replicates (r = 0.7799). In particular, 4 of 5 replicates showed a reduction in ERK phosphorylation levels in midgut epithelia of mosquitoes that overexpressed the catalytically active but docking deficient MEK (pMEK5) relative to levels in mosquitoes that overexpressed catalytically active MEK (pMEK2; P = 0.06). We reasoned that a mean reduction of approximately 23% in phosphorylated ERK levels for 4 of 5 replicates, although marginally not significant, could be biologically significant given our previous observations that incomplete inhibition of TGF-β1-induced midgut ERK phosphorylation by the MEK inhibitor PD98059 [ 21 ] could result in significant inhibition of P. falciparum growth in A. stephensi . Figure 5 D-site mutations were correlated with repressed midgut ERK phosphorylation levels in A. gambiae compared to mosquitoes transformed with catalytically active MEK. A , Representative western blot of ERK phosphorylation in midgut epithelia from transformed mosquitoes examined at 2 h after blood feeding. Values below the blots indicate relative fold change in pERK levels in the midgut for each group of A. gambiae . Phosphorylated ERK levels were normalized to GAPDH and then to pERK levels in wtMEK, which is set at 1.00. B , Fold changes in ERK phosphorylation levels are indicated as trendlines between samples from matched treatment groups for pMEK2 and pMEK5 overexpression. Note that in one replicate, relative values of phosphorylation for pMEK2 and pMEK5 transformed A. stephensi were below the level of controls (e.g., < 1), but nonetheless revealed the same trend of decreasing phosphorylation. A Pearson's r test was performed to assess the relationship between docking site mutations and ERK phosphorylation levels in midgut tissue of transformed F0 females (r = 0.7799, P = 0.06). Midgut-directed overexpression of MEK alleles with D-site polymorphisms decreased P. berghei development in A. gambiae To determine whether overexpression of catalytically active MEK with D-site mutations would result in an infection phenotype similar to that induced by PD98059 inhibition of MEK [ 21 ], we allowed 3–5 d old female mosquitoes transformed for midgut-specific overexpression of wtMEK, pMEK2 or pMEK5 to feed on P. berghei -infected mice for 30 min (Figure 6 ). As expected, mosquitoes overexpressing catalytically active MEK (pMEK2) developed a significantly greater number of oocysts per midgut (42.2 ± 8.0) than did mosquitoes overexpressing wtMEK (14.1 ± 1.2 oocysts; P = 0.03). Oocyst counts in mosquitoes overexpressing wtMEK were not significantly different from non-transformed (NT) mosquitoes ( P = 0.11). The introduction of D-site mutations (pMEK5) reduced oocyst development (28.0 ± 5.8 oocysts) relative to mosquitoes transformed with pMEK2 ( P = 0.07) to levels that were not different from mosquitoes overexpressing wtMEK ( P = 0.11) and non-transformed mosquitoes ( P = 0.48). These results confirm our previous observations that MEK-ERK signaling regulates parasite development and indicate that this regulation is dependent on MEK docking functionality. Figure 6 Midgut-directed overexpression of MEK alleles with D-site polymorphisms decreased P. berghei oocyst numbers in A. gambiae females in vivo. We allowed 3–5 d old female mosquitoes transformed to overexpress wtMEK, pMEK2 or pMEK5 to feed on P. berghei -infected mice. Twelve days after infection, mosquito midguts were dissected and P. berghei oocysts were counted. The experiment was replicated three times. There were no significant differences in NT mean oocyst counts among replicates (one-way ANOVA), so replicates were combined for analysis. Midgut-specific overexpression of catalytically active MEK (pMEK2) resulted in a significant increase in the number of oocysts per midgut compared to mosquitoes overexpressing wtMEK ( P = 0.03). Mutation of the key residues in the D-site (pMEK5), however, resulted in oocyst development that was not significantly different from NT females (P = 0.48) and females transformed with wtMEK ( P = 0.11). S243E and S247D mutations in the catalytic core of A. gambiae MEK mimicked kinase activation in Sua5B cells in vitro The substitution mutations in A. gambiae MEK (S243E and S247D; Figure 1 , Table 1 ) resulted in negatively charged residues that mimic phosphorylation in the absence of exogenous stimuli and, therefore, mimicked kinase activation as described for the analogous mammalian MEK mutations S218E/S222D [ 30 , 31 ]. Specifically, A. gambiae Sua5B cells that were transfected with pMEK1 (S243E) or with pMEK2 (S243E/S247D) had 50-70% higher levels of ERK phosphorylation relative to cells transfected with wtMEK plasmid, which encoded the unaltered MEK allele (Figure 2 A). ERK phosphorylation levels were approximately 20% higher, albeit not significantly, in cells transfected with pMEK2 relative to cells transfected with pMEK1 (Figure 2 A), suggesting a modest additive effect of the activating mutations [ 30 , 31 ]. These increases in ERK phosphorylation were comparable to human TGF-β1-induced ERK phosphorylation in A. gambiae cells (70% to 2.5-fold above control) [ 3 ], but were substantially lower than those observed following analogous overexpression studies in mammalian cells. In particular, overexpression of human MEK S218E/S222D in human kidney 293 or monkey kidney COS-7 cells increased ERK activity by more than 100-fold above wild type MEK levels [ 30 , 31 ]. However, background ERK phosphorylation in the absence of stimulation in both 293 and COS-7 cells is nearly undetectable [ 30 , 31 ], whereas previously observed basal ERK phosphorylation levels in A. gambiae cells in the absence of treatment [ 3 ] are nearly comparable to levels following transfection with wtMEK (Figure 2 B). Figure 2 SNPs within MEK D-site and catalytic domain alter Anopheles gambiae ERK phosphorylation in vitro . A , ERK phosphorylation (pERK) levels in Sua5B cells transfected with pMEK1-5 were first normalized to total ERK, and then to pERK levels in control cells transfected with the unaltered allele (wtMEK; set here as 1.0). The introduction of S243E and S243E/S247D mutations in pMEK1 and pMEK2, respectively, increased pERK levels, although not significantly, in transfected cells relative to control cells. However, pERK levels in cells transfected with catalytically active MEK K3M/K6M (pMEK5) were significantly lower than levels observed in cells transfected with catalytically active pMEK2 (ANOVA, Bonferroni's test for pairwise comparisons, P < 0.05). Data are represented as means ± SEMs (N = 5). B , Representative western blots showing phosphorylated ERK, total ERK, and FLAG in Sua5B cells transfected with plasmids as in A. Detection of FLAG confirmed MEK overexpression. D-site mutations in catalytically active A. gambiae MEK reduced ERK phosphorylation in Sua5B cells in vitro Based on functional interactions of MEK and ERK in mammalian cells [ 16 ], we predicted that conserved lysine residues K3 and K6 encoded in the A. gambiae MEK D-site should interact directly with two conserved aspartic acids in the CD domain of ERK (Figure 3 ). As such, MEK S243E/S247D is catalytically active, but the addition of K3M and K6M mutations would be expected to block the interaction of activated MEK with ERK and, hence, block ERK activation (Table 1 ). While not significant, overexpression of catalytically active MEK K3M (pMEK3) or catalytically active MEK K6M (pMEK4) in A. gambiae Sua5B cells reduced ERK phosphorylation relative to cells overexpressing catalytically active MEK (pMEK2; Figure 2 A). Furthermore, overexpression of catalytically active MEK K3M/K6M (pMEK5) resulted in a significant reduction in ERK phosphorylation relative to cells that were transfected with pMEK2 (Figure 2 A), suggesting that D-site lysine residues in A. gambiae are essential for functional docking and phosphorylation of ERK by MEK. Figure 3 MEK-ERK signaling transmission is dependent on a docking interaction between key residues. A , The A. gambiae MEK (Ag-MEK) D-site shares similarities with human MEK D-sites (Hs-MEK1 and Hs-MEK2). The key D-site residues (blue boxes) are predicted to interact with aspartic acid residues within the CD-domain of ERK (green boxes). A. gambiae ERK [Genbank: XP_319983] and human ERK2 [Genbank: NP_002736] protein sequences were aligned using the MUSCLE method with default settings in Geneious [ 26 ]. B , A schematic illustration of MEK-ERK protein interaction. The key lysine residues within the MEK D-site interact with the aspartic acids within the CD-domain of ERK. Following the binding of the two proteins, MEK phosphorylates threonine and tyrosine within the activation motif of ERK. Transovarially acquired pMEK2 and pMEK5 resulted in midgut-biased transgene overexpression in F0 mosquitoes Consumption of blood initiate vitellogenesis in the female mosquito during which the fat body produces yolk protein precursors that are absorbed by the developing oocytes [ 32 , 33 ]. Peng et al . [ 27 ] exploited this physiology to develop a "vertical DNA vector delivery method" to transiently manipulate gene expression in F0 offspring of plasmid-injected female mosquitoes. We used this strategy to investigate whether MEK D-site mutations could alter ERK phosphorylation in the A. gambiae midgut. Specifically, pMEK2 and pMEK5 plasmids were microinjected into the hemocoels of vitellogenic female mosquitoes that had fed on blood 16–24 h earlier. Following injection, mosquitoes were allowed to oviposit and eggs (F0) were collected for rearing. To confirm midgut-specific overexpression of the variant MEK alleles, 4 d old F0 adult female offspring of plasmid-injected A. gambiae were allowed to feed on blood for 30 min. Age-matched F0 offspring from mosquitoes from the same cohorts that were not injected with plasmid and, hence, not transformed were fed alongside transformed F0 females as controls to assess specificity of transcript detection. At 2 h post-feeding, midguts from six female A. gambiae in each group were dissected into PBS. TaqMan qRT-PCR with probes specific to exogenous variant MEK alleles revealed no detectable signals from midguts or carcasses of non-transformed F0 females; hence, relative levels of endogenous MEK mRNA in non-transformed females (NT) were used for comparison to variant MEK allele expression in transformed F0 females (pMEK2, pMEK5; Figure 4 ). From replicated F0 cohorts, midgut expression of mRNAs encoding catalytically active MEK (pMEK2) and catalytically active MEK K3M/K6M (pMEK5) were detected at levels four- and three-fold higher ( P < 0.05) than endogenous MEK mRNA levels in non-transformed mosquitoes (Figure 4 ). Surprisingly, expression of exogenous variant MEK alleles in the carcass appeared to be equivalent to endogenous MEK mRNA levels in the same tissue in non-transformed F0 females (compare carcass levels of pMEK2 and pMEK5 to carcass levels of endogenous MEK in non-transformed females; Figure 4 ). While variant MEK allele probes specifically detected transcripts in transformed mosquitoes, we cannot exclude the possibility that some level of endogenous MEK mRNA is also detected by these probes, which would explain signal detection in the carcass of transformed F0 females despite use of a midgut-specific promoter [ 29 , 34 ]. Figure 4 Midgut-specific overexpression of exogenous variant MEK alleles was confirmed by qRT-PCR. Total RNA was isolated from the midguts or carcasses of non-transformed (NT) female mosquitoes and female mosquitoes transformed with either pMEK2 or pMEK5 for qRT-PCR with probes that distinguished between exogenous variant and endogenous MEK alleles. Relative to levels of endogenous MEK mRNA in NT females, expression levels of exogenous variant MEK allele were four- or three-fold higher ( P < 0.05) in midguts of females transformed with plasmids encoding catalytically active MEK (pMEK2) or catalytically active MEK K3M/K6M (pMEK5), respectively. Variant MEK allele expression levels in the carcass of transformed mosquitoes were comparable to endogenous MEK allele expression levels in NT female mosquitoes. Five independent mosquito cohorts were used in this experiment. Midgut-directed overexpression of MEK alleles with D-site polymorphisms decreased ERK phosphorylation Based on midgut-biased variant MEK mRNA expression in transformed F0 mosquitoes (Figure 4 ), we examined this tissue from transformed F0 females for relative levels of ERK phosphorylation. At 2 h post-blood meal, 30–45 midguts from each group were dissected, pooled and processed for immunoblotting (Figure 5 A). As shown in Figure 5 B, the response to pMEK2 transformation was correlated with the response to pMEK5 across replicates (r = 0.7799). In particular, 4 of 5 replicates showed a reduction in ERK phosphorylation levels in midgut epithelia of mosquitoes that overexpressed the catalytically active but docking deficient MEK (pMEK5) relative to levels in mosquitoes that overexpressed catalytically active MEK (pMEK2; P = 0.06). We reasoned that a mean reduction of approximately 23% in phosphorylated ERK levels for 4 of 5 replicates, although marginally not significant, could be biologically significant given our previous observations that incomplete inhibition of TGF-β1-induced midgut ERK phosphorylation by the MEK inhibitor PD98059 [ 21 ] could result in significant inhibition of P. falciparum growth in A. stephensi . Figure 5 D-site mutations were correlated with repressed midgut ERK phosphorylation levels in A. gambiae compared to mosquitoes transformed with catalytically active MEK. A , Representative western blot of ERK phosphorylation in midgut epithelia from transformed mosquitoes examined at 2 h after blood feeding. Values below the blots indicate relative fold change in pERK levels in the midgut for each group of A. gambiae . Phosphorylated ERK levels were normalized to GAPDH and then to pERK levels in wtMEK, which is set at 1.00. B , Fold changes in ERK phosphorylation levels are indicated as trendlines between samples from matched treatment groups for pMEK2 and pMEK5 overexpression. Note that in one replicate, relative values of phosphorylation for pMEK2 and pMEK5 transformed A. stephensi were below the level of controls (e.g., < 1), but nonetheless revealed the same trend of decreasing phosphorylation. A Pearson's r test was performed to assess the relationship between docking site mutations and ERK phosphorylation levels in midgut tissue of transformed F0 females (r = 0.7799, P = 0.06). Midgut-directed overexpression of MEK alleles with D-site polymorphisms decreased P. berghei development in A. gambiae To determine whether overexpression of catalytically active MEK with D-site mutations would result in an infection phenotype similar to that induced by PD98059 inhibition of MEK [ 21 ], we allowed 3–5 d old female mosquitoes transformed for midgut-specific overexpression of wtMEK, pMEK2 or pMEK5 to feed on P. berghei -infected mice for 30 min (Figure 6 ). As expected, mosquitoes overexpressing catalytically active MEK (pMEK2) developed a significantly greater number of oocysts per midgut (42.2 ± 8.0) than did mosquitoes overexpressing wtMEK (14.1 ± 1.2 oocysts; P = 0.03). Oocyst counts in mosquitoes overexpressing wtMEK were not significantly different from non-transformed (NT) mosquitoes ( P = 0.11). The introduction of D-site mutations (pMEK5) reduced oocyst development (28.0 ± 5.8 oocysts) relative to mosquitoes transformed with pMEK2 ( P = 0.07) to levels that were not different from mosquitoes overexpressing wtMEK ( P = 0.11) and non-transformed mosquitoes ( P = 0.48). These results confirm our previous observations that MEK-ERK signaling regulates parasite development and indicate that this regulation is dependent on MEK docking functionality. Figure 6 Midgut-directed overexpression of MEK alleles with D-site polymorphisms decreased P. berghei oocyst numbers in A. gambiae females in vivo. We allowed 3–5 d old female mosquitoes transformed to overexpress wtMEK, pMEK2 or pMEK5 to feed on P. berghei -infected mice. Twelve days after infection, mosquito midguts were dissected and P. berghei oocysts were counted. The experiment was replicated three times. There were no significant differences in NT mean oocyst counts among replicates (one-way ANOVA), so replicates were combined for analysis. Midgut-specific overexpression of catalytically active MEK (pMEK2) resulted in a significant increase in the number of oocysts per midgut compared to mosquitoes overexpressing wtMEK ( P = 0.03). Mutation of the key residues in the D-site (pMEK5), however, resulted in oocyst development that was not significantly different from NT females (P = 0.48) and females transformed with wtMEK ( P = 0.11). Discussion In this study, we used a vertical DNA vector delivery method adapted from Peng et al . [ 27 ] to demonstrate that mutations in the D-site of MEK disrupt downstream ERK phosphorylation in A. gambiae cells in vitro and in vivo . Further, these mutations can alter the success of malaria parasite infection in transformed F0 mosquitoes. Specifically, K3M and K6M mutations in the D-site (pMEK5; Table 1 ) significantly repressed ERK phosphorylation relative to cells or tissue in which catalytically active MEK was overexpressed (pMEK2; Figures 2 , 5 ). These data suggest that the MEK D-site has conserved functionality in an insect of public health importance. Because of the critical involvement of MEK-ERK signaling in a number of chronic diseases and cancer, there has been significant interest in therapeutic approaches to disrupt the docking interaction of MEK and ERK, including the use of small molecule inhibitors [ 35 ], the introduction of mutations into the MEK D-site, and transfection of short blocking peptides that bind to the CD domain of ERK [ 36 ]. In mammals, disruption of the MEK D-site can also facilitate immune evasion by pathogens. For example, the anthrax lethal factor (ALF) of Bacillus anthracis impairs host cell immune activation during early infection through cleavage of the MEK D-site by anthrax lethal protease [ 37 - 39 ]. In the context of cancer, ALF-mediated cleavage of the D-site regulates both cell survival and growth. Specifically, ALF-mediated inhibition of MAPK signaling can trigger melanoma cell apoptosis and suppress the progression of renal cell carcinoma [ 40 - 42 ]. From these studies, we suggest that enhanced disruption of the D-site of MEK as a strategy to inhibit disease-associated MEK-ERK signaling could be applied to the development of transmission blocking strategies for malaria. Our engineered mutations in the MEK D-site did not appear to exert a dominant negative effect, but induced an incomplete albeit functional block of ERK phosphorylation, much like the use of small molecule MEK inhibitors. Based on these observations, we suggest that mutated MEK did not wholly outcompete endogenous MEK to block signal transmission. Alternatively, inhibition may have been more effective than was apparent because of feedback regulation. In particular, the MEK inhibitor PD98059 can interfere with ERK-dependent negative feedback regulation. Specifically, the Grb2-SOS complex is recruited to activate membrane-bound Ras, but ERK phosphorylation of SOS causes the complex to dissociate [ 43 ]. Following treatment with PD98059, ERK-dependent SOS phosphorylation is blocked, resulting in prolonged Ras activation in insulin- and epidermal growth factor (EGF)-treated human insulin receptor expressing rat cells [ 44 ]. Thus, blocking the signaling interaction of MEK and ERK can impair feedback regulation to partially restore ERK phosphorylation. In addition to canonical signaling through Ras-Raf-MEK, networked signaling pathways can provide alternative targets for manipulation of ERK phosphorylation. For example, treatment of human breast cancer T47D cells with the MEK inhibitor U0126 in combination with the phosphoinositide 3-kinase/Akt inhibitor wortmannin synergistically suppressed EGF-induced ERK activation relative to treatment with U0126 alone [ 45 ]. Similarly, overexpression of phosphatase and tensin homolog (PTEN), which functions as an endogenous inhibitor of Akt, reduced basal levels of midgut ERK phosphorylation relative to control A. stephensi , but had no effect on insulin-induced ERK activation relative to controls [ 34 ], suggesting that inhibition of Akt-dependent ERK signaling could be targeted to additively reduce ERK activation in the mosquito. The activation of endogenous ERK inhibitors has also proved successful in suppressing ERK activation with measurable biological effects. For example, overexpression of MAPK phosphatase MKP-3, which specifically targets ERK for dephosphorylation, induced hepatic gluconeogenesis and increased fasting blood glucose levels in lean mice, suggesting that MKP-3 could be targeted therapeutically for type 2 diabetes [ 46 ]. Similarly, Drosophila melanogaster MKP-3 is an endogenous regulator of ERK phosphorylation that is indispensable to fly embryonic development [ 47 ], indicating that genetic manipulation of MKP-3 can provide highly conserved control of important biological responses to ERK phosphorylation. Finally, the MKPs are an intense focus of development of therapeutic small molecule inhibitors and activators given the critical roles for MAPKs in various chronic and inflammatory human diseases [ 48 ], suggesting that analogous discovery and applications for enhancing MKP activity in mosquitoes are both possible and likely to be successful. Naturally occurring SNPs within A. gambiae immune genes have been found to be associated with parasite infection, including TOLL5B and ILP3 [ 49 ] as well as Sp SNAKElike and TOLL6 [ 50 ]. Indeed, it has been proposed that a breakdown in mosquito innate immunity is responsible for susceptibility to parasite infection [ 51 ], raising the possibility of undertaking studies searching for naturally occurring mutations in immune signaling genes. Most recently, Li et al. [ 52 ] identified nonsynonymous SNPs in A. gambiae adenosine deaminase ( AgADA ) , fibrinogen-related protein 1 ( FREP1 ) and fibrinogen-related protein 30 ( FBN30 ). RNAi-mediated silencing of FREP1 resulted in a significant decrease in infection prevalence while FBN30 transcript ablation increased infection intensity two-fold relative to controls [ 52 ]. While these association studies support the role of natural genetic variation in the control of Plasmodium development, mutations such as these have thus far been used only to identify target genes for which to query the effects of RNAi-mediated silencing on infection phenotype. However, many immune gene products require protein-protein interaction to form complexes or post-translational modification to mediate specific cellular functions. As such, the work presented here indicates that functional SNP studies can be extended to determine the mechanisms by which coding sequence mutations specifically impact infection phenotype. Conclusion We have established proof-of-principle for the functional analysis of SNPs on protein function and infection susceptibility in A. gambiae . In particular, we have demonstrated that engineered mutations in A. gambiae MEK recapitulate the effects of small molecule inhibition of MEK-ERK signaling in mosquito cells and on parasite infection [ 20 , 21 ]. In addition to proof-of-principle, the interruption of MEK-ERK signaling via engineered D-site mutations can be translated for the development of transgenic A. gambiae that are resistant to malaria parasite development and transmission. Competing interests The authors declare that they have no competing interests. Authors' contributions AAB and SL conceived and designed the experiments of the study. MC and ACB performed mutagenesis to generate the SNP-encoding MEK plasmids. AAB carried out the experiments in vitro and KC carried out the experiments in vivo . SL and LS conducted statistical analyses. SL and LS prepared the manuscript with AAB. All authors read and approved the final version of the manuscript. Acknowledgments This project support was provided by the National Institutes of Health National Institute of Allergy and Infectious Diseases R01 AI078183 (to SL), R01 AI080799 (to SL), T32 AI074550 (to AAB, LS). We thank Dr. Que Lan and her laboratory personnel (University of Wisconsin) for their patience in training us in the technique of plasmid microinjection and we regret that Dr. Lan's untimely passing prevented us from sharing our success with her.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3615472/

Effective localized collection and identification of airborne species through electrodynamic precipitation and SERS-based detection

Various nanostructured sensor designs currently aim to achieve or claim single molecular detection by a reduction of the active sensor size. However, a reduction of the sensor size has the negative effect of reducing the capture probability considering the diffusion-based analyte transport commonly used. Here we introduce and apply a localized programmable electrodynamic precipitation concept as an alternative to diffusion. The process provides higher collection rates of airborne species and detection at lower concentration. As an example, we compare an identical nanostructured surfaced-enhanced Raman spectroscopy sensor with and without localized delivery and find that the sensitivity and detection time is improved by at least two orders of magnitudes. Localized collection in an active-matrix array-like fashion is also tested, yielding hybrid molecular arrays on a single chip over a broad range of molecular weights, including small benzenethiol (110.18 Da) and 4-fluorobenzenethiol (128.17 Da), or large macromolecules such as anti-mouse IgG (~150 kDa). Results Advanced collection, spotting and detection Figure 1 depicts the investigated design elements and test structures, which were evaluated to spot and collect airborne species on a nanostructured SERS gas sensor substrate. Figure 1a contrasts the state-of-the-art diffusion-based delivery concept with basic electrostatic precipitation, where an external bias and charged molecules are tested to increase the collection efficiency. Figure 1c adds an additional design element—a charged photoresist layer with a circular opening; the goal of this structure is to form an electrodynamic nanolens to funnel and concentrate the airborne species at a predetermined location on the nanostructured sensor surface. The use of a gas-phase nanolens focusing effect has been reported recently in the context of inorganic nanoparticles 23 24 25 26 that contained several 100,000 atoms, which contrasts with the small molecules tested here that contain less than 20 atoms. The general idea of a nanolens is to use a charged resist, which influences the trajectory of charged material. The resist serves as insulation and blocks charge dissipation. The opening to the conductor provides the only location where a charged material flux can be established under steady-state conditions. Figure 1d depicts an additional modification to achieve programmability of more than one analyte. The approach uses electrically separated metallic domains (yellow cap layers) underneath the nanolens array. The fabrication of the depicted test structures is detailed in the methods section. In short, we used a Langmuir–Blodgett method 27 to apply a closely packed layer of silica spheres, 200 nm in diameter, over extended areas on the glass surface. Next, e-beam evaporation is used to coat the top with 20 and 180 nm of chromium and silver, respectively. This yields the SERS sensing surface commonly known in the literature as AgFON standard. The silver film provides a conductive layer. This conductive layer allows for the application of an external bias voltage, which is used to evaluate if a field driven approach can increase the collection efficiency of charge molecules when compared with prior concepts 14 15 , where the rate of absorption was driven by diffusion and the substrate was left floating. Prior methods were able to detect BTs at a concentration of 6 p.p.m. in 1 s. The second modification ( Fig. 1c ) adds the depicted 500-nm thick spin-coated photoresist layer whereby a 1 μm in diameter and 3 μm pitched hole pattern is defined by photolithography. The final SERS gas sensor substrate ( Fig. 1d ) is composed of two silver domains instead of one. The two domains are separated using a 50-μm wide region of uncoated silica spheres, which was masked by placing a capillary onto the surface prior metallization. All other parameters were left the same. The deposition on the individual domains is controlled using external bias voltages in the range of −50 to −200 V, whereby the electrometers records the deposition current in the range of 1–5 nA. The flux is recorded using electrometers (Keithley 6517A) marked with the letter 'A'. Domains are turned ON and OFF sequentially for selected periods of time (10 s to 1 min) to collect molecular ions including BT + and 4-FBT + on domain 1 and 2, respectively. We choose these two molecules as they have a well-characterized and known Raman scattering signal 14 28 . The process is, however, not limited to these types of molecules or molecular weights. Results for anti-mouse IgG proteins (Sigma-Aldrich, F-0257) will be presented as well. Electrospray testing environment Figure 2 depicts the details of the testing environment where the analytes are evaporated and charged using an electrospray ionization standard and where programmable analyte collection is achieved using biased domain electrodes. Electrospray ionization is chosen as it provides a controlled environment to produce airborne analytes of almost any type and concentration. Electrospray ionization systems are used in mass spectroscopy in the fields of chemical-warfare agent detection 29 and proteomics 30 . They require exceptionally small amounts of diluted analytes. For example at a common flow rate, only 50 nl is drawn through the capillary per minute. For a review, we refer to Cloupeau and Prunet-Foch 31 and Ganan-Calvo et al. 32 . In brief, we constructed a system based on a commercially available electrospray ionization system (TSI Inc., Model 3480). It consists of a high-voltage source, pressure regulator, pressure chamber, capillary and electrospray chamber. The pressure chambers house a centrifuge vial, a high-voltage platinum electrode, and a fused silica capillary, which carries the solution out into the electrospray chamber using 1.25 atm pressure. A positive electrospray voltage is increased until the extruded liquid (50 nl per min) forms a cone shape, known as cone-jet mode 33 , which leads to rapid evaporation in close proximity to the orifice and an aerosol containing charged molecules (light green for 4-FBT, and light blue for BT). Figure 2a depicts the first analyte collection sequence where BT ions (BT + ) are collected on domain 1 which is turned ON by applying a negative voltage V d =−100 V to the domain electrode. Domain 2 is switched into a floating state, whereby the domain electrode is disconnected to prevent dissipation of charge, which in turn blocks the collection on domain 2. The following conditions were used to prepare the depicted ionized aerosol containing charged BTs. The starting point is a BT solution (liquid density=1.073 g/ml, molecular weight=110 g mol −1 ), which is diluted in a 1:1 volume ratio with ethanol; second, the solution is sprayed at a rate of 50 nl/min, which translates to 2.43 × 10 −7 N A BT molecules per minute, where N A is Avogadro's constant; third, it is mixed with 1 liter per minute nitrogen carrier gas at 1.25 atm by adding 4.1 × 10 −2 N A nitrogen molecules per minute to the mixture; at this stage the analyte is diluted down to 5.9 p.p.m. In positive ion mode, the process produces positively charged molecules in addition to neutral molecules 34 . Of importance are BT ions (C 6 H 5 SH 2 + ), 4-FBT (C 6 H 4 FSH 2 + ), solvent ions (C 2 H 5 OH 2 + ) and nitrogen ions (N 2 + ). Figure 2b illustrates a second analyte collection sequence where 4-FBT + is collected on domain 2 and where domain 1 is left floating. The preparation approach for 4-fluorothiophenol (4-FBT) followed the same procedure as described before in the case of BT. The illustration in Fig. 2 under-represents the amount of neutral species for clarity. However, it is important to mention that most gas molecules are neutral and that the fraction of charged material is small. For example, a typical electrospray current is 100 nA, which is measured using the ampere metre marked with the letter 'A', which is connected to the platinum electrode that is immersed in the vial; 100 nA electrospray current translates into an ion current flux of 6.23 × 10 −11 N A elementary charges per minute. This ion flux is three orders of magnitudes smaller than the previously calculated 2.43 × 10 −7 N A BT molecules and nine orders of magnitudes smaller than 4.1 × 10 −2 N A nitrogen gas molecules contained in 1 l per min carrier gas. In other words, the gas mixture is composed of both charged (<1.5 p.p.b.) and neutral BT (<5.9 p.p.m.) molecules. The gas mixture exits the enclosed system through a 0.5 mm in diameter orifice and expands until it reaches the substrate, which is placed 5 cm away. The p.p.b. and p.p.m. estimates are upper limits and the actual concentration of charged and neutral molecules is smaller due to downstream mixture and charge exchange processes, which will be discussed further in the discussion section. Collection based on different mechanisms Figure 3 compares the recorded SERS signals using the floating, biased plate and biased nanolens collection approach after the test substrates were exposed for 30 s. The gas mixture exiting the electrospray system containing both charged (<1.5 p.p.b.) and neutral BT (<5.9 p.p.m.) molecules in nitrogen. All SERS spectra in Fig. 3c were recorded under identical exposure and recording conditions, which is important as this allows a relative comparison of the signal intensity for a sensor system with and without charge directed collection. For a standard SERS substrate, hotspots are randomly located on the surface ( Fig. 3b ) and it is hard to point out where the molecule is. The nanolens-based collection approach eliminates this uncertainty as molecules are collected at predetermined points ( Fig. 3c ). This helps in the data collection. In the particular case, all SERS spectra were recorded as an average over the indicated white dashed areas; no voltage is applied during the recording of the SERS data. The spectrum for the unbiased case shows a weak signal and the detection of the uncharged BT is difficult at 6 p.p.m. in our system; the characteristic peak at 1,075 cm −1 is recorded with 0.4 counts per unit area (inset of Fig. 3c ), which means very few BT molecules are collected on this substrate. The signal increases to 103 counts per unit area for the biased AgFON substrate and 246 counts per unit area for the biased AgFON substrate with integrated nanolens array, which represents a factor of 615 comparing the biased nanolens array with the unbiased AgFON standard. We repeated this experiment five times using separate substrates. The nanolens-based collection region produced the highest counts in each case; the intensity factor varied by 6% standard deviation (STD) between the experiments. The recorded increase of 615 is even more impressive if one considers that it is caused by 3,900 times fewer charged analyte molecules (1.5 p.p.b.) than neutral ones (5.9 p.p.m.). In other words, trace amounts of charged molecules at a concentration of 1.5 p.p.b. (parts per billion) lead to 246 counts per unit area, while 5.9 p.p.m. (parts per million) of neutral particles contribute to 0.4 counts, which means that the capture efficiencies of charged molecules is 2.4 × 10 6 times larger than neutral ones. The six orders of magnitude-higher capture efficiencies of charged molecules when compared with neutral one is an important metric, as it provides a route to higher sensitivity in any gas sensor application that presently uses diffusion as a mechanism of transport. Adding additional system components to charge the analyte to assist collection should yield similar gains. Localized nanolens enabled collection approach Figure 4a depict the Raman microscopy intensity map at 1,075 cm −1 Raman shift and spectra for the nanolens enabled collection approach as a function of collection time. The nanolens approach supports a more automated image processing that enables the elimination of the searching and hand picking of hotspots, which is a common practice in SERS-related measurements. Instead, we used a standard array of detection windows outlined with the dashed lines and averaging over these areas to record the spectra. The characteristic peaks at 1,001 cm −1 , 1,075 cm −1 and 1,573 cm −1 begin to emerge after a 10-s long exposure to charged BTs. For short periods of time, the signal increases roughly linear with exposure time and begins to level off after 60 s. For example, counts at the 1,001 cm −1 Raman shift are 170 (1 × ), 320 (1.9 × ), 540 (3.2 × ) for 10 s, 20 s, 60 s, respectively. This nonlinear behaviour and saturation for prolonged capture times can be explained by excessive packing of molecules, which are not as tightly coupled to the plasmonic surface 35 . For the same reason the signal-to-noise-ratio increases at first with exposure time before it levels off. Spot and detect different molecules on a single substrate Figure 5 illustrates experimental results where programmable domain electrodes are used to spot different molecules with different molecular weights, specifically BT (110.18 Da), 4-FBT (128.17 Da) and anti-mouse IgG (~150 kDa). The small molecules displayed characteristic Raman peaks that enable the recognition in the anticipated domains. As an example, domain 1 shows the presence of a peak at 1,001 cm −1 , which is known to be a dominant peak for the BT, which is not the case in 4-FBT in domain 2. Both molecules share a peak at 1,075 cm −1 , which represents a stretching mode of the aromatic ring. We would like to note that the intensity of the SERS signal depends on both the analyte amount and the localized plasmonic resonance. Plasmon resonances will only occur in the Ag layer and not on the photoresist surface. In other words, the absence of a SERS signal on the photoresist surface is not sufficient to conclude that there are no molecules on the photoresist. The previously presented increases in signal counts by a factor of 615 ( Fig. 3 ) comparing the biased nanolens array with an unbiased surface can, however, only be explained by an increased localized collection process. To further validate that material is only collected in the opening, we decided to use a fluorescence-based detection scheme and a much larger molecule in hope to image the location 36 using fluorescence microscopy. Specifically we tested anti-mouse IgG that is tagged with conventional fluorescence markers (fluorescein isothiocyanate, green). Figure 5d depicts the results. The fluorescence-based detection scheme decouples the detection mechanism from the plasmon resonant Ag layer. The intensity of the green fluorescence on the Ag surface and the absence of fluorescence on the photoresist surface can now be used as a measure of the selectivity with which the localized analyte delivery takes place. No detectable quantities of the IgG molecules are found on the photoresist and collection is observed only in the centre of the opening. Moreover, because IgG is three orders of magnitudes larger than the thiol molecules in this study, it was possible to resolve the location of physical analyte collection by scanning electron microscope. The molecules are confined to a 200 nm in diameter region, which is smaller than the 1 μm in diameter opening. Advanced collection, spotting and detection Figure 1 depicts the investigated design elements and test structures, which were evaluated to spot and collect airborne species on a nanostructured SERS gas sensor substrate. Figure 1a contrasts the state-of-the-art diffusion-based delivery concept with basic electrostatic precipitation, where an external bias and charged molecules are tested to increase the collection efficiency. Figure 1c adds an additional design element—a charged photoresist layer with a circular opening; the goal of this structure is to form an electrodynamic nanolens to funnel and concentrate the airborne species at a predetermined location on the nanostructured sensor surface. The use of a gas-phase nanolens focusing effect has been reported recently in the context of inorganic nanoparticles 23 24 25 26 that contained several 100,000 atoms, which contrasts with the small molecules tested here that contain less than 20 atoms. The general idea of a nanolens is to use a charged resist, which influences the trajectory of charged material. The resist serves as insulation and blocks charge dissipation. The opening to the conductor provides the only location where a charged material flux can be established under steady-state conditions. Figure 1d depicts an additional modification to achieve programmability of more than one analyte. The approach uses electrically separated metallic domains (yellow cap layers) underneath the nanolens array. The fabrication of the depicted test structures is detailed in the methods section. In short, we used a Langmuir–Blodgett method 27 to apply a closely packed layer of silica spheres, 200 nm in diameter, over extended areas on the glass surface. Next, e-beam evaporation is used to coat the top with 20 and 180 nm of chromium and silver, respectively. This yields the SERS sensing surface commonly known in the literature as AgFON standard. The silver film provides a conductive layer. This conductive layer allows for the application of an external bias voltage, which is used to evaluate if a field driven approach can increase the collection efficiency of charge molecules when compared with prior concepts 14 15 , where the rate of absorption was driven by diffusion and the substrate was left floating. Prior methods were able to detect BTs at a concentration of 6 p.p.m. in 1 s. The second modification ( Fig. 1c ) adds the depicted 500-nm thick spin-coated photoresist layer whereby a 1 μm in diameter and 3 μm pitched hole pattern is defined by photolithography. The final SERS gas sensor substrate ( Fig. 1d ) is composed of two silver domains instead of one. The two domains are separated using a 50-μm wide region of uncoated silica spheres, which was masked by placing a capillary onto the surface prior metallization. All other parameters were left the same. The deposition on the individual domains is controlled using external bias voltages in the range of −50 to −200 V, whereby the electrometers records the deposition current in the range of 1–5 nA. The flux is recorded using electrometers (Keithley 6517A) marked with the letter 'A'. Domains are turned ON and OFF sequentially for selected periods of time (10 s to 1 min) to collect molecular ions including BT + and 4-FBT + on domain 1 and 2, respectively. We choose these two molecules as they have a well-characterized and known Raman scattering signal 14 28 . The process is, however, not limited to these types of molecules or molecular weights. Results for anti-mouse IgG proteins (Sigma-Aldrich, F-0257) will be presented as well. Electrospray testing environment Figure 2 depicts the details of the testing environment where the analytes are evaporated and charged using an electrospray ionization standard and where programmable analyte collection is achieved using biased domain electrodes. Electrospray ionization is chosen as it provides a controlled environment to produce airborne analytes of almost any type and concentration. Electrospray ionization systems are used in mass spectroscopy in the fields of chemical-warfare agent detection 29 and proteomics 30 . They require exceptionally small amounts of diluted analytes. For example at a common flow rate, only 50 nl is drawn through the capillary per minute. For a review, we refer to Cloupeau and Prunet-Foch 31 and Ganan-Calvo et al. 32 . In brief, we constructed a system based on a commercially available electrospray ionization system (TSI Inc., Model 3480). It consists of a high-voltage source, pressure regulator, pressure chamber, capillary and electrospray chamber. The pressure chambers house a centrifuge vial, a high-voltage platinum electrode, and a fused silica capillary, which carries the solution out into the electrospray chamber using 1.25 atm pressure. A positive electrospray voltage is increased until the extruded liquid (50 nl per min) forms a cone shape, known as cone-jet mode 33 , which leads to rapid evaporation in close proximity to the orifice and an aerosol containing charged molecules (light green for 4-FBT, and light blue for BT). Figure 2a depicts the first analyte collection sequence where BT ions (BT + ) are collected on domain 1 which is turned ON by applying a negative voltage V d =−100 V to the domain electrode. Domain 2 is switched into a floating state, whereby the domain electrode is disconnected to prevent dissipation of charge, which in turn blocks the collection on domain 2. The following conditions were used to prepare the depicted ionized aerosol containing charged BTs. The starting point is a BT solution (liquid density=1.073 g/ml, molecular weight=110 g mol −1 ), which is diluted in a 1:1 volume ratio with ethanol; second, the solution is sprayed at a rate of 50 nl/min, which translates to 2.43 × 10 −7 N A BT molecules per minute, where N A is Avogadro's constant; third, it is mixed with 1 liter per minute nitrogen carrier gas at 1.25 atm by adding 4.1 × 10 −2 N A nitrogen molecules per minute to the mixture; at this stage the analyte is diluted down to 5.9 p.p.m. In positive ion mode, the process produces positively charged molecules in addition to neutral molecules 34 . Of importance are BT ions (C 6 H 5 SH 2 + ), 4-FBT (C 6 H 4 FSH 2 + ), solvent ions (C 2 H 5 OH 2 + ) and nitrogen ions (N 2 + ). Figure 2b illustrates a second analyte collection sequence where 4-FBT + is collected on domain 2 and where domain 1 is left floating. The preparation approach for 4-fluorothiophenol (4-FBT) followed the same procedure as described before in the case of BT. The illustration in Fig. 2 under-represents the amount of neutral species for clarity. However, it is important to mention that most gas molecules are neutral and that the fraction of charged material is small. For example, a typical electrospray current is 100 nA, which is measured using the ampere metre marked with the letter 'A', which is connected to the platinum electrode that is immersed in the vial; 100 nA electrospray current translates into an ion current flux of 6.23 × 10 −11 N A elementary charges per minute. This ion flux is three orders of magnitudes smaller than the previously calculated 2.43 × 10 −7 N A BT molecules and nine orders of magnitudes smaller than 4.1 × 10 −2 N A nitrogen gas molecules contained in 1 l per min carrier gas. In other words, the gas mixture is composed of both charged (<1.5 p.p.b.) and neutral BT (<5.9 p.p.m.) molecules. The gas mixture exits the enclosed system through a 0.5 mm in diameter orifice and expands until it reaches the substrate, which is placed 5 cm away. The p.p.b. and p.p.m. estimates are upper limits and the actual concentration of charged and neutral molecules is smaller due to downstream mixture and charge exchange processes, which will be discussed further in the discussion section. Collection based on different mechanisms Figure 3 compares the recorded SERS signals using the floating, biased plate and biased nanolens collection approach after the test substrates were exposed for 30 s. The gas mixture exiting the electrospray system containing both charged (<1.5 p.p.b.) and neutral BT (<5.9 p.p.m.) molecules in nitrogen. All SERS spectra in Fig. 3c were recorded under identical exposure and recording conditions, which is important as this allows a relative comparison of the signal intensity for a sensor system with and without charge directed collection. For a standard SERS substrate, hotspots are randomly located on the surface ( Fig. 3b ) and it is hard to point out where the molecule is. The nanolens-based collection approach eliminates this uncertainty as molecules are collected at predetermined points ( Fig. 3c ). This helps in the data collection. In the particular case, all SERS spectra were recorded as an average over the indicated white dashed areas; no voltage is applied during the recording of the SERS data. The spectrum for the unbiased case shows a weak signal and the detection of the uncharged BT is difficult at 6 p.p.m. in our system; the characteristic peak at 1,075 cm −1 is recorded with 0.4 counts per unit area (inset of Fig. 3c ), which means very few BT molecules are collected on this substrate. The signal increases to 103 counts per unit area for the biased AgFON substrate and 246 counts per unit area for the biased AgFON substrate with integrated nanolens array, which represents a factor of 615 comparing the biased nanolens array with the unbiased AgFON standard. We repeated this experiment five times using separate substrates. The nanolens-based collection region produced the highest counts in each case; the intensity factor varied by 6% standard deviation (STD) between the experiments. The recorded increase of 615 is even more impressive if one considers that it is caused by 3,900 times fewer charged analyte molecules (1.5 p.p.b.) than neutral ones (5.9 p.p.m.). In other words, trace amounts of charged molecules at a concentration of 1.5 p.p.b. (parts per billion) lead to 246 counts per unit area, while 5.9 p.p.m. (parts per million) of neutral particles contribute to 0.4 counts, which means that the capture efficiencies of charged molecules is 2.4 × 10 6 times larger than neutral ones. The six orders of magnitude-higher capture efficiencies of charged molecules when compared with neutral one is an important metric, as it provides a route to higher sensitivity in any gas sensor application that presently uses diffusion as a mechanism of transport. Adding additional system components to charge the analyte to assist collection should yield similar gains. Localized nanolens enabled collection approach Figure 4a depict the Raman microscopy intensity map at 1,075 cm −1 Raman shift and spectra for the nanolens enabled collection approach as a function of collection time. The nanolens approach supports a more automated image processing that enables the elimination of the searching and hand picking of hotspots, which is a common practice in SERS-related measurements. Instead, we used a standard array of detection windows outlined with the dashed lines and averaging over these areas to record the spectra. The characteristic peaks at 1,001 cm −1 , 1,075 cm −1 and 1,573 cm −1 begin to emerge after a 10-s long exposure to charged BTs. For short periods of time, the signal increases roughly linear with exposure time and begins to level off after 60 s. For example, counts at the 1,001 cm −1 Raman shift are 170 (1 × ), 320 (1.9 × ), 540 (3.2 × ) for 10 s, 20 s, 60 s, respectively. This nonlinear behaviour and saturation for prolonged capture times can be explained by excessive packing of molecules, which are not as tightly coupled to the plasmonic surface 35 . For the same reason the signal-to-noise-ratio increases at first with exposure time before it levels off. Spot and detect different molecules on a single substrate Figure 5 illustrates experimental results where programmable domain electrodes are used to spot different molecules with different molecular weights, specifically BT (110.18 Da), 4-FBT (128.17 Da) and anti-mouse IgG (~150 kDa). The small molecules displayed characteristic Raman peaks that enable the recognition in the anticipated domains. As an example, domain 1 shows the presence of a peak at 1,001 cm −1 , which is known to be a dominant peak for the BT, which is not the case in 4-FBT in domain 2. Both molecules share a peak at 1,075 cm −1 , which represents a stretching mode of the aromatic ring. We would like to note that the intensity of the SERS signal depends on both the analyte amount and the localized plasmonic resonance. Plasmon resonances will only occur in the Ag layer and not on the photoresist surface. In other words, the absence of a SERS signal on the photoresist surface is not sufficient to conclude that there are no molecules on the photoresist. The previously presented increases in signal counts by a factor of 615 ( Fig. 3 ) comparing the biased nanolens array with an unbiased surface can, however, only be explained by an increased localized collection process. To further validate that material is only collected in the opening, we decided to use a fluorescence-based detection scheme and a much larger molecule in hope to image the location 36 using fluorescence microscopy. Specifically we tested anti-mouse IgG that is tagged with conventional fluorescence markers (fluorescein isothiocyanate, green). Figure 5d depicts the results. The fluorescence-based detection scheme decouples the detection mechanism from the plasmon resonant Ag layer. The intensity of the green fluorescence on the Ag surface and the absence of fluorescence on the photoresist surface can now be used as a measure of the selectivity with which the localized analyte delivery takes place. No detectable quantities of the IgG molecules are found on the photoresist and collection is observed only in the centre of the opening. Moreover, because IgG is three orders of magnitudes larger than the thiol molecules in this study, it was possible to resolve the location of physical analyte collection by scanning electron microscope. The molecules are confined to a 200 nm in diameter region, which is smaller than the 1 μm in diameter opening. Discussion The guided nanolens collection process and the results in Fig. 5d lead to the question of how the required fringing field is established and why continued deposition is only observed in the opening to the conductor. Our current hypothesis and understanding is that the deposition process is a self-equilibrating electrodynamic process whereby the electric field distribution evolves over time. In the initial stage of the experiment, ions respond to the external bias voltage that is applied to the domain electrodes. The smallest ions N 2 + have the highest mobility and arrive first at the sample surface. This transient response results in a sheath of space charge on the sample surface depicted as '+' on the red coloured photoresist layer within the illustrations, which alters the potential distribution. The potential distribution equilibrates and leads to a potential funnel where the analyte deposits in the centre of the opening. Steady-state charge dissipation can only occur in the opening and leads to a measurable flux of positive gas ions that includes the targeted molecules under a negative substrate bias, which is directly recorded using the electrometers (Keithley 6517A). An open discussion point relates to the ultimate sensitivity that may be achieved. The relative sensitivity increase in the case of the SERS sensor was 615 comparing the biased nanolens array with the unbiased AgFON standard, whereby the signal was caused by 3,900 times fewer charged analyte molecules than neutral ones. This yielded the 2.4 × 10 6 times larger capture efficiency for the charged molecules when compared with the neutral ones. This value has not be optimized and is based on the 1 μm in diameter and 3 μm pitched hole pattern shown in Fig. 1 ; higher values can be anticipated but would require optimization of the opening size, pitch, domain size and domain potential. For example a smaller pitch and higher potential should allow further increases. A corona charging method could also be employed to increase the charge ratio. Another discussion point relates to the actual concentration of the charged and neutral molecules at the sensor surface. The gas mixture that exits the enclosed electrospray system through the orifice plate contains roughly 5.9 p.p.m. (parts per million) neutral and 1.5 p.p.b. (parts per billion) charged analyte molecules, which was previously discussed in the paragraph describing Fig. 2 . However, additional dilution with the surrounding atmosphere will take place before the analyte reaches the substrate, which is placed 5 cm away in an otherwise open environment. The resulting additional dilution ratio can be calculated using a steady-state Gaussian plume model 37 38 where the downstream concentration as a function of distance depends upon the diameter of the orifice plate (0.5 mm) and cone shape where half-angle of the cone is ~9° (ref. 39 ) in our case yielding a cone diameter of 15.84 mm at the substrate location. Following the procedure published by Wein et al. 38 an additional analyte dilution factor of 1,003 will take place due to downstream mixing, which leads to ~1.49 p.p.t. (parts per trillion) charged analyte molecules and 5.88 p.p.b. (parts per billion) neutral analyte molecules at the sensor location. This is highly sensitive in absolute terms, which needs to be verified in further studies before this claim should be made. As a result, we used the upstream concentration at the orifice plate with 1.5 p.p.b. charged analyte molecules and 5.9 p.p.m. neutral ones to provide a conservative measure and a relative comparison. The overall conclusion remains as the recorded increase of 615 is caused by 'at least' 3,900 times fewer charged analyte molecules than neutral ones leading to an increased capture efficiencies of 'at least' 2.4 × 10 6 . The words 'at least' account for the fact that additional dilution takes place and that further gains through optimization of the opening size, pitch, domain size and domain potential can be anticipated. The reported process can also be used as a programmable selected area deposition or surface treatment method with molecular ions. The sequence and amount can be mixed and matched with a lateral resolution that is several orders of magnitudes higher than what is possible using existing methods that are based on mechanical shutters or high-precision contact-printing robots. The ability to fabricate hybrid molecular arrays with control over material sequence, composition and lateral distribution on a single substrate within a single process could potentially be used in other applications, which include, proteomics and cell research, pharmaceutical screening processes, panel immunoassays and molecular electronics 20 21 40 . In conclusion, various nanostructured sensors currently aim to claim single molecular detection by a reduction of the active sensor size. An equally important challenge, however, can be found in the question 'whether the analyte will ever find the nanometre sized surface'. The smaller the sensor, the less likely the probability for a molecule to attach to the sensor surface considering a diffusion-based detection system. The reduction in the size of the active sensor will ultimately require research on methods that enable localized delivery. The reported electrodynamic collection concept is a first step in this direction. Methods Materials All chemicals were reagent grade and used as received. Surfactant-free, silica nanosphere suspensions (200 nm, 4 wt %) were acquired from Bangs Laboratories, Inc. BT and 4-FBT were purchased from Sigma-Aldrich (Milwaukee, WI). NH4OH, H2O2, and H2SO4 were purchased from Fisher Scientific (Fairlawn, VA). AgFON fabrication Glass slides (75 mm long, 50 mm wide, 1 mm thick) were first pretreated in piranha etch at 120 °C for 30 min, and then in 5:1:1 ratio of H 2 O:NH 4 OH:H 2 O 2 for 30 min to make the surface hydrophilic. The 200 nm monodisperse silica nanosphere suspension (4% silica spheres by weight in water) was further diluted in ethanol (1:1 volume ratio). The ethanol served as a spreading agent, which we found to help in the next step. The 2 μl suspension was carefully dropped onto the water surface which yields a surface layer of silica beads. The Langmuir–Blodgett method was used to compact the beads and to transfer the beads to the target slide. After drying the surface for 30 min, the AgFON standard substrate was completed through e-beam evaporation of 20 nm chromium and 180 nm silver to form the plasmonic cap layer. The silver thickness and deposition rate were measured by a 6 MHz quartz crystal microbalance from INFICON (East Syracuse, NY). Nanolens array The nanolens array is composed of a patterned photoresist layer. Photoresist (Microposit 1805, Shipley) was spin coated at 3,000 rpm for 40 s on an AgFON substrate. After soft-bake at 105 °C for 1 min, the substrate was photolithographically patterned with the 96 mJ cm −2 ultraviolet light and developed in a developer (Microposit 351:H 2 O=1:5) for 25 s. A 15 s oxygen plasma cleaning step was used to remove any residues. The substrates were thoroughly rinsed with deionized water and blown dry with high purity nitrogen (99.998%) before they were used. The electrodynamic nanolens pattern was composed of 1 μm diameter holes on a 3-μm pitch. In the initial stage, the photoresist becomes charged and equilibrates as a consequence of the ionic deposition process. The charged resist deflects and guides charged ions into the opening. Programmable nanolens array The programmable nanolens array used the same substrate and same photoresist film as described before. The only difference is that the Cr/Ag film is defined into separated domains to allow for the application of separate bias voltages to control the deposition in each domain. The domains are separated by a 50-μm wide region, which was not coated with silver. The region was masked using a 50 μm in diameter capillary during the e-beam evaporation of 20 nm chromium and 180 nm silver to form electrically separated plasmonic cap layers. Characterization A scanning electron microscope (JEOL 6500) was used to examine the surface morphology of AgFON substrates and measure the size. SERS spectra and two-dimensional confocal microscopy scanning images were acquired using a confocal Raman microscope system (Witec Alpha 300R) equipped with a 100 × objective lens (Nikon 100 × , 0.90 NA in air). A fibre-optic interfaced 514 nm argon ion laser was used as the laser source. The laser power was maintained constant and set to ~2 mW within one experimental set of measurements. The lateral imaging resolution of the confocal system considering the wavelength, and numerical aperture of the system is ~300 nm. The scattered light was analysed using a 600 mm −1 spectrometer grating with a spectral resolution of about 3 cm −1 . The collection time for each raster spot constant was 1 s. SERS spectra were collected from multiple spots across the substrate and from multiple substrates. The reflectance absorption spectrum was analysed using an optical fibre VIS-NIR spectrophotometer (Ocean Optics, USB4000 VIS-NIR spectrometer, QR400-7-UV–vis reflection probe). The reflectance absorption spectrum of AgFON was collected and used for the chosen wavelength (514.5 nm). Materials All chemicals were reagent grade and used as received. Surfactant-free, silica nanosphere suspensions (200 nm, 4 wt %) were acquired from Bangs Laboratories, Inc. BT and 4-FBT were purchased from Sigma-Aldrich (Milwaukee, WI). NH4OH, H2O2, and H2SO4 were purchased from Fisher Scientific (Fairlawn, VA). AgFON fabrication Glass slides (75 mm long, 50 mm wide, 1 mm thick) were first pretreated in piranha etch at 120 °C for 30 min, and then in 5:1:1 ratio of H 2 O:NH 4 OH:H 2 O 2 for 30 min to make the surface hydrophilic. The 200 nm monodisperse silica nanosphere suspension (4% silica spheres by weight in water) was further diluted in ethanol (1:1 volume ratio). The ethanol served as a spreading agent, which we found to help in the next step. The 2 μl suspension was carefully dropped onto the water surface which yields a surface layer of silica beads. The Langmuir–Blodgett method was used to compact the beads and to transfer the beads to the target slide. After drying the surface for 30 min, the AgFON standard substrate was completed through e-beam evaporation of 20 nm chromium and 180 nm silver to form the plasmonic cap layer. The silver thickness and deposition rate were measured by a 6 MHz quartz crystal microbalance from INFICON (East Syracuse, NY). Nanolens array The nanolens array is composed of a patterned photoresist layer. Photoresist (Microposit 1805, Shipley) was spin coated at 3,000 rpm for 40 s on an AgFON substrate. After soft-bake at 105 °C for 1 min, the substrate was photolithographically patterned with the 96 mJ cm −2 ultraviolet light and developed in a developer (Microposit 351:H 2 O=1:5) for 25 s. A 15 s oxygen plasma cleaning step was used to remove any residues. The substrates were thoroughly rinsed with deionized water and blown dry with high purity nitrogen (99.998%) before they were used. The electrodynamic nanolens pattern was composed of 1 μm diameter holes on a 3-μm pitch. In the initial stage, the photoresist becomes charged and equilibrates as a consequence of the ionic deposition process. The charged resist deflects and guides charged ions into the opening. Programmable nanolens array The programmable nanolens array used the same substrate and same photoresist film as described before. The only difference is that the Cr/Ag film is defined into separated domains to allow for the application of separate bias voltages to control the deposition in each domain. The domains are separated by a 50-μm wide region, which was not coated with silver. The region was masked using a 50 μm in diameter capillary during the e-beam evaporation of 20 nm chromium and 180 nm silver to form electrically separated plasmonic cap layers. Characterization A scanning electron microscope (JEOL 6500) was used to examine the surface morphology of AgFON substrates and measure the size. SERS spectra and two-dimensional confocal microscopy scanning images were acquired using a confocal Raman microscope system (Witec Alpha 300R) equipped with a 100 × objective lens (Nikon 100 × , 0.90 NA in air). A fibre-optic interfaced 514 nm argon ion laser was used as the laser source. The laser power was maintained constant and set to ~2 mW within one experimental set of measurements. The lateral imaging resolution of the confocal system considering the wavelength, and numerical aperture of the system is ~300 nm. The scattered light was analysed using a 600 mm −1 spectrometer grating with a spectral resolution of about 3 cm −1 . The collection time for each raster spot constant was 1 s. SERS spectra were collected from multiple spots across the substrate and from multiple substrates. The reflectance absorption spectrum was analysed using an optical fibre VIS-NIR spectrophotometer (Ocean Optics, USB4000 VIS-NIR spectrometer, QR400-7-UV–vis reflection probe). The reflectance absorption spectrum of AgFON was collected and used for the chosen wavelength (514.5 nm). Author contributions E.C.L. and H.O.J. designed research; E.C.L. performed research; E.C.L. and H.O.J. Analysed data; J.F., S.C.P. and F.W.J. contributed to the data analysis and interpretation; and E.C.L. and H.O.J. wrote the paper. Additional information How to cite this article: Lin, E.C. et al. Effective localized collection and identification of airborne species through electrodynamic precipitation and SERS-based detection. Nat. Commun. 4:1636 doi: 10.1038/ncomms2590 (2013).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10265212/

Expanding the potential therapeutic options of hemoperfusion in the era of improved sorbent biocompatibility

Hemoperfusion has been considered a promising adjuvant treatment for chronic diseases and some acute states when specific removal of pathogenic factors from the bloodstream is desired. Over the years, advances in adsorption materials (e.g., new synthetic polymers, biomimetic coating, and matrixes with novel structures) have renewed scientific interest and expanded the potential therapeutic indications of hemoperfusion. There is growing evidence to suggest a prominent place for hemoperfusion as an adjuvant treatment in the setting of sepsis or severe coronavirus disease 2019 and as a therapeutic option for chronic complications associated with accumulated uremic toxins in patients with end-stage renal disease. This literature review will describe the principles, therapeutic perspectives, and the emerging role of hemoperfusion as a complementary therapy for patients with kidney disease. Introduction The removal of unwanted plasma solutes and pathogens can be life-saving under certain conditions, such as sepsis, intoxication, and organ failure. Thus, the unique ability of hemoperfusion (HP) to adsorb specific molecules with large molecular weight (MW) and/or a high protein-binding affinity could explain why HP has been allured as a promising treatment for several diseases [ 1 ]. Whereas poisoning was once considered the classical indication of HP, advances in sorbents' biocompatibility and design have helped to expand its potential clinical indications to the treatment of inflammatory conditions (e.g., sepsis, pancreatitis, and hepatitis), autoimmune diseases, and chronic uremic symptoms [ 2 ]. The European Uremic Toxin Group classifies uremic toxins into three groups: small water-soluble toxins with an MW of 500 à (50 nm) (macroporous), 20 to 500 à (mesoporous), and 90% to 95% [ 56 ]. However, in a multicenter randomized trial comparing conventional care with CytoSorb in ventilated patients with sepsis and either acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), no significant differences in interleukin (IL)-6 concentration were observed [ 57 ]. In a recent randomized controlled trial (RCT; the REMOVE trial), the authors failed to demonstrate a reduction in postoperative organ dysfunction or 30-day mortality with intraoperative use of CytoSorb in patients undergoing cardiac surgery for infective endocarditis. Even though CytoSorb achieved a lower level of plasma key cytokines, no clinical benefit was obtained [ 58 ]. HP with the Jafron HA cartridges (Jafron Biomedical Company) has also been tested in acute respiratory failure caused by sepsis, with prominent results concerning hemodynamic parameters, respiratory function, and mortality within 28 days of hospitalization [ 59 ]. The HA 330 cartridge has an electrically porous resin that specifically removes cytokines, complements, and other endotoxins with MWs of 10 to 60 kDa. HA 330-based HP was studied in multiple cohorts in the context of inflammatory conditions such as sepsis, ALI, hepatitis, and pancreatitis [ 2 ]. In a small nonrandomized study, intensive care unit (ICU) mortality and length of ICU stay were found to be better in septic patients receiving HA 330-based HP compared to those given standard therapy, albeit with no effect on mortality [ 60 ]. Encouraging results come from a case series of children with sepsis and underlying hematological disorders receiving HA 330-based HP as an adjunctive treatment to counterbalance the cytokine storm [ 61 ]. In another study, patients with ALI induced by extrapulmonary sepsis were randomized to HA 330-based HP or standard therapy. In the HP group, significant reductions in the duration of mechanical ventilation and ICU stay and the ICU mortality rate were observed. Improved respiration parameters were also observed and correlated with the significant removal of inflammatory cytokines (tumor necrosis factor [TNF] and IL-1). In a recent study by Chu et al. [ 62 ], the combination of the same cartridge with pulse high-volume HF in patients experiencing septic shock led to beneficial effects on cardiovascular physiology and greater decreases in IL-6, IL-10, and TNF-α concentration when compared to patients who received continuous venous-venous HF. Finally, the AN69-based oXiris membrane (Baxter Inc.), which is a heparin-grafted membrane specifically designed for cytokine and endotoxin adsorption, alongside RRT, presents three layers: 1) AN69 copolymer hydrogel structure that adsorbs cytokines and removes solutes via convection through membrane pores, 2) a multilayer structure of polyethyleneimine that adsorbs endotoxin and offers better biocompatibility, and 3) a heparin graft that reduces local thrombogenicity [ 63 ]. In vitro comparison of oXiris with Toraymyxin and CytoSorb revealed similar efficacies in lipopolysaccharide clearance and inflammatory mediator clearance, respectively [ 64 ]. However, there are a limited number of studies to support its action in septic shock compared to the above-mentioned products [ 65 – 67 ]. Poisoning Extracorporeal therapies for drug or chemical intoxication are indicated when there is life-threatening toxicity, an inadequate response to standard supportive measures, or the poison's endogenous clearance is 90% to 95% [ 56 ]. However, in a multicenter randomized trial comparing conventional care with CytoSorb in ventilated patients with sepsis and either acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), no significant differences in interleukin (IL)-6 concentration were observed [ 57 ]. In a recent randomized controlled trial (RCT; the REMOVE trial), the authors failed to demonstrate a reduction in postoperative organ dysfunction or 30-day mortality with intraoperative use of CytoSorb in patients undergoing cardiac surgery for infective endocarditis. Even though CytoSorb achieved a lower level of plasma key cytokines, no clinical benefit was obtained [ 58 ]. HP with the Jafron HA cartridges (Jafron Biomedical Company) has also been tested in acute respiratory failure caused by sepsis, with prominent results concerning hemodynamic parameters, respiratory function, and mortality within 28 days of hospitalization [ 59 ]. The HA 330 cartridge has an electrically porous resin that specifically removes cytokines, complements, and other endotoxins with MWs of 10 to 60 kDa. HA 330-based HP was studied in multiple cohorts in the context of inflammatory conditions such as sepsis, ALI, hepatitis, and pancreatitis [ 2 ]. In a small nonrandomized study, intensive care unit (ICU) mortality and length of ICU stay were found to be better in septic patients receiving HA 330-based HP compared to those given standard therapy, albeit with no effect on mortality [ 60 ]. Encouraging results come from a case series of children with sepsis and underlying hematological disorders receiving HA 330-based HP as an adjunctive treatment to counterbalance the cytokine storm [ 61 ]. In another study, patients with ALI induced by extrapulmonary sepsis were randomized to HA 330-based HP or standard therapy. In the HP group, significant reductions in the duration of mechanical ventilation and ICU stay and the ICU mortality rate were observed. Improved respiration parameters were also observed and correlated with the significant removal of inflammatory cytokines (tumor necrosis factor [TNF] and IL-1). In a recent study by Chu et al. [ 62 ], the combination of the same cartridge with pulse high-volume HF in patients experiencing septic shock led to beneficial effects on cardiovascular physiology and greater decreases in IL-6, IL-10, and TNF-α concentration when compared to patients who received continuous venous-venous HF. Finally, the AN69-based oXiris membrane (Baxter Inc.), which is a heparin-grafted membrane specifically designed for cytokine and endotoxin adsorption, alongside RRT, presents three layers: 1) AN69 copolymer hydrogel structure that adsorbs cytokines and removes solutes via convection through membrane pores, 2) a multilayer structure of polyethyleneimine that adsorbs endotoxin and offers better biocompatibility, and 3) a heparin graft that reduces local thrombogenicity [ 63 ]. In vitro comparison of oXiris with Toraymyxin and CytoSorb revealed similar efficacies in lipopolysaccharide clearance and inflammatory mediator clearance, respectively [ 64 ]. However, there are a limited number of studies to support its action in septic shock compared to the above-mentioned products [ 65 – 67 ]. Viral infections, including severe acute respiratory syndrome coronavirus 2 Uncontrolled cytokine response was considered the hallmark of severe COVID-19 during the first months of the pandemic [ 68 ]. Several extracorporeal blood-purification techniques have been used in COVID-19 patients to restore "immune homeostasis" by removing inflammatory molecules [ 69 ]. Recently, experts' recommendations state that, in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and cytokine release syndrome, cytokine-removal strategies should be reserved for COVID-19 patients with evidence of high levels of circulating cytokines like IL-6 and IL-8, a biochemically determined inflammatory status, a high SOFA (Sequential Organ Failure Assessment) score, clinical symptoms of hemodynamic instability requiring vasopressors, and initial signs of immune dysregulation or coagulation disorders [ 69 ]. Polymyxin-based HP is indicated in the early phase for suspected sepsis (indicated by a high procalcitonin level and/or positive bacterial culture) or an elevated endotoxin level proven by activity assay. If HP is indicated for cytokine removal, sessions with CytoSorb or HA 380 might follow. In fact, the U.S. Food and Drug Administration (FDA) has approved four blood-purification devices to treat COVID-19, including 1) CytoSorb, 2) the Seraph 100 Microbind Affinity Blood Filter (ExThera Medical Corporation), 3) the oXiris Filter, and 4) the Spectra Optia Apheresis System (Terumo BCT) [ 70 ]. The first case of CytoSorb use in conjunction with CRRT in a critically ill patient with COVID-19 was reported by Rizvi et al. [ 71 ], underlining a plausible contribution to early improvement in inflammatory markers. Other case-control and retrospective studies followed, highlighting a potentially beneficial role of adjuvant HP with CytoSorb in the early phase of COVID-19 in terms of cytokine reductions (mainly IL-6 levels), a better clinical course with less need for vasoactive agents, and the improvement of respiratory distress. However, data on mortality rates were inconsistent [ 72 – 77 ]. Indeed, in a recent prospective, randomized pilot study with 50 COVID-19 patients receiving CytoSorb for 3 to 7 days or standard therapy, HP did not improve the resolution of vasoplegic shock (primary outcome) or the predefined secondary endpoints, which included mortality, IL-6 concentration, and catecholamine requirement [ 78 ]. Ongoing randomized trials and a large registry of CytoSorb therapy in COVID-19 ICU patients (NCT04391920) aim to enrich the current literature regarding the role of CytoSorb as a potential therapy in severe COVID-19 [ 70 ]. HP with the Seraph 100 Microbind Affinity Blood Filter, a biomimetic adsorber that has been shown to bind pathogens, including SARS-CoV-2, from the blood using ultra-high MW adsorptive beads [ 79 ], received Emergency Use Authorization for severe COVID-19 from the FDA. Olson et al. [ 80 ] were the first to report its use in COVID-19 patients with ARDS and septic shock who required mechanical ventilation. Rapid improvement in vasopressor needs, overall circulatory dysfunction as well as C-reactive protein and IL-6 levels were noticed following the initiation of HP. Similar results were documented by Sandoval et al. [ 81 ] who used Seraph 100 in four elderly, multimorbid ESRD patients on HD with severe COVID-19. Data from the COSA (COVID-19 patients treated with the Seraph 100 Microbind Affinity filter) registry support that Seraph 100 treatment is easy to deploy either as a stand-alone HP treatment or in combination with RRT. The observed mortality rate was lower than that calculated by established scores, but the data are limited due to the lack of a control group [ 82 ]. Initial data from an observational retrospective study (PURIFY-OBS-1; NCT04606498) suggest improvements in the survival of severely ill COVID-19 patients treated with Seraph 100. Evidence of significant reductions in inflammatory markers and improved hemodynamics, organ function, and clinical outcomes with oXiris comes mostly from case series, the oXirisNet registry, and small observational studies [ 83 – 85 ]. An RCT (oXAKI-COV Study; NCT04597034) is ongoing and aims to demonstrate the clinical efficacy of AN69-oXiris compared to the AN69 standard membrane in decreasing vasopressor requirements to sustain a stable mean arterial pressure in critically ill patients with COVID-19 and AKI requiring CRRT. Finally, the Spectra Optia Apheresis System provides therapeutic apheresis in combination with HP with the Depuro D2000 adsorption cartridge. The Depuro D2000 cartridge is composed of activated uncoated coconut shell charcoal and the non-ionic resins Amberlite XAD-7HP and Amberchrom GC300C, and its placement downstream in the apheresis circuit allows for cytokine removal with subsequent return of the treated plasma to the patient. Its use as a rescue therapy for cardiogenic shock due to stress-cardiomyopathy in severe COVID-19 has been reported only in a single patient by Faqihi et al. [ 86 ]. An ongoing large multicenter single-arm clinical trial (Plasma Adsorption in Patients With Confirmed COVID-19; NCT04358003) of the United States is expected to provide information about the effects of the D2000 cartridge with the Optia protocol on morbidity and mortality rates of COVID-19 patients admitted to the ICU. Maximizing toxin removal and clinical benefits in patients with end-stage renal disease ESRD has been increasingly recognized as an inflammatory state with protein-bound uremic toxins (PBUTs) and middle molecules like B2M being key factors and inducing various cardiovascular complications. Therefore there is a rationale for the increasing research on synergic approaches that combine HP with other dialytic techniques to achieve complementary elimination of metabolites and effectively prevent and treat complications and improve clinical outcomes [ 6 , 87 ]. Regarding overall survival, a systematic review and meta-analysis showed that the combination of HD with HP improves survival rates [ 88 ]. Important ameliorations of blood pressure—even in dialysis patients with refractory hypertension—and left ventricular mass index, reduced dosages of epoetin, and higher hemoglobin levels, have been reported when HD with HP are combined compared to HD alone [ 89 – 91 ]. Considering the more pronounced decrease in levels of myocardial enzymes associated with the combination of HP and HD, it was speculated that their concurrent use can lighten the cardiovascular burden and protect the myocardium [ 92 ]. Besides, the improvement of microinflammatory indicators associated with the combination of these therapies could partially explain the lower incidence of cardiocerebrovascular events and the improvement of anemia in patients who had received both HP and HD treatment [ 93 ]. Along the same lines, in a study by Raine et al. [ 94 ], apart from the greater reduction in inflammation markers, an important improvement in the indices of nutritional status occurred in the HD plus HP group. Greater benefits in terms of B2M and PTH reductions have been shown by several studies when HP and HD were combined. Hence, there are potential to improve secondary hyperparathyroidism, pruritus, and dialysis-related amyloidosis [ 95 – 97 ]. Interestingly, based on the reported relationship between the intestinal environment and renal disease, HP combined with dialysis showed encouraging results with respect to the potential improvement of microbiota disorders. Indeed, significantly higher levels of beneficial bacteria like Lactobacillus acidophilus and lower levels of harmful bacteria such as Escherichia coli were reported in colony distributions of patients receiving HP combined with HD plus hemodiafiltration compared to patients receiving HD plus HF [ 98 ]. Research is now focusing on promising sorbent materials—such as a divinylbenzene sorbent coated with polyvinylpyrrolidone (DVB-PVP) and cellulose with hexadecyl chains—which show a high adsorption ability of PBUTs or hydrophobic cytokines. A synergistic effect on the reduction of PBUTs was recently demonstrated during HD therapy combined with DVB-PVP resins and symbiotic formulation [ 99 ]. Moreover, improved sleep disturbance and sleep efficiency accompanied by an increase in nocturnal melatonin levels were reported with HP therapy (1–2 times/wk) for 2 years [ 100 ]. Finally, there is some evidence that the combination of HP with HD can improve the life quality of ESRD patients [ 97 , 101 ]. Symptoms like skin itching, fatigue, sleep quality, and sexual function were significantly improved by adding HP, probably due to the greater clearance of middle and large molecular toxins such as PTH and B2M [ 88 ]. Experimental indications of sorbent use in systemic diseases with kidney involvement Some interesting results have arisen from case series of patients with systemic autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and vasculitis with and without renal involvement [ 102 , 103 ]. Improvements in renal function and dialysis independence following HP sessions in combination with chemotherapy have also been reported in a patient with cast nephropathy [ 104 ]. Finally, AKI can occur as a side effect of medications used in autoimmune disease; thus, HP could also be of value in this context. A recent small case series of patients with high-dose methotrexate-induced AKI showed a possibly positive effect of using charcoal HP as a rescue therapy until glucarpidase is available [ 105 ]. Conclusion Whereas HP was once only indicated for treating poisoning from certain substances, emerging evidence suggests that other indications might be also considered. Advances in the biocompatibility of new cartridges and the selective removal of key molecules in different clinical settings and diseases like sepsis, hepatitis, and SARS-CoV-2 infection have been considered as the triggering force in that direction. With the increasing research interest in the removal of PBUTs and their involvement in CKD-related systemic complications, HP is also regaining its place as a vital accessory to dialysis treatment. Despite this progress, current clinical use of HP remains limited, with possible reasons including the cost of performance, local practice or physician preference, a lack of consensus clinical guidelines and established indications for HP, and the absence of consistent data derived from RCTs. In conclusion, the role of HP remains a point of discussion until its clinical effectiveness can be verified by further positive RCTs. Although in this era of disease-targeting treatments new indications are being investigated, efforts to better evaluate the applicability of HP and to shed light on the role of HP in current clinical practice are needed.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393193/

FURIN inhibition reduces vascular remodeling and atherosclerotic lesion progression in mice

Atherosclerotic coronary artery disease (CAD) is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large CAD case-control cohort, we previously identified proprotein convertase subtilisin/kexin family member 3 ( FURIN ) as a member of several CAD-associated pathways. Objective: To determine the role of FURIN in atherosclerosis. Approach and results: In vitro , FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo , administration of an irreversible inhibitor of FURIN, α−1-PDX, to hyperlipidemic Ldlr −/− mice resulted in lower atherosclerotic lesion area, and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers were observed. Matrix metallopeptidase 2 (MMP2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate, was significantly reduced in the aorta of inhibitor treated mice. To determine FURIN's role in vascular endothelial function, we administered α−1-PDX to Apoe −/− mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness, and lower plaque cellularity, smooth muscle cell, macrophage and inflammatory marker content, suggesting protection against vascular remodelling. Over-expression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions: We show that systemic inhibition of FURIN in mice decreases vascular remodelling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice. Objective: To determine the role of FURIN in atherosclerosis. Approach and results: In vitro , FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo , administration of an irreversible inhibitor of FURIN, α−1-PDX, to hyperlipidemic Ldlr −/− mice resulted in lower atherosclerotic lesion area, and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers were observed. Matrix metallopeptidase 2 (MMP2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate, was significantly reduced in the aorta of inhibitor treated mice. To determine FURIN's role in vascular endothelial function, we administered α−1-PDX to Apoe −/− mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness, and lower plaque cellularity, smooth muscle cell, macrophage and inflammatory marker content, suggesting protection against vascular remodelling. Over-expression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions: We show that systemic inhibition of FURIN in mice decreases vascular remodelling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219350/

Evolution and dynamics of megaplasmids with genome sizes larger than 100 kb in the Bacillus cereus group

Background Plasmids play a crucial role in the evolution of bacterial genomes by mediating horizontal gene transfer. However, the origin and evolution of most plasmids remains unclear, especially for megaplasmids. Strains of the Bacillus cereus group contain up to 13 plasmids with genome sizes ranging from 2 kb to 600 kb, and thus can be used to study plasmid dynamics and evolution. Results This work studied the origin and evolution of 31 B. cereus group megaplasmids (>100 kb) focusing on the most conserved regions on plasmids, minireplicons. Sixty-five putative minireplicons were identified and classified to six types on the basis of proteins that are essential for replication. Twenty-nine of the 31 megaplasmids contained two or more minireplicons. Phylogenetic analysis of the protein sequences showed that different minireplicons on the same megaplasmid have different evolutionary histories. Therefore, we speculated that these megaplasmids are the results of fusion of smaller plasmids. All plasmids of a bacterial strain must be compatible. In megaplasmids of the B. cereus group, individual minireplicons of different megaplasmids in the same strain belong to different types or subtypes. Thus, the subtypes of each minireplicon they contain may determine the incompatibilities of megaplasmids. A broader analysis of all 1285 bacterial plasmids with putative known minireplicons whose complete genome sequences were available from GenBank revealed that 34% (443 plasmids) of the plasmids have two or more minireplicons. This indicates that plasmid fusion events are general among bacterial plasmids. Conclusions Megaplasmids of B. cereus group are fusion of smaller plasmids, and the fusion of plasmids likely occurs frequently in the B. cereus group and in other bacterial taxa. Plasmid fusion may be one of the major mechanisms for formation of novel megaplasmids in the evolution of bacteria. Background Plasmids play a crucial role in the evolution of bacterial genomes by mediating horizontal gene transfer. However, the origin and evolution of most plasmids remains unclear, especially for megaplasmids. Strains of the Bacillus cereus group contain up to 13 plasmids with genome sizes ranging from 2 kb to 600 kb, and thus can be used to study plasmid dynamics and evolution. Results This work studied the origin and evolution of 31 B. cereus group megaplasmids (>100 kb) focusing on the most conserved regions on plasmids, minireplicons. Sixty-five putative minireplicons were identified and classified to six types on the basis of proteins that are essential for replication. Twenty-nine of the 31 megaplasmids contained two or more minireplicons. Phylogenetic analysis of the protein sequences showed that different minireplicons on the same megaplasmid have different evolutionary histories. Therefore, we speculated that these megaplasmids are the results of fusion of smaller plasmids. All plasmids of a bacterial strain must be compatible. In megaplasmids of the B. cereus group, individual minireplicons of different megaplasmids in the same strain belong to different types or subtypes. Thus, the subtypes of each minireplicon they contain may determine the incompatibilities of megaplasmids. A broader analysis of all 1285 bacterial plasmids with putative known minireplicons whose complete genome sequences were available from GenBank revealed that 34% (443 plasmids) of the plasmids have two or more minireplicons. This indicates that plasmid fusion events are general among bacterial plasmids. Conclusions Megaplasmids of B. cereus group are fusion of smaller plasmids, and the fusion of plasmids likely occurs frequently in the B. cereus group and in other bacterial taxa. Plasmid fusion may be one of the major mechanisms for formation of novel megaplasmids in the evolution of bacteria. Background Horizontal gene transfer (HGT) is the major driving force of bacterial evolution [ 1 ]. Plasmids play important roles in this process via their conjugative capability [ 2 ]. Additionally, plasmids harbor genes involved in niche specific processes, and are important for bacterial adaptation to changing environmental conditions [ 3 , 4 ]. As plasmids can transfer frequently among different bacterial strains, they display strain-dependent distributions. Some bacterial strains containing no plasmids, while others have many; sometimes more than 20 [ 5 ]. Moreover, the same host can harbor plasmids with a wide size range. For example, B. thuringiensis strain CT-43 has 10 plasmids ranging from 6 kb to 300 kb [ 6 ]. However, the origin and evolution of these plasmids remains unclear. To date, studies on the evolution and dynamics have mainly focused on plasmids that have broad host ranges and harbor antibiotic-resistance (AR) genes, for example the plasmids of the incompatibility groups IncW [ 7 ], IncU [ 8 ], IncP [ 9 , 10 ] and PromA [ 11 ]. These plasmids usually have small genome sizes and few of them are larger than 100 kb. Information on the evolution and dynamics of plasmids that have relatively narrow host range is scarce, especially for megaplasmids larger than 100 kb. It was therefore the aim of this study to elucidate the origin, evolution and dynamics of megaplasmids with relatively narrow host range using the Bacillus cereus group as a model. The B. cereus group includes B. anthracis , the causative agent of anthrax and a potential biological weapon; B. cereus , a ubiquitous soil bacterium and foodborne pathogen; B. thuringiensis , which produces insecticidal crystal proteins; and four additional species, B. cytotoxicus , B. mycoides , B. pseudomycoides , and B. weihenstephanensis [ 12 , 13 ]. Strains of this group typically contain several plasmids, with some strains containing more than 10 [ 6 , 14 , 15 ]. Plasmids of this group are crucial for the phenotype and virotype of strains. B. anthracis, B. cereus , and B. thuringiensis were defined mainly on the basis of plasmid-encoded phenotypic features [ 16 - 18 ]. Usually, these plasmids are larger than 100 kb. Indeed, most strains of this group contain one or more megaplasmids larger than 100 kb. Only two of these megaplasmids have been studied in depth. One is pXO1 (182 kb) from B. anthracis which harbors two minireplicons that support replication of the plasmid: repX [ 19 ] and pXO1-14/pXO1-16 [ 20 ]. A minireplicon represents the smallest replication region that supports plasmid replication, and contains the origin of replication and genes encoding replication proteins. The origin of replication of plasmid is a particular sequence in a plasmid genome at which replication is initiated. The other well-studied megaplasmid is B. thuringiensis plasmid pBtoxis (128 kb), whose minireplicon consists of two genes: orf156 and orf157 [ 21 ]. The availability of more than 30 sequences of megaplasmids in genomes of the B. cereus group allows the investigation of their evolution and dynamics. We collected the genome sequences of plasmids for bioinformatic analyses. First, we studied the distribution of minireplicons for all the megaplasmids. Second, we studied the relationships among different megaplasmids from the same host strain, and from all strains of the B. cereus group. We also studied the distributions of known minireplicons among all plasmids outside of the B. cereus group whose genome sequences were available. Results and discussion Six types of minireplicon exist in the megaplasmids of the B. cereus group The minireplicons are the core part of plasmids and drive plasmid replication and propagation. Their diversity and evolution directly reflects the dynamics and evolution of plasmids [ 7 , 10 , 22 , 23 ]. Strains in the B. cereus group are rich in plasmid content, with plasmid numbers ranging from zero to 13 and sizes ranging from 2 kb to 600 kb [ 6 , 15 , 24 ]. Thus, the B. cereus group is an ideal model to study plasmid dynamics and evolution. This study aimed to characterize the origin, evolution and dynamics of megaplasmids with genome sizes larger than 100 kb by studying the distribution and evolution of their minireplicons. We collected sequences of 56 plasmids with genome sizes ranging from 20 kb to 600 kb (Additional file 1 : Table S1), including 31 megaplasmids larger than 100 kb. Among these megaplasmids, 65 putative minireplicons were identified and could be classified into six types (Table 1 ). Two of the six types contain two replication essential protein coding genes. One type of minireplicon, which was first reported to support the replication of B. thuringiensis plasmid pBtoxis [ 21 ], tubZ/tubR , encodes TubZ/TubR proteins, which are FtsZ-like (TubZ) and DNA-binding proteins (TubR), respectively. The second type of minireplicon, which was first reported to support the replication of B. anthracis plasmid pXO1, encodes essential proteins belonging to the replication initiator protein (pXO1-16) and DNA-binding protein (pXO1-14) groups, respectively [ 20 , 21 ]. The other four types ( ori44 , ori60 , rep26 and repA_N ) encode four different essential single replication proteins, respectively [ 23 , 25 ]. Among these six types of minireplicon, tubZ/tubR , pXO1-14/pXO1-16 and rep26 only exist in megaplasmids, whereas the other three occur in both megaplasmids and plasmids smaller than 100 kb. Table 1 Type and number of minireplicons on plasmids larger than 20 kb Plasmid Number of minireplicon Minireplicon type Plasmid size (bp) pAH1134_566 3 rep466 , pXO1-16/pXO1-14 (2) 565,964 pE33L466 3 rep466 , rep26 , pXO1-16/pXO1-14 466,370 pBMB431 2 rep466 b , pXO1-16/pXO1-14 431,971 pBWB401 5 ori44 , repA_N , rep466 , pXO1-14/pXO1-16 (2) 417,054 pBMB400 2 rep466 , pXO1-16/pXO1-14 416,210 pBMB171 3 rep466 , rep26 , pXO1-16/pXO1-14 (2) 312,963 pBMB302 2 orf156/orf157 , pXO1-16/pXO1-14 302,255 pBMB293 2 orf156/orf157 , pXO1-16/pXO1-14 293,574 p03BB108_282 1 rep228 a 282,009 pCT281 2 orf156/orf157 , pXO1-16/pXO1-14 281,231 pAH820_272 2 repX , pXO1-16/pXO1-14 272,145 pPER272 2 repX , pXO1-16/pXO1-14 272,145 pAH187_270 2 repX , pXO1-16/pXO1-14 270,082 pH308197_258 2 repX , pXO1-16/pXO1-14 258,484 pBc239 2 repX , pXO1-16/pXO1-14 239,246 p03BB108_239 2 rep466 , pXO1-16/pXO1-14 238,933 pBMB228 2 rep228 , pXO1-16/pXO1-14 228,003 pG9842_209 2 rep228 , pXO1-16/pXO1-14 209,488 pBC210 2 rep466 , rep26 , pXO1-16/pXO1-14 209,385 pBc10987 2 repX , pXO1-16/pXO1-14 208,369 pBCXO1 2 repX , pXO1-16/pXO1-14 190,861 pBMB26 2 rep26 c , pXO1-16/pXO1-14 187,880 pCI-XO1 2 repX , pXO1-16/pXO1-14 181,907 pXO1 2 repX , pXO1-16/pXO1-14 181,677 p03BB102_179 2 repX , pXO1-16/pXO1-14 179,680 pBMB28 2 ori44 , rep466 139,013 pG9842_140 - NA 140,001 pBMB137 2 ori44 , ori60 137,573 pBtoxis 2 orf156/orf157 , pXO1-16/pXO1-14 127,923 pCT127 2 ori60 , repA_N 127,885 pBMB95 1 ori60 95,983 pXO2 1 repA 94,829 pCI-XO2 1 repA 94,469 p03BB108_86 1 repA 85,879 pCT83 1 ori44 83,590 pBT9727 1 repA 77,112 pBWB402 1 repA 75,107 pBMB74 1 repA 74,480 pH308197_73 1 ori60 72,792 pCT72 1 ori43 72,074 pAW63 1 repA 71,777 pBMB67 1 ori43 67,159 pBMB65 1 ori44 65,873 pBWB403 - NA 64,977 pBMB64 1 ori43 64,522 pLVP1401 - NA 56,149 pALH1 - NA 55,939 pFR55 1 ori44 55,712 pE33L54 - NA 53,501 pBWB404 - NA 52,830 pBc53 1 ori60 52,766 pCT51 - NA 51,488 pBMB46 - NA 46,634 pAH187_45 1 ori44 45,173 p03BB108_42 1 ori60 42,470 pH308197_29 - NA 29,189 a and b : The two new types of TubZ/TubR minireplicon identified in this study (supporting methods and results). c : Unpublished, validated by our group. (2): Number of minireplicons of the same type on the same plasmid. NA: no reported minireplicon. The minireplicon tubZ/tubR is distributed widely among the megaplasmids and is found in 26 of the 31 megaplasmids (Table 1 ). A phylogenetic tree was constructed based on the 26 TubZ protein sequences (Figure 1 A). Four different clades were formed and were supported by high bootstrap values (100%). Coincidentally, four of the TubZ proteins for which a function in replication was validated, RepX in plasmid pXO1 [ 19 ], ORF156 in plasmid pBtoxis [ 21 ], Rep228-TubZ in plasmid pBMB228 and Rep466-TubZ in plasmid pBMB28 are located in the four different clades, respectively. The replication function of Rep228-TubZ and Rep446-TubZ were validated in this study (see Additional file 2 ). We divided all 26 tubZ/tubR minireplicons into four subtypes: repX -like, orf156 / orf157 -like, rep228 -like and rep466 -like. Among the four subtypes, only minireplicon repX -like encodes an orphan TubZ protein, while the other three encode not only TubZ proteins, but also TubR proteins. TubR proteins from different subtypes show no similarity to each other. However, when the gene sequences of TubR within each subtype were inspected, we found that the topologies of the phylogenetic trees showed similarities to those of the corresponding TubZ trees (Figures 1 B, C and D), respectively. The DNA sequences of the origins of replication are rich in A + T and usually contain direct or invert repeats were additionally examined. The four minireplicon subtypes of tubZ/tubR have four different secondary structures of them, with different direct or inverted repeats (Additional file 3 : Figure S4). We therefore suggest that for each subtype of tubZ/tubR minireplicon, their TubZ, TubR proteins and the corresponding origin of replication underwent a concerted evolution. Figure 1 Phylogenetic trees constructed using the ML method based on TubZ (A) protein sequences and tubR DNA sequences (B for rep466 -like, C for rep228 -like and D for orf156/orf157 -like) from plasmids of the B. cereus group species. The four subtrees in (A) represent the four subtypes of TubZ/TubR minireplicons. Plasmids from the same strain are marked in the same color. The number at each branch point represents the percentage of bootstrap support calculated from 1,000 replicates, and only those values higher than 50 are shown. The minireplicon pXO1-14/pXO1-16 was found in 24 megaplasmids and three of these megaplasmids harbor two copies. When comparing the pXO1-14-like and pXO1-16-like protein sequences encoded by those 27 minireplicons, the sequence identities among pXO1-14-like proteins ranged from 40% to 100%. However, the pXO1-16-like proteins showed significantly greater conservation (P 65%. When phylogenetic trees based on these two families of protein sequences were constructed (Figure 2 ), the topologies of the two trees were incongruent, except for some in-group topologies. For the pXO1-14-like tree, four major subgroups were supported by high bootstrap values (Figure 2 A). However, two subgroups were identified for pXO1-16 (Figure 2 B). This indicates that genes encoding the pXO1-14-like and pXO1-16-like proteins evolved independently and multiple recombination events have occurred in this minireplicon. Figure 2 Phylogenetic trees constructed using the ML method based on pXO1-14 (A) and pXO1-16 (B) -like protein sequences from pXO1-14/pXO1-16 -like minireplicons from B. cereus group species. When there are two or more similar sequences on the same plasmid, their accession numbers are to the right of the plasmid name. Numbers at each branch point represent the percentage of bootstrap support calculated from 1000 replicates, and only those values greater than 50 are shown. The other four minireplicons contain one essential protein each. Minireplicon rep26 was found in four megaplasmids larger than 100 kb. In contrast, ori44 , ori60 and repA_N were found in both megaplasmids and plasmids smaller than 100 kb and are more widely distributed in smaller plasmids. For example, minireplicon ori44 occurs in eight plasmids, only two of which are larger than 100 kb, while minireplicon ori60 is contained by six plasmids, only one of which is larger than 100 kb. Although the two plasmids containing repA_N in this study are larger than 100 kb, plasmids from other Gram-positive bacteria that contain this type of minireplicon are usually smaller than 100 kb [ 23 ]. Megaplasmids larger than 100 kb contain two or more minireplicons in B. cereus group Twenty-nine of the 31 megaplasmids larger than 100 kb contain two types of minireplicons (Table 1 ). Among them, 26 contain pXO1-14/pXO1-16 -like minireplicons and one subtype of minireplicon tubZ/tubR . The other three have different combinations of minireplicons. The 127 kb plasmid pCT127 contains minireplicon ori60 and repA_N , while the 139 kb plasmid pBMB28 contains ori44 and rep466 , and the 137 kb plasmid pBMB137 contains ori44 and ori60 . However, there are only two exceptions, pG9842_140 (140 kb) and p03BB108_282 (282 kb). No validated minireplicon was identified in plasmid pG9842_140, which indicates that it may contain novel minireplicon(s). The sequence of plasmid p03BB108_282 is incomplete. It remains thus unclear whether the single identified minireplicon rep228 supports replication of this plasmid or whether it contains an unidentified (novel) minireplicon. Indeed, most plasmids larger than 100 kb harbor two or more minireplicons (Figure 3 ), whereas plasmids smaller than 100 kb usually harbor only one. Moreover, three of five megaplasmids larger than 400 kb have three or more minireplicons. In the 417 kb plasmid pBWB401; there are five coexisting minireplicons of four different types. Figure 3 Plot of minireplicon number against plasmid size. Plasmids with a genome size larger than 100 kb contain two or more minireplicons. In plasmids with more than one minireplicon, it is not known whether all the minireplicons are functional for plasmid replication, partitioning and maintenance. For plasmid pXO1, early studies confirmed that two different types of minireplicons, pXO1-14/pXO1-16 [ 20 ] and repX [ 19 ], are functional for its replication. Recently, both the repX and pXO1-14/pXO1-16 minireplicons were proven to independently support replication of the full-length pXO1 plasmid, with pXO1-14/pXO1-16 being more effective than repX [ 26 ]. Moreover, a 4848-bp DNA fragment within minireplicon pXO1-14/pXO1-16 can be used to deprive plasmid pXO1 from B. anthracis using plasmid incompatibility [ 27 ]. This suggests that minireplicon pXO1-14/pXO1-16 is predominantly used for plasmid replication. We therefore speculate that when there is more than one minireplicon on the same plasmid, some of them are more relevant than others. However, how these minireplicons cooperate with each other is not clear. Minireplicons are conserved during the evolutionary history of a plasmid; however, it would be interesting to determine the evolutionary relationship of multiple minireplicons on the same plasmid. To investigate this, we conducted a comparative analysis between minireplicons tubZ/tubR and pXO1-14/pXO1-16 . First, we considered the relative position of the two minireplicons on the same plasmid. When there are only two types of minireplicon on one plasmid, the distance between the minireplicons ranged from 20 to 40 kb, and the distance between two minireplicons on larger plasmids is not larger (Spearman's r = 0.17, P = 0.41). Multiple minireplicons are frequently clustered in a certain region of the plasmid, which can be recognized as the core region for replication and maintenance. Second, we compared the three phylogenetic trees that were constructed based on protein sequences of TubZ, pXO1-14-like and pXO1-16-like (Figures 1 A and 2 ). The major topologies of these trees were inconsistent. The pXO1-14 or pXO1-16 trees cannot support the classification of four subtypes of TubZ. Plasmids with the same subtype of tubZ/tubR were usually found to have different subtypes of pXO1-14/pXO1-16 . This indicates that different minireplicons on the same plasmid evolved independently. Megaplasmids may be formed by fusion of smaller plasmids in B. cereus group As mentioned above, two or more different putative minireplicons generally occur in the same megaplasmids in B. cereus group. This may indicate that these megaplasmids have resulted from the integration of two or more smaller plasmids. Minireplicons of the four tubZ/tubR subtypes and pXO1-14/pXO1-16 were not found in plasmids with only one minireplicon. Of the megaplasmids whose genome sequences were available, we observed that minireplicon pXO1-14/pXO1-16 frequently coexists with one of the four tubZ/tubR subtypes. These megaplasmids may share similar origins and are probably the result of a fusion between an ancestral pXO1-14/pXO1-16 -like plasmid and an ancestral tubZ/tubR plasmid early in evolutionary history. For other megaplasmids, such as those containing ori44 , ori60 and repA_N , the minireplicons they contained were also found on smaller plasmids which usually have only one minireplicon. These minireplicons thus exist as sole replicon for small plasmids and as one of several minireplicons on megaplasmids. Direct evidence for this situation is provided by comparing pBMB137 of B. thuringiensis YBT-1520 to pBMB65 and pBMB95 of B. thuringiensis HD1. Plasmid pBMB137 has a genome size of 137,573 bp and contains the minireplicons ori44 and ori60. B. thuringiensis HD1 harbours the 65 kb plasmid pBMB65 with minireplicon ori44 , and the 95 kb plasmid pBMB95, with minireplicon ori60 . The genome sequence of pBMB137 can be divided into two fragments, one of which is virtually identical to pBMB65, and the other shows a high level of similarity to pBMB95 (Figure 4 ). Unlike the ancestral event that formed the pXO1-like plasmids, this fusion is a recent event as the separate and smaller plasmids are maintained by some strains while others maintain with the integrated megaplasmid. Figure 4 Comparison of the genomes of pBMB137, pBMB65 and pBMB95. From the inside: pBMB137, pBMB65 and pBMB95. Analysis of the relationships between minireplicon types and plasmid sizes revealed that plasmids with one minireplicon are usually smaller than 100 kb. However, when two or more minireplicons were present on the same plasmid, the genome size could exceed that of either of the presumed original plasmids, usually larger than 100 kb (Table 1 ). For example, plasmids containing minireplicon ori44 only have genome sizes from 45 to 85 kb. In contrast, other plasmids that combine ori44 and one or more additional minireplicons usually have a genome size larger than 100 kb, even up to 417 kb for the plasmid pBWB401. This indicates that by integrating different minireplicons into a single plasmid, the new plasmid is capable of carrying more genes. Larger plasmids have lower copy numbers than smaller ones [ 15 ]. Formation of larger plasmids by fusion of smaller plasmids thus reduces the amount of DNA that is required for similar plasmid genome sizes. This could provide an evolutionary advantage by reducing the energy requirement for plasmid synthesis and maintenance. Moreover, plasmids with some minireplicons have very low copy numbers, and additional minireplicons are needed to support them to replicate effectively. For example, plasmid pXO1 with only the repX minireplicon was reported to have copy numbers ranging from 0.8 to 1. This indicates that this minireplicon cannot effectively support plasmid replication [ 26 ]. If the plasmid contains another minireplicon, pXO1-14/pXO-16, in addition to the repX minireplicon, copy numbers ranging from 3 to 3.6 were observed and the plasmid is stably inherited. For those minireplicons that support effective plasmid replication, there may be dynamic equilibrium between the existence of small plasmids with individual minireplicons and the integration into megaplasmids with multiple minireplicons on plasmids. The selective forces driving plasmid evolution includes factors that determine the fitness of plasmids evolving as autonomous genetic elements as well factors that determine the added ecological fitness of the bacterial host. Ecological determinants that shape maintenance of small plasmids with one replicon or the integration into megaplasmids with multiple replicons are not clear. Compatibility groups of megaplasmids may depend on each of their minireplicons at the subtype level Megaplasmids contain more than one minireplicon and many strains contain more than one such megaplasmid, therefore, several minireplicons co-exist in the same host. To determine the compatibility of different minireplicons, we investigated the patterns of coexistence of minireplicons tubZ/tubR and pXO1-14/pXO1-16 . As shown in Figure 1 and Additional file 3 : Figure S4, each tubZ/tubR from the same strain belongs to one of the four subtypes, with different tubZ s, tubR s and putative origins of replication. For example, the two tubZ/tubR s on the two megaplasmids pBC210 and pBCXO1 of B. cereus G9241 belong to the repX and rep228 subtypes, respectively. Many strains have more than one pXO1-14/pXO1-16 minireplicon. In most cases, each of their encoded pXO1-14 or pXO1-16 proteins from a certain strain was found to belong to different subgroups. For example, pXO1-14-like proteins encoded by plasmids pBC210 and pBCXO1 in B. cereus G9241 belong to subgroups I and III, respectively (Figure 2 A). Their two corresponding pXO1-16-like proteins are also allocated to the two different subgroups, as shown by the pXO1-16-like protein tree (Figure 2 B). In other instances, different plasmids in the same host contain pXO1-14-like or pXO-16-like proteins but only one of the two belongs to the same subgroup. For example, the two pXO1-16-like proteins of pBMB293 and pBMB400 from B. thuringiensis YBT-1520 are located on different branches of the same subgroup (Figure 2 B), while the corresponding pXO1-14-like proteins show greater diversity and were allocated into subgroups II and IV, respectively. There was only one instance where different pXO1-14 and pXO1-16-like proteins from different minireplicons in the same strain were grouped together. Plasmid pAH1134_566 contains two pXO-14/16-like minireplicons and both proteins belong to the same subgroup. This may result from gene duplication and indicates that minireplicons of the same subgroups are compatible if they are located on the same plasmid. All plasmids of a bacterial strain must be compatible. The minireplicon tubZ/tubR has four subtypes; thus, there may be four natural incompatibility groups for tubZ/tubR -containing megaplasmids in the B. cereus group. Different groups have different TubZs, TubRs and putative origins of replication. For pXO1-14/pXO1-16 -like minireplicons, as the two essential proteins they encode do not have a concerted evolution, the putative incompatibility groups appear to be determined by the subgroup types of pXO1-14 or/and pXO1-16. Many plasmids contain both of these minireplicons; however, details regarding the coexistence of these plasmids are not clear. Integrated events are general among plasmids during their evolutionary histories Plasmids outside of the B. cereus group with more than one minireplicon have been reported, and the most frequently mentioned were plasmids belonging to incompatibility group F (IncF). Most plasmids of this group harbor two or more minireplicons, suggesting that plasmids fusion events occurred in the evolutionary histories of these plasmids [ 28 ]. The direct example is plasmid pIP1206, which may have resulted from recombination between pRSB107 and a pAPEC-O1-ColBM-like plasmid. Among its 151 open reading frames, 56 (37%) were also present in pRSB107 and 44 (29%) in pAPEC-O1-ColBM (24) [ 29 ]. In addition to analyzing plasmids of the B. cereus group, we analyzed the putative fusion events among all bacterial plasmids by studying distribution of putative minireplicons they contained. We analyzed the 3340 bacterial plasmids for which genome sequences are available. Of the 1285 plasmids with putative known minireplicons (Additional file 4 : Table S3), 34% (443 plasmids) have two or more of them (Figure 5 A), indicating that plasmids fusion events are general among these plasmids. Of these 443 plasmids, 78% (345 plasmids) and 17% (75 plasmids) have 2 and 3 minireplicons, respectively. This indicates that plasmids fusion events frequently happened between two or three plasmids but rarely occur between more than three plasmids. Moreover, we compared the genome sizes between plasmids with two or more minireplicons and those with only one. Plasmids with two or more minireplicons are significant larger than plasmids with only one minireplicon (Figure 5 B, P = 1.4e -7 , Mann–Whitney test). This indicates that integrating different plasmids into a single plasmid to form larger plasmids is general during the evolution of plasmids. Figure 5 Integrated events are general among plasmids during their evolutionary histories. (A) One third of all the plasmids analyzed contain two or more minireplicons, (B) Plasmids with two or more minireplicons are larger than those with only one (P = 1.4e -7 , Mann–Whitney test), (C) One third of the selected plasmids analyzed contain two or more minireplicons, (D) Plasmids in the selected dataset with two or more minireplicons are larger than those with only one (P = 1.035e -06 , Mann–Whitney test). In order to reduce the effect of data bias on the results, we used a subset of the plasmid sequence data to repeat the analysis. For each species that has plasmid genome sequences reported, we chose all plasmids from one strain whose plasmid number is the largest in that species. Analysis of this subset of the plasmid genome sequences confirmed the results obtained with the entire data set. Among the 771 plasmids with putative minireplicons (Additional file 5 : Table S4), one third of the plasmids have two or more minireplicons (Figure 5 C) and plasmids with two or more minireplicons are larger than those with only one minireplicon (Figure 5 D, P = 1.035e -06 , Mann–Whitney test). Six types of minireplicon exist in the megaplasmids of the B. cereus group The minireplicons are the core part of plasmids and drive plasmid replication and propagation. Their diversity and evolution directly reflects the dynamics and evolution of plasmids [ 7 , 10 , 22 , 23 ]. Strains in the B. cereus group are rich in plasmid content, with plasmid numbers ranging from zero to 13 and sizes ranging from 2 kb to 600 kb [ 6 , 15 , 24 ]. Thus, the B. cereus group is an ideal model to study plasmid dynamics and evolution. This study aimed to characterize the origin, evolution and dynamics of megaplasmids with genome sizes larger than 100 kb by studying the distribution and evolution of their minireplicons. We collected sequences of 56 plasmids with genome sizes ranging from 20 kb to 600 kb (Additional file 1 : Table S1), including 31 megaplasmids larger than 100 kb. Among these megaplasmids, 65 putative minireplicons were identified and could be classified into six types (Table 1 ). Two of the six types contain two replication essential protein coding genes. One type of minireplicon, which was first reported to support the replication of B. thuringiensis plasmid pBtoxis [ 21 ], tubZ/tubR , encodes TubZ/TubR proteins, which are FtsZ-like (TubZ) and DNA-binding proteins (TubR), respectively. The second type of minireplicon, which was first reported to support the replication of B. anthracis plasmid pXO1, encodes essential proteins belonging to the replication initiator protein (pXO1-16) and DNA-binding protein (pXO1-14) groups, respectively [ 20 , 21 ]. The other four types ( ori44 , ori60 , rep26 and repA_N ) encode four different essential single replication proteins, respectively [ 23 , 25 ]. Among these six types of minireplicon, tubZ/tubR , pXO1-14/pXO1-16 and rep26 only exist in megaplasmids, whereas the other three occur in both megaplasmids and plasmids smaller than 100 kb. Table 1 Type and number of minireplicons on plasmids larger than 20 kb Plasmid Number of minireplicon Minireplicon type Plasmid size (bp) pAH1134_566 3 rep466 , pXO1-16/pXO1-14 (2) 565,964 pE33L466 3 rep466 , rep26 , pXO1-16/pXO1-14 466,370 pBMB431 2 rep466 b , pXO1-16/pXO1-14 431,971 pBWB401 5 ori44 , repA_N , rep466 , pXO1-14/pXO1-16 (2) 417,054 pBMB400 2 rep466 , pXO1-16/pXO1-14 416,210 pBMB171 3 rep466 , rep26 , pXO1-16/pXO1-14 (2) 312,963 pBMB302 2 orf156/orf157 , pXO1-16/pXO1-14 302,255 pBMB293 2 orf156/orf157 , pXO1-16/pXO1-14 293,574 p03BB108_282 1 rep228 a 282,009 pCT281 2 orf156/orf157 , pXO1-16/pXO1-14 281,231 pAH820_272 2 repX , pXO1-16/pXO1-14 272,145 pPER272 2 repX , pXO1-16/pXO1-14 272,145 pAH187_270 2 repX , pXO1-16/pXO1-14 270,082 pH308197_258 2 repX , pXO1-16/pXO1-14 258,484 pBc239 2 repX , pXO1-16/pXO1-14 239,246 p03BB108_239 2 rep466 , pXO1-16/pXO1-14 238,933 pBMB228 2 rep228 , pXO1-16/pXO1-14 228,003 pG9842_209 2 rep228 , pXO1-16/pXO1-14 209,488 pBC210 2 rep466 , rep26 , pXO1-16/pXO1-14 209,385 pBc10987 2 repX , pXO1-16/pXO1-14 208,369 pBCXO1 2 repX , pXO1-16/pXO1-14 190,861 pBMB26 2 rep26 c , pXO1-16/pXO1-14 187,880 pCI-XO1 2 repX , pXO1-16/pXO1-14 181,907 pXO1 2 repX , pXO1-16/pXO1-14 181,677 p03BB102_179 2 repX , pXO1-16/pXO1-14 179,680 pBMB28 2 ori44 , rep466 139,013 pG9842_140 - NA 140,001 pBMB137 2 ori44 , ori60 137,573 pBtoxis 2 orf156/orf157 , pXO1-16/pXO1-14 127,923 pCT127 2 ori60 , repA_N 127,885 pBMB95 1 ori60 95,983 pXO2 1 repA 94,829 pCI-XO2 1 repA 94,469 p03BB108_86 1 repA 85,879 pCT83 1 ori44 83,590 pBT9727 1 repA 77,112 pBWB402 1 repA 75,107 pBMB74 1 repA 74,480 pH308197_73 1 ori60 72,792 pCT72 1 ori43 72,074 pAW63 1 repA 71,777 pBMB67 1 ori43 67,159 pBMB65 1 ori44 65,873 pBWB403 - NA 64,977 pBMB64 1 ori43 64,522 pLVP1401 - NA 56,149 pALH1 - NA 55,939 pFR55 1 ori44 55,712 pE33L54 - NA 53,501 pBWB404 - NA 52,830 pBc53 1 ori60 52,766 pCT51 - NA 51,488 pBMB46 - NA 46,634 pAH187_45 1 ori44 45,173 p03BB108_42 1 ori60 42,470 pH308197_29 - NA 29,189 a and b : The two new types of TubZ/TubR minireplicon identified in this study (supporting methods and results). c : Unpublished, validated by our group. (2): Number of minireplicons of the same type on the same plasmid. NA: no reported minireplicon. The minireplicon tubZ/tubR is distributed widely among the megaplasmids and is found in 26 of the 31 megaplasmids (Table 1 ). A phylogenetic tree was constructed based on the 26 TubZ protein sequences (Figure 1 A). Four different clades were formed and were supported by high bootstrap values (100%). Coincidentally, four of the TubZ proteins for which a function in replication was validated, RepX in plasmid pXO1 [ 19 ], ORF156 in plasmid pBtoxis [ 21 ], Rep228-TubZ in plasmid pBMB228 and Rep466-TubZ in plasmid pBMB28 are located in the four different clades, respectively. The replication function of Rep228-TubZ and Rep446-TubZ were validated in this study (see Additional file 2 ). We divided all 26 tubZ/tubR minireplicons into four subtypes: repX -like, orf156 / orf157 -like, rep228 -like and rep466 -like. Among the four subtypes, only minireplicon repX -like encodes an orphan TubZ protein, while the other three encode not only TubZ proteins, but also TubR proteins. TubR proteins from different subtypes show no similarity to each other. However, when the gene sequences of TubR within each subtype were inspected, we found that the topologies of the phylogenetic trees showed similarities to those of the corresponding TubZ trees (Figures 1 B, C and D), respectively. The DNA sequences of the origins of replication are rich in A + T and usually contain direct or invert repeats were additionally examined. The four minireplicon subtypes of tubZ/tubR have four different secondary structures of them, with different direct or inverted repeats (Additional file 3 : Figure S4). We therefore suggest that for each subtype of tubZ/tubR minireplicon, their TubZ, TubR proteins and the corresponding origin of replication underwent a concerted evolution. Figure 1 Phylogenetic trees constructed using the ML method based on TubZ (A) protein sequences and tubR DNA sequences (B for rep466 -like, C for rep228 -like and D for orf156/orf157 -like) from plasmids of the B. cereus group species. The four subtrees in (A) represent the four subtypes of TubZ/TubR minireplicons. Plasmids from the same strain are marked in the same color. The number at each branch point represents the percentage of bootstrap support calculated from 1,000 replicates, and only those values higher than 50 are shown. The minireplicon pXO1-14/pXO1-16 was found in 24 megaplasmids and three of these megaplasmids harbor two copies. When comparing the pXO1-14-like and pXO1-16-like protein sequences encoded by those 27 minireplicons, the sequence identities among pXO1-14-like proteins ranged from 40% to 100%. However, the pXO1-16-like proteins showed significantly greater conservation (P 65%. When phylogenetic trees based on these two families of protein sequences were constructed (Figure 2 ), the topologies of the two trees were incongruent, except for some in-group topologies. For the pXO1-14-like tree, four major subgroups were supported by high bootstrap values (Figure 2 A). However, two subgroups were identified for pXO1-16 (Figure 2 B). This indicates that genes encoding the pXO1-14-like and pXO1-16-like proteins evolved independently and multiple recombination events have occurred in this minireplicon. Figure 2 Phylogenetic trees constructed using the ML method based on pXO1-14 (A) and pXO1-16 (B) -like protein sequences from pXO1-14/pXO1-16 -like minireplicons from B. cereus group species. When there are two or more similar sequences on the same plasmid, their accession numbers are to the right of the plasmid name. Numbers at each branch point represent the percentage of bootstrap support calculated from 1000 replicates, and only those values greater than 50 are shown. The other four minireplicons contain one essential protein each. Minireplicon rep26 was found in four megaplasmids larger than 100 kb. In contrast, ori44 , ori60 and repA_N were found in both megaplasmids and plasmids smaller than 100 kb and are more widely distributed in smaller plasmids. For example, minireplicon ori44 occurs in eight plasmids, only two of which are larger than 100 kb, while minireplicon ori60 is contained by six plasmids, only one of which is larger than 100 kb. Although the two plasmids containing repA_N in this study are larger than 100 kb, plasmids from other Gram-positive bacteria that contain this type of minireplicon are usually smaller than 100 kb [ 23 ]. Megaplasmids larger than 100 kb contain two or more minireplicons in B. cereus group Twenty-nine of the 31 megaplasmids larger than 100 kb contain two types of minireplicons (Table 1 ). Among them, 26 contain pXO1-14/pXO1-16 -like minireplicons and one subtype of minireplicon tubZ/tubR . The other three have different combinations of minireplicons. The 127 kb plasmid pCT127 contains minireplicon ori60 and repA_N , while the 139 kb plasmid pBMB28 contains ori44 and rep466 , and the 137 kb plasmid pBMB137 contains ori44 and ori60 . However, there are only two exceptions, pG9842_140 (140 kb) and p03BB108_282 (282 kb). No validated minireplicon was identified in plasmid pG9842_140, which indicates that it may contain novel minireplicon(s). The sequence of plasmid p03BB108_282 is incomplete. It remains thus unclear whether the single identified minireplicon rep228 supports replication of this plasmid or whether it contains an unidentified (novel) minireplicon. Indeed, most plasmids larger than 100 kb harbor two or more minireplicons (Figure 3 ), whereas plasmids smaller than 100 kb usually harbor only one. Moreover, three of five megaplasmids larger than 400 kb have three or more minireplicons. In the 417 kb plasmid pBWB401; there are five coexisting minireplicons of four different types. Figure 3 Plot of minireplicon number against plasmid size. Plasmids with a genome size larger than 100 kb contain two or more minireplicons. In plasmids with more than one minireplicon, it is not known whether all the minireplicons are functional for plasmid replication, partitioning and maintenance. For plasmid pXO1, early studies confirmed that two different types of minireplicons, pXO1-14/pXO1-16 [ 20 ] and repX [ 19 ], are functional for its replication. Recently, both the repX and pXO1-14/pXO1-16 minireplicons were proven to independently support replication of the full-length pXO1 plasmid, with pXO1-14/pXO1-16 being more effective than repX [ 26 ]. Moreover, a 4848-bp DNA fragment within minireplicon pXO1-14/pXO1-16 can be used to deprive plasmid pXO1 from B. anthracis using plasmid incompatibility [ 27 ]. This suggests that minireplicon pXO1-14/pXO1-16 is predominantly used for plasmid replication. We therefore speculate that when there is more than one minireplicon on the same plasmid, some of them are more relevant than others. However, how these minireplicons cooperate with each other is not clear. Minireplicons are conserved during the evolutionary history of a plasmid; however, it would be interesting to determine the evolutionary relationship of multiple minireplicons on the same plasmid. To investigate this, we conducted a comparative analysis between minireplicons tubZ/tubR and pXO1-14/pXO1-16 . First, we considered the relative position of the two minireplicons on the same plasmid. When there are only two types of minireplicon on one plasmid, the distance between the minireplicons ranged from 20 to 40 kb, and the distance between two minireplicons on larger plasmids is not larger (Spearman's r = 0.17, P = 0.41). Multiple minireplicons are frequently clustered in a certain region of the plasmid, which can be recognized as the core region for replication and maintenance. Second, we compared the three phylogenetic trees that were constructed based on protein sequences of TubZ, pXO1-14-like and pXO1-16-like (Figures 1 A and 2 ). The major topologies of these trees were inconsistent. The pXO1-14 or pXO1-16 trees cannot support the classification of four subtypes of TubZ. Plasmids with the same subtype of tubZ/tubR were usually found to have different subtypes of pXO1-14/pXO1-16 . This indicates that different minireplicons on the same plasmid evolved independently. Megaplasmids may be formed by fusion of smaller plasmids in B. cereus group As mentioned above, two or more different putative minireplicons generally occur in the same megaplasmids in B. cereus group. This may indicate that these megaplasmids have resulted from the integration of two or more smaller plasmids. Minireplicons of the four tubZ/tubR subtypes and pXO1-14/pXO1-16 were not found in plasmids with only one minireplicon. Of the megaplasmids whose genome sequences were available, we observed that minireplicon pXO1-14/pXO1-16 frequently coexists with one of the four tubZ/tubR subtypes. These megaplasmids may share similar origins and are probably the result of a fusion between an ancestral pXO1-14/pXO1-16 -like plasmid and an ancestral tubZ/tubR plasmid early in evolutionary history. For other megaplasmids, such as those containing ori44 , ori60 and repA_N , the minireplicons they contained were also found on smaller plasmids which usually have only one minireplicon. These minireplicons thus exist as sole replicon for small plasmids and as one of several minireplicons on megaplasmids. Direct evidence for this situation is provided by comparing pBMB137 of B. thuringiensis YBT-1520 to pBMB65 and pBMB95 of B. thuringiensis HD1. Plasmid pBMB137 has a genome size of 137,573 bp and contains the minireplicons ori44 and ori60. B. thuringiensis HD1 harbours the 65 kb plasmid pBMB65 with minireplicon ori44 , and the 95 kb plasmid pBMB95, with minireplicon ori60 . The genome sequence of pBMB137 can be divided into two fragments, one of which is virtually identical to pBMB65, and the other shows a high level of similarity to pBMB95 (Figure 4 ). Unlike the ancestral event that formed the pXO1-like plasmids, this fusion is a recent event as the separate and smaller plasmids are maintained by some strains while others maintain with the integrated megaplasmid. Figure 4 Comparison of the genomes of pBMB137, pBMB65 and pBMB95. From the inside: pBMB137, pBMB65 and pBMB95. Analysis of the relationships between minireplicon types and plasmid sizes revealed that plasmids with one minireplicon are usually smaller than 100 kb. However, when two or more minireplicons were present on the same plasmid, the genome size could exceed that of either of the presumed original plasmids, usually larger than 100 kb (Table 1 ). For example, plasmids containing minireplicon ori44 only have genome sizes from 45 to 85 kb. In contrast, other plasmids that combine ori44 and one or more additional minireplicons usually have a genome size larger than 100 kb, even up to 417 kb for the plasmid pBWB401. This indicates that by integrating different minireplicons into a single plasmid, the new plasmid is capable of carrying more genes. Larger plasmids have lower copy numbers than smaller ones [ 15 ]. Formation of larger plasmids by fusion of smaller plasmids thus reduces the amount of DNA that is required for similar plasmid genome sizes. This could provide an evolutionary advantage by reducing the energy requirement for plasmid synthesis and maintenance. Moreover, plasmids with some minireplicons have very low copy numbers, and additional minireplicons are needed to support them to replicate effectively. For example, plasmid pXO1 with only the repX minireplicon was reported to have copy numbers ranging from 0.8 to 1. This indicates that this minireplicon cannot effectively support plasmid replication [ 26 ]. If the plasmid contains another minireplicon, pXO1-14/pXO-16, in addition to the repX minireplicon, copy numbers ranging from 3 to 3.6 were observed and the plasmid is stably inherited. For those minireplicons that support effective plasmid replication, there may be dynamic equilibrium between the existence of small plasmids with individual minireplicons and the integration into megaplasmids with multiple minireplicons on plasmids. The selective forces driving plasmid evolution includes factors that determine the fitness of plasmids evolving as autonomous genetic elements as well factors that determine the added ecological fitness of the bacterial host. Ecological determinants that shape maintenance of small plasmids with one replicon or the integration into megaplasmids with multiple replicons are not clear. Compatibility groups of megaplasmids may depend on each of their minireplicons at the subtype level Megaplasmids contain more than one minireplicon and many strains contain more than one such megaplasmid, therefore, several minireplicons co-exist in the same host. To determine the compatibility of different minireplicons, we investigated the patterns of coexistence of minireplicons tubZ/tubR and pXO1-14/pXO1-16 . As shown in Figure 1 and Additional file 3 : Figure S4, each tubZ/tubR from the same strain belongs to one of the four subtypes, with different tubZ s, tubR s and putative origins of replication. For example, the two tubZ/tubR s on the two megaplasmids pBC210 and pBCXO1 of B. cereus G9241 belong to the repX and rep228 subtypes, respectively. Many strains have more than one pXO1-14/pXO1-16 minireplicon. In most cases, each of their encoded pXO1-14 or pXO1-16 proteins from a certain strain was found to belong to different subgroups. For example, pXO1-14-like proteins encoded by plasmids pBC210 and pBCXO1 in B. cereus G9241 belong to subgroups I and III, respectively (Figure 2 A). Their two corresponding pXO1-16-like proteins are also allocated to the two different subgroups, as shown by the pXO1-16-like protein tree (Figure 2 B). In other instances, different plasmids in the same host contain pXO1-14-like or pXO-16-like proteins but only one of the two belongs to the same subgroup. For example, the two pXO1-16-like proteins of pBMB293 and pBMB400 from B. thuringiensis YBT-1520 are located on different branches of the same subgroup (Figure 2 B), while the corresponding pXO1-14-like proteins show greater diversity and were allocated into subgroups II and IV, respectively. There was only one instance where different pXO1-14 and pXO1-16-like proteins from different minireplicons in the same strain were grouped together. Plasmid pAH1134_566 contains two pXO-14/16-like minireplicons and both proteins belong to the same subgroup. This may result from gene duplication and indicates that minireplicons of the same subgroups are compatible if they are located on the same plasmid. All plasmids of a bacterial strain must be compatible. The minireplicon tubZ/tubR has four subtypes; thus, there may be four natural incompatibility groups for tubZ/tubR -containing megaplasmids in the B. cereus group. Different groups have different TubZs, TubRs and putative origins of replication. For pXO1-14/pXO1-16 -like minireplicons, as the two essential proteins they encode do not have a concerted evolution, the putative incompatibility groups appear to be determined by the subgroup types of pXO1-14 or/and pXO1-16. Many plasmids contain both of these minireplicons; however, details regarding the coexistence of these plasmids are not clear. Integrated events are general among plasmids during their evolutionary histories Plasmids outside of the B. cereus group with more than one minireplicon have been reported, and the most frequently mentioned were plasmids belonging to incompatibility group F (IncF). Most plasmids of this group harbor two or more minireplicons, suggesting that plasmids fusion events occurred in the evolutionary histories of these plasmids [ 28 ]. The direct example is plasmid pIP1206, which may have resulted from recombination between pRSB107 and a pAPEC-O1-ColBM-like plasmid. Among its 151 open reading frames, 56 (37%) were also present in pRSB107 and 44 (29%) in pAPEC-O1-ColBM (24) [ 29 ]. In addition to analyzing plasmids of the B. cereus group, we analyzed the putative fusion events among all bacterial plasmids by studying distribution of putative minireplicons they contained. We analyzed the 3340 bacterial plasmids for which genome sequences are available. Of the 1285 plasmids with putative known minireplicons (Additional file 4 : Table S3), 34% (443 plasmids) have two or more of them (Figure 5 A), indicating that plasmids fusion events are general among these plasmids. Of these 443 plasmids, 78% (345 plasmids) and 17% (75 plasmids) have 2 and 3 minireplicons, respectively. This indicates that plasmids fusion events frequently happened between two or three plasmids but rarely occur between more than three plasmids. Moreover, we compared the genome sizes between plasmids with two or more minireplicons and those with only one. Plasmids with two or more minireplicons are significant larger than plasmids with only one minireplicon (Figure 5 B, P = 1.4e -7 , Mann–Whitney test). This indicates that integrating different plasmids into a single plasmid to form larger plasmids is general during the evolution of plasmids. Figure 5 Integrated events are general among plasmids during their evolutionary histories. (A) One third of all the plasmids analyzed contain two or more minireplicons, (B) Plasmids with two or more minireplicons are larger than those with only one (P = 1.4e -7 , Mann–Whitney test), (C) One third of the selected plasmids analyzed contain two or more minireplicons, (D) Plasmids in the selected dataset with two or more minireplicons are larger than those with only one (P = 1.035e -06 , Mann–Whitney test). In order to reduce the effect of data bias on the results, we used a subset of the plasmid sequence data to repeat the analysis. For each species that has plasmid genome sequences reported, we chose all plasmids from one strain whose plasmid number is the largest in that species. Analysis of this subset of the plasmid genome sequences confirmed the results obtained with the entire data set. Among the 771 plasmids with putative minireplicons (Additional file 5 : Table S4), one third of the plasmids have two or more minireplicons (Figure 5 C) and plasmids with two or more minireplicons are larger than those with only one minireplicon (Figure 5 D, P = 1.035e -06 , Mann–Whitney test). Conclusions We found that megaplasmids in the B. cereus group larger than 100 kb contain two or more minireplicons. Minireplicons on the same plasmid usually have distinct evolutionary histories. We hypothesize that these megaplasmids are fusions of smaller plasmids. About one third of the plasmids out of the B. cereus group have multiple minireplicons. This indicates that plasmids fusion events occur generally during the plasmids evolutionary histories and plasmids fusion may be an important mechanism for the formation of megaplasmids. Methods Sequence collection The genome sequences of 45 plasmids of the B. cereus group were retrieved from GenBank ( http://www.ncbi.nlm.nih.gov ) and those of 11 unpublished plasmids sequenced by our group were used in the analyses. The genome sizes of these 56 plasmids ranged from ≈20 kb to ≈566 kb (Additional file 1 : Table S1). All of these data (Dataset 1) were obtained by October 20, 2012. To study the minireplicons across all of the prokaryotic species, we collected all 3340 plasmid genome sequences from Genbank ftp site ( ftp://ftp.ncbi.nlm.nih.gov/genomes/Plasmids/ ). These data (Dataset 2) were obtained by February 10, 2013. Replication essential protein sequences and minireplicons prediction TubZ protein sequences were obtained from Dataset 1 using the hmmsearch command of the hmmer version 3.0 software [ 30 ], with an e-value<0.001, and the model Tubulin/FtsZ family (PF00091) were obtained from the Pfam database [ 31 ]. Other types of replicated protein sequences were obtained by BLASTP analysis [ 32 ] using various types of reported replication protein sequences from B. cereus group plasmids as query sequences and the non-redundant protein sequences from Dataset 1 as the database. A minireplicon was approved when all of the essential elements, including one or two genes encoding replication essential proteins and the DNA fragment containing origin of replication, were predicted. We looked for replicated protein sequences from Dataset 2 by two methods; one was searching the keywords such as "replication protein", "Rep protein" or "Primase" from the annotation files, and the other one was using hmmsearch command of hmmer software with the models associated with plasmid replication (Additional file 6 : Table S2) which were downloaded from Pfam database [ 31 ]. Then we combined the results from both of the above methods, and checked these results based on public information. Minireplicons were approved when all the essential elements were predicted. All of the 1285 plasmids with putative minireplicon were showed in Additional file 4 : Table S3. Sequence alignment and phylogenetic analysis Protein sequences for different minireplicon of B. cereus group were aligned using Muscle [ 33 ]. The most disordered regions were eliminated using G-blocks [ 34 ]. The evolutionary models that best fit these sequences were determined by ProtTest version 3.0 [ 35 ], and Maximum Likelihood (ML) phylogenetic trees were generated by PhyML software version 3.0 [ 36 ], using the best fitted models (JTT + G + F for pXO1-14 and pXO1-16, LG + G + F for TubZ). Bootstrap supports were calculated as a percent of 1000 replicates. As the identity levels of TubR protein sequences among each type are very high, we collected the DNA sequences from them. Each type of tubR DNA sequences was aligned by Muscle and a ML tree was constructed using PhyML based on the model determined by ModelTest [ 37 ]. All the phylogenetic trees were deposited in treeBASE [ 38 ]. All statistical analyses were carried out using in-house Perl scripts and R 2.15.1 [ 39 ]. Sequence collection The genome sequences of 45 plasmids of the B. cereus group were retrieved from GenBank ( http://www.ncbi.nlm.nih.gov ) and those of 11 unpublished plasmids sequenced by our group were used in the analyses. The genome sizes of these 56 plasmids ranged from ≈20 kb to ≈566 kb (Additional file 1 : Table S1). All of these data (Dataset 1) were obtained by October 20, 2012. To study the minireplicons across all of the prokaryotic species, we collected all 3340 plasmid genome sequences from Genbank ftp site ( ftp://ftp.ncbi.nlm.nih.gov/genomes/Plasmids/ ). These data (Dataset 2) were obtained by February 10, 2013. Replication essential protein sequences and minireplicons prediction TubZ protein sequences were obtained from Dataset 1 using the hmmsearch command of the hmmer version 3.0 software [ 30 ], with an e-value<0.001, and the model Tubulin/FtsZ family (PF00091) were obtained from the Pfam database [ 31 ]. Other types of replicated protein sequences were obtained by BLASTP analysis [ 32 ] using various types of reported replication protein sequences from B. cereus group plasmids as query sequences and the non-redundant protein sequences from Dataset 1 as the database. A minireplicon was approved when all of the essential elements, including one or two genes encoding replication essential proteins and the DNA fragment containing origin of replication, were predicted. We looked for replicated protein sequences from Dataset 2 by two methods; one was searching the keywords such as "replication protein", "Rep protein" or "Primase" from the annotation files, and the other one was using hmmsearch command of hmmer software with the models associated with plasmid replication (Additional file 6 : Table S2) which were downloaded from Pfam database [ 31 ]. Then we combined the results from both of the above methods, and checked these results based on public information. Minireplicons were approved when all the essential elements were predicted. All of the 1285 plasmids with putative minireplicon were showed in Additional file 4 : Table S3. Sequence alignment and phylogenetic analysis Protein sequences for different minireplicon of B. cereus group were aligned using Muscle [ 33 ]. The most disordered regions were eliminated using G-blocks [ 34 ]. The evolutionary models that best fit these sequences were determined by ProtTest version 3.0 [ 35 ], and Maximum Likelihood (ML) phylogenetic trees were generated by PhyML software version 3.0 [ 36 ], using the best fitted models (JTT + G + F for pXO1-14 and pXO1-16, LG + G + F for TubZ). Bootstrap supports were calculated as a percent of 1000 replicates. As the identity levels of TubR protein sequences among each type are very high, we collected the DNA sequences from them. Each type of tubR DNA sequences was aligned by Muscle and a ML tree was constructed using PhyML based on the model determined by ModelTest [ 37 ]. All the phylogenetic trees were deposited in treeBASE [ 38 ]. All statistical analyses were carried out using in-house Perl scripts and R 2.15.1 [ 39 ]. Competing interests The authors declare no financial or non-financial competing interests. Authors' contributions SM and ZJS designed the study with help from PDH and RLF; ZJS performed the analysis; ZJS and SM wrote the manuscript. All authors approved the final version of the manuscript. Supplementary Material Additional file 1: Table S1 Plasmids analyzed in this study. Click here for file Additional file 2 Supplementary methods and results. Click here for file Additional file 3: Figure S4 Alignments of putative origins of replication of the four TubZ/TubR-like minireplicons. Click here for file Additional file 4: Table S3 Plasmid and replication-associated protein information of the 1285 plasmids with putative minireplicon. Click here for file Additional file 5: Table S4 Plasmid and replication-associated protein information of the 771 plasmids selected by host specific with putative minireplicon. Click here for file Additional file 6: Table S2 Models associated with plasmid replication used in this study. Click here for file Acknowledgments We want to thank Michael Gänzle from University of Alberta for his critical reading of the manuscript. This work was supported by grants from the National High Technology Research and Development Program (863) of China (2011AA10A203), China 948 Program of Ministry of Agriculture (2011-G25), the National Basic Research Program (973) of China (2009CB118902), the National Natural Science Foundation of China (31170047 and 31000020), the international scientific cooperation of Hubei province (2011BFA019), and the foundmental research fund for the central university (2011PY056).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3260641/

A CMOS Time-Resolved Fluorescence Lifetime Analysis Micro-System

We describe a CMOS-based micro-system for time-resolved fluorescence lifetime analysis. It comprises a 16 × 4 array of single-photon avalanche diodes (SPADs) fabricated in 0.35 μm high-voltage CMOS technology with in-pixel time-gated photon counting circuitry and a second device incorporating an 8 × 8 AlInGaN blue micro-pixellated light-emitting diode (micro-LED) array bump-bonded to an equivalent array of LED drivers realized in a standard low-voltage 0.35 μm CMOS technology, capable of producing excitation pulses with a width of 777 ps (FWHM). This system replaces instrumentation based on lasers, photomultiplier tubes, bulk optics and discrete electronics with a PC-based micro-system. Demonstrator lifetime measurements of colloidal quantum dot and Rhodamine samples are presented. 1. Introduction Fluorescence based analysis is a fundamental research technique used in the life sciences. However, conventional fluorescence intensity measurements are prone to misinterpretation due to illumination and fluorophore concentration non-uniformities. Thus, there is a growing interest in time-resolved fluorescence detection, whereby the characteristic fluorescence decay time-constant (or lifetime) in response to an impulse excitation source is measured. The sensitivity of a sample's lifetime properties to the micro-environment provides an extremely powerful analysis tool. However, current fluorescence lifetime analysis equipment tends to be bulky, delicate and expensive, thereby restricting its use to research laboratories. Progress in miniaturization of biological and chemical analysis instrumentation is creating low-cost, robust and portable diagnostic tools capable of high-throughput, with reduced reagent quantities and analysis times. Such devices will enable point-of-care or in-the-field diagnostics. In this paper, we report an integrated fluorescence lifetime analysis system capable of sub-nano second precision with the core of the instrument measuring less than 1 cm 3 , something hitherto impossible with existing approaches. To accomplish this, recent advances in the development of AlInGaN micro-LEDs and high sensitivity CMOS detectors have been exploited [ 1 , 2 ]. CMOS technology is key to both detection and excitation in our system providing compact, low cost, high speed electronic signal -processing circuitry for the photodetectors and vertically integrated drivers for the micro-LEDs. Furthermore, we demonstrate an array of pixellated fluorescence analysis sites with potential for multiplexed, high-throughput sensors, with reduced alignment tolerances. Combined with recent advances in on-chip, real-time lifetime computation [ 3 , 4 ] this work represents as significant step towards practical, micro-scale lifetime sensors, without the need for additional external hardware or sophisticated software post-processing. 2. Background 2.1. Fluroescence Lifetime Fluorophores have associated with them an exponential fluorescent decay transient after the removal of the excitation source, which defines their characteristic lifetime [ 5 ]. Due to the random nature of fluorescence emission, a fluorescent sample's associated lifetime is the average time the molecules in a sample spend in the excited state before photon emission occurs. A sample's fluorescence lifetime, τ, is determined by the rate at which the sample leaves the excited state ( Equation 1 ). The transition can occur via two mechanisms, either by fluorescence emission (at rate Γ) or by competing non-radiative processes (represented collectively as K nt ): (1) τ = 1 Γ + Σ K n t A fluorophore's quantum yield (Θ) is the ratio of emitted photons to the number of absorbed photons. This can be represented by Equation 2 : (2) Θ = Γ Γ + Σ K n t For a given excitation light intensity, a fluorophore's brightness (molecular brightness, q) can be calculated if the molecular absorption coefficient (ε) is known, Equation 3 : (3) q = ɛ × Θ The absorption coefficient of a fluorophore is usually constant; therefore, changes in a fluorophore's brightness can usually be attributed to changes in the sample's quantum efficiency. Therefore, from Equations 2 and 3 , if the fluorescence intensity changes this will usually result in a change in sample lifetime. Due to the fact fluorescence intensity is a composite property of a sample, dependent on sample quantity and concentration as well as instrument set-up, it is very sensitive to sample variation and is subject to interference from scattered light. This makes the observation of small intensity changes very difficult. Conversely, fluorescence lifetime is an intrinsic fluorophore property, independent of sample volume and concentration. Lifetime analysis is also less sensitive to instrument setup. Fluorescence lifetime is therefore a more robust analysis method compared to intensity measurement, capable of observing subtle changes in sample conditions [ 6 ]. The rate of non-radiative recombination is dictated by the fluorophore's electron structure and its interaction with the environment. Non-radiative decay mechanisms include [ 7 ]: Inter-system crossing Collisional or static quenching Solvent effects Resonance energy transfer. Fluorescence intensity is related to lifetime according to Equation 4 (for a mono-exponentially decaying sample). The equation assumes that the sample has been excited by an infinitely sharp (δ-function) light pulse. The time-dependent intensity at time t, I(t), is given by: (4) I ( t ) = I 0 exp ( − t τ ) Fluorescence lifetime is independent of fluorophore concentration but dependent on the sample's local environment. Thus, lifetime detection allows precise quantitative data about both fluorophore distribution and local environment to be obtained, while avoiding the problems related to fluorescence intensity imaging such as photo-bleaching [ 8 ]. Fluorescence lifetime detection can also be used to differentiate between fluorophores with overlapping spectra, but exhibiting different decay characteristics. Typical fluorescence decay times of organic compounds fall between a few hundreds of picoseconds and several nanoseconds. There are a number of different imaging experiments for which time-resolved detection can be used; these include, multiple fluorophore labeling [ 9 ], quantitative detection of ion concentrations and oxygen and energy transfer characteristics using fluorescence resonance energy transfer (FRET) [ 10 ]. There are two predominantly used techniques for measuring the fluorescence lifetime of a sample: the frequency-domain and time-domain methods. In the frequency domain a sample is excited by an intensity modulated light source. This results in the fluorescence emission being modulated at the same frequency, but with a phase shift due to the intensity decay law ( Equation 4 ) of the sample [ 7 , 11 ] and a reduction in the modulation depth. In the time domain the intensity decay of a fluorescent sample is directly measured as a function of time, following absorption of a short excitation pulse ( Figure 1 ). The design and application of bio-chips and micro-devices that can perform analysis for biomedical applications rapidly and inexpensively in a miniaturized environment has been the focus of much research [ 12 , 13 ]. A need for the development of simple, robust, cost-effective medical devices capable of rapidly screening for multiple diseases and to monitor pathogens has been identified as a key step in the fight against infectious diseases, especially in developing areas [ 14 ]. The miniaturization of diagnostic devices has the potential to increase throughput and reduce the cost of a wide range of diagnostic tests [ 15 ]. Furthermore, micro-scale systems often require reduced reagent quantities, resulting in reduced operating costs. The aim of much research into device miniaturization is to produce a point-of-care device, capable of performing sample analysis quickly and easily at a patient's bed-side or in a doctor's surgery [ 16 , 17 ]. Drug discovery is an area of research that could benefit from high-throughput miniaturized devices [ 18 ]. There is also on-going research into the development of implantable in vivo analysis devices [ 19 ]. Micro-analytical systems have been developed for the analysis of a wide range of analytes including oxygen [ 20 ], glucose, chemical and biological agents [ 21 ] as well as fluorophores and biological samples such as DNA [ 22 ]. One of the key challenges in the development of such devices is the integration of the different technologies required to produce a functional device. In a fluorescence-based device this would include sample excitation and detection elements alongside a sample handling mechanism such as micro-fluidics [ 23 ]. 2.2. Excitation Sources Traditionally, fluorescence excitation is achieved using laser sources or mercury or halogen lamps. Fluorescence analysis systems often contain several sources of different wavelength in order to allow samples of different excitation wavelength to be analysed. Arc and incandescent lamps are commonly used excitation light sources due to their broadband continuous emission, but their size, low efficiency and low stability make them unsuitable for miniaturized portable analysis systems. Gas discharge lamps have also been used for fluorescence excitation; these devices operate in a free-running mode and are different to control. Furthermore, the high supply voltage which they require (>5 kV) is difficult to provide in a compact format. Currently, the standard excitation source for time-domain fluorescence lifetime analysis is the pulsed laser diode. Available over the full visible wavelength spectrum these devices provide a low cost solution, relative to the femto-second Ti:Sapphire laser, to pulsed sample excitation. Once placed within a cooling heat sink these devices are therefore significantly larger than devices based on CMOS technologies (which are in the order of a few millimeters squared). In 1995, Araki and Misawa [ 24 ] demonstrated the use of commercially available blue InGaN/AlGaN LEDs for fluorescence lifetime measurements. Driven by an external RLC (resistor, inductor, capacitor) circuit and controlled by an avalanche transistor, these devices generated 4ns wide optical pulses with a 10 kHz repetition rate and a peak optical power of 40mW. In order to operate the avalanche transistor required a 300 V collector voltage. In addition, the inclusion of an inductive component in the drive circuit makes its realization in an integrated microelectronic circuit difficult. Using a high-gain photomultiplier tube and TCSPC hardware, accurate fluorescence lifetime measurements of Quinine-Sulfate are presented using these LED devices as an excitation source. This demonstrated how pulsed LEDs were suitable for consideration as a light source in time-domain fluorescence analysis. Fluorescence lifetime analysis using micro-LED excitation was demonstrated in [ 25 ] 64 × 64 matrix-addressable LED array driven by external hardware with a pulse width of 2 ns was used to excite a sample of rhodamine-123, with the subsequent fluorescence decay being captured by a commercially available photomultiplier tube (PMT). These InGaN/GaN devices measured 20 μm in diameter and were capable of producing 40 nW average optical power with a 4 V bias. Lifetime measurements of rhodamine-123 excited with a blue (460 nm) micro-LED and capture using a fast photomultiplier are presented. Being matrix-addressable the intersection of a row and column signal will activate and array element. As array sizes increases this creates potential fan-out problems. Furthermore, each row signal must supply current to all elements in that row; this limits pulse capabilities due to the associated slow RC time constants. This issue can be addressed by active address logic or by providing each element with a local driver circuit. The development of low-cost, miniaturized excitation sources for a full optical lab-on-a-chip is often neglected. Several groups have demonstrated fluorescence excitation using vertical cavity semiconductor devices [ 12 , 26 ] where they have been integrated into a micro-analytical device. These devices, however do not allow drive electronics and signal processing circuitry to be included on the same substrate. 2.3. Detectors Photon counting applications require detectors of single-photon sensitivity, these include: micro-channel plate PMTs, high-speed amplified PMTs, discrete photodiodes and avalanche photodiodes. These devices tend to be discrete components, requiring separate power supplies and a communication interface. Furthermore, they tend to be physically large and delicate (especially PMT devices). PMT devices are also sensitive to magnetic fields making difficult their integration into medical devices such as magnetic resonance imagers (MRI). A number of groups have demonstrated micro-scale fluorescence detection using a variety of different detectors. In [ 27 ] Patounakis et al. demonstrate CMOS detection of fluorescence lifetime decays using conventional CMOS photodiodes and on-chip signal processing circuitry. These devices rely on the integration of photodiode current to estimate photon intensity and does not display single-photon sensitivity. There has been significant progress in recent years in the development of CMOS image sensors, mainly driven by the demand from the mobile telephone market. Originally developed for the CCD image sensors, the pinned photodiode has now been utilized in CMOS image sensors, offering reduced dark current and transfer noise. In [ 28 ] a CMOS image sensor, aimed specifically at fluorescence lifetime imaging, with a 256 × 256 pinned photodiode array is implemented in a 0.18 μm image sensor specific CMOS process. A novel two-stage charge transfer pixel structure allows excitation and background photons to be subtracted from the detected signal leaving only signal due to fluorescence emission. Similar to the work presented in [ 27 ], fluorescence decay data is calculated by varying the time at which the photodiode is switched from passing charge to the drain node to storing charge for readout. Recent developments in the design of CMOS compatible single-photon avalanche diodes [ 1 ] allow extremely sensitive detectors to be integrated alongside signal processing circuitry. In order to gather photon arrival time data, from which fluorescence lifetime can be extracted, a number of circuit techniques have been proposed. These include; on-chip time-to-digital converters [ 29 ] and in-pixel time-gated counters [ 30 ]. Single-photon avalanche diodes offer micro-scale single-photon detection capabilities and their ability to capture fluorescence data has been well-documented [ 30 , 31 ], and [ 32 ]. They offer a number of other significant advantages; including being robust devices which are not destroyed by high light levels, insensitive to magnetic fields and are relatively easy to manufacture [ 33 ]. Despite growing interest in fully integrated CMOS based SPAD systems, SPAD detectors based on other semiconductor materials have also become more widespread. Despite the inability to integrate electronics on the same substrate as the detection element, these devices are often packaged alongside a second external quenching device [ 34 , 35 ]. The advantage of non-CMOS based devices is that the wavelength sensitivity of the device is no longer constrained by the junction depth and bandgap of silicon and can be tailored to individual applications. This can lead to SPAD detectors capable of detection in the near infra-red [ 36 , 37 ]. Unfortunately, these devices cannot take advantage of the large scale production capabilities and investment that has been made in silicon-based CMOS technology and do not offer a low cost solution to single-photon counting. 2.4. Miniaturisation In [ 15 ], a micro-system integrating a GaN thin-film LED alongside a CdS distributed Bragg reflector (DBR) filter, a PDMS microfluidic channel and Si PIN photodetector is presented. As this system was intended for intensity analysis, LED operation is DC and is driven by external hardware. Despite having a silicon substrate, this system includes no signal processing or LED control circuitry. The use of a microfluidic channel allows the sample of interest to be easily introduced into the micro-system. This device employs a planar topology, with the excitation and detection elements located on the same substrate, allowing the micro-fluidic device to be easily placed on top of the system with just 2 mm of separation between the sample and the detector. Similar work is presented in [ 25 ], whereby a VCSEL excitation source emitting at 773 nm has been integrated alongside emission filters and PIN photodetectors. As in [ 30 ], this device is intended for fluorescence intensity analysis and the VCSEL light source was not designed for short pulse excitation. Based on III-V materials the inclusion of CMOS electronics in this system is not possible. 2.1. Fluroescence Lifetime Fluorophores have associated with them an exponential fluorescent decay transient after the removal of the excitation source, which defines their characteristic lifetime [ 5 ]. Due to the random nature of fluorescence emission, a fluorescent sample's associated lifetime is the average time the molecules in a sample spend in the excited state before photon emission occurs. A sample's fluorescence lifetime, τ, is determined by the rate at which the sample leaves the excited state ( Equation 1 ). The transition can occur via two mechanisms, either by fluorescence emission (at rate Γ) or by competing non-radiative processes (represented collectively as K nt ): (1) τ = 1 Γ + Σ K n t A fluorophore's quantum yield (Θ) is the ratio of emitted photons to the number of absorbed photons. This can be represented by Equation 2 : (2) Θ = Γ Γ + Σ K n t For a given excitation light intensity, a fluorophore's brightness (molecular brightness, q) can be calculated if the molecular absorption coefficient (ε) is known, Equation 3 : (3) q = ɛ × Θ The absorption coefficient of a fluorophore is usually constant; therefore, changes in a fluorophore's brightness can usually be attributed to changes in the sample's quantum efficiency. Therefore, from Equations 2 and 3 , if the fluorescence intensity changes this will usually result in a change in sample lifetime. Due to the fact fluorescence intensity is a composite property of a sample, dependent on sample quantity and concentration as well as instrument set-up, it is very sensitive to sample variation and is subject to interference from scattered light. This makes the observation of small intensity changes very difficult. Conversely, fluorescence lifetime is an intrinsic fluorophore property, independent of sample volume and concentration. Lifetime analysis is also less sensitive to instrument setup. Fluorescence lifetime is therefore a more robust analysis method compared to intensity measurement, capable of observing subtle changes in sample conditions [ 6 ]. The rate of non-radiative recombination is dictated by the fluorophore's electron structure and its interaction with the environment. Non-radiative decay mechanisms include [ 7 ]: Inter-system crossing Collisional or static quenching Solvent effects Resonance energy transfer. Fluorescence intensity is related to lifetime according to Equation 4 (for a mono-exponentially decaying sample). The equation assumes that the sample has been excited by an infinitely sharp (δ-function) light pulse. The time-dependent intensity at time t, I(t), is given by: (4) I ( t ) = I 0 exp ( − t τ ) Fluorescence lifetime is independent of fluorophore concentration but dependent on the sample's local environment. Thus, lifetime detection allows precise quantitative data about both fluorophore distribution and local environment to be obtained, while avoiding the problems related to fluorescence intensity imaging such as photo-bleaching [ 8 ]. Fluorescence lifetime detection can also be used to differentiate between fluorophores with overlapping spectra, but exhibiting different decay characteristics. Typical fluorescence decay times of organic compounds fall between a few hundreds of picoseconds and several nanoseconds. There are a number of different imaging experiments for which time-resolved detection can be used; these include, multiple fluorophore labeling [ 9 ], quantitative detection of ion concentrations and oxygen and energy transfer characteristics using fluorescence resonance energy transfer (FRET) [ 10 ]. There are two predominantly used techniques for measuring the fluorescence lifetime of a sample: the frequency-domain and time-domain methods. In the frequency domain a sample is excited by an intensity modulated light source. This results in the fluorescence emission being modulated at the same frequency, but with a phase shift due to the intensity decay law ( Equation 4 ) of the sample [ 7 , 11 ] and a reduction in the modulation depth. In the time domain the intensity decay of a fluorescent sample is directly measured as a function of time, following absorption of a short excitation pulse ( Figure 1 ). The design and application of bio-chips and micro-devices that can perform analysis for biomedical applications rapidly and inexpensively in a miniaturized environment has been the focus of much research [ 12 , 13 ]. A need for the development of simple, robust, cost-effective medical devices capable of rapidly screening for multiple diseases and to monitor pathogens has been identified as a key step in the fight against infectious diseases, especially in developing areas [ 14 ]. The miniaturization of diagnostic devices has the potential to increase throughput and reduce the cost of a wide range of diagnostic tests [ 15 ]. Furthermore, micro-scale systems often require reduced reagent quantities, resulting in reduced operating costs. The aim of much research into device miniaturization is to produce a point-of-care device, capable of performing sample analysis quickly and easily at a patient's bed-side or in a doctor's surgery [ 16 , 17 ]. Drug discovery is an area of research that could benefit from high-throughput miniaturized devices [ 18 ]. There is also on-going research into the development of implantable in vivo analysis devices [ 19 ]. Micro-analytical systems have been developed for the analysis of a wide range of analytes including oxygen [ 20 ], glucose, chemical and biological agents [ 21 ] as well as fluorophores and biological samples such as DNA [ 22 ]. One of the key challenges in the development of such devices is the integration of the different technologies required to produce a functional device. In a fluorescence-based device this would include sample excitation and detection elements alongside a sample handling mechanism such as micro-fluidics [ 23 ]. 2.2. Excitation Sources Traditionally, fluorescence excitation is achieved using laser sources or mercury or halogen lamps. Fluorescence analysis systems often contain several sources of different wavelength in order to allow samples of different excitation wavelength to be analysed. Arc and incandescent lamps are commonly used excitation light sources due to their broadband continuous emission, but their size, low efficiency and low stability make them unsuitable for miniaturized portable analysis systems. Gas discharge lamps have also been used for fluorescence excitation; these devices operate in a free-running mode and are different to control. Furthermore, the high supply voltage which they require (>5 kV) is difficult to provide in a compact format. Currently, the standard excitation source for time-domain fluorescence lifetime analysis is the pulsed laser diode. Available over the full visible wavelength spectrum these devices provide a low cost solution, relative to the femto-second Ti:Sapphire laser, to pulsed sample excitation. Once placed within a cooling heat sink these devices are therefore significantly larger than devices based on CMOS technologies (which are in the order of a few millimeters squared). In 1995, Araki and Misawa [ 24 ] demonstrated the use of commercially available blue InGaN/AlGaN LEDs for fluorescence lifetime measurements. Driven by an external RLC (resistor, inductor, capacitor) circuit and controlled by an avalanche transistor, these devices generated 4ns wide optical pulses with a 10 kHz repetition rate and a peak optical power of 40mW. In order to operate the avalanche transistor required a 300 V collector voltage. In addition, the inclusion of an inductive component in the drive circuit makes its realization in an integrated microelectronic circuit difficult. Using a high-gain photomultiplier tube and TCSPC hardware, accurate fluorescence lifetime measurements of Quinine-Sulfate are presented using these LED devices as an excitation source. This demonstrated how pulsed LEDs were suitable for consideration as a light source in time-domain fluorescence analysis. Fluorescence lifetime analysis using micro-LED excitation was demonstrated in [ 25 ] 64 × 64 matrix-addressable LED array driven by external hardware with a pulse width of 2 ns was used to excite a sample of rhodamine-123, with the subsequent fluorescence decay being captured by a commercially available photomultiplier tube (PMT). These InGaN/GaN devices measured 20 μm in diameter and were capable of producing 40 nW average optical power with a 4 V bias. Lifetime measurements of rhodamine-123 excited with a blue (460 nm) micro-LED and capture using a fast photomultiplier are presented. Being matrix-addressable the intersection of a row and column signal will activate and array element. As array sizes increases this creates potential fan-out problems. Furthermore, each row signal must supply current to all elements in that row; this limits pulse capabilities due to the associated slow RC time constants. This issue can be addressed by active address logic or by providing each element with a local driver circuit. The development of low-cost, miniaturized excitation sources for a full optical lab-on-a-chip is often neglected. Several groups have demonstrated fluorescence excitation using vertical cavity semiconductor devices [ 12 , 26 ] where they have been integrated into a micro-analytical device. These devices, however do not allow drive electronics and signal processing circuitry to be included on the same substrate. 2.3. Detectors Photon counting applications require detectors of single-photon sensitivity, these include: micro-channel plate PMTs, high-speed amplified PMTs, discrete photodiodes and avalanche photodiodes. These devices tend to be discrete components, requiring separate power supplies and a communication interface. Furthermore, they tend to be physically large and delicate (especially PMT devices). PMT devices are also sensitive to magnetic fields making difficult their integration into medical devices such as magnetic resonance imagers (MRI). A number of groups have demonstrated micro-scale fluorescence detection using a variety of different detectors. In [ 27 ] Patounakis et al. demonstrate CMOS detection of fluorescence lifetime decays using conventional CMOS photodiodes and on-chip signal processing circuitry. These devices rely on the integration of photodiode current to estimate photon intensity and does not display single-photon sensitivity. There has been significant progress in recent years in the development of CMOS image sensors, mainly driven by the demand from the mobile telephone market. Originally developed for the CCD image sensors, the pinned photodiode has now been utilized in CMOS image sensors, offering reduced dark current and transfer noise. In [ 28 ] a CMOS image sensor, aimed specifically at fluorescence lifetime imaging, with a 256 × 256 pinned photodiode array is implemented in a 0.18 μm image sensor specific CMOS process. A novel two-stage charge transfer pixel structure allows excitation and background photons to be subtracted from the detected signal leaving only signal due to fluorescence emission. Similar to the work presented in [ 27 ], fluorescence decay data is calculated by varying the time at which the photodiode is switched from passing charge to the drain node to storing charge for readout. Recent developments in the design of CMOS compatible single-photon avalanche diodes [ 1 ] allow extremely sensitive detectors to be integrated alongside signal processing circuitry. In order to gather photon arrival time data, from which fluorescence lifetime can be extracted, a number of circuit techniques have been proposed. These include; on-chip time-to-digital converters [ 29 ] and in-pixel time-gated counters [ 30 ]. Single-photon avalanche diodes offer micro-scale single-photon detection capabilities and their ability to capture fluorescence data has been well-documented [ 30 , 31 ], and [ 32 ]. They offer a number of other significant advantages; including being robust devices which are not destroyed by high light levels, insensitive to magnetic fields and are relatively easy to manufacture [ 33 ]. Despite growing interest in fully integrated CMOS based SPAD systems, SPAD detectors based on other semiconductor materials have also become more widespread. Despite the inability to integrate electronics on the same substrate as the detection element, these devices are often packaged alongside a second external quenching device [ 34 , 35 ]. The advantage of non-CMOS based devices is that the wavelength sensitivity of the device is no longer constrained by the junction depth and bandgap of silicon and can be tailored to individual applications. This can lead to SPAD detectors capable of detection in the near infra-red [ 36 , 37 ]. Unfortunately, these devices cannot take advantage of the large scale production capabilities and investment that has been made in silicon-based CMOS technology and do not offer a low cost solution to single-photon counting. 2.4. Miniaturisation In [ 15 ], a micro-system integrating a GaN thin-film LED alongside a CdS distributed Bragg reflector (DBR) filter, a PDMS microfluidic channel and Si PIN photodetector is presented. As this system was intended for intensity analysis, LED operation is DC and is driven by external hardware. Despite having a silicon substrate, this system includes no signal processing or LED control circuitry. The use of a microfluidic channel allows the sample of interest to be easily introduced into the micro-system. This device employs a planar topology, with the excitation and detection elements located on the same substrate, allowing the micro-fluidic device to be easily placed on top of the system with just 2 mm of separation between the sample and the detector. Similar work is presented in [ 25 ], whereby a VCSEL excitation source emitting at 773 nm has been integrated alongside emission filters and PIN photodetectors. As in [ 30 ], this device is intended for fluorescence intensity analysis and the VCSEL light source was not designed for short pulse excitation. Based on III-V materials the inclusion of CMOS electronics in this system is not possible. 3. Device Implementation In this paper we present a micro-system that incorporates pixellated excitation and detection devices in a two-chip "sandwich" structure ( Figure 2 ). Combining the excitation source with a photodetector, on-chip driving electronics and lifetime signal processing circuitry, our devices represent a highly integrated lab-on-a-chip (LoC) system. Pixellation of detector and emitter arrays at 200 μm pitch are compatible with inkjet-spotted, multiplexed assay formats. The 777 ps optical pulse width is the shortest reported pulse for a CMOS-driven micro-LED device emitting at 450 nm and is suitable for excitation of commonly used, short lifetime fluorophores such as Rhodamine and Fluoroscein. Furthermore, the inclusion of an optical filter reduces measurement error caused by the detection of scattered excitation light. 3.1. Excitation Array Sample excitation is achieved using an 8 × 8 array of 72 μm diameter AlInGaN blue micro-pixellated light-emitting diodes (micro-LEDs) fabricated from "standard" InGaN/GaN quantum well blue LED wafers (planer n- and p- type GaN layers) grown on c -plane sapphire substrates by metal organic chemical vapor deposition [ 38 ]. This micro-LED array is bump-bonded to an equivalent array of LED driver circuits realized in a standard low-voltage 0.35 μm CMOS technology ( Figure 3 ). Each array element is individually addressable, with a dedicated driver circuit per micro-LED element. The wavelength spectra of the CMOS driven blue micro-LED device peaks at a wavelength of 450 nm. Each element of the CMOS driver array measures 200 μm × 200 μm with a 200 μm pitch. A pixel contains a dedicated driver circuit, driving a full metal bond-stack to which the micro-LED array was bump-bonded ( Figure 4 ). All driver input signals were based on 3.3 V logic before being level-shifted to a higher user-definable voltage (LED_VDD), to a maximum of 5 V. This allows standard 3.3 V logic to be used for the addressing and control logic in the pixel before the signal level is increased to LED_VDD (requiring the use of physically larger transistors capable of handling 5 V). The driver circuit ( Figure 5 ) is capable of producing optical pulses of user–definable width variable from 47.48 ns down to 777 ps, FWHM (±180 ps estimated measurement error, based on PMT RMS jitter), Figure 6 . By placing a square-wave signal on INPUT_SIG, the delay through inverter I1 defines the pulse width. The inverter delay can be adjusted via the gate voltage (VBMC2) of the current starving NMOS transistor M1. The level-shifted DC, pulsed, or square wave signal is then passed to an output buffer designed using transistors capable of handling up to 5 V. To minimize load capacitance on the input signal while maximizing the drive strength of the circuit, an output buffer comprising a chain of inverters of increasing transistor width/length ratios has been implemented. An on-chip voltage controlled oscillator (VCO) has also been implemented within the 8 × 8 driver array. This circuit is capable of producing a square wave signal with a tunable frequency range from 7 MHz to 800 MHz. The design features fine and course adjustment of the VCO frequency. The core frequency of the VCO is defined by the number of elements in the ring oscillator and the delay through each of these elements. Current starving transistors are placed within the ring oscillator and the gate voltage of these transistors is defined off-chip, thus allowing fine adjustment of the core ring oscillator frequency. The output of the ring oscillator is then passed to a digital divider circuit capable of dividing the input signal by 0, 4, 16 or 64 and hence producing a course selection of lower frequency signals. The VCO output could be used as the input signal to the drivers of the main array, defining the repetition rate of a square wave or pulsed input signal. By producing a square wave input signal on-chip the need for an off-chip clock (such as a crystal oscillator) has been removed, potentially reducing system size and cost. The performance of the micro-LED excitation array is summarized in Table 1 . 2.2. Detection Array A compact micro-system for time-resolved fluorescence was achieved by making use of CMOS technology's ability to integrate signal processing circuitry on the same chip as a sensor array, thereby allowing detector data to be directly processed. We describe how time domain, time-gated fluorescence lifetime analysis has been implemented on a CMOS chip. Using this method, the sample of interest is excited by a pulsed light source. The subsequent lifetime decay is captured within a series of two or more gated count windows. Using the count values obtained in each window a histogram of the fluorescence decay curve can be generated ( Figure 7 ). A fluorescence lifetime is then obtained by applying a lifetime extraction algorithm to the histogram data. A SPAD detector has been implemented which allows single photon detection through the action of avalanche breakdown in a p+/deep n-tub photodiode, reverse biased above its breakdown voltage (Geiger mode). These are situated in a 16 × 4 array pitch-matched to the micro-LEDs, allowing histogram and lifetime analysis without the need for external photon counting hardware and significantly reducing the amount of data to be broadcast off-chip. Direct observation of SPAD output pulses is also possible from an array of addressable SPADs situated directly within the micro-LEDs for confirmation of the integrated lifetime analysis techniques. A CMOS time-resolved analysis system has been designed, consisting of a fully addressable array of 16 × 4 array of SPADs integrated with on-chip signal processing and timing circuits. Each pixel measured 100 μm × 200 μm. The pixels incorporated two 9-bit ripple up-down counters with a novel time-gating mechanism allowing fully programmable scanning of time resolved events over a 48 ns range with a 408 ps resolution. The device was controlled by a FPGA and photon count histograms were captured and displayed by a PC. Figure 8 shows a system block diagram. Both the SPAD counter array and the micro-LED array were based on this architecture. By processing raw SPAD data locally within each pixel, the amount of data that would otherwise have to broadcast across the chip and potentially off-chip is minimized. Within each pixel two 9-bit up/down ripple counter circuits were implemented, these were designed using toggle (T-type) flip-flops (FF). The SPAD pulses provided the asynchronous clock to the first T-type FF in the counter ( Figure 9 ). A ripple counter was chosen to minimize the clock loading, since no synchronous count behaviour is required. An up-down counter was used to allow background light compensation although this was not implemented. Time-gated operation is accomplished by providing the toggle input of the first T-type FF in the counter with short pulses, which are generated within the pixel from delayed versions of the 3.68 MHz system clock broadcast to the array from the on-chip timing generator. The 9-bit word-length of each counter circuit allows 512 counts to be gathered before it is necessary to read-out the counter data. Two counters allow direct on-chip implementation of the two-gate RLD lifetime extraction method. Photon collection efficiency is improved by enabling the counters in immediate succession during the two time gate bins within one clock period. The timing generator consists of a 120-element tapped delay line composed of current limited buffers. The buffer unit delay is 408 ps with 44 ps RMS jitter at 3.3 V at room temperature. Three delayed versions of the 3.68 MHz system clock are generated; each delayed output can be selected independently under the control of a latched shift register. Time-gate widths can be selected from 408 ps to 48 ns with a resolution of 408 ps. Each element of the delay line consists of a two-inverter buffer with an in-line current starving transistor. The gate bias of the current starving transistor was passed off-chip, allowing the user to control the delay through each element in the delay chain. This allows the user to extend the maximum length of the delay generator at the expense of minimum time-gate width. The delay line generates three delayed versions of the system clock. The time delay between these three signals is user definable, by selecting the element of the tapped delay line which outputs the delayed clock. These three delayed clock signals are then broadcast globally across the chip to each pixel in the array. Circuitry within each pixel then generates time gates of width equal to the time delay between the signals. A schematic of the circuitry used to achieve this and a timing diagram of the process is shown Figure 10 . By using the difference between two signals broadcast to each pixel via the same route, jitter in the enable signal is minimised, as is the bandwidth requirement of the clock bus drivers. The delay setup by the tapped delay line is user definable via PC control of the FPGA. In this way, the time gates can be easily modified to fit the sample of interest. 3.1. Excitation Array Sample excitation is achieved using an 8 × 8 array of 72 μm diameter AlInGaN blue micro-pixellated light-emitting diodes (micro-LEDs) fabricated from "standard" InGaN/GaN quantum well blue LED wafers (planer n- and p- type GaN layers) grown on c -plane sapphire substrates by metal organic chemical vapor deposition [ 38 ]. This micro-LED array is bump-bonded to an equivalent array of LED driver circuits realized in a standard low-voltage 0.35 μm CMOS technology ( Figure 3 ). Each array element is individually addressable, with a dedicated driver circuit per micro-LED element. The wavelength spectra of the CMOS driven blue micro-LED device peaks at a wavelength of 450 nm. Each element of the CMOS driver array measures 200 μm × 200 μm with a 200 μm pitch. A pixel contains a dedicated driver circuit, driving a full metal bond-stack to which the micro-LED array was bump-bonded ( Figure 4 ). All driver input signals were based on 3.3 V logic before being level-shifted to a higher user-definable voltage (LED_VDD), to a maximum of 5 V. This allows standard 3.3 V logic to be used for the addressing and control logic in the pixel before the signal level is increased to LED_VDD (requiring the use of physically larger transistors capable of handling 5 V). The driver circuit ( Figure 5 ) is capable of producing optical pulses of user–definable width variable from 47.48 ns down to 777 ps, FWHM (±180 ps estimated measurement error, based on PMT RMS jitter), Figure 6 . By placing a square-wave signal on INPUT_SIG, the delay through inverter I1 defines the pulse width. The inverter delay can be adjusted via the gate voltage (VBMC2) of the current starving NMOS transistor M1. The level-shifted DC, pulsed, or square wave signal is then passed to an output buffer designed using transistors capable of handling up to 5 V. To minimize load capacitance on the input signal while maximizing the drive strength of the circuit, an output buffer comprising a chain of inverters of increasing transistor width/length ratios has been implemented. An on-chip voltage controlled oscillator (VCO) has also been implemented within the 8 × 8 driver array. This circuit is capable of producing a square wave signal with a tunable frequency range from 7 MHz to 800 MHz. The design features fine and course adjustment of the VCO frequency. The core frequency of the VCO is defined by the number of elements in the ring oscillator and the delay through each of these elements. Current starving transistors are placed within the ring oscillator and the gate voltage of these transistors is defined off-chip, thus allowing fine adjustment of the core ring oscillator frequency. The output of the ring oscillator is then passed to a digital divider circuit capable of dividing the input signal by 0, 4, 16 or 64 and hence producing a course selection of lower frequency signals. The VCO output could be used as the input signal to the drivers of the main array, defining the repetition rate of a square wave or pulsed input signal. By producing a square wave input signal on-chip the need for an off-chip clock (such as a crystal oscillator) has been removed, potentially reducing system size and cost. The performance of the micro-LED excitation array is summarized in Table 1 . 2.2. Detection Array A compact micro-system for time-resolved fluorescence was achieved by making use of CMOS technology's ability to integrate signal processing circuitry on the same chip as a sensor array, thereby allowing detector data to be directly processed. We describe how time domain, time-gated fluorescence lifetime analysis has been implemented on a CMOS chip. Using this method, the sample of interest is excited by a pulsed light source. The subsequent lifetime decay is captured within a series of two or more gated count windows. Using the count values obtained in each window a histogram of the fluorescence decay curve can be generated ( Figure 7 ). A fluorescence lifetime is then obtained by applying a lifetime extraction algorithm to the histogram data. A SPAD detector has been implemented which allows single photon detection through the action of avalanche breakdown in a p+/deep n-tub photodiode, reverse biased above its breakdown voltage (Geiger mode). These are situated in a 16 × 4 array pitch-matched to the micro-LEDs, allowing histogram and lifetime analysis without the need for external photon counting hardware and significantly reducing the amount of data to be broadcast off-chip. Direct observation of SPAD output pulses is also possible from an array of addressable SPADs situated directly within the micro-LEDs for confirmation of the integrated lifetime analysis techniques. A CMOS time-resolved analysis system has been designed, consisting of a fully addressable array of 16 × 4 array of SPADs integrated with on-chip signal processing and timing circuits. Each pixel measured 100 μm × 200 μm. The pixels incorporated two 9-bit ripple up-down counters with a novel time-gating mechanism allowing fully programmable scanning of time resolved events over a 48 ns range with a 408 ps resolution. The device was controlled by a FPGA and photon count histograms were captured and displayed by a PC. Figure 8 shows a system block diagram. Both the SPAD counter array and the micro-LED array were based on this architecture. By processing raw SPAD data locally within each pixel, the amount of data that would otherwise have to broadcast across the chip and potentially off-chip is minimized. Within each pixel two 9-bit up/down ripple counter circuits were implemented, these were designed using toggle (T-type) flip-flops (FF). The SPAD pulses provided the asynchronous clock to the first T-type FF in the counter ( Figure 9 ). A ripple counter was chosen to minimize the clock loading, since no synchronous count behaviour is required. An up-down counter was used to allow background light compensation although this was not implemented. Time-gated operation is accomplished by providing the toggle input of the first T-type FF in the counter with short pulses, which are generated within the pixel from delayed versions of the 3.68 MHz system clock broadcast to the array from the on-chip timing generator. The 9-bit word-length of each counter circuit allows 512 counts to be gathered before it is necessary to read-out the counter data. Two counters allow direct on-chip implementation of the two-gate RLD lifetime extraction method. Photon collection efficiency is improved by enabling the counters in immediate succession during the two time gate bins within one clock period. The timing generator consists of a 120-element tapped delay line composed of current limited buffers. The buffer unit delay is 408 ps with 44 ps RMS jitter at 3.3 V at room temperature. Three delayed versions of the 3.68 MHz system clock are generated; each delayed output can be selected independently under the control of a latched shift register. Time-gate widths can be selected from 408 ps to 48 ns with a resolution of 408 ps. Each element of the delay line consists of a two-inverter buffer with an in-line current starving transistor. The gate bias of the current starving transistor was passed off-chip, allowing the user to control the delay through each element in the delay chain. This allows the user to extend the maximum length of the delay generator at the expense of minimum time-gate width. The delay line generates three delayed versions of the system clock. The time delay between these three signals is user definable, by selecting the element of the tapped delay line which outputs the delayed clock. These three delayed clock signals are then broadcast globally across the chip to each pixel in the array. Circuitry within each pixel then generates time gates of width equal to the time delay between the signals. A schematic of the circuitry used to achieve this and a timing diagram of the process is shown Figure 10 . By using the difference between two signals broadcast to each pixel via the same route, jitter in the enable signal is minimised, as is the bandwidth requirement of the clock bus drivers. The delay setup by the tapped delay line is user definable via PC control of the FPGA. In this way, the time gates can be easily modified to fit the sample of interest. 4. System Configuration A dedicated PCB daughter card was designed, with the micro-LED device situated on the under-side of the PCB, facing the SPAD detector chip located on a FPGA test board. Electrical connection to the daughter card was made via stacked header pins. This technique allowed the distance between the micro-LED device and the SPAD detector chip to be adjusted. The excitation and detection arrays have a minimum separation of 3 mm. Both devices shared the same core power supplies and ground connections. These supplies and all other bias supplies, apart from the negative SPAD detector, were generated on the test board PCB and derived from the 5V supply of the USB connection. The negative supply required by the SPAD detector was generated by an external power supply. The devices shared a single FPGA situated on the test board (Opal Kelly, XEM3010), which generated the digital input signals to both devices. An optical filter and the sample of interest were placed between the devices. A plastic holder was designed to house these two elements. This holder provided a light tight enclosure for the packaged SPAD chip, an optical filter, a sample held in a micro-cavity slide and a packaged micro-LED device. Figure 11 shows the configuration of the two-chip system. The output from the on-chip VCO, situated on the micro-LED driver, defines the repetition rate of the LED device. This signal is passed off-chip (to a SMA connector on the daughter card) and is used as the synchronization input to a time-correlated single photon counting (TCSPC) module (Becker and Hickl, SPC-130), or the detector on-chip timing generator circuit ( Figure 12 ). Using this method, the excitation and detection elements of the system can be accurately synchronized. A 514 nm long pass filter (Semrock, LP02-514RU-25) was chosen to separate the excitation light from the fluorescence emission. This allows a range of fluorophores with emission spectra greater than 514 nm to be evaluated while maximizing the rejection of excitation light. 4. Results To assess the sensitivity of the SPAD detector and in-pixel counters, fluorescence lifetime analysis of a series of quantum dot samples of different concentrations was conducted. Quantum dot samples with an emission wavelength of 548 nm were prepared at concentrations of 50, 25, 10, 1, 0.1 and 0.01 μM. 45 μL of each sample was loaded into a single cavity (15 mm diameter) glass microscope slide (Fisher Scientific, UK, MNK-140-010A) and sealed with a 0.12 mm thick cover-slip. A Nikon TE2000-U Microscope was used, with a ×20 objective and a PicoQuant 467 nm pulsed diode laser light source. The SPAD detector was placed at a side output port of the microscope. The IRF was obtained using a sample of Ludox to scatter the excitation light. An overview of the experimental setup is provided in Table 2 . SPAD output pulses were processed using an external, commercially available TCSPC module (Becker and Hickl, SPC-130), Figure 13 and using the in-pixel, time-gated counter circuits, Figure 14 . The maximum number of counts in the peak channel of the decay curve increments appropriately according to the sample concentration and the decay curves remain parallel as they all represent the same sample lifetime. It was found that the SPADs were sensitive to approximately 0.01 μM. A reduction in the concentration of the quantum dot sample correlated closely with a reduction in the number of photon counts per second. Table 3 below, summarizes the extracted lifetimes from the decay curves presented in Figures 12 and 13 . Measurement error is based on 114 ps RMS SPAD jitter plus 4ps RMS TCSPC module jitter or 44 ps on-chip time-gate jitter. These lifetimes show how there is good agreement between the values captured using external TCSPC hardware and on-chip time-gated counters. The exception to this is the 0.01 μM sample, captured using TCSPC. At this low concentration only a small portion of the decay can be observed above the noise floor. This severely limits the fitting range that can be chosen for the lifetime extraction algorithm and can lead to skewed results. Measurements of fluorescence decay curves using the two-chip micro-system and external TCSPC hardware were obtained using quantum dots in a toluene solution (concentration = 57 μM) and Rhodamine 6G (concentration = 250 μM) and Rhodamine B (concentration = 100 μM) in water, Figure 15 . Analysis of these decay curves yielded lifetime estimations of 13.81 ns, 4.36 ns and 1.34 ns for the quantum dot sample, Rhodamine 6G and Rhodamine B samples, respectively (±122 ps estimated measurement error, based on RMS SPAD and time-gate jitter). These results were performed with an LED excitation pulse width of 910 ps (FWHM) and using a sample volume of 45 μL. This is consistent with lifetimes reported in the literature [ 39 , 40 ]. Furthermore, quantum dot lifetimes are consistent with those measured using a conventional microscope system, confirming the ability of the micro-system to accurately resolve fluorescence lifetime data. 5. Conclusions We have presented a micro-scale, CMOS-based single-photon sensitive detection system capable of sensing short lifetime fluorophores without lasers, PMTs or phoron counting acquisition cards. The limit of detection of the SPAD detector and in-pixel circuitry was found to be less than 10 nM and lifetimes could be captured with a resolution of 408 ps (minimum time-gate). The micro-LED driver is capable of producing optical pulses of 300 ps in width (FWHM) and a maximum DC optical output power of 550 μW. We expect further improvements to this detection limit and acquisition time by inclusion of micro-optics to collimate the LED [ 41 , 42 ] and microlenses to recover detector fill factor [ 43 ]. Improved packaging to reduce vertical height between the chips and inclusion of microfluidic channels for sample delivery are necessary developments towards a complete, low-cost, portable chemical/bio-diagnostic device.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613519/

Role of Visible Light-Activated Photocatalyst on the Reduction of Anthrax Spore-Induced Mortality in Mice

Background Photocatalysis of titanium dioxide (TiO 2 ) substrates is primarily induced by ultraviolet light irradiation. Anion-doped TiO 2 substrates were shown to exhibit photocatalytic activities under visible-light illumination, relative environmentally-friendly materials. Their anti-spore activity against Bacillus anthracis , however, remains to be investigated. We evaluated these visible-light activated photocatalysts on the reduction of anthrax spore-induced pathogenesis. Methodology/Principal Findings Standard plating method was used to determine the inactivation of anthrax spore by visible light-induced photocatalysis. Mouse models were further employed to investigate the suppressive effects of the photocatalysis on anthrax toxin- and spore-mediated mortality. We found that anti-spore activities of visible light illuminated nitrogen- or carbon-doped titania thin films significantly reduced viability of anthrax spores. Even though the spore-killing efficiency is only approximately 25%, our data indicate that spores from photocatalyzed groups but not untreated groups have a less survival rate after macrophage clearance. In addition, the photocatalysis could directly inactivate lethal toxin, the major virulence factor of B. anthracis . In agreement with these results, we found that the photocatalyzed spores have tenfold less potency to induce mortality in mice. These data suggest that the photocatalysis might injury the spores through inactivating spore components. Conclusion/Significance Photocatalysis induced injuries of the spores might be more important than direct killing of spores to reduce pathogenicity in the host. Background Photocatalysis of titanium dioxide (TiO 2 ) substrates is primarily induced by ultraviolet light irradiation. Anion-doped TiO 2 substrates were shown to exhibit photocatalytic activities under visible-light illumination, relative environmentally-friendly materials. Their anti-spore activity against Bacillus anthracis , however, remains to be investigated. We evaluated these visible-light activated photocatalysts on the reduction of anthrax spore-induced pathogenesis. Methodology/Principal Findings Standard plating method was used to determine the inactivation of anthrax spore by visible light-induced photocatalysis. Mouse models were further employed to investigate the suppressive effects of the photocatalysis on anthrax toxin- and spore-mediated mortality. We found that anti-spore activities of visible light illuminated nitrogen- or carbon-doped titania thin films significantly reduced viability of anthrax spores. Even though the spore-killing efficiency is only approximately 25%, our data indicate that spores from photocatalyzed groups but not untreated groups have a less survival rate after macrophage clearance. In addition, the photocatalysis could directly inactivate lethal toxin, the major virulence factor of B. anthracis . In agreement with these results, we found that the photocatalyzed spores have tenfold less potency to induce mortality in mice. These data suggest that the photocatalysis might injury the spores through inactivating spore components. Conclusion/Significance Photocatalysis induced injuries of the spores might be more important than direct killing of spores to reduce pathogenicity in the host. Introduction Naturally occurring anthrax is a disease acquired following contact with anthrax-infected animals or anthrax-contaminated animal products. The disease most commonly occurs in herbivores, which are infected by ingesting spores from the soil. For centuries, anthrax has caused disease in animals and, uncommonly, serious illness in humans throughout the world [1] . Research on anthrax as a biological weapon began more than 80 years ago [2] . Recently, the anthrax-letter attacks further evidenced this emerging terrorist threat, leading to renewed attention to the importance of prophylaxis, prevention and handling for anthrax [3] . Treatments or agents commonly cited to inactivate anthrax spores include heat, formaldehyde, hypochlorite solutions, chlorine dioxide, and radiation [4] . However, most of these treatments and reagents are hazardous to humans that limit their usage in public environments only after detecting the contamination sources, rather than prevention. Thus, a safer disinfection technique, which could exert a continuous antimicrobial effect in our living environment, would be highly desirable. Here, we present a visible light inducible photocatalyst which might provide a complementary and possibly alternative approach to meet this need. Photocatalytic titanium dioxide (TiO 2 ) substrates have been shown to eliminate organic compounds and to function as disinfectants [5] . Upon ultraviolet (UV) light excitation, the photon energy excites valence electrons and generates pairs of electrons and holes (electron-vacancy in the valence band) that react with atmospheric water and oxygen to yield reactive oxygen species (ROS) such as hydroxyl radicals ( . OH) and superoxide anions (O 2 − ) [6] . Electron holes, . OH and O 2 − are extremely reactive and could react with cellular components and function as biocides [5] . Since pure TiO 2 photocatalyst is effective only upon irradiation by UV-light at levels that would induce serious damage to human cells, the potential applications of TiO 2 substrates for use in our living environments are greatly restricted. Recently, anion-containing anatase TiO 2 photocatalysts have been identified, which are activated by illumination with visible light [7] , [8] , offering the potential to overcome this problem [9] . Previously, we demonstrated that nitrogen-doped TiO 2 [TiO 2 (N)] photocatalyst could significantly eliminate Escherichia coli and other mild pathogens [9] . However, its antimicrobial activity and the antimicrobial mechanism against biological weapons, such as Bacillus anthracis , has not yet been reported. In this study, the anti-spore activity of TiO 2 (N) photocatalysts against B. anthracis was compared with several other bacillus species including Bacillus subtilis , Bacillus thuringiensis , and Bacillus cereus . Among these bacteria, B. subtilis is broadly distributed worldwide where it mainly inhabits the upper layers of soil [10] , [11] ; B. thuringiensis is an insect pathogen [12] ; and B. cereus causes broad clinical infections including local infections, septicemia, central nervous system infections, respiratory infections, endocarditis, pericarditis and food poisoning [13] . In this study, visible light-mediated photocatalysis was found to inactive 25%–40% spores of B. subtilis, B. thuringiensis, B. cereus and B. anthracis . Even though the efficiency of bacterial-killing is less than 1 log CFU, intriguingly, animal experiment revealed that the photocatalysis reduced more than ten times potency of B. anthracis spores to induce mortality in mice. To further investigate the underlining mechanism, here we analyzed photocatalyst-mediated inactivation of anthrax lethal toxin (LT) and the survival rate of spore after the macrophage clearance in vitro . Results UV-Vis absorption spectroscopic analysis To investigate the physical properties of pure, carbon- and nitrogen- doped TiO 2 films, respectively, X-ray diffraction (XRD) patterns and Raman spectra (data not shown) were obtained. Our results showed that all the films were anatase phase but the intensity of XRD and Raman peaks was slightly decreased in carbon and nitrogen doped films, indicating that the carbon/nitrogen incorporation induced decreasing crystallinity. The UV-Vis spectra of pure, carbon and nitrogen doped TiO 2 films are shown in Fig. 1 . The carbon and nitrogen substitution of oxygen in TiO 2 caused the absorbance edge of TiO 2 to shift to the higher wavelength region. The pure TiO 2 film absorption edge which was at 380 nm gradually red-shifted to ∼425 nm and ∼565 nm in carbon and nitrogen doped TiO 2 films, respectively. This shift in the visible region is the result of incorporation of carbon and nitrogen into the TiO 2 network to form Ti-C and Ti-N bonds. Substitutional carbon or nitrogen atoms introduce new states (C2p or N2p) close to the valence band edge of TiO 2 (i.e. O2p states). As a result of this the valence band edge shifts to higher energy compared with the reference TiO 2 and the band gap narrows. The energy shift of the valence band depends on the overlap of carbon states and O2p states. A higher doping concentration of carbon or nitrogen results in higher energy shift due to significant overlap of carbon or nitrogen and oxygen states and this leads to a narrower band gap in the compound [14] . 10.1371/journal.pone.0004167.g001 Figure 1 UV-Vis absorption spectrum analysis. UV-Vis absorption spectra of pure, carbon- and nitrogen-doped TiO 2 thin films that used in this study were shown. Both doped samples absorbed light extending into the visible (>380 nm) region. Bactericidal activities of TiO 2 photocatalysts against Bacillus subtilis Before the spore experiments, living bacteria were used to test the photocatalysis system. Since B. anthracis is a hazardous microorganism, we first used B. subtilis as a surrogate to determine the bactericidal activity of nitrogen-doped [TiO 2 (N)] and carbon-doped TiO 2 [TiO 2 (C)]. We placed 1×10 4 CFU B. subtilis on different substrates including cover glass (silica, without TiO 2 coating), and silica substrates coated with thin films of TiO 2 , TiO 2 (N), and TiO 2 (C). These preparations were then illuminated with visible light and the levels of surviving bacteria were quantified as previously described [9] . We found that TiO 2 (N) exhibited a significantly better performance to reduce the number of surviving B. subtilis bacteria when compared to TiO 2 and TiO 2 (C) ( Fig. 2 , ** P 380 nm) region. Bactericidal activities of TiO 2 photocatalysts against Bacillus subtilis Before the spore experiments, living bacteria were used to test the photocatalysis system. Since B. anthracis is a hazardous microorganism, we first used B. subtilis as a surrogate to determine the bactericidal activity of nitrogen-doped [TiO 2 (N)] and carbon-doped TiO 2 [TiO 2 (C)]. We placed 1×10 4 CFU B. subtilis on different substrates including cover glass (silica, without TiO 2 coating), and silica substrates coated with thin films of TiO 2 , TiO 2 (N), and TiO 2 (C). These preparations were then illuminated with visible light and the levels of surviving bacteria were quantified as previously described [9] . We found that TiO 2 (N) exhibited a significantly better performance to reduce the number of surviving B. subtilis bacteria when compared to TiO 2 and TiO 2 (C) ( Fig. 2 , ** P <0.01). 10.1371/journal.pone.0004167.g002 Figure 2 Bactericidal activity against B. subtilis . Visible-light induced bactericidal activities of TiO 2 –related substrates against B. subtilis after illumination at 4°C. Illumination was carried out at a light density of 3×10 4 lux (90 mW/cm 2 ) for either 1 or 5 min. "Without illumination" indicates experiments conducted in a dark room without illumination. ** P <0.01, compared to both respective cover glass groups and without visible light illumination TiO 2 (N) groups. To obtain dose dependent and kinetic data for B. subtilis on photocatalytic substrates, we further analyzed the effects of visible-light illumination at various distances (5 cm, 10 cm, 20 cm, and with respective illumination intensities of 3×10 4 , 1.2×10 3 , and 3×10 2 lux) or at various time points ( Fig. 3 ). The results showed that TiO 2 and TiO 2 (C) substrates had no detectable bacterial-killing effect, while TiO 2 (N) contained significantly greater bactericidal activity, by which it induced nearly a 1 log CFU reduction under 3×10 4 lux visible-light illumination for 25 minutes ( Fig. 3A, 3B , * P <0.05; ** P <0.01, compared to respective TiO 2 groups). Although prolonged illuminations tended to increase the bactericidal effect of TiO 2 (C) substrates (25 min, Fig. 3B ), the killing efficiency was still not statistically significant as compared with the TiO 2 groups. 10.1371/journal.pone.0004167.g003 Figure 3 Dose dependency and kinetics. Dose dependent (A) and kinetic (B) analyses of the bactericidal activity of TiO 2 –related substrates against B. subtilis after visible light illumination were shown. Illumination was carried out either at different light densities for 25 min (A) or at a light density of 3×10 4 lux (90 mW/cm 2 ) for different time periods (B). For each illumination condition, the surviving bacteria on the TiO 2 groups were normalized to 100%. * P <0.05 and ** P <0.01 compared to the respective TiO 2 groups. Anti-spore activities of TiO 2 (N) against Bacillus species Photocatalyst-mediated killing was performed to determine the bactericidal effect of photocatalysis on B. cereus, B. thuringiensis and B. anthracis . Compared to TiO 2 thin films, we found that TiO 2 (N) thin films were significantly more effective in killing the living B. cereus , B. thuringiensis and B. anthracis bacteria under visible light illumination ( Fig. 4A , * P <0.05, ** P <0.01). 10.1371/journal.pone.0004167.g004 Figure 4 Elimination of living bacteria (A) and spores (B). Bacteria B. subtilis , B. thuringiensis , B. cereus and B. anthracis were placed on TiO 2 and TiO 2 (N) substrates, respectively. All surviving bacteria (A) or spores (B) in the TiO 2 groups were normalized to 100%. The relative percentages of surviving pathogens in the TiO2 (N) groups are shown. The illumination intensity was 3×10 4 lux (90 mW/cm 2 ) and the reaction time was 25 minutes. * P <0.05 and ** P <0.01 compared to respective TiO 2 groups. In spore experiments, TiO 2 (N) also exhibited a better anti-spore activity than TiO 2 thin films, although this activity was less efficient (25–40% killing/inactivation) ( Fig. 4B , * P <0.05) compared to the results obtained in living bacteria experiments ( Fig. 4A ). Treatments of photocatalyzed spores and LT to mice A mouse model was further used to investigate whether these photocatalyzed spores are less pathogenic. Before the photocatalyst experiment, we determined anthrax spore mediated mortality in mice. We found that 50% mortality required a single inoculation of 1×10 6 CFU anthrax spores, and the mortality reached to 100% when 7.5×10 6 CFU spores were used ( Fig. 5A ). In photocatalyst experiment, we found that visible-light induced photocatalysis on TiO 2 (N) but not pure TiO 2 thin-films significantly attenuated the ability of anthrax spores (1×10 7 CFU, before photocatalysis) to cause mortality in mice ( Fig. 5B , n = 6). Notably, the mortality of mice in TiO 2 (N) groups was lower than those given treatments of 1×10 6 CFU spores without photocatalysis [ Fig. 5B , TiO 2 (N), mortality 33.3%, vs. Fig. 5A , 1 ×10 6 groups, mortality 50%]. According to the killing efficiency estimated in spore-killing experiments (approximately 25%, Fig. 4B ), around 7.5×10 6 CFU spores should be remained viable, and theoretically the mortality of mouse should have reached up to 100% ( Fig. 5A , 7 .5×10 6 CFU groups). As a result, the reduced pathogenicity of photocatalyzed spores could not be simply attributed to the reduction of viable spores. To explain this phenomenon, we hypothesized that photocatalysis on TiO 2 (N) might not only eliminate 25% of the viable population but also injure the remaining spores through inactivating bacterial components. Such injuries could be resolved during germination of spores on cultural dishes but the repair process could not be accomplished in time to enable the bacteria escape from phagocytic clearance in mice. To investigate whether photocatalysis could inactivate protein components of B. anthracis , anthrax lethal toxin (LT), an example of bacterial proteins and the major virulence factor, was subjected to visible light activated photocatalysis on TiO 2 and TiO 2 (N) substrates. As expected, compared to untreated LT, photocatalysis of LT on TiO 2 (N) but not on TiO 2 substrates significantly reduced the potency of LT to induce mortality in mice ( Fig. 5C ). To investigate whether LT reduced its cytotoxicity after photocatalysis, a cell culture model was used. We found that LT could induce significant cell death of macrophage J774A.1 cells before but not after being subjected to TiO 2 (N)-mediated photocatalysis ( Fig. 6A , ** P <0.01). Western blot analysis revealed that the intact forms of both purified lethal factor (LF) and protective antigen (PA) molecules, two components of LT [15] , [16] , did not decrease after photocatalysis ( Fig. 6B ). These data might suggest that protein degradation dose not play major role in the inactivation of LT. 10.1371/journal.pone.0004167.g005 Figure 5 B. anthracis spore and LT caused mortality. Mortality of C57BL/6J mice after intravenous injection of different doses (0 to 1×10 7 CFU) of B. anthracis spores within one-week interval is revealed (A) (n = 8). Aliquots of B. anthracis spores (1×10 7 CFU) was subjected to photocatalysis on TiO 2 and TiO 2 (N) photocatalysts, respectively; spores in TiO 2 (N) groups induced less mortality in mice (△) compared to untreated (○) or TiO 2 (▾) groups (B) (n = 6). Aliquots of anthrax LT (500 µg PA : LF = 5∶1) was subjected to photocatalysis on TiO 2 and TiO 2 (N) photocatalysts, respectively; LT (100 µg/g) in TiO 2 (N) groups (△) induced less mortality in mice compared to untreated (○) or TiO 2 (▾) groups (C) (n = 6). 10.1371/journal.pone.0004167.g006 Figure 6 Cytotoxicity and Western blot analysis of photocatalyzed LT. Macrophage J774A.1 cells were subjected to LT treatments for three hours, surviving cells of untreated groups were adjusted to 100% (A). Columns designated TiO 2 or TiO 2 (N) represent that LT was pretreated with photocatalysis on TiO 2 or TiO 2 (N) substrates, respectively, before treated to J774A.1 cells. Columns designated "+L" or "−L" represent experimental conditions with or without light illumination, respectively. ** P <0.01, compared to LT, TiO 2 -L and TiO 2 (N)+L groups (A). Western blot measurements of intact LF and PA levels (arrows) of purified LT before and after visible light induced photocatalysis; band intensities of individual LF and PA samples are shown, and respective untreated groups were normalized to 100% (B). In vitro phagocytic clearance analysis Anthrax spore can multiply in phagocytes [17] . To investigate whether photocatalysis might injure the spores and make them vulnerable for the clearance by phagocytes and further handicapped the bacterial amplification within phagocytes, photocatalyzed anthrax spores were then treated to macrophage J774A.1 cells. We found that spores in light illuminated-TiO 2 (N) groups were not significantly multiplied in phagocytes within 24 hours ( Fig. 7A , TiO 2 (N)+L 1 hr vs. 24 hr). By contrast, untreated spores, or spores from groups without light illumination, or spores from illuminated-TiO 2 groups were all significantly multiplied 3–4 fold within 24 hours ( Fig. 7A , untreated/TiO 2 -L/TiO 2 (N)-L/TiO 2 +L, 1 hr vs. 24 hr, * P <0.05, ** P <0.01). Since low level of surviving bacteria in the 24-hour groups might be also possible attributed to the low phagocytic efficiency, to investigate whether photocatalysis might make these spores hard to be engulfed by phagocytes, we analyzed the remaining viable spores in the macrophage-culture medium. We found that the viable spores in the medium showed no significantly differences between each groups ( Fig. 7B , 1 hr vs. 2 hr, 3 hr and 24 hr, all groups compared to each other), indicating that the low level of surviving bacteria in the TiO 2 (N)+L/24 hr groups is not attributed to the low phagocytic efficiency ( Fig. 7A ). These results suggest that visible-light induced photocatalysis on TiO 2 (N) substrates handicapped the amplification of anthrax in phagocytes. 10.1371/journal.pone.0004167.g007 Figure 7 Surviving spores after clearance by macrophages. Anthrax spores were treated to J774A.1 macrophage cells (MOI: 0.01 spores/cell). Surviving bacteria (CFU) that harvested from macrophage cell lysate (A) or macrophage cell cultural medium (B) were shown. Columns designated TiO 2 or TiO 2 (N) represent anthrax spores were pretreated with photocatalysis on TiO 2 and TiO 2 (N) substrates, respectively. Columns designated "+L" or "−L" represent experimental conditions with or without light illumination, respectively. * P <0.05, ** P <0.01, compared to 1 hr groups of respective conditions (A). Discussion The antibacterial property of photocatalysts was primarily induced under UV irradiation [5] , [18] , [19] , and more recently by visible-light illuminated conditions [9] , [20] – [25] . These studies provide valuable observations for the bactericidal activity of photocatalysts. Since these researches were mainly using laboratory E. coli strains as experimental materials, the potential application to apply on the eradication of spores of pathogenic bacteria, and especially the capability and the mechanism to reduce their pathogenicity were rarely discussed. Here we used spore forming bacillus bacteria as model systems to study the anti-spore activity of the visible-light photocatalyst. Since B. anthracis is a hazardous microorganism, in this study we first used B. subtilis , a bacillus bacterium with natural habitat in the soil that is not harmful to humans, as a surrogate for B. anthracis . We found that TiO 2 (N) exerted anti-spore effects against B. subtilis and all our tested Bacillus species including B. cereus , B. thuringiensis and B. anthracis , with similar killing-efficiencies among these bacteria. These results suggest that B. subtilis , B. cereus , B. thuringiensis might be useful as surrogates for further photocatalyst-mediated anti-anthrax research. Even though the spore-killing efficiency is not good enough as compared with other well-developed methods [4] , the contrast between the low spore-killing and a relatively high reduction of pathogenicity, inspired us to further investigate the underlining mechanism, by which it led us to find that the photocatalysis could inactivate the LT and handicapped the spore to multiply in the phagocytes. This is a finding not yet been reported previously. The molecular target of photocatalyst-induced damage on bacteria is rarely discussed. It is shown that bacterial membrane lipid components likely to be the cellular target of photocatalyst induced ROS [5] . Using living E. coli as a model system, the authors found that TiO 2 -mediated photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of bacterial membranes and then further led to respiratory activity loss and cell death [5] . Unlike vegetative cells, spores contain strong resistance to almost all antibacterial agents [4] , [26] , [27] , as first observed by Koch over 100 years ago that B. anthracis spores could survive boiling. In the century that followed, it was learned that the protein components involving not only the composition of high density spore coat, a multilayered structure surrounding the spore that attributed to the resistance, but also the sensing responses to the renewed presence of nutrients in the environment, the condition under which the spore can convert to a growing cell through a process called germination [17] , [27] , [28] . Thus, the inactivation of a spore might be not just by disrupting the lipid components; deteriorated protein function could be involved as well. Photocatalysis-mediated protein dysfunction is rarely discussed. Evidence from enzyme-linked immunosorbent assays (ELISA) indicates that photocatalysis could affect the antigenic property of hepatitis B virus surface antigen and reduce its binding to specific antibody [29] . In this present study, we demonstrate the first time that photocatalysis could inactivate the bacterial exotoxin LT efficiently. Anthrax LT is a major virulence factor beneficial for the bacterium to establish initial infections in macrophages [30] , [31] . Our Western blotting analysis revealed that both PA and LF, two components of LT, remained intact after photoreactions, indicating protein degradation does not play a major role in the inactivation. Although evidences indicate that LT is not expressed in anthrax spores [32] , [33] , since both protein toxin and spores are sensitive to photocatalysis, it seems likely that some of the spore proteins might be also sensitive to the photoreactions. Further investigation to identify the specific protein is needed. Taken together, this study demonstrated that TiO 2 (N) substrates could inactivate both spores and toxin of B. anthracis under illumination by ordinary light sources such as incandescent lamps. Our results suggest that the suppressed amplification of B. anthracis in phagocytes might be more important than the direct killing for photocatalysts to reduce the pathogenicity of the spores. These concepts might provide a new prospect to develop next generation antimicrobial agents. Materials and Methods Preparation of TiO 2 substrates Three types of films, TiO 2 , TiO 2−x C x and TiO 2−x N x, were prepared in an ion-assisted electron-beam evaporation system (Branchy Vacuum Technology Co., Ltd., Taoyuan, Taiwan). The distance between the rotating substrate holder and the electron-beam evaporation source was 550 mm. The chamber was evacuated with a mechanical pump (ALCATEL-2033SD, LACO Technologies, Salt Lake City, UT, USA) and a cryopump (Cryo-Torr8®, ULVAC Cryogenics, Chigasaki City, Kanagawa Prefecture, Japan) to a base pressure below 2.7×10 −4 Pa. The substrates used were polished Si (100), quartz and glass coupons, which were sputter-etched with argon ions (Ar + ) for 5 minutes prior to the deposition to remove any residual surface pollutants. The substrate temperature was maintained at 300°C with a quartz lamp. The TiO 2 films were deposited in oxygen atmosphere (6.7 ×10 −3 Pa) using rutile TiO 2 (99.99%) as a source material. The nitrogen flow for TiO 2−x N x films was 15 standard cm 3 min −1 through the ion gun at a constant pumping speed and the chamber pressure was 4.4 ×10 −2 Pa. The carbon dioxide gas flow for TiO 2−x C x films was 7 standard cm3 min-1 and the chamber pressure was 2.6×10 −2 Pa. The ion gun beam current of 10 mA and voltage of −1000 V was maintained by a Commonwealth Scientific IBS controller. Sufficient energy and current of the ion beam are critical to incorporate significant dopant concentration in the film. Without ion bombardment, it is difficult for the dopant to compete with the oxygen for incorporation into anatase titania. The deposition rate was adjusted to 0.2 nm·s −1 using a quartz crystal monitor for all films deposited at a thickness of 1.2 µm. The three types of films were prepared under the optimized conditions for their categories of anatase crystallinity and dopant concentration [34] , [35] . The structure and crystallinity of the films were investigated using a Rigaku D/MAX-2500V 18 kW low angle X-ray diffractometer (XRD) (Rigaku, Shibuya-Ku, Tokyo, Japan) operating with Cu-K α radiation at 40 kV and 150 mA and a Renishaw 1000B Raman spectrometer equipped with a charge-coupled detector (CCD) and a CW 532 nm wave length diode pump solid state (DPSS) laser as the excitation source (Renishaw plc, Representative Office, Nantun District, Taichung, Taiwan). The UV-Vis absorption spectra were recorded on a Hitachi 3300H spectrophotometer (Hitachi Taiwan, Taipei, Taiwan). Bacterial strains and culture B. anthracis (ATCC 14186), which contains both pXO1 and pXO2 plasmids that express functional lethal toxin (LT) and edema toxin (ET), was grown and maintained as previously described [16] , [36] , [37] . B. cereus (ATCC 13061) and B. thuringiensis (ATCC 35646) were maintained and cultured in nutrient agar or nutrient broth at 30°C [38] , [39] , and B. subtilis (ATCC 39090) was maintained and cultured in trypticase soy agar or broth at 37°C [40] . All bacteria were stored in 50% medium and 50% glycerol solution in freezers at −80°C before use. To reactivate bacteria from frozen stocks, 25 µl bacterial stock solution was transferred to a test tube containing 5 ml of freshly prepared culture medium and then incubated at 30°C or 37°C under agitation overnight (16–18 hr). Spores of B. anthracis were prepared as previously described [41] , [42] . Overnight tryptic soy broth cultures of B. anthracis were diluted to about 10 7 CFU/ml in phosphate-buffered saline, and 0.1-ml aliquots were inoculated onto blood agar plates. The agar plates were incubated at 25–37°C until 90–99% phase-bright spores were observed by phase-contrast light microscopy (see below). Spores were harvested and washed with cold sterile distilled ionized (DI) water as previously described [41] and stored in DI water at 4°C until use for up to 2 weeks, changing the water at least once a week, or in the freezer at −20°C for up to a month. The quality of spores was determined by two complementary criteria previously established to validate the presence of dormant spores [42] . The criteria consisted in the evaluation of (i) the absence of vegetative cells (rods) determined by microscopic examination as described, and (ii) the survival of spores in hydrochloric acid (2.5 N). Spore preparations of B. subtilis , B. cereus and B. thuringiensis were followed a similar protocol. Photocatalytic reaction and detection of viable bacteria In this study, bacterial concentrations were either determined by the standard plating method or inferred from optical density readings at 600 nm (OD 600 ). For each Bacillus species, a factor for converting the OD 600 values of the bacterial culture to concentration (CFU/ml) was calculated as follows. A fresh bacterial culture was diluted by factors of 10 −1 to 10 −7 , and OD 600 of these dilutions was measured. Bacterial concentrations of these dilutions were determined by the standard plating method. The OD 600 values were plotted against the bacterial concentration log values, and the conversion factors for the particular bacteria were calculated from three independent measurements. For example, the conversion factor for B. subtilis was calculated to be 1×10 8 CFU/ml per OD 600 by this method. In order to determine the bactericidal effects of the TiO 2 -related substrates, 200 µl of bacterial overnight culture was transferred into 5 ml of culture medium and incubated at 37°C until an OD 600 of 0.3 to 0.6 (log phase) was reached. The bacterial concentrations were calculated using the previously determined conversion factor for the bacteria, and the cultures were diluted to 1×10 5 CFU/ml with culture medium. One hundred microliters (1×10 4 CFU) was then applied to an area of approximately 1 cm 2 of the different TiO 2 -related substrates using a plastic yellow tip. The bacteria substrates were then placed under an incandescent lamp (Classictone incandescent lamp, 60W, Philips, Taiwan) for photocatalytic reaction. A light meter (model LX-102, Lutron Electronic Enterprises, Taiwan) was used to record the illumination density. In the following photocatalysis experiments, the bacteria solution was supplied by 5–10 µl additional distil water every 5 minutes to maintain approximately 100 µl of total volume. After the photocatalyst-killing for 25 minutes, the bacteria containing solution approximately 85 µl was recovered from the photocatalyst substrates using a tip, additional 60 µl fresh medium was used to wash the remaining bacteria on the photocatalyst substrates. Two bacterial containing solutions were mixed, diluted and placed on agar plates. To test whether spores have different efficiency to adhere on TiO 2 substrates that might influence the photocatalysis result, we analyze the spore recovery rates from TiO 2 , TiO 2 (C) and TiO 2 , TiO 2 (N) substrates. We found that the recovery rates are similar (data not shown), which has no statistic significant among these groups. In the dose-dependence experiments, illumination was carried out for 5 min at distances of 5, 10, and 15 cm from the lamp, corresponding to the illumination densities of 3×10 4 , 1.2×10 3 , and 3×10 2 lux (lumen/m 2 )(90, 30, and 10 mW/cm 2 ), respectively. In the kinetic analysis experiments, illumination was carried out for 1, 5, 10, 15, and 25 min at a distance of 5 cm, corresponding to an illumination density of 3×10 4 lux (90 mW/cm 2 ). Unless specified, illumination was carried out in a 4°C cold room to prevent over-heating of the photocatalyst substrates and prevent drying. After illumination, the bacterial solutions were recovered from the photocatalyst substrates, and an aliquot of fresh culture medium was used to collect the residual bacteria on the substrates. The two bacterial solutions were pooled to make a total volume of 150 µl. The bacterial concentration was determined by the standard plating method immediately after the bacterial collection, and the percentage of surviving bacteria was calculated. In spore experiments, 1×10 4 CFU (1×10 5 CFU/ml in 100 µl) were used, and the procedures followed the same protocols as in the live bacteria experiments. Mouse model Six to 8 week - old C57BL/6J mice were purchased from the National Experimental Animal Center (Taipei, Taiwan) [37] , [43] . The methods used in bacteremia experiments were modified from previous descriptions [9] . Mortality of C57BL/6J mice from various anthrax spores treatments (from 1×10 6 to 1×10 7 ) was recorded within one week to serve as reference points. To determine the anti-bacterial effect of TiO 2 (N), each mouse received an intravenous injection of 1×10 5 CFU spores of B. anthracis , a lethal dose for mice, with or without pretreatment by photocatalysis on TiO 2 (N) or TiO 2 substrates (3×10 4 lux, 10 minutes at 4°C). In the lethal toxin (LT) experiments, mortality of mice after a lethal dose of LT was performed based on previously described methods [37] . Each mouse received an intravenous injection of LT (100 µg/g, LF:PA = 1∶5), a lethal dose for mice, with or without pretreatment of photocatalysis on TiO 2 (N) or TiO 2 substrates (3×10 4 lux, 10 minutes at 4°C or 25°C). The mortality of mice was then recorded. During the photocatalysis reaction, the distance between lamps with bacteria- or LT-containing photocatalyst substrates was 5 cm, corresponding to an illumination density of 3×10 4 lux (or 90 mW/cm 2 ). Relative protein levels of PA and LF in the LT mixtures used in animal experiments were detected by Western blot using rabbit polyclonal anti-PA and anti-LF antibodies, and then probed by secondary horseradish peroxidase-conjugated goat anti-rabbit immunoglobulins [16] , [44] , [45] . The gel intensities of PA and LF were measured using Image J software (version 1.32; National Institutes of Health, USA). The Animal Care and Use Committee of Tzu-Chi University approved the protocol of the mice experiments. Cytotoxicity analysis Cytotoxicity of LT was measured following a previously described method [16] . In brief, a cytotoxic dose of LT (10 mg/L, LF:PA = 1∶5) with or without photocatalysis pretreatment on TiO 2 and TiO 2 (N) thin film was used to treat mouse macrophage J774A.1 cells. Three hours after the LT treatments, cell viability of J774A.1 cells were measured using a WST-1 kit (Roche, Mannheim, Germany), following the instructions provided by the manufacturer. Photocatalysis of purified LT was carried out as described in mouse experiments (3×10 4 lux, 10 min at 4°C). Phagocytosis analysis Anthrax spores is normal saline (100 µl, 1×10 5 CFU/ml) were placed on cover glass, TiO 2 and TiO 2 (N) coated thin films [9] , respectively. The spore-photocatalyst mixtures (100 µl) were then illuminated with visible light (Classictone incandescent lamp, 60W, Philips; 90 mW/cm 2 ; lamp-target distance 10 cm) for 30 minutes. After illumination, the spore containing solutions (85 µl) were recovered from the photocatalyst substrates, and an aliquot of normal saline (60 µl) was used to collect the residual spores on the substrates. The two spore solutions were pooled to make a total volume of 145 µl. This spore solution was then added into one well of a six-well cell culture dish that containing confluent murine macrophage J774A.1 cells (1×10 6 cells/well) (MOI: 0.01 spores/cell). After phagocytosis was carried out for one, two and three hours, respectively, culture medium was removed, and 200 µl cell lysis buffer (100mM Tris-HCl [pH 7.4], 10mM MgCl 2 , 100mM NaCl, 0.2% sucrose, 0.5% Triton X-100) that was modified from previous literatures [46] , [47] , was then added to release the cell-engulfed or cell-bound spores. Additional 100 µl fresh medium was used to further collect the residual spores on the dishes. Two spore containing solutions were mixed and placed on agar plates. Cell culture medium (DMEM) without antibiotics and serum supplements was used in this analysis. Statistical analysis All results were calculated from data of at least three independent experiments. A t -test was used to assess the significance of differences in results of anti-microbial effects. A P value of less than 0.05 ( P <0.05) was considered statistically significant. The statistical tests were carried out and output to graphs using Microsoft Excel (Microsoft Taiwan, Taipei, Taiwan) and SigmaPlot (Systat Software, Point Richmond, CA, USA) software. Preparation of TiO 2 substrates Three types of films, TiO 2 , TiO 2−x C x and TiO 2−x N x, were prepared in an ion-assisted electron-beam evaporation system (Branchy Vacuum Technology Co., Ltd., Taoyuan, Taiwan). The distance between the rotating substrate holder and the electron-beam evaporation source was 550 mm. The chamber was evacuated with a mechanical pump (ALCATEL-2033SD, LACO Technologies, Salt Lake City, UT, USA) and a cryopump (Cryo-Torr8®, ULVAC Cryogenics, Chigasaki City, Kanagawa Prefecture, Japan) to a base pressure below 2.7×10 −4 Pa. The substrates used were polished Si (100), quartz and glass coupons, which were sputter-etched with argon ions (Ar + ) for 5 minutes prior to the deposition to remove any residual surface pollutants. The substrate temperature was maintained at 300°C with a quartz lamp. The TiO 2 films were deposited in oxygen atmosphere (6.7 ×10 −3 Pa) using rutile TiO 2 (99.99%) as a source material. The nitrogen flow for TiO 2−x N x films was 15 standard cm 3 min −1 through the ion gun at a constant pumping speed and the chamber pressure was 4.4 ×10 −2 Pa. The carbon dioxide gas flow for TiO 2−x C x films was 7 standard cm3 min-1 and the chamber pressure was 2.6×10 −2 Pa. The ion gun beam current of 10 mA and voltage of −1000 V was maintained by a Commonwealth Scientific IBS controller. Sufficient energy and current of the ion beam are critical to incorporate significant dopant concentration in the film. Without ion bombardment, it is difficult for the dopant to compete with the oxygen for incorporation into anatase titania. The deposition rate was adjusted to 0.2 nm·s −1 using a quartz crystal monitor for all films deposited at a thickness of 1.2 µm. The three types of films were prepared under the optimized conditions for their categories of anatase crystallinity and dopant concentration [34] , [35] . The structure and crystallinity of the films were investigated using a Rigaku D/MAX-2500V 18 kW low angle X-ray diffractometer (XRD) (Rigaku, Shibuya-Ku, Tokyo, Japan) operating with Cu-K α radiation at 40 kV and 150 mA and a Renishaw 1000B Raman spectrometer equipped with a charge-coupled detector (CCD) and a CW 532 nm wave length diode pump solid state (DPSS) laser as the excitation source (Renishaw plc, Representative Office, Nantun District, Taichung, Taiwan). The UV-Vis absorption spectra were recorded on a Hitachi 3300H spectrophotometer (Hitachi Taiwan, Taipei, Taiwan). Bacterial strains and culture B. anthracis (ATCC 14186), which contains both pXO1 and pXO2 plasmids that express functional lethal toxin (LT) and edema toxin (ET), was grown and maintained as previously described [16] , [36] , [37] . B. cereus (ATCC 13061) and B. thuringiensis (ATCC 35646) were maintained and cultured in nutrient agar or nutrient broth at 30°C [38] , [39] , and B. subtilis (ATCC 39090) was maintained and cultured in trypticase soy agar or broth at 37°C [40] . All bacteria were stored in 50% medium and 50% glycerol solution in freezers at −80°C before use. To reactivate bacteria from frozen stocks, 25 µl bacterial stock solution was transferred to a test tube containing 5 ml of freshly prepared culture medium and then incubated at 30°C or 37°C under agitation overnight (16–18 hr). Spores of B. anthracis were prepared as previously described [41] , [42] . Overnight tryptic soy broth cultures of B. anthracis were diluted to about 10 7 CFU/ml in phosphate-buffered saline, and 0.1-ml aliquots were inoculated onto blood agar plates. The agar plates were incubated at 25–37°C until 90–99% phase-bright spores were observed by phase-contrast light microscopy (see below). Spores were harvested and washed with cold sterile distilled ionized (DI) water as previously described [41] and stored in DI water at 4°C until use for up to 2 weeks, changing the water at least once a week, or in the freezer at −20°C for up to a month. The quality of spores was determined by two complementary criteria previously established to validate the presence of dormant spores [42] . The criteria consisted in the evaluation of (i) the absence of vegetative cells (rods) determined by microscopic examination as described, and (ii) the survival of spores in hydrochloric acid (2.5 N). Spore preparations of B. subtilis , B. cereus and B. thuringiensis were followed a similar protocol. Photocatalytic reaction and detection of viable bacteria In this study, bacterial concentrations were either determined by the standard plating method or inferred from optical density readings at 600 nm (OD 600 ). For each Bacillus species, a factor for converting the OD 600 values of the bacterial culture to concentration (CFU/ml) was calculated as follows. A fresh bacterial culture was diluted by factors of 10 −1 to 10 −7 , and OD 600 of these dilutions was measured. Bacterial concentrations of these dilutions were determined by the standard plating method. The OD 600 values were plotted against the bacterial concentration log values, and the conversion factors for the particular bacteria were calculated from three independent measurements. For example, the conversion factor for B. subtilis was calculated to be 1×10 8 CFU/ml per OD 600 by this method. In order to determine the bactericidal effects of the TiO 2 -related substrates, 200 µl of bacterial overnight culture was transferred into 5 ml of culture medium and incubated at 37°C until an OD 600 of 0.3 to 0.6 (log phase) was reached. The bacterial concentrations were calculated using the previously determined conversion factor for the bacteria, and the cultures were diluted to 1×10 5 CFU/ml with culture medium. One hundred microliters (1×10 4 CFU) was then applied to an area of approximately 1 cm 2 of the different TiO 2 -related substrates using a plastic yellow tip. The bacteria substrates were then placed under an incandescent lamp (Classictone incandescent lamp, 60W, Philips, Taiwan) for photocatalytic reaction. A light meter (model LX-102, Lutron Electronic Enterprises, Taiwan) was used to record the illumination density. In the following photocatalysis experiments, the bacteria solution was supplied by 5–10 µl additional distil water every 5 minutes to maintain approximately 100 µl of total volume. After the photocatalyst-killing for 25 minutes, the bacteria containing solution approximately 85 µl was recovered from the photocatalyst substrates using a tip, additional 60 µl fresh medium was used to wash the remaining bacteria on the photocatalyst substrates. Two bacterial containing solutions were mixed, diluted and placed on agar plates. To test whether spores have different efficiency to adhere on TiO 2 substrates that might influence the photocatalysis result, we analyze the spore recovery rates from TiO 2 , TiO 2 (C) and TiO 2 , TiO 2 (N) substrates. We found that the recovery rates are similar (data not shown), which has no statistic significant among these groups. In the dose-dependence experiments, illumination was carried out for 5 min at distances of 5, 10, and 15 cm from the lamp, corresponding to the illumination densities of 3×10 4 , 1.2×10 3 , and 3×10 2 lux (lumen/m 2 )(90, 30, and 10 mW/cm 2 ), respectively. In the kinetic analysis experiments, illumination was carried out for 1, 5, 10, 15, and 25 min at a distance of 5 cm, corresponding to an illumination density of 3×10 4 lux (90 mW/cm 2 ). Unless specified, illumination was carried out in a 4°C cold room to prevent over-heating of the photocatalyst substrates and prevent drying. After illumination, the bacterial solutions were recovered from the photocatalyst substrates, and an aliquot of fresh culture medium was used to collect the residual bacteria on the substrates. The two bacterial solutions were pooled to make a total volume of 150 µl. The bacterial concentration was determined by the standard plating method immediately after the bacterial collection, and the percentage of surviving bacteria was calculated. In spore experiments, 1×10 4 CFU (1×10 5 CFU/ml in 100 µl) were used, and the procedures followed the same protocols as in the live bacteria experiments. Mouse model Six to 8 week - old C57BL/6J mice were purchased from the National Experimental Animal Center (Taipei, Taiwan) [37] , [43] . The methods used in bacteremia experiments were modified from previous descriptions [9] . Mortality of C57BL/6J mice from various anthrax spores treatments (from 1×10 6 to 1×10 7 ) was recorded within one week to serve as reference points. To determine the anti-bacterial effect of TiO 2 (N), each mouse received an intravenous injection of 1×10 5 CFU spores of B. anthracis , a lethal dose for mice, with or without pretreatment by photocatalysis on TiO 2 (N) or TiO 2 substrates (3×10 4 lux, 10 minutes at 4°C). In the lethal toxin (LT) experiments, mortality of mice after a lethal dose of LT was performed based on previously described methods [37] . Each mouse received an intravenous injection of LT (100 µg/g, LF:PA = 1∶5), a lethal dose for mice, with or without pretreatment of photocatalysis on TiO 2 (N) or TiO 2 substrates (3×10 4 lux, 10 minutes at 4°C or 25°C). The mortality of mice was then recorded. During the photocatalysis reaction, the distance between lamps with bacteria- or LT-containing photocatalyst substrates was 5 cm, corresponding to an illumination density of 3×10 4 lux (or 90 mW/cm 2 ). Relative protein levels of PA and LF in the LT mixtures used in animal experiments were detected by Western blot using rabbit polyclonal anti-PA and anti-LF antibodies, and then probed by secondary horseradish peroxidase-conjugated goat anti-rabbit immunoglobulins [16] , [44] , [45] . The gel intensities of PA and LF were measured using Image J software (version 1.32; National Institutes of Health, USA). The Animal Care and Use Committee of Tzu-Chi University approved the protocol of the mice experiments. Cytotoxicity analysis Cytotoxicity of LT was measured following a previously described method [16] . In brief, a cytotoxic dose of LT (10 mg/L, LF:PA = 1∶5) with or without photocatalysis pretreatment on TiO 2 and TiO 2 (N) thin film was used to treat mouse macrophage J774A.1 cells. Three hours after the LT treatments, cell viability of J774A.1 cells were measured using a WST-1 kit (Roche, Mannheim, Germany), following the instructions provided by the manufacturer. Photocatalysis of purified LT was carried out as described in mouse experiments (3×10 4 lux, 10 min at 4°C). Phagocytosis analysis Anthrax spores is normal saline (100 µl, 1×10 5 CFU/ml) were placed on cover glass, TiO 2 and TiO 2 (N) coated thin films [9] , respectively. The spore-photocatalyst mixtures (100 µl) were then illuminated with visible light (Classictone incandescent lamp, 60W, Philips; 90 mW/cm 2 ; lamp-target distance 10 cm) for 30 minutes. After illumination, the spore containing solutions (85 µl) were recovered from the photocatalyst substrates, and an aliquot of normal saline (60 µl) was used to collect the residual spores on the substrates. The two spore solutions were pooled to make a total volume of 145 µl. This spore solution was then added into one well of a six-well cell culture dish that containing confluent murine macrophage J774A.1 cells (1×10 6 cells/well) (MOI: 0.01 spores/cell). After phagocytosis was carried out for one, two and three hours, respectively, culture medium was removed, and 200 µl cell lysis buffer (100mM Tris-HCl [pH 7.4], 10mM MgCl 2 , 100mM NaCl, 0.2% sucrose, 0.5% Triton X-100) that was modified from previous literatures [46] , [47] , was then added to release the cell-engulfed or cell-bound spores. Additional 100 µl fresh medium was used to further collect the residual spores on the dishes. Two spore containing solutions were mixed and placed on agar plates. Cell culture medium (DMEM) without antibiotics and serum supplements was used in this analysis. Statistical analysis All results were calculated from data of at least three independent experiments. A t -test was used to assess the significance of differences in results of anti-microbial effects. A P value of less than 0.05 ( P <0.05) was considered statistically significant. The statistical tests were carried out and output to graphs using Microsoft Excel (Microsoft Taiwan, Taipei, Taiwan) and SigmaPlot (Systat Software, Point Richmond, CA, USA) software.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8869745/

Tomatoes: An Extensive Review of the Associated Health Impacts of Tomatoes and Factors That Can Affect Their Cultivation

Simple Summary The research outlined in this review paper discusses potential health benefits associated with a diet enriched with tomatoes and tomato products. This includes details of previous studies investigating the anticancer properties of tomatoes, protection against cardiovascular and neurodegenerative diseases and diabetes, maintenance of a healthy gut microbiome, and improved skin health, fertility, immune response, and exercise recovery. The specific parts of a tomato fruit that contribute these health benefits are also outlined. The potential disadvantages to a tomato-rich diet are detailed, especially the consumption of supplements that contain compounds found in tomatoes, such as lycopene. This review also discusses how the cultivation of tomato plants can affect the nutritional value of the fruit harvested. Different environmental growing conditions such as light intensity, growing media, and temperature are explained in terms of the impact they have on the quality of fruit, its nutrient content, and hence the potential health benefits acquired from eating the fruit. Abstract This review outlines the health benefits associated with the regular consumption of tomatoes and tomato products. The first section provides a detailed account of the horticultural techniques that can impact the quality of the fruit and its nutritional properties, including water availability, light intensity, temperature, and growing media. The next section provides information on the components of tomato that are likely to contribute to its health effects. The review then details some of the health benefits associated with tomato consumption, including anticancer properties, cardiovascular and neurodegenerative diseases and skin health. This review also discusses the impact tomatoes can have on the gut microbiome and associated health benefits, including reducing the risk of inflammatory bowel diseases. Other health benefits of eating tomatoes are also discussed in relation to effects on diabetes, the immune response, exercise recovery, and fertility. Finally, this review also addresses the negative effects that can occur as a result of overconsumption of tomato products and lycopene supplements. Simple Summary The research outlined in this review paper discusses potential health benefits associated with a diet enriched with tomatoes and tomato products. This includes details of previous studies investigating the anticancer properties of tomatoes, protection against cardiovascular and neurodegenerative diseases and diabetes, maintenance of a healthy gut microbiome, and improved skin health, fertility, immune response, and exercise recovery. The specific parts of a tomato fruit that contribute these health benefits are also outlined. The potential disadvantages to a tomato-rich diet are detailed, especially the consumption of supplements that contain compounds found in tomatoes, such as lycopene. This review also discusses how the cultivation of tomato plants can affect the nutritional value of the fruit harvested. Different environmental growing conditions such as light intensity, growing media, and temperature are explained in terms of the impact they have on the quality of fruit, its nutrient content, and hence the potential health benefits acquired from eating the fruit. Abstract This review outlines the health benefits associated with the regular consumption of tomatoes and tomato products. The first section provides a detailed account of the horticultural techniques that can impact the quality of the fruit and its nutritional properties, including water availability, light intensity, temperature, and growing media. The next section provides information on the components of tomato that are likely to contribute to its health effects. The review then details some of the health benefits associated with tomato consumption, including anticancer properties, cardiovascular and neurodegenerative diseases and skin health. This review also discusses the impact tomatoes can have on the gut microbiome and associated health benefits, including reducing the risk of inflammatory bowel diseases. Other health benefits of eating tomatoes are also discussed in relation to effects on diabetes, the immune response, exercise recovery, and fertility. Finally, this review also addresses the negative effects that can occur as a result of overconsumption of tomato products and lycopene supplements. 1. Introduction Tomatoes ( Solanum lycopersicum ) are a good source of phytochemicals and nutrients such as lycopene, potassium, iron, folate, and vitamin C [ 1 , 2 ]. Besides lycopene and vitamin C, tomatoes provide other antioxidants, such as beta-carotene, and phenolic compounds, such as flavonoids, hydroxycinnamic acid, chlorogenic, homovanillic acid, and ferulic acid [ 1 , 2 , 3 ]. Tomatoes can make an important contribution to a healthy diet and can be consumed raw or cooked while still maintaining their nutritive value [ 1 ]. Over 80% of all commercially grown tomatoes are consumed as processed products such as juice, soup, and ketchup [ 4 ]. A diet rich in tomatoes and tomato products is known to offer several health benefits and many of these benefits are attributed to their antioxidant content [ 1 , 5 , 6 ]. This review will discuss the impact of growing conditions on the tomato cultivar as well as their health-related properties. The potential health benefits of tomatoes discussed in this review include anticancer properties of lycopene in relation to its anti-angiogenic properties, the reduction in insulin-like growth factor (IGF) in blood, and the modulation of cellular pathways that lead to cancer. Anticancer properties of other components of tomatoes, including its fibre, vitamin C, and phenolic constituent ferulic acid, have also been discussed. Tomatoes may also help to reduce the risk of cardiovascular disease, with studies showing associations between tomato consumption and a reduction in hypertension and the risk of atherosclerosis. Observational and experimental studies highlighting neuroprotection and a role in diabetes-associated oxidative stress have been mentioned. The association between tomato consumption and skin health, in particular the protection against atopic dermatitis, is discussed. This is followed by the impact tomatoes have on the gut microbiome and how this may lead to a reduced risk of liver inflammatory disease and inflammatory bowel diseases. Further potential health benefits of tomatoes are then discussed, such as improved exercise recovery and decreased muscle damage after physical exertion, immune system modulation, and reduced risk of infertility. 2. Factors Affecting Tomato Crop Cultivation and Its Nutritional Value Tomato cultivation is a major industry, and global production in 2018 was estimated at 182 million tons [ 7 ] in 2018, rising to 186 million tons in 2020 [ 8 ]. It is known that growing conditions such as water availability can impact the growth, metabolism, and yield of plants [ 9 ]. The key limiting factors to consider in crop growth are water availability, temperature, salinity, and contaminants [ 10 ]. Greenhouse systems allow control over many factors in tomato cultivation, including light intensity, temperature, and humidity [ 11 ]. Water availability affects plant growth, rate of photosynthesis, fruit production, and quality of tomato crop [ 11 , 12 ]. Due to this, the use of plant fertigation in combination with a drip irrigation system is becoming increasingly common in tomato cultivation [ 13 ]. These systems are beneficial not only for the regular and reliable watering of tomatoes but also for the application of a controlled dosage of fertiliser added at regulated times in the growth stage [ 13 ]. A 2010 study focusing on the effects of drought in tomato plants grew five varieties of cherry tomato plants—Kosaco, Josefina, Katalina, Salome, and Zarina—and subjected them to 50% of a standard watering regime compared to a control with 100% [ 12 ]. This study found that the Zarina cultivar was the most tolerant to stress, showing greater biomass, leaf relative water content, relative growth rate, and a higher antioxidant activity [ 12 ]. The authors concluded that tomato plants show genotypic differences to oxidative stress caused by drought and suggested that the Zarina cultivar be used in any studies aiming to improve the growth of drought-tolerant plants [ 12 ]. It is, however, worth noting that drought- and heat-induced stress reduces the growth and yield of tomato crops but increases their carotenoid content and antioxidant enzyme activity, likely due to raised oxidative stress induced by such conditions [ 14 ]. It has been reported that the rate of plant photosynthesis can be impacted by drought [ 15 ]. The stress created by drought may cause an energy imbalance in which the energy absorbed through photosynthesising complexes is more than photosystem II can dissipate [ 16 ]. It has been suggested that this excess energy is dissipated in cells by the conversion of O 2 into reactive oxygen species (ROS), resulting in plants synthesising antioxidants such as superoxide dismutase [ 17 , 18 , 19 ]. The accumulation of antioxidants and phytochemicals in tomato fruit is also heavily impacted by the environmental conditions (light intensity, water availability, temperature, growing media salinity) that the fruit is grown in [ 20 ]. A 2009 trial compared tomatoes grown in Ireland with tomatoes grown in Spain to investigate if geographical location impacts carotenoid content in four different tomato types (cherry, plum, round, and on the vine) [ 21 ]. The authors concluded that the geographical location rather than the type of tomato had a bigger impact on the bioaccessibility (bioavailability after consumption of tomatoes) of carotenoids in the fruit [ 21 ]. The bioaccessibility of carotenoids such as lycopene is an important factor for the health benefits gained by eating this fruit. Vitamin C content in fresh tomatoes increases to a maximum and then decreases during the ripening process [ 22 ]. It was reported by Abushita et al. that salad tomatoes grown in field conditions contained 15–21 mg/100 g fresh weight (FW) of vitamin C compared to a range of industrial grades of tomatoes with an average vitamin C value of 19 mg/100 g FW [ 23 ]. As vitamin C has been linked to immune modulation [ 24 ], this implies that the growing conditions of tomato fruit could impact the immune benefits associated with it. Another factor to consider in the cultivation of tomatoes is the temperature. Higher temperatures are known to affect photosynthesis as they can cause damage to the photosynthetic apparatus, leading to the inhibition of photosystem II [ 25 , 26 ]. It has also been reported that high temperatures reduce photosynthesis through the inhibition of the ribulose-1,5-bisphosphate carboxylase in the Calvin cycle, leading to an inactivation of carbon dioxide (CO 2 ) fixation [ 27 ]. A 2005 study focussed on the effects of high temperatures in tomato cultivation; researchers exposed a group of Campbell-28 variety plants to heat shock treatment of 45 °C for 2 h and measured gas exchange, chlorophyll fluorescence, and electrolyte leakage [ 25 ]. This study concluded that the heat shock treatment resulted in reductions in the net photosynthetic rate of the plants due to changes in the Calvin cycle and in photosystem II functioning [ 25 ]. Temperature impacts the distribution of photoassimilates (biological compounds formed by assimilation using light-dependent reactions) between the fruit and the rest of the tomato plant [ 28 ]. At higher temperatures, photoassimilate accumulation in fruits is increased, impacting vegetative growth of the tomato plant [ 21 , 29 ]. The temperature of the growing environment also affects water distribution in the plant, the cellular structures affecting the quality of the fruit (such as size and colour), and fruit development [ 21 , 30 , 31 ]. The type and number of phenolic compounds found in tomato fruit are known to vary greatly with plant genotype, fruit storage, and light intensity during cultivation [ 8 , 32 ]. A 2006 study grew two tomato cultivars under two different conditions: one designed to transmit ambient solar UV radiation in the range 290–400 nm, the other designed to block UV radiation below 380 nm [ 33 ]. The phenolic content of these tomatoes was tested using high-pressure liquid chromatography and a colorimetric Folin–Ciocalteu assay, and the results indicated that the higher wavelength and intensity of UV radiation exposure of the tomato plants during cultivation significantly increased the phenolic levels of the fruit level [ 33 ], which are beneficial to health. The growing media used for tomato cultivation is also known to impact the growth and health of the plants and the resulting fruit. In tomato greenhouse production, soilless cultivation systems are in place using solid substrates [ 34 ] such as peat, bark, rockwool, synthetic foams, and perlite [ 35 ]. Sphagnum peat moss, harvested from wetland ecosystems, is a common growing medium in horticulture due to its high nutrient exchange capacity [ 36 ]. The physical properties of the substrates, such as pore size, tortuosity, and continuity, are determined by substrate particle size and shape and can affect the availability of water and air [ 37 ]. A study in 2004 tested seven substrates in greenhouse tomato cultivation: rockwool, fresh spruce sawdust, spruce wood shaving, composted spruce bark, fine blond peat, and mixtures of 66% fine blond peat +33% composted spruce bark and 33% fine blond peat +66% composted spruce bark [ 37 ]. Substrate performance was assessed according to water retention, hydraulic conductivity, pore tortuosity, and gas diffusivity [ 37 ]. While the physical properties of these substrates varied greatly, yield was not related to these properties, and if irrigation is adjusted for the physical properties of each substrate, then all tested substrates can be utilised for tomato greenhouse cultivation [ 37 ]. A study carried out in 2017 investigated three different growing media—rockwool, coconut coir, and peat–vermiculite to understand how they affected tomato plant growth, fruit yield, and quality [ 38 ]. Tomato plants grown with coconut coir had an increased photosynthesis rate, individual fruit weight, and total fruit yield [ 39 ]. This study observed that coconut coir significantly increased potassium and sulphur uptake compared to tomato plants grown on rockwool, and an increased phosphorus and sulphur uptake compared to peat–vermiculite growing media [ 38 ]. A 2015 study assessed the impact of growing media on the nutritional quality of tomato fruit by growing tomato plants with a compost prepared using effective microorganisms (EM)—a combination of microbial inoculants that stimulate plant growth [ 39 ]. The authors showed that the EM supplement not only improved plant growth and fruit yield but also lycopene content, antioxidant activity, and defence enzyme activity compared to the control [ 39 ]. A 2021 study compared soil-based growing media with hydroponic growing systems using rockwool with either drip-feed irrigation or deep-water culture for the cultivation of tomato plants [ 40 ]. This study observed that tomato plants grown with the two hydroponic systems were more water efficient and had a lower transpiration rate, requiring less water than tomato plants grown in soil [ 39 ]. It was also observed that the total lycopene and β-carotene fruit content was highest in the deep-water culture system [ 40 ]. In a 2020 study, 20 tomato varieties grown in medium and high levels of soil salinity were examined for their lycopene, vitamin C, total phenolic content, and total antioxidant capacity, and it was reported that tomato plants with a tolerance to higher soil salinities produce fruit with increased levels of antioxidants, such as phenolic compounds, and carotenoids, such as lycopene [ 41 ]. This suggests that the salinity of tomato plant growing media can directly impact the nutritional quality associated with the fruit, and hence the health benefits associated with this fruit. It should be noted that inorganic substrates such as rockwool and perlite require large amounts of energy to manufacture and are not biodegradable, making them less sustainable than other substrates [ 34 ]. Peat is another substrate known to be unsustainable when utilised for crop cultivation [ 42 ]. The harvesting of peat has negative effects on wetland ecosystems, including the loss of peat bogs, which have a major role as carbon sinks [ 43 ]. Research is currently focused on the use of sustainable substrates such as wood fibres, bark, or recycled waste products from industries for the sustainable cultivation of tomatoes and other crops [ 42 , 44 , 45 , 46 , 47 ]. A sustainable growing media amendment under investigation is chitin and chitosan, which are waste products of the shellfish industry [ 48 ]. Studies have been carried out assessing the potential benefits these waste products have on the production of various crops [ 49 , 50 , 51 , 52 , 53 ]. A 2004 study observed an approximate 20% increase in yield for two out of three tomato trials with chitosan applied to soils and leaves as 2.5–5 mL/L solutions [ 53 ]. In all three tomato trials chitosan application resulted in significant control of powdery mildew, a fungi which weakens a plant and causes fruit to prematurely ripen [ 53 ]. It is common practice for tomatoes to be harvested at the mature green stage for ripening in transit [ 54 ], and this can impact the levels of antioxidants such as lycopene, which are synthesised during ripening [ 21 ]. However, unlike carotenoids such as lycopene and β-carotene, the vitamin C levels of tomatoes are reported to be lower in tomatoes picked at the fully ripened stage compared to those picked at the mature green stage and ripened off the vine [ 55 ]. In summary, the antioxidant and phytochemical content of tomatoes can be influenced by environmental conditions, including light intensity, water availability, temperature, and growing media as well as the ripeness stage, and all this can have an impact on their potential health effects. 3. Tomato Constituents for Health Tomato fruit is a fleshy berry of varying sizes and colours [ 56 ]. The fruit is composed mostly of water (>90%), with very little protein or fat, and around 3% carbohydrates (glucose and fructose) [ 56 ]. The nutrients obtained from an average round tomato and how these relate to the recommended daily intakes per person is described in Figure 1 . Tomato fruit has a pericarp, which includes an outer layer of exocarp and inner layers of mesocarp and endocarp [ 57 ]. The fruit exocarp (epidermis) consists of a thin cuticle with no stomata, the phenolic content of which increases during fruit growth [ 57 , 58 ]. Tomato cuticle is mostly composed of a lipid polymer known as cutin, and waxes, which are complex and variable [ 59 ]. The mesocarp contains fruit vascular tissue connected to pedicel vascular tissue [ 57 ]. Vascular tissue is located in the centre of tomato fruit, supplying seeds with necessary water and minerals, and is also parallel to the fruit surface [ 57 ]. Within the unicellular endocarp boundary are seed-containing cavities derived from carpels, known as locules [ 57 , 60 ]. The number of locules within a fruit can vary, changing the size and shape of the fruit [ 60 ]. Locules are divided by a septum, with seeds bound to an elongated axial placenta [ 61 ]. Tomato seeds are known to contain steroidal saponins called lycoperosides, particularly lycoperoside H, which are believed to exert anti-inflammatory effects [ 62 , 63 ]. A study carried out by Moretti et al. analysed the chemical composition of different sections of tomatoes [ 64 ]. It was observed that vitamin C content is highest in the locule tissue (228.90 ± 5.44 mg/kg) compared to the pericarp tissue (194.90 ± 2.13 mg/kg) [ 64 ]. This study also found both total carotenoid and total chlorophyll levels to be higher in pericarp (108.03 ± 2.22 mg/kg and 0.40 ± 0.03 mg/kg, respectively) than locule tissue (87.84 ± 2.23 mg/kg and 0.33 ± 0.06 mg/kg, respectively) [ 64 ]. The presence of oxalic acid in tomatoes has been linked to renal disease, especially renal stones; however, it is worth noting that the oxalic acid content of tomatoes is reported to be between 5–11 mg per 100 g FW [ 65 ]. The oxalic acid content is suggested to increase with the ripeness of the fruit [ 66 ], and one of the suggested mechanisms for this increase is due to the conversion of ascorbic acid to oxalic acid as the fruit ripens. Cooking tomatoes, especially boiling fresh tomatoes, has been suggested to reduce their oxalic acid content [ 67 ]. Figure 1 Infographic representing the nutrients obtained from an average round tomato and how these relate to the daily recommended intakes [ 68 , 69 ]. The health-beneficial properties of tomatoes are studied the most in relation to their role in cancer prevention. Not only cancer but several other age-related diseases such as cardiovascular, diabetes, and Alzheimer's as well as skin health, fertility, and exercise recovery can be influenced by constituents of tomatoes. There are several reviews that have addressed anticancer and cardioprotective properties of tomatoes but most of them focused these effects on the lycopene constituent. Tomatoes have a range of other nutrients that could confer their biological properties, as shown in Table 1 . The aim of this review is to provide comprehensive literature on the health-related properties of tomatoes that can be attributed not just to lycopene but also to their other constituents. 4. Health Effects 4.1. Tomatoes and Cancer Pathology Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020 [ 142 ]. Schwingshackl et al. discussed the effects of a tomato-rich Mediterranean diet on the risk of overall cancer mortality [ 143 ]. This paper observed that, in a clinical trial, a Mediterranean diet was found to reduce cancer incidence by 61% and also stated that a "healthy diet" can prevent approximately 30% of cancers [ 143 ]. A review by Farinetti et al. studied the benefits of the Mediterranean diet on colorectal cancer, with lycopene in particular as an important component of this diet, including polyphenols from olive oil and red wine resveratrol, which act to inhibit molecular cancer pathways in vitro [ 119 ]. The health benefits from tomatoes are enhanced as part of the Mediterranean diet as lycopene is more readily absorbed in the intestines when it has been dissolved in olive oil and heated [ 119 ]. Lycopene and β-carotene are two important carotenoids found in tomatoes and both have been suggested to confer the anticancer properties of the fruit. Lycopene, a red pigment found in tomatoes and tomato products, has antioxidant and free radical scavenging activity, and is known to be the most effective singlet oxygen quencher among the natural carotenoids [ 5 , 95 , 144 ]. The human body absorbs a significant proportion (23–24%) of ingested lycopene that proceeds to circulate and accumulate in blood plasma, liver, and other tissues with a half-life of 12–33 days [ 145 ]. Among the various plausible beneficial effects of lycopene, its anticancer properties have been studied the most. These suggestions initially stemmed from epidemiological [ 146 , 147 , 148 , 149 ] studies and were later supported by several experimental studies [ 77 , 78 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 ]. Various anticancer mechanisms of lycopene include the modulation of gene functions and apoptosis, increasing gap junction communications, anti-angiogenic effects [ 146 , 150 , 159 ], and antioxidant, anti-inflammatory, and anti-lipid peroxidation activities [ 160 , 161 , 162 , 163 ]. Due to their antioxidant properties, lycopene and other carotenoids are suggested to protect against carcinogenesis by preventing oxidative damage in DNA and proteins through antioxidant mechanisms [ 164 ]. It has been observed that the cleavage of lycopene via in vitro oxidation at random conjugated double bonds in the molecule forms monocarbonyl compounds [ 151 , 165 ]. Zhang et al. [ 151 ] showed that the products of lycopene oxidation can induce apoptosis in cancer cells. This was further investigated by Arathi et al. [ 152 ] who extracted and autoxidised lycopene from ripened tomatoes and used the products in in vitro cell culture assays to assess the toxicity and apoptosis-inducing ability in various cancer cells. This study found that there were several unknown metabolites or oxidation products of lycopene that may be involved in the inhibition of cancer cell proliferation through modulating cell cycle progression [ 152 ]. This study also demonstrated that chemically induced lycopene oxidation products were a key component in the induction of apoptosis in cancer cells [ 152 ]. A review published in 2020 by Przybylska discussed the anticancer properties of lycopene, particularly in prostate cancer [ 153 ]. This paper evaluated lycopene's effects on prostate cancer, discussed in later sections of this review, as well as breast cancer, the second most prevalent cancer in the world [ 153 ]. Przybylska states that lycopene consumption can reduce the blood concentration of insulin-like growth factor 1 (IGF-1) via the stimulation of synthesis of a protein that binds IGF-1 [ 153 ]. It has been shown that IGF-1 is an important factor in the development of breast cancer in pre-menopausal women, and therefore lycopene's reduction in this growth factor may reduce the risk of this cancer [ 154 ]. This paper further discusses how lycopene inhibits the proliferation of oestrogen-dependent/-independent cancer cells through multiple mechanisms, including inhibiting the activation of genes responsible for the cell cycle or protein-1-responsive genes [ 153 , 166 ]. Another 2020 paper by Saini et al. reviewed the anticancer properties of lycopene [ 78 ] and concluded that the antioxidant abilities of lycopene via a reduction in ROS in cells play a key role in the anticancer properties of this carotenoid. The phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway has been of interest in cancer biology for decades [ 155 ]. Mutations or aberrations to this pathway are found in many cancers, and the inhibition of PI3K presents a therapeutic target for a range of tumour types [ 156 ]. AKT is known to promote cell growth and survival and is further upregulated in breast, prostate, and other forms of cancer [ 156 ]. AKT plays a part in tumour-induced angiogenesis as AKT is activated downstream of vascular endothelial growth factor (VEGF), promoting cell growth and angiogenesis, which is critical for the survival of tumour cells [ 155 , 159 ]. A study by Tang et al. [ 157 ] investigated the inhibitory effects of lycopene on the AKT signalling pathway in HT-29 human colon cancer cells [ 157 ]. It was observed that the proliferation of HT-29 colon cancer cells was inhibited by lycopene in a dose-dependent manner. This study concluded that lycopene treatments may inhibit the PI3K–AKT pathway and further demonstrated the involvement of this pathway in tumour development [ 157 ]. Downstream signalling through the PI3K–AKT pathway increases the expression of transcription factor hypoxia-inducible factor-1 (HIF-1) which upregulates the expression of VEGF [ 155 ]. Therefore, it can be speculated that the suppression of this pathway could prevent tumour development [ 167 , 168 ]. VEGF is also the fundamental regulator in cellular signalling of angiogenesis, which supplies tumour cells with blood supply [ 169 ]. Two studies using human umbilical vein endothelial cells (HUVEC) demonstrated anti-angiogenic effects of lycopene, and one of these studies showed that lycopene also inhibited angiogenesis in freshly dissected rat aorta cells at physiologically relevant concentrations of 1–2 μmol/L [ 159 ]. In another study, lycopene was shown to inhibit angiogenesis both in vitro and in vivo by inhibiting the MMP-2/uPA system through VEGFR2-mediated PI3K–AKT and ERK/p38 signalling pathways [ 168 ]. A prospective study highlighted that angiogenic potential, a biomarker of lethal cancer, was lower in individuals who had been consuming tomato products for a longer period of time [ 169 ]. The PI3K–AKT pathway also activates oncogenic signalling pathways via the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Wnt/β-catenin [ 169 ]. NF-κB influences cell growth, proliferation, and metabolism [ 170 ] and is known to play a key role in the development of cancers [ 171 ]. NF-κB dimers are pro-survival transcription factors and are usually cytoplasmic due to interactions with the inhibitors of kappa B (IkBs); they therefore remain transcriptionally inactive [ 171 , 172 ]. NF-κB activation may result from different signalling pathways triggered by a variety of cytokines, or growth factors, and involves the phosphorylation and proteasome-dependent degradation of IkBs [ 171 , 173 ]. NF-κB activation leads to nuclear translocation followed by the transcription of target genes involved in the oncogenic pathway [ 171 ]. NF-κB is known to be active in several tumour cell types, including leukaemia, breast, and prostate [ 174 ]. A study by Assar et al. [ 77 ] studied the effects that dietary lycopene would have on several points along this oncogenic pathway. This experiment examined the effects in two human cancer cell lines, prostate (PC3) and breast (MDA-MB-231), in the absence and presence of lycopene at concentrations of 0.5–5 µM [ 77 ]. This study not only conducted MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-4-sulfophenyl)-2H-tetrazolium) cell growth assay and Western blots but also NF-κB-responsive gene activation reporter assays to monitor the pathway's activity in real-time [ 77 ]. This study concluded that lycopene inhibits the NF-κB pathway at different stages for both breast and prostate cancer cells in vitro [ 78 ]. NF-κB and the Wnt/β-catenin signalling pathways cross-regulate each other's activities and functions. The Wnt/β-catenin signalling pathway is involved in cell proliferation and can lead to cancer development [ 172 ]. This pathway is upregulated by inflammation and oxidative stress, which can lead to a variety of cancers [ 150 ]. Therefore, it can be suggested that a reduction in ROS caused by lycopene or other antioxidants found in tomatoes leads to the inhibition of Wnt/β-catenin signalling. The Wnt/β-catenin signalling pathway is associated with colorectal cancer [ 169 ]. A 2019 study by Kim et al. [ 150 ] explored the mechanism by which lycopene can influence cancer cell growth through the induction of apoptosis in human gastric cancer AGS cells. Various apoptotic indices such as cell viability, DNA fragmentation, and ROS concentrations were examined in the gastric cancer cells [ 150 ], and the authors concluded that lycopene at 0.3% final concentration led to the induction of apoptosis by inhibiting Wnt/β-catenin signalling, stopping the nuclear translocation of β-catenin and suppressing the expression of specific cell survival genes. Furthermore, a study by Preet et al. [ 158 ] tested the effect of lycopene on human breast cancer cell lines by measuring protein compounds associated with the Wnt/β-catenin signalling pathway and cancer cell viability. Preet et al. [ 158 ] showed that lycopene treatment in combination with quinacrine (a derivative of the naturally occurring compound quinine) inhibited the proliferation of breast cancer cells. It was concluded that the reduced proliferation of the breast cancer cells was a result of the inhibition of the Wnt/β-catenin signalling pathway [ 158 ]. Lycopene is the key antioxidant found in tomatoes and is the focus of many cancer studies. However, tomatoes also contain β-carotene. β-Carotene is a provitamin and is converted into retinol—a compound needed for vision [ 9 ]; it has been the focus of many studies that conclude that it is associated with anticancer activities, including inducing cancer cell apoptosis and reducing cancer cell proliferation [ 174 , 175 ]. Tomatoes also contain a diverse array of other potentially chemo-preventive compounds that are not the primary focus of current research, including vitamins and phenolic constituents [ 176 ]. For example, vitamin C is thought to reduce the risk of stomach carcinogenesis by controlling levels of ROS that can lead to DNA damage, or by stopping the development of carcinogenic nitrosamines introduced as part of the diet [ 133 , 177 ]. The effectiveness of vitamin C as an anticancer agent was debated until a 2011 study investigated the impacts of vitamin C on the human body, which concluded that, when ingested, vitamin C blood concentrations are highly controlled by renal reabsorption [ 178 ]. It was concluded that at a pharmacological dose administered intravenously, the blood plasma levels of this nutrient can be raised to 25–30 mmol/L, a concentration that has been shown to be cytotoxic to cancer cells [ 178 ]. Ferulic acid, a phenolic acid found in tomatoes, is an effective antioxidant and is suggested to have anticancer properties [ 112 , 113 ]. One study investigated the effects of 24 h treatment of Caco-2 colon cancer cells with 150 µmol/L ferulic acid and found that 517 genes were significantly affected [ 114 ]. The treatment delayed cell cycle progression in the S phase via the upregulation of genes involved in centrosome assembly and the S phase checkpoint [ 114 ]. Tomato peel and seeds are composed of 60% dietary fibre [ 179 ]. When fibre is metabolised by intestinal microbiota to form short-chain fatty acids such as butyric and acetic acids, cancerous colonocytes cannot use these components as a source of energy and they accumulate, inhibiting the action of histone deacetylases in these cells [ 180 , 181 ]. As a result, the epigenetic regulation of gene expression in these cells is changed, reducing cell proliferation and increasing apoptosis [ 181 ]. It can be concluded that a tomato-rich diet could increase human blood lycopene levels, and this has many potential anticancer properties ( Table 2 ). However, some healthcare professionals argue that lycopene may not be the only cancer lowering constituent of tomatoes, and perhaps it is a biomarker of tomatoes that, due to an array of constituents, confer anticancer properties [ 78 , 182 ]. 4.2. Tomato's Specific Influence on Prostate Cancer Prostate cancer is the second most common cancer found in men worldwide [ 183 , 184 ]. A study by Giovannucci et al. [ 185 ] investigated dietary carotenoids and prostate cancer risk. Questionnaires were used to find trends between diet and the risk of prostate cancer, and it was found that the only carotenoid associated with a decreased risk of prostate cancer was lycopene [ 185 ]. Of the four tomato-based items high in lycopene that were listed (tomato sauce, tomatoes, tomato juice, and pizza), all except tomato juice were associated with a significantly lower risk of prostate cancer [ 185 ]. More recently, a 2018 review by Rowles et al. compared the results from 30 different articles discussing tomato consumption and prostate cancer [ 183 ]. This review concluded that there was a significant inverse association between tomato consumption and the risk of prostate cancer [ 183 ]. In tomato-rich diets, lycopene is one of the most abundant carotenoids found to be accumulated in blood and tissues, reaching plasma concentrations of up to 1.8 µmol/L [ 184 , 186 ]. Lycopene has been shown to accumulate in several tissues, including the liver and the prostate [ 76 , 184 ]. A review by Rao and Agarwal in 1999 compared lycopene accumulation in major organs and found that the prostate accumulated 0.8 nmol lycopene/g tissue, the adrenal glands 1.9–21.60 nmol/g, and the testes 4.34–21.36 nmol/g [ 5 ]. In 2002, a study was conducted on 60 men with adenocarcinoma of the prostate (clinical stages T1 or T2) in which their diet was supplemented with lycopene-rich pasta sauces and other meals rich in lycopene for a three-week period [ 187 ]. Blood samples showed increased serum lycopene, from baseline 0.638 μM to 1.258 μM, and increased prostate lycopene, from 0.279 nmol/g tissue prior to the trial to 0.82 ± 0.119 nmol/g after the intervention [ 187 ]. However, this study does conclude that the impact of this uptake of lycopene on prostate cells needs further research [ 187 ]. The level of insulin-like growth factor 1 (IGF-1) in the human body is associated with prostate cancer due to the mitogenic and antiapoptotic effects on prostate epithelial cells [ 188 , 189 , 190 ]. Diet is known to influence the level of IGF-1 in the human body [ 189 , 190 ]. Diets primarily containing red meats and dairy products were shown to increase the levels of IGF-1, whereas diets containing high amounts of fruits and vegetables, particularly tomato-containing products, were found to associate with lower levels of IGF-1 [ 189 , 190 , 191 ]. However, studies by Chan [ 192 ] and Graydon [ 193 ] found that lycopene supplementation had no significant effect on the IGF-1 levels of male subjects with and without prostate cancer. In 2019, Applegate, Rowles, and Erdman carried out a systematic review on the impact lycopene has on prostate cancer [ 194 ]. This was focused on androgen activity, which is associated with prostate cancer growth as androgen-regulated, prostate-specific antigen (PSA) is higher in serum samples taken from men diagnosed with prostate cancer [ 194 , 195 , 196 ]. The review suggested that lycopene reduced androgen metabolism and signalling, one of the main factors influencing prostate cancer growth and progression [ 194 ]. Obermüller-Jevic et al. [ 197 ] observed that human prostate epithelial cells treated with 5 μmol/L lycopene showed no expression of cyclin D1. A similar effect on cell growth inhibition was observed in human breast and endometrial cancer cell lines with lycopene. Cyclin D1 is a regulatory subunit of cyclin-dependent kinases CDK4 and CDK 6, which allows cells to transit from the G1 phase of the cell cycle to the S phase and is synthesised in the G1 phase that accumulates in the nucleus [ 197 , 198 , 199 , 200 ]. Wertz et al. [ 198 ] provided a detailed review of the mode of action of lycopene and highlighted that the inhibition of cell growth by lycopene involves the downregulation of cyclin D1, but not of cyclin E, and leads to cell cycle arrest at the G0/G1 phase. In the absence of functioning cyclin D1 in the G1 phase, cell cycle progression is halted, and the cell proliferation rate is reduced [ 199 ]. Gap junctions are intracellular channels formed by connexin proteins, joining cells and allowing the passage of nutrients and intracellular signalling molecules [ 201 ]. In a healthy prostate, basal cells use connexin 43 gap junctions in communication, and luminal cells use connexin 32 gap junctions [ 201 ]. It has been reported that in differentiated prostate cancer there is decreased expression of both channels [ 201 ]. Overall, lycopene treatments have shown an upregulation of connexin 43 expression and enhanced the gap junction channel communication in mouse fibroblast cells and prostate gland cells [ 198 ]. Through the upregulation of connexin 43 and an increased gap junction channel communication, lycopene inhibits carcinogen-induced neoplastic transformation in cell culture [ 198 , 201 ]. This review details the potential anticancer properties associated with the consumption of tomatoes. Most research focuses on lycopene as the primary anticancer agent in tomatoes ( Table 2 ). Lycopene is also the focus of many published reviews that do not discuss other naturally occurring tomato compounds with anticancer associations [ 77 , 152 , 202 ]. This review, however, details not only the anticancer properties of lycopene but also vitamin C, β-carotene, ferulic acid, and the dietary fibre incorporated in tomato tissues. This highlights the importance of how the suggested anticancer properties associated with tomatoes may not derive solely from lycopene but from a combination of anticancer compounds naturally occurring in this fruit. biology-11-00239-t002_Table 2 Table 2 Main findings of the effect of tomato compounds on cancers. Biological Property Studied Type of Study (In Vitro/In Vivo) Main Findings References Antioxidant and anticancer activity In vitro study with human prostate cancer (PC-3) and human breast adenocarcinoma (MCF-7) cell lines. Cell viability assay showed chemically induced lycopene oxidised products (1–50 µM) were a key component in cancer cell apoptosis. [ 152 ] In vitro study with HL-60 human promyelocytic leukaemia cells. Products of lycopene oxidation, identified by spectral analyses, were added to HL-60 cell suspension as a 1% ( v / v ) concentration. This treatment was shown to induce apoptosis in leukaemia cells, shown using flow cytometry to evaluate the ratio of apoptotic cell death. [ 151 ] Anti-angiogenic role in cancer cells In vitro study testing human umbilical vein endothelial cells (HUVEC) and rat aortic rings. Lycopene inhibited angiogenesis in HUVEC and rat aortic rings at physiologically relevant concentrations (1–2 μmol/L) when angiogenesis was analysed using phase-contrast microscopy. [ 159 ] In vitro and in vivo study testing human umbilical vein endothelial cells (HUVEC). Lycopene (0, 1, 5, 10 µM) was shown to inhibit angiogenesis of HUVEC cells in vitro and in vivo by inhibiting MMP-2/uPA system through VEGFR2-mediated PI3K–Akt and ERK/p38 signalling pathways. Cell proliferation assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, cell migration assessed with Millipore QCM™ Endothelial Migration Assay Kit. [ 168 ] Longitudinal cohort study. Lycopene used as a marker of tomato intake and higher intake inversely correlated with total, and the aggressive nature of prostate cancer. The reduced severity of cancer and lesser degree of angiogenesis were reported only in individuals who consumed a tomato-rich diet for a long time period but not in those whose intake recently increased. Tissue microarrays and immunohistochemistry were used to assess tumour biomarker expression. [ 169 ] Modulation of molecular pathways in cancer cells In vitro study with HT-29 human colon cancer cells. Lycopene treatment (0, 2, 5, 10 µM) was shown to inhibit the PI3K–AKT signalling pathway in colon cancer cells, demonstrating its effects on tumour development via angiogenesis inhibition. Assessment of cell proliferation using MTT assay and gene expression investigated using transient transfection and luciferase reporter assays. [ 157 ] Ex vivo and in vivo study testing human umbilical vein endothelial cells (HUVEC) and rat aortic rings. Lycopene (400 μg/mouse) reduced angiogenesis cell signalling through inhibition of the VEGF cell signalling pathway. Anti-angiogenic activity of lycopene confirmed by ex vivo rat aortic ring and in vivo chorioallantoic membrane assays. [ 167 ] In vitro study with human prostate (PC-3) and breast (MDA-MB-231) cancer cell lines. Lycopene (0.5–5 µM) inhibited different stages of the NF-κB cell signalling pathway in both cancer cell lines in vitro as seen in Western blots and NF-κB-responsive gene activation reporter assays. [ 77 ] In vitro study in human gastric cancer (AGS) cells. Lycopene at 0.3% was shown to induce apoptosis by inhibiting Wnt/β-catenin signalling, stopping the nuclear translocation of β-catenin and suppressing the expression of specific cell survival genes AGS cells. Cell viability, DNA fragmentation, and ROS concentrations were examined in these cells. [ 150 ] Cytotoxicity and cancer cell growth In vitro study testing human prostate epithelial cells (PrEC). PrEC treated with lycopene (up to 5 μmol/L) showed no expression of cyclin D1 in vitro. This regulatory subunit of kinases essential to the cancer cell cycle, resulting in reduced cancer cell cycle progression. High-performance liquid chromatography (HPLC) analysis, a thymidine incorporation assay, and flow cytometry were carried out to assess the impact of lycopene. [ 199 ] In vitro study testing human prostate (PC-3) and breast (MDA-MB-231) cancer cell lines. PC-3 and MDA-MB-231 cancer cell lines were tested in vitro in the absence and presence of lycopene at concentrations of 0.5–5 µM. MTS cell growth assays, Western blots, and NF-κB-responsive gene activation reporter assays showed that lycopene inhibits the NF-kB pathway at different stages in both cell lines. [ 77 ] In vitro study treating Caco-2 colon cancer cells. Treatment of Caco-2 colon cancer cells with 150 μmol/L dietary fibre ferulic acid delayed cell cycle progression in the S phase. Gene expression was analysed with cDNA microarray technique. [ 115 ] Cancer cell apoptosis In vitro study testing human prostate cells (PC-3). Flow cytometry analysis showed 27–32% apoptosis in PC-3 when supplemented with (10–50 μM) β-carotene. [ 174 ] Gap junction communication in cancer cells In vitro study with rat liver epithelial WB-F344 cells. Incubation of WB-F344 cells with oxidation products of lycopene (0.2% v / v ) improved the gap junction communication in dye transfer assay using microinjection of the fluorescent dye Lucifer Yellow CH. [ 203 ] 4.3. Cardioprotective Effects of Tomatoes A tomato-rich diet has been linked to a reduction in the risk of heart disease. Song et al. reviewed 14 eligible studies and found a significant inverse association between lycopene intake and coronary heart disease [ 204 ]. Another meta-analysis reviewed 25 studies and reported that high lycopene consumption and lycopene serum concentrations reduced the overall mortality by 37%, cardiovascular disease by 14%, and stroke by 23% [ 205 ]. A randomised, cross-over controlled trial in healthy participants examined the effect of a single dose of raw tomatoes, tomato sauce, or tomato sauce plus refined olive oil on biomarkers of cardiovascular disease [ 206 ]. The results showed all three interventions reduced plasma cholesterol and triglycerides and raised plasma high-density lipoprotein (HDL) cholesterol and interleukin-10 concentrations. Tomato sauce plus olive oil produced the maximum effect, likely due to the increased bioavailability of lycopene as oil is known to improve this. The authors indicated that including tomatoes as a regular part of a diet may help to prevent postprandial lipemia by reducing blood triglyceride levels, and in doing so, reduce the risk of developing atherosclerosis [ 206 ]. An increase in triglyceride levels can lead to the production of small, dense low-density lipoprotein (LDL), which is highly atherogenic [ 207 , 208 ]. It is worth noting that the fat-soluble pigment lycopene is released from tomato cell wall protein–carotenoid complexes during food preparation, therefore the bioavailability of lycopene is higher with cooked tomatoes and tomato products such as juices and sauces than fresh tomatoes, and daily consumption of such tomato products significantly reduces blood LDL cholesterol levels in adults [ 209 ]. In a recent cross-over study, feeding of tomato sauce from vine-ripened tomatoes at 150 mL/day for 6 weeks was compared with sterol-enriched yoghurt and both interventions reduced LDL cholesterol by 12% and 15%, respectively [ 210 ]. Heart disease is a collective term that includes hypertension and atherosclerosis. Hypertension is one of the most common chronic diseases worldwide, with accompanying risks including cardiovascular disease (CVD) and kidney disease [ 6 ]. In a study conducted by Engelhard et al. [ 211 ], patients with grade-1 hypertension were found to have significantly lower systolic and diastolic blood pressure after short-term treatment with 250 mg tomato extract Lyc-O-Mato. In a double-blind placebo study of grade-1 hypertension patients, both systolic and diastolic blood pressure were significantly lower after treatment with tomato extracts [ 211 ]. 𝛾-Aminobutyric acid (GABA), a neurotransmitter present in the sympathetic nervous system, is known to lower systolic blood pressure [ 212 , 213 ], and tomatoes have been shown to contain high levels of GABA [ 214 ]. GABA has been reported to lower the blood pressure of hypertensive patients but not of normotensive individuals [ 168 ]. Daily supplementation of 80 mg of GABA has been found to reduce blood pressure in adults with mild hypertension [ 215 ]. A study carried out in 2008 analysed tomato varieties and found that they had an average GABA content of 50.3 mg/100 g fresh weight [ 216 ]. Many of the antioxidants found in tomatoes, including lycopene, beta-carotene, and vitamin C, protect vascular cells and lipoproteins from oxidation and thus prevent the formation of atherosclerosis [ 134 , 217 ]. Low-density lipoprotein (LDL) oxidation is a well-known factor in genesis [ 218 ] and the progression of atherosclerosis, a process that leads to the narrowing of arteries due to a build-up of cholesterol in subendothelial space. Oxidised LDL is believed to be important in the formation of atherosclerosis and, therefore, vascular diseases. Oxidised LDL increases the expression of pro-inflammatory cytokines, which promote the adhesion of white blood cells to the blood vessel wall [ 219 ]. This can lead to the transmigration of the adhered cells into the innermost layer of the vessel where they are transformed into macrophages, which rapidly accumulate oxidised LDL [ 219 ]. These cells are often the origin of atherosclerotic lesions, which form in artery walls and potentially lead to coronary heart disease and heart attacks [ 134 , 219 ]. Chopra et al. found that increased intake of fruits and vegetables, especially red coloured ones, improves the ex vivo resistance of LDL to oxidation [ 220 ]. In another human study, a 3-week low-tomato diet followed by a 3-week high-tomato diet (400 mL tomato juice and 30 mg tomato ketchup daily) led to a reduction in LDL cholesterol levels and increased ex vivo resistance of LDL to oxidation in normocholesterolaemic participants [ 209 ]. Interestingly, a study conducted in 2000 showed that the regular intake of tomato juice is associated with an increase in blood vitamin E levels [ 134 ]. Lycopene and beta-carotene are known to more effectively inhibit LDL oxidation in the presence of vitamin E [ 134 , 209 , 219 ]. Blood platelets respond to vascular damage by binding to the subendothelial matrix, eventually leading to atherosclerotic lesions, thrombus formation, and vascular events. Platelets are therefore considered as the driving force to myocardial infarction and ischaemic stroke [ 221 , 222 , 223 ]. Tomatoes have been shown to have platelet anti-aggregatory properties. In a double-blind, randomised trial, the dietary supplementation of adults 40–70 years old, these being healthy individuals, with tomato extract was shown to reduce ex vivo platelet aggregation induced by both ADP and collagen [ 224 ]. Although initially carotenoids lycopene and beta-carotene were suggested to contribute to the anti-aggregatory properties of tomatoes, later studies suggested that the anti-platelet factor of tomatoes was due to water-soluble, heat-stable compounds that are concentrated in the jelly substance surrounding the seeds [ 225 , 226 ]. It has been suggested that a diet containing anti-platelet compounds such as these have the potential of reducing lipid levels and lowering blood pressure and can reduce the risk of ischaemic heart disease and strokes by up to 80% in middle-aged individuals [ 227 , 228 ]. Many studies have shown tomato extracts to have platelet anti-aggregatory activity in vitro and in vivo and possibly preventing thrombus formation [ 222 , 224 , 225 , 226 , 229 , 230 , 231 , 232 ]. A study by Zhang et al. [ 233 ] investigated the impact of water-soluble tomato concentrate (WSTC) on the platelet aggregation in Sprague Dawley rats. This study found that WSTC inhibited adenosine diphosphate (ADP)-induced platelet aggregation in vitro and ex vivo in the rats without affecting their coagulation system [ 233 ]. Platelet aggregation relies on fibrinogen binding to the calcium-dependent glycoprotein (GP) IIb/IIIa complexes found on platelets [ 234 ]. When platelets are activated by ADP, these GP IIb/IIIa complexes bind with fibrinogen, leading to many platelets assembling and connecting to the same fibrinogen strands and forming a clot [ 235 ]. Zhang et al. [ 233 ] found that WSTC increased cytoskeleton stability and led to the inhibition of platelet aggregation. There are suggestions that people should adjust their diet to reduce cardiovascular risk and prevent any potential side effects, such as headaches or dizziness, nausea, and increased bleeding, including nose bleeds, associated with anti-platelet medications [ 236 ]. Inflammation plays an important role in atherosclerosis, a process associated with lipid accumulation in the artery wall [ 237 ]. Postprandial lipemia, characterised by an increase in triglyceride-rich lipoproteins, is a condition known to trigger inflammation and atherogenesis in humans [ 238 ]. The transcription factor NF-κB, previously mentioned in relation to cancer development, is also a modulator of inflammation in the liver [ 169 ]. The liver is involved in the uptake, formation, and exportation of lipoprotein and is thus an essential component of lipid metabolism [ 239 ]. Therefore, inflammation and liver damage can have a detrimental impact on lipid metabolism in mammals. Sahin et al. [ 169 ] supplemented a rat diet with tomato powder and found that this resulted in reduced liver damage caused by age-associated inflammation and oxidative stress through the inhibition of the NF-κB pathway. The activation of NF-κB in cultures of endothelial and smooth muscle cells with inflammatory stimuli is also suggested to have a role in atherosclerosis formation [ 240 , 241 ]. NF-κB activation has also been observed in previous studies [ 241 , 242 ] and genes expressed in atherosclerotic plaques are regulated by NF-κB [ 242 ]. There is strong evidence that a reduction in inflammation can reduce the risk of atherosclerosis caused by NF-κB activation but no research to date has investigated the impact of tomato extract on atherosclerosis caused by NF-κB activation. It is worth noting that tomato powder supplementation of rats has been shown to inhibit the NF-κB pathway in the liver of animals [ 169 ]. Vascular endothelium plays an important role in the constriction and dilation of blood vessels, and its dysfunction is suggested as an early, reversible precursor of atherosclerosis. In a randomised controlled trial in post-menopausal women, the effect of 70 g tomato puree ingestion was examined on endothelial-dependent, flow-mediated dilation (FMD) and endothelial-independent, nitro-mediated dilation of the brachial artery using high-resolution ultrasound. The effects after 24 h and 7-day intake were examined [ 243 ]. Although a significant increase in plasma lycopene was observed after 7 days, it did not affect endothelium-dependent or -independent dilation of the brachial artery. Similar findings were reported by another group, where 80 g of tomato paste puree per day for 7 days failed to affect the flow-mediated dilation of the brachial artery after a standardised fat meal, however, the incorporation of tomato paste produced a significant improvement in the haemodynamic changes, such as reduction in diastolic blood pressure, increase in brachial artery diameter, and decrease in stiffness index [ 244 ]. Overall, the potential benefits of a tomato-enriched diet are associated with lowering of blood pressure; anti-platelet, anti-inflammatory, and anti-apoptotic activity; and lipid lowering, as well as the inhibition of LDL oxidation; the latter is believed to be key player in the pathophysiology of atherosclerosis, a hallmark of cardiovascular disease. It is worth noting that the role of a tomato-rich diet on cardiovascular disease was further evaluated in a recent meta-analysis by Rosato et al. [ 245 ]. This paper described the positive impact of the Mediterranean diet on cardiovascular disease in particular foods such as olive oil, fresh fruits and vegetables, nuts, legumes, and fish, but not red meats [ 245 ]. It is stressed that it is the combination of elements in the Mediterranean diet, including the phytochemicals found in fresh fruits and vegetables such as tomatoes, that results in these positive impacts, and these elements on their own would not have the same impact [ 143 ]. Although a tomato-enriched diet has been shown to influence several key mechanisms that are important in vascular pathology, and population-based studies show an inverse correlation between their intake and cardiovascular disease, it is unlikely to affect a multifactorial disease such as CVD when used in isolation. However, if incorporated as a component of a healthy diet, it should provide an additive effect when combined with other cardioprotective nutrients in food. Recent studies have shown that the beneficial effects of tomato compounds are not limited to cardiovascular diseases and cancer but have also been reported in neurological disorders and diabetes mellitus (DM) [ 74 , 78 , 109 , 246 , 247 ]. 4.4. Neurodegenerative Disorders Neurodegenerative diseases are associated with the degeneration of the nervous system over a long period of time and, among these, Alzheimer's, Parkinson's, and cerebral ischaemia associated with stroke are the most common neurodegenerative diseases. Neuroinflammation, oxidative stress, and apoptosis are important hallmarks of these diseases. The majority of cases of stroke are due to cerebral ischaemia, and population-based studies have shown a negative association between tomato, especially lycopene intake, and incidence of stroke [ 205 , 248 , 249 ]. In rats, a tomato pomace powder pre-treatment at doses of 2, 10, and 50 mg/kg protected various areas of the brain, including hippocampus, striatum, and cerebral cortex affected by experimental cerebral ischaemia induced by permanent occlusion of the middle cerebral artery [ 250 ]. The tomato pomace powder pre-treatment of animals also increased the activities of antioxidant enzymes glutathione peroxidase and superoxide dismutase and decreased the lipid peroxidation product malondialdehyde in the hippocampus and cerebral cortex area of the brain. In Alzheimer's disease (AD), neurofibrillary tangles (aggregates of tau protein), a reduction in neurotropic factors, and amyloid-β plaque are present. Amyloid-β accumulation can induce apoptosis both via extrinsic death receptor-mediated and intrinsic mitochondria-mediated pathways, and lycopene has been shown to inhibit these pathways. In human neuroblastoma SH-SY5Y cells, the pre-treatment of cells with 0.2 and 0.5 μmol/L lycopene for one hour, followed by 24 h stimulation with amyloid-β (20 μM), significantly inhibited apoptosis through its dose-related effects on Bax/Bcl-2 and cleavage of pro-caspase-3 [ 251 ]. In addition, lycopene pre-treatment reduced amyloid-β induced oxidative stress, mitochondrial dysfunction, and NF-κB activation in the cells. Lycopene has also been shown to improve the production of brain-derived neurotrophic factor (BDNF) during neurotoxic challenges [ 252 ]. Lycopene supplementation of male Wistar rats at a dose of 5 mL/kg body weight for 21 days reduced neuro-inflammation, oxidative damage to mitochondria, and apoptosis, and improved memory retention and restoration of BDNF level in β-A1-42 treated rats [ 252 ]. Yu et al. also provided evidence that dietary lycopene supplementation could improve cognitive performance in tau transgenic mice expressing P301L mutation [ 253 ]. Likewise, Zhao et al. demonstrated that lycopene supplementation could reduce oxidative stress and neuroinflammation and improve cognitive impairment in aged CD-1 mice [ 254 ]. A recent review provided details of in vitro and animal studies describing the neuroprotective role of lycopene and related this to its antioxidant activity, the inhibition of a redox-sensitive transcription factor NF-kB, a reduced expression of amyloid-β and its precursor, a reduction in neuroinflammation, an improvement in mitochondrial function, memory, and learning as well as a restoration of antioxidant defence [ 77 ]. Vascular dementia is associated with cerebral ischaemia and a recent study examined the effects of supplementation with lycopene at a dose of 50, 100 and 200 mg/kg body weight every other day for two months in a vascular dementia model in rats. At 100 mg/kg dose, lycopene supplementation reduced the oxidative stress, improved the antioxidant enzyme superoxide dismutase and glutathione peroxidase levels in the hippocampus and improved the learning-memory ability of animals [ 255 ]. In human studies, lower antioxidant status, especially vitamin C, lycopene, vitamin E, and antioxidant enzymes superoxide dismutase and glutathione peroxidase, and high levels of markers of oxidative stress have been reported in the plasma [ 256 , 257 , 258 , 259 ] as well as cerebrospinal fluid [ 259 , 260 , 261 ] of AD patients. In the Nurses Health study, participants aged ≥70 years were followed up for 4 years, and a decline in cognitive function was reported to be slower with high lycopene intake but not with vitamin C, β-carotene, and vitamin E intakes [ 262 ]. A recent study that followed up elderly patients over 5–6 years reported that plasma antioxidants such as vitamin E isomers (alpha- and gamma-tocopherol), retinol, and carotenoids were not significantly associated with a reduced risk of dementia or AD [ 263 ]. The lack of association could be related to the age group of 70–75 years that was studied. AD is a chronic disease and may have already been present in the cohort that was included in the study; therefore, the study population was not likely to be a suitable age group to examine the associations. The prevalence rate of AD is less than 1% before 65 years of age, and it increases to 10% after 65 years of age. The brain, due to its high oxygen consumption, high polyunsaturated fatty acids, and transition metals ion content, is highly susceptible to oxidative stress, and antioxidants such as vitamin C, carotenoids, and flavonoids present in tomatoes are therefore a likely candidate for the protection offered by this fruit against neurodegenerative disease. Future follow-up studies should be conducted in 60–65-year-old individuals to confirm whether tomato or its constituents, carotenoid, vitamin C, and flavonoid, intake can slow down cognitive decline with aging. Parkinson's, a neurodegenerative disease, is also associated with oxidative stress and neuronal apoptosis, and its pathology is also likely to be influenced by antioxidant and anti-apoptotic dietary components. The motor disability seen in Parkinson's is suggested to be due to the degeneration of dopaminergic neurons leading to a decrease in dopamine (DA) in the striatum. Supplementation with 20% ( w / w ) lyophilised tomato powder for 4 weeks before methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced Parkinson's disease (PD) in mice was reported to prevent a striatal decrease in the DA levels [ 264 ]. In another study in mice, 7-day pre-treatment with lycopene at doses of 5, 10, and 20 mg/kg was examined on MPTP-induced PD, and treatment was found to reduce MPTP-induced oxidative stress, apoptosis, and depletion of dopamine in the striatum [ 265 ]. Likewise, vitamin C feeding of mice at a dose of 15 mg/kg body for 3 days before intraperitoneal injection of MPTP at 20 mg/kg reduced neuroinflammation and dopaminergic neuronal degradation in the striatum and improved the locomotor inability caused by the neurotoxin [ 266 ]. Overall, pathological changes seen in AD, PD, and cerebral ischaemia have been shown to be ameliorated with lycopene and tomato extract in studies that were conducted in vitro as well as in animal studies. Few human studies in AD and PD patients have indicated a protective role; however, studies are limited and not conclusive likely due to the age group that has been studied in observational as well as intervention studies. Further human studies for AD- and PD-related investigations in the age group 60–65 years old are likely to provide a better insight into the protective role that a tomato-enriched diet may offer against these neurodegenerative diseases. 4.5. Diabetes Carotenoids may play a role in reducing the risk of insulin resistance and the development of diabetes, and an inverse association has been reported between plasma β-carotene, lycopene, and glucose intolerance in newly diagnosed patients [ 267 ] and on glycated haemoglobin levels in older type 2 diabetes patients [ 268 , 269 , 270 ]. Type 2 diabetes is described as a multifactorial metabolic syndrome associated with oxidative stress, inflammation, hyperglycaemia, and hyperlipidaemia [ 271 ]. Tomato constituents have antioxidant and anti-inflammatory properties. Studies have examined hypoglycaemic, hypolipidemic, anti-inflammatory, and antioxidant effects of tomatoes, especially lycopene. In experimental diabetic rats, lycopene at a dose of 10 mg/kg/day for 28 days significantly reduced the increase in blood glucose and glycated haemoglobin (HbA1c) levels induced by streptozotocin (STZ) [ 272 ]. In another study, male albino Sprague Dawley rats were fed a high-fat diet for 4 weeks followed by intraperitoneal injection of STZ at 25 mg/kg. The effects of lycopene administration at 10 and 20 mg lycopene per kg body weight/day for 10 days was examined on the fasting blood glucose, lipids, and glycosylated haemoglobin levels, and a reversal to normality of these parameters was seen with lycopene supplementation [ 273 ]. A recent case-control study reported that lycopene intake positively correlated with peripheral antioxidant activity, antioxidant enzymes superoxide dismutase, and glutathione peroxidase levels and negatively correlated with fasting blood glucose and glycated haemoglobin (HbA1c) levels in patients with type 2 diabetes [ 274 ]. A detailed account of evidence from in vitro, animal, and human studies (cross-sectional, prospective, and two randomised controlled trials) suggesting a preventive role of lycopene and tomato-enriched diet against diabetes is provided by Zhu et al. [ 80 ]. Figueriredo et al. [ 275 ] reported that a combination of lycopene with metformin had an additive effect on improvements in postprandial blood glucose levels, dyslipidaemia, and antioxidant status. The investigation was performed in rats, and STZ-induced diabetic rats were treated with lycopene (45 mg/kg) and metformin (250 mg/kg) alone and in combination for 35 days. There is ample evidence from animal studies suggesting a decrease in diabetes-induced hyperglycaemia, dyslipidaemia, and oxidative stress. There is, however, limited evidence from human intervention trials. Shidfar et al. [ 276 ] fed type 2 diabetic patients with 200 g raw tomatoes/day for 8 weeks. No significant effect was observed in blood glucose levels. However, there was a significant improvement in both systolic and diastolic blood pressure as well as improvements in apoprotein A-1 (ApoA-1) levels. Diabetic patients are at increased risk of cardiovascular disease and the ApoA-1 constituent protein of high-density lipoprotein is important for the anti-atherogenic properties of HDL. Bose and Agarwal [ 277 ] reported that the supplementation of diabetic patients with cooked tomatoes improved the antioxidant defence and plasma lipid peroxidation products but failed to affect the lipid profile and HbA1c levels. It is important to note that the majority of studies that show hypoglycaemic effects of lycopene were carried out using pure compounds, and concentrations were much higher than likely to be achieved by the amount of tomato products that were used by Shidfar et al. [ 276 ] and Bose and Agarwal [ 277 ] for human studies. Zidani et al. [ 278 ] reported that 12 weeks' supplementation of mice with 46 and 84 mg of lycopene/kg of food provided by tomato peel extract significantly reduced insulin resistance caused by the high-fat diet. Both type 2 diabetes and gestational diabetes (GD) are associated with insulin resistance. In the case of GD, it is caused by a hormonal change during pregnancy. Tomatoes have a low glycaemic index, and therefore can be considered as a potential fruit of choice for pregnant women. Very few studies have examined the association or effect of tomato-rich diets on gestational diabetes. A cross-sectional study that used food frequency questionnaires to estimate the food/nutrient intake of its participants highlighted that high lycopene protected against gestational diabetes-associated hyperglycaemia in women and suggested that intake can offer a protective effect against GD [ 279 ]. To date, anti-diabetic effects in animal studies have either tested the effects of lycopene or tomato extract. Tomatoes contain glycoalkaloid esculeoside A, and its concentration is four times higher than that of lycopene. Yang et al. [ 100 ] suggested that esculeoside A can be considered as a functional supplement for diabetes. In their study, wild-type C57BLKS mice were used and esculeoside A (100 mg/kg) administration by gavage for 56 days was found to lead to a reduction in fasting blood glucose levels and improved glucose tolerance in mice [ 100 ]. Both type 2 diabetes and gestational diabetes are on the rise and can be prevented with diet and lifestyle interventions [ 280 , 281 ]. It is well known that synthetic hypoglycaemic medications that are used for type 2 diabetes induce side effects. Further investigations of foods such as tomato products either on their own or in combination with other hypoglycaemic foods are warranted to confirm if the findings of animal studies can be replicated in humans, as the evidence from randomised controlled trials is limited and inconclusive at present. 4.6. Tomato Fruit for Skin Health The properties of tomatoes are not limited to disease prevention. Studies have provided evidence of the beneficial effects of dietary tomato and its supplements for improved skin health [ 282 , 283 , 284 ]. The principle of oral photoprotection provided by antioxidants to prevent the harmful effects from UV radiation has gained popularity over the last decade [ 282 , 283 , 284 ]. The benefits and hazards of solar ultraviolet (UV) radiation are well documented and include the effects of solar exposure on skin cancer, malignant melanoma, immune suppression, photoaging, photosensitivity, and diseases in the eye [ 85 , 86 , 285 , 286 , 287 ]. Acute UV radiation has been linked to skin burns, oedema, abnormal pigmentation, and photokeratitis, and long-term exposure increases the risk of photoaging and malignant tumours [ 85 , 86 , 284 , 286 , 287 ]. Three types of UV rays are produced by sunlight, UVA, UVB, and UVC [ 86 , 284 , 288 ]. UVA rays have the longest wavelength, followed by UVB, while UVC rays have the shortest wavelength. UVC has the strongest mutagenicity, followed by UVB, while UVA is considered a weak mutagen [ 85 , 284 , 287 ]. However, all UVC rays are absorbed by the Earth's ozone layer, therefore, exposure is unlikely, except through an artificial source such as a laser [ 85 , 287 ]. UVA rays can penetrate the dermis and the subcutaneous tissue area [ 84 , 284 , 287 ]. UVB rays can reach the epidermis and have the capacity to interact with DNA [ 84 , 284 , 289 ]. The underlying mechanism involves the production of ROS by UV radiation, which hinders DNA replication and transcription and results in destructive oxidative stress, the activation of the arachidonic acid pathway, and the mediation of inflammatory responses [ 85 , 287 ]. Studies by Groten et al. and Aust et al. provided evidence that carotenoid-containing supplements could significantly protect against UVB-induced erythema by reducing oxidative stress [ 282 , 288 ]. Baswan et al. reported similar findings regarding carotenoid supplementation against both UVB-induced erythema and UVA-induced pigmentation [ 289 ]. Grether-Beck et al. examined the capacity of carotenoids, including lycopene-rich tomato nutrient complex (TNC) and lutein, to protect against UVA/B and UVA1 radiation at a molecular level [ 283 ]. Analysis of the mRNA expression of key genes involved in solar radiation-induced skin damage, including heme-oxygenase 1 (HO1), matrix metalloproteinase 1 (MMP1), and intercellular adhesion molecule 1 (ICAM1), revealed that UVB/A and UVA1 radiation significantly upregulated steady-state levels of HO-1, ICAM-1, and MMP-1 mRNA in the skin of healthy volunteers who did not receive the supplement. Moreover, TNC and lutein treatment significantly inhibited UVB/A and UVA1 radiation-induced gene expression [ 283 ]. Calniquer et al. assessed the effect of a combination of carotenoids and polyphenols (tomato extract with rosemary extract) on the response of skin cells to UV irradiation [ 290 ]. The results demonstrated that carotenoids and polyphenols worked in synergy and that combining these compounds was more effective in balancing UV-induced skin cell damage than using them separately [ 290 ]. Vitamin C is another compound found in tomatoes that contributes to immune modulation [ 24 ]. When applied topically, it is known to be actively taken up by epidermal and dermal skin cells using sodium-dependent vitamin C transporter isoforms (SVCT1 and SVCT2) [ 137 ]. These cells are involved in the production of collagen fibres and therefore are essential to the function of the skin as a barrier against pathogens [ 136 ]. The use of natural ingredients such as tomato extract or tomato seed oil in cosmetic products for skin care and health has also received popularity over the last few years [ 85 , 86 , 87 , 121 , 284 , 287 , 291 , 292 , 293 , 294 , 295 , 296 , 297 ]. Tomato seed oil has been extensively used in the production of cosmetic and personal care products such as anti-aging serums, body butter, sunscreens, and skin lightening cream due to its high linoleic acid, lecithin, antioxidant, and natural UV protection attributes [ 284 ]. Furthermore, there is increasing evidence that a diet rich in antioxidants may significantly influence the course of certain skin diseases such as atopic dermatitis, acne, and psoriasis [ 285 , 291 , 298 ]. Multiple in vitro studies in mice have revealed that polyphenols including quercetin and gallic acid present in tomatoes may be an alternative for the development of cosmetics that could be used to treat acne vulgaris [ 108 , 299 , 300 , 301 ]. Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease that can affect up to 25% of children within a diverse paediatric population [ 302 ]. Symptoms can include itching and scratching, dry skin, patchy eczema, exudation, and skin thickening and discolouration [ 302 ]. Although the mechanisms of the pathogenesis of AD have not been fully elucidated, the chronically inflamed skin of patients with AD plays a key role in pathogenesis, with the overproduction of ROS and a decrease in antioxidant defence [ 302 ]. Sapuntsova et al. have reported that levels of ROS were significantly higher in the skin biopsies in AD patients compared to those of controls [ 303 ]. Lycoperoside H, an anti-inflammatory component present in the seed part of tomatoes, was shown by Takeda et al. to relieve symptoms of AD in transgenic mice expressing IL-33 driven by a keratin-14 promoter (IL33tg) [ 62 ]. Other reported benefits of tomato compounds for skin health include the protection against tick bites and heavy metal toxicity [ 64 , 121 , 292 , 296 , 299 ]. Boulanger et al. showed that natural skin repellents made from eucalyptus, tomato, and coconut can protect against tick-borne infections such as Lyme disease [ 63 ]. A study by Tito et al. demonstrated that an active ingredient derived from Lycopersicon esculentum tomato cultured stem cells protected skin cells against heavy metal toxicity [ 292 ]. The mechanism of action involves the preservation of nuclear DNA integrity from heavy metal damage by inducing the genes responsible for DNA repair and protection and also involves the neutralisation of the effect of heavy metals on collagen degradation by inhibiting collagenase expression and inducing the synthesis of new collagen [ 292 ]. Additionally, in a study involving Indian women, Nutrova, a blend of collagen peptides and natural antioxidants from tomatoes, green tea, and grapes, has been shown to significantly reduce wrinkles, skin roughness, and hyperpigmentation while improving skin hydration and firmness [ 296 ]. Similarly, another study of 4000 women showed that a diet reported as high in potassium and vitamins A and C correlated to fewer wrinkles in patients' skin [ 291 ]. It can be concluded that incorporating tomatoes into a diet could have benefits to a person's skin health. The suggested benefits include protection against UV radiation through antioxidant properties [ 284 ], treatment of skin inflammatory conditions such as AD [ 62 ], and protection against tick bites and heavy metal toxicity [ 63 , 292 ]. 4.7. Tomatoes, Gut Microbiome, and Inflammation "Microbiome" is a term used to describe a community of colonising microorganisms such as fungi, viruses, and bacteria found in a particular environment [ 304 ]. Microbial populations reside throughout the human body, including the stomach and intestines, and are increasingly described as a key link between genetic and environmental impacts that affect an individual's health [ 304 , 305 ]. The gut microbiome is defined as all microorganisms found in the gastrointestinal tract consisting of bacteria, archaea, viruses, and eukaryotic microbes, and can have as many as 100 trillion cells [ 305 , 306 ]. These bacteria are known to vary in composition depending on the lifestyle, genetics, and diet of the host [ 305 ]. The composition of the gut microbiome is implicated in the development of liver inflammatory disease and liver cancer [ 307 ]. In a 2018 study by Xia et al. [ 308 ], mice were fed a high-fat diet (HFD) supplemented with a liver-specific carcinogen (DEN) along with a tomato powder rich in lycopene, which has previously been shown to inhibit HFD-induced liver disease [ 309 ]. The tomato powder significantly increased both diversity and richness of the gut microbiota in all mice [ 308 ]. The gut microbiome contains both Gram-positive and Gram-negative bacteria, and it has been shown that an increase in Gram-negative bacteria in the gut can lead to an increase in the level of hepatotoxic compounds, such as lipopolysaccharides (LPS) [ 310 ]. LPS are major components of the outer membrane of Gram-negative bacteria and are known to induce inflammation through induction of Toll-like receptor 4, which can lead to cell proliferation and a reduction in apoptosis [ 129 , 310 ]. In 2018, Xia et al. reported that feeding mice tomato powder reduced the relative abundance of the gut Gram-negative bacteria by reducing levels of gut LPS in the mice, lowering the risk of inflammation [ 308 ]. Increasing the diversity and richness of the gut microbiome through tomato powder supplementation was found by the authors to regulate inflammation, lipid metabolism, and the circadian clock in the liver [ 308 ]. Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions of the gastrointestinal tract and include Crohn's disease and ulcerative colitis [ 308 ]. Both of these conditions are affected by a number of factors, including abnormal gut microbiota [ 311 ]. A study by Scarano et al. (2018) [ 129 ] developed a bronze tomato line that was shown to have 30–50% higher levels of flavanols and anthocyanins when compared to other tomato lines. These tomatoes were freeze-dried, ground into powder, and incorporated into the diets of mice with induced chronic colitis [ 129 ]. The study found that a diet enriched with 1% bronze tomato fruit powder promoted a change in microbiota composition, moderately inhibiting inflammatory responses in the mice and thus reducing intestinal damage caused by chronic colitis [ 129 ]. This was continued in a follow-up study using the same bronze tomato line, which demonstrated the beneficial effects of this variety of tomato on intestinal inflammation and showed changes in the gut microbiome, especially an increase in Flavobacterium and Lactobacillus and a reduction in Oscillospira [ 130 ]. In summary, the composition of the gut microbiome is affected by the consumption of tomatoes, and this has implications for human health. Tomato powder has been shown to significantly increase the diversity and richness of the gut microbiome in mice, preventing a build-up of Gram-negative bacteria that produce hepatotoxic compounds, which can cause liver inflammatory disease and cancer [ 308 , 310 ]. Furthermore, freeze-dried tomatoes high in flavanols and anthocyanins have been shown to alter the composition of the gut microbiome by increasing Flavobacterium and Lactobacillus and decreasing Oscillospira populations, resulting in reduced inflammatory responses in mice and preventing intestinal damage by chronic colitis [ 129 , 130 ]. It is worth noting that most evidence shown to date is from animal studies, and it remains to be seen whether tomato product intervention can be beneficial for conditions associated with gut dysbiosis. 4.8. Tomatoes and Exercise Recovery Exercise and increased muscle activity result in higher amounts of ROS due to increased ATP production and oxygen utilisation [ 312 ]. ROS are highly reactive and can damage macromolecules such as proteins, DNA, and lipids. ROS such as peroxides and superoxides can be damaging to cells if concentrations are excessive [ 313 , 314 ]. Regular exercise builds resistance of the body against oxidative stress by upregulating the expression of genes that synthesise antioxidant enzymes such as superoxide dismutase [ 312 ]. Antioxidants such as vitamins, terpenoids, and phenolics can inhibit oxidative stress and neutralise ROS [ 314 , 315 , 316 ]. Antioxidants found naturally in plant tissues, including tomato fruit, can provide the protection against ROS. Previous studies have indicated that lycopene and lycopene metabolites can have a positive effect on recovery from exercise-induced physiological stress [ 144 , 317 ]. Lycopene is known to be the most effective singlet oxygen scavenger, exhibiting quenching rates multiple times greater than any other carotenoid [ 317 , 318 ]. Creatinine phosphokinase (CPK) and lactate dehydrogenase (LDH) are associated with ATP and NADH conversion in muscle cells and can be monitored as markers of muscle damage in individuals undergoing exercise sessions [ 319 ]. A study by Tsitsimpikou et al. [ 319 ] tested whether the administration of 100 g of tomato juice could improve the recovery of anaerobically trained athletes. The study found that a 2-month administration of tomato juice, post exercise, led to a significant decrease in LDH and CPK levels compared to a carbohydrate supplementation beverage [ 319 ]. Another study carried out by Nieman et al. [ 318 ] administered a "tomato complex" containing lycopene, phytoene, and phytofluene (T-LPP) to endurance runners for a 4-week period and monitored inflammation, muscle damage, and oxidative stress post exercise and during recovery from a two-hour running session. Myoglobin is an iron- and oxygen-binding protein found in muscle tissue that is translocated to the blood compartment after low-level muscle injury caused by exercising and can be used as a marker for muscle injury [ 318 ]. Nieman et al. [ 318 ] found a significant increase in plasma carotenoid levels and a reduction in myoglobin, suggesting possible reductions in muscle injury as a result of consuming the "tomato complex" supplement following a 2 h running session. Tomato products and supplementation with their constituents are suggested to reduce muscle damage caused during anaerobic exercise as well as reduce oxidative stress during aerobic exercise, as shown by Harms-Ringdahl et al. [ 320 ]. In their study, 15 healthy and untrained participants engaged in 20 min of aerobic exercise on a bicycle after receiving 150 mL of tomato juice for 5 weeks, followed by 5 weeks without tomato juice, and for the final intervention they received tomato juice for another 5 weeks [ 320 ]. The blood samples were collected before and after each intervention, and results showed that tomato juice intake significantly suppressed 8-oxodG (a marker of oxidative damage) levels produced with the physical activity [ 320 ]. Therefore, there is evidence that tomato products can reduce oxidative stress and muscle damage caused by physical exertion and can be considered as a workout drink [ 320 ]. 4.9. Tomatoes and the Immune System Tomatoes and tomato products are suggested to affect the immune system [ 321 ], and current literature relates this to their lycopene, β-carotene, and vitamin C content. In a human study, supplementation with tomato products (tomato sauce, tomato puree, and raw tomatoes) providing 8 mg lycopene, 0.5 mg β-carotene, and 11 mg vitamin C for 3 weeks was reported to produce a significant increase in plasma levels of lycopene, β-carotene, and vitamin C; however, only lycopene and vitamin C levels increased in the lymphocyte [ 322 ]. Supplementation also reduced ex vivo oxidative damage to the DNA of lymphocytes [ 322 ]. How vitamin C can affect the immune system has been reviewed by Van Gorkam et al. who describe that, although the data on the effects of vitamin C on B lymphocytes are limited and inconclusive, vitamin C increases the proliferation of T-lymphocytes and natural killer (NK) cells [ 323 ]. T-lymphocytes play a key role in cell-mediated, cytotoxic adaptive immunity, and natural killer (NK) cells provide rapid cytolytic responses to virus-infected cells and tumour cells [ 324 , 325 ]. Few studies in human subjects reported that supplementation with β-carotene stimulates the proliferation of lymphocytes [ 326 , 327 ] and enhances the lytic activity of NK cells [ 5 , 327 ]. A study carried out by Watzl et al. [ 321 ] supplemented human subjects with tomato juice and carrot juice—both of which are known for their high β-carotene content. This study found that supplementation with the juices significantly increased lymphocyte proliferation and enhanced the lytic activity of natural killer cells [ 321 ]. No significant differences were observed between the effects of either juice, indicating a similar effect on the immune response, or that other compounds present in both juices resulted in the observed effects [ 321 ]. Naringenin is a flavanone (a subclass of flavonoids) that has also been shown to have immune-modulating functions [ 328 , 329 ]. In an in vitro study, Niu and colleagues demonstrated that naringenin could inhibit T cell activity by various mechanisms, such as lowering the secretion of specific T cell cytokines and affecting T cell proliferation [ 329 ]. Further investigation revealed that that inhibition of cell proliferation was triggered by delayed degradation of the cyclin-dependent kinase inhibitor p27kip1 and the downregulation of retinoblastoma protein phosphorylation in activated T cells, resulting in a T cell cycle arrest at G0/G1 phase [ 329 ]. Other findings indicated that the T cell-suppressive effects could be attributed to the capacity of naringenin to interfere with the interleukin-2/interleukin-2 receptor (IL-2/IL-2R)-mediated signalling pathway and STAT5 phosphorylation in activated T cells [ 329 ]. The immune-modulating effects of lycopene have been hypothesised through their antioxidant activity and their effects on lymphocyte proliferation and on improving cell–cell communication [ 330 , 331 ]. A 2017 study tested 40 mice divided into five groups: an ambient air control; a vehicle control group receiving 200 µL of sunflower oil; a group exposed to cigarette smoke; and two groups administered lycopene diluted in sunflower oil (25 or 50 mg/kg/day) prior to cigarette smoke exposure [ 332 ]. The 5-day testing period resulted in an increase in the number of lymphocytes in lycopene-treated groups compared to other treatments [ 332 ]. This study suggested that the increase in lymphocytes was a result of lycopene activating the adaptive immune response [ 333 ], and the latter is known to be vital in pathogenic defence. However, further studies are warranted to fully understand lycopene's direct impact on the adaptive immune system in humans [ 334 ]. As one of the most popular world crops, the tomato has also been considered as an edible vaccine for a wide variety of diseases, including malaria, coronavirus (COVID-19), human papillomavirus infections, human immunodeficiency virus infections, shigellosis, cholera, anthrax, and hepatitis B [ 97 , 335 , 336 , 337 , 338 , 339 ]. The main objectives of edible vaccines are to democratize preventive vaccination, especially in developing countries, and to better control potential outbreaks such as coronavirus disease. Traditional vaccine development requires more time and high cost, while the development of an edible vaccine in a plant expression system provides an efficient mode of oral delivery and bypasses the assistance of a medical professional to perform injections. It is also economically sustainable, with higher scale production. However, there are several hurdles to overcome, such as the immunogenicity of an oral vaccine, the stability of the vaccine in the gastrointestinal tract, the variability of the expression of antigens in plants, and the effects associated with the consumption of genetically modified plants on health [ 336 , 339 ]. Shchelkunov et al. designed an oral vaccine against hepatitis B and human immunodeficiency viruses using tomato fruits, which was administered to experimental mice [ 337 ]. Examination of serum and stool samples of the test animals revealed high levels of HIV- and HBV-specific antibodies [ 337 ]. Salyaev et al. investigated the duration of the mucosal immune response in mice after administration of this vaccine in a subsequent study [ 340 ]. Results showed a steady increase in the immune response, with a peak observed between 6 and 11 months post-administration followed by a gradual decrease in the levels of antibodies until they became undetectable after 19 months [ 340 ]. Evidence from in vitro, animal, and a few human studies describes a significant increase in lycopene and vitamin C content of lymphocytes, improvements in T cell mediated immunity, and the lytic activity of NK cells, and there is also a suggestion of the use of tomato fruit as an edible vaccine. However, even though plant-based vaccines offer a promising alternative, their clinical development remains challenging, and further research is required in human clinical studies [ 336 , 339 ]. 4.10. Tomatoes and Fertility Infertility is a disease of the male or female reproductive system characterised by the inability to accomplish a pregnancy following at least 12 months of regular unprotected sexual intercourse [ 341 , 342 , 343 ]. According to statistics, 48 million couples and 186 million individuals live with infertility globally [ 341 ], and male factors account for at least 50% of all infertility cases worldwide [ 343 ]. Oxidative stress (OS), which arises from an imbalance between ROS and protective antioxidants, can affect the entire reproductive lifespan of men and women [ 344 ] and has been shown to be a major cause of reproductive dysfunction [ 344 , 345 ]. The positive effects of antioxidants in female fertility have also been described [ 346 , 347 , 348 , 349 ]. OS has also been recognised as one of the main mediators of female infertility and has been associated with various reproductive pathologies, including endometriosis, preeclampsia, spontaneous abortion, and unexplained infertility [ 347 , 348 ]. Studies have shown the presence of ROS in the ovaries, fallopian tubes, and embryos of women with idiopathic fertility [ 348 ]. Additionally, ROS have been shown to play a role in the regulation of ovarian steroid biosynthesis and secretion, primordial follicle recruitment, and ovulation, and they can also affect the fertilisation process and post-fertilisation events, although the underlying molecular mechanisms have not been fully elucidated [ 346 ]. The current literature on folate and fertility endpoints indicates that a high intake of folic acid in the preconception period may increase pregnancy success rates. Upadhyaya et al. showed that folate levels in red-ripe tomato fruits could range from 14 to 46 μg/100 g FW [ 131 ]. Furthermore, a lack of vitamin C seems to be associated with an increased risk of preeclampsia, and some studies have shown that vitamin supplements could lower the risk of preeclampsia in normal or underweight women [ 346 ]. Some studies have demonstrated that oral administration of multivitamins including folic acid and vitamins C, D, and E can increase fertility [ 346 ]. Yu et al. reported that β-carotene has a similar antioxidant potential to folic acid and could also improve the oocyte development and maturation and ovarian function in mice [ 348 ]. Therefore, there is some indirect evidence on the role that a tomato-enriched diet may play in female fertility; however, to date, no studies have specifically examined the effects of a tomato-enriched diet on OS-related effects on female fertility. According to research, between 30 and 80% of male infertility cases are caused by OS and a decreased level of seminal total antioxidant capacity [ 342 , 345 , 350 ]. Evidence shows that the semen from infertile men has a lower antioxidant capacity and high levels of ROS compared to fertile men [ 345 , 346 ]. As a source of antioxidants, tomato's constituents and their supplement counterparts may be important for reducing OS and improving semen parameters, including sperm concentration, motility, morphology, and fertility rate [ 341 , 343 , 351 ]. In a human study, tomato soup consumption at 400 g/day significantly increased seminal plasma levels of lycopene, though the effects on plasma antioxidant levels failed to reach significance [ 352 ]. As potent antioxidants, the role of carotenoids in fertility has been extensively investigated [ 345 , 348 , 353 , 354 , 355 ]. Williams et al. examined the effect of lactolycopene, a combination of lycopene with whey protein, which protects lycopene from digestion, on sperm quality in a randomised placebo-controlled trial [ 354 ]. Findings suggested that a dose of 14 mg/d lactolycopene over the course of 12 weeks improved the sperm motility and morphology in healthy individuals [ 354 ]. Another study by Yamamoto et al. reported similar findings regarding lycopene in a study involving three groups of male infertile patients [ 356 ]. On a daily basis, the first group was given 190 g of tomato juice (containing 30 mg lycopene, 38 mg vitamin C, and 3 mg vitamin E), the second group received antioxidant capsules (containing vitamin C 600 mg, vitamin E 200 mg, and glutathione 300 mg), and the third group was given the placebo [ 356 ]. The consumption of tomato juice over the course of 12 weeks significantly increased the plasma lycopene level and sperm motility compared to the control group [ 356 ]. The group that received the antioxidant capsule, however, showed no significant improvement in semen parameters, suggesting that the increase in plasma lycopene seen in the tomato juice group improved male fertility [ 356 ]. Research on the polyphenols, flavonoids, and vitamins of tomatoes, including vitamin E, quercetin, and naringenin, indicates that these compounds may also play important roles in the enhancement of semen quality, including sperm concentration, motility, vitality, and structural integrity [ 139 , 341 , 342 , 345 , 349 , 351 , 357 ]. Although other findings are conflicting, according to Aitken et al., at high doses quercetin can have adverse effects on spermatozoa [ 341 , 358 ]. Sabetian et al. provided evidence that oral synthetic vitamin E (400 IU/day) for eight weeks could improve semen parameters and pregnancy rates by neutralising free radical activity and protecting cellular membranes of sperm, which are particularly vulnerable to oxidative damage [ 139 , 346 , 350 ]. Similarly, in vitro studies in rats and boars have reported the protective effects of quercetin and naringenin on semen [ 359 , 360 ]. Moretti et al. reported that quercetin and naringenin can protect spermatozoa by inhibiting lipid peroxidation in human sperm [ 361 ]. Vitamin C, a constituent of tomatoes, has been reported to be present in high concentrations in seminal plasma, and it is established that increasing the concentration of vitamin C in seminal plasma protects against DNA damage [ 345 , 346 ]. Greco et al. conducted a trial involving infertile men treated with both vitamin E and vitamin C [ 362 ]. After 8 weeks, the levels of DNA damage were significantly reduced in the treatment group ( p < 0.001). However, vitamin E and C intake did not seem to have a significant effect on major semen parameters [ 362 ]. ROS can be detrimental for fertility both in women and men. Tomato constituents as well as the consumption of tomato products have been suggested to play an important role in fertility. However, the fertility related role of tomato products has only been studied in men, and human intervention with tomato products was shown to increase lycopene levels in the seminal fluids of men and improve sperm motility but failed to improve the antioxidant activity. There are some studies that show an increase in antioxidant activity of seminal plasma with vitamin C and naringenin, which are known to be constituents of tomatoes; however, the current literature also suggests that the individual bioactive compounds of tomato may not have the same mechanisms of action in vivo as their food counterparts [ 129 ]. This is likely due to the synergistic action of nutrients when consumed in food rather than individual constituents. Overall, the role of tomato products in fertility requires further investigation to confirm the dose and length of time that is likely to be beneficial for infertility issues both in men and women. Table 3 provides a summary of the main findings of studies that have indicated a beneficial role of tomatoes and their constituents on age-related chronic diseases as well as fertility- and exercise-induced physiological stress. 4.1. Tomatoes and Cancer Pathology Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020 [ 142 ]. Schwingshackl et al. discussed the effects of a tomato-rich Mediterranean diet on the risk of overall cancer mortality [ 143 ]. This paper observed that, in a clinical trial, a Mediterranean diet was found to reduce cancer incidence by 61% and also stated that a "healthy diet" can prevent approximately 30% of cancers [ 143 ]. A review by Farinetti et al. studied the benefits of the Mediterranean diet on colorectal cancer, with lycopene in particular as an important component of this diet, including polyphenols from olive oil and red wine resveratrol, which act to inhibit molecular cancer pathways in vitro [ 119 ]. The health benefits from tomatoes are enhanced as part of the Mediterranean diet as lycopene is more readily absorbed in the intestines when it has been dissolved in olive oil and heated [ 119 ]. Lycopene and β-carotene are two important carotenoids found in tomatoes and both have been suggested to confer the anticancer properties of the fruit. Lycopene, a red pigment found in tomatoes and tomato products, has antioxidant and free radical scavenging activity, and is known to be the most effective singlet oxygen quencher among the natural carotenoids [ 5 , 95 , 144 ]. The human body absorbs a significant proportion (23–24%) of ingested lycopene that proceeds to circulate and accumulate in blood plasma, liver, and other tissues with a half-life of 12–33 days [ 145 ]. Among the various plausible beneficial effects of lycopene, its anticancer properties have been studied the most. These suggestions initially stemmed from epidemiological [ 146 , 147 , 148 , 149 ] studies and were later supported by several experimental studies [ 77 , 78 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 ]. Various anticancer mechanisms of lycopene include the modulation of gene functions and apoptosis, increasing gap junction communications, anti-angiogenic effects [ 146 , 150 , 159 ], and antioxidant, anti-inflammatory, and anti-lipid peroxidation activities [ 160 , 161 , 162 , 163 ]. Due to their antioxidant properties, lycopene and other carotenoids are suggested to protect against carcinogenesis by preventing oxidative damage in DNA and proteins through antioxidant mechanisms [ 164 ]. It has been observed that the cleavage of lycopene via in vitro oxidation at random conjugated double bonds in the molecule forms monocarbonyl compounds [ 151 , 165 ]. Zhang et al. [ 151 ] showed that the products of lycopene oxidation can induce apoptosis in cancer cells. This was further investigated by Arathi et al. [ 152 ] who extracted and autoxidised lycopene from ripened tomatoes and used the products in in vitro cell culture assays to assess the toxicity and apoptosis-inducing ability in various cancer cells. This study found that there were several unknown metabolites or oxidation products of lycopene that may be involved in the inhibition of cancer cell proliferation through modulating cell cycle progression [ 152 ]. This study also demonstrated that chemically induced lycopene oxidation products were a key component in the induction of apoptosis in cancer cells [ 152 ]. A review published in 2020 by Przybylska discussed the anticancer properties of lycopene, particularly in prostate cancer [ 153 ]. This paper evaluated lycopene's effects on prostate cancer, discussed in later sections of this review, as well as breast cancer, the second most prevalent cancer in the world [ 153 ]. Przybylska states that lycopene consumption can reduce the blood concentration of insulin-like growth factor 1 (IGF-1) via the stimulation of synthesis of a protein that binds IGF-1 [ 153 ]. It has been shown that IGF-1 is an important factor in the development of breast cancer in pre-menopausal women, and therefore lycopene's reduction in this growth factor may reduce the risk of this cancer [ 154 ]. This paper further discusses how lycopene inhibits the proliferation of oestrogen-dependent/-independent cancer cells through multiple mechanisms, including inhibiting the activation of genes responsible for the cell cycle or protein-1-responsive genes [ 153 , 166 ]. Another 2020 paper by Saini et al. reviewed the anticancer properties of lycopene [ 78 ] and concluded that the antioxidant abilities of lycopene via a reduction in ROS in cells play a key role in the anticancer properties of this carotenoid. The phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway has been of interest in cancer biology for decades [ 155 ]. Mutations or aberrations to this pathway are found in many cancers, and the inhibition of PI3K presents a therapeutic target for a range of tumour types [ 156 ]. AKT is known to promote cell growth and survival and is further upregulated in breast, prostate, and other forms of cancer [ 156 ]. AKT plays a part in tumour-induced angiogenesis as AKT is activated downstream of vascular endothelial growth factor (VEGF), promoting cell growth and angiogenesis, which is critical for the survival of tumour cells [ 155 , 159 ]. A study by Tang et al. [ 157 ] investigated the inhibitory effects of lycopene on the AKT signalling pathway in HT-29 human colon cancer cells [ 157 ]. It was observed that the proliferation of HT-29 colon cancer cells was inhibited by lycopene in a dose-dependent manner. This study concluded that lycopene treatments may inhibit the PI3K–AKT pathway and further demonstrated the involvement of this pathway in tumour development [ 157 ]. Downstream signalling through the PI3K–AKT pathway increases the expression of transcription factor hypoxia-inducible factor-1 (HIF-1) which upregulates the expression of VEGF [ 155 ]. Therefore, it can be speculated that the suppression of this pathway could prevent tumour development [ 167 , 168 ]. VEGF is also the fundamental regulator in cellular signalling of angiogenesis, which supplies tumour cells with blood supply [ 169 ]. Two studies using human umbilical vein endothelial cells (HUVEC) demonstrated anti-angiogenic effects of lycopene, and one of these studies showed that lycopene also inhibited angiogenesis in freshly dissected rat aorta cells at physiologically relevant concentrations of 1–2 μmol/L [ 159 ]. In another study, lycopene was shown to inhibit angiogenesis both in vitro and in vivo by inhibiting the MMP-2/uPA system through VEGFR2-mediated PI3K–AKT and ERK/p38 signalling pathways [ 168 ]. A prospective study highlighted that angiogenic potential, a biomarker of lethal cancer, was lower in individuals who had been consuming tomato products for a longer period of time [ 169 ]. The PI3K–AKT pathway also activates oncogenic signalling pathways via the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Wnt/β-catenin [ 169 ]. NF-κB influences cell growth, proliferation, and metabolism [ 170 ] and is known to play a key role in the development of cancers [ 171 ]. NF-κB dimers are pro-survival transcription factors and are usually cytoplasmic due to interactions with the inhibitors of kappa B (IkBs); they therefore remain transcriptionally inactive [ 171 , 172 ]. NF-κB activation may result from different signalling pathways triggered by a variety of cytokines, or growth factors, and involves the phosphorylation and proteasome-dependent degradation of IkBs [ 171 , 173 ]. NF-κB activation leads to nuclear translocation followed by the transcription of target genes involved in the oncogenic pathway [ 171 ]. NF-κB is known to be active in several tumour cell types, including leukaemia, breast, and prostate [ 174 ]. A study by Assar et al. [ 77 ] studied the effects that dietary lycopene would have on several points along this oncogenic pathway. This experiment examined the effects in two human cancer cell lines, prostate (PC3) and breast (MDA-MB-231), in the absence and presence of lycopene at concentrations of 0.5–5 µM [ 77 ]. This study not only conducted MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-4-sulfophenyl)-2H-tetrazolium) cell growth assay and Western blots but also NF-κB-responsive gene activation reporter assays to monitor the pathway's activity in real-time [ 77 ]. This study concluded that lycopene inhibits the NF-κB pathway at different stages for both breast and prostate cancer cells in vitro [ 78 ]. NF-κB and the Wnt/β-catenin signalling pathways cross-regulate each other's activities and functions. The Wnt/β-catenin signalling pathway is involved in cell proliferation and can lead to cancer development [ 172 ]. This pathway is upregulated by inflammation and oxidative stress, which can lead to a variety of cancers [ 150 ]. Therefore, it can be suggested that a reduction in ROS caused by lycopene or other antioxidants found in tomatoes leads to the inhibition of Wnt/β-catenin signalling. The Wnt/β-catenin signalling pathway is associated with colorectal cancer [ 169 ]. A 2019 study by Kim et al. [ 150 ] explored the mechanism by which lycopene can influence cancer cell growth through the induction of apoptosis in human gastric cancer AGS cells. Various apoptotic indices such as cell viability, DNA fragmentation, and ROS concentrations were examined in the gastric cancer cells [ 150 ], and the authors concluded that lycopene at 0.3% final concentration led to the induction of apoptosis by inhibiting Wnt/β-catenin signalling, stopping the nuclear translocation of β-catenin and suppressing the expression of specific cell survival genes. Furthermore, a study by Preet et al. [ 158 ] tested the effect of lycopene on human breast cancer cell lines by measuring protein compounds associated with the Wnt/β-catenin signalling pathway and cancer cell viability. Preet et al. [ 158 ] showed that lycopene treatment in combination with quinacrine (a derivative of the naturally occurring compound quinine) inhibited the proliferation of breast cancer cells. It was concluded that the reduced proliferation of the breast cancer cells was a result of the inhibition of the Wnt/β-catenin signalling pathway [ 158 ]. Lycopene is the key antioxidant found in tomatoes and is the focus of many cancer studies. However, tomatoes also contain β-carotene. β-Carotene is a provitamin and is converted into retinol—a compound needed for vision [ 9 ]; it has been the focus of many studies that conclude that it is associated with anticancer activities, including inducing cancer cell apoptosis and reducing cancer cell proliferation [ 174 , 175 ]. Tomatoes also contain a diverse array of other potentially chemo-preventive compounds that are not the primary focus of current research, including vitamins and phenolic constituents [ 176 ]. For example, vitamin C is thought to reduce the risk of stomach carcinogenesis by controlling levels of ROS that can lead to DNA damage, or by stopping the development of carcinogenic nitrosamines introduced as part of the diet [ 133 , 177 ]. The effectiveness of vitamin C as an anticancer agent was debated until a 2011 study investigated the impacts of vitamin C on the human body, which concluded that, when ingested, vitamin C blood concentrations are highly controlled by renal reabsorption [ 178 ]. It was concluded that at a pharmacological dose administered intravenously, the blood plasma levels of this nutrient can be raised to 25–30 mmol/L, a concentration that has been shown to be cytotoxic to cancer cells [ 178 ]. Ferulic acid, a phenolic acid found in tomatoes, is an effective antioxidant and is suggested to have anticancer properties [ 112 , 113 ]. One study investigated the effects of 24 h treatment of Caco-2 colon cancer cells with 150 µmol/L ferulic acid and found that 517 genes were significantly affected [ 114 ]. The treatment delayed cell cycle progression in the S phase via the upregulation of genes involved in centrosome assembly and the S phase checkpoint [ 114 ]. Tomato peel and seeds are composed of 60% dietary fibre [ 179 ]. When fibre is metabolised by intestinal microbiota to form short-chain fatty acids such as butyric and acetic acids, cancerous colonocytes cannot use these components as a source of energy and they accumulate, inhibiting the action of histone deacetylases in these cells [ 180 , 181 ]. As a result, the epigenetic regulation of gene expression in these cells is changed, reducing cell proliferation and increasing apoptosis [ 181 ]. It can be concluded that a tomato-rich diet could increase human blood lycopene levels, and this has many potential anticancer properties ( Table 2 ). However, some healthcare professionals argue that lycopene may not be the only cancer lowering constituent of tomatoes, and perhaps it is a biomarker of tomatoes that, due to an array of constituents, confer anticancer properties [ 78 , 182 ]. 4.2. Tomato's Specific Influence on Prostate Cancer Prostate cancer is the second most common cancer found in men worldwide [ 183 , 184 ]. A study by Giovannucci et al. [ 185 ] investigated dietary carotenoids and prostate cancer risk. Questionnaires were used to find trends between diet and the risk of prostate cancer, and it was found that the only carotenoid associated with a decreased risk of prostate cancer was lycopene [ 185 ]. Of the four tomato-based items high in lycopene that were listed (tomato sauce, tomatoes, tomato juice, and pizza), all except tomato juice were associated with a significantly lower risk of prostate cancer [ 185 ]. More recently, a 2018 review by Rowles et al. compared the results from 30 different articles discussing tomato consumption and prostate cancer [ 183 ]. This review concluded that there was a significant inverse association between tomato consumption and the risk of prostate cancer [ 183 ]. In tomato-rich diets, lycopene is one of the most abundant carotenoids found to be accumulated in blood and tissues, reaching plasma concentrations of up to 1.8 µmol/L [ 184 , 186 ]. Lycopene has been shown to accumulate in several tissues, including the liver and the prostate [ 76 , 184 ]. A review by Rao and Agarwal in 1999 compared lycopene accumulation in major organs and found that the prostate accumulated 0.8 nmol lycopene/g tissue, the adrenal glands 1.9–21.60 nmol/g, and the testes 4.34–21.36 nmol/g [ 5 ]. In 2002, a study was conducted on 60 men with adenocarcinoma of the prostate (clinical stages T1 or T2) in which their diet was supplemented with lycopene-rich pasta sauces and other meals rich in lycopene for a three-week period [ 187 ]. Blood samples showed increased serum lycopene, from baseline 0.638 μM to 1.258 μM, and increased prostate lycopene, from 0.279 nmol/g tissue prior to the trial to 0.82 ± 0.119 nmol/g after the intervention [ 187 ]. However, this study does conclude that the impact of this uptake of lycopene on prostate cells needs further research [ 187 ]. The level of insulin-like growth factor 1 (IGF-1) in the human body is associated with prostate cancer due to the mitogenic and antiapoptotic effects on prostate epithelial cells [ 188 , 189 , 190 ]. Diet is known to influence the level of IGF-1 in the human body [ 189 , 190 ]. Diets primarily containing red meats and dairy products were shown to increase the levels of IGF-1, whereas diets containing high amounts of fruits and vegetables, particularly tomato-containing products, were found to associate with lower levels of IGF-1 [ 189 , 190 , 191 ]. However, studies by Chan [ 192 ] and Graydon [ 193 ] found that lycopene supplementation had no significant effect on the IGF-1 levels of male subjects with and without prostate cancer. In 2019, Applegate, Rowles, and Erdman carried out a systematic review on the impact lycopene has on prostate cancer [ 194 ]. This was focused on androgen activity, which is associated with prostate cancer growth as androgen-regulated, prostate-specific antigen (PSA) is higher in serum samples taken from men diagnosed with prostate cancer [ 194 , 195 , 196 ]. The review suggested that lycopene reduced androgen metabolism and signalling, one of the main factors influencing prostate cancer growth and progression [ 194 ]. Obermüller-Jevic et al. [ 197 ] observed that human prostate epithelial cells treated with 5 μmol/L lycopene showed no expression of cyclin D1. A similar effect on cell growth inhibition was observed in human breast and endometrial cancer cell lines with lycopene. Cyclin D1 is a regulatory subunit of cyclin-dependent kinases CDK4 and CDK 6, which allows cells to transit from the G1 phase of the cell cycle to the S phase and is synthesised in the G1 phase that accumulates in the nucleus [ 197 , 198 , 199 , 200 ]. Wertz et al. [ 198 ] provided a detailed review of the mode of action of lycopene and highlighted that the inhibition of cell growth by lycopene involves the downregulation of cyclin D1, but not of cyclin E, and leads to cell cycle arrest at the G0/G1 phase. In the absence of functioning cyclin D1 in the G1 phase, cell cycle progression is halted, and the cell proliferation rate is reduced [ 199 ]. Gap junctions are intracellular channels formed by connexin proteins, joining cells and allowing the passage of nutrients and intracellular signalling molecules [ 201 ]. In a healthy prostate, basal cells use connexin 43 gap junctions in communication, and luminal cells use connexin 32 gap junctions [ 201 ]. It has been reported that in differentiated prostate cancer there is decreased expression of both channels [ 201 ]. Overall, lycopene treatments have shown an upregulation of connexin 43 expression and enhanced the gap junction channel communication in mouse fibroblast cells and prostate gland cells [ 198 ]. Through the upregulation of connexin 43 and an increased gap junction channel communication, lycopene inhibits carcinogen-induced neoplastic transformation in cell culture [ 198 , 201 ]. This review details the potential anticancer properties associated with the consumption of tomatoes. Most research focuses on lycopene as the primary anticancer agent in tomatoes ( Table 2 ). Lycopene is also the focus of many published reviews that do not discuss other naturally occurring tomato compounds with anticancer associations [ 77 , 152 , 202 ]. This review, however, details not only the anticancer properties of lycopene but also vitamin C, β-carotene, ferulic acid, and the dietary fibre incorporated in tomato tissues. This highlights the importance of how the suggested anticancer properties associated with tomatoes may not derive solely from lycopene but from a combination of anticancer compounds naturally occurring in this fruit. biology-11-00239-t002_Table 2 Table 2 Main findings of the effect of tomato compounds on cancers. Biological Property Studied Type of Study (In Vitro/In Vivo) Main Findings References Antioxidant and anticancer activity In vitro study with human prostate cancer (PC-3) and human breast adenocarcinoma (MCF-7) cell lines. Cell viability assay showed chemically induced lycopene oxidised products (1–50 µM) were a key component in cancer cell apoptosis. [ 152 ] In vitro study with HL-60 human promyelocytic leukaemia cells. Products of lycopene oxidation, identified by spectral analyses, were added to HL-60 cell suspension as a 1% ( v / v ) concentration. This treatment was shown to induce apoptosis in leukaemia cells, shown using flow cytometry to evaluate the ratio of apoptotic cell death. [ 151 ] Anti-angiogenic role in cancer cells In vitro study testing human umbilical vein endothelial cells (HUVEC) and rat aortic rings. Lycopene inhibited angiogenesis in HUVEC and rat aortic rings at physiologically relevant concentrations (1–2 μmol/L) when angiogenesis was analysed using phase-contrast microscopy. [ 159 ] In vitro and in vivo study testing human umbilical vein endothelial cells (HUVEC). Lycopene (0, 1, 5, 10 µM) was shown to inhibit angiogenesis of HUVEC cells in vitro and in vivo by inhibiting MMP-2/uPA system through VEGFR2-mediated PI3K–Akt and ERK/p38 signalling pathways. Cell proliferation assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, cell migration assessed with Millipore QCM™ Endothelial Migration Assay Kit. [ 168 ] Longitudinal cohort study. Lycopene used as a marker of tomato intake and higher intake inversely correlated with total, and the aggressive nature of prostate cancer. The reduced severity of cancer and lesser degree of angiogenesis were reported only in individuals who consumed a tomato-rich diet for a long time period but not in those whose intake recently increased. Tissue microarrays and immunohistochemistry were used to assess tumour biomarker expression. [ 169 ] Modulation of molecular pathways in cancer cells In vitro study with HT-29 human colon cancer cells. Lycopene treatment (0, 2, 5, 10 µM) was shown to inhibit the PI3K–AKT signalling pathway in colon cancer cells, demonstrating its effects on tumour development via angiogenesis inhibition. Assessment of cell proliferation using MTT assay and gene expression investigated using transient transfection and luciferase reporter assays. [ 157 ] Ex vivo and in vivo study testing human umbilical vein endothelial cells (HUVEC) and rat aortic rings. Lycopene (400 μg/mouse) reduced angiogenesis cell signalling through inhibition of the VEGF cell signalling pathway. Anti-angiogenic activity of lycopene confirmed by ex vivo rat aortic ring and in vivo chorioallantoic membrane assays. [ 167 ] In vitro study with human prostate (PC-3) and breast (MDA-MB-231) cancer cell lines. Lycopene (0.5–5 µM) inhibited different stages of the NF-κB cell signalling pathway in both cancer cell lines in vitro as seen in Western blots and NF-κB-responsive gene activation reporter assays. [ 77 ] In vitro study in human gastric cancer (AGS) cells. Lycopene at 0.3% was shown to induce apoptosis by inhibiting Wnt/β-catenin signalling, stopping the nuclear translocation of β-catenin and suppressing the expression of specific cell survival genes AGS cells. Cell viability, DNA fragmentation, and ROS concentrations were examined in these cells. [ 150 ] Cytotoxicity and cancer cell growth In vitro study testing human prostate epithelial cells (PrEC). PrEC treated with lycopene (up to 5 μmol/L) showed no expression of cyclin D1 in vitro. This regulatory subunit of kinases essential to the cancer cell cycle, resulting in reduced cancer cell cycle progression. High-performance liquid chromatography (HPLC) analysis, a thymidine incorporation assay, and flow cytometry were carried out to assess the impact of lycopene. [ 199 ] In vitro study testing human prostate (PC-3) and breast (MDA-MB-231) cancer cell lines. PC-3 and MDA-MB-231 cancer cell lines were tested in vitro in the absence and presence of lycopene at concentrations of 0.5–5 µM. MTS cell growth assays, Western blots, and NF-κB-responsive gene activation reporter assays showed that lycopene inhibits the NF-kB pathway at different stages in both cell lines. [ 77 ] In vitro study treating Caco-2 colon cancer cells. Treatment of Caco-2 colon cancer cells with 150 μmol/L dietary fibre ferulic acid delayed cell cycle progression in the S phase. Gene expression was analysed with cDNA microarray technique. [ 115 ] Cancer cell apoptosis In vitro study testing human prostate cells (PC-3). Flow cytometry analysis showed 27–32% apoptosis in PC-3 when supplemented with (10–50 μM) β-carotene. [ 174 ] Gap junction communication in cancer cells In vitro study with rat liver epithelial WB-F344 cells. Incubation of WB-F344 cells with oxidation products of lycopene (0.2% v / v ) improved the gap junction communication in dye transfer assay using microinjection of the fluorescent dye Lucifer Yellow CH. [ 203 ] 4.3. Cardioprotective Effects of Tomatoes A tomato-rich diet has been linked to a reduction in the risk of heart disease. Song et al. reviewed 14 eligible studies and found a significant inverse association between lycopene intake and coronary heart disease [ 204 ]. Another meta-analysis reviewed 25 studies and reported that high lycopene consumption and lycopene serum concentrations reduced the overall mortality by 37%, cardiovascular disease by 14%, and stroke by 23% [ 205 ]. A randomised, cross-over controlled trial in healthy participants examined the effect of a single dose of raw tomatoes, tomato sauce, or tomato sauce plus refined olive oil on biomarkers of cardiovascular disease [ 206 ]. The results showed all three interventions reduced plasma cholesterol and triglycerides and raised plasma high-density lipoprotein (HDL) cholesterol and interleukin-10 concentrations. Tomato sauce plus olive oil produced the maximum effect, likely due to the increased bioavailability of lycopene as oil is known to improve this. The authors indicated that including tomatoes as a regular part of a diet may help to prevent postprandial lipemia by reducing blood triglyceride levels, and in doing so, reduce the risk of developing atherosclerosis [ 206 ]. An increase in triglyceride levels can lead to the production of small, dense low-density lipoprotein (LDL), which is highly atherogenic [ 207 , 208 ]. It is worth noting that the fat-soluble pigment lycopene is released from tomato cell wall protein–carotenoid complexes during food preparation, therefore the bioavailability of lycopene is higher with cooked tomatoes and tomato products such as juices and sauces than fresh tomatoes, and daily consumption of such tomato products significantly reduces blood LDL cholesterol levels in adults [ 209 ]. In a recent cross-over study, feeding of tomato sauce from vine-ripened tomatoes at 150 mL/day for 6 weeks was compared with sterol-enriched yoghurt and both interventions reduced LDL cholesterol by 12% and 15%, respectively [ 210 ]. Heart disease is a collective term that includes hypertension and atherosclerosis. Hypertension is one of the most common chronic diseases worldwide, with accompanying risks including cardiovascular disease (CVD) and kidney disease [ 6 ]. In a study conducted by Engelhard et al. [ 211 ], patients with grade-1 hypertension were found to have significantly lower systolic and diastolic blood pressure after short-term treatment with 250 mg tomato extract Lyc-O-Mato. In a double-blind placebo study of grade-1 hypertension patients, both systolic and diastolic blood pressure were significantly lower after treatment with tomato extracts [ 211 ]. 𝛾-Aminobutyric acid (GABA), a neurotransmitter present in the sympathetic nervous system, is known to lower systolic blood pressure [ 212 , 213 ], and tomatoes have been shown to contain high levels of GABA [ 214 ]. GABA has been reported to lower the blood pressure of hypertensive patients but not of normotensive individuals [ 168 ]. Daily supplementation of 80 mg of GABA has been found to reduce blood pressure in adults with mild hypertension [ 215 ]. A study carried out in 2008 analysed tomato varieties and found that they had an average GABA content of 50.3 mg/100 g fresh weight [ 216 ]. Many of the antioxidants found in tomatoes, including lycopene, beta-carotene, and vitamin C, protect vascular cells and lipoproteins from oxidation and thus prevent the formation of atherosclerosis [ 134 , 217 ]. Low-density lipoprotein (LDL) oxidation is a well-known factor in genesis [ 218 ] and the progression of atherosclerosis, a process that leads to the narrowing of arteries due to a build-up of cholesterol in subendothelial space. Oxidised LDL is believed to be important in the formation of atherosclerosis and, therefore, vascular diseases. Oxidised LDL increases the expression of pro-inflammatory cytokines, which promote the adhesion of white blood cells to the blood vessel wall [ 219 ]. This can lead to the transmigration of the adhered cells into the innermost layer of the vessel where they are transformed into macrophages, which rapidly accumulate oxidised LDL [ 219 ]. These cells are often the origin of atherosclerotic lesions, which form in artery walls and potentially lead to coronary heart disease and heart attacks [ 134 , 219 ]. Chopra et al. found that increased intake of fruits and vegetables, especially red coloured ones, improves the ex vivo resistance of LDL to oxidation [ 220 ]. In another human study, a 3-week low-tomato diet followed by a 3-week high-tomato diet (400 mL tomato juice and 30 mg tomato ketchup daily) led to a reduction in LDL cholesterol levels and increased ex vivo resistance of LDL to oxidation in normocholesterolaemic participants [ 209 ]. Interestingly, a study conducted in 2000 showed that the regular intake of tomato juice is associated with an increase in blood vitamin E levels [ 134 ]. Lycopene and beta-carotene are known to more effectively inhibit LDL oxidation in the presence of vitamin E [ 134 , 209 , 219 ]. Blood platelets respond to vascular damage by binding to the subendothelial matrix, eventually leading to atherosclerotic lesions, thrombus formation, and vascular events. Platelets are therefore considered as the driving force to myocardial infarction and ischaemic stroke [ 221 , 222 , 223 ]. Tomatoes have been shown to have platelet anti-aggregatory properties. In a double-blind, randomised trial, the dietary supplementation of adults 40–70 years old, these being healthy individuals, with tomato extract was shown to reduce ex vivo platelet aggregation induced by both ADP and collagen [ 224 ]. Although initially carotenoids lycopene and beta-carotene were suggested to contribute to the anti-aggregatory properties of tomatoes, later studies suggested that the anti-platelet factor of tomatoes was due to water-soluble, heat-stable compounds that are concentrated in the jelly substance surrounding the seeds [ 225 , 226 ]. It has been suggested that a diet containing anti-platelet compounds such as these have the potential of reducing lipid levels and lowering blood pressure and can reduce the risk of ischaemic heart disease and strokes by up to 80% in middle-aged individuals [ 227 , 228 ]. Many studies have shown tomato extracts to have platelet anti-aggregatory activity in vitro and in vivo and possibly preventing thrombus formation [ 222 , 224 , 225 , 226 , 229 , 230 , 231 , 232 ]. A study by Zhang et al. [ 233 ] investigated the impact of water-soluble tomato concentrate (WSTC) on the platelet aggregation in Sprague Dawley rats. This study found that WSTC inhibited adenosine diphosphate (ADP)-induced platelet aggregation in vitro and ex vivo in the rats without affecting their coagulation system [ 233 ]. Platelet aggregation relies on fibrinogen binding to the calcium-dependent glycoprotein (GP) IIb/IIIa complexes found on platelets [ 234 ]. When platelets are activated by ADP, these GP IIb/IIIa complexes bind with fibrinogen, leading to many platelets assembling and connecting to the same fibrinogen strands and forming a clot [ 235 ]. Zhang et al. [ 233 ] found that WSTC increased cytoskeleton stability and led to the inhibition of platelet aggregation. There are suggestions that people should adjust their diet to reduce cardiovascular risk and prevent any potential side effects, such as headaches or dizziness, nausea, and increased bleeding, including nose bleeds, associated with anti-platelet medications [ 236 ]. Inflammation plays an important role in atherosclerosis, a process associated with lipid accumulation in the artery wall [ 237 ]. Postprandial lipemia, characterised by an increase in triglyceride-rich lipoproteins, is a condition known to trigger inflammation and atherogenesis in humans [ 238 ]. The transcription factor NF-κB, previously mentioned in relation to cancer development, is also a modulator of inflammation in the liver [ 169 ]. The liver is involved in the uptake, formation, and exportation of lipoprotein and is thus an essential component of lipid metabolism [ 239 ]. Therefore, inflammation and liver damage can have a detrimental impact on lipid metabolism in mammals. Sahin et al. [ 169 ] supplemented a rat diet with tomato powder and found that this resulted in reduced liver damage caused by age-associated inflammation and oxidative stress through the inhibition of the NF-κB pathway. The activation of NF-κB in cultures of endothelial and smooth muscle cells with inflammatory stimuli is also suggested to have a role in atherosclerosis formation [ 240 , 241 ]. NF-κB activation has also been observed in previous studies [ 241 , 242 ] and genes expressed in atherosclerotic plaques are regulated by NF-κB [ 242 ]. There is strong evidence that a reduction in inflammation can reduce the risk of atherosclerosis caused by NF-κB activation but no research to date has investigated the impact of tomato extract on atherosclerosis caused by NF-κB activation. It is worth noting that tomato powder supplementation of rats has been shown to inhibit the NF-κB pathway in the liver of animals [ 169 ]. Vascular endothelium plays an important role in the constriction and dilation of blood vessels, and its dysfunction is suggested as an early, reversible precursor of atherosclerosis. In a randomised controlled trial in post-menopausal women, the effect of 70 g tomato puree ingestion was examined on endothelial-dependent, flow-mediated dilation (FMD) and endothelial-independent, nitro-mediated dilation of the brachial artery using high-resolution ultrasound. The effects after 24 h and 7-day intake were examined [ 243 ]. Although a significant increase in plasma lycopene was observed after 7 days, it did not affect endothelium-dependent or -independent dilation of the brachial artery. Similar findings were reported by another group, where 80 g of tomato paste puree per day for 7 days failed to affect the flow-mediated dilation of the brachial artery after a standardised fat meal, however, the incorporation of tomato paste produced a significant improvement in the haemodynamic changes, such as reduction in diastolic blood pressure, increase in brachial artery diameter, and decrease in stiffness index [ 244 ]. Overall, the potential benefits of a tomato-enriched diet are associated with lowering of blood pressure; anti-platelet, anti-inflammatory, and anti-apoptotic activity; and lipid lowering, as well as the inhibition of LDL oxidation; the latter is believed to be key player in the pathophysiology of atherosclerosis, a hallmark of cardiovascular disease. It is worth noting that the role of a tomato-rich diet on cardiovascular disease was further evaluated in a recent meta-analysis by Rosato et al. [ 245 ]. This paper described the positive impact of the Mediterranean diet on cardiovascular disease in particular foods such as olive oil, fresh fruits and vegetables, nuts, legumes, and fish, but not red meats [ 245 ]. It is stressed that it is the combination of elements in the Mediterranean diet, including the phytochemicals found in fresh fruits and vegetables such as tomatoes, that results in these positive impacts, and these elements on their own would not have the same impact [ 143 ]. Although a tomato-enriched diet has been shown to influence several key mechanisms that are important in vascular pathology, and population-based studies show an inverse correlation between their intake and cardiovascular disease, it is unlikely to affect a multifactorial disease such as CVD when used in isolation. However, if incorporated as a component of a healthy diet, it should provide an additive effect when combined with other cardioprotective nutrients in food. Recent studies have shown that the beneficial effects of tomato compounds are not limited to cardiovascular diseases and cancer but have also been reported in neurological disorders and diabetes mellitus (DM) [ 74 , 78 , 109 , 246 , 247 ]. 4.4. Neurodegenerative Disorders Neurodegenerative diseases are associated with the degeneration of the nervous system over a long period of time and, among these, Alzheimer's, Parkinson's, and cerebral ischaemia associated with stroke are the most common neurodegenerative diseases. Neuroinflammation, oxidative stress, and apoptosis are important hallmarks of these diseases. The majority of cases of stroke are due to cerebral ischaemia, and population-based studies have shown a negative association between tomato, especially lycopene intake, and incidence of stroke [ 205 , 248 , 249 ]. In rats, a tomato pomace powder pre-treatment at doses of 2, 10, and 50 mg/kg protected various areas of the brain, including hippocampus, striatum, and cerebral cortex affected by experimental cerebral ischaemia induced by permanent occlusion of the middle cerebral artery [ 250 ]. The tomato pomace powder pre-treatment of animals also increased the activities of antioxidant enzymes glutathione peroxidase and superoxide dismutase and decreased the lipid peroxidation product malondialdehyde in the hippocampus and cerebral cortex area of the brain. In Alzheimer's disease (AD), neurofibrillary tangles (aggregates of tau protein), a reduction in neurotropic factors, and amyloid-β plaque are present. Amyloid-β accumulation can induce apoptosis both via extrinsic death receptor-mediated and intrinsic mitochondria-mediated pathways, and lycopene has been shown to inhibit these pathways. In human neuroblastoma SH-SY5Y cells, the pre-treatment of cells with 0.2 and 0.5 μmol/L lycopene for one hour, followed by 24 h stimulation with amyloid-β (20 μM), significantly inhibited apoptosis through its dose-related effects on Bax/Bcl-2 and cleavage of pro-caspase-3 [ 251 ]. In addition, lycopene pre-treatment reduced amyloid-β induced oxidative stress, mitochondrial dysfunction, and NF-κB activation in the cells. Lycopene has also been shown to improve the production of brain-derived neurotrophic factor (BDNF) during neurotoxic challenges [ 252 ]. Lycopene supplementation of male Wistar rats at a dose of 5 mL/kg body weight for 21 days reduced neuro-inflammation, oxidative damage to mitochondria, and apoptosis, and improved memory retention and restoration of BDNF level in β-A1-42 treated rats [ 252 ]. Yu et al. also provided evidence that dietary lycopene supplementation could improve cognitive performance in tau transgenic mice expressing P301L mutation [ 253 ]. Likewise, Zhao et al. demonstrated that lycopene supplementation could reduce oxidative stress and neuroinflammation and improve cognitive impairment in aged CD-1 mice [ 254 ]. A recent review provided details of in vitro and animal studies describing the neuroprotective role of lycopene and related this to its antioxidant activity, the inhibition of a redox-sensitive transcription factor NF-kB, a reduced expression of amyloid-β and its precursor, a reduction in neuroinflammation, an improvement in mitochondrial function, memory, and learning as well as a restoration of antioxidant defence [ 77 ]. Vascular dementia is associated with cerebral ischaemia and a recent study examined the effects of supplementation with lycopene at a dose of 50, 100 and 200 mg/kg body weight every other day for two months in a vascular dementia model in rats. At 100 mg/kg dose, lycopene supplementation reduced the oxidative stress, improved the antioxidant enzyme superoxide dismutase and glutathione peroxidase levels in the hippocampus and improved the learning-memory ability of animals [ 255 ]. In human studies, lower antioxidant status, especially vitamin C, lycopene, vitamin E, and antioxidant enzymes superoxide dismutase and glutathione peroxidase, and high levels of markers of oxidative stress have been reported in the plasma [ 256 , 257 , 258 , 259 ] as well as cerebrospinal fluid [ 259 , 260 , 261 ] of AD patients. In the Nurses Health study, participants aged ≥70 years were followed up for 4 years, and a decline in cognitive function was reported to be slower with high lycopene intake but not with vitamin C, β-carotene, and vitamin E intakes [ 262 ]. A recent study that followed up elderly patients over 5–6 years reported that plasma antioxidants such as vitamin E isomers (alpha- and gamma-tocopherol), retinol, and carotenoids were not significantly associated with a reduced risk of dementia or AD [ 263 ]. The lack of association could be related to the age group of 70–75 years that was studied. AD is a chronic disease and may have already been present in the cohort that was included in the study; therefore, the study population was not likely to be a suitable age group to examine the associations. The prevalence rate of AD is less than 1% before 65 years of age, and it increases to 10% after 65 years of age. The brain, due to its high oxygen consumption, high polyunsaturated fatty acids, and transition metals ion content, is highly susceptible to oxidative stress, and antioxidants such as vitamin C, carotenoids, and flavonoids present in tomatoes are therefore a likely candidate for the protection offered by this fruit against neurodegenerative disease. Future follow-up studies should be conducted in 60–65-year-old individuals to confirm whether tomato or its constituents, carotenoid, vitamin C, and flavonoid, intake can slow down cognitive decline with aging. Parkinson's, a neurodegenerative disease, is also associated with oxidative stress and neuronal apoptosis, and its pathology is also likely to be influenced by antioxidant and anti-apoptotic dietary components. The motor disability seen in Parkinson's is suggested to be due to the degeneration of dopaminergic neurons leading to a decrease in dopamine (DA) in the striatum. Supplementation with 20% ( w / w ) lyophilised tomato powder for 4 weeks before methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced Parkinson's disease (PD) in mice was reported to prevent a striatal decrease in the DA levels [ 264 ]. In another study in mice, 7-day pre-treatment with lycopene at doses of 5, 10, and 20 mg/kg was examined on MPTP-induced PD, and treatment was found to reduce MPTP-induced oxidative stress, apoptosis, and depletion of dopamine in the striatum [ 265 ]. Likewise, vitamin C feeding of mice at a dose of 15 mg/kg body for 3 days before intraperitoneal injection of MPTP at 20 mg/kg reduced neuroinflammation and dopaminergic neuronal degradation in the striatum and improved the locomotor inability caused by the neurotoxin [ 266 ]. Overall, pathological changes seen in AD, PD, and cerebral ischaemia have been shown to be ameliorated with lycopene and tomato extract in studies that were conducted in vitro as well as in animal studies. Few human studies in AD and PD patients have indicated a protective role; however, studies are limited and not conclusive likely due to the age group that has been studied in observational as well as intervention studies. Further human studies for AD- and PD-related investigations in the age group 60–65 years old are likely to provide a better insight into the protective role that a tomato-enriched diet may offer against these neurodegenerative diseases. 4.5. Diabetes Carotenoids may play a role in reducing the risk of insulin resistance and the development of diabetes, and an inverse association has been reported between plasma β-carotene, lycopene, and glucose intolerance in newly diagnosed patients [ 267 ] and on glycated haemoglobin levels in older type 2 diabetes patients [ 268 , 269 , 270 ]. Type 2 diabetes is described as a multifactorial metabolic syndrome associated with oxidative stress, inflammation, hyperglycaemia, and hyperlipidaemia [ 271 ]. Tomato constituents have antioxidant and anti-inflammatory properties. Studies have examined hypoglycaemic, hypolipidemic, anti-inflammatory, and antioxidant effects of tomatoes, especially lycopene. In experimental diabetic rats, lycopene at a dose of 10 mg/kg/day for 28 days significantly reduced the increase in blood glucose and glycated haemoglobin (HbA1c) levels induced by streptozotocin (STZ) [ 272 ]. In another study, male albino Sprague Dawley rats were fed a high-fat diet for 4 weeks followed by intraperitoneal injection of STZ at 25 mg/kg. The effects of lycopene administration at 10 and 20 mg lycopene per kg body weight/day for 10 days was examined on the fasting blood glucose, lipids, and glycosylated haemoglobin levels, and a reversal to normality of these parameters was seen with lycopene supplementation [ 273 ]. A recent case-control study reported that lycopene intake positively correlated with peripheral antioxidant activity, antioxidant enzymes superoxide dismutase, and glutathione peroxidase levels and negatively correlated with fasting blood glucose and glycated haemoglobin (HbA1c) levels in patients with type 2 diabetes [ 274 ]. A detailed account of evidence from in vitro, animal, and human studies (cross-sectional, prospective, and two randomised controlled trials) suggesting a preventive role of lycopene and tomato-enriched diet against diabetes is provided by Zhu et al. [ 80 ]. Figueriredo et al. [ 275 ] reported that a combination of lycopene with metformin had an additive effect on improvements in postprandial blood glucose levels, dyslipidaemia, and antioxidant status. The investigation was performed in rats, and STZ-induced diabetic rats were treated with lycopene (45 mg/kg) and metformin (250 mg/kg) alone and in combination for 35 days. There is ample evidence from animal studies suggesting a decrease in diabetes-induced hyperglycaemia, dyslipidaemia, and oxidative stress. There is, however, limited evidence from human intervention trials. Shidfar et al. [ 276 ] fed type 2 diabetic patients with 200 g raw tomatoes/day for 8 weeks. No significant effect was observed in blood glucose levels. However, there was a significant improvement in both systolic and diastolic blood pressure as well as improvements in apoprotein A-1 (ApoA-1) levels. Diabetic patients are at increased risk of cardiovascular disease and the ApoA-1 constituent protein of high-density lipoprotein is important for the anti-atherogenic properties of HDL. Bose and Agarwal [ 277 ] reported that the supplementation of diabetic patients with cooked tomatoes improved the antioxidant defence and plasma lipid peroxidation products but failed to affect the lipid profile and HbA1c levels. It is important to note that the majority of studies that show hypoglycaemic effects of lycopene were carried out using pure compounds, and concentrations were much higher than likely to be achieved by the amount of tomato products that were used by Shidfar et al. [ 276 ] and Bose and Agarwal [ 277 ] for human studies. Zidani et al. [ 278 ] reported that 12 weeks' supplementation of mice with 46 and 84 mg of lycopene/kg of food provided by tomato peel extract significantly reduced insulin resistance caused by the high-fat diet. Both type 2 diabetes and gestational diabetes (GD) are associated with insulin resistance. In the case of GD, it is caused by a hormonal change during pregnancy. Tomatoes have a low glycaemic index, and therefore can be considered as a potential fruit of choice for pregnant women. Very few studies have examined the association or effect of tomato-rich diets on gestational diabetes. A cross-sectional study that used food frequency questionnaires to estimate the food/nutrient intake of its participants highlighted that high lycopene protected against gestational diabetes-associated hyperglycaemia in women and suggested that intake can offer a protective effect against GD [ 279 ]. To date, anti-diabetic effects in animal studies have either tested the effects of lycopene or tomato extract. Tomatoes contain glycoalkaloid esculeoside A, and its concentration is four times higher than that of lycopene. Yang et al. [ 100 ] suggested that esculeoside A can be considered as a functional supplement for diabetes. In their study, wild-type C57BLKS mice were used and esculeoside A (100 mg/kg) administration by gavage for 56 days was found to lead to a reduction in fasting blood glucose levels and improved glucose tolerance in mice [ 100 ]. Both type 2 diabetes and gestational diabetes are on the rise and can be prevented with diet and lifestyle interventions [ 280 , 281 ]. It is well known that synthetic hypoglycaemic medications that are used for type 2 diabetes induce side effects. Further investigations of foods such as tomato products either on their own or in combination with other hypoglycaemic foods are warranted to confirm if the findings of animal studies can be replicated in humans, as the evidence from randomised controlled trials is limited and inconclusive at present. 4.6. Tomato Fruit for Skin Health The properties of tomatoes are not limited to disease prevention. Studies have provided evidence of the beneficial effects of dietary tomato and its supplements for improved skin health [ 282 , 283 , 284 ]. The principle of oral photoprotection provided by antioxidants to prevent the harmful effects from UV radiation has gained popularity over the last decade [ 282 , 283 , 284 ]. The benefits and hazards of solar ultraviolet (UV) radiation are well documented and include the effects of solar exposure on skin cancer, malignant melanoma, immune suppression, photoaging, photosensitivity, and diseases in the eye [ 85 , 86 , 285 , 286 , 287 ]. Acute UV radiation has been linked to skin burns, oedema, abnormal pigmentation, and photokeratitis, and long-term exposure increases the risk of photoaging and malignant tumours [ 85 , 86 , 284 , 286 , 287 ]. Three types of UV rays are produced by sunlight, UVA, UVB, and UVC [ 86 , 284 , 288 ]. UVA rays have the longest wavelength, followed by UVB, while UVC rays have the shortest wavelength. UVC has the strongest mutagenicity, followed by UVB, while UVA is considered a weak mutagen [ 85 , 284 , 287 ]. However, all UVC rays are absorbed by the Earth's ozone layer, therefore, exposure is unlikely, except through an artificial source such as a laser [ 85 , 287 ]. UVA rays can penetrate the dermis and the subcutaneous tissue area [ 84 , 284 , 287 ]. UVB rays can reach the epidermis and have the capacity to interact with DNA [ 84 , 284 , 289 ]. The underlying mechanism involves the production of ROS by UV radiation, which hinders DNA replication and transcription and results in destructive oxidative stress, the activation of the arachidonic acid pathway, and the mediation of inflammatory responses [ 85 , 287 ]. Studies by Groten et al. and Aust et al. provided evidence that carotenoid-containing supplements could significantly protect against UVB-induced erythema by reducing oxidative stress [ 282 , 288 ]. Baswan et al. reported similar findings regarding carotenoid supplementation against both UVB-induced erythema and UVA-induced pigmentation [ 289 ]. Grether-Beck et al. examined the capacity of carotenoids, including lycopene-rich tomato nutrient complex (TNC) and lutein, to protect against UVA/B and UVA1 radiation at a molecular level [ 283 ]. Analysis of the mRNA expression of key genes involved in solar radiation-induced skin damage, including heme-oxygenase 1 (HO1), matrix metalloproteinase 1 (MMP1), and intercellular adhesion molecule 1 (ICAM1), revealed that UVB/A and UVA1 radiation significantly upregulated steady-state levels of HO-1, ICAM-1, and MMP-1 mRNA in the skin of healthy volunteers who did not receive the supplement. Moreover, TNC and lutein treatment significantly inhibited UVB/A and UVA1 radiation-induced gene expression [ 283 ]. Calniquer et al. assessed the effect of a combination of carotenoids and polyphenols (tomato extract with rosemary extract) on the response of skin cells to UV irradiation [ 290 ]. The results demonstrated that carotenoids and polyphenols worked in synergy and that combining these compounds was more effective in balancing UV-induced skin cell damage than using them separately [ 290 ]. Vitamin C is another compound found in tomatoes that contributes to immune modulation [ 24 ]. When applied topically, it is known to be actively taken up by epidermal and dermal skin cells using sodium-dependent vitamin C transporter isoforms (SVCT1 and SVCT2) [ 137 ]. These cells are involved in the production of collagen fibres and therefore are essential to the function of the skin as a barrier against pathogens [ 136 ]. The use of natural ingredients such as tomato extract or tomato seed oil in cosmetic products for skin care and health has also received popularity over the last few years [ 85 , 86 , 87 , 121 , 284 , 287 , 291 , 292 , 293 , 294 , 295 , 296 , 297 ]. Tomato seed oil has been extensively used in the production of cosmetic and personal care products such as anti-aging serums, body butter, sunscreens, and skin lightening cream due to its high linoleic acid, lecithin, antioxidant, and natural UV protection attributes [ 284 ]. Furthermore, there is increasing evidence that a diet rich in antioxidants may significantly influence the course of certain skin diseases such as atopic dermatitis, acne, and psoriasis [ 285 , 291 , 298 ]. Multiple in vitro studies in mice have revealed that polyphenols including quercetin and gallic acid present in tomatoes may be an alternative for the development of cosmetics that could be used to treat acne vulgaris [ 108 , 299 , 300 , 301 ]. Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease that can affect up to 25% of children within a diverse paediatric population [ 302 ]. Symptoms can include itching and scratching, dry skin, patchy eczema, exudation, and skin thickening and discolouration [ 302 ]. Although the mechanisms of the pathogenesis of AD have not been fully elucidated, the chronically inflamed skin of patients with AD plays a key role in pathogenesis, with the overproduction of ROS and a decrease in antioxidant defence [ 302 ]. Sapuntsova et al. have reported that levels of ROS were significantly higher in the skin biopsies in AD patients compared to those of controls [ 303 ]. Lycoperoside H, an anti-inflammatory component present in the seed part of tomatoes, was shown by Takeda et al. to relieve symptoms of AD in transgenic mice expressing IL-33 driven by a keratin-14 promoter (IL33tg) [ 62 ]. Other reported benefits of tomato compounds for skin health include the protection against tick bites and heavy metal toxicity [ 64 , 121 , 292 , 296 , 299 ]. Boulanger et al. showed that natural skin repellents made from eucalyptus, tomato, and coconut can protect against tick-borne infections such as Lyme disease [ 63 ]. A study by Tito et al. demonstrated that an active ingredient derived from Lycopersicon esculentum tomato cultured stem cells protected skin cells against heavy metal toxicity [ 292 ]. The mechanism of action involves the preservation of nuclear DNA integrity from heavy metal damage by inducing the genes responsible for DNA repair and protection and also involves the neutralisation of the effect of heavy metals on collagen degradation by inhibiting collagenase expression and inducing the synthesis of new collagen [ 292 ]. Additionally, in a study involving Indian women, Nutrova, a blend of collagen peptides and natural antioxidants from tomatoes, green tea, and grapes, has been shown to significantly reduce wrinkles, skin roughness, and hyperpigmentation while improving skin hydration and firmness [ 296 ]. Similarly, another study of 4000 women showed that a diet reported as high in potassium and vitamins A and C correlated to fewer wrinkles in patients' skin [ 291 ]. It can be concluded that incorporating tomatoes into a diet could have benefits to a person's skin health. The suggested benefits include protection against UV radiation through antioxidant properties [ 284 ], treatment of skin inflammatory conditions such as AD [ 62 ], and protection against tick bites and heavy metal toxicity [ 63 , 292 ]. 4.7. Tomatoes, Gut Microbiome, and Inflammation "Microbiome" is a term used to describe a community of colonising microorganisms such as fungi, viruses, and bacteria found in a particular environment [ 304 ]. Microbial populations reside throughout the human body, including the stomach and intestines, and are increasingly described as a key link between genetic and environmental impacts that affect an individual's health [ 304 , 305 ]. The gut microbiome is defined as all microorganisms found in the gastrointestinal tract consisting of bacteria, archaea, viruses, and eukaryotic microbes, and can have as many as 100 trillion cells [ 305 , 306 ]. These bacteria are known to vary in composition depending on the lifestyle, genetics, and diet of the host [ 305 ]. The composition of the gut microbiome is implicated in the development of liver inflammatory disease and liver cancer [ 307 ]. In a 2018 study by Xia et al. [ 308 ], mice were fed a high-fat diet (HFD) supplemented with a liver-specific carcinogen (DEN) along with a tomato powder rich in lycopene, which has previously been shown to inhibit HFD-induced liver disease [ 309 ]. The tomato powder significantly increased both diversity and richness of the gut microbiota in all mice [ 308 ]. The gut microbiome contains both Gram-positive and Gram-negative bacteria, and it has been shown that an increase in Gram-negative bacteria in the gut can lead to an increase in the level of hepatotoxic compounds, such as lipopolysaccharides (LPS) [ 310 ]. LPS are major components of the outer membrane of Gram-negative bacteria and are known to induce inflammation through induction of Toll-like receptor 4, which can lead to cell proliferation and a reduction in apoptosis [ 129 , 310 ]. In 2018, Xia et al. reported that feeding mice tomato powder reduced the relative abundance of the gut Gram-negative bacteria by reducing levels of gut LPS in the mice, lowering the risk of inflammation [ 308 ]. Increasing the diversity and richness of the gut microbiome through tomato powder supplementation was found by the authors to regulate inflammation, lipid metabolism, and the circadian clock in the liver [ 308 ]. Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions of the gastrointestinal tract and include Crohn's disease and ulcerative colitis [ 308 ]. Both of these conditions are affected by a number of factors, including abnormal gut microbiota [ 311 ]. A study by Scarano et al. (2018) [ 129 ] developed a bronze tomato line that was shown to have 30–50% higher levels of flavanols and anthocyanins when compared to other tomato lines. These tomatoes were freeze-dried, ground into powder, and incorporated into the diets of mice with induced chronic colitis [ 129 ]. The study found that a diet enriched with 1% bronze tomato fruit powder promoted a change in microbiota composition, moderately inhibiting inflammatory responses in the mice and thus reducing intestinal damage caused by chronic colitis [ 129 ]. This was continued in a follow-up study using the same bronze tomato line, which demonstrated the beneficial effects of this variety of tomato on intestinal inflammation and showed changes in the gut microbiome, especially an increase in Flavobacterium and Lactobacillus and a reduction in Oscillospira [ 130 ]. In summary, the composition of the gut microbiome is affected by the consumption of tomatoes, and this has implications for human health. Tomato powder has been shown to significantly increase the diversity and richness of the gut microbiome in mice, preventing a build-up of Gram-negative bacteria that produce hepatotoxic compounds, which can cause liver inflammatory disease and cancer [ 308 , 310 ]. Furthermore, freeze-dried tomatoes high in flavanols and anthocyanins have been shown to alter the composition of the gut microbiome by increasing Flavobacterium and Lactobacillus and decreasing Oscillospira populations, resulting in reduced inflammatory responses in mice and preventing intestinal damage by chronic colitis [ 129 , 130 ]. It is worth noting that most evidence shown to date is from animal studies, and it remains to be seen whether tomato product intervention can be beneficial for conditions associated with gut dysbiosis. 4.8. Tomatoes and Exercise Recovery Exercise and increased muscle activity result in higher amounts of ROS due to increased ATP production and oxygen utilisation [ 312 ]. ROS are highly reactive and can damage macromolecules such as proteins, DNA, and lipids. ROS such as peroxides and superoxides can be damaging to cells if concentrations are excessive [ 313 , 314 ]. Regular exercise builds resistance of the body against oxidative stress by upregulating the expression of genes that synthesise antioxidant enzymes such as superoxide dismutase [ 312 ]. Antioxidants such as vitamins, terpenoids, and phenolics can inhibit oxidative stress and neutralise ROS [ 314 , 315 , 316 ]. Antioxidants found naturally in plant tissues, including tomato fruit, can provide the protection against ROS. Previous studies have indicated that lycopene and lycopene metabolites can have a positive effect on recovery from exercise-induced physiological stress [ 144 , 317 ]. Lycopene is known to be the most effective singlet oxygen scavenger, exhibiting quenching rates multiple times greater than any other carotenoid [ 317 , 318 ]. Creatinine phosphokinase (CPK) and lactate dehydrogenase (LDH) are associated with ATP and NADH conversion in muscle cells and can be monitored as markers of muscle damage in individuals undergoing exercise sessions [ 319 ]. A study by Tsitsimpikou et al. [ 319 ] tested whether the administration of 100 g of tomato juice could improve the recovery of anaerobically trained athletes. The study found that a 2-month administration of tomato juice, post exercise, led to a significant decrease in LDH and CPK levels compared to a carbohydrate supplementation beverage [ 319 ]. Another study carried out by Nieman et al. [ 318 ] administered a "tomato complex" containing lycopene, phytoene, and phytofluene (T-LPP) to endurance runners for a 4-week period and monitored inflammation, muscle damage, and oxidative stress post exercise and during recovery from a two-hour running session. Myoglobin is an iron- and oxygen-binding protein found in muscle tissue that is translocated to the blood compartment after low-level muscle injury caused by exercising and can be used as a marker for muscle injury [ 318 ]. Nieman et al. [ 318 ] found a significant increase in plasma carotenoid levels and a reduction in myoglobin, suggesting possible reductions in muscle injury as a result of consuming the "tomato complex" supplement following a 2 h running session. Tomato products and supplementation with their constituents are suggested to reduce muscle damage caused during anaerobic exercise as well as reduce oxidative stress during aerobic exercise, as shown by Harms-Ringdahl et al. [ 320 ]. In their study, 15 healthy and untrained participants engaged in 20 min of aerobic exercise on a bicycle after receiving 150 mL of tomato juice for 5 weeks, followed by 5 weeks without tomato juice, and for the final intervention they received tomato juice for another 5 weeks [ 320 ]. The blood samples were collected before and after each intervention, and results showed that tomato juice intake significantly suppressed 8-oxodG (a marker of oxidative damage) levels produced with the physical activity [ 320 ]. Therefore, there is evidence that tomato products can reduce oxidative stress and muscle damage caused by physical exertion and can be considered as a workout drink [ 320 ]. 4.9. Tomatoes and the Immune System Tomatoes and tomato products are suggested to affect the immune system [ 321 ], and current literature relates this to their lycopene, β-carotene, and vitamin C content. In a human study, supplementation with tomato products (tomato sauce, tomato puree, and raw tomatoes) providing 8 mg lycopene, 0.5 mg β-carotene, and 11 mg vitamin C for 3 weeks was reported to produce a significant increase in plasma levels of lycopene, β-carotene, and vitamin C; however, only lycopene and vitamin C levels increased in the lymphocyte [ 322 ]. Supplementation also reduced ex vivo oxidative damage to the DNA of lymphocytes [ 322 ]. How vitamin C can affect the immune system has been reviewed by Van Gorkam et al. who describe that, although the data on the effects of vitamin C on B lymphocytes are limited and inconclusive, vitamin C increases the proliferation of T-lymphocytes and natural killer (NK) cells [ 323 ]. T-lymphocytes play a key role in cell-mediated, cytotoxic adaptive immunity, and natural killer (NK) cells provide rapid cytolytic responses to virus-infected cells and tumour cells [ 324 , 325 ]. Few studies in human subjects reported that supplementation with β-carotene stimulates the proliferation of lymphocytes [ 326 , 327 ] and enhances the lytic activity of NK cells [ 5 , 327 ]. A study carried out by Watzl et al. [ 321 ] supplemented human subjects with tomato juice and carrot juice—both of which are known for their high β-carotene content. This study found that supplementation with the juices significantly increased lymphocyte proliferation and enhanced the lytic activity of natural killer cells [ 321 ]. No significant differences were observed between the effects of either juice, indicating a similar effect on the immune response, or that other compounds present in both juices resulted in the observed effects [ 321 ]. Naringenin is a flavanone (a subclass of flavonoids) that has also been shown to have immune-modulating functions [ 328 , 329 ]. In an in vitro study, Niu and colleagues demonstrated that naringenin could inhibit T cell activity by various mechanisms, such as lowering the secretion of specific T cell cytokines and affecting T cell proliferation [ 329 ]. Further investigation revealed that that inhibition of cell proliferation was triggered by delayed degradation of the cyclin-dependent kinase inhibitor p27kip1 and the downregulation of retinoblastoma protein phosphorylation in activated T cells, resulting in a T cell cycle arrest at G0/G1 phase [ 329 ]. Other findings indicated that the T cell-suppressive effects could be attributed to the capacity of naringenin to interfere with the interleukin-2/interleukin-2 receptor (IL-2/IL-2R)-mediated signalling pathway and STAT5 phosphorylation in activated T cells [ 329 ]. The immune-modulating effects of lycopene have been hypothesised through their antioxidant activity and their effects on lymphocyte proliferation and on improving cell–cell communication [ 330 , 331 ]. A 2017 study tested 40 mice divided into five groups: an ambient air control; a vehicle control group receiving 200 µL of sunflower oil; a group exposed to cigarette smoke; and two groups administered lycopene diluted in sunflower oil (25 or 50 mg/kg/day) prior to cigarette smoke exposure [ 332 ]. The 5-day testing period resulted in an increase in the number of lymphocytes in lycopene-treated groups compared to other treatments [ 332 ]. This study suggested that the increase in lymphocytes was a result of lycopene activating the adaptive immune response [ 333 ], and the latter is known to be vital in pathogenic defence. However, further studies are warranted to fully understand lycopene's direct impact on the adaptive immune system in humans [ 334 ]. As one of the most popular world crops, the tomato has also been considered as an edible vaccine for a wide variety of diseases, including malaria, coronavirus (COVID-19), human papillomavirus infections, human immunodeficiency virus infections, shigellosis, cholera, anthrax, and hepatitis B [ 97 , 335 , 336 , 337 , 338 , 339 ]. The main objectives of edible vaccines are to democratize preventive vaccination, especially in developing countries, and to better control potential outbreaks such as coronavirus disease. Traditional vaccine development requires more time and high cost, while the development of an edible vaccine in a plant expression system provides an efficient mode of oral delivery and bypasses the assistance of a medical professional to perform injections. It is also economically sustainable, with higher scale production. However, there are several hurdles to overcome, such as the immunogenicity of an oral vaccine, the stability of the vaccine in the gastrointestinal tract, the variability of the expression of antigens in plants, and the effects associated with the consumption of genetically modified plants on health [ 336 , 339 ]. Shchelkunov et al. designed an oral vaccine against hepatitis B and human immunodeficiency viruses using tomato fruits, which was administered to experimental mice [ 337 ]. Examination of serum and stool samples of the test animals revealed high levels of HIV- and HBV-specific antibodies [ 337 ]. Salyaev et al. investigated the duration of the mucosal immune response in mice after administration of this vaccine in a subsequent study [ 340 ]. Results showed a steady increase in the immune response, with a peak observed between 6 and 11 months post-administration followed by a gradual decrease in the levels of antibodies until they became undetectable after 19 months [ 340 ]. Evidence from in vitro, animal, and a few human studies describes a significant increase in lycopene and vitamin C content of lymphocytes, improvements in T cell mediated immunity, and the lytic activity of NK cells, and there is also a suggestion of the use of tomato fruit as an edible vaccine. However, even though plant-based vaccines offer a promising alternative, their clinical development remains challenging, and further research is required in human clinical studies [ 336 , 339 ]. 4.10. Tomatoes and Fertility Infertility is a disease of the male or female reproductive system characterised by the inability to accomplish a pregnancy following at least 12 months of regular unprotected sexual intercourse [ 341 , 342 , 343 ]. According to statistics, 48 million couples and 186 million individuals live with infertility globally [ 341 ], and male factors account for at least 50% of all infertility cases worldwide [ 343 ]. Oxidative stress (OS), which arises from an imbalance between ROS and protective antioxidants, can affect the entire reproductive lifespan of men and women [ 344 ] and has been shown to be a major cause of reproductive dysfunction [ 344 , 345 ]. The positive effects of antioxidants in female fertility have also been described [ 346 , 347 , 348 , 349 ]. OS has also been recognised as one of the main mediators of female infertility and has been associated with various reproductive pathologies, including endometriosis, preeclampsia, spontaneous abortion, and unexplained infertility [ 347 , 348 ]. Studies have shown the presence of ROS in the ovaries, fallopian tubes, and embryos of women with idiopathic fertility [ 348 ]. Additionally, ROS have been shown to play a role in the regulation of ovarian steroid biosynthesis and secretion, primordial follicle recruitment, and ovulation, and they can also affect the fertilisation process and post-fertilisation events, although the underlying molecular mechanisms have not been fully elucidated [ 346 ]. The current literature on folate and fertility endpoints indicates that a high intake of folic acid in the preconception period may increase pregnancy success rates. Upadhyaya et al. showed that folate levels in red-ripe tomato fruits could range from 14 to 46 μg/100 g FW [ 131 ]. Furthermore, a lack of vitamin C seems to be associated with an increased risk of preeclampsia, and some studies have shown that vitamin supplements could lower the risk of preeclampsia in normal or underweight women [ 346 ]. Some studies have demonstrated that oral administration of multivitamins including folic acid and vitamins C, D, and E can increase fertility [ 346 ]. Yu et al. reported that β-carotene has a similar antioxidant potential to folic acid and could also improve the oocyte development and maturation and ovarian function in mice [ 348 ]. Therefore, there is some indirect evidence on the role that a tomato-enriched diet may play in female fertility; however, to date, no studies have specifically examined the effects of a tomato-enriched diet on OS-related effects on female fertility. According to research, between 30 and 80% of male infertility cases are caused by OS and a decreased level of seminal total antioxidant capacity [ 342 , 345 , 350 ]. Evidence shows that the semen from infertile men has a lower antioxidant capacity and high levels of ROS compared to fertile men [ 345 , 346 ]. As a source of antioxidants, tomato's constituents and their supplement counterparts may be important for reducing OS and improving semen parameters, including sperm concentration, motility, morphology, and fertility rate [ 341 , 343 , 351 ]. In a human study, tomato soup consumption at 400 g/day significantly increased seminal plasma levels of lycopene, though the effects on plasma antioxidant levels failed to reach significance [ 352 ]. As potent antioxidants, the role of carotenoids in fertility has been extensively investigated [ 345 , 348 , 353 , 354 , 355 ]. Williams et al. examined the effect of lactolycopene, a combination of lycopene with whey protein, which protects lycopene from digestion, on sperm quality in a randomised placebo-controlled trial [ 354 ]. Findings suggested that a dose of 14 mg/d lactolycopene over the course of 12 weeks improved the sperm motility and morphology in healthy individuals [ 354 ]. Another study by Yamamoto et al. reported similar findings regarding lycopene in a study involving three groups of male infertile patients [ 356 ]. On a daily basis, the first group was given 190 g of tomato juice (containing 30 mg lycopene, 38 mg vitamin C, and 3 mg vitamin E), the second group received antioxidant capsules (containing vitamin C 600 mg, vitamin E 200 mg, and glutathione 300 mg), and the third group was given the placebo [ 356 ]. The consumption of tomato juice over the course of 12 weeks significantly increased the plasma lycopene level and sperm motility compared to the control group [ 356 ]. The group that received the antioxidant capsule, however, showed no significant improvement in semen parameters, suggesting that the increase in plasma lycopene seen in the tomato juice group improved male fertility [ 356 ]. Research on the polyphenols, flavonoids, and vitamins of tomatoes, including vitamin E, quercetin, and naringenin, indicates that these compounds may also play important roles in the enhancement of semen quality, including sperm concentration, motility, vitality, and structural integrity [ 139 , 341 , 342 , 345 , 349 , 351 , 357 ]. Although other findings are conflicting, according to Aitken et al., at high doses quercetin can have adverse effects on spermatozoa [ 341 , 358 ]. Sabetian et al. provided evidence that oral synthetic vitamin E (400 IU/day) for eight weeks could improve semen parameters and pregnancy rates by neutralising free radical activity and protecting cellular membranes of sperm, which are particularly vulnerable to oxidative damage [ 139 , 346 , 350 ]. Similarly, in vitro studies in rats and boars have reported the protective effects of quercetin and naringenin on semen [ 359 , 360 ]. Moretti et al. reported that quercetin and naringenin can protect spermatozoa by inhibiting lipid peroxidation in human sperm [ 361 ]. Vitamin C, a constituent of tomatoes, has been reported to be present in high concentrations in seminal plasma, and it is established that increasing the concentration of vitamin C in seminal plasma protects against DNA damage [ 345 , 346 ]. Greco et al. conducted a trial involving infertile men treated with both vitamin E and vitamin C [ 362 ]. After 8 weeks, the levels of DNA damage were significantly reduced in the treatment group ( p < 0.001). However, vitamin E and C intake did not seem to have a significant effect on major semen parameters [ 362 ]. ROS can be detrimental for fertility both in women and men. Tomato constituents as well as the consumption of tomato products have been suggested to play an important role in fertility. However, the fertility related role of tomato products has only been studied in men, and human intervention with tomato products was shown to increase lycopene levels in the seminal fluids of men and improve sperm motility but failed to improve the antioxidant activity. There are some studies that show an increase in antioxidant activity of seminal plasma with vitamin C and naringenin, which are known to be constituents of tomatoes; however, the current literature also suggests that the individual bioactive compounds of tomato may not have the same mechanisms of action in vivo as their food counterparts [ 129 ]. This is likely due to the synergistic action of nutrients when consumed in food rather than individual constituents. Overall, the role of tomato products in fertility requires further investigation to confirm the dose and length of time that is likely to be beneficial for infertility issues both in men and women. Table 3 provides a summary of the main findings of studies that have indicated a beneficial role of tomatoes and their constituents on age-related chronic diseases as well as fertility- and exercise-induced physiological stress. 5. Detrimental Effects of Tomatoes In contrast to the above-mentioned beneficial effects of a tomato-enriched diet, it is also important to report the potentially detrimental effects that tomato-enriched diets can have on human health. Examples of these detrimental effects include heartburn, irritable bowel syndrome, urinary problems, exposure to pollutants (pesticides, soil herbicides, atmospheric gaseous pollutants, and ethylene gas), migraines, body aches related to glycoalkaloids, anaphylactic reactions, lycopenodermia (an orange or red discolouration of the skin), renal calculi, hepatitis A, and Salmonella sp. infections [ 363 , 364 , 365 , 366 , 367 , 368 , 369 , 370 , 371 , 372 , 373 , 374 ]. A cohort study has revealed that the intake of fruits and vegetables with higher levels of pesticide residue contamination has been associated with poorer semen quality and a lower probability of live birth among couples undergoing fertility treatment [ 370 ]. Another investigation showed that pesticides residues on tomatoes may cause harmful health effects and constitute a threat particularly to children's health [ 369 ]. Concerning heavy metal toxicity, it is acknowledged that some metals have the capacity to translocate into plant shoots and accumulate in given plant organs, including roots, stems, leaves, and fruits. More specifically, tomatoes grown in contaminated soil constitute a significant health risk due to a higher potential heavy metal uptake and therefore a higher toxicity [ 372 ]. From tomato crop in Quito markets (Ecuador), Romero-Estévez and his team highlighted that levels of lead in tomatoes were near or exceeded the threshold value (0.100 mg/kg) from four markets (0.209, 0.162, 0.110, 0.099 mg/kg), suggesting a possible risk of lead toxicity from tomato consumption [ 372 ]. Similar studies also underline the importance of monitoring the content of heavy metals in tomatoes due to their ability to accumulate in the human body and the health risks that they can pose after long-term exposure, even with small doses [ 371 ]. Not only is the presence of heavy metal a health risk but it can also adversely impact the levels of nutrients in tomatoes, including lycopene and ascorbic acid [ 371 , 372 , 373 ]. Another risk involves outbreaks of human Salmonella infections and hepatitis A. Three outbreaks of Salmonella infections associated with eating Roma tomatoes were detected in the United States and Canada in the summer of 2004 [ 363 ]. Between 2005 and 2006, multistate outbreaks of Salmonella infections were associated with tomatoes in restaurants in the United States [ 364 ]. In addition, three other hepatitis A outbreaks were associated with eating semi-dried tomatoes: in Australia in 2009 and in the Netherlands and in France in 2010 [ 365 , 367 , 374 ]. The health effects of carotenoids in tomatoes and associated supplements have been extensively discussed, especially lycopene [ 368 , 375 ]. Several studies have reported conflicting findings for the effect of lycopene supplementation on cardiovascular risk factors and cancer [ 375 , 376 ]. Lycopene supplementation is contra-indicated for patients on blood thinners and blood-pressure-lowering medications due to its anti-platelet effect [ 377 , 378 , 379 ] as it might increase the risks of bruising and bleeding. Recent studies have provided evidence that β-carotene supplements can increase the risk of lung cancer in smokers [ 73 ]. Beta-carotene supplements also increased the risk of other cancers [ 73 ]. These examples support that the artificial supplements of naturally occurring constituents of tomatoes are likely to work in synergy and that their beneficial properties should not be attributed to one compound alone, although further research would be required to establish these facts [ 376 , 380 , 381 , 382 ]. The examples above also provide evidence that most nutrients in fresh fruits and vegetables do not exhibit the same properties as their supplement counterparts that can cause adverse effects [ 73 , 182 , 375 , 380 , 381 , 382 ]. So far, research has not proven antioxidant supplements to be beneficial in preventing diseases [ 375 ]. Consequently, dietary guidelines recommend the regular consumption of fruits and vegetables as part of a balanced diet in order to reap the full benefits of antioxidants in tomatoes. However, one could argue that the bioavailability of beneficial compounds in tomato varies depending on processing and cooking methods, and that one would have to regularly consume tomato in various forms (raw, cooked, boiled, etc.) to access the full range of positive effects [ 73 , 380 , 381 , 382 , 383 , 384 , 385 ]. To illustrate, on one hand, thermal processing can significantly increase the bioavailability of carotenoids and phenolics in tomatoes [ 386 , 387 , 388 ]. In vitro studies have revealed that pulsed electric fields without heat can increase lycopene bioavailability by up to 40%, and when combining it with thermal treatment, by up to 238%, as compared to raw tomato juice [ 387 ]. On the other hand, thermal processing can adversely affect the content of other compounds in tomatoes, including water-soluble vitamins and minerals [ 383 , 384 , 385 ]. Similarly, one could debate whether nutrients from natural unprocessed foods are enough to meet the requirements of human daily intake and confer protective effects against certain diseases, especially when considering the environmental challenges faced by society such as soil erosion and nutrient depletion [ 389 , 390 ], and whether the development of novel genetic engineering and selective breeding techniques could be advantageous [ 126 , 391 , 392 , 393 , 394 ]. 6. Conclusions In conclusion, a tomato-rich diet is associated with a diverse range of health benefits, including anticancer properties, reducing the risk of cardiovascular, neurodegenerative, and bowel diseases, and improving skin health, exercise recovery, and immune response. Several factors, including cultivation, processing, the amount consumed, and bioavailability, are likely to influence the overall biological effects of tomatoes seen in the body. The majority of research to date has focused on the biological properties of lycopene. However, there are a number of other bioactive compounds in tomatoes that confer cardiovascular, anticancer, and skin health properties. The synergistic effects of all tomato constituents are likely to outweigh the benefits of tomato's individual constituents, such as lycopene, and any health benefits of tomatoes should be considered in the wider context of a balanced and healthy diet.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152208/

Alterations in Blood Components

Cellular blood components include leukocytes, erythrocytes, and platelets. Leukocyte subtypes are neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Red blood cell components include mature erythrocytes and reticulocytes. Increases or decreases in cell counts/concentrations for each of these blood components may occur in response to many stimuli or pathologic conditions, including stress, inflammation, infectious agents, neoplasia, and the toxic effects of endogenous, environmental, or pharmaceutical compounds. Changes may be limited to a few blood components or may be observed in all blood components depending on the stimulus. Morphologic alterations in blood components may also be observed in conjunction with alterations in blood cell counts. Specific examples of conditions that cause alterations in blood components are described, as well as the mechanisms by which many of these specific alterations occur. Many xenobiotics are able to cause similar alterations in blood components through the same or comparable mechanisms, and examples of xenobiotic-induced alterations in blood components are provided. Glossary ACD Anemia of chronic disease AIHA/AHA Autoimmune hemolytic anemia AML Acute myeloid leukemia ATP Adenosine triphosphate BFU-E Burst-forming unit-erythroid BFU-Mk Burst-forming unit-megakaryocyte C3a Complement 3a C5a Complement 5a CALR Calreticulin encoding gene CARPA Complement activation-related pseudoallergy CD Cluster of differentiation CFU-E Colony-forming unit-erythroid CFU-GM Colony-forming unit-granulocyte macrophage CFU-Mk Colony-forming unit-megakaryocyte CHCM Corpuscular mean hemoglobin concentration COPD Chronic obstructive pulmonary disease CUBAM Ileal cubilin and amnionless complex receptor CXCL12 C-X-C motif chemokine ligand 12 DAT Direct antiglobulin test DIC Disseminated intravascular coagulation EGLN1 Egl-9 family hypoxia-inducible factor 1 gene EPO Erythropoietin FAD Flavin adenine dinucleotide Fe 2 + Ferrous iron Fe 3 + Ferric iron FeLV Feline leukemia virus FIV Feline immunodeficiency virus G6PD Glucose-6-phosphate dehydrogenase G-CSF Granulocyte colony-stimulating factor GATA2 GATA binding protein 2 GM-CSF Granulocyte-macrophage colony-stimulating factor HIF-1 Hypoxia-inducible factor 1 HIV Human immunodeficiency virus HUS Hemolytic uremic syndrome IL Interleukin IMHA Immune-mediated hemolytic anemia IMT Immune-mediated thrombocytopenia INFγ Interferon gamma ITP immune-mediated thrombocytopenic purpura JAK Janus kinase LNK Lymphocyte-specific adaptor protein M-CSF Macrophage colony-stimulating factor MCHC Mean corpuscular hemoglobin concentration MCV Mean corpuscular volume MPL Thrombopoietin receptor encoding gene MPV Mean platelet volume NK Natural killer nRBC Nucleated red blood cell NSAID Nonsteroidal antiinflammatory drug P 450 Cytochrome P 450 PARR Antigen receptor rearrangement PCR Polymerase chain reaction PF4 Platelet factor 4 PFCP Primary familial and congenital polycythemia PFK Phosphofructokinase PIMA Precursor-targeted immune-mediated anemia PK Pyruvate kinase PRCA Pure red cell aplasia RAG Recombination activating gene RANTES Regulated on activation, normal T-cell expressed and secreted SCF Stem cell factor SCID Severe combined immunodeficiency SIV Simian immunodeficiency virus SLE Systemic lupus erythematosus TGFα Transforming growth factor alpha TGFβ Transforming growth factor beta T H 1 T-helper cell type 1 T H 2 T-helper cell type 2 TNFα Tissue necrosis factor alpha TPO Thrombopoietin TTP Thrombotic thrombocytopenic purpura VHL von Hippel-Lindau tumor suppressor 12.11.1 Introduction An increasing number of xenobiotics are associated with alterations in the hematopoietic system. These alterations encompass a variety of direct and indirect effects, many of which are associated with the intended pharmacology of the compound but may also be related to off-target effects. Alterations in the hematopoietic system are commonly reflected by changes in the peripheral blood components. Blood components include leukocytes, erythrocytes, and platelets. Leukocyte subtypes in humans and common laboratory species include neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts. The erythrocyte component includes both total erythrocyte and reticulocyte counts. The complete blood count (CBC) is the standard method for analyzing blood leukocyte, erythrocyte, and platelet components. Absolute concentrations (cells μL − 1 ), conventionally referred to as absolute counts in nonclinical toxicity studies, should be used to evaluate and interpret changes in blood components because relative percentages do not account for the total numbers of cells present and may be misleading. Interpretation of xenobiotic-related alterations in blood components should be made with the consideration of the inherent biological variation in CBC endpoints observed in common laboratory species, and an understanding of the various mechanisms underlying increased or decreased blood component counts. There is considerable overlap in the mechanisms of blood cell alterations in both naturally occurring and xenobiotic-induced changes. The focus of this article is to review more commonly observed alterations in blood leukocyte, erythrocyte, and platelet components. 12.11.2 Leukocytes Common patterns of alterations in blood leukocyte components are summarized in Table 1 . Table 1 Classic patterns of alterations in blood leukocyte components Epinephrine Glucocorticoid Inflammatory patterns Bone marrow suppression Overwhelming Acute Chronic Segmented neutrophils ↑ ↑ ↓ to ↓↓↓ c ↑ to ↑↑↑ c ↑ to ↑↑↑ ↓ to ↓↓↓ Band neutrophils - - a - to ↑↑ c ↑ to ↑↑ c - to ↑ - Lymphocytes ↑ ↓ ↓ to ↓↓ ↓ to ↓↓ - to ↑↑ d ↓ to ↓↓ Monocytes - to ↑ - to ↑ - to ↓ - to ↑↑ - to ↑↑ ↓ to ↓↓↓ Eosinophils - to ↓ ↓ b - to ↓ b ↓ b to ↑ - to ↑ ↓ b Basophils - - to ↓ b - to ↓ b ↓ b to ↑ ↓ b to ↑ ↓ b Patterns described in this table indicate classic or expected changes in blood leukocyte counts due to these relatively common processes. However, duration of these processes, variations in the underlying causes of these conditions, and superimposition of multiple processes may result in differences between expected patterns of leukocyte changes and actual changes observed in an individual. -, no apparent change; ↑, mild increase; ↑↑, moderate increase; ↑↑↑, marked increase; ↓, mild decrease; ↓↓, moderate decrease; ↓↓↓, marked decrease. a May be slightly increased in some cases. b Due to low eosinophil and basophil counts in health, decreases may be difficult to identify. c Morphologic changes indicative of rapid neutropoiesis (Döhle bodies, cytoplasmic vacuolation, and cytoplasmic basophilia) may be observed. d A proportion of blood lymphocytes may appear reactive. 12.11.2.1 Neutrophils The production of neutrophils, or granulopoiesis, occurs predominantly in the bone marrow, although extramedullary hematopoiesis may contribute, particularly in rats and mice, and if increased tissue demand for granulocytes or bone marrow disease is present. Stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-6 promotes the differentiation of common myeloid progenitor cells to granulocyte/monocyte progenitor cells, and subsequent stimulation with granulocyte colony-stimulating factor (G-CSF) promotes differentiation, proliferation, and maturation of granulocytes from myeloblasts to mature segmented neutrophils ( Radin and Wellman, 2010 ). Granulocyte pools in the bone marrow can be divided into proliferating (mitotic) and maturing (postmitotic) pools. The proliferating pool encompasses the most immature myeloid precursors: myeloblasts, promyelocytes, and myelocytes. The maturing pool includes the later stages of metamyelocytes, band neutrophils, and mature segmented neutrophils. The mature segmented neutrophil population within the maturing pool is also considered the bone marrow storage pool, and acts as a reserve store of neutrophils that replenish blood stores following normal turnover and can be quickly mobilized to the blood in times of increased tissue demand. In health, mammalian blood neutrophils are mature, segmented neutrophils. They are distributed in two pools within the blood vessels, the circulating pool and the marginating pool. Circulating pool neutrophils flow freely through the vessel lumen and are sampled during blood collection. Marginating pool neutrophils are in contact with endothelial cells and temporarily adhere to or roll along the vessel wall. Neutrophils may shift between the circulating and marginating pools. The lifespan of a neutrophil in circulation is short (approximately 7 h half-life in blood), and ultimately neutrophils migrate into tissues where they survive for a few days ( Smith, 2016 ). Neutrophils are part of the innate immune system and primarily function as phagocytes to remove and destroy invading pathogens, but also can secrete proinflammatory and chemotactic mediators to enhance responses of both the innate and adaptive immune systems. In blood, neutrophils are typically the most numerous leukocyte in humans, rhesus monkeys, African green monkeys, and dogs. However, many New World monkeys tend to have lymphocyte counts greater than neutrophil counts ( Provencher Bolliger et al., 2010 ), and cynomolgus monkeys tend to initially have predominantly lymphocytes in circulation when they are young but shift to predominantly neutrophils with maturity ( Sugimoto et al., 1986 ). Neutrophil counts are lower than lymphocyte counts in rats and mice. Blood neutrophil counts may increase or decrease depending on the stimulus or insult. Clinically, increases in neutrophil counts above expected values in health (usually conveyed in a reference interval) may be referred to as neutrophilia or granulocytosis, while decreases in neutrophil counts may be called neutropenia, granulocytopenia, or, if severe, agranulocytosis. However, in nonclinical toxicology studies, the convention is to not use such clinical terminology to describe alterations caused by a test article. Referring to changes as increases or decreases in neutrophil counts is the standard for nonclinical toxicology studies, and this terminology is used throughout this article. However, clinical terms are also provided for reference. 12.11.2.1.1 Increases in neutrophil counts (neutrophilia, granulocytosis) 12.11.2.1.1.1 Catecholamine-induced Acute, transient increases (typically  50,000 cells μL − 1 , typically due to a marked neutrophilia with a left shift that remains orderly and may or may not have morphologic changes indicative of rapid granulopoiesis ( Schultze, 2010 , Sakka et al., 2006 ). Extreme neutrophilia typically has > 100,000 cells μL − 1 and evidence of a left shift. The terms leukemoid reaction and extreme neutrophilia are most appropriately applied retrospectively, after the possibility for hematopoietic neoplasia has been excluded. Differentiation of a leukemoid response or extreme neutrophilia from chronic myelogenous leukemia or chronic neutrophilic leukemia includes CBC, blood smear, and bone marrow evaluations in most species, and may also include leukocyte alkaline phosphatase activity, immunophenotyping, cytogenetic analysis (e.g., evaluation for bcr–abl translocation), serum G-CSF, and clonality evaluations in humans ( Schultze, 2010 , Sakka et al., 2006 ). Leukemoid reactions have been associated with carcinomas of various origins, including renal and pulmonary carcinomas, Hodgkin's lymphoma, melanoma, and sarcomas, and may be attributable to aberrant production of proinflammatory mediators by the neoplasm, such as G-CSF, GM-CSF, or IL-6 ( Sakka et al., 2006 ). Leukemoid reactions have also been reported in F334/N rats affected by large granular cell leukemia ( Car et al., 2006 ). However, leukemoid reactions may also be associated with infectious processes, including disseminated tuberculosis, Clostridium difficile colitis, severe shigellosis ( Sakka et al., 2006 ), chronic localized suppurative lesions such as pyometra, pleuritis, and internal abscesses ( Schultze, 2010 , Stockham and Scott, 2008a ). Leukemoid reactions may also be seen secondary to severe hemorrhage or immune-mediated hemolytic anemia ( Schultze, 2010 , Sakka et al., 2006 ). 12.11.2.1.1.4 Inherited leukocyte adhesion deficiencies Increases in neutrophil counts associated with deficiencies in leukocyte adhesion molecules may manifest as a leukemoid response or extreme neutrophilia. Adhesion molecules expressed on neutrophils are responsible for neutrophil margination, rolling along vessel walls, and emigration into tissues. L-selectin (CD62L) mediates low-affinity initial binding of leukocyte to endothelial cells, while integrins, including CD11b/CD18 (Mac-1), mediate firm adhesion to endothelial cells and ligands in the extracellular matrix ( Muller, 2012 ). Neutrophils constitutively express CD11b/CD18. A deficiency of this integrin (leukocyte adhesion deficiency [LAD] type 1) results in the failure of neutrophils to emigrate to tissues, and may result in severe, recurrent bacterial infections ( Arnaout, 1990 ). LAD type 2 is due to an inherited disorder of fucose metabolism, resulting in the lack of selectin ligands expressed on neutrophils and therefore results in immunodeficiency from a failure of selectin-mediated neutrophil rolling along vessel walls ( Marquardt et al., 1999 ). Leukocyte adhesion deficiencies have been reported in humans, dogs, mice, and Holstein cattle ( Arnaout, 1990 , Marquardt et al., 1999 , Gu et al., 2004 ). 12.11.2.1.1.5 Neoplasia Neoplasms involving hematopoietic cells naturally occur with relatively low frequency. In general, such neoplastic processes may be observed as background findings in rats and mice during longer toxicity studies (e.g., carcinogenicity studies), but are uncommon in nonrodent species during toxicity studies ( Smith et al, 2002 ). Increases in neutrophil counts may be observed as part of the neoplastic processes of chronic myeloid leukemia (CML) or chronic neutrophilic leukemia (CNL). Leukocytosis in CML is ≥ 25,000 cells μL − 1 with increases in all stages of neutrophilic myeloid cells in blood, which are typically accompanied by increases in monocyte counts, eosinophil counts, basophil counts, platelet counts, and possibly increases in nucleated erythroid precursors ( Sawyers, 1999 ). In contrast to a leukemoid response, the left shift associated with CML tends to be less orderly, with greater numbers of earlier stages of myeloid cells in circulation along with the band and segmented neutrophils. Morphologic features indicative of dysplasia may be seen, and cytoplasmic features indicative of rapid granulopoiesis may be observed in neutrophils associated with CML. Bone marrow findings include hypercellularity with increased myeloid to erythroid ratios where myeloblasts and promyelocytes make up  80% segmented and band neutrophils ( Uppal and Gong, 2015 ). These neutrophils may or may not have cytoplasmic changes indicative of rapid neutropoiesis. Unlike CML, the increases in neutrophil counts associated with CNL are typically not associated with morphologic features of dysplasia, the presence of earlier stages of myeloid cells (i.e. myeloblasts, promyelocytes, myelocytes, and metamyelocytes), increases in monocyte, eosinophil, or basophil counts, or increases in nucleated erythroid precursors ( Uppal and Gong, 2015 ). Bone marrow changes associated with CNL include hypercellularity with increased myeloid to erythroid ratios, where the myelocyte through band neutrophil stages are increased without apparent increases in myeloblasts or promyelocytes ( Uppal and Gong, 2015 ). As such, differentiation of CNL from leukemoid reactions or extreme neutrophilia may be difficult, and affected patients should be carefully evaluated to exclude causes of neutrophilia not associated with hematopoietic malignancy. 12.11.2.1.1.6 Xenobiotic-induced There are numerous xenobiotics that can increase blood neutrophil counts, typically through similar mechanisms as described for naturally occurring increases in neutrophil counts. Several examples of these compounds are described later. Administration of exogenous catecholamines, such as epinephrine, or some adrenergic agonists has similar effects as those mediated by endogenous catecholamines. Shifting of neutrophils from the marginating to the circulating pools is responsible for the increases in blood neutrophil counts, potentially from altered adhesion molecule expression or hemodynamic forces. Leukocyte subtypes have different receptors modulating their adrenergic effects, and neutrophils appear to be primarily affected by α-adrenergic receptors ( Benschop et al., 1996 ). Exogenous glucocorticoids, such as prednisolone, dexamethasone, and betamethasone, will mediate increases in neutrophil counts through similar mechanisms as endogenous glucocorticoids. In brief, these increases in neutrophil counts are due to shifting of neutrophils from the marginating to circulating pool, release of neutrophils from the bone marrow storage pool, and expansion of granulopoiesis in the bone marrow. The dose and duration of the glucocorticoid therapy may alter the proportional contribution of each of these effects to the increases in neutrophil counts. Heparin-induced increases in neutrophil counts are reported in a small percentage of patients receiving heparin therapy, and the mechanisms are likely multifactorial. Heparin may cause shifting of neutrophils to the circulating pool from the marginating pool due to decreased expression of selectins ( Zhang et al., 2012 ). Heparin-related release of CXCL12, a chemokine that is constitutively expressed by bone marrow stromal cells and plays a role in bone marrow neutrophil retention and leukocyte trafficking, may alter bone marrow and circulating CXCL12 gradients and promote release of neutrophils from the bone marrow storage pool, contributing to the increases in circulating neutrophil counts ( Zhang et al., 2012 ). Administration of G-CSF or GM-CSF may be used as a rescue therapy following chemotherapy that causes neutropenia and compromises the immune system's ability to respond to infectious agents. While the goal for treatment in chemotherapy patients is to increase blood neutrophil counts to normal levels and not to achieve increased neutrophil counts, repeated or high dose administration of G-CSF or GM-CSF-related compounds to healthy laboratory animals results in markedly increased neutrophil counts. Typically these changes are associated with a left shift and cytoplasmic changes indicative of rapid neutropoiesis. Dysplastic changes may also occur, most commonly recognized by the presence of unusually large neutrophils or hypersegmented neutrophils in circulation. Several xenobiotics appear to induce increases in neutrophil counts by stimulating increases in endogenous G-CSF or an inflammatory stimulus. Early in the course of treatment with clozapine, an antipsychotic agent, rats have been shown to have spikes in G-CSF with concomitant increases in bone marrow granulopoiesis and release of less mature neutrophils into circulation ( Lobach and Uetrecht, 2014 ). However, other xenobiotics may directly cause an inflammatory stimulus and increase in endogenous proinflammatory stimuli, such as G-CSF and IL-1, which result in increases in neutrophil counts. For example, lithium, which has been used in treatment of bipolar disorder and in combination with antidepressants, also appears to cause increased neutrophil counts due to increased G-CSF ( Focosi et al., 2009 ). Xenobiotic-induced increases in neutrophil counts may be observed in conjunction with several cutaneous drug reactions that result in inflammatory stimuli. Acute generalized exanthematous pustulosis is a drug-related skin reaction that generally occurs within 2 days of drug administration but resolves spontaneously within 15 days ( Roujeau, 2005 ). Increases in eosinophil counts may or may not be observed along with the increases in neutrophil counts. Xenobiotics classically associated with this syndrome include diltiazem and antibiotics such as aminopenicillins and pristinamycine ( Roujeau, 2005 ). Acute febrile dermatosis (Sweet's syndrome) has also been associated with elevations in circulating neutrophil counts, and drug-induced forms of this condition have been variably linked to a variety of compounds, including GM-CSF, trimethoprim–sulfamethoxazole, minocycline, celecoxib, furosemide, and azathioprine ( Rochet et al., 2013 , Saavedra et al., 2006 ). 12.11.2.1.2 Decreases in neutrophil counts (neutropenia, granulocytopenia, agranulocytosis) 12.11.2.1.2.1 Overwhelming or severe acute inflammation Although inflammation often causes increases in neutrophil counts, an overwhelming or severe acute inflammatory stimulus may result in decreases in neutrophil counts. Such an inflammatory stimulus, sometimes also referred to as peracute inflammation, is commonly observed with bacterial sepsis. Decreases in neutrophil counts are a result of emigration to tissues that exceed the bone marrow capacity to release neutrophils. Proinflammatory mediators and chemoattractants stimulate increased neutrophil margination, firm adhesion to endothelial cells, and emigration into tissue, which shortens neutrophil circulating half-lives and depletes the circulating neutrophil pool. As neutrophils are released from the bone marrow and the storage pool is depleted, recruitment of immature neutrophils into circulation results in a left shift in which the number of immature forms exceeds the number of mature segmented neutrophils (degenerative left shift). If the stimulus persists long enough, granulocytic hyperplasia of the bone marrow will occur and the numbers of circulating neutrophils will increase and shift toward greater numbers of mature than immature neutrophils. 12.11.2.1.2.2 Endotoxemia Decreases in neutrophil counts may be observed as a result of Gram-negative bacterial infections that release endotoxin. Endotoxemia has been demonstrated to increase expression of the integrin CD11b/CD18 and decreased expression of L-selectin on neutrophils ( Wagner and Roth, 1999 ), mediating a shift from circulating to marginating pools with firm adhesion to endothelial cells. There is evidence that this upregulation of CD18 contributes to neutrophil vascular sequestration in the lungs and liver, but there is also evidence for other mechanisms, such as cytoskeletal changes that alter neutrophil deformability, causing the initial vascular sequestration of neutrophils that is consistently observed with endotoxemia ( Wagner and Roth, 1999 ). The decreases in neutrophil counts associated with endotoxemia tend to be rapid but transient, and detection of these decreases in neutrophil counts will depend on timing of the blood collection in relation to endotoxemic and other concurrent or subsequent proinflammatory stimuli. 12.11.2.1.2.3 Viral-induced Decreases in neutrophil counts may be associated with viral infections, although viral-specific mechanisms vary. Decreased neutrophil counts associated with parvovirus likely represent both a primary effect due to infection causing death of rapidly dividing hematopoietic precursors in the bone marrow, as well as a secondary increase in tissue demand from disease and loss of integrity of the intestinal tract. Decreases in neutrophil counts associated with human immunodeficiency virus (HIV), infectious hepatitis, and infectious mononucleosis in people have also been associated with decreased or ineffective neutrophil production due to infection of hematopoietic precursors ( Dale and Welte, 2016 , Lima et al., 2006 ). With measles and dengue virus, decreases in neutrophil counts have been observed with endothelial cell alterations leading to increased adhesion of neutrophils and therefore a shift from the circulating to the marginating pool ( Dale and Welte, 2016 ). Decreases in blood neutrophil counts may also result from decreased neutrophil production, as in acquired viral immunodeficiency. Immunodeficiency syndromes with decreased neutrophil counts have been reported with infections by HIV in people, simian immunodeficiency virus (SIV) or simian betaretrovirus in monkeys, or feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) in cats ( Levine et al., 2006 , Israel and Plaisance, 1991 , Magden et al., 2015 , Gill et al., 2012 , Gleich and Hartmann, 2009 ). 12.11.2.1.2.4 Immune-mediated Primary or idiopathic immune-mediated destruction of mature neutrophils or neutrophil precursors will result in decreases in neutrophil counts, and has been reported in humans and most laboratory species. Antineutrophil antibodies are most commonly IgG and less commonly IgM class. Opsonization of neutrophil membranes may lead to leukoagglutination and neutrophil sequestration in sites including the spleen, liver, and lymph nodes, with phagocytosis by macrophages. Some antineutrophil antibodies may cause direct cytotoxicity through either complement-mediated or complement-independent mechanisms ( Chickering and Prasse, 1981 ). In humans, immune-mediated neutrophil destruction due to antibody-dependent lymphocyte cytotoxicity has been reported ( Logue et al., 1978 ). Systemic lupus erythematosus (SLE) is an autoimmune condition that has been associated with decreases in neutrophil counts. These decreases in blood neutrophil counts appear to be relatively common with SLE, and are usually observed in conjunction with other manifestations of SLE, such as arthritis, skin lesions, or neurologic disorders ( Stone, 2005 ). There is an association between decreases in neutrophil counts and the presence of autoantibodies in SLE ( Hsieh et al., 2003 , Budman and Steinberg, 1977 ), which lead to destruction or death of neutrophils. However, SLE-related anti-G-CSF autoantibodies have also been observed, causing decreased blood neutrophil counts due to decreased production rather than or in addition to direct neutrophil destruction or death ( Hellmich et al., 2002 ). Most cases of primary or idiopathic aplastic anemia result from underlying immune-mediated destruction of uncommitted or early lineage-committed hematopoietic stem cells ( Young et al., 2006 ). Aplastic anemia is characterized by pancytopenia (marked decreases in all blood component counts) and hematopoietic cell hypocellularity in bone marrow. Although most commonly associated with immune-mediated destruction of hematopoietic precursors, marked depletion of bone marrow hematopoietic cells or aplastic anemia may also occur with severe nutritional deficiencies associated with anorexia nervosa ( Abella et al., 2002 ) and 75% feed restriction in rats ( Moriyama et al., 2008 , Levin et al., 1993 ). 12.11.2.1.2.5 Cobalamin (B 12 ) and folate (B 9 ) Deficiencies in cobalamin and folate cause disruption of DNA synthesis that results in megaloblastic hematopoiesis, commonly associated with megaloblastic anemia and occasionally with decreases in neutrophil counts and/or pancytopenia ( Dale and Welte, 2016 , Green, 2016 ). Cobalamin or folate deficiencies may be due to malnutrition, malabsorption from gastrointestinal disease or dysfunction, or genetic deficiencies of transport proteins, such as intrinsic factor, transcobalamin, or R-factor for cobalamin, receptors for intestinal absorption, such as the CUBAM receptor, or metabolizing enzymes, such as methylenetetrahydrofolate reductase for folate ( Fowler, 1998 , Whitehead, 2006 , Montagle and Tauro, 1995 , Green, 2016 , Quadros, 2010 ). During megaloblastic hematopoiesis, asynchronous cytoplasmic and nuclear maturation may result in giant granulocytes with irregular nuclear chromatin patterns, such as giant metamyelocytes ( Green, 2016 ), or hypersegmented neutrophils ( Thompson et al., 1989 ). These conditions can often be managed with supplementation of the deficient vitamin. A differential for cobalamin or folate deficiency-induced decreases in blood neutrophil counts is copper deficiency, which can have a similar clinical manifestation ( Lazarchick, 2012 ). 12.11.2.1.2.6 Myelophthisis, myelofibrosis, and myelonecrosis Conditions that efface the bone marrow cavities or displace hematopoietic cells (myelophthisis) may result in decreased production of all hematopoietic cell lines, including neutrophils. Myeloproliferative syndromes ( Tefferi and Vardiman, 2009 ), many leukemic diseases ( Talcott et al., 1992 , Lamy and Loughran, 1999 ), lymphoproliferative neoplasia ( Schultze, 2010 ), or metastatic carcinoma ( Makoni and Laber, 2004 ) can cause sufficient bone marrow overcrowding to decrease the production of neutrophils. Fungal infections resulting in granulomatous inflammation of the bone marrow, such as can be observed with disseminated histoplasmosis, may also result in decreased neutrophil production. Disruption of the bone marrow cavities and hematopoietic stem cells from myelofibrosis or myelonecrosis may also be associated with decreased blood neutrophil counts ( Stockham and Scott, 2008a ). 12.11.2.1.2.7 Xenobiotic-induced Xenobiotic-induced decreases in neutrophil counts may be caused by immune-mediated or nonimmune-mediated mechanisms. The incidences of these events tend to be low with most implicated xenobiotics, with the exception of chemotherapeutic agents. Examples of both immune-mediated and nonimmune-mediated mechanisms are described later. Similar to primary immune-mediated decreases in neutrophil counts, xenobiotic-induced immune-mediated decreases in neutrophil counts are commonly associated with IgG or IgM antineutrophil antibodies, although some drugs may induce both ( Salama et al., 1989 ). Decreases in neutrophil counts by this mechanism tend to occur rapidly, within a few hours to 2 days after exposure ( Schwartzberg, 2006 ). Some xenobiotics mediate their effects by acting as haptens, which induce an antibody response targeting an antigen formed by the xenobiotic–neutrophil combination. Drugs reported to act as haptens include penicillin, gold-based compounds, aminopyrine, and propylthiouracil ( Salama et al., 1989 , Murphy et al., 1985 ). These xenobiotics induce neutrophil destruction in a drug-dependent manner, and discontinuation of treatment generally results in resolution of blood neutrophil counts within 7 days ( Schwartzberg, 2006 ). Some xenobiotics, including propylthiouracil and quinidine sulfate, cause the formation of immune complexes that can subsequently bind and destroy neutrophils, even if the xenobiotic is no longer present ( Schwartzberg, 2006 , Bhatt and Saleem, 2004 , Eisner et al., 1977 ). In addition, some xenobiotics, such as propylthiouracil, may also cause the formation of antineutrophil antibodies resulting in complement-mediated neutrophil destruction ( Akamizu et al., 2002 ). Similar to idiopathic aplastic anemia, xenobiotic-induced aplastic anemia is most commonly associated with immune-mediated destruction of uncommitted or early hematopoietic stem cells, although direct cytotoxicity, such as with chemotherapeutic agents, may also lead to aplastic anemia. Several xenobiotics, including chloramphenicol, anticonvulsants such as phenytoin and carbamazepine, gold-based compounds, and phenylbutazone have been associated with aplastic anemia ( Bloom and Brandt, 2008 ). Numerous xenobiotics have also been linked to SLE, with decreases in blood neutrophil counts in some cases. In xenobiotic-induced SLE, decreased neutrophil counts reflect production of autoantibodies and subsequent neutrophil destruction, similar to nonxenobiotic-induced SLE. Xenobiotics associated with SLE include anticonvulsants such as phenothiazines, chlorpromazine, and valproate, antibiotics such as penicillin, streptomycin, tetracycline, griseofulvin, and sulphonamides, and miscellaneous xenobiotics such as captopril, phenylbutazone, and lovastatin ( Stone, 2005 , Mutasim and Adams, 2000 ). In laboratory species used in nonclinical toxicology studies, transient xenobiotic-induced decreases in blood neutrophil counts have been associated with anaphylactoid reactions termed complement activation-related pseudoallergy (CARPA). These nonhypersensitive reactions are mediated instead by activation of the complement cascade, leading to the production of the anaphylatoxins C3a and C5a. CARPA typically occurs after the first dose of xenobiotic with decreasing severity or resolution following additional doses. Xenobiotics implicated in CARPA reactions include radiocontrast media, drug-carrying liposomes and lipid complexes, and some solvents with amphiphilic emulsifiers, such as Cremophor EL ( Szebeni, 2005 ). Nonimmune-mediated decreases in neutrophil counts may be observed with bone marrow suppression, which is associated with many xenobiotics, particularly chemotherapeutic agents. Chemotherapeutic-related decreases in neutrophil counts are often a result of direct cytotoxicity or suppressed proliferation of the rapidly dividing granulocytic precursors in the bone marrow. Examples of chemotherapeutic classes associated with decreased blood neutrophil counts include agents that cause direct DNA damage, such as platinum-containing agents (e.g., cisplatin, carboplatin) and classical alkylating agents (e.g., cyclophosphamide, melphalan, busulfan); mitotic spindle inhibitors (e.g., paclitaxel, docetaxel, vinblastine, vincristine); topoisomerase inhibitors (e.g., etoposide, doxorubicin); and antimetabolites (e.g., methotrexate, 6-mercaptopurine, 5-fluorouracil) ( Bhatt and Saleem, 2004 , Weiss, 2010 , Wailoo et al., 2009 ). Decreases in neutrophil counts are expected with chemotherapeutic treatment, and can be easily monitored through serial CBCs. If severe enough to increase the risk of life-threatening infections clinically, treatment with empiric antibiotics or G-CSF-like compounds may be considered. Suppression of granulopoiesis or direct granulocyte toxicity is also reported with some nonchemotherapeutic agents. Dose-dependent inhibition of colony-forming units of granulocytes and macrophages in the bone marrow has been reported with several anticonvulsant drugs, including valproic acid and carbamazepine, and beta-lactam antibiotics ( Schwartzberg, 2006 , Irvine et al., 1994 , Watts et al., 1990 , Neftel et al., 1985 ). Several other anticonvulsant drugs, including ethosuximide ( Mintzer et al., 2009 ) and phenytoin ( Sharafuddin et al., 1991 ), have been reported to cause direct toxic effects on granulocyte precursors. Antipsychotic agents, including clozapine and chlorpromazine, may also cause direct cytotoxic effects: neutrophil metabolism of clozapine has been demonstrated to form an unstable intracellular metabolite, nitrenium ion, which depletes glutathione and makes the neutrophils susceptible to oxidative damage and apoptosis ( Williams et al., 2000 ); chlorpromazine may cause cytotoxicity through the inhibition of thymidine and uracil uptake by neutrophils ( Pisciotta and Kaldahl, 1962 ). Thienopyridine inhibitors of platelet aggregation, including clopidogrel and ticlopidine, have also been associated with direct neutrophil toxicity resulting from mitochondrial toxicity by metabolites generated through myeloperoxidase metabolism of the parent compounds ( Maseneni et al., 2012 , Maseneni et al., 2013 ). 12.11.2.2 Lymphocytes The production of lymphocytes, or lymphopoiesis, progresses from pluripotent hematopoietic stem cells that differentiate into common lymphoid progenitor cells, and further differentiate into B-cells, T-cells, and natural killer (NK) cells. B-cell development begins in the fetal liver and transitions to bone marrow postnatally, where the cells undergo proliferation and differentiation, followed by migration to the peripheral lymphoid tissues. B-cell development requires a variety of soluble factors, including IL-3, IL-4, IL-11, INFγ, and TGFβ ( Burkhard, 2010 ). T-cell precursors migrate to the thymus during embryonic development, where they undergo proliferation, differentiation, and both positive and negative selection. T-cell development requires IL-7 stimulation ( Burkhard, 2010 ). NK-cell development, which requires IL-15 stimulation, occurs mostly in the fetal liver and thymus, as well as in the bone marrow after birth ( Burkhard, 2010 ). In health, blood lymphocytes are predominantly T-cells. Similar to neutrophils, blood lymphocytes are divided into circulating and marginating pools, with cells frequently shifting between these pools. Lymphocytes in the blood travel to lymph nodes, where they exit the blood through high endothelial venules and enter the lymph node cortices. Lymphocytes that migrate through the lymph nodes leave through efferent lymphatic vessels, from which they return to the blood. Blood lymphocytes may also emigrate to other tissues if chemotactic stimuli are present. In tissues, lymphocytes may proliferate, die, or migrate back into blood. Lymphocyte life spans are highly variable depending on the cell type and function, and some lymphocytes may live for years ( Stockham and Scott, 2008a ). Lymphocytes are the most numerous blood leukocytes in rats and mice, and are typically present in greater numbers than neutrophils in young cynomolgus monkeys and dogs. Cynomolgus monkeys used in nonclinical toxicology studies are often young or peripubertal, with greater lymphocyte than neutrophil counts. However, in both cynomolgus monkeys and dogs, there is a shift of blood leukocyte populations to a predominance of neutrophils with maturation, and nonclinical toxicology studies may include animals having either hematologic pattern. In adults of species with neutrophils as the most numerous blood leukocyte, lymphocytes are typically the second most numerous. 12.11.2.2.1 Increases in lymphocyte counts (lymphocytosis) 12.11.2.2.1.1 Age-related Lymphocytes are common as the predominant blood leukocyte of neonate and juvenile animals, even in species with predominant neutrophils in the circulation of adults. Recognition of this apparent "lymphocytosis" of young animals is important, as comparison of lymphocyte counts in young animals with adult historical control data may give the appearance of increased lymphocyte counts. Species in which "lymphocytosis" in young animals has been described include dogs and cats ( Stockham and Scott, 2008a ) and cynomolgus monkeys ( Sugimoto et al., 1986 ). As the shift from predominantly lymphocytes to predominantly neutrophils in circulation occurs around 4 to 5 years old in cynomolgus monkeys ( Sugimoto et al., 1986 ), it is not uncommon for nonclinical toxicology studies to include individuals with both lymphocyte and neutrophil-predominant leukograms. 12.11.2.2.1.2 Catecholamine-induced Similar to increases in neutrophil counts, increases in endogenous or exogenous catecholamines associated with excitement, fear, or exercise result in transient increases in lymphocyte counts. Catecholamine-induced increases in lymphocyte counts are associated with rapid shifts from the marginating to circulating lymphocyte pool, which is thought to be due to both decreased lymphocyte adhesion to endothelial cells and increased blood flow ( Benschop et al., 1996 ). Release of lymphocytes from the spleen in response to catecholamine stimulation likely also contributes to the increase in blood lymphocyte counts, but lymph nodes and bone marrow do not appear to be significant contributors ( Benschop et al., 1996 ). There are species differences in the response of lymphocytes to catecholamines, and the resultant increases in lymphocyte tend to be more common in monkeys and cats, while less common in adult dogs ( Smith et al., 2002 , Schultze, 2010 ). 12.11.2.2.1.3 Decreased Glucocorticoids Glucocorticoids have a negative effect on blood lymphocyte counts due to their effects on lymphocyte distribution in the body and suppression of lymphopoieis. Hypoadrenocorticism (Addison's disease), in which the adrenal glands are unable to maintain normal concentrations of glucocorticoids, may be associated with increases in blood lymphocyte counts due to the loss of the inhibitory effects of endogenous glucocorticoids ( Oelkers, 1996 , Stockham and Scott, 2008a , Avery and Avery, 2007 ). 12.11.2.2.1.4 Inflammation Acute inflammatory processes tend to cause decreases in lymphocyte counts, but some acute infectious processes, particularly several viral infections, may cause increases in lymphocyte counts. However, increases in lymphocyte counts are commonly observed with chronic inflammatory processes. Chronic stimulation of lymphocytes with antigens or cytokines results in the increased production of lymphocytes with release into the blood, causing the increases in blood lymphocyte counts. Reactive lymphocytes may be observed in blood accompanying inflammation-induced increases in lymphocytes counts. Reactive lymphocytes have a spectrum of morphologic changes that include increased cytoplasmic basophilia, large cells with mildly increased amounts of cytoplasm (lower nuclear to cytoplasmic ratios), variable patterns of chromatin clumping, and variable numbers of visible nucleoli. Occasionally, reactive lymphocytes may have paranuclear cytoplasmic clearing, giving a plasmacytoid appearance. Infectious mononucleosis syndromes in people are a relatively common cause of an inflammatory or reactive increase in lymphocyte counts that are usually acute ( Vasu and Caligiuri, 2016 ). Chronic infections leading to increase in blood lymphocyte counts may include visceral leishmaniasis, parasitic infections such as strongyloidiasis, and leprosy ( Vasu and Caligiuri, 2016 , Rai et al., 2008 , Myers et al., 2000 ). Several chronic infections, including ehrlichiosis, Rocky Mountain spotted fever, leishmaniasis, trypanosomiasis, and brucellosis, have been associated with increases in blood lymphocyte counts in dogs ( Schultze, 2010 ). 12.11.2.2.1.5 Neoplasia Increases in blood lymphocyte counts associated with lymphoproliferative neoplasia may represent either lymphocytic leukemia or the leukemic phase of lymphoma. A normal circulating lymphocyte population should be heterogeneous, with predominantly small lymphocytes and fewer intermediate to large lymphocytes. Increased blood lymphocyte counts with a loss of heterogeneity in the blood lymphocyte population or predominantly a monomorphic intermediate to large lymphocyte population are key features for diagnosing lymphoproliferative neoplasia. With the exception of chronic lymphocytic leukemia, which is characterized by increased numbers of small lymphocytes with few or subtle morphologic alterations, circulating lymphocytes often have abnormal morphologic features that may be observed microscopically. Abnormal morphologic features of the leukemic lymphocyte population may include increased cytoplasmic basophilia, increased amounts of cytoplasm with altered nuclear to cytoplasmic ratios, irregular clumping of nuclear chromatin, indentation or lobulation of nuclei, variably sized but typically prominent nucleoli, or multiple nucleoli. Some of these morphologic features are similar to those observed in reactive lymphocytes, but these two processes may be distinguished by the overall heterogeneity of the lymphocyte population and proportion of the lymphocyte population with these morphologic alterations. Additional testing, such as flow cytometry for phenotyping or PCR for antigen receptor rearrangement (PARR), also aids in the diagnosis or characterization of lymphoproliferative neoplasia. Lymphoproliferative neoplasia is observed as a relatively common background finding in older rats and mice during nonclinical toxicology studies ( Frith et al., 1993 ). Although less common, it may also be observed in low frequencies in older monkeys. Monkeys with concurrent infection with species-specific lymphocryptoviruses and immunosuppression have been reported to have virus-related lymphoproliferative neoplasias ( Magden et al., 2015 ). Lymphocryptoviruses are in the Gammaherpesvirinae subfamily and are related to Epstein-Barr virus, which has been associated with lymphoproliferative neoplasia in people but may aberrantly infect New World monkeys ( Magden et al., 2015 , Thorley-Lawson and Gross, 2004 ). Increases in blood lymphocyte counts may also occur secondary to nonlymphoproliferative neoplasms. Increases in polyclonal T-cells have been reported in patients with malignant thymoma, while increases in reactive lymphocytes have been reported with AML and systemic mastocytosis ( Vasu and Caligiuri, 2016 ). 12.11.2.2.1.6 Xenobiotic-induced Xenobiotic-induced increases in blood lymphocyte counts are relatively uncommon, with most reports associated with an administration of catecholamines or rare idiosyncratic hypersensitivity-type reactions. Administration of exogenous catecholamines, such as epinephrine, or adrenergic agonists has similar effects on blood lymphocyte counts as those mediated by endogenous catecholamines. Rapid shifting of lymphocytes from marginating to circulating blood pools as well as mobilization of lymphocytes from the spleen contributes to the increases in lymphocyte counts. Lymphocytes appear to have their adrenergic effects primarily mediated by β 2 -adrenergic receptors ( Benschop et al., 1996 ). Increases in blood lymphocyte counts with the presence of atypical lymphocytes in circulation have been associated with drug reaction with eosinophilia and systemic symptoms (DRESS), a form of drug-related hypersensitivity. Several anticonvulsant drugs, including phenobarbital and phenytoin, allopurinol, minocycline, sulfonamides, gold salts, and dapsone have been associated with DRESS ( Roujeau, 2005 , Callot et al., 1996 ). Treatment of CML and chronic lymphocytic leukemia with dasatinib and ibrutinib, respectively, has been associated with increases in blood lymphocytes counts ( Vasu and Caligiuri, 2016 ). Dasatinib-related increases in lymphocyte counts may be related to expansion of T-cell or NK-cell populations and increases in IL-2R, INF-γ, and IL-6, with reported favorable outcome to treatment, while ibrutinib-related increases in lymphocyte counts may be related to and increased efflux of lymphocytes from lymphoid tissues ( Vasu and Caligiuri, 2016 ). 12.11.2.2.2 Decreases in lymphocyte counts (lymphopenia) 12.11.2.2.2.1 Glucocorticoid-induced Endogenous glucocorticoids may be increased with chronic stress or hyperadrenocorticism, and decreases in lymphocyte counts tend to be the most prominent and consistent glucocorticoid-mediated leukocyte change across species. Glucocorticoids induce decreases in blood lymphocyte counts through several mechanisms. In addition to a rapid shift of lymphocytes from the circulating to marginating and tissue pools, there is evidence for both lymphocyte redistribution from blood to bone marrow ( Fauci, 1975 ) and decreased efflux of lymphocytes from lymphoid tissues ( Bloemena et al., 1990 ) contributing to the shift of lymphocytes to tissue pools. With long-term increases in glucocorticoid concentrations, lymphotoxicity may be observed due to an increased activation of apoptotic pathways ( Garvy et al., 1993 , Tuckermann et al., 2005 ). In rats, feed restriction has been associated with decreases in blood lymphocyte counts and lymphocyte depletion in various lymphoid tissues ( Moriyama et al., 2008 ), possibly associated with prolonged stress and therefore a glucocorticoid-mediated effect. Indirect test article-related effects mediated by altered food consumption are important to consider in nonclinical toxicology studies, in which test articles may cause direct or indirect hematologic effects. Interpretation of these changes, including stress-associated effects, must be made cautiously and thoughtfully, using a weight of evidence approach. 12.11.2.2.2.2 Inflammation Decreases in lymphocyte counts are typically observed with acute inflammation. These decreases are likely due to increased margination and emigration of lymphocytes to the site of inflammation, increased migration of lymphocytes to lymphoid tissues, and decreased efflux of lymphocytes out of lymphoid tissues ( Stockham and Scott, 2008a ). Stress associated with illness or acute inflammation may also contribute by glucocorticoid-induced mechanisms ( Stockham and Scott, 2008a , Schultze, 2010 ). Many infectious agents may cause a decrease in lymphocyte counts due to inflammation. Infectious agents associated with decreases in lymphocyte counts include viruses such as coronavirus, parvovirus, West Nile virus, hepatitis viruses, and influenza ( Vasu and Caligiuri, 2016 , Schultze, 2010 ); acute systemic bacterial infections; as well as tuberculosis, typhoid fever, and bacterial pneumonias ( Vasu and Caligiuri, 2016 , Magden et al., 2015 ). The acute phase of malaria may also be associated with decreases in lymphocyte counts ( Vasu and Caligiuri, 2016 ). 12.11.2.2.2.3 Viral-induced Infection with immunodeficiency viruses, including human, simian, and feline immunodeficiency viruses, may result in destruction of both infected and noninfected lymphocytes. HIV directly infects CD4 + T-cells via the CD4 molecule; infected cells then migrate to lymphoid tissues where the virus replicates and infects more CD4 + T-cells ( Chinen and Shearer, 2010 ). HIV-mediated lymphocyte destruction is likely multifactorial; HIV may be cytotoxic, directly induce T-cell apoptosis, induce T-cell death through a nonspecific immune response, and cause T-cell death by stimulating autophagocytic pathways ( Chinen and Shearer, 2010 , Stump and VandeWoude, 2007 ). SIV and FIV tropism for T-cells is also mediated by receptors expressed by CD4 + T-cells ( Stump and VandeWoude, 2007 ). Simian betaretrovirus also causes decreases in blood lymphocyte counts due to infection and eventual depletion of both B-cells and T-cells, although infection of nonlymphoid cells also occurs ( Montiel, 2010 ). 12.11.2.2.2.4 Immune-mediated Immune-mediated destruction of lymphocytes is uncommon. When occurring in the autoimmune disease SLE, such immune-mediated decreases in lymphocyte counts typically occur with concurrent cutaneous, arthritic, or neurologic disorders ( Stone, 2005 ), and may be the result of autoantibodies causing lymphocyte destruction or death through apoptosis ( Lu et al., 2012 , Massardo et al., 2009 , Noguchi et al., 1992 , Budman and Steinberg, 1977 ). 12.11.2.2.2.5 Inherited causes Although rare, some inherited immunodeficiency syndromes cause blood lymphocyte counts to be decreased. One example, severe combined immunodeficiency (SCID), has been reported in humans, dogs, mice, and horses ( Suter, 2010 , Notarangelo, 2010 , Meek et al., 2001 , Felsberg et al., 1992 , Custer et al., 1985 ). SCID may be inherited through autosomal recessive or X-linked recessive patterns, and causes consistent decreases in T-cells, with concurrent decreases in B-cells or NK-cells in some forms of the disease. SCID in humans is caused by a variety of mechanisms, including: adenosine deaminase deficiency resulting in early cell death due to metabolite accumulation; common gamma chain or janus kinase 3 (JAK3) mutations that cause decreased survival of T-cell precursors due to defective cytokine signaling; recombinase-activating gene 1 (RAG1) or RAG2 mutations that cause defective V(D)J rearrangement of B-cell and T-cell receptors; and mutations in CD3 or CD45 that cause defects in T-cell receptor signaling ( Suter, 2010 , Notarangelo, 2010 ). SCID in Jack Russell terriers, Arabian foals, and mice has been demonstrated to be caused by defective V(D)J recombination due to loss of DNA-dependent protein kinase ( Meek et al., 2001 ). X-linked SCID has been described in both Bassett Hounds and Welsh Corgi dogs ( Suter, 2010 ). Other inherited immunodeficiency syndromes resulting in decreases in blood lymphocyte counts include reticular dysgenesis, ataxia-telangiectasia, and Wiskott-Aldrich syndrome ( Vasu and Caligiuri, 2016 ). 12.11.2.2.2.6 Loss of lymph fluid Although uncommon, disorders causing chronic loss of lymphocyte-rich lymph fluid lead to body depletion of lymphocytes and decrease in blood lymphocyte counts. Examples of such conditions include protein-losing enteropathy, lymphangiectasia, ulcerative enteritis, or repeated iatrogenic removal of chylothoracic fluid ( Vasu and Caligiuri, 2016 , Schultze, 2010 , Stockham and Scott, 2008a ). 12.11.2.2.2.7 Neoplasia Although lymphoproliferative neoplasia may be associated with increases in lymphocyte counts as previously discussed, lymphoma and lymphocytic leukemia may also be associated with decreases in lymphocyte counts including from altered lymphocyte recirculation patterns or decreased production of nonneoplasic lymphocytes secondary to lymphoid organ damage ( Mitrovic et al., 2012 , Schultze, 2010 , Stockham and Scott, 2008a ). 12.11.2.2.2.8 Xenobiotic-induced Lymphoid suppression is a common finding with many xenobiotics and may be associated with decreases in blood lymphocyte counts. Mechanisms through which these decreases occur may either be part of the expected pharmacology of these compounds or may represent an off-target effect. Several examples of xenobiotic-induced decreases in lymphocyte counts are described here. Administration of exogenous glucocorticoids, for antiinflammatory or immunosuppressive purposes, will result in decreases in blood lymphocyte counts. The mechanisms for this are the same as those for endogenous glucocorticoids, and include altered blood and tissue pool distribution, decreased efflux of lymphocytes from lymphoid tissues, and increased lymphocyte apoptosis with prolonged glucocorticoid exposure. Other immunosuppressive agents that cause decreased blood lymphocyte counts include cyclophosphamide, methotrexate, purine nucleoside analogs, and azathioprine. Cyclophosphamide has been associated with profound decrease in blood lymphocyte counts through its effects on all lymphocyte subtypes ( Gergely, 1999 ). Methotrexate causes decreases in circulating CD4 + and CD8 + T-cells, while cladribine, a purine nucleoside analog, causes apoptosis of lymphocytes and has been reported to affect both CD4 + and CD8 + T-cells ( Gergely, 1999 ). Azothiaprine-induced decreases in blood lymphocyte counts appear to be due to long-term administration at high dose levels ( Johnson et al., 1995 , Gergely, 1999 ). As discussed previously, numerous xenobiotics have been associated with drug-induced SLE in people. Decreases in lymphocytes in these cases are likely due to the production of autoantibodies with subsequent lymphocyte destruction, similar to nonxenobiotic-induced SLE. Xenobiotics associated with SLE include several anticonvulsants such as phenothiazines, chlorpromazine, and valproate, several antibiotics such as penicillin, streptomycin, tetracycline, griseofulvin, and sulphonamides, and miscellaneous xenobiotics such as captopril, phenylbutazone, and lovastatin ( Stone, 2005 , Mutasim and Adams, 2000 ). Chemotherapeutic agents are also frequently associated with decreases in lymphocyte counts, which may precede episodes of febrile neutropenia ( Gergely, 1999 ). Carboplatin, dacarbazine, and paclitaxel have been reported to induce decreases in CD4 + T-cells, while epirubicin and mitomycin appear to affect CD8 + T-cells to a greater degree than CD4 + T-cells, and pentostatin affects both B-cell and T-cell populations ( Gergely, 1999 ). Antilymphocyte monoclonal antibodies have been used to treat autoimmune diseases as well as to cause immunosuppression to prevent acute transplant rejection. Examples of these monoclonal antibodies include Muromonab CD3 (OKT3) and CAMPATH-1H ( Vial et al., 2002 , Gergely, 1999 ). Other classes of drugs reported to cause decrease lymphocyte counts are varied: pesticides including organochloride pesticides such as pentachlorophenol, organotin compounds, and organophosphates ( Corsini et al., 2013 ); thienopyridines, such as clopidogrel and ticlopidine, which can cause direct lymphotoxicity at high concentrations ( Maseneni et al., 2013 ); the histamine H2 receptor antagonist cimetidine; the anticonvulsant carbamazepine; imidazoles used to treat fungal infections; and opioids such as morphine ( Gergely, 1999 ). 12.11.2.3 Monocytes Bone marrow common myeloid progenitors differentiate into the monocytic lineage under stimulation by stem cell factor, GM-CSF, macrophage-colony-stimulating factor (M-CSF), IL-6, IL-1, and IL-3 ( Papenfuss, 2010 ). Monoblasts further differentiate to promonocytes and then to monocytes. Blood monocytes are distributed in both circulating and marginating pools, and marginating pool monocytes can emigrate into tissues. Circulating half-life of monocytes in mice has been reported to range from 24 to 60 h ( Provencher Bolliger et al., 2010 ). Macrophages and dendritic cells can arise from monocytes or monocyte precursors depending on the local tissue microenvironment and cytokine stimulation ( Papenfuss, 2010 ), forming the mononuclear phagocytic system. Although present in low numbers, monocytes represent the third most numerous blood leukocytes in health, after neutrophils and lymphocytes. They are typically the largest of the leukocytes in routine blood films. Monocytes play a major role in resolution of infectious processes, particularly those involving larger or more complex organisms, such as fungi and protozoa, and in the clearance of other foreign material from the body. Following cardiac blood collection in mice, tissue macrophages may be inadvertently collected and may rarely be observed during blood smear evaluation. 12.11.2.3.1 Increases in monocyte counts (monocytosis) 12.11.2.3.1.1 Catecholamine-induced Although less commonly observed than increases in neutrophil or lymphocyte counts, blood monocyte counts may be modestly increased under the stimulation of endogenous catecholamines. This effect is most likely mediated by rapid shifting of monocytes from the marginating pool to the circulating pool ( Everds et al., 2013a ). 12.11.2.3.1.2 Glucocorticoid-induced Endogenous glucocorticoids classically cause increases in blood monocyte counts. However, these increases in monocyte counts are less consistently observed than decreases in lymphocyte counts or increases in neutrophil counts, and no perceptible change in monocyte counts may occur ( Hall, 2013 , Everds et al., 2013a ). There have also been reports of decreases in monocyte counts attributable to endogenous glucocorticoids, which may be associated with decreased production with prolonged glucocorticoid exposure ( Thompson and van Furth, 1973 ), or may be transient and followed by an increased in monocyte counts ( Steer et al., 1997 , Rinehart et al., 1975 ). 12.11.2.3.1.3 Inflammation Increases in blood monocyte counts occur with both acute and chronic inflammation, and such inflammatory increases in monocytes may be associated with both infectious and noninfectious etiologies. Tuberculosis and other mycobacterial infections are commonly associated with increases in monocyte counts which may represent an increased tissue demand for macrophages ( Lichtman, 2016a ). Increases in monocyte counts have also been associated with bacterial endocarditis and sepsis, osteomyelitis, various pyogranulomatous diseases, candidiasis, viral infections including cytomegalovirus and influenza, and several parasitic diseases including pneumocystosis, Entopolypoides macacai infection in old world monkeys and apes, and dirofilariasis in dogs ( Lichtman, 2016a , Magden et al., 2015 , Schultze, 2010 ). However, increases in monocyte counts may be less commonly related to malaria, leishmaniasis, and rickettsial diseases in people than previously thought ( Lichtman, 2016a ). Noninfectious causes of increases in blood monocyte counts include inflammatory bowel disease, ulcerative gastritis, myocardial infarction, and parturition ( Lichtman, 2016a ). 12.11.2.3.1.4 Immune disorders Numerous immune disorders are associated with increases in blood monocyte counts. Immune-mediated destruction of erythrocytes or neutrophils often has concurrent increases in monocyte counts ( Schultze, 2010 , Stockham and Scott, 2008a ). The increase in monocyte count associated with immune-mediated neutropenia may be due to cytokine stimulation of the common precursor of both granulocytes and monocytes ( Stockham and Scott, 2008a ). Rebound increases in neutrophil counts during recovery from agranulocytosis often have concurrent increases in monocyte counts ( Lichtman, 2016a , Schultze, 2010 ), which may also be due to stimulation of the common granulocyte and monocyte precursor. Systemic lupus erythematosus ( Budman and Steinberg, 1977 ), rheumatoid arthritis ( Buchan et al., 1985 ), sarcoidosis ( Goodwin et al., 1979 ), and other connective tissue diseases ( Lichtman, 2016a ) may also cause increases in monocyte counts. 12.11.2.3.1.5 Neoplasia Hematopoietic malignancies involving the monocytic lineage are rare, but are commonly associated with increased blood monocyte counts. These hematopoietic neoplasms include myelodysplastic syndromes (e.g., chronic myelomonocytic leukemia), acute monocytic leukemia, acute myelomonocytic leukemia, dendritic cell leukemia, and malignant histiocytosis ( Schultze, 2010 , Tefferi and Vardiman, 2009 , Villeneuve et al., 2008 , Sun et al., 2007 , Lichtman and Segel, 2005 , Castoldi and Rigolin, 2001 , Rigolin et al., 1997 , Laurencet et al., 1994 ). Blood monocytes associated with these hematopoietic neoplasms frequently have abnormal morphologic features and may be accompanied by monoblasts or promonocytes in circulation. Increases in monocyte counts related to neoplasia are not limited to hematopoietic neoplasms of the monocytic lineage. Increases in monocyte counts have also been described in association with a wide spectrum of lymphoproliferative neoplasms, soft tissue sarcomas, hemangiosarcomas, chondrosarcomas, rectal polyps, and colon cancer ( Lichtman, 2016a , Schultze, 2010 , Melichar et al., 2001 , Ruka et al., 2001 ). In one study, over half of the patients with solid tumor malignancies were reported to have concurrent increases in monocyte counts, which were independent of tumor metastasis ( Barrett, 1970 ). 12.11.2.3.1.6 Xenobiotic-induced Administration of exogenous glucocorticoids classically causes increases in monocyte counts, although this effect may be inconsistently observed and no change in blood monocyte counts may occur. Some studies have demonstrated that transient decreases in monocyte counts occur immediately following administration of exogenous glucocorticoids with subsequent increases in monocyte counts ( Rinehart et al., 1975 , Steer et al., 1997 ), but others have only reported increases in monocyte counts with exogenous glucocorticoid administration ( Barker et al., 2012 ). Administration of exogenous cytokines may also result in increases in blood monocyte counts. Such increases in blood monocyte counts have been observed with G-CSF ( Ranaghan et al., 1998 , Liu et al., 2004 ), GM-CSF ( Schmitz et al., 1994 ), M-CSF ( Weiner et al., 1994 , Cole et al., 1994 ), or IL-10 administration ( Chernoff et al., 1995 ). M-CSF administration-related increases in monocyte counts have been associated with concurrent, dose-limiting decreases in platelet counts ( Weiner et al., 1994 , Cole et al., 1994 ). Increases in monocyte counts have also been reported in a few patients associated with pseudolymphoma syndrome caused by therapy with several anticonvulsants, including phenytoin, phenobarbital, and valproic acid ( Choi et al., 2003 ). 12.11.2.3.2 Decreases in monocyte counts (monocytopenia) Decreases in monocyte counts may be difficult to detect due to the relatively low blood monocyte counts in humans and most laboratory animal species, particularly if reference intervals or historical control ranges, rather than values from a concurrent control group, are being used for determining if changes in blood monocyte counts are present. In nonclinical toxicology studies, comparison to concurrent controls or pretest may enable detection of more subtle decreases in monocyte counts. 12.11.2.3.2.1 Immune-mediated Immune-mediated decreases in monocyte counts are typically not observed in isolation and are associated with causes of pancytopenia, such as aplastic anemia. With aplastic anemia, destruction of early hematopoietic precursors results in loss of production of most or all hematopoietic cell lineages and subsequent development of severe decreases in multiple blood component counts. Aplastic anemia can also occur with nonimmune-mediated conditions, such as anorexia nervosa ( Abella et al., 2002 ). 12.11.2.3.2.2 Inherited causes Uncommonly there are cases of inherited marked decreases in blood monocyte counts. In humans, an autosomal dominant inheritance pattern has been associated with decreased monocyte counts with a resulting increased susceptibility to mycobacteria and a variety of other infectious agents ( Vinh et al., 2010 ). Mutations in GATA2, a transcription factor that regulates hematopoietic cell gene expression and integrity, have been reported as a cause for autosomal dominant decreases in blood monocyte counts ( Camargo et al., 2013 , Hsu et al., 2011 ). 12.11.2.3.2.3 Neoplastic Decreases in blood monocyte counts may occur secondary to hematologic malignancies or metastatic nonhematology malignancies that efface the bone marrow. Neoplastic myelophthisis results in decreased production of normal hematopoietic cells and therefore blood component counts, including monocytes. Examples of such reported conditions include hairy cell leukemia ( den Ottolander et al., 1983 , Ratain et al., 1985 ) and chronic lymphocytic leukemia ( De Rossi et al., 1991 ). 12.11.2.3.2.4 Xenobiotic-induced Xenobiotic-induced bone marrow suppression can often cause decreases in blood monocyte counts in combination with decreases in other blood component counts. Causes include chemotherapeutic agents, such as discussed in xenobiotic-induced decreases in neutrophil counts, due to direct hematopoietic precursor cell toxicity and xenobiotics associated with aplastic anemia, such as chloramphenicol. Monocyte cytotoxicity has been reported with methylmetacrylate monomer used in joint replacement surgery ( Dahl et al., 1994 ). Lindane, a pesticide, has been reported to cause CFU-GM cytotoxicity ( Parent-Massin and Thouvenot, 1993 ). 12.11.2.4 Eosinophils Eosinophils share a common early myeloid precursor with neutrophils. Early proliferation of the eosinophil lineage appears to be largely due to stimulation of myeloid precursors with GM-CSF and IL-3, while IL-5 plays a critical role in terminal eosinophil differentiation and maturation ( Sanderson, 1992 ). Similar to other granulocytes, eosinophils are distributed into proliferating and maturing pools in the bone marrow, and into circulating and marginating pools in the blood. The bone marrow is the primary site of eosinophil production, although rat eosinophils migrate and complete their maturation in the spleen ( Young and Meadows, 2010 ). Early proliferating bone marrow eosinophils are usually indistinguishable microscopically from other early myeloid precursors because their characteristic granules do not begin to form until the late promyelocyte stage ( Radin and Wellman, 2010 ). The time for production of mature eosinophils from the myeloblast stage is 2–6 days and the half-life of mature eosinophils in circulation ranges from less than 1 to 24 h, although both of these transit times vary by species ( Young and Meadows, 2010 ). Eosinophils migrate into tissue from circulation, where they live for about 2 days unless stimulation occurs ( Young and Meadows, 2010 ). Eosinophil counts are normally low in most species, which can make detection of decreases in eosinophil counts difficult. Similar to detecting decreases in monocyte counts, comparison of treated groups with concurrent controls or pretest values in nonclinical toxicology studies will aid in the identification of changes in eosinophil counts. Eosinophils are morphologically distinct from other leukocytes due to their large, pink-staining cytoplasmic granules; however, species differences in granule size, shape, and staining properties exist. Blood eosinophil counts in health generally only exceed basophil counts. 12.11.2.4.1 Increases in eosinophil counts (eosinophilia) 12.11.2.4.1.1 Decreased glucocorticoids Although uncommon, decreases in endogenous glucocorticoid concentrations due to hypoadrenocorticism have been associated with mild increases in blood eosinophil counts ( Wardlaw, 2016 , Stockham and Scott, 2008a ). This effect is most likely due to the loss of glucocorticoid-associated shifting of blood eosinophils to the marginating pool as well as the lack of proapoptotic stimulation of glucocorticoids on eosinophil precursors. 12.11.2.4.1.2 Inflammation and Hypersensitivity Both acute and chronic inflammatory stimulation may result in increases in blood eosinophil counts along with increases in neutrophil, lymphocyte, and/or monocyte counts. However, inflammatory processes that stimulate primarily an eosinophil response may also occur. IL-5, eotaxin, and RANTES are cytokines and chemokines that selectively stimulate eosinophil responses ( Sampson, 2000 ). Some of the most common inflammatory processes involving eosinophils include parasite and allergy-induced inflammation. Parasitism is considered the most common cause of increases in blood eosinophil counts worldwide ( Wardlaw, 2016 ), of which helminth (e.g., nematode, trematode, or cestode) infections are the major cause ( Tefferi et al., 2006 , Leder and Weller, 2000 ). Inflammatory increases in eosinophil counts in response to helminths are cytokine (e.g., IL-5) mediated ( Valent, 2009 ), but IgE and histamine release from mast cells, anaphylatoxin (e.g., C5a) production from complement activation, helper T-cell activation, and direct stimulation of eosinophils with helminthic antigens also play a role in eosinophil recruitment ( Wardlaw, 2016 , Leder and Weller, 2000 , McEwen, 1992 ). Helminth migration through host tissues is a key factor in stimulating increases in blood eosinophils and tissue eosinophilic inflammation, and helminths that remain confined to the intestinal lumen may not result in an eosinophil response ( Leder and Weller, 2000 ). The degree of the eosinophil response and increases in blood eosinophil counts are also dependent on parasite burden, maturation, and distribution in tissues ( Leder and Weller, 2000 ). Ascariasis, strongyloidiasis, trichinosis, filariasis, and ancylosomiasis have all been associated with increases in eosinophil counts in humans ( Wardlaw, 2016 , Tefferi et al., 2006 , Leder and Weller, 2000 ), and many of these may also be observed in common laboratory species ( Schultze, 2010 , Magden et al., 2015 , Korenaga et al., 1991 , Ogilvie et al., 1980 ). Helminth infection in most purpose-bred laboratory animals is uncommonly observed during nonclinical toxicology studies due to breeding and housing facility biosecurity measures, but several less commonly used large animal species from other sources, such as farm pigs, calves, and sheep, have helminth infections more frequently observed. Infection with nonhelminth parasites may also cause increases in blood eosinophil counts. Ectoparasites, including fleas and ticks, have been associated with increase in eosinophil counts in dogs and cats and may be due to arthropod-related allergic or hypersensitivity reactions ( Schultze, 2010 , Valenciano et al., 2010 , Stockham and Scott, 2008a ). Several protozoal infections, including isosporiasis ( Jongwutiwes et al., 2002 , Junod, 1987 ) and toxoplasmosis ( Grant and Klein, 1987 ), may result in increases in blood eosinophil counts. However, protozoal agents that infect erythrocytes, such as Plasmodium and Babesia species, are generally not expected to result in altered blood eosinophil counts ( Tefferi et al., 2006 , Stockham and Scott, 2008a ). Some bacterial infections, including borreliosis ( Granter et al., 1996 ) and rickettsiosis ( Wardlaw, 2016 ), and several viral infections, including herpes virus and HIV ( Wardlaw, 2016 , Tietz et al., 1997 ), have also been associated with increases in blood eosinophil counts. Fungal infections that cause allergic inflammation, such as coccidiomycosis, candidiasis, and aspergillosis ( Wardlaw, 2016 ), may also cause increases in blood eosinophil counts. Allergic inflammation is another common cause of increases in blood eosinophil counts. Allergic conditions associated with increases in blood eosinophil counts include asthma, atopic dermatitis, and allergic rhinitis although increases in eosinophil counts with these conditions are usually mild ( Wardlaw, 2016 ). Allergic inflammation associated with immediate release of IgE is considered a type I hypersensitivity reaction. Some allergen-induced inflammation may be attributable to type IV (delayed-type or cell-mediated) hypersensitivity following T H activation with subsequent eosinophil recruitment. However, some differences have been observed between atopic dermatitis and classic type IV hypersensitivity ( Gaga et al., 1991 ), so not all T H -mediated allergic inflammation may represent a true type IV hypersensitivity reaction. As with most forms of inflammatory increases in blood eosinophil counts, production of cytokines and chemokines, such as IL-5 and eotaxin, appear to play a major role. In allergic asthma, activated T-helper type 2 (T H 2) cells release or stimulate the release of these cytokines and chemokines, resulting in recruitment and activation of eosinophils ( Rosenberg et al., 2013 ). Sensitization of the airways to ovalbumin with subsequent challenge in mice has mimicked many of the clinical and pathological features of allergic asthma, including the interaction of T H 2 cells and eosinophils ( Rosenberg et al., 2013 ). However, asthma encompasses a heterogeneous set of phenotypes and not all forms demonstrate clinical improvement in response to therapies targeting IL-5 or eosinophils ( Wegmann, 2011 ). While asthma is uncommon in most laboratory animal species, it can be experimentally induced in mice and may occur naturally in cats. Activation of T H 2 and T H 1 cells have been proposed to contribute to the pathology of atopic dermatitis, with the T H 2 activation being of particular relevance to the development of increases in blood eosinophil counts ( Grewe et al., 1998 ), similar to allergic asthma. Activation of T H 2 cells also plays a role in cytokine and chemokine elaboration with eosinophil recruitment in allergic rhinitis, although effects on eosinophils in allergic rhinitis are also mediated by histamine and IgE release from mast cells and histamine release from basophils ( Borish, 2003 ). Inflammatory increases in blood eosinophil counts can also be associated with paraneoplastic syndromes, likely related to increases in IL-5, which may be liberated by activated T H cells or directly by the neoplasm. Lymphoma, including both T- and B-cell lymphomas, is a common cause of paraneoplastic increases in eosinophil counts in multiple species, including humans, dogs, cats, and horses ( Wardlaw, 2016 , Davis and Rothenberg, 2014 , Valent, 2009 , Schultze, 2010 , Valenciano et al., 2010 , Stockham and Scott, 2008a , Marchetti et al., 2005 , Cave et al., 2004 , Duckett and Matthews, 1997 ). However, many nonlymphoid tumors have also been associated with paraneoplastic increases in blood eosinophil counts, including mammary carcinoma, hepatocellular carcinoma, squamous cell carcinoma, thymoma, nonsmall-cell lung cancer, and mast cell diseases including systemic mastocytosis and mast cell leukemia ( Schultze, 2010 , Balian et al., 2001 , Walter et al., 2002 , Pandit et al., 2007 , Valent, 2009 ). 12.11.2.4.1.3 Neoplastic Myeloid neoplasms can result in clonal eosinophil expansion and increases in blood eosinophil counts expansion ( Tefferi et al., 2006 ). Neoplastic increases in eosinophil counts have been associated with acute eosinophilic leukemia, chronic eosinophilic leukemia, chronic myeloid leukemia, and myelodysplastic syndrome ( Wardlaw, 2016 , Tefferi et al., 2006 , Schultze, 2010 ). Clonal increases in eosinophil counts may be difficult to distinguish from idiopathic hypereosinophilic syndrome, and cytogenetic analysis may be necessary; numerous cytogenetic abnormalities have been reported with clonal increases in eosinophil counts ( Tefferi et al., 2006 ). 12.11.2.4.1.4 Idiopathic There are numerous reports of idiopathic increases in blood eosinophil counts. Such conditions include eosinophilic esophagitis, eosinophilic gastroenteritis, eosinophilic myositis, eosinophilic cellulitis, and eosinophilic pneumonitis in people, dogs, and/or cats ( Wardlaw, 2016 , Stockham and Scott, 2008a ). Hypereosinophilic syndrome (HES) in people is another condition that falls under the umbrella of idiopathic increases in eosinophil counts. In HES, chronic increases in eosinophil counts are observed without evidence of an underlying causative condition. This condition is associated with marked tissue infiltration and eventual organ damage and failure ( Wardlaw, 2016 , Rosenberg et al., 2013 ). However, some patients with HES eventually develop either a lymphoid or myeloid neoplasm ( Wardlaw, 2016 ). 12.11.2.4.1.5 Spurious In mice, automated hematology analyzer-generated blood eosinophil counts may be falsely elevated by large platelet clumps present in the specimen ( O'Connell et al., 2015 ). Platelet clumping in mice is extremely common, and blood smear evaluation is often necessary to confirm the automated leukocyte differential count. 12.11.2.4.1.6 Xenobiotic-induced Beta adrenergic blocking agents may be associated with modest increases in eosinophil counts, and administration of propranolol has been demonstrated to prevent catecholamine-induced decreases in eosinophil counts ( Reed et al., 1970 , Koch-Weser, 1968 ). The antibiotic tetracycline has been associated with increased eosinophil counts in dogs ( Domina et al., 1997 ) and humans ( Ho et al., 1979 ). Therapeutic administration of IL-2 for renal cell carcinoma has also been reported to cause increased blood eosinophil counts ( Wardlaw, 2016 ). Administration of G-CSF and GM-CSF will also cause increases in blood eosinophil counts due to stimulation of common myeloid precursor proliferation. However, increases in eosinophil counts with these compounds will be small in comparison with the increases in blood neutrophil counts. Numerous reactions to xenobiotics can also cause increases in blood eosinophil counts. Acute generalized exanthematous pustulosis due to drugs such as aminopenicillins and diltiazem, as discussed with xenobiotic-induced increases in neutrophil counts, may be associated with concurrent increases in eosinophil counts ( Roujeau, 2005 ). Drug reaction with eosinophilia and systemic syndromes (DRESS) is a predominantly cutaneous manifestation of a drug hypersensitivity reaction. Numerous compounds have been associated with DRESS, including several anticonvulsant drugs, such as phenobarbital and phenytoin, allopurinol, minocycline, sulfonamides, gold salts, dapsone, and spironolactone ( Roujeau, 2005 ; Callot et al., 1996 , Tefferi et al., 2006 , Ghislain et al., 2004 ). 12.11.2.4.2 Decreases in eosinophil counts (eosinopenia) 12.11.2.4.2.1 Catecholamine-induced In contrast to neutrophil, lymphocyte, and monocyte counts, blood eosinophil counts decrease in response to increased endogenous catecholamines. These effects may be inconsistent and difficult to detect due to timing of blood collection relative to the rapid changes in eosinophil counts and the normally low blood eosinophil counts. The β-adrenergic effects of epinephrine are believed to be the cause of the decreases in blood eosinophil counts ( Koch-Weser, 1968 ). Catecholamines may also cause decreased release of eosinophils from bone marrow ( McEwen, 1992 ). 12.11.2.4.2.2 Glucocorticoid-induced Decreases in blood eosinophil counts are a classic feature of a glucocorticoid leukogram occurring in conjunction with decreases in lymphocyte counts and usually with increases in neutrophil counts. Blood eosinophils appear to be particularly responsive to the effects of glucocorticoids, and concurrent decreases in blood lymphocyte and eosinophil counts in common laboratory species used in nonclinical toxicology studies are a good indicator of stress ( Hall, 2013 ). Glucocorticoids cause shifts in blood eosinophils from the circulating to the marginating pool as well as decreased release of eosinophils from the bone marrow ( McEwen, 1992 ), and may also contribute to decreases in blood eosinophil counts from inhibition of prosurvival stimulation and direct induction of apoptosis ( Druilhe et al., 2003 , Wallen et al., 1991 ). 12.11.2.4.2.3 Inflammation Severe acute or overwhelming inflammation, such as associated with sepsis, may cause eosinopenia in conjunction with neutropenia. Studies in mice have demonstrated that the decrease in blood eosinophils associated with severe acute inflammation occurs more rapidly than increases in glucocorticoids ( Bass, 1975 ), and injection of material from an inflammatory exudate to adrenalectomized mice still resulted in deceases in eosinophil counts ( Bass, 1977 ), indicating the mechanism of eosinophil decrease in acute inflammation is independent of adrenal function. It is believed that acute inflammatory decreases in blood eosinophil counts are due to shifting of eosinophils from the circulating to the marginating pool and subsequent egress into tissues in response to chemotactic stimuli. Acute inflammation associated with fungal and viral infections also tends to cause decreases in eosinophil counts ( Leder and Weller, 2000 ). 12.11.2.4.2.4 Xenobiotic-induced Although decreases in eosinophil counts are relatively uncommon with the exception of the administration of exogenous glucocorticoid-based xenobiotics, they may also be observed in cases of xenobiotic-induced bone marrow suppression and aplastic anemia. In these situations, the decreases in eosinophil counts do not occur in isolation but are generally observed with concurrent decreases in neutrophil, lymphocyte, and/or monocyte counts. Xenobiotic causes of bone marrow suppression classically include chemotherapeutic agents, while xenobiotics that can sporadically be associated with aplastic anemia include chloramphenicol and anticonvulsants such as phenytoin. Other xenobiotics associated with bone marrow suppression or aplastic anemia are described in more detail in the previous leukocyte subtype sections. 12.11.2.5 Basophils Basophils develop in the bone marrow from uncommitted myeloid progenitor cells that differentiate into committed basophil progenitors. However, intermediate stages in basophil production have not been definitively identified, and there is evidence that basophils may share a common precursor with eosinophil, mast cells, or megakaryocytes ( Radin and Wellman, 2010 , Arock et al., 2002 ). Stimulation with IL-3 plays a major role in the terminal differentiation of basophils, while GM-CSF and IL-5 also play a role in basophil differentiation ( Arock et al., 2002 , Mayer et al., 1989 ). There is also some evidence for stem cell factor (SCF) and IL-4 stimulation in basophil differentiation ( Pohlman, 2010 , Favre et al., 1990 ). As studies in mice have shown that normal blood basophil counts may be maintained in the absence of IL-3, a required role for IL-3 in basophil production is not apparent ( Lantz et al., 1998 ). Specific factors leading to terminal differentiation have not been identified for the basophil lineage, and basophil differentiation may in fact represent a default leukocyte differentiation pathway ( Arock et al., 2002 ). In blood, basophils are distributed into circulating and marginating pools, similar to other granulocytes. The circulating half-life of basophils is short (about 6 h), and they rapidly migrate into tissues where they have a much longer survival (up to 2 weeks) ( Hirai et al., 1997 , Pohlman, 2010 ). Basophils are the least numerous leukocyte in blood, and in health usually compose approximately 0.5% or less of the blood leukocyte differential ( Pohlman, 2010 , Galli et al., 2016 ). Automated hematology analyzer differentials may provide low estimates of actual blood basophil counts in humans, and flow cytometric methods may provide a more accurate estimate ( Meintker et al., 2013 , Amundsen et al., 2012 , Ducrest et al., 2005 ). Automated hematology analyzer counts in dogs and cats have been demonstrated to be inaccurate ( Pohlman, 2010 , Lilliehöök and Tvedten, 2011 , Tvedten and Lilliehöök, 2011 ). However, basophils in rabbits appear to be detected with automated methods ( Lilliehöök and Tvedten, 2011 ). Due to the evidence for low or inaccurate basophil counts in humans, dogs, and cats, it is unclear how accurate automated basophil counts are in nonhuman primates and rodents used in nonclinical toxicology studies. 12.11.2.5.1 Increases in basophil counts (basophilia) 12.11.2.5.1.1 Inflammation Increases in blood basophil counts may be associated with inflammatory stimuli, although decreases in basophil counts are more commonly observed ( Galli et al., 2016 ). Increases in basophil counts have been associated with infectious, allergic, and paraneoplastic inflammatory conditions. Parasitism is a relatively frequent cause of inflammatory increases in basophil counts, which are almost always observed in conjunction with increases in blood eosinophil counts. Many endoparasites, predominantly helminths with tissue exposure or migration, and ectoparasites, including a variety of arthropods, have been associated with concurrent increase in eosinophil and basophil counts ( Schultze, 2010 , Pohlman, 2010 , Voehringer, 2009 , Falcone et al., 2001 , Brown and Rosalsky, 1984 , Roth and Levy, 1980 , Ogilvie et al., 1980 ). Infectious agents other than parasites have also been reported to cause increases in basophil counts. Several viral etiologies associated with increases in basophil counts in humans include influenza, chickenpox, and smallpox viruses ( Galli et al., 2016 ). Several bacterial infections may also cause increases in blood basophil counts, including tuberculosis ( Galli et al., 2016 ) and infection with Helicobacter pylori ( Karttunen et al., 1996 ). Allergic inflammation that involves IgE and/or causes increases in eosinophil counts typically also causes increases in blood basophil counts. Immediate or delayed hypersensitivity may cause increases in basophil counts, although immediate hypersensitivity has also been associated with decreases in basophil counts in some cases; whether basophil counts increase or decrease may represent a distinction between allergic sensitization and an immediate allergic reaction ( Shelley and Parnes, 1965 ). Food or inhalant allergies, such as ragweed pollen ( Otsuka et al., 1986 ), can frequently cause increases in blood basophil counts ( Galli et al., 2016 , Pohlman, 2010 ). Allergic inflammation also occurs with insect stings or bites ( Stockham and Scott, 2008a ) and probably H . pylori infection ( Karttunen et al., 1996 ). Paraneoplastic increases in basophil counts have been associated with several neoplastic processes, including disseminated mast cell neoplasia ( Schultze, 2010 , Stockham and Scott, 2008a ) and carcinomas ( Galli et al., 2016 ). Increases in blood basophil counts may also be observed with lymphomatoid granulomatosis ( Schultze, 2010 , Stockham and Scott, 2008a ), and myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis ( Galli et al., 2016 , Stockham and Scott, 2008a ). Other miscellaneous causes of inflammatory increases in blood basophil counts include ulcerative colitis ( Juhlin, 1963a ), the systemic mast cell disorder urticaria pigmentosa ( Asboe-Hansen, 1960 ), juvenile rheumatoid arthritis ( Athreya et al., 1975 ), and immunological responses causing acute rejection of some tissue grafts ( Tikkanen et al., 2001 ). 12.11.2.5.1.2 Endocrinopathy Several endocrinopathies have been associated with increases in blood basophil counts. These endocrinopathies include hypothyroidism (myxedema) and diabetes mellitus ( Galli et al., 2016 , Shelley and Parnes, 1965 ). It has been suggested that the increases in basophil counts are secondary to hyperlipidemia associated with the endocrinopathies, but supporting mechanistic evidence is scant ( Pohlman, 2010 , Schultze, 2010 ). 12.11.2.5.1.3 Neoplasia Although relatively rare, chronic myelogenous leukemia is frequently associated with increases in blood basophil counts ( Spiers et al., 1977 ), in which the blood basophils have been demonstrated through cytogenetic analysis to arise from the neoplastic clone ( Goh and Anderson, 1979 ). However, there is some suggestion that chronic basophilic leukemia may be a separate process than basophilic chronic myeloid or granulocytic leukemia ( Pardanani et al., 2003 ). Chronic leukemia associated with clonal increases in blood basophil counts also has the potential to undergo blast transformation. Acute basophilic leukemia may also occur but is rare ( Duchayne et al., 1999 ). Other forms of acute myelogenous leukemia may also be associated with increases in basophil counts ( Galli et al., 2016 ). 12.11.2.5.1.4 Xenobiotic-induced Administration of G-CSF or GM-CSF may cause modest increases in blood basophil counts along with the more pronounced increases in neutrophil and eosinophil counts through stimulation of granulocyte production. Administration of phenylhydrazine as a rat model of hemolysis has been associated with increases in blood leukocyte counts, including basophil counts ( Criswell et al., 2000 ). Perhaps the most common xenobiotic-induced increases in blood basophil counts occur as a hypersensitivity or allergic reaction, and reports have included associated administration of heparin, penicillin, and novobiocin ( Schultze, 2010 , Shelley, 1963 ). 12.11.2.5.2 Decreases in basophil counts (basopenia) The lower reference limit of historical control data in common laboratory species may include basophil counts of 0 cells μL − 1 . Due to these normally low blood basophil counts in health, decreases in basophil counts may not be detectable or recognizable. However, the following sections list some conditions in which decreased basophil counts have been reported. 12.11.2.5.2.1 Glucocorticoid-induced Increases in glucocorticoid concentrations, particularly if prolonged, cause decreases in blood basophil counts ( Shelley and Parnes, 1965 , Juhlin, 1963b , Boseila, 1963 ). Similar to glucocorticoid-induced decreases in eosinophil counts, there is likely shifting of blood basophils from the circulating to marginating pool. There is also evidence that glucocorticoids cause migration of basophils from circulation into tissues and decrease recirculation from tissue back into blood ( Wald et al., 1991 ). Direct lytic effects on blood or tissue basophils and suppression of basophil production in the bone marrow may also contribute ( Boseila, 1963 ). Myocardial infarction, which has been associated with decreases in blood basophil counts, may be mediated by effects of chronic stress secondary to the ischemic event ( Juhlin, 1963c ). 12.11.2.5.2.2 Inflammation and hypersensitivity Severe acute or overwhelming inflammation can lead to a decrease in basophil counts along with decreases in other blood leukocyte counts. A relationship between endotoxemia and decreases in basophil counts has been supported by reductions in blood basophil counts in rabbits administered endotoxin from Salmonella typhi ( Goncharova and Krylova, 1967 ). Also, inflammatory leukocytosis not associated with overwhelming inflammation is often associated with decreases in basophil counts ( Galli et al., 2016 ). Type I hypersensitivity reactions, which are mediated by rapid IgE release, are commonly associated with decreases in both eosinophil and basophil counts. However, type IV hypersensitivity (cell-mediated) causing histamine release from mast cells may also be associated with decreases in basophil counts. Such hypersensitivity-related decreases in basophil counts can be observed with anaphylaxis and urticaria ( Galli et al., 2016 , Grattan et al., 1997 , Grattan et al., 2003 , Shelley and Juhlin, 1961 ). 12.11.2.5.2.3 Endocrinopathy Hyperthyroidism (thyrotoxicosis) is reported to cause decreases in blood basophil counts ( Shelley and Parnes, 1965 , Juhlin, 1963c ). However, the mechanism of this decrease has not been clearly demonstrated. 12.11.2.5.2.4 Xenobiotic-induced Exogenous administration of glucocorticoids will result in decreases in blood basophil counts, similar to decreases caused by endogenous glucocorticoids. Also, administration of thyroid hormones or thyroid stimulating hormone to healthy individuals has been reported to cause decreases in blood basophil counts, consistent with the decreases in basophil counts observed with naturally occurring hyperthyroidism ( Boseila, 1963 ). Acute hypersensitivity reactions to xenobiotics may cause decreases in blood basophil counts. Xenobiotic-induced urticaria, angioedema, and anaphylactic reactions are IgE-mediated type I hypersensitivity reactions, and have been associated with angiotensin-converting enzyme (ACE) inhibitors and various NSAIDs ( Roujeau, 2005 ). Due to the IgE-mediated nature of these reactions, these xenobiotics would have the potential to cause concurrent decreases in blood basophil counts, although IgE-related increases in basophil counts could also occur as described previously. Menthol has also been associated with urticaria and decreases in basophil counts ( Papa and Shelley, 1964 ). Decreases in blood basophil counts may also occur along with decreases in other leukocyte counts associated with xenobiotic-induced bone marrow suppression and aplastic anemia. As a class, chemotherapeutic agents may cause bone marrow suppression resulting in decreases in multiple leukocyte lineages in blood, including basophils. Xenobiotics implicated in causing aplastic anemia include chloramphenicol, anticonvulsants such as phenytoin and carbamazepine, gold-based compounds, penicillamine, and phenylbutazone ( Bloom and Brandt, 2008 , Kaufman et al., 1996 ). 12.11.2.6 Large Unclassified or Other Cells Some automated hematology analyzers, such as the Siemens Healthcare ADVIA systems, include a "large unclassified cell" (LUC) or "other" cell category in the leukocyte differential. These cells are generally large with no or minimal myeloperoxidase activity, and do not fall within the predefined species' gating parameters for typically identified leukocyte subtypes. These cells do not represent a distinct cell type, but are most commonly large and/or reactive lymphocytes or monocytes, and increases in LUC counts may be observed with any conditions resulting in increases in blood lymphocyte or monocyte counts. In species where automated basophil counts are not reliable, increases in blood basophil counts may also appear as an increase in LUC counts ( Lilliehöök and Tvedten, 2011 ). Acute leukemia, typified by increases in hematopoietic blast cells in bone marrow and circulation, almost always results in increases in LUC counts. In the presence of high LUC counts, blood smear evaluation should be performed to assess the leukocyte differential and morphologic appearance of the blood leukocytes. Occasionally there may be mast cells observed in the blood smears of dogs, cats, or laboratory rodents. Mast cells in blood (mastocytemia) typically occur in low in numbers that do not significantly affect the automated leukocyte differential. Such mastocytemia may occur along with an inflammatory response. However, systemic mastocytosis or mast cell leukemia may cause notable increases in blood mast cells, and mast cell neoplasia is the most common cause of circulating mast cells in cats ( Skeldon et al., 2010 ). Blood smear evaluation is required for enumerating mast cells as part of a leukocyte differentiation. 12.11.2.1 Neutrophils The production of neutrophils, or granulopoiesis, occurs predominantly in the bone marrow, although extramedullary hematopoiesis may contribute, particularly in rats and mice, and if increased tissue demand for granulocytes or bone marrow disease is present. Stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-6 promotes the differentiation of common myeloid progenitor cells to granulocyte/monocyte progenitor cells, and subsequent stimulation with granulocyte colony-stimulating factor (G-CSF) promotes differentiation, proliferation, and maturation of granulocytes from myeloblasts to mature segmented neutrophils ( Radin and Wellman, 2010 ). Granulocyte pools in the bone marrow can be divided into proliferating (mitotic) and maturing (postmitotic) pools. The proliferating pool encompasses the most immature myeloid precursors: myeloblasts, promyelocytes, and myelocytes. The maturing pool includes the later stages of metamyelocytes, band neutrophils, and mature segmented neutrophils. The mature segmented neutrophil population within the maturing pool is also considered the bone marrow storage pool, and acts as a reserve store of neutrophils that replenish blood stores following normal turnover and can be quickly mobilized to the blood in times of increased tissue demand. In health, mammalian blood neutrophils are mature, segmented neutrophils. They are distributed in two pools within the blood vessels, the circulating pool and the marginating pool. Circulating pool neutrophils flow freely through the vessel lumen and are sampled during blood collection. Marginating pool neutrophils are in contact with endothelial cells and temporarily adhere to or roll along the vessel wall. Neutrophils may shift between the circulating and marginating pools. The lifespan of a neutrophil in circulation is short (approximately 7 h half-life in blood), and ultimately neutrophils migrate into tissues where they survive for a few days ( Smith, 2016 ). Neutrophils are part of the innate immune system and primarily function as phagocytes to remove and destroy invading pathogens, but also can secrete proinflammatory and chemotactic mediators to enhance responses of both the innate and adaptive immune systems. In blood, neutrophils are typically the most numerous leukocyte in humans, rhesus monkeys, African green monkeys, and dogs. However, many New World monkeys tend to have lymphocyte counts greater than neutrophil counts ( Provencher Bolliger et al., 2010 ), and cynomolgus monkeys tend to initially have predominantly lymphocytes in circulation when they are young but shift to predominantly neutrophils with maturity ( Sugimoto et al., 1986 ). Neutrophil counts are lower than lymphocyte counts in rats and mice. Blood neutrophil counts may increase or decrease depending on the stimulus or insult. Clinically, increases in neutrophil counts above expected values in health (usually conveyed in a reference interval) may be referred to as neutrophilia or granulocytosis, while decreases in neutrophil counts may be called neutropenia, granulocytopenia, or, if severe, agranulocytosis. However, in nonclinical toxicology studies, the convention is to not use such clinical terminology to describe alterations caused by a test article. Referring to changes as increases or decreases in neutrophil counts is the standard for nonclinical toxicology studies, and this terminology is used throughout this article. However, clinical terms are also provided for reference. 12.11.2.1.1 Increases in neutrophil counts (neutrophilia, granulocytosis) 12.11.2.1.1.1 Catecholamine-induced Acute, transient increases (typically  50,000 cells μL − 1 , typically due to a marked neutrophilia with a left shift that remains orderly and may or may not have morphologic changes indicative of rapid granulopoiesis ( Schultze, 2010 , Sakka et al., 2006 ). Extreme neutrophilia typically has > 100,000 cells μL − 1 and evidence of a left shift. The terms leukemoid reaction and extreme neutrophilia are most appropriately applied retrospectively, after the possibility for hematopoietic neoplasia has been excluded. Differentiation of a leukemoid response or extreme neutrophilia from chronic myelogenous leukemia or chronic neutrophilic leukemia includes CBC, blood smear, and bone marrow evaluations in most species, and may also include leukocyte alkaline phosphatase activity, immunophenotyping, cytogenetic analysis (e.g., evaluation for bcr–abl translocation), serum G-CSF, and clonality evaluations in humans ( Schultze, 2010 , Sakka et al., 2006 ). Leukemoid reactions have been associated with carcinomas of various origins, including renal and pulmonary carcinomas, Hodgkin's lymphoma, melanoma, and sarcomas, and may be attributable to aberrant production of proinflammatory mediators by the neoplasm, such as G-CSF, GM-CSF, or IL-6 ( Sakka et al., 2006 ). Leukemoid reactions have also been reported in F334/N rats affected by large granular cell leukemia ( Car et al., 2006 ). However, leukemoid reactions may also be associated with infectious processes, including disseminated tuberculosis, Clostridium difficile colitis, severe shigellosis ( Sakka et al., 2006 ), chronic localized suppurative lesions such as pyometra, pleuritis, and internal abscesses ( Schultze, 2010 , Stockham and Scott, 2008a ). Leukemoid reactions may also be seen secondary to severe hemorrhage or immune-mediated hemolytic anemia ( Schultze, 2010 , Sakka et al., 2006 ). 12.11.2.1.1.4 Inherited leukocyte adhesion deficiencies Increases in neutrophil counts associated with deficiencies in leukocyte adhesion molecules may manifest as a leukemoid response or extreme neutrophilia. Adhesion molecules expressed on neutrophils are responsible for neutrophil margination, rolling along vessel walls, and emigration into tissues. L-selectin (CD62L) mediates low-affinity initial binding of leukocyte to endothelial cells, while integrins, including CD11b/CD18 (Mac-1), mediate firm adhesion to endothelial cells and ligands in the extracellular matrix ( Muller, 2012 ). Neutrophils constitutively express CD11b/CD18. A deficiency of this integrin (leukocyte adhesion deficiency [LAD] type 1) results in the failure of neutrophils to emigrate to tissues, and may result in severe, recurrent bacterial infections ( Arnaout, 1990 ). LAD type 2 is due to an inherited disorder of fucose metabolism, resulting in the lack of selectin ligands expressed on neutrophils and therefore results in immunodeficiency from a failure of selectin-mediated neutrophil rolling along vessel walls ( Marquardt et al., 1999 ). Leukocyte adhesion deficiencies have been reported in humans, dogs, mice, and Holstein cattle ( Arnaout, 1990 , Marquardt et al., 1999 , Gu et al., 2004 ). 12.11.2.1.1.5 Neoplasia Neoplasms involving hematopoietic cells naturally occur with relatively low frequency. In general, such neoplastic processes may be observed as background findings in rats and mice during longer toxicity studies (e.g., carcinogenicity studies), but are uncommon in nonrodent species during toxicity studies ( Smith et al, 2002 ). Increases in neutrophil counts may be observed as part of the neoplastic processes of chronic myeloid leukemia (CML) or chronic neutrophilic leukemia (CNL). Leukocytosis in CML is ≥ 25,000 cells μL − 1 with increases in all stages of neutrophilic myeloid cells in blood, which are typically accompanied by increases in monocyte counts, eosinophil counts, basophil counts, platelet counts, and possibly increases in nucleated erythroid precursors ( Sawyers, 1999 ). In contrast to a leukemoid response, the left shift associated with CML tends to be less orderly, with greater numbers of earlier stages of myeloid cells in circulation along with the band and segmented neutrophils. Morphologic features indicative of dysplasia may be seen, and cytoplasmic features indicative of rapid granulopoiesis may be observed in neutrophils associated with CML. Bone marrow findings include hypercellularity with increased myeloid to erythroid ratios where myeloblasts and promyelocytes make up  80% segmented and band neutrophils ( Uppal and Gong, 2015 ). These neutrophils may or may not have cytoplasmic changes indicative of rapid neutropoiesis. Unlike CML, the increases in neutrophil counts associated with CNL are typically not associated with morphologic features of dysplasia, the presence of earlier stages of myeloid cells (i.e. myeloblasts, promyelocytes, myelocytes, and metamyelocytes), increases in monocyte, eosinophil, or basophil counts, or increases in nucleated erythroid precursors ( Uppal and Gong, 2015 ). Bone marrow changes associated with CNL include hypercellularity with increased myeloid to erythroid ratios, where the myelocyte through band neutrophil stages are increased without apparent increases in myeloblasts or promyelocytes ( Uppal and Gong, 2015 ). As such, differentiation of CNL from leukemoid reactions or extreme neutrophilia may be difficult, and affected patients should be carefully evaluated to exclude causes of neutrophilia not associated with hematopoietic malignancy. 12.11.2.1.1.6 Xenobiotic-induced There are numerous xenobiotics that can increase blood neutrophil counts, typically through similar mechanisms as described for naturally occurring increases in neutrophil counts. Several examples of these compounds are described later. Administration of exogenous catecholamines, such as epinephrine, or some adrenergic agonists has similar effects as those mediated by endogenous catecholamines. Shifting of neutrophils from the marginating to the circulating pools is responsible for the increases in blood neutrophil counts, potentially from altered adhesion molecule expression or hemodynamic forces. Leukocyte subtypes have different receptors modulating their adrenergic effects, and neutrophils appear to be primarily affected by α-adrenergic receptors ( Benschop et al., 1996 ). Exogenous glucocorticoids, such as prednisolone, dexamethasone, and betamethasone, will mediate increases in neutrophil counts through similar mechanisms as endogenous glucocorticoids. In brief, these increases in neutrophil counts are due to shifting of neutrophils from the marginating to circulating pool, release of neutrophils from the bone marrow storage pool, and expansion of granulopoiesis in the bone marrow. The dose and duration of the glucocorticoid therapy may alter the proportional contribution of each of these effects to the increases in neutrophil counts. Heparin-induced increases in neutrophil counts are reported in a small percentage of patients receiving heparin therapy, and the mechanisms are likely multifactorial. Heparin may cause shifting of neutrophils to the circulating pool from the marginating pool due to decreased expression of selectins ( Zhang et al., 2012 ). Heparin-related release of CXCL12, a chemokine that is constitutively expressed by bone marrow stromal cells and plays a role in bone marrow neutrophil retention and leukocyte trafficking, may alter bone marrow and circulating CXCL12 gradients and promote release of neutrophils from the bone marrow storage pool, contributing to the increases in circulating neutrophil counts ( Zhang et al., 2012 ). Administration of G-CSF or GM-CSF may be used as a rescue therapy following chemotherapy that causes neutropenia and compromises the immune system's ability to respond to infectious agents. While the goal for treatment in chemotherapy patients is to increase blood neutrophil counts to normal levels and not to achieve increased neutrophil counts, repeated or high dose administration of G-CSF or GM-CSF-related compounds to healthy laboratory animals results in markedly increased neutrophil counts. Typically these changes are associated with a left shift and cytoplasmic changes indicative of rapid neutropoiesis. Dysplastic changes may also occur, most commonly recognized by the presence of unusually large neutrophils or hypersegmented neutrophils in circulation. Several xenobiotics appear to induce increases in neutrophil counts by stimulating increases in endogenous G-CSF or an inflammatory stimulus. Early in the course of treatment with clozapine, an antipsychotic agent, rats have been shown to have spikes in G-CSF with concomitant increases in bone marrow granulopoiesis and release of less mature neutrophils into circulation ( Lobach and Uetrecht, 2014 ). However, other xenobiotics may directly cause an inflammatory stimulus and increase in endogenous proinflammatory stimuli, such as G-CSF and IL-1, which result in increases in neutrophil counts. For example, lithium, which has been used in treatment of bipolar disorder and in combination with antidepressants, also appears to cause increased neutrophil counts due to increased G-CSF ( Focosi et al., 2009 ). Xenobiotic-induced increases in neutrophil counts may be observed in conjunction with several cutaneous drug reactions that result in inflammatory stimuli. Acute generalized exanthematous pustulosis is a drug-related skin reaction that generally occurs within 2 days of drug administration but resolves spontaneously within 15 days ( Roujeau, 2005 ). Increases in eosinophil counts may or may not be observed along with the increases in neutrophil counts. Xenobiotics classically associated with this syndrome include diltiazem and antibiotics such as aminopenicillins and pristinamycine ( Roujeau, 2005 ). Acute febrile dermatosis (Sweet's syndrome) has also been associated with elevations in circulating neutrophil counts, and drug-induced forms of this condition have been variably linked to a variety of compounds, including GM-CSF, trimethoprim–sulfamethoxazole, minocycline, celecoxib, furosemide, and azathioprine ( Rochet et al., 2013 , Saavedra et al., 2006 ). 12.11.2.1.2 Decreases in neutrophil counts (neutropenia, granulocytopenia, agranulocytosis) 12.11.2.1.2.1 Overwhelming or severe acute inflammation Although inflammation often causes increases in neutrophil counts, an overwhelming or severe acute inflammatory stimulus may result in decreases in neutrophil counts. Such an inflammatory stimulus, sometimes also referred to as peracute inflammation, is commonly observed with bacterial sepsis. Decreases in neutrophil counts are a result of emigration to tissues that exceed the bone marrow capacity to release neutrophils. Proinflammatory mediators and chemoattractants stimulate increased neutrophil margination, firm adhesion to endothelial cells, and emigration into tissue, which shortens neutrophil circulating half-lives and depletes the circulating neutrophil pool. As neutrophils are released from the bone marrow and the storage pool is depleted, recruitment of immature neutrophils into circulation results in a left shift in which the number of immature forms exceeds the number of mature segmented neutrophils (degenerative left shift). If the stimulus persists long enough, granulocytic hyperplasia of the bone marrow will occur and the numbers of circulating neutrophils will increase and shift toward greater numbers of mature than immature neutrophils. 12.11.2.1.2.2 Endotoxemia Decreases in neutrophil counts may be observed as a result of Gram-negative bacterial infections that release endotoxin. Endotoxemia has been demonstrated to increase expression of the integrin CD11b/CD18 and decreased expression of L-selectin on neutrophils ( Wagner and Roth, 1999 ), mediating a shift from circulating to marginating pools with firm adhesion to endothelial cells. There is evidence that this upregulation of CD18 contributes to neutrophil vascular sequestration in the lungs and liver, but there is also evidence for other mechanisms, such as cytoskeletal changes that alter neutrophil deformability, causing the initial vascular sequestration of neutrophils that is consistently observed with endotoxemia ( Wagner and Roth, 1999 ). The decreases in neutrophil counts associated with endotoxemia tend to be rapid but transient, and detection of these decreases in neutrophil counts will depend on timing of the blood collection in relation to endotoxemic and other concurrent or subsequent proinflammatory stimuli. 12.11.2.1.2.3 Viral-induced Decreases in neutrophil counts may be associated with viral infections, although viral-specific mechanisms vary. Decreased neutrophil counts associated with parvovirus likely represent both a primary effect due to infection causing death of rapidly dividing hematopoietic precursors in the bone marrow, as well as a secondary increase in tissue demand from disease and loss of integrity of the intestinal tract. Decreases in neutrophil counts associated with human immunodeficiency virus (HIV), infectious hepatitis, and infectious mononucleosis in people have also been associated with decreased or ineffective neutrophil production due to infection of hematopoietic precursors ( Dale and Welte, 2016 , Lima et al., 2006 ). With measles and dengue virus, decreases in neutrophil counts have been observed with endothelial cell alterations leading to increased adhesion of neutrophils and therefore a shift from the circulating to the marginating pool ( Dale and Welte, 2016 ). Decreases in blood neutrophil counts may also result from decreased neutrophil production, as in acquired viral immunodeficiency. Immunodeficiency syndromes with decreased neutrophil counts have been reported with infections by HIV in people, simian immunodeficiency virus (SIV) or simian betaretrovirus in monkeys, or feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) in cats ( Levine et al., 2006 , Israel and Plaisance, 1991 , Magden et al., 2015 , Gill et al., 2012 , Gleich and Hartmann, 2009 ). 12.11.2.1.2.4 Immune-mediated Primary or idiopathic immune-mediated destruction of mature neutrophils or neutrophil precursors will result in decreases in neutrophil counts, and has been reported in humans and most laboratory species. Antineutrophil antibodies are most commonly IgG and less commonly IgM class. Opsonization of neutrophil membranes may lead to leukoagglutination and neutrophil sequestration in sites including the spleen, liver, and lymph nodes, with phagocytosis by macrophages. Some antineutrophil antibodies may cause direct cytotoxicity through either complement-mediated or complement-independent mechanisms ( Chickering and Prasse, 1981 ). In humans, immune-mediated neutrophil destruction due to antibody-dependent lymphocyte cytotoxicity has been reported ( Logue et al., 1978 ). Systemic lupus erythematosus (SLE) is an autoimmune condition that has been associated with decreases in neutrophil counts. These decreases in blood neutrophil counts appear to be relatively common with SLE, and are usually observed in conjunction with other manifestations of SLE, such as arthritis, skin lesions, or neurologic disorders ( Stone, 2005 ). There is an association between decreases in neutrophil counts and the presence of autoantibodies in SLE ( Hsieh et al., 2003 , Budman and Steinberg, 1977 ), which lead to destruction or death of neutrophils. However, SLE-related anti-G-CSF autoantibodies have also been observed, causing decreased blood neutrophil counts due to decreased production rather than or in addition to direct neutrophil destruction or death ( Hellmich et al., 2002 ). Most cases of primary or idiopathic aplastic anemia result from underlying immune-mediated destruction of uncommitted or early lineage-committed hematopoietic stem cells ( Young et al., 2006 ). Aplastic anemia is characterized by pancytopenia (marked decreases in all blood component counts) and hematopoietic cell hypocellularity in bone marrow. Although most commonly associated with immune-mediated destruction of hematopoietic precursors, marked depletion of bone marrow hematopoietic cells or aplastic anemia may also occur with severe nutritional deficiencies associated with anorexia nervosa ( Abella et al., 2002 ) and 75% feed restriction in rats ( Moriyama et al., 2008 , Levin et al., 1993 ). 12.11.2.1.2.5 Cobalamin (B 12 ) and folate (B 9 ) Deficiencies in cobalamin and folate cause disruption of DNA synthesis that results in megaloblastic hematopoiesis, commonly associated with megaloblastic anemia and occasionally with decreases in neutrophil counts and/or pancytopenia ( Dale and Welte, 2016 , Green, 2016 ). Cobalamin or folate deficiencies may be due to malnutrition, malabsorption from gastrointestinal disease or dysfunction, or genetic deficiencies of transport proteins, such as intrinsic factor, transcobalamin, or R-factor for cobalamin, receptors for intestinal absorption, such as the CUBAM receptor, or metabolizing enzymes, such as methylenetetrahydrofolate reductase for folate ( Fowler, 1998 , Whitehead, 2006 , Montagle and Tauro, 1995 , Green, 2016 , Quadros, 2010 ). During megaloblastic hematopoiesis, asynchronous cytoplasmic and nuclear maturation may result in giant granulocytes with irregular nuclear chromatin patterns, such as giant metamyelocytes ( Green, 2016 ), or hypersegmented neutrophils ( Thompson et al., 1989 ). These conditions can often be managed with supplementation of the deficient vitamin. A differential for cobalamin or folate deficiency-induced decreases in blood neutrophil counts is copper deficiency, which can have a similar clinical manifestation ( Lazarchick, 2012 ). 12.11.2.1.2.6 Myelophthisis, myelofibrosis, and myelonecrosis Conditions that efface the bone marrow cavities or displace hematopoietic cells (myelophthisis) may result in decreased production of all hematopoietic cell lines, including neutrophils. Myeloproliferative syndromes ( Tefferi and Vardiman, 2009 ), many leukemic diseases ( Talcott et al., 1992 , Lamy and Loughran, 1999 ), lymphoproliferative neoplasia ( Schultze, 2010 ), or metastatic carcinoma ( Makoni and Laber, 2004 ) can cause sufficient bone marrow overcrowding to decrease the production of neutrophils. Fungal infections resulting in granulomatous inflammation of the bone marrow, such as can be observed with disseminated histoplasmosis, may also result in decreased neutrophil production. Disruption of the bone marrow cavities and hematopoietic stem cells from myelofibrosis or myelonecrosis may also be associated with decreased blood neutrophil counts ( Stockham and Scott, 2008a ). 12.11.2.1.2.7 Xenobiotic-induced Xenobiotic-induced decreases in neutrophil counts may be caused by immune-mediated or nonimmune-mediated mechanisms. The incidences of these events tend to be low with most implicated xenobiotics, with the exception of chemotherapeutic agents. Examples of both immune-mediated and nonimmune-mediated mechanisms are described later. Similar to primary immune-mediated decreases in neutrophil counts, xenobiotic-induced immune-mediated decreases in neutrophil counts are commonly associated with IgG or IgM antineutrophil antibodies, although some drugs may induce both ( Salama et al., 1989 ). Decreases in neutrophil counts by this mechanism tend to occur rapidly, within a few hours to 2 days after exposure ( Schwartzberg, 2006 ). Some xenobiotics mediate their effects by acting as haptens, which induce an antibody response targeting an antigen formed by the xenobiotic–neutrophil combination. Drugs reported to act as haptens include penicillin, gold-based compounds, aminopyrine, and propylthiouracil ( Salama et al., 1989 , Murphy et al., 1985 ). These xenobiotics induce neutrophil destruction in a drug-dependent manner, and discontinuation of treatment generally results in resolution of blood neutrophil counts within 7 days ( Schwartzberg, 2006 ). Some xenobiotics, including propylthiouracil and quinidine sulfate, cause the formation of immune complexes that can subsequently bind and destroy neutrophils, even if the xenobiotic is no longer present ( Schwartzberg, 2006 , Bhatt and Saleem, 2004 , Eisner et al., 1977 ). In addition, some xenobiotics, such as propylthiouracil, may also cause the formation of antineutrophil antibodies resulting in complement-mediated neutrophil destruction ( Akamizu et al., 2002 ). Similar to idiopathic aplastic anemia, xenobiotic-induced aplastic anemia is most commonly associated with immune-mediated destruction of uncommitted or early hematopoietic stem cells, although direct cytotoxicity, such as with chemotherapeutic agents, may also lead to aplastic anemia. Several xenobiotics, including chloramphenicol, anticonvulsants such as phenytoin and carbamazepine, gold-based compounds, and phenylbutazone have been associated with aplastic anemia ( Bloom and Brandt, 2008 ). Numerous xenobiotics have also been linked to SLE, with decreases in blood neutrophil counts in some cases. In xenobiotic-induced SLE, decreased neutrophil counts reflect production of autoantibodies and subsequent neutrophil destruction, similar to nonxenobiotic-induced SLE. Xenobiotics associated with SLE include anticonvulsants such as phenothiazines, chlorpromazine, and valproate, antibiotics such as penicillin, streptomycin, tetracycline, griseofulvin, and sulphonamides, and miscellaneous xenobiotics such as captopril, phenylbutazone, and lovastatin ( Stone, 2005 , Mutasim and Adams, 2000 ). In laboratory species used in nonclinical toxicology studies, transient xenobiotic-induced decreases in blood neutrophil counts have been associated with anaphylactoid reactions termed complement activation-related pseudoallergy (CARPA). These nonhypersensitive reactions are mediated instead by activation of the complement cascade, leading to the production of the anaphylatoxins C3a and C5a. CARPA typically occurs after the first dose of xenobiotic with decreasing severity or resolution following additional doses. Xenobiotics implicated in CARPA reactions include radiocontrast media, drug-carrying liposomes and lipid complexes, and some solvents with amphiphilic emulsifiers, such as Cremophor EL ( Szebeni, 2005 ). Nonimmune-mediated decreases in neutrophil counts may be observed with bone marrow suppression, which is associated with many xenobiotics, particularly chemotherapeutic agents. Chemotherapeutic-related decreases in neutrophil counts are often a result of direct cytotoxicity or suppressed proliferation of the rapidly dividing granulocytic precursors in the bone marrow. Examples of chemotherapeutic classes associated with decreased blood neutrophil counts include agents that cause direct DNA damage, such as platinum-containing agents (e.g., cisplatin, carboplatin) and classical alkylating agents (e.g., cyclophosphamide, melphalan, busulfan); mitotic spindle inhibitors (e.g., paclitaxel, docetaxel, vinblastine, vincristine); topoisomerase inhibitors (e.g., etoposide, doxorubicin); and antimetabolites (e.g., methotrexate, 6-mercaptopurine, 5-fluorouracil) ( Bhatt and Saleem, 2004 , Weiss, 2010 , Wailoo et al., 2009 ). Decreases in neutrophil counts are expected with chemotherapeutic treatment, and can be easily monitored through serial CBCs. If severe enough to increase the risk of life-threatening infections clinically, treatment with empiric antibiotics or G-CSF-like compounds may be considered. Suppression of granulopoiesis or direct granulocyte toxicity is also reported with some nonchemotherapeutic agents. Dose-dependent inhibition of colony-forming units of granulocytes and macrophages in the bone marrow has been reported with several anticonvulsant drugs, including valproic acid and carbamazepine, and beta-lactam antibiotics ( Schwartzberg, 2006 , Irvine et al., 1994 , Watts et al., 1990 , Neftel et al., 1985 ). Several other anticonvulsant drugs, including ethosuximide ( Mintzer et al., 2009 ) and phenytoin ( Sharafuddin et al., 1991 ), have been reported to cause direct toxic effects on granulocyte precursors. Antipsychotic agents, including clozapine and chlorpromazine, may also cause direct cytotoxic effects: neutrophil metabolism of clozapine has been demonstrated to form an unstable intracellular metabolite, nitrenium ion, which depletes glutathione and makes the neutrophils susceptible to oxidative damage and apoptosis ( Williams et al., 2000 ); chlorpromazine may cause cytotoxicity through the inhibition of thymidine and uracil uptake by neutrophils ( Pisciotta and Kaldahl, 1962 ). Thienopyridine inhibitors of platelet aggregation, including clopidogrel and ticlopidine, have also been associated with direct neutrophil toxicity resulting from mitochondrial toxicity by metabolites generated through myeloperoxidase metabolism of the parent compounds ( Maseneni et al., 2012 , Maseneni et al., 2013 ). 12.11.2.1.1 Increases in neutrophil counts (neutrophilia, granulocytosis) 12.11.2.1.1.1 Catecholamine-induced Acute, transient increases (typically  50,000 cells μL − 1 , typically due to a marked neutrophilia with a left shift that remains orderly and may or may not have morphologic changes indicative of rapid granulopoiesis ( Schultze, 2010 , Sakka et al., 2006 ). Extreme neutrophilia typically has > 100,000 cells μL − 1 and evidence of a left shift. The terms leukemoid reaction and extreme neutrophilia are most appropriately applied retrospectively, after the possibility for hematopoietic neoplasia has been excluded. Differentiation of a leukemoid response or extreme neutrophilia from chronic myelogenous leukemia or chronic neutrophilic leukemia includes CBC, blood smear, and bone marrow evaluations in most species, and may also include leukocyte alkaline phosphatase activity, immunophenotyping, cytogenetic analysis (e.g., evaluation for bcr–abl translocation), serum G-CSF, and clonality evaluations in humans ( Schultze, 2010 , Sakka et al., 2006 ). Leukemoid reactions have been associated with carcinomas of various origins, including renal and pulmonary carcinomas, Hodgkin's lymphoma, melanoma, and sarcomas, and may be attributable to aberrant production of proinflammatory mediators by the neoplasm, such as G-CSF, GM-CSF, or IL-6 ( Sakka et al., 2006 ). Leukemoid reactions have also been reported in F334/N rats affected by large granular cell leukemia ( Car et al., 2006 ). However, leukemoid reactions may also be associated with infectious processes, including disseminated tuberculosis, Clostridium difficile colitis, severe shigellosis ( Sakka et al., 2006 ), chronic localized suppurative lesions such as pyometra, pleuritis, and internal abscesses ( Schultze, 2010 , Stockham and Scott, 2008a ). Leukemoid reactions may also be seen secondary to severe hemorrhage or immune-mediated hemolytic anemia ( Schultze, 2010 , Sakka et al., 2006 ). 12.11.2.1.1.4 Inherited leukocyte adhesion deficiencies Increases in neutrophil counts associated with deficiencies in leukocyte adhesion molecules may manifest as a leukemoid response or extreme neutrophilia. Adhesion molecules expressed on neutrophils are responsible for neutrophil margination, rolling along vessel walls, and emigration into tissues. L-selectin (CD62L) mediates low-affinity initial binding of leukocyte to endothelial cells, while integrins, including CD11b/CD18 (Mac-1), mediate firm adhesion to endothelial cells and ligands in the extracellular matrix ( Muller, 2012 ). Neutrophils constitutively express CD11b/CD18. A deficiency of this integrin (leukocyte adhesion deficiency [LAD] type 1) results in the failure of neutrophils to emigrate to tissues, and may result in severe, recurrent bacterial infections ( Arnaout, 1990 ). LAD type 2 is due to an inherited disorder of fucose metabolism, resulting in the lack of selectin ligands expressed on neutrophils and therefore results in immunodeficiency from a failure of selectin-mediated neutrophil rolling along vessel walls ( Marquardt et al., 1999 ). Leukocyte adhesion deficiencies have been reported in humans, dogs, mice, and Holstein cattle ( Arnaout, 1990 , Marquardt et al., 1999 , Gu et al., 2004 ). 12.11.2.1.1.5 Neoplasia Neoplasms involving hematopoietic cells naturally occur with relatively low frequency. In general, such neoplastic processes may be observed as background findings in rats and mice during longer toxicity studies (e.g., carcinogenicity studies), but are uncommon in nonrodent species during toxicity studies ( Smith et al, 2002 ). Increases in neutrophil counts may be observed as part of the neoplastic processes of chronic myeloid leukemia (CML) or chronic neutrophilic leukemia (CNL). Leukocytosis in CML is ≥ 25,000 cells μL − 1 with increases in all stages of neutrophilic myeloid cells in blood, which are typically accompanied by increases in monocyte counts, eosinophil counts, basophil counts, platelet counts, and possibly increases in nucleated erythroid precursors ( Sawyers, 1999 ). In contrast to a leukemoid response, the left shift associated with CML tends to be less orderly, with greater numbers of earlier stages of myeloid cells in circulation along with the band and segmented neutrophils. Morphologic features indicative of dysplasia may be seen, and cytoplasmic features indicative of rapid granulopoiesis may be observed in neutrophils associated with CML. Bone marrow findings include hypercellularity with increased myeloid to erythroid ratios where myeloblasts and promyelocytes make up  80% segmented and band neutrophils ( Uppal and Gong, 2015 ). These neutrophils may or may not have cytoplasmic changes indicative of rapid neutropoiesis. Unlike CML, the increases in neutrophil counts associated with CNL are typically not associated with morphologic features of dysplasia, the presence of earlier stages of myeloid cells (i.e. myeloblasts, promyelocytes, myelocytes, and metamyelocytes), increases in monocyte, eosinophil, or basophil counts, or increases in nucleated erythroid precursors ( Uppal and Gong, 2015 ). Bone marrow changes associated with CNL include hypercellularity with increased myeloid to erythroid ratios, where the myelocyte through band neutrophil stages are increased without apparent increases in myeloblasts or promyelocytes ( Uppal and Gong, 2015 ). As such, differentiation of CNL from leukemoid reactions or extreme neutrophilia may be difficult, and affected patients should be carefully evaluated to exclude causes of neutrophilia not associated with hematopoietic malignancy. 12.11.2.1.1.6 Xenobiotic-induced There are numerous xenobiotics that can increase blood neutrophil counts, typically through similar mechanisms as described for naturally occurring increases in neutrophil counts. Several examples of these compounds are described later. Administration of exogenous catecholamines, such as epinephrine, or some adrenergic agonists has similar effects as those mediated by endogenous catecholamines. Shifting of neutrophils from the marginating to the circulating pools is responsible for the increases in blood neutrophil counts, potentially from altered adhesion molecule expression or hemodynamic forces. Leukocyte subtypes have different receptors modulating their adrenergic effects, and neutrophils appear to be primarily affected by α-adrenergic receptors ( Benschop et al., 1996 ). Exogenous glucocorticoids, such as prednisolone, dexamethasone, and betamethasone, will mediate increases in neutrophil counts through similar mechanisms as endogenous glucocorticoids. In brief, these increases in neutrophil counts are due to shifting of neutrophils from the marginating to circulating pool, release of neutrophils from the bone marrow storage pool, and expansion of granulopoiesis in the bone marrow. The dose and duration of the glucocorticoid therapy may alter the proportional contribution of each of these effects to the increases in neutrophil counts. Heparin-induced increases in neutrophil counts are reported in a small percentage of patients receiving heparin therapy, and the mechanisms are likely multifactorial. Heparin may cause shifting of neutrophils to the circulating pool from the marginating pool due to decreased expression of selectins ( Zhang et al., 2012 ). Heparin-related release of CXCL12, a chemokine that is constitutively expressed by bone marrow stromal cells and plays a role in bone marrow neutrophil retention and leukocyte trafficking, may alter bone marrow and circulating CXCL12 gradients and promote release of neutrophils from the bone marrow storage pool, contributing to the increases in circulating neutrophil counts ( Zhang et al., 2012 ). Administration of G-CSF or GM-CSF may be used as a rescue therapy following chemotherapy that causes neutropenia and compromises the immune system's ability to respond to infectious agents. While the goal for treatment in chemotherapy patients is to increase blood neutrophil counts to normal levels and not to achieve increased neutrophil counts, repeated or high dose administration of G-CSF or GM-CSF-related compounds to healthy laboratory animals results in markedly increased neutrophil counts. Typically these changes are associated with a left shift and cytoplasmic changes indicative of rapid neutropoiesis. Dysplastic changes may also occur, most commonly recognized by the presence of unusually large neutrophils or hypersegmented neutrophils in circulation. Several xenobiotics appear to induce increases in neutrophil counts by stimulating increases in endogenous G-CSF or an inflammatory stimulus. Early in the course of treatment with clozapine, an antipsychotic agent, rats have been shown to have spikes in G-CSF with concomitant increases in bone marrow granulopoiesis and release of less mature neutrophils into circulation ( Lobach and Uetrecht, 2014 ). However, other xenobiotics may directly cause an inflammatory stimulus and increase in endogenous proinflammatory stimuli, such as G-CSF and IL-1, which result in increases in neutrophil counts. For example, lithium, which has been used in treatment of bipolar disorder and in combination with antidepressants, also appears to cause increased neutrophil counts due to increased G-CSF ( Focosi et al., 2009 ). Xenobiotic-induced increases in neutrophil counts may be observed in conjunction with several cutaneous drug reactions that result in inflammatory stimuli. Acute generalized exanthematous pustulosis is a drug-related skin reaction that generally occurs within 2 days of drug administration but resolves spontaneously within 15 days ( Roujeau, 2005 ). Increases in eosinophil counts may or may not be observed along with the increases in neutrophil counts. Xenobiotics classically associated with this syndrome include diltiazem and antibiotics such as aminopenicillins and pristinamycine ( Roujeau, 2005 ). Acute febrile dermatosis (Sweet's syndrome) has also been associated with elevations in circulating neutrophil counts, and drug-induced forms of this condition have been variably linked to a variety of compounds, including GM-CSF, trimethoprim–sulfamethoxazole, minocycline, celecoxib, furosemide, and azathioprine ( Rochet et al., 2013 , Saavedra et al., 2006 ). 12.11.2.1.1.1 Catecholamine-induced Acute, transient increases (typically  50,000 cells μL − 1 , typically due to a marked neutrophilia with a left shift that remains orderly and may or may not have morphologic changes indicative of rapid granulopoiesis ( Schultze, 2010 , Sakka et al., 2006 ). Extreme neutrophilia typically has > 100,000 cells μL − 1 and evidence of a left shift. The terms leukemoid reaction and extreme neutrophilia are most appropriately applied retrospectively, after the possibility for hematopoietic neoplasia has been excluded. Differentiation of a leukemoid response or extreme neutrophilia from chronic myelogenous leukemia or chronic neutrophilic leukemia includes CBC, blood smear, and bone marrow evaluations in most species, and may also include leukocyte alkaline phosphatase activity, immunophenotyping, cytogenetic analysis (e.g., evaluation for bcr–abl translocation), serum G-CSF, and clonality evaluations in humans ( Schultze, 2010 , Sakka et al., 2006 ). Leukemoid reactions have been associated with carcinomas of various origins, including renal and pulmonary carcinomas, Hodgkin's lymphoma, melanoma, and sarcomas, and may be attributable to aberrant production of proinflammatory mediators by the neoplasm, such as G-CSF, GM-CSF, or IL-6 ( Sakka et al., 2006 ). Leukemoid reactions have also been reported in F334/N rats affected by large granular cell leukemia ( Car et al., 2006 ). However, leukemoid reactions may also be associated with infectious processes, including disseminated tuberculosis, Clostridium difficile colitis, severe shigellosis ( Sakka et al., 2006 ), chronic localized suppurative lesions such as pyometra, pleuritis, and internal abscesses ( Schultze, 2010 , Stockham and Scott, 2008a ). Leukemoid reactions may also be seen secondary to severe hemorrhage or immune-mediated hemolytic anemia ( Schultze, 2010 , Sakka et al., 2006 ). 12.11.2.1.1.4 Inherited leukocyte adhesion deficiencies Increases in neutrophil counts associated with deficiencies in leukocyte adhesion molecules may manifest as a leukemoid response or extreme neutrophilia. Adhesion molecules expressed on neutrophils are responsible for neutrophil margination, rolling along vessel walls, and emigration into tissues. L-selectin (CD62L) mediates low-affinity initial binding of leukocyte to endothelial cells, while integrins, including CD11b/CD18 (Mac-1), mediate firm adhesion to endothelial cells and ligands in the extracellular matrix ( Muller, 2012 ). Neutrophils constitutively express CD11b/CD18. A deficiency of this integrin (leukocyte adhesion deficiency [LAD] type 1) results in the failure of neutrophils to emigrate to tissues, and may result in severe, recurrent bacterial infections ( Arnaout, 1990 ). LAD type 2 is due to an inherited disorder of fucose metabolism, resulting in the lack of selectin ligands expressed on neutrophils and therefore results in immunodeficiency from a failure of selectin-mediated neutrophil rolling along vessel walls ( Marquardt et al., 1999 ). Leukocyte adhesion deficiencies have been reported in humans, dogs, mice, and Holstein cattle ( Arnaout, 1990 , Marquardt et al., 1999 , Gu et al., 2004 ). 12.11.2.1.1.5 Neoplasia Neoplasms involving hematopoietic cells naturally occur with relatively low frequency. In general, such neoplastic processes may be observed as background findings in rats and mice during longer toxicity studies (e.g., carcinogenicity studies), but are uncommon in nonrodent species during toxicity studies ( Smith et al, 2002 ). Increases in neutrophil counts may be observed as part of the neoplastic processes of chronic myeloid leukemia (CML) or chronic neutrophilic leukemia (CNL). Leukocytosis in CML is ≥ 25,000 cells μL − 1 with increases in all stages of neutrophilic myeloid cells in blood, which are typically accompanied by increases in monocyte counts, eosinophil counts, basophil counts, platelet counts, and possibly increases in nucleated erythroid precursors ( Sawyers, 1999 ). In contrast to a leukemoid response, the left shift associated with CML tends to be less orderly, with greater numbers of earlier stages of myeloid cells in circulation along with the band and segmented neutrophils. Morphologic features indicative of dysplasia may be seen, and cytoplasmic features indicative of rapid granulopoiesis may be observed in neutrophils associated with CML. Bone marrow findings include hypercellularity with increased myeloid to erythroid ratios where myeloblasts and promyelocytes make up  80% segmented and band neutrophils ( Uppal and Gong, 2015 ). These neutrophils may or may not have cytoplasmic changes indicative of rapid neutropoiesis. Unlike CML, the increases in neutrophil counts associated with CNL are typically not associated with morphologic features of dysplasia, the presence of earlier stages of myeloid cells (i.e. myeloblasts, promyelocytes, myelocytes, and metamyelocytes), increases in monocyte, eosinophil, or basophil counts, or increases in nucleated erythroid precursors ( Uppal and Gong, 2015 ). Bone marrow changes associated with CNL include hypercellularity with increased myeloid to erythroid ratios, where the myelocyte through band neutrophil stages are increased without apparent increases in myeloblasts or promyelocytes ( Uppal and Gong, 2015 ). As such, differentiation of CNL from leukemoid reactions or extreme neutrophilia may be difficult, and affected patients should be carefully evaluated to exclude causes of neutrophilia not associated with hematopoietic malignancy. 12.11.2.1.1.6 Xenobiotic-induced There are numerous xenobiotics that can increase blood neutrophil counts, typically through similar mechanisms as described for naturally occurring increases in neutrophil counts. Several examples of these compounds are described later. Administration of exogenous catecholamines, such as epinephrine, or some adrenergic agonists has similar effects as those mediated by endogenous catecholamines. Shifting of neutrophils from the marginating to the circulating pools is responsible for the increases in blood neutrophil counts, potentially from altered adhesion molecule expression or hemodynamic forces. Leukocyte subtypes have different receptors modulating their adrenergic effects, and neutrophils appear to be primarily affected by α-adrenergic receptors ( Benschop et al., 1996 ). Exogenous glucocorticoids, such as prednisolone, dexamethasone, and betamethasone, will mediate increases in neutrophil counts through similar mechanisms as endogenous glucocorticoids. In brief, these increases in neutrophil counts are due to shifting of neutrophils from the marginating to circulating pool, release of neutrophils from the bone marrow storage pool, and expansion of granulopoiesis in the bone marrow. The dose and duration of the glucocorticoid therapy may alter the proportional contribution of each of these effects to the increases in neutrophil counts. Heparin-induced increases in neutrophil counts are reported in a small percentage of patients receiving heparin therapy, and the mechanisms are likely multifactorial. Heparin may cause shifting of neutrophils to the circulating pool from the marginating pool due to decreased expression of selectins ( Zhang et al., 2012 ). Heparin-related release of CXCL12, a chemokine that is constitutively expressed by bone marrow stromal cells and plays a role in bone marrow neutrophil retention and leukocyte trafficking, may alter bone marrow and circulating CXCL12 gradients and promote release of neutrophils from the bone marrow storage pool, contributing to the increases in circulating neutrophil counts ( Zhang et al., 2012 ). Administration of G-CSF or GM-CSF may be used as a rescue therapy following chemotherapy that causes neutropenia and compromises the immune system's ability to respond to infectious agents. While the goal for treatment in chemotherapy patients is to increase blood neutrophil counts to normal levels and not to achieve increased neutrophil counts, repeated or high dose administration of G-CSF or GM-CSF-related compounds to healthy laboratory animals results in markedly increased neutrophil counts. Typically these changes are associated with a left shift and cytoplasmic changes indicative of rapid neutropoiesis. Dysplastic changes may also occur, most commonly recognized by the presence of unusually large neutrophils or hypersegmented neutrophils in circulation. Several xenobiotics appear to induce increases in neutrophil counts by stimulating increases in endogenous G-CSF or an inflammatory stimulus. Early in the course of treatment with clozapine, an antipsychotic agent, rats have been shown to have spikes in G-CSF with concomitant increases in bone marrow granulopoiesis and release of less mature neutrophils into circulation ( Lobach and Uetrecht, 2014 ). However, other xenobiotics may directly cause an inflammatory stimulus and increase in endogenous proinflammatory stimuli, such as G-CSF and IL-1, which result in increases in neutrophil counts. For example, lithium, which has been used in treatment of bipolar disorder and in combination with antidepressants, also appears to cause increased neutrophil counts due to increased G-CSF ( Focosi et al., 2009 ). Xenobiotic-induced increases in neutrophil counts may be observed in conjunction with several cutaneous drug reactions that result in inflammatory stimuli. Acute generalized exanthematous pustulosis is a drug-related skin reaction that generally occurs within 2 days of drug administration but resolves spontaneously within 15 days ( Roujeau, 2005 ). Increases in eosinophil counts may or may not be observed along with the increases in neutrophil counts. Xenobiotics classically associated with this syndrome include diltiazem and antibiotics such as aminopenicillins and pristinamycine ( Roujeau, 2005 ). Acute febrile dermatosis (Sweet's syndrome) has also been associated with elevations in circulating neutrophil counts, and drug-induced forms of this condition have been variably linked to a variety of compounds, including GM-CSF, trimethoprim–sulfamethoxazole, minocycline, celecoxib, furosemide, and azathioprine ( Rochet et al., 2013 , Saavedra et al., 2006 ). 12.11.2.1.2 Decreases in neutrophil counts (neutropenia, granulocytopenia, agranulocytosis) 12.11.2.1.2.1 Overwhelming or severe acute inflammation Although inflammation often causes increases in neutrophil counts, an overwhelming or severe acute inflammatory stimulus may result in decreases in neutrophil counts. Such an inflammatory stimulus, sometimes also referred to as peracute inflammation, is commonly observed with bacterial sepsis. Decreases in neutrophil counts are a result of emigration to tissues that exceed the bone marrow capacity to release neutrophils. Proinflammatory mediators and chemoattractants stimulate increased neutrophil margination, firm adhesion to endothelial cells, and emigration into tissue, which shortens neutrophil circulating half-lives and depletes the circulating neutrophil pool. As neutrophils are released from the bone marrow and the storage pool is depleted, recruitment of immature neutrophils into circulation results in a left shift in which the number of immature forms exceeds the number of mature segmented neutrophils (degenerative left shift). If the stimulus persists long enough, granulocytic hyperplasia of the bone marrow will occur and the numbers of circulating neutrophils will increase and shift toward greater numbers of mature than immature neutrophils. 12.11.2.1.2.2 Endotoxemia Decreases in neutrophil counts may be observed as a result of Gram-negative bacterial infections that release endotoxin. Endotoxemia has been demonstrated to increase expression of the integrin CD11b/CD18 and decreased expression of L-selectin on neutrophils ( Wagner and Roth, 1999 ), mediating a shift from circulating to marginating pools with firm adhesion to endothelial cells. There is evidence that this upregulation of CD18 contributes to neutrophil vascular sequestration in the lungs and liver, but there is also evidence for other mechanisms, such as cytoskeletal changes that alter neutrophil deformability, causing the initial vascular sequestration of neutrophils that is consistently observed with endotoxemia ( Wagner and Roth, 1999 ). The decreases in neutrophil counts associated with endotoxemia tend to be rapid but transient, and detection of these decreases in neutrophil counts will depend on timing of the blood collection in relation to endotoxemic and other concurrent or subsequent proinflammatory stimuli. 12.11.2.1.2.3 Viral-induced Decreases in neutrophil counts may be associated with viral infections, although viral-specific mechanisms vary. Decreased neutrophil counts associated with parvovirus likely represent both a primary effect due to infection causing death of rapidly dividing hematopoietic precursors in the bone marrow, as well as a secondary increase in tissue demand from disease and loss of integrity of the intestinal tract. Decreases in neutrophil counts associated with human immunodeficiency virus (HIV), infectious hepatitis, and infectious mononucleosis in people have also been associated with decreased or ineffective neutrophil production due to infection of hematopoietic precursors ( Dale and Welte, 2016 , Lima et al., 2006 ). With measles and dengue virus, decreases in neutrophil counts have been observed with endothelial cell alterations leading to increased adhesion of neutrophils and therefore a shift from the circulating to the marginating pool ( Dale and Welte, 2016 ). Decreases in blood neutrophil counts may also result from decreased neutrophil production, as in acquired viral immunodeficiency. Immunodeficiency syndromes with decreased neutrophil counts have been reported with infections by HIV in people, simian immunodeficiency virus (SIV) or simian betaretrovirus in monkeys, or feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) in cats ( Levine et al., 2006 , Israel and Plaisance, 1991 , Magden et al., 2015 , Gill et al., 2012 , Gleich and Hartmann, 2009 ). 12.11.2.1.2.4 Immune-mediated Primary or idiopathic immune-mediated destruction of mature neutrophils or neutrophil precursors will result in decreases in neutrophil counts, and has been reported in humans and most laboratory species. Antineutrophil antibodies are most commonly IgG and less commonly IgM class. Opsonization of neutrophil membranes may lead to leukoagglutination and neutrophil sequestration in sites including the spleen, liver, and lymph nodes, with phagocytosis by macrophages. Some antineutrophil antibodies may cause direct cytotoxicity through either complement-mediated or complement-independent mechanisms ( Chickering and Prasse, 1981 ). In humans, immune-mediated neutrophil destruction due to antibody-dependent lymphocyte cytotoxicity has been reported ( Logue et al., 1978 ). Systemic lupus erythematosus (SLE) is an autoimmune condition that has been associated with decreases in neutrophil counts. These decreases in blood neutrophil counts appear to be relatively common with SLE, and are usually observed in conjunction with other manifestations of SLE, such as arthritis, skin lesions, or neurologic disorders ( Stone, 2005 ). There is an association between decreases in neutrophil counts and the presence of autoantibodies in SLE ( Hsieh et al., 2003 , Budman and Steinberg, 1977 ), which lead to destruction or death of neutrophils. However, SLE-related anti-G-CSF autoantibodies have also been observed, causing decreased blood neutrophil counts due to decreased production rather than or in addition to direct neutrophil destruction or death ( Hellmich et al., 2002 ). Most cases of primary or idiopathic aplastic anemia result from underlying immune-mediated destruction of uncommitted or early lineage-committed hematopoietic stem cells ( Young et al., 2006 ). Aplastic anemia is characterized by pancytopenia (marked decreases in all blood component counts) and hematopoietic cell hypocellularity in bone marrow. Although most commonly associated with immune-mediated destruction of hematopoietic precursors, marked depletion of bone marrow hematopoietic cells or aplastic anemia may also occur with severe nutritional deficiencies associated with anorexia nervosa ( Abella et al., 2002 ) and 75% feed restriction in rats ( Moriyama et al., 2008 , Levin et al., 1993 ). 12.11.2.1.2.5 Cobalamin (B 12 ) and folate (B 9 ) Deficiencies in cobalamin and folate cause disruption of DNA synthesis that results in megaloblastic hematopoiesis, commonly associated with megaloblastic anemia and occasionally with decreases in neutrophil counts and/or pancytopenia ( Dale and Welte, 2016 , Green, 2016 ). Cobalamin or folate deficiencies may be due to malnutrition, malabsorption from gastrointestinal disease or dysfunction, or genetic deficiencies of transport proteins, such as intrinsic factor, transcobalamin, or R-factor for cobalamin, receptors for intestinal absorption, such as the CUBAM receptor, or metabolizing enzymes, such as methylenetetrahydrofolate reductase for folate ( Fowler, 1998 , Whitehead, 2006 , Montagle and Tauro, 1995 , Green, 2016 , Quadros, 2010 ). During megaloblastic hematopoiesis, asynchronous cytoplasmic and nuclear maturation may result in giant granulocytes with irregular nuclear chromatin patterns, such as giant metamyelocytes ( Green, 2016 ), or hypersegmented neutrophils ( Thompson et al., 1989 ). These conditions can often be managed with supplementation of the deficient vitamin. A differential for cobalamin or folate deficiency-induced decreases in blood neutrophil counts is copper deficiency, which can have a similar clinical manifestation ( Lazarchick, 2012 ). 12.11.2.1.2.6 Myelophthisis, myelofibrosis, and myelonecrosis Conditions that efface the bone marrow cavities or displace hematopoietic cells (myelophthisis) may result in decreased production of all hematopoietic cell lines, including neutrophils. Myeloproliferative syndromes ( Tefferi and Vardiman, 2009 ), many leukemic diseases ( Talcott et al., 1992 , Lamy and Loughran, 1999 ), lymphoproliferative neoplasia ( Schultze, 2010 ), or metastatic carcinoma ( Makoni and Laber, 2004 ) can cause sufficient bone marrow overcrowding to decrease the production of neutrophils. Fungal infections resulting in granulomatous inflammation of the bone marrow, such as can be observed with disseminated histoplasmosis, may also result in decreased neutrophil production. Disruption of the bone marrow cavities and hematopoietic stem cells from myelofibrosis or myelonecrosis may also be associated with decreased blood neutrophil counts ( Stockham and Scott, 2008a ). 12.11.2.1.2.7 Xenobiotic-induced Xenobiotic-induced decreases in neutrophil counts may be caused by immune-mediated or nonimmune-mediated mechanisms. The incidences of these events tend to be low with most implicated xenobiotics, with the exception of chemotherapeutic agents. Examples of both immune-mediated and nonimmune-mediated mechanisms are described later. Similar to primary immune-mediated decreases in neutrophil counts, xenobiotic-induced immune-mediated decreases in neutrophil counts are commonly associated with IgG or IgM antineutrophil antibodies, although some drugs may induce both ( Salama et al., 1989 ). Decreases in neutrophil counts by this mechanism tend to occur rapidly, within a few hours to 2 days after exposure ( Schwartzberg, 2006 ). Some xenobiotics mediate their effects by acting as haptens, which induce an antibody response targeting an antigen formed by the xenobiotic–neutrophil combination. Drugs reported to act as haptens include penicillin, gold-based compounds, aminopyrine, and propylthiouracil ( Salama et al., 1989 , Murphy et al., 1985 ). These xenobiotics induce neutrophil destruction in a drug-dependent manner, and discontinuation of treatment generally results in resolution of blood neutrophil counts within 7 days ( Schwartzberg, 2006 ). Some xenobiotics, including propylthiouracil and quinidine sulfate, cause the formation of immune complexes that can subsequently bind and destroy neutrophils, even if the xenobiotic is no longer present ( Schwartzberg, 2006 , Bhatt and Saleem, 2004 , Eisner et al., 1977 ). In addition, some xenobiotics, such as propylthiouracil, may also cause the formation of antineutrophil antibodies resulting in complement-mediated neutrophil destruction ( Akamizu et al., 2002 ). Similar to idiopathic aplastic anemia, xenobiotic-induced aplastic anemia is most commonly associated with immune-mediated destruction of uncommitted or early hematopoietic stem cells, although direct cytotoxicity, such as with chemotherapeutic agents, may also lead to aplastic anemia. Several xenobiotics, including chloramphenicol, anticonvulsants such as phenytoin and carbamazepine, gold-based compounds, and phenylbutazone have been associated with aplastic anemia ( Bloom and Brandt, 2008 ). Numerous xenobiotics have also been linked to SLE, with decreases in blood neutrophil counts in some cases. In xenobiotic-induced SLE, decreased neutrophil counts reflect production of autoantibodies and subsequent neutrophil destruction, similar to nonxenobiotic-induced SLE. Xenobiotics associated with SLE include anticonvulsants such as phenothiazines, chlorpromazine, and valproate, antibiotics such as penicillin, streptomycin, tetracycline, griseofulvin, and sulphonamides, and miscellaneous xenobiotics such as captopril, phenylbutazone, and lovastatin ( Stone, 2005 , Mutasim and Adams, 2000 ). In laboratory species used in nonclinical toxicology studies, transient xenobiotic-induced decreases in blood neutrophil counts have been associated with anaphylactoid reactions termed complement activation-related pseudoallergy (CARPA). These nonhypersensitive reactions are mediated instead by activation of the complement cascade, leading to the production of the anaphylatoxins C3a and C5a. CARPA typically occurs after the first dose of xenobiotic with decreasing severity or resolution following additional doses. Xenobiotics implicated in CARPA reactions include radiocontrast media, drug-carrying liposomes and lipid complexes, and some solvents with amphiphilic emulsifiers, such as Cremophor EL ( Szebeni, 2005 ). Nonimmune-mediated decreases in neutrophil counts may be observed with bone marrow suppression, which is associated with many xenobiotics, particularly chemotherapeutic agents. Chemotherapeutic-related decreases in neutrophil counts are often a result of direct cytotoxicity or suppressed proliferation of the rapidly dividing granulocytic precursors in the bone marrow. Examples of chemotherapeutic classes associated with decreased blood neutrophil counts include agents that cause direct DNA damage, such as platinum-containing agents (e.g., cisplatin, carboplatin) and classical alkylating agents (e.g., cyclophosphamide, melphalan, busulfan); mitotic spindle inhibitors (e.g., paclitaxel, docetaxel, vinblastine, vincristine); topoisomerase inhibitors (e.g., etoposide, doxorubicin); and antimetabolites (e.g., methotrexate, 6-mercaptopurine, 5-fluorouracil) ( Bhatt and Saleem, 2004 , Weiss, 2010 , Wailoo et al., 2009 ). Decreases in neutrophil counts are expected with chemotherapeutic treatment, and can be easily monitored through serial CBCs. If severe enough to increase the risk of life-threatening infections clinically, treatment with empiric antibiotics or G-CSF-like compounds may be considered. Suppression of granulopoiesis or direct granulocyte toxicity is also reported with some nonchemotherapeutic agents. Dose-dependent inhibition of colony-forming units of granulocytes and macrophages in the bone marrow has been reported with several anticonvulsant drugs, including valproic acid and carbamazepine, and beta-lactam antibiotics ( Schwartzberg, 2006 , Irvine et al., 1994 , Watts et al., 1990 , Neftel et al., 1985 ). Several other anticonvulsant drugs, including ethosuximide ( Mintzer et al., 2009 ) and phenytoin ( Sharafuddin et al., 1991 ), have been reported to cause direct toxic effects on granulocyte precursors. Antipsychotic agents, including clozapine and chlorpromazine, may also cause direct cytotoxic effects: neutrophil metabolism of clozapine has been demonstrated to form an unstable intracellular metabolite, nitrenium ion, which depletes glutathione and makes the neutrophils susceptible to oxidative damage and apoptosis ( Williams et al., 2000 ); chlorpromazine may cause cytotoxicity through the inhibition of thymidine and uracil uptake by neutrophils ( Pisciotta and Kaldahl, 1962 ). Thienopyridine inhibitors of platelet aggregation, including clopidogrel and ticlopidine, have also been associated with direct neutrophil toxicity resulting from mitochondrial toxicity by metabolites generated through myeloperoxidase metabolism of the parent compounds ( Maseneni et al., 2012 , Maseneni et al., 2013 ). 12.11.2.1.2.1 Overwhelming or severe acute inflammation Although inflammation often causes increases in neutrophil counts, an overwhelming or severe acute inflammatory stimulus may result in decreases in neutrophil counts. Such an inflammatory stimulus, sometimes also referred to as peracute inflammation, is commonly observed with bacterial sepsis. Decreases in neutrophil counts are a result of emigration to tissues that exceed the bone marrow capacity to release neutrophils. Proinflammatory mediators and chemoattractants stimulate increased neutrophil margination, firm adhesion to endothelial cells, and emigration into tissue, which shortens neutrophil circulating half-lives and depletes the circulating neutrophil pool. As neutrophils are released from the bone marrow and the storage pool is depleted, recruitment of immature neutrophils into circulation results in a left shift in which the number of immature forms exceeds the number of mature segmented neutrophils (degenerative left shift). If the stimulus persists long enough, granulocytic hyperplasia of the bone marrow will occur and the numbers of circulating neutrophils will increase and shift toward greater numbers of mature than immature neutrophils. 12.11.2.1.2.2 Endotoxemia Decreases in neutrophil counts may be observed as a result of Gram-negative bacterial infections that release endotoxin. Endotoxemia has been demonstrated to increase expression of the integrin CD11b/CD18 and decreased expression of L-selectin on neutrophils ( Wagner and Roth, 1999 ), mediating a shift from circulating to marginating pools with firm adhesion to endothelial cells. There is evidence that this upregulation of CD18 contributes to neutrophil vascular sequestration in the lungs and liver, but there is also evidence for other mechanisms, such as cytoskeletal changes that alter neutrophil deformability, causing the initial vascular sequestration of neutrophils that is consistently observed with endotoxemia ( Wagner and Roth, 1999 ). The decreases in neutrophil counts associated with endotoxemia tend to be rapid but transient, and detection of these decreases in neutrophil counts will depend on timing of the blood collection in relation to endotoxemic and other concurrent or subsequent proinflammatory stimuli. 12.11.2.1.2.3 Viral-induced Decreases in neutrophil counts may be associated with viral infections, although viral-specific mechanisms vary. Decreased neutrophil counts associated with parvovirus likely represent both a primary effect due to infection causing death of rapidly dividing hematopoietic precursors in the bone marrow, as well as a secondary increase in tissue demand from disease and loss of integrity of the intestinal tract. Decreases in neutrophil counts associated with human immunodeficiency virus (HIV), infectious hepatitis, and infectious mononucleosis in people have also been associated with decreased or ineffective neutrophil production due to infection of hematopoietic precursors ( Dale and Welte, 2016 , Lima et al., 2006 ). With measles and dengue virus, decreases in neutrophil counts have been observed with endothelial cell alterations leading to increased adhesion of neutrophils and therefore a shift from the circulating to the marginating pool ( Dale and Welte, 2016 ). Decreases in blood neutrophil counts may also result from decreased neutrophil production, as in acquired viral immunodeficiency. Immunodeficiency syndromes with decreased neutrophil counts have been reported with infections by HIV in people, simian immunodeficiency virus (SIV) or simian betaretrovirus in monkeys, or feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) in cats ( Levine et al., 2006 , Israel and Plaisance, 1991 , Magden et al., 2015 , Gill et al., 2012 , Gleich and Hartmann, 2009 ). 12.11.2.1.2.4 Immune-mediated Primary or idiopathic immune-mediated destruction of mature neutrophils or neutrophil precursors will result in decreases in neutrophil counts, and has been reported in humans and most laboratory species. Antineutrophil antibodies are most commonly IgG and less commonly IgM class. Opsonization of neutrophil membranes may lead to leukoagglutination and neutrophil sequestration in sites including the spleen, liver, and lymph nodes, with phagocytosis by macrophages. Some antineutrophil antibodies may cause direct cytotoxicity through either complement-mediated or complement-independent mechanisms ( Chickering and Prasse, 1981 ). In humans, immune-mediated neutrophil destruction due to antibody-dependent lymphocyte cytotoxicity has been reported ( Logue et al., 1978 ). Systemic lupus erythematosus (SLE) is an autoimmune condition that has been associated with decreases in neutrophil counts. These decreases in blood neutrophil counts appear to be relatively common with SLE, and are usually observed in conjunction with other manifestations of SLE, such as arthritis, skin lesions, or neurologic disorders ( Stone, 2005 ). There is an association between decreases in neutrophil counts and the presence of autoantibodies in SLE ( Hsieh et al., 2003 , Budman and Steinberg, 1977 ), which lead to destruction or death of neutrophils. However, SLE-related anti-G-CSF autoantibodies have also been observed, causing decreased blood neutrophil counts due to decreased production rather than or in addition to direct neutrophil destruction or death ( Hellmich et al., 2002 ). Most cases of primary or idiopathic aplastic anemia result from underlying immune-mediated destruction of uncommitted or early lineage-committed hematopoietic stem cells ( Young et al., 2006 ). Aplastic anemia is characterized by pancytopenia (marked decreases in all blood component counts) and hematopoietic cell hypocellularity in bone marrow. Although most commonly associated with immune-mediated destruction of hematopoietic precursors, marked depletion of bone marrow hematopoietic cells or aplastic anemia may also occur with severe nutritional deficiencies associated with anorexia nervosa ( Abella et al., 2002 ) and 75% feed restriction in rats ( Moriyama et al., 2008 , Levin et al., 1993 ). 12.11.2.1.2.5 Cobalamin (B 12 ) and folate (B 9 ) Deficiencies in cobalamin and folate cause disruption of DNA synthesis that results in megaloblastic hematopoiesis, commonly associated with megaloblastic anemia and occasionally with decreases in neutrophil counts and/or pancytopenia ( Dale and Welte, 2016 , Green, 2016 ). Cobalamin or folate deficiencies may be due to malnutrition, malabsorption from gastrointestinal disease or dysfunction, or genetic deficiencies of transport proteins, such as intrinsic factor, transcobalamin, or R-factor for cobalamin, receptors for intestinal absorption, such as the CUBAM receptor, or metabolizing enzymes, such as methylenetetrahydrofolate reductase for folate ( Fowler, 1998 , Whitehead, 2006 , Montagle and Tauro, 1995 , Green, 2016 , Quadros, 2010 ). During megaloblastic hematopoiesis, asynchronous cytoplasmic and nuclear maturation may result in giant granulocytes with irregular nuclear chromatin patterns, such as giant metamyelocytes ( Green, 2016 ), or hypersegmented neutrophils ( Thompson et al., 1989 ). These conditions can often be managed with supplementation of the deficient vitamin. A differential for cobalamin or folate deficiency-induced decreases in blood neutrophil counts is copper deficiency, which can have a similar clinical manifestation ( Lazarchick, 2012 ). 12.11.2.1.2.6 Myelophthisis, myelofibrosis, and myelonecrosis Conditions that efface the bone marrow cavities or displace hematopoietic cells (myelophthisis) may result in decreased production of all hematopoietic cell lines, including neutrophils. Myeloproliferative syndromes ( Tefferi and Vardiman, 2009 ), many leukemic diseases ( Talcott et al., 1992 , Lamy and Loughran, 1999 ), lymphoproliferative neoplasia ( Schultze, 2010 ), or metastatic carcinoma ( Makoni and Laber, 2004 ) can cause sufficient bone marrow overcrowding to decrease the production of neutrophils. Fungal infections resulting in granulomatous inflammation of the bone marrow, such as can be observed with disseminated histoplasmosis, may also result in decreased neutrophil production. Disruption of the bone marrow cavities and hematopoietic stem cells from myelofibrosis or myelonecrosis may also be associated with decreased blood neutrophil counts ( Stockham and Scott, 2008a ). 12.11.2.1.2.7 Xenobiotic-induced Xenobiotic-induced decreases in neutrophil counts may be caused by immune-mediated or nonimmune-mediated mechanisms. The incidences of these events tend to be low with most implicated xenobiotics, with the exception of chemotherapeutic agents. Examples of both immune-mediated and nonimmune-mediated mechanisms are described later. Similar to primary immune-mediated decreases in neutrophil counts, xenobiotic-induced immune-mediated decreases in neutrophil counts are commonly associated with IgG or IgM antineutrophil antibodies, although some drugs may induce both ( Salama et al., 1989 ). Decreases in neutrophil counts by this mechanism tend to occur rapidly, within a few hours to 2 days after exposure ( Schwartzberg, 2006 ). Some xenobiotics mediate their effects by acting as haptens, which induce an antibody response targeting an antigen formed by the xenobiotic–neutrophil combination. Drugs reported to act as haptens include penicillin, gold-based compounds, aminopyrine, and propylthiouracil ( Salama et al., 1989 , Murphy et al., 1985 ). These xenobiotics induce neutrophil destruction in a drug-dependent manner, and discontinuation of treatment generally results in resolution of blood neutrophil counts within 7 days ( Schwartzberg, 2006 ). Some xenobiotics, including propylthiouracil and quinidine sulfate, cause the formation of immune complexes that can subsequently bind and destroy neutrophils, even if the xenobiotic is no longer present ( Schwartzberg, 2006 , Bhatt and Saleem, 2004 , Eisner et al., 1977 ). In addition, some xenobiotics, such as propylthiouracil, may also cause the formation of antineutrophil antibodies resulting in complement-mediated neutrophil destruction ( Akamizu et al., 2002 ). Similar to idiopathic aplastic anemia, xenobiotic-induced aplastic anemia is most commonly associated with immune-mediated destruction of uncommitted or early hematopoietic stem cells, although direct cytotoxicity, such as with chemotherapeutic agents, may also lead to aplastic anemia. Several xenobiotics, including chloramphenicol, anticonvulsants such as phenytoin and carbamazepine, gold-based compounds, and phenylbutazone have been associated with aplastic anemia ( Bloom and Brandt, 2008 ). Numerous xenobiotics have also been linked to SLE, with decreases in blood neutrophil counts in some cases. In xenobiotic-induced SLE, decreased neutrophil counts reflect production of autoantibodies and subsequent neutrophil destruction, similar to nonxenobiotic-induced SLE. Xenobiotics associated with SLE include anticonvulsants such as phenothiazines, chlorpromazine, and valproate, antibiotics such as penicillin, streptomycin, tetracycline, griseofulvin, and sulphonamides, and miscellaneous xenobiotics such as captopril, phenylbutazone, and lovastatin ( Stone, 2005 , Mutasim and Adams, 2000 ). In laboratory species used in nonclinical toxicology studies, transient xenobiotic-induced decreases in blood neutrophil counts have been associated with anaphylactoid reactions termed complement activation-related pseudoallergy (CARPA). These nonhypersensitive reactions are mediated instead by activation of the complement cascade, leading to the production of the anaphylatoxins C3a and C5a. CARPA typically occurs after the first dose of xenobiotic with decreasing severity or resolution following additional doses. Xenobiotics implicated in CARPA reactions include radiocontrast media, drug-carrying liposomes and lipid complexes, and some solvents with amphiphilic emulsifiers, such as Cremophor EL ( Szebeni, 2005 ). Nonimmune-mediated decreases in neutrophil counts may be observed with bone marrow suppression, which is associated with many xenobiotics, particularly chemotherapeutic agents. Chemotherapeutic-related decreases in neutrophil counts are often a result of direct cytotoxicity or suppressed proliferation of the rapidly dividing granulocytic precursors in the bone marrow. Examples of chemotherapeutic classes associated with decreased blood neutrophil counts include agents that cause direct DNA damage, such as platinum-containing agents (e.g., cisplatin, carboplatin) and classical alkylating agents (e.g., cyclophosphamide, melphalan, busulfan); mitotic spindle inhibitors (e.g., paclitaxel, docetaxel, vinblastine, vincristine); topoisomerase inhibitors (e.g., etoposide, doxorubicin); and antimetabolites (e.g., methotrexate, 6-mercaptopurine, 5-fluorouracil) ( Bhatt and Saleem, 2004 , Weiss, 2010 , Wailoo et al., 2009 ). Decreases in neutrophil counts are expected with chemotherapeutic treatment, and can be easily monitored through serial CBCs. If severe enough to increase the risk of life-threatening infections clinically, treatment with empiric antibiotics or G-CSF-like compounds may be considered. Suppression of granulopoiesis or direct granulocyte toxicity is also reported with some nonchemotherapeutic agents. Dose-dependent inhibition of colony-forming units of granulocytes and macrophages in the bone marrow has been reported with several anticonvulsant drugs, including valproic acid and carbamazepine, and beta-lactam antibiotics ( Schwartzberg, 2006 , Irvine et al., 1994 , Watts et al., 1990 , Neftel et al., 1985 ). Several other anticonvulsant drugs, including ethosuximide ( Mintzer et al., 2009 ) and phenytoin ( Sharafuddin et al., 1991 ), have been reported to cause direct toxic effects on granulocyte precursors. Antipsychotic agents, including clozapine and chlorpromazine, may also cause direct cytotoxic effects: neutrophil metabolism of clozapine has been demonstrated to form an unstable intracellular metabolite, nitrenium ion, which depletes glutathione and makes the neutrophils susceptible to oxidative damage and apoptosis ( Williams et al., 2000 ); chlorpromazine may cause cytotoxicity through the inhibition of thymidine and uracil uptake by neutrophils ( Pisciotta and Kaldahl, 1962 ). Thienopyridine inhibitors of platelet aggregation, including clopidogrel and ticlopidine, have also been associated with direct neutrophil toxicity resulting from mitochondrial toxicity by metabolites generated through myeloperoxidase metabolism of the parent compounds ( Maseneni et al., 2012 , Maseneni et al., 2013 ). 12.11.2.2 Lymphocytes The production of lymphocytes, or lymphopoiesis, progresses from pluripotent hematopoietic stem cells that differentiate into common lymphoid progenitor cells, and further differentiate into B-cells, T-cells, and natural killer (NK) cells. B-cell development begins in the fetal liver and transitions to bone marrow postnatally, where the cells undergo proliferation and differentiation, followed by migration to the peripheral lymphoid tissues. B-cell development requires a variety of soluble factors, including IL-3, IL-4, IL-11, INFγ, and TGFβ ( Burkhard, 2010 ). T-cell precursors migrate to the thymus during embryonic development, where they undergo proliferation, differentiation, and both positive and negative selection. T-cell development requires IL-7 stimulation ( Burkhard, 2010 ). NK-cell development, which requires IL-15 stimulation, occurs mostly in the fetal liver and thymus, as well as in the bone marrow after birth ( Burkhard, 2010 ). In health, blood lymphocytes are predominantly T-cells. Similar to neutrophils, blood lymphocytes are divided into circulating and marginating pools, with cells frequently shifting between these pools. Lymphocytes in the blood travel to lymph nodes, where they exit the blood through high endothelial venules and enter the lymph node cortices. Lymphocytes that migrate through the lymph nodes leave through efferent lymphatic vessels, from which they return to the blood. Blood lymphocytes may also emigrate to other tissues if chemotactic stimuli are present. In tissues, lymphocytes may proliferate, die, or migrate back into blood. Lymphocyte life spans are highly variable depending on the cell type and function, and some lymphocytes may live for years ( Stockham and Scott, 2008a ). Lymphocytes are the most numerous blood leukocytes in rats and mice, and are typically present in greater numbers than neutrophils in young cynomolgus monkeys and dogs. Cynomolgus monkeys used in nonclinical toxicology studies are often young or peripubertal, with greater lymphocyte than neutrophil counts. However, in both cynomolgus monkeys and dogs, there is a shift of blood leukocyte populations to a predominance of neutrophils with maturation, and nonclinical toxicology studies may include animals having either hematologic pattern. In adults of species with neutrophils as the most numerous blood leukocyte, lymphocytes are typically the second most numerous. 12.11.2.2.1 Increases in lymphocyte counts (lymphocytosis) 12.11.2.2.1.1 Age-related Lymphocytes are common as the predominant blood leukocyte of neonate and juvenile animals, even in species with predominant neutrophils in the circulation of adults. Recognition of this apparent "lymphocytosis" of young animals is important, as comparison of lymphocyte counts in young animals with adult historical control data may give the appearance of increased lymphocyte counts. Species in which "lymphocytosis" in young animals has been described include dogs and cats ( Stockham and Scott, 2008a ) and cynomolgus monkeys ( Sugimoto et al., 1986 ). As the shift from predominantly lymphocytes to predominantly neutrophils in circulation occurs around 4 to 5 years old in cynomolgus monkeys ( Sugimoto et al., 1986 ), it is not uncommon for nonclinical toxicology studies to include individuals with both lymphocyte and neutrophil-predominant leukograms. 12.11.2.2.1.2 Catecholamine-induced Similar to increases in neutrophil counts, increases in endogenous or exogenous catecholamines associated with excitement, fear, or exercise result in transient increases in lymphocyte counts. Catecholamine-induced increases in lymphocyte counts are associated with rapid shifts from the marginating to circulating lymphocyte pool, which is thought to be due to both decreased lymphocyte adhesion to endothelial cells and increased blood flow ( Benschop et al., 1996 ). Release of lymphocytes from the spleen in response to catecholamine stimulation likely also contributes to the increase in blood lymphocyte counts, but lymph nodes and bone marrow do not appear to be significant contributors ( Benschop et al., 1996 ). There are species differences in the response of lymphocytes to catecholamines, and the resultant increases in lymphocyte tend to be more common in monkeys and cats, while less common in adult dogs ( Smith et al., 2002 , Schultze, 2010 ). 12.11.2.2.1.3 Decreased Glucocorticoids Glucocorticoids have a negative effect on blood lymphocyte counts due to their effects on lymphocyte distribution in the body and suppression of lymphopoieis. Hypoadrenocorticism (Addison's disease), in which the adrenal glands are unable to maintain normal concentrations of glucocorticoids, may be associated with increases in blood lymphocyte counts due to the loss of the inhibitory effects of endogenous glucocorticoids ( Oelkers, 1996 , Stockham and Scott, 2008a , Avery and Avery, 2007 ). 12.11.2.2.1.4 Inflammation Acute inflammatory processes tend to cause decreases in lymphocyte counts, but some acute infectious processes, particularly several viral infections, may cause increases in lymphocyte counts. However, increases in lymphocyte counts are commonly observed with chronic inflammatory processes. Chronic stimulation of lymphocytes with antigens or cytokines results in the increased production of lymphocytes with release into the blood, causing the increases in blood lymphocyte counts. Reactive lymphocytes may be observed in blood accompanying inflammation-induced increases in lymphocytes counts. Reactive lymphocytes have a spectrum of morphologic changes that include increased cytoplasmic basophilia, large cells with mildly increased amounts of cytoplasm (lower nuclear to cytoplasmic ratios), variable patterns of chromatin clumping, and variable numbers of visible nucleoli. Occasionally, reactive lymphocytes may have paranuclear cytoplasmic clearing, giving a plasmacytoid appearance. Infectious mononucleosis syndromes in people are a relatively common cause of an inflammatory or reactive increase in lymphocyte counts that are usually acute ( Vasu and Caligiuri, 2016 ). Chronic infections leading to increase in blood lymphocyte counts may include visceral leishmaniasis, parasitic infections such as strongyloidiasis, and leprosy ( Vasu and Caligiuri, 2016 , Rai et al., 2008 , Myers et al., 2000 ). Several chronic infections, including ehrlichiosis, Rocky Mountain spotted fever, leishmaniasis, trypanosomiasis, and brucellosis, have been associated with increases in blood lymphocyte counts in dogs ( Schultze, 2010 ). 12.11.2.2.1.5 Neoplasia Increases in blood lymphocyte counts associated with lymphoproliferative neoplasia may represent either lymphocytic leukemia or the leukemic phase of lymphoma. A normal circulating lymphocyte population should be heterogeneous, with predominantly small lymphocytes and fewer intermediate to large lymphocytes. Increased blood lymphocyte counts with a loss of heterogeneity in the blood lymphocyte population or predominantly a monomorphic intermediate to large lymphocyte population are key features for diagnosing lymphoproliferative neoplasia. With the exception of chronic lymphocytic leukemia, which is characterized by increased numbers of small lymphocytes with few or subtle morphologic alterations, circulating lymphocytes often have abnormal morphologic features that may be observed microscopically. Abnormal morphologic features of the leukemic lymphocyte population may include increased cytoplasmic basophilia, increased amounts of cytoplasm with altered nuclear to cytoplasmic ratios, irregular clumping of nuclear chromatin, indentation or lobulation of nuclei, variably sized but typically prominent nucleoli, or multiple nucleoli. Some of these morphologic features are similar to those observed in reactive lymphocytes, but these two processes may be distinguished by the overall heterogeneity of the lymphocyte population and proportion of the lymphocyte population with these morphologic alterations. Additional testing, such as flow cytometry for phenotyping or PCR for antigen receptor rearrangement (PARR), also aids in the diagnosis or characterization of lymphoproliferative neoplasia. Lymphoproliferative neoplasia is observed as a relatively common background finding in older rats and mice during nonclinical toxicology studies ( Frith et al., 1993 ). Although less common, it may also be observed in low frequencies in older monkeys. Monkeys with concurrent infection with species-specific lymphocryptoviruses and immunosuppression have been reported to have virus-related lymphoproliferative neoplasias ( Magden et al., 2015 ). Lymphocryptoviruses are in the Gammaherpesvirinae subfamily and are related to Epstein-Barr virus, which has been associated with lymphoproliferative neoplasia in people but may aberrantly infect New World monkeys ( Magden et al., 2015 , Thorley-Lawson and Gross, 2004 ). Increases in blood lymphocyte counts may also occur secondary to nonlymphoproliferative neoplasms. Increases in polyclonal T-cells have been reported in patients with malignant thymoma, while increases in reactive lymphocytes have been reported with AML and systemic mastocytosis ( Vasu and Caligiuri, 2016 ). 12.11.2.2.1.6 Xenobiotic-induced Xenobiotic-induced increases in blood lymphocyte counts are relatively uncommon, with most reports associated with an administration of catecholamines or rare idiosyncratic hypersensitivity-type reactions. Administration of exogenous catecholamines, such as epinephrine, or adrenergic agonists has similar effects on blood lymphocyte counts as those mediated by endogenous catecholamines. Rapid shifting of lymphocytes from marginating to circulating blood pools as well as mobilization of lymphocytes from the spleen contributes to the increases in lymphocyte counts. Lymphocytes appear to have their adrenergic effects primarily mediated by β 2 -adrenergic receptors ( Benschop et al., 1996 ). Increases in blood lymphocyte counts with the presence of atypical lymphocytes in circulation have been associated with drug reaction with eosinophilia and systemic symptoms (DRESS), a form of drug-related hypersensitivity. Several anticonvulsant drugs, including phenobarbital and phenytoin, allopurinol, minocycline, sulfonamides, gold salts, and dapsone have been associated with DRESS ( Roujeau, 2005 , Callot et al., 1996 ). Treatment of CML and chronic lymphocytic leukemia with dasatinib and ibrutinib, respectively, has been associated with increases in blood lymphocytes counts ( Vasu and Caligiuri, 2016 ). Dasatinib-related increases in lymphocyte counts may be related to expansion of T-cell or NK-cell populations and increases in IL-2R, INF-γ, and IL-6, with reported favorable outcome to treatment, while ibrutinib-related increases in lymphocyte counts may be related to and increased efflux of lymphocytes from lymphoid tissues ( Vasu and Caligiuri, 2016 ). 12.11.2.2.2 Decreases in lymphocyte counts (lymphopenia) 12.11.2.2.2.1 Glucocorticoid-induced Endogenous glucocorticoids may be increased with chronic stress or hyperadrenocorticism, and decreases in lymphocyte counts tend to be the most prominent and consistent glucocorticoid-mediated leukocyte change across species. Glucocorticoids induce decreases in blood lymphocyte counts through several mechanisms. In addition to a rapid shift of lymphocytes from the circulating to marginating and tissue pools, there is evidence for both lymphocyte redistribution from blood to bone marrow ( Fauci, 1975 ) and decreased efflux of lymphocytes from lymphoid tissues ( Bloemena et al., 1990 ) contributing to the shift of lymphocytes to tissue pools. With long-term increases in glucocorticoid concentrations, lymphotoxicity may be observed due to an increased activation of apoptotic pathways ( Garvy et al., 1993 , Tuckermann et al., 2005 ). In rats, feed restriction has been associated with decreases in blood lymphocyte counts and lymphocyte depletion in various lymphoid tissues ( Moriyama et al., 2008 ), possibly associated with prolonged stress and therefore a glucocorticoid-mediated effect. Indirect test article-related effects mediated by altered food consumption are important to consider in nonclinical toxicology studies, in which test articles may cause direct or indirect hematologic effects. Interpretation of these changes, including stress-associated effects, must be made cautiously and thoughtfully, using a weight of evidence approach. 12.11.2.2.2.2 Inflammation Decreases in lymphocyte counts are typically observed with acute inflammation. These decreases are likely due to increased margination and emigration of lymphocytes to the site of inflammation, increased migration of lymphocytes to lymphoid tissues, and decreased efflux of lymphocytes out of lymphoid tissues ( Stockham and Scott, 2008a ). Stress associated with illness or acute inflammation may also contribute by glucocorticoid-induced mechanisms ( Stockham and Scott, 2008a , Schultze, 2010 ). Many infectious agents may cause a decrease in lymphocyte counts due to inflammation. Infectious agents associated with decreases in lymphocyte counts include viruses such as coronavirus, parvovirus, West Nile virus, hepatitis viruses, and influenza ( Vasu and Caligiuri, 2016 , Schultze, 2010 ); acute systemic bacterial infections; as well as tuberculosis, typhoid fever, and bacterial pneumonias ( Vasu and Caligiuri, 2016 , Magden et al., 2015 ). The acute phase of malaria may also be associated with decreases in lymphocyte counts ( Vasu and Caligiuri, 2016 ). 12.11.2.2.2.3 Viral-induced Infection with immunodeficiency viruses, including human, simian, and feline immunodeficiency viruses, may result in destruction of both infected and noninfected lymphocytes. HIV directly infects CD4 + T-cells via the CD4 molecule; infected cells then migrate to lymphoid tissues where the virus replicates and infects more CD4 + T-cells ( Chinen and Shearer, 2010 ). HIV-mediated lymphocyte destruction is likely multifactorial; HIV may be cytotoxic, directly induce T-cell apoptosis, induce T-cell death through a nonspecific immune response, and cause T-cell death by stimulating autophagocytic pathways ( Chinen and Shearer, 2010 , Stump and VandeWoude, 2007 ). SIV and FIV tropism for T-cells is also mediated by receptors expressed by CD4 + T-cells ( Stump and VandeWoude, 2007 ). Simian betaretrovirus also causes decreases in blood lymphocyte counts due to infection and eventual depletion of both B-cells and T-cells, although infection of nonlymphoid cells also occurs ( Montiel, 2010 ). 12.11.2.2.2.4 Immune-mediated Immune-mediated destruction of lymphocytes is uncommon. When occurring in the autoimmune disease SLE, such immune-mediated decreases in lymphocyte counts typically occur with concurrent cutaneous, arthritic, or neurologic disorders ( Stone, 2005 ), and may be the result of autoantibodies causing lymphocyte destruction or death through apoptosis ( Lu et al., 2012 , Massardo et al., 2009 , Noguchi et al., 1992 , Budman and Steinberg, 1977 ). 12.11.2.2.2.5 Inherited causes Although rare, some inherited immunodeficiency syndromes cause blood lymphocyte counts to be decreased. One example, severe combined immunodeficiency (SCID), has been reported in humans, dogs, mice, and horses ( Suter, 2010 , Notarangelo, 2010 , Meek et al., 2001 , Felsberg et al., 1992 , Custer et al., 1985 ). SCID may be inherited through autosomal recessive or X-linked recessive patterns, and causes consistent decreases in T-cells, with concurrent decreases in B-cells or NK-cells in some forms of the disease. SCID in humans is caused by a variety of mechanisms, including: adenosine deaminase deficiency resulting in early cell death due to metabolite accumulation; common gamma chain or janus kinase 3 (JAK3) mutations that cause decreased survival of T-cell precursors due to defective cytokine signaling; recombinase-activating gene 1 (RAG1) or RAG2 mutations that cause defective V(D)J rearrangement of B-cell and T-cell receptors; and mutations in CD3 or CD45 that cause defects in T-cell receptor signaling ( Suter, 2010 , Notarangelo, 2010 ). SCID in Jack Russell terriers, Arabian foals, and mice has been demonstrated to be caused by defective V(D)J recombination due to loss of DNA-dependent protein kinase ( Meek et al., 2001 ). X-linked SCID has been described in both Bassett Hounds and Welsh Corgi dogs ( Suter, 2010 ). Other inherited immunodeficiency syndromes resulting in decreases in blood lymphocyte counts include reticular dysgenesis, ataxia-telangiectasia, and Wiskott-Aldrich syndrome ( Vasu and Caligiuri, 2016 ). 12.11.2.2.2.6 Loss of lymph fluid Although uncommon, disorders causing chronic loss of lymphocyte-rich lymph fluid lead to body depletion of lymphocytes and decrease in blood lymphocyte counts. Examples of such conditions include protein-losing enteropathy, lymphangiectasia, ulcerative enteritis, or repeated iatrogenic removal of chylothoracic fluid ( Vasu and Caligiuri, 2016 , Schultze, 2010 , Stockham and Scott, 2008a ). 12.11.2.2.2.7 Neoplasia Although lymphoproliferative neoplasia may be associated with increases in lymphocyte counts as previously discussed, lymphoma and lymphocytic leukemia may also be associated with decreases in lymphocyte counts including from altered lymphocyte recirculation patterns or decreased production of nonneoplasic lymphocytes secondary to lymphoid organ damage ( Mitrovic et al., 2012 , Schultze, 2010 , Stockham and Scott, 2008a ). 12.11.2.2.2.8 Xenobiotic-induced Lymphoid suppression is a common finding with many xenobiotics and may be associated with decreases in blood lymphocyte counts. Mechanisms through which these decreases occur may either be part of the expected pharmacology of these compounds or may represent an off-target effect. Several examples of xenobiotic-induced decreases in lymphocyte counts are described here. Administration of exogenous glucocorticoids, for antiinflammatory or immunosuppressive purposes, will result in decreases in blood lymphocyte counts. The mechanisms for this are the same as those for endogenous glucocorticoids, and include altered blood and tissue pool distribution, decreased efflux of lymphocytes from lymphoid tissues, and increased lymphocyte apoptosis with prolonged glucocorticoid exposure. Other immunosuppressive agents that cause decreased blood lymphocyte counts include cyclophosphamide, methotrexate, purine nucleoside analogs, and azathioprine. Cyclophosphamide has been associated with profound decrease in blood lymphocyte counts through its effects on all lymphocyte subtypes ( Gergely, 1999 ). Methotrexate causes decreases in circulating CD4 + and CD8 + T-cells, while cladribine, a purine nucleoside analog, causes apoptosis of lymphocytes and has been reported to affect both CD4 + and CD8 + T-cells ( Gergely, 1999 ). Azothiaprine-induced decreases in blood lymphocyte counts appear to be due to long-term administration at high dose levels ( Johnson et al., 1995 , Gergely, 1999 ). As discussed previously, numerous xenobiotics have been associated with drug-induced SLE in people. Decreases in lymphocytes in these cases are likely due to the production of autoantibodies with subsequent lymphocyte destruction, similar to nonxenobiotic-induced SLE. Xenobiotics associated with SLE include several anticonvulsants such as phenothiazines, chlorpromazine, and valproate, several antibiotics such as penicillin, streptomycin, tetracycline, griseofulvin, and sulphonamides, and miscellaneous xenobiotics such as captopril, phenylbutazone, and lovastatin ( Stone, 2005 , Mutasim and Adams, 2000 ). Chemotherapeutic agents are also frequently associated with decreases in lymphocyte counts, which may precede episodes of febrile neutropenia ( Gergely, 1999 ). Carboplatin, dacarbazine, and paclitaxel have been reported to induce decreases in CD4 + T-cells, while epirubicin and mitomycin appear to affect CD8 + T-cells to a greater degree than CD4 + T-cells, and pentostatin affects both B-cell and T-cell populations ( Gergely, 1999 ). Antilymphocyte monoclonal antibodies have been used to treat autoimmune diseases as well as to cause immunosuppression to prevent acute transplant rejection. Examples of these monoclonal antibodies include Muromonab CD3 (OKT3) and CAMPATH-1H ( Vial et al., 2002 , Gergely, 1999 ). Other classes of drugs reported to cause decrease lymphocyte counts are varied: pesticides including organochloride pesticides such as pentachlorophenol, organotin compounds, and organophosphates ( Corsini et al., 2013 ); thienopyridines, such as clopidogrel and ticlopidine, which can cause direct lymphotoxicity at high concentrations ( Maseneni et al., 2013 ); the histamine H2 receptor antagonist cimetidine; the anticonvulsant carbamazepine; imidazoles used to treat fungal infections; and opioids such as morphine ( Gergely, 1999 ). 12.11.2.2.1 Increases in lymphocyte counts (lymphocytosis) 12.11.2.2.1.1 Age-related Lymphocytes are common as the predominant blood leukocyte of neonate and juvenile animals, even in species with predominant neutrophils in the circulation of adults. Recognition of this apparent "lymphocytosis" of young animals is important, as comparison of lymphocyte counts in young animals with adult historical control data may give the appearance of increased lymphocyte counts. Species in which "lymphocytosis" in young animals has been described include dogs and cats ( Stockham and Scott, 2008a ) and cynomolgus monkeys ( Sugimoto et al., 1986 ). As the shift from predominantly lymphocytes to predominantly neutrophils in circulation occurs around 4 to 5 years old in cynomolgus monkeys ( Sugimoto et al., 1986 ), it is not uncommon for nonclinical toxicology studies to include individuals with both lymphocyte and neutrophil-predominant leukograms. 12.11.2.2.1.2 Catecholamine-induced Similar to increases in neutrophil counts, increases in endogenous or exogenous catecholamines associated with excitement, fear, or exercise result in transient increases in lymphocyte counts. Catecholamine-induced increases in lymphocyte counts are associated with rapid shifts from the marginating to circulating lymphocyte pool, which is thought to be due to both decreased lymphocyte adhesion to endothelial cells and increased blood flow ( Benschop et al., 1996 ). Release of lymphocytes from the spleen in response to catecholamine stimulation likely also contributes to the increase in blood lymphocyte counts, but lymph nodes and bone marrow do not appear to be significant contributors ( Benschop et al., 1996 ). There are species differences in the response of lymphocytes to catecholamines, and the resultant increases in lymphocyte tend to be more common in monkeys and cats, while less common in adult dogs ( Smith et al., 2002 , Schultze, 2010 ). 12.11.2.2.1.3 Decreased Glucocorticoids Glucocorticoids have a negative effect on blood lymphocyte counts due to their effects on lymphocyte distribution in the body and suppression of lymphopoieis. Hypoadrenocorticism (Addison's disease), in which the adrenal glands are unable to maintain normal concentrations of glucocorticoids, may be associated with increases in blood lymphocyte counts due to the loss of the inhibitory effects of endogenous glucocorticoids ( Oelkers, 1996 , Stockham and Scott, 2008a , Avery and Avery, 2007 ). 12.11.2.2.1.4 Inflammation Acute inflammatory processes tend to cause decreases in lymphocyte counts, but some acute infectious processes, particularly several viral infections, may cause increases in lymphocyte counts. However, increases in lymphocyte counts are commonly observed with chronic inflammatory processes. Chronic stimulation of lymphocytes with antigens or cytokines results in the increased production of lymphocytes with release into the blood, causing the increases in blood lymphocyte counts. Reactive lymphocytes may be observed in blood accompanying inflammation-induced increases in lymphocytes counts. Reactive lymphocytes have a spectrum of morphologic changes that include increased cytoplasmic basophilia, large cells with mildly increased amounts of cytoplasm (lower nuclear to cytoplasmic ratios), variable patterns of chromatin clumping, and variable numbers of visible nucleoli. Occasionally, reactive lymphocytes may have paranuclear cytoplasmic clearing, giving a plasmacytoid appearance. Infectious mononucleosis syndromes in people are a relatively common cause of an inflammatory or reactive increase in lymphocyte counts that are usually acute ( Vasu and Caligiuri, 2016 ). Chronic infections leading to increase in blood lymphocyte counts may include visceral leishmaniasis, parasitic infections such as strongyloidiasis, and leprosy ( Vasu and Caligiuri, 2016 , Rai et al., 2008 , Myers et al., 2000 ). Several chronic infections, including ehrlichiosis, Rocky Mountain spotted fever, leishmaniasis, trypanosomiasis, and brucellosis, have been associated with increases in blood lymphocyte counts in dogs ( Schultze, 2010 ). 12.11.2.2.1.5 Neoplasia Increases in blood lymphocyte counts associated with lymphoproliferative neoplasia may represent either lymphocytic leukemia or the leukemic phase of lymphoma. A normal circulating lymphocyte population should be heterogeneous, with predominantly small lymphocytes and fewer intermediate to large lymphocytes. Increased blood lymphocyte counts with a loss of heterogeneity in the blood lymphocyte population or predominantly a monomorphic intermediate to large lymphocyte population are key features for diagnosing lymphoproliferative neoplasia. With the exception of chronic lymphocytic leukemia, which is characterized by increased numbers of small lymphocytes with few or subtle morphologic alterations, circulating lymphocytes often have abnormal morphologic features that may be observed microscopically. Abnormal morphologic features of the leukemic lymphocyte population may include increased cytoplasmic basophilia, increased amounts of cytoplasm with altered nuclear to cytoplasmic ratios, irregular clumping of nuclear chromatin, indentation or lobulation of nuclei, variably sized but typically prominent nucleoli, or multiple nucleoli. Some of these morphologic features are similar to those observed in reactive lymphocytes, but these two processes may be distinguished by the overall heterogeneity of the lymphocyte population and proportion of the lymphocyte population with these morphologic alterations. Additional testing, such as flow cytometry for phenotyping or PCR for antigen receptor rearrangement (PARR), also aids in the diagnosis or characterization of lymphoproliferative neoplasia. Lymphoproliferative neoplasia is observed as a relatively common background finding in older rats and mice during nonclinical toxicology studies ( Frith et al., 1993 ). Although less common, it may also be observed in low frequencies in older monkeys. Monkeys with concurrent infection with species-specific lymphocryptoviruses and immunosuppression have been reported to have virus-related lymphoproliferative neoplasias ( Magden et al., 2015 ). Lymphocryptoviruses are in the Gammaherpesvirinae subfamily and are related to Epstein-Barr virus, which has been associated with lymphoproliferative neoplasia in people but may aberrantly infect New World monkeys ( Magden et al., 2015 , Thorley-Lawson and Gross, 2004 ). Increases in blood lymphocyte counts may also occur secondary to nonlymphoproliferative neoplasms. Increases in polyclonal T-cells have been reported in patients with malignant thymoma, while increases in reactive lymphocytes have been reported with AML and systemic mastocytosis ( Vasu and Caligiuri, 2016 ). 12.11.2.2.1.6 Xenobiotic-induced Xenobiotic-induced increases in blood lymphocyte counts are relatively uncommon, with most reports associated with an administration of catecholamines or rare idiosyncratic hypersensitivity-type reactions. Administration of exogenous catecholamines, such as epinephrine, or adrenergic agonists has similar effects on blood lymphocyte counts as those mediated by endogenous catecholamines. Rapid shifting of lymphocytes from marginating to circulating blood pools as well as mobilization of lymphocytes from the spleen contributes to the increases in lymphocyte counts. Lymphocytes appear to have their adrenergic effects primarily mediated by β 2 -adrenergic receptors ( Benschop et al., 1996 ). Increases in blood lymphocyte counts with the presence of atypical lymphocytes in circulation have been associated with drug reaction with eosinophilia and systemic symptoms (DRESS), a form of drug-related hypersensitivity. Several anticonvulsant drugs, including phenobarbital and phenytoin, allopurinol, minocycline, sulfonamides, gold salts, and dapsone have been associated with DRESS ( Roujeau, 2005 , Callot et al., 1996 ). Treatment of CML and chronic lymphocytic leukemia with dasatinib and ibrutinib, respectively, has been associated with increases in blood lymphocytes counts ( Vasu and Caligiuri, 2016 ). Dasatinib-related increases in lymphocyte counts may be related to expansion of T-cell or NK-cell populations and increases in IL-2R, INF-γ, and IL-6, with reported favorable outcome to treatment, while ibrutinib-related increases in lymphocyte counts may be related to and increased efflux of lymphocytes from lymphoid tissues ( Vasu and Caligiuri, 2016 ). 12.11.2.2.1.1 Age-related Lymphocytes are common as the predominant blood leukocyte of neonate and juvenile animals, even in species with predominant neutrophils in the circulation of adults. Recognition of this apparent "lymphocytosis" of young animals is important, as comparison of lymphocyte counts in young animals with adult historical control data may give the appearance of increased lymphocyte counts. Species in which "lymphocytosis" in young animals has been described include dogs and cats ( Stockham and Scott, 2008a ) and cynomolgus monkeys ( Sugimoto et al., 1986 ). As the shift from predominantly lymphocytes to predominantly neutrophils in circulation occurs around 4 to 5 years old in cynomolgus monkeys ( Sugimoto et al., 1986 ), it is not uncommon for nonclinical toxicology studies to include individuals with both lymphocyte and neutrophil-predominant leukograms. 12.11.2.2.1.2 Catecholamine-induced Similar to increases in neutrophil counts, increases in endogenous or exogenous catecholamines associated with excitement, fear, or exercise result in transient increases in lymphocyte counts. Catecholamine-induced increases in lymphocyte counts are associated with rapid shifts from the marginating to circulating lymphocyte pool, which is thought to be due to both decreased lymphocyte adhesion to endothelial cells and increased blood flow ( Benschop et al., 1996 ). Release of lymphocytes from the spleen in response to catecholamine stimulation likely also contributes to the increase in blood lymphocyte counts, but lymph nodes and bone marrow do not appear to be significant contributors ( Benschop et al., 1996 ). There are species differences in the response of lymphocytes to catecholamines, and the resultant increases in lymphocyte tend to be more common in monkeys and cats, while less common in adult dogs ( Smith et al., 2002 , Schultze, 2010 ). 12.11.2.2.1.3 Decreased Glucocorticoids Glucocorticoids have a negative effect on blood lymphocyte counts due to their effects on lymphocyte distribution in the body and suppression of lymphopoieis. Hypoadrenocorticism (Addison's disease), in which the adrenal glands are unable to maintain normal concentrations of glucocorticoids, may be associated with increases in blood lymphocyte counts due to the loss of the inhibitory effects of endogenous glucocorticoids ( Oelkers, 1996 , Stockham and Scott, 2008a , Avery and Avery, 2007 ). 12.11.2.2.1.4 Inflammation Acute inflammatory processes tend to cause decreases in lymphocyte counts, but some acute infectious processes, particularly several viral infections, may cause increases in lymphocyte counts. However, increases in lymphocyte counts are commonly observed with chronic inflammatory processes. Chronic stimulation of lymphocytes with antigens or cytokines results in the increased production of lymphocytes with release into the blood, causing the increases in blood lymphocyte counts. Reactive lymphocytes may be observed in blood accompanying inflammation-induced increases in lymphocytes counts. Reactive lymphocytes have a spectrum of morphologic changes that include increased cytoplasmic basophilia, large cells with mildly increased amounts of cytoplasm (lower nuclear to cytoplasmic ratios), variable patterns of chromatin clumping, and variable numbers of visible nucleoli. Occasionally, reactive lymphocytes may have paranuclear cytoplasmic clearing, giving a plasmacytoid appearance. Infectious mononucleosis syndromes in people are a relatively common cause of an inflammatory or reactive increase in lymphocyte counts that are usually acute ( Vasu and Caligiuri, 2016 ). Chronic infections leading to increase in blood lymphocyte counts may include visceral leishmaniasis, parasitic infections such as strongyloidiasis, and leprosy ( Vasu and Caligiuri, 2016 , Rai et al., 2008 , Myers et al., 2000 ). Several chronic infections, including ehrlichiosis, Rocky Mountain spotted fever, leishmaniasis, trypanosomiasis, and brucellosis, have been associated with increases in blood lymphocyte counts in dogs ( Schultze, 2010 ). 12.11.2.2.1.5 Neoplasia Increases in blood lymphocyte counts associated with lymphoproliferative neoplasia may represent either lymphocytic leukemia or the leukemic phase of lymphoma. A normal circulating lymphocyte population should be heterogeneous, with predominantly small lymphocytes and fewer intermediate to large lymphocytes. Increased blood lymphocyte counts with a loss of heterogeneity in the blood lymphocyte population or predominantly a monomorphic intermediate to large lymphocyte population are key features for diagnosing lymphoproliferative neoplasia. With the exception of chronic lymphocytic leukemia, which is characterized by increased numbers of small lymphocytes with few or subtle morphologic alterations, circulating lymphocytes often have abnormal morphologic features that may be observed microscopically. Abnormal morphologic features of the leukemic lymphocyte population may include increased cytoplasmic basophilia, increased amounts of cytoplasm with altered nuclear to cytoplasmic ratios, irregular clumping of nuclear chromatin, indentation or lobulation of nuclei, variably sized but typically prominent nucleoli, or multiple nucleoli. Some of these morphologic features are similar to those observed in reactive lymphocytes, but these two processes may be distinguished by the overall heterogeneity of the lymphocyte population and proportion of the lymphocyte population with these morphologic alterations. Additional testing, such as flow cytometry for phenotyping or PCR for antigen receptor rearrangement (PARR), also aids in the diagnosis or characterization of lymphoproliferative neoplasia. Lymphoproliferative neoplasia is observed as a relatively common background finding in older rats and mice during nonclinical toxicology studies ( Frith et al., 1993 ). Although less common, it may also be observed in low frequencies in older monkeys. Monkeys with concurrent infection with species-specific lymphocryptoviruses and immunosuppression have been reported to have virus-related lymphoproliferative neoplasias ( Magden et al., 2015 ). Lymphocryptoviruses are in the Gammaherpesvirinae subfamily and are related to Epstein-Barr virus, which has been associated with lymphoproliferative neoplasia in people but may aberrantly infect New World monkeys ( Magden et al., 2015 , Thorley-Lawson and Gross, 2004 ). Increases in blood lymphocyte counts may also occur secondary to nonlymphoproliferative neoplasms. Increases in polyclonal T-cells have been reported in patients with malignant thymoma, while increases in reactive lymphocytes have been reported with AML and systemic mastocytosis ( Vasu and Caligiuri, 2016 ). 12.11.2.2.1.6 Xenobiotic-induced Xenobiotic-induced increases in blood lymphocyte counts are relatively uncommon, with most reports associated with an administration of catecholamines or rare idiosyncratic hypersensitivity-type reactions. Administration of exogenous catecholamines, such as epinephrine, or adrenergic agonists has similar effects on blood lymphocyte counts as those mediated by endogenous catecholamines. Rapid shifting of lymphocytes from marginating to circulating blood pools as well as mobilization of lymphocytes from the spleen contributes to the increases in lymphocyte counts. Lymphocytes appear to have their adrenergic effects primarily mediated by β 2 -adrenergic receptors ( Benschop et al., 1996 ). Increases in blood lymphocyte counts with the presence of atypical lymphocytes in circulation have been associated with drug reaction with eosinophilia and systemic symptoms (DRESS), a form of drug-related hypersensitivity. Several anticonvulsant drugs, including phenobarbital and phenytoin, allopurinol, minocycline, sulfonamides, gold salts, and dapsone have been associated with DRESS ( Roujeau, 2005 , Callot et al., 1996 ). Treatment of CML and chronic lymphocytic leukemia with dasatinib and ibrutinib, respectively, has been associated with increases in blood lymphocytes counts ( Vasu and Caligiuri, 2016 ). Dasatinib-related increases in lymphocyte counts may be related to expansion of T-cell or NK-cell populations and increases in IL-2R, INF-γ, and IL-6, with reported favorable outcome to treatment, while ibrutinib-related increases in lymphocyte counts may be related to and increased efflux of lymphocytes from lymphoid tissues ( Vasu and Caligiuri, 2016 ). 12.11.2.2.2 Decreases in lymphocyte counts (lymphopenia) 12.11.2.2.2.1 Glucocorticoid-induced Endogenous glucocorticoids may be increased with chronic stress or hyperadrenocorticism, and decreases in lymphocyte counts tend to be the most prominent and consistent glucocorticoid-mediated leukocyte change across species. Glucocorticoids induce decreases in blood lymphocyte counts through several mechanisms. In addition to a rapid shift of lymphocytes from the circulating to marginating and tissue pools, there is evidence for both lymphocyte redistribution from blood to bone marrow ( Fauci, 1975 ) and decreased efflux of lymphocytes from lymphoid tissues ( Bloemena et al., 1990 ) contributing to the shift of lymphocytes to tissue pools. With long-term increases in glucocorticoid concentrations, lymphotoxicity may be observed due to an increased activation of apoptotic pathways ( Garvy et al., 1993 , Tuckermann et al., 2005 ). In rats, feed restriction has been associated with decreases in blood lymphocyte counts and lymphocyte depletion in various lymphoid tissues ( Moriyama et al., 2008 ), possibly associated with prolonged stress and therefore a glucocorticoid-mediated effect. Indirect test article-related effects mediated by altered food consumption are important to consider in nonclinical toxicology studies, in which test articles may cause direct or indirect hematologic effects. Interpretation of these changes, including stress-associated effects, must be made cautiously and thoughtfully, using a weight of evidence approach. 12.11.2.2.2.2 Inflammation Decreases in lymphocyte counts are typically observed with acute inflammation. These decreases are likely due to increased margination and emigration of lymphocytes to the site of inflammation, increased migration of lymphocytes to lymphoid tissues, and decreased efflux of lymphocytes out of lymphoid tissues ( Stockham and Scott, 2008a ). Stress associated with illness or acute inflammation may also contribute by glucocorticoid-induced mechanisms ( Stockham and Scott, 2008a , Schultze, 2010 ). Many infectious agents may cause a decrease in lymphocyte counts due to inflammation. Infectious agents associated with decreases in lymphocyte counts include viruses such as coronavirus, parvovirus, West Nile virus, hepatitis viruses, and influenza ( Vasu and Caligiuri, 2016 , Schultze, 2010 ); acute systemic bacterial infections; as well as tuberculosis, typhoid fever, and bacterial pneumonias ( Vasu and Caligiuri, 2016 , Magden et al., 2015 ). The acute phase of malaria may also be associated with decreases in lymphocyte counts ( Vasu and Caligiuri, 2016 ). 12.11.2.2.2.3 Viral-induced Infection with immunodeficiency viruses, including human, simian, and feline immunodeficiency viruses, may result in destruction of both infected and noninfected lymphocytes. HIV directly infects CD4 + T-cells via the CD4 molecule; infected cells then migrate to lymphoid tissues where the virus replicates and infects more CD4 + T-cells ( Chinen and Shearer, 2010 ). HIV-mediated lymphocyte destruction is likely multifactorial; HIV may be cytotoxic, directly induce T-cell apoptosis, induce T-cell death through a nonspecific immune response, and cause T-cell death by stimulating autophagocytic pathways ( Chinen and Shearer, 2010 , Stump and VandeWoude, 2007 ). SIV and FIV tropism for T-cells is also mediated by receptors expressed by CD4 + T-cells ( Stump and VandeWoude, 2007 ). Simian betaretrovirus also causes decreases in blood lymphocyte counts due to infection and eventual depletion of both B-cells and T-cells, although infection of nonlymphoid cells also occurs ( Montiel, 2010 ). 12.11.2.2.2.4 Immune-mediated Immune-mediated destruction of lymphocytes is uncommon. When occurring in the autoimmune disease SLE, such immune-mediated decreases in lymphocyte counts typically occur with concurrent cutaneous, arthritic, or neurologic disorders ( Stone, 2005 ), and may be the result of autoantibodies causing lymphocyte destruction or death through apoptosis ( Lu et al., 2012 , Massardo et al., 2009 , Noguchi et al., 1992 , Budman and Steinberg, 1977 ). 12.11.2.2.2.5 Inherited causes Although rare, some inherited immunodeficiency syndromes cause blood lymphocyte counts to be decreased. One example, severe combined immunodeficiency (SCID), has been reported in humans, dogs, mice, and horses ( Suter, 2010 , Notarangelo, 2010 , Meek et al., 2001 , Felsberg et al., 1992 , Custer et al., 1985 ). SCID may be inherited through autosomal recessive or X-linked recessive patterns, and causes consistent decreases in T-cells, with concurrent decreases in B-cells or NK-cells in some forms of the disease. SCID in humans is caused by a variety of mechanisms, including: adenosine deaminase deficiency resulting in early cell death due to metabolite accumulation; common gamma chain or janus kinase 3 (JAK3) mutations that cause decreased survival of T-cell precursors due to defective cytokine signaling; recombinase-activating gene 1 (RAG1) or RAG2 mutations that cause defective V(D)J rearrangement of B-cell and T-cell receptors; and mutations in CD3 or CD45 that cause defects in T-cell receptor signaling ( Suter, 2010 , Notarangelo, 2010 ). SCID in Jack Russell terriers, Arabian foals, and mice has been demonstrated to be caused by defective V(D)J recombination due to loss of DNA-dependent protein kinase ( Meek et al., 2001 ). X-linked SCID has been described in both Bassett Hounds and Welsh Corgi dogs ( Suter, 2010 ). Other inherited immunodeficiency syndromes resulting in decreases in blood lymphocyte counts include reticular dysgenesis, ataxia-telangiectasia, and Wiskott-Aldrich syndrome ( Vasu and Caligiuri, 2016 ). 12.11.2.2.2.6 Loss of lymph fluid Although uncommon, disorders causing chronic loss of lymphocyte-rich lymph fluid lead to body depletion of lymphocytes and decrease in blood lymphocyte counts. Examples of such conditions include protein-losing enteropathy, lymphangiectasia, ulcerative enteritis, or repeated iatrogenic removal of chylothoracic fluid ( Vasu and Caligiuri, 2016 , Schultze, 2010 , Stockham and Scott, 2008a ). 12.11.2.2.2.7 Neoplasia Although lymphoproliferative neoplasia may be associated with increases in lymphocyte counts as previously discussed, lymphoma and lymphocytic leukemia may also be associated with decreases in lymphocyte counts including from altered lymphocyte recirculation patterns or decreased production of nonneoplasic lymphocytes secondary to lymphoid organ damage ( Mitrovic et al., 2012 , Schultze, 2010 , Stockham and Scott, 2008a ). 12.11.2.2.2.8 Xenobiotic-induced Lymphoid suppression is a common finding with many xenobiotics and may be associated with decreases in blood lymphocyte counts. Mechanisms through which these decreases occur may either be part of the expected pharmacology of these compounds or may represent an off-target effect. Several examples of xenobiotic-induced decreases in lymphocyte counts are described here. Administration of exogenous glucocorticoids, for antiinflammatory or immunosuppressive purposes, will result in decreases in blood lymphocyte counts. The mechanisms for this are the same as those for endogenous glucocorticoids, and include altered blood and tissue pool distribution, decreased efflux of lymphocytes from lymphoid tissues, and increased lymphocyte apoptosis with prolonged glucocorticoid exposure. Other immunosuppressive agents that cause decreased blood lymphocyte counts include cyclophosphamide, methotrexate, purine nucleoside analogs, and azathioprine. Cyclophosphamide has been associated with profound decrease in blood lymphocyte counts through its effects on all lymphocyte subtypes ( Gergely, 1999 ). Methotrexate causes decreases in circulating CD4 + and CD8 + T-cells, while cladribine, a purine nucleoside analog, causes apoptosis of lymphocytes and has been reported to affect both CD4 + and CD8 + T-cells ( Gergely, 1999 ). Azothiaprine-induced decreases in blood lymphocyte counts appear to be due to long-term administration at high dose levels ( Johnson et al., 1995 , Gergely, 1999 ). As discussed previously, numerous xenobiotics have been associated with drug-induced SLE in people. Decreases in lymphocytes in these cases are likely due to the production of autoantibodies with subsequent lymphocyte destruction, similar to nonxenobiotic-induced SLE. Xenobiotics associated with SLE include several anticonvulsants such as phenothiazines, chlorpromazine, and valproate, several antibiotics such as penicillin, streptomycin, tetracycline, griseofulvin, and sulphonamides, and miscellaneous xenobiotics such as captopril, phenylbutazone, and lovastatin ( Stone, 2005 , Mutasim and Adams, 2000 ). Chemotherapeutic agents are also frequently associated with decreases in lymphocyte counts, which may precede episodes of febrile neutropenia ( Gergely, 1999 ). Carboplatin, dacarbazine, and paclitaxel have been reported to induce decreases in CD4 + T-cells, while epirubicin and mitomycin appear to affect CD8 + T-cells to a greater degree than CD4 + T-cells, and pentostatin affects both B-cell and T-cell populations ( Gergely, 1999 ). Antilymphocyte monoclonal antibodies have been used to treat autoimmune diseases as well as to cause immunosuppression to prevent acute transplant rejection. Examples of these monoclonal antibodies include Muromonab CD3 (OKT3) and CAMPATH-1H ( Vial et al., 2002 , Gergely, 1999 ). Other classes of drugs reported to cause decrease lymphocyte counts are varied: pesticides including organochloride pesticides such as pentachlorophenol, organotin compounds, and organophosphates ( Corsini et al., 2013 ); thienopyridines, such as clopidogrel and ticlopidine, which can cause direct lymphotoxicity at high concentrations ( Maseneni et al., 2013 ); the histamine H2 receptor antagonist cimetidine; the anticonvulsant carbamazepine; imidazoles used to treat fungal infections; and opioids such as morphine ( Gergely, 1999 ). 12.11.2.2.2.1 Glucocorticoid-induced Endogenous glucocorticoids may be increased with chronic stress or hyperadrenocorticism, and decreases in lymphocyte counts tend to be the most prominent and consistent glucocorticoid-mediated leukocyte change across species. Glucocorticoids induce decreases in blood lymphocyte counts through several mechanisms. In addition to a rapid shift of lymphocytes from the circulating to marginating and tissue pools, there is evidence for both lymphocyte redistribution from blood to bone marrow ( Fauci, 1975 ) and decreased efflux of lymphocytes from lymphoid tissues ( Bloemena et al., 1990 ) contributing to the shift of lymphocytes to tissue pools. With long-term increases in glucocorticoid concentrations, lymphotoxicity may be observed due to an increased activation of apoptotic pathways ( Garvy et al., 1993 , Tuckermann et al., 2005 ). In rats, feed restriction has been associated with decreases in blood lymphocyte counts and lymphocyte depletion in various lymphoid tissues ( Moriyama et al., 2008 ), possibly associated with prolonged stress and therefore a glucocorticoid-mediated effect. Indirect test article-related effects mediated by altered food consumption are important to consider in nonclinical toxicology studies, in which test articles may cause direct or indirect hematologic effects. Interpretation of these changes, including stress-associated effects, must be made cautiously and thoughtfully, using a weight of evidence approach. 12.11.2.2.2.2 Inflammation Decreases in lymphocyte counts are typically observed with acute inflammation. These decreases are likely due to increased margination and emigration of lymphocytes to the site of inflammation, increased migration of lymphocytes to lymphoid tissues, and decreased efflux of lymphocytes out of lymphoid tissues ( Stockham and Scott, 2008a ). Stress associated with illness or acute inflammation may also contribute by glucocorticoid-induced mechanisms ( Stockham and Scott, 2008a , Schultze, 2010 ). Many infectious agents may cause a decrease in lymphocyte counts due to inflammation. Infectious agents associated with decreases in lymphocyte counts include viruses such as coronavirus, parvovirus, West Nile virus, hepatitis viruses, and influenza ( Vasu and Caligiuri, 2016 , Schultze, 2010 ); acute systemic bacterial infections; as well as tuberculosis, typhoid fever, and bacterial pneumonias ( Vasu and Caligiuri, 2016 , Magden et al., 2015 ). The acute phase of malaria may also be associated with decreases in lymphocyte counts ( Vasu and Caligiuri, 2016 ). 12.11.2.2.2.3 Viral-induced Infection with immunodeficiency viruses, including human, simian, and feline immunodeficiency viruses, may result in destruction of both infected and noninfected lymphocytes. HIV directly infects CD4 + T-cells via the CD4 molecule; infected cells then migrate to lymphoid tissues where the virus replicates and infects more CD4 + T-cells ( Chinen and Shearer, 2010 ). HIV-mediated lymphocyte destruction is likely multifactorial; HIV may be cytotoxic, directly induce T-cell apoptosis, induce T-cell death through a nonspecific immune response, and cause T-cell death by stimulating autophagocytic pathways ( Chinen and Shearer, 2010 , Stump and VandeWoude, 2007 ). SIV and FIV tropism for T-cells is also mediated by receptors expressed by CD4 + T-cells ( Stump and VandeWoude, 2007 ). Simian betaretrovirus also causes decreases in blood lymphocyte counts due to infection and eventual depletion of both B-cells and T-cells, although infection of nonlymphoid cells also occurs ( Montiel, 2010 ). 12.11.2.2.2.4 Immune-mediated Immune-mediated destruction of lymphocytes is uncommon. When occurring in the autoimmune disease SLE, such immune-mediated decreases in lymphocyte counts typically occur with concurrent cutaneous, arthritic, or neurologic disorders ( Stone, 2005 ), and may be the result of autoantibodies causing lymphocyte destruction or death through apoptosis ( Lu et al., 2012 , Massardo et al., 2009 , Noguchi et al., 1992 , Budman and Steinberg, 1977 ). 12.11.2.2.2.5 Inherited causes Although rare, some inherited immunodeficiency syndromes cause blood lymphocyte counts to be decreased. One example, severe combined immunodeficiency (SCID), has been reported in humans, dogs, mice, and horses ( Suter, 2010 , Notarangelo, 2010 , Meek et al., 2001 , Felsberg et al., 1992 , Custer et al., 1985 ). SCID may be inherited through autosomal recessive or X-linked recessive patterns, and causes consistent decreases in T-cells, with concurrent decreases in B-cells or NK-cells in some forms of the disease. SCID in humans is caused by a variety of mechanisms, including: adenosine deaminase deficiency resulting in early cell death due to metabolite accumulation; common gamma chain or janus kinase 3 (JAK3) mutations that cause decreased survival of T-cell precursors due to defective cytokine signaling; recombinase-activating gene 1 (RAG1) or RAG2 mutations that cause defective V(D)J rearrangement of B-cell and T-cell receptors; and mutations in CD3 or CD45 that cause defects in T-cell receptor signaling ( Suter, 2010 , Notarangelo, 2010 ). SCID in Jack Russell terriers, Arabian foals, and mice has been demonstrated to be caused by defective V(D)J recombination due to loss of DNA-dependent protein kinase ( Meek et al., 2001 ). X-linked SCID has been described in both Bassett Hounds and Welsh Corgi dogs ( Suter, 2010 ). Other inherited immunodeficiency syndromes resulting in decreases in blood lymphocyte counts include reticular dysgenesis, ataxia-telangiectasia, and Wiskott-Aldrich syndrome ( Vasu and Caligiuri, 2016 ). 12.11.2.2.2.6 Loss of lymph fluid Although uncommon, disorders causing chronic loss of lymphocyte-rich lymph fluid lead to body depletion of lymphocytes and decrease in blood lymphocyte counts. Examples of such conditions include protein-losing enteropathy, lymphangiectasia, ulcerative enteritis, or repeated iatrogenic removal of chylothoracic fluid ( Vasu and Caligiuri, 2016 , Schultze, 2010 , Stockham and Scott, 2008a ). 12.11.2.2.2.7 Neoplasia Although lymphoproliferative neoplasia may be associated with increases in lymphocyte counts as previously discussed, lymphoma and lymphocytic leukemia may also be associated with decreases in lymphocyte counts including from altered lymphocyte recirculation patterns or decreased production of nonneoplasic lymphocytes secondary to lymphoid organ damage ( Mitrovic et al., 2012 , Schultze, 2010 , Stockham and Scott, 2008a ). 12.11.2.2.2.8 Xenobiotic-induced Lymphoid suppression is a common finding with many xenobiotics and may be associated with decreases in blood lymphocyte counts. Mechanisms through which these decreases occur may either be part of the expected pharmacology of these compounds or may represent an off-target effect. Several examples of xenobiotic-induced decreases in lymphocyte counts are described here. Administration of exogenous glucocorticoids, for antiinflammatory or immunosuppressive purposes, will result in decreases in blood lymphocyte counts. The mechanisms for this are the same as those for endogenous glucocorticoids, and include altered blood and tissue pool distribution, decreased efflux of lymphocytes from lymphoid tissues, and increased lymphocyte apoptosis with prolonged glucocorticoid exposure. Other immunosuppressive agents that cause decreased blood lymphocyte counts include cyclophosphamide, methotrexate, purine nucleoside analogs, and azathioprine. Cyclophosphamide has been associated with profound decrease in blood lymphocyte counts through its effects on all lymphocyte subtypes ( Gergely, 1999 ). Methotrexate causes decreases in circulating CD4 + and CD8 + T-cells, while cladribine, a purine nucleoside analog, causes apoptosis of lymphocytes and has been reported to affect both CD4 + and CD8 + T-cells ( Gergely, 1999 ). Azothiaprine-induced decreases in blood lymphocyte counts appear to be due to long-term administration at high dose levels ( Johnson et al., 1995 , Gergely, 1999 ). As discussed previously, numerous xenobiotics have been associated with drug-induced SLE in people. Decreases in lymphocytes in these cases are likely due to the production of autoantibodies with subsequent lymphocyte destruction, similar to nonxenobiotic-induced SLE. Xenobiotics associated with SLE include several anticonvulsants such as phenothiazines, chlorpromazine, and valproate, several antibiotics such as penicillin, streptomycin, tetracycline, griseofulvin, and sulphonamides, and miscellaneous xenobiotics such as captopril, phenylbutazone, and lovastatin ( Stone, 2005 , Mutasim and Adams, 2000 ). Chemotherapeutic agents are also frequently associated with decreases in lymphocyte counts, which may precede episodes of febrile neutropenia ( Gergely, 1999 ). Carboplatin, dacarbazine, and paclitaxel have been reported to induce decreases in CD4 + T-cells, while epirubicin and mitomycin appear to affect CD8 + T-cells to a greater degree than CD4 + T-cells, and pentostatin affects both B-cell and T-cell populations ( Gergely, 1999 ). Antilymphocyte monoclonal antibodies have been used to treat autoimmune diseases as well as to cause immunosuppression to prevent acute transplant rejection. Examples of these monoclonal antibodies include Muromonab CD3 (OKT3) and CAMPATH-1H ( Vial et al., 2002 , Gergely, 1999 ). Other classes of drugs reported to cause decrease lymphocyte counts are varied: pesticides including organochloride pesticides such as pentachlorophenol, organotin compounds, and organophosphates ( Corsini et al., 2013 ); thienopyridines, such as clopidogrel and ticlopidine, which can cause direct lymphotoxicity at high concentrations ( Maseneni et al., 2013 ); the histamine H2 receptor antagonist cimetidine; the anticonvulsant carbamazepine; imidazoles used to treat fungal infections; and opioids such as morphine ( Gergely, 1999 ). 12.11.2.3 Monocytes Bone marrow common myeloid progenitors differentiate into the monocytic lineage under stimulation by stem cell factor, GM-CSF, macrophage-colony-stimulating factor (M-CSF), IL-6, IL-1, and IL-3 ( Papenfuss, 2010 ). Monoblasts further differentiate to promonocytes and then to monocytes. Blood monocytes are distributed in both circulating and marginating pools, and marginating pool monocytes can emigrate into tissues. Circulating half-life of monocytes in mice has been reported to range from 24 to 60 h ( Provencher Bolliger et al., 2010 ). Macrophages and dendritic cells can arise from monocytes or monocyte precursors depending on the local tissue microenvironment and cytokine stimulation ( Papenfuss, 2010 ), forming the mononuclear phagocytic system. Although present in low numbers, monocytes represent the third most numerous blood leukocytes in health, after neutrophils and lymphocytes. They are typically the largest of the leukocytes in routine blood films. Monocytes play a major role in resolution of infectious processes, particularly those involving larger or more complex organisms, such as fungi and protozoa, and in the clearance of other foreign material from the body. Following cardiac blood collection in mice, tissue macrophages may be inadvertently collected and may rarely be observed during blood smear evaluation. 12.11.2.3.1 Increases in monocyte counts (monocytosis) 12.11.2.3.1.1 Catecholamine-induced Although less commonly observed than increases in neutrophil or lymphocyte counts, blood monocyte counts may be modestly increased under the stimulation of endogenous catecholamines. This effect is most likely mediated by rapid shifting of monocytes from the marginating pool to the circulating pool ( Everds et al., 2013a ). 12.11.2.3.1.2 Glucocorticoid-induced Endogenous glucocorticoids classically cause increases in blood monocyte counts. However, these increases in monocyte counts are less consistently observed than decreases in lymphocyte counts or increases in neutrophil counts, and no perceptible change in monocyte counts may occur ( Hall, 2013 , Everds et al., 2013a ). There have also been reports of decreases in monocyte counts attributable to endogenous glucocorticoids, which may be associated with decreased production with prolonged glucocorticoid exposure ( Thompson and van Furth, 1973 ), or may be transient and followed by an increased in monocyte counts ( Steer et al., 1997 , Rinehart et al., 1975 ). 12.11.2.3.1.3 Inflammation Increases in blood monocyte counts occur with both acute and chronic inflammation, and such inflammatory increases in monocytes may be associated with both infectious and noninfectious etiologies. Tuberculosis and other mycobacterial infections are commonly associated with increases in monocyte counts which may represent an increased tissue demand for macrophages ( Lichtman, 2016a ). Increases in monocyte counts have also been associated with bacterial endocarditis and sepsis, osteomyelitis, various pyogranulomatous diseases, candidiasis, viral infections including cytomegalovirus and influenza, and several parasitic diseases including pneumocystosis, Entopolypoides macacai infection in old world monkeys and apes, and dirofilariasis in dogs ( Lichtman, 2016a , Magden et al., 2015 , Schultze, 2010 ). However, increases in monocyte counts may be less commonly related to malaria, leishmaniasis, and rickettsial diseases in people than previously thought ( Lichtman, 2016a ). Noninfectious causes of increases in blood monocyte counts include inflammatory bowel disease, ulcerative gastritis, myocardial infarction, and parturition ( Lichtman, 2016a ). 12.11.2.3.1.4 Immune disorders Numerous immune disorders are associated with increases in blood monocyte counts. Immune-mediated destruction of erythrocytes or neutrophils often has concurrent increases in monocyte counts ( Schultze, 2010 , Stockham and Scott, 2008a ). The increase in monocyte count associated with immune-mediated neutropenia may be due to cytokine stimulation of the common precursor of both granulocytes and monocytes ( Stockham and Scott, 2008a ). Rebound increases in neutrophil counts during recovery from agranulocytosis often have concurrent increases in monocyte counts ( Lichtman, 2016a , Schultze, 2010 ), which may also be due to stimulation of the common granulocyte and monocyte precursor. Systemic lupus erythematosus ( Budman and Steinberg, 1977 ), rheumatoid arthritis ( Buchan et al., 1985 ), sarcoidosis ( Goodwin et al., 1979 ), and other connective tissue diseases ( Lichtman, 2016a ) may also cause increases in monocyte counts. 12.11.2.3.1.5 Neoplasia Hematopoietic malignancies involving the monocytic lineage are rare, but are commonly associated with increased blood monocyte counts. These hematopoietic neoplasms include myelodysplastic syndromes (e.g., chronic myelomonocytic leukemia), acute monocytic leukemia, acute myelomonocytic leukemia, dendritic cell leukemia, and malignant histiocytosis ( Schultze, 2010 , Tefferi and Vardiman, 2009 , Villeneuve et al., 2008 , Sun et al., 2007 , Lichtman and Segel, 2005 , Castoldi and Rigolin, 2001 , Rigolin et al., 1997 , Laurencet et al., 1994 ). Blood monocytes associated with these hematopoietic neoplasms frequently have abnormal morphologic features and may be accompanied by monoblasts or promonocytes in circulation. Increases in monocyte counts related to neoplasia are not limited to hematopoietic neoplasms of the monocytic lineage. Increases in monocyte counts have also been described in association with a wide spectrum of lymphoproliferative neoplasms, soft tissue sarcomas, hemangiosarcomas, chondrosarcomas, rectal polyps, and colon cancer ( Lichtman, 2016a , Schultze, 2010 , Melichar et al., 2001 , Ruka et al., 2001 ). In one study, over half of the patients with solid tumor malignancies were reported to have concurrent increases in monocyte counts, which were independent of tumor metastasis ( Barrett, 1970 ). 12.11.2.3.1.6 Xenobiotic-induced Administration of exogenous glucocorticoids classically causes increases in monocyte counts, although this effect may be inconsistently observed and no change in blood monocyte counts may occur. Some studies have demonstrated that transient decreases in monocyte counts occur immediately following administration of exogenous glucocorticoids with subsequent increases in monocyte counts ( Rinehart et al., 1975 , Steer et al., 1997 ), but others have only reported increases in monocyte counts with exogenous glucocorticoid administration ( Barker et al., 2012 ). Administration of exogenous cytokines may also result in increases in blood monocyte counts. Such increases in blood monocyte counts have been observed with G-CSF ( Ranaghan et al., 1998 , Liu et al., 2004 ), GM-CSF ( Schmitz et al., 1994 ), M-CSF ( Weiner et al., 1994 , Cole et al., 1994 ), or IL-10 administration ( Chernoff et al., 1995 ). M-CSF administration-related increases in monocyte counts have been associated with concurrent, dose-limiting decreases in platelet counts ( Weiner et al., 1994 , Cole et al., 1994 ). Increases in monocyte counts have also been reported in a few patients associated with pseudolymphoma syndrome caused by therapy with several anticonvulsants, including phenytoin, phenobarbital, and valproic acid ( Choi et al., 2003 ). 12.11.2.3.2 Decreases in monocyte counts (monocytopenia) Decreases in monocyte counts may be difficult to detect due to the relatively low blood monocyte counts in humans and most laboratory animal species, particularly if reference intervals or historical control ranges, rather than values from a concurrent control group, are being used for determining if changes in blood monocyte counts are present. In nonclinical toxicology studies, comparison to concurrent controls or pretest may enable detection of more subtle decreases in monocyte counts. 12.11.2.3.2.1 Immune-mediated Immune-mediated decreases in monocyte counts are typically not observed in isolation and are associated with causes of pancytopenia, such as aplastic anemia. With aplastic anemia, destruction of early hematopoietic precursors results in loss of production of most or all hematopoietic cell lineages and subsequent development of severe decreases in multiple blood component counts. Aplastic anemia can also occur with nonimmune-mediated conditions, such as anorexia nervosa ( Abella et al., 2002 ). 12.11.2.3.2.2 Inherited causes Uncommonly there are cases of inherited marked decreases in blood monocyte counts. In humans, an autosomal dominant inheritance pattern has been associated with decreased monocyte counts with a resulting increased susceptibility to mycobacteria and a variety of other infectious agents ( Vinh et al., 2010 ). Mutations in GATA2, a transcription factor that regulates hematopoietic cell gene expression and integrity, have been reported as a cause for autosomal dominant decreases in blood monocyte counts ( Camargo et al., 2013 , Hsu et al., 2011 ). 12.11.2.3.2.3 Neoplastic Decreases in blood monocyte counts may occur secondary to hematologic malignancies or metastatic nonhematology malignancies that efface the bone marrow. Neoplastic myelophthisis results in decreased production of normal hematopoietic cells and therefore blood component counts, including monocytes. Examples of such reported conditions include hairy cell leukemia ( den Ottolander et al., 1983 , Ratain et al., 1985 ) and chronic lymphocytic leukemia ( De Rossi et al., 1991 ). 12.11.2.3.2.4 Xenobiotic-induced Xenobiotic-induced bone marrow suppression can often cause decreases in blood monocyte counts in combination with decreases in other blood component counts. Causes include chemotherapeutic agents, such as discussed in xenobiotic-induced decreases in neutrophil counts, due to direct hematopoietic precursor cell toxicity and xenobiotics associated with aplastic anemia, such as chloramphenicol. Monocyte cytotoxicity has been reported with methylmetacrylate monomer used in joint replacement surgery ( Dahl et al., 1994 ). Lindane, a pesticide, has been reported to cause CFU-GM cytotoxicity ( Parent-Massin and Thouvenot, 1993 ). 12.11.2.3.1 Increases in monocyte counts (monocytosis) 12.11.2.3.1.1 Catecholamine-induced Although less commonly observed than increases in neutrophil or lymphocyte counts, blood monocyte counts may be modestly increased under the stimulation of endogenous catecholamines. This effect is most likely mediated by rapid shifting of monocytes from the marginating pool to the circulating pool ( Everds et al., 2013a ). 12.11.2.3.1.2 Glucocorticoid-induced Endogenous glucocorticoids classically cause increases in blood monocyte counts. However, these increases in monocyte counts are less consistently observed than decreases in lymphocyte counts or increases in neutrophil counts, and no perceptible change in monocyte counts may occur ( Hall, 2013 , Everds et al., 2013a ). There have also been reports of decreases in monocyte counts attributable to endogenous glucocorticoids, which may be associated with decreased production with prolonged glucocorticoid exposure ( Thompson and van Furth, 1973 ), or may be transient and followed by an increased in monocyte counts ( Steer et al., 1997 , Rinehart et al., 1975 ). 12.11.2.3.1.3 Inflammation Increases in blood monocyte counts occur with both acute and chronic inflammation, and such inflammatory increases in monocytes may be associated with both infectious and noninfectious etiologies. Tuberculosis and other mycobacterial infections are commonly associated with increases in monocyte counts which may represent an increased tissue demand for macrophages ( Lichtman, 2016a ). Increases in monocyte counts have also been associated with bacterial endocarditis and sepsis, osteomyelitis, various pyogranulomatous diseases, candidiasis, viral infections including cytomegalovirus and influenza, and several parasitic diseases including pneumocystosis, Entopolypoides macacai infection in old world monkeys and apes, and dirofilariasis in dogs ( Lichtman, 2016a , Magden et al., 2015 , Schultze, 2010 ). However, increases in monocyte counts may be less commonly related to malaria, leishmaniasis, and rickettsial diseases in people than previously thought ( Lichtman, 2016a ). Noninfectious causes of increases in blood monocyte counts include inflammatory bowel disease, ulcerative gastritis, myocardial infarction, and parturition ( Lichtman, 2016a ). 12.11.2.3.1.4 Immune disorders Numerous immune disorders are associated with increases in blood monocyte counts. Immune-mediated destruction of erythrocytes or neutrophils often has concurrent increases in monocyte counts ( Schultze, 2010 , Stockham and Scott, 2008a ). The increase in monocyte count associated with immune-mediated neutropenia may be due to cytokine stimulation of the common precursor of both granulocytes and monocytes ( Stockham and Scott, 2008a ). Rebound increases in neutrophil counts during recovery from agranulocytosis often have concurrent increases in monocyte counts ( Lichtman, 2016a , Schultze, 2010 ), which may also be due to stimulation of the common granulocyte and monocyte precursor. Systemic lupus erythematosus ( Budman and Steinberg, 1977 ), rheumatoid arthritis ( Buchan et al., 1985 ), sarcoidosis ( Goodwin et al., 1979 ), and other connective tissue diseases ( Lichtman, 2016a ) may also cause increases in monocyte counts. 12.11.2.3.1.5 Neoplasia Hematopoietic malignancies involving the monocytic lineage are rare, but are commonly associated with increased blood monocyte counts. These hematopoietic neoplasms include myelodysplastic syndromes (e.g., chronic myelomonocytic leukemia), acute monocytic leukemia, acute myelomonocytic leukemia, dendritic cell leukemia, and malignant histiocytosis ( Schultze, 2010 , Tefferi and Vardiman, 2009 , Villeneuve et al., 2008 , Sun et al., 2007 , Lichtman and Segel, 2005 , Castoldi and Rigolin, 2001 , Rigolin et al., 1997 , Laurencet et al., 1994 ). Blood monocytes associated with these hematopoietic neoplasms frequently have abnormal morphologic features and may be accompanied by monoblasts or promonocytes in circulation. Increases in monocyte counts related to neoplasia are not limited to hematopoietic neoplasms of the monocytic lineage. Increases in monocyte counts have also been described in association with a wide spectrum of lymphoproliferative neoplasms, soft tissue sarcomas, hemangiosarcomas, chondrosarcomas, rectal polyps, and colon cancer ( Lichtman, 2016a , Schultze, 2010 , Melichar et al., 2001 , Ruka et al., 2001 ). In one study, over half of the patients with solid tumor malignancies were reported to have concurrent increases in monocyte counts, which were independent of tumor metastasis ( Barrett, 1970 ). 12.11.2.3.1.6 Xenobiotic-induced Administration of exogenous glucocorticoids classically causes increases in monocyte counts, although this effect may be inconsistently observed and no change in blood monocyte counts may occur. Some studies have demonstrated that transient decreases in monocyte counts occur immediately following administration of exogenous glucocorticoids with subsequent increases in monocyte counts ( Rinehart et al., 1975 , Steer et al., 1997 ), but others have only reported increases in monocyte counts with exogenous glucocorticoid administration ( Barker et al., 2012 ). Administration of exogenous cytokines may also result in increases in blood monocyte counts. Such increases in blood monocyte counts have been observed with G-CSF ( Ranaghan et al., 1998 , Liu et al., 2004 ), GM-CSF ( Schmitz et al., 1994 ), M-CSF ( Weiner et al., 1994 , Cole et al., 1994 ), or IL-10 administration ( Chernoff et al., 1995 ). M-CSF administration-related increases in monocyte counts have been associated with concurrent, dose-limiting decreases in platelet counts ( Weiner et al., 1994 , Cole et al., 1994 ). Increases in monocyte counts have also been reported in a few patients associated with pseudolymphoma syndrome caused by therapy with several anticonvulsants, including phenytoin, phenobarbital, and valproic acid ( Choi et al., 2003 ). 12.11.2.3.1.1 Catecholamine-induced Although less commonly observed than increases in neutrophil or lymphocyte counts, blood monocyte counts may be modestly increased under the stimulation of endogenous catecholamines. This effect is most likely mediated by rapid shifting of monocytes from the marginating pool to the circulating pool ( Everds et al., 2013a ). 12.11.2.3.1.2 Glucocorticoid-induced Endogenous glucocorticoids classically cause increases in blood monocyte counts. However, these increases in monocyte counts are less consistently observed than decreases in lymphocyte counts or increases in neutrophil counts, and no perceptible change in monocyte counts may occur ( Hall, 2013 , Everds et al., 2013a ). There have also been reports of decreases in monocyte counts attributable to endogenous glucocorticoids, which may be associated with decreased production with prolonged glucocorticoid exposure ( Thompson and van Furth, 1973 ), or may be transient and followed by an increased in monocyte counts ( Steer et al., 1997 , Rinehart et al., 1975 ). 12.11.2.3.1.3 Inflammation Increases in blood monocyte counts occur with both acute and chronic inflammation, and such inflammatory increases in monocytes may be associated with both infectious and noninfectious etiologies. Tuberculosis and other mycobacterial infections are commonly associated with increases in monocyte counts which may represent an increased tissue demand for macrophages ( Lichtman, 2016a ). Increases in monocyte counts have also been associated with bacterial endocarditis and sepsis, osteomyelitis, various pyogranulomatous diseases, candidiasis, viral infections including cytomegalovirus and influenza, and several parasitic diseases including pneumocystosis, Entopolypoides macacai infection in old world monkeys and apes, and dirofilariasis in dogs ( Lichtman, 2016a , Magden et al., 2015 , Schultze, 2010 ). However, increases in monocyte counts may be less commonly related to malaria, leishmaniasis, and rickettsial diseases in people than previously thought ( Lichtman, 2016a ). Noninfectious causes of increases in blood monocyte counts include inflammatory bowel disease, ulcerative gastritis, myocardial infarction, and parturition ( Lichtman, 2016a ). 12.11.2.3.1.4 Immune disorders Numerous immune disorders are associated with increases in blood monocyte counts. Immune-mediated destruction of erythrocytes or neutrophils often has concurrent increases in monocyte counts ( Schultze, 2010 , Stockham and Scott, 2008a ). The increase in monocyte count associated with immune-mediated neutropenia may be due to cytokine stimulation of the common precursor of both granulocytes and monocytes ( Stockham and Scott, 2008a ). Rebound increases in neutrophil counts during recovery from agranulocytosis often have concurrent increases in monocyte counts ( Lichtman, 2016a , Schultze, 2010 ), which may also be due to stimulation of the common granulocyte and monocyte precursor. Systemic lupus erythematosus ( Budman and Steinberg, 1977 ), rheumatoid arthritis ( Buchan et al., 1985 ), sarcoidosis ( Goodwin et al., 1979 ), and other connective tissue diseases ( Lichtman, 2016a ) may also cause increases in monocyte counts. 12.11.2.3.1.5 Neoplasia Hematopoietic malignancies involving the monocytic lineage are rare, but are commonly associated with increased blood monocyte counts. These hematopoietic neoplasms include myelodysplastic syndromes (e.g., chronic myelomonocytic leukemia), acute monocytic leukemia, acute myelomonocytic leukemia, dendritic cell leukemia, and malignant histiocytosis ( Schultze, 2010 , Tefferi and Vardiman, 2009 , Villeneuve et al., 2008 , Sun et al., 2007 , Lichtman and Segel, 2005 , Castoldi and Rigolin, 2001 , Rigolin et al., 1997 , Laurencet et al., 1994 ). Blood monocytes associated with these hematopoietic neoplasms frequently have abnormal morphologic features and may be accompanied by monoblasts or promonocytes in circulation. Increases in monocyte counts related to neoplasia are not limited to hematopoietic neoplasms of the monocytic lineage. Increases in monocyte counts have also been described in association with a wide spectrum of lymphoproliferative neoplasms, soft tissue sarcomas, hemangiosarcomas, chondrosarcomas, rectal polyps, and colon cancer ( Lichtman, 2016a , Schultze, 2010 , Melichar et al., 2001 , Ruka et al., 2001 ). In one study, over half of the patients with solid tumor malignancies were reported to have concurrent increases in monocyte counts, which were independent of tumor metastasis ( Barrett, 1970 ). 12.11.2.3.1.6 Xenobiotic-induced Administration of exogenous glucocorticoids classically causes increases in monocyte counts, although this effect may be inconsistently observed and no change in blood monocyte counts may occur. Some studies have demonstrated that transient decreases in monocyte counts occur immediately following administration of exogenous glucocorticoids with subsequent increases in monocyte counts ( Rinehart et al., 1975 , Steer et al., 1997 ), but others have only reported increases in monocyte counts with exogenous glucocorticoid administration ( Barker et al., 2012 ). Administration of exogenous cytokines may also result in increases in blood monocyte counts. Such increases in blood monocyte counts have been observed with G-CSF ( Ranaghan et al., 1998 , Liu et al., 2004 ), GM-CSF ( Schmitz et al., 1994 ), M-CSF ( Weiner et al., 1994 , Cole et al., 1994 ), or IL-10 administration ( Chernoff et al., 1995 ). M-CSF administration-related increases in monocyte counts have been associated with concurrent, dose-limiting decreases in platelet counts ( Weiner et al., 1994 , Cole et al., 1994 ). Increases in monocyte counts have also been reported in a few patients associated with pseudolymphoma syndrome caused by therapy with several anticonvulsants, including phenytoin, phenobarbital, and valproic acid ( Choi et al., 2003 ). 12.11.2.3.2 Decreases in monocyte counts (monocytopenia) Decreases in monocyte counts may be difficult to detect due to the relatively low blood monocyte counts in humans and most laboratory animal species, particularly if reference intervals or historical control ranges, rather than values from a concurrent control group, are being used for determining if changes in blood monocyte counts are present. In nonclinical toxicology studies, comparison to concurrent controls or pretest may enable detection of more subtle decreases in monocyte counts. 12.11.2.3.2.1 Immune-mediated Immune-mediated decreases in monocyte counts are typically not observed in isolation and are associated with causes of pancytopenia, such as aplastic anemia. With aplastic anemia, destruction of early hematopoietic precursors results in loss of production of most or all hematopoietic cell lineages and subsequent development of severe decreases in multiple blood component counts. Aplastic anemia can also occur with nonimmune-mediated conditions, such as anorexia nervosa ( Abella et al., 2002 ). 12.11.2.3.2.2 Inherited causes Uncommonly there are cases of inherited marked decreases in blood monocyte counts. In humans, an autosomal dominant inheritance pattern has been associated with decreased monocyte counts with a resulting increased susceptibility to mycobacteria and a variety of other infectious agents ( Vinh et al., 2010 ). Mutations in GATA2, a transcription factor that regulates hematopoietic cell gene expression and integrity, have been reported as a cause for autosomal dominant decreases in blood monocyte counts ( Camargo et al., 2013 , Hsu et al., 2011 ). 12.11.2.3.2.3 Neoplastic Decreases in blood monocyte counts may occur secondary to hematologic malignancies or metastatic nonhematology malignancies that efface the bone marrow. Neoplastic myelophthisis results in decreased production of normal hematopoietic cells and therefore blood component counts, including monocytes. Examples of such reported conditions include hairy cell leukemia ( den Ottolander et al., 1983 , Ratain et al., 1985 ) and chronic lymphocytic leukemia ( De Rossi et al., 1991 ). 12.11.2.3.2.4 Xenobiotic-induced Xenobiotic-induced bone marrow suppression can often cause decreases in blood monocyte counts in combination with decreases in other blood component counts. Causes include chemotherapeutic agents, such as discussed in xenobiotic-induced decreases in neutrophil counts, due to direct hematopoietic precursor cell toxicity and xenobiotics associated with aplastic anemia, such as chloramphenicol. Monocyte cytotoxicity has been reported with methylmetacrylate monomer used in joint replacement surgery ( Dahl et al., 1994 ). Lindane, a pesticide, has been reported to cause CFU-GM cytotoxicity ( Parent-Massin and Thouvenot, 1993 ). 12.11.2.3.2.1 Immune-mediated Immune-mediated decreases in monocyte counts are typically not observed in isolation and are associated with causes of pancytopenia, such as aplastic anemia. With aplastic anemia, destruction of early hematopoietic precursors results in loss of production of most or all hematopoietic cell lineages and subsequent development of severe decreases in multiple blood component counts. Aplastic anemia can also occur with nonimmune-mediated conditions, such as anorexia nervosa ( Abella et al., 2002 ). 12.11.2.3.2.2 Inherited causes Uncommonly there are cases of inherited marked decreases in blood monocyte counts. In humans, an autosomal dominant inheritance pattern has been associated with decreased monocyte counts with a resulting increased susceptibility to mycobacteria and a variety of other infectious agents ( Vinh et al., 2010 ). Mutations in GATA2, a transcription factor that regulates hematopoietic cell gene expression and integrity, have been reported as a cause for autosomal dominant decreases in blood monocyte counts ( Camargo et al., 2013 , Hsu et al., 2011 ). 12.11.2.3.2.3 Neoplastic Decreases in blood monocyte counts may occur secondary to hematologic malignancies or metastatic nonhematology malignancies that efface the bone marrow. Neoplastic myelophthisis results in decreased production of normal hematopoietic cells and therefore blood component counts, including monocytes. Examples of such reported conditions include hairy cell leukemia ( den Ottolander et al., 1983 , Ratain et al., 1985 ) and chronic lymphocytic leukemia ( De Rossi et al., 1991 ). 12.11.2.3.2.4 Xenobiotic-induced Xenobiotic-induced bone marrow suppression can often cause decreases in blood monocyte counts in combination with decreases in other blood component counts. Causes include chemotherapeutic agents, such as discussed in xenobiotic-induced decreases in neutrophil counts, due to direct hematopoietic precursor cell toxicity and xenobiotics associated with aplastic anemia, such as chloramphenicol. Monocyte cytotoxicity has been reported with methylmetacrylate monomer used in joint replacement surgery ( Dahl et al., 1994 ). Lindane, a pesticide, has been reported to cause CFU-GM cytotoxicity ( Parent-Massin and Thouvenot, 1993 ). 12.11.2.4 Eosinophils Eosinophils share a common early myeloid precursor with neutrophils. Early proliferation of the eosinophil lineage appears to be largely due to stimulation of myeloid precursors with GM-CSF and IL-3, while IL-5 plays a critical role in terminal eosinophil differentiation and maturation ( Sanderson, 1992 ). Similar to other granulocytes, eosinophils are distributed into proliferating and maturing pools in the bone marrow, and into circulating and marginating pools in the blood. The bone marrow is the primary site of eosinophil production, although rat eosinophils migrate and complete their maturation in the spleen ( Young and Meadows, 2010 ). Early proliferating bone marrow eosinophils are usually indistinguishable microscopically from other early myeloid precursors because their characteristic granules do not begin to form until the late promyelocyte stage ( Radin and Wellman, 2010 ). The time for production of mature eosinophils from the myeloblast stage is 2–6 days and the half-life of mature eosinophils in circulation ranges from less than 1 to 24 h, although both of these transit times vary by species ( Young and Meadows, 2010 ). Eosinophils migrate into tissue from circulation, where they live for about 2 days unless stimulation occurs ( Young and Meadows, 2010 ). Eosinophil counts are normally low in most species, which can make detection of decreases in eosinophil counts difficult. Similar to detecting decreases in monocyte counts, comparison of treated groups with concurrent controls or pretest values in nonclinical toxicology studies will aid in the identification of changes in eosinophil counts. Eosinophils are morphologically distinct from other leukocytes due to their large, pink-staining cytoplasmic granules; however, species differences in granule size, shape, and staining properties exist. Blood eosinophil counts in health generally only exceed basophil counts. 12.11.2.4.1 Increases in eosinophil counts (eosinophilia) 12.11.2.4.1.1 Decreased glucocorticoids Although uncommon, decreases in endogenous glucocorticoid concentrations due to hypoadrenocorticism have been associated with mild increases in blood eosinophil counts ( Wardlaw, 2016 , Stockham and Scott, 2008a ). This effect is most likely due to the loss of glucocorticoid-associated shifting of blood eosinophils to the marginating pool as well as the lack of proapoptotic stimulation of glucocorticoids on eosinophil precursors. 12.11.2.4.1.2 Inflammation and Hypersensitivity Both acute and chronic inflammatory stimulation may result in increases in blood eosinophil counts along with increases in neutrophil, lymphocyte, and/or monocyte counts. However, inflammatory processes that stimulate primarily an eosinophil response may also occur. IL-5, eotaxin, and RANTES are cytokines and chemokines that selectively stimulate eosinophil responses ( Sampson, 2000 ). Some of the most common inflammatory processes involving eosinophils include parasite and allergy-induced inflammation. Parasitism is considered the most common cause of increases in blood eosinophil counts worldwide ( Wardlaw, 2016 ), of which helminth (e.g., nematode, trematode, or cestode) infections are the major cause ( Tefferi et al., 2006 , Leder and Weller, 2000 ). Inflammatory increases in eosinophil counts in response to helminths are cytokine (e.g., IL-5) mediated ( Valent, 2009 ), but IgE and histamine release from mast cells, anaphylatoxin (e.g., C5a) production from complement activation, helper T-cell activation, and direct stimulation of eosinophils with helminthic antigens also play a role in eosinophil recruitment ( Wardlaw, 2016 , Leder and Weller, 2000 , McEwen, 1992 ). Helminth migration through host tissues is a key factor in stimulating increases in blood eosinophils and tissue eosinophilic inflammation, and helminths that remain confined to the intestinal lumen may not result in an eosinophil response ( Leder and Weller, 2000 ). The degree of the eosinophil response and increases in blood eosinophil counts are also dependent on parasite burden, maturation, and distribution in tissues ( Leder and Weller, 2000 ). Ascariasis, strongyloidiasis, trichinosis, filariasis, and ancylosomiasis have all been associated with increases in eosinophil counts in humans ( Wardlaw, 2016 , Tefferi et al., 2006 , Leder and Weller, 2000 ), and many of these may also be observed in common laboratory species ( Schultze, 2010 , Magden et al., 2015 , Korenaga et al., 1991 , Ogilvie et al., 1980 ). Helminth infection in most purpose-bred laboratory animals is uncommonly observed during nonclinical toxicology studies due to breeding and housing facility biosecurity measures, but several less commonly used large animal species from other sources, such as farm pigs, calves, and sheep, have helminth infections more frequently observed. Infection with nonhelminth parasites may also cause increases in blood eosinophil counts. Ectoparasites, including fleas and ticks, have been associated with increase in eosinophil counts in dogs and cats and may be due to arthropod-related allergic or hypersensitivity reactions ( Schultze, 2010 , Valenciano et al., 2010 , Stockham and Scott, 2008a ). Several protozoal infections, including isosporiasis ( Jongwutiwes et al., 2002 , Junod, 1987 ) and toxoplasmosis ( Grant and Klein, 1987 ), may result in increases in blood eosinophil counts. However, protozoal agents that infect erythrocytes, such as Plasmodium and Babesia species, are generally not expected to result in altered blood eosinophil counts ( Tefferi et al., 2006 , Stockham and Scott, 2008a ). Some bacterial infections, including borreliosis ( Granter et al., 1996 ) and rickettsiosis ( Wardlaw, 2016 ), and several viral infections, including herpes virus and HIV ( Wardlaw, 2016 , Tietz et al., 1997 ), have also been associated with increases in blood eosinophil counts. Fungal infections that cause allergic inflammation, such as coccidiomycosis, candidiasis, and aspergillosis ( Wardlaw, 2016 ), may also cause increases in blood eosinophil counts. Allergic inflammation is another common cause of increases in blood eosinophil counts. Allergic conditions associated with increases in blood eosinophil counts include asthma, atopic dermatitis, and allergic rhinitis although increases in eosinophil counts with these conditions are usually mild ( Wardlaw, 2016 ). Allergic inflammation associated with immediate release of IgE is considered a type I hypersensitivity reaction. Some allergen-induced inflammation may be attributable to type IV (delayed-type or cell-mediated) hypersensitivity following T H activation with subsequent eosinophil recruitment. However, some differences have been observed between atopic dermatitis and classic type IV hypersensitivity ( Gaga et al., 1991 ), so not all T H -mediated allergic inflammation may represent a true type IV hypersensitivity reaction. As with most forms of inflammatory increases in blood eosinophil counts, production of cytokines and chemokines, such as IL-5 and eotaxin, appear to play a major role. In allergic asthma, activated T-helper type 2 (T H 2) cells release or stimulate the release of these cytokines and chemokines, resulting in recruitment and activation of eosinophils ( Rosenberg et al., 2013 ). Sensitization of the airways to ovalbumin with subsequent challenge in mice has mimicked many of the clinical and pathological features of allergic asthma, including the interaction of T H 2 cells and eosinophils ( Rosenberg et al., 2013 ). However, asthma encompasses a heterogeneous set of phenotypes and not all forms demonstrate clinical improvement in response to therapies targeting IL-5 or eosinophils ( Wegmann, 2011 ). While asthma is uncommon in most laboratory animal species, it can be experimentally induced in mice and may occur naturally in cats. Activation of T H 2 and T H 1 cells have been proposed to contribute to the pathology of atopic dermatitis, with the T H 2 activation being of particular relevance to the development of increases in blood eosinophil counts ( Grewe et al., 1998 ), similar to allergic asthma. Activation of T H 2 cells also plays a role in cytokine and chemokine elaboration with eosinophil recruitment in allergic rhinitis, although effects on eosinophils in allergic rhinitis are also mediated by histamine and IgE release from mast cells and histamine release from basophils ( Borish, 2003 ). Inflammatory increases in blood eosinophil counts can also be associated with paraneoplastic syndromes, likely related to increases in IL-5, which may be liberated by activated T H cells or directly by the neoplasm. Lymphoma, including both T- and B-cell lymphomas, is a common cause of paraneoplastic increases in eosinophil counts in multiple species, including humans, dogs, cats, and horses ( Wardlaw, 2016 , Davis and Rothenberg, 2014 , Valent, 2009 , Schultze, 2010 , Valenciano et al., 2010 , Stockham and Scott, 2008a , Marchetti et al., 2005 , Cave et al., 2004 , Duckett and Matthews, 1997 ). However, many nonlymphoid tumors have also been associated with paraneoplastic increases in blood eosinophil counts, including mammary carcinoma, hepatocellular carcinoma, squamous cell carcinoma, thymoma, nonsmall-cell lung cancer, and mast cell diseases including systemic mastocytosis and mast cell leukemia ( Schultze, 2010 , Balian et al., 2001 , Walter et al., 2002 , Pandit et al., 2007 , Valent, 2009 ). 12.11.2.4.1.3 Neoplastic Myeloid neoplasms can result in clonal eosinophil expansion and increases in blood eosinophil counts expansion ( Tefferi et al., 2006 ). Neoplastic increases in eosinophil counts have been associated with acute eosinophilic leukemia, chronic eosinophilic leukemia, chronic myeloid leukemia, and myelodysplastic syndrome ( Wardlaw, 2016 , Tefferi et al., 2006 , Schultze, 2010 ). Clonal increases in eosinophil counts may be difficult to distinguish from idiopathic hypereosinophilic syndrome, and cytogenetic analysis may be necessary; numerous cytogenetic abnormalities have been reported with clonal increases in eosinophil counts ( Tefferi et al., 2006 ). 12.11.2.4.1.4 Idiopathic There are numerous reports of idiopathic increases in blood eosinophil counts. Such conditions include eosinophilic esophagitis, eosinophilic gastroenteritis, eosinophilic myositis, eosinophilic cellulitis, and eosinophilic pneumonitis in people, dogs, and/or cats ( Wardlaw, 2016 , Stockham and Scott, 2008a ). Hypereosinophilic syndrome (HES) in people is another condition that falls under the umbrella of idiopathic increases in eosinophil counts. In HES, chronic increases in eosinophil counts are observed without evidence of an underlying causative condition. This condition is associated with marked tissue infiltration and eventual organ damage and failure ( Wardlaw, 2016 , Rosenberg et al., 2013 ). However, some patients with HES eventually develop either a lymphoid or myeloid neoplasm ( Wardlaw, 2016 ). 12.11.2.4.1.5 Spurious In mice, automated hematology analyzer-generated blood eosinophil counts may be falsely elevated by large platelet clumps present in the specimen ( O'Connell et al., 2015 ). Platelet clumping in mice is extremely common, and blood smear evaluation is often necessary to confirm the automated leukocyte differential count. 12.11.2.4.1.6 Xenobiotic-induced Beta adrenergic blocking agents may be associated with modest increases in eosinophil counts, and administration of propranolol has been demonstrated to prevent catecholamine-induced decreases in eosinophil counts ( Reed et al., 1970 , Koch-Weser, 1968 ). The antibiotic tetracycline has been associated with increased eosinophil counts in dogs ( Domina et al., 1997 ) and humans ( Ho et al., 1979 ). Therapeutic administration of IL-2 for renal cell carcinoma has also been reported to cause increased blood eosinophil counts ( Wardlaw, 2016 ). Administration of G-CSF and GM-CSF will also cause increases in blood eosinophil counts due to stimulation of common myeloid precursor proliferation. However, increases in eosinophil counts with these compounds will be small in comparison with the increases in blood neutrophil counts. Numerous reactions to xenobiotics can also cause increases in blood eosinophil counts. Acute generalized exanthematous pustulosis due to drugs such as aminopenicillins and diltiazem, as discussed with xenobiotic-induced increases in neutrophil counts, may be associated with concurrent increases in eosinophil counts ( Roujeau, 2005 ). Drug reaction with eosinophilia and systemic syndromes (DRESS) is a predominantly cutaneous manifestation of a drug hypersensitivity reaction. Numerous compounds have been associated with DRESS, including several anticonvulsant drugs, such as phenobarbital and phenytoin, allopurinol, minocycline, sulfonamides, gold salts, dapsone, and spironolactone ( Roujeau, 2005 ; Callot et al., 1996 , Tefferi et al., 2006 , Ghislain et al., 2004 ). 12.11.2.4.2 Decreases in eosinophil counts (eosinopenia) 12.11.2.4.2.1 Catecholamine-induced In contrast to neutrophil, lymphocyte, and monocyte counts, blood eosinophil counts decrease in response to increased endogenous catecholamines. These effects may be inconsistent and difficult to detect due to timing of blood collection relative to the rapid changes in eosinophil counts and the normally low blood eosinophil counts. The β-adrenergic effects of epinephrine are believed to be the cause of the decreases in blood eosinophil counts ( Koch-Weser, 1968 ). Catecholamines may also cause decreased release of eosinophils from bone marrow ( McEwen, 1992 ). 12.11.2.4.2.2 Glucocorticoid-induced Decreases in blood eosinophil counts are a classic feature of a glucocorticoid leukogram occurring in conjunction with decreases in lymphocyte counts and usually with increases in neutrophil counts. Blood eosinophils appear to be particularly responsive to the effects of glucocorticoids, and concurrent decreases in blood lymphocyte and eosinophil counts in common laboratory species used in nonclinical toxicology studies are a good indicator of stress ( Hall, 2013 ). Glucocorticoids cause shifts in blood eosinophils from the circulating to the marginating pool as well as decreased release of eosinophils from the bone marrow ( McEwen, 1992 ), and may also contribute to decreases in blood eosinophil counts from inhibition of prosurvival stimulation and direct induction of apoptosis ( Druilhe et al., 2003 , Wallen et al., 1991 ). 12.11.2.4.2.3 Inflammation Severe acute or overwhelming inflammation, such as associated with sepsis, may cause eosinopenia in conjunction with neutropenia. Studies in mice have demonstrated that the decrease in blood eosinophils associated with severe acute inflammation occurs more rapidly than increases in glucocorticoids ( Bass, 1975 ), and injection of material from an inflammatory exudate to adrenalectomized mice still resulted in deceases in eosinophil counts ( Bass, 1977 ), indicating the mechanism of eosinophil decrease in acute inflammation is independent of adrenal function. It is believed that acute inflammatory decreases in blood eosinophil counts are due to shifting of eosinophils from the circulating to the marginating pool and subsequent egress into tissues in response to chemotactic stimuli. Acute inflammation associated with fungal and viral infections also tends to cause decreases in eosinophil counts ( Leder and Weller, 2000 ). 12.11.2.4.2.4 Xenobiotic-induced Although decreases in eosinophil counts are relatively uncommon with the exception of the administration of exogenous glucocorticoid-based xenobiotics, they may also be observed in cases of xenobiotic-induced bone marrow suppression and aplastic anemia. In these situations, the decreases in eosinophil counts do not occur in isolation but are generally observed with concurrent decreases in neutrophil, lymphocyte, and/or monocyte counts. Xenobiotic causes of bone marrow suppression classically include chemotherapeutic agents, while xenobiotics that can sporadically be associated with aplastic anemia include chloramphenicol and anticonvulsants such as phenytoin. Other xenobiotics associated with bone marrow suppression or aplastic anemia are described in more detail in the previous leukocyte subtype sections. 12.11.2.4.1 Increases in eosinophil counts (eosinophilia) 12.11.2.4.1.1 Decreased glucocorticoids Although uncommon, decreases in endogenous glucocorticoid concentrations due to hypoadrenocorticism have been associated with mild increases in blood eosinophil counts ( Wardlaw, 2016 , Stockham and Scott, 2008a ). This effect is most likely due to the loss of glucocorticoid-associated shifting of blood eosinophils to the marginating pool as well as the lack of proapoptotic stimulation of glucocorticoids on eosinophil precursors. 12.11.2.4.1.2 Inflammation and Hypersensitivity Both acute and chronic inflammatory stimulation may result in increases in blood eosinophil counts along with increases in neutrophil, lymphocyte, and/or monocyte counts. However, inflammatory processes that stimulate primarily an eosinophil response may also occur. IL-5, eotaxin, and RANTES are cytokines and chemokines that selectively stimulate eosinophil responses ( Sampson, 2000 ). Some of the most common inflammatory processes involving eosinophils include parasite and allergy-induced inflammation. Parasitism is considered the most common cause of increases in blood eosinophil counts worldwide ( Wardlaw, 2016 ), of which helminth (e.g., nematode, trematode, or cestode) infections are the major cause ( Tefferi et al., 2006 , Leder and Weller, 2000 ). Inflammatory increases in eosinophil counts in response to helminths are cytokine (e.g., IL-5) mediated ( Valent, 2009 ), but IgE and histamine release from mast cells, anaphylatoxin (e.g., C5a) production from complement activation, helper T-cell activation, and direct stimulation of eosinophils with helminthic antigens also play a role in eosinophil recruitment ( Wardlaw, 2016 , Leder and Weller, 2000 , McEwen, 1992 ). Helminth migration through host tissues is a key factor in stimulating increases in blood eosinophils and tissue eosinophilic inflammation, and helminths that remain confined to the intestinal lumen may not result in an eosinophil response ( Leder and Weller, 2000 ). The degree of the eosinophil response and increases in blood eosinophil counts are also dependent on parasite burden, maturation, and distribution in tissues ( Leder and Weller, 2000 ). Ascariasis, strongyloidiasis, trichinosis, filariasis, and ancylosomiasis have all been associated with increases in eosinophil counts in humans ( Wardlaw, 2016 , Tefferi et al., 2006 , Leder and Weller, 2000 ), and many of these may also be observed in common laboratory species ( Schultze, 2010 , Magden et al., 2015 , Korenaga et al., 1991 , Ogilvie et al., 1980 ). Helminth infection in most purpose-bred laboratory animals is uncommonly observed during nonclinical toxicology studies due to breeding and housing facility biosecurity measures, but several less commonly used large animal species from other sources, such as farm pigs, calves, and sheep, have helminth infections more frequently observed. Infection with nonhelminth parasites may also cause increases in blood eosinophil counts. Ectoparasites, including fleas and ticks, have been associated with increase in eosinophil counts in dogs and cats and may be due to arthropod-related allergic or hypersensitivity reactions ( Schultze, 2010 , Valenciano et al., 2010 , Stockham and Scott, 2008a ). Several protozoal infections, including isosporiasis ( Jongwutiwes et al., 2002 , Junod, 1987 ) and toxoplasmosis ( Grant and Klein, 1987 ), may result in increases in blood eosinophil counts. However, protozoal agents that infect erythrocytes, such as Plasmodium and Babesia species, are generally not expected to result in altered blood eosinophil counts ( Tefferi et al., 2006 , Stockham and Scott, 2008a ). Some bacterial infections, including borreliosis ( Granter et al., 1996 ) and rickettsiosis ( Wardlaw, 2016 ), and several viral infections, including herpes virus and HIV ( Wardlaw, 2016 , Tietz et al., 1997 ), have also been associated with increases in blood eosinophil counts. Fungal infections that cause allergic inflammation, such as coccidiomycosis, candidiasis, and aspergillosis ( Wardlaw, 2016 ), may also cause increases in blood eosinophil counts. Allergic inflammation is another common cause of increases in blood eosinophil counts. Allergic conditions associated with increases in blood eosinophil counts include asthma, atopic dermatitis, and allergic rhinitis although increases in eosinophil counts with these conditions are usually mild ( Wardlaw, 2016 ). Allergic inflammation associated with immediate release of IgE is considered a type I hypersensitivity reaction. Some allergen-induced inflammation may be attributable to type IV (delayed-type or cell-mediated) hypersensitivity following T H activation with subsequent eosinophil recruitment. However, some differences have been observed between atopic dermatitis and classic type IV hypersensitivity ( Gaga et al., 1991 ), so not all T H -mediated allergic inflammation may represent a true type IV hypersensitivity reaction. As with most forms of inflammatory increases in blood eosinophil counts, production of cytokines and chemokines, such as IL-5 and eotaxin, appear to play a major role. In allergic asthma, activated T-helper type 2 (T H 2) cells release or stimulate the release of these cytokines and chemokines, resulting in recruitment and activation of eosinophils ( Rosenberg et al., 2013 ). Sensitization of the airways to ovalbumin with subsequent challenge in mice has mimicked many of the clinical and pathological features of allergic asthma, including the interaction of T H 2 cells and eosinophils ( Rosenberg et al., 2013 ). However, asthma encompasses a heterogeneous set of phenotypes and not all forms demonstrate clinical improvement in response to therapies targeting IL-5 or eosinophils ( Wegmann, 2011 ). While asthma is uncommon in most laboratory animal species, it can be experimentally induced in mice and may occur naturally in cats. Activation of T H 2 and T H 1 cells have been proposed to contribute to the pathology of atopic dermatitis, with the T H 2 activation being of particular relevance to the development of increases in blood eosinophil counts ( Grewe et al., 1998 ), similar to allergic asthma. Activation of T H 2 cells also plays a role in cytokine and chemokine elaboration with eosinophil recruitment in allergic rhinitis, although effects on eosinophils in allergic rhinitis are also mediated by histamine and IgE release from mast cells and histamine release from basophils ( Borish, 2003 ). Inflammatory increases in blood eosinophil counts can also be associated with paraneoplastic syndromes, likely related to increases in IL-5, which may be liberated by activated T H cells or directly by the neoplasm. Lymphoma, including both T- and B-cell lymphomas, is a common cause of paraneoplastic increases in eosinophil counts in multiple species, including humans, dogs, cats, and horses ( Wardlaw, 2016 , Davis and Rothenberg, 2014 , Valent, 2009 , Schultze, 2010 , Valenciano et al., 2010 , Stockham and Scott, 2008a , Marchetti et al., 2005 , Cave et al., 2004 , Duckett and Matthews, 1997 ). However, many nonlymphoid tumors have also been associated with paraneoplastic increases in blood eosinophil counts, including mammary carcinoma, hepatocellular carcinoma, squamous cell carcinoma, thymoma, nonsmall-cell lung cancer, and mast cell diseases including systemic mastocytosis and mast cell leukemia ( Schultze, 2010 , Balian et al., 2001 , Walter et al., 2002 , Pandit et al., 2007 , Valent, 2009 ). 12.11.2.4.1.3 Neoplastic Myeloid neoplasms can result in clonal eosinophil expansion and increases in blood eosinophil counts expansion ( Tefferi et al., 2006 ). Neoplastic increases in eosinophil counts have been associated with acute eosinophilic leukemia, chronic eosinophilic leukemia, chronic myeloid leukemia, and myelodysplastic syndrome ( Wardlaw, 2016 , Tefferi et al., 2006 , Schultze, 2010 ). Clonal increases in eosinophil counts may be difficult to distinguish from idiopathic hypereosinophilic syndrome, and cytogenetic analysis may be necessary; numerous cytogenetic abnormalities have been reported with clonal increases in eosinophil counts ( Tefferi et al., 2006 ). 12.11.2.4.1.4 Idiopathic There are numerous reports of idiopathic increases in blood eosinophil counts. Such conditions include eosinophilic esophagitis, eosinophilic gastroenteritis, eosinophilic myositis, eosinophilic cellulitis, and eosinophilic pneumonitis in people, dogs, and/or cats ( Wardlaw, 2016 , Stockham and Scott, 2008a ). Hypereosinophilic syndrome (HES) in people is another condition that falls under the umbrella of idiopathic increases in eosinophil counts. In HES, chronic increases in eosinophil counts are observed without evidence of an underlying causative condition. This condition is associated with marked tissue infiltration and eventual organ damage and failure ( Wardlaw, 2016 , Rosenberg et al., 2013 ). However, some patients with HES eventually develop either a lymphoid or myeloid neoplasm ( Wardlaw, 2016 ). 12.11.2.4.1.5 Spurious In mice, automated hematology analyzer-generated blood eosinophil counts may be falsely elevated by large platelet clumps present in the specimen ( O'Connell et al., 2015 ). Platelet clumping in mice is extremely common, and blood smear evaluation is often necessary to confirm the automated leukocyte differential count. 12.11.2.4.1.6 Xenobiotic-induced Beta adrenergic blocking agents may be associated with modest increases in eosinophil counts, and administration of propranolol has been demonstrated to prevent catecholamine-induced decreases in eosinophil counts ( Reed et al., 1970 , Koch-Weser, 1968 ). The antibiotic tetracycline has been associated with increased eosinophil counts in dogs ( Domina et al., 1997 ) and humans ( Ho et al., 1979 ). Therapeutic administration of IL-2 for renal cell carcinoma has also been reported to cause increased blood eosinophil counts ( Wardlaw, 2016 ). Administration of G-CSF and GM-CSF will also cause increases in blood eosinophil counts due to stimulation of common myeloid precursor proliferation. However, increases in eosinophil counts with these compounds will be small in comparison with the increases in blood neutrophil counts. Numerous reactions to xenobiotics can also cause increases in blood eosinophil counts. Acute generalized exanthematous pustulosis due to drugs such as aminopenicillins and diltiazem, as discussed with xenobiotic-induced increases in neutrophil counts, may be associated with concurrent increases in eosinophil counts ( Roujeau, 2005 ). Drug reaction with eosinophilia and systemic syndromes (DRESS) is a predominantly cutaneous manifestation of a drug hypersensitivity reaction. Numerous compounds have been associated with DRESS, including several anticonvulsant drugs, such as phenobarbital and phenytoin, allopurinol, minocycline, sulfonamides, gold salts, dapsone, and spironolactone ( Roujeau, 2005 ; Callot et al., 1996 , Tefferi et al., 2006 , Ghislain et al., 2004 ). 12.11.2.4.1.1 Decreased glucocorticoids Although uncommon, decreases in endogenous glucocorticoid concentrations due to hypoadrenocorticism have been associated with mild increases in blood eosinophil counts ( Wardlaw, 2016 , Stockham and Scott, 2008a ). This effect is most likely due to the loss of glucocorticoid-associated shifting of blood eosinophils to the marginating pool as well as the lack of proapoptotic stimulation of glucocorticoids on eosinophil precursors. 12.11.2.4.1.2 Inflammation and Hypersensitivity Both acute and chronic inflammatory stimulation may result in increases in blood eosinophil counts along with increases in neutrophil, lymphocyte, and/or monocyte counts. However, inflammatory processes that stimulate primarily an eosinophil response may also occur. IL-5, eotaxin, and RANTES are cytokines and chemokines that selectively stimulate eosinophil responses ( Sampson, 2000 ). Some of the most common inflammatory processes involving eosinophils include parasite and allergy-induced inflammation. Parasitism is considered the most common cause of increases in blood eosinophil counts worldwide ( Wardlaw, 2016 ), of which helminth (e.g., nematode, trematode, or cestode) infections are the major cause ( Tefferi et al., 2006 , Leder and Weller, 2000 ). Inflammatory increases in eosinophil counts in response to helminths are cytokine (e.g., IL-5) mediated ( Valent, 2009 ), but IgE and histamine release from mast cells, anaphylatoxin (e.g., C5a) production from complement activation, helper T-cell activation, and direct stimulation of eosinophils with helminthic antigens also play a role in eosinophil recruitment ( Wardlaw, 2016 , Leder and Weller, 2000 , McEwen, 1992 ). Helminth migration through host tissues is a key factor in stimulating increases in blood eosinophils and tissue eosinophilic inflammation, and helminths that remain confined to the intestinal lumen may not result in an eosinophil response ( Leder and Weller, 2000 ). The degree of the eosinophil response and increases in blood eosinophil counts are also dependent on parasite burden, maturation, and distribution in tissues ( Leder and Weller, 2000 ). Ascariasis, strongyloidiasis, trichinosis, filariasis, and ancylosomiasis have all been associated with increases in eosinophil counts in humans ( Wardlaw, 2016 , Tefferi et al., 2006 , Leder and Weller, 2000 ), and many of these may also be observed in common laboratory species ( Schultze, 2010 , Magden et al., 2015 , Korenaga et al., 1991 , Ogilvie et al., 1980 ). Helminth infection in most purpose-bred laboratory animals is uncommonly observed during nonclinical toxicology studies due to breeding and housing facility biosecurity measures, but several less commonly used large animal species from other sources, such as farm pigs, calves, and sheep, have helminth infections more frequently observed. Infection with nonhelminth parasites may also cause increases in blood eosinophil counts. Ectoparasites, including fleas and ticks, have been associated with increase in eosinophil counts in dogs and cats and may be due to arthropod-related allergic or hypersensitivity reactions ( Schultze, 2010 , Valenciano et al., 2010 , Stockham and Scott, 2008a ). Several protozoal infections, including isosporiasis ( Jongwutiwes et al., 2002 , Junod, 1987 ) and toxoplasmosis ( Grant and Klein, 1987 ), may result in increases in blood eosinophil counts. However, protozoal agents that infect erythrocytes, such as Plasmodium and Babesia species, are generally not expected to result in altered blood eosinophil counts ( Tefferi et al., 2006 , Stockham and Scott, 2008a ). Some bacterial infections, including borreliosis ( Granter et al., 1996 ) and rickettsiosis ( Wardlaw, 2016 ), and several viral infections, including herpes virus and HIV ( Wardlaw, 2016 , Tietz et al., 1997 ), have also been associated with increases in blood eosinophil counts. Fungal infections that cause allergic inflammation, such as coccidiomycosis, candidiasis, and aspergillosis ( Wardlaw, 2016 ), may also cause increases in blood eosinophil counts. Allergic inflammation is another common cause of increases in blood eosinophil counts. Allergic conditions associated with increases in blood eosinophil counts include asthma, atopic dermatitis, and allergic rhinitis although increases in eosinophil counts with these conditions are usually mild ( Wardlaw, 2016 ). Allergic inflammation associated with immediate release of IgE is considered a type I hypersensitivity reaction. Some allergen-induced inflammation may be attributable to type IV (delayed-type or cell-mediated) hypersensitivity following T H activation with subsequent eosinophil recruitment. However, some differences have been observed between atopic dermatitis and classic type IV hypersensitivity ( Gaga et al., 1991 ), so not all T H -mediated allergic inflammation may represent a true type IV hypersensitivity reaction. As with most forms of inflammatory increases in blood eosinophil counts, production of cytokines and chemokines, such as IL-5 and eotaxin, appear to play a major role. In allergic asthma, activated T-helper type 2 (T H 2) cells release or stimulate the release of these cytokines and chemokines, resulting in recruitment and activation of eosinophils ( Rosenberg et al., 2013 ). Sensitization of the airways to ovalbumin with subsequent challenge in mice has mimicked many of the clinical and pathological features of allergic asthma, including the interaction of T H 2 cells and eosinophils ( Rosenberg et al., 2013 ). However, asthma encompasses a heterogeneous set of phenotypes and not all forms demonstrate clinical improvement in response to therapies targeting IL-5 or eosinophils ( Wegmann, 2011 ). While asthma is uncommon in most laboratory animal species, it can be experimentally induced in mice and may occur naturally in cats. Activation of T H 2 and T H 1 cells have been proposed to contribute to the pathology of atopic dermatitis, with the T H 2 activation being of particular relevance to the development of increases in blood eosinophil counts ( Grewe et al., 1998 ), similar to allergic asthma. Activation of T H 2 cells also plays a role in cytokine and chemokine elaboration with eosinophil recruitment in allergic rhinitis, although effects on eosinophils in allergic rhinitis are also mediated by histamine and IgE release from mast cells and histamine release from basophils ( Borish, 2003 ). Inflammatory increases in blood eosinophil counts can also be associated with paraneoplastic syndromes, likely related to increases in IL-5, which may be liberated by activated T H cells or directly by the neoplasm. Lymphoma, including both T- and B-cell lymphomas, is a common cause of paraneoplastic increases in eosinophil counts in multiple species, including humans, dogs, cats, and horses ( Wardlaw, 2016 , Davis and Rothenberg, 2014 , Valent, 2009 , Schultze, 2010 , Valenciano et al., 2010 , Stockham and Scott, 2008a , Marchetti et al., 2005 , Cave et al., 2004 , Duckett and Matthews, 1997 ). However, many nonlymphoid tumors have also been associated with paraneoplastic increases in blood eosinophil counts, including mammary carcinoma, hepatocellular carcinoma, squamous cell carcinoma, thymoma, nonsmall-cell lung cancer, and mast cell diseases including systemic mastocytosis and mast cell leukemia ( Schultze, 2010 , Balian et al., 2001 , Walter et al., 2002 , Pandit et al., 2007 , Valent, 2009 ). 12.11.2.4.1.3 Neoplastic Myeloid neoplasms can result in clonal eosinophil expansion and increases in blood eosinophil counts expansion ( Tefferi et al., 2006 ). Neoplastic increases in eosinophil counts have been associated with acute eosinophilic leukemia, chronic eosinophilic leukemia, chronic myeloid leukemia, and myelodysplastic syndrome ( Wardlaw, 2016 , Tefferi et al., 2006 , Schultze, 2010 ). Clonal increases in eosinophil counts may be difficult to distinguish from idiopathic hypereosinophilic syndrome, and cytogenetic analysis may be necessary; numerous cytogenetic abnormalities have been reported with clonal increases in eosinophil counts ( Tefferi et al., 2006 ). 12.11.2.4.1.4 Idiopathic There are numerous reports of idiopathic increases in blood eosinophil counts. Such conditions include eosinophilic esophagitis, eosinophilic gastroenteritis, eosinophilic myositis, eosinophilic cellulitis, and eosinophilic pneumonitis in people, dogs, and/or cats ( Wardlaw, 2016 , Stockham and Scott, 2008a ). Hypereosinophilic syndrome (HES) in people is another condition that falls under the umbrella of idiopathic increases in eosinophil counts. In HES, chronic increases in eosinophil counts are observed without evidence of an underlying causative condition. This condition is associated with marked tissue infiltration and eventual organ damage and failure ( Wardlaw, 2016 , Rosenberg et al., 2013 ). However, some patients with HES eventually develop either a lymphoid or myeloid neoplasm ( Wardlaw, 2016 ). 12.11.2.4.1.5 Spurious In mice, automated hematology analyzer-generated blood eosinophil counts may be falsely elevated by large platelet clumps present in the specimen ( O'Connell et al., 2015 ). Platelet clumping in mice is extremely common, and blood smear evaluation is often necessary to confirm the automated leukocyte differential count. 12.11.2.4.1.6 Xenobiotic-induced Beta adrenergic blocking agents may be associated with modest increases in eosinophil counts, and administration of propranolol has been demonstrated to prevent catecholamine-induced decreases in eosinophil counts ( Reed et al., 1970 , Koch-Weser, 1968 ). The antibiotic tetracycline has been associated with increased eosinophil counts in dogs ( Domina et al., 1997 ) and humans ( Ho et al., 1979 ). Therapeutic administration of IL-2 for renal cell carcinoma has also been reported to cause increased blood eosinophil counts ( Wardlaw, 2016 ). Administration of G-CSF and GM-CSF will also cause increases in blood eosinophil counts due to stimulation of common myeloid precursor proliferation. However, increases in eosinophil counts with these compounds will be small in comparison with the increases in blood neutrophil counts. Numerous reactions to xenobiotics can also cause increases in blood eosinophil counts. Acute generalized exanthematous pustulosis due to drugs such as aminopenicillins and diltiazem, as discussed with xenobiotic-induced increases in neutrophil counts, may be associated with concurrent increases in eosinophil counts ( Roujeau, 2005 ). Drug reaction with eosinophilia and systemic syndromes (DRESS) is a predominantly cutaneous manifestation of a drug hypersensitivity reaction. Numerous compounds have been associated with DRESS, including several anticonvulsant drugs, such as phenobarbital and phenytoin, allopurinol, minocycline, sulfonamides, gold salts, dapsone, and spironolactone ( Roujeau, 2005 ; Callot et al., 1996 , Tefferi et al., 2006 , Ghislain et al., 2004 ). 12.11.2.4.2 Decreases in eosinophil counts (eosinopenia) 12.11.2.4.2.1 Catecholamine-induced In contrast to neutrophil, lymphocyte, and monocyte counts, blood eosinophil counts decrease in response to increased endogenous catecholamines. These effects may be inconsistent and difficult to detect due to timing of blood collection relative to the rapid changes in eosinophil counts and the normally low blood eosinophil counts. The β-adrenergic effects of epinephrine are believed to be the cause of the decreases in blood eosinophil counts ( Koch-Weser, 1968 ). Catecholamines may also cause decreased release of eosinophils from bone marrow ( McEwen, 1992 ). 12.11.2.4.2.2 Glucocorticoid-induced Decreases in blood eosinophil counts are a classic feature of a glucocorticoid leukogram occurring in conjunction with decreases in lymphocyte counts and usually with increases in neutrophil counts. Blood eosinophils appear to be particularly responsive to the effects of glucocorticoids, and concurrent decreases in blood lymphocyte and eosinophil counts in common laboratory species used in nonclinical toxicology studies are a good indicator of stress ( Hall, 2013 ). Glucocorticoids cause shifts in blood eosinophils from the circulating to the marginating pool as well as decreased release of eosinophils from the bone marrow ( McEwen, 1992 ), and may also contribute to decreases in blood eosinophil counts from inhibition of prosurvival stimulation and direct induction of apoptosis ( Druilhe et al., 2003 , Wallen et al., 1991 ). 12.11.2.4.2.3 Inflammation Severe acute or overwhelming inflammation, such as associated with sepsis, may cause eosinopenia in conjunction with neutropenia. Studies in mice have demonstrated that the decrease in blood eosinophils associated with severe acute inflammation occurs more rapidly than increases in glucocorticoids ( Bass, 1975 ), and injection of material from an inflammatory exudate to adrenalectomized mice still resulted in deceases in eosinophil counts ( Bass, 1977 ), indicating the mechanism of eosinophil decrease in acute inflammation is independent of adrenal function. It is believed that acute inflammatory decreases in blood eosinophil counts are due to shifting of eosinophils from the circulating to the marginating pool and subsequent egress into tissues in response to chemotactic stimuli. Acute inflammation associated with fungal and viral infections also tends to cause decreases in eosinophil counts ( Leder and Weller, 2000 ). 12.11.2.4.2.4 Xenobiotic-induced Although decreases in eosinophil counts are relatively uncommon with the exception of the administration of exogenous glucocorticoid-based xenobiotics, they may also be observed in cases of xenobiotic-induced bone marrow suppression and aplastic anemia. In these situations, the decreases in eosinophil counts do not occur in isolation but are generally observed with concurrent decreases in neutrophil, lymphocyte, and/or monocyte counts. Xenobiotic causes of bone marrow suppression classically include chemotherapeutic agents, while xenobiotics that can sporadically be associated with aplastic anemia include chloramphenicol and anticonvulsants such as phenytoin. Other xenobiotics associated with bone marrow suppression or aplastic anemia are described in more detail in the previous leukocyte subtype sections. 12.11.2.4.2.1 Catecholamine-induced In contrast to neutrophil, lymphocyte, and monocyte counts, blood eosinophil counts decrease in response to increased endogenous catecholamines. These effects may be inconsistent and difficult to detect due to timing of blood collection relative to the rapid changes in eosinophil counts and the normally low blood eosinophil counts. The β-adrenergic effects of epinephrine are believed to be the cause of the decreases in blood eosinophil counts ( Koch-Weser, 1968 ). Catecholamines may also cause decreased release of eosinophils from bone marrow ( McEwen, 1992 ). 12.11.2.4.2.2 Glucocorticoid-induced Decreases in blood eosinophil counts are a classic feature of a glucocorticoid leukogram occurring in conjunction with decreases in lymphocyte counts and usually with increases in neutrophil counts. Blood eosinophils appear to be particularly responsive to the effects of glucocorticoids, and concurrent decreases in blood lymphocyte and eosinophil counts in common laboratory species used in nonclinical toxicology studies are a good indicator of stress ( Hall, 2013 ). Glucocorticoids cause shifts in blood eosinophils from the circulating to the marginating pool as well as decreased release of eosinophils from the bone marrow ( McEwen, 1992 ), and may also contribute to decreases in blood eosinophil counts from inhibition of prosurvival stimulation and direct induction of apoptosis ( Druilhe et al., 2003 , Wallen et al., 1991 ). 12.11.2.4.2.3 Inflammation Severe acute or overwhelming inflammation, such as associated with sepsis, may cause eosinopenia in conjunction with neutropenia. Studies in mice have demonstrated that the decrease in blood eosinophils associated with severe acute inflammation occurs more rapidly than increases in glucocorticoids ( Bass, 1975 ), and injection of material from an inflammatory exudate to adrenalectomized mice still resulted in deceases in eosinophil counts ( Bass, 1977 ), indicating the mechanism of eosinophil decrease in acute inflammation is independent of adrenal function. It is believed that acute inflammatory decreases in blood eosinophil counts are due to shifting of eosinophils from the circulating to the marginating pool and subsequent egress into tissues in response to chemotactic stimuli. Acute inflammation associated with fungal and viral infections also tends to cause decreases in eosinophil counts ( Leder and Weller, 2000 ). 12.11.2.4.2.4 Xenobiotic-induced Although decreases in eosinophil counts are relatively uncommon with the exception of the administration of exogenous glucocorticoid-based xenobiotics, they may also be observed in cases of xenobiotic-induced bone marrow suppression and aplastic anemia. In these situations, the decreases in eosinophil counts do not occur in isolation but are generally observed with concurrent decreases in neutrophil, lymphocyte, and/or monocyte counts. Xenobiotic causes of bone marrow suppression classically include chemotherapeutic agents, while xenobiotics that can sporadically be associated with aplastic anemia include chloramphenicol and anticonvulsants such as phenytoin. Other xenobiotics associated with bone marrow suppression or aplastic anemia are described in more detail in the previous leukocyte subtype sections. 12.11.2.5 Basophils Basophils develop in the bone marrow from uncommitted myeloid progenitor cells that differentiate into committed basophil progenitors. However, intermediate stages in basophil production have not been definitively identified, and there is evidence that basophils may share a common precursor with eosinophil, mast cells, or megakaryocytes ( Radin and Wellman, 2010 , Arock et al., 2002 ). Stimulation with IL-3 plays a major role in the terminal differentiation of basophils, while GM-CSF and IL-5 also play a role in basophil differentiation ( Arock et al., 2002 , Mayer et al., 1989 ). There is also some evidence for stem cell factor (SCF) and IL-4 stimulation in basophil differentiation ( Pohlman, 2010 , Favre et al., 1990 ). As studies in mice have shown that normal blood basophil counts may be maintained in the absence of IL-3, a required role for IL-3 in basophil production is not apparent ( Lantz et al., 1998 ). Specific factors leading to terminal differentiation have not been identified for the basophil lineage, and basophil differentiation may in fact represent a default leukocyte differentiation pathway ( Arock et al., 2002 ). In blood, basophils are distributed into circulating and marginating pools, similar to other granulocytes. The circulating half-life of basophils is short (about 6 h), and they rapidly migrate into tissues where they have a much longer survival (up to 2 weeks) ( Hirai et al., 1997 , Pohlman, 2010 ). Basophils are the least numerous leukocyte in blood, and in health usually compose approximately 0.5% or less of the blood leukocyte differential ( Pohlman, 2010 , Galli et al., 2016 ). Automated hematology analyzer differentials may provide low estimates of actual blood basophil counts in humans, and flow cytometric methods may provide a more accurate estimate ( Meintker et al., 2013 , Amundsen et al., 2012 , Ducrest et al., 2005 ). Automated hematology analyzer counts in dogs and cats have been demonstrated to be inaccurate ( Pohlman, 2010 , Lilliehöök and Tvedten, 2011 , Tvedten and Lilliehöök, 2011 ). However, basophils in rabbits appear to be detected with automated methods ( Lilliehöök and Tvedten, 2011 ). Due to the evidence for low or inaccurate basophil counts in humans, dogs, and cats, it is unclear how accurate automated basophil counts are in nonhuman primates and rodents used in nonclinical toxicology studies. 12.11.2.5.1 Increases in basophil counts (basophilia) 12.11.2.5.1.1 Inflammation Increases in blood basophil counts may be associated with inflammatory stimuli, although decreases in basophil counts are more commonly observed ( Galli et al., 2016 ). Increases in basophil counts have been associated with infectious, allergic, and paraneoplastic inflammatory conditions. Parasitism is a relatively frequent cause of inflammatory increases in basophil counts, which are almost always observed in conjunction with increases in blood eosinophil counts. Many endoparasites, predominantly helminths with tissue exposure or migration, and ectoparasites, including a variety of arthropods, have been associated with concurrent increase in eosinophil and basophil counts ( Schultze, 2010 , Pohlman, 2010 , Voehringer, 2009 , Falcone et al., 2001 , Brown and Rosalsky, 1984 , Roth and Levy, 1980 , Ogilvie et al., 1980 ). Infectious agents other than parasites have also been reported to cause increases in basophil counts. Several viral etiologies associated with increases in basophil counts in humans include influenza, chickenpox, and smallpox viruses ( Galli et al., 2016 ). Several bacterial infections may also cause increases in blood basophil counts, including tuberculosis ( Galli et al., 2016 ) and infection with Helicobacter pylori ( Karttunen et al., 1996 ). Allergic inflammation that involves IgE and/or causes increases in eosinophil counts typically also causes increases in blood basophil counts. Immediate or delayed hypersensitivity may cause increases in basophil counts, although immediate hypersensitivity has also been associated with decreases in basophil counts in some cases; whether basophil counts increase or decrease may represent a distinction between allergic sensitization and an immediate allergic reaction ( Shelley and Parnes, 1965 ). Food or inhalant allergies, such as ragweed pollen ( Otsuka et al., 1986 ), can frequently cause increases in blood basophil counts ( Galli et al., 2016 , Pohlman, 2010 ). Allergic inflammation also occurs with insect stings or bites ( Stockham and Scott, 2008a ) and probably H . pylori infection ( Karttunen et al., 1996 ). Paraneoplastic increases in basophil counts have been associated with several neoplastic processes, including disseminated mast cell neoplasia ( Schultze, 2010 , Stockham and Scott, 2008a ) and carcinomas ( Galli et al., 2016 ). Increases in blood basophil counts may also be observed with lymphomatoid granulomatosis ( Schultze, 2010 , Stockham and Scott, 2008a ), and myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis ( Galli et al., 2016 , Stockham and Scott, 2008a ). Other miscellaneous causes of inflammatory increases in blood basophil counts include ulcerative colitis ( Juhlin, 1963a ), the systemic mast cell disorder urticaria pigmentosa ( Asboe-Hansen, 1960 ), juvenile rheumatoid arthritis ( Athreya et al., 1975 ), and immunological responses causing acute rejection of some tissue grafts ( Tikkanen et al., 2001 ). 12.11.2.5.1.2 Endocrinopathy Several endocrinopathies have been associated with increases in blood basophil counts. These endocrinopathies include hypothyroidism (myxedema) and diabetes mellitus ( Galli et al., 2016 , Shelley and Parnes, 1965 ). It has been suggested that the increases in basophil counts are secondary to hyperlipidemia associated with the endocrinopathies, but supporting mechanistic evidence is scant ( Pohlman, 2010 , Schultze, 2010 ). 12.11.2.5.1.3 Neoplasia Although relatively rare, chronic myelogenous leukemia is frequently associated with increases in blood basophil counts ( Spiers et al., 1977 ), in which the blood basophils have been demonstrated through cytogenetic analysis to arise from the neoplastic clone ( Goh and Anderson, 1979 ). However, there is some suggestion that chronic basophilic leukemia may be a separate process than basophilic chronic myeloid or granulocytic leukemia ( Pardanani et al., 2003 ). Chronic leukemia associated with clonal increases in blood basophil counts also has the potential to undergo blast transformation. Acute basophilic leukemia may also occur but is rare ( Duchayne et al., 1999 ). Other forms of acute myelogenous leukemia may also be associated with increases in basophil counts ( Galli et al., 2016 ). 12.11.2.5.1.4 Xenobiotic-induced Administration of G-CSF or GM-CSF may cause modest increases in blood basophil counts along with the more pronounced increases in neutrophil and eosinophil counts through stimulation of granulocyte production. Administration of phenylhydrazine as a rat model of hemolysis has been associated with increases in blood leukocyte counts, including basophil counts ( Criswell et al., 2000 ). Perhaps the most common xenobiotic-induced increases in blood basophil counts occur as a hypersensitivity or allergic reaction, and reports have included associated administration of heparin, penicillin, and novobiocin ( Schultze, 2010 , Shelley, 1963 ). 12.11.2.5.2 Decreases in basophil counts (basopenia) The lower reference limit of historical control data in common laboratory species may include basophil counts of 0 cells μL − 1 . Due to these normally low blood basophil counts in health, decreases in basophil counts may not be detectable or recognizable. However, the following sections list some conditions in which decreased basophil counts have been reported. 12.11.2.5.2.1 Glucocorticoid-induced Increases in glucocorticoid concentrations, particularly if prolonged, cause decreases in blood basophil counts ( Shelley and Parnes, 1965 , Juhlin, 1963b , Boseila, 1963 ). Similar to glucocorticoid-induced decreases in eosinophil counts, there is likely shifting of blood basophils from the circulating to marginating pool. There is also evidence that glucocorticoids cause migration of basophils from circulation into tissues and decrease recirculation from tissue back into blood ( Wald et al., 1991 ). Direct lytic effects on blood or tissue basophils and suppression of basophil production in the bone marrow may also contribute ( Boseila, 1963 ). Myocardial infarction, which has been associated with decreases in blood basophil counts, may be mediated by effects of chronic stress secondary to the ischemic event ( Juhlin, 1963c ). 12.11.2.5.2.2 Inflammation and hypersensitivity Severe acute or overwhelming inflammation can lead to a decrease in basophil counts along with decreases in other blood leukocyte counts. A relationship between endotoxemia and decreases in basophil counts has been supported by reductions in blood basophil counts in rabbits administered endotoxin from Salmonella typhi ( Goncharova and Krylova, 1967 ). Also, inflammatory leukocytosis not associated with overwhelming inflammation is often associated with decreases in basophil counts ( Galli et al., 2016 ). Type I hypersensitivity reactions, which are mediated by rapid IgE release, are commonly associated with decreases in both eosinophil and basophil counts. However, type IV hypersensitivity (cell-mediated) causing histamine release from mast cells may also be associated with decreases in basophil counts. Such hypersensitivity-related decreases in basophil counts can be observed with anaphylaxis and urticaria ( Galli et al., 2016 , Grattan et al., 1997 , Grattan et al., 2003 , Shelley and Juhlin, 1961 ). 12.11.2.5.2.3 Endocrinopathy Hyperthyroidism (thyrotoxicosis) is reported to cause decreases in blood basophil counts ( Shelley and Parnes, 1965 , Juhlin, 1963c ). However, the mechanism of this decrease has not been clearly demonstrated. 12.11.2.5.2.4 Xenobiotic-induced Exogenous administration of glucocorticoids will result in decreases in blood basophil counts, similar to decreases caused by endogenous glucocorticoids. Also, administration of thyroid hormones or thyroid stimulating hormone to healthy individuals has been reported to cause decreases in blood basophil counts, consistent with the decreases in basophil counts observed with naturally occurring hyperthyroidism ( Boseila, 1963 ). Acute hypersensitivity reactions to xenobiotics may cause decreases in blood basophil counts. Xenobiotic-induced urticaria, angioedema, and anaphylactic reactions are IgE-mediated type I hypersensitivity reactions, and have been associated with angiotensin-converting enzyme (ACE) inhibitors and various NSAIDs ( Roujeau, 2005 ). Due to the IgE-mediated nature of these reactions, these xenobiotics would have the potential to cause concurrent decreases in blood basophil counts, although IgE-related increases in basophil counts could also occur as described previously. Menthol has also been associated with urticaria and decreases in basophil counts ( Papa and Shelley, 1964 ). Decreases in blood basophil counts may also occur along with decreases in other leukocyte counts associated with xenobiotic-induced bone marrow suppression and aplastic anemia. As a class, chemotherapeutic agents may cause bone marrow suppression resulting in decreases in multiple leukocyte lineages in blood, including basophils. Xenobiotics implicated in causing aplastic anemia include chloramphenicol, anticonvulsants such as phenytoin and carbamazepine, gold-based compounds, penicillamine, and phenylbutazone ( Bloom and Brandt, 2008 , Kaufman et al., 1996 ). 12.11.2.5.1 Increases in basophil counts (basophilia) 12.11.2.5.1.1 Inflammation Increases in blood basophil counts may be associated with inflammatory stimuli, although decreases in basophil counts are more commonly observed ( Galli et al., 2016 ). Increases in basophil counts have been associated with infectious, allergic, and paraneoplastic inflammatory conditions. Parasitism is a relatively frequent cause of inflammatory increases in basophil counts, which are almost always observed in conjunction with increases in blood eosinophil counts. Many endoparasites, predominantly helminths with tissue exposure or migration, and ectoparasites, including a variety of arthropods, have been associated with concurrent increase in eosinophil and basophil counts ( Schultze, 2010 , Pohlman, 2010 , Voehringer, 2009 , Falcone et al., 2001 , Brown and Rosalsky, 1984 , Roth and Levy, 1980 , Ogilvie et al., 1980 ). Infectious agents other than parasites have also been reported to cause increases in basophil counts. Several viral etiologies associated with increases in basophil counts in humans include influenza, chickenpox, and smallpox viruses ( Galli et al., 2016 ). Several bacterial infections may also cause increases in blood basophil counts, including tuberculosis ( Galli et al., 2016 ) and infection with Helicobacter pylori ( Karttunen et al., 1996 ). Allergic inflammation that involves IgE and/or causes increases in eosinophil counts typically also causes increases in blood basophil counts. Immediate or delayed hypersensitivity may cause increases in basophil counts, although immediate hypersensitivity has also been associated with decreases in basophil counts in some cases; whether basophil counts increase or decrease may represent a distinction between allergic sensitization and an immediate allergic reaction ( Shelley and Parnes, 1965 ). Food or inhalant allergies, such as ragweed pollen ( Otsuka et al., 1986 ), can frequently cause increases in blood basophil counts ( Galli et al., 2016 , Pohlman, 2010 ). Allergic inflammation also occurs with insect stings or bites ( Stockham and Scott, 2008a ) and probably H . pylori infection ( Karttunen et al., 1996 ). Paraneoplastic increases in basophil counts have been associated with several neoplastic processes, including disseminated mast cell neoplasia ( Schultze, 2010 , Stockham and Scott, 2008a ) and carcinomas ( Galli et al., 2016 ). Increases in blood basophil counts may also be observed with lymphomatoid granulomatosis ( Schultze, 2010 , Stockham and Scott, 2008a ), and myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis ( Galli et al., 2016 , Stockham and Scott, 2008a ). Other miscellaneous causes of inflammatory increases in blood basophil counts include ulcerative colitis ( Juhlin, 1963a ), the systemic mast cell disorder urticaria pigmentosa ( Asboe-Hansen, 1960 ), juvenile rheumatoid arthritis ( Athreya et al., 1975 ), and immunological responses causing acute rejection of some tissue grafts ( Tikkanen et al., 2001 ). 12.11.2.5.1.2 Endocrinopathy Several endocrinopathies have been associated with increases in blood basophil counts. These endocrinopathies include hypothyroidism (myxedema) and diabetes mellitus ( Galli et al., 2016 , Shelley and Parnes, 1965 ). It has been suggested that the increases in basophil counts are secondary to hyperlipidemia associated with the endocrinopathies, but supporting mechanistic evidence is scant ( Pohlman, 2010 , Schultze, 2010 ). 12.11.2.5.1.3 Neoplasia Although relatively rare, chronic myelogenous leukemia is frequently associated with increases in blood basophil counts ( Spiers et al., 1977 ), in which the blood basophils have been demonstrated through cytogenetic analysis to arise from the neoplastic clone ( Goh and Anderson, 1979 ). However, there is some suggestion that chronic basophilic leukemia may be a separate process than basophilic chronic myeloid or granulocytic leukemia ( Pardanani et al., 2003 ). Chronic leukemia associated with clonal increases in blood basophil counts also has the potential to undergo blast transformation. Acute basophilic leukemia may also occur but is rare ( Duchayne et al., 1999 ). Other forms of acute myelogenous leukemia may also be associated with increases in basophil counts ( Galli et al., 2016 ). 12.11.2.5.1.4 Xenobiotic-induced Administration of G-CSF or GM-CSF may cause modest increases in blood basophil counts along with the more pronounced increases in neutrophil and eosinophil counts through stimulation of granulocyte production. Administration of phenylhydrazine as a rat model of hemolysis has been associated with increases in blood leukocyte counts, including basophil counts ( Criswell et al., 2000 ). Perhaps the most common xenobiotic-induced increases in blood basophil counts occur as a hypersensitivity or allergic reaction, and reports have included associated administration of heparin, penicillin, and novobiocin ( Schultze, 2010 , Shelley, 1963 ). 12.11.2.5.1.1 Inflammation Increases in blood basophil counts may be associated with inflammatory stimuli, although decreases in basophil counts are more commonly observed ( Galli et al., 2016 ). Increases in basophil counts have been associated with infectious, allergic, and paraneoplastic inflammatory conditions. Parasitism is a relatively frequent cause of inflammatory increases in basophil counts, which are almost always observed in conjunction with increases in blood eosinophil counts. Many endoparasites, predominantly helminths with tissue exposure or migration, and ectoparasites, including a variety of arthropods, have been associated with concurrent increase in eosinophil and basophil counts ( Schultze, 2010 , Pohlman, 2010 , Voehringer, 2009 , Falcone et al., 2001 , Brown and Rosalsky, 1984 , Roth and Levy, 1980 , Ogilvie et al., 1980 ). Infectious agents other than parasites have also been reported to cause increases in basophil counts. Several viral etiologies associated with increases in basophil counts in humans include influenza, chickenpox, and smallpox viruses ( Galli et al., 2016 ). Several bacterial infections may also cause increases in blood basophil counts, including tuberculosis ( Galli et al., 2016 ) and infection with Helicobacter pylori ( Karttunen et al., 1996 ). Allergic inflammation that involves IgE and/or causes increases in eosinophil counts typically also causes increases in blood basophil counts. Immediate or delayed hypersensitivity may cause increases in basophil counts, although immediate hypersensitivity has also been associated with decreases in basophil counts in some cases; whether basophil counts increase or decrease may represent a distinction between allergic sensitization and an immediate allergic reaction ( Shelley and Parnes, 1965 ). Food or inhalant allergies, such as ragweed pollen ( Otsuka et al., 1986 ), can frequently cause increases in blood basophil counts ( Galli et al., 2016 , Pohlman, 2010 ). Allergic inflammation also occurs with insect stings or bites ( Stockham and Scott, 2008a ) and probably H . pylori infection ( Karttunen et al., 1996 ). Paraneoplastic increases in basophil counts have been associated with several neoplastic processes, including disseminated mast cell neoplasia ( Schultze, 2010 , Stockham and Scott, 2008a ) and carcinomas ( Galli et al., 2016 ). Increases in blood basophil counts may also be observed with lymphomatoid granulomatosis ( Schultze, 2010 , Stockham and Scott, 2008a ), and myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis ( Galli et al., 2016 , Stockham and Scott, 2008a ). Other miscellaneous causes of inflammatory increases in blood basophil counts include ulcerative colitis ( Juhlin, 1963a ), the systemic mast cell disorder urticaria pigmentosa ( Asboe-Hansen, 1960 ), juvenile rheumatoid arthritis ( Athreya et al., 1975 ), and immunological responses causing acute rejection of some tissue grafts ( Tikkanen et al., 2001 ). 12.11.2.5.1.2 Endocrinopathy Several endocrinopathies have been associated with increases in blood basophil counts. These endocrinopathies include hypothyroidism (myxedema) and diabetes mellitus ( Galli et al., 2016 , Shelley and Parnes, 1965 ). It has been suggested that the increases in basophil counts are secondary to hyperlipidemia associated with the endocrinopathies, but supporting mechanistic evidence is scant ( Pohlman, 2010 , Schultze, 2010 ). 12.11.2.5.1.3 Neoplasia Although relatively rare, chronic myelogenous leukemia is frequently associated with increases in blood basophil counts ( Spiers et al., 1977 ), in which the blood basophils have been demonstrated through cytogenetic analysis to arise from the neoplastic clone ( Goh and Anderson, 1979 ). However, there is some suggestion that chronic basophilic leukemia may be a separate process than basophilic chronic myeloid or granulocytic leukemia ( Pardanani et al., 2003 ). Chronic leukemia associated with clonal increases in blood basophil counts also has the potential to undergo blast transformation. Acute basophilic leukemia may also occur but is rare ( Duchayne et al., 1999 ). Other forms of acute myelogenous leukemia may also be associated with increases in basophil counts ( Galli et al., 2016 ). 12.11.2.5.1.4 Xenobiotic-induced Administration of G-CSF or GM-CSF may cause modest increases in blood basophil counts along with the more pronounced increases in neutrophil and eosinophil counts through stimulation of granulocyte production. Administration of phenylhydrazine as a rat model of hemolysis has been associated with increases in blood leukocyte counts, including basophil counts ( Criswell et al., 2000 ). Perhaps the most common xenobiotic-induced increases in blood basophil counts occur as a hypersensitivity or allergic reaction, and reports have included associated administration of heparin, penicillin, and novobiocin ( Schultze, 2010 , Shelley, 1963 ). 12.11.2.5.2 Decreases in basophil counts (basopenia) The lower reference limit of historical control data in common laboratory species may include basophil counts of 0 cells μL − 1 . Due to these normally low blood basophil counts in health, decreases in basophil counts may not be detectable or recognizable. However, the following sections list some conditions in which decreased basophil counts have been reported. 12.11.2.5.2.1 Glucocorticoid-induced Increases in glucocorticoid concentrations, particularly if prolonged, cause decreases in blood basophil counts ( Shelley and Parnes, 1965 , Juhlin, 1963b , Boseila, 1963 ). Similar to glucocorticoid-induced decreases in eosinophil counts, there is likely shifting of blood basophils from the circulating to marginating pool. There is also evidence that glucocorticoids cause migration of basophils from circulation into tissues and decrease recirculation from tissue back into blood ( Wald et al., 1991 ). Direct lytic effects on blood or tissue basophils and suppression of basophil production in the bone marrow may also contribute ( Boseila, 1963 ). Myocardial infarction, which has been associated with decreases in blood basophil counts, may be mediated by effects of chronic stress secondary to the ischemic event ( Juhlin, 1963c ). 12.11.2.5.2.2 Inflammation and hypersensitivity Severe acute or overwhelming inflammation can lead to a decrease in basophil counts along with decreases in other blood leukocyte counts. A relationship between endotoxemia and decreases in basophil counts has been supported by reductions in blood basophil counts in rabbits administered endotoxin from Salmonella typhi ( Goncharova and Krylova, 1967 ). Also, inflammatory leukocytosis not associated with overwhelming inflammation is often associated with decreases in basophil counts ( Galli et al., 2016 ). Type I hypersensitivity reactions, which are mediated by rapid IgE release, are commonly associated with decreases in both eosinophil and basophil counts. However, type IV hypersensitivity (cell-mediated) causing histamine release from mast cells may also be associated with decreases in basophil counts. Such hypersensitivity-related decreases in basophil counts can be observed with anaphylaxis and urticaria ( Galli et al., 2016 , Grattan et al., 1997 , Grattan et al., 2003 , Shelley and Juhlin, 1961 ). 12.11.2.5.2.3 Endocrinopathy Hyperthyroidism (thyrotoxicosis) is reported to cause decreases in blood basophil counts ( Shelley and Parnes, 1965 , Juhlin, 1963c ). However, the mechanism of this decrease has not been clearly demonstrated. 12.11.2.5.2.4 Xenobiotic-induced Exogenous administration of glucocorticoids will result in decreases in blood basophil counts, similar to decreases caused by endogenous glucocorticoids. Also, administration of thyroid hormones or thyroid stimulating hormone to healthy individuals has been reported to cause decreases in blood basophil counts, consistent with the decreases in basophil counts observed with naturally occurring hyperthyroidism ( Boseila, 1963 ). Acute hypersensitivity reactions to xenobiotics may cause decreases in blood basophil counts. Xenobiotic-induced urticaria, angioedema, and anaphylactic reactions are IgE-mediated type I hypersensitivity reactions, and have been associated with angiotensin-converting enzyme (ACE) inhibitors and various NSAIDs ( Roujeau, 2005 ). Due to the IgE-mediated nature of these reactions, these xenobiotics would have the potential to cause concurrent decreases in blood basophil counts, although IgE-related increases in basophil counts could also occur as described previously. Menthol has also been associated with urticaria and decreases in basophil counts ( Papa and Shelley, 1964 ). Decreases in blood basophil counts may also occur along with decreases in other leukocyte counts associated with xenobiotic-induced bone marrow suppression and aplastic anemia. As a class, chemotherapeutic agents may cause bone marrow suppression resulting in decreases in multiple leukocyte lineages in blood, including basophils. Xenobiotics implicated in causing aplastic anemia include chloramphenicol, anticonvulsants such as phenytoin and carbamazepine, gold-based compounds, penicillamine, and phenylbutazone ( Bloom and Brandt, 2008 , Kaufman et al., 1996 ). 12.11.2.5.2.1 Glucocorticoid-induced Increases in glucocorticoid concentrations, particularly if prolonged, cause decreases in blood basophil counts ( Shelley and Parnes, 1965 , Juhlin, 1963b , Boseila, 1963 ). Similar to glucocorticoid-induced decreases in eosinophil counts, there is likely shifting of blood basophils from the circulating to marginating pool. There is also evidence that glucocorticoids cause migration of basophils from circulation into tissues and decrease recirculation from tissue back into blood ( Wald et al., 1991 ). Direct lytic effects on blood or tissue basophils and suppression of basophil production in the bone marrow may also contribute ( Boseila, 1963 ). Myocardial infarction, which has been associated with decreases in blood basophil counts, may be mediated by effects of chronic stress secondary to the ischemic event ( Juhlin, 1963c ). 12.11.2.5.2.2 Inflammation and hypersensitivity Severe acute or overwhelming inflammation can lead to a decrease in basophil counts along with decreases in other blood leukocyte counts. A relationship between endotoxemia and decreases in basophil counts has been supported by reductions in blood basophil counts in rabbits administered endotoxin from Salmonella typhi ( Goncharova and Krylova, 1967 ). Also, inflammatory leukocytosis not associated with overwhelming inflammation is often associated with decreases in basophil counts ( Galli et al., 2016 ). Type I hypersensitivity reactions, which are mediated by rapid IgE release, are commonly associated with decreases in both eosinophil and basophil counts. However, type IV hypersensitivity (cell-mediated) causing histamine release from mast cells may also be associated with decreases in basophil counts. Such hypersensitivity-related decreases in basophil counts can be observed with anaphylaxis and urticaria ( Galli et al., 2016 , Grattan et al., 1997 , Grattan et al., 2003 , Shelley and Juhlin, 1961 ). 12.11.2.5.2.3 Endocrinopathy Hyperthyroidism (thyrotoxicosis) is reported to cause decreases in blood basophil counts ( Shelley and Parnes, 1965 , Juhlin, 1963c ). However, the mechanism of this decrease has not been clearly demonstrated. 12.11.2.5.2.4 Xenobiotic-induced Exogenous administration of glucocorticoids will result in decreases in blood basophil counts, similar to decreases caused by endogenous glucocorticoids. Also, administration of thyroid hormones or thyroid stimulating hormone to healthy individuals has been reported to cause decreases in blood basophil counts, consistent with the decreases in basophil counts observed with naturally occurring hyperthyroidism ( Boseila, 1963 ). Acute hypersensitivity reactions to xenobiotics may cause decreases in blood basophil counts. Xenobiotic-induced urticaria, angioedema, and anaphylactic reactions are IgE-mediated type I hypersensitivity reactions, and have been associated with angiotensin-converting enzyme (ACE) inhibitors and various NSAIDs ( Roujeau, 2005 ). Due to the IgE-mediated nature of these reactions, these xenobiotics would have the potential to cause concurrent decreases in blood basophil counts, although IgE-related increases in basophil counts could also occur as described previously. Menthol has also been associated with urticaria and decreases in basophil counts ( Papa and Shelley, 1964 ). Decreases in blood basophil counts may also occur along with decreases in other leukocyte counts associated with xenobiotic-induced bone marrow suppression and aplastic anemia. As a class, chemotherapeutic agents may cause bone marrow suppression resulting in decreases in multiple leukocyte lineages in blood, including basophils. Xenobiotics implicated in causing aplastic anemia include chloramphenicol, anticonvulsants such as phenytoin and carbamazepine, gold-based compounds, penicillamine, and phenylbutazone ( Bloom and Brandt, 2008 , Kaufman et al., 1996 ). 12.11.2.6 Large Unclassified or Other Cells Some automated hematology analyzers, such as the Siemens Healthcare ADVIA systems, include a "large unclassified cell" (LUC) or "other" cell category in the leukocyte differential. These cells are generally large with no or minimal myeloperoxidase activity, and do not fall within the predefined species' gating parameters for typically identified leukocyte subtypes. These cells do not represent a distinct cell type, but are most commonly large and/or reactive lymphocytes or monocytes, and increases in LUC counts may be observed with any conditions resulting in increases in blood lymphocyte or monocyte counts. In species where automated basophil counts are not reliable, increases in blood basophil counts may also appear as an increase in LUC counts ( Lilliehöök and Tvedten, 2011 ). Acute leukemia, typified by increases in hematopoietic blast cells in bone marrow and circulation, almost always results in increases in LUC counts. In the presence of high LUC counts, blood smear evaluation should be performed to assess the leukocyte differential and morphologic appearance of the blood leukocytes. Occasionally there may be mast cells observed in the blood smears of dogs, cats, or laboratory rodents. Mast cells in blood (mastocytemia) typically occur in low in numbers that do not significantly affect the automated leukocyte differential. Such mastocytemia may occur along with an inflammatory response. However, systemic mastocytosis or mast cell leukemia may cause notable increases in blood mast cells, and mast cell neoplasia is the most common cause of circulating mast cells in cats ( Skeldon et al., 2010 ). Blood smear evaluation is required for enumerating mast cells as part of a leukocyte differentiation. 12.11.3 Erythrocytes The earliest erythrocyte production, or primitive erythropoiesis, occurs in the yolk sac initially and eventually also in the liver during fetal development. Later in fetal development, erythrocyte production switches to predominantly the bone marrow, which is considered definitive erythropoiesis ( Harvey, 2012 ). In neonates, foci of extramedullary hematopoiesis may be observed within the liver histologically. In rats and mice, erythropoiesis within the spleen often significantly contributes to the maintenance of normal blood erythrocyte content. Increases in erythropoiesis in these species may be accompanied by increased extramedullary hematopoiesis in the spleen without appreciable changes in the bone marrow when observed histologically. Increased splenic hematopoiesis in these species may be sufficient to cause detectable changes in organ weight values. Erythropoiesis is largely under the control of stimulation with erythropoietin (EPO), although stem cell factor (SCF), IL-3, and thrombopoietin (TPO) may also play a role in early erythrocyte differentiation and insulin-like growth factor (IGF)-1 may contribute to later erythropoiesis ( Olver, 2010 ). Erythrocyte differentiation begins with pluripotent hematopoietic stem cells that differentiate into common myeloid progenitor cells, which further differentiate into megakaryocyte–erythrocyte progenitor cells. Under stimulation with EPO, megakaryocyte–erythrocyte progenitors differentiate into the first committed erythroid progenitor, the burst-forming unit erythrocyte (BFU-E), which then differentiates into colony-forming unit erythrocyte (CFU-E) precursors. The next stage in erythroid development is the rubriblast, which is the earliest erythroid progenitor that may be identified with light microscopy. Erythroid cell maturation then progresses through prorubricyte, basophilic rubricyte, polychromatophilic rubricyte, and metarubricyte stages. As these stages progress, the nuclear chromatin becomes more condensed and the nucleus becomes pyknotic, coinciding with increased hemoglobin production and accumulation within the cytoplasm and simultaneously decreased RNA production. At this point, pyknotic nuclei are extruded from the cell to form reticulocytes, which are released from the bone marrow. Proliferative bone marrow erythrocyte pools include rubriblasts through basophilic rubricytes, while maturing bone marrow erythrocyte pools include polychromatophilic rubricytes and reticulocytes. Reticulocytes may mature into erythrocytes either in the spleen or the blood. Sometimes reticulocytes or mature erythrocytes may contain small remnants or fragments of their nuclei, called Howell-Jolly bodies. These may be observed in low numbers in circulation, but passage blood through the spleen typically results in their removal. Most species have a sinusoidal splenic architecture, but cats have nonsinusoidal splenic architecture and are less efficient at removal of Howell-Jolly bodies, so more circulating erythrocytes with Howell-Jolly bodies may be observed in healthy cats than in other species. Occasionally metarubricytes may also be released from the bone marrow and observed in circulation, although nucleus extrusion typically occurs during splenic passage of these cells. Also, the splenic reticuloendothelial system has a major role in removing damages or senescent erythrocytes from circulation. The major role of erythrocytes is to carry oxygen, which binds to hemoglobin, from the lungs to tissues. Altered tissue demands for oxygen can increase or decrease the production of EPO and therefore erythropoiesis. In the adult, EPO is produced by the kidney in response to hypoxia. The circulating blood volume is composed of about 40%–45% erythrocytes ( Bloom and Brandt, 2008 ), but there are usually many noncirculating erythrocytes present within the splenic red pulp. Automated hematology analyzers provide indications of blood erythrocyte counts, blood hemoglobin concentration, and hematocrit, which are collectively indicative of red cell mass. In health, the vast majority of blood erythrocytes are mature erythrocytes, with only a very small proportion of reticulocytes, except in rodents. Rodents normally have mildly greater reticulocyte counts relative to most other common laboratory species due to their higher erythrocyte turnover; gerbils tend to have the shortest erythrocyte lifespans and therefore the highest reticulocyte counts ( Zimmerman et al., 2010 ). Erythrocyte lifespan in circulation is species-dependent. Human erythrocytes have an average lifespan of about 120 days ( Thiagarajan and Prchal, 2016 ), while macaque erythrocytes have a lifespan of approximately 100 days ( Provencher Bolliger et al., 2010 ). Canine and feline erythrocyte lifespans are approximately 100 and 70 days, respectively ( Stockham and Scott, 2008b ). In contrast, rat erythrocytes have an estimated lifespan of approximately 60 days ( Van Putten and Croon, 1958 ) although there is some strain-related variability ( Derelanko, 1987 ), while mouse erythrocyte lifespans are even shorter, with an estimate of about 41 days ( Van Putten and Croon, 1958 ). Mongolian gerbil erythrocyte lifespans have an estimate of 9–10 days ( Zimmerman et al., 2010 ). Classic patterns of alterations in red blood cell parameters are summarized in Table 2 . Table 2 Classic patterns of alterations in red blood cell components and related endpoints Erythrocytosis Regenerative anemia Iron deficiency Nonregenerative anemia Relative Primary Secondary Blood loss IV hemolysis EV hemolysis ACD Bone marrow suppression RBC ↑ ↑ to ↑↑↑ ↑ to ↑↑↑ ↓ to ↓↓↓ ↓ to ↓↓↓ ↓ to ↓↓ ↓ to ↓↓↓ ↓ ↓ to ↓↓↓ Retic a - ↑ to ↑↑ ↑ to ↑↑ - to ↑↑ - to ↑↑ - to ↑↑ - to ↓ d - to ↓ ↓ to ↓↓↓ MCV - - to ↑ - to ↑ - to ↑ - to ↑ - to ↑ ↓ - - MCHC - - to ↓ - to ↓ - to ↓ ↓ to ↑ b - to ↓ - to ↓ - - Total bilirubin - - - - - to ↑↑ ↑ to ↑↑ - - - Free Hgb - - - - - to ↑↑ c - - - - Patterns described in this table indicate classic or expected changes in red blood cell components and associated endpoints due to these processes. However, duration of these processes, variations in the underlying causes of these conditions, and superimposition of multiple processes may result in differences between expected patterns of leukocyte changes and actual changes observed in an individual. -, no apparent change; ↑, mild increase; ↑↑, moderate increase; ↑↑↑, marked increase; ↓, mild decrease; ↓↓, moderate decrease; ↓↓↓, marked decrease. RBC , red blood cells; Retic , reticulocytes; MCV , mean corpuscular volume; MCHC , mean corpuscular hemoglobin concentration; Hgb , hemoglobin; IV , intravascular; EV , extravascular; ACD , anemia of chronic disease. a The magnitude of reticulocyte count increases generally reflects the magnitude of red cell mass decreases; all decreases in red cell mass are initially preregenerative without apparent increases in reticulocyte counts. b When overwhelming intravascular hemolysis causes increases in plasma or serum free hemoglobin, MCHC may be artifactually increased, but MCHC is more commonly decreased due to increases in reticulocyte counts. c Increases in plasma or serum free hemoglobin are only expected to occur with overwhelming intravascular hemolysis. d Chronic iron deficiency anemia is typically nonregenerative with decreases in reticulocyte counts, but increases in reticulocyte counts may be observed following transfusion or iron supplementation. 12.11.3.1 Increases in Red Cell Mass (Erythrocytosis) 12.11.3.1.1 Relative increases in red cell mass Relative increases in red cell mass, or hemoconcentration, most commonly occur due to dehydration and splenic contraction. In contrast to the absolute or "true" increases in red cell mass that result from proliferation of erythroid precursors in the bone marrow and/or spleen, relative increases are transient and consist of changes to total blood volume or shifting or noncirculating erythrocytes into circulation resulting in increases in red cell mass, which can rapidly resolve. 12.11.3.1.1.1 Dehydration Dehydration is a relatively common cause of secondary increases in red cell mass. Dehydration results in depletion of the water content of blood, and a relative increase in the other blood components, including cells (hemoconcentration). Due to the high number of erythrocytes present, increases in red cell mass are detectable, whereas increases in leukocyte subtypes are often not observed. These increases in red cell mass are usually observed in conjunction with increases in urea nitrogen and/or creatinine (prerenal azotemia) with concurrent decreases in urine volume and increases in urine specific gravity, as well as proportional increases in albumin and globulin. If evaluating plasma, fibrinogen may also be increased. In nonclinical toxicology studies in rodents, decreased food consumption is often associated with concurrent decreases in water intake, resulting in increases in red cell mass from subclinical or clinical dehydration. Resolution of these secondary increases in red cell mass will occur with adequate rehydration. 12.11.3.1.1.2 Catecholamine-induced Increases in circulating catecholamine levels in response to fright, excitement, or acute stress can result in increases in red cell mass due to splenic contraction. Noncirculating erythrocytes stored within the red pulp of the spleen are expelled, resulting in increased circulating red cell mass. Such increases in red cell mass are transient and resolve as splenic relaxation occurs following a decline in circulating catecholamine levels. Catecholamine-induced splenic contraction-associated relative increases in red cell mass are most commonly observed at pretest collections in nonclinical toxicology studies utilizing nonhuman primates, dogs, or cats, particularly the first pretest collection if multiple collections are performed. It is typically not observed at subsequent collections as the animal becomes acclimated to the housing, handling/restraint, phlebotomy, and other study-related procedures. 12.11.3.1.1.3 Xenobiotic-induced Xenobiotic-related causes of relative increases in red cell mass are relatively uncommon, with the exception of dehydration associated with decreased food consumption in rodents utilized in nonclinical toxicology studies, as described earlier. Diuretics, such as furosemide or spironolactone, which result in increased elimination of water into urine, may result in hemoconcentration due to dehydration ( Mintzer et al., 2009 ). 12.11.3.1.2 Secondary increases in red cell mass Secondary increases in red cell mass are dependent on stimulating factors and are not autonomous, in contrast to primary increases in red cell mass. These secondary increases in red cell mass are most commonly associated with increases in EPO concentrations due to hypoxia, and are therefore considered appropriate. However, hypoxia-independent (inappropriate) mechanisms may also cause secondary increases in red cell mass and are also described later. 12.11.3.1.2.1 Hypoxia-dependent (appropriate) Increases in EPO production occur in response to hypoxia primarily from the kidney, although there is also some evidence that hypoxia may also stimulate production of EPO by the liver ( Rankin et al., 2007 ). Under normal oxygenation states, hypoxia-inducible factor (HIF) subunits are polyubiquitinated by a von Hipple-Lindau (VHL) tumor suppressor E3 ligase complex, which results in proteosomal degradation of HIF ( Jaakkola et al., 2001 ). Binding of the VHL E3 ligase complex to HIF requires hydroxylation of a proline residue in the VHL protein, a process that requires both oxygen and iron ( Ivan et al., 2001 ). In hypoxic states, HIF is not ubiquitinated and HIF subunits translocate to the nucleus, forming a transcription factor for genes, including the erythropoietin gene. Increases in circulating EPO concentrations as a result of hypoxia cause an appropriate increase in erythropoiesis and subsequent increase in red cell mass, which should improve delivery of oxygen to tissues. Sustained tissue hypoxia can result from high altitude residence, cardiac disease causing poor tissue or lung perfusion, or prolonged or chronic pulmonary diseases that impair oxygenation, such as COPD secondary to chronic smoking or obstructive sleep apnea in humans ( Prchal, 2016 , Stockham and Scott, 2008b ). Other conditions that may cause hypoxia-induced increases in red cell mass include mutations leading to hemoglobin with high affinity for oxygen, carboxyhemoglobin formation with heavy smoking, and erythrocyte enzyme deficiencies leading to methemoglobinemia, such as cytochrome b 5 reductase deficiency may also result in hypoxia-induced increases in red cell mass ( Prchal, 2016 ). Erythrocyte enzyme deficiencies resulting in methemoglobinemia have been reported in veterinary species ( Stockham and Scott, 2008b ), but hemoglobinopathies, conditions characterized by abnormal hemoglobin, have not yet been reported in domestic animals ( Randolph et al., 2010 ). 12.11.3.1.2.2 Hypoxia-independent (inappropriate) Secondary but hypoxia-independent increases in red cell mass are associated with increases in circulating EPO levels. However, increased EPO in these cases are attributable to autonomous production of EPO rather than hypoxia response. Reported associations include renal diseases, renal or nonrenal neoplasms, or rare dysfunctions of the oxygen sensing pathway. Nonneoplasic renal diseases associated with increased EPO production include hydronephrosis, renal cysts, and polycystic renal disease ( Prchal, 2016 ). These renal diseases may be associated with local tissue hypoxia ( Randolph et al., 2010 ), but are not associated with systemic hypoxia. Similarly, increases in red cell mass may be observed in humans following renal transplantation ( Prchal, 2016 ). Autonomous production of EPO by neoplasms has been associated with both benign and malignant tumors. Renal adenoma, renal carcinoma, and sarcoma of the kidney have been reported to cause increases in red cell mass ( Ways et al., 1961 ). Renal lymphoma has also been associated with increases in red cell mass ( Durno et al., 2011 ). Nonrenal tumors with inappropriate EPO production include hepatoma, hamartoma of the liver, leiomyosarcoma, schwannoma, and pheochromocytoma ( Stockham and Scott, 2008b , LevGur and Levie, 1995 , Muta et al., 1994 , Shulkin et al., 1987 , Josephs et al., 1961 ). VHL syndrome, which follows an autosomal dominant pattern of inheritance, may predispose affected people to developing renal or nonrenal neoplasms that can autonomously produce EPO ( Prchal, 2016 ). There are also rare inherited conditions that cause defects in oxygen sensing pathways described in humans. These include Chuvash polycythemia, which follows an autosomal recessive inheritance pattern, and EGLN1 gene mutations, which cause a deficiency in proline hydroxylase ( Prchal, 2016 ). High cobalt concentrations may also inhibit the oxygen sensing pathway by preventing binding of the VHL E3 ligase to HIF ( Yuan et al., 2003 , Schuster et al., 1989 ). 12.11.3.1.2.3 Endocrinopathies Increases in red cell mass associated with endocrinopathies are generally mild and do not result in overt clinical signs. Hyperthyroidism causes a sustained increase in tissue demand for oxygen, leading to hypoxia, increased EPO production, and consequent increases in red cell mass ( Stockham and Scott, 2008b ). Acromegaly, caused by an increase in growth hormone concentrations, has also been associated with increases in red cell mass, particularly in cats ( Randolph et al., 2010 ). Hyperadrenocorticism or adrenal neoplasms that produce androgens or aldosterone may also be associated with increases in red cell mass ( Prchal, 2016 , Ghio et al., 1981 , Mann et al., 1967 ). 12.11.3.1.2.4 Xenobiotic-induced Xenobiotic-induced increases in red cell mass are uncommon. Administration of recombinant erythropoietin and anabolic steroids has been reported to cause increases in red cell mass ( Mintzer et al., 2009 ). For example, increases in red cell mass have been associated with testosterone administration ( Gardner et al., 1968 ). Theoretically, excess administration of thyroid hormones could also cause increases in red cell mass. 12.11.3.1.3 Primary increases in red cell mass Primary, or erythropoietin-independent, increases in red cell mass are associated with myelodysplastic conditions with autonomous production of erythrocytes. Due to the autonomous nature of the proliferations, primary increases in red cell mass are also termed inappropriate as they are not dependent on EPO stimulation. Causes of the more common secondary increases in red cell mass should be excluded prior to the diagnosis of a primary increase in red cell mass. Measurement of EPO concentrations may be useful clinically in people, but assays that quantify EPO are not readily available for most veterinary species. Primary increases in red cell mass are uncommon, and are typically not observed in common laboratory species during nonclinical toxicology studies. 12.11.3.1.3.1 Polycythemia vera Polycythemia vera is categorized as a chronic myeloproliferative disorder. Neoplastic transformation, from an acquired somatic mutation ( Prchal, 2016 ), of a hematopoietic progenitor cell results in clonal and autonomous expansion of hematopoietic cells, including erythrocytes. Eventually the clonal expansion is sufficient to suppress normal hematopoiesis ( Prchal and Prchal, 2016 ). Increases in red cell mass are the prototypical findings, but concurrent increases in leukocyte and platelet counts that arise from the neoplastic clone are often also be observed in people ( Pearson, 2001 ), although these findings are typically not observed in dogs or cats ( Randolph et al., 2010 ). The most common causative somatic mutation of polycythemia vera in humans is a mutation of JAK2, a kinase that plays a role in intracellular proliferative signaling ( Prchal, 2016 ). However, forms of polycythemia vera or "idiopathic erythrocytosis" without JAK2 mutations have been observed and associated with mutations in lymphocyte-specific adaptor protein (LNK) that inhibits JAK2 phosphorylation ( Lasho et al., 2010 ). Serum EPO concentrations are expected to be low in patients with polycythemia vera. In domestic dogs and cats, middle-aged female dogs and male cats tend to be most commonly affected ( Randolph et al., 2010 ). 12.11.3.1.3.2 Primary familial and congenital polycythemia Similar to polycythemia vera, primary familial and congenital polycythemia (PFCP) is also caused by autonomous erythroid proliferation despite low serum EPO. However, PFCP is associated with nonclonal erythroid proliferation from an inherited mutation that has an autosomal dominant pattern of inheritance ( Prchal et al., 1985 ). Identified mutations definitively associated with PFCP result in the truncation of the EPO receptor with a loss of the negative regulatory domain, causing constitutive activity of the signaling pathway promoting erythrocyte proliferation ( Prchal and Prchal, 2016 ). 12.11.3.2 Decreases in Red Cell Mass (Anemia) Decreases in red cell mass, or anemia, are a relatively common finding in humans and common laboratory species. Decreases in red cell mass are further categorized by concurrent changes in reticulocyte counts, which provide an indication of bone marrow responsiveness and may help to differentiate among possible mechanisms. Decreases in red cell mass with concurrent increases in reticulocyte counts indicate a regenerative erythroid bone marrow response, where normal or low reticulocyte counts may represent a preregenerative, suppressed, or ineffective erythroid response. 12.11.3.2.1 Decreases in red cell mass with increases in reticulocyte counts (regenerative anemia) Decreases in red cell mass with concurrent increases in reticulocyte counts (reticulocytosis) indicate a regenerative erythroid response by the bone marrow, or by extramedullary hematopoiesis in the spleen of rodents. An increase in reticulocyte count is typically first observed 3–4 days after an acute drop in red cell mass due to bone marrow erythrocyte production and transit time, and peak responses generally occur around 7–14 days depending on the species ( Stockham and Scott, 2008b ). The regenerative erythroid response is considered appropriate if the increases in reticulocyte counts reflect the magnitude of the decreases in red cell mass; in other words, a mild decrease in red cell mass is expected to result in a mild increase in reticulocyte count, while a moderate to marked decrease in red cell mass should have a concurrent moderate to marked increase in reticulocyte count. The regenerative erythroid response is considered inappropriate if there is an inconsistency between the magnitude of the decrease in red cell mass and the magnitude of the increase in reticulocyte count. For example, a marked decrease in red cell mass with only a mild increase in reticulocyte count a week after the insult would be considered an inappropriate regenerative erythroid response. Increases in blood reticulocyte counts may be associated with concurrent changes in mean corpuscular volume (MCV; an indicator of erythrocyte size) and mean corpuscular hemoglobin concentration (MCHC; an indicator of erythrocyte hemoglobin content), two red blood cell indices provided by most automated hematology analyzers. Due to the reticulocyte's increased volume relative to mature erythrocytes, increases in blood reticulocyte counts may cause increases in MCV (macrocytosis) and decreases in MCHC (hypochromasia). Regenerative anemias with increases in MCV and decreases in MCHC and/or CHCM may also be classified by these indices as macrocytic, hypochromic anemias. The decrease in MCHC does not necessarily reflect less hemoglobin content per cell, but is a consequence of reduced concentration of hemoglobin due to the larger cytoplasmic volume of reticulocytes. MCHC, which is a calculated endpoint, may be artificially increased when free plasma hemoglobin is present due to intravascular hemolysis. Some automated hematology analyzers also provide the corpuscular mean hemoglobin content (CHCM) which provides a mean of direct measurements of cellular hemoglobin concentration and is therefore resistant to interference from free plasma hemoglobin. Regenerative erythroid responses with increases in blood reticulocyte counts may be associated with several morphologic findings observed during blood smear evaluation. Most commonly, increases in polychromatophils (polychromasia) are observed with Wright-Giemsa or modified Wright stains. Polychromatophils are erythrocytes that stain blue–purple in color due to the combined effects of blue-staining RNA content typical of reticulocytes and pink-staining hemoglobin. As reticulocytes mature and lose RNA, a visual difference in staining cannot longer be detected between late reticulocytes and mature erythrocytes. However, staining of blood with a vital dye such as New Methylene Blue permits differentiation between aggregate and punctate-type reticulocytes. Polychromasia usually correlates well with increases in reticulocytes in most species, except cats ( Stockham and Scott, 2008b ). In cats, aggregate-type but not punctate-type reticulocytes correlate with polychromasia and are considered clinically relevant, and differentiation of these two with manual reticulocyte counts should be performed ( Harvey, 2012 , Stockham and Scott, 2008b ). Reticulocytes or erythrocytes with few small, punctate dark blue-gray inclusions may be observed during a regenerative erythroid response. These inclusions contain iron and may be called Pappenheimer bodies or siderotic inclusions. Due to the rapid production and release of erythrocytes during a regenerative erythroid response, there may also be increase in nucleated red blood cells or erythrocytes with Howell-Jolly bodies. Nucleated red blood cells are usually present in low numbers, but if 10 or more are observed per 100 leukocytes, the automated total leukocyte count will be falsely increased and should be corrected using published equations ( Stockham and Scott, 2008a ). In some species, particularly cows and sheep, erythrocytes with basophilic stippling may also be observed in circulation during a regenerative erythroid response. Hemolysis and blood loss are the two main categories of decreases in red cell mass with appropriate increases in reticulocyte counts. 12.11.3.2.1.1 Hemolysis Destruction of mature erythrocytes is called hemolysis. Hemolysis may occur either intravascularly or extravascularly. With intravascular hemolysis, erythrocyte destruction occurs within the blood and results in hemoglobinemia, or free hemoglobin within plasma. Ghost erythrocytes, or the remnant membranes of erythrocytes that no longer contain cytoplasm or hemoglobin, may be observed with intravascular hemolysis. Consequent hemoglobinuria, or free hemoglobin in the urine, is rare and only occurs in cases of massive intravascular hemolysis that overwhelm the normal pathways that clear free hemoglobin from the blood. In contrast, extravascular hemolysis does not occur within the blood, but rather occurs in the spleen, liver, or bone marrow, where resident macrophages phagocytose erythrocytes and destroy them intracellularly. Extravascular hemolysis does not result in free plasma hemoglobin or hemoglobinuria. Both types of hemolysis may be associated with increases in total bilirubin concentrations where unconjugated (indirect) bilirubin usually exceeds conjugated (direct) bilirubin, and may result in plasma or serum icterus (yellow discoloration) or bilirubinuria (bilirubin present in urine). However, not all cases of hemolysis are clearly either intravascular or extravascular, and both forms of hemolysis may contribute in some conditions. 12.11.3.2.1.1.1 Infectious There are numerous protozoal, bacterial, and viral diseases that can be associated with hemolysis. Mechanisms by which infectious agents cause erythrocyte destruction are varied, and may include direct infection of erythrocytes, elaboration of toxins such as hemolysin, or stimulation of an immune-mediated response against infected cells ( Berkowitz, 1991 ). Several examples of infectious agents that cause hemolytic anemia are discussed later. Direct infection of erythrocytes with protozoal Plasmodium species, the causative agent of malaria that is transmitted by mosquitoes, is a relatively common cause of hemolysis in humans, but may also be observed in nonhuman primates used in nonclinical toxicology studies. Humans are infected by one of five different Plasmodium species: P . falciparum , P . vivax , P . knowlesi , P . malariae , or P . ovale , although only P . falciparum and P . vivax are commonly associated with severe hemolysis ( Lichtman, 2016b ). Macaques are most commonly infected with P . inui or P . knowlesi , although the cynomolgus monkey appears to be more resistant to disease from these infections than the rhesus monkey ( Ameri, 2010 ). Infection with P . cynomolgi , P . fieldi , or P . fragile may also occur in macaques ( Magden et al., 2015 ). Although it is uncommon to include macaques infected with Plasmodium species during a nonclinical toxicity study due to current screening practices and pretest evaluations, rare animals with decreases in red cell mass and increases in reticulocyte counts and intraerythrocytic Plasmodium organisms have been observed. Rats and mice may be infected with Plasmodium berghei ( Holloway et al., 1995 , Sadun et al., 1965 ). Plasmodium berghei has a specific tropism for reticulocytes rather than mature erythrocytes Plasmodium species that infect humans ( Car et al., 2006 , Cromer et al., 2006 ). Concurrent increases in reticulocyte counts may occur in early stages or disease or with recrudescence of parasitemia and hemolysis. Hemolysis is associated with clearance of parasitized erythrocytes from circulation predominantly by splenic macrophages ( Lichtman, 2016b ), although accumulation of hemin, an iron-containing porphyrin, which can directly stimulate apoptotic erythrocyte death (eryptosis) ( Gatidis et al., 2009 ), oxidative damage to erythrocyte membranes ( Clark and Hunt, 1983 ), and increased osmotic fragility ( George et al., 1967 ) may all contribute to hemolysis. However, late-stage infections in humans and rodents have also been associated with inappropriate or decreased reticulocyte counts indicative of suppressed erythropoiesis despite decreases in red cell mass from hemolysis ( Lichtman, 2016b , Cromer et al., 2006 ). Babesia species are tick-borne protozoal organisms that directly infect erythrocytes in most species, including humans, nonhuman primates, dogs, and cats. Babesia species appear as intracellular oval to pyriform organisms. Babesia microti and Babesia divergens may infect humans in North America and Europe, respectively, and cause moderate hemolytic anemia from intraerythrocytic replication and subsequent erythrocyte lysis ( Lichtman, 2016b , Kjemtrup and Conrad, 2000 ). Babesia pitheci has been reported to infect both old and new world monkeys and cause anemia ( Magden et al., 2015 ). B . canis , a large babesial species, and B . gibsoni , a small babesial species, infect dogs, while cats may be infected by the small babesial organisms B . felis and B . cati ( Stockham and Scott, 2008b , Penzhorn et al., 2004 ). These organisms are generally not of concern in purpose-bred animals used in nonclinical toxicology studies. Bartonella bacilliformis in people and the hemotrophic mycoplasmas (hemoplasmas) in dogs and cats (formerly Haemobartonella species) and swine (formerly Eperythrozoon species) are organisms that parasitize erythrocytes, but these organisms remain extracellular in shallow depressions of the erythrocyte membrane. These organisms are typically round-, rod-, or ring-shaped and may be observed individually or in chains on erythrocyte surfaces. Hemolysis with these organisms may be immune-mediate and associated either with binding of antibodies to parasite antigens or antigens exposed on the erythrocyte secondary to parasite-induced membrane changes ( Stockham and Scott, 2008b ). Clostridium perfringens (formerly Clostridium welchii ) infection in humans is an example of a bacterial cause of hemolysis. During intestinal overgrowth or septicemia, C . perfringens type A elaborates an α toxin that has lecithinase C activity, resulting in membrane phospholipid breakdown and release of lysolethicins, which have potent hemolytic capabilities ( Lichtman, 2016b , Songer, 1996 ). C . perfringens α toxin release is usually associated with severe intravascular hemolysis with both hemoglobinemia and hemoglobinuria. However, in veterinary species, C . perfringens -related hemolysis is typically limited to ruminants and horses ( Stockham and Scott, 2008b ), and is unlikely to be observed in the common species used in nonclinical toxicology studies. Infection of humans with Mycoplasma pneumoniae has also been associated with hemolytic decreases in red cell mass, although most cases of M . pneumoniae infection are asymptomatic. Hemolysis with this organism is attributable to stimulation of autoimmune erythrocyte destruction with agglutination of erythrocytes ( Khan and Yassin, 2009 ). Several viral organisms in humans have also been reported to cause decreases in red cell mass due to hemolysis. Viral causes of hemolysis are commonly associated with autoimmune mechanisms, and include infection with Epstein-Barr virus ( Palanduz et al., 2002 ), hepatitis A, B, and C viruses ( Kanematsu et al., 1996 , Chao et al., 2001 ), cytomegalovirus ( Murray et al., 2001 ), and HIV ( Koduri et al., 2002 ), although HIV infection is also commonly associated with decreases in reticulocyte counts rather than the expected increases secondary to hemolysis, indicative of concurrent suppressed erythropoiesis ( Telen et al., 1990 ). 12.11.3.2.1.1.2 Oxidative Another major cause of decreases in red cell mass due to hemolysis is oxidative damage to erythrocytes. Under normal conditions, ferrous iron (Fe 2 + ) in hemoglobin binds to and dissociates from oxygen as it delivers oxygen from the lungs to the tissues. At times, this binding and dissociation results in the formation of ferric iron (Fe 3 + ) in hemoglobin (methemoglobin) as well as superoxide (O 2 − ). Superoxide is a free radical with potent oxidative capacity that may cause cellular damage. Cytochrome b 5 -reductase is an intraerythrocytic enzyme that converts methemoglobin back to hemoglobin. Superoxide dismutase converts superoxide to hydrogen peroxide (H 2 O 2 ), which also may produce oxidative damage to cells. Further metabolism of hydrogen peroxide by catalase or glutathione peroxidase protects cells from oxidative damage. These pathways are usually sufficient to address the normal low-level formation of methemoglobin and superoxide, but methemoglobin can increase and impair delivery of oxygen to tissues and superoxide can accumulate and cause oxidative damage if these pathways are overwhelmed or defective. Oxidative damage to erythrocytes may affect the lipid membranes, cytoskeleton, or hemoglobin. Peroxidation of internal membrane lipids or cytoskeletal components of erythrocytes results in the fusion of portions of the membrane with consequent shifting of the cytoplasm and hemoglobin to one side of the cells. Erythrocytes with this morphologic change are called eccentrocytes. Oxidative damage that causes the formation of eccentrocytes may result in hemolysis due to increased clearance of eccentrocytes by splenic macrophages due to trapping of rigid erythrocytes in splenic sinusoids or spontaneous rupture in blood due to the increased fragility of eccentrocytes ( Stockham and Scott, 2008b ). Oxidative damage to exposed cysteine sulfhydryl groups on hemoglobin results in hemoglobin denaturation and decreased solubility ( Bloom and Brandt, 2008 ). Denatured hemoglobin may then precipitate and aggregate within the erythrocyte, forming small, pale-staining round structures that bind to the erythrocyte membrane and tend to protrude from the surface of the erythrocyte. These aggregates of denatured hemoglobin are called Heinz bodies. Cats appear to be particularly sensitive to the formation of Heinz bodies because of an increased number of reactive sulfhydryl groups in hemoglobin relative to other species ( Christopher et al., 1990 ), and may be more rapidly observed on blood smear evaluation due to the nonsinusoidal architecture of the feline spleen that results in decreased clearance of Heinz bodies from circulation. Similar to eccentrocytes, erythrocytes with Heinz bodies may undergo hemolysis due to increased clearance by splenic macrophages following trapping in splenic sinusoids due to decreased erythrocyte deformability and spontaneous rupture due to increased fragility from membrane damage; immune-mediated clearance may also occur and is believed to result from binding of hemochromes to and subsequent redistribution of band 3, an erythrocyte membrane structural protein, which may then be recognized by autologous antibodies ( Winterbourn, 1990 ). There are many conditions that may cause oxidative damage and result in eccentrocytosis, Heinz body formation, or even both simultaneously. Diabetes mellitus may cause either morphologic change, and diabetic ketoacidosis appears to be associated with an increased susceptibility and incidence of oxidative erythrocyte damage ( Desnoyers, 2010 , Caldin et al., 2005 , Christopher et al., 1995 ). Inherited deficiencies in erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and flavin adenine dinucleotide (FAD) have also been associated with erythrocyte oxidative damage, eccentrocyte or Heinz body formation, and hemolysis or a predisposition for these events due to the loss of protective antioxidant pathways ( Chan et al., 1982 , Harvey, 2006 ). Lymphoma has also been associated with Heinz body formation in cats ( Christopher, 1989 ) and eccentrocytes formation in dogs ( Caldin et al., 2005 ). In dogs and cats, ingestion of Allium species, particularly onions, garlic, and Chinese chive, may cause erythrocyte oxidative damage with formation of eccentrocytes and/or Heinz bodies ( Caldin et al., 2005 , Yamato et al., 2005 , Robertson et al., 1998 ). Ingestion of zinc in dogs ( Bexfield et al., 2007 ) and exposure to skunk musk ( Fierro et al., 2013 ) have also been reported to cause hemolysis due to Heinz body formation. 12.11.3.2.1.1.3 Fragmentation Physical trauma to erythrocytes results in hemolysis due to erythrocyte fragmentation and lysis. Sometimes this type of hemolysis is referred to as microangiopathic hemolytic anemia. Morphologic changes to erythrocytes occur as a result of physical trauma. Schistocytes (also called schizocytes or erythrocyte fragments), keratocytes (also called helmet cells), prekeratocytes (also called blister cells), or even spherocytes or microspherocytes may be observed. Schistocytes are very small, usually irregularly shaped fragments that can break off erythrocytes when physical trauma occurs. Keratocytes have one to two variably sized projections or horns adjacent to a small flattened region of the erythrocyte surface, while prekeratocytes appear to be precursors that have small loops of erythrocyte cytoplasm extending from the surface and surrounding a small hole in the cell. Spherocytes and microspherocytes are spherical cells that appear smaller and have more intensely pink-staining cytoplasm than normal mature erythrocytes. Spherocytes and microspherocytes may be formed during physical trauma as fragments are broken off, resulting in less membrane surface area in the parent erythrocyte surrounding a similar volume (spherocytes) or smaller volume (microspherocytes) as the parent erythrocyte. The physical trauma to erythrocytes that causes fragmentation or microangiopathic hemolysis may result from consumptive coagulopathies, either local or disseminated (DIC), with fibrin or thrombus formation in the vasculature that impedes the passage of erythrocytes through the vessel lumen, creating both turbulence and physical obstruction of blood flow. Local coagulopathy or DIC may occur secondary to trauma, infections with sepsis, or neoplasia ( Baker and Moake, 2016 , Toh and Dennis, 2003 ). Microangiopathic hemolysis due to neoplasia is most commonly associated with malignant rather than benign neoplasms and with metastatic disease or neoplasic emboli rather than primary tumors, with the exception of primary vascular neoplasms ( Susano et al., 1994 , Kupers et al., 1975 , Lohrmann et al., 1973 ). Infectious agents may also lead to fragmentation of erythrocytes, and some Leptospirosis interrogans serovars associated with vasculitis ( Stockham and Scott, 2008b ), Brucella species infection ( Yaramis et al., 2001 ), and cutaneous anthrax ( Freedman et al., 2002 ) have been reported to cause microangiopathic hemolysis. In children, fragmentation hemolysis associated with thrombotic microangiopathy may occur with Shigella dysenteriae type 1 and some Escherichia coli infections ( Pisoni et al., 2001 ). Hemolysis from erythrocyte fragmentation may also occur with HIV infection ( Maslo et al., 1997 ). Decreases in red cell mass with increases in reticulocyte counts from erythrocyte fragmentation may also occur secondary to cardiac or other conditions that alter hemodynamics and increase turbulent blood flow. For example, subaortic stenosis ( Solanki and Sheikh, 1978 ), intraluminal aortic grafts ( Sayar et al., 2006 ), uncorrected cardiac valvular disease ( Marsh and Lewis, 1969 ), prosthetic valves ( Crexells et al., 1972 ), and hypertrophic obstructive cardiomyopathy ( Kubo et al., 2010 ) have all been reported to cause hemolysis from erythrocyte fragmentation. Increased turbulence associated with hypertension may also cause decreases in red cell mass from fragmentation, and has been associated with pulmonary hypertension ( Baker and Moake, 2016 ) and malignant systemic hypertension ( Capelli et al., 1966 ). 12.11.3.2.1.1.4 Immune-mediated Autoimmune hemolytic anemia (AIHA or AHA) described in humans or immune-mediated hemolytic anemia (IMHA) described in most common laboratory species is a cause of hemolysis, and may be primary or idiopathic, but may also be secondary to conditions such as infections as discussed previously. Primary or idiopathic AIHA/IMHA is discussed here. Primary AIHA has no underlying detectable cause and is an immune-mediated condition that produces antibodies targeting erythrocyte antigens. These antierythrocyte antibodies tend to be very specific for a single erythrocyte antigen in a given case ( Packman, 2016 ). These autoantibodies may be classified as warm antibodies, which are usually IgG, or cold antibodies, which are usually IgM ( Stockham and Scott, 2008b ). Immune-mediated AIHA may be associated with erythrocyte morphologic changes that include agglutination and spherocytes. Agglutination may be observed grossly as red speckling along the inside of the specimen tube as blood is gently moved within the tube. If agglutination is present, blood smears may have a "reverse smear" appearance with the densest region of the smear observed at the feathered edge rather than the edge where the drop of blood was initially placed. Microscopically agglutination appears as grape-like clusters of erythrocytes. Spherocytes are erythrocytes that are spherical instead of having the normal biconcave disc shape. While spherocytes appear smaller and stain more intensely pink that unaffected mature erythrocytes, they have the same volume as unaffected erythrocytes. Loss of erythrocyte membrane occurs when macrophages begin to phagocytize antibody-bound erythrocytes, leading to decreased erythrocyte surface area without an appreciable change in volume, forcing erythrocytes to form spheres. Hence, spherocytosis alone will not result in an altered MCV. Of the most common laboratory species, dogs tend to have the most pronounced central pallor of normal mature erythrocytes, making microscopic identification of spherocytes easiest in the dog. Hemolysis in AIHA is largely attributable to extravascular hemolysis due to phagocytosis of antibody-bound erythrocytes by tissue macrophages. Macrophages or monocytes containing phagocytized erythrocytes may be rarely observed in blood smears of laboratory species with immune-mediated hemolysis. However, antibody-mediated complement activation or increased fragility of spherocytes may result in direct intravascular lysis or rupture of erythrocytes ( Packman, 2016 ). Evaluation of patients for the presence of antierythrocyte antibodies may be performed using the direct antiglobulin test (DAT; also called the Coombs' test) or by flow cytometry. AIHA has rarely been observed in association with lymphoproliferative neoplasia, such as chronic lymphocytic leukemia. Antierythrocyte antibodies in chronic lymphocytic leukemia are predominantly IgG with few cases of IgM reported ( Mauro et al., 2000 ). IMHA may occasionally be observed following blood transfusion ( Garratty, 2004 ). This may occur in response to alloantigens, and would not technically be considered autoimmune ( Stockham and Scott, 2008b ). Posttransfusion immune-mediated hemolysis may also be observed when the host has autoantibodies that bind the donor erythrocytes and cause immune-mediated destruction. However, crossmatching of host and donor erythrocytes and plasma is able to prevent many cases with incompatible transfusion-related AIHA. AIHA and IMHA commonly have concurrent inflammatory increases in leukocyte subtype counts, characterized mainly by neutrophilia that may or may not have a left shift with cytoplasmic changes indicative of rapid neutropoiesis. 12.11.3.2.1.1.5 Inherited Some phenotypes of sickle cell disease are associated with hemolysis. The mechanism of hemolysis in sickle cell disease is likely multifactorial and not associated with a single pathogenesis. There is evidence for oxidative damage to erythrocytes ( Lachant et al., 1983 ), which may contribute to the hemolysis observed with sickle cell disease. However, hemoglobin polymerization leads to erythrocyte deformation and may lead to decreased flexibility of erythrocytes and veno-occlusive disorders ( Bookchin and Lew, 1996 ). Decreased flexibility or deformability of erythrocytes may contribute directly to increased cell fragility and rupture or promote clearance of deformed erythrocytes by splenic macrophages, while veno-occlusive disease has the potential to cause decreases in red cell mass through physical trauma and fragmentation. However, there is also evidence that in some severe cases of sickle cell disease there may be an increase in reticulocyte counts that are inappropriate for the magnitude of the decrease in red cell mass, suggesting a concurrent mechanism causing suppressed or ineffective erythropoiesis ( Wu et al., 2005 , Bookchin and Lew, 1996 ). Oxidative stress on erythroid precursors may also contribute to ineffective erythropoiesis in some severe cases of sickle cell disease ( Fibach and Rachmilewitz, 2008 ). Several metabolic defects of erythrocyte metabolism may also be associated with decreases in red cell mass and increases in reticulocyte counts. Deficiencies in erythrocyte pathways of glycolysis may result in decreased ATP concentrations that lead to erythrocyte membrane dysfunctions with shortened erythrocyte lifespan and occasionally hemolysis ( Stockham and Scott, 2008b ). Pyruvate kinase (PK) is the enzyme that catalyzes the last step in aerobic glycolysis. Deficiencies of PK that result in hemolysis have been reported in humans ( Baronciani and Beutler, 1993 ), dogs including beagles ( Harvey et al., 1977 , Giger et al., 1991 , Prasse et al., 1975 ), and a few breeds of cats ( Kohn and Fumi, 2008 ). Phosphofructokinase (PFK) catalyzes the rate-limiting step of the glycolysis pathway. Deficiencies in PFK have also been described in humans ( Etiemble et al., 1976 ) and dogs ( Giger et al., 1985 ). Respiratory alkalosis, which may be observed following intense exercise, is associated with acute hemolytic crises in patients with PFK deficiencies ( Giger et al., 1985 ). The association of inherited G6PD and FAD deficiencies with hemolysis resulting from oxidative damage is discussed earlier. In brief, G6PD and FAD play a role in the antioxidant pathways of erythrocytes. Deficiencies of G6PD and FAD may result in increased oxidative damage to erythrocytes and subsequent hemolysis. Collectively, the porphyrias are a group of enzymatic defects in the heme synthesis pathway. Porphyrias may be congenital or, more commonly, acquired. In these conditions, the accumulation of porphyrins, the precursors of heme, within erythrocytes leads to hemolysis. The mechanism of hemolysis may be related to lysis of erythrocytes following exposure to light (photolysis) in superficial vasculature, or by direct erythrocyte membrane damage due to the lipid soluble nature of porphyrins or following porphyrin absorption of ultraviolet light and excitation ( Phillips and Anderson, 2016 , Kaneko, 2008 ). 12.11.3.2.1.1.6 Neoplastic Various neoplastic conditions may be associated with hemolysis. Malignant metastatic neoplasms or primary vascular neoplasms may result in fragmentation hemolysis by physical trauma to erythrocytes, as previously discussed. However, neoplastic conditions may also rarely be associated with phagocytosis and destruction of erythrocytes, or hemophagocytic syndrome. Hemophagocytic syndromes have been associated with T-cell lymphoma ( Gonzalez et al., 1991 ), NK-cell leukemia ( Kobayashi et al., 1996 ), hemophagocytic histiocytic sarcoma ( Moore et al., 2006 ), and various hematological neoplasias ( Majluf-Cruz et al., 1998 ). 12.11.3.2.1.1.7 Xenobiotic-induced Many xenobiotics are capable of causing hemolysis, and may cause hemolysis through oxidative, fragmentation, or immune-mediated mechanisms. Examples of each are discussed here. Many of the agents that cause oxidative erythrocyte injury contain aromatic structures that can be metabolized, mostly commonly by cytochrome P 450, to free radicals ( Bradberry, 2003 , Edwards and Fuller, 1996 ), which overwhelm the normal protective antioxidant pathways of erythrocytes leading to both direct erythrocyte oxidative injury and oxidation of hemoglobin sulfhydryl groups resulting in methemoglobin formation. A few specific aromatic compounds that have been associated with free radical formation include dapsone, phenacetin, and anthracyclines such as doxorubicin ( Edwards and Fuller, 1996 , Coleman et al., 1991 , Handa and Sato, 1975 , Easley and Condon, 1974 ). Phenacetin has also been associated with the formation of Heinz bodies ( Boelsterli et al., 1983 ). In dogs and cats, acetaminophen (paracetamol) may be metabolized to a minor reactive metabolite that causes oxidative damage to erythrocytes resulting in hemolysis and the formation of Heinz bodies and/or eccentrocytes, although methemoglobinemia has also been observed in cats ( Desnoyers, 2010 , Wallace et al., 2002 , Mariani and Fulton, 2001 , Aronson and Drobatz, 1996 ). Xenobiotics that cause methemoglobinemia can also cause indirect oxidative damage through the peroxidation activity of methemoglobin itself ( Edwards and Fuller, 1996 ). In some cases, oxygenated hemoglobin may act as a peroxidase and cause the metabolism of a xenobiotic to a reactive compound that causes erythrocyte oxidative damage and conversion of oxyhemoglobin to methemoglobin. Examples of xenobiotics that cause oxidative damage through this mechanism are phenylhydrazine and primaquine ( Edwards and Fuller, 1996 ). Vitamin K administration in dogs can also cause oxidative erythrocyte damage through this mechanism ( Fernandez et al., 1984 ). Some chemical agents may cause oxidative damage by directly oxidizing hemoglobin sulfhydryl groups or through direct oxidation of erythrocyte cytoskeletal proteins. Arsine gas, predominantly an environmental toxin, appears to mediate its hemolytic effects through erythrocyte membrane oxidation ( Rael et al., 2000 ), although studies in mice have also demonstrated the formation of Heinz bodies following exposure ( Blair et al., 1990 ), suggesting an oxidative effect on hemoglobin as well. Many xenobiotics may also cause hemolysis through their association with microangiopathy, most commonly as part of the thrombotic microangiopathy syndrome, which is associated with fragmentation hemolysis and decreases in platelet counts. Drug-induced endothelial injury, including from direct and antibody or immune complex-mediated mechanisms, plays a major role in the pathogenesis of thrombotic microangiopathy ( Pisoni et al., 2001 ). Endothelial damage may be propagated by leukocyte adhesion and release of granule contents or reactive oxygen species, platelet activation and aggregation, and complement activation ( Pisoni et al, 2001 ). Drugs implicated in thrombotic microangiopathy include chemotherapeutic agents include xenobiotics from a wide variety of chemotherapeutic classes. Examples of chemotherapeutics associated with thrombotic microangiopathy include mitomycin C ( Cantrell et al., 1985 ), cisplatin ( Palmisano et al., 1998 ), estramustine phosphate ( Tassinari et al., 1999 ), gemcitabine ( Nackaerts et al., 1998 ), and daunorubicin ( Byrnes et al., 1986 ). Nonchemotherapeutic agents that have been reported to cause thrombotic microangiopathy include immunomodulators such as cyclosporine and tacrolimus ( Katznelson et al., 1994 , Trimarchi et al., 1999 ), simvastatin ( McCarthy et al., 1998 ), and inhibitors of platelet aggregation including ticlopidine and clopidogrel ( Bennett et al., 1998 , Bennett et al., 2000 ). Immune-mediated mechanisms of hemolysis have also been reported following exposure to numerous xenobiotics. Xenobiotics may induce antibodies by binding to the erythrocyte membrane and acting as haptens. These antibodies are considered drug-dependent as they only mediate hemolysis when the drug is present. Penicillin is the prototypical xenobiotic that acts as a hapten to generate drug-dependent antibodies, and typically induces an IgG response ( Ferner, 2012 , Petz et al., 1966 ). Semisynthetic penicillins, some cephalosporins, and tetracycline have also been reported to cause drug-dependent antibody-mediated hemolysis ( Garratty, 2010 , Tuffs and Manoharan, 1986 , Seldon et al., 1982 , Großjohann et al., 2004 , Gallagher et al., 1992 , Branch et al., 1985 , Simpson et al., 1985 ). Xenobiotics may also induce the production of antierythrocyte antibodies that mediate hemolysis even when the drug is no longer present, also called drug-independent antibodies or autoantibodies. In this type of hemolysis, xenobiotic exposure stimulates production of an antibody that can bind to native erythrocyte antigens even in the absence of the drug. This type of immune-mediated xenobiotic-induced hemolysis is classically caused by α-methyldopa, and is characterized by predominantly an IgG response ( Packman, 2016 ). However, nucleoside purine analogs such as cladribine and fludarabine have also been associated with hemolysis due to production of autoimmune antibodies ( Garratty, 2010 , Mintzer et al., 2009 , Hamblin, 2006 ). A third mechanism by which xenobiotics may cause immune-mediated hemolysis is through a complex interaction of the drug, a drug binding site on erythrocytes, and an antibody. This mechanism is considered the ternary complex mechanism, but has previously, and perhaps less accurately, been called an immune complex or innocent bystander mechanism ( Packman, 2016 ). Quinidine is the prototypical drug that causes hemolysis via this mechanism. Quinidine may be associated with either IgM or IgG antibodies and predominantly causes complement-mediated lysis of erythrocytes or clearance of complement-coated erythrocytes by tissue macrophages ( Packman, 2016 ). Ceftriaxone has also been reported to cause hemolysis through this mechanism ( Arndt and Garratty, 2005 ). Xenobiotic-induced immune-mediated hemolysis may not be limited to one of the three mechanisms described earlier, and a combination of these mechanisms may occur in some patients. For example, the NSAID diclofenac may cause hemolysis through both drug-dependent and drug-independent mechanisms ( Salama et al., 1996 ). Carboplatin has been reported to cause hemolysis through all three immune-mediated mechanisms ( Marani et al., 1996 ). Other compounds may cause hemolysis through mechanisms other than oxidative, microangiopathic, or immune-mediated. For example, although the primary effect of lead toxicity is impairment of heme synthesis, lead may also cause hemolysis. The mechanism of lead-induced hemolysis has not been fully determined, but interference with the erythrocyte membrane sodium/potassium transporter may be involved ( Bloom and Brandt, 2008 ). Copper toxicity causes hemolysis as well, possibly through inhibition of many enzymes involved in glycolysis resulting in decreased intracellular ATP ( Boulard et al., 1972 ). Envenomation from multiple animals is reported to cause hemolysis. Envenomation by snakes, such as rattlesnakes and coral snakes, can cause hemolysis through phospholipase A2 activity, which may cause direct hemolysis or liberate hemolysins such as lysolethicin, or through complement-mediated hemolysis ( Arce-Bejarano et al., 2014 , Tambourgi and van den Berg, 2014 , Walton et al., 1997 ). 12.11.3.2.1.2 Blood loss 12.11.3.2.1.2.1 Hemorrhage Hemorrhage may cause internal or external blood loss. Due to the loss of whole blood during hemorrhage, decreases in red cell mass are usually accompanied by proportionate decreases in albumin and globulin concentrations. The decreases in plasma proteins tend to be less pronounced with internal hemorrhage because the lost proteins may be resorbed in lymph and returned to blood ( Stockham and Scott, 2008b ). Cases of internal hemorrhage are typically not associated with iron deficiency. However, prolonged external blood loss may cause depletion of total body iron. Iron deficiency anemia is characterized by small erythrocytes with a decrease in MCV and erythrocytes that contain less hemoglobin with a decrease in MCHC, and may be classified as a microcytic, hypochromic anemia. Hemoglobin synthesis plays a role in inhibiting erythrocyte division, and when sufficient iron is not available for heme production, there is loss of the inhibitory effect resulting in more cell divisions and microcytes ( Stohlman et al., 1963 ). Hypochromasia of the erythrocytes is due to the lower than normal hemoglobin content due to decreased production of heme. Morphologic erythrocyte changes that accompany iron deficiency anemia include visual microcytosis and hypochromasia, keratocytes and schistocytes from physical damage to the more fragile erythrocytes, and sometimes codocytes (also called target cells) that have a thin rim of pink-staining hemoglobin and a small central area of hemoglobin with a ring of pallor in between, typical of erythrocytes with less hemoglobin present relative to the amount of membrane. In chronic iron deficiency, increases in reticulocyte counts and microscopic polychromasia may be lower than expected for a regenerative anemia due to loss of RNA during the extended maturation phase of erythrocyte production caused by decreased hemoglobin content ( Burkhard et al., 2001 ). Direct damage to blood vessels from trauma is a relatively common cause of acute external or internal blood loss. Traumatic rupture of the spleen may also cause significant acute internal blood loss. Decreases in red cell mass due to acute hemorrhage are typically due to dilution of remaining blood from shifting of intracellular fluid to extracellular fluid in an attempt to preserve blood volume and therefore tissue perfusion ( Stockham and Scott, 2008b ). Dilution of red cell mass may also be observed following administration of intravenous fluids to replace blood volume. A detectable increase in reticulocyte count is expected 3–4 days following the acute event in a patient with normally functioning bone marrow. Damage to blood vessels that results in hemorrhage also may occur secondary to ulcerative or neoplastic conditions. In dogs, rupture of splenic hemangiosarcoma is a common cause of internal blood loss into the abdomen (hemoabdomen). Ulceration of the gastrointestinal system may lead to blood loss into feces, which can be observed as black, tarry feces (melena) if the ulceration occurs in the small intestines or as frank blood if the ulceration occurs in the large intestines. In humans and nonhuman primate species with true menstrual cycles, including Old World monkeys and great apes ( Provencher Bolliger et al., 2010 ), decreases in red cell mass are uncommon but may be observed from menses-related blood loss. In women, heavy blood loss, abnormal cycling, or uterine neoplasms may lead to sufficient blood loss to cause decreases in red cell mass and potentially even iron deficiency ( Van Voorhis, 2009 , Goel and Gupta, 2007 ). In cynomolgus monkeys, decreases in red cell mass have been occasionally observed in females with prolonged menses ( Perigard et al., 2016 ). Coagulation disorders may also be associated with either internal or external hemorrhage. Primary deficiencies in coagulation factors or von Willebrand factor may be inherited causes of hemorrhage. Deficiencies in coagulation factors that lead to hemorrhage sufficient to cause decreases in red cell mass include hemophilia A (factor VIII deficiency) and hemophilia B (factor IX deficiency); deficiencies in factor XI and von Willebrand factor are usually mild and are often not associated with notable hemorrhage ( Bolton-Maggs and Pasi, 2003 ). Hemorrhage may be secondary to marked decreases in platelet counts from consumptive coagulopathies secondary to infectious or neoplastic processes. Although not truly hemorrhage, external blood loss can occur from repeated phlebotomy. Decreases in red cell mass may be acutely observed following collection of blood from donors for transfusion, and regular donors have a risk of developing iron deficiency from repeated external blood loss ( Cable et al., 2011 ). Repeated phlebotomy is a common occurrence in nonclinical toxicology studies, particularly in dogs and nonhuman primates, although rats may also occasionally undergo repeated blood collections. Blood is collected through the studies mainly for toxicokinetic or pharmacokinetic analysis, but also for analysis of hematology, coagulation, and clinical chemistry profiles. Decreases in red cell mass with increases in reticulocyte counts of similar magnitude relative to pretest values across all treatment groups, including controls, are a common procedure-related phenomenon in nonclinical toxicology studies and should be distinguished from a true test article-related effect. 12.11.3.2.1.2.2 Parasitism Both external and internal parasites may contribute to blood loss. Hookworms are a major internal parasite associated with chronic blood loss, and may lead to iron deficiency with prolonged infections ( Stoltzfus et al., 1997 ). However, whipworm infection and schistosomiasis may also be associated with blood loss, the latter being associated with blood loss through the urinary system ( Farid et al., 1969 ). Heavy infestation of animals with arthropods that take blood meals, such as ticks, some lice, and fleas, may also cause sufficient blood loss to result in decreases in red cell mass ( Stockham and Scott, 2008b ). 12.11.3.2.1.2.3 Xenobiotic-induced Xenobiotic-induced blood loss is relatively uncommon but can occur. Classically, hemorrhage into the intestinal tract can result from ulceration associated with chronic NSAID or coxib administration ( Laine et al., 2003 , Langman et al., 1999 , Bjarnason et al., 1987 ). Also, prolonged or high dose administration of anticoagulants, such as warfarin or heparin, can result in hemorrhage-related decreases in red cell mass ( Levine et al., 2001 ). Ingestion of rodenticides, including brodifacoum chlorophacinone, has been reported to cause marked hemorrhage in humans and many other nonrodent species ( Berny et al., 2010 , Palmer et al., 1999 , Sheafor and Couto, 1999 ). Xenobiotic-induced marked decreases in platelet counts may also be associated with hemorrhage and are discussed in more detail later. However, chemotherapeutics that cause bone marrow suppression can be associated with spontaneous or postvenipuncture hemorrhage. Occasional idiopathic decreases in platelet counts have also been observed following xenobiotic administration and are most likely attributable to immune-mediated destruction; some examples of implicated xenobiotics are quinine, trimethoprim–sulfamethoxazole, anticonvulsants such as phenytoin and carbamazepine, unfractionated or low molecular weight heparin, and rituximab ( Aster and Bougie, 2007 ). 12.11.3.2.2 Decreases in red cell mass with "normal" or low reticulocyte counts (nonregenerative anemia) 12.11.3.2.2.1 Preregenerative Depending on the timing of the insult causing the decreases in red cell mass, reticulocyte counts within reference interval may represent a preregenerative anemia rather than suppressed erythropoiesis. Production of erythrocytes by the bone marrow requires at least 3–4 days, and a peak increase in blood reticulocyte count occurs about 7–14 days following the insult ( Stockham and Scott, 2008b ). If it an individual with decreased red cell mass and reticulocyte counts that are within the reference interval and it is unclear if the individual has a preregenerative anemia or suppressed erythropoiesis, repeating a CBC several days later may help clarify which process is occurring. 12.11.3.2.2.2 Infectious Acute Chagas disease, caused by infection with Trypanosoma cruzi , has been reported to cause decreases in red cell mass in humans and monkeys ( de Titto and Araujo, 1988 , Rosner et al., 1988 , Seah et al., 1974 ). In experimentally infected Cebus paella monkeys, the acute phase of Chagas disease was reported to cause normocytic, normochromic anemia ( Rosner et al., 1988 ), typical of a nonregenerative anemia. Experimentally infected mice demonstrated bone marrow suppression with decreases in red cell mass as well as decreases in leukocyte and platelet counts ( Marcondes et al., 2000 ). Although rarely encountered in nonclinical toxicology studies, monkeys held in the southwestern United States may become infected with T . cruzi prior to distribution ( Magden et al., 2015 ). During parasitemia, trypomastigotes may be observed in peripheral blood smears. Viral infections may also cause decreases in red cell mass without concurrent increases in reticulocyte counts. Parvoviruses may cause decreases in red cell mass from direct infection of erythroid precursor resulting in decreased erythrocyte production, as well as decreased erythrocyte lifespans. Parvovirus may result in transient pure red cell aplasia (PRCA) in humans ( Van Horn et al., 1986 ). Cell-mediated suppression of erythropoiesis resulting in PRCA has also been reported with viral hepatitis ( Wilson et al., 1980 ) and Epstein-Barr virus infection ( Socinski et al., 1984 ). Although HIV infection can result in decreases in red cell mass through various mechanisms, direct infection of erythroid precursors appears to contribute to suppressed erythropoiesis ( Evans and Scadden, 2000 ). In cats, a membrane protein of feline leukemia virus has been associated with decreased growth of CFU-E ( Wellman et al., 1984 ). Flavivirus infection, such as dengue, may also result in decreases in red cell mass and reticulocyte counts through bone marrow suppression ( La Russa and Innis, 1995 ). 12.11.3.2.2.3 Chronic disease Anemia of chronic disease (ACD) is a relatively common cause of anemia, and anemia associated with inflammatory disease is included in ACD. The decreases in red cell mass observed with ACD are generally mild, and are generally normocytic, normochromic, indicating no changes in MCV or MCHC, respectively. ACD may occur through shortening of erythrocyte lifespans, alterations in iron metabolism, a blunted response of erythroid precursors to EPO, and decreased EPO production. Altered erythrocyte lifespans in patients with ACD may be related to increased macrophagic clearance of erythrocytes from circulation through unknown mechanisms ( Ganz, 2016 ). This type of mechanism has been associated with several chronic infections, including tuberculosis and endocarditis ( Weiss, 2002 ). More commonly, ACD is associated with impaired iron mobilization with low iron concentrations in serum or plasma despite adequate iron stores ( Means, 2000 ). Impaired mobilization of iron results from IL-6 induction of hepcidin that results in sequestration of iron in macrophages and decreased intestinal iron update ( Ganz, 2003 ), IL-1 stimulation of increased synthesis of ferritin which may bind to iron and impair delivery of iron to erythroid precursors ( Rogers et al., 1994 ), and with decreased expression and impaired internalization of the transferrin receptor ( Means, 2000 ). ACD from impaired iron metabolism is associated with numerous inflammatory, infectious, and even neoplastic conditions. ACD may also cause altered EPO responsiveness or decreased EPO production. Decreased responsiveness of erythroid precursors to EPO is cytokine-mediated, and has been associated with increases in TNFα, IL-1, and interferons ( Johnson et al., 1989 , Johnson et al., 1990 , Raefsky et al., 1985 ) that may commonly be associated with inflammatory conditions. Decreased EPO production may also be cytokine-mediated, and has been reported with increases in TGFβ, TNFα, and IL-1 ( Faquin et al., 1992 , Jelkmann et al., 1992 ). However, chronic renal disease may also result directly in impaired EPO production and decreased production of erythrocytes ( Sato and Yanagita, 2013 ). 12.11.3.2.2.4 Immune-mediated Immune-mediated destruction of erythroid precursors in the bone marrow results in decreases in red cell mass with concurrent decreases in reticulocyte counts. The immune-mediated conditions discussed here may represent a spectrum of disease associated with immune destruction of various stages of erythroid precursors rather than unrelated entities. 12.11.3.2.2.4.1 Autoimmune hemolytic anemia with decreases in reticulocyte counts Autoimmune hemolytic anemia with antibodies that target antigens on mid- to late-stage erythroid precursors ranging from rubricytes to metarubricytes results in AIHA with a decrease in reticulocyte count, which may also be called immune-mediated nonregenerative anemia or precursor-targeted immune-mediated anemia (PIMA). AIHA with reticulocytopenia is generally a normocytic, normochromic anemia. Bone marrow examination may reveal erythroid hyperplasia or maturation arrest ( Weiss, 2008 ) with pyramidal expansion of erythroid precursors at stages earlier than the targeted stage, indicative of ineffective erythropoiesis. This may be less apparent with autoantibodies that recognize more immature stages of erythroid precursors. Bone marrow evaluation may also reveal rubriphagocytosis, or erythroid precursors phagocytized by macrophages. The stage of phagocytized precursor depends on the stage or stages expressing the targeted antigen. 12.11.3.2.2.4.2 Pure red cell aplasia In patients affected by PRCA, there are marked decreases in reticulocyte counts along with variable decreases in red cell mass. Bone marrow examination typically reveals an absence of erythroid precursors (erythroid aplasia) or low numbers of the earliest stages of erythroid precursors (erythroid hypoplasia) ( Young, 2016 ). PRCA in people may be caused by antibodies that bind antigens on the earliest erythroid precursors or even antibodies that bind EPO and prevent EPO-dependent erythropoiesis, but it has also been attributed to clonal T-cell disorders ( Stockham and Scott, 2008b ). PRCA in dogs has been associated with IgG that inhibit erythropoiesis ( Weiss, 1986 ). PRCA may also be caused by inherited genetic defect in people. Inherited PRCA in people is called Diamond-Blackfan anemia, and often has an autosomal dominant inheritance pattern with defects in genes encoding ribosomal proteins ( Young, 2016 ). Macrocytosis, or increased numbers of large erythrocytes with increases in MCV, may be observed and is consistent with impaired EPO-dependent erythropoiesis ( Young, 2016 , Ohene-Abuakwa et al., 2005 ). 12.11.3.2.2.4.3 Aplastic anemia Aplastic anemia is a condition associated with decreases in all cellular blood components (pancytopenia), including decreases in red cell mass with concurrent decreases in reticulocyte counts. Upon examination, the bone marrow classically had severe hypocellularity of hematopoietic cells or an absence of hematopoietic precursors the marrow cavities filled by mostly adipocytes and some stromal elements. Aplastic anemia is thought to be most commonly immune-mediated ( Young et al., 2006 ), and may be frequently associated with cytotoxic T-cells that become autoreactive ( Segel and Lichtman, 2016 ). However, there are also cases of inherited aplastic anemia, most commonly Fanconi anemia associated with genetic mutations that impair DNA repair resulting in pancytopenia developing around 5–10 years of age in people ( Segel and Lichtman, 2016 ). A form of aplastic anemia associated with bone marrow depletion or hypocellularity of hematopoietic tissue and gelatinous transformation of marrow cavity fat has been reported with anorexia nervosa in people ( Abella et al., 2002 ) and with severe food restriction in rats ( Moriyama et al., 2008 ). 12.11.3.2.2.5 Nutritional deficiencies In addition to aplastic anemia associated with anorexia nervosa and severe food restriction, other nutritional deficiencies have been associated with ineffective erythropoiesis leading to decreases in red cell mass with decreases in reticulocyte counts. Iron deficiency and deficiencies of the B vitamins folate and cobalamin are examples of these nutritional deficiencies. Chronic iron deficiency results in impaired hematopoiesis due to the inability to synthesize sufficient hemoglobin, which may lead to a decrease in reticulocyte production. Deficiencies in folate and cobalamin also cause ineffective erythropoiesis due to defects in DNA synthesis, as discussed with folate and cobalamin deficiencies as a cause of deceases in neutrophil counts. In people, folate and cobalamin deficiencies result in megaloblastic anemia, characterized by larger than normal erythroid precursors in the bone marrow that have more cytoplasm with lower nuclear to cytoplasmic ratios than in normal erythroid precursors and asynchronous cytoplasmic and nuclear maturation ( Green, 2016 ). Megaloblastic erythrocytes may also be observed in circulation, and basophilic stippling or Howell-Jolly bodies may also be observed ( Green, 2016 ). In people, anemia attributable to a deficiency in cobalamin (vitamin B 12 ) may also be called pernicious anemia. In dogs and cats, megaloblastic erythroid cells may be observed in the bone marrow but may not be observed in blood ( Stockham and Scott, 2008b ). 12.11.3.2.2.6 Endocrinopathy Several endocrinopathies have also been associated with decreases in red cell mass with decreases in reticulocyte counts, including hypothyroidism, hypoadrenocorticism, and hyperestrogenism. In cases of hypothyroidism, several mechanisms may be contributing to the decreases in red cell mass. Decreased folate or cobalamin concentrations secondary to the hypothyroidism leading to ineffective erythropoiesis, decreased tissue oxygen demand leading to decreased EPO and lower baseline red cell mass, and ACD may contribute to the mild decreases in red cell mass observed with hypothyroidism ( Ottesen et al., 1995 , Hines et al., 1968 , Stockham and Scott, 2008b , Mehmet et al., 2012 ). Mild decreases in red cell mass without apparent changes in reticulocyte counts have been associated with hypoadrenocorticism. This may be due to a decrease in glucocorticoids, and the loss of the apparent proerythropoietic stimulation of glucocorticoids ( Stockham and Scott, 2008b ). Hyperestrogenism, which occurs with some ovarian or testicular neoplasms, may result in bone marrow toxicity and suppression of erythropoiesis, particularly in dogs ( Sontas et al., 2009 ). 12.11.3.2.2.7 Neoplasia Neoplasia may result in suppressed erythropoiesis. This may be due to neoplasia-related inflammation and cytokine release leading to ACD. However, granulocytic leukemia or lymphoproliferative neoplasia involving the bone marrow may result in crowding or effacement of the bone marrow cavities with impaired erythropoiesis that results in decreases in red cell mass with concurrent decreases in reticulocyte counts. Hematopoietic neoplasia involving the erythroid lineage usually results in atypical erythrocyte production that can lead to decreases in red cell mass and reticulocyte counts; however, nucleated erythrocytes with evidence of dysplasia may be observed in blood. Similar to hematopoietic neoplasms that efface the bone marrow, metastatic neoplasia, often carcinomas, may also cause myelophthisis and result in decreased erythropoiesis. 12.11.3.2.2.8 Xenobiotic-induced There are many xenobiotics that can cause decreases in red cell mass with concurrent decreases in reticulocyte counts. Bone marrow suppression that affects the erythroid lineage is commonly observed with chemotherapeutics in general. For example, agents that are directly cytotoxic to hematopoietic precursors, that inhibit mitotic spindle formation, and antimetabolites that alter folate metabolism may all result in suppression of erythropoiesis. However, development of parvovirus-induced PRCA has been reported as a consequence of chemotherapeutic administration ( Song et al., 2002 , Rao et al., 1994 ). PRCA has occasionally been linked to xenobiotic treatment. A wide variety of xenobiotics from many different classes have been reported to cause PRCA. Examples of xenobiotics reportedly associated with PCRA include sulfonamides, allopurinol, procainamide, gold-containing compounds, rifampin, and chloroquine ( Young, 2016 , Mintzer et al., 2009 ). However, causality is often difficult to prove, and most associations are limited to low numbers of case reports. One study evaluated reports of PRCA associated with administration of 30 different xenobiotics, but causality was only attributed to treatment with azathioprine, isoniazid, and phenytoin ( Thompson and Gales, 1996 ). PRCA due to the development of anti-EPO antibodies may follow the administration of recombinant EPO in humans ( Casadevall et al., 2002 ) and EPO gene therapy in monkeys ( Gao et al., 2004 ). Administration of recombinant EPO to dogs has also led to the development of anti-EPO antibodies and PRCA ( Randolph et al., 2004 ). Aplastic anemia has also been linked to administration of xenobiotics. Classically, chloramphenicol is reported to sporadically cause aplastic anemia ( Segel and Lichtman, 2016 ). However, antithyroid compounds, sulfonamides including trimethoprim sulfamethoxazole, beta-lactams, the diuretic furosemide, gold-containing compounds, penicillamine, and anticonvulsants including carbamazepine and phenacetin have all been reported in association with aplastic anemia ( Mintzer et al., 2009 , Kaufman et al., 1996 ). Aplastic anemia has also been attributed to environmental or occupational exposure to benzene ( Smith, 1996 ). In a case of aplastic anemia in a dog, griseofulvin administration was suspected to be the cause of the aplastic anemia ( Brazzell and Weiss, 2006 ). Decreases in red cell mass with concurrent decreases in reticulocyte counts have occurred with prolonged or repeated high dose administration of G-CSF or GM-CSF-based xenobiotics in nonclinical toxicology studies, particularly in rodents. Impaired erythropoiesis in these cases occurs due to the massive expansion of myeloid precursors within the bone marrow. Extreme myeloid hyperplasia with continued stimulation results in overcrowding of the marrow cavities with less physical space available for erythroid production. 12.11.3.1 Increases in Red Cell Mass (Erythrocytosis) 12.11.3.1.1 Relative increases in red cell mass Relative increases in red cell mass, or hemoconcentration, most commonly occur due to dehydration and splenic contraction. In contrast to the absolute or "true" increases in red cell mass that result from proliferation of erythroid precursors in the bone marrow and/or spleen, relative increases are transient and consist of changes to total blood volume or shifting or noncirculating erythrocytes into circulation resulting in increases in red cell mass, which can rapidly resolve. 12.11.3.1.1.1 Dehydration Dehydration is a relatively common cause of secondary increases in red cell mass. Dehydration results in depletion of the water content of blood, and a relative increase in the other blood components, including cells (hemoconcentration). Due to the high number of erythrocytes present, increases in red cell mass are detectable, whereas increases in leukocyte subtypes are often not observed. These increases in red cell mass are usually observed in conjunction with increases in urea nitrogen and/or creatinine (prerenal azotemia) with concurrent decreases in urine volume and increases in urine specific gravity, as well as proportional increases in albumin and globulin. If evaluating plasma, fibrinogen may also be increased. In nonclinical toxicology studies in rodents, decreased food consumption is often associated with concurrent decreases in water intake, resulting in increases in red cell mass from subclinical or clinical dehydration. Resolution of these secondary increases in red cell mass will occur with adequate rehydration. 12.11.3.1.1.2 Catecholamine-induced Increases in circulating catecholamine levels in response to fright, excitement, or acute stress can result in increases in red cell mass due to splenic contraction. Noncirculating erythrocytes stored within the red pulp of the spleen are expelled, resulting in increased circulating red cell mass. Such increases in red cell mass are transient and resolve as splenic relaxation occurs following a decline in circulating catecholamine levels. Catecholamine-induced splenic contraction-associated relative increases in red cell mass are most commonly observed at pretest collections in nonclinical toxicology studies utilizing nonhuman primates, dogs, or cats, particularly the first pretest collection if multiple collections are performed. It is typically not observed at subsequent collections as the animal becomes acclimated to the housing, handling/restraint, phlebotomy, and other study-related procedures. 12.11.3.1.1.3 Xenobiotic-induced Xenobiotic-related causes of relative increases in red cell mass are relatively uncommon, with the exception of dehydration associated with decreased food consumption in rodents utilized in nonclinical toxicology studies, as described earlier. Diuretics, such as furosemide or spironolactone, which result in increased elimination of water into urine, may result in hemoconcentration due to dehydration ( Mintzer et al., 2009 ). 12.11.3.1.2 Secondary increases in red cell mass Secondary increases in red cell mass are dependent on stimulating factors and are not autonomous, in contrast to primary increases in red cell mass. These secondary increases in red cell mass are most commonly associated with increases in EPO concentrations due to hypoxia, and are therefore considered appropriate. However, hypoxia-independent (inappropriate) mechanisms may also cause secondary increases in red cell mass and are also described later. 12.11.3.1.2.1 Hypoxia-dependent (appropriate) Increases in EPO production occur in response to hypoxia primarily from the kidney, although there is also some evidence that hypoxia may also stimulate production of EPO by the liver ( Rankin et al., 2007 ). Under normal oxygenation states, hypoxia-inducible factor (HIF) subunits are polyubiquitinated by a von Hipple-Lindau (VHL) tumor suppressor E3 ligase complex, which results in proteosomal degradation of HIF ( Jaakkola et al., 2001 ). Binding of the VHL E3 ligase complex to HIF requires hydroxylation of a proline residue in the VHL protein, a process that requires both oxygen and iron ( Ivan et al., 2001 ). In hypoxic states, HIF is not ubiquitinated and HIF subunits translocate to the nucleus, forming a transcription factor for genes, including the erythropoietin gene. Increases in circulating EPO concentrations as a result of hypoxia cause an appropriate increase in erythropoiesis and subsequent increase in red cell mass, which should improve delivery of oxygen to tissues. Sustained tissue hypoxia can result from high altitude residence, cardiac disease causing poor tissue or lung perfusion, or prolonged or chronic pulmonary diseases that impair oxygenation, such as COPD secondary to chronic smoking or obstructive sleep apnea in humans ( Prchal, 2016 , Stockham and Scott, 2008b ). Other conditions that may cause hypoxia-induced increases in red cell mass include mutations leading to hemoglobin with high affinity for oxygen, carboxyhemoglobin formation with heavy smoking, and erythrocyte enzyme deficiencies leading to methemoglobinemia, such as cytochrome b 5 reductase deficiency may also result in hypoxia-induced increases in red cell mass ( Prchal, 2016 ). Erythrocyte enzyme deficiencies resulting in methemoglobinemia have been reported in veterinary species ( Stockham and Scott, 2008b ), but hemoglobinopathies, conditions characterized by abnormal hemoglobin, have not yet been reported in domestic animals ( Randolph et al., 2010 ). 12.11.3.1.2.2 Hypoxia-independent (inappropriate) Secondary but hypoxia-independent increases in red cell mass are associated with increases in circulating EPO levels. However, increased EPO in these cases are attributable to autonomous production of EPO rather than hypoxia response. Reported associations include renal diseases, renal or nonrenal neoplasms, or rare dysfunctions of the oxygen sensing pathway. Nonneoplasic renal diseases associated with increased EPO production include hydronephrosis, renal cysts, and polycystic renal disease ( Prchal, 2016 ). These renal diseases may be associated with local tissue hypoxia ( Randolph et al., 2010 ), but are not associated with systemic hypoxia. Similarly, increases in red cell mass may be observed in humans following renal transplantation ( Prchal, 2016 ). Autonomous production of EPO by neoplasms has been associated with both benign and malignant tumors. Renal adenoma, renal carcinoma, and sarcoma of the kidney have been reported to cause increases in red cell mass ( Ways et al., 1961 ). Renal lymphoma has also been associated with increases in red cell mass ( Durno et al., 2011 ). Nonrenal tumors with inappropriate EPO production include hepatoma, hamartoma of the liver, leiomyosarcoma, schwannoma, and pheochromocytoma ( Stockham and Scott, 2008b , LevGur and Levie, 1995 , Muta et al., 1994 , Shulkin et al., 1987 , Josephs et al., 1961 ). VHL syndrome, which follows an autosomal dominant pattern of inheritance, may predispose affected people to developing renal or nonrenal neoplasms that can autonomously produce EPO ( Prchal, 2016 ). There are also rare inherited conditions that cause defects in oxygen sensing pathways described in humans. These include Chuvash polycythemia, which follows an autosomal recessive inheritance pattern, and EGLN1 gene mutations, which cause a deficiency in proline hydroxylase ( Prchal, 2016 ). High cobalt concentrations may also inhibit the oxygen sensing pathway by preventing binding of the VHL E3 ligase to HIF ( Yuan et al., 2003 , Schuster et al., 1989 ). 12.11.3.1.2.3 Endocrinopathies Increases in red cell mass associated with endocrinopathies are generally mild and do not result in overt clinical signs. Hyperthyroidism causes a sustained increase in tissue demand for oxygen, leading to hypoxia, increased EPO production, and consequent increases in red cell mass ( Stockham and Scott, 2008b ). Acromegaly, caused by an increase in growth hormone concentrations, has also been associated with increases in red cell mass, particularly in cats ( Randolph et al., 2010 ). Hyperadrenocorticism or adrenal neoplasms that produce androgens or aldosterone may also be associated with increases in red cell mass ( Prchal, 2016 , Ghio et al., 1981 , Mann et al., 1967 ). 12.11.3.1.2.4 Xenobiotic-induced Xenobiotic-induced increases in red cell mass are uncommon. Administration of recombinant erythropoietin and anabolic steroids has been reported to cause increases in red cell mass ( Mintzer et al., 2009 ). For example, increases in red cell mass have been associated with testosterone administration ( Gardner et al., 1968 ). Theoretically, excess administration of thyroid hormones could also cause increases in red cell mass. 12.11.3.1.3 Primary increases in red cell mass Primary, or erythropoietin-independent, increases in red cell mass are associated with myelodysplastic conditions with autonomous production of erythrocytes. Due to the autonomous nature of the proliferations, primary increases in red cell mass are also termed inappropriate as they are not dependent on EPO stimulation. Causes of the more common secondary increases in red cell mass should be excluded prior to the diagnosis of a primary increase in red cell mass. Measurement of EPO concentrations may be useful clinically in people, but assays that quantify EPO are not readily available for most veterinary species. Primary increases in red cell mass are uncommon, and are typically not observed in common laboratory species during nonclinical toxicology studies. 12.11.3.1.3.1 Polycythemia vera Polycythemia vera is categorized as a chronic myeloproliferative disorder. Neoplastic transformation, from an acquired somatic mutation ( Prchal, 2016 ), of a hematopoietic progenitor cell results in clonal and autonomous expansion of hematopoietic cells, including erythrocytes. Eventually the clonal expansion is sufficient to suppress normal hematopoiesis ( Prchal and Prchal, 2016 ). Increases in red cell mass are the prototypical findings, but concurrent increases in leukocyte and platelet counts that arise from the neoplastic clone are often also be observed in people ( Pearson, 2001 ), although these findings are typically not observed in dogs or cats ( Randolph et al., 2010 ). The most common causative somatic mutation of polycythemia vera in humans is a mutation of JAK2, a kinase that plays a role in intracellular proliferative signaling ( Prchal, 2016 ). However, forms of polycythemia vera or "idiopathic erythrocytosis" without JAK2 mutations have been observed and associated with mutations in lymphocyte-specific adaptor protein (LNK) that inhibits JAK2 phosphorylation ( Lasho et al., 2010 ). Serum EPO concentrations are expected to be low in patients with polycythemia vera. In domestic dogs and cats, middle-aged female dogs and male cats tend to be most commonly affected ( Randolph et al., 2010 ). 12.11.3.1.3.2 Primary familial and congenital polycythemia Similar to polycythemia vera, primary familial and congenital polycythemia (PFCP) is also caused by autonomous erythroid proliferation despite low serum EPO. However, PFCP is associated with nonclonal erythroid proliferation from an inherited mutation that has an autosomal dominant pattern of inheritance ( Prchal et al., 1985 ). Identified mutations definitively associated with PFCP result in the truncation of the EPO receptor with a loss of the negative regulatory domain, causing constitutive activity of the signaling pathway promoting erythrocyte proliferation ( Prchal and Prchal, 2016 ). 12.11.3.1.1 Relative increases in red cell mass Relative increases in red cell mass, or hemoconcentration, most commonly occur due to dehydration and splenic contraction. In contrast to the absolute or "true" increases in red cell mass that result from proliferation of erythroid precursors in the bone marrow and/or spleen, relative increases are transient and consist of changes to total blood volume or shifting or noncirculating erythrocytes into circulation resulting in increases in red cell mass, which can rapidly resolve. 12.11.3.1.1.1 Dehydration Dehydration is a relatively common cause of secondary increases in red cell mass. Dehydration results in depletion of the water content of blood, and a relative increase in the other blood components, including cells (hemoconcentration). Due to the high number of erythrocytes present, increases in red cell mass are detectable, whereas increases in leukocyte subtypes are often not observed. These increases in red cell mass are usually observed in conjunction with increases in urea nitrogen and/or creatinine (prerenal azotemia) with concurrent decreases in urine volume and increases in urine specific gravity, as well as proportional increases in albumin and globulin. If evaluating plasma, fibrinogen may also be increased. In nonclinical toxicology studies in rodents, decreased food consumption is often associated with concurrent decreases in water intake, resulting in increases in red cell mass from subclinical or clinical dehydration. Resolution of these secondary increases in red cell mass will occur with adequate rehydration. 12.11.3.1.1.2 Catecholamine-induced Increases in circulating catecholamine levels in response to fright, excitement, or acute stress can result in increases in red cell mass due to splenic contraction. Noncirculating erythrocytes stored within the red pulp of the spleen are expelled, resulting in increased circulating red cell mass. Such increases in red cell mass are transient and resolve as splenic relaxation occurs following a decline in circulating catecholamine levels. Catecholamine-induced splenic contraction-associated relative increases in red cell mass are most commonly observed at pretest collections in nonclinical toxicology studies utilizing nonhuman primates, dogs, or cats, particularly the first pretest collection if multiple collections are performed. It is typically not observed at subsequent collections as the animal becomes acclimated to the housing, handling/restraint, phlebotomy, and other study-related procedures. 12.11.3.1.1.3 Xenobiotic-induced Xenobiotic-related causes of relative increases in red cell mass are relatively uncommon, with the exception of dehydration associated with decreased food consumption in rodents utilized in nonclinical toxicology studies, as described earlier. Diuretics, such as furosemide or spironolactone, which result in increased elimination of water into urine, may result in hemoconcentration due to dehydration ( Mintzer et al., 2009 ). 12.11.3.1.1.1 Dehydration Dehydration is a relatively common cause of secondary increases in red cell mass. Dehydration results in depletion of the water content of blood, and a relative increase in the other blood components, including cells (hemoconcentration). Due to the high number of erythrocytes present, increases in red cell mass are detectable, whereas increases in leukocyte subtypes are often not observed. These increases in red cell mass are usually observed in conjunction with increases in urea nitrogen and/or creatinine (prerenal azotemia) with concurrent decreases in urine volume and increases in urine specific gravity, as well as proportional increases in albumin and globulin. If evaluating plasma, fibrinogen may also be increased. In nonclinical toxicology studies in rodents, decreased food consumption is often associated with concurrent decreases in water intake, resulting in increases in red cell mass from subclinical or clinical dehydration. Resolution of these secondary increases in red cell mass will occur with adequate rehydration. 12.11.3.1.1.2 Catecholamine-induced Increases in circulating catecholamine levels in response to fright, excitement, or acute stress can result in increases in red cell mass due to splenic contraction. Noncirculating erythrocytes stored within the red pulp of the spleen are expelled, resulting in increased circulating red cell mass. Such increases in red cell mass are transient and resolve as splenic relaxation occurs following a decline in circulating catecholamine levels. Catecholamine-induced splenic contraction-associated relative increases in red cell mass are most commonly observed at pretest collections in nonclinical toxicology studies utilizing nonhuman primates, dogs, or cats, particularly the first pretest collection if multiple collections are performed. It is typically not observed at subsequent collections as the animal becomes acclimated to the housing, handling/restraint, phlebotomy, and other study-related procedures. 12.11.3.1.1.3 Xenobiotic-induced Xenobiotic-related causes of relative increases in red cell mass are relatively uncommon, with the exception of dehydration associated with decreased food consumption in rodents utilized in nonclinical toxicology studies, as described earlier. Diuretics, such as furosemide or spironolactone, which result in increased elimination of water into urine, may result in hemoconcentration due to dehydration ( Mintzer et al., 2009 ). 12.11.3.1.2 Secondary increases in red cell mass Secondary increases in red cell mass are dependent on stimulating factors and are not autonomous, in contrast to primary increases in red cell mass. These secondary increases in red cell mass are most commonly associated with increases in EPO concentrations due to hypoxia, and are therefore considered appropriate. However, hypoxia-independent (inappropriate) mechanisms may also cause secondary increases in red cell mass and are also described later. 12.11.3.1.2.1 Hypoxia-dependent (appropriate) Increases in EPO production occur in response to hypoxia primarily from the kidney, although there is also some evidence that hypoxia may also stimulate production of EPO by the liver ( Rankin et al., 2007 ). Under normal oxygenation states, hypoxia-inducible factor (HIF) subunits are polyubiquitinated by a von Hipple-Lindau (VHL) tumor suppressor E3 ligase complex, which results in proteosomal degradation of HIF ( Jaakkola et al., 2001 ). Binding of the VHL E3 ligase complex to HIF requires hydroxylation of a proline residue in the VHL protein, a process that requires both oxygen and iron ( Ivan et al., 2001 ). In hypoxic states, HIF is not ubiquitinated and HIF subunits translocate to the nucleus, forming a transcription factor for genes, including the erythropoietin gene. Increases in circulating EPO concentrations as a result of hypoxia cause an appropriate increase in erythropoiesis and subsequent increase in red cell mass, which should improve delivery of oxygen to tissues. Sustained tissue hypoxia can result from high altitude residence, cardiac disease causing poor tissue or lung perfusion, or prolonged or chronic pulmonary diseases that impair oxygenation, such as COPD secondary to chronic smoking or obstructive sleep apnea in humans ( Prchal, 2016 , Stockham and Scott, 2008b ). Other conditions that may cause hypoxia-induced increases in red cell mass include mutations leading to hemoglobin with high affinity for oxygen, carboxyhemoglobin formation with heavy smoking, and erythrocyte enzyme deficiencies leading to methemoglobinemia, such as cytochrome b 5 reductase deficiency may also result in hypoxia-induced increases in red cell mass ( Prchal, 2016 ). Erythrocyte enzyme deficiencies resulting in methemoglobinemia have been reported in veterinary species ( Stockham and Scott, 2008b ), but hemoglobinopathies, conditions characterized by abnormal hemoglobin, have not yet been reported in domestic animals ( Randolph et al., 2010 ). 12.11.3.1.2.2 Hypoxia-independent (inappropriate) Secondary but hypoxia-independent increases in red cell mass are associated with increases in circulating EPO levels. However, increased EPO in these cases are attributable to autonomous production of EPO rather than hypoxia response. Reported associations include renal diseases, renal or nonrenal neoplasms, or rare dysfunctions of the oxygen sensing pathway. Nonneoplasic renal diseases associated with increased EPO production include hydronephrosis, renal cysts, and polycystic renal disease ( Prchal, 2016 ). These renal diseases may be associated with local tissue hypoxia ( Randolph et al., 2010 ), but are not associated with systemic hypoxia. Similarly, increases in red cell mass may be observed in humans following renal transplantation ( Prchal, 2016 ). Autonomous production of EPO by neoplasms has been associated with both benign and malignant tumors. Renal adenoma, renal carcinoma, and sarcoma of the kidney have been reported to cause increases in red cell mass ( Ways et al., 1961 ). Renal lymphoma has also been associated with increases in red cell mass ( Durno et al., 2011 ). Nonrenal tumors with inappropriate EPO production include hepatoma, hamartoma of the liver, leiomyosarcoma, schwannoma, and pheochromocytoma ( Stockham and Scott, 2008b , LevGur and Levie, 1995 , Muta et al., 1994 , Shulkin et al., 1987 , Josephs et al., 1961 ). VHL syndrome, which follows an autosomal dominant pattern of inheritance, may predispose affected people to developing renal or nonrenal neoplasms that can autonomously produce EPO ( Prchal, 2016 ). There are also rare inherited conditions that cause defects in oxygen sensing pathways described in humans. These include Chuvash polycythemia, which follows an autosomal recessive inheritance pattern, and EGLN1 gene mutations, which cause a deficiency in proline hydroxylase ( Prchal, 2016 ). High cobalt concentrations may also inhibit the oxygen sensing pathway by preventing binding of the VHL E3 ligase to HIF ( Yuan et al., 2003 , Schuster et al., 1989 ). 12.11.3.1.2.3 Endocrinopathies Increases in red cell mass associated with endocrinopathies are generally mild and do not result in overt clinical signs. Hyperthyroidism causes a sustained increase in tissue demand for oxygen, leading to hypoxia, increased EPO production, and consequent increases in red cell mass ( Stockham and Scott, 2008b ). Acromegaly, caused by an increase in growth hormone concentrations, has also been associated with increases in red cell mass, particularly in cats ( Randolph et al., 2010 ). Hyperadrenocorticism or adrenal neoplasms that produce androgens or aldosterone may also be associated with increases in red cell mass ( Prchal, 2016 , Ghio et al., 1981 , Mann et al., 1967 ). 12.11.3.1.2.4 Xenobiotic-induced Xenobiotic-induced increases in red cell mass are uncommon. Administration of recombinant erythropoietin and anabolic steroids has been reported to cause increases in red cell mass ( Mintzer et al., 2009 ). For example, increases in red cell mass have been associated with testosterone administration ( Gardner et al., 1968 ). Theoretically, excess administration of thyroid hormones could also cause increases in red cell mass. 12.11.3.1.2.1 Hypoxia-dependent (appropriate) Increases in EPO production occur in response to hypoxia primarily from the kidney, although there is also some evidence that hypoxia may also stimulate production of EPO by the liver ( Rankin et al., 2007 ). Under normal oxygenation states, hypoxia-inducible factor (HIF) subunits are polyubiquitinated by a von Hipple-Lindau (VHL) tumor suppressor E3 ligase complex, which results in proteosomal degradation of HIF ( Jaakkola et al., 2001 ). Binding of the VHL E3 ligase complex to HIF requires hydroxylation of a proline residue in the VHL protein, a process that requires both oxygen and iron ( Ivan et al., 2001 ). In hypoxic states, HIF is not ubiquitinated and HIF subunits translocate to the nucleus, forming a transcription factor for genes, including the erythropoietin gene. Increases in circulating EPO concentrations as a result of hypoxia cause an appropriate increase in erythropoiesis and subsequent increase in red cell mass, which should improve delivery of oxygen to tissues. Sustained tissue hypoxia can result from high altitude residence, cardiac disease causing poor tissue or lung perfusion, or prolonged or chronic pulmonary diseases that impair oxygenation, such as COPD secondary to chronic smoking or obstructive sleep apnea in humans ( Prchal, 2016 , Stockham and Scott, 2008b ). Other conditions that may cause hypoxia-induced increases in red cell mass include mutations leading to hemoglobin with high affinity for oxygen, carboxyhemoglobin formation with heavy smoking, and erythrocyte enzyme deficiencies leading to methemoglobinemia, such as cytochrome b 5 reductase deficiency may also result in hypoxia-induced increases in red cell mass ( Prchal, 2016 ). Erythrocyte enzyme deficiencies resulting in methemoglobinemia have been reported in veterinary species ( Stockham and Scott, 2008b ), but hemoglobinopathies, conditions characterized by abnormal hemoglobin, have not yet been reported in domestic animals ( Randolph et al., 2010 ). 12.11.3.1.2.2 Hypoxia-independent (inappropriate) Secondary but hypoxia-independent increases in red cell mass are associated with increases in circulating EPO levels. However, increased EPO in these cases are attributable to autonomous production of EPO rather than hypoxia response. Reported associations include renal diseases, renal or nonrenal neoplasms, or rare dysfunctions of the oxygen sensing pathway. Nonneoplasic renal diseases associated with increased EPO production include hydronephrosis, renal cysts, and polycystic renal disease ( Prchal, 2016 ). These renal diseases may be associated with local tissue hypoxia ( Randolph et al., 2010 ), but are not associated with systemic hypoxia. Similarly, increases in red cell mass may be observed in humans following renal transplantation ( Prchal, 2016 ). Autonomous production of EPO by neoplasms has been associated with both benign and malignant tumors. Renal adenoma, renal carcinoma, and sarcoma of the kidney have been reported to cause increases in red cell mass ( Ways et al., 1961 ). Renal lymphoma has also been associated with increases in red cell mass ( Durno et al., 2011 ). Nonrenal tumors with inappropriate EPO production include hepatoma, hamartoma of the liver, leiomyosarcoma, schwannoma, and pheochromocytoma ( Stockham and Scott, 2008b , LevGur and Levie, 1995 , Muta et al., 1994 , Shulkin et al., 1987 , Josephs et al., 1961 ). VHL syndrome, which follows an autosomal dominant pattern of inheritance, may predispose affected people to developing renal or nonrenal neoplasms that can autonomously produce EPO ( Prchal, 2016 ). There are also rare inherited conditions that cause defects in oxygen sensing pathways described in humans. These include Chuvash polycythemia, which follows an autosomal recessive inheritance pattern, and EGLN1 gene mutations, which cause a deficiency in proline hydroxylase ( Prchal, 2016 ). High cobalt concentrations may also inhibit the oxygen sensing pathway by preventing binding of the VHL E3 ligase to HIF ( Yuan et al., 2003 , Schuster et al., 1989 ). 12.11.3.1.2.3 Endocrinopathies Increases in red cell mass associated with endocrinopathies are generally mild and do not result in overt clinical signs. Hyperthyroidism causes a sustained increase in tissue demand for oxygen, leading to hypoxia, increased EPO production, and consequent increases in red cell mass ( Stockham and Scott, 2008b ). Acromegaly, caused by an increase in growth hormone concentrations, has also been associated with increases in red cell mass, particularly in cats ( Randolph et al., 2010 ). Hyperadrenocorticism or adrenal neoplasms that produce androgens or aldosterone may also be associated with increases in red cell mass ( Prchal, 2016 , Ghio et al., 1981 , Mann et al., 1967 ). 12.11.3.1.2.4 Xenobiotic-induced Xenobiotic-induced increases in red cell mass are uncommon. Administration of recombinant erythropoietin and anabolic steroids has been reported to cause increases in red cell mass ( Mintzer et al., 2009 ). For example, increases in red cell mass have been associated with testosterone administration ( Gardner et al., 1968 ). Theoretically, excess administration of thyroid hormones could also cause increases in red cell mass. 12.11.3.1.3 Primary increases in red cell mass Primary, or erythropoietin-independent, increases in red cell mass are associated with myelodysplastic conditions with autonomous production of erythrocytes. Due to the autonomous nature of the proliferations, primary increases in red cell mass are also termed inappropriate as they are not dependent on EPO stimulation. Causes of the more common secondary increases in red cell mass should be excluded prior to the diagnosis of a primary increase in red cell mass. Measurement of EPO concentrations may be useful clinically in people, but assays that quantify EPO are not readily available for most veterinary species. Primary increases in red cell mass are uncommon, and are typically not observed in common laboratory species during nonclinical toxicology studies. 12.11.3.1.3.1 Polycythemia vera Polycythemia vera is categorized as a chronic myeloproliferative disorder. Neoplastic transformation, from an acquired somatic mutation ( Prchal, 2016 ), of a hematopoietic progenitor cell results in clonal and autonomous expansion of hematopoietic cells, including erythrocytes. Eventually the clonal expansion is sufficient to suppress normal hematopoiesis ( Prchal and Prchal, 2016 ). Increases in red cell mass are the prototypical findings, but concurrent increases in leukocyte and platelet counts that arise from the neoplastic clone are often also be observed in people ( Pearson, 2001 ), although these findings are typically not observed in dogs or cats ( Randolph et al., 2010 ). The most common causative somatic mutation of polycythemia vera in humans is a mutation of JAK2, a kinase that plays a role in intracellular proliferative signaling ( Prchal, 2016 ). However, forms of polycythemia vera or "idiopathic erythrocytosis" without JAK2 mutations have been observed and associated with mutations in lymphocyte-specific adaptor protein (LNK) that inhibits JAK2 phosphorylation ( Lasho et al., 2010 ). Serum EPO concentrations are expected to be low in patients with polycythemia vera. In domestic dogs and cats, middle-aged female dogs and male cats tend to be most commonly affected ( Randolph et al., 2010 ). 12.11.3.1.3.2 Primary familial and congenital polycythemia Similar to polycythemia vera, primary familial and congenital polycythemia (PFCP) is also caused by autonomous erythroid proliferation despite low serum EPO. However, PFCP is associated with nonclonal erythroid proliferation from an inherited mutation that has an autosomal dominant pattern of inheritance ( Prchal et al., 1985 ). Identified mutations definitively associated with PFCP result in the truncation of the EPO receptor with a loss of the negative regulatory domain, causing constitutive activity of the signaling pathway promoting erythrocyte proliferation ( Prchal and Prchal, 2016 ). 12.11.3.1.3.1 Polycythemia vera Polycythemia vera is categorized as a chronic myeloproliferative disorder. Neoplastic transformation, from an acquired somatic mutation ( Prchal, 2016 ), of a hematopoietic progenitor cell results in clonal and autonomous expansion of hematopoietic cells, including erythrocytes. Eventually the clonal expansion is sufficient to suppress normal hematopoiesis ( Prchal and Prchal, 2016 ). Increases in red cell mass are the prototypical findings, but concurrent increases in leukocyte and platelet counts that arise from the neoplastic clone are often also be observed in people ( Pearson, 2001 ), although these findings are typically not observed in dogs or cats ( Randolph et al., 2010 ). The most common causative somatic mutation of polycythemia vera in humans is a mutation of JAK2, a kinase that plays a role in intracellular proliferative signaling ( Prchal, 2016 ). However, forms of polycythemia vera or "idiopathic erythrocytosis" without JAK2 mutations have been observed and associated with mutations in lymphocyte-specific adaptor protein (LNK) that inhibits JAK2 phosphorylation ( Lasho et al., 2010 ). Serum EPO concentrations are expected to be low in patients with polycythemia vera. In domestic dogs and cats, middle-aged female dogs and male cats tend to be most commonly affected ( Randolph et al., 2010 ). 12.11.3.1.3.2 Primary familial and congenital polycythemia Similar to polycythemia vera, primary familial and congenital polycythemia (PFCP) is also caused by autonomous erythroid proliferation despite low serum EPO. However, PFCP is associated with nonclonal erythroid proliferation from an inherited mutation that has an autosomal dominant pattern of inheritance ( Prchal et al., 1985 ). Identified mutations definitively associated with PFCP result in the truncation of the EPO receptor with a loss of the negative regulatory domain, causing constitutive activity of the signaling pathway promoting erythrocyte proliferation ( Prchal and Prchal, 2016 ). 12.11.3.2 Decreases in Red Cell Mass (Anemia) Decreases in red cell mass, or anemia, are a relatively common finding in humans and common laboratory species. Decreases in red cell mass are further categorized by concurrent changes in reticulocyte counts, which provide an indication of bone marrow responsiveness and may help to differentiate among possible mechanisms. Decreases in red cell mass with concurrent increases in reticulocyte counts indicate a regenerative erythroid bone marrow response, where normal or low reticulocyte counts may represent a preregenerative, suppressed, or ineffective erythroid response. 12.11.3.2.1 Decreases in red cell mass with increases in reticulocyte counts (regenerative anemia) Decreases in red cell mass with concurrent increases in reticulocyte counts (reticulocytosis) indicate a regenerative erythroid response by the bone marrow, or by extramedullary hematopoiesis in the spleen of rodents. An increase in reticulocyte count is typically first observed 3–4 days after an acute drop in red cell mass due to bone marrow erythrocyte production and transit time, and peak responses generally occur around 7–14 days depending on the species ( Stockham and Scott, 2008b ). The regenerative erythroid response is considered appropriate if the increases in reticulocyte counts reflect the magnitude of the decreases in red cell mass; in other words, a mild decrease in red cell mass is expected to result in a mild increase in reticulocyte count, while a moderate to marked decrease in red cell mass should have a concurrent moderate to marked increase in reticulocyte count. The regenerative erythroid response is considered inappropriate if there is an inconsistency between the magnitude of the decrease in red cell mass and the magnitude of the increase in reticulocyte count. For example, a marked decrease in red cell mass with only a mild increase in reticulocyte count a week after the insult would be considered an inappropriate regenerative erythroid response. Increases in blood reticulocyte counts may be associated with concurrent changes in mean corpuscular volume (MCV; an indicator of erythrocyte size) and mean corpuscular hemoglobin concentration (MCHC; an indicator of erythrocyte hemoglobin content), two red blood cell indices provided by most automated hematology analyzers. Due to the reticulocyte's increased volume relative to mature erythrocytes, increases in blood reticulocyte counts may cause increases in MCV (macrocytosis) and decreases in MCHC (hypochromasia). Regenerative anemias with increases in MCV and decreases in MCHC and/or CHCM may also be classified by these indices as macrocytic, hypochromic anemias. The decrease in MCHC does not necessarily reflect less hemoglobin content per cell, but is a consequence of reduced concentration of hemoglobin due to the larger cytoplasmic volume of reticulocytes. MCHC, which is a calculated endpoint, may be artificially increased when free plasma hemoglobin is present due to intravascular hemolysis. Some automated hematology analyzers also provide the corpuscular mean hemoglobin content (CHCM) which provides a mean of direct measurements of cellular hemoglobin concentration and is therefore resistant to interference from free plasma hemoglobin. Regenerative erythroid responses with increases in blood reticulocyte counts may be associated with several morphologic findings observed during blood smear evaluation. Most commonly, increases in polychromatophils (polychromasia) are observed with Wright-Giemsa or modified Wright stains. Polychromatophils are erythrocytes that stain blue–purple in color due to the combined effects of blue-staining RNA content typical of reticulocytes and pink-staining hemoglobin. As reticulocytes mature and lose RNA, a visual difference in staining cannot longer be detected between late reticulocytes and mature erythrocytes. However, staining of blood with a vital dye such as New Methylene Blue permits differentiation between aggregate and punctate-type reticulocytes. Polychromasia usually correlates well with increases in reticulocytes in most species, except cats ( Stockham and Scott, 2008b ). In cats, aggregate-type but not punctate-type reticulocytes correlate with polychromasia and are considered clinically relevant, and differentiation of these two with manual reticulocyte counts should be performed ( Harvey, 2012 , Stockham and Scott, 2008b ). Reticulocytes or erythrocytes with few small, punctate dark blue-gray inclusions may be observed during a regenerative erythroid response. These inclusions contain iron and may be called Pappenheimer bodies or siderotic inclusions. Due to the rapid production and release of erythrocytes during a regenerative erythroid response, there may also be increase in nucleated red blood cells or erythrocytes with Howell-Jolly bodies. Nucleated red blood cells are usually present in low numbers, but if 10 or more are observed per 100 leukocytes, the automated total leukocyte count will be falsely increased and should be corrected using published equations ( Stockham and Scott, 2008a ). In some species, particularly cows and sheep, erythrocytes with basophilic stippling may also be observed in circulation during a regenerative erythroid response. Hemolysis and blood loss are the two main categories of decreases in red cell mass with appropriate increases in reticulocyte counts. 12.11.3.2.1.1 Hemolysis Destruction of mature erythrocytes is called hemolysis. Hemolysis may occur either intravascularly or extravascularly. With intravascular hemolysis, erythrocyte destruction occurs within the blood and results in hemoglobinemia, or free hemoglobin within plasma. Ghost erythrocytes, or the remnant membranes of erythrocytes that no longer contain cytoplasm or hemoglobin, may be observed with intravascular hemolysis. Consequent hemoglobinuria, or free hemoglobin in the urine, is rare and only occurs in cases of massive intravascular hemolysis that overwhelm the normal pathways that clear free hemoglobin from the blood. In contrast, extravascular hemolysis does not occur within the blood, but rather occurs in the spleen, liver, or bone marrow, where resident macrophages phagocytose erythrocytes and destroy them intracellularly. Extravascular hemolysis does not result in free plasma hemoglobin or hemoglobinuria. Both types of hemolysis may be associated with increases in total bilirubin concentrations where unconjugated (indirect) bilirubin usually exceeds conjugated (direct) bilirubin, and may result in plasma or serum icterus (yellow discoloration) or bilirubinuria (bilirubin present in urine). However, not all cases of hemolysis are clearly either intravascular or extravascular, and both forms of hemolysis may contribute in some conditions. 12.11.3.2.1.1.1 Infectious There are numerous protozoal, bacterial, and viral diseases that can be associated with hemolysis. Mechanisms by which infectious agents cause erythrocyte destruction are varied, and may include direct infection of erythrocytes, elaboration of toxins such as hemolysin, or stimulation of an immune-mediated response against infected cells ( Berkowitz, 1991 ). Several examples of infectious agents that cause hemolytic anemia are discussed later. Direct infection of erythrocytes with protozoal Plasmodium species, the causative agent of malaria that is transmitted by mosquitoes, is a relatively common cause of hemolysis in humans, but may also be observed in nonhuman primates used in nonclinical toxicology studies. Humans are infected by one of five different Plasmodium species: P . falciparum , P . vivax , P . knowlesi , P . malariae , or P . ovale , although only P . falciparum and P . vivax are commonly associated with severe hemolysis ( Lichtman, 2016b ). Macaques are most commonly infected with P . inui or P . knowlesi , although the cynomolgus monkey appears to be more resistant to disease from these infections than the rhesus monkey ( Ameri, 2010 ). Infection with P . cynomolgi , P . fieldi , or P . fragile may also occur in macaques ( Magden et al., 2015 ). Although it is uncommon to include macaques infected with Plasmodium species during a nonclinical toxicity study due to current screening practices and pretest evaluations, rare animals with decreases in red cell mass and increases in reticulocyte counts and intraerythrocytic Plasmodium organisms have been observed. Rats and mice may be infected with Plasmodium berghei ( Holloway et al., 1995 , Sadun et al., 1965 ). Plasmodium berghei has a specific tropism for reticulocytes rather than mature erythrocytes Plasmodium species that infect humans ( Car et al., 2006 , Cromer et al., 2006 ). Concurrent increases in reticulocyte counts may occur in early stages or disease or with recrudescence of parasitemia and hemolysis. Hemolysis is associated with clearance of parasitized erythrocytes from circulation predominantly by splenic macrophages ( Lichtman, 2016b ), although accumulation of hemin, an iron-containing porphyrin, which can directly stimulate apoptotic erythrocyte death (eryptosis) ( Gatidis et al., 2009 ), oxidative damage to erythrocyte membranes ( Clark and Hunt, 1983 ), and increased osmotic fragility ( George et al., 1967 ) may all contribute to hemolysis. However, late-stage infections in humans and rodents have also been associated with inappropriate or decreased reticulocyte counts indicative of suppressed erythropoiesis despite decreases in red cell mass from hemolysis ( Lichtman, 2016b , Cromer et al., 2006 ). Babesia species are tick-borne protozoal organisms that directly infect erythrocytes in most species, including humans, nonhuman primates, dogs, and cats. Babesia species appear as intracellular oval to pyriform organisms. Babesia microti and Babesia divergens may infect humans in North America and Europe, respectively, and cause moderate hemolytic anemia from intraerythrocytic replication and subsequent erythrocyte lysis ( Lichtman, 2016b , Kjemtrup and Conrad, 2000 ). Babesia pitheci has been reported to infect both old and new world monkeys and cause anemia ( Magden et al., 2015 ). B . canis , a large babesial species, and B . gibsoni , a small babesial species, infect dogs, while cats may be infected by the small babesial organisms B . felis and B . cati ( Stockham and Scott, 2008b , Penzhorn et al., 2004 ). These organisms are generally not of concern in purpose-bred animals used in nonclinical toxicology studies. Bartonella bacilliformis in people and the hemotrophic mycoplasmas (hemoplasmas) in dogs and cats (formerly Haemobartonella species) and swine (formerly Eperythrozoon species) are organisms that parasitize erythrocytes, but these organisms remain extracellular in shallow depressions of the erythrocyte membrane. These organisms are typically round-, rod-, or ring-shaped and may be observed individually or in chains on erythrocyte surfaces. Hemolysis with these organisms may be immune-mediate and associated either with binding of antibodies to parasite antigens or antigens exposed on the erythrocyte secondary to parasite-induced membrane changes ( Stockham and Scott, 2008b ). Clostridium perfringens (formerly Clostridium welchii ) infection in humans is an example of a bacterial cause of hemolysis. During intestinal overgrowth or septicemia, C . perfringens type A elaborates an α toxin that has lecithinase C activity, resulting in membrane phospholipid breakdown and release of lysolethicins, which have potent hemolytic capabilities ( Lichtman, 2016b , Songer, 1996 ). C . perfringens α toxin release is usually associated with severe intravascular hemolysis with both hemoglobinemia and hemoglobinuria. However, in veterinary species, C . perfringens -related hemolysis is typically limited to ruminants and horses ( Stockham and Scott, 2008b ), and is unlikely to be observed in the common species used in nonclinical toxicology studies. Infection of humans with Mycoplasma pneumoniae has also been associated with hemolytic decreases in red cell mass, although most cases of M . pneumoniae infection are asymptomatic. Hemolysis with this organism is attributable to stimulation of autoimmune erythrocyte destruction with agglutination of erythrocytes ( Khan and Yassin, 2009 ). Several viral organisms in humans have also been reported to cause decreases in red cell mass due to hemolysis. Viral causes of hemolysis are commonly associated with autoimmune mechanisms, and include infection with Epstein-Barr virus ( Palanduz et al., 2002 ), hepatitis A, B, and C viruses ( Kanematsu et al., 1996 , Chao et al., 2001 ), cytomegalovirus ( Murray et al., 2001 ), and HIV ( Koduri et al., 2002 ), although HIV infection is also commonly associated with decreases in reticulocyte counts rather than the expected increases secondary to hemolysis, indicative of concurrent suppressed erythropoiesis ( Telen et al., 1990 ). 12.11.3.2.1.1.2 Oxidative Another major cause of decreases in red cell mass due to hemolysis is oxidative damage to erythrocytes. Under normal conditions, ferrous iron (Fe 2 + ) in hemoglobin binds to and dissociates from oxygen as it delivers oxygen from the lungs to the tissues. At times, this binding and dissociation results in the formation of ferric iron (Fe 3 + ) in hemoglobin (methemoglobin) as well as superoxide (O 2 − ). Superoxide is a free radical with potent oxidative capacity that may cause cellular damage. Cytochrome b 5 -reductase is an intraerythrocytic enzyme that converts methemoglobin back to hemoglobin. Superoxide dismutase converts superoxide to hydrogen peroxide (H 2 O 2 ), which also may produce oxidative damage to cells. Further metabolism of hydrogen peroxide by catalase or glutathione peroxidase protects cells from oxidative damage. These pathways are usually sufficient to address the normal low-level formation of methemoglobin and superoxide, but methemoglobin can increase and impair delivery of oxygen to tissues and superoxide can accumulate and cause oxidative damage if these pathways are overwhelmed or defective. Oxidative damage to erythrocytes may affect the lipid membranes, cytoskeleton, or hemoglobin. Peroxidation of internal membrane lipids or cytoskeletal components of erythrocytes results in the fusion of portions of the membrane with consequent shifting of the cytoplasm and hemoglobin to one side of the cells. Erythrocytes with this morphologic change are called eccentrocytes. Oxidative damage that causes the formation of eccentrocytes may result in hemolysis due to increased clearance of eccentrocytes by splenic macrophages due to trapping of rigid erythrocytes in splenic sinusoids or spontaneous rupture in blood due to the increased fragility of eccentrocytes ( Stockham and Scott, 2008b ). Oxidative damage to exposed cysteine sulfhydryl groups on hemoglobin results in hemoglobin denaturation and decreased solubility ( Bloom and Brandt, 2008 ). Denatured hemoglobin may then precipitate and aggregate within the erythrocyte, forming small, pale-staining round structures that bind to the erythrocyte membrane and tend to protrude from the surface of the erythrocyte. These aggregates of denatured hemoglobin are called Heinz bodies. Cats appear to be particularly sensitive to the formation of Heinz bodies because of an increased number of reactive sulfhydryl groups in hemoglobin relative to other species ( Christopher et al., 1990 ), and may be more rapidly observed on blood smear evaluation due to the nonsinusoidal architecture of the feline spleen that results in decreased clearance of Heinz bodies from circulation. Similar to eccentrocytes, erythrocytes with Heinz bodies may undergo hemolysis due to increased clearance by splenic macrophages following trapping in splenic sinusoids due to decreased erythrocyte deformability and spontaneous rupture due to increased fragility from membrane damage; immune-mediated clearance may also occur and is believed to result from binding of hemochromes to and subsequent redistribution of band 3, an erythrocyte membrane structural protein, which may then be recognized by autologous antibodies ( Winterbourn, 1990 ). There are many conditions that may cause oxidative damage and result in eccentrocytosis, Heinz body formation, or even both simultaneously. Diabetes mellitus may cause either morphologic change, and diabetic ketoacidosis appears to be associated with an increased susceptibility and incidence of oxidative erythrocyte damage ( Desnoyers, 2010 , Caldin et al., 2005 , Christopher et al., 1995 ). Inherited deficiencies in erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and flavin adenine dinucleotide (FAD) have also been associated with erythrocyte oxidative damage, eccentrocyte or Heinz body formation, and hemolysis or a predisposition for these events due to the loss of protective antioxidant pathways ( Chan et al., 1982 , Harvey, 2006 ). Lymphoma has also been associated with Heinz body formation in cats ( Christopher, 1989 ) and eccentrocytes formation in dogs ( Caldin et al., 2005 ). In dogs and cats, ingestion of Allium species, particularly onions, garlic, and Chinese chive, may cause erythrocyte oxidative damage with formation of eccentrocytes and/or Heinz bodies ( Caldin et al., 2005 , Yamato et al., 2005 , Robertson et al., 1998 ). Ingestion of zinc in dogs ( Bexfield et al., 2007 ) and exposure to skunk musk ( Fierro et al., 2013 ) have also been reported to cause hemolysis due to Heinz body formation. 12.11.3.2.1.1.3 Fragmentation Physical trauma to erythrocytes results in hemolysis due to erythrocyte fragmentation and lysis. Sometimes this type of hemolysis is referred to as microangiopathic hemolytic anemia. Morphologic changes to erythrocytes occur as a result of physical trauma. Schistocytes (also called schizocytes or erythrocyte fragments), keratocytes (also called helmet cells), prekeratocytes (also called blister cells), or even spherocytes or microspherocytes may be observed. Schistocytes are very small, usually irregularly shaped fragments that can break off erythrocytes when physical trauma occurs. Keratocytes have one to two variably sized projections or horns adjacent to a small flattened region of the erythrocyte surface, while prekeratocytes appear to be precursors that have small loops of erythrocyte cytoplasm extending from the surface and surrounding a small hole in the cell. Spherocytes and microspherocytes are spherical cells that appear smaller and have more intensely pink-staining cytoplasm than normal mature erythrocytes. Spherocytes and microspherocytes may be formed during physical trauma as fragments are broken off, resulting in less membrane surface area in the parent erythrocyte surrounding a similar volume (spherocytes) or smaller volume (microspherocytes) as the parent erythrocyte. The physical trauma to erythrocytes that causes fragmentation or microangiopathic hemolysis may result from consumptive coagulopathies, either local or disseminated (DIC), with fibrin or thrombus formation in the vasculature that impedes the passage of erythrocytes through the vessel lumen, creating both turbulence and physical obstruction of blood flow. Local coagulopathy or DIC may occur secondary to trauma, infections with sepsis, or neoplasia ( Baker and Moake, 2016 , Toh and Dennis, 2003 ). Microangiopathic hemolysis due to neoplasia is most commonly associated with malignant rather than benign neoplasms and with metastatic disease or neoplasic emboli rather than primary tumors, with the exception of primary vascular neoplasms ( Susano et al., 1994 , Kupers et al., 1975 , Lohrmann et al., 1973 ). Infectious agents may also lead to fragmentation of erythrocytes, and some Leptospirosis interrogans serovars associated with vasculitis ( Stockham and Scott, 2008b ), Brucella species infection ( Yaramis et al., 2001 ), and cutaneous anthrax ( Freedman et al., 2002 ) have been reported to cause microangiopathic hemolysis. In children, fragmentation hemolysis associated with thrombotic microangiopathy may occur with Shigella dysenteriae type 1 and some Escherichia coli infections ( Pisoni et al., 2001 ). Hemolysis from erythrocyte fragmentation may also occur with HIV infection ( Maslo et al., 1997 ). Decreases in red cell mass with increases in reticulocyte counts from erythrocyte fragmentation may also occur secondary to cardiac or other conditions that alter hemodynamics and increase turbulent blood flow. For example, subaortic stenosis ( Solanki and Sheikh, 1978 ), intraluminal aortic grafts ( Sayar et al., 2006 ), uncorrected cardiac valvular disease ( Marsh and Lewis, 1969 ), prosthetic valves ( Crexells et al., 1972 ), and hypertrophic obstructive cardiomyopathy ( Kubo et al., 2010 ) have all been reported to cause hemolysis from erythrocyte fragmentation. Increased turbulence associated with hypertension may also cause decreases in red cell mass from fragmentation, and has been associated with pulmonary hypertension ( Baker and Moake, 2016 ) and malignant systemic hypertension ( Capelli et al., 1966 ). 12.11.3.2.1.1.4 Immune-mediated Autoimmune hemolytic anemia (AIHA or AHA) described in humans or immune-mediated hemolytic anemia (IMHA) described in most common laboratory species is a cause of hemolysis, and may be primary or idiopathic, but may also be secondary to conditions such as infections as discussed previously. Primary or idiopathic AIHA/IMHA is discussed here. Primary AIHA has no underlying detectable cause and is an immune-mediated condition that produces antibodies targeting erythrocyte antigens. These antierythrocyte antibodies tend to be very specific for a single erythrocyte antigen in a given case ( Packman, 2016 ). These autoantibodies may be classified as warm antibodies, which are usually IgG, or cold antibodies, which are usually IgM ( Stockham and Scott, 2008b ). Immune-mediated AIHA may be associated with erythrocyte morphologic changes that include agglutination and spherocytes. Agglutination may be observed grossly as red speckling along the inside of the specimen tube as blood is gently moved within the tube. If agglutination is present, blood smears may have a "reverse smear" appearance with the densest region of the smear observed at the feathered edge rather than the edge where the drop of blood was initially placed. Microscopically agglutination appears as grape-like clusters of erythrocytes. Spherocytes are erythrocytes that are spherical instead of having the normal biconcave disc shape. While spherocytes appear smaller and stain more intensely pink that unaffected mature erythrocytes, they have the same volume as unaffected erythrocytes. Loss of erythrocyte membrane occurs when macrophages begin to phagocytize antibody-bound erythrocytes, leading to decreased erythrocyte surface area without an appreciable change in volume, forcing erythrocytes to form spheres. Hence, spherocytosis alone will not result in an altered MCV. Of the most common laboratory species, dogs tend to have the most pronounced central pallor of normal mature erythrocytes, making microscopic identification of spherocytes easiest in the dog. Hemolysis in AIHA is largely attributable to extravascular hemolysis due to phagocytosis of antibody-bound erythrocytes by tissue macrophages. Macrophages or monocytes containing phagocytized erythrocytes may be rarely observed in blood smears of laboratory species with immune-mediated hemolysis. However, antibody-mediated complement activation or increased fragility of spherocytes may result in direct intravascular lysis or rupture of erythrocytes ( Packman, 2016 ). Evaluation of patients for the presence of antierythrocyte antibodies may be performed using the direct antiglobulin test (DAT; also called the Coombs' test) or by flow cytometry. AIHA has rarely been observed in association with lymphoproliferative neoplasia, such as chronic lymphocytic leukemia. Antierythrocyte antibodies in chronic lymphocytic leukemia are predominantly IgG with few cases of IgM reported ( Mauro et al., 2000 ). IMHA may occasionally be observed following blood transfusion ( Garratty, 2004 ). This may occur in response to alloantigens, and would not technically be considered autoimmune ( Stockham and Scott, 2008b ). Posttransfusion immune-mediated hemolysis may also be observed when the host has autoantibodies that bind the donor erythrocytes and cause immune-mediated destruction. However, crossmatching of host and donor erythrocytes and plasma is able to prevent many cases with incompatible transfusion-related AIHA. AIHA and IMHA commonly have concurrent inflammatory increases in leukocyte subtype counts, characterized mainly by neutrophilia that may or may not have a left shift with cytoplasmic changes indicative of rapid neutropoiesis. 12.11.3.2.1.1.5 Inherited Some phenotypes of sickle cell disease are associated with hemolysis. The mechanism of hemolysis in sickle cell disease is likely multifactorial and not associated with a single pathogenesis. There is evidence for oxidative damage to erythrocytes ( Lachant et al., 1983 ), which may contribute to the hemolysis observed with sickle cell disease. However, hemoglobin polymerization leads to erythrocyte deformation and may lead to decreased flexibility of erythrocytes and veno-occlusive disorders ( Bookchin and Lew, 1996 ). Decreased flexibility or deformability of erythrocytes may contribute directly to increased cell fragility and rupture or promote clearance of deformed erythrocytes by splenic macrophages, while veno-occlusive disease has the potential to cause decreases in red cell mass through physical trauma and fragmentation. However, there is also evidence that in some severe cases of sickle cell disease there may be an increase in reticulocyte counts that are inappropriate for the magnitude of the decrease in red cell mass, suggesting a concurrent mechanism causing suppressed or ineffective erythropoiesis ( Wu et al., 2005 , Bookchin and Lew, 1996 ). Oxidative stress on erythroid precursors may also contribute to ineffective erythropoiesis in some severe cases of sickle cell disease ( Fibach and Rachmilewitz, 2008 ). Several metabolic defects of erythrocyte metabolism may also be associated with decreases in red cell mass and increases in reticulocyte counts. Deficiencies in erythrocyte pathways of glycolysis may result in decreased ATP concentrations that lead to erythrocyte membrane dysfunctions with shortened erythrocyte lifespan and occasionally hemolysis ( Stockham and Scott, 2008b ). Pyruvate kinase (PK) is the enzyme that catalyzes the last step in aerobic glycolysis. Deficiencies of PK that result in hemolysis have been reported in humans ( Baronciani and Beutler, 1993 ), dogs including beagles ( Harvey et al., 1977 , Giger et al., 1991 , Prasse et al., 1975 ), and a few breeds of cats ( Kohn and Fumi, 2008 ). Phosphofructokinase (PFK) catalyzes the rate-limiting step of the glycolysis pathway. Deficiencies in PFK have also been described in humans ( Etiemble et al., 1976 ) and dogs ( Giger et al., 1985 ). Respiratory alkalosis, which may be observed following intense exercise, is associated with acute hemolytic crises in patients with PFK deficiencies ( Giger et al., 1985 ). The association of inherited G6PD and FAD deficiencies with hemolysis resulting from oxidative damage is discussed earlier. In brief, G6PD and FAD play a role in the antioxidant pathways of erythrocytes. Deficiencies of G6PD and FAD may result in increased oxidative damage to erythrocytes and subsequent hemolysis. Collectively, the porphyrias are a group of enzymatic defects in the heme synthesis pathway. Porphyrias may be congenital or, more commonly, acquired. In these conditions, the accumulation of porphyrins, the precursors of heme, within erythrocytes leads to hemolysis. The mechanism of hemolysis may be related to lysis of erythrocytes following exposure to light (photolysis) in superficial vasculature, or by direct erythrocyte membrane damage due to the lipid soluble nature of porphyrins or following porphyrin absorption of ultraviolet light and excitation ( Phillips and Anderson, 2016 , Kaneko, 2008 ). 12.11.3.2.1.1.6 Neoplastic Various neoplastic conditions may be associated with hemolysis. Malignant metastatic neoplasms or primary vascular neoplasms may result in fragmentation hemolysis by physical trauma to erythrocytes, as previously discussed. However, neoplastic conditions may also rarely be associated with phagocytosis and destruction of erythrocytes, or hemophagocytic syndrome. Hemophagocytic syndromes have been associated with T-cell lymphoma ( Gonzalez et al., 1991 ), NK-cell leukemia ( Kobayashi et al., 1996 ), hemophagocytic histiocytic sarcoma ( Moore et al., 2006 ), and various hematological neoplasias ( Majluf-Cruz et al., 1998 ). 12.11.3.2.1.1.7 Xenobiotic-induced Many xenobiotics are capable of causing hemolysis, and may cause hemolysis through oxidative, fragmentation, or immune-mediated mechanisms. Examples of each are discussed here. Many of the agents that cause oxidative erythrocyte injury contain aromatic structures that can be metabolized, mostly commonly by cytochrome P 450, to free radicals ( Bradberry, 2003 , Edwards and Fuller, 1996 ), which overwhelm the normal protective antioxidant pathways of erythrocytes leading to both direct erythrocyte oxidative injury and oxidation of hemoglobin sulfhydryl groups resulting in methemoglobin formation. A few specific aromatic compounds that have been associated with free radical formation include dapsone, phenacetin, and anthracyclines such as doxorubicin ( Edwards and Fuller, 1996 , Coleman et al., 1991 , Handa and Sato, 1975 , Easley and Condon, 1974 ). Phenacetin has also been associated with the formation of Heinz bodies ( Boelsterli et al., 1983 ). In dogs and cats, acetaminophen (paracetamol) may be metabolized to a minor reactive metabolite that causes oxidative damage to erythrocytes resulting in hemolysis and the formation of Heinz bodies and/or eccentrocytes, although methemoglobinemia has also been observed in cats ( Desnoyers, 2010 , Wallace et al., 2002 , Mariani and Fulton, 2001 , Aronson and Drobatz, 1996 ). Xenobiotics that cause methemoglobinemia can also cause indirect oxidative damage through the peroxidation activity of methemoglobin itself ( Edwards and Fuller, 1996 ). In some cases, oxygenated hemoglobin may act as a peroxidase and cause the metabolism of a xenobiotic to a reactive compound that causes erythrocyte oxidative damage and conversion of oxyhemoglobin to methemoglobin. Examples of xenobiotics that cause oxidative damage through this mechanism are phenylhydrazine and primaquine ( Edwards and Fuller, 1996 ). Vitamin K administration in dogs can also cause oxidative erythrocyte damage through this mechanism ( Fernandez et al., 1984 ). Some chemical agents may cause oxidative damage by directly oxidizing hemoglobin sulfhydryl groups or through direct oxidation of erythrocyte cytoskeletal proteins. Arsine gas, predominantly an environmental toxin, appears to mediate its hemolytic effects through erythrocyte membrane oxidation ( Rael et al., 2000 ), although studies in mice have also demonstrated the formation of Heinz bodies following exposure ( Blair et al., 1990 ), suggesting an oxidative effect on hemoglobin as well. Many xenobiotics may also cause hemolysis through their association with microangiopathy, most commonly as part of the thrombotic microangiopathy syndrome, which is associated with fragmentation hemolysis and decreases in platelet counts. Drug-induced endothelial injury, including from direct and antibody or immune complex-mediated mechanisms, plays a major role in the pathogenesis of thrombotic microangiopathy ( Pisoni et al., 2001 ). Endothelial damage may be propagated by leukocyte adhesion and release of granule contents or reactive oxygen species, platelet activation and aggregation, and complement activation ( Pisoni et al, 2001 ). Drugs implicated in thrombotic microangiopathy include chemotherapeutic agents include xenobiotics from a wide variety of chemotherapeutic classes. Examples of chemotherapeutics associated with thrombotic microangiopathy include mitomycin C ( Cantrell et al., 1985 ), cisplatin ( Palmisano et al., 1998 ), estramustine phosphate ( Tassinari et al., 1999 ), gemcitabine ( Nackaerts et al., 1998 ), and daunorubicin ( Byrnes et al., 1986 ). Nonchemotherapeutic agents that have been reported to cause thrombotic microangiopathy include immunomodulators such as cyclosporine and tacrolimus ( Katznelson et al., 1994 , Trimarchi et al., 1999 ), simvastatin ( McCarthy et al., 1998 ), and inhibitors of platelet aggregation including ticlopidine and clopidogrel ( Bennett et al., 1998 , Bennett et al., 2000 ). Immune-mediated mechanisms of hemolysis have also been reported following exposure to numerous xenobiotics. Xenobiotics may induce antibodies by binding to the erythrocyte membrane and acting as haptens. These antibodies are considered drug-dependent as they only mediate hemolysis when the drug is present. Penicillin is the prototypical xenobiotic that acts as a hapten to generate drug-dependent antibodies, and typically induces an IgG response ( Ferner, 2012 , Petz et al., 1966 ). Semisynthetic penicillins, some cephalosporins, and tetracycline have also been reported to cause drug-dependent antibody-mediated hemolysis ( Garratty, 2010 , Tuffs and Manoharan, 1986 , Seldon et al., 1982 , Großjohann et al., 2004 , Gallagher et al., 1992 , Branch et al., 1985 , Simpson et al., 1985 ). Xenobiotics may also induce the production of antierythrocyte antibodies that mediate hemolysis even when the drug is no longer present, also called drug-independent antibodies or autoantibodies. In this type of hemolysis, xenobiotic exposure stimulates production of an antibody that can bind to native erythrocyte antigens even in the absence of the drug. This type of immune-mediated xenobiotic-induced hemolysis is classically caused by α-methyldopa, and is characterized by predominantly an IgG response ( Packman, 2016 ). However, nucleoside purine analogs such as cladribine and fludarabine have also been associated with hemolysis due to production of autoimmune antibodies ( Garratty, 2010 , Mintzer et al., 2009 , Hamblin, 2006 ). A third mechanism by which xenobiotics may cause immune-mediated hemolysis is through a complex interaction of the drug, a drug binding site on erythrocytes, and an antibody. This mechanism is considered the ternary complex mechanism, but has previously, and perhaps less accurately, been called an immune complex or innocent bystander mechanism ( Packman, 2016 ). Quinidine is the prototypical drug that causes hemolysis via this mechanism. Quinidine may be associated with either IgM or IgG antibodies and predominantly causes complement-mediated lysis of erythrocytes or clearance of complement-coated erythrocytes by tissue macrophages ( Packman, 2016 ). Ceftriaxone has also been reported to cause hemolysis through this mechanism ( Arndt and Garratty, 2005 ). Xenobiotic-induced immune-mediated hemolysis may not be limited to one of the three mechanisms described earlier, and a combination of these mechanisms may occur in some patients. For example, the NSAID diclofenac may cause hemolysis through both drug-dependent and drug-independent mechanisms ( Salama et al., 1996 ). Carboplatin has been reported to cause hemolysis through all three immune-mediated mechanisms ( Marani et al., 1996 ). Other compounds may cause hemolysis through mechanisms other than oxidative, microangiopathic, or immune-mediated. For example, although the primary effect of lead toxicity is impairment of heme synthesis, lead may also cause hemolysis. The mechanism of lead-induced hemolysis has not been fully determined, but interference with the erythrocyte membrane sodium/potassium transporter may be involved ( Bloom and Brandt, 2008 ). Copper toxicity causes hemolysis as well, possibly through inhibition of many enzymes involved in glycolysis resulting in decreased intracellular ATP ( Boulard et al., 1972 ). Envenomation from multiple animals is reported to cause hemolysis. Envenomation by snakes, such as rattlesnakes and coral snakes, can cause hemolysis through phospholipase A2 activity, which may cause direct hemolysis or liberate hemolysins such as lysolethicin, or through complement-mediated hemolysis ( Arce-Bejarano et al., 2014 , Tambourgi and van den Berg, 2014 , Walton et al., 1997 ). 12.11.3.2.1.2 Blood loss 12.11.3.2.1.2.1 Hemorrhage Hemorrhage may cause internal or external blood loss. Due to the loss of whole blood during hemorrhage, decreases in red cell mass are usually accompanied by proportionate decreases in albumin and globulin concentrations. The decreases in plasma proteins tend to be less pronounced with internal hemorrhage because the lost proteins may be resorbed in lymph and returned to blood ( Stockham and Scott, 2008b ). Cases of internal hemorrhage are typically not associated with iron deficiency. However, prolonged external blood loss may cause depletion of total body iron. Iron deficiency anemia is characterized by small erythrocytes with a decrease in MCV and erythrocytes that contain less hemoglobin with a decrease in MCHC, and may be classified as a microcytic, hypochromic anemia. Hemoglobin synthesis plays a role in inhibiting erythrocyte division, and when sufficient iron is not available for heme production, there is loss of the inhibitory effect resulting in more cell divisions and microcytes ( Stohlman et al., 1963 ). Hypochromasia of the erythrocytes is due to the lower than normal hemoglobin content due to decreased production of heme. Morphologic erythrocyte changes that accompany iron deficiency anemia include visual microcytosis and hypochromasia, keratocytes and schistocytes from physical damage to the more fragile erythrocytes, and sometimes codocytes (also called target cells) that have a thin rim of pink-staining hemoglobin and a small central area of hemoglobin with a ring of pallor in between, typical of erythrocytes with less hemoglobin present relative to the amount of membrane. In chronic iron deficiency, increases in reticulocyte counts and microscopic polychromasia may be lower than expected for a regenerative anemia due to loss of RNA during the extended maturation phase of erythrocyte production caused by decreased hemoglobin content ( Burkhard et al., 2001 ). Direct damage to blood vessels from trauma is a relatively common cause of acute external or internal blood loss. Traumatic rupture of the spleen may also cause significant acute internal blood loss. Decreases in red cell mass due to acute hemorrhage are typically due to dilution of remaining blood from shifting of intracellular fluid to extracellular fluid in an attempt to preserve blood volume and therefore tissue perfusion ( Stockham and Scott, 2008b ). Dilution of red cell mass may also be observed following administration of intravenous fluids to replace blood volume. A detectable increase in reticulocyte count is expected 3–4 days following the acute event in a patient with normally functioning bone marrow. Damage to blood vessels that results in hemorrhage also may occur secondary to ulcerative or neoplastic conditions. In dogs, rupture of splenic hemangiosarcoma is a common cause of internal blood loss into the abdomen (hemoabdomen). Ulceration of the gastrointestinal system may lead to blood loss into feces, which can be observed as black, tarry feces (melena) if the ulceration occurs in the small intestines or as frank blood if the ulceration occurs in the large intestines. In humans and nonhuman primate species with true menstrual cycles, including Old World monkeys and great apes ( Provencher Bolliger et al., 2010 ), decreases in red cell mass are uncommon but may be observed from menses-related blood loss. In women, heavy blood loss, abnormal cycling, or uterine neoplasms may lead to sufficient blood loss to cause decreases in red cell mass and potentially even iron deficiency ( Van Voorhis, 2009 , Goel and Gupta, 2007 ). In cynomolgus monkeys, decreases in red cell mass have been occasionally observed in females with prolonged menses ( Perigard et al., 2016 ). Coagulation disorders may also be associated with either internal or external hemorrhage. Primary deficiencies in coagulation factors or von Willebrand factor may be inherited causes of hemorrhage. Deficiencies in coagulation factors that lead to hemorrhage sufficient to cause decreases in red cell mass include hemophilia A (factor VIII deficiency) and hemophilia B (factor IX deficiency); deficiencies in factor XI and von Willebrand factor are usually mild and are often not associated with notable hemorrhage ( Bolton-Maggs and Pasi, 2003 ). Hemorrhage may be secondary to marked decreases in platelet counts from consumptive coagulopathies secondary to infectious or neoplastic processes. Although not truly hemorrhage, external blood loss can occur from repeated phlebotomy. Decreases in red cell mass may be acutely observed following collection of blood from donors for transfusion, and regular donors have a risk of developing iron deficiency from repeated external blood loss ( Cable et al., 2011 ). Repeated phlebotomy is a common occurrence in nonclinical toxicology studies, particularly in dogs and nonhuman primates, although rats may also occasionally undergo repeated blood collections. Blood is collected through the studies mainly for toxicokinetic or pharmacokinetic analysis, but also for analysis of hematology, coagulation, and clinical chemistry profiles. Decreases in red cell mass with increases in reticulocyte counts of similar magnitude relative to pretest values across all treatment groups, including controls, are a common procedure-related phenomenon in nonclinical toxicology studies and should be distinguished from a true test article-related effect. 12.11.3.2.1.2.2 Parasitism Both external and internal parasites may contribute to blood loss. Hookworms are a major internal parasite associated with chronic blood loss, and may lead to iron deficiency with prolonged infections ( Stoltzfus et al., 1997 ). However, whipworm infection and schistosomiasis may also be associated with blood loss, the latter being associated with blood loss through the urinary system ( Farid et al., 1969 ). Heavy infestation of animals with arthropods that take blood meals, such as ticks, some lice, and fleas, may also cause sufficient blood loss to result in decreases in red cell mass ( Stockham and Scott, 2008b ). 12.11.3.2.1.2.3 Xenobiotic-induced Xenobiotic-induced blood loss is relatively uncommon but can occur. Classically, hemorrhage into the intestinal tract can result from ulceration associated with chronic NSAID or coxib administration ( Laine et al., 2003 , Langman et al., 1999 , Bjarnason et al., 1987 ). Also, prolonged or high dose administration of anticoagulants, such as warfarin or heparin, can result in hemorrhage-related decreases in red cell mass ( Levine et al., 2001 ). Ingestion of rodenticides, including brodifacoum chlorophacinone, has been reported to cause marked hemorrhage in humans and many other nonrodent species ( Berny et al., 2010 , Palmer et al., 1999 , Sheafor and Couto, 1999 ). Xenobiotic-induced marked decreases in platelet counts may also be associated with hemorrhage and are discussed in more detail later. However, chemotherapeutics that cause bone marrow suppression can be associated with spontaneous or postvenipuncture hemorrhage. Occasional idiopathic decreases in platelet counts have also been observed following xenobiotic administration and are most likely attributable to immune-mediated destruction; some examples of implicated xenobiotics are quinine, trimethoprim–sulfamethoxazole, anticonvulsants such as phenytoin and carbamazepine, unfractionated or low molecular weight heparin, and rituximab ( Aster and Bougie, 2007 ). 12.11.3.2.2 Decreases in red cell mass with "normal" or low reticulocyte counts (nonregenerative anemia) 12.11.3.2.2.1 Preregenerative Depending on the timing of the insult causing the decreases in red cell mass, reticulocyte counts within reference interval may represent a preregenerative anemia rather than suppressed erythropoiesis. Production of erythrocytes by the bone marrow requires at least 3–4 days, and a peak increase in blood reticulocyte count occurs about 7–14 days following the insult ( Stockham and Scott, 2008b ). If it an individual with decreased red cell mass and reticulocyte counts that are within the reference interval and it is unclear if the individual has a preregenerative anemia or suppressed erythropoiesis, repeating a CBC several days later may help clarify which process is occurring. 12.11.3.2.2.2 Infectious Acute Chagas disease, caused by infection with Trypanosoma cruzi , has been reported to cause decreases in red cell mass in humans and monkeys ( de Titto and Araujo, 1988 , Rosner et al., 1988 , Seah et al., 1974 ). In experimentally infected Cebus paella monkeys, the acute phase of Chagas disease was reported to cause normocytic, normochromic anemia ( Rosner et al., 1988 ), typical of a nonregenerative anemia. Experimentally infected mice demonstrated bone marrow suppression with decreases in red cell mass as well as decreases in leukocyte and platelet counts ( Marcondes et al., 2000 ). Although rarely encountered in nonclinical toxicology studies, monkeys held in the southwestern United States may become infected with T . cruzi prior to distribution ( Magden et al., 2015 ). During parasitemia, trypomastigotes may be observed in peripheral blood smears. Viral infections may also cause decreases in red cell mass without concurrent increases in reticulocyte counts. Parvoviruses may cause decreases in red cell mass from direct infection of erythroid precursor resulting in decreased erythrocyte production, as well as decreased erythrocyte lifespans. Parvovirus may result in transient pure red cell aplasia (PRCA) in humans ( Van Horn et al., 1986 ). Cell-mediated suppression of erythropoiesis resulting in PRCA has also been reported with viral hepatitis ( Wilson et al., 1980 ) and Epstein-Barr virus infection ( Socinski et al., 1984 ). Although HIV infection can result in decreases in red cell mass through various mechanisms, direct infection of erythroid precursors appears to contribute to suppressed erythropoiesis ( Evans and Scadden, 2000 ). In cats, a membrane protein of feline leukemia virus has been associated with decreased growth of CFU-E ( Wellman et al., 1984 ). Flavivirus infection, such as dengue, may also result in decreases in red cell mass and reticulocyte counts through bone marrow suppression ( La Russa and Innis, 1995 ). 12.11.3.2.2.3 Chronic disease Anemia of chronic disease (ACD) is a relatively common cause of anemia, and anemia associated with inflammatory disease is included in ACD. The decreases in red cell mass observed with ACD are generally mild, and are generally normocytic, normochromic, indicating no changes in MCV or MCHC, respectively. ACD may occur through shortening of erythrocyte lifespans, alterations in iron metabolism, a blunted response of erythroid precursors to EPO, and decreased EPO production. Altered erythrocyte lifespans in patients with ACD may be related to increased macrophagic clearance of erythrocytes from circulation through unknown mechanisms ( Ganz, 2016 ). This type of mechanism has been associated with several chronic infections, including tuberculosis and endocarditis ( Weiss, 2002 ). More commonly, ACD is associated with impaired iron mobilization with low iron concentrations in serum or plasma despite adequate iron stores ( Means, 2000 ). Impaired mobilization of iron results from IL-6 induction of hepcidin that results in sequestration of iron in macrophages and decreased intestinal iron update ( Ganz, 2003 ), IL-1 stimulation of increased synthesis of ferritin which may bind to iron and impair delivery of iron to erythroid precursors ( Rogers et al., 1994 ), and with decreased expression and impaired internalization of the transferrin receptor ( Means, 2000 ). ACD from impaired iron metabolism is associated with numerous inflammatory, infectious, and even neoplastic conditions. ACD may also cause altered EPO responsiveness or decreased EPO production. Decreased responsiveness of erythroid precursors to EPO is cytokine-mediated, and has been associated with increases in TNFα, IL-1, and interferons ( Johnson et al., 1989 , Johnson et al., 1990 , Raefsky et al., 1985 ) that may commonly be associated with inflammatory conditions. Decreased EPO production may also be cytokine-mediated, and has been reported with increases in TGFβ, TNFα, and IL-1 ( Faquin et al., 1992 , Jelkmann et al., 1992 ). However, chronic renal disease may also result directly in impaired EPO production and decreased production of erythrocytes ( Sato and Yanagita, 2013 ). 12.11.3.2.2.4 Immune-mediated Immune-mediated destruction of erythroid precursors in the bone marrow results in decreases in red cell mass with concurrent decreases in reticulocyte counts. The immune-mediated conditions discussed here may represent a spectrum of disease associated with immune destruction of various stages of erythroid precursors rather than unrelated entities. 12.11.3.2.2.4.1 Autoimmune hemolytic anemia with decreases in reticulocyte counts Autoimmune hemolytic anemia with antibodies that target antigens on mid- to late-stage erythroid precursors ranging from rubricytes to metarubricytes results in AIHA with a decrease in reticulocyte count, which may also be called immune-mediated nonregenerative anemia or precursor-targeted immune-mediated anemia (PIMA). AIHA with reticulocytopenia is generally a normocytic, normochromic anemia. Bone marrow examination may reveal erythroid hyperplasia or maturation arrest ( Weiss, 2008 ) with pyramidal expansion of erythroid precursors at stages earlier than the targeted stage, indicative of ineffective erythropoiesis. This may be less apparent with autoantibodies that recognize more immature stages of erythroid precursors. Bone marrow evaluation may also reveal rubriphagocytosis, or erythroid precursors phagocytized by macrophages. The stage of phagocytized precursor depends on the stage or stages expressing the targeted antigen. 12.11.3.2.2.4.2 Pure red cell aplasia In patients affected by PRCA, there are marked decreases in reticulocyte counts along with variable decreases in red cell mass. Bone marrow examination typically reveals an absence of erythroid precursors (erythroid aplasia) or low numbers of the earliest stages of erythroid precursors (erythroid hypoplasia) ( Young, 2016 ). PRCA in people may be caused by antibodies that bind antigens on the earliest erythroid precursors or even antibodies that bind EPO and prevent EPO-dependent erythropoiesis, but it has also been attributed to clonal T-cell disorders ( Stockham and Scott, 2008b ). PRCA in dogs has been associated with IgG that inhibit erythropoiesis ( Weiss, 1986 ). PRCA may also be caused by inherited genetic defect in people. Inherited PRCA in people is called Diamond-Blackfan anemia, and often has an autosomal dominant inheritance pattern with defects in genes encoding ribosomal proteins ( Young, 2016 ). Macrocytosis, or increased numbers of large erythrocytes with increases in MCV, may be observed and is consistent with impaired EPO-dependent erythropoiesis ( Young, 2016 , Ohene-Abuakwa et al., 2005 ). 12.11.3.2.2.4.3 Aplastic anemia Aplastic anemia is a condition associated with decreases in all cellular blood components (pancytopenia), including decreases in red cell mass with concurrent decreases in reticulocyte counts. Upon examination, the bone marrow classically had severe hypocellularity of hematopoietic cells or an absence of hematopoietic precursors the marrow cavities filled by mostly adipocytes and some stromal elements. Aplastic anemia is thought to be most commonly immune-mediated ( Young et al., 2006 ), and may be frequently associated with cytotoxic T-cells that become autoreactive ( Segel and Lichtman, 2016 ). However, there are also cases of inherited aplastic anemia, most commonly Fanconi anemia associated with genetic mutations that impair DNA repair resulting in pancytopenia developing around 5–10 years of age in people ( Segel and Lichtman, 2016 ). A form of aplastic anemia associated with bone marrow depletion or hypocellularity of hematopoietic tissue and gelatinous transformation of marrow cavity fat has been reported with anorexia nervosa in people ( Abella et al., 2002 ) and with severe food restriction in rats ( Moriyama et al., 2008 ). 12.11.3.2.2.5 Nutritional deficiencies In addition to aplastic anemia associated with anorexia nervosa and severe food restriction, other nutritional deficiencies have been associated with ineffective erythropoiesis leading to decreases in red cell mass with decreases in reticulocyte counts. Iron deficiency and deficiencies of the B vitamins folate and cobalamin are examples of these nutritional deficiencies. Chronic iron deficiency results in impaired hematopoiesis due to the inability to synthesize sufficient hemoglobin, which may lead to a decrease in reticulocyte production. Deficiencies in folate and cobalamin also cause ineffective erythropoiesis due to defects in DNA synthesis, as discussed with folate and cobalamin deficiencies as a cause of deceases in neutrophil counts. In people, folate and cobalamin deficiencies result in megaloblastic anemia, characterized by larger than normal erythroid precursors in the bone marrow that have more cytoplasm with lower nuclear to cytoplasmic ratios than in normal erythroid precursors and asynchronous cytoplasmic and nuclear maturation ( Green, 2016 ). Megaloblastic erythrocytes may also be observed in circulation, and basophilic stippling or Howell-Jolly bodies may also be observed ( Green, 2016 ). In people, anemia attributable to a deficiency in cobalamin (vitamin B 12 ) may also be called pernicious anemia. In dogs and cats, megaloblastic erythroid cells may be observed in the bone marrow but may not be observed in blood ( Stockham and Scott, 2008b ). 12.11.3.2.2.6 Endocrinopathy Several endocrinopathies have also been associated with decreases in red cell mass with decreases in reticulocyte counts, including hypothyroidism, hypoadrenocorticism, and hyperestrogenism. In cases of hypothyroidism, several mechanisms may be contributing to the decreases in red cell mass. Decreased folate or cobalamin concentrations secondary to the hypothyroidism leading to ineffective erythropoiesis, decreased tissue oxygen demand leading to decreased EPO and lower baseline red cell mass, and ACD may contribute to the mild decreases in red cell mass observed with hypothyroidism ( Ottesen et al., 1995 , Hines et al., 1968 , Stockham and Scott, 2008b , Mehmet et al., 2012 ). Mild decreases in red cell mass without apparent changes in reticulocyte counts have been associated with hypoadrenocorticism. This may be due to a decrease in glucocorticoids, and the loss of the apparent proerythropoietic stimulation of glucocorticoids ( Stockham and Scott, 2008b ). Hyperestrogenism, which occurs with some ovarian or testicular neoplasms, may result in bone marrow toxicity and suppression of erythropoiesis, particularly in dogs ( Sontas et al., 2009 ). 12.11.3.2.2.7 Neoplasia Neoplasia may result in suppressed erythropoiesis. This may be due to neoplasia-related inflammation and cytokine release leading to ACD. However, granulocytic leukemia or lymphoproliferative neoplasia involving the bone marrow may result in crowding or effacement of the bone marrow cavities with impaired erythropoiesis that results in decreases in red cell mass with concurrent decreases in reticulocyte counts. Hematopoietic neoplasia involving the erythroid lineage usually results in atypical erythrocyte production that can lead to decreases in red cell mass and reticulocyte counts; however, nucleated erythrocytes with evidence of dysplasia may be observed in blood. Similar to hematopoietic neoplasms that efface the bone marrow, metastatic neoplasia, often carcinomas, may also cause myelophthisis and result in decreased erythropoiesis. 12.11.3.2.2.8 Xenobiotic-induced There are many xenobiotics that can cause decreases in red cell mass with concurrent decreases in reticulocyte counts. Bone marrow suppression that affects the erythroid lineage is commonly observed with chemotherapeutics in general. For example, agents that are directly cytotoxic to hematopoietic precursors, that inhibit mitotic spindle formation, and antimetabolites that alter folate metabolism may all result in suppression of erythropoiesis. However, development of parvovirus-induced PRCA has been reported as a consequence of chemotherapeutic administration ( Song et al., 2002 , Rao et al., 1994 ). PRCA has occasionally been linked to xenobiotic treatment. A wide variety of xenobiotics from many different classes have been reported to cause PRCA. Examples of xenobiotics reportedly associated with PCRA include sulfonamides, allopurinol, procainamide, gold-containing compounds, rifampin, and chloroquine ( Young, 2016 , Mintzer et al., 2009 ). However, causality is often difficult to prove, and most associations are limited to low numbers of case reports. One study evaluated reports of PRCA associated with administration of 30 different xenobiotics, but causality was only attributed to treatment with azathioprine, isoniazid, and phenytoin ( Thompson and Gales, 1996 ). PRCA due to the development of anti-EPO antibodies may follow the administration of recombinant EPO in humans ( Casadevall et al., 2002 ) and EPO gene therapy in monkeys ( Gao et al., 2004 ). Administration of recombinant EPO to dogs has also led to the development of anti-EPO antibodies and PRCA ( Randolph et al., 2004 ). Aplastic anemia has also been linked to administration of xenobiotics. Classically, chloramphenicol is reported to sporadically cause aplastic anemia ( Segel and Lichtman, 2016 ). However, antithyroid compounds, sulfonamides including trimethoprim sulfamethoxazole, beta-lactams, the diuretic furosemide, gold-containing compounds, penicillamine, and anticonvulsants including carbamazepine and phenacetin have all been reported in association with aplastic anemia ( Mintzer et al., 2009 , Kaufman et al., 1996 ). Aplastic anemia has also been attributed to environmental or occupational exposure to benzene ( Smith, 1996 ). In a case of aplastic anemia in a dog, griseofulvin administration was suspected to be the cause of the aplastic anemia ( Brazzell and Weiss, 2006 ). Decreases in red cell mass with concurrent decreases in reticulocyte counts have occurred with prolonged or repeated high dose administration of G-CSF or GM-CSF-based xenobiotics in nonclinical toxicology studies, particularly in rodents. Impaired erythropoiesis in these cases occurs due to the massive expansion of myeloid precursors within the bone marrow. Extreme myeloid hyperplasia with continued stimulation results in overcrowding of the marrow cavities with less physical space available for erythroid production. 12.11.3.2.1 Decreases in red cell mass with increases in reticulocyte counts (regenerative anemia) Decreases in red cell mass with concurrent increases in reticulocyte counts (reticulocytosis) indicate a regenerative erythroid response by the bone marrow, or by extramedullary hematopoiesis in the spleen of rodents. An increase in reticulocyte count is typically first observed 3–4 days after an acute drop in red cell mass due to bone marrow erythrocyte production and transit time, and peak responses generally occur around 7–14 days depending on the species ( Stockham and Scott, 2008b ). The regenerative erythroid response is considered appropriate if the increases in reticulocyte counts reflect the magnitude of the decreases in red cell mass; in other words, a mild decrease in red cell mass is expected to result in a mild increase in reticulocyte count, while a moderate to marked decrease in red cell mass should have a concurrent moderate to marked increase in reticulocyte count. The regenerative erythroid response is considered inappropriate if there is an inconsistency between the magnitude of the decrease in red cell mass and the magnitude of the increase in reticulocyte count. For example, a marked decrease in red cell mass with only a mild increase in reticulocyte count a week after the insult would be considered an inappropriate regenerative erythroid response. Increases in blood reticulocyte counts may be associated with concurrent changes in mean corpuscular volume (MCV; an indicator of erythrocyte size) and mean corpuscular hemoglobin concentration (MCHC; an indicator of erythrocyte hemoglobin content), two red blood cell indices provided by most automated hematology analyzers. Due to the reticulocyte's increased volume relative to mature erythrocytes, increases in blood reticulocyte counts may cause increases in MCV (macrocytosis) and decreases in MCHC (hypochromasia). Regenerative anemias with increases in MCV and decreases in MCHC and/or CHCM may also be classified by these indices as macrocytic, hypochromic anemias. The decrease in MCHC does not necessarily reflect less hemoglobin content per cell, but is a consequence of reduced concentration of hemoglobin due to the larger cytoplasmic volume of reticulocytes. MCHC, which is a calculated endpoint, may be artificially increased when free plasma hemoglobin is present due to intravascular hemolysis. Some automated hematology analyzers also provide the corpuscular mean hemoglobin content (CHCM) which provides a mean of direct measurements of cellular hemoglobin concentration and is therefore resistant to interference from free plasma hemoglobin. Regenerative erythroid responses with increases in blood reticulocyte counts may be associated with several morphologic findings observed during blood smear evaluation. Most commonly, increases in polychromatophils (polychromasia) are observed with Wright-Giemsa or modified Wright stains. Polychromatophils are erythrocytes that stain blue–purple in color due to the combined effects of blue-staining RNA content typical of reticulocytes and pink-staining hemoglobin. As reticulocytes mature and lose RNA, a visual difference in staining cannot longer be detected between late reticulocytes and mature erythrocytes. However, staining of blood with a vital dye such as New Methylene Blue permits differentiation between aggregate and punctate-type reticulocytes. Polychromasia usually correlates well with increases in reticulocytes in most species, except cats ( Stockham and Scott, 2008b ). In cats, aggregate-type but not punctate-type reticulocytes correlate with polychromasia and are considered clinically relevant, and differentiation of these two with manual reticulocyte counts should be performed ( Harvey, 2012 , Stockham and Scott, 2008b ). Reticulocytes or erythrocytes with few small, punctate dark blue-gray inclusions may be observed during a regenerative erythroid response. These inclusions contain iron and may be called Pappenheimer bodies or siderotic inclusions. Due to the rapid production and release of erythrocytes during a regenerative erythroid response, there may also be increase in nucleated red blood cells or erythrocytes with Howell-Jolly bodies. Nucleated red blood cells are usually present in low numbers, but if 10 or more are observed per 100 leukocytes, the automated total leukocyte count will be falsely increased and should be corrected using published equations ( Stockham and Scott, 2008a ). In some species, particularly cows and sheep, erythrocytes with basophilic stippling may also be observed in circulation during a regenerative erythroid response. Hemolysis and blood loss are the two main categories of decreases in red cell mass with appropriate increases in reticulocyte counts. 12.11.3.2.1.1 Hemolysis Destruction of mature erythrocytes is called hemolysis. Hemolysis may occur either intravascularly or extravascularly. With intravascular hemolysis, erythrocyte destruction occurs within the blood and results in hemoglobinemia, or free hemoglobin within plasma. Ghost erythrocytes, or the remnant membranes of erythrocytes that no longer contain cytoplasm or hemoglobin, may be observed with intravascular hemolysis. Consequent hemoglobinuria, or free hemoglobin in the urine, is rare and only occurs in cases of massive intravascular hemolysis that overwhelm the normal pathways that clear free hemoglobin from the blood. In contrast, extravascular hemolysis does not occur within the blood, but rather occurs in the spleen, liver, or bone marrow, where resident macrophages phagocytose erythrocytes and destroy them intracellularly. Extravascular hemolysis does not result in free plasma hemoglobin or hemoglobinuria. Both types of hemolysis may be associated with increases in total bilirubin concentrations where unconjugated (indirect) bilirubin usually exceeds conjugated (direct) bilirubin, and may result in plasma or serum icterus (yellow discoloration) or bilirubinuria (bilirubin present in urine). However, not all cases of hemolysis are clearly either intravascular or extravascular, and both forms of hemolysis may contribute in some conditions. 12.11.3.2.1.1.1 Infectious There are numerous protozoal, bacterial, and viral diseases that can be associated with hemolysis. Mechanisms by which infectious agents cause erythrocyte destruction are varied, and may include direct infection of erythrocytes, elaboration of toxins such as hemolysin, or stimulation of an immune-mediated response against infected cells ( Berkowitz, 1991 ). Several examples of infectious agents that cause hemolytic anemia are discussed later. Direct infection of erythrocytes with protozoal Plasmodium species, the causative agent of malaria that is transmitted by mosquitoes, is a relatively common cause of hemolysis in humans, but may also be observed in nonhuman primates used in nonclinical toxicology studies. Humans are infected by one of five different Plasmodium species: P . falciparum , P . vivax , P . knowlesi , P . malariae , or P . ovale , although only P . falciparum and P . vivax are commonly associated with severe hemolysis ( Lichtman, 2016b ). Macaques are most commonly infected with P . inui or P . knowlesi , although the cynomolgus monkey appears to be more resistant to disease from these infections than the rhesus monkey ( Ameri, 2010 ). Infection with P . cynomolgi , P . fieldi , or P . fragile may also occur in macaques ( Magden et al., 2015 ). Although it is uncommon to include macaques infected with Plasmodium species during a nonclinical toxicity study due to current screening practices and pretest evaluations, rare animals with decreases in red cell mass and increases in reticulocyte counts and intraerythrocytic Plasmodium organisms have been observed. Rats and mice may be infected with Plasmodium berghei ( Holloway et al., 1995 , Sadun et al., 1965 ). Plasmodium berghei has a specific tropism for reticulocytes rather than mature erythrocytes Plasmodium species that infect humans ( Car et al., 2006 , Cromer et al., 2006 ). Concurrent increases in reticulocyte counts may occur in early stages or disease or with recrudescence of parasitemia and hemolysis. Hemolysis is associated with clearance of parasitized erythrocytes from circulation predominantly by splenic macrophages ( Lichtman, 2016b ), although accumulation of hemin, an iron-containing porphyrin, which can directly stimulate apoptotic erythrocyte death (eryptosis) ( Gatidis et al., 2009 ), oxidative damage to erythrocyte membranes ( Clark and Hunt, 1983 ), and increased osmotic fragility ( George et al., 1967 ) may all contribute to hemolysis. However, late-stage infections in humans and rodents have also been associated with inappropriate or decreased reticulocyte counts indicative of suppressed erythropoiesis despite decreases in red cell mass from hemolysis ( Lichtman, 2016b , Cromer et al., 2006 ). Babesia species are tick-borne protozoal organisms that directly infect erythrocytes in most species, including humans, nonhuman primates, dogs, and cats. Babesia species appear as intracellular oval to pyriform organisms. Babesia microti and Babesia divergens may infect humans in North America and Europe, respectively, and cause moderate hemolytic anemia from intraerythrocytic replication and subsequent erythrocyte lysis ( Lichtman, 2016b , Kjemtrup and Conrad, 2000 ). Babesia pitheci has been reported to infect both old and new world monkeys and cause anemia ( Magden et al., 2015 ). B . canis , a large babesial species, and B . gibsoni , a small babesial species, infect dogs, while cats may be infected by the small babesial organisms B . felis and B . cati ( Stockham and Scott, 2008b , Penzhorn et al., 2004 ). These organisms are generally not of concern in purpose-bred animals used in nonclinical toxicology studies. Bartonella bacilliformis in people and the hemotrophic mycoplasmas (hemoplasmas) in dogs and cats (formerly Haemobartonella species) and swine (formerly Eperythrozoon species) are organisms that parasitize erythrocytes, but these organisms remain extracellular in shallow depressions of the erythrocyte membrane. These organisms are typically round-, rod-, or ring-shaped and may be observed individually or in chains on erythrocyte surfaces. Hemolysis with these organisms may be immune-mediate and associated either with binding of antibodies to parasite antigens or antigens exposed on the erythrocyte secondary to parasite-induced membrane changes ( Stockham and Scott, 2008b ). Clostridium perfringens (formerly Clostridium welchii ) infection in humans is an example of a bacterial cause of hemolysis. During intestinal overgrowth or septicemia, C . perfringens type A elaborates an α toxin that has lecithinase C activity, resulting in membrane phospholipid breakdown and release of lysolethicins, which have potent hemolytic capabilities ( Lichtman, 2016b , Songer, 1996 ). C . perfringens α toxin release is usually associated with severe intravascular hemolysis with both hemoglobinemia and hemoglobinuria. However, in veterinary species, C . perfringens -related hemolysis is typically limited to ruminants and horses ( Stockham and Scott, 2008b ), and is unlikely to be observed in the common species used in nonclinical toxicology studies. Infection of humans with Mycoplasma pneumoniae has also been associated with hemolytic decreases in red cell mass, although most cases of M . pneumoniae infection are asymptomatic. Hemolysis with this organism is attributable to stimulation of autoimmune erythrocyte destruction with agglutination of erythrocytes ( Khan and Yassin, 2009 ). Several viral organisms in humans have also been reported to cause decreases in red cell mass due to hemolysis. Viral causes of hemolysis are commonly associated with autoimmune mechanisms, and include infection with Epstein-Barr virus ( Palanduz et al., 2002 ), hepatitis A, B, and C viruses ( Kanematsu et al., 1996 , Chao et al., 2001 ), cytomegalovirus ( Murray et al., 2001 ), and HIV ( Koduri et al., 2002 ), although HIV infection is also commonly associated with decreases in reticulocyte counts rather than the expected increases secondary to hemolysis, indicative of concurrent suppressed erythropoiesis ( Telen et al., 1990 ). 12.11.3.2.1.1.2 Oxidative Another major cause of decreases in red cell mass due to hemolysis is oxidative damage to erythrocytes. Under normal conditions, ferrous iron (Fe 2 + ) in hemoglobin binds to and dissociates from oxygen as it delivers oxygen from the lungs to the tissues. At times, this binding and dissociation results in the formation of ferric iron (Fe 3 + ) in hemoglobin (methemoglobin) as well as superoxide (O 2 − ). Superoxide is a free radical with potent oxidative capacity that may cause cellular damage. Cytochrome b 5 -reductase is an intraerythrocytic enzyme that converts methemoglobin back to hemoglobin. Superoxide dismutase converts superoxide to hydrogen peroxide (H 2 O 2 ), which also may produce oxidative damage to cells. Further metabolism of hydrogen peroxide by catalase or glutathione peroxidase protects cells from oxidative damage. These pathways are usually sufficient to address the normal low-level formation of methemoglobin and superoxide, but methemoglobin can increase and impair delivery of oxygen to tissues and superoxide can accumulate and cause oxidative damage if these pathways are overwhelmed or defective. Oxidative damage to erythrocytes may affect the lipid membranes, cytoskeleton, or hemoglobin. Peroxidation of internal membrane lipids or cytoskeletal components of erythrocytes results in the fusion of portions of the membrane with consequent shifting of the cytoplasm and hemoglobin to one side of the cells. Erythrocytes with this morphologic change are called eccentrocytes. Oxidative damage that causes the formation of eccentrocytes may result in hemolysis due to increased clearance of eccentrocytes by splenic macrophages due to trapping of rigid erythrocytes in splenic sinusoids or spontaneous rupture in blood due to the increased fragility of eccentrocytes ( Stockham and Scott, 2008b ). Oxidative damage to exposed cysteine sulfhydryl groups on hemoglobin results in hemoglobin denaturation and decreased solubility ( Bloom and Brandt, 2008 ). Denatured hemoglobin may then precipitate and aggregate within the erythrocyte, forming small, pale-staining round structures that bind to the erythrocyte membrane and tend to protrude from the surface of the erythrocyte. These aggregates of denatured hemoglobin are called Heinz bodies. Cats appear to be particularly sensitive to the formation of Heinz bodies because of an increased number of reactive sulfhydryl groups in hemoglobin relative to other species ( Christopher et al., 1990 ), and may be more rapidly observed on blood smear evaluation due to the nonsinusoidal architecture of the feline spleen that results in decreased clearance of Heinz bodies from circulation. Similar to eccentrocytes, erythrocytes with Heinz bodies may undergo hemolysis due to increased clearance by splenic macrophages following trapping in splenic sinusoids due to decreased erythrocyte deformability and spontaneous rupture due to increased fragility from membrane damage; immune-mediated clearance may also occur and is believed to result from binding of hemochromes to and subsequent redistribution of band 3, an erythrocyte membrane structural protein, which may then be recognized by autologous antibodies ( Winterbourn, 1990 ). There are many conditions that may cause oxidative damage and result in eccentrocytosis, Heinz body formation, or even both simultaneously. Diabetes mellitus may cause either morphologic change, and diabetic ketoacidosis appears to be associated with an increased susceptibility and incidence of oxidative erythrocyte damage ( Desnoyers, 2010 , Caldin et al., 2005 , Christopher et al., 1995 ). Inherited deficiencies in erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and flavin adenine dinucleotide (FAD) have also been associated with erythrocyte oxidative damage, eccentrocyte or Heinz body formation, and hemolysis or a predisposition for these events due to the loss of protective antioxidant pathways ( Chan et al., 1982 , Harvey, 2006 ). Lymphoma has also been associated with Heinz body formation in cats ( Christopher, 1989 ) and eccentrocytes formation in dogs ( Caldin et al., 2005 ). In dogs and cats, ingestion of Allium species, particularly onions, garlic, and Chinese chive, may cause erythrocyte oxidative damage with formation of eccentrocytes and/or Heinz bodies ( Caldin et al., 2005 , Yamato et al., 2005 , Robertson et al., 1998 ). Ingestion of zinc in dogs ( Bexfield et al., 2007 ) and exposure to skunk musk ( Fierro et al., 2013 ) have also been reported to cause hemolysis due to Heinz body formation. 12.11.3.2.1.1.3 Fragmentation Physical trauma to erythrocytes results in hemolysis due to erythrocyte fragmentation and lysis. Sometimes this type of hemolysis is referred to as microangiopathic hemolytic anemia. Morphologic changes to erythrocytes occur as a result of physical trauma. Schistocytes (also called schizocytes or erythrocyte fragments), keratocytes (also called helmet cells), prekeratocytes (also called blister cells), or even spherocytes or microspherocytes may be observed. Schistocytes are very small, usually irregularly shaped fragments that can break off erythrocytes when physical trauma occurs. Keratocytes have one to two variably sized projections or horns adjacent to a small flattened region of the erythrocyte surface, while prekeratocytes appear to be precursors that have small loops of erythrocyte cytoplasm extending from the surface and surrounding a small hole in the cell. Spherocytes and microspherocytes are spherical cells that appear smaller and have more intensely pink-staining cytoplasm than normal mature erythrocytes. Spherocytes and microspherocytes may be formed during physical trauma as fragments are broken off, resulting in less membrane surface area in the parent erythrocyte surrounding a similar volume (spherocytes) or smaller volume (microspherocytes) as the parent erythrocyte. The physical trauma to erythrocytes that causes fragmentation or microangiopathic hemolysis may result from consumptive coagulopathies, either local or disseminated (DIC), with fibrin or thrombus formation in the vasculature that impedes the passage of erythrocytes through the vessel lumen, creating both turbulence and physical obstruction of blood flow. Local coagulopathy or DIC may occur secondary to trauma, infections with sepsis, or neoplasia ( Baker and Moake, 2016 , Toh and Dennis, 2003 ). Microangiopathic hemolysis due to neoplasia is most commonly associated with malignant rather than benign neoplasms and with metastatic disease or neoplasic emboli rather than primary tumors, with the exception of primary vascular neoplasms ( Susano et al., 1994 , Kupers et al., 1975 , Lohrmann et al., 1973 ). Infectious agents may also lead to fragmentation of erythrocytes, and some Leptospirosis interrogans serovars associated with vasculitis ( Stockham and Scott, 2008b ), Brucella species infection ( Yaramis et al., 2001 ), and cutaneous anthrax ( Freedman et al., 2002 ) have been reported to cause microangiopathic hemolysis. In children, fragmentation hemolysis associated with thrombotic microangiopathy may occur with Shigella dysenteriae type 1 and some Escherichia coli infections ( Pisoni et al., 2001 ). Hemolysis from erythrocyte fragmentation may also occur with HIV infection ( Maslo et al., 1997 ). Decreases in red cell mass with increases in reticulocyte counts from erythrocyte fragmentation may also occur secondary to cardiac or other conditions that alter hemodynamics and increase turbulent blood flow. For example, subaortic stenosis ( Solanki and Sheikh, 1978 ), intraluminal aortic grafts ( Sayar et al., 2006 ), uncorrected cardiac valvular disease ( Marsh and Lewis, 1969 ), prosthetic valves ( Crexells et al., 1972 ), and hypertrophic obstructive cardiomyopathy ( Kubo et al., 2010 ) have all been reported to cause hemolysis from erythrocyte fragmentation. Increased turbulence associated with hypertension may also cause decreases in red cell mass from fragmentation, and has been associated with pulmonary hypertension ( Baker and Moake, 2016 ) and malignant systemic hypertension ( Capelli et al., 1966 ). 12.11.3.2.1.1.4 Immune-mediated Autoimmune hemolytic anemia (AIHA or AHA) described in humans or immune-mediated hemolytic anemia (IMHA) described in most common laboratory species is a cause of hemolysis, and may be primary or idiopathic, but may also be secondary to conditions such as infections as discussed previously. Primary or idiopathic AIHA/IMHA is discussed here. Primary AIHA has no underlying detectable cause and is an immune-mediated condition that produces antibodies targeting erythrocyte antigens. These antierythrocyte antibodies tend to be very specific for a single erythrocyte antigen in a given case ( Packman, 2016 ). These autoantibodies may be classified as warm antibodies, which are usually IgG, or cold antibodies, which are usually IgM ( Stockham and Scott, 2008b ). Immune-mediated AIHA may be associated with erythrocyte morphologic changes that include agglutination and spherocytes. Agglutination may be observed grossly as red speckling along the inside of the specimen tube as blood is gently moved within the tube. If agglutination is present, blood smears may have a "reverse smear" appearance with the densest region of the smear observed at the feathered edge rather than the edge where the drop of blood was initially placed. Microscopically agglutination appears as grape-like clusters of erythrocytes. Spherocytes are erythrocytes that are spherical instead of having the normal biconcave disc shape. While spherocytes appear smaller and stain more intensely pink that unaffected mature erythrocytes, they have the same volume as unaffected erythrocytes. Loss of erythrocyte membrane occurs when macrophages begin to phagocytize antibody-bound erythrocytes, leading to decreased erythrocyte surface area without an appreciable change in volume, forcing erythrocytes to form spheres. Hence, spherocytosis alone will not result in an altered MCV. Of the most common laboratory species, dogs tend to have the most pronounced central pallor of normal mature erythrocytes, making microscopic identification of spherocytes easiest in the dog. Hemolysis in AIHA is largely attributable to extravascular hemolysis due to phagocytosis of antibody-bound erythrocytes by tissue macrophages. Macrophages or monocytes containing phagocytized erythrocytes may be rarely observed in blood smears of laboratory species with immune-mediated hemolysis. However, antibody-mediated complement activation or increased fragility of spherocytes may result in direct intravascular lysis or rupture of erythrocytes ( Packman, 2016 ). Evaluation of patients for the presence of antierythrocyte antibodies may be performed using the direct antiglobulin test (DAT; also called the Coombs' test) or by flow cytometry. AIHA has rarely been observed in association with lymphoproliferative neoplasia, such as chronic lymphocytic leukemia. Antierythrocyte antibodies in chronic lymphocytic leukemia are predominantly IgG with few cases of IgM reported ( Mauro et al., 2000 ). IMHA may occasionally be observed following blood transfusion ( Garratty, 2004 ). This may occur in response to alloantigens, and would not technically be considered autoimmune ( Stockham and Scott, 2008b ). Posttransfusion immune-mediated hemolysis may also be observed when the host has autoantibodies that bind the donor erythrocytes and cause immune-mediated destruction. However, crossmatching of host and donor erythrocytes and plasma is able to prevent many cases with incompatible transfusion-related AIHA. AIHA and IMHA commonly have concurrent inflammatory increases in leukocyte subtype counts, characterized mainly by neutrophilia that may or may not have a left shift with cytoplasmic changes indicative of rapid neutropoiesis. 12.11.3.2.1.1.5 Inherited Some phenotypes of sickle cell disease are associated with hemolysis. The mechanism of hemolysis in sickle cell disease is likely multifactorial and not associated with a single pathogenesis. There is evidence for oxidative damage to erythrocytes ( Lachant et al., 1983 ), which may contribute to the hemolysis observed with sickle cell disease. However, hemoglobin polymerization leads to erythrocyte deformation and may lead to decreased flexibility of erythrocytes and veno-occlusive disorders ( Bookchin and Lew, 1996 ). Decreased flexibility or deformability of erythrocytes may contribute directly to increased cell fragility and rupture or promote clearance of deformed erythrocytes by splenic macrophages, while veno-occlusive disease has the potential to cause decreases in red cell mass through physical trauma and fragmentation. However, there is also evidence that in some severe cases of sickle cell disease there may be an increase in reticulocyte counts that are inappropriate for the magnitude of the decrease in red cell mass, suggesting a concurrent mechanism causing suppressed or ineffective erythropoiesis ( Wu et al., 2005 , Bookchin and Lew, 1996 ). Oxidative stress on erythroid precursors may also contribute to ineffective erythropoiesis in some severe cases of sickle cell disease ( Fibach and Rachmilewitz, 2008 ). Several metabolic defects of erythrocyte metabolism may also be associated with decreases in red cell mass and increases in reticulocyte counts. Deficiencies in erythrocyte pathways of glycolysis may result in decreased ATP concentrations that lead to erythrocyte membrane dysfunctions with shortened erythrocyte lifespan and occasionally hemolysis ( Stockham and Scott, 2008b ). Pyruvate kinase (PK) is the enzyme that catalyzes the last step in aerobic glycolysis. Deficiencies of PK that result in hemolysis have been reported in humans ( Baronciani and Beutler, 1993 ), dogs including beagles ( Harvey et al., 1977 , Giger et al., 1991 , Prasse et al., 1975 ), and a few breeds of cats ( Kohn and Fumi, 2008 ). Phosphofructokinase (PFK) catalyzes the rate-limiting step of the glycolysis pathway. Deficiencies in PFK have also been described in humans ( Etiemble et al., 1976 ) and dogs ( Giger et al., 1985 ). Respiratory alkalosis, which may be observed following intense exercise, is associated with acute hemolytic crises in patients with PFK deficiencies ( Giger et al., 1985 ). The association of inherited G6PD and FAD deficiencies with hemolysis resulting from oxidative damage is discussed earlier. In brief, G6PD and FAD play a role in the antioxidant pathways of erythrocytes. Deficiencies of G6PD and FAD may result in increased oxidative damage to erythrocytes and subsequent hemolysis. Collectively, the porphyrias are a group of enzymatic defects in the heme synthesis pathway. Porphyrias may be congenital or, more commonly, acquired. In these conditions, the accumulation of porphyrins, the precursors of heme, within erythrocytes leads to hemolysis. The mechanism of hemolysis may be related to lysis of erythrocytes following exposure to light (photolysis) in superficial vasculature, or by direct erythrocyte membrane damage due to the lipid soluble nature of porphyrins or following porphyrin absorption of ultraviolet light and excitation ( Phillips and Anderson, 2016 , Kaneko, 2008 ). 12.11.3.2.1.1.6 Neoplastic Various neoplastic conditions may be associated with hemolysis. Malignant metastatic neoplasms or primary vascular neoplasms may result in fragmentation hemolysis by physical trauma to erythrocytes, as previously discussed. However, neoplastic conditions may also rarely be associated with phagocytosis and destruction of erythrocytes, or hemophagocytic syndrome. Hemophagocytic syndromes have been associated with T-cell lymphoma ( Gonzalez et al., 1991 ), NK-cell leukemia ( Kobayashi et al., 1996 ), hemophagocytic histiocytic sarcoma ( Moore et al., 2006 ), and various hematological neoplasias ( Majluf-Cruz et al., 1998 ). 12.11.3.2.1.1.7 Xenobiotic-induced Many xenobiotics are capable of causing hemolysis, and may cause hemolysis through oxidative, fragmentation, or immune-mediated mechanisms. Examples of each are discussed here. Many of the agents that cause oxidative erythrocyte injury contain aromatic structures that can be metabolized, mostly commonly by cytochrome P 450, to free radicals ( Bradberry, 2003 , Edwards and Fuller, 1996 ), which overwhelm the normal protective antioxidant pathways of erythrocytes leading to both direct erythrocyte oxidative injury and oxidation of hemoglobin sulfhydryl groups resulting in methemoglobin formation. A few specific aromatic compounds that have been associated with free radical formation include dapsone, phenacetin, and anthracyclines such as doxorubicin ( Edwards and Fuller, 1996 , Coleman et al., 1991 , Handa and Sato, 1975 , Easley and Condon, 1974 ). Phenacetin has also been associated with the formation of Heinz bodies ( Boelsterli et al., 1983 ). In dogs and cats, acetaminophen (paracetamol) may be metabolized to a minor reactive metabolite that causes oxidative damage to erythrocytes resulting in hemolysis and the formation of Heinz bodies and/or eccentrocytes, although methemoglobinemia has also been observed in cats ( Desnoyers, 2010 , Wallace et al., 2002 , Mariani and Fulton, 2001 , Aronson and Drobatz, 1996 ). Xenobiotics that cause methemoglobinemia can also cause indirect oxidative damage through the peroxidation activity of methemoglobin itself ( Edwards and Fuller, 1996 ). In some cases, oxygenated hemoglobin may act as a peroxidase and cause the metabolism of a xenobiotic to a reactive compound that causes erythrocyte oxidative damage and conversion of oxyhemoglobin to methemoglobin. Examples of xenobiotics that cause oxidative damage through this mechanism are phenylhydrazine and primaquine ( Edwards and Fuller, 1996 ). Vitamin K administration in dogs can also cause oxidative erythrocyte damage through this mechanism ( Fernandez et al., 1984 ). Some chemical agents may cause oxidative damage by directly oxidizing hemoglobin sulfhydryl groups or through direct oxidation of erythrocyte cytoskeletal proteins. Arsine gas, predominantly an environmental toxin, appears to mediate its hemolytic effects through erythrocyte membrane oxidation ( Rael et al., 2000 ), although studies in mice have also demonstrated the formation of Heinz bodies following exposure ( Blair et al., 1990 ), suggesting an oxidative effect on hemoglobin as well. Many xenobiotics may also cause hemolysis through their association with microangiopathy, most commonly as part of the thrombotic microangiopathy syndrome, which is associated with fragmentation hemolysis and decreases in platelet counts. Drug-induced endothelial injury, including from direct and antibody or immune complex-mediated mechanisms, plays a major role in the pathogenesis of thrombotic microangiopathy ( Pisoni et al., 2001 ). Endothelial damage may be propagated by leukocyte adhesion and release of granule contents or reactive oxygen species, platelet activation and aggregation, and complement activation ( Pisoni et al, 2001 ). Drugs implicated in thrombotic microangiopathy include chemotherapeutic agents include xenobiotics from a wide variety of chemotherapeutic classes. Examples of chemotherapeutics associated with thrombotic microangiopathy include mitomycin C ( Cantrell et al., 1985 ), cisplatin ( Palmisano et al., 1998 ), estramustine phosphate ( Tassinari et al., 1999 ), gemcitabine ( Nackaerts et al., 1998 ), and daunorubicin ( Byrnes et al., 1986 ). Nonchemotherapeutic agents that have been reported to cause thrombotic microangiopathy include immunomodulators such as cyclosporine and tacrolimus ( Katznelson et al., 1994 , Trimarchi et al., 1999 ), simvastatin ( McCarthy et al., 1998 ), and inhibitors of platelet aggregation including ticlopidine and clopidogrel ( Bennett et al., 1998 , Bennett et al., 2000 ). Immune-mediated mechanisms of hemolysis have also been reported following exposure to numerous xenobiotics. Xenobiotics may induce antibodies by binding to the erythrocyte membrane and acting as haptens. These antibodies are considered drug-dependent as they only mediate hemolysis when the drug is present. Penicillin is the prototypical xenobiotic that acts as a hapten to generate drug-dependent antibodies, and typically induces an IgG response ( Ferner, 2012 , Petz et al., 1966 ). Semisynthetic penicillins, some cephalosporins, and tetracycline have also been reported to cause drug-dependent antibody-mediated hemolysis ( Garratty, 2010 , Tuffs and Manoharan, 1986 , Seldon et al., 1982 , Großjohann et al., 2004 , Gallagher et al., 1992 , Branch et al., 1985 , Simpson et al., 1985 ). Xenobiotics may also induce the production of antierythrocyte antibodies that mediate hemolysis even when the drug is no longer present, also called drug-independent antibodies or autoantibodies. In this type of hemolysis, xenobiotic exposure stimulates production of an antibody that can bind to native erythrocyte antigens even in the absence of the drug. This type of immune-mediated xenobiotic-induced hemolysis is classically caused by α-methyldopa, and is characterized by predominantly an IgG response ( Packman, 2016 ). However, nucleoside purine analogs such as cladribine and fludarabine have also been associated with hemolysis due to production of autoimmune antibodies ( Garratty, 2010 , Mintzer et al., 2009 , Hamblin, 2006 ). A third mechanism by which xenobiotics may cause immune-mediated hemolysis is through a complex interaction of the drug, a drug binding site on erythrocytes, and an antibody. This mechanism is considered the ternary complex mechanism, but has previously, and perhaps less accurately, been called an immune complex or innocent bystander mechanism ( Packman, 2016 ). Quinidine is the prototypical drug that causes hemolysis via this mechanism. Quinidine may be associated with either IgM or IgG antibodies and predominantly causes complement-mediated lysis of erythrocytes or clearance of complement-coated erythrocytes by tissue macrophages ( Packman, 2016 ). Ceftriaxone has also been reported to cause hemolysis through this mechanism ( Arndt and Garratty, 2005 ). Xenobiotic-induced immune-mediated hemolysis may not be limited to one of the three mechanisms described earlier, and a combination of these mechanisms may occur in some patients. For example, the NSAID diclofenac may cause hemolysis through both drug-dependent and drug-independent mechanisms ( Salama et al., 1996 ). Carboplatin has been reported to cause hemolysis through all three immune-mediated mechanisms ( Marani et al., 1996 ). Other compounds may cause hemolysis through mechanisms other than oxidative, microangiopathic, or immune-mediated. For example, although the primary effect of lead toxicity is impairment of heme synthesis, lead may also cause hemolysis. The mechanism of lead-induced hemolysis has not been fully determined, but interference with the erythrocyte membrane sodium/potassium transporter may be involved ( Bloom and Brandt, 2008 ). Copper toxicity causes hemolysis as well, possibly through inhibition of many enzymes involved in glycolysis resulting in decreased intracellular ATP ( Boulard et al., 1972 ). Envenomation from multiple animals is reported to cause hemolysis. Envenomation by snakes, such as rattlesnakes and coral snakes, can cause hemolysis through phospholipase A2 activity, which may cause direct hemolysis or liberate hemolysins such as lysolethicin, or through complement-mediated hemolysis ( Arce-Bejarano et al., 2014 , Tambourgi and van den Berg, 2014 , Walton et al., 1997 ). 12.11.3.2.1.2 Blood loss 12.11.3.2.1.2.1 Hemorrhage Hemorrhage may cause internal or external blood loss. Due to the loss of whole blood during hemorrhage, decreases in red cell mass are usually accompanied by proportionate decreases in albumin and globulin concentrations. The decreases in plasma proteins tend to be less pronounced with internal hemorrhage because the lost proteins may be resorbed in lymph and returned to blood ( Stockham and Scott, 2008b ). Cases of internal hemorrhage are typically not associated with iron deficiency. However, prolonged external blood loss may cause depletion of total body iron. Iron deficiency anemia is characterized by small erythrocytes with a decrease in MCV and erythrocytes that contain less hemoglobin with a decrease in MCHC, and may be classified as a microcytic, hypochromic anemia. Hemoglobin synthesis plays a role in inhibiting erythrocyte division, and when sufficient iron is not available for heme production, there is loss of the inhibitory effect resulting in more cell divisions and microcytes ( Stohlman et al., 1963 ). Hypochromasia of the erythrocytes is due to the lower than normal hemoglobin content due to decreased production of heme. Morphologic erythrocyte changes that accompany iron deficiency anemia include visual microcytosis and hypochromasia, keratocytes and schistocytes from physical damage to the more fragile erythrocytes, and sometimes codocytes (also called target cells) that have a thin rim of pink-staining hemoglobin and a small central area of hemoglobin with a ring of pallor in between, typical of erythrocytes with less hemoglobin present relative to the amount of membrane. In chronic iron deficiency, increases in reticulocyte counts and microscopic polychromasia may be lower than expected for a regenerative anemia due to loss of RNA during the extended maturation phase of erythrocyte production caused by decreased hemoglobin content ( Burkhard et al., 2001 ). Direct damage to blood vessels from trauma is a relatively common cause of acute external or internal blood loss. Traumatic rupture of the spleen may also cause significant acute internal blood loss. Decreases in red cell mass due to acute hemorrhage are typically due to dilution of remaining blood from shifting of intracellular fluid to extracellular fluid in an attempt to preserve blood volume and therefore tissue perfusion ( Stockham and Scott, 2008b ). Dilution of red cell mass may also be observed following administration of intravenous fluids to replace blood volume. A detectable increase in reticulocyte count is expected 3–4 days following the acute event in a patient with normally functioning bone marrow. Damage to blood vessels that results in hemorrhage also may occur secondary to ulcerative or neoplastic conditions. In dogs, rupture of splenic hemangiosarcoma is a common cause of internal blood loss into the abdomen (hemoabdomen). Ulceration of the gastrointestinal system may lead to blood loss into feces, which can be observed as black, tarry feces (melena) if the ulceration occurs in the small intestines or as frank blood if the ulceration occurs in the large intestines. In humans and nonhuman primate species with true menstrual cycles, including Old World monkeys and great apes ( Provencher Bolliger et al., 2010 ), decreases in red cell mass are uncommon but may be observed from menses-related blood loss. In women, heavy blood loss, abnormal cycling, or uterine neoplasms may lead to sufficient blood loss to cause decreases in red cell mass and potentially even iron deficiency ( Van Voorhis, 2009 , Goel and Gupta, 2007 ). In cynomolgus monkeys, decreases in red cell mass have been occasionally observed in females with prolonged menses ( Perigard et al., 2016 ). Coagulation disorders may also be associated with either internal or external hemorrhage. Primary deficiencies in coagulation factors or von Willebrand factor may be inherited causes of hemorrhage. Deficiencies in coagulation factors that lead to hemorrhage sufficient to cause decreases in red cell mass include hemophilia A (factor VIII deficiency) and hemophilia B (factor IX deficiency); deficiencies in factor XI and von Willebrand factor are usually mild and are often not associated with notable hemorrhage ( Bolton-Maggs and Pasi, 2003 ). Hemorrhage may be secondary to marked decreases in platelet counts from consumptive coagulopathies secondary to infectious or neoplastic processes. Although not truly hemorrhage, external blood loss can occur from repeated phlebotomy. Decreases in red cell mass may be acutely observed following collection of blood from donors for transfusion, and regular donors have a risk of developing iron deficiency from repeated external blood loss ( Cable et al., 2011 ). Repeated phlebotomy is a common occurrence in nonclinical toxicology studies, particularly in dogs and nonhuman primates, although rats may also occasionally undergo repeated blood collections. Blood is collected through the studies mainly for toxicokinetic or pharmacokinetic analysis, but also for analysis of hematology, coagulation, and clinical chemistry profiles. Decreases in red cell mass with increases in reticulocyte counts of similar magnitude relative to pretest values across all treatment groups, including controls, are a common procedure-related phenomenon in nonclinical toxicology studies and should be distinguished from a true test article-related effect. 12.11.3.2.1.2.2 Parasitism Both external and internal parasites may contribute to blood loss. Hookworms are a major internal parasite associated with chronic blood loss, and may lead to iron deficiency with prolonged infections ( Stoltzfus et al., 1997 ). However, whipworm infection and schistosomiasis may also be associated with blood loss, the latter being associated with blood loss through the urinary system ( Farid et al., 1969 ). Heavy infestation of animals with arthropods that take blood meals, such as ticks, some lice, and fleas, may also cause sufficient blood loss to result in decreases in red cell mass ( Stockham and Scott, 2008b ). 12.11.3.2.1.2.3 Xenobiotic-induced Xenobiotic-induced blood loss is relatively uncommon but can occur. Classically, hemorrhage into the intestinal tract can result from ulceration associated with chronic NSAID or coxib administration ( Laine et al., 2003 , Langman et al., 1999 , Bjarnason et al., 1987 ). Also, prolonged or high dose administration of anticoagulants, such as warfarin or heparin, can result in hemorrhage-related decreases in red cell mass ( Levine et al., 2001 ). Ingestion of rodenticides, including brodifacoum chlorophacinone, has been reported to cause marked hemorrhage in humans and many other nonrodent species ( Berny et al., 2010 , Palmer et al., 1999 , Sheafor and Couto, 1999 ). Xenobiotic-induced marked decreases in platelet counts may also be associated with hemorrhage and are discussed in more detail later. However, chemotherapeutics that cause bone marrow suppression can be associated with spontaneous or postvenipuncture hemorrhage. Occasional idiopathic decreases in platelet counts have also been observed following xenobiotic administration and are most likely attributable to immune-mediated destruction; some examples of implicated xenobiotics are quinine, trimethoprim–sulfamethoxazole, anticonvulsants such as phenytoin and carbamazepine, unfractionated or low molecular weight heparin, and rituximab ( Aster and Bougie, 2007 ). 12.11.3.2.1.1 Hemolysis Destruction of mature erythrocytes is called hemolysis. Hemolysis may occur either intravascularly or extravascularly. With intravascular hemolysis, erythrocyte destruction occurs within the blood and results in hemoglobinemia, or free hemoglobin within plasma. Ghost erythrocytes, or the remnant membranes of erythrocytes that no longer contain cytoplasm or hemoglobin, may be observed with intravascular hemolysis. Consequent hemoglobinuria, or free hemoglobin in the urine, is rare and only occurs in cases of massive intravascular hemolysis that overwhelm the normal pathways that clear free hemoglobin from the blood. In contrast, extravascular hemolysis does not occur within the blood, but rather occurs in the spleen, liver, or bone marrow, where resident macrophages phagocytose erythrocytes and destroy them intracellularly. Extravascular hemolysis does not result in free plasma hemoglobin or hemoglobinuria. Both types of hemolysis may be associated with increases in total bilirubin concentrations where unconjugated (indirect) bilirubin usually exceeds conjugated (direct) bilirubin, and may result in plasma or serum icterus (yellow discoloration) or bilirubinuria (bilirubin present in urine). However, not all cases of hemolysis are clearly either intravascular or extravascular, and both forms of hemolysis may contribute in some conditions. 12.11.3.2.1.1.1 Infectious There are numerous protozoal, bacterial, and viral diseases that can be associated with hemolysis. Mechanisms by which infectious agents cause erythrocyte destruction are varied, and may include direct infection of erythrocytes, elaboration of toxins such as hemolysin, or stimulation of an immune-mediated response against infected cells ( Berkowitz, 1991 ). Several examples of infectious agents that cause hemolytic anemia are discussed later. Direct infection of erythrocytes with protozoal Plasmodium species, the causative agent of malaria that is transmitted by mosquitoes, is a relatively common cause of hemolysis in humans, but may also be observed in nonhuman primates used in nonclinical toxicology studies. Humans are infected by one of five different Plasmodium species: P . falciparum , P . vivax , P . knowlesi , P . malariae , or P . ovale , although only P . falciparum and P . vivax are commonly associated with severe hemolysis ( Lichtman, 2016b ). Macaques are most commonly infected with P . inui or P . knowlesi , although the cynomolgus monkey appears to be more resistant to disease from these infections than the rhesus monkey ( Ameri, 2010 ). Infection with P . cynomolgi , P . fieldi , or P . fragile may also occur in macaques ( Magden et al., 2015 ). Although it is uncommon to include macaques infected with Plasmodium species during a nonclinical toxicity study due to current screening practices and pretest evaluations, rare animals with decreases in red cell mass and increases in reticulocyte counts and intraerythrocytic Plasmodium organisms have been observed. Rats and mice may be infected with Plasmodium berghei ( Holloway et al., 1995 , Sadun et al., 1965 ). Plasmodium berghei has a specific tropism for reticulocytes rather than mature erythrocytes Plasmodium species that infect humans ( Car et al., 2006 , Cromer et al., 2006 ). Concurrent increases in reticulocyte counts may occur in early stages or disease or with recrudescence of parasitemia and hemolysis. Hemolysis is associated with clearance of parasitized erythrocytes from circulation predominantly by splenic macrophages ( Lichtman, 2016b ), although accumulation of hemin, an iron-containing porphyrin, which can directly stimulate apoptotic erythrocyte death (eryptosis) ( Gatidis et al., 2009 ), oxidative damage to erythrocyte membranes ( Clark and Hunt, 1983 ), and increased osmotic fragility ( George et al., 1967 ) may all contribute to hemolysis. However, late-stage infections in humans and rodents have also been associated with inappropriate or decreased reticulocyte counts indicative of suppressed erythropoiesis despite decreases in red cell mass from hemolysis ( Lichtman, 2016b , Cromer et al., 2006 ). Babesia species are tick-borne protozoal organisms that directly infect erythrocytes in most species, including humans, nonhuman primates, dogs, and cats. Babesia species appear as intracellular oval to pyriform organisms. Babesia microti and Babesia divergens may infect humans in North America and Europe, respectively, and cause moderate hemolytic anemia from intraerythrocytic replication and subsequent erythrocyte lysis ( Lichtman, 2016b , Kjemtrup and Conrad, 2000 ). Babesia pitheci has been reported to infect both old and new world monkeys and cause anemia ( Magden et al., 2015 ). B . canis , a large babesial species, and B . gibsoni , a small babesial species, infect dogs, while cats may be infected by the small babesial organisms B . felis and B . cati ( Stockham and Scott, 2008b , Penzhorn et al., 2004 ). These organisms are generally not of concern in purpose-bred animals used in nonclinical toxicology studies. Bartonella bacilliformis in people and the hemotrophic mycoplasmas (hemoplasmas) in dogs and cats (formerly Haemobartonella species) and swine (formerly Eperythrozoon species) are organisms that parasitize erythrocytes, but these organisms remain extracellular in shallow depressions of the erythrocyte membrane. These organisms are typically round-, rod-, or ring-shaped and may be observed individually or in chains on erythrocyte surfaces. Hemolysis with these organisms may be immune-mediate and associated either with binding of antibodies to parasite antigens or antigens exposed on the erythrocyte secondary to parasite-induced membrane changes ( Stockham and Scott, 2008b ). Clostridium perfringens (formerly Clostridium welchii ) infection in humans is an example of a bacterial cause of hemolysis. During intestinal overgrowth or septicemia, C . perfringens type A elaborates an α toxin that has lecithinase C activity, resulting in membrane phospholipid breakdown and release of lysolethicins, which have potent hemolytic capabilities ( Lichtman, 2016b , Songer, 1996 ). C . perfringens α toxin release is usually associated with severe intravascular hemolysis with both hemoglobinemia and hemoglobinuria. However, in veterinary species, C . perfringens -related hemolysis is typically limited to ruminants and horses ( Stockham and Scott, 2008b ), and is unlikely to be observed in the common species used in nonclinical toxicology studies. Infection of humans with Mycoplasma pneumoniae has also been associated with hemolytic decreases in red cell mass, although most cases of M . pneumoniae infection are asymptomatic. Hemolysis with this organism is attributable to stimulation of autoimmune erythrocyte destruction with agglutination of erythrocytes ( Khan and Yassin, 2009 ). Several viral organisms in humans have also been reported to cause decreases in red cell mass due to hemolysis. Viral causes of hemolysis are commonly associated with autoimmune mechanisms, and include infection with Epstein-Barr virus ( Palanduz et al., 2002 ), hepatitis A, B, and C viruses ( Kanematsu et al., 1996 , Chao et al., 2001 ), cytomegalovirus ( Murray et al., 2001 ), and HIV ( Koduri et al., 2002 ), although HIV infection is also commonly associated with decreases in reticulocyte counts rather than the expected increases secondary to hemolysis, indicative of concurrent suppressed erythropoiesis ( Telen et al., 1990 ). 12.11.3.2.1.1.2 Oxidative Another major cause of decreases in red cell mass due to hemolysis is oxidative damage to erythrocytes. Under normal conditions, ferrous iron (Fe 2 + ) in hemoglobin binds to and dissociates from oxygen as it delivers oxygen from the lungs to the tissues. At times, this binding and dissociation results in the formation of ferric iron (Fe 3 + ) in hemoglobin (methemoglobin) as well as superoxide (O 2 − ). Superoxide is a free radical with potent oxidative capacity that may cause cellular damage. Cytochrome b 5 -reductase is an intraerythrocytic enzyme that converts methemoglobin back to hemoglobin. Superoxide dismutase converts superoxide to hydrogen peroxide (H 2 O 2 ), which also may produce oxidative damage to cells. Further metabolism of hydrogen peroxide by catalase or glutathione peroxidase protects cells from oxidative damage. These pathways are usually sufficient to address the normal low-level formation of methemoglobin and superoxide, but methemoglobin can increase and impair delivery of oxygen to tissues and superoxide can accumulate and cause oxidative damage if these pathways are overwhelmed or defective. Oxidative damage to erythrocytes may affect the lipid membranes, cytoskeleton, or hemoglobin. Peroxidation of internal membrane lipids or cytoskeletal components of erythrocytes results in the fusion of portions of the membrane with consequent shifting of the cytoplasm and hemoglobin to one side of the cells. Erythrocytes with this morphologic change are called eccentrocytes. Oxidative damage that causes the formation of eccentrocytes may result in hemolysis due to increased clearance of eccentrocytes by splenic macrophages due to trapping of rigid erythrocytes in splenic sinusoids or spontaneous rupture in blood due to the increased fragility of eccentrocytes ( Stockham and Scott, 2008b ). Oxidative damage to exposed cysteine sulfhydryl groups on hemoglobin results in hemoglobin denaturation and decreased solubility ( Bloom and Brandt, 2008 ). Denatured hemoglobin may then precipitate and aggregate within the erythrocyte, forming small, pale-staining round structures that bind to the erythrocyte membrane and tend to protrude from the surface of the erythrocyte. These aggregates of denatured hemoglobin are called Heinz bodies. Cats appear to be particularly sensitive to the formation of Heinz bodies because of an increased number of reactive sulfhydryl groups in hemoglobin relative to other species ( Christopher et al., 1990 ), and may be more rapidly observed on blood smear evaluation due to the nonsinusoidal architecture of the feline spleen that results in decreased clearance of Heinz bodies from circulation. Similar to eccentrocytes, erythrocytes with Heinz bodies may undergo hemolysis due to increased clearance by splenic macrophages following trapping in splenic sinusoids due to decreased erythrocyte deformability and spontaneous rupture due to increased fragility from membrane damage; immune-mediated clearance may also occur and is believed to result from binding of hemochromes to and subsequent redistribution of band 3, an erythrocyte membrane structural protein, which may then be recognized by autologous antibodies ( Winterbourn, 1990 ). There are many conditions that may cause oxidative damage and result in eccentrocytosis, Heinz body formation, or even both simultaneously. Diabetes mellitus may cause either morphologic change, and diabetic ketoacidosis appears to be associated with an increased susceptibility and incidence of oxidative erythrocyte damage ( Desnoyers, 2010 , Caldin et al., 2005 , Christopher et al., 1995 ). Inherited deficiencies in erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and flavin adenine dinucleotide (FAD) have also been associated with erythrocyte oxidative damage, eccentrocyte or Heinz body formation, and hemolysis or a predisposition for these events due to the loss of protective antioxidant pathways ( Chan et al., 1982 , Harvey, 2006 ). Lymphoma has also been associated with Heinz body formation in cats ( Christopher, 1989 ) and eccentrocytes formation in dogs ( Caldin et al., 2005 ). In dogs and cats, ingestion of Allium species, particularly onions, garlic, and Chinese chive, may cause erythrocyte oxidative damage with formation of eccentrocytes and/or Heinz bodies ( Caldin et al., 2005 , Yamato et al., 2005 , Robertson et al., 1998 ). Ingestion of zinc in dogs ( Bexfield et al., 2007 ) and exposure to skunk musk ( Fierro et al., 2013 ) have also been reported to cause hemolysis due to Heinz body formation. 12.11.3.2.1.1.3 Fragmentation Physical trauma to erythrocytes results in hemolysis due to erythrocyte fragmentation and lysis. Sometimes this type of hemolysis is referred to as microangiopathic hemolytic anemia. Morphologic changes to erythrocytes occur as a result of physical trauma. Schistocytes (also called schizocytes or erythrocyte fragments), keratocytes (also called helmet cells), prekeratocytes (also called blister cells), or even spherocytes or microspherocytes may be observed. Schistocytes are very small, usually irregularly shaped fragments that can break off erythrocytes when physical trauma occurs. Keratocytes have one to two variably sized projections or horns adjacent to a small flattened region of the erythrocyte surface, while prekeratocytes appear to be precursors that have small loops of erythrocyte cytoplasm extending from the surface and surrounding a small hole in the cell. Spherocytes and microspherocytes are spherical cells that appear smaller and have more intensely pink-staining cytoplasm than normal mature erythrocytes. Spherocytes and microspherocytes may be formed during physical trauma as fragments are broken off, resulting in less membrane surface area in the parent erythrocyte surrounding a similar volume (spherocytes) or smaller volume (microspherocytes) as the parent erythrocyte. The physical trauma to erythrocytes that causes fragmentation or microangiopathic hemolysis may result from consumptive coagulopathies, either local or disseminated (DIC), with fibrin or thrombus formation in the vasculature that impedes the passage of erythrocytes through the vessel lumen, creating both turbulence and physical obstruction of blood flow. Local coagulopathy or DIC may occur secondary to trauma, infections with sepsis, or neoplasia ( Baker and Moake, 2016 , Toh and Dennis, 2003 ). Microangiopathic hemolysis due to neoplasia is most commonly associated with malignant rather than benign neoplasms and with metastatic disease or neoplasic emboli rather than primary tumors, with the exception of primary vascular neoplasms ( Susano et al., 1994 , Kupers et al., 1975 , Lohrmann et al., 1973 ). Infectious agents may also lead to fragmentation of erythrocytes, and some Leptospirosis interrogans serovars associated with vasculitis ( Stockham and Scott, 2008b ), Brucella species infection ( Yaramis et al., 2001 ), and cutaneous anthrax ( Freedman et al., 2002 ) have been reported to cause microangiopathic hemolysis. In children, fragmentation hemolysis associated with thrombotic microangiopathy may occur with Shigella dysenteriae type 1 and some Escherichia coli infections ( Pisoni et al., 2001 ). Hemolysis from erythrocyte fragmentation may also occur with HIV infection ( Maslo et al., 1997 ). Decreases in red cell mass with increases in reticulocyte counts from erythrocyte fragmentation may also occur secondary to cardiac or other conditions that alter hemodynamics and increase turbulent blood flow. For example, subaortic stenosis ( Solanki and Sheikh, 1978 ), intraluminal aortic grafts ( Sayar et al., 2006 ), uncorrected cardiac valvular disease ( Marsh and Lewis, 1969 ), prosthetic valves ( Crexells et al., 1972 ), and hypertrophic obstructive cardiomyopathy ( Kubo et al., 2010 ) have all been reported to cause hemolysis from erythrocyte fragmentation. Increased turbulence associated with hypertension may also cause decreases in red cell mass from fragmentation, and has been associated with pulmonary hypertension ( Baker and Moake, 2016 ) and malignant systemic hypertension ( Capelli et al., 1966 ). 12.11.3.2.1.1.4 Immune-mediated Autoimmune hemolytic anemia (AIHA or AHA) described in humans or immune-mediated hemolytic anemia (IMHA) described in most common laboratory species is a cause of hemolysis, and may be primary or idiopathic, but may also be secondary to conditions such as infections as discussed previously. Primary or idiopathic AIHA/IMHA is discussed here. Primary AIHA has no underlying detectable cause and is an immune-mediated condition that produces antibodies targeting erythrocyte antigens. These antierythrocyte antibodies tend to be very specific for a single erythrocyte antigen in a given case ( Packman, 2016 ). These autoantibodies may be classified as warm antibodies, which are usually IgG, or cold antibodies, which are usually IgM ( Stockham and Scott, 2008b ). Immune-mediated AIHA may be associated with erythrocyte morphologic changes that include agglutination and spherocytes. Agglutination may be observed grossly as red speckling along the inside of the specimen tube as blood is gently moved within the tube. If agglutination is present, blood smears may have a "reverse smear" appearance with the densest region of the smear observed at the feathered edge rather than the edge where the drop of blood was initially placed. Microscopically agglutination appears as grape-like clusters of erythrocytes. Spherocytes are erythrocytes that are spherical instead of having the normal biconcave disc shape. While spherocytes appear smaller and stain more intensely pink that unaffected mature erythrocytes, they have the same volume as unaffected erythrocytes. Loss of erythrocyte membrane occurs when macrophages begin to phagocytize antibody-bound erythrocytes, leading to decreased erythrocyte surface area without an appreciable change in volume, forcing erythrocytes to form spheres. Hence, spherocytosis alone will not result in an altered MCV. Of the most common laboratory species, dogs tend to have the most pronounced central pallor of normal mature erythrocytes, making microscopic identification of spherocytes easiest in the dog. Hemolysis in AIHA is largely attributable to extravascular hemolysis due to phagocytosis of antibody-bound erythrocytes by tissue macrophages. Macrophages or monocytes containing phagocytized erythrocytes may be rarely observed in blood smears of laboratory species with immune-mediated hemolysis. However, antibody-mediated complement activation or increased fragility of spherocytes may result in direct intravascular lysis or rupture of erythrocytes ( Packman, 2016 ). Evaluation of patients for the presence of antierythrocyte antibodies may be performed using the direct antiglobulin test (DAT; also called the Coombs' test) or by flow cytometry. AIHA has rarely been observed in association with lymphoproliferative neoplasia, such as chronic lymphocytic leukemia. Antierythrocyte antibodies in chronic lymphocytic leukemia are predominantly IgG with few cases of IgM reported ( Mauro et al., 2000 ). IMHA may occasionally be observed following blood transfusion ( Garratty, 2004 ). This may occur in response to alloantigens, and would not technically be considered autoimmune ( Stockham and Scott, 2008b ). Posttransfusion immune-mediated hemolysis may also be observed when the host has autoantibodies that bind the donor erythrocytes and cause immune-mediated destruction. However, crossmatching of host and donor erythrocytes and plasma is able to prevent many cases with incompatible transfusion-related AIHA. AIHA and IMHA commonly have concurrent inflammatory increases in leukocyte subtype counts, characterized mainly by neutrophilia that may or may not have a left shift with cytoplasmic changes indicative of rapid neutropoiesis. 12.11.3.2.1.1.5 Inherited Some phenotypes of sickle cell disease are associated with hemolysis. The mechanism of hemolysis in sickle cell disease is likely multifactorial and not associated with a single pathogenesis. There is evidence for oxidative damage to erythrocytes ( Lachant et al., 1983 ), which may contribute to the hemolysis observed with sickle cell disease. However, hemoglobin polymerization leads to erythrocyte deformation and may lead to decreased flexibility of erythrocytes and veno-occlusive disorders ( Bookchin and Lew, 1996 ). Decreased flexibility or deformability of erythrocytes may contribute directly to increased cell fragility and rupture or promote clearance of deformed erythrocytes by splenic macrophages, while veno-occlusive disease has the potential to cause decreases in red cell mass through physical trauma and fragmentation. However, there is also evidence that in some severe cases of sickle cell disease there may be an increase in reticulocyte counts that are inappropriate for the magnitude of the decrease in red cell mass, suggesting a concurrent mechanism causing suppressed or ineffective erythropoiesis ( Wu et al., 2005 , Bookchin and Lew, 1996 ). Oxidative stress on erythroid precursors may also contribute to ineffective erythropoiesis in some severe cases of sickle cell disease ( Fibach and Rachmilewitz, 2008 ). Several metabolic defects of erythrocyte metabolism may also be associated with decreases in red cell mass and increases in reticulocyte counts. Deficiencies in erythrocyte pathways of glycolysis may result in decreased ATP concentrations that lead to erythrocyte membrane dysfunctions with shortened erythrocyte lifespan and occasionally hemolysis ( Stockham and Scott, 2008b ). Pyruvate kinase (PK) is the enzyme that catalyzes the last step in aerobic glycolysis. Deficiencies of PK that result in hemolysis have been reported in humans ( Baronciani and Beutler, 1993 ), dogs including beagles ( Harvey et al., 1977 , Giger et al., 1991 , Prasse et al., 1975 ), and a few breeds of cats ( Kohn and Fumi, 2008 ). Phosphofructokinase (PFK) catalyzes the rate-limiting step of the glycolysis pathway. Deficiencies in PFK have also been described in humans ( Etiemble et al., 1976 ) and dogs ( Giger et al., 1985 ). Respiratory alkalosis, which may be observed following intense exercise, is associated with acute hemolytic crises in patients with PFK deficiencies ( Giger et al., 1985 ). The association of inherited G6PD and FAD deficiencies with hemolysis resulting from oxidative damage is discussed earlier. In brief, G6PD and FAD play a role in the antioxidant pathways of erythrocytes. Deficiencies of G6PD and FAD may result in increased oxidative damage to erythrocytes and subsequent hemolysis. Collectively, the porphyrias are a group of enzymatic defects in the heme synthesis pathway. Porphyrias may be congenital or, more commonly, acquired. In these conditions, the accumulation of porphyrins, the precursors of heme, within erythrocytes leads to hemolysis. The mechanism of hemolysis may be related to lysis of erythrocytes following exposure to light (photolysis) in superficial vasculature, or by direct erythrocyte membrane damage due to the lipid soluble nature of porphyrins or following porphyrin absorption of ultraviolet light and excitation ( Phillips and Anderson, 2016 , Kaneko, 2008 ). 12.11.3.2.1.1.6 Neoplastic Various neoplastic conditions may be associated with hemolysis. Malignant metastatic neoplasms or primary vascular neoplasms may result in fragmentation hemolysis by physical trauma to erythrocytes, as previously discussed. However, neoplastic conditions may also rarely be associated with phagocytosis and destruction of erythrocytes, or hemophagocytic syndrome. Hemophagocytic syndromes have been associated with T-cell lymphoma ( Gonzalez et al., 1991 ), NK-cell leukemia ( Kobayashi et al., 1996 ), hemophagocytic histiocytic sarcoma ( Moore et al., 2006 ), and various hematological neoplasias ( Majluf-Cruz et al., 1998 ). 12.11.3.2.1.1.7 Xenobiotic-induced Many xenobiotics are capable of causing hemolysis, and may cause hemolysis through oxidative, fragmentation, or immune-mediated mechanisms. Examples of each are discussed here. Many of the agents that cause oxidative erythrocyte injury contain aromatic structures that can be metabolized, mostly commonly by cytochrome P 450, to free radicals ( Bradberry, 2003 , Edwards and Fuller, 1996 ), which overwhelm the normal protective antioxidant pathways of erythrocytes leading to both direct erythrocyte oxidative injury and oxidation of hemoglobin sulfhydryl groups resulting in methemoglobin formation. A few specific aromatic compounds that have been associated with free radical formation include dapsone, phenacetin, and anthracyclines such as doxorubicin ( Edwards and Fuller, 1996 , Coleman et al., 1991 , Handa and Sato, 1975 , Easley and Condon, 1974 ). Phenacetin has also been associated with the formation of Heinz bodies ( Boelsterli et al., 1983 ). In dogs and cats, acetaminophen (paracetamol) may be metabolized to a minor reactive metabolite that causes oxidative damage to erythrocytes resulting in hemolysis and the formation of Heinz bodies and/or eccentrocytes, although methemoglobinemia has also been observed in cats ( Desnoyers, 2010 , Wallace et al., 2002 , Mariani and Fulton, 2001 , Aronson and Drobatz, 1996 ). Xenobiotics that cause methemoglobinemia can also cause indirect oxidative damage through the peroxidation activity of methemoglobin itself ( Edwards and Fuller, 1996 ). In some cases, oxygenated hemoglobin may act as a peroxidase and cause the metabolism of a xenobiotic to a reactive compound that causes erythrocyte oxidative damage and conversion of oxyhemoglobin to methemoglobin. Examples of xenobiotics that cause oxidative damage through this mechanism are phenylhydrazine and primaquine ( Edwards and Fuller, 1996 ). Vitamin K administration in dogs can also cause oxidative erythrocyte damage through this mechanism ( Fernandez et al., 1984 ). Some chemical agents may cause oxidative damage by directly oxidizing hemoglobin sulfhydryl groups or through direct oxidation of erythrocyte cytoskeletal proteins. Arsine gas, predominantly an environmental toxin, appears to mediate its hemolytic effects through erythrocyte membrane oxidation ( Rael et al., 2000 ), although studies in mice have also demonstrated the formation of Heinz bodies following exposure ( Blair et al., 1990 ), suggesting an oxidative effect on hemoglobin as well. Many xenobiotics may also cause hemolysis through their association with microangiopathy, most commonly as part of the thrombotic microangiopathy syndrome, which is associated with fragmentation hemolysis and decreases in platelet counts. Drug-induced endothelial injury, including from direct and antibody or immune complex-mediated mechanisms, plays a major role in the pathogenesis of thrombotic microangiopathy ( Pisoni et al., 2001 ). Endothelial damage may be propagated by leukocyte adhesion and release of granule contents or reactive oxygen species, platelet activation and aggregation, and complement activation ( Pisoni et al, 2001 ). Drugs implicated in thrombotic microangiopathy include chemotherapeutic agents include xenobiotics from a wide variety of chemotherapeutic classes. Examples of chemotherapeutics associated with thrombotic microangiopathy include mitomycin C ( Cantrell et al., 1985 ), cisplatin ( Palmisano et al., 1998 ), estramustine phosphate ( Tassinari et al., 1999 ), gemcitabine ( Nackaerts et al., 1998 ), and daunorubicin ( Byrnes et al., 1986 ). Nonchemotherapeutic agents that have been reported to cause thrombotic microangiopathy include immunomodulators such as cyclosporine and tacrolimus ( Katznelson et al., 1994 , Trimarchi et al., 1999 ), simvastatin ( McCarthy et al., 1998 ), and inhibitors of platelet aggregation including ticlopidine and clopidogrel ( Bennett et al., 1998 , Bennett et al., 2000 ). Immune-mediated mechanisms of hemolysis have also been reported following exposure to numerous xenobiotics. Xenobiotics may induce antibodies by binding to the erythrocyte membrane and acting as haptens. These antibodies are considered drug-dependent as they only mediate hemolysis when the drug is present. Penicillin is the prototypical xenobiotic that acts as a hapten to generate drug-dependent antibodies, and typically induces an IgG response ( Ferner, 2012 , Petz et al., 1966 ). Semisynthetic penicillins, some cephalosporins, and tetracycline have also been reported to cause drug-dependent antibody-mediated hemolysis ( Garratty, 2010 , Tuffs and Manoharan, 1986 , Seldon et al., 1982 , Großjohann et al., 2004 , Gallagher et al., 1992 , Branch et al., 1985 , Simpson et al., 1985 ). Xenobiotics may also induce the production of antierythrocyte antibodies that mediate hemolysis even when the drug is no longer present, also called drug-independent antibodies or autoantibodies. In this type of hemolysis, xenobiotic exposure stimulates production of an antibody that can bind to native erythrocyte antigens even in the absence of the drug. This type of immune-mediated xenobiotic-induced hemolysis is classically caused by α-methyldopa, and is characterized by predominantly an IgG response ( Packman, 2016 ). However, nucleoside purine analogs such as cladribine and fludarabine have also been associated with hemolysis due to production of autoimmune antibodies ( Garratty, 2010 , Mintzer et al., 2009 , Hamblin, 2006 ). A third mechanism by which xenobiotics may cause immune-mediated hemolysis is through a complex interaction of the drug, a drug binding site on erythrocytes, and an antibody. This mechanism is considered the ternary complex mechanism, but has previously, and perhaps less accurately, been called an immune complex or innocent bystander mechanism ( Packman, 2016 ). Quinidine is the prototypical drug that causes hemolysis via this mechanism. Quinidine may be associated with either IgM or IgG antibodies and predominantly causes complement-mediated lysis of erythrocytes or clearance of complement-coated erythrocytes by tissue macrophages ( Packman, 2016 ). Ceftriaxone has also been reported to cause hemolysis through this mechanism ( Arndt and Garratty, 2005 ). Xenobiotic-induced immune-mediated hemolysis may not be limited to one of the three mechanisms described earlier, and a combination of these mechanisms may occur in some patients. For example, the NSAID diclofenac may cause hemolysis through both drug-dependent and drug-independent mechanisms ( Salama et al., 1996 ). Carboplatin has been reported to cause hemolysis through all three immune-mediated mechanisms ( Marani et al., 1996 ). Other compounds may cause hemolysis through mechanisms other than oxidative, microangiopathic, or immune-mediated. For example, although the primary effect of lead toxicity is impairment of heme synthesis, lead may also cause hemolysis. The mechanism of lead-induced hemolysis has not been fully determined, but interference with the erythrocyte membrane sodium/potassium transporter may be involved ( Bloom and Brandt, 2008 ). Copper toxicity causes hemolysis as well, possibly through inhibition of many enzymes involved in glycolysis resulting in decreased intracellular ATP ( Boulard et al., 1972 ). Envenomation from multiple animals is reported to cause hemolysis. Envenomation by snakes, such as rattlesnakes and coral snakes, can cause hemolysis through phospholipase A2 activity, which may cause direct hemolysis or liberate hemolysins such as lysolethicin, or through complement-mediated hemolysis ( Arce-Bejarano et al., 2014 , Tambourgi and van den Berg, 2014 , Walton et al., 1997 ). 12.11.3.2.1.1.1 Infectious There are numerous protozoal, bacterial, and viral diseases that can be associated with hemolysis. Mechanisms by which infectious agents cause erythrocyte destruction are varied, and may include direct infection of erythrocytes, elaboration of toxins such as hemolysin, or stimulation of an immune-mediated response against infected cells ( Berkowitz, 1991 ). Several examples of infectious agents that cause hemolytic anemia are discussed later. Direct infection of erythrocytes with protozoal Plasmodium species, the causative agent of malaria that is transmitted by mosquitoes, is a relatively common cause of hemolysis in humans, but may also be observed in nonhuman primates used in nonclinical toxicology studies. Humans are infected by one of five different Plasmodium species: P . falciparum , P . vivax , P . knowlesi , P . malariae , or P . ovale , although only P . falciparum and P . vivax are commonly associated with severe hemolysis ( Lichtman, 2016b ). Macaques are most commonly infected with P . inui or P . knowlesi , although the cynomolgus monkey appears to be more resistant to disease from these infections than the rhesus monkey ( Ameri, 2010 ). Infection with P . cynomolgi , P . fieldi , or P . fragile may also occur in macaques ( Magden et al., 2015 ). Although it is uncommon to include macaques infected with Plasmodium species during a nonclinical toxicity study due to current screening practices and pretest evaluations, rare animals with decreases in red cell mass and increases in reticulocyte counts and intraerythrocytic Plasmodium organisms have been observed. Rats and mice may be infected with Plasmodium berghei ( Holloway et al., 1995 , Sadun et al., 1965 ). Plasmodium berghei has a specific tropism for reticulocytes rather than mature erythrocytes Plasmodium species that infect humans ( Car et al., 2006 , Cromer et al., 2006 ). Concurrent increases in reticulocyte counts may occur in early stages or disease or with recrudescence of parasitemia and hemolysis. Hemolysis is associated with clearance of parasitized erythrocytes from circulation predominantly by splenic macrophages ( Lichtman, 2016b ), although accumulation of hemin, an iron-containing porphyrin, which can directly stimulate apoptotic erythrocyte death (eryptosis) ( Gatidis et al., 2009 ), oxidative damage to erythrocyte membranes ( Clark and Hunt, 1983 ), and increased osmotic fragility ( George et al., 1967 ) may all contribute to hemolysis. However, late-stage infections in humans and rodents have also been associated with inappropriate or decreased reticulocyte counts indicative of suppressed erythropoiesis despite decreases in red cell mass from hemolysis ( Lichtman, 2016b , Cromer et al., 2006 ). Babesia species are tick-borne protozoal organisms that directly infect erythrocytes in most species, including humans, nonhuman primates, dogs, and cats. Babesia species appear as intracellular oval to pyriform organisms. Babesia microti and Babesia divergens may infect humans in North America and Europe, respectively, and cause moderate hemolytic anemia from intraerythrocytic replication and subsequent erythrocyte lysis ( Lichtman, 2016b , Kjemtrup and Conrad, 2000 ). Babesia pitheci has been reported to infect both old and new world monkeys and cause anemia ( Magden et al., 2015 ). B . canis , a large babesial species, and B . gibsoni , a small babesial species, infect dogs, while cats may be infected by the small babesial organisms B . felis and B . cati ( Stockham and Scott, 2008b , Penzhorn et al., 2004 ). These organisms are generally not of concern in purpose-bred animals used in nonclinical toxicology studies. Bartonella bacilliformis in people and the hemotrophic mycoplasmas (hemoplasmas) in dogs and cats (formerly Haemobartonella species) and swine (formerly Eperythrozoon species) are organisms that parasitize erythrocytes, but these organisms remain extracellular in shallow depressions of the erythrocyte membrane. These organisms are typically round-, rod-, or ring-shaped and may be observed individually or in chains on erythrocyte surfaces. Hemolysis with these organisms may be immune-mediate and associated either with binding of antibodies to parasite antigens or antigens exposed on the erythrocyte secondary to parasite-induced membrane changes ( Stockham and Scott, 2008b ). Clostridium perfringens (formerly Clostridium welchii ) infection in humans is an example of a bacterial cause of hemolysis. During intestinal overgrowth or septicemia, C . perfringens type A elaborates an α toxin that has lecithinase C activity, resulting in membrane phospholipid breakdown and release of lysolethicins, which have potent hemolytic capabilities ( Lichtman, 2016b , Songer, 1996 ). C . perfringens α toxin release is usually associated with severe intravascular hemolysis with both hemoglobinemia and hemoglobinuria. However, in veterinary species, C . perfringens -related hemolysis is typically limited to ruminants and horses ( Stockham and Scott, 2008b ), and is unlikely to be observed in the common species used in nonclinical toxicology studies. Infection of humans with Mycoplasma pneumoniae has also been associated with hemolytic decreases in red cell mass, although most cases of M . pneumoniae infection are asymptomatic. Hemolysis with this organism is attributable to stimulation of autoimmune erythrocyte destruction with agglutination of erythrocytes ( Khan and Yassin, 2009 ). Several viral organisms in humans have also been reported to cause decreases in red cell mass due to hemolysis. Viral causes of hemolysis are commonly associated with autoimmune mechanisms, and include infection with Epstein-Barr virus ( Palanduz et al., 2002 ), hepatitis A, B, and C viruses ( Kanematsu et al., 1996 , Chao et al., 2001 ), cytomegalovirus ( Murray et al., 2001 ), and HIV ( Koduri et al., 2002 ), although HIV infection is also commonly associated with decreases in reticulocyte counts rather than the expected increases secondary to hemolysis, indicative of concurrent suppressed erythropoiesis ( Telen et al., 1990 ). 12.11.3.2.1.1.2 Oxidative Another major cause of decreases in red cell mass due to hemolysis is oxidative damage to erythrocytes. Under normal conditions, ferrous iron (Fe 2 + ) in hemoglobin binds to and dissociates from oxygen as it delivers oxygen from the lungs to the tissues. At times, this binding and dissociation results in the formation of ferric iron (Fe 3 + ) in hemoglobin (methemoglobin) as well as superoxide (O 2 − ). Superoxide is a free radical with potent oxidative capacity that may cause cellular damage. Cytochrome b 5 -reductase is an intraerythrocytic enzyme that converts methemoglobin back to hemoglobin. Superoxide dismutase converts superoxide to hydrogen peroxide (H 2 O 2 ), which also may produce oxidative damage to cells. Further metabolism of hydrogen peroxide by catalase or glutathione peroxidase protects cells from oxidative damage. These pathways are usually sufficient to address the normal low-level formation of methemoglobin and superoxide, but methemoglobin can increase and impair delivery of oxygen to tissues and superoxide can accumulate and cause oxidative damage if these pathways are overwhelmed or defective. Oxidative damage to erythrocytes may affect the lipid membranes, cytoskeleton, or hemoglobin. Peroxidation of internal membrane lipids or cytoskeletal components of erythrocytes results in the fusion of portions of the membrane with consequent shifting of the cytoplasm and hemoglobin to one side of the cells. Erythrocytes with this morphologic change are called eccentrocytes. Oxidative damage that causes the formation of eccentrocytes may result in hemolysis due to increased clearance of eccentrocytes by splenic macrophages due to trapping of rigid erythrocytes in splenic sinusoids or spontaneous rupture in blood due to the increased fragility of eccentrocytes ( Stockham and Scott, 2008b ). Oxidative damage to exposed cysteine sulfhydryl groups on hemoglobin results in hemoglobin denaturation and decreased solubility ( Bloom and Brandt, 2008 ). Denatured hemoglobin may then precipitate and aggregate within the erythrocyte, forming small, pale-staining round structures that bind to the erythrocyte membrane and tend to protrude from the surface of the erythrocyte. These aggregates of denatured hemoglobin are called Heinz bodies. Cats appear to be particularly sensitive to the formation of Heinz bodies because of an increased number of reactive sulfhydryl groups in hemoglobin relative to other species ( Christopher et al., 1990 ), and may be more rapidly observed on blood smear evaluation due to the nonsinusoidal architecture of the feline spleen that results in decreased clearance of Heinz bodies from circulation. Similar to eccentrocytes, erythrocytes with Heinz bodies may undergo hemolysis due to increased clearance by splenic macrophages following trapping in splenic sinusoids due to decreased erythrocyte deformability and spontaneous rupture due to increased fragility from membrane damage; immune-mediated clearance may also occur and is believed to result from binding of hemochromes to and subsequent redistribution of band 3, an erythrocyte membrane structural protein, which may then be recognized by autologous antibodies ( Winterbourn, 1990 ). There are many conditions that may cause oxidative damage and result in eccentrocytosis, Heinz body formation, or even both simultaneously. Diabetes mellitus may cause either morphologic change, and diabetic ketoacidosis appears to be associated with an increased susceptibility and incidence of oxidative erythrocyte damage ( Desnoyers, 2010 , Caldin et al., 2005 , Christopher et al., 1995 ). Inherited deficiencies in erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and flavin adenine dinucleotide (FAD) have also been associated with erythrocyte oxidative damage, eccentrocyte or Heinz body formation, and hemolysis or a predisposition for these events due to the loss of protective antioxidant pathways ( Chan et al., 1982 , Harvey, 2006 ). Lymphoma has also been associated with Heinz body formation in cats ( Christopher, 1989 ) and eccentrocytes formation in dogs ( Caldin et al., 2005 ). In dogs and cats, ingestion of Allium species, particularly onions, garlic, and Chinese chive, may cause erythrocyte oxidative damage with formation of eccentrocytes and/or Heinz bodies ( Caldin et al., 2005 , Yamato et al., 2005 , Robertson et al., 1998 ). Ingestion of zinc in dogs ( Bexfield et al., 2007 ) and exposure to skunk musk ( Fierro et al., 2013 ) have also been reported to cause hemolysis due to Heinz body formation. 12.11.3.2.1.1.3 Fragmentation Physical trauma to erythrocytes results in hemolysis due to erythrocyte fragmentation and lysis. Sometimes this type of hemolysis is referred to as microangiopathic hemolytic anemia. Morphologic changes to erythrocytes occur as a result of physical trauma. Schistocytes (also called schizocytes or erythrocyte fragments), keratocytes (also called helmet cells), prekeratocytes (also called blister cells), or even spherocytes or microspherocytes may be observed. Schistocytes are very small, usually irregularly shaped fragments that can break off erythrocytes when physical trauma occurs. Keratocytes have one to two variably sized projections or horns adjacent to a small flattened region of the erythrocyte surface, while prekeratocytes appear to be precursors that have small loops of erythrocyte cytoplasm extending from the surface and surrounding a small hole in the cell. Spherocytes and microspherocytes are spherical cells that appear smaller and have more intensely pink-staining cytoplasm than normal mature erythrocytes. Spherocytes and microspherocytes may be formed during physical trauma as fragments are broken off, resulting in less membrane surface area in the parent erythrocyte surrounding a similar volume (spherocytes) or smaller volume (microspherocytes) as the parent erythrocyte. The physical trauma to erythrocytes that causes fragmentation or microangiopathic hemolysis may result from consumptive coagulopathies, either local or disseminated (DIC), with fibrin or thrombus formation in the vasculature that impedes the passage of erythrocytes through the vessel lumen, creating both turbulence and physical obstruction of blood flow. Local coagulopathy or DIC may occur secondary to trauma, infections with sepsis, or neoplasia ( Baker and Moake, 2016 , Toh and Dennis, 2003 ). Microangiopathic hemolysis due to neoplasia is most commonly associated with malignant rather than benign neoplasms and with metastatic disease or neoplasic emboli rather than primary tumors, with the exception of primary vascular neoplasms ( Susano et al., 1994 , Kupers et al., 1975 , Lohrmann et al., 1973 ). Infectious agents may also lead to fragmentation of erythrocytes, and some Leptospirosis interrogans serovars associated with vasculitis ( Stockham and Scott, 2008b ), Brucella species infection ( Yaramis et al., 2001 ), and cutaneous anthrax ( Freedman et al., 2002 ) have been reported to cause microangiopathic hemolysis. In children, fragmentation hemolysis associated with thrombotic microangiopathy may occur with Shigella dysenteriae type 1 and some Escherichia coli infections ( Pisoni et al., 2001 ). Hemolysis from erythrocyte fragmentation may also occur with HIV infection ( Maslo et al., 1997 ). Decreases in red cell mass with increases in reticulocyte counts from erythrocyte fragmentation may also occur secondary to cardiac or other conditions that alter hemodynamics and increase turbulent blood flow. For example, subaortic stenosis ( Solanki and Sheikh, 1978 ), intraluminal aortic grafts ( Sayar et al., 2006 ), uncorrected cardiac valvular disease ( Marsh and Lewis, 1969 ), prosthetic valves ( Crexells et al., 1972 ), and hypertrophic obstructive cardiomyopathy ( Kubo et al., 2010 ) have all been reported to cause hemolysis from erythrocyte fragmentation. Increased turbulence associated with hypertension may also cause decreases in red cell mass from fragmentation, and has been associated with pulmonary hypertension ( Baker and Moake, 2016 ) and malignant systemic hypertension ( Capelli et al., 1966 ). 12.11.3.2.1.1.4 Immune-mediated Autoimmune hemolytic anemia (AIHA or AHA) described in humans or immune-mediated hemolytic anemia (IMHA) described in most common laboratory species is a cause of hemolysis, and may be primary or idiopathic, but may also be secondary to conditions such as infections as discussed previously. Primary or idiopathic AIHA/IMHA is discussed here. Primary AIHA has no underlying detectable cause and is an immune-mediated condition that produces antibodies targeting erythrocyte antigens. These antierythrocyte antibodies tend to be very specific for a single erythrocyte antigen in a given case ( Packman, 2016 ). These autoantibodies may be classified as warm antibodies, which are usually IgG, or cold antibodies, which are usually IgM ( Stockham and Scott, 2008b ). Immune-mediated AIHA may be associated with erythrocyte morphologic changes that include agglutination and spherocytes. Agglutination may be observed grossly as red speckling along the inside of the specimen tube as blood is gently moved within the tube. If agglutination is present, blood smears may have a "reverse smear" appearance with the densest region of the smear observed at the feathered edge rather than the edge where the drop of blood was initially placed. Microscopically agglutination appears as grape-like clusters of erythrocytes. Spherocytes are erythrocytes that are spherical instead of having the normal biconcave disc shape. While spherocytes appear smaller and stain more intensely pink that unaffected mature erythrocytes, they have the same volume as unaffected erythrocytes. Loss of erythrocyte membrane occurs when macrophages begin to phagocytize antibody-bound erythrocytes, leading to decreased erythrocyte surface area without an appreciable change in volume, forcing erythrocytes to form spheres. Hence, spherocytosis alone will not result in an altered MCV. Of the most common laboratory species, dogs tend to have the most pronounced central pallor of normal mature erythrocytes, making microscopic identification of spherocytes easiest in the dog. Hemolysis in AIHA is largely attributable to extravascular hemolysis due to phagocytosis of antibody-bound erythrocytes by tissue macrophages. Macrophages or monocytes containing phagocytized erythrocytes may be rarely observed in blood smears of laboratory species with immune-mediated hemolysis. However, antibody-mediated complement activation or increased fragility of spherocytes may result in direct intravascular lysis or rupture of erythrocytes ( Packman, 2016 ). Evaluation of patients for the presence of antierythrocyte antibodies may be performed using the direct antiglobulin test (DAT; also called the Coombs' test) or by flow cytometry. AIHA has rarely been observed in association with lymphoproliferative neoplasia, such as chronic lymphocytic leukemia. Antierythrocyte antibodies in chronic lymphocytic leukemia are predominantly IgG with few cases of IgM reported ( Mauro et al., 2000 ). IMHA may occasionally be observed following blood transfusion ( Garratty, 2004 ). This may occur in response to alloantigens, and would not technically be considered autoimmune ( Stockham and Scott, 2008b ). Posttransfusion immune-mediated hemolysis may also be observed when the host has autoantibodies that bind the donor erythrocytes and cause immune-mediated destruction. However, crossmatching of host and donor erythrocytes and plasma is able to prevent many cases with incompatible transfusion-related AIHA. AIHA and IMHA commonly have concurrent inflammatory increases in leukocyte subtype counts, characterized mainly by neutrophilia that may or may not have a left shift with cytoplasmic changes indicative of rapid neutropoiesis. 12.11.3.2.1.1.5 Inherited Some phenotypes of sickle cell disease are associated with hemolysis. The mechanism of hemolysis in sickle cell disease is likely multifactorial and not associated with a single pathogenesis. There is evidence for oxidative damage to erythrocytes ( Lachant et al., 1983 ), which may contribute to the hemolysis observed with sickle cell disease. However, hemoglobin polymerization leads to erythrocyte deformation and may lead to decreased flexibility of erythrocytes and veno-occlusive disorders ( Bookchin and Lew, 1996 ). Decreased flexibility or deformability of erythrocytes may contribute directly to increased cell fragility and rupture or promote clearance of deformed erythrocytes by splenic macrophages, while veno-occlusive disease has the potential to cause decreases in red cell mass through physical trauma and fragmentation. However, there is also evidence that in some severe cases of sickle cell disease there may be an increase in reticulocyte counts that are inappropriate for the magnitude of the decrease in red cell mass, suggesting a concurrent mechanism causing suppressed or ineffective erythropoiesis ( Wu et al., 2005 , Bookchin and Lew, 1996 ). Oxidative stress on erythroid precursors may also contribute to ineffective erythropoiesis in some severe cases of sickle cell disease ( Fibach and Rachmilewitz, 2008 ). Several metabolic defects of erythrocyte metabolism may also be associated with decreases in red cell mass and increases in reticulocyte counts. Deficiencies in erythrocyte pathways of glycolysis may result in decreased ATP concentrations that lead to erythrocyte membrane dysfunctions with shortened erythrocyte lifespan and occasionally hemolysis ( Stockham and Scott, 2008b ). Pyruvate kinase (PK) is the enzyme that catalyzes the last step in aerobic glycolysis. Deficiencies of PK that result in hemolysis have been reported in humans ( Baronciani and Beutler, 1993 ), dogs including beagles ( Harvey et al., 1977 , Giger et al., 1991 , Prasse et al., 1975 ), and a few breeds of cats ( Kohn and Fumi, 2008 ). Phosphofructokinase (PFK) catalyzes the rate-limiting step of the glycolysis pathway. Deficiencies in PFK have also been described in humans ( Etiemble et al., 1976 ) and dogs ( Giger et al., 1985 ). Respiratory alkalosis, which may be observed following intense exercise, is associated with acute hemolytic crises in patients with PFK deficiencies ( Giger et al., 1985 ). The association of inherited G6PD and FAD deficiencies with hemolysis resulting from oxidative damage is discussed earlier. In brief, G6PD and FAD play a role in the antioxidant pathways of erythrocytes. Deficiencies of G6PD and FAD may result in increased oxidative damage to erythrocytes and subsequent hemolysis. Collectively, the porphyrias are a group of enzymatic defects in the heme synthesis pathway. Porphyrias may be congenital or, more commonly, acquired. In these conditions, the accumulation of porphyrins, the precursors of heme, within erythrocytes leads to hemolysis. The mechanism of hemolysis may be related to lysis of erythrocytes following exposure to light (photolysis) in superficial vasculature, or by direct erythrocyte membrane damage due to the lipid soluble nature of porphyrins or following porphyrin absorption of ultraviolet light and excitation ( Phillips and Anderson, 2016 , Kaneko, 2008 ). 12.11.3.2.1.1.6 Neoplastic Various neoplastic conditions may be associated with hemolysis. Malignant metastatic neoplasms or primary vascular neoplasms may result in fragmentation hemolysis by physical trauma to erythrocytes, as previously discussed. However, neoplastic conditions may also rarely be associated with phagocytosis and destruction of erythrocytes, or hemophagocytic syndrome. Hemophagocytic syndromes have been associated with T-cell lymphoma ( Gonzalez et al., 1991 ), NK-cell leukemia ( Kobayashi et al., 1996 ), hemophagocytic histiocytic sarcoma ( Moore et al., 2006 ), and various hematological neoplasias ( Majluf-Cruz et al., 1998 ). 12.11.3.2.1.1.7 Xenobiotic-induced Many xenobiotics are capable of causing hemolysis, and may cause hemolysis through oxidative, fragmentation, or immune-mediated mechanisms. Examples of each are discussed here. Many of the agents that cause oxidative erythrocyte injury contain aromatic structures that can be metabolized, mostly commonly by cytochrome P 450, to free radicals ( Bradberry, 2003 , Edwards and Fuller, 1996 ), which overwhelm the normal protective antioxidant pathways of erythrocytes leading to both direct erythrocyte oxidative injury and oxidation of hemoglobin sulfhydryl groups resulting in methemoglobin formation. A few specific aromatic compounds that have been associated with free radical formation include dapsone, phenacetin, and anthracyclines such as doxorubicin ( Edwards and Fuller, 1996 , Coleman et al., 1991 , Handa and Sato, 1975 , Easley and Condon, 1974 ). Phenacetin has also been associated with the formation of Heinz bodies ( Boelsterli et al., 1983 ). In dogs and cats, acetaminophen (paracetamol) may be metabolized to a minor reactive metabolite that causes oxidative damage to erythrocytes resulting in hemolysis and the formation of Heinz bodies and/or eccentrocytes, although methemoglobinemia has also been observed in cats ( Desnoyers, 2010 , Wallace et al., 2002 , Mariani and Fulton, 2001 , Aronson and Drobatz, 1996 ). Xenobiotics that cause methemoglobinemia can also cause indirect oxidative damage through the peroxidation activity of methemoglobin itself ( Edwards and Fuller, 1996 ). In some cases, oxygenated hemoglobin may act as a peroxidase and cause the metabolism of a xenobiotic to a reactive compound that causes erythrocyte oxidative damage and conversion of oxyhemoglobin to methemoglobin. Examples of xenobiotics that cause oxidative damage through this mechanism are phenylhydrazine and primaquine ( Edwards and Fuller, 1996 ). Vitamin K administration in dogs can also cause oxidative erythrocyte damage through this mechanism ( Fernandez et al., 1984 ). Some chemical agents may cause oxidative damage by directly oxidizing hemoglobin sulfhydryl groups or through direct oxidation of erythrocyte cytoskeletal proteins. Arsine gas, predominantly an environmental toxin, appears to mediate its hemolytic effects through erythrocyte membrane oxidation ( Rael et al., 2000 ), although studies in mice have also demonstrated the formation of Heinz bodies following exposure ( Blair et al., 1990 ), suggesting an oxidative effect on hemoglobin as well. Many xenobiotics may also cause hemolysis through their association with microangiopathy, most commonly as part of the thrombotic microangiopathy syndrome, which is associated with fragmentation hemolysis and decreases in platelet counts. Drug-induced endothelial injury, including from direct and antibody or immune complex-mediated mechanisms, plays a major role in the pathogenesis of thrombotic microangiopathy ( Pisoni et al., 2001 ). Endothelial damage may be propagated by leukocyte adhesion and release of granule contents or reactive oxygen species, platelet activation and aggregation, and complement activation ( Pisoni et al, 2001 ). Drugs implicated in thrombotic microangiopathy include chemotherapeutic agents include xenobiotics from a wide variety of chemotherapeutic classes. Examples of chemotherapeutics associated with thrombotic microangiopathy include mitomycin C ( Cantrell et al., 1985 ), cisplatin ( Palmisano et al., 1998 ), estramustine phosphate ( Tassinari et al., 1999 ), gemcitabine ( Nackaerts et al., 1998 ), and daunorubicin ( Byrnes et al., 1986 ). Nonchemotherapeutic agents that have been reported to cause thrombotic microangiopathy include immunomodulators such as cyclosporine and tacrolimus ( Katznelson et al., 1994 , Trimarchi et al., 1999 ), simvastatin ( McCarthy et al., 1998 ), and inhibitors of platelet aggregation including ticlopidine and clopidogrel ( Bennett et al., 1998 , Bennett et al., 2000 ). Immune-mediated mechanisms of hemolysis have also been reported following exposure to numerous xenobiotics. Xenobiotics may induce antibodies by binding to the erythrocyte membrane and acting as haptens. These antibodies are considered drug-dependent as they only mediate hemolysis when the drug is present. Penicillin is the prototypical xenobiotic that acts as a hapten to generate drug-dependent antibodies, and typically induces an IgG response ( Ferner, 2012 , Petz et al., 1966 ). Semisynthetic penicillins, some cephalosporins, and tetracycline have also been reported to cause drug-dependent antibody-mediated hemolysis ( Garratty, 2010 , Tuffs and Manoharan, 1986 , Seldon et al., 1982 , Großjohann et al., 2004 , Gallagher et al., 1992 , Branch et al., 1985 , Simpson et al., 1985 ). Xenobiotics may also induce the production of antierythrocyte antibodies that mediate hemolysis even when the drug is no longer present, also called drug-independent antibodies or autoantibodies. In this type of hemolysis, xenobiotic exposure stimulates production of an antibody that can bind to native erythrocyte antigens even in the absence of the drug. This type of immune-mediated xenobiotic-induced hemolysis is classically caused by α-methyldopa, and is characterized by predominantly an IgG response ( Packman, 2016 ). However, nucleoside purine analogs such as cladribine and fludarabine have also been associated with hemolysis due to production of autoimmune antibodies ( Garratty, 2010 , Mintzer et al., 2009 , Hamblin, 2006 ). A third mechanism by which xenobiotics may cause immune-mediated hemolysis is through a complex interaction of the drug, a drug binding site on erythrocytes, and an antibody. This mechanism is considered the ternary complex mechanism, but has previously, and perhaps less accurately, been called an immune complex or innocent bystander mechanism ( Packman, 2016 ). Quinidine is the prototypical drug that causes hemolysis via this mechanism. Quinidine may be associated with either IgM or IgG antibodies and predominantly causes complement-mediated lysis of erythrocytes or clearance of complement-coated erythrocytes by tissue macrophages ( Packman, 2016 ). Ceftriaxone has also been reported to cause hemolysis through this mechanism ( Arndt and Garratty, 2005 ). Xenobiotic-induced immune-mediated hemolysis may not be limited to one of the three mechanisms described earlier, and a combination of these mechanisms may occur in some patients. For example, the NSAID diclofenac may cause hemolysis through both drug-dependent and drug-independent mechanisms ( Salama et al., 1996 ). Carboplatin has been reported to cause hemolysis through all three immune-mediated mechanisms ( Marani et al., 1996 ). Other compounds may cause hemolysis through mechanisms other than oxidative, microangiopathic, or immune-mediated. For example, although the primary effect of lead toxicity is impairment of heme synthesis, lead may also cause hemolysis. The mechanism of lead-induced hemolysis has not been fully determined, but interference with the erythrocyte membrane sodium/potassium transporter may be involved ( Bloom and Brandt, 2008 ). Copper toxicity causes hemolysis as well, possibly through inhibition of many enzymes involved in glycolysis resulting in decreased intracellular ATP ( Boulard et al., 1972 ). Envenomation from multiple animals is reported to cause hemolysis. Envenomation by snakes, such as rattlesnakes and coral snakes, can cause hemolysis through phospholipase A2 activity, which may cause direct hemolysis or liberate hemolysins such as lysolethicin, or through complement-mediated hemolysis ( Arce-Bejarano et al., 2014 , Tambourgi and van den Berg, 2014 , Walton et al., 1997 ). 12.11.3.2.1.2 Blood loss 12.11.3.2.1.2.1 Hemorrhage Hemorrhage may cause internal or external blood loss. Due to the loss of whole blood during hemorrhage, decreases in red cell mass are usually accompanied by proportionate decreases in albumin and globulin concentrations. The decreases in plasma proteins tend to be less pronounced with internal hemorrhage because the lost proteins may be resorbed in lymph and returned to blood ( Stockham and Scott, 2008b ). Cases of internal hemorrhage are typically not associated with iron deficiency. However, prolonged external blood loss may cause depletion of total body iron. Iron deficiency anemia is characterized by small erythrocytes with a decrease in MCV and erythrocytes that contain less hemoglobin with a decrease in MCHC, and may be classified as a microcytic, hypochromic anemia. Hemoglobin synthesis plays a role in inhibiting erythrocyte division, and when sufficient iron is not available for heme production, there is loss of the inhibitory effect resulting in more cell divisions and microcytes ( Stohlman et al., 1963 ). Hypochromasia of the erythrocytes is due to the lower than normal hemoglobin content due to decreased production of heme. Morphologic erythrocyte changes that accompany iron deficiency anemia include visual microcytosis and hypochromasia, keratocytes and schistocytes from physical damage to the more fragile erythrocytes, and sometimes codocytes (also called target cells) that have a thin rim of pink-staining hemoglobin and a small central area of hemoglobin with a ring of pallor in between, typical of erythrocytes with less hemoglobin present relative to the amount of membrane. In chronic iron deficiency, increases in reticulocyte counts and microscopic polychromasia may be lower than expected for a regenerative anemia due to loss of RNA during the extended maturation phase of erythrocyte production caused by decreased hemoglobin content ( Burkhard et al., 2001 ). Direct damage to blood vessels from trauma is a relatively common cause of acute external or internal blood loss. Traumatic rupture of the spleen may also cause significant acute internal blood loss. Decreases in red cell mass due to acute hemorrhage are typically due to dilution of remaining blood from shifting of intracellular fluid to extracellular fluid in an attempt to preserve blood volume and therefore tissue perfusion ( Stockham and Scott, 2008b ). Dilution of red cell mass may also be observed following administration of intravenous fluids to replace blood volume. A detectable increase in reticulocyte count is expected 3–4 days following the acute event in a patient with normally functioning bone marrow. Damage to blood vessels that results in hemorrhage also may occur secondary to ulcerative or neoplastic conditions. In dogs, rupture of splenic hemangiosarcoma is a common cause of internal blood loss into the abdomen (hemoabdomen). Ulceration of the gastrointestinal system may lead to blood loss into feces, which can be observed as black, tarry feces (melena) if the ulceration occurs in the small intestines or as frank blood if the ulceration occurs in the large intestines. In humans and nonhuman primate species with true menstrual cycles, including Old World monkeys and great apes ( Provencher Bolliger et al., 2010 ), decreases in red cell mass are uncommon but may be observed from menses-related blood loss. In women, heavy blood loss, abnormal cycling, or uterine neoplasms may lead to sufficient blood loss to cause decreases in red cell mass and potentially even iron deficiency ( Van Voorhis, 2009 , Goel and Gupta, 2007 ). In cynomolgus monkeys, decreases in red cell mass have been occasionally observed in females with prolonged menses ( Perigard et al., 2016 ). Coagulation disorders may also be associated with either internal or external hemorrhage. Primary deficiencies in coagulation factors or von Willebrand factor may be inherited causes of hemorrhage. Deficiencies in coagulation factors that lead to hemorrhage sufficient to cause decreases in red cell mass include hemophilia A (factor VIII deficiency) and hemophilia B (factor IX deficiency); deficiencies in factor XI and von Willebrand factor are usually mild and are often not associated with notable hemorrhage ( Bolton-Maggs and Pasi, 2003 ). Hemorrhage may be secondary to marked decreases in platelet counts from consumptive coagulopathies secondary to infectious or neoplastic processes. Although not truly hemorrhage, external blood loss can occur from repeated phlebotomy. Decreases in red cell mass may be acutely observed following collection of blood from donors for transfusion, and regular donors have a risk of developing iron deficiency from repeated external blood loss ( Cable et al., 2011 ). Repeated phlebotomy is a common occurrence in nonclinical toxicology studies, particularly in dogs and nonhuman primates, although rats may also occasionally undergo repeated blood collections. Blood is collected through the studies mainly for toxicokinetic or pharmacokinetic analysis, but also for analysis of hematology, coagulation, and clinical chemistry profiles. Decreases in red cell mass with increases in reticulocyte counts of similar magnitude relative to pretest values across all treatment groups, including controls, are a common procedure-related phenomenon in nonclinical toxicology studies and should be distinguished from a true test article-related effect. 12.11.3.2.1.2.2 Parasitism Both external and internal parasites may contribute to blood loss. Hookworms are a major internal parasite associated with chronic blood loss, and may lead to iron deficiency with prolonged infections ( Stoltzfus et al., 1997 ). However, whipworm infection and schistosomiasis may also be associated with blood loss, the latter being associated with blood loss through the urinary system ( Farid et al., 1969 ). Heavy infestation of animals with arthropods that take blood meals, such as ticks, some lice, and fleas, may also cause sufficient blood loss to result in decreases in red cell mass ( Stockham and Scott, 2008b ). 12.11.3.2.1.2.3 Xenobiotic-induced Xenobiotic-induced blood loss is relatively uncommon but can occur. Classically, hemorrhage into the intestinal tract can result from ulceration associated with chronic NSAID or coxib administration ( Laine et al., 2003 , Langman et al., 1999 , Bjarnason et al., 1987 ). Also, prolonged or high dose administration of anticoagulants, such as warfarin or heparin, can result in hemorrhage-related decreases in red cell mass ( Levine et al., 2001 ). Ingestion of rodenticides, including brodifacoum chlorophacinone, has been reported to cause marked hemorrhage in humans and many other nonrodent species ( Berny et al., 2010 , Palmer et al., 1999 , Sheafor and Couto, 1999 ). Xenobiotic-induced marked decreases in platelet counts may also be associated with hemorrhage and are discussed in more detail later. However, chemotherapeutics that cause bone marrow suppression can be associated with spontaneous or postvenipuncture hemorrhage. Occasional idiopathic decreases in platelet counts have also been observed following xenobiotic administration and are most likely attributable to immune-mediated destruction; some examples of implicated xenobiotics are quinine, trimethoprim–sulfamethoxazole, anticonvulsants such as phenytoin and carbamazepine, unfractionated or low molecular weight heparin, and rituximab ( Aster and Bougie, 2007 ). 12.11.3.2.1.2.1 Hemorrhage Hemorrhage may cause internal or external blood loss. Due to the loss of whole blood during hemorrhage, decreases in red cell mass are usually accompanied by proportionate decreases in albumin and globulin concentrations. The decreases in plasma proteins tend to be less pronounced with internal hemorrhage because the lost proteins may be resorbed in lymph and returned to blood ( Stockham and Scott, 2008b ). Cases of internal hemorrhage are typically not associated with iron deficiency. However, prolonged external blood loss may cause depletion of total body iron. Iron deficiency anemia is characterized by small erythrocytes with a decrease in MCV and erythrocytes that contain less hemoglobin with a decrease in MCHC, and may be classified as a microcytic, hypochromic anemia. Hemoglobin synthesis plays a role in inhibiting erythrocyte division, and when sufficient iron is not available for heme production, there is loss of the inhibitory effect resulting in more cell divisions and microcytes ( Stohlman et al., 1963 ). Hypochromasia of the erythrocytes is due to the lower than normal hemoglobin content due to decreased production of heme. Morphologic erythrocyte changes that accompany iron deficiency anemia include visual microcytosis and hypochromasia, keratocytes and schistocytes from physical damage to the more fragile erythrocytes, and sometimes codocytes (also called target cells) that have a thin rim of pink-staining hemoglobin and a small central area of hemoglobin with a ring of pallor in between, typical of erythrocytes with less hemoglobin present relative to the amount of membrane. In chronic iron deficiency, increases in reticulocyte counts and microscopic polychromasia may be lower than expected for a regenerative anemia due to loss of RNA during the extended maturation phase of erythrocyte production caused by decreased hemoglobin content ( Burkhard et al., 2001 ). Direct damage to blood vessels from trauma is a relatively common cause of acute external or internal blood loss. Traumatic rupture of the spleen may also cause significant acute internal blood loss. Decreases in red cell mass due to acute hemorrhage are typically due to dilution of remaining blood from shifting of intracellular fluid to extracellular fluid in an attempt to preserve blood volume and therefore tissue perfusion ( Stockham and Scott, 2008b ). Dilution of red cell mass may also be observed following administration of intravenous fluids to replace blood volume. A detectable increase in reticulocyte count is expected 3–4 days following the acute event in a patient with normally functioning bone marrow. Damage to blood vessels that results in hemorrhage also may occur secondary to ulcerative or neoplastic conditions. In dogs, rupture of splenic hemangiosarcoma is a common cause of internal blood loss into the abdomen (hemoabdomen). Ulceration of the gastrointestinal system may lead to blood loss into feces, which can be observed as black, tarry feces (melena) if the ulceration occurs in the small intestines or as frank blood if the ulceration occurs in the large intestines. In humans and nonhuman primate species with true menstrual cycles, including Old World monkeys and great apes ( Provencher Bolliger et al., 2010 ), decreases in red cell mass are uncommon but may be observed from menses-related blood loss. In women, heavy blood loss, abnormal cycling, or uterine neoplasms may lead to sufficient blood loss to cause decreases in red cell mass and potentially even iron deficiency ( Van Voorhis, 2009 , Goel and Gupta, 2007 ). In cynomolgus monkeys, decreases in red cell mass have been occasionally observed in females with prolonged menses ( Perigard et al., 2016 ). Coagulation disorders may also be associated with either internal or external hemorrhage. Primary deficiencies in coagulation factors or von Willebrand factor may be inherited causes of hemorrhage. Deficiencies in coagulation factors that lead to hemorrhage sufficient to cause decreases in red cell mass include hemophilia A (factor VIII deficiency) and hemophilia B (factor IX deficiency); deficiencies in factor XI and von Willebrand factor are usually mild and are often not associated with notable hemorrhage ( Bolton-Maggs and Pasi, 2003 ). Hemorrhage may be secondary to marked decreases in platelet counts from consumptive coagulopathies secondary to infectious or neoplastic processes. Although not truly hemorrhage, external blood loss can occur from repeated phlebotomy. Decreases in red cell mass may be acutely observed following collection of blood from donors for transfusion, and regular donors have a risk of developing iron deficiency from repeated external blood loss ( Cable et al., 2011 ). Repeated phlebotomy is a common occurrence in nonclinical toxicology studies, particularly in dogs and nonhuman primates, although rats may also occasionally undergo repeated blood collections. Blood is collected through the studies mainly for toxicokinetic or pharmacokinetic analysis, but also for analysis of hematology, coagulation, and clinical chemistry profiles. Decreases in red cell mass with increases in reticulocyte counts of similar magnitude relative to pretest values across all treatment groups, including controls, are a common procedure-related phenomenon in nonclinical toxicology studies and should be distinguished from a true test article-related effect. 12.11.3.2.1.2.2 Parasitism Both external and internal parasites may contribute to blood loss. Hookworms are a major internal parasite associated with chronic blood loss, and may lead to iron deficiency with prolonged infections ( Stoltzfus et al., 1997 ). However, whipworm infection and schistosomiasis may also be associated with blood loss, the latter being associated with blood loss through the urinary system ( Farid et al., 1969 ). Heavy infestation of animals with arthropods that take blood meals, such as ticks, some lice, and fleas, may also cause sufficient blood loss to result in decreases in red cell mass ( Stockham and Scott, 2008b ). 12.11.3.2.1.2.3 Xenobiotic-induced Xenobiotic-induced blood loss is relatively uncommon but can occur. Classically, hemorrhage into the intestinal tract can result from ulceration associated with chronic NSAID or coxib administration ( Laine et al., 2003 , Langman et al., 1999 , Bjarnason et al., 1987 ). Also, prolonged or high dose administration of anticoagulants, such as warfarin or heparin, can result in hemorrhage-related decreases in red cell mass ( Levine et al., 2001 ). Ingestion of rodenticides, including brodifacoum chlorophacinone, has been reported to cause marked hemorrhage in humans and many other nonrodent species ( Berny et al., 2010 , Palmer et al., 1999 , Sheafor and Couto, 1999 ). Xenobiotic-induced marked decreases in platelet counts may also be associated with hemorrhage and are discussed in more detail later. However, chemotherapeutics that cause bone marrow suppression can be associated with spontaneous or postvenipuncture hemorrhage. Occasional idiopathic decreases in platelet counts have also been observed following xenobiotic administration and are most likely attributable to immune-mediated destruction; some examples of implicated xenobiotics are quinine, trimethoprim–sulfamethoxazole, anticonvulsants such as phenytoin and carbamazepine, unfractionated or low molecular weight heparin, and rituximab ( Aster and Bougie, 2007 ). 12.11.3.2.2 Decreases in red cell mass with "normal" or low reticulocyte counts (nonregenerative anemia) 12.11.3.2.2.1 Preregenerative Depending on the timing of the insult causing the decreases in red cell mass, reticulocyte counts within reference interval may represent a preregenerative anemia rather than suppressed erythropoiesis. Production of erythrocytes by the bone marrow requires at least 3–4 days, and a peak increase in blood reticulocyte count occurs about 7–14 days following the insult ( Stockham and Scott, 2008b ). If it an individual with decreased red cell mass and reticulocyte counts that are within the reference interval and it is unclear if the individual has a preregenerative anemia or suppressed erythropoiesis, repeating a CBC several days later may help clarify which process is occurring. 12.11.3.2.2.2 Infectious Acute Chagas disease, caused by infection with Trypanosoma cruzi , has been reported to cause decreases in red cell mass in humans and monkeys ( de Titto and Araujo, 1988 , Rosner et al., 1988 , Seah et al., 1974 ). In experimentally infected Cebus paella monkeys, the acute phase of Chagas disease was reported to cause normocytic, normochromic anemia ( Rosner et al., 1988 ), typical of a nonregenerative anemia. Experimentally infected mice demonstrated bone marrow suppression with decreases in red cell mass as well as decreases in leukocyte and platelet counts ( Marcondes et al., 2000 ). Although rarely encountered in nonclinical toxicology studies, monkeys held in the southwestern United States may become infected with T . cruzi prior to distribution ( Magden et al., 2015 ). During parasitemia, trypomastigotes may be observed in peripheral blood smears. Viral infections may also cause decreases in red cell mass without concurrent increases in reticulocyte counts. Parvoviruses may cause decreases in red cell mass from direct infection of erythroid precursor resulting in decreased erythrocyte production, as well as decreased erythrocyte lifespans. Parvovirus may result in transient pure red cell aplasia (PRCA) in humans ( Van Horn et al., 1986 ). Cell-mediated suppression of erythropoiesis resulting in PRCA has also been reported with viral hepatitis ( Wilson et al., 1980 ) and Epstein-Barr virus infection ( Socinski et al., 1984 ). Although HIV infection can result in decreases in red cell mass through various mechanisms, direct infection of erythroid precursors appears to contribute to suppressed erythropoiesis ( Evans and Scadden, 2000 ). In cats, a membrane protein of feline leukemia virus has been associated with decreased growth of CFU-E ( Wellman et al., 1984 ). Flavivirus infection, such as dengue, may also result in decreases in red cell mass and reticulocyte counts through bone marrow suppression ( La Russa and Innis, 1995 ). 12.11.3.2.2.3 Chronic disease Anemia of chronic disease (ACD) is a relatively common cause of anemia, and anemia associated with inflammatory disease is included in ACD. The decreases in red cell mass observed with ACD are generally mild, and are generally normocytic, normochromic, indicating no changes in MCV or MCHC, respectively. ACD may occur through shortening of erythrocyte lifespans, alterations in iron metabolism, a blunted response of erythroid precursors to EPO, and decreased EPO production. Altered erythrocyte lifespans in patients with ACD may be related to increased macrophagic clearance of erythrocytes from circulation through unknown mechanisms ( Ganz, 2016 ). This type of mechanism has been associated with several chronic infections, including tuberculosis and endocarditis ( Weiss, 2002 ). More commonly, ACD is associated with impaired iron mobilization with low iron concentrations in serum or plasma despite adequate iron stores ( Means, 2000 ). Impaired mobilization of iron results from IL-6 induction of hepcidin that results in sequestration of iron in macrophages and decreased intestinal iron update ( Ganz, 2003 ), IL-1 stimulation of increased synthesis of ferritin which may bind to iron and impair delivery of iron to erythroid precursors ( Rogers et al., 1994 ), and with decreased expression and impaired internalization of the transferrin receptor ( Means, 2000 ). ACD from impaired iron metabolism is associated with numerous inflammatory, infectious, and even neoplastic conditions. ACD may also cause altered EPO responsiveness or decreased EPO production. Decreased responsiveness of erythroid precursors to EPO is cytokine-mediated, and has been associated with increases in TNFα, IL-1, and interferons ( Johnson et al., 1989 , Johnson et al., 1990 , Raefsky et al., 1985 ) that may commonly be associated with inflammatory conditions. Decreased EPO production may also be cytokine-mediated, and has been reported with increases in TGFβ, TNFα, and IL-1 ( Faquin et al., 1992 , Jelkmann et al., 1992 ). However, chronic renal disease may also result directly in impaired EPO production and decreased production of erythrocytes ( Sato and Yanagita, 2013 ). 12.11.3.2.2.4 Immune-mediated Immune-mediated destruction of erythroid precursors in the bone marrow results in decreases in red cell mass with concurrent decreases in reticulocyte counts. The immune-mediated conditions discussed here may represent a spectrum of disease associated with immune destruction of various stages of erythroid precursors rather than unrelated entities. 12.11.3.2.2.4.1 Autoimmune hemolytic anemia with decreases in reticulocyte counts Autoimmune hemolytic anemia with antibodies that target antigens on mid- to late-stage erythroid precursors ranging from rubricytes to metarubricytes results in AIHA with a decrease in reticulocyte count, which may also be called immune-mediated nonregenerative anemia or precursor-targeted immune-mediated anemia (PIMA). AIHA with reticulocytopenia is generally a normocytic, normochromic anemia. Bone marrow examination may reveal erythroid hyperplasia or maturation arrest ( Weiss, 2008 ) with pyramidal expansion of erythroid precursors at stages earlier than the targeted stage, indicative of ineffective erythropoiesis. This may be less apparent with autoantibodies that recognize more immature stages of erythroid precursors. Bone marrow evaluation may also reveal rubriphagocytosis, or erythroid precursors phagocytized by macrophages. The stage of phagocytized precursor depends on the stage or stages expressing the targeted antigen. 12.11.3.2.2.4.2 Pure red cell aplasia In patients affected by PRCA, there are marked decreases in reticulocyte counts along with variable decreases in red cell mass. Bone marrow examination typically reveals an absence of erythroid precursors (erythroid aplasia) or low numbers of the earliest stages of erythroid precursors (erythroid hypoplasia) ( Young, 2016 ). PRCA in people may be caused by antibodies that bind antigens on the earliest erythroid precursors or even antibodies that bind EPO and prevent EPO-dependent erythropoiesis, but it has also been attributed to clonal T-cell disorders ( Stockham and Scott, 2008b ). PRCA in dogs has been associated with IgG that inhibit erythropoiesis ( Weiss, 1986 ). PRCA may also be caused by inherited genetic defect in people. Inherited PRCA in people is called Diamond-Blackfan anemia, and often has an autosomal dominant inheritance pattern with defects in genes encoding ribosomal proteins ( Young, 2016 ). Macrocytosis, or increased numbers of large erythrocytes with increases in MCV, may be observed and is consistent with impaired EPO-dependent erythropoiesis ( Young, 2016 , Ohene-Abuakwa et al., 2005 ). 12.11.3.2.2.4.3 Aplastic anemia Aplastic anemia is a condition associated with decreases in all cellular blood components (pancytopenia), including decreases in red cell mass with concurrent decreases in reticulocyte counts. Upon examination, the bone marrow classically had severe hypocellularity of hematopoietic cells or an absence of hematopoietic precursors the marrow cavities filled by mostly adipocytes and some stromal elements. Aplastic anemia is thought to be most commonly immune-mediated ( Young et al., 2006 ), and may be frequently associated with cytotoxic T-cells that become autoreactive ( Segel and Lichtman, 2016 ). However, there are also cases of inherited aplastic anemia, most commonly Fanconi anemia associated with genetic mutations that impair DNA repair resulting in pancytopenia developing around 5–10 years of age in people ( Segel and Lichtman, 2016 ). A form of aplastic anemia associated with bone marrow depletion or hypocellularity of hematopoietic tissue and gelatinous transformation of marrow cavity fat has been reported with anorexia nervosa in people ( Abella et al., 2002 ) and with severe food restriction in rats ( Moriyama et al., 2008 ). 12.11.3.2.2.5 Nutritional deficiencies In addition to aplastic anemia associated with anorexia nervosa and severe food restriction, other nutritional deficiencies have been associated with ineffective erythropoiesis leading to decreases in red cell mass with decreases in reticulocyte counts. Iron deficiency and deficiencies of the B vitamins folate and cobalamin are examples of these nutritional deficiencies. Chronic iron deficiency results in impaired hematopoiesis due to the inability to synthesize sufficient hemoglobin, which may lead to a decrease in reticulocyte production. Deficiencies in folate and cobalamin also cause ineffective erythropoiesis due to defects in DNA synthesis, as discussed with folate and cobalamin deficiencies as a cause of deceases in neutrophil counts. In people, folate and cobalamin deficiencies result in megaloblastic anemia, characterized by larger than normal erythroid precursors in the bone marrow that have more cytoplasm with lower nuclear to cytoplasmic ratios than in normal erythroid precursors and asynchronous cytoplasmic and nuclear maturation ( Green, 2016 ). Megaloblastic erythrocytes may also be observed in circulation, and basophilic stippling or Howell-Jolly bodies may also be observed ( Green, 2016 ). In people, anemia attributable to a deficiency in cobalamin (vitamin B 12 ) may also be called pernicious anemia. In dogs and cats, megaloblastic erythroid cells may be observed in the bone marrow but may not be observed in blood ( Stockham and Scott, 2008b ). 12.11.3.2.2.6 Endocrinopathy Several endocrinopathies have also been associated with decreases in red cell mass with decreases in reticulocyte counts, including hypothyroidism, hypoadrenocorticism, and hyperestrogenism. In cases of hypothyroidism, several mechanisms may be contributing to the decreases in red cell mass. Decreased folate or cobalamin concentrations secondary to the hypothyroidism leading to ineffective erythropoiesis, decreased tissue oxygen demand leading to decreased EPO and lower baseline red cell mass, and ACD may contribute to the mild decreases in red cell mass observed with hypothyroidism ( Ottesen et al., 1995 , Hines et al., 1968 , Stockham and Scott, 2008b , Mehmet et al., 2012 ). Mild decreases in red cell mass without apparent changes in reticulocyte counts have been associated with hypoadrenocorticism. This may be due to a decrease in glucocorticoids, and the loss of the apparent proerythropoietic stimulation of glucocorticoids ( Stockham and Scott, 2008b ). Hyperestrogenism, which occurs with some ovarian or testicular neoplasms, may result in bone marrow toxicity and suppression of erythropoiesis, particularly in dogs ( Sontas et al., 2009 ). 12.11.3.2.2.7 Neoplasia Neoplasia may result in suppressed erythropoiesis. This may be due to neoplasia-related inflammation and cytokine release leading to ACD. However, granulocytic leukemia or lymphoproliferative neoplasia involving the bone marrow may result in crowding or effacement of the bone marrow cavities with impaired erythropoiesis that results in decreases in red cell mass with concurrent decreases in reticulocyte counts. Hematopoietic neoplasia involving the erythroid lineage usually results in atypical erythrocyte production that can lead to decreases in red cell mass and reticulocyte counts; however, nucleated erythrocytes with evidence of dysplasia may be observed in blood. Similar to hematopoietic neoplasms that efface the bone marrow, metastatic neoplasia, often carcinomas, may also cause myelophthisis and result in decreased erythropoiesis. 12.11.3.2.2.8 Xenobiotic-induced There are many xenobiotics that can cause decreases in red cell mass with concurrent decreases in reticulocyte counts. Bone marrow suppression that affects the erythroid lineage is commonly observed with chemotherapeutics in general. For example, agents that are directly cytotoxic to hematopoietic precursors, that inhibit mitotic spindle formation, and antimetabolites that alter folate metabolism may all result in suppression of erythropoiesis. However, development of parvovirus-induced PRCA has been reported as a consequence of chemotherapeutic administration ( Song et al., 2002 , Rao et al., 1994 ). PRCA has occasionally been linked to xenobiotic treatment. A wide variety of xenobiotics from many different classes have been reported to cause PRCA. Examples of xenobiotics reportedly associated with PCRA include sulfonamides, allopurinol, procainamide, gold-containing compounds, rifampin, and chloroquine ( Young, 2016 , Mintzer et al., 2009 ). However, causality is often difficult to prove, and most associations are limited to low numbers of case reports. One study evaluated reports of PRCA associated with administration of 30 different xenobiotics, but causality was only attributed to treatment with azathioprine, isoniazid, and phenytoin ( Thompson and Gales, 1996 ). PRCA due to the development of anti-EPO antibodies may follow the administration of recombinant EPO in humans ( Casadevall et al., 2002 ) and EPO gene therapy in monkeys ( Gao et al., 2004 ). Administration of recombinant EPO to dogs has also led to the development of anti-EPO antibodies and PRCA ( Randolph et al., 2004 ). Aplastic anemia has also been linked to administration of xenobiotics. Classically, chloramphenicol is reported to sporadically cause aplastic anemia ( Segel and Lichtman, 2016 ). However, antithyroid compounds, sulfonamides including trimethoprim sulfamethoxazole, beta-lactams, the diuretic furosemide, gold-containing compounds, penicillamine, and anticonvulsants including carbamazepine and phenacetin have all been reported in association with aplastic anemia ( Mintzer et al., 2009 , Kaufman et al., 1996 ). Aplastic anemia has also been attributed to environmental or occupational exposure to benzene ( Smith, 1996 ). In a case of aplastic anemia in a dog, griseofulvin administration was suspected to be the cause of the aplastic anemia ( Brazzell and Weiss, 2006 ). Decreases in red cell mass with concurrent decreases in reticulocyte counts have occurred with prolonged or repeated high dose administration of G-CSF or GM-CSF-based xenobiotics in nonclinical toxicology studies, particularly in rodents. Impaired erythropoiesis in these cases occurs due to the massive expansion of myeloid precursors within the bone marrow. Extreme myeloid hyperplasia with continued stimulation results in overcrowding of the marrow cavities with less physical space available for erythroid production. 12.11.3.2.2.1 Preregenerative Depending on the timing of the insult causing the decreases in red cell mass, reticulocyte counts within reference interval may represent a preregenerative anemia rather than suppressed erythropoiesis. Production of erythrocytes by the bone marrow requires at least 3–4 days, and a peak increase in blood reticulocyte count occurs about 7–14 days following the insult ( Stockham and Scott, 2008b ). If it an individual with decreased red cell mass and reticulocyte counts that are within the reference interval and it is unclear if the individual has a preregenerative anemia or suppressed erythropoiesis, repeating a CBC several days later may help clarify which process is occurring. 12.11.3.2.2.2 Infectious Acute Chagas disease, caused by infection with Trypanosoma cruzi , has been reported to cause decreases in red cell mass in humans and monkeys ( de Titto and Araujo, 1988 , Rosner et al., 1988 , Seah et al., 1974 ). In experimentally infected Cebus paella monkeys, the acute phase of Chagas disease was reported to cause normocytic, normochromic anemia ( Rosner et al., 1988 ), typical of a nonregenerative anemia. Experimentally infected mice demonstrated bone marrow suppression with decreases in red cell mass as well as decreases in leukocyte and platelet counts ( Marcondes et al., 2000 ). Although rarely encountered in nonclinical toxicology studies, monkeys held in the southwestern United States may become infected with T . cruzi prior to distribution ( Magden et al., 2015 ). During parasitemia, trypomastigotes may be observed in peripheral blood smears. Viral infections may also cause decreases in red cell mass without concurrent increases in reticulocyte counts. Parvoviruses may cause decreases in red cell mass from direct infection of erythroid precursor resulting in decreased erythrocyte production, as well as decreased erythrocyte lifespans. Parvovirus may result in transient pure red cell aplasia (PRCA) in humans ( Van Horn et al., 1986 ). Cell-mediated suppression of erythropoiesis resulting in PRCA has also been reported with viral hepatitis ( Wilson et al., 1980 ) and Epstein-Barr virus infection ( Socinski et al., 1984 ). Although HIV infection can result in decreases in red cell mass through various mechanisms, direct infection of erythroid precursors appears to contribute to suppressed erythropoiesis ( Evans and Scadden, 2000 ). In cats, a membrane protein of feline leukemia virus has been associated with decreased growth of CFU-E ( Wellman et al., 1984 ). Flavivirus infection, such as dengue, may also result in decreases in red cell mass and reticulocyte counts through bone marrow suppression ( La Russa and Innis, 1995 ). 12.11.3.2.2.3 Chronic disease Anemia of chronic disease (ACD) is a relatively common cause of anemia, and anemia associated with inflammatory disease is included in ACD. The decreases in red cell mass observed with ACD are generally mild, and are generally normocytic, normochromic, indicating no changes in MCV or MCHC, respectively. ACD may occur through shortening of erythrocyte lifespans, alterations in iron metabolism, a blunted response of erythroid precursors to EPO, and decreased EPO production. Altered erythrocyte lifespans in patients with ACD may be related to increased macrophagic clearance of erythrocytes from circulation through unknown mechanisms ( Ganz, 2016 ). This type of mechanism has been associated with several chronic infections, including tuberculosis and endocarditis ( Weiss, 2002 ). More commonly, ACD is associated with impaired iron mobilization with low iron concentrations in serum or plasma despite adequate iron stores ( Means, 2000 ). Impaired mobilization of iron results from IL-6 induction of hepcidin that results in sequestration of iron in macrophages and decreased intestinal iron update ( Ganz, 2003 ), IL-1 stimulation of increased synthesis of ferritin which may bind to iron and impair delivery of iron to erythroid precursors ( Rogers et al., 1994 ), and with decreased expression and impaired internalization of the transferrin receptor ( Means, 2000 ). ACD from impaired iron metabolism is associated with numerous inflammatory, infectious, and even neoplastic conditions. ACD may also cause altered EPO responsiveness or decreased EPO production. Decreased responsiveness of erythroid precursors to EPO is cytokine-mediated, and has been associated with increases in TNFα, IL-1, and interferons ( Johnson et al., 1989 , Johnson et al., 1990 , Raefsky et al., 1985 ) that may commonly be associated with inflammatory conditions. Decreased EPO production may also be cytokine-mediated, and has been reported with increases in TGFβ, TNFα, and IL-1 ( Faquin et al., 1992 , Jelkmann et al., 1992 ). However, chronic renal disease may also result directly in impaired EPO production and decreased production of erythrocytes ( Sato and Yanagita, 2013 ). 12.11.3.2.2.4 Immune-mediated Immune-mediated destruction of erythroid precursors in the bone marrow results in decreases in red cell mass with concurrent decreases in reticulocyte counts. The immune-mediated conditions discussed here may represent a spectrum of disease associated with immune destruction of various stages of erythroid precursors rather than unrelated entities. 12.11.3.2.2.4.1 Autoimmune hemolytic anemia with decreases in reticulocyte counts Autoimmune hemolytic anemia with antibodies that target antigens on mid- to late-stage erythroid precursors ranging from rubricytes to metarubricytes results in AIHA with a decrease in reticulocyte count, which may also be called immune-mediated nonregenerative anemia or precursor-targeted immune-mediated anemia (PIMA). AIHA with reticulocytopenia is generally a normocytic, normochromic anemia. Bone marrow examination may reveal erythroid hyperplasia or maturation arrest ( Weiss, 2008 ) with pyramidal expansion of erythroid precursors at stages earlier than the targeted stage, indicative of ineffective erythropoiesis. This may be less apparent with autoantibodies that recognize more immature stages of erythroid precursors. Bone marrow evaluation may also reveal rubriphagocytosis, or erythroid precursors phagocytized by macrophages. The stage of phagocytized precursor depends on the stage or stages expressing the targeted antigen. 12.11.3.2.2.4.2 Pure red cell aplasia In patients affected by PRCA, there are marked decreases in reticulocyte counts along with variable decreases in red cell mass. Bone marrow examination typically reveals an absence of erythroid precursors (erythroid aplasia) or low numbers of the earliest stages of erythroid precursors (erythroid hypoplasia) ( Young, 2016 ). PRCA in people may be caused by antibodies that bind antigens on the earliest erythroid precursors or even antibodies that bind EPO and prevent EPO-dependent erythropoiesis, but it has also been attributed to clonal T-cell disorders ( Stockham and Scott, 2008b ). PRCA in dogs has been associated with IgG that inhibit erythropoiesis ( Weiss, 1986 ). PRCA may also be caused by inherited genetic defect in people. Inherited PRCA in people is called Diamond-Blackfan anemia, and often has an autosomal dominant inheritance pattern with defects in genes encoding ribosomal proteins ( Young, 2016 ). Macrocytosis, or increased numbers of large erythrocytes with increases in MCV, may be observed and is consistent with impaired EPO-dependent erythropoiesis ( Young, 2016 , Ohene-Abuakwa et al., 2005 ). 12.11.3.2.2.4.3 Aplastic anemia Aplastic anemia is a condition associated with decreases in all cellular blood components (pancytopenia), including decreases in red cell mass with concurrent decreases in reticulocyte counts. Upon examination, the bone marrow classically had severe hypocellularity of hematopoietic cells or an absence of hematopoietic precursors the marrow cavities filled by mostly adipocytes and some stromal elements. Aplastic anemia is thought to be most commonly immune-mediated ( Young et al., 2006 ), and may be frequently associated with cytotoxic T-cells that become autoreactive ( Segel and Lichtman, 2016 ). However, there are also cases of inherited aplastic anemia, most commonly Fanconi anemia associated with genetic mutations that impair DNA repair resulting in pancytopenia developing around 5–10 years of age in people ( Segel and Lichtman, 2016 ). A form of aplastic anemia associated with bone marrow depletion or hypocellularity of hematopoietic tissue and gelatinous transformation of marrow cavity fat has been reported with anorexia nervosa in people ( Abella et al., 2002 ) and with severe food restriction in rats ( Moriyama et al., 2008 ). 12.11.3.2.2.4.1 Autoimmune hemolytic anemia with decreases in reticulocyte counts Autoimmune hemolytic anemia with antibodies that target antigens on mid- to late-stage erythroid precursors ranging from rubricytes to metarubricytes results in AIHA with a decrease in reticulocyte count, which may also be called immune-mediated nonregenerative anemia or precursor-targeted immune-mediated anemia (PIMA). AIHA with reticulocytopenia is generally a normocytic, normochromic anemia. Bone marrow examination may reveal erythroid hyperplasia or maturation arrest ( Weiss, 2008 ) with pyramidal expansion of erythroid precursors at stages earlier than the targeted stage, indicative of ineffective erythropoiesis. This may be less apparent with autoantibodies that recognize more immature stages of erythroid precursors. Bone marrow evaluation may also reveal rubriphagocytosis, or erythroid precursors phagocytized by macrophages. The stage of phagocytized precursor depends on the stage or stages expressing the targeted antigen. 12.11.3.2.2.4.2 Pure red cell aplasia In patients affected by PRCA, there are marked decreases in reticulocyte counts along with variable decreases in red cell mass. Bone marrow examination typically reveals an absence of erythroid precursors (erythroid aplasia) or low numbers of the earliest stages of erythroid precursors (erythroid hypoplasia) ( Young, 2016 ). PRCA in people may be caused by antibodies that bind antigens on the earliest erythroid precursors or even antibodies that bind EPO and prevent EPO-dependent erythropoiesis, but it has also been attributed to clonal T-cell disorders ( Stockham and Scott, 2008b ). PRCA in dogs has been associated with IgG that inhibit erythropoiesis ( Weiss, 1986 ). PRCA may also be caused by inherited genetic defect in people. Inherited PRCA in people is called Diamond-Blackfan anemia, and often has an autosomal dominant inheritance pattern with defects in genes encoding ribosomal proteins ( Young, 2016 ). Macrocytosis, or increased numbers of large erythrocytes with increases in MCV, may be observed and is consistent with impaired EPO-dependent erythropoiesis ( Young, 2016 , Ohene-Abuakwa et al., 2005 ). 12.11.3.2.2.4.3 Aplastic anemia Aplastic anemia is a condition associated with decreases in all cellular blood components (pancytopenia), including decreases in red cell mass with concurrent decreases in reticulocyte counts. Upon examination, the bone marrow classically had severe hypocellularity of hematopoietic cells or an absence of hematopoietic precursors the marrow cavities filled by mostly adipocytes and some stromal elements. Aplastic anemia is thought to be most commonly immune-mediated ( Young et al., 2006 ), and may be frequently associated with cytotoxic T-cells that become autoreactive ( Segel and Lichtman, 2016 ). However, there are also cases of inherited aplastic anemia, most commonly Fanconi anemia associated with genetic mutations that impair DNA repair resulting in pancytopenia developing around 5–10 years of age in people ( Segel and Lichtman, 2016 ). A form of aplastic anemia associated with bone marrow depletion or hypocellularity of hematopoietic tissue and gelatinous transformation of marrow cavity fat has been reported with anorexia nervosa in people ( Abella et al., 2002 ) and with severe food restriction in rats ( Moriyama et al., 2008 ). 12.11.3.2.2.5 Nutritional deficiencies In addition to aplastic anemia associated with anorexia nervosa and severe food restriction, other nutritional deficiencies have been associated with ineffective erythropoiesis leading to decreases in red cell mass with decreases in reticulocyte counts. Iron deficiency and deficiencies of the B vitamins folate and cobalamin are examples of these nutritional deficiencies. Chronic iron deficiency results in impaired hematopoiesis due to the inability to synthesize sufficient hemoglobin, which may lead to a decrease in reticulocyte production. Deficiencies in folate and cobalamin also cause ineffective erythropoiesis due to defects in DNA synthesis, as discussed with folate and cobalamin deficiencies as a cause of deceases in neutrophil counts. In people, folate and cobalamin deficiencies result in megaloblastic anemia, characterized by larger than normal erythroid precursors in the bone marrow that have more cytoplasm with lower nuclear to cytoplasmic ratios than in normal erythroid precursors and asynchronous cytoplasmic and nuclear maturation ( Green, 2016 ). Megaloblastic erythrocytes may also be observed in circulation, and basophilic stippling or Howell-Jolly bodies may also be observed ( Green, 2016 ). In people, anemia attributable to a deficiency in cobalamin (vitamin B 12 ) may also be called pernicious anemia. In dogs and cats, megaloblastic erythroid cells may be observed in the bone marrow but may not be observed in blood ( Stockham and Scott, 2008b ). 12.11.3.2.2.6 Endocrinopathy Several endocrinopathies have also been associated with decreases in red cell mass with decreases in reticulocyte counts, including hypothyroidism, hypoadrenocorticism, and hyperestrogenism. In cases of hypothyroidism, several mechanisms may be contributing to the decreases in red cell mass. Decreased folate or cobalamin concentrations secondary to the hypothyroidism leading to ineffective erythropoiesis, decreased tissue oxygen demand leading to decreased EPO and lower baseline red cell mass, and ACD may contribute to the mild decreases in red cell mass observed with hypothyroidism ( Ottesen et al., 1995 , Hines et al., 1968 , Stockham and Scott, 2008b , Mehmet et al., 2012 ). Mild decreases in red cell mass without apparent changes in reticulocyte counts have been associated with hypoadrenocorticism. This may be due to a decrease in glucocorticoids, and the loss of the apparent proerythropoietic stimulation of glucocorticoids ( Stockham and Scott, 2008b ). Hyperestrogenism, which occurs with some ovarian or testicular neoplasms, may result in bone marrow toxicity and suppression of erythropoiesis, particularly in dogs ( Sontas et al., 2009 ). 12.11.3.2.2.7 Neoplasia Neoplasia may result in suppressed erythropoiesis. This may be due to neoplasia-related inflammation and cytokine release leading to ACD. However, granulocytic leukemia or lymphoproliferative neoplasia involving the bone marrow may result in crowding or effacement of the bone marrow cavities with impaired erythropoiesis that results in decreases in red cell mass with concurrent decreases in reticulocyte counts. Hematopoietic neoplasia involving the erythroid lineage usually results in atypical erythrocyte production that can lead to decreases in red cell mass and reticulocyte counts; however, nucleated erythrocytes with evidence of dysplasia may be observed in blood. Similar to hematopoietic neoplasms that efface the bone marrow, metastatic neoplasia, often carcinomas, may also cause myelophthisis and result in decreased erythropoiesis. 12.11.3.2.2.8 Xenobiotic-induced There are many xenobiotics that can cause decreases in red cell mass with concurrent decreases in reticulocyte counts. Bone marrow suppression that affects the erythroid lineage is commonly observed with chemotherapeutics in general. For example, agents that are directly cytotoxic to hematopoietic precursors, that inhibit mitotic spindle formation, and antimetabolites that alter folate metabolism may all result in suppression of erythropoiesis. However, development of parvovirus-induced PRCA has been reported as a consequence of chemotherapeutic administration ( Song et al., 2002 , Rao et al., 1994 ). PRCA has occasionally been linked to xenobiotic treatment. A wide variety of xenobiotics from many different classes have been reported to cause PRCA. Examples of xenobiotics reportedly associated with PCRA include sulfonamides, allopurinol, procainamide, gold-containing compounds, rifampin, and chloroquine ( Young, 2016 , Mintzer et al., 2009 ). However, causality is often difficult to prove, and most associations are limited to low numbers of case reports. One study evaluated reports of PRCA associated with administration of 30 different xenobiotics, but causality was only attributed to treatment with azathioprine, isoniazid, and phenytoin ( Thompson and Gales, 1996 ). PRCA due to the development of anti-EPO antibodies may follow the administration of recombinant EPO in humans ( Casadevall et al., 2002 ) and EPO gene therapy in monkeys ( Gao et al., 2004 ). Administration of recombinant EPO to dogs has also led to the development of anti-EPO antibodies and PRCA ( Randolph et al., 2004 ). Aplastic anemia has also been linked to administration of xenobiotics. Classically, chloramphenicol is reported to sporadically cause aplastic anemia ( Segel and Lichtman, 2016 ). However, antithyroid compounds, sulfonamides including trimethoprim sulfamethoxazole, beta-lactams, the diuretic furosemide, gold-containing compounds, penicillamine, and anticonvulsants including carbamazepine and phenacetin have all been reported in association with aplastic anemia ( Mintzer et al., 2009 , Kaufman et al., 1996 ). Aplastic anemia has also been attributed to environmental or occupational exposure to benzene ( Smith, 1996 ). In a case of aplastic anemia in a dog, griseofulvin administration was suspected to be the cause of the aplastic anemia ( Brazzell and Weiss, 2006 ). Decreases in red cell mass with concurrent decreases in reticulocyte counts have occurred with prolonged or repeated high dose administration of G-CSF or GM-CSF-based xenobiotics in nonclinical toxicology studies, particularly in rodents. Impaired erythropoiesis in these cases occurs due to the massive expansion of myeloid precursors within the bone marrow. Extreme myeloid hyperplasia with continued stimulation results in overcrowding of the marrow cavities with less physical space available for erythroid production. 12.11.4 Platelets The production of platelets from megakaryocytes (thrombopoiesis) and the production of megakaryocytes (megakaryopoiesis) occur mainly in the bone marrow of adult animals. Common myeloid progenitors differentiate into megakaryocyte-erythroid progenitors. Further differentiation results in formation of the earliest committed megakaryocytic cell, the burst-forming unit-megakaryocyte (BFU-Mk), which further differentiates into the colony-forming unit-megakaryocyte (CFU-Mk). Subsequent stages of differentiation are megakaryoblasts, followed by promegakaryocytes, then megakaryocytes. These latter stages may be recognized during light microscopic evaluation of bone marrow. Extensions of megakaryocyte cytoplasm (proplatelets) enter sinuses, or the microvasculature of the bone marrow. Within sinuses, these proplatelets are detached from the megakaryocyte by the shear forces of blood, after which they are further fragmented into platelets ( Harvey, 2012 , Junt et al., 2007 ). Thrombopoiesis, as well as megakaryopoiesis, is predominantly stimulated by TPO. However, SCF, stromal cell-derived factor 1 (SDF-1), IL-3, G-CSF, and GM-CSF may all contributed to platelet production ( Boudreaux, 2010 ). There are large species-based variations in platelet counts in health; rodents generally have the highest platelet counts of the common laboratory species, which may exceed 1,000,000 platelets μL − 1 , while nonhuman primates and dogs generally have lower but highly variable platelet counts. The circulating lifespan of platelets is approximately 5–9 days, and up to 30% of circulating platelets may be transiently contained by the spleen ( Russell, 2010 ). Clearance of senescent platelets from circulation is mainly due to phagocytosis by splenic macrophages. 12.11.4.1 Increases in Platelet Counts (Thrombocytosis) 12.11.4.1.1 Catecholamine-induced Increases in circulating catecholamine concentrations, such as epinephrine, from fright or excitement can result in mobilization of platelets from the spleen into circulation, usually through splenic contraction. The increases in platelet counts from catecholamine-induced redistribution are generally transient and resolve with splenic relaxation and decreases in catecholamine concentrations back to basal levels. Strenuous exercise may also cause redistribution of splenic platelets due to α-adrenergic stimulation, resulting in increases in blood platelet counts ( Chamberlain et al., 1990 ). 12.11.4.1.2 Inflammation or reactive Inflammatory or reactive increases in platelet counts are typically secondary to a process that causes general bone marrow stimulation, resulting in increased circulating TPO and therefore increased thrombopoiesis. These increases in platelets are not clonal. Increases in IL-6 may occur as the result of inflammatory or immune stimulation of myriad etiologies, but may also increase as part of a paraneoplastic syndrome in association with malignant neoplasia of nonhemic origin, including renal cell carcinoma, ovarian neoplasia, primary lung cancer, and gastrointestinal neoplasia ( Blay et al., 1993 , Stone et al., 2012 , Yanagawa et al., 1995 , Kabir et al., 1995 , Lin et al., 2014 ). Increased IL-6 concentrations have been demonstrated to increase liver production of TPO ( Kaser et al., 2001 ), which stimulates thrombopoiesis with consequent increases in blood platelet counts. Iron deficiency with decreases in red cell mass has been associated with reactive increases in platelet counts in many, but not all, cases ( Stockham and Scott, 2008c ). The mechanism of the increases in platelet counts is unclear ( Dan, 2005 ). Some studies have demonstrated that there are no detectable increases in TPO or IL-6 in iron deficiency anemia ( Akan et al., 2000 ), and that although EPO is increased with iron deficiency anemia, cross-reactivity of TPO and EPO does not explain the reactive thrombocytosis ( Geddis and Kaushansky, 2003 ). Transient increases in blood platelet counts have been reported following splenectomy. Splenectomy has been associated with increases in circulating TPO levels ( Ichikawa et al., 1998 ), resulting in increased platelet product ion and the observed increases in platelet counts. Increases in TPO also occur during instances of decreases in platelet counts, such as observed with immune-mediated platelet destruction, bone marrow suppression, or blood loss, as discussed later. Following resolution of the cause of decreased blood platelet counts, TPO stimulation of increased production may cause a transient increases in blood platelet counts, or rebound thrombocytosis, prior to normalization of platelet counts ( Stockham and Scott, 2008c ). 12.11.4.1.3 Neoplastic Neoplastic processes that cause primary (nonreactive) increases in blood platelet counts are characterized by clonal expansions of megakaryocytes and therefore platelets. Hemic neoplasia that may result in clonal increases in platelet counts include acute megakaryoblastic leukemia and chronic myeloproliferative disease such as primary or essential thrombocythemia. However, clonal increases in platelet counts are associated with increases in TPO greater than increases in TPO observed with reactive thrombocytosis, so TPO-mediated thrombopoiesis may also play a role in clonal increases in platelet counts ( Wang et al., 1998 ). In acute megakaryoblastic leukemia (AML M7), bone marrow contains ≥ 30% megakaryoblasts, and many cells of the megakaryocytic lineage have cytoplasmic blebs ( Gassmann and Löffler, 1995 ). Myelofibrosis is frequently also observed ( Tallman et al., 2000 ). Acute megakaryoblastic leukemia has been associated with increases in platelet counts, but decreases in platelet counts have also been described ( Stockham and Scott, 2008c ). A majority of cases of acute megakaryoblastic leukemia in both adults and children are associated with chromosomal abnormalities, often chromosomal translocations ( Duchayne et al., 2003 , Lion et al., 1992 ). Primary thrombocythemia has been associated with marked increases in platelet counts, but bone marrow megakaryoblasts are < 30% in contrast to acute megakaryoblastic leukemia. Evidence of platelet dysplasia may be observed on microscopic evaluation of blood smears; these morphologic changes may include hypogranular platelets, large platelets associated with an increase in mean platelet volume (MPV), or pleomorphic platelets ( Stockham and Scott, 2008c ). Similar to polycythemia vera, primary thrombocythemia has been associated with activating mutations in JAK2 ( Levine et al., 2005 ). However, primary thrombocythemia may also be associated with mutations in the TPO receptor gene, MPL , and the calreticulin gene, CALR ( Beer and Green, 2016 ). 12.11.4.1.4 Xenobiotic-induced Administration of exogenous catecholamines has been reported to cause increases in blood platelet counts, and the mechanism is primarily from splenic contraction and a transient increased in platelet counts. However, platelet pools from pulmonary circulation may also contribute. For example, administration of epinephrine to dogs resulted in dose-responsive increases in blood platelet counts that were believed to be the result of adrenaline-induced mobilization of platelets from pulmonary circulation into peripheral blood ( Bierman et al., 1952 ). Increases in platelet counts associated with xenobiotic administration are most commonly reactive, and associated with increases in IL-6 and/or TPO. Any xenobiotic that can result in an inflammatory stimulus may result in reactive increases in platelet counts, and examples of such xenobiotics are discussed in more detail in previous sections. Resolution of xenobiotic-induced bone marrow suppression may cause a transient rebound increase in platelet counts due to increased TPO and thrombopoiesis secondary to the xenobiotic-induced decreases in platelet counts. G-CSF and GM-CSF-based xenobiotic administration has been associated with increases in blood platelet counts from general bone marrow stimulation. These changes may be due to proliferation of myeloid precursors including common precursors that may then differentiate into megakaryocytes. Xenobiotic-induced increases in platelet counts have also been attributed to treatment with vinca alkaloids or miconazole, and are believed to be attributable to the ability of these xenobiotics to stimulate increased megakaryocyte production within the bone marrow ( Frye and Thompson, 1993 ). While increases in platelet counts have been reported with treatment with several antibiotic classes, including some cephalosporins, β-lactams, and penicillin, causality is difficult to prove in these cases and a reactive thrombocytosis from inflammation associated with the infectious process being treated must be considered ( Frye and Thompson, 1993 ). 12.11.4.2 Decreases in Platelet Counts (Thrombocytopenia) Marked decreases in platelet counts are a clinical concern because they may be associated with spontaneous bleeding. Animals are typically considered at risk for spontaneous bleeding when platelet counts are < 50,000 μL − 1 and at a significantly greater risk for spontaneous bleeding with < 10,000 platelets μL − 1 ( Russell, 2010 ), although hemorrhage with platelet counts < 50,000 μL − 1 may also occur following surgery or trauma, including venipuncture routinely performed during nonclinical toxicology studies. Minimal to moderate decreases in platelet counts are typically not associated with hemorrhage, and are not a major clinical concern unless there is a concurrent platelet functional defect. Increased MPV is an indicator of increased average platelet size, and may be associated with rapid or increased thrombopoiesis and indicative of a bone marrow response to decreases in platelet counts. 12.11.4.2.1 Relative Relative decreases in platelet counts occur when circulating platelet mass is not changed, but platelet clumping, redistribution, or hemodilution alter automated or estimated platelet counts. False decreases in platelet counts (pseudo-thrombocytopenia) due to platelet clumping are often present in rats, mice, and cats, although it may be observed in any species. MPV may be increased with the presence of platelet clumps, and microscopic blood smear review should be performed in all cases of decreased platelet counts to assess for the presence of platelet clumps; increases in MPV in instances of platelet clumping do not reflect a bone marrow response because a true decrease in platelet count is not present to stimulate thrombopoiesis. Resampling of blood with a clean venipuncture and use of sodium citrate as the anticoagulant may help reduce platelet clumps and result in a more accurate automated platelet count. Redistribution or sequestration of circulating platelets may cause decreases in platelet counts, which may be transient. Platelet redistribution may be observed with splenomegaly, hypersplenism, or with severe hypothermia; total platelet mass is unaffected in these cases and typically does not result in increased TPO or platelet production ( Stockham and Scott, 2008c ). Hemodilution may occur following administration of intravenous fluids or massive transfusion, and is expected to decrease all blood components to variable degrees, with the exception of any transfused blood components. Decreases in platelets from hemodilution are usually mild and may not be detected if platelet counts remain within reference intervals or historical control ranges. These changes are generally transient and resolve with redistribution of intravascular (extracellular) fluid into intracellular fluid compartments or elimination of excess fluid. 12.11.4.2.2 Loss Acute, severe loss of whole blood may result in decrease in all blood components, including platelets. Blood loss of significant magnitude to cause decreases in blood platelet counts may occur following trauma, splenic rupture from trauma or neoplasia, or from uncontrolled bleeding associated with coagulopathies, such as hemophilia A or B. However, consumption of platelets at the site(s) of hemorrhage may also contribute to the decreases in platelet counts observed with blood loss. 12.11.4.2.3 Decreased survival Decreased platelet survival may be due to increased platelet destruction and/or consumption, and is a relatively common cause of decreases in blood platelet counts. Destruction of platelets is commonly associated with immune-mediated mechanisms, including immune-mediated thrombocytopenia (IMT) and immune-mediated thrombocytopenic purpura (ITP) in humans. Most autoantibodies that cause platelet destruction are of the IgG class, although cases with IgM or IgA antiplatelet antibodies have also been reported ( Diz-Küçükkaya and López, 2016 ). Antiplatelet antibodies may directly target platelet antigens, such as the glycoprotein integrin α IIβ β 3(GPIIβ-IIIα) that plays a major role in platelet aggregation ( He et al., 1994 ), antigens exposed or formed on platelet surfaces by infectious agents, or infectious agent-targeted antibodies that cross-react with normally expressed platelet surface antigens or membrane components such as phospholipid ( Stockham and Scott, 2008c ). Antibody-bound platelets may be phagocytosed and destroyed by splenic, bone marrow, or hepatic macrophages, or may be associated with complement-mediated destruction or stimulation of phagocytosis ( Diz-Küçükkaya and López, 2016 , Russell, 2010 ). Production of antiplatelet antibodies may be unassociated with an underlying disease condition (idiopathic or primary) and is usually presumed to be an autoimmune process, or may be secondary to infectious or neoplastic processes ( Stockham and Scott, 2008c ). In sexually mature Göttingen minipigs, spontaneous thrombocytopenic purpura has been described and is believed to be caused immune-mediated platelet destruction ( Carrasco et al., 2003 ). ITP secondary to infectious disease has been reported to occur with various bacterial and viral etiologies, including Helicobacter pylori , many rickettsial diseases, HIV, cytomegalovirus, and hepatitis B and C viruses ( Diz-Küçükkaya and López, 2016 , Russell, 2010 , Cines et al., 2009 ). Lymphoproliferative neoplasia is a relatively common cause of ITP, and has been associated with chronic lymphocytic leukemia, Hodgkin lymphoma, and leukemia of large granular T-lymphocytes ( Cines et al., 2009 ). Connective tissue diseases such as SLE have also been associated with ITP ( Cines et al., 2009 ). Decreases in platelet counts due to platelet activation and consumption are commonly associated with local or disseminated consumptive coagulopathies. Localized consumptive coagulopathy may be associated with sites of hemorrhage, microangiopathy or thrombosis, or vascular neoplasia such as hemangiosarcoma ( Stockham and Scott, 2008c ). Thrombosis associated with chronic catheterization may be a cause of localized platelet consumption. DIC may be associated with infectious agents, particularly bacterial infections that cause septicemia or endotoxemia, hepatic disease, a variety of neoplastic diseases, and pancreatitis ( Stockham and Scott, 2008c ). Activation and consumption of platelets associated with decreases in platelet counts has also been observed with vasculitis and conditions associated with turbulent blood flow such as endocarditis, cardiac valvular disease, following cardiac surgery, and with arterial disease that alter or damage endothelial cells ( Russell, 2010 , Selleng et al., 2010 , Stockham and Scott, 2008c , Gregg and Goldschmidt-Clermont, 2003 ). Hemolytic uremic syndrome (HUS) is a cause of thrombotic microangiopathy that may be associated with infection with Shigella dysenteriae or Escherichia coli ( Russell, 2010 ). 12.11.4.2.4 Decreased production Bone marrow suppression that involves the megakaryocytic lineage causes decreases in platelet production and therefore decreases in platelet counts. Bone marrow suppression may occur in association with infectious agents that can directly infect hematopoietic precursors such as immunodeficiency viruses in various species, parvoviruses, distemper virus in dogs, and feline leukemia virus in cats ( Russell, 2010 , Stockham and Scott, 2008c , Scaradavou, 2002 ). Chronic ehrlichiosis in dogs has also been reported to cause bone marrow hypoplasia, although the mechanism remains unclear ( Stockham and Scott, 2008c ). However, decreases in platelet counts attributable to infectious agents may have contributions from mechanisms other than bone marrow suppression, such as peripheral consumption or immune-mediated destruction. Hyperestrogenism associated with testicular or ovarian neoplasia may also cause general bone marrow suppression, and dogs appear to be particularly sensitive to this effect ( Sontas et al., 2009 ). Causes of decreased bone marrow megakaryocytes with decreases in blood platelet counts have also been associated with mechanisms that are likely immune-mediated. Aplastic anemia, which is most commonly caused by immune-mediated mechanisms ( Young et al., 2006 ), is associated with generalized bone marrow hypoplasia involving all hematopoietic lineages, including megakaryocytes. Amegakaryocytic thrombocytopenic purpura in humans may be inherited or acquired, and acquired forms commonly occur through immune-mediated mechanisms ( Diz-Küçükkaya and López, 2016 ). Inherited or congenital forms cause marked decreases in blood platelet counts with an absence of megakaryocytes in the bone marrow. This form is frequently associated with mutations in the TPO receptor gene, MPL , and may progress to aplastic anemia ( Germeshausen et al., 2006 , Van Den Oudenrijn et al., 2000 ). Acquired amegakaryocytic thrombocytopenic purpura is likely associated with immune-mediated decreases in bone marrow megakaryocytes ( Tristano, 2005 ), and antibodies that bind TPO or the TPO receptor have also been reported to cause acquired amegakaryocytic thrombocytopenic purpura ( Shiozaki et al., 2000 , Katsumata et al., 2003 ). Generalized bone marrow disease, such as replacement of normal bone marrow hematopoietic tissue with hemic or nonhemic neoplasia, severe granulomatous inflammation effacing normal bone marrow tissue, myelofibrosis, or bone marrow necrosis, will result in decreases in megakaryocytes and thrombopoiesis with subsequent decreases in blood platelet counts ( Russell, 2010 , Stockham and Scott, 2008c ). 12.11.4.2.5 Xenobiotic-induced Blood loss associated with xenobiotics has been reported with coagulopathies due to abnormal vitamin K recycling and function of vitamin K-dependent coagulation factors. Drugs implicated in abnormal vitamin K synthesis include warfarin, rodenticides such as brodifacoum, and some broad-spectrum antibiotics ( Bloom and Brandt, 2008 ). With severe, acute loss of whole blood, all blood components, including platelets, will be decreased. If other mechanisms of xenobiotic-induced decreases in platelet counts, as discussed later, cause severe enough decreases in blood platelet counts (e.g., < 50,000 platelets μL − 1 ), then blood loss could be secondary to the decreases in platelets. Thrombotic microangiopathy syndrome, which may include thrombotic thrombocytopenic purpura (TTP) and HUS, may cause peripheral consumption and/or destruction of platelets with the development of decreases in platelet counts. Xenobiotic-induced endothelial injury leads to platelet activation and aggregation ( Pisoni et al., 2001 ). Several chemotherapeutic agents associated with thrombotic microangiopathy syndrome include mitomycin C ( Cantrell et al., 1985 ), cisplatin ( Palmisano et al., 1998 ), estramustine phosphate ( Tassinari et al., 1999 ), gemcitabine ( Nackaerts et al., 1998 ), and daunorubicin ( Byrnes et al., 1986 ). Nonchemotherapeutic agents, including immunomodulators such as cyclosporine and tacrolimus ( Katznelson et al., 1994 , Trimarchi et al., 1999 ), simvastatin ( McCarthy et al., 1998 ), and inhibitors of platelet aggregation including ticlopidine and clopidogrel ( Bennett et al., 1998 , Bennett et al., 2000 ), have also been associated with thrombotic microangiopathy syndrome. Immune-mediated destruction of platelets is a relatively common cause of xenobiotic-induced decreases in blood platelet counts. Antiplatelet antibodies may be formed through various mechanisms. Penicillin and some cephalosporins cause platelet destruction through hapten-type or drug-dependent antibody production, while quinidine and some NSAIDS can cause immune-mediated platelet destruction through the formation of antibodies that only bind platelets when the soluble drug is present ( Aster and Bougie, 2007 ). Some drugs that inhibit the platelet glycoprotein α IIβ β 3(GPIIβ-IIIα) may lead to platelet expression of a new antigen due to conformation changes to the glycoprotein complexes that can then be bound by antibodies; such drugs include tirofiban, roxifiban, and eptifibatide ( Aster and Bougie, 2007 , Aster, 2005 ). Abciximab has been reported to cause drug-specific antibodies that cause decreases in platelet counts ( Aster, 2005 ). Xenobiotic-induced production of autoantibodies that bind platelets, or drug-independent antibodies, has been attributed to procainamide and gold-based compound administration ( Aster and Bougie, 2007 ). In humans, immune-complex type antibodies are classically associated with heparin therapy, and are due to the interaction of heparin, a platelet granule component, and platelet factor 4 (PF4) ( Aster and Bougie, 2007 , Visentin and Liu, 2007 , Arepally and Ortel, 2006 ). Bone marrow suppression is caused by numerous xenobiotics, most notably chemotherapeutic agents. Chemotherapeutic agents associated with bone marrow suppression include compounds from many classes, including alkylating agents such as busulfan, antimetabolites that impair DNA synthesis such as methotrexate and mercaptopurine, antibiotics such as doxorubicin, and mitotic spindle inhibitors such as the vinca alkaloids vinblastine and vincristine ( Carey, 2003 , Weiss, 2010 , Stockham and Scott, 2008c ). Idiosyncratic myelosuppression may also occur with numerous nonchemotherapeutic xenobiotics, and has been reported with antithyroid drugs such as methimazole, anticonvulsants including felbamate and carbamazepine, antipsychotic agents such as clozapine, cardiovascular drugs such as methyldopa and captopril, antibiotics including trimethoprim–sulfamethoxazole and chloroquine, and other xenobiotic agents including gold-based compounds, diclofenac, and allopurinol ( Carey, 2003 ). Xenobiotic-induced aplastic anemia is most commonly associated with immune-mediated destruction of uncommitted or early hematopoietic stem cells, although direct cytotoxicity, such as with chemotherapeutic agents, may also lead to aplastic anemia. Several xenobiotics, including chloramphenicol, anticonvulsants such as phenytoin and carbamazepine, gold-based compounds, and phenylbutazone have been associated with aplastic anemia ( Bloom and Brandt, 2008 ). Miscellaneous causes of xenobiotic-induced decreases in blood platelet counts include nonimmune-mediated destruction reported to occur with desmopressin ( Bloom and Brandt, 2008 ) and idiosyncratic reactions associated with CARPA ( Patkó and Szebeni, 2015 ) or administration of some antisense oligonucleotides ( Frazier, 2015 ). Idiosyncratic decreases in platelet counts have also been reported as an off-target effect of human monoclonal antibodies during nonclinical toxicology studies ( Everds et al., 2013b ). 12.11.4.1 Increases in Platelet Counts (Thrombocytosis) 12.11.4.1.1 Catecholamine-induced Increases in circulating catecholamine concentrations, such as epinephrine, from fright or excitement can result in mobilization of platelets from the spleen into circulation, usually through splenic contraction. The increases in platelet counts from catecholamine-induced redistribution are generally transient and resolve with splenic relaxation and decreases in catecholamine concentrations back to basal levels. Strenuous exercise may also cause redistribution of splenic platelets due to α-adrenergic stimulation, resulting in increases in blood platelet counts ( Chamberlain et al., 1990 ). 12.11.4.1.2 Inflammation or reactive Inflammatory or reactive increases in platelet counts are typically secondary to a process that causes general bone marrow stimulation, resulting in increased circulating TPO and therefore increased thrombopoiesis. These increases in platelets are not clonal. Increases in IL-6 may occur as the result of inflammatory or immune stimulation of myriad etiologies, but may also increase as part of a paraneoplastic syndrome in association with malignant neoplasia of nonhemic origin, including renal cell carcinoma, ovarian neoplasia, primary lung cancer, and gastrointestinal neoplasia ( Blay et al., 1993 , Stone et al., 2012 , Yanagawa et al., 1995 , Kabir et al., 1995 , Lin et al., 2014 ). Increased IL-6 concentrations have been demonstrated to increase liver production of TPO ( Kaser et al., 2001 ), which stimulates thrombopoiesis with consequent increases in blood platelet counts. Iron deficiency with decreases in red cell mass has been associated with reactive increases in platelet counts in many, but not all, cases ( Stockham and Scott, 2008c ). The mechanism of the increases in platelet counts is unclear ( Dan, 2005 ). Some studies have demonstrated that there are no detectable increases in TPO or IL-6 in iron deficiency anemia ( Akan et al., 2000 ), and that although EPO is increased with iron deficiency anemia, cross-reactivity of TPO and EPO does not explain the reactive thrombocytosis ( Geddis and Kaushansky, 2003 ). Transient increases in blood platelet counts have been reported following splenectomy. Splenectomy has been associated with increases in circulating TPO levels ( Ichikawa et al., 1998 ), resulting in increased platelet product ion and the observed increases in platelet counts. Increases in TPO also occur during instances of decreases in platelet counts, such as observed with immune-mediated platelet destruction, bone marrow suppression, or blood loss, as discussed later. Following resolution of the cause of decreased blood platelet counts, TPO stimulation of increased production may cause a transient increases in blood platelet counts, or rebound thrombocytosis, prior to normalization of platelet counts ( Stockham and Scott, 2008c ). 12.11.4.1.3 Neoplastic Neoplastic processes that cause primary (nonreactive) increases in blood platelet counts are characterized by clonal expansions of megakaryocytes and therefore platelets. Hemic neoplasia that may result in clonal increases in platelet counts include acute megakaryoblastic leukemia and chronic myeloproliferative disease such as primary or essential thrombocythemia. However, clonal increases in platelet counts are associated with increases in TPO greater than increases in TPO observed with reactive thrombocytosis, so TPO-mediated thrombopoiesis may also play a role in clonal increases in platelet counts ( Wang et al., 1998 ). In acute megakaryoblastic leukemia (AML M7), bone marrow contains ≥ 30% megakaryoblasts, and many cells of the megakaryocytic lineage have cytoplasmic blebs ( Gassmann and Löffler, 1995 ). Myelofibrosis is frequently also observed ( Tallman et al., 2000 ). Acute megakaryoblastic leukemia has been associated with increases in platelet counts, but decreases in platelet counts have also been described ( Stockham and Scott, 2008c ). A majority of cases of acute megakaryoblastic leukemia in both adults and children are associated with chromosomal abnormalities, often chromosomal translocations ( Duchayne et al., 2003 , Lion et al., 1992 ). Primary thrombocythemia has been associated with marked increases in platelet counts, but bone marrow megakaryoblasts are < 30% in contrast to acute megakaryoblastic leukemia. Evidence of platelet dysplasia may be observed on microscopic evaluation of blood smears; these morphologic changes may include hypogranular platelets, large platelets associated with an increase in mean platelet volume (MPV), or pleomorphic platelets ( Stockham and Scott, 2008c ). Similar to polycythemia vera, primary thrombocythemia has been associated with activating mutations in JAK2 ( Levine et al., 2005 ). However, primary thrombocythemia may also be associated with mutations in the TPO receptor gene, MPL , and the calreticulin gene, CALR ( Beer and Green, 2016 ). 12.11.4.1.4 Xenobiotic-induced Administration of exogenous catecholamines has been reported to cause increases in blood platelet counts, and the mechanism is primarily from splenic contraction and a transient increased in platelet counts. However, platelet pools from pulmonary circulation may also contribute. For example, administration of epinephrine to dogs resulted in dose-responsive increases in blood platelet counts that were believed to be the result of adrenaline-induced mobilization of platelets from pulmonary circulation into peripheral blood ( Bierman et al., 1952 ). Increases in platelet counts associated with xenobiotic administration are most commonly reactive, and associated with increases in IL-6 and/or TPO. Any xenobiotic that can result in an inflammatory stimulus may result in reactive increases in platelet counts, and examples of such xenobiotics are discussed in more detail in previous sections. Resolution of xenobiotic-induced bone marrow suppression may cause a transient rebound increase in platelet counts due to increased TPO and thrombopoiesis secondary to the xenobiotic-induced decreases in platelet counts. G-CSF and GM-CSF-based xenobiotic administration has been associated with increases in blood platelet counts from general bone marrow stimulation. These changes may be due to proliferation of myeloid precursors including common precursors that may then differentiate into megakaryocytes. Xenobiotic-induced increases in platelet counts have also been attributed to treatment with vinca alkaloids or miconazole, and are believed to be attributable to the ability of these xenobiotics to stimulate increased megakaryocyte production within the bone marrow ( Frye and Thompson, 1993 ). While increases in platelet counts have been reported with treatment with several antibiotic classes, including some cephalosporins, β-lactams, and penicillin, causality is difficult to prove in these cases and a reactive thrombocytosis from inflammation associated with the infectious process being treated must be considered ( Frye and Thompson, 1993 ). 12.11.4.1.1 Catecholamine-induced Increases in circulating catecholamine concentrations, such as epinephrine, from fright or excitement can result in mobilization of platelets from the spleen into circulation, usually through splenic contraction. The increases in platelet counts from catecholamine-induced redistribution are generally transient and resolve with splenic relaxation and decreases in catecholamine concentrations back to basal levels. Strenuous exercise may also cause redistribution of splenic platelets due to α-adrenergic stimulation, resulting in increases in blood platelet counts ( Chamberlain et al., 1990 ). 12.11.4.1.2 Inflammation or reactive Inflammatory or reactive increases in platelet counts are typically secondary to a process that causes general bone marrow stimulation, resulting in increased circulating TPO and therefore increased thrombopoiesis. These increases in platelets are not clonal. Increases in IL-6 may occur as the result of inflammatory or immune stimulation of myriad etiologies, but may also increase as part of a paraneoplastic syndrome in association with malignant neoplasia of nonhemic origin, including renal cell carcinoma, ovarian neoplasia, primary lung cancer, and gastrointestinal neoplasia ( Blay et al., 1993 , Stone et al., 2012 , Yanagawa et al., 1995 , Kabir et al., 1995 , Lin et al., 2014 ). Increased IL-6 concentrations have been demonstrated to increase liver production of TPO ( Kaser et al., 2001 ), which stimulates thrombopoiesis with consequent increases in blood platelet counts. Iron deficiency with decreases in red cell mass has been associated with reactive increases in platelet counts in many, but not all, cases ( Stockham and Scott, 2008c ). The mechanism of the increases in platelet counts is unclear ( Dan, 2005 ). Some studies have demonstrated that there are no detectable increases in TPO or IL-6 in iron deficiency anemia ( Akan et al., 2000 ), and that although EPO is increased with iron deficiency anemia, cross-reactivity of TPO and EPO does not explain the reactive thrombocytosis ( Geddis and Kaushansky, 2003 ). Transient increases in blood platelet counts have been reported following splenectomy. Splenectomy has been associated with increases in circulating TPO levels ( Ichikawa et al., 1998 ), resulting in increased platelet product ion and the observed increases in platelet counts. Increases in TPO also occur during instances of decreases in platelet counts, such as observed with immune-mediated platelet destruction, bone marrow suppression, or blood loss, as discussed later. Following resolution of the cause of decreased blood platelet counts, TPO stimulation of increased production may cause a transient increases in blood platelet counts, or rebound thrombocytosis, prior to normalization of platelet counts ( Stockham and Scott, 2008c ). 12.11.4.1.3 Neoplastic Neoplastic processes that cause primary (nonreactive) increases in blood platelet counts are characterized by clonal expansions of megakaryocytes and therefore platelets. Hemic neoplasia that may result in clonal increases in platelet counts include acute megakaryoblastic leukemia and chronic myeloproliferative disease such as primary or essential thrombocythemia. However, clonal increases in platelet counts are associated with increases in TPO greater than increases in TPO observed with reactive thrombocytosis, so TPO-mediated thrombopoiesis may also play a role in clonal increases in platelet counts ( Wang et al., 1998 ). In acute megakaryoblastic leukemia (AML M7), bone marrow contains ≥ 30% megakaryoblasts, and many cells of the megakaryocytic lineage have cytoplasmic blebs ( Gassmann and Löffler, 1995 ). Myelofibrosis is frequently also observed ( Tallman et al., 2000 ). Acute megakaryoblastic leukemia has been associated with increases in platelet counts, but decreases in platelet counts have also been described ( Stockham and Scott, 2008c ). A majority of cases of acute megakaryoblastic leukemia in both adults and children are associated with chromosomal abnormalities, often chromosomal translocations ( Duchayne et al., 2003 , Lion et al., 1992 ). Primary thrombocythemia has been associated with marked increases in platelet counts, but bone marrow megakaryoblasts are < 30% in contrast to acute megakaryoblastic leukemia. Evidence of platelet dysplasia may be observed on microscopic evaluation of blood smears; these morphologic changes may include hypogranular platelets, large platelets associated with an increase in mean platelet volume (MPV), or pleomorphic platelets ( Stockham and Scott, 2008c ). Similar to polycythemia vera, primary thrombocythemia has been associated with activating mutations in JAK2 ( Levine et al., 2005 ). However, primary thrombocythemia may also be associated with mutations in the TPO receptor gene, MPL , and the calreticulin gene, CALR ( Beer and Green, 2016 ). 12.11.4.1.4 Xenobiotic-induced Administration of exogenous catecholamines has been reported to cause increases in blood platelet counts, and the mechanism is primarily from splenic contraction and a transient increased in platelet counts. However, platelet pools from pulmonary circulation may also contribute. For example, administration of epinephrine to dogs resulted in dose-responsive increases in blood platelet counts that were believed to be the result of adrenaline-induced mobilization of platelets from pulmonary circulation into peripheral blood ( Bierman et al., 1952 ). Increases in platelet counts associated with xenobiotic administration are most commonly reactive, and associated with increases in IL-6 and/or TPO. Any xenobiotic that can result in an inflammatory stimulus may result in reactive increases in platelet counts, and examples of such xenobiotics are discussed in more detail in previous sections. Resolution of xenobiotic-induced bone marrow suppression may cause a transient rebound increase in platelet counts due to increased TPO and thrombopoiesis secondary to the xenobiotic-induced decreases in platelet counts. G-CSF and GM-CSF-based xenobiotic administration has been associated with increases in blood platelet counts from general bone marrow stimulation. These changes may be due to proliferation of myeloid precursors including common precursors that may then differentiate into megakaryocytes. Xenobiotic-induced increases in platelet counts have also been attributed to treatment with vinca alkaloids or miconazole, and are believed to be attributable to the ability of these xenobiotics to stimulate increased megakaryocyte production within the bone marrow ( Frye and Thompson, 1993 ). While increases in platelet counts have been reported with treatment with several antibiotic classes, including some cephalosporins, β-lactams, and penicillin, causality is difficult to prove in these cases and a reactive thrombocytosis from inflammation associated with the infectious process being treated must be considered ( Frye and Thompson, 1993 ). 12.11.4.2 Decreases in Platelet Counts (Thrombocytopenia) Marked decreases in platelet counts are a clinical concern because they may be associated with spontaneous bleeding. Animals are typically considered at risk for spontaneous bleeding when platelet counts are < 50,000 μL − 1 and at a significantly greater risk for spontaneous bleeding with < 10,000 platelets μL − 1 ( Russell, 2010 ), although hemorrhage with platelet counts < 50,000 μL − 1 may also occur following surgery or trauma, including venipuncture routinely performed during nonclinical toxicology studies. Minimal to moderate decreases in platelet counts are typically not associated with hemorrhage, and are not a major clinical concern unless there is a concurrent platelet functional defect. Increased MPV is an indicator of increased average platelet size, and may be associated with rapid or increased thrombopoiesis and indicative of a bone marrow response to decreases in platelet counts. 12.11.4.2.1 Relative Relative decreases in platelet counts occur when circulating platelet mass is not changed, but platelet clumping, redistribution, or hemodilution alter automated or estimated platelet counts. False decreases in platelet counts (pseudo-thrombocytopenia) due to platelet clumping are often present in rats, mice, and cats, although it may be observed in any species. MPV may be increased with the presence of platelet clumps, and microscopic blood smear review should be performed in all cases of decreased platelet counts to assess for the presence of platelet clumps; increases in MPV in instances of platelet clumping do not reflect a bone marrow response because a true decrease in platelet count is not present to stimulate thrombopoiesis. Resampling of blood with a clean venipuncture and use of sodium citrate as the anticoagulant may help reduce platelet clumps and result in a more accurate automated platelet count. Redistribution or sequestration of circulating platelets may cause decreases in platelet counts, which may be transient. Platelet redistribution may be observed with splenomegaly, hypersplenism, or with severe hypothermia; total platelet mass is unaffected in these cases and typically does not result in increased TPO or platelet production ( Stockham and Scott, 2008c ). Hemodilution may occur following administration of intravenous fluids or massive transfusion, and is expected to decrease all blood components to variable degrees, with the exception of any transfused blood components. Decreases in platelets from hemodilution are usually mild and may not be detected if platelet counts remain within reference intervals or historical control ranges. These changes are generally transient and resolve with redistribution of intravascular (extracellular) fluid into intracellular fluid compartments or elimination of excess fluid. 12.11.4.2.2 Loss Acute, severe loss of whole blood may result in decrease in all blood components, including platelets. Blood loss of significant magnitude to cause decreases in blood platelet counts may occur following trauma, splenic rupture from trauma or neoplasia, or from uncontrolled bleeding associated with coagulopathies, such as hemophilia A or B. However, consumption of platelets at the site(s) of hemorrhage may also contribute to the decreases in platelet counts observed with blood loss. 12.11.4.2.3 Decreased survival Decreased platelet survival may be due to increased platelet destruction and/or consumption, and is a relatively common cause of decreases in blood platelet counts. Destruction of platelets is commonly associated with immune-mediated mechanisms, including immune-mediated thrombocytopenia (IMT) and immune-mediated thrombocytopenic purpura (ITP) in humans. Most autoantibodies that cause platelet destruction are of the IgG class, although cases with IgM or IgA antiplatelet antibodies have also been reported ( Diz-Küçükkaya and López, 2016 ). Antiplatelet antibodies may directly target platelet antigens, such as the glycoprotein integrin α IIβ β 3(GPIIβ-IIIα) that plays a major role in platelet aggregation ( He et al., 1994 ), antigens exposed or formed on platelet surfaces by infectious agents, or infectious agent-targeted antibodies that cross-react with normally expressed platelet surface antigens or membrane components such as phospholipid ( Stockham and Scott, 2008c ). Antibody-bound platelets may be phagocytosed and destroyed by splenic, bone marrow, or hepatic macrophages, or may be associated with complement-mediated destruction or stimulation of phagocytosis ( Diz-Küçükkaya and López, 2016 , Russell, 2010 ). Production of antiplatelet antibodies may be unassociated with an underlying disease condition (idiopathic or primary) and is usually presumed to be an autoimmune process, or may be secondary to infectious or neoplastic processes ( Stockham and Scott, 2008c ). In sexually mature Göttingen minipigs, spontaneous thrombocytopenic purpura has been described and is believed to be caused immune-mediated platelet destruction ( Carrasco et al., 2003 ). ITP secondary to infectious disease has been reported to occur with various bacterial and viral etiologies, including Helicobacter pylori , many rickettsial diseases, HIV, cytomegalovirus, and hepatitis B and C viruses ( Diz-Küçükkaya and López, 2016 , Russell, 2010 , Cines et al., 2009 ). Lymphoproliferative neoplasia is a relatively common cause of ITP, and has been associated with chronic lymphocytic leukemia, Hodgkin lymphoma, and leukemia of large granular T-lymphocytes ( Cines et al., 2009 ). Connective tissue diseases such as SLE have also been associated with ITP ( Cines et al., 2009 ). Decreases in platelet counts due to platelet activation and consumption are commonly associated with local or disseminated consumptive coagulopathies. Localized consumptive coagulopathy may be associated with sites of hemorrhage, microangiopathy or thrombosis, or vascular neoplasia such as hemangiosarcoma ( Stockham and Scott, 2008c ). Thrombosis associated with chronic catheterization may be a cause of localized platelet consumption. DIC may be associated with infectious agents, particularly bacterial infections that cause septicemia or endotoxemia, hepatic disease, a variety of neoplastic diseases, and pancreatitis ( Stockham and Scott, 2008c ). Activation and consumption of platelets associated with decreases in platelet counts has also been observed with vasculitis and conditions associated with turbulent blood flow such as endocarditis, cardiac valvular disease, following cardiac surgery, and with arterial disease that alter or damage endothelial cells ( Russell, 2010 , Selleng et al., 2010 , Stockham and Scott, 2008c , Gregg and Goldschmidt-Clermont, 2003 ). Hemolytic uremic syndrome (HUS) is a cause of thrombotic microangiopathy that may be associated with infection with Shigella dysenteriae or Escherichia coli ( Russell, 2010 ). 12.11.4.2.4 Decreased production Bone marrow suppression that involves the megakaryocytic lineage causes decreases in platelet production and therefore decreases in platelet counts. Bone marrow suppression may occur in association with infectious agents that can directly infect hematopoietic precursors such as immunodeficiency viruses in various species, parvoviruses, distemper virus in dogs, and feline leukemia virus in cats ( Russell, 2010 , Stockham and Scott, 2008c , Scaradavou, 2002 ). Chronic ehrlichiosis in dogs has also been reported to cause bone marrow hypoplasia, although the mechanism remains unclear ( Stockham and Scott, 2008c ). However, decreases in platelet counts attributable to infectious agents may have contributions from mechanisms other than bone marrow suppression, such as peripheral consumption or immune-mediated destruction. Hyperestrogenism associated with testicular or ovarian neoplasia may also cause general bone marrow suppression, and dogs appear to be particularly sensitive to this effect ( Sontas et al., 2009 ). Causes of decreased bone marrow megakaryocytes with decreases in blood platelet counts have also been associated with mechanisms that are likely immune-mediated. Aplastic anemia, which is most commonly caused by immune-mediated mechanisms ( Young et al., 2006 ), is associated with generalized bone marrow hypoplasia involving all hematopoietic lineages, including megakaryocytes. Amegakaryocytic thrombocytopenic purpura in humans may be inherited or acquired, and acquired forms commonly occur through immune-mediated mechanisms ( Diz-Küçükkaya and López, 2016 ). Inherited or congenital forms cause marked decreases in blood platelet counts with an absence of megakaryocytes in the bone marrow. This form is frequently associated with mutations in the TPO receptor gene, MPL , and may progress to aplastic anemia ( Germeshausen et al., 2006 , Van Den Oudenrijn et al., 2000 ). Acquired amegakaryocytic thrombocytopenic purpura is likely associated with immune-mediated decreases in bone marrow megakaryocytes ( Tristano, 2005 ), and antibodies that bind TPO or the TPO receptor have also been reported to cause acquired amegakaryocytic thrombocytopenic purpura ( Shiozaki et al., 2000 , Katsumata et al., 2003 ). Generalized bone marrow disease, such as replacement of normal bone marrow hematopoietic tissue with hemic or nonhemic neoplasia, severe granulomatous inflammation effacing normal bone marrow tissue, myelofibrosis, or bone marrow necrosis, will result in decreases in megakaryocytes and thrombopoiesis with subsequent decreases in blood platelet counts ( Russell, 2010 , Stockham and Scott, 2008c ). 12.11.4.2.5 Xenobiotic-induced Blood loss associated with xenobiotics has been reported with coagulopathies due to abnormal vitamin K recycling and function of vitamin K-dependent coagulation factors. Drugs implicated in abnormal vitamin K synthesis include warfarin, rodenticides such as brodifacoum, and some broad-spectrum antibiotics ( Bloom and Brandt, 2008 ). With severe, acute loss of whole blood, all blood components, including platelets, will be decreased. If other mechanisms of xenobiotic-induced decreases in platelet counts, as discussed later, cause severe enough decreases in blood platelet counts (e.g., < 50,000 platelets μL − 1 ), then blood loss could be secondary to the decreases in platelets. Thrombotic microangiopathy syndrome, which may include thrombotic thrombocytopenic purpura (TTP) and HUS, may cause peripheral consumption and/or destruction of platelets with the development of decreases in platelet counts. Xenobiotic-induced endothelial injury leads to platelet activation and aggregation ( Pisoni et al., 2001 ). Several chemotherapeutic agents associated with thrombotic microangiopathy syndrome include mitomycin C ( Cantrell et al., 1985 ), cisplatin ( Palmisano et al., 1998 ), estramustine phosphate ( Tassinari et al., 1999 ), gemcitabine ( Nackaerts et al., 1998 ), and daunorubicin ( Byrnes et al., 1986 ). Nonchemotherapeutic agents, including immunomodulators such as cyclosporine and tacrolimus ( Katznelson et al., 1994 , Trimarchi et al., 1999 ), simvastatin ( McCarthy et al., 1998 ), and inhibitors of platelet aggregation including ticlopidine and clopidogrel ( Bennett et al., 1998 , Bennett et al., 2000 ), have also been associated with thrombotic microangiopathy syndrome. Immune-mediated destruction of platelets is a relatively common cause of xenobiotic-induced decreases in blood platelet counts. Antiplatelet antibodies may be formed through various mechanisms. Penicillin and some cephalosporins cause platelet destruction through hapten-type or drug-dependent antibody production, while quinidine and some NSAIDS can cause immune-mediated platelet destruction through the formation of antibodies that only bind platelets when the soluble drug is present ( Aster and Bougie, 2007 ). Some drugs that inhibit the platelet glycoprotein α IIβ β 3(GPIIβ-IIIα) may lead to platelet expression of a new antigen due to conformation changes to the glycoprotein complexes that can then be bound by antibodies; such drugs include tirofiban, roxifiban, and eptifibatide ( Aster and Bougie, 2007 , Aster, 2005 ). Abciximab has been reported to cause drug-specific antibodies that cause decreases in platelet counts ( Aster, 2005 ). Xenobiotic-induced production of autoantibodies that bind platelets, or drug-independent antibodies, has been attributed to procainamide and gold-based compound administration ( Aster and Bougie, 2007 ). In humans, immune-complex type antibodies are classically associated with heparin therapy, and are due to the interaction of heparin, a platelet granule component, and platelet factor 4 (PF4) ( Aster and Bougie, 2007 , Visentin and Liu, 2007 , Arepally and Ortel, 2006 ). Bone marrow suppression is caused by numerous xenobiotics, most notably chemotherapeutic agents. Chemotherapeutic agents associated with bone marrow suppression include compounds from many classes, including alkylating agents such as busulfan, antimetabolites that impair DNA synthesis such as methotrexate and mercaptopurine, antibiotics such as doxorubicin, and mitotic spindle inhibitors such as the vinca alkaloids vinblastine and vincristine ( Carey, 2003 , Weiss, 2010 , Stockham and Scott, 2008c ). Idiosyncratic myelosuppression may also occur with numerous nonchemotherapeutic xenobiotics, and has been reported with antithyroid drugs such as methimazole, anticonvulsants including felbamate and carbamazepine, antipsychotic agents such as clozapine, cardiovascular drugs such as methyldopa and captopril, antibiotics including trimethoprim–sulfamethoxazole and chloroquine, and other xenobiotic agents including gold-based compounds, diclofenac, and allopurinol ( Carey, 2003 ). Xenobiotic-induced aplastic anemia is most commonly associated with immune-mediated destruction of uncommitted or early hematopoietic stem cells, although direct cytotoxicity, such as with chemotherapeutic agents, may also lead to aplastic anemia. Several xenobiotics, including chloramphenicol, anticonvulsants such as phenytoin and carbamazepine, gold-based compounds, and phenylbutazone have been associated with aplastic anemia ( Bloom and Brandt, 2008 ). Miscellaneous causes of xenobiotic-induced decreases in blood platelet counts include nonimmune-mediated destruction reported to occur with desmopressin ( Bloom and Brandt, 2008 ) and idiosyncratic reactions associated with CARPA ( Patkó and Szebeni, 2015 ) or administration of some antisense oligonucleotides ( Frazier, 2015 ). Idiosyncratic decreases in platelet counts have also been reported as an off-target effect of human monoclonal antibodies during nonclinical toxicology studies ( Everds et al., 2013b ). 12.11.4.2.1 Relative Relative decreases in platelet counts occur when circulating platelet mass is not changed, but platelet clumping, redistribution, or hemodilution alter automated or estimated platelet counts. False decreases in platelet counts (pseudo-thrombocytopenia) due to platelet clumping are often present in rats, mice, and cats, although it may be observed in any species. MPV may be increased with the presence of platelet clumps, and microscopic blood smear review should be performed in all cases of decreased platelet counts to assess for the presence of platelet clumps; increases in MPV in instances of platelet clumping do not reflect a bone marrow response because a true decrease in platelet count is not present to stimulate thrombopoiesis. Resampling of blood with a clean venipuncture and use of sodium citrate as the anticoagulant may help reduce platelet clumps and result in a more accurate automated platelet count. Redistribution or sequestration of circulating platelets may cause decreases in platelet counts, which may be transient. Platelet redistribution may be observed with splenomegaly, hypersplenism, or with severe hypothermia; total platelet mass is unaffected in these cases and typically does not result in increased TPO or platelet production ( Stockham and Scott, 2008c ). Hemodilution may occur following administration of intravenous fluids or massive transfusion, and is expected to decrease all blood components to variable degrees, with the exception of any transfused blood components. Decreases in platelets from hemodilution are usually mild and may not be detected if platelet counts remain within reference intervals or historical control ranges. These changes are generally transient and resolve with redistribution of intravascular (extracellular) fluid into intracellular fluid compartments or elimination of excess fluid. 12.11.4.2.2 Loss Acute, severe loss of whole blood may result in decrease in all blood components, including platelets. Blood loss of significant magnitude to cause decreases in blood platelet counts may occur following trauma, splenic rupture from trauma or neoplasia, or from uncontrolled bleeding associated with coagulopathies, such as hemophilia A or B. However, consumption of platelets at the site(s) of hemorrhage may also contribute to the decreases in platelet counts observed with blood loss. 12.11.4.2.3 Decreased survival Decreased platelet survival may be due to increased platelet destruction and/or consumption, and is a relatively common cause of decreases in blood platelet counts. Destruction of platelets is commonly associated with immune-mediated mechanisms, including immune-mediated thrombocytopenia (IMT) and immune-mediated thrombocytopenic purpura (ITP) in humans. Most autoantibodies that cause platelet destruction are of the IgG class, although cases with IgM or IgA antiplatelet antibodies have also been reported ( Diz-Küçükkaya and López, 2016 ). Antiplatelet antibodies may directly target platelet antigens, such as the glycoprotein integrin α IIβ β 3(GPIIβ-IIIα) that plays a major role in platelet aggregation ( He et al., 1994 ), antigens exposed or formed on platelet surfaces by infectious agents, or infectious agent-targeted antibodies that cross-react with normally expressed platelet surface antigens or membrane components such as phospholipid ( Stockham and Scott, 2008c ). Antibody-bound platelets may be phagocytosed and destroyed by splenic, bone marrow, or hepatic macrophages, or may be associated with complement-mediated destruction or stimulation of phagocytosis ( Diz-Küçükkaya and López, 2016 , Russell, 2010 ). Production of antiplatelet antibodies may be unassociated with an underlying disease condition (idiopathic or primary) and is usually presumed to be an autoimmune process, or may be secondary to infectious or neoplastic processes ( Stockham and Scott, 2008c ). In sexually mature Göttingen minipigs, spontaneous thrombocytopenic purpura has been described and is believed to be caused immune-mediated platelet destruction ( Carrasco et al., 2003 ). ITP secondary to infectious disease has been reported to occur with various bacterial and viral etiologies, including Helicobacter pylori , many rickettsial diseases, HIV, cytomegalovirus, and hepatitis B and C viruses ( Diz-Küçükkaya and López, 2016 , Russell, 2010 , Cines et al., 2009 ). Lymphoproliferative neoplasia is a relatively common cause of ITP, and has been associated with chronic lymphocytic leukemia, Hodgkin lymphoma, and leukemia of large granular T-lymphocytes ( Cines et al., 2009 ). Connective tissue diseases such as SLE have also been associated with ITP ( Cines et al., 2009 ). Decreases in platelet counts due to platelet activation and consumption are commonly associated with local or disseminated consumptive coagulopathies. Localized consumptive coagulopathy may be associated with sites of hemorrhage, microangiopathy or thrombosis, or vascular neoplasia such as hemangiosarcoma ( Stockham and Scott, 2008c ). Thrombosis associated with chronic catheterization may be a cause of localized platelet consumption. DIC may be associated with infectious agents, particularly bacterial infections that cause septicemia or endotoxemia, hepatic disease, a variety of neoplastic diseases, and pancreatitis ( Stockham and Scott, 2008c ). Activation and consumption of platelets associated with decreases in platelet counts has also been observed with vasculitis and conditions associated with turbulent blood flow such as endocarditis, cardiac valvular disease, following cardiac surgery, and with arterial disease that alter or damage endothelial cells ( Russell, 2010 , Selleng et al., 2010 , Stockham and Scott, 2008c , Gregg and Goldschmidt-Clermont, 2003 ). Hemolytic uremic syndrome (HUS) is a cause of thrombotic microangiopathy that may be associated with infection with Shigella dysenteriae or Escherichia coli ( Russell, 2010 ). 12.11.4.2.4 Decreased production Bone marrow suppression that involves the megakaryocytic lineage causes decreases in platelet production and therefore decreases in platelet counts. Bone marrow suppression may occur in association with infectious agents that can directly infect hematopoietic precursors such as immunodeficiency viruses in various species, parvoviruses, distemper virus in dogs, and feline leukemia virus in cats ( Russell, 2010 , Stockham and Scott, 2008c , Scaradavou, 2002 ). Chronic ehrlichiosis in dogs has also been reported to cause bone marrow hypoplasia, although the mechanism remains unclear ( Stockham and Scott, 2008c ). However, decreases in platelet counts attributable to infectious agents may have contributions from mechanisms other than bone marrow suppression, such as peripheral consumption or immune-mediated destruction. Hyperestrogenism associated with testicular or ovarian neoplasia may also cause general bone marrow suppression, and dogs appear to be particularly sensitive to this effect ( Sontas et al., 2009 ). Causes of decreased bone marrow megakaryocytes with decreases in blood platelet counts have also been associated with mechanisms that are likely immune-mediated. Aplastic anemia, which is most commonly caused by immune-mediated mechanisms ( Young et al., 2006 ), is associated with generalized bone marrow hypoplasia involving all hematopoietic lineages, including megakaryocytes. Amegakaryocytic thrombocytopenic purpura in humans may be inherited or acquired, and acquired forms commonly occur through immune-mediated mechanisms ( Diz-Küçükkaya and López, 2016 ). Inherited or congenital forms cause marked decreases in blood platelet counts with an absence of megakaryocytes in the bone marrow. This form is frequently associated with mutations in the TPO receptor gene, MPL , and may progress to aplastic anemia ( Germeshausen et al., 2006 , Van Den Oudenrijn et al., 2000 ). Acquired amegakaryocytic thrombocytopenic purpura is likely associated with immune-mediated decreases in bone marrow megakaryocytes ( Tristano, 2005 ), and antibodies that bind TPO or the TPO receptor have also been reported to cause acquired amegakaryocytic thrombocytopenic purpura ( Shiozaki et al., 2000 , Katsumata et al., 2003 ). Generalized bone marrow disease, such as replacement of normal bone marrow hematopoietic tissue with hemic or nonhemic neoplasia, severe granulomatous inflammation effacing normal bone marrow tissue, myelofibrosis, or bone marrow necrosis, will result in decreases in megakaryocytes and thrombopoiesis with subsequent decreases in blood platelet counts ( Russell, 2010 , Stockham and Scott, 2008c ). 12.11.4.2.5 Xenobiotic-induced Blood loss associated with xenobiotics has been reported with coagulopathies due to abnormal vitamin K recycling and function of vitamin K-dependent coagulation factors. Drugs implicated in abnormal vitamin K synthesis include warfarin, rodenticides such as brodifacoum, and some broad-spectrum antibiotics ( Bloom and Brandt, 2008 ). With severe, acute loss of whole blood, all blood components, including platelets, will be decreased. If other mechanisms of xenobiotic-induced decreases in platelet counts, as discussed later, cause severe enough decreases in blood platelet counts (e.g., < 50,000 platelets μL − 1 ), then blood loss could be secondary to the decreases in platelets. Thrombotic microangiopathy syndrome, which may include thrombotic thrombocytopenic purpura (TTP) and HUS, may cause peripheral consumption and/or destruction of platelets with the development of decreases in platelet counts. Xenobiotic-induced endothelial injury leads to platelet activation and aggregation ( Pisoni et al., 2001 ). Several chemotherapeutic agents associated with thrombotic microangiopathy syndrome include mitomycin C ( Cantrell et al., 1985 ), cisplatin ( Palmisano et al., 1998 ), estramustine phosphate ( Tassinari et al., 1999 ), gemcitabine ( Nackaerts et al., 1998 ), and daunorubicin ( Byrnes et al., 1986 ). Nonchemotherapeutic agents, including immunomodulators such as cyclosporine and tacrolimus ( Katznelson et al., 1994 , Trimarchi et al., 1999 ), simvastatin ( McCarthy et al., 1998 ), and inhibitors of platelet aggregation including ticlopidine and clopidogrel ( Bennett et al., 1998 , Bennett et al., 2000 ), have also been associated with thrombotic microangiopathy syndrome. Immune-mediated destruction of platelets is a relatively common cause of xenobiotic-induced decreases in blood platelet counts. Antiplatelet antibodies may be formed through various mechanisms. Penicillin and some cephalosporins cause platelet destruction through hapten-type or drug-dependent antibody production, while quinidine and some NSAIDS can cause immune-mediated platelet destruction through the formation of antibodies that only bind platelets when the soluble drug is present ( Aster and Bougie, 2007 ). Some drugs that inhibit the platelet glycoprotein α IIβ β 3(GPIIβ-IIIα) may lead to platelet expression of a new antigen due to conformation changes to the glycoprotein complexes that can then be bound by antibodies; such drugs include tirofiban, roxifiban, and eptifibatide ( Aster and Bougie, 2007 , Aster, 2005 ). Abciximab has been reported to cause drug-specific antibodies that cause decreases in platelet counts ( Aster, 2005 ). Xenobiotic-induced production of autoantibodies that bind platelets, or drug-independent antibodies, has been attributed to procainamide and gold-based compound administration ( Aster and Bougie, 2007 ). In humans, immune-complex type antibodies are classically associated with heparin therapy, and are due to the interaction of heparin, a platelet granule component, and platelet factor 4 (PF4) ( Aster and Bougie, 2007 , Visentin and Liu, 2007 , Arepally and Ortel, 2006 ). Bone marrow suppression is caused by numerous xenobiotics, most notably chemotherapeutic agents. Chemotherapeutic agents associated with bone marrow suppression include compounds from many classes, including alkylating agents such as busulfan, antimetabolites that impair DNA synthesis such as methotrexate and mercaptopurine, antibiotics such as doxorubicin, and mitotic spindle inhibitors such as the vinca alkaloids vinblastine and vincristine ( Carey, 2003 , Weiss, 2010 , Stockham and Scott, 2008c ). Idiosyncratic myelosuppression may also occur with numerous nonchemotherapeutic xenobiotics, and has been reported with antithyroid drugs such as methimazole, anticonvulsants including felbamate and carbamazepine, antipsychotic agents such as clozapine, cardiovascular drugs such as methyldopa and captopril, antibiotics including trimethoprim–sulfamethoxazole and chloroquine, and other xenobiotic agents including gold-based compounds, diclofenac, and allopurinol ( Carey, 2003 ). Xenobiotic-induced aplastic anemia is most commonly associated with immune-mediated destruction of uncommitted or early hematopoietic stem cells, although direct cytotoxicity, such as with chemotherapeutic agents, may also lead to aplastic anemia. Several xenobiotics, including chloramphenicol, anticonvulsants such as phenytoin and carbamazepine, gold-based compounds, and phenylbutazone have been associated with aplastic anemia ( Bloom and Brandt, 2008 ). Miscellaneous causes of xenobiotic-induced decreases in blood platelet counts include nonimmune-mediated destruction reported to occur with desmopressin ( Bloom and Brandt, 2008 ) and idiosyncratic reactions associated with CARPA ( Patkó and Szebeni, 2015 ) or administration of some antisense oligonucleotides ( Frazier, 2015 ). Idiosyncratic decreases in platelet counts have also been reported as an off-target effect of human monoclonal antibodies during nonclinical toxicology studies ( Everds et al., 2013b ). 12.11.5 Conclusions There are numerous causes of direct alterations in blood components, including an increasing number of xenobiotics. Alterations in the bone marrow hematopoietic systemic either independent of xenobiotics or induced by xenobiotics may also result in changes in peripheral blood cell counts. Many of the mechanisms of alterations in blood cell counts that are not related to xenobiotic administration have considerable overlap with the mechanisms through which xenobiotics cause changes, and understanding these mechanisms and their association with individual compounds or drug classes may help elucidate potential pathways through which novel xenobiotics cause alterations in blood components.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10559356/

Phage based vaccine: A novel strategy in prevention and treatment

The vaccine was first developed in 1796 by a British physician, Edward Jenner, against the smallpox virus. This invention revolutionized medical science and saved lives around the world. The production of effective vaccines requires dominant immune epitopes to elicit a robust immune response. Thus, applying bacteriophages has attracted the attention of many researchers because of their advantages in vaccine design and development. Bacteriophages are not infectious to humans and are unlikely to bind to cellular receptors and activate signaling pathways. Phages could activate both cellular and humoral immunity, which is another goal of an effective vaccine design. Also, phages act as an effective adjuvant, along with the antigens, and induce a robust immune response. Phage-based vaccines can also be administered orally because of their stability in the gastrointestinal tract, in contrast to common vaccination routes, which are intradermal, subcutaneous, or intramuscular. This review presents the current improvements in phage-based vaccines and their applications as preventive or therapeutic vaccines. 1 Introduction The vaccine innovation by Edward Jenner against smallpox [ 1 , 2 ] has revolutionized medicine during the last centuries and saved the lives of humanity and many animals [ 3 , 4 ]. The vaccines are classified into various categories based on their nature, including inactivated, live-attenuated, and recombinant vaccines [ 3 , 5 ]. Although remarkable advances have been made regarding conventional vaccines, some limitations, such as the high cost and low immune responses, have been reported. Therefore, novel strategies, such as phage-based vaccines, have been developed to overcome the limitations of classical vaccines [ 3 , 6 ]. Phage-based vaccines elicit an effective cellular and humoral immune response with no need for adjuvants and no potential risk of infection in human cells or integration into the human genome [ [6] , [7] , [8] ]. Bacteriophages are the most abundant living organisms around the world, and they have a high ability to coexist with a multitude of organisms like mammals; thus, oral administration to humans has been approved by the FDA [ [9] , [10] , [11] , [12] ]. Phages could also play various roles in drug delivery, phage therapy, biosensors, and biotechnology applications such as the identification of ligand binding sites and B-cell epitopes and the production of monoclonal antibodies in large volumes [ 6 , [13] , [14] , [15] , [16] , [17] , [18] , [19] ]. In this regard, recombinant bacteriophage technology provides a cost-effective and potent solution to the increasing demand for efficient vaccines against different pathogens. Herein, we first discuss the basics of phage biology and mechanisms of immunity against engineered phages and then review the three categories of phage-based vaccines, including Phage display vaccines, Phage- DNA vaccines, and Hybrid phage-DNA vaccines; which are under clinical trials or commercially available. 2 Bacteriophages Phages, or bacteriophages, are a class of viruses that infect archaea and bacteria, but they are incompetent for infecting eukaryotes [ 7 , 9 ]. The bacteriophages are divided into RNA phages and DNA phages in terms of genetic material, which is covered by a coating protein called a capsid. 3 1. RNA phages RNA phages in two groups Cystoviridae with double-stranded segmented RNA (ds RNAs) and Leviviridae with single-stranded RNA (ss RNAs) are classified. Leviviridae are easy to produce in large amounts, and because they have a plus-sense single-stranded RNA genome, they served as a convenient source of messenger RNA (mRNA) for studies of protein synthesis. MS2 and Qβ are examples of Leviviridae; both use F-pilus to enter E. coli cells [ 20 ]. φ6 is an example of Cystoviridae, most of which use type IV pili, and some of them use lipopolysaccharides on the bacterial cell surface to enter the bacterial cell [ 20 ]. 3.1 DNA phages 3.1.1 Non-lytic phages 3.1.1.1 Filamentous phages Filamentous phages, such as M13, are a group of viruses that are non-lytic and consist of a 6.4 Kb circular ssDNA [ 10 , 21 ]. One of the important advantages of filamentous phages is their workability and inexpensive purification, which can be purified from bacterial culture mediums with high titers [ 22 ]. In addition, M13 particles can be used as vaccine carriers because of their immunogenic potential [ 7 , 23 ]. The coating of the particles contains minor coat proteins (pIII, pVI, pVII, and pIX) and major coat proteins (pVIIIs). 2700 copies of pVIII cover the entire particle surface. Among the coat proteins, pIII and pVIII proteins are the most widely used proteins for displaying external peptides, depending on the length and sequence of the peptide in the phage display technique. Immunogenic peptides are fused with PIII or PVIII and displayed on the phage surface ( Fig. 1 ) [ 9 , 24 ]. Although pVIIIs surround the whole viral particle, short peptides of up to 8 amino acids can be fused to them; thus, it is required to maximize the presentation flexibility of the system to be able to display longer peptides on them [ 23 , [25] , [26] , [27] ] or engage pIIIs for larger peptide fragments [ [28] , [29] , [30] ]. However, the capability of pVIIIs to display a higher number of immunogenic peptides leads to an enhanced immune response. Another important advantage of filamentous phages is their stability of particles against harsh temperatures and pH variations, so the filamentous phage genome can be applied as a cloning vector for the attachment of external DNA in different sizes [ 9 , 29 ]. Fig. 1 Schematic illustration of Filamentous phages. Fig. 1 3.1.2 Lytic phages 3.1.2.1 T4 phages T4 phages are categorized as lytic viruses with a double-stranded DNA (dsDNA) genome about 169 kb in size [ 31 ]. Hoc and Soc are high-copy bacteriophage capsid proteins that can display peptides with high copies and larger than the peptides displayed by filamentous phages on their surface ( Fig. 2 ), leading to an effective immune response in mice and humans [ 11 , 32 , 33 ]. Clinical trial evidence suggests the oral administration of whole wild-type T4 is remarkably safe for humans [ 11 ]. Other advantages of T4 phages include their high immunogenicity, non-toxicity of the secreted protein, and ability to express the dual-engineered protein. Therefore, considering the advantages mentioned, T4 application as a phage display vector is more common than filamentous phages [ 34 ]. Fig. 2 Schematic illustration of T4 phages. Fig. 2 3.1.2.2 T7 phages Other lytic viruses include T7 phages. The structure of the T7 phage consists of a linear dsDNA with a size of about 40 kb and six main proteins called capsid proteins (10A and 10B), tail proteins (gp11 and gp12), tail fiber protein (gp17), and connector protein (gp8). Gp 10 A and gp 10 B are often chosen for phage display purposes ( Fig. 3 ) [ 6 , [35] , [36] , [37] ]. The carboxyl-terminus of the 10 B protein of the T7 phage has been engineered, and the recombinant T7 phage was produced to display heterologous proteins and activate the humoral and cellular immune responses [ [35] , [36] , [37] , [38] ]. The advantages of using recombinant T7 phage include high cloning capacity, high stability to maintain external gene inserts, and a high propagation rate [ 39 ]. Fig. 3 Schematic illustration of T7 phages. Fig. 3 3.1.3 Temperate phages 3.1.3.1 λ phages Lambda phages are categorized as temperate phages with a linear dsDNA genome about 48.5 kb in size ( Fig. 4 ) [ 40 ]. Lambda phages can display peptides with proper folding, higher density, and a size 2–3 times larger than what filamentous phages display. Antigens on lambda can be exposed by both the head protein pD and the tail protein pV [ 9 ]. Although filamentous phages are still one of the most common vectors for phage display, lambda phages could also be a good option for displaying complex antigens [ 9 , 41 , 42 ]. For instance, beta-galactosidase with about 400 kDa MW was properly displayed on the surface of the lambda phage with no changes in phage structure or survival [ 26 ]. Fig. 4 Schematic illustration of lambda phages. Fig. 4 3.1 DNA phages 3.1.1 Non-lytic phages 3.1.1.1 Filamentous phages Filamentous phages, such as M13, are a group of viruses that are non-lytic and consist of a 6.4 Kb circular ssDNA [ 10 , 21 ]. One of the important advantages of filamentous phages is their workability and inexpensive purification, which can be purified from bacterial culture mediums with high titers [ 22 ]. In addition, M13 particles can be used as vaccine carriers because of their immunogenic potential [ 7 , 23 ]. The coating of the particles contains minor coat proteins (pIII, pVI, pVII, and pIX) and major coat proteins (pVIIIs). 2700 copies of pVIII cover the entire particle surface. Among the coat proteins, pIII and pVIII proteins are the most widely used proteins for displaying external peptides, depending on the length and sequence of the peptide in the phage display technique. Immunogenic peptides are fused with PIII or PVIII and displayed on the phage surface ( Fig. 1 ) [ 9 , 24 ]. Although pVIIIs surround the whole viral particle, short peptides of up to 8 amino acids can be fused to them; thus, it is required to maximize the presentation flexibility of the system to be able to display longer peptides on them [ 23 , [25] , [26] , [27] ] or engage pIIIs for larger peptide fragments [ [28] , [29] , [30] ]. However, the capability of pVIIIs to display a higher number of immunogenic peptides leads to an enhanced immune response. Another important advantage of filamentous phages is their stability of particles against harsh temperatures and pH variations, so the filamentous phage genome can be applied as a cloning vector for the attachment of external DNA in different sizes [ 9 , 29 ]. Fig. 1 Schematic illustration of Filamentous phages. Fig. 1 3.1.2 Lytic phages 3.1.2.1 T4 phages T4 phages are categorized as lytic viruses with a double-stranded DNA (dsDNA) genome about 169 kb in size [ 31 ]. Hoc and Soc are high-copy bacteriophage capsid proteins that can display peptides with high copies and larger than the peptides displayed by filamentous phages on their surface ( Fig. 2 ), leading to an effective immune response in mice and humans [ 11 , 32 , 33 ]. Clinical trial evidence suggests the oral administration of whole wild-type T4 is remarkably safe for humans [ 11 ]. Other advantages of T4 phages include their high immunogenicity, non-toxicity of the secreted protein, and ability to express the dual-engineered protein. Therefore, considering the advantages mentioned, T4 application as a phage display vector is more common than filamentous phages [ 34 ]. Fig. 2 Schematic illustration of T4 phages. Fig. 2 3.1.2.2 T7 phages Other lytic viruses include T7 phages. The structure of the T7 phage consists of a linear dsDNA with a size of about 40 kb and six main proteins called capsid proteins (10A and 10B), tail proteins (gp11 and gp12), tail fiber protein (gp17), and connector protein (gp8). Gp 10 A and gp 10 B are often chosen for phage display purposes ( Fig. 3 ) [ 6 , [35] , [36] , [37] ]. The carboxyl-terminus of the 10 B protein of the T7 phage has been engineered, and the recombinant T7 phage was produced to display heterologous proteins and activate the humoral and cellular immune responses [ [35] , [36] , [37] , [38] ]. The advantages of using recombinant T7 phage include high cloning capacity, high stability to maintain external gene inserts, and a high propagation rate [ 39 ]. Fig. 3 Schematic illustration of T7 phages. Fig. 3 3.1.3 Temperate phages 3.1.3.1 λ phages Lambda phages are categorized as temperate phages with a linear dsDNA genome about 48.5 kb in size ( Fig. 4 ) [ 40 ]. Lambda phages can display peptides with proper folding, higher density, and a size 2–3 times larger than what filamentous phages display. Antigens on lambda can be exposed by both the head protein pD and the tail protein pV [ 9 ]. Although filamentous phages are still one of the most common vectors for phage display, lambda phages could also be a good option for displaying complex antigens [ 9 , 41 , 42 ]. For instance, beta-galactosidase with about 400 kDa MW was properly displayed on the surface of the lambda phage with no changes in phage structure or survival [ 26 ]. Fig. 4 Schematic illustration of lambda phages. Fig. 4 3.1.1 Non-lytic phages 3.1.1.1 Filamentous phages Filamentous phages, such as M13, are a group of viruses that are non-lytic and consist of a 6.4 Kb circular ssDNA [ 10 , 21 ]. One of the important advantages of filamentous phages is their workability and inexpensive purification, which can be purified from bacterial culture mediums with high titers [ 22 ]. In addition, M13 particles can be used as vaccine carriers because of their immunogenic potential [ 7 , 23 ]. The coating of the particles contains minor coat proteins (pIII, pVI, pVII, and pIX) and major coat proteins (pVIIIs). 2700 copies of pVIII cover the entire particle surface. Among the coat proteins, pIII and pVIII proteins are the most widely used proteins for displaying external peptides, depending on the length and sequence of the peptide in the phage display technique. Immunogenic peptides are fused with PIII or PVIII and displayed on the phage surface ( Fig. 1 ) [ 9 , 24 ]. Although pVIIIs surround the whole viral particle, short peptides of up to 8 amino acids can be fused to them; thus, it is required to maximize the presentation flexibility of the system to be able to display longer peptides on them [ 23 , [25] , [26] , [27] ] or engage pIIIs for larger peptide fragments [ [28] , [29] , [30] ]. However, the capability of pVIIIs to display a higher number of immunogenic peptides leads to an enhanced immune response. Another important advantage of filamentous phages is their stability of particles against harsh temperatures and pH variations, so the filamentous phage genome can be applied as a cloning vector for the attachment of external DNA in different sizes [ 9 , 29 ]. Fig. 1 Schematic illustration of Filamentous phages. Fig. 1 3.1.1.1 Filamentous phages Filamentous phages, such as M13, are a group of viruses that are non-lytic and consist of a 6.4 Kb circular ssDNA [ 10 , 21 ]. One of the important advantages of filamentous phages is their workability and inexpensive purification, which can be purified from bacterial culture mediums with high titers [ 22 ]. In addition, M13 particles can be used as vaccine carriers because of their immunogenic potential [ 7 , 23 ]. The coating of the particles contains minor coat proteins (pIII, pVI, pVII, and pIX) and major coat proteins (pVIIIs). 2700 copies of pVIII cover the entire particle surface. Among the coat proteins, pIII and pVIII proteins are the most widely used proteins for displaying external peptides, depending on the length and sequence of the peptide in the phage display technique. Immunogenic peptides are fused with PIII or PVIII and displayed on the phage surface ( Fig. 1 ) [ 9 , 24 ]. Although pVIIIs surround the whole viral particle, short peptides of up to 8 amino acids can be fused to them; thus, it is required to maximize the presentation flexibility of the system to be able to display longer peptides on them [ 23 , [25] , [26] , [27] ] or engage pIIIs for larger peptide fragments [ [28] , [29] , [30] ]. However, the capability of pVIIIs to display a higher number of immunogenic peptides leads to an enhanced immune response. Another important advantage of filamentous phages is their stability of particles against harsh temperatures and pH variations, so the filamentous phage genome can be applied as a cloning vector for the attachment of external DNA in different sizes [ 9 , 29 ]. Fig. 1 Schematic illustration of Filamentous phages. Fig. 1 3.1.2 Lytic phages 3.1.2.1 T4 phages T4 phages are categorized as lytic viruses with a double-stranded DNA (dsDNA) genome about 169 kb in size [ 31 ]. Hoc and Soc are high-copy bacteriophage capsid proteins that can display peptides with high copies and larger than the peptides displayed by filamentous phages on their surface ( Fig. 2 ), leading to an effective immune response in mice and humans [ 11 , 32 , 33 ]. Clinical trial evidence suggests the oral administration of whole wild-type T4 is remarkably safe for humans [ 11 ]. Other advantages of T4 phages include their high immunogenicity, non-toxicity of the secreted protein, and ability to express the dual-engineered protein. Therefore, considering the advantages mentioned, T4 application as a phage display vector is more common than filamentous phages [ 34 ]. Fig. 2 Schematic illustration of T4 phages. Fig. 2 3.1.2.2 T7 phages Other lytic viruses include T7 phages. The structure of the T7 phage consists of a linear dsDNA with a size of about 40 kb and six main proteins called capsid proteins (10A and 10B), tail proteins (gp11 and gp12), tail fiber protein (gp17), and connector protein (gp8). Gp 10 A and gp 10 B are often chosen for phage display purposes ( Fig. 3 ) [ 6 , [35] , [36] , [37] ]. The carboxyl-terminus of the 10 B protein of the T7 phage has been engineered, and the recombinant T7 phage was produced to display heterologous proteins and activate the humoral and cellular immune responses [ [35] , [36] , [37] , [38] ]. The advantages of using recombinant T7 phage include high cloning capacity, high stability to maintain external gene inserts, and a high propagation rate [ 39 ]. Fig. 3 Schematic illustration of T7 phages. Fig. 3 3.1.2.1 T4 phages T4 phages are categorized as lytic viruses with a double-stranded DNA (dsDNA) genome about 169 kb in size [ 31 ]. Hoc and Soc are high-copy bacteriophage capsid proteins that can display peptides with high copies and larger than the peptides displayed by filamentous phages on their surface ( Fig. 2 ), leading to an effective immune response in mice and humans [ 11 , 32 , 33 ]. Clinical trial evidence suggests the oral administration of whole wild-type T4 is remarkably safe for humans [ 11 ]. Other advantages of T4 phages include their high immunogenicity, non-toxicity of the secreted protein, and ability to express the dual-engineered protein. Therefore, considering the advantages mentioned, T4 application as a phage display vector is more common than filamentous phages [ 34 ]. Fig. 2 Schematic illustration of T4 phages. Fig. 2 3.1.2.2 T7 phages Other lytic viruses include T7 phages. The structure of the T7 phage consists of a linear dsDNA with a size of about 40 kb and six main proteins called capsid proteins (10A and 10B), tail proteins (gp11 and gp12), tail fiber protein (gp17), and connector protein (gp8). Gp 10 A and gp 10 B are often chosen for phage display purposes ( Fig. 3 ) [ 6 , [35] , [36] , [37] ]. The carboxyl-terminus of the 10 B protein of the T7 phage has been engineered, and the recombinant T7 phage was produced to display heterologous proteins and activate the humoral and cellular immune responses [ [35] , [36] , [37] , [38] ]. The advantages of using recombinant T7 phage include high cloning capacity, high stability to maintain external gene inserts, and a high propagation rate [ 39 ]. Fig. 3 Schematic illustration of T7 phages. Fig. 3 3.1.3 Temperate phages 3.1.3.1 λ phages Lambda phages are categorized as temperate phages with a linear dsDNA genome about 48.5 kb in size ( Fig. 4 ) [ 40 ]. Lambda phages can display peptides with proper folding, higher density, and a size 2–3 times larger than what filamentous phages display. Antigens on lambda can be exposed by both the head protein pD and the tail protein pV [ 9 ]. Although filamentous phages are still one of the most common vectors for phage display, lambda phages could also be a good option for displaying complex antigens [ 9 , 41 , 42 ]. For instance, beta-galactosidase with about 400 kDa MW was properly displayed on the surface of the lambda phage with no changes in phage structure or survival [ 26 ]. Fig. 4 Schematic illustration of lambda phages. Fig. 4 3.1.3.1 λ phages Lambda phages are categorized as temperate phages with a linear dsDNA genome about 48.5 kb in size ( Fig. 4 ) [ 40 ]. Lambda phages can display peptides with proper folding, higher density, and a size 2–3 times larger than what filamentous phages display. Antigens on lambda can be exposed by both the head protein pD and the tail protein pV [ 9 ]. Although filamentous phages are still one of the most common vectors for phage display, lambda phages could also be a good option for displaying complex antigens [ 9 , 41 , 42 ]. For instance, beta-galactosidase with about 400 kDa MW was properly displayed on the surface of the lambda phage with no changes in phage structure or survival [ 26 ]. Fig. 4 Schematic illustration of lambda phages. Fig. 4 4 Weaknesses and strengths of phage systems The M13 phage is the most frequently used phage in the phage display technique because it has non-essential regions that permit exogenous gene insertions and because exogenous peptides express themselves on the phage surface through their natural folding and function [ 43 , 44 ] he most important feature of phage M13 compared to other phages is that it can be purified and used with less effort. In fact, the host bacterium acts as a factory to produce M13 phages [ 45 , 46 ]. However, the M13 phage display is associated with limitations in the construction of the cDNA library, and the formation of a fusion protein comprising phage coat protein and exogenous peptide and the secretion of phage into the bacterial periplasmic space are also potential problems with the M13 phage display. Unlike M13 phages, which contain single-stranded DNA, T7 phages have double-stranded DNA, which makes them less prone to mutation during replication and more stable, and they no longer depend on the protein secretion pathway. The exogenous peptides are usually in the C-terminal region of the gp10B capsid protein, which does not have the problems associated with steric hindrance. Also, T7 phages have high stability and tolerate harsh conditions such as low pH and high temperatures [ 39 ]. Lambda phages have both lysogenic and lytic alternatives for their life cycle. The genome of lambda phages consists of linear dsDNA, which becomes circular for replication after infecting E. coli bacteria. The limitation of lambda phages as vectors is theire low delivery capacity of about 35–50 kb of DNA [ 47 , 48 ]. Lambda Phage is attached to the surface of E. coli cells, which have a place to transfer maltose into the cell. Therefore, to infect bacteria with Lambda phage, the culture medium must contain maltose [ 49 , 50 ] and when the phage DNA enters the bacterial cell, a circular DNA molecule is formed due to the complementary sticky 12 bp sequence at both ends of its genome. 45 min after the infection, the bacterial cells are lysed, the phages are released, and the infection spreads to nearby cells. This cycle is repeated regularly [ 49 , 51 ]. Another thing that should be considered when working with Lambda phage in the laboratory is that, due to the free sticky ends in the Lambda phage genome, under normal conditions these ends can stick together and create a large fragment. To avoid this possibility, it is recommended to heat the Lambda markers before electrophoresis if electrophoresis is necessary during the operation [ 52 ]. And for T4 lytic phages, two distinct features have made their use more common than filamentous phages: one is the ability to express dual-engineered proteins, and the other is the high immunogenicity of their capsid [ 34 ]. One of the prominent features of the T4 genome is that instead of cytosine (C), 5-hydroxymethylcytosine (HMC) is located. HMC causes the viral enzymes to distinguish between their own and the host's nucleic acids, and the virus genome is protected from the effects of its enzymes. Because the viral enzymes break the host's genome into pieces and take over all the host's facilities. Also, glycosylation happens by adding glucose to HMC, which makes the phage immune to the attack and degradation of bacterial repair systems. It is noteworthy that glycosylation is specific to eukaryotes and is not found in bacteria except for a few exceptions. But this phenomenon happens in T4 phages, which is one of the wonders of creation [ [53] , [54] , [55] , [56] , [57] , [58] , [59] , [60] ]. Additionally, some stability studies showed that T4 remained stable at ambient temperature for at least 10 weeks. As a result, the distribution of T4-based vaccines does not require a cold chain [ 61 , 62 ]. 5 Phage display technology In phage display technology, immunogenic peptides are joined to coating proteins and then shown on the surface of the phage ( Fig. 5 ) [ 7 , 21 , 63 ]. Using bacteriophages as antigen carriers leads to increased antigen half-life in peripheral blood and also enhances the immune response by facilitating the activation of T helper cells [ 64 ]. The phage display technique has been used for vaccine research and immunotherapy in the past 10 years, and hopefully it has opened new landscapes for the vaccine industry and the prevention of infectious diseases [ 21 , 65 ]. Phages that have been used to display antigens and develop phage-based vaccines are Lytic, Filamentous, and Lambda phages, which are detailed in Table 1 . Fig. 5 Schematic illustration of phage displayed antigens and phage display vaccines. Fig. 5 Table 1 Features, design, application, and main findings after administration of common phage display vaccines. Table 1 VECTOR TYPE MODEL OF STUDY PHAGE TYPE DOSAGE USED PROTEIN EFFECTS AFTER ADMINISTRATION/COMMENTS STRAIN OF E.Coli REFERENCE Anti-viral Hepatitis B virus epitope S28–39 pC89 Female BALB/c mice M13 10 μg i.d i.p pVIII MHC I limited response of HBs specific cytotoxic T cells XL1-blue [ 66 , 66 ] Anti-parasite Peptides GK1, KETc12, KETc1, and KETc7 Taenia solium cysticercosis M13KE Pig M13 2 ml,10 12 phage particle of each sc pVIII Reduction of 70% of tongue cysticercoss Reduction of 87% of total cysticerci Reduction of 54% of muscle cysticercosis TG-1 [ 15 , 15 ] Anti-viral Epitopes of the glycoprotein G of HSV-2 (gG2) n.r BALB/c mice Fd-tet n.r pVIII Upgrade of protective immunity in a mice model of HSV-2 infection K91Kan [ 25 , 25 ] Anti-cancer EGFR ICR-62 binding peptide mimotope pAK8-GVO Female BALB/c mice M13 10 12 pfu s.c pVIII Decreased tumor growth in ectopic Lewis lung carcinoma in mice Humoral immunity stimulated in mice TG-1 [ 18 , 18 ] Anti-viral Ectodomain of influenza A virus channel protein M2 (M2e) n.r Female BALB/c mice T7 10 9 pfu s.c 10 B capsid protein M2e-specific serum antibody responses Reduction of viral load due to Cytotoxic T cell response Protection against Influenza A virus BL21 [ 35 , 35 ] Hybrid Anti-cancer HLA-DR-restricted Th cell peptide epitope p23 and MAGE-A10254–262 or p23 and MAGE-A3271–279 from the HIV-1-RT pTfd8p-66 for p23 peptide Human cell system in vitro Humanized murine model in vivo fd 140 μg phage particles s.c pVIII Inhibited tumor growth due to stimulation of potent and specific cytotoxic T cell response TG1 rec O [ 67 , 67 ] Dual display of swine fever virus (CSFV) major antigenic determinant cluster mE2 and CSFV primary antigen E2 pcDSW Female BALB/c mice T4-Zh- 10 10 pfu s.c Soc C-terminus fusion and Hoc N-terminus Increased immune response High antibody production BL21 [ 68 , 68 ] Yersinia pestis (Plague) capsular F1 and calcium response V antigen pET-28b Female BALB/c mice T4 Hoc-soc- 10 μg phage particles i.m Soc Induced Th1 and Th2 response Complete protection against pneumonic plague BL21 [ 69 , 69 ] Anti-cancer Melanoma Epitope MAGE-A1161–169 pfd8wf Mice and cell lines YAC-1 and B16.F10 fd 50 μg phage particles i.p pVIII Hypersensitivity type IV Inhibition of tumor growth due to Specific cytotoxic T cell response and enhanced NK activity TG1 [ 70 , 70 ] Anti-viral HRSV G glycoprotein-epitope 173–187 Bacteriophagevector Fuse 5 BALB/c mice fd 1 mg i.p pIII Stimulation of strong immune response against RSV infection in mice K91 Kan [ 71 , 71 ] Anti-liver cancer ASPH peptides pVCDcDL1A Murine λ 10 10 pfu s.c gpD Enhanced in CD4 + and CD8 + response Secretion of Th1 and Th2 Specific cytokine Decreased Hepatocellular carcinoma growth n.r [ 42 , 42 ] Anti-cancer EGFR gene of extracellular domain of chicken xenogeneic EGFR n.r Male Kunming mice T7 2.5 × 10 13 pfu 10 B Decreased tumor growth and progression Stimulation of humoral immune response and increase in specific anti-EGFR antibodies n.r [ 72 , 72 ] Contraceptive 3 Gonadotrophin Releasing Hormone (GnRH) fragment 43 kDa n.r Male BALB/c mice T7 10 10 pfu s.c 10 B Secretion of Specific anti-GnRH antibody Spermatogenesis suppression BL21 [ 73 , 73 ] Anti-cancer Mouse Fms-like tyrosine kinase 4 (Flt4) T4-Z Mice T4 10 11 pfu s.c Soc Prevention of metastasis in Lewis lung carcinom due to stimulation of antitumoral immunity Induction of anti- Flt4 antibody BL21 [ 74 , 74 ] Anti-cancer Vascular endothelial growth factor receptor 2 (VEGFR2) pD-mVEGFR2 Mouse tumor model Male C57BL/6J T4-SGPDS 5 × 10 11 pfu s.c Soc C-terminus fusion Stimulation of anti-angiogenesis activity Stimulation of anti-tumor immunity Stimulation of CD4 + T cell–mediated effector mechanisms BL21 or HB101 [ 34 ] Anti-bacteria in vitro display Protective antigen (PA) from B. anthracis pET-15b Female CBA/J mice T4 Hoc-soc- 10 10 pfu i.m Hoc N-terminus Neutralization of anthrax lethal toxin due to the production of high levels antibodies P301 (sup-) [ 75 , 75 ] i. p: intraperitoneal; i. d: Intradermal; HRSV: Human Respiratory Syncitial Virus; s.c: subcutaneous; MHC: major histocompatibility complex; pfu: Plaque forming units; ASPH: Aspartateβ-hydroxylase; Herpes Simplex Virus (HSV); n.r.: not reported; EGFR: Epidermal Growth Factor Receptor; i.m: intramuscular; F: Filamentous; HLA-DR: Human Leukocyte Antigen-DR isotype; T4-S-GPDS: T4 bacteriophage nanoparticle surface gene-protein display system; RT: Reverse Transcriptase. 5.1 Phage display strategy for vaccine development As mentioned previously, phage display has been employed as an effective tool for vaccine design and development. This strategy could also be used to produce vectors to identify new antigens with desired biological and physicochemical properties as the first step of vaccine design, in addition to displaying antigens. In the first step of the vaccine design process, after the identification of novel antigens, phage display is employed to produce random peptides through genetic engineering, which leads to the selection of suitable epitopes for stimulation of the immune system. In an in vivo phage display system , the expression of antigens occurs during the phage infection stage [ 75 ]. There are problems with the system, such as differences in how proteins are expressed inside cells, incomplete phage assembly, and protein accumulation [ 75 ], which leads to improper folding and poor performance as a vaccine [ 10 ]. In vitro phage display systems resolved the limitations and problems of the In vivo system, and the protein fusion process was performed with correct folding and more quantity [ 10 ]. 5.1.1 Production of antigen-displaying vectors In phage display and phage-based vaccine technology, peptide antigens are displayed in fusion with the phage surface proteins. The resulting recombinant viral particles have specific immunological activity [ 76 ]. Anti-cancer phage-based vaccines, antiviral phage-based vaccines, and immunocontraceptive phage-based vaccines are examples of phage-based vaccines. Anticancer vaccines have been effective in cancer immunotherapy in recent years. Some candidate tumor antigens have been assessed as the best immunogenic antigens in anticancer phage-based vaccines, such as Epidermal Growth Factor Receptor (EGFR) [ 72 ], Fms-like tyrosine kinase 4 (Flt4), and Melanoma Antigen Gene (MAGE) [ 77 ]. The phages used in anti-cancer phage vaccines include filamentous phages in mouse and rabbit tumor models [ 28 ] and T4 and M13 phages in mouse tumor models [ 78 ]. The anticancer mechanisms used include the death of tumor cells and the reduction of viable tumor cells by inducing the infiltration of neutrophils [ 79 ], the reduction of tumor cell proliferation by stimulating the humoral immune system and the production of antibodies [ 18 ], and the degradation of tumors by the use of toll-like receptors (TLR) [ 79 ]. Among the effective phage-based antiviral vaccines, epitopes of hepatitis B, HIV, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2) [ 25 ], human respiratory syncytial virus [ 71 ], and circovirus 2 (PCV2) [ 10 ] are displayed on the phage surface. Another immunocontraceptive phage-based vaccine that has been used in mouse models for immunocastration is the gonadotropin-releasing hormone (GnRH) antigen on the T7 phage surface [ 73 ]. Table 1 shows in detail several phage-based vaccines and the main findings after their administration. 5.1.2 Antigen identification (Phage bio-panning technology) In this process, random or natural cDNA sequence variants are cloned into the phage genome to express different antigens in fusion with coat proteins on the phage surface and produce libraries of phages carrying different candidate antigens. From the libraries, phages with high affinity for desired targets are selected and their genomes sequenced, and in this way, the encoding sequence for the target antigen is identified [ 30 ]. The advantages of this method in vaccine design include rapid identification, low cost, and high efficacy [ 21 ]. For the display of antigens in M13, pIII is a better option because the fused antigens (displayed antigens) bind to the desired targets with a higher affinity [ 30 ]. The main applications of the bio-panning technique are the identification of linear and continuous epitopes with a length of 4–6 amino acids ( Fig. 6 ) [ 21 , 80 ] and mimotope (a peptide mimicking the structure of an epitope) ( Table 2 ). The application of mimotopes is more advantageous than epitopes because of the facility of synthesis, imitation of non-protein antigens such as carbohydrates and lipids, and high bioactivity [ 18 , 28 , 29 , 81 ]. The application of mimotopes was reported to identify amino acid sequences of immunogenic domains in toxins like botulinum neurotoxin A [ 82 ]. Table 2 describes some of the epitopes and mimotopes displayed on phages that have developed in vivo effective responses in mouse models. Fig. 6 Antigen identification using phage display technology includes the stages of phage library construction, phage selection, and antigen identification. Fig. 6 Table 2 Examples of identification of epitopes, antigens, and mimotopes by bio-panning technology, describing the type of vector used (phage or phagemid), the main findings such as screening antibodies, and their therapeutic or prophylactic results. Table 2 SOURCE OF ANTIGEN BACTERIOPHAGE VECTOR IDENTIFIED ANTIGEN THERAPEUTIC/PROPHYLACTIC EFFECTS IDENTIFIED ANTIBODIES REFERENCE EGFR GENE PAK-8 M13 pVIII EGFR mimotope Triple tandem repeat Decreased tumor growth Humoral response Lewis lung carcinoma tumor model High level of cytokines Anti-cancer activity Anti EGFR monoclonal antibody ICR62 [ 28 , 28 ] MYCOBACTERIUM LEPRAE M13 (pIII) Anti M.leprae epitopes High antibody titer High immunogenicity in mice Human antiserum [ 83 , 83 ] IXODES SCAPULARIS TICKS SALIVARY GLAND pHORF3 M13 Metalloprotease (MP1) No evaluated 3 biopanning rounds with human serum antibodies against salivary gland homogenate (SGH) [ 84 , 84 ] LEISHMANIA INFANTUM (SYN. L. CHAGASI) M13 Peptide 5 Protective effect vs. L. infantum in mice model High immunogenicity Polyclonal IgGs from L. infantum infected dogs [ 85 , 85 ] SALMONELLA TYPHIMURIUM pHORF3 M13 Novel immunogenic antigens High immunogenicity Serum from infected pigs [ 86 , 86 ] TRICHINELLA SPIRALLIS M13 (pIII) Peptide 8F7 High levels of IgG1 Reduction of larvae in vaccinated mice mAb 8F12 [ 19 , 19 ] 5.1 Phage display strategy for vaccine development As mentioned previously, phage display has been employed as an effective tool for vaccine design and development. This strategy could also be used to produce vectors to identify new antigens with desired biological and physicochemical properties as the first step of vaccine design, in addition to displaying antigens. In the first step of the vaccine design process, after the identification of novel antigens, phage display is employed to produce random peptides through genetic engineering, which leads to the selection of suitable epitopes for stimulation of the immune system. In an in vivo phage display system , the expression of antigens occurs during the phage infection stage [ 75 ]. There are problems with the system, such as differences in how proteins are expressed inside cells, incomplete phage assembly, and protein accumulation [ 75 ], which leads to improper folding and poor performance as a vaccine [ 10 ]. In vitro phage display systems resolved the limitations and problems of the In vivo system, and the protein fusion process was performed with correct folding and more quantity [ 10 ]. 5.1.1 Production of antigen-displaying vectors In phage display and phage-based vaccine technology, peptide antigens are displayed in fusion with the phage surface proteins. The resulting recombinant viral particles have specific immunological activity [ 76 ]. Anti-cancer phage-based vaccines, antiviral phage-based vaccines, and immunocontraceptive phage-based vaccines are examples of phage-based vaccines. Anticancer vaccines have been effective in cancer immunotherapy in recent years. Some candidate tumor antigens have been assessed as the best immunogenic antigens in anticancer phage-based vaccines, such as Epidermal Growth Factor Receptor (EGFR) [ 72 ], Fms-like tyrosine kinase 4 (Flt4), and Melanoma Antigen Gene (MAGE) [ 77 ]. The phages used in anti-cancer phage vaccines include filamentous phages in mouse and rabbit tumor models [ 28 ] and T4 and M13 phages in mouse tumor models [ 78 ]. The anticancer mechanisms used include the death of tumor cells and the reduction of viable tumor cells by inducing the infiltration of neutrophils [ 79 ], the reduction of tumor cell proliferation by stimulating the humoral immune system and the production of antibodies [ 18 ], and the degradation of tumors by the use of toll-like receptors (TLR) [ 79 ]. Among the effective phage-based antiviral vaccines, epitopes of hepatitis B, HIV, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2) [ 25 ], human respiratory syncytial virus [ 71 ], and circovirus 2 (PCV2) [ 10 ] are displayed on the phage surface. Another immunocontraceptive phage-based vaccine that has been used in mouse models for immunocastration is the gonadotropin-releasing hormone (GnRH) antigen on the T7 phage surface [ 73 ]. Table 1 shows in detail several phage-based vaccines and the main findings after their administration. 5.1.2 Antigen identification (Phage bio-panning technology) In this process, random or natural cDNA sequence variants are cloned into the phage genome to express different antigens in fusion with coat proteins on the phage surface and produce libraries of phages carrying different candidate antigens. From the libraries, phages with high affinity for desired targets are selected and their genomes sequenced, and in this way, the encoding sequence for the target antigen is identified [ 30 ]. The advantages of this method in vaccine design include rapid identification, low cost, and high efficacy [ 21 ]. For the display of antigens in M13, pIII is a better option because the fused antigens (displayed antigens) bind to the desired targets with a higher affinity [ 30 ]. The main applications of the bio-panning technique are the identification of linear and continuous epitopes with a length of 4–6 amino acids ( Fig. 6 ) [ 21 , 80 ] and mimotope (a peptide mimicking the structure of an epitope) ( Table 2 ). The application of mimotopes is more advantageous than epitopes because of the facility of synthesis, imitation of non-protein antigens such as carbohydrates and lipids, and high bioactivity [ 18 , 28 , 29 , 81 ]. The application of mimotopes was reported to identify amino acid sequences of immunogenic domains in toxins like botulinum neurotoxin A [ 82 ]. Table 2 describes some of the epitopes and mimotopes displayed on phages that have developed in vivo effective responses in mouse models. Fig. 6 Antigen identification using phage display technology includes the stages of phage library construction, phage selection, and antigen identification. Fig. 6 Table 2 Examples of identification of epitopes, antigens, and mimotopes by bio-panning technology, describing the type of vector used (phage or phagemid), the main findings such as screening antibodies, and their therapeutic or prophylactic results. Table 2 SOURCE OF ANTIGEN BACTERIOPHAGE VECTOR IDENTIFIED ANTIGEN THERAPEUTIC/PROPHYLACTIC EFFECTS IDENTIFIED ANTIBODIES REFERENCE EGFR GENE PAK-8 M13 pVIII EGFR mimotope Triple tandem repeat Decreased tumor growth Humoral response Lewis lung carcinoma tumor model High level of cytokines Anti-cancer activity Anti EGFR monoclonal antibody ICR62 [ 28 , 28 ] MYCOBACTERIUM LEPRAE M13 (pIII) Anti M.leprae epitopes High antibody titer High immunogenicity in mice Human antiserum [ 83 , 83 ] IXODES SCAPULARIS TICKS SALIVARY GLAND pHORF3 M13 Metalloprotease (MP1) No evaluated 3 biopanning rounds with human serum antibodies against salivary gland homogenate (SGH) [ 84 , 84 ] LEISHMANIA INFANTUM (SYN. L. CHAGASI) M13 Peptide 5 Protective effect vs. L. infantum in mice model High immunogenicity Polyclonal IgGs from L. infantum infected dogs [ 85 , 85 ] SALMONELLA TYPHIMURIUM pHORF3 M13 Novel immunogenic antigens High immunogenicity Serum from infected pigs [ 86 , 86 ] TRICHINELLA SPIRALLIS M13 (pIII) Peptide 8F7 High levels of IgG1 Reduction of larvae in vaccinated mice mAb 8F12 [ 19 , 19 ] 5.1.1 Production of antigen-displaying vectors In phage display and phage-based vaccine technology, peptide antigens are displayed in fusion with the phage surface proteins. The resulting recombinant viral particles have specific immunological activity [ 76 ]. Anti-cancer phage-based vaccines, antiviral phage-based vaccines, and immunocontraceptive phage-based vaccines are examples of phage-based vaccines. Anticancer vaccines have been effective in cancer immunotherapy in recent years. Some candidate tumor antigens have been assessed as the best immunogenic antigens in anticancer phage-based vaccines, such as Epidermal Growth Factor Receptor (EGFR) [ 72 ], Fms-like tyrosine kinase 4 (Flt4), and Melanoma Antigen Gene (MAGE) [ 77 ]. The phages used in anti-cancer phage vaccines include filamentous phages in mouse and rabbit tumor models [ 28 ] and T4 and M13 phages in mouse tumor models [ 78 ]. The anticancer mechanisms used include the death of tumor cells and the reduction of viable tumor cells by inducing the infiltration of neutrophils [ 79 ], the reduction of tumor cell proliferation by stimulating the humoral immune system and the production of antibodies [ 18 ], and the degradation of tumors by the use of toll-like receptors (TLR) [ 79 ]. Among the effective phage-based antiviral vaccines, epitopes of hepatitis B, HIV, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2) [ 25 ], human respiratory syncytial virus [ 71 ], and circovirus 2 (PCV2) [ 10 ] are displayed on the phage surface. Another immunocontraceptive phage-based vaccine that has been used in mouse models for immunocastration is the gonadotropin-releasing hormone (GnRH) antigen on the T7 phage surface [ 73 ]. Table 1 shows in detail several phage-based vaccines and the main findings after their administration. 5.1.2 Antigen identification (Phage bio-panning technology) In this process, random or natural cDNA sequence variants are cloned into the phage genome to express different antigens in fusion with coat proteins on the phage surface and produce libraries of phages carrying different candidate antigens. From the libraries, phages with high affinity for desired targets are selected and their genomes sequenced, and in this way, the encoding sequence for the target antigen is identified [ 30 ]. The advantages of this method in vaccine design include rapid identification, low cost, and high efficacy [ 21 ]. For the display of antigens in M13, pIII is a better option because the fused antigens (displayed antigens) bind to the desired targets with a higher affinity [ 30 ]. The main applications of the bio-panning technique are the identification of linear and continuous epitopes with a length of 4–6 amino acids ( Fig. 6 ) [ 21 , 80 ] and mimotope (a peptide mimicking the structure of an epitope) ( Table 2 ). The application of mimotopes is more advantageous than epitopes because of the facility of synthesis, imitation of non-protein antigens such as carbohydrates and lipids, and high bioactivity [ 18 , 28 , 29 , 81 ]. The application of mimotopes was reported to identify amino acid sequences of immunogenic domains in toxins like botulinum neurotoxin A [ 82 ]. Table 2 describes some of the epitopes and mimotopes displayed on phages that have developed in vivo effective responses in mouse models. Fig. 6 Antigen identification using phage display technology includes the stages of phage library construction, phage selection, and antigen identification. Fig. 6 Table 2 Examples of identification of epitopes, antigens, and mimotopes by bio-panning technology, describing the type of vector used (phage or phagemid), the main findings such as screening antibodies, and their therapeutic or prophylactic results. Table 2 SOURCE OF ANTIGEN BACTERIOPHAGE VECTOR IDENTIFIED ANTIGEN THERAPEUTIC/PROPHYLACTIC EFFECTS IDENTIFIED ANTIBODIES REFERENCE EGFR GENE PAK-8 M13 pVIII EGFR mimotope Triple tandem repeat Decreased tumor growth Humoral response Lewis lung carcinoma tumor model High level of cytokines Anti-cancer activity Anti EGFR monoclonal antibody ICR62 [ 28 , 28 ] MYCOBACTERIUM LEPRAE M13 (pIII) Anti M.leprae epitopes High antibody titer High immunogenicity in mice Human antiserum [ 83 , 83 ] IXODES SCAPULARIS TICKS SALIVARY GLAND pHORF3 M13 Metalloprotease (MP1) No evaluated 3 biopanning rounds with human serum antibodies against salivary gland homogenate (SGH) [ 84 , 84 ] LEISHMANIA INFANTUM (SYN. L. CHAGASI) M13 Peptide 5 Protective effect vs. L. infantum in mice model High immunogenicity Polyclonal IgGs from L. infantum infected dogs [ 85 , 85 ] SALMONELLA TYPHIMURIUM pHORF3 M13 Novel immunogenic antigens High immunogenicity Serum from infected pigs [ 86 , 86 ] TRICHINELLA SPIRALLIS M13 (pIII) Peptide 8F7 High levels of IgG1 Reduction of larvae in vaccinated mice mAb 8F12 [ 19 , 19 ] 6 Disadvantages of using the phage display technique Phages cannot proliferate and grow in eukaryotic hosts because in prokaryotic hosts, post-translational modifications such as glycosylation of proteins and formation of disulfide bonds are not performed, and because folding is directly related to the function of a protein, the desired function of a complex protein such as complex antibodies or some active epitopes on the phage surface is not possible. Also, although phage particles are potentially harmless to humans and animals, the oral route of administration can infect intestinal bacteria and cause dysbiosis, as well as release endotoxins from infectious bacteria, increasing the risk of damage to the host. This issue can be solved using non-lytic phages [ 76 , 87 , 88 ]. 7 Bacteriophage-delivered DNA vaccines The limitations of DNA vaccines, including low immunogenicity and the need for adjuvants, were solved through the application of delivery vectors and bacteriophage-DNA vaccines. Phage particles are strong and appropriate adjuvants, so they are regarded as a good vehicle for delivering DNA. The bacteriophage-DNA vaccine comprises a eukaryotic expression cassette that encodes specific antigens and is under the control of target-specific promoters [ 76 , 89 ]. The expression cassette should contain regulatory elements for proper antigen expression and folding ( Fig. 7 ) [ 6 ]. The most commonly used phages for this purpose are lambda phages, but filamentous phages have also been used [ 90 , 91 ]. One of the significant features of filamentous phage-DNA vaccines is that the vectors can carry several gene copies simultaneously, which leads to the stimulation of immune responses against several different antigens simultaneously using only one delivery vector [ 90 ]. Bacteriophage-delivered DNA vaccines have advantages over other types of vaccines, including easy production, low cost, high safety, mass production, high stability (resistant to enzymatic digestion, high temperatures, and pH changes), the ability to carry large DNA fragments (up to 20 kb in lambda phage), and the ability to elicit an effective immune response by properly presenting antigens to immune cells [ 10 , 42 , 76 , 89 , 92 ]. Table 3 describes the bacteriophage-DNA vaccines currently developed. Fig. 7 Schematic illustration of phage DNA vaccines. Fig. 7 Table 3 Description of antigen, carrier phage type, DNA cloning vector, post-administration effects, selected promoter, model of study, and dose of some antiviral phage DNA vaccines. Table 3 Vaccine Phage type Promoter DNA Cloning Vector Model of Study Dosage Effects after administration Reference Anti-viral Small surface antigen (HBsAg) of hepatitis B Λ n.r pRcCMVHBs(S) λ-gt11 Rabbits 4 × 10 10 pfu Strong antibody response (IgG, IgM) [ 89 , 89 ] Anti-viral Human papillomavirus (HPV)-16 E7 λ ZAP CMV n.r C57BL / 6 mice 2 × 10 12 particles Decreased tumor size [ 41 , 41 ] Anti-viral Hepatitis B Surface antigen (HB) Λ CMV λ-gt11 Mice and rabbits Mice: 5 × 10 9 pfu Rabbits: 4 × 10 10 HBsAg pfu Anti HB response [ 92 , 92 ] Anti-viral Herpes simplex virus 1 (HSV-1 ) glycoprotein D M13 Human cytomegalovirus immediate-early pcDNA3-gD plasmid Mice 1.4 × 10 15 pfu Anti-HSV-1 neutralizing antibodies and CytotoxicT cell response [ 90 , 90 ] pfu: Plaque forming units, CMV: Cytomegalovirus, n.r.: not reported. 8 Hybrid bacteriophage vaccines Hybrid phage-DNA vaccines are a combination of phage-display vaccines and bacteriophage-delivered DNA vaccines. High-affinity peptides to Antigen-Presenting Cells (APCs) or the antigen itself are displayed on the phage surface, and at the same time, they harbor an eukaryotic expression cassette that contains the DNA sequences encoding specific antigens ( Fig. 8 ) [ 6 ]. The feature enhances effective humoral and cellular immune responses [ 76 ]. In 2008, a double-hybrid filamentous phage (fd) was developed whose co-displayed peptides were recognized by the Major Histocompatibility Complex (MHC) class I and class II cell surface receptors and epitopes from MAGE antigen with the aim of enhancing T cell-based antitumor activity. Therefore, hybrid phage-DNA vaccines were proposed as a good choice for more effective anticancer vaccines [ 67 ]. Fig. 8 Schematic illustration of Hybrid phage vaccines. Fig. 8 9 Immunity against a phage-based vaccine The most crucial aim of vaccines is to elicit the immune system's response against pathogens such as bacteria, viruses, parasites, and fungi; therefore, it is vital to find the best vaccine that efficiently activates both immune systems' arms. After bacteriophage discovery, the interaction between phages and the immune system of mammalian cells was precisely studied [ [93] , [94] , [95] ]. The crosstalk between the immune system and phage can be studied from two distinct perspectives: phage immunomodulatory activity and phage immunogenicity ( Fig. 9 ). Immunomodulatory phage activity is a nonspecific effect of phages to induce an innate immune response and also help to increase specific immune responses via the natural ability of phages to elicit the adaptive immune system [ 93 , 96 ]. Fig. 9 A schematic picture of how phage peptide vaccines stimulate immunity. Phages that are used in phage vaccines are engineered to display peptides designed to stimulate the immune system. These phage vaccines activate innate immune responses by macrophages, granulocytes, and complement proteins. Phage components including CpG motifs on DNA and LPS stimulate several PRRs, such as TLRs 3, 4, 7, 8, 9, and 13, on the other hand, cytokines like IL6 - IL12, etc. are produced by phagocytes to induce adaptive immune responses. Displayed recombinant protein is presented by APCs such as dendritic cells (DCs) via Major Histocompatibility Complex Class II (MHC II) to T helper cells (Th), causing to activate memory B and T cells. Fig. 9 9.1 The phage's immunomodulatory activity 9.1.1 Innate immune system and cytokine secretion Innate immunity is the body's first line of defense against pathogens. Intrinsic immunity includes phagocytes (macrophages and dendritic cells), granulocytes (eosinophils, basophils, neutrophils, mast cells, and NK cells), and complement proteins, which contribute to phagocytosis [ 93 , 97 , 98 ]. Mammalian cells express receptors called pattern recognition receptors (PRPs), which either bind to specific signals such as pathogen-associated molecular patterns (PAMPs) or identify damage-associated molecular patterns (DAMPs), such as specific motifs on the genome of foreign agents or proteins released from pathogens. One of the principal PRPs is toll-like receptors (TLRs), of which, TLR3, 7, 8, 9, 13 identify the viral genome and activate the innate immune system. Bacteriophages trigger immune responses via extracellular, endosomal, or cytoplasmic PRPs. Following the phage 's entrance via transcytosis, the phage is exposed to macrophages, dendritic cells, and mast cells. Moreover, the interaction of phages with immune cells is possible due to the circulation of phages in the blood after their entrance into the spleen. The unmethylated deoxycytidylate phosphate deoxyguanylate (CpG) sites in the phage genome act as PAMPs detected by TLR9 and activate downstream signaling pathways leading to the secretion of inflammatory cytokines. The inflammatory cytokines recruit the immune cells to the infection site and thus enhance the immune responses [ 93 , 94 , 96 , [99] , [100] , [101] ]. 9.2 The phage 's immunogenicity 9.2.1 Humoral immunity Phages express immunogenic peptides or mimotopes on their surfaces that lead to direct or indirect activation of B cells by Th2 cells. Several studies have shown that the injection of M13 phage into the mouse models induced an efficient IgG antibody response even in the absence of excipients [ 96 , 102 ]. Researchers have analyzed the antibody responses induced by phage M13 in mouse models through MyD88 deletion to investigate the adaptive immune response. The authors have studied the initial IgG responses induced by the M13 phage in mice on day 13 after phage administration. They reported that, in contrast to wild-type mice, the mice carrying the MyD88 deletion did not respond to IgG antibodies. Results showed that phage M13 triggered an efficient antibody response consisting of IgG2b, 2c, and 3, through MyD88 [ 93 , 103 ]. 9.2.2 Cell immunity T cells, including both CD8 + and CD4 + T cell subsets, play essential roles in viral infections and in preventing the growth and metastasis of tumor cells. The activation of CD8 + T cells and engagement of MHCI are of great importance to fighting against viral infections and one of the goals of vaccine design. Although the efficacy of phages on the activation of cellular immunity is based on a broader range of studies, it has been shown that the epitopes of phages can activate cellular immunity as well. For instance, the expression of the rt2 peptide, derived from the HIV reverse transcriptase enzyme on phage f2, activates cytotoxic T lymphocytes (CTLs) in human cells. As mentioned above, the phage 's entrance as a foreign particle triggers the antigen-presenting cell (APS) to process and present the epitopes to T cells [ 101 , [103] , [104] , [105] ]. Filamentous bacteriophages displaying peptide epitopes can be administered by oral or parenteral means. Although the parenteral administration exhibits robust immunogenic features the same as the oral or intranasal administration, the amount of serum IgA and IgG was higher in the oral and parenteral administrations when compared to the intranasal administration [ 106 , 107 ]. In another aerosol-based strategy, a short sequence of the pIII protein was used to target and transport the phage particles into the systemic circulation. The results revealed that the intranasal route of immunization might exhibit biologically significant advantages. Moreover, inhalation is needle-free and non-invasive, eliminating the requirement for specialized medical staff. Additionally, rapid access to the upper and lower respiratory tracts reduces viral shedding and subsequently lowers transmission [ 108 ]. 9.1 The phage's immunomodulatory activity 9.1.1 Innate immune system and cytokine secretion Innate immunity is the body's first line of defense against pathogens. Intrinsic immunity includes phagocytes (macrophages and dendritic cells), granulocytes (eosinophils, basophils, neutrophils, mast cells, and NK cells), and complement proteins, which contribute to phagocytosis [ 93 , 97 , 98 ]. Mammalian cells express receptors called pattern recognition receptors (PRPs), which either bind to specific signals such as pathogen-associated molecular patterns (PAMPs) or identify damage-associated molecular patterns (DAMPs), such as specific motifs on the genome of foreign agents or proteins released from pathogens. One of the principal PRPs is toll-like receptors (TLRs), of which, TLR3, 7, 8, 9, 13 identify the viral genome and activate the innate immune system. Bacteriophages trigger immune responses via extracellular, endosomal, or cytoplasmic PRPs. Following the phage 's entrance via transcytosis, the phage is exposed to macrophages, dendritic cells, and mast cells. Moreover, the interaction of phages with immune cells is possible due to the circulation of phages in the blood after their entrance into the spleen. The unmethylated deoxycytidylate phosphate deoxyguanylate (CpG) sites in the phage genome act as PAMPs detected by TLR9 and activate downstream signaling pathways leading to the secretion of inflammatory cytokines. The inflammatory cytokines recruit the immune cells to the infection site and thus enhance the immune responses [ 93 , 94 , 96 , [99] , [100] , [101] ]. 9.1.1 Innate immune system and cytokine secretion Innate immunity is the body's first line of defense against pathogens. Intrinsic immunity includes phagocytes (macrophages and dendritic cells), granulocytes (eosinophils, basophils, neutrophils, mast cells, and NK cells), and complement proteins, which contribute to phagocytosis [ 93 , 97 , 98 ]. Mammalian cells express receptors called pattern recognition receptors (PRPs), which either bind to specific signals such as pathogen-associated molecular patterns (PAMPs) or identify damage-associated molecular patterns (DAMPs), such as specific motifs on the genome of foreign agents or proteins released from pathogens. One of the principal PRPs is toll-like receptors (TLRs), of which, TLR3, 7, 8, 9, 13 identify the viral genome and activate the innate immune system. Bacteriophages trigger immune responses via extracellular, endosomal, or cytoplasmic PRPs. Following the phage 's entrance via transcytosis, the phage is exposed to macrophages, dendritic cells, and mast cells. Moreover, the interaction of phages with immune cells is possible due to the circulation of phages in the blood after their entrance into the spleen. The unmethylated deoxycytidylate phosphate deoxyguanylate (CpG) sites in the phage genome act as PAMPs detected by TLR9 and activate downstream signaling pathways leading to the secretion of inflammatory cytokines. The inflammatory cytokines recruit the immune cells to the infection site and thus enhance the immune responses [ 93 , 94 , 96 , [99] , [100] , [101] ]. 9.2 The phage 's immunogenicity 9.2.1 Humoral immunity Phages express immunogenic peptides or mimotopes on their surfaces that lead to direct or indirect activation of B cells by Th2 cells. Several studies have shown that the injection of M13 phage into the mouse models induced an efficient IgG antibody response even in the absence of excipients [ 96 , 102 ]. Researchers have analyzed the antibody responses induced by phage M13 in mouse models through MyD88 deletion to investigate the adaptive immune response. The authors have studied the initial IgG responses induced by the M13 phage in mice on day 13 after phage administration. They reported that, in contrast to wild-type mice, the mice carrying the MyD88 deletion did not respond to IgG antibodies. Results showed that phage M13 triggered an efficient antibody response consisting of IgG2b, 2c, and 3, through MyD88 [ 93 , 103 ]. 9.2.2 Cell immunity T cells, including both CD8 + and CD4 + T cell subsets, play essential roles in viral infections and in preventing the growth and metastasis of tumor cells. The activation of CD8 + T cells and engagement of MHCI are of great importance to fighting against viral infections and one of the goals of vaccine design. Although the efficacy of phages on the activation of cellular immunity is based on a broader range of studies, it has been shown that the epitopes of phages can activate cellular immunity as well. For instance, the expression of the rt2 peptide, derived from the HIV reverse transcriptase enzyme on phage f2, activates cytotoxic T lymphocytes (CTLs) in human cells. As mentioned above, the phage 's entrance as a foreign particle triggers the antigen-presenting cell (APS) to process and present the epitopes to T cells [ 101 , [103] , [104] , [105] ]. Filamentous bacteriophages displaying peptide epitopes can be administered by oral or parenteral means. Although the parenteral administration exhibits robust immunogenic features the same as the oral or intranasal administration, the amount of serum IgA and IgG was higher in the oral and parenteral administrations when compared to the intranasal administration [ 106 , 107 ]. In another aerosol-based strategy, a short sequence of the pIII protein was used to target and transport the phage particles into the systemic circulation. The results revealed that the intranasal route of immunization might exhibit biologically significant advantages. Moreover, inhalation is needle-free and non-invasive, eliminating the requirement for specialized medical staff. Additionally, rapid access to the upper and lower respiratory tracts reduces viral shedding and subsequently lowers transmission [ 108 ]. 9.2.1 Humoral immunity Phages express immunogenic peptides or mimotopes on their surfaces that lead to direct or indirect activation of B cells by Th2 cells. Several studies have shown that the injection of M13 phage into the mouse models induced an efficient IgG antibody response even in the absence of excipients [ 96 , 102 ]. Researchers have analyzed the antibody responses induced by phage M13 in mouse models through MyD88 deletion to investigate the adaptive immune response. The authors have studied the initial IgG responses induced by the M13 phage in mice on day 13 after phage administration. They reported that, in contrast to wild-type mice, the mice carrying the MyD88 deletion did not respond to IgG antibodies. Results showed that phage M13 triggered an efficient antibody response consisting of IgG2b, 2c, and 3, through MyD88 [ 93 , 103 ]. 9.2.2 Cell immunity T cells, including both CD8 + and CD4 + T cell subsets, play essential roles in viral infections and in preventing the growth and metastasis of tumor cells. The activation of CD8 + T cells and engagement of MHCI are of great importance to fighting against viral infections and one of the goals of vaccine design. Although the efficacy of phages on the activation of cellular immunity is based on a broader range of studies, it has been shown that the epitopes of phages can activate cellular immunity as well. For instance, the expression of the rt2 peptide, derived from the HIV reverse transcriptase enzyme on phage f2, activates cytotoxic T lymphocytes (CTLs) in human cells. As mentioned above, the phage 's entrance as a foreign particle triggers the antigen-presenting cell (APS) to process and present the epitopes to T cells [ 101 , [103] , [104] , [105] ]. Filamentous bacteriophages displaying peptide epitopes can be administered by oral or parenteral means. Although the parenteral administration exhibits robust immunogenic features the same as the oral or intranasal administration, the amount of serum IgA and IgG was higher in the oral and parenteral administrations when compared to the intranasal administration [ 106 , 107 ]. In another aerosol-based strategy, a short sequence of the pIII protein was used to target and transport the phage particles into the systemic circulation. The results revealed that the intranasal route of immunization might exhibit biologically significant advantages. Moreover, inhalation is needle-free and non-invasive, eliminating the requirement for specialized medical staff. Additionally, rapid access to the upper and lower respiratory tracts reduces viral shedding and subsequently lowers transmission [ 108 ]. 10 Application of the phage vaccine 10.1 Anti-parasite phage-based vaccines Various studies evaluated the application of phage-based vaccines against parasitic diseases. Bacteriophages were first applied to stimulate the immune response against parasite peptides in 1988. In this study, repeated sequences of the circumsporozoite (CS) gene of the human malaria parasite, Plasmodium falciparum, were inserted into the pIII gene of the filamentous bacteriophage, f1. Immune responses against the CS protein were reported upon administration of the engineered phage in mice and rabbit models ( Table 4 ) [ 109 ]. As a cysticercosis vaccination, three Taenia solium antigens were produced on pVIII phage M13 (S3Pvac). A randomized trial on a pig population was used to compare the effectiveness of S3Pvac-Phage with a placebo. Tongue examination and necropsy results from three to five months after oral administration of the vaccine revealed that vaccination resulted in a 70% decrease in the frequency of tongue cysticercosis, a 54% decrease in muscle cysticercosis, and an 87% decrease in the total number of cysticercoses ( Table 4 ) [ 15 ]. A 28 kDa glutathione S-transferase (Sm28GST) antigen from the human parasite Schistosoma mansoni was produced by Kakuturu V. N. Rao et al., in 2003. IgG2b, IgG3, and IgM antibody levels were shown to be considerably greater in the sera of inoculated animals compared to pre-immune sera in subsequent immunization trials. The levels of IgG1 and IgG2a, however, did not significantly differ from pre-immune values. Anti-M13 antibodies were used to measure the levels of anti-phage antibodies in the serum samples, and results showed that these antibodies were present in all inoculated animals two weeks following immunization. Six weeks after vaccination, 100 S. mansoni cercariae were given to each mouse as a challenge. Only around 30% of the population was protected after a single challenge. This may be due to the fact that Sm28GST is not the only antigen present in irradiation cercariae that contributes to the development of protective immune responses [ 110 ]. Another study tested the capacity of fd phages against infection with the human protozoan Trypanosoma cruzi , the etiologic agent of Chagas Disease. In this study, the OVA257–264 peptide or the T. cruzi immunodominant peptides, PA8 and TSKB20, were inserted into the fd phage. The results demonstrated fd phages as a potent delivery system, activating both humoral and cellular-mediated responses against T. cruzi infection through a TLR9-dependent mechanism ( Table 4 ) [ 111 ]. Table 4 Application of phage-based vaccines. Table 4 Vaccine application Model of study Main Effects of Phage-Based Vaccine Administration and Comments Phage used Protein of pathogen that displayed Protein Used for Phage Display reference Parasites Plasmodium falciparum mice and rabbits Elicitation of responses to the fusion protein f1 phage Repeat regions of the circumsporozoite (CS) protein gene pIII [ 109 ] Taenia solium Pig Induced antigen-specific cellular immune responses in pigs M13 phage Three peptides (KETc1, KETc12, GK1) and a recombinant antigen KETc7 pVIII [ 63 ] Trypanosoma cruzi C57BL/6 (B6) and Tlr9−/− mice Induces anti-PA8 and antiTSKB20 IgG production, expansion of Ag-specifc IFN-γ, TNF-α, and Granzyme B-producing CD8 + T cells, as well as in vivo Ag-specifc cytotoxic responses f1 phage OVA257–264 peptide or the T. cruzi-immunodominant peptides PA8 and TSKB20 pVIII [ 111 ] Viruses hepatitis B BALB:c (H-2d) mice Inducing MHC class I restricted cytotoxic T lymphocytes response in vivo Filamentous phage Epitope S28–39 pVIII [ 112 ] SARS-COV-2 Swiss Webster or BALB/c Enhanced a systemic and specific spike (S) protein-specific antibody response Filamentous phage Epitopes of the SARS-CoV-2 spike (S) protein and signal prptide pVIII and pIII [ 108 ] FMDV BALB/C mice Induce high levels of IFN-γ levels in mice 31 with little effect on IL-4 levels and produce high levels of anti- 33 FMD antibodies T7 phage The capsid protein VP1 of the 22 OHM-02 strain, and the recombinant VP1 phage was termed OHM-T7 [ 113 ] HIV 6–8 week-female BALB/c mice The displayed p24 was highly immunogenic in mice in the absence of any external adjuvant, eliciting strong p24-specific antibodies, as well as Th1 and Th2 cellular responses with a bias toward the Th2 response T4 phage HIV antigens, p24-gag, Nef, and an engineered gp41 C-peptide trimer Hoc-capsid [ 114 ] porcine Circovirus 2 (PCV2) pigs The LDP-D-CAP elicited both cellular and humoral immune responses Phage lambda Capsid protein (LDP-D-CAP) Carboxyl-terminal of lambda head protein D [ 115 ] herpes simplex virus 1(HSV-1) 6–8 week-female BALB/c mice In both arms of immune responses induced by recombinant filamentous phage inoculation Recombinant phage Glycoprotein D [ 116 ] Respiratory Syncytial Virus 5–6 week old BALB/C mice Inducing a high level of circulating RSV-specific antibodies Fd phage Epitope 173–187 from the glycoprotein G pIII [ 117 ] Bacteria Bacillus anthracis rabbit Activated strong anthrax- and plague-specific immune responses specially T-helper 1 and 2 T4 phage anthrax protective antigen (PA) (83 kDa) The small outer capsid protein Soc (9 kDa) [ 118 ] Yersinia pestis rabbit Activated strong anthrax- and plague-specific immune responses specially T-helper 1 and 2 T4 phage The mutated (mut) capsular antigen F1 and the low-calcium-response V antigen of the type 3 secretion system The small outer capsid protein Soc (9 kDa) [ 118 ] Fungies Sporothrix globosa 6- to 10-week-old BALB/c mice Induce Gp70-specific antibody production in mice, which can in turn bind with Gp70 and treat the infection Phage vector fuse-55 Epitope peptide (kpvqhalltplgldr) of Gp70 pIII [ 119 ] Candida albicans 6–8 week-old female BALB/c mice Produce strong immune response as rSap2 and generate antibodies that can bind Sap2 and CA to inhibit the CA infection and increases the survival rate of CA-infected mice Filamentous phage aspartyl proteinases 2 (Sap2) pVIII [ 120 ] Other application Contraceptive vaccine Male BALB/c mice Specific anti-GnRH antibody faster than conventional vaccine Spermatogenesis suppression T7 phage Gonadotrophin Releasing Hormone (GnRH) fragment 43 kDa 10 B [ 121 ] cocaine addiction vaccine Rat Filamentous phage Cocaine-binding proteins (antibody-displaying) pVIII [ 122 ] A potential phage-based vaccine candidate's immune response against R. microplus (the cattle tick) was assessed. The R. microplus Bm86 protein epitopes, Sbm7462, and a truncated pIII protein were displayed on the M13 phage to develop the vaccine. In an ex vivo experiment, the Sbm7462 phage vaccination induced the maturation of bovine monocyte-derived dendritic cells. Peripheral blood mononuclear cells (PBMC) from the spleen proliferated and produced antibodies against the Bm86 and Sbm7462 antigens in subcutaneously vaccinated mice. So according to the findings of the study on cattle tick antigen, phage-based vaccines induced antibody production in such a way that was higher on day 25 than on day 12 after injection [ 123 ]. 10.2 Anti-viral phage-based vaccines There are many diseases in animals and humans of viral origin, and phage vaccines have also been successfully developed against them. Hepatitis B was the target of the first phage vaccination against viruses. In order to develop a Hepatitis B hybrid phage vaccine, the hepatitis B virus S28-39 epitope was cloned into the pC89 phagemid vector in frame with the major envelope protein (pVIII) of M13. Studies on the immunization of mice revealed that the injection can cause certain cytotoxic T lymphocyte (CTL) responses ( Table 4 ) [ 66 ]. Additionally, the hepatitis B core antigen gene (HBcAg) was inserted into the minor envelope protein (pIII) gene of M13. Following vaccination, ELISA results revealed that recombinant phages and recombinant phages prepared in incomplete Freund's adjuvant (IFA) were both effective vaccines that caused powerful immunological reactions. Phages alone or in combination with IFA did not significantly alter the immune response, demonstrating that the phages themselves are antigenic and trigger an immunological response [ 124 ]. Soc and Hoc have been employed to express antigens on the surface of T4 since they are not required for phage infectivity [ 125 ]. Via Hoc-capsid interactions, HIV antigens, p24-gag, Nef, and a modified C-peptide trimer of gp41 are presented on the T4 phage capsid surface. The human immunodeficiency virus (HIV) genes were attached to the 5′ or 3′ end of the Hoc gene to achieve in-frame integration. All of the accessible capsid binding sites were effectively saturated by the efficient presentation of single or several antigens. In the absence of any external adjuvant, p24 exhibited in mice was very immunogenic and produced potent p24-specific antibodies as well as Th1 and Th2 cell responses, with a preference for Th2 responses ( Table 4 ) [ 114 ]. Li and colleagues discovered that influenza virus 3M2e proteins exhibited on T4 nanoparticles create extraordinarily high levels of 3M2e-specific IgG antibodies in the absence of any adjuvant, while 3M2e coupled to RB69 Soc only causes low amounts of 3M2e-specific IgG antibodies [ 126 ]. Among other viruses that have been used for this purpose are Herpes simplex virus 1 (HSV-1) ( Table 4 ) [ 116 ], Circovirus 2 (PCV2) ( Table 4 ) [ 115 ], FMDV ( Table 4 ) [ 113 ], Zika virus , and RSV ( Table 4 ) [ 117 ], which have been displayed on various phages and elicited a strong antibody response in mice when compared to non-immunized mice. Recent studies have also used phage vaccines to prevent coronavirus species, for instance, by displaying the S protein's epitope 4 of SARS-CoV2 on protein VIII of the Filamentous phage display vector f88-4, while the capsid protein pIII included the peptide CAKSMGDIVC. The transport peptide CAKSMGDIVC can identify lung epithelial cells in the lung. Recombinant phages were successfully applied to activate an antigen-specific humoral response against SARS-CoV 2 , thus representing an appropriate candidate for vaccine development against COVID-19 ( Table 4 ) [ 108 ]. Furthermore, vaccines using antigens other than the S protein from SARS-CoV-2 have been developed. While the nucleocapsid protein (NP) was inside the phage, the ectodomain trimers of the S protein were produced on the T4 phage to develop T4-CoV-2. A SARS-CoV-2 vaccine administered intravenously boosted the immune response on mucosal surfaces in comparison. The intramuscular injection, however, did not show this. Moreover, this was noticed in transgenic mice for the human angiotensin-converting enzyme (hACE2). These mice were resistant to the original SARS-CoV-2 and its delta form up to a lethal dose. The vaccine did not harm the microbiome and was stable at room temperature [ 61 ]. Zhu et al. created a platform to develop various SARS-CoV-2 vaccine candidates utilizing CRISPR editing of the T4 phage. Several phage segments were constructed using various SARS-CoV-2 antigens (such as the S, E, and NP proteins). Mice were shielded from SARS-CoV-2 infection by T4 adorned with S-trimers, which generate particular antibodies and prevent the binding of RBD to ACE-2 [ 127 ]. The safety of ABNCoV2, a vaccine based on VLPs of bacteriophage AP205 adorned with RBD of SARS-CoV-2 generated in S2 Drosophila cells, was assessed in healthy volunteers during a clinical phase I/II experiment (NCT04839146) [ 128 ]. Clinical trials.gov has not yet published the study's results, although it ended on February 25, 2022. ABNCoV2 is also the subject of an open-label phase 2 trial in Germany (NCT05077267; EUCTR2021-001393-31). Adults who had previously received the SARS-CoV-2 vaccine underwent a phase three trial (NCT05329220) to assess the immunogenicity, safety, and tolerability of ABNCoV2 [ 128 ]. 10.3 Anti-bacterial phage-based vaccines One of the most critical issues in bacterial infections is antibiotic resistance due to excessive use of antibiotics. The phage-based vaccine is introduced as a reliable approach to overcoming bacterial antibiotic resistance. The phage-based vaccines have been developed against Bacillus anthracis and Yersinia pestis's antigens, the causative agents of anthrax and plaque, respectively. This phage-based vaccine elicited strong anti-anthrax and plague-specific immune responses, especially T-helper 1 and 2 cellular immune responses, which are essential for eradicating bacterial infection ( Table 4 ). Administration of phage-based vaccines in animal models (mice, rats, and rabbits) infected with lethal doses of both anthrax lethal toxin and Y. pestis CO92 bacteria revealed complete protection. Therefore, phages like T4 could be applied with nanoparticles to formulate multivalent vaccines against high-risk pathogens and emerging infections [ 109 , 118 ]. Chlamydia trachomatis , a bacterium found in the mouth, genital tract, cervix, and urethra of adults, was studied as a candidate for a phage vaccine. The Q-CT584 vaccine was developed by chemically attaching epitopes 70–77 and 154–164 of CT584 to bacteriophage Q VLPs. CT584, a protein that binds to the type three secretion system (T3SS) involved in invasion, intracellular survival, and cell egress, was selected. Mice immunized intramuscularly with Q-CT584 and then challenged with C. trachomatis produced large amounts of IgG that prevented infection [ 129 ]. Another example of a phage vaccine against bacteria is cholera disease. Cholera is an acute, secretory diarrheal disease caused by the highly pathogenic bacterium Vibrio cholerae . Only the serogroups O1 and O139 may result in epidemic cholera out of the more than 200 serogroups. By chemically joining the antigen O-specific polysaccharide (OSP) isolated from V. cholerae O1 El Tor Inaba to VLP produced from phage Q, a vaccine (Q-OSP) against V. cholera was developed. When mice were immunized with the Q-OSP formula, high and enduring levels of IgG antibodies were produced against OSP. These antibodies recognized the natural LPS from V. cholerae O1 El Tor Inaba. Moreover, the live bacteria could be killed by antibodies in sera from mice that had received complement vaccinations [ 130 ]. 10.4 Anti-fungal phage-based vaccines One of the applications of phage is the induction of an immune response against fungal infections. Although there is still no approved antifungal phage-based vaccine for humans, there have been several successful studies regarding anti-fungal bacteriophage vaccines. Candida albicans ( Table 4 ) [ 120 ] and Sporothrix globose ( Table 4 ) [ 119 ] are the fungal agents that the phage vaccines have developed against. Immunization with recombinant phage increased the survival rate of mice following each fungal infection, separately. 10.5 Anti-cancer phage-based vaccines Cancer vaccines, regarded as promising tools for cancer treatment, have recently received increased attention. The expression of antigens fused to phage surface proteins in the phage display system is intended to induce tumor-specific T-cell responses [ 93 ]. Cross_presentation is a strategy by which phages can induce antigen presentation mediated by MHC-I and MHC-II molecules. The cross-presentation is also extremely useful in developing cancer immunotherapies, as cytotoxic T lymphocyte (CTL) activation by MHC-I recognition is critical for tumor cell death [ 131 , 132 ]. In recent years, many tumor-associated antigens that CTLs recognize have been identified and characterized. Cancer/testis (C/T) antigens are the fastest-growing group of tumor-associated antigens, expressed in various types of tumors but not in normal tissues [ 133 , 134 ]. As a result of their tumor-specificity, C/T antigens are known as appropriate candidates for cancer vaccines [ 135 ]. Sartorius and colleagues used the filamentous fd phage to co-express T helper (Th) cell-specific epitopes and C/T antigens to elicit Th-dependent CTL responses. The recombinant phage could elicit potent, specific CTL responses in vitro and in vivo. The filamentous phages enhance the immunogenicity of tumor-associated antigens while simultaneously slowing tumor growth [ 136 ]. In a similar way, another study used recombinant M13 phage as a vaccine for Lewis lung carcinoma. The pVIII coat protein showed an epidermal growth factor receptor (EGFR) mimotope. This study demonstrated that recombinant phage vaccines could significantly induce immune responses and elicit specific antibodies against cancer cells [ 18 ]. Non-filamentous phages such as T4 and T7 have also been used as vaccines to improve cancer therapy after exposure to appropriate peptides. For instance, T4 phage was used to express vascular endothelial growth factor (VEGF), and subsequently, the engineered age was applied as a vaccine against Lewis lung carcinoma in a mouse model. The T4-mVEGFR2 phage could effectively suppress angiogenesis and have significant anti-tumor activity. Furthermore, the T7 phage displays the five fragments of the EGFR mimotope, which were fused with the T7 phage's 10B coat protein via genetic engineering. The results showed that the EGFR was successfully expressed on the surface of the T7 phage, and thus the recombinant phage could effectively inhibit tumor growth in the BALB/c mouse model [ 32 ]. The discovery of tumor-specific antigens opens the way for the successful development of phage-displayed vaccines for cancer prevention. Shadidi et al. for example, used a proteomics-based method to identify breast cancer tumor antigens and created vaccines by displaying the identified tumor antigens on the T7 phage and measuring the immune responses in mice after oral administration. Their findings showed that the phage's surface display of tumor antigens could effectively elicit immune responses, thus introducing the engineered phage as a promising mucosal cancer vaccine [ 137 ]. n the fd phage display system, a tumor-specific antigen epitope called melanoma antigen A1161169 was joined to the pVIII coat protein of the fd phage. An in vivo tumor protection assay confirmed that the hybrid phage effectively inhibited tumor growth. These studies clearly showed the potency of phages for engineering an effective vaccine and improving cancer therapy [ 70 ]. Recently, an immunogenic bacteriophage-based vaccine with HER2/neu overexpression stimulated CTL activity. This study revealed the phage nanoparticles containing GP2 as a fused peptide to the gpD phage capsid protein, elicited a strong CTL response, and protected mice against HER2/neu-positive tumors [ 138 ]. The Δ16HER2 is a HER2 splice variant and the transforming isoform in HER2-positive breast cancer. Δ16HER2 has been shown to promote breast cancer aggressiveness and is a leading factor in drug resistance. However, due to tolerogenic mechanisms against the human HER2 self-antigen, a situation frequently observed in HER2+ patients, the vaccines failed to elicit immunological protection in Δ16HER2 transgenic mice. An engineered bacteriophage with immunogenic Δ16HER2 epitopes was used as an anticancer vaccine in a recent study. Through the disruption of immunological tolerance, these phage-based vaccinations were able to elicit a protective anti- Δ16HER2 humoral response. Altogether, these findings support the practice of phage-based anti-HER2/Δ16HER2 vaccination as a safe and effective immunotherapy strategy for HER2-positive breast cancers [ 139 ]. Unexpectedly, many promising preclinical investigations that could have led to clinical trials have not been repeated, and we are unsure of the reason for this. Hence, more work is needed to advance this technology, which has a lot of benefits and great promise, to the next stage of clinical trials, where it can be shown effective for usage in both humans and animals. The use of phage-based vaccinations is advantageous because several clinical trials have demonstrated that phages are both safe for humans and animals. The encouraging findings of Roehnisch in 2014 serve as a solid springboard for expanding the use of phage-based vaccines in clinical trials, at least for those based on M13 that have demonstrated safety [ 140 ]. This viewpoint is positive since it suggests that phages may eventually be used in vaccinations, which is possible based on the information presented above [ 62 ]. 10.6 Other types of diseases or conditions Another application of phage-based vaccines is in population control using the immune contraceptive vaccine, which induces an adaptive immune response against the reproductive system and causes temporary infertility. For instance, the gonadotrophin-releasing hormone (GnRH) antigen was displayed on the surface of T7 phages in a mouse model leading to immune castration [ 121 ]. Vaccine targeting could also be applied to drug addiction by using bacteriophages that display antibodies that block the effects of various drugs, such as cocaine [ 122 ]. A group of neurodegenerative illnesses known as tauopathies includes frontotemporal dementia (FTD) and Alzheimer's disease (AD). Tauopathies are brought on by neurofibrillary tangles (NFTs), which are formed in neurons by hyperphosphorylated pathogenic tau (pTau). Tau may therefore be a useful target for vaccination to prevent these illnesses. The development of pT181-Q, a phage-based vaccine against tauopathies, involved conjugating a tau peptide to bacteriophage Q's VLPs. Animals given the pT181-Q vaccine produced a strong IgG reaction against pT181. In mice immunized with pT181-Q, less pTau accumulated in the brain and hippocampus. The amount of circulating CD3 + T-cells and neuroinflammation in the brain both decreased [ 141 ]. Another application of phage vaccines is in heart disease. A higher risk of cardiovascular disease is linked to low-density lipoprotein cholesterol, which contains three checkpoint proteins (PCSK9, ApoB, and CETP). The three proteins were displayed on the bacteriophage Q coat protein VLPs to develop a phage-based vaccination. Immunized mice produced IgG1 and IgG2b immunoglobulin isotypes against PCSK9, ApoB, and CETP. The immunizations also lower the levels of these proteins, which lowers the plasma's overall cholesterol level [ 142 ]. 10.1 Anti-parasite phage-based vaccines Various studies evaluated the application of phage-based vaccines against parasitic diseases. Bacteriophages were first applied to stimulate the immune response against parasite peptides in 1988. In this study, repeated sequences of the circumsporozoite (CS) gene of the human malaria parasite, Plasmodium falciparum, were inserted into the pIII gene of the filamentous bacteriophage, f1. Immune responses against the CS protein were reported upon administration of the engineered phage in mice and rabbit models ( Table 4 ) [ 109 ]. As a cysticercosis vaccination, three Taenia solium antigens were produced on pVIII phage M13 (S3Pvac). A randomized trial on a pig population was used to compare the effectiveness of S3Pvac-Phage with a placebo. Tongue examination and necropsy results from three to five months after oral administration of the vaccine revealed that vaccination resulted in a 70% decrease in the frequency of tongue cysticercosis, a 54% decrease in muscle cysticercosis, and an 87% decrease in the total number of cysticercoses ( Table 4 ) [ 15 ]. A 28 kDa glutathione S-transferase (Sm28GST) antigen from the human parasite Schistosoma mansoni was produced by Kakuturu V. N. Rao et al., in 2003. IgG2b, IgG3, and IgM antibody levels were shown to be considerably greater in the sera of inoculated animals compared to pre-immune sera in subsequent immunization trials. The levels of IgG1 and IgG2a, however, did not significantly differ from pre-immune values. Anti-M13 antibodies were used to measure the levels of anti-phage antibodies in the serum samples, and results showed that these antibodies were present in all inoculated animals two weeks following immunization. Six weeks after vaccination, 100 S. mansoni cercariae were given to each mouse as a challenge. Only around 30% of the population was protected after a single challenge. This may be due to the fact that Sm28GST is not the only antigen present in irradiation cercariae that contributes to the development of protective immune responses [ 110 ]. Another study tested the capacity of fd phages against infection with the human protozoan Trypanosoma cruzi , the etiologic agent of Chagas Disease. In this study, the OVA257–264 peptide or the T. cruzi immunodominant peptides, PA8 and TSKB20, were inserted into the fd phage. The results demonstrated fd phages as a potent delivery system, activating both humoral and cellular-mediated responses against T. cruzi infection through a TLR9-dependent mechanism ( Table 4 ) [ 111 ]. Table 4 Application of phage-based vaccines. Table 4 Vaccine application Model of study Main Effects of Phage-Based Vaccine Administration and Comments Phage used Protein of pathogen that displayed Protein Used for Phage Display reference Parasites Plasmodium falciparum mice and rabbits Elicitation of responses to the fusion protein f1 phage Repeat regions of the circumsporozoite (CS) protein gene pIII [ 109 ] Taenia solium Pig Induced antigen-specific cellular immune responses in pigs M13 phage Three peptides (KETc1, KETc12, GK1) and a recombinant antigen KETc7 pVIII [ 63 ] Trypanosoma cruzi C57BL/6 (B6) and Tlr9−/− mice Induces anti-PA8 and antiTSKB20 IgG production, expansion of Ag-specifc IFN-γ, TNF-α, and Granzyme B-producing CD8 + T cells, as well as in vivo Ag-specifc cytotoxic responses f1 phage OVA257–264 peptide or the T. cruzi-immunodominant peptides PA8 and TSKB20 pVIII [ 111 ] Viruses hepatitis B BALB:c (H-2d) mice Inducing MHC class I restricted cytotoxic T lymphocytes response in vivo Filamentous phage Epitope S28–39 pVIII [ 112 ] SARS-COV-2 Swiss Webster or BALB/c Enhanced a systemic and specific spike (S) protein-specific antibody response Filamentous phage Epitopes of the SARS-CoV-2 spike (S) protein and signal prptide pVIII and pIII [ 108 ] FMDV BALB/C mice Induce high levels of IFN-γ levels in mice 31 with little effect on IL-4 levels and produce high levels of anti- 33 FMD antibodies T7 phage The capsid protein VP1 of the 22 OHM-02 strain, and the recombinant VP1 phage was termed OHM-T7 [ 113 ] HIV 6–8 week-female BALB/c mice The displayed p24 was highly immunogenic in mice in the absence of any external adjuvant, eliciting strong p24-specific antibodies, as well as Th1 and Th2 cellular responses with a bias toward the Th2 response T4 phage HIV antigens, p24-gag, Nef, and an engineered gp41 C-peptide trimer Hoc-capsid [ 114 ] porcine Circovirus 2 (PCV2) pigs The LDP-D-CAP elicited both cellular and humoral immune responses Phage lambda Capsid protein (LDP-D-CAP) Carboxyl-terminal of lambda head protein D [ 115 ] herpes simplex virus 1(HSV-1) 6–8 week-female BALB/c mice In both arms of immune responses induced by recombinant filamentous phage inoculation Recombinant phage Glycoprotein D [ 116 ] Respiratory Syncytial Virus 5–6 week old BALB/C mice Inducing a high level of circulating RSV-specific antibodies Fd phage Epitope 173–187 from the glycoprotein G pIII [ 117 ] Bacteria Bacillus anthracis rabbit Activated strong anthrax- and plague-specific immune responses specially T-helper 1 and 2 T4 phage anthrax protective antigen (PA) (83 kDa) The small outer capsid protein Soc (9 kDa) [ 118 ] Yersinia pestis rabbit Activated strong anthrax- and plague-specific immune responses specially T-helper 1 and 2 T4 phage The mutated (mut) capsular antigen F1 and the low-calcium-response V antigen of the type 3 secretion system The small outer capsid protein Soc (9 kDa) [ 118 ] Fungies Sporothrix globosa 6- to 10-week-old BALB/c mice Induce Gp70-specific antibody production in mice, which can in turn bind with Gp70 and treat the infection Phage vector fuse-55 Epitope peptide (kpvqhalltplgldr) of Gp70 pIII [ 119 ] Candida albicans 6–8 week-old female BALB/c mice Produce strong immune response as rSap2 and generate antibodies that can bind Sap2 and CA to inhibit the CA infection and increases the survival rate of CA-infected mice Filamentous phage aspartyl proteinases 2 (Sap2) pVIII [ 120 ] Other application Contraceptive vaccine Male BALB/c mice Specific anti-GnRH antibody faster than conventional vaccine Spermatogenesis suppression T7 phage Gonadotrophin Releasing Hormone (GnRH) fragment 43 kDa 10 B [ 121 ] cocaine addiction vaccine Rat Filamentous phage Cocaine-binding proteins (antibody-displaying) pVIII [ 122 ] A potential phage-based vaccine candidate's immune response against R. microplus (the cattle tick) was assessed. The R. microplus Bm86 protein epitopes, Sbm7462, and a truncated pIII protein were displayed on the M13 phage to develop the vaccine. In an ex vivo experiment, the Sbm7462 phage vaccination induced the maturation of bovine monocyte-derived dendritic cells. Peripheral blood mononuclear cells (PBMC) from the spleen proliferated and produced antibodies against the Bm86 and Sbm7462 antigens in subcutaneously vaccinated mice. So according to the findings of the study on cattle tick antigen, phage-based vaccines induced antibody production in such a way that was higher on day 25 than on day 12 after injection [ 123 ]. 10.2 Anti-viral phage-based vaccines There are many diseases in animals and humans of viral origin, and phage vaccines have also been successfully developed against them. Hepatitis B was the target of the first phage vaccination against viruses. In order to develop a Hepatitis B hybrid phage vaccine, the hepatitis B virus S28-39 epitope was cloned into the pC89 phagemid vector in frame with the major envelope protein (pVIII) of M13. Studies on the immunization of mice revealed that the injection can cause certain cytotoxic T lymphocyte (CTL) responses ( Table 4 ) [ 66 ]. Additionally, the hepatitis B core antigen gene (HBcAg) was inserted into the minor envelope protein (pIII) gene of M13. Following vaccination, ELISA results revealed that recombinant phages and recombinant phages prepared in incomplete Freund's adjuvant (IFA) were both effective vaccines that caused powerful immunological reactions. Phages alone or in combination with IFA did not significantly alter the immune response, demonstrating that the phages themselves are antigenic and trigger an immunological response [ 124 ]. Soc and Hoc have been employed to express antigens on the surface of T4 since they are not required for phage infectivity [ 125 ]. Via Hoc-capsid interactions, HIV antigens, p24-gag, Nef, and a modified C-peptide trimer of gp41 are presented on the T4 phage capsid surface. The human immunodeficiency virus (HIV) genes were attached to the 5′ or 3′ end of the Hoc gene to achieve in-frame integration. All of the accessible capsid binding sites were effectively saturated by the efficient presentation of single or several antigens. In the absence of any external adjuvant, p24 exhibited in mice was very immunogenic and produced potent p24-specific antibodies as well as Th1 and Th2 cell responses, with a preference for Th2 responses ( Table 4 ) [ 114 ]. Li and colleagues discovered that influenza virus 3M2e proteins exhibited on T4 nanoparticles create extraordinarily high levels of 3M2e-specific IgG antibodies in the absence of any adjuvant, while 3M2e coupled to RB69 Soc only causes low amounts of 3M2e-specific IgG antibodies [ 126 ]. Among other viruses that have been used for this purpose are Herpes simplex virus 1 (HSV-1) ( Table 4 ) [ 116 ], Circovirus 2 (PCV2) ( Table 4 ) [ 115 ], FMDV ( Table 4 ) [ 113 ], Zika virus , and RSV ( Table 4 ) [ 117 ], which have been displayed on various phages and elicited a strong antibody response in mice when compared to non-immunized mice. Recent studies have also used phage vaccines to prevent coronavirus species, for instance, by displaying the S protein's epitope 4 of SARS-CoV2 on protein VIII of the Filamentous phage display vector f88-4, while the capsid protein pIII included the peptide CAKSMGDIVC. The transport peptide CAKSMGDIVC can identify lung epithelial cells in the lung. Recombinant phages were successfully applied to activate an antigen-specific humoral response against SARS-CoV 2 , thus representing an appropriate candidate for vaccine development against COVID-19 ( Table 4 ) [ 108 ]. Furthermore, vaccines using antigens other than the S protein from SARS-CoV-2 have been developed. While the nucleocapsid protein (NP) was inside the phage, the ectodomain trimers of the S protein were produced on the T4 phage to develop T4-CoV-2. A SARS-CoV-2 vaccine administered intravenously boosted the immune response on mucosal surfaces in comparison. The intramuscular injection, however, did not show this. Moreover, this was noticed in transgenic mice for the human angiotensin-converting enzyme (hACE2). These mice were resistant to the original SARS-CoV-2 and its delta form up to a lethal dose. The vaccine did not harm the microbiome and was stable at room temperature [ 61 ]. Zhu et al. created a platform to develop various SARS-CoV-2 vaccine candidates utilizing CRISPR editing of the T4 phage. Several phage segments were constructed using various SARS-CoV-2 antigens (such as the S, E, and NP proteins). Mice were shielded from SARS-CoV-2 infection by T4 adorned with S-trimers, which generate particular antibodies and prevent the binding of RBD to ACE-2 [ 127 ]. The safety of ABNCoV2, a vaccine based on VLPs of bacteriophage AP205 adorned with RBD of SARS-CoV-2 generated in S2 Drosophila cells, was assessed in healthy volunteers during a clinical phase I/II experiment (NCT04839146) [ 128 ]. Clinical trials.gov has not yet published the study's results, although it ended on February 25, 2022. ABNCoV2 is also the subject of an open-label phase 2 trial in Germany (NCT05077267; EUCTR2021-001393-31). Adults who had previously received the SARS-CoV-2 vaccine underwent a phase three trial (NCT05329220) to assess the immunogenicity, safety, and tolerability of ABNCoV2 [ 128 ]. 10.3 Anti-bacterial phage-based vaccines One of the most critical issues in bacterial infections is antibiotic resistance due to excessive use of antibiotics. The phage-based vaccine is introduced as a reliable approach to overcoming bacterial antibiotic resistance. The phage-based vaccines have been developed against Bacillus anthracis and Yersinia pestis's antigens, the causative agents of anthrax and plaque, respectively. This phage-based vaccine elicited strong anti-anthrax and plague-specific immune responses, especially T-helper 1 and 2 cellular immune responses, which are essential for eradicating bacterial infection ( Table 4 ). Administration of phage-based vaccines in animal models (mice, rats, and rabbits) infected with lethal doses of both anthrax lethal toxin and Y. pestis CO92 bacteria revealed complete protection. Therefore, phages like T4 could be applied with nanoparticles to formulate multivalent vaccines against high-risk pathogens and emerging infections [ 109 , 118 ]. Chlamydia trachomatis , a bacterium found in the mouth, genital tract, cervix, and urethra of adults, was studied as a candidate for a phage vaccine. The Q-CT584 vaccine was developed by chemically attaching epitopes 70–77 and 154–164 of CT584 to bacteriophage Q VLPs. CT584, a protein that binds to the type three secretion system (T3SS) involved in invasion, intracellular survival, and cell egress, was selected. Mice immunized intramuscularly with Q-CT584 and then challenged with C. trachomatis produced large amounts of IgG that prevented infection [ 129 ]. Another example of a phage vaccine against bacteria is cholera disease. Cholera is an acute, secretory diarrheal disease caused by the highly pathogenic bacterium Vibrio cholerae . Only the serogroups O1 and O139 may result in epidemic cholera out of the more than 200 serogroups. By chemically joining the antigen O-specific polysaccharide (OSP) isolated from V. cholerae O1 El Tor Inaba to VLP produced from phage Q, a vaccine (Q-OSP) against V. cholera was developed. When mice were immunized with the Q-OSP formula, high and enduring levels of IgG antibodies were produced against OSP. These antibodies recognized the natural LPS from V. cholerae O1 El Tor Inaba. Moreover, the live bacteria could be killed by antibodies in sera from mice that had received complement vaccinations [ 130 ]. 10.4 Anti-fungal phage-based vaccines One of the applications of phage is the induction of an immune response against fungal infections. Although there is still no approved antifungal phage-based vaccine for humans, there have been several successful studies regarding anti-fungal bacteriophage vaccines. Candida albicans ( Table 4 ) [ 120 ] and Sporothrix globose ( Table 4 ) [ 119 ] are the fungal agents that the phage vaccines have developed against. Immunization with recombinant phage increased the survival rate of mice following each fungal infection, separately. 10.5 Anti-cancer phage-based vaccines Cancer vaccines, regarded as promising tools for cancer treatment, have recently received increased attention. The expression of antigens fused to phage surface proteins in the phage display system is intended to induce tumor-specific T-cell responses [ 93 ]. Cross_presentation is a strategy by which phages can induce antigen presentation mediated by MHC-I and MHC-II molecules. The cross-presentation is also extremely useful in developing cancer immunotherapies, as cytotoxic T lymphocyte (CTL) activation by MHC-I recognition is critical for tumor cell death [ 131 , 132 ]. In recent years, many tumor-associated antigens that CTLs recognize have been identified and characterized. Cancer/testis (C/T) antigens are the fastest-growing group of tumor-associated antigens, expressed in various types of tumors but not in normal tissues [ 133 , 134 ]. As a result of their tumor-specificity, C/T antigens are known as appropriate candidates for cancer vaccines [ 135 ]. Sartorius and colleagues used the filamentous fd phage to co-express T helper (Th) cell-specific epitopes and C/T antigens to elicit Th-dependent CTL responses. The recombinant phage could elicit potent, specific CTL responses in vitro and in vivo. The filamentous phages enhance the immunogenicity of tumor-associated antigens while simultaneously slowing tumor growth [ 136 ]. In a similar way, another study used recombinant M13 phage as a vaccine for Lewis lung carcinoma. The pVIII coat protein showed an epidermal growth factor receptor (EGFR) mimotope. This study demonstrated that recombinant phage vaccines could significantly induce immune responses and elicit specific antibodies against cancer cells [ 18 ]. Non-filamentous phages such as T4 and T7 have also been used as vaccines to improve cancer therapy after exposure to appropriate peptides. For instance, T4 phage was used to express vascular endothelial growth factor (VEGF), and subsequently, the engineered age was applied as a vaccine against Lewis lung carcinoma in a mouse model. The T4-mVEGFR2 phage could effectively suppress angiogenesis and have significant anti-tumor activity. Furthermore, the T7 phage displays the five fragments of the EGFR mimotope, which were fused with the T7 phage's 10B coat protein via genetic engineering. The results showed that the EGFR was successfully expressed on the surface of the T7 phage, and thus the recombinant phage could effectively inhibit tumor growth in the BALB/c mouse model [ 32 ]. The discovery of tumor-specific antigens opens the way for the successful development of phage-displayed vaccines for cancer prevention. Shadidi et al. for example, used a proteomics-based method to identify breast cancer tumor antigens and created vaccines by displaying the identified tumor antigens on the T7 phage and measuring the immune responses in mice after oral administration. Their findings showed that the phage's surface display of tumor antigens could effectively elicit immune responses, thus introducing the engineered phage as a promising mucosal cancer vaccine [ 137 ]. n the fd phage display system, a tumor-specific antigen epitope called melanoma antigen A1161169 was joined to the pVIII coat protein of the fd phage. An in vivo tumor protection assay confirmed that the hybrid phage effectively inhibited tumor growth. These studies clearly showed the potency of phages for engineering an effective vaccine and improving cancer therapy [ 70 ]. Recently, an immunogenic bacteriophage-based vaccine with HER2/neu overexpression stimulated CTL activity. This study revealed the phage nanoparticles containing GP2 as a fused peptide to the gpD phage capsid protein, elicited a strong CTL response, and protected mice against HER2/neu-positive tumors [ 138 ]. The Δ16HER2 is a HER2 splice variant and the transforming isoform in HER2-positive breast cancer. Δ16HER2 has been shown to promote breast cancer aggressiveness and is a leading factor in drug resistance. However, due to tolerogenic mechanisms against the human HER2 self-antigen, a situation frequently observed in HER2+ patients, the vaccines failed to elicit immunological protection in Δ16HER2 transgenic mice. An engineered bacteriophage with immunogenic Δ16HER2 epitopes was used as an anticancer vaccine in a recent study. Through the disruption of immunological tolerance, these phage-based vaccinations were able to elicit a protective anti- Δ16HER2 humoral response. Altogether, these findings support the practice of phage-based anti-HER2/Δ16HER2 vaccination as a safe and effective immunotherapy strategy for HER2-positive breast cancers [ 139 ]. Unexpectedly, many promising preclinical investigations that could have led to clinical trials have not been repeated, and we are unsure of the reason for this. Hence, more work is needed to advance this technology, which has a lot of benefits and great promise, to the next stage of clinical trials, where it can be shown effective for usage in both humans and animals. The use of phage-based vaccinations is advantageous because several clinical trials have demonstrated that phages are both safe for humans and animals. The encouraging findings of Roehnisch in 2014 serve as a solid springboard for expanding the use of phage-based vaccines in clinical trials, at least for those based on M13 that have demonstrated safety [ 140 ]. This viewpoint is positive since it suggests that phages may eventually be used in vaccinations, which is possible based on the information presented above [ 62 ]. 10.6 Other types of diseases or conditions Another application of phage-based vaccines is in population control using the immune contraceptive vaccine, which induces an adaptive immune response against the reproductive system and causes temporary infertility. For instance, the gonadotrophin-releasing hormone (GnRH) antigen was displayed on the surface of T7 phages in a mouse model leading to immune castration [ 121 ]. Vaccine targeting could also be applied to drug addiction by using bacteriophages that display antibodies that block the effects of various drugs, such as cocaine [ 122 ]. A group of neurodegenerative illnesses known as tauopathies includes frontotemporal dementia (FTD) and Alzheimer's disease (AD). Tauopathies are brought on by neurofibrillary tangles (NFTs), which are formed in neurons by hyperphosphorylated pathogenic tau (pTau). Tau may therefore be a useful target for vaccination to prevent these illnesses. The development of pT181-Q, a phage-based vaccine against tauopathies, involved conjugating a tau peptide to bacteriophage Q's VLPs. Animals given the pT181-Q vaccine produced a strong IgG reaction against pT181. In mice immunized with pT181-Q, less pTau accumulated in the brain and hippocampus. The amount of circulating CD3 + T-cells and neuroinflammation in the brain both decreased [ 141 ]. Another application of phage vaccines is in heart disease. A higher risk of cardiovascular disease is linked to low-density lipoprotein cholesterol, which contains three checkpoint proteins (PCSK9, ApoB, and CETP). The three proteins were displayed on the bacteriophage Q coat protein VLPs to develop a phage-based vaccination. Immunized mice produced IgG1 and IgG2b immunoglobulin isotypes against PCSK9, ApoB, and CETP. The immunizations also lower the levels of these proteins, which lowers the plasma's overall cholesterol level [ 142 ]. 11 Conclusion and future perspective Phage vaccines may provide the key to developing innovative methods for battling cancer and other bacterial and viral infections. The global abundance of phages and our increased understanding of how to exploit them will undoubtedly lead to the emergence of new phage display systems. This overview discusses the most recent developments in the field of phage-based vaccines and their capacity to be used as a preventative and therapeutic platform for a variety of diseases. It also covers the potential directions for advancing this field. Although there have been some breakthroughs with phage-based vaccinations, limitations still need to be resolved to improve this field. Without a doubt, phage display cannot overcome all hurdles that stand in the way of the development of vaccines and is unable to address all issues that may arise in the way of vaccine design and production. Different phage-based vaccine platforms have a promising future, and it can be claimed that phage display's importance will only increase over the next few years. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2042517/

The Allometry of Host-Pathogen Interactions

Background Understanding the mechanisms that control rates of disease progression in humans and other species is an important area of research relevant to epidemiology and to translating studies in small laboratory animals to humans. Body size and metabolic rate influence a great number of biological rates and times. We hypothesize that body size and metabolic rate affect rates of pathogenesis, specifically the times between infection and first symptoms or death. Methods and Principal Findings We conducted a literature search to find estimates of the time from infection to first symptoms ( t S ) and to death ( t D ) for five pathogens infecting a variety of bird and mammal hosts. A broad sampling of diseases (1 bacterial, 1 prion, 3 viruses) indicates that pathogenesis is controlled by the scaling of host metabolism. We find that the time for symptoms to appear is a constant fraction of time to death in all but one disease. Our findings also predict that many population-level attributes of disease dynamics are likely to be expressed as dimensionless quantities that are independent of host body size. Conclusions and Significance Our results show that much variability in host pathogenesis can be described by simple power functions consistent with the scaling of host metabolic rate. Assessing how disease progression is controlled by geometric relationships will be important for future research. To our knowledge this is the first study to report the allometric scaling of host/pathogen interactions. Background Understanding the mechanisms that control rates of disease progression in humans and other species is an important area of research relevant to epidemiology and to translating studies in small laboratory animals to humans. Body size and metabolic rate influence a great number of biological rates and times. We hypothesize that body size and metabolic rate affect rates of pathogenesis, specifically the times between infection and first symptoms or death. Methods and Principal Findings We conducted a literature search to find estimates of the time from infection to first symptoms ( t S ) and to death ( t D ) for five pathogens infecting a variety of bird and mammal hosts. A broad sampling of diseases (1 bacterial, 1 prion, 3 viruses) indicates that pathogenesis is controlled by the scaling of host metabolism. We find that the time for symptoms to appear is a constant fraction of time to death in all but one disease. Our findings also predict that many population-level attributes of disease dynamics are likely to be expressed as dimensionless quantities that are independent of host body size. Conclusions and Significance Our results show that much variability in host pathogenesis can be described by simple power functions consistent with the scaling of host metabolic rate. Assessing how disease progression is controlled by geometric relationships will be important for future research. To our knowledge this is the first study to report the allometric scaling of host/pathogen interactions. Introduction Most emerging infectious diseases that cause human epidemics (e.g. HIV, Influenza, West Nile Virus, Ebola) evolved in other animal hosts [1] , [2] . However, little theory exists that enables the translation of our knowledge about pathogenesis, rates of evolution, vaccination strategies or epidemiology in these zoonotic diseases to their behavior in human hosts. One important observation is that the rate pathogens spread through populations appears to be influenced by the rate of spread through individual hosts [3] , [4] . Therefore, understanding the time-course of pathogenesis in an individual, including the length of the latency and infection periods, could aid in parameterization of epidemiological models. A comparative approach to studying disease progression in different animal hosts may also elucidate how diseases affect human health. Pathogenesis is a complex phenomenon that results from several aspects of host-disease interactions [5] . There are four main determinants of pathogenesis: ( i ) the interaction of the disease with the target tissue, ( ii ) the ability of the infection to cause cell death or cytopathology, ( iii ) the host immune response to infection, and ( iv ) immunopathology (e.g, T-cell and antibody responses). Although we know much about the physiological mechanisms for each of these host-disease interactions, there is still no easy answer for how pathogen infection 'causes' disease in a host [5] . Here we take a scaling approach to understand variation in the pace of pathogenesis. Specifically, what controls the scaling of pathogenesis times within and across diseases? Here we focus on the role of host body size and metabolic rate in influencing the scaling of pathogenesis. There is a rich literature documenting how the body size of an animal influences its structure, function, and life history [6] – [11] . The overwhelming importance of body size has been eloquently summarized by George Bartholomew who stated, "It is only a slight overstatement to say that the most important attribute of an animal, both physiological and ecologically, is its size. Size constrains virtually every aspect of structure and function and strongly influences the nature of most inter- and intraspecific interactions. Body mass is the most widely used predictor of physiological rates." [12] . However, little is known about the influence of host body size on pathogenesis in the context of the scaling of host-pathogen interactions. As we outline below, it is reasonable to expect that host body size and metabolic rate must constrain rates of pathogenesis. The Metabolic Scaling Theory (MST) for pathogenesis Our hypothesis that host body size and ultimately host metabolic rate constrains rates of pathogenesis is based on recent theoretical developments for the scaling of biological rates and times [8] , [10] , [11] . Metabolic Scaling Theory (MST) predicts that physiological times and cellular metabolic rates are ultimately controlled by the scaling of the geometry of fractal-like vascular networks. This work secondarily hypothesizes that the scaling of physiological rates and times are governed by quarter-power scaling exponents. Specifically, quarter-power exponents in biology are the result of natural selection on vascular networks to minimize the scaling of internal transport times while maximizing the scaling of resource exchange surfaces (lung surface area, gut surface area etc). This work predicts a fractal-like vascular network design that, when scaled with the size of the organism in which it is contained, will lead to rates and times scaling as the 1/4 power of organism size. If rates of disease progression are ultimately constrained by network geometry and host metabolism, then pathogenesis will vary, or scale, with host body mass raised to a 1/4 power. A number of studies have pointed to the fundamental importance of metabolism, or body size, in controlling the rates and timings of biological phenomena [9] – [11] , [13] . This work has its roots in fundamental work by Kleiber [7] . Kleiber showed that whole organism metabolic rate, B , scaled allometrically (with an exponent less than 1) so that B = B 0 ⋠M 3/4 . Many subsequent studies supported this finding in a variety of taxa [6] , [9] , [14] , [15] , although others have questioned the generality of the 3/4 exponent [16] , [17] . If B ∝ M 3/4 then the cellular or mass-specific metabolic rate, [ [9 and refs therein,] [14] ]. The theoretically predicted and empirically observed M −1/4 decease in metabolism appears regulated by a similar M −1/4 decrease in the amount of metabolic machinery. For example, the membrane surface area and number of mitochondria, concentration of ATP, number of cytochrome oxidase molecules, etc, all decrease with increasing host body size to the approximate −1/4 power [13] , [18] – [20] . Since all other cellular rates are constrained by the metabolic rate of the cell, then the theory suggests that the rate of DNA synthesis, protein synthesis, immune response, and cellular turnover should also scale with M −1/4 , and biological times, T , should scale as the inverse of those rates, or as M 1/4 [9] . Cellular rates are relevant to pathogenesis because they control the rates at which pathogens enter a host, replicate, spread through the host body and cause disease. Thus, according to the MST the pace of host-pathogen interactions (e.g. pathogenesis) is set by rate of host metabolism. The host metabolic rate influences pathogenesis by ( i ) constraining the rate of growth of pathogens that rely on host metabolic machinery (in much the same way as it limits the rate of growth of host [21] ) as well as ( ii ) influencing the rate of the immune response of the host. In fact, cellular-mediated immunity appears to scale with body size and associated life history traits [22] . Thus, host metabolic rate influences the rate of pathogenesis since the ability of a pathogen to invade and replicate within a host may be driven by physiological rates and times of the host. Specifically, the times associated with pathogenesis are related to M by t = c ⋠M b where c is a constant particular to the time of interest, and b = 1/4. Since mass specific metabolism ( B/M) , scales as M −1/4 , then we expect rates of pathogenesis to scale with M −1/4 and times associated with pathogenesis to scale with M 1/4 . We assess these functional predictions with empirical data compiled from the literature. As far as we know, this is the first study to examine how pathogenesis varies as a function of host body size. For a sampling of pathogens (one bacteria, three viruses, and one prion), we show how variation in host size, M , influences variation in the timing of pathogenesis. We focus on the time from inoculation to first symptoms ( t S ) and to death ( t D ). The MST predicts that both t S and t D will scale as the 1/4 power of mammalian body size, giving: (1) and (2) The terms, c 1 and c 2 , in Eqs. 1 and 2 are scaling constants. The simplest model would have the values of c 1 and c 2 independent of M . Nevertheless, differences in their values reflect important interactions between host and pathogen and may be different for the pathogenesis of different diseases. We can combine Eqs. 1 and 2 to predict the relationship between t D and t S : (3) The values of c 1 and c 2 likely reflect the timing of the host immune response and additional physiological responses to infection. These values may also be influenced by host body temperature, taxonomic group or other factors that alter host metabolism [10] . As c 2 > c 1 , is the ratio between time to death and time to symptoms. Variability in this quotient between diseases would indicate proportional differences in the timing of between time until first symptom and time until death between diseases. However, similarity in this quotient between diseases would indicate generality in the proportional rates between diseases. Since our study is limited to homeotherms in similar taxonomic groups (mammals, and in the case of West Nile Virus, birds), within a particular pathogen, we expect c 1 and c 2 to be constant. If correct, then Eqs. 2–3 predict the timing of pathogenesis should be fundamentally set by host metabolism. However, for a given disease, the paces of various pathological events are predicted to be directly proportional, or isometric, to one another, so that t S ∝ t D . Further, the ratio of pathogenesis times should be independent of body mass, so that . Alternative Hypotheses We use MST to predict that t S and t D scale with M 1/4 . We contrast these predictions with the null hypothesis that t S and t D are independent of host body size ( M ). However, it is important to note alternative hypotheses that relate t S and t D to M . For example, symptoms or death may occur when some fraction of the number of cells in the organism has been infected. Since the number of cells is isometric with body mass [23] , then t S and t D would be predicted to be linear with M . Alternatively the relationship between host mass and the timing of pathogenesis could be a geometric relationship controlled by body length or internal transport distances (i.e., in rabies or PRV, the distance the pathogen must travel from the site of infection to the brain). In simple Euclidean geometry, body length scales with body mass to the 1/3 power [e.g., 24]. The fractal network geometry of MST predicts that internal transport lengths show the same scaling as biological times; both scale with M 1/4 . Here we specifically test the MST 1/4 power predictions, but we note which of the alternative predictions ( M 1 , and M 1/3 ) are consistent with the data. The Metabolic Scaling Theory (MST) for pathogenesis Our hypothesis that host body size and ultimately host metabolic rate constrains rates of pathogenesis is based on recent theoretical developments for the scaling of biological rates and times [8] , [10] , [11] . Metabolic Scaling Theory (MST) predicts that physiological times and cellular metabolic rates are ultimately controlled by the scaling of the geometry of fractal-like vascular networks. This work secondarily hypothesizes that the scaling of physiological rates and times are governed by quarter-power scaling exponents. Specifically, quarter-power exponents in biology are the result of natural selection on vascular networks to minimize the scaling of internal transport times while maximizing the scaling of resource exchange surfaces (lung surface area, gut surface area etc). This work predicts a fractal-like vascular network design that, when scaled with the size of the organism in which it is contained, will lead to rates and times scaling as the 1/4 power of organism size. If rates of disease progression are ultimately constrained by network geometry and host metabolism, then pathogenesis will vary, or scale, with host body mass raised to a 1/4 power. A number of studies have pointed to the fundamental importance of metabolism, or body size, in controlling the rates and timings of biological phenomena [9] – [11] , [13] . This work has its roots in fundamental work by Kleiber [7] . Kleiber showed that whole organism metabolic rate, B , scaled allometrically (with an exponent less than 1) so that B = B 0 ⋠M 3/4 . Many subsequent studies supported this finding in a variety of taxa [6] , [9] , [14] , [15] , although others have questioned the generality of the 3/4 exponent [16] , [17] . If B ∝ M 3/4 then the cellular or mass-specific metabolic rate, [ [9 and refs therein,] [14] ]. The theoretically predicted and empirically observed M −1/4 decease in metabolism appears regulated by a similar M −1/4 decrease in the amount of metabolic machinery. For example, the membrane surface area and number of mitochondria, concentration of ATP, number of cytochrome oxidase molecules, etc, all decrease with increasing host body size to the approximate −1/4 power [13] , [18] – [20] . Since all other cellular rates are constrained by the metabolic rate of the cell, then the theory suggests that the rate of DNA synthesis, protein synthesis, immune response, and cellular turnover should also scale with M −1/4 , and biological times, T , should scale as the inverse of those rates, or as M 1/4 [9] . Cellular rates are relevant to pathogenesis because they control the rates at which pathogens enter a host, replicate, spread through the host body and cause disease. Thus, according to the MST the pace of host-pathogen interactions (e.g. pathogenesis) is set by rate of host metabolism. The host metabolic rate influences pathogenesis by ( i ) constraining the rate of growth of pathogens that rely on host metabolic machinery (in much the same way as it limits the rate of growth of host [21] ) as well as ( ii ) influencing the rate of the immune response of the host. In fact, cellular-mediated immunity appears to scale with body size and associated life history traits [22] . Thus, host metabolic rate influences the rate of pathogenesis since the ability of a pathogen to invade and replicate within a host may be driven by physiological rates and times of the host. Specifically, the times associated with pathogenesis are related to M by t = c ⋠M b where c is a constant particular to the time of interest, and b = 1/4. Since mass specific metabolism ( B/M) , scales as M −1/4 , then we expect rates of pathogenesis to scale with M −1/4 and times associated with pathogenesis to scale with M 1/4 . We assess these functional predictions with empirical data compiled from the literature. As far as we know, this is the first study to examine how pathogenesis varies as a function of host body size. For a sampling of pathogens (one bacteria, three viruses, and one prion), we show how variation in host size, M , influences variation in the timing of pathogenesis. We focus on the time from inoculation to first symptoms ( t S ) and to death ( t D ). The MST predicts that both t S and t D will scale as the 1/4 power of mammalian body size, giving: (1) and (2) The terms, c 1 and c 2 , in Eqs. 1 and 2 are scaling constants. The simplest model would have the values of c 1 and c 2 independent of M . Nevertheless, differences in their values reflect important interactions between host and pathogen and may be different for the pathogenesis of different diseases. We can combine Eqs. 1 and 2 to predict the relationship between t D and t S : (3) The values of c 1 and c 2 likely reflect the timing of the host immune response and additional physiological responses to infection. These values may also be influenced by host body temperature, taxonomic group or other factors that alter host metabolism [10] . As c 2 > c 1 , is the ratio between time to death and time to symptoms. Variability in this quotient between diseases would indicate proportional differences in the timing of between time until first symptom and time until death between diseases. However, similarity in this quotient between diseases would indicate generality in the proportional rates between diseases. Since our study is limited to homeotherms in similar taxonomic groups (mammals, and in the case of West Nile Virus, birds), within a particular pathogen, we expect c 1 and c 2 to be constant. If correct, then Eqs. 2–3 predict the timing of pathogenesis should be fundamentally set by host metabolism. However, for a given disease, the paces of various pathological events are predicted to be directly proportional, or isometric, to one another, so that t S ∝ t D . Further, the ratio of pathogenesis times should be independent of body mass, so that . Alternative Hypotheses We use MST to predict that t S and t D scale with M 1/4 . We contrast these predictions with the null hypothesis that t S and t D are independent of host body size ( M ). However, it is important to note alternative hypotheses that relate t S and t D to M . For example, symptoms or death may occur when some fraction of the number of cells in the organism has been infected. Since the number of cells is isometric with body mass [23] , then t S and t D would be predicted to be linear with M . Alternatively the relationship between host mass and the timing of pathogenesis could be a geometric relationship controlled by body length or internal transport distances (i.e., in rabies or PRV, the distance the pathogen must travel from the site of infection to the brain). In simple Euclidean geometry, body length scales with body mass to the 1/3 power [e.g., 24]. The fractal network geometry of MST predicts that internal transport lengths show the same scaling as biological times; both scale with M 1/4 . Here we specifically test the MST 1/4 power predictions, but we note which of the alternative predictions ( M 1 , and M 1/3 ) are consistent with the data. Results We assessed the MST hypothesis by assembling data on the scaling of the timings of pathogenesis for five diseases. We collected data on t S and t D and M for a variety of pathogens infecting mammalian and bird species. Each of these diseases infect mammalian hosts that range in body size, M , by several orders of magnitude (e.g., Table 1 ). For the most part, empirical data support predictions made by the MST. In all five pathogens, there is a significant positive correlation between the timing of pathogenesis and M ( Figure 1 ). For PRV, the scaling slope was positive, but was significantly lower than the predicted value of 1/4. For the remaining diseases, all slopes overlapped the predicted value of 1/4 ( Table 1 ). The relationship between t S and t D had a slope close to 1 ( Figure 2 ). Our results were not consistent with the alternative hypothesis that the timing of pathogenesis and M are isometric (slope of 1). However, the following diseases were consistent with the geometric hypothesis that pathogenesis times scale with mass to the 1/3: anthrax ( t S , t D ), rabies ( t S , t D ), TSE ( t S ) ( Table 1 ). 10.1371/journal.pone.0001130.g001 Figure 1 Time (days) from inoculation to (a) death and (b) 1 st symptom versus mammalian body mass for Pseudorabies virus (PRV), Anthrax, Rabies, West Nile Virus (WNV), and Transmissible Spongiform Encephalopathy (TSE). 10.1371/journal.pone.0001130.g002 Figure 2 Time (days) from inoculation to death ( t D ) versus time from inoculation to 1 st symptom ( t S ) for Pseudorabies Virus (PRV), Anthrax, and Rabies for a large range of mammalian body sizes, plotted with the 1∶1 line. 10.1371/journal.pone.0001130.t001 Table 1 Slope and intercept (2.5%, 97.5% values), R 2 , p values, and body mass range (kg) for t S (time from inoculation to 1st symptom), t D (time from inoculation to death), and t S vs. t D for each disease * . Slope Intercept R 2 p Mass Range t S Anthrax 0.18 (0.11, 0.32) ab 0.17 (0, 0.33) c 0.90 0.013 0.55–442 PRV 0.12 (0.07, 0.21) a 0.43 (0.30, 0.55) c 0.33 0.051 0.022–442 Rabies 0.33 (0.21, 0.54) b 0.93 (0.57, 1.29) 0.28 0.037 0.022–4545 TSE 0.22 (0.15, 0.32) ab 2.35 (2.20, 2.52) 0.76 0.001 0.015–500 t D Anthrax 0.22 (0.12, 0.39) a 0.29 (0.08, 0.51) c 0.62 0.021 0.022–442 PRV 0.11(0.08, 0.16) b 0.59 (0.53, 0.66) d 0.69 <0.001 0.022–450 Rabies 0.26 (0.15, 0.44) a 1.2 (0.84, 1.5) e 0.35 0.041 0.022–4545 WNV 0.17 (0.10, 0.28) ab 0.95 (0.84, 1.1) e 0.51 0.014 0.02–200 t S vs t D Anthrax 1.17 (0.65, 2.12) a 0.05 (−0.26, 0.37) b 0.88 0.018 0.55–442 PRV 0.83 (0.46, 1.48) a 0.23 (−0.06, 0.53) b 0.34 0.058 0.022–442 Rabies 0.82 (0.63, 1.06) a 0.34 (−0.01, 0.68) b 0.86 <0.001 0.022–4545 * PRV: Pseudorabies Virus, TSE: Transmissible Spongiform Encephalopathy, WNV: West Nile Virus Significant p values (<0.05) denote slopes that differ from 0. Bolded slope values do not differ from 0.25. Slope and intercept values that differ among diseases (but within each time category) have different super-scripted letters. The intercept value for t S is c 1 and for t D is c 2 (Eqns 1, 2). There was variation in the scaling constants for each disease ( Table 1 ), as exemplified in Figure 1 where the scaling slope was very similar but the intercepts (which gives c 1 ) differed. The values of c 1 and c 2 ranged from 0.64 to 4.4. For example, for PRV, the time until death, t D , was 2.8 days for a 21 g mammal, whereas in Rabies the same size mammal was characterized by t D of 8.6 days. However, there was much less variation in the ratio of as shown in Figure 3 , where 86% of the values fell between 0.8–1.8 ( Figure 3 ). 10.1371/journal.pone.0001130.g003 Figure 3 The frequency of values for Pseudorabies Virus, Rabies, and Anthrax across a large range of mammalian body sizes. Discussion Our results are generally consistent with the MST, where the timing of pathogenesis is controlled by host cellular metabolic rate. That is, the progression of disease to symptoms and to death slows as a function of M 1/4 . Variation in t S and t D for each disease appears to scale with host body size with exponents consistent with the scaling of host metabolism. Observed relationships all scale with exponents very close and often indistinguishable from the predicted value of 1/4 ( Figure 1 ). As indexed by the fitted allometric intercepts, each disease differs in the relative timing of t S and t D (i.e. host-pathogen interactions differ in their value of c 2 and possibly c 1 ). A plot of t S vs. t D across the diverse diseases studied reveals that the timing of pathogenesis for each disease, remarkably, falls on the same function that is approximately isometric (slope of 1) ( Figure 2 ). Such invariance indicates that the allometric value of the ratio (see Eq. 3) is the same invariant quantity for each of the diseases studied here. We also provide a histogram of to show this ratio typically has a mean value of 1.6 (standard deviation 0.80) ( Figure 3 ) and does not change systematically with M . This implies a relationship, general among these diseases, whereby the time to the first sign of infection is a constant proportion of the time to death–a constant that is conserved across each of the diseases studied here. The histogram of shows a long tail ( Figure 3 ); perhaps these outliers are influenced by host immune response, medial care in humans, or specific host-pathogen interactions. Further investigation of pathogenesis in these mammals (cat, human, camelid, and elephant) may shed more light on mechanisms of allometric pathogenesis. It would also be interesting to understand how variation in evolutionary forces on these organisms affects host-pathogen interactions. The scaling for PRV appears to not follow the predicted pattern of timing of pathogenesis as strongly as the other four diseases. PRV has a positive trend in the scaling relationship with significant slopes but they are more-shallow than predicted. It is unclear why PRV differs from the other diseases. Nevertheless, our model provides a baseline to begin to explore why PRV may deviate from the exact predictions of the MST. Explaining the causes of variation around the regression lines in Figures 1 is a natural, and we believe, fruitful next step to this analysis. Our results also indicate that disease allometry across diverse populations may be characterized by invariant dimensionless quantities. Because mammalian life-span and population doubling time scale as t LS = c 3 ⋠M 1/4 and t P = c 4 ⋠M 1/4 , respectively [9] , where c 3 and c 4 are allometric constants with units of time, and if t D = c 2 ⋠M 1/4 , then the values for both and are equal to: (4) (5) Note, both X 1 and X 2 are dimensionless ratios invariant of mammalian body size. Thus, remarkably, across all mammals the fraction of adult lifespan or population cycle influenced by a given disease is an approximately constant value independent of mammalian body size. We have shown that the scaling of times associated with pathogenesis is consistent with the scaling of host metabolic rate, supporting the MST. We have suggested that such scaling could result if pathogen growth and replication are directly limited by the cellular metabolic rates of the hosts. We are not aware of any other model(s) that would lead to functional relationships of t S and t D that are power-functions of body mass with exponents near 1/4. However, it is possible that the observed scaling could be an indirect result of metabolic rate. For example, host immune and other physiological responses to pathogens may cause the observed scaling, rather than the rate at which pathogens replicate, or the scaling may represent some combination of factors. It is also possible that pathogens may evolve latency periods in order to maximize their fitness given the population dynamics of the host. Evidence of this is seen in the evolution of t S in TSE. When laboratory mice are infected with TSE from larger animals (sheep or cows), t S is initially several times longer than after the infection has persisted in mouse populations for several generations. This effect is known as the 'species barrier' (Gardash'yan 1976, Nonno and Trevitt 2006; in supplementary material). Thus, when TSE is transmitted to a new, smaller, species, it evolves a faster t S after just a few generations. We would like to note that an extensive survey of the veterinary and disease literature (see Supplementary Information) revealed only five diseases that allowed for sufficient body size variation and with enough reported values of pathogenesis times, and only three of those gave both time to symptoms and time to death. In future pathogenesis studies, we urge researchers to carefully report associated pathogenesis times as this will greatly increase the range of studies available for disease allometry, and greatly improve the ability to discriminate between MST and other hypotheses, such as the geometric hypothesis (scaling exponent of 1/3). While data were available for a large range of mammal body sizes (see Table 1 ), data were unavailable for animals at either extreme of the spectrum of body masses, such as shrews and whales. MST makes theoretical predictions for these animals. For example, in whales, experimental infection with disease would be very difficult. Our model, however, indicates that we would expect pathogenesis times for a blue whale to be about 1.5 orders of magnitude longer than for a 1 kg mammal. Our results suggest that a comparative approach to pathogenesis is valuable, and that MST gives novel theoretical predictions for understanding the pace and progression of disease. While there is variation in the scaling relationships we show, there are clearly systematic and allometric (slopes less than 1) relationships between times of pathogenesis and body size. Our initial survey indicates that the observed scaling exponents are consistent with the scaling of host metabolic rate (MST). These results support the notion that the scaling of metabolism fundamentally constrains rates of pathogenesis. Furthermore, our results have important implications for epidemic models that often assume that the timing of and dynamics of pathogenesis is independent of host body size, metabolism, or pathogen transport times [3] . Our findings also suggest that a focus on the fundamental role of how the scaling of host metabolism influences the pace of pathogenesis could contribute to a mechanistic understanding of pathogenesis, and in turn, a foundation for predictive diagnostics, effective vaccination and therapy. Materials and Methods Empirical Data Data were gathered from an extensive literature search, the references for which are supplied as supporting online material ( Text S1 ). The data incorporated four measures of host-pathogen interactions including: t D , the time to death of the host from inoculation with the pathogen (as indexed by either the time when an individual animal died or the time at which 50% of the experimental population died from a lethal dose or LD 50 ); t S , the time to first sign of infection from inoculation; and [ P ] the concentration of pathogen particles injected during the reported study ( Table S1 ). Data stem from in situ experiments. Values of pathogenesis times were reported in the original citations listed in the supplementary information ( Text S1 ). In general, each study reported the observed time of first infection, sign of infection, and death. Studies reported values for a single individual or for a population. When data were assembled from population observations the recorded times were average values. Our literature survey revealed three diseases for which t D , and t S were measured for a sufficient number of mammalian hosts that span a sufficiently wide range of M to test the value of the scaling exponent. Note that both t D and t S were not reported for every animal ( Table S1 ). The number of animals for each disease: Pseudorabies Virus or PRV ( Herpesvirus suis) n = 16; Anthrax bacteria ( Bacillus anthracis) n = 11; Rabies virus (Lyssavirus sp.) n = 21, Transmissible Spongiform Encephalopathy (prion) n = 10, and West Nile Virus (flavivirus) n = 11. Each is extremely lethal in its host and exhibits characteristic symptoms. Mammalian hosts differed in M by approximately 5 orders of magnitude (ranging from mice to horses and bears, Table S1 ). We found data on t D (but not t S ) for West Nile Virus (WNV) and t S (but not t D ) Transmissible Spongiform Encephalopathy (TSE), diseases such as scrapie and mad cow disease that are caused by a prion pathogen. Analysis We tested Eqs. 1 and 2 using reduced major axis (RMA) regression (analysis of covariance, ANCOVA) on log transformed data [SMATR, 25]. Each data point represents t S or t D and M for a particular pathogen in a particular host species. We treated each disease as a separate regression and estimated c 1 and c 2 for each disease. We also tested whether the ratio, , was constant across all pathogens by plotting log c 1 vs log c 2 and testing whether the slope of the RMA regression equals 1, and the group slope of PRV, Anthrax, and Rabies do not differ from 1 ( p = 0.426) [26] . Since such methods do not necessarily indicate how much variation there is in that ratio [27] following Savage et al. (2006) [28] we further plot against M and provide a histogram of the values of ( Figure 3 ). We did not incorporate phylogenetic corrections [29] in this analysis because it is not feasible for the limited number of animal hosts for which we have data. Nor did we attempt to look at the scaling of pathogenesis across growing individuals of the same species, again due to lack of data. If more data become available, we encourage such analysis in future tests of the MST for pathogenesis. Empirical Data Data were gathered from an extensive literature search, the references for which are supplied as supporting online material ( Text S1 ). The data incorporated four measures of host-pathogen interactions including: t D , the time to death of the host from inoculation with the pathogen (as indexed by either the time when an individual animal died or the time at which 50% of the experimental population died from a lethal dose or LD 50 ); t S , the time to first sign of infection from inoculation; and [ P ] the concentration of pathogen particles injected during the reported study ( Table S1 ). Data stem from in situ experiments. Values of pathogenesis times were reported in the original citations listed in the supplementary information ( Text S1 ). In general, each study reported the observed time of first infection, sign of infection, and death. Studies reported values for a single individual or for a population. When data were assembled from population observations the recorded times were average values. Our literature survey revealed three diseases for which t D , and t S were measured for a sufficient number of mammalian hosts that span a sufficiently wide range of M to test the value of the scaling exponent. Note that both t D and t S were not reported for every animal ( Table S1 ). The number of animals for each disease: Pseudorabies Virus or PRV ( Herpesvirus suis) n = 16; Anthrax bacteria ( Bacillus anthracis) n = 11; Rabies virus (Lyssavirus sp.) n = 21, Transmissible Spongiform Encephalopathy (prion) n = 10, and West Nile Virus (flavivirus) n = 11. Each is extremely lethal in its host and exhibits characteristic symptoms. Mammalian hosts differed in M by approximately 5 orders of magnitude (ranging from mice to horses and bears, Table S1 ). We found data on t D (but not t S ) for West Nile Virus (WNV) and t S (but not t D ) Transmissible Spongiform Encephalopathy (TSE), diseases such as scrapie and mad cow disease that are caused by a prion pathogen. Analysis We tested Eqs. 1 and 2 using reduced major axis (RMA) regression (analysis of covariance, ANCOVA) on log transformed data [SMATR, 25]. Each data point represents t S or t D and M for a particular pathogen in a particular host species. We treated each disease as a separate regression and estimated c 1 and c 2 for each disease. We also tested whether the ratio, , was constant across all pathogens by plotting log c 1 vs log c 2 and testing whether the slope of the RMA regression equals 1, and the group slope of PRV, Anthrax, and Rabies do not differ from 1 ( p = 0.426) [26] . Since such methods do not necessarily indicate how much variation there is in that ratio [27] following Savage et al. (2006) [28] we further plot against M and provide a histogram of the values of ( Figure 3 ). We did not incorporate phylogenetic corrections [29] in this analysis because it is not feasible for the limited number of animal hosts for which we have data. Nor did we attempt to look at the scaling of pathogenesis across growing individuals of the same species, again due to lack of data. If more data become available, we encourage such analysis in future tests of the MST for pathogenesis. Supporting Information Table S1 The species, mass (kg), and time data collected from the literature. These data are from the literature listed in the supplementary material Text S1 and used in analyses across the five diseases; tD is the time to death from inoculation and tS is time to first symptom from inoculation (d). The five diseases are as follows: A = Anthrax, P = Pseudorabies Virus, R = Rabies, W = West Nile Virus, T = Transmissible Spongiform Encephalopathy. Where multiple masses are listed, the value used with each disease is noted with the letter of the disease (A, P, R, W, or T). (0.11 MB DOC) Click here for additional data file. Text S1 Literature for disease data. (0.05 MB DOC) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9639593/

A Retrospective Study on the Epidemiology of Anthrax Among Livestock from 2011 to 2020 in Awi Administrative Zone, Amhara Region, Northwest Ethiopia

Background In Ethiopia, anthrax is the second most important zoonotic disease, next to rabies. Data quantifying occurrence and distribution of animal anthrax in Awi administrative zone of Amhara region, Ethiopia, are limited. Thus, this study was conducted to describe the distribution of animal anthrax between 2011 and 2020 in Awi zone. Methods This study used secondary data of animal anthrax that occurred in the Awi zone and reported to the Regional and National Veterinary Authority between 2011 and 2020. Results A total of 1262 cases of anthrax in animals and 324 animals that died due to anthrax were reported. The highest number of anthrax cases were reported in 2012 (n = 671), sharing 48.9% of the 10-year animal anthrax reported. However, the highest number of animal death due to anthrax (n = 104) was reported in 2014. The overall case fatality rate of anthrax was 25.67% (n = 324). The highest animal anthrax cases (n = 984; 77.97%) and deaths (n = 259; 79.94%) were recorded in Bovine. The highest cases of anthrax were registered in May (n = 313), while no anthrax case was reported during December. The highest and lowest number of animal death due to anthrax were reported during July (n = 64) and January (n = 6), respectively. The highest number of anthrax cases was reported in the hot-dry season (n = 479; 37.96%) whereas the lowest was reported during the cold-dry season (n = 30; 2.38%). Conclusion The current study revealed a considerable number of animal anthrax cases and deaths in Awi zone every year. Hence, it is necessary for practicing prevention strategies including immunization programs before the peak season of anthrax outbreaks. Background In Ethiopia, anthrax is the second most important zoonotic disease, next to rabies. Data quantifying occurrence and distribution of animal anthrax in Awi administrative zone of Amhara region, Ethiopia, are limited. Thus, this study was conducted to describe the distribution of animal anthrax between 2011 and 2020 in Awi zone. Methods This study used secondary data of animal anthrax that occurred in the Awi zone and reported to the Regional and National Veterinary Authority between 2011 and 2020. Results A total of 1262 cases of anthrax in animals and 324 animals that died due to anthrax were reported. The highest number of anthrax cases were reported in 2012 (n = 671), sharing 48.9% of the 10-year animal anthrax reported. However, the highest number of animal death due to anthrax (n = 104) was reported in 2014. The overall case fatality rate of anthrax was 25.67% (n = 324). The highest animal anthrax cases (n = 984; 77.97%) and deaths (n = 259; 79.94%) were recorded in Bovine. The highest cases of anthrax were registered in May (n = 313), while no anthrax case was reported during December. The highest and lowest number of animal death due to anthrax were reported during July (n = 64) and January (n = 6), respectively. The highest number of anthrax cases was reported in the hot-dry season (n = 479; 37.96%) whereas the lowest was reported during the cold-dry season (n = 30; 2.38%). Conclusion The current study revealed a considerable number of animal anthrax cases and deaths in Awi zone every year. Hence, it is necessary for practicing prevention strategies including immunization programs before the peak season of anthrax outbreaks. Introduction Anthrax is caused by a gram-positive, endospore-forming bacterium named Bacillus anthracis . The disease affects animals, humans, and wildlife and is zoonotic, very importantly known for its occupational hazard. It has a worldwide occurrence, though the burden varies with regions and countries. 1 Even though anthrax is known to affect multiple species, 2 , 3 it principally affects domestic and wild herbivores such as cattle, sheep, goats, bison, deer, antelope, and hippos and in those species, it is usually fatal. 4 Anthrax has significant animal and public health as well as socio-economic impacts that play a substantial role in the global trade of animals and animal products. 2 This disease is among the most priority zoonotic diseases. 5 , 6 Animal owners in resource-limited settings are usually at high risk of contracting anthrax infection because of their animal handling practices. 7 Different environmental, geographical, ecological, and demographic factors determine the perseverance and transmission of B. anthracis in an area. 6 B. anthracis spores usually persist in the soil under extreme environmental and climatic conditions for long periods and are a source for re-emergence of disease and transmit to animal hosts through grazing on B. anthracis spores contaminated areas, usually by ingestion or inhalation. Humans get infected when they are exposed to infected animals or their products such as meat, animal hides, bones, and other materials 4 as well as contact with an animal that died from anthrax. 8 , 9 Anthrax is an important but neglected zoonosis in many parts of the world. 10 It is a World Organization for Animal Health (WOAH)-listed and reportable disease. 8 The disease is mainly endemic in developing countries. 11 A compiled global occurrence dataset of human, livestock, and wildlife anthrax outbreaks report revealed that a global total of 63.8 million poor livestock keepers and 1.1 billion livestock live within regions at risk for the disease. 12 In Ethiopia, anthrax is endemic, 13 , 14 occurs in the dry season every year, 15 and usually, occurs as an outbreak year after year. 14 Anthrax is one of the top five important livestock diseases 16 and the second top priority zoonotic disease, next to rabies, in Ethiopia. 5 , 17 Anthrax remains a major problem for animals and public health in Ethiopia. 13 , 15 Particularly, the Amhara Regional state is frequently affected by diseases due to a humid to sub-humid environment, weak animal health services, and a lack of awareness of the community about animal anthrax case management which leads to widespread outbreaks. 18 The most efficient ways of preventing and controlling anthrax infection in domestic herds are sustainable surveillance, annual vaccination of livestock, and proper carcass disposal management. A study on spatio-temporal analysis and environmental suitability modeling of Anthrax in the Amhara Region indicated that Awi administrative zone was suitable for Bacillus anthracis and grouped at-risk areas for the disease. 18 An important step in the implementation of anthrax control is the acquisition of data or information about the occurrence of the disease. 4 However, data are limited to quantifying the occurrence and distribution of animal anthrax in Awi zone of the Amhara regional state of Ethiopia. The analysis of this data is very crucial to design more effective intervention strategies in the regions. Therefore, this retrospective study was conducted to describe the distribution of animal anthrax during a period from 2011 to 2020 in Awi administrative zone of Amhara Region, Northwest Ethiopia. Materials and Methods Description of the Study Area The study was conducted in the Awi administrative zone of Amhara Regional State, Ethiopia ( Figure 1 ). Amhara region (9° to 13°45'N and 36° to 40°30'E) is located in the northern part of Ethiopia, bordered by the state of Sudan to the northwest. 14 The Awi administrative zone has nine districts and three towns. Geographically, it is located at an elevation of 700–2920 meters above sea level with latitude and longitude of 11°16'N and 36°50°E, respectively. The zone has three rainfall seasons: Bega, Belg, and Kiremt. The primary rainy season, Kiremt, occurs from mid-June to mid-September. It also experiences a sporadic, secondary wet season, Belg, which often has considerably less rainfall and occurs from February to May. It receives an annual rainfall ranging from 800 to 2700 mm. During the hottest seasons, the temperature may range from 25 to 30 °C, while during the coldest; it ranges from 7 to 12 °C. Currently, the estimated livestock population of the Awi administrative zone is cattle (1,137,033), sheep (551,125), goats (130,732), horses (91,932), mules (20,517), and donkeys (79,037). 19–21 Figure 1 Map of Awi administrative zone in Amhara Region, Ethiopia. This map was developed from Ethiopian's Administrative boundaries shapefile 2021 using QGIS version 3.1.1.2. Study Design, Population, and Data Collection A retrospective study design was conducted from data recorded for 10 years, from 2011 to 2020. The study population was animals that are susceptible to anthrax infection. A structured data recording sheet was prepared using a Microsoft Excel worksheet. It was then used to extract data related to animal anthrax cases and deaths in the study area. Case Definition Clinical signs of peracute anthrax in cattle, sheep, and goats are staggering, trembling, breathing difficulty, convulsions, and death. Progression of the disease is rapid and premonitory signs may go unnoticed; often animals are found dead. After death, blood discharges from the nostrils, mouth, anus, and vulva occur and the blood may fail to clot. Acute anthrax manifests itself in high fevers (up to 42 °C), excitement, increased heart rate, deepening of respiration, followed by depression, incoordination, cessation of rumination, reduction in milk production, discolored milk (blood-tinged or deep yellow), bloody discharges, respiratory distress, convulsions, abortion, and death within 48 to 72 hours. Subcutaneous swelling and edema, usually involving the ventral aspect of the neck (brisket), thorax, shoulders, perineum, and flank, are characteristic of chronic anthrax infection. 22 The clinical signs of anthrax in horses are loss of appetite, colic, enteritis, fever, trembling depression, and bloody diarrhea. Death usually occurs within 48 to 96 hours. Edematous, subcutaneous swellings appear on the throat, lower neck, floor of the thorax and abdomen, prepuce, and mammary gland. 22 Anthrax cases were identified based on the clinical signs and/or blood smears (blue square-ended rods, usually in short chains, surrounded by a pinkish-red capsule). Similarly, dead animals were found with blood oozing from the natural orifices, and the rapid swelling and decomposition of carcasses were considered as death associated with anthrax. Sources of Anthrax Data Animal cases and deaths associated with anthrax were collected retrospectively for 10 years (2011–2020) of recorded data from each district in the Awi zone. The data were extracted from recorded databases of the passive surveillance animal disease and reported from the districts to the Regional (Amhara Regional Livestock Resources Development Promotion Agency) and National Veterinary Authority (Ministry of Agriculture). Passive animal disease surveillance is an animal owner's disease reporting system in which farmers report diseases and deaths in their livestock, but the information of that report is used by the Veterinary Authority for surveillance. Data such as species of animals sick and died, the number of cases and deaths in each district, monthly animal cases and death, and the season of the year were retrieved from the record. Anthrax is a notifiable disease, all cases and deaths associated with anthrax are mandatory to report to the Regional and National Veterinary Authority. Data Management and Analysis Data collected in an excel worksheet were reorganized, cleaned, and coded. Descriptive statistics mainly proportions were calculated using excel tools. Proportions were calculated to indicate the distribution of animal anthrax (including cases and deaths) in time (ie, distribution across different months, seasons, and years), animal (ie, distribution in different animal species in the 10 years), and place (distribution in different districts of the zone, the later may represent different agro-ecologies). The trend and distributions of anthrax cases by animals, place, and time were presented using graphs, and tables. The year was categorized into four seasons: rainy (June to August), post-rainy (September to November), cold-dry (December to February), and hot-dry (March to May) seasons. The case fatality rate was determined by dividing the number of deaths due to anthrax by the number of animal anthrax cases reported. Description of the Study Area The study was conducted in the Awi administrative zone of Amhara Regional State, Ethiopia ( Figure 1 ). Amhara region (9° to 13°45'N and 36° to 40°30'E) is located in the northern part of Ethiopia, bordered by the state of Sudan to the northwest. 14 The Awi administrative zone has nine districts and three towns. Geographically, it is located at an elevation of 700–2920 meters above sea level with latitude and longitude of 11°16'N and 36°50°E, respectively. The zone has three rainfall seasons: Bega, Belg, and Kiremt. The primary rainy season, Kiremt, occurs from mid-June to mid-September. It also experiences a sporadic, secondary wet season, Belg, which often has considerably less rainfall and occurs from February to May. It receives an annual rainfall ranging from 800 to 2700 mm. During the hottest seasons, the temperature may range from 25 to 30 °C, while during the coldest; it ranges from 7 to 12 °C. Currently, the estimated livestock population of the Awi administrative zone is cattle (1,137,033), sheep (551,125), goats (130,732), horses (91,932), mules (20,517), and donkeys (79,037). 19–21 Figure 1 Map of Awi administrative zone in Amhara Region, Ethiopia. This map was developed from Ethiopian's Administrative boundaries shapefile 2021 using QGIS version 3.1.1.2. Study Design, Population, and Data Collection A retrospective study design was conducted from data recorded for 10 years, from 2011 to 2020. The study population was animals that are susceptible to anthrax infection. A structured data recording sheet was prepared using a Microsoft Excel worksheet. It was then used to extract data related to animal anthrax cases and deaths in the study area. Case Definition Clinical signs of peracute anthrax in cattle, sheep, and goats are staggering, trembling, breathing difficulty, convulsions, and death. Progression of the disease is rapid and premonitory signs may go unnoticed; often animals are found dead. After death, blood discharges from the nostrils, mouth, anus, and vulva occur and the blood may fail to clot. Acute anthrax manifests itself in high fevers (up to 42 °C), excitement, increased heart rate, deepening of respiration, followed by depression, incoordination, cessation of rumination, reduction in milk production, discolored milk (blood-tinged or deep yellow), bloody discharges, respiratory distress, convulsions, abortion, and death within 48 to 72 hours. Subcutaneous swelling and edema, usually involving the ventral aspect of the neck (brisket), thorax, shoulders, perineum, and flank, are characteristic of chronic anthrax infection. 22 The clinical signs of anthrax in horses are loss of appetite, colic, enteritis, fever, trembling depression, and bloody diarrhea. Death usually occurs within 48 to 96 hours. Edematous, subcutaneous swellings appear on the throat, lower neck, floor of the thorax and abdomen, prepuce, and mammary gland. 22 Anthrax cases were identified based on the clinical signs and/or blood smears (blue square-ended rods, usually in short chains, surrounded by a pinkish-red capsule). Similarly, dead animals were found with blood oozing from the natural orifices, and the rapid swelling and decomposition of carcasses were considered as death associated with anthrax. Sources of Anthrax Data Animal cases and deaths associated with anthrax were collected retrospectively for 10 years (2011–2020) of recorded data from each district in the Awi zone. The data were extracted from recorded databases of the passive surveillance animal disease and reported from the districts to the Regional (Amhara Regional Livestock Resources Development Promotion Agency) and National Veterinary Authority (Ministry of Agriculture). Passive animal disease surveillance is an animal owner's disease reporting system in which farmers report diseases and deaths in their livestock, but the information of that report is used by the Veterinary Authority for surveillance. Data such as species of animals sick and died, the number of cases and deaths in each district, monthly animal cases and death, and the season of the year were retrieved from the record. Anthrax is a notifiable disease, all cases and deaths associated with anthrax are mandatory to report to the Regional and National Veterinary Authority. Data Management and Analysis Data collected in an excel worksheet were reorganized, cleaned, and coded. Descriptive statistics mainly proportions were calculated using excel tools. Proportions were calculated to indicate the distribution of animal anthrax (including cases and deaths) in time (ie, distribution across different months, seasons, and years), animal (ie, distribution in different animal species in the 10 years), and place (distribution in different districts of the zone, the later may represent different agro-ecologies). The trend and distributions of anthrax cases by animals, place, and time were presented using graphs, and tables. The year was categorized into four seasons: rainy (June to August), post-rainy (September to November), cold-dry (December to February), and hot-dry (March to May) seasons. The case fatality rate was determined by dividing the number of deaths due to anthrax by the number of animal anthrax cases reported. Results A total of 1586 animal anthrax reports were found from 2011 to 2020, of which 1262 reports were cases of anthrax and 324 were animals that died due to anthrax. The highest proportion of animal anthrax cases was reported in 2012 (n = 671; 53.12%), which share 48.9% of the 10 years of animal anthrax reports. Similarly, the highest proportion of animal death due to anthrax (n = 104; 32.10%) was reported in 2014. In contrast, the lowest proportion of animal anthrax cases (n = 6; 0.48%) and deaths (n = 1; 0.31%) were recorded in 2018. In this study, the overall case fatality rate of anthrax was 25.67% (n = 324) with which the highest case fatality rate (63.64%) was recorded in 2016 followed by 2014 (46.25%) (n = 111), and 2013 (45.50%) (n = 10) as shown in Table 1 . Table 1 The Number of Cases, Deaths, and Case Fatality Rate of Anthrax in Awi Administrative Zone During 2011–2020 Year No. of Cases (%) No. of Deaths (%) Case Fatality Rate (%) 2011 107 (8.48) 34 (10.49) 31.78 2012 671 (53.12) 104 (32.10) 15.50 2013 22 (1.74) 10 (3.09) 45.50 2014 240 (19.02) 111 (34.26) 46.25 2015 97 (7.69) 28 (8.64) 28.87 2016 11 (0.87) 7 (2.16) 63.64 2017 55 (4.36) 16 (4.94) 29.10 2018 6 (0.48) 1 (0.31) 16.67 2019 35 (2.77) 6 (1.85) 17.14 2020 18 (1.43) 7 (2.16) 38.89 Total 1262 324 25.67 Note : % is the proportion from the 10-year period. Among animals, the highest animal anthrax cases were recorded in bovines (n = 984; 77.97%), followed by equine species (n = 202; 16.01%). The lowest animal anthrax cases were recorded in caprine (2%). Similarly, the highest number of animal deaths due to anthrax were recorded in bovines (n = 259; 79.94%) while the lowest was recorded (n = 15; 4.63%) in ovines. Although only two anthrax cases were recorded in caprine, death associated with anthrax had not been recorded as shown in Figure 2 . Figure 2 Distribution of animal anthrax among animal species affected in Awi administrative zone, 2011–2020. In this study, the distribution of animal anthrax cases and deaths varies among districts in Awi administrative zone of the Amhara Region, Ethiopia. The highest number of anthrax cases were reported in Banja district (n = 386) followed by Ankesha district (n = 351). The highest number of animal deaths due to anthrax were reported in Banja district (n = 68) followed by Zigem district (n = 57), while the lowest number of animal deaths was recorded in Fagita Lekoma district as shown in Figure 3 . Figure 3 Distribution of animal anthrax across districts in Awi zone, 2011–2020. According to the 10 years of recorded animal anthrax cases and deaths, the highest cases of anthrax were registered in May (n = 313) followed by July (n = 280). The lowest number of animal anthrax cases was reported during January and no anthrax case was reported in December during the 10 years. The highest number of animal death due to anthrax was reported during July (n = 64) followed by June (n = 48). In contrast, the lowest number of animal death due to anthrax was reported in January (n = 6) as illustrated in Figure 4 . Besides, the seasonal distribution of anthrax indicated that the highest number of animal anthrax cases were reported during the hot-dry season (n = 479; 37.96%) followed by the rainy season (n = 446; 35.34%). The lowest number of animal anthrax cases were recorded during the cold-dry season (n = 30; 2.38%) as shown in Figure 5 . Figure 4 Monthly distribution of animal anthrax in Awi administrative zone, 2011–2020. Figure 5 Seasonal distribution of animal anthrax in Awi administrative zone, 2011–2020. Discussion The current study revealed that from the year 2011 to 2020, a total of 1262 animal anthrax cases and 324 animal death associated with anthrax were reported in Awi administrative zone of Amhara Region, Ethiopia. In this study, the proportions of animal anthrax between 2011 and 2020 indicated that about 53.12% of animal anthrax cases were recorded in 2012. This high proportion of anthrax cases in this period might be attributed to the occurrence of the longest-dry season, 23 limited vaccination coverage, 16 and low awareness and practices to manage the disease. The lowest number of animal anthrax was recorded in 2018. This might be due to good vaccination coverage and sound case management practice by the community and professionals, and/or underreporting of cases and deaths by the local veterinary officials. Even though it is known that anthrax affects multiple species, 2 more than two-thirds of animal anthrax (77.97%) reports were recorded in bovines. Furthermore, the reported anthrax cases were very among species. These differences might be due to the grazing or browsing behavior of animals and animal owners' health-seeking behavior and attitude toward their animals. 24 , 25 Only two anthrax cases were recorded in caprine for the 10 years. This might be associated with the browsing behaviors of the goats which reduce the probability of getting the spore in the soil. This result is in line with other reports where anthrax occurs in all vertebrates but most common in cattle and sheep and less frequent in goats. 22 Close grazing of tough, scratchy feed in dry times, which results in abrasions of the oral mucosa, and confined grazing on heavily contaminated areas around water holes are the risk factors for the occurrence of anthrax outbreaks. 22 In this study, variations in the proportion of cases and death associated with anthrax among districts in Awi administrative zone were reported. The highest number of anthrax cases were recorded in Banja district (386/1262), followed by Ankesha district (351/1262), whereas the lowest number of anthrax cases were reported in Fagita Lekoma (37/1262). The highest number of animal deaths due to anthrax were reported in Banja district (68/324) followed by Zigem district (57/324). Case fatality rates were also assessed for each district and the highest (84.21%) was recorded in the Jawi district. The highest number of animal deaths due to anthrax were reported in Banja district (n = 68) followed by Zigem district (n = 57), while the lowest number of animal deaths was recorded in Fagita Lekoma district. Differences in the number of anthrax cases and deaths among districts might be due to varying climatic, soil, and temperature conditions 19 , 26 as well as carcass and environmental management practices following animal anthrax cases and deaths, 9 , 14 which will facilitate the clustering of anthrax in a specific place. 6 Although B. anthracis can be found worldwide, anthrax cases usually occur only in limited geographic regions. Outbreaks are most common in areas characterized by alkaline, calcium-rich soils, warm environments, and periodic episodes of flooding. 22 Variations in the proportions of animal anthrax cases among different months and seasons of the year were observed. The highest proportion of animal anthrax cases was reported in May (24.8%) followed by July (22.19%). These findings were in line with the nature of anthrax, where most outbreaks occur during heavy rainfall following a period of prolonged drought. 27 Whereas, the lowest proportion of animal anthrax cases were found in January, as low as approximately 5 in 1000 animals. It has been recognized that environmental and climatic drivers were important factors influencing the ecology of anthrax. 28 The seasonal distribution in this study indicated that anthrax occurs in animals in both dry and cold seasons, however, the highest proportion of animal anthrax cases was reported during the hot-dry season (37.96%) whereas the lowest was recorded during the cold-dry season. During the dry season in the study areas, it was noted that the vegetation for grazing is depleted and becomes short, which leads the animals to close grazing to the ground. This significantly increases the chance of contracting B. anthracis . During long-dry seasons of the year, vegetation were scarce or short 7 and during heavy rainy seasons, there may be soil disturbance, 2 , 29 which results in ease of exposure of animals to the spores of B. anthracis leading to high numbers of cases or probably to outbreaks. In Ethiopia, a study reported that high numbers of anthrax were recorded during rainy seasons. 30 Furthermore, longer dry/hot seasons have the potential to induce stress on animals and hence animals' innate resistance to infections will be negatively affected. In such circumstances, low doses of B. anthracis spores will get the potential to initiate infection in animals. 31 , 32 The current study revealed that during the 10 years, there was no report of animal anthrax in December. However, a recent epidemiological investigation report indicated that the index case of anthrax that occurred in December was reported. 14 Limitation Given that anthrax is a notifiable disease, all cases and deaths associated with anthrax are mandatory to report to the Regional and National Veterinary Authority. However, the animal owners may not report all animal deaths to the nearest veterinary officials, which significantly underreports animals death associated with anthrax in the regular passive surveillance system. For this retrospective study, the secondary data were obtained from the monthly animal anthrax cases and death records. The record has a limitation on the quality especially data related to transmission to animals, the environment, and case management practices. Conclusion The current study showed that a considerable number of animal anthrax cases and deaths occurred in the Awi administrative zone, with varying degrees among different species of susceptible animals, months/seasons, and districts. In this study, the highest number of cases of anthrax was registered in May and during the hot-dry season; that calls for the need for practicing prevention strategies including immunization programs before the peak season of anthrax outbreaks. Data Sharing Statement The data used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Ethical Approval Approval for retrieving and reporting the animal anthrax data reported between 2011 and 2020 was obtained from the Amhara Regional Livestock Resources Development Promotion Agency and the Ministry of Agriculture, Ethiopia. Author Contributions All authors contributed to data analysis, drafting, or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work. Disclosure The authors declare that they have no competing interests in this work.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734955/

Elderly patients and inflammatory bowel disease

The incidence and prevalence of inflammatory bowel disease (IBD) is increasing globally. Coupled with an ageing population, the number of older patients with IBD is set to increase. The clinical features and therapeutic options in young and elderly patients are comparable but there are some significant differences. The wide differential diagnosis of IBD in elderly patients may result in a delay in diagnosis. The relative dearth of data specific to elderly IBD patients often resulting from their exclusion from pivotal clinical trials and the lack of consensus guidelines have made clinical decisions somewhat challenging. In addition, age specific concerns such as co-morbidity; loco-motor and cognitive function, poly-pharmacy and its consequences need to be taken into account. In applying modern treatment paradigms to the elderly, the clinician must consider the potential for more pronounced adverse effects in this vulnerable group and set appropriate boundaries maximising benefit and minimising harm. Meanwhile, clinicians need to make personalised decisions but as evidence based as possible in the holistic, considered and optimal management of IBD in elderly patients. In this review we will cover the clinical features and therapeutic options of IBD in the elderly; as well as addressing common questions and challenges posed by its management.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC166172/

Reexamining age, race, site, and thermometer type as variables affecting temperature measurement in adults – A comparison study

Background As a result of the recent international vigilance regarding disease assessment, accurate measurement of body temperature has become increasingly important. Yet, trusted low-tech, portable mercury glass thermometers are no longer available. Thus, comparing accuracy of mercury-free thermometers with mercury devices is essential. Study purposes were 1) to examine age, race, site as variables affecting temperature measurement in adults, and 2) to compare clinical accuracy of low-tech Galinstan-in-glass device to mercury-in-glass at oral, axillary, groin, and rectal sites in adults. Methods Setting 176 bed accredited healthcare facility, rural northwest US Participants Convenience sample (N = 120) of hospitalized persons ≥ 18 years old. Instruments Temperatures (°F) measured at oral, skin (simultaneous), immediately followed by rectal sites with four each mercury-glass (BD) and Galinstan-glass (Geratherm) thermometers; 10 minute dwell times. Results Participants averaged 61.6 years (SD 17.9), 188 pounds (SD 55.3); 61% female; race: 85% White, 8.3% Native Am., 4.2% Hispanic, 1.7 % Asian, 0.8% Black. For both mercury and Galinstan-glass thermometers, within-subject temperature readings were highest rectally; followed by oral, then skin sites. Galinstan assessments demonstrated rectal sites 0.91°F > oral and ≠1.3°F > skin sites. Devices strongly correlated between and across sites. Site difference scores between devices showed greatest variability at skin sites; least at rectal site. 95% confidence intervals of difference scores by site (°F): oral (0.142 – 0.265), axilla (0.167 – 0.339), groin (0.037 – 0.321), and rectal (-0.111 – 0.111). Race correlated with age, temperature readings each site and device. Conclusion Temperature readings varied by age, race. Mercury readings correlated with Galinstan thermometer readings at all sites. Site mean differences between devices were considered clinically insignificant. Still considered the gold standard, mercury-glass thermometers may no longer be available worldwide. Therefore, mercury-free, environmentally safe low-tech Galinstan-in-glass may be an appropriate replacement. This is especially important as we face new, internationally transmitted diseases. Background As a result of the recent international vigilance regarding disease assessment, accurate measurement of body temperature has become increasingly important. Yet, trusted low-tech, portable mercury glass thermometers are no longer available. Thus, comparing accuracy of mercury-free thermometers with mercury devices is essential. Study purposes were 1) to examine age, race, site as variables affecting temperature measurement in adults, and 2) to compare clinical accuracy of low-tech Galinstan-in-glass device to mercury-in-glass at oral, axillary, groin, and rectal sites in adults. Methods Setting 176 bed accredited healthcare facility, rural northwest US Participants Convenience sample (N = 120) of hospitalized persons ≥ 18 years old. Instruments Temperatures (°F) measured at oral, skin (simultaneous), immediately followed by rectal sites with four each mercury-glass (BD) and Galinstan-glass (Geratherm) thermometers; 10 minute dwell times. Results Participants averaged 61.6 years (SD 17.9), 188 pounds (SD 55.3); 61% female; race: 85% White, 8.3% Native Am., 4.2% Hispanic, 1.7 % Asian, 0.8% Black. For both mercury and Galinstan-glass thermometers, within-subject temperature readings were highest rectally; followed by oral, then skin sites. Galinstan assessments demonstrated rectal sites 0.91°F > oral and ≠1.3°F > skin sites. Devices strongly correlated between and across sites. Site difference scores between devices showed greatest variability at skin sites; least at rectal site. 95% confidence intervals of difference scores by site (°F): oral (0.142 – 0.265), axilla (0.167 – 0.339), groin (0.037 – 0.321), and rectal (-0.111 – 0.111). Race correlated with age, temperature readings each site and device. Conclusion Temperature readings varied by age, race. Mercury readings correlated with Galinstan thermometer readings at all sites. Site mean differences between devices were considered clinically insignificant. Still considered the gold standard, mercury-glass thermometers may no longer be available worldwide. Therefore, mercury-free, environmentally safe low-tech Galinstan-in-glass may be an appropriate replacement. This is especially important as we face new, internationally transmitted diseases. Background All health services need reliable, valid, readily available and accessible body temperature assessment devices. Obviously, body temperature assessments are key diagnostic indicators. Yet, the measurement of human body temperature has recently been cause for concern. Since Wunderlich's seminal work [ 1 ], mercury has been and continues to be the "gold standard" for temperature measurement [ 2 - 5 ]. Current values used to define fever are founded on Wunderlich's classic temperature research efforts. However, the manufacture, distribution, and sales of mercury filled fever thermometers has been banned or restricted in at least ten US states and US federal legislation to restrict mercury thermometer availability nationwide, is pending. Similar restrictions are occurring globally. As increased disease detection and management efforts continue worldwide, the key physical indicator of body temperature becomes critically important [ 6 ]. Thus, study purposes were 1) to examine age, race, and site as variables affecting temperature measurement in adults, and 2) to compare the clinical accuracy of the low-tech Galinstan-in-glass device to mercury-in-glass at oral, axillary, groin, and rectal sites in persons 18 and older. Numerous variables are known to influence body temperature measures. These variables include age [ 7 - 10 ], race [ 11 , 12 ], pharmacologic agents [ 13 - 15 ], infectious agents [ 1 , 16 , 17 ], exercise [ 18 , 19 ], device dwell times [ 20 - 23 ], device type [ 1 , 20 , 24 - 28 ], and body site [ 5 , 20 , 26 , 29 - 31 ]. For this study, dwell times were consistent for all readings; study variables included age, race, device type, and body site. Chosen temperature assessment sites for this investigation were oral, axillary, groin, and rectal. The oral site is inappropriate and unsafe for patients younger than six but well accepted by the general public. Possible oral site reading errors include the influence of food/fluids, smoking, placement technique, oral seal, and hypothermic status. The non-invasive axillary skin site is commonly used and accepted with infants and generally familiar and accepted by adults. Possible assessment errors include the influence of ambient temperature, hypothermic status, fatty layers, dominant arm muscle mass, skin folds, and circulatory differences due to asymmetric thoracic cavity organ placement [ 21 , 32 , 33 ]. Other non-invasive skin sites are the groin sites, located bilaterally directly over the femoral artery in the inguinal area. Though identified as viable sites in infants and neonates [ 23 , 34 , 35 ], other than as a potential prediction of psychosexual arousal, [ 36 , 37 ] groin temperature sites have not been well investigated in adult population groups. For adults, the groin site is potentially better tolerated and less invasive than rectal. The rectal temperature site requires a thermometer placement in the patient's rectum, beyond the anal sphincter. Though not well tolerated by adults, rectal is a common site in young children and considered to be accurate in children and infants. However, rectal readings may be imprecise, especially for older adults, due to presence of stool, heavy lower extremity size, decreased rectal circulation, and mobility variations [ 21 , 38 - 40 ]. Rectal lag has been identified as the lag time or delayed response of rectal versus core body temperatures. This lag time is especially evident during core temperature fluctuations [ 41 - 43 , 21 ]. Rectal and oral sites have the disadvantage of body fluid contact. Study devices for this investigation included the low-tech, portable, lightweight, and sterilizable mercury-in-glass and Galinstan-in-glass thermometers. Low-tech glass thermometers present no concern regarding battery disposal or power source competence. Both thermometer types can be used with oral, skin, or rectal sites. Both thermometer types can be person-specific, preventing cross transfer and cross contamination of bio-hazardous materials. Mercury-in-glass thermometers may no longer be available; and if broken, become potentially toxic to patient and environment. Galinstan-in-glass is a metal alloy-in-glass thermometer that is safe to the patient/environment [ 44 ]; identified internationally as the in-glass mercury substitute thermometer. It is easy to read (both F and C measures) and has only a slightly higher cost than mercury-in-glass devices. Though mercury-free, the Galinstan-in-glass product, as with all glass thermometers, requires careful handling due to risk of accidental injury from breakage. Significance Except for small, unpublished pilot work by Smith (N = 39) [ 45 ] this investigation is the first to assess and compare the Galinstan-in-glass thermometer device in humans. A true need exists to compare this mercury substitute thermometer with the mercury device. This need is especially critical because these low-tech mercury free devices are ideal additions to hazardous materials (HAZMAT) units, emergency, and law enforcement vehicles. Significance Except for small, unpublished pilot work by Smith (N = 39) [ 45 ] this investigation is the first to assess and compare the Galinstan-in-glass thermometer device in humans. A true need exists to compare this mercury substitute thermometer with the mercury device. This need is especially critical because these low-tech mercury free devices are ideal additions to hazardous materials (HAZMAT) units, emergency, and law enforcement vehicles. Methods A descriptive, correlation design was used to determine within-subject mean differences between and among sites and instruments. Descriptive statistics described the overall sample and temperature measures. Subjects were their own controls. Alpha was set at 0.05. Sample and Setting A convenience sample (N = 120) of adult in-patients in a rural pacific northwest accredited hospital and medical center was obtained. Participants were English-speaking currently hospitalized men and women who were at least 18 years of age, mentally competent, and willing to participate. Participants had palpable femoral pulses and competent rectums. Oral temperature assessments occurred ≥ 15 minutes post oral food/fluids [ 38 ]. This was assessed by asking participants, "when did you last eat or drink anything?" Data collection took place from August to October, 2002. Human Subjects In addition to Oregon Health & Science University approval, IRB approval was also obtained through the active medical center review board. All participants signed informed consent documents prior to data collection. A copy of the consent form was given to participants immediately following written consent. A copy of all temperature data was handed to participants, after their temperatures were assessed. General Procedures All temperature measurement devices were assessed for accuracy prior to study use. A swirling water bath was used with identified temperature levels and controls; consistent with procedures used by previous researchers [ 23 , 34 ]. An ASTM mercury-in-glass thermometer with traceable certification of accuracy (against NIST standards) was used to assess water bath temperatures. All devices used read within ± 0.2°F at three different temperature settings (range between 97 – 102°F). An ambient temperature thermometer with NIST traceable certificate of compliance was also used. A count-up timer for precise time intervals was employed. Instruments Temperatures were simultaneously measured at two oral and four skin sites, immediately followed by rectal sites. Four (each) individual, sheathed mercury-in-glass and Galinstan-in-glass thermometers were used for these temperature measurements. Environmental air temperature was recorded. All temperature readings were reported in degrees Fahrenheit. An equal dwell time of ten minutes was used for each measurement to control time as a factor affecting instruments and sites [ 21 - 23 , 46 - 48 ]. Between use, thermometers were cleaned as follows: 1. Cool soap and water bath, with two-minute agitation and cold rinse, times three 2. Soak in 1:10 mixture of bleach/water minimum of 6 hours, followed by cool soap and water bath, with two minute agitation, and cold rinse [ 49 - 51 ]. The Australian Resuscitation Council has accepted that bleach can be used for cleansing of resuscitation equipment [ 51 ]. 3. Soak in full strength isopropyl alcohol minimum of 12 hours, followed by cold water rinse times two. 4. Dry on clean, dry towel Procedure The principal investigator and a trained research assistant performed all procedures. Right and left readings for oral (temperature oral), axillary (temperature axilla), groin (temperature groin), and rectal (temperature rectal) were taken using sheathed oral/skin mercury-in-glass thermometers and sheathed rectal or oral/skin or rectal Galinstan-in-glass thermometers (respectively). Participants were instructed beforehand to keep lips closed once oral thermometers were in place. Right/left device type locations for oral, axilla, and groin were random. – Oral readings were simultaneous (Galinstan on one side; mercury on the other) bilateral measures at the location of the sublingual artery (pocket of tissue at the base of the tongue, just above the sublingual artery) [ 21 , 52 ]. – Axillary readings were simultaneous (mercury on one side; Galinstan on the other) bilateral measures deep into mid-axilla [apex] [ 10 , 23 , 34 , 53 ] with client's arms adducted. Axillary and groin site thermometers were placed immediately after placement of oral. – Groin readings were simultaneous (Galinstan on one side; mercury on the other) bilateral at the location of the femoral artery. The technique for groin/femoral/inguinal temperature readings involved gentle slight abduction of the patient's leg, location of the femoral pulse, placement of the sheathed thermometer on and lateral to the pulse site, and slight adducting of the leg to create a seal [ 23 , 34 ]. – Technique for rectal included two sheathed thermometers (one mercury, one Galinstan) with water-soluble lubrication, inserted together to a depth of five cms [ 21 , 54 , 55 ]. Once the rectal thermometers were inserted, the principal investigator's gloved thumb and forefinger held the top safety grip on the mercury and top 0.5 cm of the 12.7 cm long Galinstan. All thermometers began at a reading below 96°F. This reading was chosen because the mercury-in-glass thermometers were not calibrated below 96°F. Prior to recording, both observers agreed upon all measurement readings. Upon completion of temperature assessments, the room temperature was assessed and recorded and the patient was returned to a comfortable, resting position. A carbon copy of temperature readings was given to each participant. Sample and Setting A convenience sample (N = 120) of adult in-patients in a rural pacific northwest accredited hospital and medical center was obtained. Participants were English-speaking currently hospitalized men and women who were at least 18 years of age, mentally competent, and willing to participate. Participants had palpable femoral pulses and competent rectums. Oral temperature assessments occurred ≥ 15 minutes post oral food/fluids [ 38 ]. This was assessed by asking participants, "when did you last eat or drink anything?" Data collection took place from August to October, 2002. Human Subjects In addition to Oregon Health & Science University approval, IRB approval was also obtained through the active medical center review board. All participants signed informed consent documents prior to data collection. A copy of the consent form was given to participants immediately following written consent. A copy of all temperature data was handed to participants, after their temperatures were assessed. General Procedures All temperature measurement devices were assessed for accuracy prior to study use. A swirling water bath was used with identified temperature levels and controls; consistent with procedures used by previous researchers [ 23 , 34 ]. An ASTM mercury-in-glass thermometer with traceable certification of accuracy (against NIST standards) was used to assess water bath temperatures. All devices used read within ± 0.2°F at three different temperature settings (range between 97 – 102°F). An ambient temperature thermometer with NIST traceable certificate of compliance was also used. A count-up timer for precise time intervals was employed. Instruments Temperatures were simultaneously measured at two oral and four skin sites, immediately followed by rectal sites. Four (each) individual, sheathed mercury-in-glass and Galinstan-in-glass thermometers were used for these temperature measurements. Environmental air temperature was recorded. All temperature readings were reported in degrees Fahrenheit. An equal dwell time of ten minutes was used for each measurement to control time as a factor affecting instruments and sites [ 21 - 23 , 46 - 48 ]. Between use, thermometers were cleaned as follows: 1. Cool soap and water bath, with two-minute agitation and cold rinse, times three 2. Soak in 1:10 mixture of bleach/water minimum of 6 hours, followed by cool soap and water bath, with two minute agitation, and cold rinse [ 49 - 51 ]. The Australian Resuscitation Council has accepted that bleach can be used for cleansing of resuscitation equipment [ 51 ]. 3. Soak in full strength isopropyl alcohol minimum of 12 hours, followed by cold water rinse times two. 4. Dry on clean, dry towel Procedure The principal investigator and a trained research assistant performed all procedures. Right and left readings for oral (temperature oral), axillary (temperature axilla), groin (temperature groin), and rectal (temperature rectal) were taken using sheathed oral/skin mercury-in-glass thermometers and sheathed rectal or oral/skin or rectal Galinstan-in-glass thermometers (respectively). Participants were instructed beforehand to keep lips closed once oral thermometers were in place. Right/left device type locations for oral, axilla, and groin were random. – Oral readings were simultaneous (Galinstan on one side; mercury on the other) bilateral measures at the location of the sublingual artery (pocket of tissue at the base of the tongue, just above the sublingual artery) [ 21 , 52 ]. – Axillary readings were simultaneous (mercury on one side; Galinstan on the other) bilateral measures deep into mid-axilla [apex] [ 10 , 23 , 34 , 53 ] with client's arms adducted. Axillary and groin site thermometers were placed immediately after placement of oral. – Groin readings were simultaneous (Galinstan on one side; mercury on the other) bilateral at the location of the femoral artery. The technique for groin/femoral/inguinal temperature readings involved gentle slight abduction of the patient's leg, location of the femoral pulse, placement of the sheathed thermometer on and lateral to the pulse site, and slight adducting of the leg to create a seal [ 23 , 34 ]. – Technique for rectal included two sheathed thermometers (one mercury, one Galinstan) with water-soluble lubrication, inserted together to a depth of five cms [ 21 , 54 , 55 ]. Once the rectal thermometers were inserted, the principal investigator's gloved thumb and forefinger held the top safety grip on the mercury and top 0.5 cm of the 12.7 cm long Galinstan. All thermometers began at a reading below 96°F. This reading was chosen because the mercury-in-glass thermometers were not calibrated below 96°F. Prior to recording, both observers agreed upon all measurement readings. Upon completion of temperature assessments, the room temperature was assessed and recorded and the patient was returned to a comfortable, resting position. A carbon copy of temperature readings was given to each participant. Results Description of Sample Although 120 individuals participated in this study, not all paired readings were possible for all participants because mercury-in-glass thermometers were calibrated only as low as 96 degrees F. When temperature readings fell below this level, no evaluation could be recorded and thus, paired readings and difference scores were not possible. Participants averaged 61.6 years old (SD 17.9) and 188 pounds (SD 55.3); they were 61% female. Eighty-five percent self-reported as having a White racial heritage. Native-American was the second most commonly reported racial heritage at 8.3% followed by Hispanic (4.2%) Asian (1.7%) and Black (.8%). These percentages are consistent with the racial mix within this rural community. More females than males volunteered as research participants (see Table 1 ). Table 1 Description of Sample Variable Mean SD Range Age (years) 61.6 17.9 19 94 Weight (pounds) 188 55.3 100 400 Weight (estimated Kgs) 85.4 25.12 45.5 181.8 Room Temperature (°F) 74 1.19 71.2 78.3 Culture/race White Asian Native American Hispanic Black Total Gender Male 42 0 2 2 1 47 Female 60 2 8 3 0 73 Total 102 2 10 5 1 120 Comparing temperature sites For both the mercury-in-glass and Galinstan-in-glass thermometers, temperature readings by site were as follows: R > O > Skin (axilla and groin). That is, within-subject temperature assessments were highest rectally, followed by oral, and then groin or axillary skin sites. This result was as expected [ 56 - 59 ]. To examine same-device temperature reading differences across body sites, mean difference scores were calculated along with standard deviations (see Table 2 ). Table 2 Mean temperature differences between sites (°F) for two devices Mercury-in-glass site differences Sites Mean Difference SD N To> Tg .396°F .83 119 To> Tx .397°F .75 119 Tg> Tx .000°F .88 120 Tr> Tx 1.06°F .68 117 Tr> To .648°F .62 116 Tr> Tg 1.04°F .76 117 Galinstan-in-glass site differences Sites Mean Difference SD N To> Tg .380°F .80 119 To> Tx .449°F .81 119 Tg> Tx .007°F .75 120 Tr> Tx 1.35°F .78 117 Tr> To .913°F .57 116 Tr> Tg 1.29°F .67 117 Legend: To – Temperature oral Tx – Temperature axilla Tg – Temperature groin Tr – Temperature rectal Comparing temperature devices See Table 3 for a description of within site differences between the two devices. Table 3 Mean temperature differences, SD between devices at four sites (°F) Site Mean Difference (Bias) SD N To: mercury – Galinstan .203°F .34 119 Tx: mercury – Galinstan .253°F .48 120 Tg: mercury – Galinstan .179°F .79 120 Tr: mercury – Galinstan -.05°F .31 117 Legend: To – Temperature oral Tx – Temperature axilla Tg – Temperature groin Tr – Temperature rectal As suggested by Bland and Altman [ 60 ] to further examine variability of mean temperature differences between devices, a box plot diagram was formulated (see Figure 1 ). The question is: Do measurements from different devices sufficiently agree and how different are the two methods [ 60 ]? Thus, a scatter plot of devices by site was devised (see Figure 2 ) followed by plots of the difference between devices by site against their means (see Figures 3 , 4 , 5 and 6 ). According to Bland and Altman [ 60 ] this method of plotting differences "… allows us to investigate any possible relationship between the measurement error and the true value. We do not know the true value, and the mean of the … measurements is the best estimate we have," (p. 308). Figure 1 Box plot showing mean temperature reading differences and variability between mercury-in-glass and Galinstan-in-glass devices at four sites (°F). Box plot – mean differences by site Mercury/Galinstan pairs Figure 2 Scatter plot – devices by site (°F). Scatter plot – devices by site Mercury/Galinstan pairs o – oral x-axilla g-groin r-rectal hg-Mercury-in-glass gal-Galinstan-in glass Figure 3 Scatter plot of differences, at the oral site, against their mean (°F). The mean difference (mercury minus Galinstan) is 0.20°F, SD 0.34, skewness 0.676. Oral Device differences against their means _________ mean difference, mercury-Galinstan - - - - - - 2 SDs from the mean Figure 4 Scatter plot of differences, axilla site, against their mean (°F). The mean difference (mercury minus Galinstan) is 0.25°F, SD 0.48, skewness 0.396. Axilla Device differences against their mean _________ mean difference, mercury-Galinstan - - - - - - 2 SDs from the mean Figure 5 Scatter plot of differences, at the groin site, against their mean (°F). The mean difference (mercury minus Galinstan) is 0.18°F, SD 0.79, and skewness -0.075. Groin Device differences against their means _________ mean difference, mercury-Galinstan - - - - - - 2 SDs from the mean Figure 6 Scatter plot of differences, at the rectal site, against their mean (°F). The mean difference (mercury minus Galinstan) is -0.06°F, SD 0.31, and skewness -1.04. Rectal Device differences against their means _________ mean difference, mercury-Galinstan - - - - - - 2 SDs from the mean Further examination of differences included repeatability analyses. If differences were normally distributed, the expectation was that 95% of all sample means of differences, based on this sample, would fall within this confidence interval. An analysis of the limits of agreement was computed based on the mean and two standard deviations (precision) of the difference between devices by sites [ 60 ]. According to Gardner and Altman [ 61 ] confidence intervals are a more useful and informative approach than P values because they present a range of values. For confidence intervals of difference scores, see Table 4 . However, caution is expressed due to the skewed histograms of difference scores. Thus, the assumption of Normality may be invalid [ 60 , 61 ]. Table 4 95% confidence intervals of difference scores by site Site Lower (°F) Upper (°F) Oral 0.142 0.265 Axilla 0.167 0.339 Groin 0.037 0.321 Rectal -0.111 0.111 Correlation is a quantification of the linear relationship between variables and does not examine the question of agreement. Often used to analyze thermometer reading accuracy, correlation is an estimate of how much two variables change in relation to one another. Perfect correlation does not mean perfect agreement [ 62 ]. Altman and Bland [ 63 ] identified this as a favorite approach to the comparison of two methods of measurement. Therefore, correlation statistics were established for the purpose of literature comparisons. Furthermore, human body within and between site agreement cannot be assumed. Thus, relationships between and among variables are represented with a correlation table (see Table 5 ). Table 5 Correlations between variables (two devices each site) Variable pair Pearson r Significance (2-tailed) N Oral Hg:Galinstan .929 O > Skin (axilla and groin). That is, within-subject temperature assessments were highest rectally, followed by oral, and then groin or axillary skin sites. This result was as expected [ 56 - 59 ]. To examine same-device temperature reading differences across body sites, mean difference scores were calculated along with standard deviations (see Table 2 ). Table 2 Mean temperature differences between sites (°F) for two devices Mercury-in-glass site differences Sites Mean Difference SD N To> Tg .396°F .83 119 To> Tx .397°F .75 119 Tg> Tx .000°F .88 120 Tr> Tx 1.06°F .68 117 Tr> To .648°F .62 116 Tr> Tg 1.04°F .76 117 Galinstan-in-glass site differences Sites Mean Difference SD N To> Tg .380°F .80 119 To> Tx .449°F .81 119 Tg> Tx .007°F .75 120 Tr> Tx 1.35°F .78 117 Tr> To .913°F .57 116 Tr> Tg 1.29°F .67 117 Legend: To – Temperature oral Tx – Temperature axilla Tg – Temperature groin Tr – Temperature rectal Comparing temperature devices See Table 3 for a description of within site differences between the two devices. Table 3 Mean temperature differences, SD between devices at four sites (°F) Site Mean Difference (Bias) SD N To: mercury – Galinstan .203°F .34 119 Tx: mercury – Galinstan .253°F .48 120 Tg: mercury – Galinstan .179°F .79 120 Tr: mercury – Galinstan -.05°F .31 117 Legend: To – Temperature oral Tx – Temperature axilla Tg – Temperature groin Tr – Temperature rectal As suggested by Bland and Altman [ 60 ] to further examine variability of mean temperature differences between devices, a box plot diagram was formulated (see Figure 1 ). The question is: Do measurements from different devices sufficiently agree and how different are the two methods [ 60 ]? Thus, a scatter plot of devices by site was devised (see Figure 2 ) followed by plots of the difference between devices by site against their means (see Figures 3 , 4 , 5 and 6 ). According to Bland and Altman [ 60 ] this method of plotting differences "… allows us to investigate any possible relationship between the measurement error and the true value. We do not know the true value, and the mean of the … measurements is the best estimate we have," (p. 308). Figure 1 Box plot showing mean temperature reading differences and variability between mercury-in-glass and Galinstan-in-glass devices at four sites (°F). Box plot – mean differences by site Mercury/Galinstan pairs Figure 2 Scatter plot – devices by site (°F). Scatter plot – devices by site Mercury/Galinstan pairs o – oral x-axilla g-groin r-rectal hg-Mercury-in-glass gal-Galinstan-in glass Figure 3 Scatter plot of differences, at the oral site, against their mean (°F). The mean difference (mercury minus Galinstan) is 0.20°F, SD 0.34, skewness 0.676. Oral Device differences against their means _________ mean difference, mercury-Galinstan - - - - - - 2 SDs from the mean Figure 4 Scatter plot of differences, axilla site, against their mean (°F). The mean difference (mercury minus Galinstan) is 0.25°F, SD 0.48, skewness 0.396. Axilla Device differences against their mean _________ mean difference, mercury-Galinstan - - - - - - 2 SDs from the mean Figure 5 Scatter plot of differences, at the groin site, against their mean (°F). The mean difference (mercury minus Galinstan) is 0.18°F, SD 0.79, and skewness -0.075. Groin Device differences against their means _________ mean difference, mercury-Galinstan - - - - - - 2 SDs from the mean Figure 6 Scatter plot of differences, at the rectal site, against their mean (°F). The mean difference (mercury minus Galinstan) is -0.06°F, SD 0.31, and skewness -1.04. Rectal Device differences against their means _________ mean difference, mercury-Galinstan - - - - - - 2 SDs from the mean Further examination of differences included repeatability analyses. If differences were normally distributed, the expectation was that 95% of all sample means of differences, based on this sample, would fall within this confidence interval. An analysis of the limits of agreement was computed based on the mean and two standard deviations (precision) of the difference between devices by sites [ 60 ]. According to Gardner and Altman [ 61 ] confidence intervals are a more useful and informative approach than P values because they present a range of values. For confidence intervals of difference scores, see Table 4 . However, caution is expressed due to the skewed histograms of difference scores. Thus, the assumption of Normality may be invalid [ 60 , 61 ]. Table 4 95% confidence intervals of difference scores by site Site Lower (°F) Upper (°F) Oral 0.142 0.265 Axilla 0.167 0.339 Groin 0.037 0.321 Rectal -0.111 0.111 Correlation is a quantification of the linear relationship between variables and does not examine the question of agreement. Often used to analyze thermometer reading accuracy, correlation is an estimate of how much two variables change in relation to one another. Perfect correlation does not mean perfect agreement [ 62 ]. Altman and Bland [ 63 ] identified this as a favorite approach to the comparison of two methods of measurement. Therefore, correlation statistics were established for the purpose of literature comparisons. Furthermore, human body within and between site agreement cannot be assumed. Thus, relationships between and among variables are represented with a correlation table (see Table 5 ). Table 5 Correlations between variables (two devices each site) Variable pair Pearson r Significance (2-tailed) N Oral Hg:Galinstan .929 <.001 119 Axilla Hg:Galinstan .886 <.001 120 Groin Hg:Galinstan .701 <.001 120 Rectal Hg:Galinstan .927 <.001 117 Demographic variables and mean temperature readings by site Race: Mean Oral .264 .004 119 Race: Mean Axilla .284 .002 120 Race: Mean Groin .300 .001 120 Race: Mean Rectal .227 .014 117 Race: age -.291 .001 119 Age: Weight -.269 .001 117 Age: Mean Oral -.308 .001 118 Age: Mean Axilla -.332 <.001 119 Age: Mean Groin -.077 .406 119 Age: Mean Rectal -.193 .037 117 No statistically significant correlations were found between difference scores and mean temperatures at each site. That is, variability of difference scores was not correlated with lower or higher temperature readings. Comparing demographic variables No statistically significant correlations were found for temperature readings and variables such as gender, weight, random right/left placement of devices (oral, axilla, groin), and room temperature. Race significantly correlated with age and mean temperature readings for each site. Age correlated significantly and negatively (the higher the age, the lower the weight) with weight and race (non-whites tended to be younger), as well as oral, axillary, and rectal mean temperature readings (the higher the age, the lower the reading). Discussion Discussion of site comparisons: Contradictions to the one degree Fahrenheit rule Tradition has dictated to lay and professional caregivers the one degree Fahrenheit rule. Many remember learning about the one degree Fahrenheit estimated difference: rectal site temperatures are about one degree higher than oral which is about one degree higher than axillary sites. As noted in Table 1 , this estimate "rule" could not be applied to either device. For the mercury-in-glass device, rectal was 0.64°F higher than oral, which was 0.39°F higher than groin/axillary sites. Rectal readings were only about one degree Fahrenheit higher than skin sites, not two degrees, as the "rule" would imply. Importantly, the Galinstan-in-glass readings also represented site differences that were different from the one-degree rule. Though rectal site temperature assessments were slightly less than one degree higher than oral (0.91°F above oral), skin sites were only about 1.3°F below rectal readings (not the expected two degree estimate). Discussion of device comparisons When comparing within-subject mean differences between the two low-tech temperature devices (BD mercury and Geratherm Galinstan), the smallest differences occurred at the rectal site; the largest differences were recorded at skin sites. Skin site variability concurs with findings from other studies [ 10 , 26 , 64 ]. Skin temperature sites, by their very nature, have innate differences, even with simultaneous side-to-side use and identical dwell times. Macro and microcirculation, fatty layers, muscle mass, and skin pockets may differ bilaterally. Unless the same site and time are measured, body temperature gradients become confounding factors [ 62 ]. As expected, simultaneous insertion of both thermometers into the moist and protected rectal cavity created a near identical temperature reading match. Prior to data collection, thermometers were swirling water-bath tested and those with readings greater than ± 0.2°F from water temperature were eliminated from the study. Thus, difference scores at or less than this measure can be deemed to be clinically insignificant. As noted in Table 2 , only axillary readings had a difference mean of greater than 0.2°F and this mean difference was 0.253°F. All mean difference scores were less than the described 0.2°C level reported by Fallis and Christiani [ 65 ] to be clinically significant. Mean difference scores were also less than the 0.36°F deemed clinically insignificant by authors Stephen and Sexton [ 66 ] and 0.2°C deemed clinically insignificant by Fulbrook [ 58 ] when he compared axillary and pulmonary artery temperature readings. All but the axillary mean difference scores were at or below the 0.2°F accepted variability for inclusion of human temperature assessment devices in clinical investigations [ 23 , 34 , 65 , 67 ]. Variability among mean difference scores was assessed in several ways. A box plot showing mean temperature reading differences and variability between mercury-in-glass and Galinstan-in-glass devices at four sites, was constructed (see Figure 1 ). Standard deviations were identified for difference scores by site (Table 3 ), along with a 95% confidence interval of the mean of difference readings between devices (see Table 4 ). The greatest variability between device readings by site occurred at the two skin sites. This greater variability at skin sites corresponds to findings from other researchers [ 22 , 53 , 68 ]. Ninety-five percent confidence intervals, for the mean of difference readings, were smallest at the rectal site and largest at the groin skin site. Correlations between the two thermometer types by site were strong and statistically significant. The strongest correlations were, again as expected, at the rectal and oral temperature sites. Human body asymmetry must be considered as a possible variable for groin and axilla sites. Room temperature was not a significant variable. This result is different from the work of other researchers who found that ambient temperatures significantly affected readings at various sites in infants[ 57 ] and various temperature levels in adults [ 52 ]. Discussion of demographic variables With these study participants, the fairly insignificant role played by gender differs from the work of Nagy [ 69 ], Nichols and Kucha [ 47 ] and Gillum [ 12 ]. In contrast, race was a significant variable for this study. Non-whites were more likely to be younger and have higher temperature readings at each site and with each device. Because subjects were all in-patients in an acute-care hospital setting, and the number of non-white subjects was small (N = 18; 15% of total) no conclusions should be made regarding this finding. However, the influence of race concurs with other investigations [ 11 , 12 ]; race may have a significant influence on body temperatures and needs to be studied further. Though the variable of weight was insignificant, age remained an important factor for temperature readings (both devices) at the oral, axillary, and rectal sites. The greater the age, the lower the temperature readings. This finding is consistent with the work of Frankenfield, et al. [ 8 ] and Howell [ 10 ]. Discussion of site comparisons: Contradictions to the one degree Fahrenheit rule Tradition has dictated to lay and professional caregivers the one degree Fahrenheit rule. Many remember learning about the one degree Fahrenheit estimated difference: rectal site temperatures are about one degree higher than oral which is about one degree higher than axillary sites. As noted in Table 1 , this estimate "rule" could not be applied to either device. For the mercury-in-glass device, rectal was 0.64°F higher than oral, which was 0.39°F higher than groin/axillary sites. Rectal readings were only about one degree Fahrenheit higher than skin sites, not two degrees, as the "rule" would imply. Importantly, the Galinstan-in-glass readings also represented site differences that were different from the one-degree rule. Though rectal site temperature assessments were slightly less than one degree higher than oral (0.91°F above oral), skin sites were only about 1.3°F below rectal readings (not the expected two degree estimate). Discussion of device comparisons When comparing within-subject mean differences between the two low-tech temperature devices (BD mercury and Geratherm Galinstan), the smallest differences occurred at the rectal site; the largest differences were recorded at skin sites. Skin site variability concurs with findings from other studies [ 10 , 26 , 64 ]. Skin temperature sites, by their very nature, have innate differences, even with simultaneous side-to-side use and identical dwell times. Macro and microcirculation, fatty layers, muscle mass, and skin pockets may differ bilaterally. Unless the same site and time are measured, body temperature gradients become confounding factors [ 62 ]. As expected, simultaneous insertion of both thermometers into the moist and protected rectal cavity created a near identical temperature reading match. Prior to data collection, thermometers were swirling water-bath tested and those with readings greater than ± 0.2°F from water temperature were eliminated from the study. Thus, difference scores at or less than this measure can be deemed to be clinically insignificant. As noted in Table 2 , only axillary readings had a difference mean of greater than 0.2°F and this mean difference was 0.253°F. All mean difference scores were less than the described 0.2°C level reported by Fallis and Christiani [ 65 ] to be clinically significant. Mean difference scores were also less than the 0.36°F deemed clinically insignificant by authors Stephen and Sexton [ 66 ] and 0.2°C deemed clinically insignificant by Fulbrook [ 58 ] when he compared axillary and pulmonary artery temperature readings. All but the axillary mean difference scores were at or below the 0.2°F accepted variability for inclusion of human temperature assessment devices in clinical investigations [ 23 , 34 , 65 , 67 ]. Variability among mean difference scores was assessed in several ways. A box plot showing mean temperature reading differences and variability between mercury-in-glass and Galinstan-in-glass devices at four sites, was constructed (see Figure 1 ). Standard deviations were identified for difference scores by site (Table 3 ), along with a 95% confidence interval of the mean of difference readings between devices (see Table 4 ). The greatest variability between device readings by site occurred at the two skin sites. This greater variability at skin sites corresponds to findings from other researchers [ 22 , 53 , 68 ]. Ninety-five percent confidence intervals, for the mean of difference readings, were smallest at the rectal site and largest at the groin skin site. Correlations between the two thermometer types by site were strong and statistically significant. The strongest correlations were, again as expected, at the rectal and oral temperature sites. Human body asymmetry must be considered as a possible variable for groin and axilla sites. Room temperature was not a significant variable. This result is different from the work of other researchers who found that ambient temperatures significantly affected readings at various sites in infants[ 57 ] and various temperature levels in adults [ 52 ]. Discussion of demographic variables With these study participants, the fairly insignificant role played by gender differs from the work of Nagy [ 69 ], Nichols and Kucha [ 47 ] and Gillum [ 12 ]. In contrast, race was a significant variable for this study. Non-whites were more likely to be younger and have higher temperature readings at each site and with each device. Because subjects were all in-patients in an acute-care hospital setting, and the number of non-white subjects was small (N = 18; 15% of total) no conclusions should be made regarding this finding. However, the influence of race concurs with other investigations [ 11 , 12 ]; race may have a significant influence on body temperatures and needs to be studied further. Though the variable of weight was insignificant, age remained an important factor for temperature readings (both devices) at the oral, axillary, and rectal sites. The greater the age, the lower the temperature readings. This finding is consistent with the work of Frankenfield, et al. [ 8 ] and Howell [ 10 ]. Conclusions For both mercury and Galinstan glass thermometers, rectal readings were higher than oral and oral readings were higher than skin site temperature assessments. This finding further verifies similarities between mercury and Galinstan and remains consistent with findings from other studies [ 1 , 26 , 29 , 30 ]. Mercury-in-glass fever thermometers are no longer available in many regions of the US and world. For this study and with these hospitalised participants, the mercury-free Galinstan-in-glass thermometer correlated strongly with the mercury thermometer and may be a practical alternative to mercury filled thermometer products. For adults, and with careful consideration of site differences and site competence, temperature sites to be considered include all investigated sites – rectal, oral, axillary, and groin. With proper use, non-invasive, non-mucus temperature sites may be safe alternatives to more invasive, less acceptable sites. Limitations of the study include an unblinded, convenience sample of hospitalised patients; although participants demonstrated a wide range of age, weight, and temperature readings. A further limitation is the small number of non-white participants and the greater number, by percent, of female and older volunteers. Implications Temperature assessment accuracy is critically important. False high readings may lead to expensive and painful diagnostic studies and medical interventions. False low readings may lead to greater morbidity and mortality. Accurate readings are critical to the rapid, effective, and precise patient diagnosis process. All temperatures should be reported and recorded with the added notation of site and device. Patient-specific temperature trending needs to be consistent by site. However, healthcare professionals can no longer assume the one-degree Fahrenheit rule. Product information will need to be clearly conveyed, in professional and lay literature sources, regarding mean differences among temperature sites in humans. Vital sign chart forms (electronic and paper) need to reflect temperature site as well as device, time, day, and reading in Fahrenheit or Celsius. Health care professionals will need to assist lay caregivers and self-care patients in accurately understanding, recording, and reporting body temperatures. When tracking temperature changes, site and device must be consistent. Low-tech glass thermometers present the advantage of being portable, storable, lightweight, sterilizable, low in cost, and easy to use. Battery and power source competence is never a question. Person-specific low-tech glass thermometers prevent cross contamination of biological agents and must be considered as essential. Thus, low-tech Galinstan-in-glass thermometers may be an appropriate replacement for mercury filled devices. Further Research Study findings draw attention to needed areas of research for better understanding of the influence of variables such as device, site, race, and age on body temperature assessments. Because comparisons of low-tech Galinstan-in-glass and mercury-in-glass body temperature devices are not well described in the literature, it will be essential to replicate this study for comparison of bias and 95% CI of the bias between these two devices by site. The recording of temperatures using all four sites over time, with a sample of ill and well individuals, would add to knowledge of how different variables affect temperature readings. The issue of race must be addressed as a potential body temperature variable. Furthermore, age, as identified in this study, may be a critically important indicator relative to how healthcare professionals define and treat body temperature alterations. The groin temperature site, though demonstrating greater mean difference variability and not well studied in adults, may be a good alternative to rectal site temperature assessment. This conclusion, however, cannot be made without further investigation. Use and study of low-tech Galinstan-in-glass thermometers during biological epidemics would provide additional information regarding the practical application and efficacy of this device during times of extreme international need. Further Research Study findings draw attention to needed areas of research for better understanding of the influence of variables such as device, site, race, and age on body temperature assessments. Because comparisons of low-tech Galinstan-in-glass and mercury-in-glass body temperature devices are not well described in the literature, it will be essential to replicate this study for comparison of bias and 95% CI of the bias between these two devices by site. The recording of temperatures using all four sites over time, with a sample of ill and well individuals, would add to knowledge of how different variables affect temperature readings. The issue of race must be addressed as a potential body temperature variable. Furthermore, age, as identified in this study, may be a critically important indicator relative to how healthcare professionals define and treat body temperature alterations. The groin temperature site, though demonstrating greater mean difference variability and not well studied in adults, may be a good alternative to rectal site temperature assessment. This conclusion, however, cannot be made without further investigation. Use and study of low-tech Galinstan-in-glass thermometers during biological epidemics would provide additional information regarding the practical application and efficacy of this device during times of extreme international need. List of abbreviations Oral temperature site: Oral Axillary temperature site: Axilla Groin temperature site: Groin Rectal temperature site: Rectal Becton Dickinson Corporation: BD Standard deviation: SD Number of participant readings: N Mercury: Hg Galinstan: Gal O: Oral X: Axillary G: Groin R: Rectal Competing interests Thermometer devices and sheaths were donated by BD (mercury-in-glass) and RG Medical Diagnostics (Galinstan-in-glass). Funding for this investigation was received by the Oregon Health & Science University (OHSU) from two supporting sponsors: Geratherm Medical Diagnostic Systems and RG Medical Diagnostics. Funding requests were initiated by OHSU staff. Grant sources of the study had no role in study design, data collection, data analysis/interpretation, or report writing. Authors' contributions The principal investigator (manuscript author) formulated and implemented all aspects of this study and report. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements The author of this investigation wishes to acknowledge the financial support, as received by OHSU, of two study sponsors: Geratherm Medical Diagnostic Systems and RG Medical Diagnostics. The donation of thermometer devices from BD Corporation and RG Medical Diagnostics is also acknowledged and sincerely appreciated. Thanks go to three research assistants: Deborah James, Aubrey Sharp, and Denise Stiltner. Finally, sincere appreciation is expressed to all of the staff and volunteering patients of the Merle West Medical Center, without whom this investigation could not have taken place.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378188/

Animal NLRs provide structural insights into plant NLR function

Abstract Background The plant immune system employs intracellular NLRs (nucleotide binding [NB], leucine-rich repeat [LRR]/nucleotide-binding oligomerization domain [NOD]-like receptors) to detect effector proteins secreted into the plant cell by potential pathogens. Activated plant NLRs trigger a range of immune responses, collectively known as the hypersensitive response (HR), which culminates in death of the infected cell. Plant NLRs show structural and functional resemblance to animal NLRs involved in inflammatory and innate immune responses. Therefore, knowledge of the activation and regulation of animal NLRs can help us understand the mechanism of action of plant NLRs, and vice versa. Scope This review provides an overview of the innate immune pathways in plants and animals, focusing on the available structural and biochemical information available for both plant and animal NLRs. We highlight the gap in knowledge between the animal and plant systems, in particular the lack of structural information for plant NLRs, with crystal structures only available for the N-terminal domains of plant NLRs and an integrated decoy domain, in contrast to the more complete structures available for animal NLRs. We assess the similarities and differences between plant and animal NLRs, and use the structural information on the animal NLR pair NAIP/NLRC4 to derive a plausible model for plant NLR activation. Conclusions Signalling by cooperative assembly formation (SCAF) appears to operate in most innate immunity pathways, including plant and animal NLRs. Our proposed model of plant NLR activation includes three key steps: (1) initially, the NLR exists in an inactive auto-inhibited state; (2) a combination of binding by activating elicitor and ATP leads to a structural rearrangement of the NLR; and (3) signalling occurs through cooperative assembly of the resistosome. Further studies, structural and biochemical in particular, will be required to provide additional evidence for the different features of this model and shed light on the many existing variations, e.g. helper NLRs and NLRs containing integrated decoys.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4099211/

Serum adenosine deaminase activity in cutaneous anthrax

Background Adenosine deaminase (ADA) activity has been discovered in several inflammatory conditions; however, there are no data associated with cutaneous anthrax. The aim of this study was to investigate serum ADA activity in patients with cutaneous anthrax. Material/Methods Sixteen patients with cutaneous anthrax and 17 healthy controls were enrolled. We measured ADA activity; peripheral blood leukocyte, lymphocyte, neutrophil, and monocyte counts; erythrocyte sedimentation rate; and C reactive protein levels. Results Serum ADA activity was significantly higher in patients with cutaneous anthrax than in the controls (p0.05) ( Table 1 ). Blood leukocyte counts were significantly higher in the patients with cutaneous anthrax compared with the controls (p=0.037). Although lymphocyte and neutrophil counts were higher in the patient group, there were no statistically significant differences (p>0.05). ESR and CRP levels were significantly higher in the patients with cutaneous anthrax compared with the controls (p0.05). Discussion We evaluated the serum ADA activity in patients with cutaneous anthrax. In the diagnosis of cutaneous anthrax, the clinical presentation of the disease and a history of close contact with sick animals are very important. If a patient has a typical malignant pustule or malignant edema and has had a history of contact with animals, the diagnosis may be easy. The clinical laboratory diagnosis of cutaneous anthrax is generally established by conventional microbiological methods, such as bacterial cultures and directly gram staining smears of clinical specimens [ 15 ]. However, the clinical presentation could be atypical and the patient's recollection of contact with animals may not be accurate, or the patient may neglect to supply this information. In these situations, the diagnosis of cutaneous anthrax might be difficult. PCR, immunohistochemistry, laboratory parameters (e.g., CRP, ESR, and white blood cell [WBC]), and anthracin skin tests may be helpful in the diagnosis of anthrax [ 16 , 17 ]. Inflammatory markers such as ESR and WBC count were found to be high in some patients [ 18 ]. However, the elevation of CRP levels in patients with cutaneous anthrax, as we found, has not been reported previously. ADA is a key enzyme in purine metabolism; it converts adenosine and deoxyadenosine to inosine and deoxyinosine by irreversible deamination [ 19 ]. ADA is widely distributed in human tissues (its highest activity being in lymphoid tissues), and it is primarily associated with T-lymphocyte proliferation [ 20 ]. Although ADA has been considered a nonspecific marker of T-cell activation, the precise mechanisms by which serum ADA activity is altered have not been clearly identified [ 21 ]. High levels of serum ADA have been reported in infectious diseases such as viral and bacterial pneumonia, HIV infection, extra-pulmonary and pulmonary tuberculosis, H. pylori , acute appendicitis, visceral leishmaniasis, and mononucleosis [ 11 , 12 , 22 , 23 ] and might have diagnostic value. Moreover, circulating levels of ADA have been shown to increase in several inflammatory conditions, including Behçet's disease, systemic lupus erythematosus, rheumatoid arthritis, and certain malignancies, especially those of hematopoietic origin [ 24 – 34 ]. Conclusions Although to our knowledge this is the first study to investigate serum ADA enzyme activity in patients with cutaneous anthrax, it has a major limitation. Our study design did not allow us to investigate whether ADA activity is uniquely associated with cutaneous anthrax. Ideally, a third group of sick patients, with elevated CRP or ESR, but without lymphocytes or ADA levels, could have been included to highlight the issue. However, in the current study, we observed that serum ADA enzyme activity was significantly higher in patients with cutaneous anthrax than in the healthy controls. Moreover, we found that acute cutaneous anthrax patients had increased WBC, CRP levels, and ESR compared with the control group. These results suggest that ADA contributes to the inflammation seen in cutaneous anthrax and might be used in the clinical setting.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9495858/

Alternative Routes of Administration for Therapeutic Antibodies—State of the Art

Background: For the past two decades, there has been a huge expansion in the development of therapeutic antibodies, with 6 to 10 novel entities approved each year. Around 70% of these Abs are delivered through IV injection, a mode of administration allowing rapid and systemic delivery of the drug. However, according to the evidence presented in the literature, beyond the reduction of invasiveness, a better efficacy can be achieved with local delivery. Consequently, efforts have been made toward the development of innovative methods of administration, and in the formulation and engineering of novel Abs to improve their therapeutic index. Objective: This review presents an overview of the routes of administration used to deliver Abs, different from the IV route, whether approved or in the clinical evaluation stage. We provide a description of the physical and biological fundamentals for each route of administration, highlighting their relevance with examples of clinically-relevant Abs, and discussing their strengths and limitations. Methods: We reviewed and analyzed the current literature, published as of the 1 April 2022 using MEDLINE and EMBASE databases, as well as the FDA and EMA websites. Ongoing trials were identified using clinicaltrials.gov. Publications and data were identified using a list of general keywords. Conclusions: Apart from the most commonly used IV route, topical delivery of Abs has shown clinical successes, improving drug bioavailability and efficacy while reducing side-effects. However, additional research is necessary to understand the consequences of biological barriers associated with local delivery for Ab partitioning, in order to optimize delivery methods and devices, and to adapt Ab formulation to local delivery. Novel modes of administration for Abs might in fine allow a better support to patients, especially in the context of chronic diseases, as well as a reduction of the treatment cost. 1. Introduction Over the past 30 years, therapeutic antibodies (Abs) have been found to be valuable therapeutics [ 1 ]. A total of 6 to 12 new Abs are approved by the U.S. FDA and/or the EMA each year, and new molecules are reaching clinical trials every month [ 2 ]. Therapeutic antibodies are used in the treatment of numerous diseases, including infection, cancer, and autoimmune disorders, in which they have already demonstrated their efficacy [ 3 , 4 ]. The success of Abs is due to (I) a high level of specificity and affinity to their target antigen, (II) a favorable safety profile, and (III) a unique pharmacokinetic profile, supporting a longer half-life as compared to other drugs [ 5 ]. These characteristics have allowed Abs to move rapidly from pre-clinical studies to clinical trials, as observed during the COVID-19 pandemic [ 6 ]. From the historical full-length antibody, molecular engineering has enabled the development of multiple and diverse Ab formats, including multi-specific Abs, fragments, and conjugated Abs that are now extensively evaluated in clinical trials [ 7 ]. Due to their intrinsic biological properties, Abs have a specific interconnected pharmacokinetic and pharmacodynamic profile, which influence their absorption and biodistribution after administration [ 5 ]. Abs pharmacokinetics is linked to their route of administration [ 8 ]. Historically, Abs were delivered via intravenous (IV) injection. Nowadays, the subcutaneous (SC) route is often used for chronic diseases [ 9 ]. These systemic routes have the advantage of allowing the delivery of large amounts of Abs and to enable rapid systemic bioavailability. However, one of their drawbacks is the limited distribution from the site of injection via the blood flow to the diseased organ, which may result in limited Ab amount in the vicinity of the target antigen. Ultimately, this necessitates the injection of a high dose, which may be associated with potential toxicity and cost issues. Accumulating preclinical evidence has driven researchers to reconsider Abs' route of administration in order to maximize their therapeutic index. Alternative delivery methods, addressing Abs to the disease site (e.g., delivery of Abs in the lung to treat respiratory pathologies [ 10 ], or inside a tumor [ 11 ]) have emerged and progressed to the clinical trial stage. In theory, a higher concentration of the antibody at the target site should improve the therapeutic response, while lowering the concentration in neighboring healthy tissues, resulting in reduced side effects. Here, we reviewed and analyzed the literature published as of the 1 April 2022, describing the different routes of administration used for the delivery of Abs. The IV route has not been considered in this review, being the subject of many reviews elsewhere [ 12 , 13 ] ( Figure 1 ). Each section highlights the basics of the administration route, its application, the potential hurdles, and, when applicable, describes the Abs approved or under review by the regulatory agencies [ 14 , 15 , 16 ], and the molecules in the late stages of clinical trials. The publications were identified by searching MEDLINE and EMBASE databases. Ongoing clinical trials were found on https://www.clinicaltrials.gov/ (accessed on 1 April 2022) [ 17 ]. Our research strategy was based on the use of the keywords "Ab", "mAbs" "therapeutic antibody", "monoclonal antibody" "administration", "delivery", "injection", "barriers" and "clinical trial", as general criteria, and the keywords "subcutaneous", "intramuscular", "intravitreal", "airways", "inhalation", "intra-tumoral", "peri-tumoral", "intra-articular", "oral", "intra-cerebral", "intranasal", "topical" for specific information on the route of administration. 2. Routes of Administration for Therapeutic Antibodies 2.1. The Subcutaneous Route: The Most Popular after IV Injection 2.1.1. Fundamentals Related to the SC Route After IV injection, the second most popular route for the delivery of antibodies is the subcutaneous (SC) route. It consists in the injection of Abs using a syringe and needle under the skin of patients at an angle of 90 °C, thus bypassing the barrier formed by the epidermis and dermis layers [ 18 ]. The choice of the anatomical site is important due to differences in dermal thickness which may reduce the absorption of the injected Abs. Nowadays, around 30% of the approved Abs are delivered by SC injection ( Table 1 ). If the delivery of drugs, mainly opioids, by SC administration, has been in practice since the middle of the 19th century, the administration of Abs by this route is recent. The first subcutaneous injected Ab was Adalimumab, used in the treatment of rheumatoid arthritis and approved by the FDA in 2002, and by the EMA in 2003 [ 19 ]. After this first success, and particularly since 2009, the number of marketed Abs delivered by the SC route has significantly increased. It is noteworthy that SC administration is already the standard route in the treatment of chronic diseases such as rheumatoid arthritis. Indeed, it allows self-administration and improves patients' compliance. The SC route is mainly used for the delivery of Abs targeting interleukins such as TNF-α, critically involved in the development of rheumatoid arthritis (Adalimumab, Golimumab, Certolizumab pegol) or cytokine receptors such as the IL-17a receptor, involved in the progression of psoriasis (Brodalumab, Secukinumab, Ixekizumab) [ 20 ]. The development of Abs intended for a subcutaneous injection necessitates understanding the physiology of the skin. After injection, the drug reaches the hypodermis interstitial space between the dermis and the deep fascia covering the muscle tissue. This layer is composed of adipose tissue, blood, lymph vessels, and resident immune cells such as fibroblasts and macrophages. All components are enmeshed in an extracellular matrix (ECM) network, rich in collagen, elastin, and glycosaminoglycans [ 21 ]. To pass into the systemic compartment (via either the blood capillaries or lymphatic vessels), and thus reach their target, Abs have to diffuse through the ECM, which constitutes both a physical and chemical barrier. The fate of the Ab is dictated by its size, charge, and affinity with transporters. Despite the presence of the positively charged collagen fibrils, the hypodermis interstitial space displayed an overall negative charge due to important concentrations of hyaluronic acid and chondroitin sulfate, two major glycosaminoglycans of the hypodermis ECM, which are negatively charged. The global negative charge of ECM favors the transport of negatively charged drugs thanks to electrostatic repulsion [ 22 ]. However, the majority of therapeutic Abs are positively charged. Once the ECM is traversed, drugs may enter the systemic circulation by two different mechanisms. Molecules smaller than 16 kDa diffuse directly into the bloodstream, taking advantage of the permeability of the vascular endothelium [ 22 ]. However, Abs, along with drugs with a higher molecular weight, are absorbed by convection into lymphatic vessels. Thus, the subcutaneous route is particularly interesting to target lymphoid cells and the molecules they secrete. Abs in the lymphatic vessels pass to larger lymphatics and then reach the blood vascular system, from where they diffuse throughout the body. If the development of Abs for subcutaneous injection is quite challenging, multiple factors explain the attractiveness of this route as compared to other parenteral ones. In the hypodermis, the walls in the fat lobule are thinner than those in the dermis, which facilitates the diffusion of drugs into blood capillaries [ 23 ]. Moreover, the absence of antigen-presenting cells in the hypodermis, usually present in the top layers of the skin (Langerhans cells and/or macrophages), may decrease the immunogenicity of the antibody. Thus, an increasing number of Abs delivered by SC are being developed, allowing a quicker delivery time of administration as compared to IV injection, enabling longer dosing intervals and, in fine, reducing the frequency of administration. In addition, SC administration is less invasive and painful than IV injection [ 22 ] and allows self-delivery at home [ 24 ]. Thus, the subcutaneous route may improve patient comfort and compliance, which is critical for the treatment of chronic diseases, and may be associated with a reduction in treatment costs, consuming fewer healthcare resources. 2.1.2. Abs Approved for Subcutaneous Delivery Abs approved for subcutaneous administration must be formulated at a high concentration, thus necessitating a careful control of their stability and formulation viscosity. Different strategies have been considered to ensure the efficient absorption and bioavailability of Abs after hypodermis injection. They include, but are not limited to, the increase in delivered Abs concentration (e.g., SC administration limiting the injection volume to 1–2 mL [ 25 ]), the development of specific formulations to reduce physical and chemical destabilization (e.g., the use of polysorbate preventing aggregation and particle formation [ 26 ]) and the development of novel administration devices (e.g., the autoinjectors enabling a faster delivery for larger concentrations of Abs [ 27 ]). Those strategies have led to the approval of around 40 different Abs ( Table 1 ). A major concern for SC injection is the isoelectric point (pI) of Abs, found between 7 and 9, making Abs positively charged at the physiological pH. A study by Bumbaca Yadav et al., showed that positively charged Abs present a reduced bioavailability by 31%, while their negatively charged counterparts demonstrate enhanced bioavailability up to 70% after SC administration [ 30 ]. Another study found that the reduced bioavailability of Abs delivered subcutaneously is due to their interaction with ECM components, thus limiting the amount of Ab reaching the vascular compartment [ 31 ]. Moreover, the overall negative charge of the hypodermis interstitial space increases the interaction of ECM components with water molecules resulting in a low hydraulic conductivity and limiting the subcutaneous injection volume [ 32 ]. To circumvent this serious issue, one strategy consists in combining Abs with hyaluronidase. Hyaluronidase degrades hyaluronic acid, lowering the amount of negatively charged molecules and enhancing the bioavailability of Ab after SC injection [ 33 ]. Moreover, combining Abs with hyaluronidase may facilitate bulk fluid flow and improve the pharmacokinetic profile after SC injection [ 34 ], as demonstrated in multiple clinical studies [ 35 , 36 , 37 ]. Based on these results, the regulatory agencies approved Rituximab, Trastuzumab, and Daratumumab in combination with recombinant human hyaluronidase (rHuPH20), in 2017 (Rituxan Hycela/mAbThera s.c), 2019 (Herceptin Hylecta), and 2020 (Darzalex Faspro), respectively. These encouraging results have fueled the repurposing of Abs approved for delivery by IV injection to this novel modality of administration. Notably, Tocilizumab ((Ro)-Actemra), an antibody used in the treatment of rheumatoid polyarthritis, was formulated for the SC route, in response to patient demand, and to allow a less invasive route for a treatment usually delivered monthly [ 38 ]. It is noteworthy that multiple studies have demonstrated the absence of significant differences between the IV and SC routes of administration for Abs, thus making SC a legitimate option for patients [ 37 , 38 ]. 2.1.3. Abs in Clinical Development for the SC Route The clinical development of subcutaneously-delivered Ab concerns either de novo development, expansion of the disease target, and/or new formulation for already approved Abs. A high number of those Abs are currently found in clinical trials. Here, we listed subcutaneously delivered Abs either in active phase 3 trials or under review by regulatory agencies ( Table 2 ). Novel developments include Fasinumab, a recombinant fully human IgG4, targeting the nerve growth factor (NGF) and evaluated for pain relief in patients suffering from osteoarthritis (OA). A phase 2b/3 trial showed that Fasinumab provides improvement in OA pain and motor function, even in patients that are non-responsive to analgesics [ 39 ]. The drug approval is pending an evaluation by the FDA (NCT03161093; NCT02683239). In the meantime, studies are also investigating lower doses of Fasinumab in patients with knee or hip OA. The repurposing of IV delivery approved Abs for SC application in a disease context different than the original approval is also investigated. For example, Ofatumumab (Arzerra ® , Novartis) is a monoclonal antibody targeting CD20 and causing cytotoxicity in cells expressing CD20. It was first approved in 2010 for the treatment of certain chronic lymphocytic leukaemia by IV injection, and has been reformulated (Kesimpta) for SC administration and evaluated in patients with relapsed multiple sclerosis. Two ongoing phase 3 trials, OLIKOS (NCT04486716) and ARTIOS (NCT04353492) are evaluating the efficacy, safety, and tolerability of the SC drug in patients with relapsing multiple sclerosis, all of whom are transitioning from a CD20 Ab therapy (Rituximab or Ocrelizumab), or dimethyl fumarate therapy [ 40 ]. Many Abs have been developed or repurposed as emergency treatments since the beginning of the SARS-CoV2 pandemic, and target either the virus or the host inflammatory response. Among them, REGEN-COV2 comprising Casirivimab and Imdevimab has been approved for emergency use by IV infusion and is now undergoing regulatory review, for its use by SC administration to treat and prevent SARS-CoV-2 infection in non-hospitalized individuals [ 41 ]. The first phases of its clinical investigation showed a significant efficacy with improved survival. A phase 3 trial is also in progress to evaluate its potency for the prevention of COVID-19 in immunocompromised patients (NCT05074433). 2.1.4. Conclusion and Perspectives Regarding the Subcutaneous Route The relevance of the SC route for the administration of Abs has been illustrated by several clinical successes. However, the bioavailability of Abs delivered subcutaneously remains difficult to predict. Advances in preclinical models would be necessary to investigate the fate of Abs at the SC injection site and their diffusion into the blood/lymphatic compartment. Interestingly, novel in vitro tools have been developed to predict the in vivo absorption of biopharmaceuticals after SC injection, by modeling the environmental changes an Ab will experience after its injection. Among those, Scissor (Subcutaneous Injection Site Simulator) device provides a tractable method to study the fate and the pharmacokinetics of biopharmaceuticals once in the hypodermis [ 42 , 43 ]. Nevertheless, as no in vitro model is yet accurate enough, the pharmacokinetics of Abs still relies on in vivo studies. Formulating Abs for the SC route remains challenging, as formulations need to afford high concentration with low viscosity, aggregation and immunogenicity [ 22 ]. Biotechnological platforms have been developed to support the switch from intravenous infusion to SC delivery, using proprietary excipients and proteins, allowing the reduction of ionic strength and hydrophobicity areas of the molecule, thus limiting aggregation when the Ab is highly concentrated. 2.2. The Intramuscular Route: The Favorite Choice for Infectious Diseases? 2.2.1. Basics of the Intramuscular Route If the intramuscular route (IM) is often used for the administration of vaccines, this modality is rarely considered for the delivery of Ab. The IM injection consists in administering a drug deep into the muscle mass, where the blood supply allows a rapid and better absorption than the subcutaneous route. As for the SC route, the drug is administered via a syringe and needle into the skin at an angle of 90°. However, where the SC route uses small length- and diameter-needles, IM injection requires longer needles—2.5 to 4 cm—to bypass the different layers of the skin. The volume of administration is relatively low ~2–5 mL, depending on the muscle chosen for the injection, necessitating a more concentrated product than for IV administration. The preferred injection sites are the deltoid muscle in the upper arm, the vastus lateralis found at the front of the thigh toward the outside of the leg, especially for IM injection in young children [ 44 ], the ventrogluteal muscle of the hip, the safest site for adults and children older than 7 months, and finally the dorsogluteal muscles of the buttocks, albeit less used nowadays due to potential injury to the sciatic nerve. The pharmacokinetics of a drug injected by IM follows a specific sequence: dissolution rate, solvent supply, phase transfer, and diffusion to the vascular system. Therefore, injection depth is an important parameter which influences the absorption rate of the drug after IM injection. Consequently, a too superficial injection will deliver the drug either SC, or into the fat layer, retarding the action of the drug. The absorption will also depend on the muscle mass and its vascularization. Patients with muscular atrophy see a delay in drug absorption, as well as an increase in the risk of neurovascular complications. These anatomical parameters also influence the rapidity of action of the drug [ 44 ]. 2.2.2. Ab Approved for Delivery by the IM Route One of the most threatening pathogens responsible for lower respiratory tract infection in children is the respiratory syncytial virus (RSV). Nearly every infant develops an RSV infection during their childhood. A total of 60% of them have an immune system mature enough to control the infection [ 45 ]. However, between 15 and 40% of infants, especially preterm infants, develop a more serious airway infection, which may eventually lead to bronchiolitis and pneumonia. Although no vaccine or specific RSV treatment exist to treat the infection (apart from general anti-viral therapy), a passive immunization may limit the infection in young infants with high risk factors. In 1998, the FDA approved Palivizumab (Synagis) [ 46 ], a humanized monoclonal IgG1 antibody targeting the glycoprotein F on the RSV virus, which is responsible for membrane fusion and infection of the host cell. Intramuscular injection of palivizumab is recommended for premature infants and young children with heart or lung comorbidities, and injections usually start before the expected RSV epidemic season [ 47 , 48 ]. Intramuscular delivery was chosen to facilitate delivery and limit invasive risks to the young recipients of the treatment. Aggregated clinical data have established the half-life of Palivizumab administered via intramuscular injection to be around 17–27 days, with a mean of 20 days, in infants younger than 24 months. As such, a monthly administration during the epidemic season is necessary to protect effectively against RSV. Palivizumab prophylaxis has shown a significant decrease in the hospitalization, ICU stay, and mortality [ 49 , 50 ]. Unfortunately, its high cost limits the number of children who benefit from this prophylaxis protection, especially in low-income countries [ 51 ]. Although Palivizumab is not licensed for the treatment of RSV disease, it has paved the way to the clinical use of intramuscular injection, and has shown some potential to facilitate everyday care, promoting a less invasive route for patients, in particular infants, in whom IV injection is more complicated [ 52 ]. 2.2.3. Abs in Clinical Development Delivered by the IM Route The IM injection allows a more rapid absorption and onset of action compared to SC delivery: mathematical in silico models associate the IM route with higher drug concentration on target and shorter time to reach the peak of concentration as compared to SC delivery. This route benefits from the long experience of vaccine administration. As shown below, several anti-infective Abs delivered by IM administration are close to reaching patients ( Table 3 ). In 2021, Sotrovimab (Vir Biotechnology—GlaxoSmithKline) obtained an emergency use authorization (EUA) from the FDA for the treatment of mild-to-moderate COVID-19. However, since May 2022, and due to its inefficacy against the BA.2 variant of SARS-CoV-2, its authorization has been revoked [ 53 ]. A phase 3 trial (COMET-TAIL, NCT04913675) which has established that the intramuscular injection of Sotrovimab had a similar clinical effectiveness as compared to an IV injection is still active. SYN023, a therapeutic cocktail comprising two monoclonal Abs, CTB011 and CTB012, is under development for post-exposure prophylaxis to rabies. Nowadays, the standard of care treatment after rabies infection is an on-site (wound injection) and IM administration of anti-rabies immunoglobulin (RIG), called post-exposure prophylaxis, followed by four doses of the vaccine, delivered by IM or intradermal routes. The full dose of RIG facilitates rapid protection until the immune system can produce its own immunoglobulins thanks to the vaccination. However, the current RIG products are limited and expensive, thus their use is limited in high-burden countries [ 54 ]. SYN023, administered by IM injection, targets protein residues found on human rabies virus, and displays a neutralization capacity equivalent or superior to RIGs, even at a lower dose [ 55 ]. The ongoing phase 3 trial will provide additional information regarding the efficacy of SYN023 in populations at risk of rabies infection. Ibalizumab-uiyk (Trograzo) is an anti-HIV monoclonal antibody, approved in 2018, for the treatment of patients with resistant forms of AIDS by IV injection. It targets CD4 protein expressed by T cells, inhibiting the entry of HIV. As a "first class medication" (defined by the FDA), the use of Ibalizumab-uiyk needs to be expanded across the world and for patients reluctant to receive IV injection or where the access to medical facilities and personnel qualified to administer IV injections is limited. A phase 3 is currently evaluating the IM route in comparison to IV injection, notably in terms of safety, pharmacokinetics, and limiting the spread of infection (NCT03913195). 2.2.4. Conclusions and Perspectives on the Intramuscular Route The IM injection is usually considered as a rescue route of administration when other ways to deliver drugs are not appropriate. For example, drugs inducing vein irritation or sensitive to oral digestion have been moved from IV and per os injection, respectively, to IM injection. Among other advantages, IM administration is associated with a quicker and more uniform absorption of the drug as compared to other routes, and it is considered to be as efficient and potent as IV injection, with less invasive characteristics. Even with these potential advantages over other routes of administration, the IM route is still underestimated for Abs. The development of depot injections may represent an opportunity for the expansion of the IM route. Depot injections are a slow-release form of medication usually implanted in the muscle. They are already used for neuroleptic drugs (fluphenazine), prolonging their pharmacological effect. Moreover, they allow a sustained delivery over time [ 56 ] which may be of particular interest regarding the treatment of chronic diseases. 2.3. The Intraocular Route: An Invasive Route for Ophthalmic Disorders 2.3.1. Principles Related to the Intraocular Delivery of Abs With the population aging, the prevalence of ophthalmic disorders has increased over the last 20 years and is estimated to reach several hundred million in the next decades [ 57 ]. The most prevalent age-related eye disease is macular degeneration (AMD), accounting for 196 million patients, in 2020. This continuously evolving disease affecting the retina leads to near blindness, due to the overexpression of vascular endothelial growth factor (VEGF), and has no curative treatment. During AMD, VEGF expression is abnormally increased promoting the development of microvessels, leading to micro-hemorrhage and the progression of the disease. Drug delivery to the retina is challenging, and various routes of administration have been considered to circumvent the complexity of the eye anatomy and improve the therapeutic index of drugs. The eye comprises an anterior and a posterior part. The anterior part, closer to the external environment, composed by the cornea, the conjunctiva, the ciliary body, the aqueous humor, lens, and the lachrymal system, forms a static barrier which prevents the access of foreign particles to the eye. Topical administration on this side results in low bioavailability of the drug (less than 3%) inside the eye, and an even lower in the posterior part [ 58 , 59 ]. Penetration enhancers may be required to improve topical administration [ 60 ]. The posterior part encompasses the vitreous humor, retina, sclera, and choroid. The main barrier on this side is due to the cornea, a negatively charged tissue repulsing negatively charged drugs [ 61 ]. The intra-ocular space is also protected by two additional obstacles, the blood-aqueous, and the blood-retinal barriers [ 62 ], both composed of tight junctions which limit the transfer of drugs from the blood compartment to the eye, especially considering high molecular weight biotherapeutics. In this context, the local delivery of Ab, directly in the internal parts of the eye by intravitreal injection has been developed to provide a high amount of drug in the retina and the vitreous compartments. The intravitreal administration is an invasive procedure consisting in the injection of the Ab, posterior to the limbus, thanks to an incision through the sclera at a specific angle of 30°. The only approved syringe systems for intravitreal injection are the ones already prefilled with Ab. Once the needle is in position, the Ab solution (with a limited volume, ~100–200 µL) is slowly applied into the vitreous cavity, avoiding damage to the retinal surface [ 63 ]. 2.3.2. Approved Abs by Intravitreal Administration Up to now, three Abs delivered by intravitreal injection have been approved for the treatment of AMD and equivalent ophthalmic diseases ( Table 4 ). Ranibizumab (Lucentis), a humanized Fab fragment, [ 64 ] and Brolucizumab (Beovu), a humanized single-chain antibody fragment, both targeting all isoforms of VEGF-A [ 65 ] have been approved in 2006/2007 and 2019/2020, respectively. Recently, Faricimab (Vabysmo), a bispecific antibody targeting both VEGF-A and ANG-2 [ 66 ], has been approved by the FDA, for the treatment of AMD and diabetic macular edema. All these Abs aim at blocking the neo-vascularization, which is the main pathological process associated to retinal diseases [ 67 ]. It is noteworthy that Bevacizumab (Avastin), the first anti-VEGF-A antibody approved for cancer treatment, was also considered as an off-label indication for AMD by intravitreal injection, due to its more affordable price [ 68 ]. Unfortunately, it did not attain the required professional or political consensus to obtain final approval [ 69 ]. The pharmacokinetics of these Abs has been extensively studied. In aqueous humor, Bevacizumab and Ranibizumab were shown to have a half-life of around 10 days, and 7 days, respectively [ 70 , 71 ]. Abs concentration peaked on the first day of injection, and then rapidly declined. The short intravitreal half-lives implied frequent injections to ensure optimal effect, increasing the potential risks of side-effects. Interestingly, Faricimab demonstrated longer duration of action in a phase 3 clinical trial, with a sustained efficacy over 16 weeks post-injection [ 72 ]. To better understand the PK-PD of Abs after intravitreal administration, Mazer et al. developed a mechanistic model of intravitreal pharmacodynamics of anti-VEGF in the eye and demonstrated the interrelationship between the half-life of the Ranibizumab and the in vivo VEGF kD [ 73 ]. They also showed that the ocular Ab t1/2 was proportional to the hydrodynamic radius of the antibody, and the radius of the vitreous globe. Thus, the biodistribution and absorption of Ab depends on the physiological state of the eye, with a potential decrease in Abs efficacy for older eyes, or eyes impaired with either globe abnormalities or underlying pathologies (e.g., myopia, or hypermetropia) [ 74 ]. 2.3.3. Abs in Clinical Development for Intravitreal/Intraocular Delivery The global aging of the human population accounting for an increased prevalence of AMD, or retinal vein occlusion along with the effectiveness of intravitreal administration, has paved the way for further developments of intravitreally/intraocularly-delivered Abs products ( Table 5 ). It is noteworthy that several ongoing trials are further evaluating currently approved Abs delivered by the intravitreal route ( Table 4 ) to either identify their long-term effects in patients with AMD (NCT04777201) or evaluate their efficacy in other ophthalmic diseases (NCT04740905). Notably, Faricimab, after being recently approved for AMD is currently being investigated in macular edema and branch retinal occlusion. KSI-301 is a humanized antibody against VEGF-A conjugated to a biopolymer, under investigation for the treatment of different ophthalmic disorders [ 75 , 76 ]. Apart from its excellent safety and better efficacy than the current treatment using the fusion protein Aflibercept, the phase 1 trial showed longer durability, allowing patients to achieve treatment-free intervals of 4 to 6 months. Few novel drugs delivered by intravitreal injection are currently being developed. However, biosimilars are currently under investigation. HLX04-O, a biosimilar of Bevacizumab, is a monoclonal antibody under development by Shanghai Henlius Biotech, targeting VEGF to treat AMD. Contrary to the original molecule, this biosimilar has been developed de novo for intraocular administration. Early-stage clinical trials, analyzing the safety and toxicity of the molecule delivered intravitreally, showed promising results, similar to the results obtained with Bevacizumab. As of April 2022, a Phase 3 trial was launched with the administration of HLX04-O to a first patient [ 77 ]. MW02, a recombinant anti-VEGF humanized monoclonal antibody, is under phase 2 and 3 evaluation. The trials compare its efficacy and safety to Ranibizumab (Lucentis) (NCT05297292). 2.3.4. Conclusion and Perspectives on the Intravitreal Route While anatomic barriers prevent Abs access to the eye from the blood compartment, intravitreal injection has demonstrated efficacy and has become the standard-of-care in the treatment of eye disease. Although it is invasive, it is associated with limited side-effects such as uveitis and vitreitis [ 78 ], and a higher therapeutic index. To support the development of intravitreally-delivered Abs, advances are necessary in formulation and medical devices, to allow prolonged action, longer periods between two injections, and better tolerability. For example, the Port Delivery System (PDS) has been proposed for the delivery of Ranibizumab. It consists of a device surgically implanted into the vitreous cavity, allowing continuous delivery of the Ab. It dispenses the need for frequent intravitreal injections, thereby reducing invasiveness for patients. Its clinical evaluation is on-going and the first results for patients suffering from neovascular age-related macular degeneration are promising, showing equivalent control of the disease as compared to the standard care treatment, a monthly intravitreal injection of Ranibizumab (NCT04657289). The repertoire of excipients recognized as safe by the intravitreal route is limited due to ocular toxicity. For instance, only polysorbate (80 and 20) and sugar residues have been included in the formulation of Brolucizumab, Faricimab, and Ranibizumab [ 79 , 80 ]. Similar to what is observed in SC administration, Abs diffuse poorly into the vitreous cavity due to their positive charge [ 81 ]. Multiple parameters may influence the diffusion of Abs inside the eye, including: (i) the age of the eye, which can be associated with a change in the viscosity of the vitreous humor; and (ii) the quality of the lens. Patients with cataract surgery might experience a more rapid clearance of Abs from the eye, as observed with intravitreal delivery of antibiotics [ 82 ]. Overall, intravitreal injection is quite challenging, and inter-individual variability will dictate the fate and the activity of the antibody inside the eye. Like other local routes of administration, intravitreal injection of Ab provides effective on-target treatment, while limiting systemic deleterious exposure, and thereby improving Ab therapeutic index. 2.4. Inhalation: An Alternative Route for Respiratory Diseases 2.4.1. Rationale of Delivering Abs through the Airways Respiratory diseases are a major worldwide public health issue. They represent the fourth most common cause of death worldwide, which is mainly attributable to lung cancer, lung inflammatory diseases (e.g., chronic obstructive pulmonary disease (COPD)), and lower respiratory tract infections. The increase in antimicrobial resistance, seasonal virus outbreaks, newly emerging pathogens, or atmospheric pollution have severely complicated the management of airway diseases. Owing the success of Obiltoxaximab/Raxibacumab against pulmonary anthrax and Benralizumab against asthma, Abs have emerged as powerful therapeutics to tackle multiple respiratory diseases [ 83 ]. However, Abs administration through systemic routes led to a low bioavailability in the airway compartment [ 84 , 85 ]. This may limit the efficacy of Abs to treat respiratory diseases, if their target antigen primarily acts within the respiratory organ, while exposing the rest of the body to potential side effects [ 86 ]. Oral inhalation is the gold standard route of administration for small molecules commonly used for the treatment of inflammatory diseases (asthma, COPD). It allows direct drug access—as an aerosol—to both the upper and lower respiratory tract. It is associated with a rapid onset of action and a better therapeutic index as compared to parenteral injections [ 87 , 88 ]. Abs aerosolization is generally difficult since they are highly sensitive to mechanical, thermal, and physical stresses occurring during aerosol generation. Two types of devices have been evaluated for the oral inhalation of proteins: dry-powder inhalers, which deliver solid aerosol, and nebulizers, delivering liquid-formed aerosols. So far, metered-dose inhalers are not recommended for proteins due to the use of propellants [ 89 ]. 2.4.2. Challenges Associated with the Clinical Development of Inhaled Abs Researchers and pharmaceutical companies have been interested by local delivery of Abs through the airways for the last 20 years [ 90 ]. However, the first attempts were unsuccessful [ 91 ] and may be explained by multiple factors: − Abs instability during aerosolization/spraying, resulting in aggregation and thereby potential alteration of activity and immunogenicity concerns [ 86 , 92 , 93 ]. − Relevant target antigen, which must operate within the respiratory tract and be critical in the pathophysiology of the respiratory disease − Selection of the appropriate population, which may benefit from inhaled Abs. − Biological barriers, which may impair Abs PK and activity [ 94 ]. Several inhaled Abs are in early clinical trials, after preclinical studies showing promising efficacy in animal models (for example anti-IL-13 Fab for the treatment of asthma [ 95 ]). A few inhaled Abs are in phase 2 and 3 clinical evaluation, notably for the treatment of SARS-CoV-2 infection ( Table 6 ). CSJ117 (Ecleralimab) is an inhaled TSLP inhibitory antibody fragment developed to treat moderate to severe asthma [ 96 ] and COPD. The antibody is provided as a powder in hard capsules to be delivered daily in the lungs using a dry powder inhaler. First results showed promising results, with CSJ117 having the ability to attenuate airways inflammation in asthma patients. The recent SARS-CoV-2 pandemic has brought attention to the inhalation route, and several inhaled anti-SARS-CoV-2 Ab are being tested in the clinic by oral inhalation. Among the most advanced is a combination therapy developed to tackle the emerging SARS-CoV2 mutants, showing promising results. CT-P63 and CT-P59 (Regdanvimab) are monoclonal Abs, both targeting the receptor binding domain of the spike protein of SARS-CoV-2. More than just eliciting a neutralizing response, these Abs have the property to trap viral particles within the mucus, promoting their elimination by mucociliary clearance [ 97 ]. It is noteworthy that one molecule is currently under development by nasal inhalation, to address the Ab into the nasal cavity rather than in the lungs (COVI-DROPS). 2.4.3. Conclusion and Perspectives on the Inhalation Route Although airway delivery of Abs has been demonstrated as feasible and promising in preclinical studies, the benefits of Abs inhalation have not materialized yet in the clinical setting. A major advantage of the inhalation route is on the possibility of self-administration of Abs treatment for non-hospitalized patients, thereby reducing healthcare costs, and increasing patient comfort [ 98 ]. However, groundwork is still necessary to optimize Abs design and formulation, inhalation devices, as well as improving our knowledge of the physical and biological barriers that may impair the therapeutic response to inhaled Abs. 2.5. Intra-Tumoral Administration: Overcoming the Tumor Stromal Barrier for Anti-Cancerous Abs 2.5.1. Overview of the Barriers Associated with Tumor Abs used for the treatment of cancer are systematically delivered through IV injection. Their targets cover a wide range of proteins. They will promote the blocking of signals needed for cancer cell survival/growth, induce cancer cell destruction when coupled with toxins, or mark the tumor so that the immune system will better recognize and destroy it. Recently, the development of Abs targeting immune checkpoints has changed the treatment paradigm in many cancers. Abs targeting immune checkpoints inhibitors (ICI) block the immune "off" signal induced by the tumor cell in order to prevent the immune system from destroying the cancer, thus allowing a natural anti-cancerous immune response, and a higher survival for patients [ 99 ]. Systemic anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) Ab was the first licensed ICI against advanced metastatic melanoma [ 100 ], and was then quickly followed by anti-programmed cell death ligand and protein 1 (PD-L1/PD-1) Abs. However, if ICI antibodies have shown efficacy in patients with melanoma or non-small cell lung cancers, many cancers remain refractory to them. In addition, if the systemic ICI treatment has undeniable advantages, such as predictable pharmacokinetics, it is associated with hazardous on-target/off-tumor side effects, including widespread inflammation. Thus, improving the tumor targeting of ICI Abs appears to be an evident goal. In fact, most solid tumors are poorly reachable from the systemic circulation, due to multiple surrounding barriers [ 94 ]: − Densified tumor-associated ECM with the overexpression of collagens, and fibronectin, preventing the diffusion of Abs [ 101 ]. − Abnormal growth of blood vessels in the vicinity of the tumor resulting in non-vascularized, inaccessible areas for Abs coming from the blood circulation [ 102 ]. − Disorganized vessel structure associated with blood flow resistance, impeding the transport of the drug [ 103 ]. Consequently, several strategies have been proposed to limit the side effects of Abs targeting ICI, as well as optimizing their bioavailability within the tumor environment. A novel accurate method of injection consists in the delivery of Abs intratumorally (IT), allowing access to the tumor vicinity, circumventing extracellular barriers, and in theory improving on-target efficacy. Intra-tumoral injection is performed in a clinical environment by a physician, under the guidance of imagery (e.g., ultrasound, computed tomography (CT), or endoscopy) [ 104 ]. 2.5.2. Abs in Clinical Development by Intra-Tumoral Injection Preclinical studies have established that local administration of anti-CTLA-4 Abs was able to restore an anti-tumor response, eradicating both local and distant tumors, and used a significant lower amount of Abs as compared to IV Ab [ 105 , 106 ]. Thanks to the success of those pre-clinical studies, novel intratumorally-delivered Abs are under clinical evaluation ( Table 7 ). In 2019, an anti-CD40 agonist antibody (ADC-1013) was developed and clinically evaluated in solid tumors after intra-tumoral administration (NCT02379741). The results from the phase 1 study showed a good safety profile and were associated with positive pharmacodynamic responses [ 107 ]. Ipilimumab is a human anti-CTLA4 antibody already approved for the treatment of metastatic melanoma and given by IV injection. A phase 1 study showed that Ipilimumab in combination with IL-2, given intratumorally, was well tolerated and was able to generate anti-tumoral responses in the majority of patients [ 108 ]. A phase 2 study is currently evaluating the tolerance of Ipilimumab IT and Nivolumab IV for the treatment of stage III/IV melanoma patients (NCT02857569). Moreover, a phase 2/3 trial is comparing Pembrolizumab and/or Ipilimumab in patients with advanced solid tumors after intra-tumoral injection and IV infusion. (NCT03755739). 2.5.3. Conclusion and Perspectives on the Intra-Tumoral Route Intra-tumoral administration of anti-cancerous Abs has multiple advantages, especially in reducing the harmful side effects associated with on-target/off-tumor of Abs after IV injection. In addition, local administration, within the tumor environment, may promote a superior priming of T-cells already on-site, and a multi-clonal response [ 106 ]. For now, clinical validation of intra-tumoral delivery of Abs is still in the preliminary stages. Further work is needed to optimize the dosage and the formulation according to the type and the stage of the tumor lesion. In addition, this route of administration is operator-dependent, which means that a subtle change in needle position may change the outcome of the administration. The development of new live imaging tools may improve injection reproducibility. Combinatorial approaches with intra-tumoral and intravenous treatment, or tumor ablation, are also being investigated and may represent a promising therapeutic alternative [ 109 ]. 2.6. Intra-Articular Administration: A New Hope for the Relief of Joint Pain 2.6.1. Overview of Joint Physiology Joints degeneration is one of the leading causes of permanent motor disability worldwide requiring long-term therapeutic treatment. Among them, osteoarthritis (caused by trauma to the joint cartilage), rheumatoid arthritis (autoimmune disease where immune cells attack the joints), and gout (joint inflammation due to excess of uric acid), are the most common forms [ 110 ]. Standard care includes systemic administration of analgesic and anti-inflammatory agents, including anti-TNF-α (Infliximab, Etanercept, Adalimumab) molecules and anti-IL1β Ab (Canakinumab) [ 111 ]. However, although symptoms show substantial improvements thanks to these treatments, non-responsive patients and side effects are increasing with time [ 112 ]. Efforts have been made in recent years toward the development of intra-articular (IA)-delivered treatments, with multiple benefits such as a better bioavailability and a reduced systemic exposure [ 113 ]. Intra-articular injection is performed using syringe/needle into a joint. To help the guidance of the needle, ultrasound or fluoroscopy techniques can be used. The development of drugs dedicated to IA injection necessitates understanding the anatomy and physiology of the joints, to identify the key parameters influencing the pharmacokinetics and pharmacodynamics of the drug. Synovial joints, or diarthrosis, joins bone endings and hyaline cartilage with a fibrous capsule delimitating the synovial cavity filled with synovial fluid. Synovial joints are particularly affected during the degeneration processes [ 114 ]. Cartilage is an avascular tissue composed of chondrocytes embedded in a negatively charged ECM which make this tissue impermeable to molecules bigger than 50 kDa (such as antibodies) depending upon their charge and conformation. Consequently, the cartilage is inefficiently targeted by drugs administered systemically, which first need to reach the synovial fluid, before diffusing through cartilaginous ECM. To enter the joints, the drug needs to pass through the capillary endothelium of the synovium, the ECM of the synovial intima, and the synoviocytes composing the synovial membrane. Both cellular layers are highly fenestrated, allowing high diffusion of molecules smaller than 10 kDA. For larger molecules, the fenestration will allow a size-dependent diffusion, slowing the passage of large molecules such as antibodies [ 115 ]. During inflammation, there is an increased permeability of both capillary and synovial membranes, allowing macromolecules to reach the synovial compartment. However, joint inflammation will also accelerate synovial clearance [ 116 ]; studies have revealed a mean clinical half-life of around 3 h for an anti-inflammatory antibody, not leaving enough time for an optimal action of the antibody. 2.6.2. Abs in Clinical Development for IA Administration Despite some promising features, intra-articular delivery of therapeutic antibodies remains rare. Studies performed 20 years ago have shown conflicting results regarding IA delivery of Infliximab depending on the disease, with positive outcomes in patients suffering from ankylosing spondylitis [ 117 ], while there was no positive effect in patients with acute joint inflammation [ 118 ]. In 2007, the clinical evaluation of intra-articularly-delivered Infliximab in patients suffering from intractable knee monoarthritis, reached a phase 3 clinical trial but the development was stopped in 2015 due to the insufficient recruitment of patients [ 117 ] (NCT00521963). More recently, two novel intra-articularly-delivered Abs have entered clinical evaluation ( Table 8 ). AMB-05X is a fully human antibody targeting colony-stimulating factor I (c-FMS), a protein overexpressed in many cancers, and on tumor-associated macrophages. This antibody entered a clinical trial phase 2 (NCT04731675), to treat patients suffering from tenosynovial giant cell tumor, and pigmented villonodular synovitis, two afflictions of the knee, after IA delivery. The ongoing study is investigating the safety, tolerability, and efficacy of the treatment delivered by IA injection. Canakinumab, a human anti-IL1β antibody, is currently in phase 2 of clinical evaluation, in combination with LNA043 (a protein inducing chondrogenesis and cartilage repair) for the treatment of patients with knee osteoarthritis (NCT04814368) after IA delivery. 2.6.3. Conclusion and Perspectives Regarding the Intra-Articular Route Intra-articular delivery may be of particular importance for the treatment of joint diseases (and notably rheumatoid arthritis) as around 30% of patients are resistant to biotherapies, and few novel molecules are under development. The limited half-life of antibodies reaching the inflamed joint is one of the major limitations associated with IA delivery, requiring frequent injections and thus increasing side effects, discomfort, and morbidity. Specific formulations including hydrogels [ 119 ], micro/nano particles [ 120 ], or in situ implants [ 121 ] are under consideration to improve antibody concentration on-target. A long-lasting Ab formulation through the use of biopolymers was recently developed for IA administration, showing a sustained high concentration of Abs in the synovial fluid, with minimal inflammatory side effects [ 122 ]. However, controlled-release methods are often associated with a loss of bioactivity of the drug, and/or inflammatory side effects that could accelerate the degeneration of the cartilage and the disease [ 123 ]. Additional work is needed to develop safe and active intra-articularly-delivered Abs to fight joints auto-immune diseases. 2.7. Delivery within the Central Nervous System: A Method to Bypass the Blood–Brain Barrier 2.7.1. The Blood–Brain Barrier (BBB), a Barrier for Abs The CNS is an essential part of the nervous system consisting of the brain and the spinal cord. The CNS integrates and coordinates essential functions of the body [ 124 ]. Because of its importance, the CNS is well protected, notably by the blood–brain barrier (BBB), limiting its exposure to exogenous particles carried by the blood circulatory system. The BBB is a highly selective semipermeable barrier preventing the passage of solutes from the blood into the extracellular fluid of the CNS. The BBB is composed of specialized endothelial cells sealed together with tight junctions reinforcing trans-endothelial electrical resistance, as well as the high expression of energy-dependent efflux transporter, inducing a selective passage of solutes [ 125 ]. In particular, FcRn, expressed in the microvascular endothelium and in the choroid plexus epithelium, essential components of the BBB, is involved in the reverse transcytosis of IgG, from the brain to the blood vessels. Therefore, it is estimated that less than 0.1% of systemically injected IgG enter the CNS through nonspecific pathways. Thus, to attain a therapeutic dose in the CNS, Abs have to be administered in high quantity—which may be associated with toxicity—or specific transporter pathways existing between the circulatory system and the CNS have to be used [ 126 , 127 ]. In order to circumvent these barriers, novel methods have been considered aimed at addressing the drug into the CNS by surgery, either via the intracerebroventricular, intracerebral routes or convection-enhanced delivery [ 126 , 128 ]. The intracerebral injection, the most direct method, consists in intermittent bolus injections, which are administered locally in the brain, after a surgical intervention. The intracerebroventricular technique allows the injection of drugs directly into the cerebrospinal fluid in the cerebral ventricles. Both methods use a syringe and needle system filled with the drug to be administered. Convection-enhanced delivery consists in the generation of a pressure gradient at the tip of an infusion catheter or canula (usually implanted in the cerebral tumor), allowing the delivery of drugs through the interstitial spaces of the CNS. 2.7.2. Abs in Clinical Development for Direct Delivery to the CNS There are many diseases targeting the CNS, including the neurodegenerative Parkinson or Alzheimer's disease (AD), for which the development of Abs has been considered. For example, Aducanumab, an anti-Aβ Ab was approved by the FDA for the treatment of AD, after parenteral injection [ 129 , 130 ]. Cerebral delivery of Abs is mainly being evaluated in the preclinical phases with some success [ 131 ]; only one Ab has already reached clinical trials. The antibody 131I-Omburtamab (Y-Abs Therapeutics), is a murine IgG1 recognizing CD276, used for radioimmunotherapy. This antibody is injected via the intracerebroventricular route, and phase 1 and 2 trials in patients with CNS Neuroblastoma, CNS metastases, or leptomeningeal metastases, have shown that the antibody was well tolerated and improved survival. A phase 3 trial is ongoing, evaluating the efficacy and safety of the Ab in children (NCT03275402). In the meantime, the same antibody is entering a phase 1 trial to test its efficacy, once delivered through a convention-enhanced delivery in patients with diffuse pontine gliomas profile (NCT05063357) [ 132 ]. 2.7.3. Conclusion and Perspectives on CNS Delivery The CNS is particularly well protected from the environment impeding an efficient access to therapeutic molecules. The BBB remains the major obstacle, when considering systemic infusion of therapeutic Abs, and limits CNS bioavailability. Novel methods bypassing the BBB, described above, are still in infancy and require further work to be standardized and to reduce their invasiveness. In this context, the investigation of the "nose-to-brain" route may be of particular interest. It consists of administering drugs in the olfactory region of the nose, by inhalation, from where they may transfer to the brain through the epithelial layer and via neuronal bundles that project to the olfactory bulb. The "nose-to-brain" route is under preclinical evaluation for Abs [ 133 ]. 2.1. The Subcutaneous Route: The Most Popular after IV Injection 2.1.1. Fundamentals Related to the SC Route After IV injection, the second most popular route for the delivery of antibodies is the subcutaneous (SC) route. It consists in the injection of Abs using a syringe and needle under the skin of patients at an angle of 90 °C, thus bypassing the barrier formed by the epidermis and dermis layers [ 18 ]. The choice of the anatomical site is important due to differences in dermal thickness which may reduce the absorption of the injected Abs. Nowadays, around 30% of the approved Abs are delivered by SC injection ( Table 1 ). If the delivery of drugs, mainly opioids, by SC administration, has been in practice since the middle of the 19th century, the administration of Abs by this route is recent. The first subcutaneous injected Ab was Adalimumab, used in the treatment of rheumatoid arthritis and approved by the FDA in 2002, and by the EMA in 2003 [ 19 ]. After this first success, and particularly since 2009, the number of marketed Abs delivered by the SC route has significantly increased. It is noteworthy that SC administration is already the standard route in the treatment of chronic diseases such as rheumatoid arthritis. Indeed, it allows self-administration and improves patients' compliance. The SC route is mainly used for the delivery of Abs targeting interleukins such as TNF-α, critically involved in the development of rheumatoid arthritis (Adalimumab, Golimumab, Certolizumab pegol) or cytokine receptors such as the IL-17a receptor, involved in the progression of psoriasis (Brodalumab, Secukinumab, Ixekizumab) [ 20 ]. The development of Abs intended for a subcutaneous injection necessitates understanding the physiology of the skin. After injection, the drug reaches the hypodermis interstitial space between the dermis and the deep fascia covering the muscle tissue. This layer is composed of adipose tissue, blood, lymph vessels, and resident immune cells such as fibroblasts and macrophages. All components are enmeshed in an extracellular matrix (ECM) network, rich in collagen, elastin, and glycosaminoglycans [ 21 ]. To pass into the systemic compartment (via either the blood capillaries or lymphatic vessels), and thus reach their target, Abs have to diffuse through the ECM, which constitutes both a physical and chemical barrier. The fate of the Ab is dictated by its size, charge, and affinity with transporters. Despite the presence of the positively charged collagen fibrils, the hypodermis interstitial space displayed an overall negative charge due to important concentrations of hyaluronic acid and chondroitin sulfate, two major glycosaminoglycans of the hypodermis ECM, which are negatively charged. The global negative charge of ECM favors the transport of negatively charged drugs thanks to electrostatic repulsion [ 22 ]. However, the majority of therapeutic Abs are positively charged. Once the ECM is traversed, drugs may enter the systemic circulation by two different mechanisms. Molecules smaller than 16 kDa diffuse directly into the bloodstream, taking advantage of the permeability of the vascular endothelium [ 22 ]. However, Abs, along with drugs with a higher molecular weight, are absorbed by convection into lymphatic vessels. Thus, the subcutaneous route is particularly interesting to target lymphoid cells and the molecules they secrete. Abs in the lymphatic vessels pass to larger lymphatics and then reach the blood vascular system, from where they diffuse throughout the body. If the development of Abs for subcutaneous injection is quite challenging, multiple factors explain the attractiveness of this route as compared to other parenteral ones. In the hypodermis, the walls in the fat lobule are thinner than those in the dermis, which facilitates the diffusion of drugs into blood capillaries [ 23 ]. Moreover, the absence of antigen-presenting cells in the hypodermis, usually present in the top layers of the skin (Langerhans cells and/or macrophages), may decrease the immunogenicity of the antibody. Thus, an increasing number of Abs delivered by SC are being developed, allowing a quicker delivery time of administration as compared to IV injection, enabling longer dosing intervals and, in fine, reducing the frequency of administration. In addition, SC administration is less invasive and painful than IV injection [ 22 ] and allows self-delivery at home [ 24 ]. Thus, the subcutaneous route may improve patient comfort and compliance, which is critical for the treatment of chronic diseases, and may be associated with a reduction in treatment costs, consuming fewer healthcare resources. 2.1.2. Abs Approved for Subcutaneous Delivery Abs approved for subcutaneous administration must be formulated at a high concentration, thus necessitating a careful control of their stability and formulation viscosity. Different strategies have been considered to ensure the efficient absorption and bioavailability of Abs after hypodermis injection. They include, but are not limited to, the increase in delivered Abs concentration (e.g., SC administration limiting the injection volume to 1–2 mL [ 25 ]), the development of specific formulations to reduce physical and chemical destabilization (e.g., the use of polysorbate preventing aggregation and particle formation [ 26 ]) and the development of novel administration devices (e.g., the autoinjectors enabling a faster delivery for larger concentrations of Abs [ 27 ]). Those strategies have led to the approval of around 40 different Abs ( Table 1 ). A major concern for SC injection is the isoelectric point (pI) of Abs, found between 7 and 9, making Abs positively charged at the physiological pH. A study by Bumbaca Yadav et al., showed that positively charged Abs present a reduced bioavailability by 31%, while their negatively charged counterparts demonstrate enhanced bioavailability up to 70% after SC administration [ 30 ]. Another study found that the reduced bioavailability of Abs delivered subcutaneously is due to their interaction with ECM components, thus limiting the amount of Ab reaching the vascular compartment [ 31 ]. Moreover, the overall negative charge of the hypodermis interstitial space increases the interaction of ECM components with water molecules resulting in a low hydraulic conductivity and limiting the subcutaneous injection volume [ 32 ]. To circumvent this serious issue, one strategy consists in combining Abs with hyaluronidase. Hyaluronidase degrades hyaluronic acid, lowering the amount of negatively charged molecules and enhancing the bioavailability of Ab after SC injection [ 33 ]. Moreover, combining Abs with hyaluronidase may facilitate bulk fluid flow and improve the pharmacokinetic profile after SC injection [ 34 ], as demonstrated in multiple clinical studies [ 35 , 36 , 37 ]. Based on these results, the regulatory agencies approved Rituximab, Trastuzumab, and Daratumumab in combination with recombinant human hyaluronidase (rHuPH20), in 2017 (Rituxan Hycela/mAbThera s.c), 2019 (Herceptin Hylecta), and 2020 (Darzalex Faspro), respectively. These encouraging results have fueled the repurposing of Abs approved for delivery by IV injection to this novel modality of administration. Notably, Tocilizumab ((Ro)-Actemra), an antibody used in the treatment of rheumatoid polyarthritis, was formulated for the SC route, in response to patient demand, and to allow a less invasive route for a treatment usually delivered monthly [ 38 ]. It is noteworthy that multiple studies have demonstrated the absence of significant differences between the IV and SC routes of administration for Abs, thus making SC a legitimate option for patients [ 37 , 38 ]. 2.1.3. Abs in Clinical Development for the SC Route The clinical development of subcutaneously-delivered Ab concerns either de novo development, expansion of the disease target, and/or new formulation for already approved Abs. A high number of those Abs are currently found in clinical trials. Here, we listed subcutaneously delivered Abs either in active phase 3 trials or under review by regulatory agencies ( Table 2 ). Novel developments include Fasinumab, a recombinant fully human IgG4, targeting the nerve growth factor (NGF) and evaluated for pain relief in patients suffering from osteoarthritis (OA). A phase 2b/3 trial showed that Fasinumab provides improvement in OA pain and motor function, even in patients that are non-responsive to analgesics [ 39 ]. The drug approval is pending an evaluation by the FDA (NCT03161093; NCT02683239). In the meantime, studies are also investigating lower doses of Fasinumab in patients with knee or hip OA. The repurposing of IV delivery approved Abs for SC application in a disease context different than the original approval is also investigated. For example, Ofatumumab (Arzerra ® , Novartis) is a monoclonal antibody targeting CD20 and causing cytotoxicity in cells expressing CD20. It was first approved in 2010 for the treatment of certain chronic lymphocytic leukaemia by IV injection, and has been reformulated (Kesimpta) for SC administration and evaluated in patients with relapsed multiple sclerosis. Two ongoing phase 3 trials, OLIKOS (NCT04486716) and ARTIOS (NCT04353492) are evaluating the efficacy, safety, and tolerability of the SC drug in patients with relapsing multiple sclerosis, all of whom are transitioning from a CD20 Ab therapy (Rituximab or Ocrelizumab), or dimethyl fumarate therapy [ 40 ]. Many Abs have been developed or repurposed as emergency treatments since the beginning of the SARS-CoV2 pandemic, and target either the virus or the host inflammatory response. Among them, REGEN-COV2 comprising Casirivimab and Imdevimab has been approved for emergency use by IV infusion and is now undergoing regulatory review, for its use by SC administration to treat and prevent SARS-CoV-2 infection in non-hospitalized individuals [ 41 ]. The first phases of its clinical investigation showed a significant efficacy with improved survival. A phase 3 trial is also in progress to evaluate its potency for the prevention of COVID-19 in immunocompromised patients (NCT05074433). 2.1.4. Conclusion and Perspectives Regarding the Subcutaneous Route The relevance of the SC route for the administration of Abs has been illustrated by several clinical successes. However, the bioavailability of Abs delivered subcutaneously remains difficult to predict. Advances in preclinical models would be necessary to investigate the fate of Abs at the SC injection site and their diffusion into the blood/lymphatic compartment. Interestingly, novel in vitro tools have been developed to predict the in vivo absorption of biopharmaceuticals after SC injection, by modeling the environmental changes an Ab will experience after its injection. Among those, Scissor (Subcutaneous Injection Site Simulator) device provides a tractable method to study the fate and the pharmacokinetics of biopharmaceuticals once in the hypodermis [ 42 , 43 ]. Nevertheless, as no in vitro model is yet accurate enough, the pharmacokinetics of Abs still relies on in vivo studies. Formulating Abs for the SC route remains challenging, as formulations need to afford high concentration with low viscosity, aggregation and immunogenicity [ 22 ]. Biotechnological platforms have been developed to support the switch from intravenous infusion to SC delivery, using proprietary excipients and proteins, allowing the reduction of ionic strength and hydrophobicity areas of the molecule, thus limiting aggregation when the Ab is highly concentrated. 2.1.1. Fundamentals Related to the SC Route After IV injection, the second most popular route for the delivery of antibodies is the subcutaneous (SC) route. It consists in the injection of Abs using a syringe and needle under the skin of patients at an angle of 90 °C, thus bypassing the barrier formed by the epidermis and dermis layers [ 18 ]. The choice of the anatomical site is important due to differences in dermal thickness which may reduce the absorption of the injected Abs. Nowadays, around 30% of the approved Abs are delivered by SC injection ( Table 1 ). If the delivery of drugs, mainly opioids, by SC administration, has been in practice since the middle of the 19th century, the administration of Abs by this route is recent. The first subcutaneous injected Ab was Adalimumab, used in the treatment of rheumatoid arthritis and approved by the FDA in 2002, and by the EMA in 2003 [ 19 ]. After this first success, and particularly since 2009, the number of marketed Abs delivered by the SC route has significantly increased. It is noteworthy that SC administration is already the standard route in the treatment of chronic diseases such as rheumatoid arthritis. Indeed, it allows self-administration and improves patients' compliance. The SC route is mainly used for the delivery of Abs targeting interleukins such as TNF-α, critically involved in the development of rheumatoid arthritis (Adalimumab, Golimumab, Certolizumab pegol) or cytokine receptors such as the IL-17a receptor, involved in the progression of psoriasis (Brodalumab, Secukinumab, Ixekizumab) [ 20 ]. The development of Abs intended for a subcutaneous injection necessitates understanding the physiology of the skin. After injection, the drug reaches the hypodermis interstitial space between the dermis and the deep fascia covering the muscle tissue. This layer is composed of adipose tissue, blood, lymph vessels, and resident immune cells such as fibroblasts and macrophages. All components are enmeshed in an extracellular matrix (ECM) network, rich in collagen, elastin, and glycosaminoglycans [ 21 ]. To pass into the systemic compartment (via either the blood capillaries or lymphatic vessels), and thus reach their target, Abs have to diffuse through the ECM, which constitutes both a physical and chemical barrier. The fate of the Ab is dictated by its size, charge, and affinity with transporters. Despite the presence of the positively charged collagen fibrils, the hypodermis interstitial space displayed an overall negative charge due to important concentrations of hyaluronic acid and chondroitin sulfate, two major glycosaminoglycans of the hypodermis ECM, which are negatively charged. The global negative charge of ECM favors the transport of negatively charged drugs thanks to electrostatic repulsion [ 22 ]. However, the majority of therapeutic Abs are positively charged. Once the ECM is traversed, drugs may enter the systemic circulation by two different mechanisms. Molecules smaller than 16 kDa diffuse directly into the bloodstream, taking advantage of the permeability of the vascular endothelium [ 22 ]. However, Abs, along with drugs with a higher molecular weight, are absorbed by convection into lymphatic vessels. Thus, the subcutaneous route is particularly interesting to target lymphoid cells and the molecules they secrete. Abs in the lymphatic vessels pass to larger lymphatics and then reach the blood vascular system, from where they diffuse throughout the body. If the development of Abs for subcutaneous injection is quite challenging, multiple factors explain the attractiveness of this route as compared to other parenteral ones. In the hypodermis, the walls in the fat lobule are thinner than those in the dermis, which facilitates the diffusion of drugs into blood capillaries [ 23 ]. Moreover, the absence of antigen-presenting cells in the hypodermis, usually present in the top layers of the skin (Langerhans cells and/or macrophages), may decrease the immunogenicity of the antibody. Thus, an increasing number of Abs delivered by SC are being developed, allowing a quicker delivery time of administration as compared to IV injection, enabling longer dosing intervals and, in fine, reducing the frequency of administration. In addition, SC administration is less invasive and painful than IV injection [ 22 ] and allows self-delivery at home [ 24 ]. Thus, the subcutaneous route may improve patient comfort and compliance, which is critical for the treatment of chronic diseases, and may be associated with a reduction in treatment costs, consuming fewer healthcare resources. 2.1.2. Abs Approved for Subcutaneous Delivery Abs approved for subcutaneous administration must be formulated at a high concentration, thus necessitating a careful control of their stability and formulation viscosity. Different strategies have been considered to ensure the efficient absorption and bioavailability of Abs after hypodermis injection. They include, but are not limited to, the increase in delivered Abs concentration (e.g., SC administration limiting the injection volume to 1–2 mL [ 25 ]), the development of specific formulations to reduce physical and chemical destabilization (e.g., the use of polysorbate preventing aggregation and particle formation [ 26 ]) and the development of novel administration devices (e.g., the autoinjectors enabling a faster delivery for larger concentrations of Abs [ 27 ]). Those strategies have led to the approval of around 40 different Abs ( Table 1 ). A major concern for SC injection is the isoelectric point (pI) of Abs, found between 7 and 9, making Abs positively charged at the physiological pH. A study by Bumbaca Yadav et al., showed that positively charged Abs present a reduced bioavailability by 31%, while their negatively charged counterparts demonstrate enhanced bioavailability up to 70% after SC administration [ 30 ]. Another study found that the reduced bioavailability of Abs delivered subcutaneously is due to their interaction with ECM components, thus limiting the amount of Ab reaching the vascular compartment [ 31 ]. Moreover, the overall negative charge of the hypodermis interstitial space increases the interaction of ECM components with water molecules resulting in a low hydraulic conductivity and limiting the subcutaneous injection volume [ 32 ]. To circumvent this serious issue, one strategy consists in combining Abs with hyaluronidase. Hyaluronidase degrades hyaluronic acid, lowering the amount of negatively charged molecules and enhancing the bioavailability of Ab after SC injection [ 33 ]. Moreover, combining Abs with hyaluronidase may facilitate bulk fluid flow and improve the pharmacokinetic profile after SC injection [ 34 ], as demonstrated in multiple clinical studies [ 35 , 36 , 37 ]. Based on these results, the regulatory agencies approved Rituximab, Trastuzumab, and Daratumumab in combination with recombinant human hyaluronidase (rHuPH20), in 2017 (Rituxan Hycela/mAbThera s.c), 2019 (Herceptin Hylecta), and 2020 (Darzalex Faspro), respectively. These encouraging results have fueled the repurposing of Abs approved for delivery by IV injection to this novel modality of administration. Notably, Tocilizumab ((Ro)-Actemra), an antibody used in the treatment of rheumatoid polyarthritis, was formulated for the SC route, in response to patient demand, and to allow a less invasive route for a treatment usually delivered monthly [ 38 ]. It is noteworthy that multiple studies have demonstrated the absence of significant differences between the IV and SC routes of administration for Abs, thus making SC a legitimate option for patients [ 37 , 38 ]. 2.1.3. Abs in Clinical Development for the SC Route The clinical development of subcutaneously-delivered Ab concerns either de novo development, expansion of the disease target, and/or new formulation for already approved Abs. A high number of those Abs are currently found in clinical trials. Here, we listed subcutaneously delivered Abs either in active phase 3 trials or under review by regulatory agencies ( Table 2 ). Novel developments include Fasinumab, a recombinant fully human IgG4, targeting the nerve growth factor (NGF) and evaluated for pain relief in patients suffering from osteoarthritis (OA). A phase 2b/3 trial showed that Fasinumab provides improvement in OA pain and motor function, even in patients that are non-responsive to analgesics [ 39 ]. The drug approval is pending an evaluation by the FDA (NCT03161093; NCT02683239). In the meantime, studies are also investigating lower doses of Fasinumab in patients with knee or hip OA. The repurposing of IV delivery approved Abs for SC application in a disease context different than the original approval is also investigated. For example, Ofatumumab (Arzerra ® , Novartis) is a monoclonal antibody targeting CD20 and causing cytotoxicity in cells expressing CD20. It was first approved in 2010 for the treatment of certain chronic lymphocytic leukaemia by IV injection, and has been reformulated (Kesimpta) for SC administration and evaluated in patients with relapsed multiple sclerosis. Two ongoing phase 3 trials, OLIKOS (NCT04486716) and ARTIOS (NCT04353492) are evaluating the efficacy, safety, and tolerability of the SC drug in patients with relapsing multiple sclerosis, all of whom are transitioning from a CD20 Ab therapy (Rituximab or Ocrelizumab), or dimethyl fumarate therapy [ 40 ]. Many Abs have been developed or repurposed as emergency treatments since the beginning of the SARS-CoV2 pandemic, and target either the virus or the host inflammatory response. Among them, REGEN-COV2 comprising Casirivimab and Imdevimab has been approved for emergency use by IV infusion and is now undergoing regulatory review, for its use by SC administration to treat and prevent SARS-CoV-2 infection in non-hospitalized individuals [ 41 ]. The first phases of its clinical investigation showed a significant efficacy with improved survival. A phase 3 trial is also in progress to evaluate its potency for the prevention of COVID-19 in immunocompromised patients (NCT05074433). 2.1.4. Conclusion and Perspectives Regarding the Subcutaneous Route The relevance of the SC route for the administration of Abs has been illustrated by several clinical successes. However, the bioavailability of Abs delivered subcutaneously remains difficult to predict. Advances in preclinical models would be necessary to investigate the fate of Abs at the SC injection site and their diffusion into the blood/lymphatic compartment. Interestingly, novel in vitro tools have been developed to predict the in vivo absorption of biopharmaceuticals after SC injection, by modeling the environmental changes an Ab will experience after its injection. Among those, Scissor (Subcutaneous Injection Site Simulator) device provides a tractable method to study the fate and the pharmacokinetics of biopharmaceuticals once in the hypodermis [ 42 , 43 ]. Nevertheless, as no in vitro model is yet accurate enough, the pharmacokinetics of Abs still relies on in vivo studies. Formulating Abs for the SC route remains challenging, as formulations need to afford high concentration with low viscosity, aggregation and immunogenicity [ 22 ]. Biotechnological platforms have been developed to support the switch from intravenous infusion to SC delivery, using proprietary excipients and proteins, allowing the reduction of ionic strength and hydrophobicity areas of the molecule, thus limiting aggregation when the Ab is highly concentrated. 2.2. The Intramuscular Route: The Favorite Choice for Infectious Diseases? 2.2.1. Basics of the Intramuscular Route If the intramuscular route (IM) is often used for the administration of vaccines, this modality is rarely considered for the delivery of Ab. The IM injection consists in administering a drug deep into the muscle mass, where the blood supply allows a rapid and better absorption than the subcutaneous route. As for the SC route, the drug is administered via a syringe and needle into the skin at an angle of 90°. However, where the SC route uses small length- and diameter-needles, IM injection requires longer needles—2.5 to 4 cm—to bypass the different layers of the skin. The volume of administration is relatively low ~2–5 mL, depending on the muscle chosen for the injection, necessitating a more concentrated product than for IV administration. The preferred injection sites are the deltoid muscle in the upper arm, the vastus lateralis found at the front of the thigh toward the outside of the leg, especially for IM injection in young children [ 44 ], the ventrogluteal muscle of the hip, the safest site for adults and children older than 7 months, and finally the dorsogluteal muscles of the buttocks, albeit less used nowadays due to potential injury to the sciatic nerve. The pharmacokinetics of a drug injected by IM follows a specific sequence: dissolution rate, solvent supply, phase transfer, and diffusion to the vascular system. Therefore, injection depth is an important parameter which influences the absorption rate of the drug after IM injection. Consequently, a too superficial injection will deliver the drug either SC, or into the fat layer, retarding the action of the drug. The absorption will also depend on the muscle mass and its vascularization. Patients with muscular atrophy see a delay in drug absorption, as well as an increase in the risk of neurovascular complications. These anatomical parameters also influence the rapidity of action of the drug [ 44 ]. 2.2.2. Ab Approved for Delivery by the IM Route One of the most threatening pathogens responsible for lower respiratory tract infection in children is the respiratory syncytial virus (RSV). Nearly every infant develops an RSV infection during their childhood. A total of 60% of them have an immune system mature enough to control the infection [ 45 ]. However, between 15 and 40% of infants, especially preterm infants, develop a more serious airway infection, which may eventually lead to bronchiolitis and pneumonia. Although no vaccine or specific RSV treatment exist to treat the infection (apart from general anti-viral therapy), a passive immunization may limit the infection in young infants with high risk factors. In 1998, the FDA approved Palivizumab (Synagis) [ 46 ], a humanized monoclonal IgG1 antibody targeting the glycoprotein F on the RSV virus, which is responsible for membrane fusion and infection of the host cell. Intramuscular injection of palivizumab is recommended for premature infants and young children with heart or lung comorbidities, and injections usually start before the expected RSV epidemic season [ 47 , 48 ]. Intramuscular delivery was chosen to facilitate delivery and limit invasive risks to the young recipients of the treatment. Aggregated clinical data have established the half-life of Palivizumab administered via intramuscular injection to be around 17–27 days, with a mean of 20 days, in infants younger than 24 months. As such, a monthly administration during the epidemic season is necessary to protect effectively against RSV. Palivizumab prophylaxis has shown a significant decrease in the hospitalization, ICU stay, and mortality [ 49 , 50 ]. Unfortunately, its high cost limits the number of children who benefit from this prophylaxis protection, especially in low-income countries [ 51 ]. Although Palivizumab is not licensed for the treatment of RSV disease, it has paved the way to the clinical use of intramuscular injection, and has shown some potential to facilitate everyday care, promoting a less invasive route for patients, in particular infants, in whom IV injection is more complicated [ 52 ]. 2.2.3. Abs in Clinical Development Delivered by the IM Route The IM injection allows a more rapid absorption and onset of action compared to SC delivery: mathematical in silico models associate the IM route with higher drug concentration on target and shorter time to reach the peak of concentration as compared to SC delivery. This route benefits from the long experience of vaccine administration. As shown below, several anti-infective Abs delivered by IM administration are close to reaching patients ( Table 3 ). In 2021, Sotrovimab (Vir Biotechnology—GlaxoSmithKline) obtained an emergency use authorization (EUA) from the FDA for the treatment of mild-to-moderate COVID-19. However, since May 2022, and due to its inefficacy against the BA.2 variant of SARS-CoV-2, its authorization has been revoked [ 53 ]. A phase 3 trial (COMET-TAIL, NCT04913675) which has established that the intramuscular injection of Sotrovimab had a similar clinical effectiveness as compared to an IV injection is still active. SYN023, a therapeutic cocktail comprising two monoclonal Abs, CTB011 and CTB012, is under development for post-exposure prophylaxis to rabies. Nowadays, the standard of care treatment after rabies infection is an on-site (wound injection) and IM administration of anti-rabies immunoglobulin (RIG), called post-exposure prophylaxis, followed by four doses of the vaccine, delivered by IM or intradermal routes. The full dose of RIG facilitates rapid protection until the immune system can produce its own immunoglobulins thanks to the vaccination. However, the current RIG products are limited and expensive, thus their use is limited in high-burden countries [ 54 ]. SYN023, administered by IM injection, targets protein residues found on human rabies virus, and displays a neutralization capacity equivalent or superior to RIGs, even at a lower dose [ 55 ]. The ongoing phase 3 trial will provide additional information regarding the efficacy of SYN023 in populations at risk of rabies infection. Ibalizumab-uiyk (Trograzo) is an anti-HIV monoclonal antibody, approved in 2018, for the treatment of patients with resistant forms of AIDS by IV injection. It targets CD4 protein expressed by T cells, inhibiting the entry of HIV. As a "first class medication" (defined by the FDA), the use of Ibalizumab-uiyk needs to be expanded across the world and for patients reluctant to receive IV injection or where the access to medical facilities and personnel qualified to administer IV injections is limited. A phase 3 is currently evaluating the IM route in comparison to IV injection, notably in terms of safety, pharmacokinetics, and limiting the spread of infection (NCT03913195). 2.2.4. Conclusions and Perspectives on the Intramuscular Route The IM injection is usually considered as a rescue route of administration when other ways to deliver drugs are not appropriate. For example, drugs inducing vein irritation or sensitive to oral digestion have been moved from IV and per os injection, respectively, to IM injection. Among other advantages, IM administration is associated with a quicker and more uniform absorption of the drug as compared to other routes, and it is considered to be as efficient and potent as IV injection, with less invasive characteristics. Even with these potential advantages over other routes of administration, the IM route is still underestimated for Abs. The development of depot injections may represent an opportunity for the expansion of the IM route. Depot injections are a slow-release form of medication usually implanted in the muscle. They are already used for neuroleptic drugs (fluphenazine), prolonging their pharmacological effect. Moreover, they allow a sustained delivery over time [ 56 ] which may be of particular interest regarding the treatment of chronic diseases. 2.2.1. Basics of the Intramuscular Route If the intramuscular route (IM) is often used for the administration of vaccines, this modality is rarely considered for the delivery of Ab. The IM injection consists in administering a drug deep into the muscle mass, where the blood supply allows a rapid and better absorption than the subcutaneous route. As for the SC route, the drug is administered via a syringe and needle into the skin at an angle of 90°. However, where the SC route uses small length- and diameter-needles, IM injection requires longer needles—2.5 to 4 cm—to bypass the different layers of the skin. The volume of administration is relatively low ~2–5 mL, depending on the muscle chosen for the injection, necessitating a more concentrated product than for IV administration. The preferred injection sites are the deltoid muscle in the upper arm, the vastus lateralis found at the front of the thigh toward the outside of the leg, especially for IM injection in young children [ 44 ], the ventrogluteal muscle of the hip, the safest site for adults and children older than 7 months, and finally the dorsogluteal muscles of the buttocks, albeit less used nowadays due to potential injury to the sciatic nerve. The pharmacokinetics of a drug injected by IM follows a specific sequence: dissolution rate, solvent supply, phase transfer, and diffusion to the vascular system. Therefore, injection depth is an important parameter which influences the absorption rate of the drug after IM injection. Consequently, a too superficial injection will deliver the drug either SC, or into the fat layer, retarding the action of the drug. The absorption will also depend on the muscle mass and its vascularization. Patients with muscular atrophy see a delay in drug absorption, as well as an increase in the risk of neurovascular complications. These anatomical parameters also influence the rapidity of action of the drug [ 44 ]. 2.2.2. Ab Approved for Delivery by the IM Route One of the most threatening pathogens responsible for lower respiratory tract infection in children is the respiratory syncytial virus (RSV). Nearly every infant develops an RSV infection during their childhood. A total of 60% of them have an immune system mature enough to control the infection [ 45 ]. However, between 15 and 40% of infants, especially preterm infants, develop a more serious airway infection, which may eventually lead to bronchiolitis and pneumonia. Although no vaccine or specific RSV treatment exist to treat the infection (apart from general anti-viral therapy), a passive immunization may limit the infection in young infants with high risk factors. In 1998, the FDA approved Palivizumab (Synagis) [ 46 ], a humanized monoclonal IgG1 antibody targeting the glycoprotein F on the RSV virus, which is responsible for membrane fusion and infection of the host cell. Intramuscular injection of palivizumab is recommended for premature infants and young children with heart or lung comorbidities, and injections usually start before the expected RSV epidemic season [ 47 , 48 ]. Intramuscular delivery was chosen to facilitate delivery and limit invasive risks to the young recipients of the treatment. Aggregated clinical data have established the half-life of Palivizumab administered via intramuscular injection to be around 17–27 days, with a mean of 20 days, in infants younger than 24 months. As such, a monthly administration during the epidemic season is necessary to protect effectively against RSV. Palivizumab prophylaxis has shown a significant decrease in the hospitalization, ICU stay, and mortality [ 49 , 50 ]. Unfortunately, its high cost limits the number of children who benefit from this prophylaxis protection, especially in low-income countries [ 51 ]. Although Palivizumab is not licensed for the treatment of RSV disease, it has paved the way to the clinical use of intramuscular injection, and has shown some potential to facilitate everyday care, promoting a less invasive route for patients, in particular infants, in whom IV injection is more complicated [ 52 ]. 2.2.3. Abs in Clinical Development Delivered by the IM Route The IM injection allows a more rapid absorption and onset of action compared to SC delivery: mathematical in silico models associate the IM route with higher drug concentration on target and shorter time to reach the peak of concentration as compared to SC delivery. This route benefits from the long experience of vaccine administration. As shown below, several anti-infective Abs delivered by IM administration are close to reaching patients ( Table 3 ). In 2021, Sotrovimab (Vir Biotechnology—GlaxoSmithKline) obtained an emergency use authorization (EUA) from the FDA for the treatment of mild-to-moderate COVID-19. However, since May 2022, and due to its inefficacy against the BA.2 variant of SARS-CoV-2, its authorization has been revoked [ 53 ]. A phase 3 trial (COMET-TAIL, NCT04913675) which has established that the intramuscular injection of Sotrovimab had a similar clinical effectiveness as compared to an IV injection is still active. SYN023, a therapeutic cocktail comprising two monoclonal Abs, CTB011 and CTB012, is under development for post-exposure prophylaxis to rabies. Nowadays, the standard of care treatment after rabies infection is an on-site (wound injection) and IM administration of anti-rabies immunoglobulin (RIG), called post-exposure prophylaxis, followed by four doses of the vaccine, delivered by IM or intradermal routes. The full dose of RIG facilitates rapid protection until the immune system can produce its own immunoglobulins thanks to the vaccination. However, the current RIG products are limited and expensive, thus their use is limited in high-burden countries [ 54 ]. SYN023, administered by IM injection, targets protein residues found on human rabies virus, and displays a neutralization capacity equivalent or superior to RIGs, even at a lower dose [ 55 ]. The ongoing phase 3 trial will provide additional information regarding the efficacy of SYN023 in populations at risk of rabies infection. Ibalizumab-uiyk (Trograzo) is an anti-HIV monoclonal antibody, approved in 2018, for the treatment of patients with resistant forms of AIDS by IV injection. It targets CD4 protein expressed by T cells, inhibiting the entry of HIV. As a "first class medication" (defined by the FDA), the use of Ibalizumab-uiyk needs to be expanded across the world and for patients reluctant to receive IV injection or where the access to medical facilities and personnel qualified to administer IV injections is limited. A phase 3 is currently evaluating the IM route in comparison to IV injection, notably in terms of safety, pharmacokinetics, and limiting the spread of infection (NCT03913195). 2.2.4. Conclusions and Perspectives on the Intramuscular Route The IM injection is usually considered as a rescue route of administration when other ways to deliver drugs are not appropriate. For example, drugs inducing vein irritation or sensitive to oral digestion have been moved from IV and per os injection, respectively, to IM injection. Among other advantages, IM administration is associated with a quicker and more uniform absorption of the drug as compared to other routes, and it is considered to be as efficient and potent as IV injection, with less invasive characteristics. Even with these potential advantages over other routes of administration, the IM route is still underestimated for Abs. The development of depot injections may represent an opportunity for the expansion of the IM route. Depot injections are a slow-release form of medication usually implanted in the muscle. They are already used for neuroleptic drugs (fluphenazine), prolonging their pharmacological effect. Moreover, they allow a sustained delivery over time [ 56 ] which may be of particular interest regarding the treatment of chronic diseases. 2.3. The Intraocular Route: An Invasive Route for Ophthalmic Disorders 2.3.1. Principles Related to the Intraocular Delivery of Abs With the population aging, the prevalence of ophthalmic disorders has increased over the last 20 years and is estimated to reach several hundred million in the next decades [ 57 ]. The most prevalent age-related eye disease is macular degeneration (AMD), accounting for 196 million patients, in 2020. This continuously evolving disease affecting the retina leads to near blindness, due to the overexpression of vascular endothelial growth factor (VEGF), and has no curative treatment. During AMD, VEGF expression is abnormally increased promoting the development of microvessels, leading to micro-hemorrhage and the progression of the disease. Drug delivery to the retina is challenging, and various routes of administration have been considered to circumvent the complexity of the eye anatomy and improve the therapeutic index of drugs. The eye comprises an anterior and a posterior part. The anterior part, closer to the external environment, composed by the cornea, the conjunctiva, the ciliary body, the aqueous humor, lens, and the lachrymal system, forms a static barrier which prevents the access of foreign particles to the eye. Topical administration on this side results in low bioavailability of the drug (less than 3%) inside the eye, and an even lower in the posterior part [ 58 , 59 ]. Penetration enhancers may be required to improve topical administration [ 60 ]. The posterior part encompasses the vitreous humor, retina, sclera, and choroid. The main barrier on this side is due to the cornea, a negatively charged tissue repulsing negatively charged drugs [ 61 ]. The intra-ocular space is also protected by two additional obstacles, the blood-aqueous, and the blood-retinal barriers [ 62 ], both composed of tight junctions which limit the transfer of drugs from the blood compartment to the eye, especially considering high molecular weight biotherapeutics. In this context, the local delivery of Ab, directly in the internal parts of the eye by intravitreal injection has been developed to provide a high amount of drug in the retina and the vitreous compartments. The intravitreal administration is an invasive procedure consisting in the injection of the Ab, posterior to the limbus, thanks to an incision through the sclera at a specific angle of 30°. The only approved syringe systems for intravitreal injection are the ones already prefilled with Ab. Once the needle is in position, the Ab solution (with a limited volume, ~100–200 µL) is slowly applied into the vitreous cavity, avoiding damage to the retinal surface [ 63 ]. 2.3.2. Approved Abs by Intravitreal Administration Up to now, three Abs delivered by intravitreal injection have been approved for the treatment of AMD and equivalent ophthalmic diseases ( Table 4 ). Ranibizumab (Lucentis), a humanized Fab fragment, [ 64 ] and Brolucizumab (Beovu), a humanized single-chain antibody fragment, both targeting all isoforms of VEGF-A [ 65 ] have been approved in 2006/2007 and 2019/2020, respectively. Recently, Faricimab (Vabysmo), a bispecific antibody targeting both VEGF-A and ANG-2 [ 66 ], has been approved by the FDA, for the treatment of AMD and diabetic macular edema. All these Abs aim at blocking the neo-vascularization, which is the main pathological process associated to retinal diseases [ 67 ]. It is noteworthy that Bevacizumab (Avastin), the first anti-VEGF-A antibody approved for cancer treatment, was also considered as an off-label indication for AMD by intravitreal injection, due to its more affordable price [ 68 ]. Unfortunately, it did not attain the required professional or political consensus to obtain final approval [ 69 ]. The pharmacokinetics of these Abs has been extensively studied. In aqueous humor, Bevacizumab and Ranibizumab were shown to have a half-life of around 10 days, and 7 days, respectively [ 70 , 71 ]. Abs concentration peaked on the first day of injection, and then rapidly declined. The short intravitreal half-lives implied frequent injections to ensure optimal effect, increasing the potential risks of side-effects. Interestingly, Faricimab demonstrated longer duration of action in a phase 3 clinical trial, with a sustained efficacy over 16 weeks post-injection [ 72 ]. To better understand the PK-PD of Abs after intravitreal administration, Mazer et al. developed a mechanistic model of intravitreal pharmacodynamics of anti-VEGF in the eye and demonstrated the interrelationship between the half-life of the Ranibizumab and the in vivo VEGF kD [ 73 ]. They also showed that the ocular Ab t1/2 was proportional to the hydrodynamic radius of the antibody, and the radius of the vitreous globe. Thus, the biodistribution and absorption of Ab depends on the physiological state of the eye, with a potential decrease in Abs efficacy for older eyes, or eyes impaired with either globe abnormalities or underlying pathologies (e.g., myopia, or hypermetropia) [ 74 ]. 2.3.3. Abs in Clinical Development for Intravitreal/Intraocular Delivery The global aging of the human population accounting for an increased prevalence of AMD, or retinal vein occlusion along with the effectiveness of intravitreal administration, has paved the way for further developments of intravitreally/intraocularly-delivered Abs products ( Table 5 ). It is noteworthy that several ongoing trials are further evaluating currently approved Abs delivered by the intravitreal route ( Table 4 ) to either identify their long-term effects in patients with AMD (NCT04777201) or evaluate their efficacy in other ophthalmic diseases (NCT04740905). Notably, Faricimab, after being recently approved for AMD is currently being investigated in macular edema and branch retinal occlusion. KSI-301 is a humanized antibody against VEGF-A conjugated to a biopolymer, under investigation for the treatment of different ophthalmic disorders [ 75 , 76 ]. Apart from its excellent safety and better efficacy than the current treatment using the fusion protein Aflibercept, the phase 1 trial showed longer durability, allowing patients to achieve treatment-free intervals of 4 to 6 months. Few novel drugs delivered by intravitreal injection are currently being developed. However, biosimilars are currently under investigation. HLX04-O, a biosimilar of Bevacizumab, is a monoclonal antibody under development by Shanghai Henlius Biotech, targeting VEGF to treat AMD. Contrary to the original molecule, this biosimilar has been developed de novo for intraocular administration. Early-stage clinical trials, analyzing the safety and toxicity of the molecule delivered intravitreally, showed promising results, similar to the results obtained with Bevacizumab. As of April 2022, a Phase 3 trial was launched with the administration of HLX04-O to a first patient [ 77 ]. MW02, a recombinant anti-VEGF humanized monoclonal antibody, is under phase 2 and 3 evaluation. The trials compare its efficacy and safety to Ranibizumab (Lucentis) (NCT05297292). 2.3.4. Conclusion and Perspectives on the Intravitreal Route While anatomic barriers prevent Abs access to the eye from the blood compartment, intravitreal injection has demonstrated efficacy and has become the standard-of-care in the treatment of eye disease. Although it is invasive, it is associated with limited side-effects such as uveitis and vitreitis [ 78 ], and a higher therapeutic index. To support the development of intravitreally-delivered Abs, advances are necessary in formulation and medical devices, to allow prolonged action, longer periods between two injections, and better tolerability. For example, the Port Delivery System (PDS) has been proposed for the delivery of Ranibizumab. It consists of a device surgically implanted into the vitreous cavity, allowing continuous delivery of the Ab. It dispenses the need for frequent intravitreal injections, thereby reducing invasiveness for patients. Its clinical evaluation is on-going and the first results for patients suffering from neovascular age-related macular degeneration are promising, showing equivalent control of the disease as compared to the standard care treatment, a monthly intravitreal injection of Ranibizumab (NCT04657289). The repertoire of excipients recognized as safe by the intravitreal route is limited due to ocular toxicity. For instance, only polysorbate (80 and 20) and sugar residues have been included in the formulation of Brolucizumab, Faricimab, and Ranibizumab [ 79 , 80 ]. Similar to what is observed in SC administration, Abs diffuse poorly into the vitreous cavity due to their positive charge [ 81 ]. Multiple parameters may influence the diffusion of Abs inside the eye, including: (i) the age of the eye, which can be associated with a change in the viscosity of the vitreous humor; and (ii) the quality of the lens. Patients with cataract surgery might experience a more rapid clearance of Abs from the eye, as observed with intravitreal delivery of antibiotics [ 82 ]. Overall, intravitreal injection is quite challenging, and inter-individual variability will dictate the fate and the activity of the antibody inside the eye. Like other local routes of administration, intravitreal injection of Ab provides effective on-target treatment, while limiting systemic deleterious exposure, and thereby improving Ab therapeutic index. 2.3.1. Principles Related to the Intraocular Delivery of Abs With the population aging, the prevalence of ophthalmic disorders has increased over the last 20 years and is estimated to reach several hundred million in the next decades [ 57 ]. The most prevalent age-related eye disease is macular degeneration (AMD), accounting for 196 million patients, in 2020. This continuously evolving disease affecting the retina leads to near blindness, due to the overexpression of vascular endothelial growth factor (VEGF), and has no curative treatment. During AMD, VEGF expression is abnormally increased promoting the development of microvessels, leading to micro-hemorrhage and the progression of the disease. Drug delivery to the retina is challenging, and various routes of administration have been considered to circumvent the complexity of the eye anatomy and improve the therapeutic index of drugs. The eye comprises an anterior and a posterior part. The anterior part, closer to the external environment, composed by the cornea, the conjunctiva, the ciliary body, the aqueous humor, lens, and the lachrymal system, forms a static barrier which prevents the access of foreign particles to the eye. Topical administration on this side results in low bioavailability of the drug (less than 3%) inside the eye, and an even lower in the posterior part [ 58 , 59 ]. Penetration enhancers may be required to improve topical administration [ 60 ]. The posterior part encompasses the vitreous humor, retina, sclera, and choroid. The main barrier on this side is due to the cornea, a negatively charged tissue repulsing negatively charged drugs [ 61 ]. The intra-ocular space is also protected by two additional obstacles, the blood-aqueous, and the blood-retinal barriers [ 62 ], both composed of tight junctions which limit the transfer of drugs from the blood compartment to the eye, especially considering high molecular weight biotherapeutics. In this context, the local delivery of Ab, directly in the internal parts of the eye by intravitreal injection has been developed to provide a high amount of drug in the retina and the vitreous compartments. The intravitreal administration is an invasive procedure consisting in the injection of the Ab, posterior to the limbus, thanks to an incision through the sclera at a specific angle of 30°. The only approved syringe systems for intravitreal injection are the ones already prefilled with Ab. Once the needle is in position, the Ab solution (with a limited volume, ~100–200 µL) is slowly applied into the vitreous cavity, avoiding damage to the retinal surface [ 63 ]. 2.3.2. Approved Abs by Intravitreal Administration Up to now, three Abs delivered by intravitreal injection have been approved for the treatment of AMD and equivalent ophthalmic diseases ( Table 4 ). Ranibizumab (Lucentis), a humanized Fab fragment, [ 64 ] and Brolucizumab (Beovu), a humanized single-chain antibody fragment, both targeting all isoforms of VEGF-A [ 65 ] have been approved in 2006/2007 and 2019/2020, respectively. Recently, Faricimab (Vabysmo), a bispecific antibody targeting both VEGF-A and ANG-2 [ 66 ], has been approved by the FDA, for the treatment of AMD and diabetic macular edema. All these Abs aim at blocking the neo-vascularization, which is the main pathological process associated to retinal diseases [ 67 ]. It is noteworthy that Bevacizumab (Avastin), the first anti-VEGF-A antibody approved for cancer treatment, was also considered as an off-label indication for AMD by intravitreal injection, due to its more affordable price [ 68 ]. Unfortunately, it did not attain the required professional or political consensus to obtain final approval [ 69 ]. The pharmacokinetics of these Abs has been extensively studied. In aqueous humor, Bevacizumab and Ranibizumab were shown to have a half-life of around 10 days, and 7 days, respectively [ 70 , 71 ]. Abs concentration peaked on the first day of injection, and then rapidly declined. The short intravitreal half-lives implied frequent injections to ensure optimal effect, increasing the potential risks of side-effects. Interestingly, Faricimab demonstrated longer duration of action in a phase 3 clinical trial, with a sustained efficacy over 16 weeks post-injection [ 72 ]. To better understand the PK-PD of Abs after intravitreal administration, Mazer et al. developed a mechanistic model of intravitreal pharmacodynamics of anti-VEGF in the eye and demonstrated the interrelationship between the half-life of the Ranibizumab and the in vivo VEGF kD [ 73 ]. They also showed that the ocular Ab t1/2 was proportional to the hydrodynamic radius of the antibody, and the radius of the vitreous globe. Thus, the biodistribution and absorption of Ab depends on the physiological state of the eye, with a potential decrease in Abs efficacy for older eyes, or eyes impaired with either globe abnormalities or underlying pathologies (e.g., myopia, or hypermetropia) [ 74 ]. 2.3.3. Abs in Clinical Development for Intravitreal/Intraocular Delivery The global aging of the human population accounting for an increased prevalence of AMD, or retinal vein occlusion along with the effectiveness of intravitreal administration, has paved the way for further developments of intravitreally/intraocularly-delivered Abs products ( Table 5 ). It is noteworthy that several ongoing trials are further evaluating currently approved Abs delivered by the intravitreal route ( Table 4 ) to either identify their long-term effects in patients with AMD (NCT04777201) or evaluate their efficacy in other ophthalmic diseases (NCT04740905). Notably, Faricimab, after being recently approved for AMD is currently being investigated in macular edema and branch retinal occlusion. KSI-301 is a humanized antibody against VEGF-A conjugated to a biopolymer, under investigation for the treatment of different ophthalmic disorders [ 75 , 76 ]. Apart from its excellent safety and better efficacy than the current treatment using the fusion protein Aflibercept, the phase 1 trial showed longer durability, allowing patients to achieve treatment-free intervals of 4 to 6 months. Few novel drugs delivered by intravitreal injection are currently being developed. However, biosimilars are currently under investigation. HLX04-O, a biosimilar of Bevacizumab, is a monoclonal antibody under development by Shanghai Henlius Biotech, targeting VEGF to treat AMD. Contrary to the original molecule, this biosimilar has been developed de novo for intraocular administration. Early-stage clinical trials, analyzing the safety and toxicity of the molecule delivered intravitreally, showed promising results, similar to the results obtained with Bevacizumab. As of April 2022, a Phase 3 trial was launched with the administration of HLX04-O to a first patient [ 77 ]. MW02, a recombinant anti-VEGF humanized monoclonal antibody, is under phase 2 and 3 evaluation. The trials compare its efficacy and safety to Ranibizumab (Lucentis) (NCT05297292). 2.3.4. Conclusion and Perspectives on the Intravitreal Route While anatomic barriers prevent Abs access to the eye from the blood compartment, intravitreal injection has demonstrated efficacy and has become the standard-of-care in the treatment of eye disease. Although it is invasive, it is associated with limited side-effects such as uveitis and vitreitis [ 78 ], and a higher therapeutic index. To support the development of intravitreally-delivered Abs, advances are necessary in formulation and medical devices, to allow prolonged action, longer periods between two injections, and better tolerability. For example, the Port Delivery System (PDS) has been proposed for the delivery of Ranibizumab. It consists of a device surgically implanted into the vitreous cavity, allowing continuous delivery of the Ab. It dispenses the need for frequent intravitreal injections, thereby reducing invasiveness for patients. Its clinical evaluation is on-going and the first results for patients suffering from neovascular age-related macular degeneration are promising, showing equivalent control of the disease as compared to the standard care treatment, a monthly intravitreal injection of Ranibizumab (NCT04657289). The repertoire of excipients recognized as safe by the intravitreal route is limited due to ocular toxicity. For instance, only polysorbate (80 and 20) and sugar residues have been included in the formulation of Brolucizumab, Faricimab, and Ranibizumab [ 79 , 80 ]. Similar to what is observed in SC administration, Abs diffuse poorly into the vitreous cavity due to their positive charge [ 81 ]. Multiple parameters may influence the diffusion of Abs inside the eye, including: (i) the age of the eye, which can be associated with a change in the viscosity of the vitreous humor; and (ii) the quality of the lens. Patients with cataract surgery might experience a more rapid clearance of Abs from the eye, as observed with intravitreal delivery of antibiotics [ 82 ]. Overall, intravitreal injection is quite challenging, and inter-individual variability will dictate the fate and the activity of the antibody inside the eye. Like other local routes of administration, intravitreal injection of Ab provides effective on-target treatment, while limiting systemic deleterious exposure, and thereby improving Ab therapeutic index. 2.4. Inhalation: An Alternative Route for Respiratory Diseases 2.4.1. Rationale of Delivering Abs through the Airways Respiratory diseases are a major worldwide public health issue. They represent the fourth most common cause of death worldwide, which is mainly attributable to lung cancer, lung inflammatory diseases (e.g., chronic obstructive pulmonary disease (COPD)), and lower respiratory tract infections. The increase in antimicrobial resistance, seasonal virus outbreaks, newly emerging pathogens, or atmospheric pollution have severely complicated the management of airway diseases. Owing the success of Obiltoxaximab/Raxibacumab against pulmonary anthrax and Benralizumab against asthma, Abs have emerged as powerful therapeutics to tackle multiple respiratory diseases [ 83 ]. However, Abs administration through systemic routes led to a low bioavailability in the airway compartment [ 84 , 85 ]. This may limit the efficacy of Abs to treat respiratory diseases, if their target antigen primarily acts within the respiratory organ, while exposing the rest of the body to potential side effects [ 86 ]. Oral inhalation is the gold standard route of administration for small molecules commonly used for the treatment of inflammatory diseases (asthma, COPD). It allows direct drug access—as an aerosol—to both the upper and lower respiratory tract. It is associated with a rapid onset of action and a better therapeutic index as compared to parenteral injections [ 87 , 88 ]. Abs aerosolization is generally difficult since they are highly sensitive to mechanical, thermal, and physical stresses occurring during aerosol generation. Two types of devices have been evaluated for the oral inhalation of proteins: dry-powder inhalers, which deliver solid aerosol, and nebulizers, delivering liquid-formed aerosols. So far, metered-dose inhalers are not recommended for proteins due to the use of propellants [ 89 ]. 2.4.2. Challenges Associated with the Clinical Development of Inhaled Abs Researchers and pharmaceutical companies have been interested by local delivery of Abs through the airways for the last 20 years [ 90 ]. However, the first attempts were unsuccessful [ 91 ] and may be explained by multiple factors: − Abs instability during aerosolization/spraying, resulting in aggregation and thereby potential alteration of activity and immunogenicity concerns [ 86 , 92 , 93 ]. − Relevant target antigen, which must operate within the respiratory tract and be critical in the pathophysiology of the respiratory disease − Selection of the appropriate population, which may benefit from inhaled Abs. − Biological barriers, which may impair Abs PK and activity [ 94 ]. Several inhaled Abs are in early clinical trials, after preclinical studies showing promising efficacy in animal models (for example anti-IL-13 Fab for the treatment of asthma [ 95 ]). A few inhaled Abs are in phase 2 and 3 clinical evaluation, notably for the treatment of SARS-CoV-2 infection ( Table 6 ). CSJ117 (Ecleralimab) is an inhaled TSLP inhibitory antibody fragment developed to treat moderate to severe asthma [ 96 ] and COPD. The antibody is provided as a powder in hard capsules to be delivered daily in the lungs using a dry powder inhaler. First results showed promising results, with CSJ117 having the ability to attenuate airways inflammation in asthma patients. The recent SARS-CoV-2 pandemic has brought attention to the inhalation route, and several inhaled anti-SARS-CoV-2 Ab are being tested in the clinic by oral inhalation. Among the most advanced is a combination therapy developed to tackle the emerging SARS-CoV2 mutants, showing promising results. CT-P63 and CT-P59 (Regdanvimab) are monoclonal Abs, both targeting the receptor binding domain of the spike protein of SARS-CoV-2. More than just eliciting a neutralizing response, these Abs have the property to trap viral particles within the mucus, promoting their elimination by mucociliary clearance [ 97 ]. It is noteworthy that one molecule is currently under development by nasal inhalation, to address the Ab into the nasal cavity rather than in the lungs (COVI-DROPS). 2.4.3. Conclusion and Perspectives on the Inhalation Route Although airway delivery of Abs has been demonstrated as feasible and promising in preclinical studies, the benefits of Abs inhalation have not materialized yet in the clinical setting. A major advantage of the inhalation route is on the possibility of self-administration of Abs treatment for non-hospitalized patients, thereby reducing healthcare costs, and increasing patient comfort [ 98 ]. However, groundwork is still necessary to optimize Abs design and formulation, inhalation devices, as well as improving our knowledge of the physical and biological barriers that may impair the therapeutic response to inhaled Abs. 2.4.1. Rationale of Delivering Abs through the Airways Respiratory diseases are a major worldwide public health issue. They represent the fourth most common cause of death worldwide, which is mainly attributable to lung cancer, lung inflammatory diseases (e.g., chronic obstructive pulmonary disease (COPD)), and lower respiratory tract infections. The increase in antimicrobial resistance, seasonal virus outbreaks, newly emerging pathogens, or atmospheric pollution have severely complicated the management of airway diseases. Owing the success of Obiltoxaximab/Raxibacumab against pulmonary anthrax and Benralizumab against asthma, Abs have emerged as powerful therapeutics to tackle multiple respiratory diseases [ 83 ]. However, Abs administration through systemic routes led to a low bioavailability in the airway compartment [ 84 , 85 ]. This may limit the efficacy of Abs to treat respiratory diseases, if their target antigen primarily acts within the respiratory organ, while exposing the rest of the body to potential side effects [ 86 ]. Oral inhalation is the gold standard route of administration for small molecules commonly used for the treatment of inflammatory diseases (asthma, COPD). It allows direct drug access—as an aerosol—to both the upper and lower respiratory tract. It is associated with a rapid onset of action and a better therapeutic index as compared to parenteral injections [ 87 , 88 ]. Abs aerosolization is generally difficult since they are highly sensitive to mechanical, thermal, and physical stresses occurring during aerosol generation. Two types of devices have been evaluated for the oral inhalation of proteins: dry-powder inhalers, which deliver solid aerosol, and nebulizers, delivering liquid-formed aerosols. So far, metered-dose inhalers are not recommended for proteins due to the use of propellants [ 89 ]. 2.4.2. Challenges Associated with the Clinical Development of Inhaled Abs Researchers and pharmaceutical companies have been interested by local delivery of Abs through the airways for the last 20 years [ 90 ]. However, the first attempts were unsuccessful [ 91 ] and may be explained by multiple factors: − Abs instability during aerosolization/spraying, resulting in aggregation and thereby potential alteration of activity and immunogenicity concerns [ 86 , 92 , 93 ]. − Relevant target antigen, which must operate within the respiratory tract and be critical in the pathophysiology of the respiratory disease − Selection of the appropriate population, which may benefit from inhaled Abs. − Biological barriers, which may impair Abs PK and activity [ 94 ]. Several inhaled Abs are in early clinical trials, after preclinical studies showing promising efficacy in animal models (for example anti-IL-13 Fab for the treatment of asthma [ 95 ]). A few inhaled Abs are in phase 2 and 3 clinical evaluation, notably for the treatment of SARS-CoV-2 infection ( Table 6 ). CSJ117 (Ecleralimab) is an inhaled TSLP inhibitory antibody fragment developed to treat moderate to severe asthma [ 96 ] and COPD. The antibody is provided as a powder in hard capsules to be delivered daily in the lungs using a dry powder inhaler. First results showed promising results, with CSJ117 having the ability to attenuate airways inflammation in asthma patients. The recent SARS-CoV-2 pandemic has brought attention to the inhalation route, and several inhaled anti-SARS-CoV-2 Ab are being tested in the clinic by oral inhalation. Among the most advanced is a combination therapy developed to tackle the emerging SARS-CoV2 mutants, showing promising results. CT-P63 and CT-P59 (Regdanvimab) are monoclonal Abs, both targeting the receptor binding domain of the spike protein of SARS-CoV-2. More than just eliciting a neutralizing response, these Abs have the property to trap viral particles within the mucus, promoting their elimination by mucociliary clearance [ 97 ]. It is noteworthy that one molecule is currently under development by nasal inhalation, to address the Ab into the nasal cavity rather than in the lungs (COVI-DROPS). 2.4.3. Conclusion and Perspectives on the Inhalation Route Although airway delivery of Abs has been demonstrated as feasible and promising in preclinical studies, the benefits of Abs inhalation have not materialized yet in the clinical setting. A major advantage of the inhalation route is on the possibility of self-administration of Abs treatment for non-hospitalized patients, thereby reducing healthcare costs, and increasing patient comfort [ 98 ]. However, groundwork is still necessary to optimize Abs design and formulation, inhalation devices, as well as improving our knowledge of the physical and biological barriers that may impair the therapeutic response to inhaled Abs. 2.5. Intra-Tumoral Administration: Overcoming the Tumor Stromal Barrier for Anti-Cancerous Abs 2.5.1. Overview of the Barriers Associated with Tumor Abs used for the treatment of cancer are systematically delivered through IV injection. Their targets cover a wide range of proteins. They will promote the blocking of signals needed for cancer cell survival/growth, induce cancer cell destruction when coupled with toxins, or mark the tumor so that the immune system will better recognize and destroy it. Recently, the development of Abs targeting immune checkpoints has changed the treatment paradigm in many cancers. Abs targeting immune checkpoints inhibitors (ICI) block the immune "off" signal induced by the tumor cell in order to prevent the immune system from destroying the cancer, thus allowing a natural anti-cancerous immune response, and a higher survival for patients [ 99 ]. Systemic anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) Ab was the first licensed ICI against advanced metastatic melanoma [ 100 ], and was then quickly followed by anti-programmed cell death ligand and protein 1 (PD-L1/PD-1) Abs. However, if ICI antibodies have shown efficacy in patients with melanoma or non-small cell lung cancers, many cancers remain refractory to them. In addition, if the systemic ICI treatment has undeniable advantages, such as predictable pharmacokinetics, it is associated with hazardous on-target/off-tumor side effects, including widespread inflammation. Thus, improving the tumor targeting of ICI Abs appears to be an evident goal. In fact, most solid tumors are poorly reachable from the systemic circulation, due to multiple surrounding barriers [ 94 ]: − Densified tumor-associated ECM with the overexpression of collagens, and fibronectin, preventing the diffusion of Abs [ 101 ]. − Abnormal growth of blood vessels in the vicinity of the tumor resulting in non-vascularized, inaccessible areas for Abs coming from the blood circulation [ 102 ]. − Disorganized vessel structure associated with blood flow resistance, impeding the transport of the drug [ 103 ]. Consequently, several strategies have been proposed to limit the side effects of Abs targeting ICI, as well as optimizing their bioavailability within the tumor environment. A novel accurate method of injection consists in the delivery of Abs intratumorally (IT), allowing access to the tumor vicinity, circumventing extracellular barriers, and in theory improving on-target efficacy. Intra-tumoral injection is performed in a clinical environment by a physician, under the guidance of imagery (e.g., ultrasound, computed tomography (CT), or endoscopy) [ 104 ]. 2.5.2. Abs in Clinical Development by Intra-Tumoral Injection Preclinical studies have established that local administration of anti-CTLA-4 Abs was able to restore an anti-tumor response, eradicating both local and distant tumors, and used a significant lower amount of Abs as compared to IV Ab [ 105 , 106 ]. Thanks to the success of those pre-clinical studies, novel intratumorally-delivered Abs are under clinical evaluation ( Table 7 ). In 2019, an anti-CD40 agonist antibody (ADC-1013) was developed and clinically evaluated in solid tumors after intra-tumoral administration (NCT02379741). The results from the phase 1 study showed a good safety profile and were associated with positive pharmacodynamic responses [ 107 ]. Ipilimumab is a human anti-CTLA4 antibody already approved for the treatment of metastatic melanoma and given by IV injection. A phase 1 study showed that Ipilimumab in combination with IL-2, given intratumorally, was well tolerated and was able to generate anti-tumoral responses in the majority of patients [ 108 ]. A phase 2 study is currently evaluating the tolerance of Ipilimumab IT and Nivolumab IV for the treatment of stage III/IV melanoma patients (NCT02857569). Moreover, a phase 2/3 trial is comparing Pembrolizumab and/or Ipilimumab in patients with advanced solid tumors after intra-tumoral injection and IV infusion. (NCT03755739). 2.5.3. Conclusion and Perspectives on the Intra-Tumoral Route Intra-tumoral administration of anti-cancerous Abs has multiple advantages, especially in reducing the harmful side effects associated with on-target/off-tumor of Abs after IV injection. In addition, local administration, within the tumor environment, may promote a superior priming of T-cells already on-site, and a multi-clonal response [ 106 ]. For now, clinical validation of intra-tumoral delivery of Abs is still in the preliminary stages. Further work is needed to optimize the dosage and the formulation according to the type and the stage of the tumor lesion. In addition, this route of administration is operator-dependent, which means that a subtle change in needle position may change the outcome of the administration. The development of new live imaging tools may improve injection reproducibility. Combinatorial approaches with intra-tumoral and intravenous treatment, or tumor ablation, are also being investigated and may represent a promising therapeutic alternative [ 109 ]. 2.5.1. Overview of the Barriers Associated with Tumor Abs used for the treatment of cancer are systematically delivered through IV injection. Their targets cover a wide range of proteins. They will promote the blocking of signals needed for cancer cell survival/growth, induce cancer cell destruction when coupled with toxins, or mark the tumor so that the immune system will better recognize and destroy it. Recently, the development of Abs targeting immune checkpoints has changed the treatment paradigm in many cancers. Abs targeting immune checkpoints inhibitors (ICI) block the immune "off" signal induced by the tumor cell in order to prevent the immune system from destroying the cancer, thus allowing a natural anti-cancerous immune response, and a higher survival for patients [ 99 ]. Systemic anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) Ab was the first licensed ICI against advanced metastatic melanoma [ 100 ], and was then quickly followed by anti-programmed cell death ligand and protein 1 (PD-L1/PD-1) Abs. However, if ICI antibodies have shown efficacy in patients with melanoma or non-small cell lung cancers, many cancers remain refractory to them. In addition, if the systemic ICI treatment has undeniable advantages, such as predictable pharmacokinetics, it is associated with hazardous on-target/off-tumor side effects, including widespread inflammation. Thus, improving the tumor targeting of ICI Abs appears to be an evident goal. In fact, most solid tumors are poorly reachable from the systemic circulation, due to multiple surrounding barriers [ 94 ]: − Densified tumor-associated ECM with the overexpression of collagens, and fibronectin, preventing the diffusion of Abs [ 101 ]. − Abnormal growth of blood vessels in the vicinity of the tumor resulting in non-vascularized, inaccessible areas for Abs coming from the blood circulation [ 102 ]. − Disorganized vessel structure associated with blood flow resistance, impeding the transport of the drug [ 103 ]. Consequently, several strategies have been proposed to limit the side effects of Abs targeting ICI, as well as optimizing their bioavailability within the tumor environment. A novel accurate method of injection consists in the delivery of Abs intratumorally (IT), allowing access to the tumor vicinity, circumventing extracellular barriers, and in theory improving on-target efficacy. Intra-tumoral injection is performed in a clinical environment by a physician, under the guidance of imagery (e.g., ultrasound, computed tomography (CT), or endoscopy) [ 104 ]. 2.5.2. Abs in Clinical Development by Intra-Tumoral Injection Preclinical studies have established that local administration of anti-CTLA-4 Abs was able to restore an anti-tumor response, eradicating both local and distant tumors, and used a significant lower amount of Abs as compared to IV Ab [ 105 , 106 ]. Thanks to the success of those pre-clinical studies, novel intratumorally-delivered Abs are under clinical evaluation ( Table 7 ). In 2019, an anti-CD40 agonist antibody (ADC-1013) was developed and clinically evaluated in solid tumors after intra-tumoral administration (NCT02379741). The results from the phase 1 study showed a good safety profile and were associated with positive pharmacodynamic responses [ 107 ]. Ipilimumab is a human anti-CTLA4 antibody already approved for the treatment of metastatic melanoma and given by IV injection. A phase 1 study showed that Ipilimumab in combination with IL-2, given intratumorally, was well tolerated and was able to generate anti-tumoral responses in the majority of patients [ 108 ]. A phase 2 study is currently evaluating the tolerance of Ipilimumab IT and Nivolumab IV for the treatment of stage III/IV melanoma patients (NCT02857569). Moreover, a phase 2/3 trial is comparing Pembrolizumab and/or Ipilimumab in patients with advanced solid tumors after intra-tumoral injection and IV infusion. (NCT03755739). 2.5.3. Conclusion and Perspectives on the Intra-Tumoral Route Intra-tumoral administration of anti-cancerous Abs has multiple advantages, especially in reducing the harmful side effects associated with on-target/off-tumor of Abs after IV injection. In addition, local administration, within the tumor environment, may promote a superior priming of T-cells already on-site, and a multi-clonal response [ 106 ]. For now, clinical validation of intra-tumoral delivery of Abs is still in the preliminary stages. Further work is needed to optimize the dosage and the formulation according to the type and the stage of the tumor lesion. In addition, this route of administration is operator-dependent, which means that a subtle change in needle position may change the outcome of the administration. The development of new live imaging tools may improve injection reproducibility. Combinatorial approaches with intra-tumoral and intravenous treatment, or tumor ablation, are also being investigated and may represent a promising therapeutic alternative [ 109 ]. 2.6. Intra-Articular Administration: A New Hope for the Relief of Joint Pain 2.6.1. Overview of Joint Physiology Joints degeneration is one of the leading causes of permanent motor disability worldwide requiring long-term therapeutic treatment. Among them, osteoarthritis (caused by trauma to the joint cartilage), rheumatoid arthritis (autoimmune disease where immune cells attack the joints), and gout (joint inflammation due to excess of uric acid), are the most common forms [ 110 ]. Standard care includes systemic administration of analgesic and anti-inflammatory agents, including anti-TNF-α (Infliximab, Etanercept, Adalimumab) molecules and anti-IL1β Ab (Canakinumab) [ 111 ]. However, although symptoms show substantial improvements thanks to these treatments, non-responsive patients and side effects are increasing with time [ 112 ]. Efforts have been made in recent years toward the development of intra-articular (IA)-delivered treatments, with multiple benefits such as a better bioavailability and a reduced systemic exposure [ 113 ]. Intra-articular injection is performed using syringe/needle into a joint. To help the guidance of the needle, ultrasound or fluoroscopy techniques can be used. The development of drugs dedicated to IA injection necessitates understanding the anatomy and physiology of the joints, to identify the key parameters influencing the pharmacokinetics and pharmacodynamics of the drug. Synovial joints, or diarthrosis, joins bone endings and hyaline cartilage with a fibrous capsule delimitating the synovial cavity filled with synovial fluid. Synovial joints are particularly affected during the degeneration processes [ 114 ]. Cartilage is an avascular tissue composed of chondrocytes embedded in a negatively charged ECM which make this tissue impermeable to molecules bigger than 50 kDa (such as antibodies) depending upon their charge and conformation. Consequently, the cartilage is inefficiently targeted by drugs administered systemically, which first need to reach the synovial fluid, before diffusing through cartilaginous ECM. To enter the joints, the drug needs to pass through the capillary endothelium of the synovium, the ECM of the synovial intima, and the synoviocytes composing the synovial membrane. Both cellular layers are highly fenestrated, allowing high diffusion of molecules smaller than 10 kDA. For larger molecules, the fenestration will allow a size-dependent diffusion, slowing the passage of large molecules such as antibodies [ 115 ]. During inflammation, there is an increased permeability of both capillary and synovial membranes, allowing macromolecules to reach the synovial compartment. However, joint inflammation will also accelerate synovial clearance [ 116 ]; studies have revealed a mean clinical half-life of around 3 h for an anti-inflammatory antibody, not leaving enough time for an optimal action of the antibody. 2.6.2. Abs in Clinical Development for IA Administration Despite some promising features, intra-articular delivery of therapeutic antibodies remains rare. Studies performed 20 years ago have shown conflicting results regarding IA delivery of Infliximab depending on the disease, with positive outcomes in patients suffering from ankylosing spondylitis [ 117 ], while there was no positive effect in patients with acute joint inflammation [ 118 ]. In 2007, the clinical evaluation of intra-articularly-delivered Infliximab in patients suffering from intractable knee monoarthritis, reached a phase 3 clinical trial but the development was stopped in 2015 due to the insufficient recruitment of patients [ 117 ] (NCT00521963). More recently, two novel intra-articularly-delivered Abs have entered clinical evaluation ( Table 8 ). AMB-05X is a fully human antibody targeting colony-stimulating factor I (c-FMS), a protein overexpressed in many cancers, and on tumor-associated macrophages. This antibody entered a clinical trial phase 2 (NCT04731675), to treat patients suffering from tenosynovial giant cell tumor, and pigmented villonodular synovitis, two afflictions of the knee, after IA delivery. The ongoing study is investigating the safety, tolerability, and efficacy of the treatment delivered by IA injection. Canakinumab, a human anti-IL1β antibody, is currently in phase 2 of clinical evaluation, in combination with LNA043 (a protein inducing chondrogenesis and cartilage repair) for the treatment of patients with knee osteoarthritis (NCT04814368) after IA delivery. 2.6.3. Conclusion and Perspectives Regarding the Intra-Articular Route Intra-articular delivery may be of particular importance for the treatment of joint diseases (and notably rheumatoid arthritis) as around 30% of patients are resistant to biotherapies, and few novel molecules are under development. The limited half-life of antibodies reaching the inflamed joint is one of the major limitations associated with IA delivery, requiring frequent injections and thus increasing side effects, discomfort, and morbidity. Specific formulations including hydrogels [ 119 ], micro/nano particles [ 120 ], or in situ implants [ 121 ] are under consideration to improve antibody concentration on-target. A long-lasting Ab formulation through the use of biopolymers was recently developed for IA administration, showing a sustained high concentration of Abs in the synovial fluid, with minimal inflammatory side effects [ 122 ]. However, controlled-release methods are often associated with a loss of bioactivity of the drug, and/or inflammatory side effects that could accelerate the degeneration of the cartilage and the disease [ 123 ]. Additional work is needed to develop safe and active intra-articularly-delivered Abs to fight joints auto-immune diseases. 2.6.1. Overview of Joint Physiology Joints degeneration is one of the leading causes of permanent motor disability worldwide requiring long-term therapeutic treatment. Among them, osteoarthritis (caused by trauma to the joint cartilage), rheumatoid arthritis (autoimmune disease where immune cells attack the joints), and gout (joint inflammation due to excess of uric acid), are the most common forms [ 110 ]. Standard care includes systemic administration of analgesic and anti-inflammatory agents, including anti-TNF-α (Infliximab, Etanercept, Adalimumab) molecules and anti-IL1β Ab (Canakinumab) [ 111 ]. However, although symptoms show substantial improvements thanks to these treatments, non-responsive patients and side effects are increasing with time [ 112 ]. Efforts have been made in recent years toward the development of intra-articular (IA)-delivered treatments, with multiple benefits such as a better bioavailability and a reduced systemic exposure [ 113 ]. Intra-articular injection is performed using syringe/needle into a joint. To help the guidance of the needle, ultrasound or fluoroscopy techniques can be used. The development of drugs dedicated to IA injection necessitates understanding the anatomy and physiology of the joints, to identify the key parameters influencing the pharmacokinetics and pharmacodynamics of the drug. Synovial joints, or diarthrosis, joins bone endings and hyaline cartilage with a fibrous capsule delimitating the synovial cavity filled with synovial fluid. Synovial joints are particularly affected during the degeneration processes [ 114 ]. Cartilage is an avascular tissue composed of chondrocytes embedded in a negatively charged ECM which make this tissue impermeable to molecules bigger than 50 kDa (such as antibodies) depending upon their charge and conformation. Consequently, the cartilage is inefficiently targeted by drugs administered systemically, which first need to reach the synovial fluid, before diffusing through cartilaginous ECM. To enter the joints, the drug needs to pass through the capillary endothelium of the synovium, the ECM of the synovial intima, and the synoviocytes composing the synovial membrane. Both cellular layers are highly fenestrated, allowing high diffusion of molecules smaller than 10 kDA. For larger molecules, the fenestration will allow a size-dependent diffusion, slowing the passage of large molecules such as antibodies [ 115 ]. During inflammation, there is an increased permeability of both capillary and synovial membranes, allowing macromolecules to reach the synovial compartment. However, joint inflammation will also accelerate synovial clearance [ 116 ]; studies have revealed a mean clinical half-life of around 3 h for an anti-inflammatory antibody, not leaving enough time for an optimal action of the antibody. 2.6.2. Abs in Clinical Development for IA Administration Despite some promising features, intra-articular delivery of therapeutic antibodies remains rare. Studies performed 20 years ago have shown conflicting results regarding IA delivery of Infliximab depending on the disease, with positive outcomes in patients suffering from ankylosing spondylitis [ 117 ], while there was no positive effect in patients with acute joint inflammation [ 118 ]. In 2007, the clinical evaluation of intra-articularly-delivered Infliximab in patients suffering from intractable knee monoarthritis, reached a phase 3 clinical trial but the development was stopped in 2015 due to the insufficient recruitment of patients [ 117 ] (NCT00521963). More recently, two novel intra-articularly-delivered Abs have entered clinical evaluation ( Table 8 ). AMB-05X is a fully human antibody targeting colony-stimulating factor I (c-FMS), a protein overexpressed in many cancers, and on tumor-associated macrophages. This antibody entered a clinical trial phase 2 (NCT04731675), to treat patients suffering from tenosynovial giant cell tumor, and pigmented villonodular synovitis, two afflictions of the knee, after IA delivery. The ongoing study is investigating the safety, tolerability, and efficacy of the treatment delivered by IA injection. Canakinumab, a human anti-IL1β antibody, is currently in phase 2 of clinical evaluation, in combination with LNA043 (a protein inducing chondrogenesis and cartilage repair) for the treatment of patients with knee osteoarthritis (NCT04814368) after IA delivery. 2.6.3. Conclusion and Perspectives Regarding the Intra-Articular Route Intra-articular delivery may be of particular importance for the treatment of joint diseases (and notably rheumatoid arthritis) as around 30% of patients are resistant to biotherapies, and few novel molecules are under development. The limited half-life of antibodies reaching the inflamed joint is one of the major limitations associated with IA delivery, requiring frequent injections and thus increasing side effects, discomfort, and morbidity. Specific formulations including hydrogels [ 119 ], micro/nano particles [ 120 ], or in situ implants [ 121 ] are under consideration to improve antibody concentration on-target. A long-lasting Ab formulation through the use of biopolymers was recently developed for IA administration, showing a sustained high concentration of Abs in the synovial fluid, with minimal inflammatory side effects [ 122 ]. However, controlled-release methods are often associated with a loss of bioactivity of the drug, and/or inflammatory side effects that could accelerate the degeneration of the cartilage and the disease [ 123 ]. Additional work is needed to develop safe and active intra-articularly-delivered Abs to fight joints auto-immune diseases. 2.7. Delivery within the Central Nervous System: A Method to Bypass the Blood–Brain Barrier 2.7.1. The Blood–Brain Barrier (BBB), a Barrier for Abs The CNS is an essential part of the nervous system consisting of the brain and the spinal cord. The CNS integrates and coordinates essential functions of the body [ 124 ]. Because of its importance, the CNS is well protected, notably by the blood–brain barrier (BBB), limiting its exposure to exogenous particles carried by the blood circulatory system. The BBB is a highly selective semipermeable barrier preventing the passage of solutes from the blood into the extracellular fluid of the CNS. The BBB is composed of specialized endothelial cells sealed together with tight junctions reinforcing trans-endothelial electrical resistance, as well as the high expression of energy-dependent efflux transporter, inducing a selective passage of solutes [ 125 ]. In particular, FcRn, expressed in the microvascular endothelium and in the choroid plexus epithelium, essential components of the BBB, is involved in the reverse transcytosis of IgG, from the brain to the blood vessels. Therefore, it is estimated that less than 0.1% of systemically injected IgG enter the CNS through nonspecific pathways. Thus, to attain a therapeutic dose in the CNS, Abs have to be administered in high quantity—which may be associated with toxicity—or specific transporter pathways existing between the circulatory system and the CNS have to be used [ 126 , 127 ]. In order to circumvent these barriers, novel methods have been considered aimed at addressing the drug into the CNS by surgery, either via the intracerebroventricular, intracerebral routes or convection-enhanced delivery [ 126 , 128 ]. The intracerebral injection, the most direct method, consists in intermittent bolus injections, which are administered locally in the brain, after a surgical intervention. The intracerebroventricular technique allows the injection of drugs directly into the cerebrospinal fluid in the cerebral ventricles. Both methods use a syringe and needle system filled with the drug to be administered. Convection-enhanced delivery consists in the generation of a pressure gradient at the tip of an infusion catheter or canula (usually implanted in the cerebral tumor), allowing the delivery of drugs through the interstitial spaces of the CNS. 2.7.2. Abs in Clinical Development for Direct Delivery to the CNS There are many diseases targeting the CNS, including the neurodegenerative Parkinson or Alzheimer's disease (AD), for which the development of Abs has been considered. For example, Aducanumab, an anti-Aβ Ab was approved by the FDA for the treatment of AD, after parenteral injection [ 129 , 130 ]. Cerebral delivery of Abs is mainly being evaluated in the preclinical phases with some success [ 131 ]; only one Ab has already reached clinical trials. The antibody 131I-Omburtamab (Y-Abs Therapeutics), is a murine IgG1 recognizing CD276, used for radioimmunotherapy. This antibody is injected via the intracerebroventricular route, and phase 1 and 2 trials in patients with CNS Neuroblastoma, CNS metastases, or leptomeningeal metastases, have shown that the antibody was well tolerated and improved survival. A phase 3 trial is ongoing, evaluating the efficacy and safety of the Ab in children (NCT03275402). In the meantime, the same antibody is entering a phase 1 trial to test its efficacy, once delivered through a convention-enhanced delivery in patients with diffuse pontine gliomas profile (NCT05063357) [ 132 ]. 2.7.3. Conclusion and Perspectives on CNS Delivery The CNS is particularly well protected from the environment impeding an efficient access to therapeutic molecules. The BBB remains the major obstacle, when considering systemic infusion of therapeutic Abs, and limits CNS bioavailability. Novel methods bypassing the BBB, described above, are still in infancy and require further work to be standardized and to reduce their invasiveness. In this context, the investigation of the "nose-to-brain" route may be of particular interest. It consists of administering drugs in the olfactory region of the nose, by inhalation, from where they may transfer to the brain through the epithelial layer and via neuronal bundles that project to the olfactory bulb. The "nose-to-brain" route is under preclinical evaluation for Abs [ 133 ]. 2.7.1. The Blood–Brain Barrier (BBB), a Barrier for Abs The CNS is an essential part of the nervous system consisting of the brain and the spinal cord. The CNS integrates and coordinates essential functions of the body [ 124 ]. Because of its importance, the CNS is well protected, notably by the blood–brain barrier (BBB), limiting its exposure to exogenous particles carried by the blood circulatory system. The BBB is a highly selective semipermeable barrier preventing the passage of solutes from the blood into the extracellular fluid of the CNS. The BBB is composed of specialized endothelial cells sealed together with tight junctions reinforcing trans-endothelial electrical resistance, as well as the high expression of energy-dependent efflux transporter, inducing a selective passage of solutes [ 125 ]. In particular, FcRn, expressed in the microvascular endothelium and in the choroid plexus epithelium, essential components of the BBB, is involved in the reverse transcytosis of IgG, from the brain to the blood vessels. Therefore, it is estimated that less than 0.1% of systemically injected IgG enter the CNS through nonspecific pathways. Thus, to attain a therapeutic dose in the CNS, Abs have to be administered in high quantity—which may be associated with toxicity—or specific transporter pathways existing between the circulatory system and the CNS have to be used [ 126 , 127 ]. In order to circumvent these barriers, novel methods have been considered aimed at addressing the drug into the CNS by surgery, either via the intracerebroventricular, intracerebral routes or convection-enhanced delivery [ 126 , 128 ]. The intracerebral injection, the most direct method, consists in intermittent bolus injections, which are administered locally in the brain, after a surgical intervention. The intracerebroventricular technique allows the injection of drugs directly into the cerebrospinal fluid in the cerebral ventricles. Both methods use a syringe and needle system filled with the drug to be administered. Convection-enhanced delivery consists in the generation of a pressure gradient at the tip of an infusion catheter or canula (usually implanted in the cerebral tumor), allowing the delivery of drugs through the interstitial spaces of the CNS. 2.7.2. Abs in Clinical Development for Direct Delivery to the CNS There are many diseases targeting the CNS, including the neurodegenerative Parkinson or Alzheimer's disease (AD), for which the development of Abs has been considered. For example, Aducanumab, an anti-Aβ Ab was approved by the FDA for the treatment of AD, after parenteral injection [ 129 , 130 ]. Cerebral delivery of Abs is mainly being evaluated in the preclinical phases with some success [ 131 ]; only one Ab has already reached clinical trials. The antibody 131I-Omburtamab (Y-Abs Therapeutics), is a murine IgG1 recognizing CD276, used for radioimmunotherapy. This antibody is injected via the intracerebroventricular route, and phase 1 and 2 trials in patients with CNS Neuroblastoma, CNS metastases, or leptomeningeal metastases, have shown that the antibody was well tolerated and improved survival. A phase 3 trial is ongoing, evaluating the efficacy and safety of the Ab in children (NCT03275402). In the meantime, the same antibody is entering a phase 1 trial to test its efficacy, once delivered through a convention-enhanced delivery in patients with diffuse pontine gliomas profile (NCT05063357) [ 132 ]. 2.7.3. Conclusion and Perspectives on CNS Delivery The CNS is particularly well protected from the environment impeding an efficient access to therapeutic molecules. The BBB remains the major obstacle, when considering systemic infusion of therapeutic Abs, and limits CNS bioavailability. Novel methods bypassing the BBB, described above, are still in infancy and require further work to be standardized and to reduce their invasiveness. In this context, the investigation of the "nose-to-brain" route may be of particular interest. It consists of administering drugs in the olfactory region of the nose, by inhalation, from where they may transfer to the brain through the epithelial layer and via neuronal bundles that project to the olfactory bulb. The "nose-to-brain" route is under preclinical evaluation for Abs [ 133 ]. 3. Future Perspectives If the pharmacokinetic and pharmacodynamic properties of therapeutic antibodies depend on their format and their target, they also depend on their route of administration. In fact, depending on the location of the Ab's target, alternative modes of administration are being explored and developed to optimize Ab deposition in the vicinity of its target. The choice of route of administration is thus a critical factor for efficient Ab-based therapy and needs to be considered in the early stages of the development concomitantly with the appropriate formats, formulations, and devices ( Table 9 ). If among the alternative routes some have progressed quite well to clinical trials, others are still at the very beginning of the research process. 3.1. Oral Delivery: Protects Abs from the Harsh Environment of the Intestinal Tract Some Abs have been approved over the years for the treatment of intestinal diseases, such as inflammatory bowel disease (IBD). However, their delivery through parenteral administration has displayed several limitations, such as non-responding patients, or the increase of anti-drug antibodies limiting the effect of the Abs. Oral delivery has been used for centuries and is the most accessible and the least invasive route for drug administration. Specific issues associated with the gastro-intestinal (GI) tract have to be considered to ensure efficient drug activity. The intestinal mucosa, in contact with external environment, maintains a state of homeostasis that sustains tolerance and detects and eliminates exogenous materials or pathogens. Within each part of the gastrointestinal tract, specific cellular and extracellular barriers exist, such as the harsh pH conditions, which denature Abs, and the presence of multiple proteases that may degrade proteins [ 134 ]. Studies have shown that less than 20% of Abs administered orally were still immunologically active after proteolytic digestion by gastric enzymes [ 135 ]. Thus, oral administration of Abs necessitates their protection them from the harsh conditions of intestinal transit. Among the most advanced studies, AVX-470 is an orally delivered Ab targeting TNF, used to treat IBD. A phase 1 study concluded that the drug was safe and well-tolerated, and suggested a beneficial effect (NCT01759056) [ 136 ]. However, a high amount of AVX-470 was required because the majority of the antibody was degraded. This highlights the importance of protecting the antibody from the environment encountered in the GI tract. One protection strategy may consist in encapsulating the Ab in either nanoparticles or enteric coated capsules, a strategy evaluated in a phase 1 trial for Foralumab—an anti-CD3 Ab—given to patients with active Crohn's disease [ 137 ] (NCT05028946). Another strategy consists in decreasing the gastric acidic pH by combining the Ab with a proton pump inhibitor. An anti-CD3 (OKT3) was combined with omeprazole in patients suffering moderate to severe ulcerative colitis [ 138 ]. The six patients who received both OKT3 and omeprazole orally showed a promising reduction in inflammatory genes expression in peripheral blood cells, associated with low side effects. Although studies have shown consistently promising results, it is important to better understand the fate of Abs once ingested. Strategies to bypass Abs degradation and uphold their activity remain to be investigated, and may also include the rational selection of the best isotypes which could survive to the extreme gastro-intestinal environment [ 139 ]. 3.2. Skin Administration: A Topical Delivery of Ab in Wounds Abs offer interesting opportunities to treat local skin diseases or wounds. While Abs may be administered directly to open wounds and allow direct healing, the treatment of blisters or scars would require their administration on intact skin [ 140 ]. However, when applied topically, antibodies showed low skin penetration, which may be explained by the hydrophobic nature of the keratin—essential and main component of the skin—and the hydrophilic nature of antibodies, and their size, limiting cellular and transcellular diffusion [ 141 ]. Thus, a high dose of antibody should be required to achieve a therapeutic dose inside the tissue. Moreover, for topical administration of drugs, gels or creams are recommended, which imply complex formulations of Abs to ensure their stability. Several strategies using cell-penetrating peptides, physical penetration enhancer, or injection with microneedles have been tested, but none of them has entered clinical trials. 3.1. Oral Delivery: Protects Abs from the Harsh Environment of the Intestinal Tract Some Abs have been approved over the years for the treatment of intestinal diseases, such as inflammatory bowel disease (IBD). However, their delivery through parenteral administration has displayed several limitations, such as non-responding patients, or the increase of anti-drug antibodies limiting the effect of the Abs. Oral delivery has been used for centuries and is the most accessible and the least invasive route for drug administration. Specific issues associated with the gastro-intestinal (GI) tract have to be considered to ensure efficient drug activity. The intestinal mucosa, in contact with external environment, maintains a state of homeostasis that sustains tolerance and detects and eliminates exogenous materials or pathogens. Within each part of the gastrointestinal tract, specific cellular and extracellular barriers exist, such as the harsh pH conditions, which denature Abs, and the presence of multiple proteases that may degrade proteins [ 134 ]. Studies have shown that less than 20% of Abs administered orally were still immunologically active after proteolytic digestion by gastric enzymes [ 135 ]. Thus, oral administration of Abs necessitates their protection them from the harsh conditions of intestinal transit. Among the most advanced studies, AVX-470 is an orally delivered Ab targeting TNF, used to treat IBD. A phase 1 study concluded that the drug was safe and well-tolerated, and suggested a beneficial effect (NCT01759056) [ 136 ]. However, a high amount of AVX-470 was required because the majority of the antibody was degraded. This highlights the importance of protecting the antibody from the environment encountered in the GI tract. One protection strategy may consist in encapsulating the Ab in either nanoparticles or enteric coated capsules, a strategy evaluated in a phase 1 trial for Foralumab—an anti-CD3 Ab—given to patients with active Crohn's disease [ 137 ] (NCT05028946). Another strategy consists in decreasing the gastric acidic pH by combining the Ab with a proton pump inhibitor. An anti-CD3 (OKT3) was combined with omeprazole in patients suffering moderate to severe ulcerative colitis [ 138 ]. The six patients who received both OKT3 and omeprazole orally showed a promising reduction in inflammatory genes expression in peripheral blood cells, associated with low side effects. Although studies have shown consistently promising results, it is important to better understand the fate of Abs once ingested. Strategies to bypass Abs degradation and uphold their activity remain to be investigated, and may also include the rational selection of the best isotypes which could survive to the extreme gastro-intestinal environment [ 139 ]. 3.2. Skin Administration: A Topical Delivery of Ab in Wounds Abs offer interesting opportunities to treat local skin diseases or wounds. While Abs may be administered directly to open wounds and allow direct healing, the treatment of blisters or scars would require their administration on intact skin [ 140 ]. However, when applied topically, antibodies showed low skin penetration, which may be explained by the hydrophobic nature of the keratin—essential and main component of the skin—and the hydrophilic nature of antibodies, and their size, limiting cellular and transcellular diffusion [ 141 ]. Thus, a high dose of antibody should be required to achieve a therapeutic dose inside the tissue. Moreover, for topical administration of drugs, gels or creams are recommended, which imply complex formulations of Abs to ensure their stability. Several strategies using cell-penetrating peptides, physical penetration enhancer, or injection with microneedles have been tested, but none of them has entered clinical trials.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8988924/

Application of big data in COVID-19 epidemic

The scientific research centered on generating new data by performing basic experiments to answer specific questions related to any infectious diseases. The application of big data in the area of infectious diseases has introduced a number of changes in the information accumulation models using analytics. Therefore this chapter discusses the concept of big data for guaranteeing better expansion and research against coronavirus disease 2019 (COVID-19). The chapter examines how large-volume medical data can help clarify and elucidate COVID-19 disease patterns. Also, the chapter conveys the benefits of big data analytics during COVID-19. The hope of using big data in COVID-19 will have a great impact on the quality of outbreak care that can be delivered to patients across socioeconomic and geographic boundaries. 1 Introduction Data science can simply be referred to as a method of formatting data that comprises an emerging approach of acquiring, storing, and analyzing data to produce a meaningful and an efficient way of extracting basic information for decision making [ 1 , 2 ]. Data science cuts across almost all the fields that use scientific approaches, procedure systems, and computer algorithms to obtain knowledgeable information and perceptions from both structured and unstructured data [ [3] , [4] , [5] ]. Thus the main goal remains to be the improvement of understanding from various data types [ 6 , 7 ]. The field of data science is associated with the computer science field, but a keen observation of the two fields reveals that they differ slightly with some characteristics. The field of computer science deals with the development of computer programs and procedures for data processing and management, while data science applies to any form of a manual or computerized method of analyzing data. Data science involves gathering, investigating, and producing of data for analysis that is thoroughly associated with the field of mathematics and statistics [ [8] , [9] , [10] ]. The advancement in information technology increases the amount of data used by the modern establishment, thereby making data science an essential tool for maintaining data in any organization [ [10] , [11] , [12] ]. For instance, an organization with a huge amount of user data needs data science to effectively improve methods to collect, accumulate, and process the data. Various systematic techniques may be used by the company to process and obtain resourceful outcomes about the user data. Data mining and big data are used interrelated with data science. To comprehend and process actual data concepts, data science is of great importance, as it incorporates the field of statistics and machine learning with their various procedures [ 13 , 14 ]. The study of information science, computer science, mathematics, and statistics with their concepts and methods are all embedded in the field of data science [ 14 , 15 ]. The 21st century is an age of big data affecting every aspect of human life, including biology and medicine [ 16 , 17 ]. The move from paper medical records to electronic health record (EHR) systems has resulted in an unprecedented increase in data [ 18 , 19 ]. Big data thus offers a great opportunity for doctors, epidemiologists, and specialists in health policy to make evidence-driven decisions that will eventually enhance patient care [ 20 , 21 ]. "Big data is not only a modern reality for the biomedical scientist but an imperative that needs to be fully grasped and used in the search for new knowledge" [ [22] , [23] , [24] ]. The coronavirus pandemic is a global health issue that threatens the peace of the entire world since the New Year 2020 [ 25 ]. The number of confirmed cases of people who contracted the disease increases daily, but the death rate recorded is low compared with the recovery cases, which makes the disease less deadly but more infectious (2,367,959 cases in 213 countries, with over 160,450 deaths as of April 20, 2020) ( https://covid19.who.int/ ). The daily increase of this infectious disease has become a huge challenge to the socioeconomic development of each country across the globe. The Secretary-General of the United Nation has advised that urgent measures should be taken by governments in various countries to contain the outspread of the infectious disease [ [26] , [27] , [28] , [29] , [30] ]. One of the goals of the United Nations Sustainable Development is to discuss economic, social, and environmental concerns between 2015 and 2030 and change toward maintainable growth [ [31] , [32] , [33] ]. The UN Sustainable Development Goals (SDGs) include 17 aims and 169 objectives. Three (3) SDG focuses on maintaining lives and encouraging well-being for all ages, including the SDG 3.3 specifically targeting the end of AIDS, tuberculosis, malaria, and abandoned tropical illnesses, as well as the fight against hepatitis, waterborne diseases, and other transmittable infections, by 2030 [ 31 , 32 ]. The coronavirus disease 2019 (COVID-19) epidemic directly challenges the accomplishment of the abovementioned health objectives and also affects the achievement of economic and social development goals. The transmission features of the COVID-19 epidemic have still not been properly recognized in the sense of global environmental changes [ 32 , 34 ]. Furthermore, the pace of global development, increased population size, more common and nuanced encounters, and lack of medical security in developing countries all contribute to the complexities of COVID-19 prevention and regulation. COVID-19 seems to have a greater impact on the global economy when compared with the severe acute respiratory syndrome (SARS) that occurred in 2013. The bone of contention is how to fight the spread of infectious diseases and how this one is different from the 2003 epidemic. Most countries depended on the observation of classic steps to monitor diseases and public health to contain the COVID-19 pandemic, similar to those used with SARS in 2003 [ 35 ]. They cover from drastic quarantine actions in China (for example, locking down over 60 million people in Hubei province) to extensive contact monitoring with hundreds of contact tracers (for example, Singapore, Hong Kong, South Korea). Nevertheless, these interventions may not be successful in tackling the COVID-19 scale by 2020. From Table 8.1 and Fig. 8.1 , as of May 17, 2020, a total of 4,798,545 confirmed cases and 316,505 deaths were reported across 218 countries, thus showing that there is a need for a better way of prevention of this novel pandemic. Table 8.1 The confirmed and death cases by continents. Continents Confirmed cases Death cases Europe 1,780,768 162,839 North America 1,684,258 102,919 Asia 798,308 24,734 South America 440,182 23,117 Africa 86,381 2777 Oceania 8648 119 Figure 8.1 The confirmed and death cases across continents. The 12 countries with the highest number of confirmed cases are shown in Table 8.2 ; the United States of America has 1,409,452 (29%) of the total confirmed cases around the globe, the Russian Federation has 281,751, and the United Kingdom came third with 240,165. Interestingly, six of these countries are from Europe, Asia, and America, with three countries each, while the rest are not among the 12 leading countries. Table 8.2 The 12 countries with the highest number of confirmed cases in the world. Country name Total cases Percentage (%) The United States of America 1,409,452 42 Russian Federation 281,751 8 The United Kingdom 240,165 7 Spain 230,698 7 Italy 224,760 7 Brazil 218,223 6 Germany 174,355 5 Turkey 148,067 4 France 140,008 4 Iran 120,198 4 India 90,927 3 Peru 84,495 3 TOTAL 3,363,099 100 This chapter, therefore, discusses the possible application of big data and analytics to improve conventional public health approaches to tackle COVID-19 to control, manage, identify, and avoid COVID-19 and reduce the effect on health implicitly associated with COVID-19. The remaining part of this chapter is organized as follows: Section 2 discusses the growth of data in healthcare, the challenges, and the importance of big data in COVID-19. Section 3 presents the big data privacy and ethical challenges in COVID-19. Section 4 discusses big data analytics in the COVID-19 epidemic. Finally, Section 5 concludes the chapter. 2 The growth of data in healthcare The mutual collaboration that exists in the healthcare sector results in the generation of a huge amount of data from various sources [ [36] , [37] , [38] ]. This has a result of huge figures of varied sections and departments including physicians of different disciplines, ranging from nurses, pathologists, radiologists, and laboratory technologists, which collaborate efforts to achieve unified goals toward bringing medical costs and mistakes down to the barest minimum, as well as to provide excellent and standardized healthcare services. Various stakeholders that work collaboratively in the health sector get data from different sources. This source includes data from patient observation, imaging reports from scans, interview and test results, insurance and bills, summaries of patient discharges, reports from pharmacists, case notes from physicians, admission notes from hospitals, feedback from social media, and journals of medical articles. Data in the healthcare sector are usually huge and not easy to handle [ 39 , 40 ]. This results from the enormous way by which data grows in the healthcare sector, the rate at which data are produced, and the variety of data in the healthcare system [ 36 , 37 ]. The rate at which data are captured, stored, analyzed, and retrieved in the healthcare sector has changed rapidly from the aged paper-based storage technique to the use of digital techniques and methods [ 37 , 41 ]. On the other hand, the complication of data makes processing and analysis of data by the age-long traditional method very difficult and uneasy to handle. However, the vast volume, as well as the complexity, of these data makes it difficult for the data to be processed and analyzed by traditional approaches and techniques. Therefore the application of advanced technologies, which includes virtualization and cloud computing, allows for huge and effective data processing in the healthcare system, thereby rapidly turning the healthcare system into a big data industry. Nevertheless, in these modern days, the improvement in the information and communication technologies (ICT) brings the advancement of varied data from new sources in the healthcare system ( Fig. 8.2 ). Figure 8.2 An integrated big data conceptual model for infectious diseases [ 42 ]. This source includes the Global Positioning System (GPS), data from gene sequence, file logs, devices that identify radio frequency (RFID), smart meters, and posts from social media. The increasing rate with which data is being produced from various sources brings about an increase in the amount of data in the healthcare system [ 37 , 43 ]. Thus their results give tedious means of storing, processing, and analyzing data with the agelong traditional method of data processing applications [ 44 ]. Nevertheless, modern methods and tools, as well as advanced computing technologies, are used to store up, manage, and explore values from large and varying data in the healthcare system in a real-time manner [ 37 , 43 , 44 ]. Therefore the healthcare system has now become a big data industry. Big data now brings huge opportunities to the healthcare system [ 45 ]. Improvement in information technology and data computing has greatly changed population-based research by encouraging easy access to a huge amount of data. Sometimes such database links are referred to as "big data" [ 46 , 47 ]. In order to make efficient use of these data for research in clinical health or public health, the researchers need to widen research further than the traditional surveillance model, as operating with big data differs from focusing on performing narrow analysis, treatment-oriented clinical data. Therefore leveraging on big data to reflect accurately on the heterogeneous population it represents becomes expedient [ 37 , 43 ]. This endeavor needs a swift research environment that can adopt a quick advancement in computing technology to an all-time combined data while making use of new methods to reduce complexity [ 37 , 47 ]. Also, trends and patterns that make it easy to diagnose and treat patients are revealed by big data. As big data in today's digital world will be crucial to handed the COVID-19 outbreak, the criteria for effective collection of data and analysis on a global scale need to be clear. The chapter claims the use of prediction and surveillance of digitally accessible data and algorithms. For example, it is of vital importance in the battle against the COVID-19 pandemic to recognize people who have traveled to places where the disease has spread or has been identified and isolate the contacts of infected people. However, it is equally important to use these data and algorithms responsibly, in compliance with the data protection regulations and with due regard for privacy and confidentiality. The inability to do so would weaken public confidence, which would cause people less inclined to obey recommendations or guidelines from public health and more likely to have worse health outcomes [ 48 ]. Hence the exploitation of big data in COVID-19 will bring about improved care with minimum cost and good satisfaction to patients. 2.1 Challenges of big data in COVID-19 The ability to combine sophisticated epidemiologic models and new big data sources has been discussed [ 49 , 50 ]. For example, the use of smart-phones, social networks, or public health response satellites at different stages, mainly in the situation of disease forecasts, for making a decision [ 50 , 51 ]. These new sources of data include significant, real-time data on tourism visits that spread infection and spatial shifts in vulnerable residents that probably up till now were hard to report on timescales related to a rapidly fast-growing epidemic [ 50 ]. With increasing flexibility and growing universal connectivity, this information will be crucial for monitoring and containment planning [ 52 ]. By principle, with correct data sharing protocols in place, accurate, up-to-date disease predictions can be generated that are guided using this data stream. The success of big data in fighting epidemics depends on various organizations, both public and private, and the government is needed to be in contact frequently [ 50 , 51 ]. Privacy issues for daily use of new data channels need to be addressed, specifically the most suitable way to vigorously integrate such data streams to confirm individual confidentiality. Yet even if security is tackled, the transformation of new methods into a policymaking context raises additional systemic challenges [ 50 , 51 ]. Big data mining provides many tempting opportunities [ 43 , 53 ]. Nonetheless, when investigating big data sets and mining meaning and expertise from these facts extracted, researchers and professionals face many challenges [ 43 , [53] , [54] , [55] ]. The complications at various levels include data collection, storing, scanning, sharing, reviewing, handling, and watching. Furthermore, security and privacy issues particularly arise in distributed applications that are powered by data. The surge of knowledge and distributed sources also surpass our harnessing capability indeed, although the scale of big data continually grows exponentially, the actual technical ability to accomplish and explore large datasets is only in the comparatively lower rates of data petabytes, exabytes, and zettabytes [ 43 ]. In this chapter, we address in detail some technical issues that are still open to research. While working with big data, data scientists face a lot of obstacles. One obstacle is the compilation, integration, and storage of tremendous datasets generated from disseminated sources, with far less needed hardware and software [ 56 , 57 ]. The management of big data results in another huge problem. Effective big data management is essential to promote the abstraction of accurate information to maximize investment [ 58 , 59 ]. Proper management of data is the basis for big data analytics [ 60 ]. Another difficulty is to synchronize with an organization's internal infrastructure's external data sources, and big data dispersed systems (sensors, apps, databases, networks, etc.) [ 43 ]. Most of the time, an analysis of the data produced within organizations is not sufficient. We need to go one step further to integrate internal data with external data sources, to gain useful insight and information. External data may include the sources from third parties, market fluctuation details, weather forecasts and traffic situations, social network data, comments from clients, and resident input. This will aid to optimize the power of analytic models used in the analysis, for example. The computer design and performance are an important question. Indeed, CPU output is known to double every one and half years according to Moore's Law and diskette drive output is also expanding at the same time. The input and output operations, however, do not follow the same form of results (i.e., spontaneous input and output speeds increased moderately, while systematic input and output speeds developed slowly through thickness) [ 57 ]. Thus this varied device capacity will delay data access and affect the efficiency and scalability of big data applications. Machine learning is a very active area of study in artificial intelligence (AI) and pattern recognition nowadays. This plays a significant part in applications of probabilistic learning, such as computer vision, voice recognition, and the interpretation of natural languages [ 61 ]. Currently, the ability of computer-learning techniques and functionality engineering algorithms to process raw natural data is limited [ 62 ]. Deep learning is, on the contrary, more effective in solving data processing and learning complications contained in large datasets. This helps extract a huge size of unsupervised and unstructured raw data automatically. Dynamic policies learning consists of incremental and group learning that are important approaches to learn from a large stream of data with meaningful perception [ 63 ]. Big data and data stream uses the concept of collaborative and incremental learning. They deal with numerous challenges that arise during the learning of data, such as data accessibility and restricted resources. They make use of user profiling and stock forecasting applications. The application of incremental learning to data produces a faster means of prediction when dealing with the new data stream. Hence, using the incremental algorithm is highly recommended when the drift concept is not available. On the other hand, it is highly recommended to use ensemble algorithms to obtain accurate outcomes with large drift concept. Also, when dealing with a modest data stream or real-time processing, the incremental algorithm should be considered for use. Conversely, in the case of a complex data stream, an ensemble algorithm is considered the best. Another big problem is analogic reasoning: fundamentally, disease forecasting is unpredictable. In the narrative on big data and AI, there is often an underlying assumption about the statistical covariates or cell phone data and difficult models of simulation that avoid the need to gather basic epidemiologic details. Nevertheless, for the evolving COVID-19 epidemic, emphasizing this opinion still lacks a reliable fact on situation counts and biotic processes that drive an outbreak of the disease, let only the behavioral reactions of infected individuals, and make it difficult to rapidly adjust or understand precise intricate prototypes on spatiotemporal scales applicable in making a verdict. Hopefully, the best effective systems would continue to be simple [ 64 , 65 ] equally out of necessity for versatile modules that provide quick responses, given the controversy surrounding emergency epidemiologic studies and because simple models are more easily available to interpret and communicate [ 66 ]. Clear knowledge of both the importance and weaknesses of typical outputs is a precondition for their successful implementation but is often absent. Because policymakers have not yet reached an in-depth modeling experience, the lack of consistent message threatens to lead to two destructive results: (1) assume that the method would be misinformed without skepticism and choices or (2) ignore off-hand modeling and neglect the proof that we must control epidemics most efficiently. During epidemics, verdicts have to be taken rapidly, on a patchy basis, and inaccurate data models could be effective tools for guiding them. Considering the abovementioned obstacles, computing capacity, novel methods, and novel data are steadily advancing sources that give authentic courage for improved monitoring and development of valuable predicting systems. The novel database available to us comprises not only inactive experimental large data streams from cell phones but also comprehensive ecological data and local sensor information from embedded devices, internet search information, pathogenic genomic data that can be produced quickly informing the response during an outbreak, [ 67 ] and crowd-sourced methods to track emergencies changing rapidly [ 68 ]. Information sharing systems and structured aggregation strategies are being established to secure personal data privacy [ 69 ], and internet access enables rapid data transmission and collaboration between geographically distant responding teams. Methodologically, effective modeling methods that combine multiple predictions to reduce uncertainty are being built [ 65 , 70 ]. In reaction to the outbreak of COVID-19, an innovative, collaborative approach has unfolded between academic organizations, for example, between Twitter and other channels to exchange, evaluate, and publicly debate the effects of new research as it arises (Ironically, the control of misinformation that is now proliferating on social networks during a crisis is likely to become one of the most significant concerns for containing the disease.). Unless the abovementioned problems are resolved, these technologies will remain dislocated and impracticable. It is promising that all these problems may be strengthened by transferring most of the funding and knowledge focus to those communities that require most help during epidemics. The unpredictability of the COVID-19 outbreak and the progressive technologic mechanisms of these methods will require agile, dispersed groups of people covering the systematic and functioning dimensions of the epidemic response using novel big data methods to supplement and explain the limited spread, sociogeographic data that are necessary for the progress of important predictions. Provincial or small groups with investigative experience and current connections with government and industry stakeholders can contribute to scalable modeling strategies that exploit broad curated and international scientific database skills, building on partnerships formed if no new or ongoing outbreaks occur. This strategy may likewise be of help to mitigate some policy concerns related to data sharing, particularly through sensitive information not being shared publicly. To achieve this idea, significant continuous investment is required to finance individuals to fill what currently represents a significant void in the analytics process, especially in some countries with low and middle wages. Big data can be created from a lot of sources, such as social network sites, smartphones, IoT sensors, and publicly available data [ 71 ], in different formats such as text or video. Big data applies to patient care details in the sense of COVID-19, such as medical records, X-ray reports, case history, list of doctors and nurses, and information about the epidemic location. Big data has proved its capability to support fighting infectious diseases like COVID-19 [ 72 , 73 ]. 2.2 Importance of big data in COVID-19 The possibility of fast-spreading diseases that cause uncontrolled death and a bad influence on the economies and sustainability of countries in the world has stretched the importance and needs for developing a quantitative framework to provide support for making nearly real-time decisions in the public health system. According to a case study, the 2003 SARS outbreak that originated in China and spread to 29 countries became a healthcare-acquired infection in various regions by August 2003 [ 74 , 75 ]. The 2009 pandemic known as influenza (H1N1) originated in Mexico and spread rapidly all over the world through airline network and infected 20 countries, with maximum hit on the travelers coming from Mexico within a little time of the disease outbreak [ 76 ]. The socioeconomic implication and outcome of the outbreak of a disease related to 2009 (influenza A [H1N1]) were projected to be a huge amount ranging between $360 billion and $4 trillion [ 77 ], which was the calculation for the outbreak in the first year. Significantly, the SARS outbreak at Wuhan, China, in 2019 was calculated and projected to be 2,034,802 confirmed cases and 123,150 mortalities across the globe. Ever since the outbreak of COVID-19 in China, people who traveled globally from Wuhan transferred the disease to various countries of the world. The outbreak of SARS had significant effects on the people and economy all over the world. The effects include restrictions and bans on traveling, total closure of shops and markets resulting in a huge loss on earnings and proceeds, fear of contracting the disease from the infected person, and unpleasant side effects on world tourism at large, as many flights were canceled or suspended. Express and huge damage on government finances in various countries as agricultural activities were greatly hampered resulting in food shortage, and so many other effects resulted from the outbreak. Therefore it becomes imperative to make an immediate response from all angles to fight and contain the virus efficiently. Big data entails the innovative processing of both new and existing data to proffer meaningful solutions that are of huge business benefits [ [78] , [79] , [80] ]. However, processing large data, or varied data, will only be a mere technologic solution until it is associated with business objectives and goals. Big data promises more lights to be shed on complicated facts and information on the dynamics of how infectious diseases are transmitted and how to develop new modeling and analytic tools that have far been hidden due to lack of smooth and workable data. More progress will be made in the area of disease forecast when a huge amount of information on epidemics required for modeling is available. Big data will be the best in accomplishing this goal. Improved consistency in reporting diseases and case definition is therefore expedient within time and space. Data obtained from news media sources can give a perfect estimation and evaluation of how the disease is transmitted. Thus it is of high importance, especially when comprehensive and well-examined data are not available. In a country with low and middle income, and where studies on disease transmission details are scarce, resources based on internet surveys will provide an opportunity for a detailed study and correct assessment on how the disease is transmitted, for instance, in the crisis of an infectious disease like COVID-19 when real-time assessment is expedient. A well-calibrated computational model tool that is useful in assessing and analyzing the epidemic spread signifies a potent tool to sustain the process of decision-making at a time of emergency epidemic outbreaks. Epidemic models are progressively used to generate predictions of the spatial-temporal progression of a disease outbreak at several spatial scales and for easy access to possible impacts of the various strategies of the intervention [ 81 ]. The ability to produce innovative facts or information is greatly expanded by big data. The costs applicable to answer various questions retrospectively and prospectively by gathering ordered and structured data are exorbitant. Acquisition of finer data in a computerized format is achieved by analyzing unstructured data that are within EHR systems by using various computational techniques such as traditional language processors that extract clinical concepts from free documents. Analyzing the unstructured data contained within her systems using computational techniques (e.g., natural language processing to extract medical concepts from free-text documents) permits finer data acquisition in an automated fashion. For example, computerized detection within her systems with the use of natural language processing is better used to detect complications that arise after the operation when compared to patient well-being indicators based on discharge coding [ 82 ]. Big data provides the possibility to generate an observable indication base for clinically related questions, which may not be achievable but will be helpful with generalizability issues. Generalizability issues restrict the application of conclusions that are derived from random trials carried out on a very narrow scale of participants to patients that show dissimilar characteristics. Big data helps disseminate knowledge. Many healthcare practitioners strive to be updated with current evidential proof and support that guide clinical practices. The computerization of medical journalism to a great extent has enhanced access; conversely, with great numbers of studies, translation of knowledge becomes difficult. If at all a physician has enough relevant data and guiding principles, organizing information to formulate a realistic approach for the treatment of patients with numerous chronic illnesses becomes extremely difficult. Analyzing existing EHR systems to generate a template to guide in making clinical decisions is the only way by which this problem may be solved [ 83 ]. This technique is exploited in the alliance involving Memorial Sloan Kettering Cancer Center and IBM's Watson supercomputer to aid in the diagnosis and proposed treatment for cancer patients. The difference between the big data method and traditional decision support tools is that with the big data technique, suggestions are obtained through analysis of patients' data in real time instead of depending solely on the use of rule-based decision trees. For instance, longitudinal analytic data have been proven to be efficient for forecasting future diagnostic risks and domestic abuse in patients [ 83 , 84 ]. With data-driven clinical decision support tools, the cost is greatly minimized and proper care standardization is guaranteed. Physicians can get information that guides them on the diagnostic and therapy options that are provided by esteemed colleagues encountering related patient profiles with the use of big data on the cloud. The healthcare system can be transformed by getting information directly across to patients and equipping them to take part in more active roles. In the future, clinical reports can also reside with the patients, compared to the existing model where health records of patients are stored with healthcare professionals, thereby making the patients be in a passive situation. Big data provides the opportunity to progress the clinical records by connecting the conventional health-related data such as family history and medication list to other private data located on other sites such as education, income, neighborhood, military service [ 83 , 85 ], exercise regimens, diet habits, and forms of entertainment, all that can be easily accessed without the need to interview the patient with a comprehensive list of questions. Thus big data provides an opportunity to collaborate with the conventional medical model with social determinants of health in a patient-directed fashion. The initiatives of public health to reduce obesity and smoking can be efficiently delivered by focusing the message on the most appropriate set of people based on the profiles on their social media. With the opportunity to use analytic capabilities, system biology can be incorporated. Big data assist in translating personalized medicine initiatives into clinical practice (e.g., genomics) with data [ 83 , 86 ]. The Genomics Network and Electronic Medical Records achieve this with the use of natural language processing to phenotype patients, in an attempt to simplify genomics research. With big data, patients are opportune to access correct and updated information. This helps patients to comprehend their choices, take decisions about their care, and also better their lifestyle to protect them from chronic diseases. Big data makes it easy to detect and identify/diagnose diseases at an early stage. Thus the right decisions on how to treat a certain disease efficiently and on time are guaranteed. Hence death rate and the rate of contracting diseases are greatly reduced. Big data aids the quality level of care given to patients by making sure that decisions are focused on a huge amount of related and updated data. Big data is promising in the area of identifying public health intervention targeted through the analysis of large volume and varied data and improved subsequent interventions by the use of large-velocity feedback mechanisms [ 87 ]. The quantity of data generated from the existence of the human race to 2003 can now be obtained in a few minutes [ 25 ]. Also, improved computational models, such as machine learning-based models, have revealed enormous possibilities in tracing the source or forecasting the spread of a novel disease in the nearest future [ 88 , 89 ]. Therefore leveraging big data and intelligent analytics becomes expedient in order to enjoy their use in COVID-19 outbreak and public health. 2.1 Challenges of big data in COVID-19 The ability to combine sophisticated epidemiologic models and new big data sources has been discussed [ 49 , 50 ]. For example, the use of smart-phones, social networks, or public health response satellites at different stages, mainly in the situation of disease forecasts, for making a decision [ 50 , 51 ]. These new sources of data include significant, real-time data on tourism visits that spread infection and spatial shifts in vulnerable residents that probably up till now were hard to report on timescales related to a rapidly fast-growing epidemic [ 50 ]. With increasing flexibility and growing universal connectivity, this information will be crucial for monitoring and containment planning [ 52 ]. By principle, with correct data sharing protocols in place, accurate, up-to-date disease predictions can be generated that are guided using this data stream. The success of big data in fighting epidemics depends on various organizations, both public and private, and the government is needed to be in contact frequently [ 50 , 51 ]. Privacy issues for daily use of new data channels need to be addressed, specifically the most suitable way to vigorously integrate such data streams to confirm individual confidentiality. Yet even if security is tackled, the transformation of new methods into a policymaking context raises additional systemic challenges [ 50 , 51 ]. Big data mining provides many tempting opportunities [ 43 , 53 ]. Nonetheless, when investigating big data sets and mining meaning and expertise from these facts extracted, researchers and professionals face many challenges [ 43 , [53] , [54] , [55] ]. The complications at various levels include data collection, storing, scanning, sharing, reviewing, handling, and watching. Furthermore, security and privacy issues particularly arise in distributed applications that are powered by data. The surge of knowledge and distributed sources also surpass our harnessing capability indeed, although the scale of big data continually grows exponentially, the actual technical ability to accomplish and explore large datasets is only in the comparatively lower rates of data petabytes, exabytes, and zettabytes [ 43 ]. In this chapter, we address in detail some technical issues that are still open to research. While working with big data, data scientists face a lot of obstacles. One obstacle is the compilation, integration, and storage of tremendous datasets generated from disseminated sources, with far less needed hardware and software [ 56 , 57 ]. The management of big data results in another huge problem. Effective big data management is essential to promote the abstraction of accurate information to maximize investment [ 58 , 59 ]. Proper management of data is the basis for big data analytics [ 60 ]. Another difficulty is to synchronize with an organization's internal infrastructure's external data sources, and big data dispersed systems (sensors, apps, databases, networks, etc.) [ 43 ]. Most of the time, an analysis of the data produced within organizations is not sufficient. We need to go one step further to integrate internal data with external data sources, to gain useful insight and information. External data may include the sources from third parties, market fluctuation details, weather forecasts and traffic situations, social network data, comments from clients, and resident input. This will aid to optimize the power of analytic models used in the analysis, for example. The computer design and performance are an important question. Indeed, CPU output is known to double every one and half years according to Moore's Law and diskette drive output is also expanding at the same time. The input and output operations, however, do not follow the same form of results (i.e., spontaneous input and output speeds increased moderately, while systematic input and output speeds developed slowly through thickness) [ 57 ]. Thus this varied device capacity will delay data access and affect the efficiency and scalability of big data applications. Machine learning is a very active area of study in artificial intelligence (AI) and pattern recognition nowadays. This plays a significant part in applications of probabilistic learning, such as computer vision, voice recognition, and the interpretation of natural languages [ 61 ]. Currently, the ability of computer-learning techniques and functionality engineering algorithms to process raw natural data is limited [ 62 ]. Deep learning is, on the contrary, more effective in solving data processing and learning complications contained in large datasets. This helps extract a huge size of unsupervised and unstructured raw data automatically. Dynamic policies learning consists of incremental and group learning that are important approaches to learn from a large stream of data with meaningful perception [ 63 ]. Big data and data stream uses the concept of collaborative and incremental learning. They deal with numerous challenges that arise during the learning of data, such as data accessibility and restricted resources. They make use of user profiling and stock forecasting applications. The application of incremental learning to data produces a faster means of prediction when dealing with the new data stream. Hence, using the incremental algorithm is highly recommended when the drift concept is not available. On the other hand, it is highly recommended to use ensemble algorithms to obtain accurate outcomes with large drift concept. Also, when dealing with a modest data stream or real-time processing, the incremental algorithm should be considered for use. Conversely, in the case of a complex data stream, an ensemble algorithm is considered the best. Another big problem is analogic reasoning: fundamentally, disease forecasting is unpredictable. In the narrative on big data and AI, there is often an underlying assumption about the statistical covariates or cell phone data and difficult models of simulation that avoid the need to gather basic epidemiologic details. Nevertheless, for the evolving COVID-19 epidemic, emphasizing this opinion still lacks a reliable fact on situation counts and biotic processes that drive an outbreak of the disease, let only the behavioral reactions of infected individuals, and make it difficult to rapidly adjust or understand precise intricate prototypes on spatiotemporal scales applicable in making a verdict. Hopefully, the best effective systems would continue to be simple [ 64 , 65 ] equally out of necessity for versatile modules that provide quick responses, given the controversy surrounding emergency epidemiologic studies and because simple models are more easily available to interpret and communicate [ 66 ]. Clear knowledge of both the importance and weaknesses of typical outputs is a precondition for their successful implementation but is often absent. Because policymakers have not yet reached an in-depth modeling experience, the lack of consistent message threatens to lead to two destructive results: (1) assume that the method would be misinformed without skepticism and choices or (2) ignore off-hand modeling and neglect the proof that we must control epidemics most efficiently. During epidemics, verdicts have to be taken rapidly, on a patchy basis, and inaccurate data models could be effective tools for guiding them. Considering the abovementioned obstacles, computing capacity, novel methods, and novel data are steadily advancing sources that give authentic courage for improved monitoring and development of valuable predicting systems. The novel database available to us comprises not only inactive experimental large data streams from cell phones but also comprehensive ecological data and local sensor information from embedded devices, internet search information, pathogenic genomic data that can be produced quickly informing the response during an outbreak, [ 67 ] and crowd-sourced methods to track emergencies changing rapidly [ 68 ]. Information sharing systems and structured aggregation strategies are being established to secure personal data privacy [ 69 ], and internet access enables rapid data transmission and collaboration between geographically distant responding teams. Methodologically, effective modeling methods that combine multiple predictions to reduce uncertainty are being built [ 65 , 70 ]. In reaction to the outbreak of COVID-19, an innovative, collaborative approach has unfolded between academic organizations, for example, between Twitter and other channels to exchange, evaluate, and publicly debate the effects of new research as it arises (Ironically, the control of misinformation that is now proliferating on social networks during a crisis is likely to become one of the most significant concerns for containing the disease.). Unless the abovementioned problems are resolved, these technologies will remain dislocated and impracticable. It is promising that all these problems may be strengthened by transferring most of the funding and knowledge focus to those communities that require most help during epidemics. The unpredictability of the COVID-19 outbreak and the progressive technologic mechanisms of these methods will require agile, dispersed groups of people covering the systematic and functioning dimensions of the epidemic response using novel big data methods to supplement and explain the limited spread, sociogeographic data that are necessary for the progress of important predictions. Provincial or small groups with investigative experience and current connections with government and industry stakeholders can contribute to scalable modeling strategies that exploit broad curated and international scientific database skills, building on partnerships formed if no new or ongoing outbreaks occur. This strategy may likewise be of help to mitigate some policy concerns related to data sharing, particularly through sensitive information not being shared publicly. To achieve this idea, significant continuous investment is required to finance individuals to fill what currently represents a significant void in the analytics process, especially in some countries with low and middle wages. Big data can be created from a lot of sources, such as social network sites, smartphones, IoT sensors, and publicly available data [ 71 ], in different formats such as text or video. Big data applies to patient care details in the sense of COVID-19, such as medical records, X-ray reports, case history, list of doctors and nurses, and information about the epidemic location. Big data has proved its capability to support fighting infectious diseases like COVID-19 [ 72 , 73 ]. 2.2 Importance of big data in COVID-19 The possibility of fast-spreading diseases that cause uncontrolled death and a bad influence on the economies and sustainability of countries in the world has stretched the importance and needs for developing a quantitative framework to provide support for making nearly real-time decisions in the public health system. According to a case study, the 2003 SARS outbreak that originated in China and spread to 29 countries became a healthcare-acquired infection in various regions by August 2003 [ 74 , 75 ]. The 2009 pandemic known as influenza (H1N1) originated in Mexico and spread rapidly all over the world through airline network and infected 20 countries, with maximum hit on the travelers coming from Mexico within a little time of the disease outbreak [ 76 ]. The socioeconomic implication and outcome of the outbreak of a disease related to 2009 (influenza A [H1N1]) were projected to be a huge amount ranging between $360 billion and $4 trillion [ 77 ], which was the calculation for the outbreak in the first year. Significantly, the SARS outbreak at Wuhan, China, in 2019 was calculated and projected to be 2,034,802 confirmed cases and 123,150 mortalities across the globe. Ever since the outbreak of COVID-19 in China, people who traveled globally from Wuhan transferred the disease to various countries of the world. The outbreak of SARS had significant effects on the people and economy all over the world. The effects include restrictions and bans on traveling, total closure of shops and markets resulting in a huge loss on earnings and proceeds, fear of contracting the disease from the infected person, and unpleasant side effects on world tourism at large, as many flights were canceled or suspended. Express and huge damage on government finances in various countries as agricultural activities were greatly hampered resulting in food shortage, and so many other effects resulted from the outbreak. Therefore it becomes imperative to make an immediate response from all angles to fight and contain the virus efficiently. Big data entails the innovative processing of both new and existing data to proffer meaningful solutions that are of huge business benefits [ [78] , [79] , [80] ]. However, processing large data, or varied data, will only be a mere technologic solution until it is associated with business objectives and goals. Big data promises more lights to be shed on complicated facts and information on the dynamics of how infectious diseases are transmitted and how to develop new modeling and analytic tools that have far been hidden due to lack of smooth and workable data. More progress will be made in the area of disease forecast when a huge amount of information on epidemics required for modeling is available. Big data will be the best in accomplishing this goal. Improved consistency in reporting diseases and case definition is therefore expedient within time and space. Data obtained from news media sources can give a perfect estimation and evaluation of how the disease is transmitted. Thus it is of high importance, especially when comprehensive and well-examined data are not available. In a country with low and middle income, and where studies on disease transmission details are scarce, resources based on internet surveys will provide an opportunity for a detailed study and correct assessment on how the disease is transmitted, for instance, in the crisis of an infectious disease like COVID-19 when real-time assessment is expedient. A well-calibrated computational model tool that is useful in assessing and analyzing the epidemic spread signifies a potent tool to sustain the process of decision-making at a time of emergency epidemic outbreaks. Epidemic models are progressively used to generate predictions of the spatial-temporal progression of a disease outbreak at several spatial scales and for easy access to possible impacts of the various strategies of the intervention [ 81 ]. The ability to produce innovative facts or information is greatly expanded by big data. The costs applicable to answer various questions retrospectively and prospectively by gathering ordered and structured data are exorbitant. Acquisition of finer data in a computerized format is achieved by analyzing unstructured data that are within EHR systems by using various computational techniques such as traditional language processors that extract clinical concepts from free documents. Analyzing the unstructured data contained within her systems using computational techniques (e.g., natural language processing to extract medical concepts from free-text documents) permits finer data acquisition in an automated fashion. For example, computerized detection within her systems with the use of natural language processing is better used to detect complications that arise after the operation when compared to patient well-being indicators based on discharge coding [ 82 ]. Big data provides the possibility to generate an observable indication base for clinically related questions, which may not be achievable but will be helpful with generalizability issues. Generalizability issues restrict the application of conclusions that are derived from random trials carried out on a very narrow scale of participants to patients that show dissimilar characteristics. Big data helps disseminate knowledge. Many healthcare practitioners strive to be updated with current evidential proof and support that guide clinical practices. The computerization of medical journalism to a great extent has enhanced access; conversely, with great numbers of studies, translation of knowledge becomes difficult. If at all a physician has enough relevant data and guiding principles, organizing information to formulate a realistic approach for the treatment of patients with numerous chronic illnesses becomes extremely difficult. Analyzing existing EHR systems to generate a template to guide in making clinical decisions is the only way by which this problem may be solved [ 83 ]. This technique is exploited in the alliance involving Memorial Sloan Kettering Cancer Center and IBM's Watson supercomputer to aid in the diagnosis and proposed treatment for cancer patients. The difference between the big data method and traditional decision support tools is that with the big data technique, suggestions are obtained through analysis of patients' data in real time instead of depending solely on the use of rule-based decision trees. For instance, longitudinal analytic data have been proven to be efficient for forecasting future diagnostic risks and domestic abuse in patients [ 83 , 84 ]. With data-driven clinical decision support tools, the cost is greatly minimized and proper care standardization is guaranteed. Physicians can get information that guides them on the diagnostic and therapy options that are provided by esteemed colleagues encountering related patient profiles with the use of big data on the cloud. The healthcare system can be transformed by getting information directly across to patients and equipping them to take part in more active roles. In the future, clinical reports can also reside with the patients, compared to the existing model where health records of patients are stored with healthcare professionals, thereby making the patients be in a passive situation. Big data provides the opportunity to progress the clinical records by connecting the conventional health-related data such as family history and medication list to other private data located on other sites such as education, income, neighborhood, military service [ 83 , 85 ], exercise regimens, diet habits, and forms of entertainment, all that can be easily accessed without the need to interview the patient with a comprehensive list of questions. Thus big data provides an opportunity to collaborate with the conventional medical model with social determinants of health in a patient-directed fashion. The initiatives of public health to reduce obesity and smoking can be efficiently delivered by focusing the message on the most appropriate set of people based on the profiles on their social media. With the opportunity to use analytic capabilities, system biology can be incorporated. Big data assist in translating personalized medicine initiatives into clinical practice (e.g., genomics) with data [ 83 , 86 ]. The Genomics Network and Electronic Medical Records achieve this with the use of natural language processing to phenotype patients, in an attempt to simplify genomics research. With big data, patients are opportune to access correct and updated information. This helps patients to comprehend their choices, take decisions about their care, and also better their lifestyle to protect them from chronic diseases. Big data makes it easy to detect and identify/diagnose diseases at an early stage. Thus the right decisions on how to treat a certain disease efficiently and on time are guaranteed. Hence death rate and the rate of contracting diseases are greatly reduced. Big data aids the quality level of care given to patients by making sure that decisions are focused on a huge amount of related and updated data. Big data is promising in the area of identifying public health intervention targeted through the analysis of large volume and varied data and improved subsequent interventions by the use of large-velocity feedback mechanisms [ 87 ]. The quantity of data generated from the existence of the human race to 2003 can now be obtained in a few minutes [ 25 ]. Also, improved computational models, such as machine learning-based models, have revealed enormous possibilities in tracing the source or forecasting the spread of a novel disease in the nearest future [ 88 , 89 ]. Therefore leveraging big data and intelligent analytics becomes expedient in order to enjoy their use in COVID-19 outbreak and public health. 3 Big data privacy and ethical challenges in COVID-19 The latest outbreak of COVID-19 has brought the difficulty of securing personal data to a head in a transnational sense [ 90 ]; this is because COVID-19 spreads fast with the international travel of people [ 91 ]. Many countries require international travelers to disclose their personal information such as the name, gender, date of birth, travel history, purpose of travel, and residence, among others, and impose quarantine requirements accordingly [ 92 ]. Using a genuine case where the Chinese media secretly published the sensitive information of a foreign traveler, the article describes that multiple patterns for LEX cause emerged at each point of dispute-of-law analysis: (1) the European Union, the United States, and China vary in characterizing the right to personal data; (2) the expanding centralized approach to relevant legislation lies in the fact that all three territories either find the law on personal information privacy to be a contractual law or follow linking factors leading to the law of the forum; and (3) actively support the de-Americanization of meaningful data privacy legislation. The patterns and their mechanisms have important consequences in the application of regulations for transnational information [ 90 ]. Evolving contagious diseases such as HIV/AIDS, SARS, and pandemic influenza, and the 2001 anthrax attacks, have proven that we appear vulnerable to health risks from contagious diseases [ [93] , [94] , [95] ]. The key expert recommendation for two decades has been the value of improving global public health surveillance to provide an early warning [ 94 , 95 ]. Based on digital information sources, including data from smartphones and other digital equipment, the outbreaks triggered by new findings are of special importance in infectious diseases [ 25 , 93 , 95 ], for which the survey data and accurate predictions are indeed limited. Recent research shows the likelihood of predicting the spreading of COVID-19 by integrating Government Aviation Guide data with WeChat App data on human accessibility and other digital platforms run by the Chinese technology giant Tencent [ 96 ]. Smartphone data also demonstrated possibilities for modeling regional cholera outbreak in Haiti during the 2010 outbreak [ 97 ], while using big data analysis demonstrated efficacy during the Ebola crisis in the year 2014–16 [ 98 ]. Moreover, the big data gathering of cellular data from users around the world, particularly mobile data archives and social network accounts, also raised questions about security and data integrity during those epidemics. In 2014, the GSM organization (a sector body that serves the benefits of major mobile network operators) urged privacy issues to issue privacy rules for the use of cell phone data to respond to an Ebola outbreak [ 25 , 99 ]. In the information-intensive world of 2020, these issues can easily be compounded by pervasive data points and automated surveillance devices. China is the area hardest hit with COVID-19 and has reportedly used omnipresent transmitter data and safety monitoring software to avoid disease spread [ 25 , 100 ]. The New York Times reports [ 101 ] that how these data are reviewed and recycled for monitoring reasons is not clear. For instance, the document said that Alipay Health Code, an Alibaba-backed government-run app that helps decision on who is to be isolated for COVID-19, also seemed to share details with the police [ 25 , 101 ]. The European Republic in Italy with the highest figure of COVID-19 incidents, the remote data-privacy agency was advised to issue a statement [ 102 ] on March 2, 2020, to explain the requirements of permissible data usage for containment and prevention. In its declaration, the expert cautioned against data gathering and analysis by a noninstitutional body that violates privacy [ 102 , 103 ]. A couple of days later, the European Data Security Committee published a press release on the idea of securing people information if used to counter COVID-19 and highlighted relevant research of the General Data Protection Regulation, which include legal reasons for the handling of people information in the form of outbreaks [ 90 ]. For instance, the article enables the compilation of people information "for purposes of general interest in the field of health research, including guarding toward severe cross-border challenges to health," given that such analysis is directly proportional to the purpose sought, reverences the principle of the right to data privacy, and safety the rights and liberties of the person concerned. Big data is recognized as important tools for COVID-19 pandemic management in this era of technologic advancement; there must be specific criteria for effective gathering and analysis of data on a global scale. They claim that the usage of technologically accessible data and techniques for forecasting and tracking, e.g., describing people who have traveled to places where the infection has transmitted or has been identified and quarantining the people contacted, is of vital importance in combating the COVID-19 outbreak. However, it is equally necessary to use these data and technologies responsibly by data privacy legislation and with proper regard for safety and security. Failure to do so will weaken public confidence, causing people less likely to obey advice or guidelines on global health and more likely to have worse patient outcomes [ 48 ]. Practices of conscientious data management can guide both collection of data and information processing. The concept of proportionality will apply to the sharing of information from the persons affected, which means the collection of data must be proportionate to the severity of the hazard to public health, be partial to what is required to accomplish particular public health goals, and be systematically proven. For example, obtaining access to personal contact device data tracking purposes may be acceptable if it happens inside defined limits; if it has a valid reason, such as alert and quarantine those who have had contact with the infected person or the virus itself; and if no less-intrusive option, such as using anonymized mobile tracking data, is appropriate for that reason. Also, health monitoring "do for yourself," as the Italian data protection agency has called it, must be resisted. Data product quality standards are required at the data analysis stage [ 25 , 104 ]. Deficiencies in data protection, which are normal when data from digital personal devices are used, can lead to minor errors in one or more factors [ 105 , 106 ], which in turn may have an enormous impact on predictive large-scale models. Also, privacy violations, inadequate or unsuccessful deidentification, and prejudices in databases can become major causes of public health mistrust [ 107 , 108 ]. Data protection issues are not only of a technologic aspect but also of a legal and political nature [ 109 , 110 ]. For purposes tracing the index or infected cases, demanding or guaranteeing access to personal devices may be more successful than simply exploiting anonymous mobile tracking data. Taiwanese studies display a compelling method to exploit big data analysis to react to COVID-19 without fostering public distrust. To improve classifying incidents, Taiwanese agencies have combined their national health insurance database with customs database travel history. Certain tools have also been used for surveillance purposes, such as QR code scanning and electronic monitoring. Such interventions were paired with techniques for official communications including regular health checks and support for those under lockdown [ 111 ]. More nations are planning to use big data and emerging technology in the battle against the evolving COVID-19 outbreak, so if used appropriately, data and algorithms are one of the main resources in our bow [ 25 ]. 4 Big data analytics in COVID-19 epidemic Big data became a big dilemma a few years ago. In the 2000s, big data overwhelmed the technologies of storage and CPU systems by the abundance of terabytes of data required to be stored. The IT industries face the problem of data scalability as the need to store a large volume of data skyrockets [ 112 , 113 ]. The rapid rate at which health information technologies advance leads to the need for big data in the hospital environments and the healthcare system at large. However, the extraction of meaningful information from the highly loaded datasets is still inadequate and very limited. The decision-support tools that are required for efficient handling of rich and large-scale healthcare records, optimizing operational dynamics in the healthcare environment, and extracting relevant knowledge and information with regard to the health conditions of patients and availability of additional and efficient healthcare services are seriously lacking in the practice of general medicine. The healthcare system grows from not being able to handle big data to wasting huge budgets on its collection and analysis, respectively. As of now, the healthcare system is leveraging big data to extract essential information that was not acknowledged in the past. The healthcare system can now study big data to have a better understanding of the current situation on health and still be able to track evolving aspects such as patient behavior by using advanced and sophisticated analytic tools. To gain more insight into better healthcare intelligence and facts, it is helpful to tap into data that has never been tapped before. Most of this untapped information will be very new to you as they come from devices, sensors, third parties, social media, and web applications. Data are obtained incessantly in a real-time manner from some big data sources. When put together, one can easily detect that the big data not only is about a huge volume of data but also focuses on astonishingly diverse data types that can be delivered at varying frequencies and speeds. Hence it is noteworthy that we now have the coming together of two technical entities. The first entity is big data that has been used for enormous and detailed information. Second, the advanced and more sophisticated analytics, which comprises of a collection of various types of tools, that includes tools that are based on natural language processing, data mining, AI, statistics, predictive analytics, and many more. When all of this is put together, you get what is called the big data analytics, which is the hottest new practice today in Heath Intelligence [ [113] , [114] , [115] ]. The availability of massive data presents unlimited opportunities to visualize, manage, analyze, and extract meaningful information from huge, varied, dispersed, and heterogeneous datasets that enable making better medical decisions and improved performance in the healthcare system. Across all corners of the healthcare system, big data is inspiring a reflective transformation. The manner with which biomedical research has been carried out and how the delivery of healthcare services has been managed across the globe are the new transformations in informatics and analytics research. The analysis of big data in COVID-19 involves several tools and sophisticated technologies as illustrated in Fig. 8.3 . Big data can provide healthcare intelligence for the COVID-19 outbreak, thereby being useful for governments, organizations, and healthcare policymakers for smart and quick response during this pandemic. The big data if not analyzed become a liability and unrealistic to the owners and users. For intelligent prediction, forecasts, and decision-making, big data analytics involves mining and extracting useful knowledge. Scalability, visualization, and computation of data are the challenges in big data analytics, thus the flow in the amount of data increases the information security risk. Figure 8.3 Big data analytics. Making better medical decisions, enhanced patient management, monitoring systems, and efficient public health supervision are progressively viewed by the government, the general public, and the medical community as a major key to support the improvement of and reduce the cost of the healthcare arena [ 49 , 114 ]. The operations of advanced analytic methods on big data are known as big data analytics. With the gigantic statistical samples that big data provides, an improved result of the analytic tool is also enhanced. Various tools intended for statistical analysis and data mining are liable to be optimized for large amounts of datasets [ 113 ]. It is a general rule that the accuracy of statistics and other products of the analysis depends and increases with how large the data sample is [ 113 , 116 ]. The latest generation of tools for data visualization and analytics for in-database functions similarly operates and functions on big data [ 113 , 116 , 117 ]. Big data analytics simply is the procedure, techniques, and technologies used in analyzing big data to discover useful information through the use of nontraditional and advanced methods [ 118 , 119 ]. It is designed in such a way that it increases accuracy and scalability when compared to the conventional methods, such as the regression-based models and many other statistical models. AI, among other advanced techniques, has been acknowledged as a very significant development in the roles it plays in various application domains [ 120 ], together with other disciplines related to public health [ 121 ]. In a vacuum, big data is worthless. Its prospective usefulness can only be tapped when it is adopted for decision-making. Organizations require an effective means of revolving large volumes of fast-growing and varied data into useful insights to facilitate the making of evidence-based decisions. The general techniques for extracting useful information from big data can be classified into five different stages [ 1 , 44 ]; Fig. 8.4 shows the five stages that depict the two major analytics and management subprocesses. Data management comprises of methods and technologies that support the means of acquiring, storing, preparing, and retrieving data for analysis. Conversely, analytics describes the technique used in analyzing and obtaining intelligence from big data. Hence big data analytics is seen as a subprocess in the overall technique of extracting insights from big data. Figure 8.4 Processes for extracting insights from big data. By combining and efficiently using big data in a digitalized manner, the healthcare system and organizations that range from single-physician workstations and multiprovider groups to big hospital networks and responsible healthcare organizations stand to gain many benefits [ 118 , 122 ]. The prospective advantages of big data analytics in COVID-19 consist of early disease detection, which can then be treated effortlessly and efficiently; maintaining precise individual and public health; and easy detection of healthcare frauds speedily and effectively [ 118 ]. Addressing numerous questions is made possible with big data analytics. Predictions and estimations are based on a huge amount of chronological data, which includes how long a patient will stay, numbers of patients who will prefer an elective surgery, other hospital-acquired illness/the rate of disease progression, disease causal factors, and risks of disease advancement in patients [ 118 ]. Yearly, big data analytics will facilitate over $300 billion in total savings in the US healthcare system. Two-thirds of such reductions amount to approximately 8% in the national healthcare expenses. Two biggest areas of prospective savings with $165 billion and $108 billion savings are medical operations and research and development (R&D), respectively [ 118 , 123 ]. Big data will possibly assist in cost reduction and ineffectiveness in three areas. Clinical operations: relative efficient research to establish added medically related and cost-efficient ways of diagnosing and treating patients. R&D: (1) the use of predictive modeling to reduce the error rate and construct faster, compact, and more centered R&D pipeline in drugs and devices; (2) statistical tools and algorithms to progress medical trial design and patient enrollment to match treatments to patients efficiently by reducing trial failures and encouraging advanced treatments to patients, thus reducing trial failures and bringing new treatments to the market; and (3) to examine medical trials and patient records to detect and follow-up on indications and adverse effects before the products reach the market. The speedy examination of various symptoms in diseases, and tracking of disease outbreak in real-time have really help in public healthcare systems. The BDA has speedy improved the production and development of vaccines for COVID-19 outbreak. For example, the conversion of huge amount of data to a meaningful information has been used to predict, forecast, and diagnose various infectious diseases and in the development of vaccine for the advantages of the population at large [ 118 , 123 ]. Hence big data analytics has a high ability to innovate the approach so that healthcare providers can use complicated technologies to provide insight from their medical and many other repositories for data to ensure making informed decisions regarding the COVID-19 epidemic and the outbreak of any associated infectious disease. 5 Conclusion The December 2019 COVID-19 is known as a pandemic because it has cut across the globe. It has become a great threat to global health. It has caused close to 2.5 million infections and 180,578 deaths in almost 213 countries by April 22, 2020, and the daily increase in this infectious disease has become a huge challenge to the socioeconomic development of each country across the globe. The application of big data offers possibilities for forecasting and analyzing viral behavior for directing healthcare in individual nations to enhance their readiness for the COVID-19 outbreak. This can be achieved using different global databases, for instance, the Official Aviation Guide, the Tencent Location Services (Shenzhen, China), and the Wuhan Municipal Transportation Management Bureau conducted a model analysis of "now-casting" and prediction of COVID-19 development inside and outside China that could be used by public health planning and controlling authorities around the world [ 124 ]. Likewise, the WHO International Health Regulations, the State Parties Annual Reporting Guide, the Joint External Assessment Reports, and the Infectious Disease Vulnerability Index were used to evaluate the readiness and vulnerability of African countries to combat COVID-19, which will help raise awareness among the health authorities in Africa to better plan for the viral outbreak [ 48 , 125 ]. Big data potentially provide several promising solutions to help combat the COVID-19 epidemic. The use of big data with different analytic tools will help understand COVID-19 in terms of outbreak monitoring, virus development, disease control, and the manufacturing of vaccines. Big data coupled with intelligence-based applications can create complex prediction models using coronavirus data streams to estimate the outbreak, thus allowing health authorities to track the spread of coronavirus and plan for effective preventive measures. Big data models also support future forecasting of the COVID-19 outbreak through their ability to combine enormous quantities of data for prevention and treatment. Moreover, big data analytics from a variety of real-world sources, including infected patients, can help implement large-scale COVID-19 investigations to develop comprehensive treatment solutions with high reliability [ 126 , 127 ]. This would also help healthcare providers to understand the virus development for better response to the various treatment and diagnoses. As one of the most effective solutions to combat the COVID-19 pandemic, early treatment and prediction are of importance. Hence the capability of big data in the fight against the COVID-19 outbreak can be useful and divided into four main areas of application: outbreak virus spread, monitoring forecasting, diagnosis/treatment of coronavirus, and discovering vaccines/drugs. In response to the COVID-19 virus globally, different traditional approaches were dedicated to improve future forecasting, prevention, and treatment, with other alternatives. However, these approaches are typically costly and time-consuming, have a low positive result rate, and require different materials, equipment, and resources. Moreover, most countries are suffering from a lack of testing kits because of the limitation on budget and techniques. Thus these standard methods are not suitable to meet the requirements of fast detection and tracking during the COVID-19 pandemic. Therefore the use of connected devices, along with big data, is an easy and low-cost approach for COVID-19 detection. In the COVID-19 pandemic battle, developing efficient diagnostic and treatment methods plays an important role in mitigating the impact of the COVID-19 virus.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182894/

Orthologues of Bacillus subtilis Spore Crust Proteins Have a Structural Role in the Bacillus megaterium QM B1551 Spore Exosporium

When starved of nutrients, some bacterial species develop metabolically dormant spores that can persist in a viable state in the environment for several years. The outermost layers of spores are of particular interest since (i) these represent the primary site for interaction with the environment and (ii) the protein constituents may have biotechnological applications. The outermost layer, or exosporium, in Bacillus megaterium QM B1551 spores is of interest, as it is morphologically distinct from the exosporia of spores of the pathogenic Bacillus cereus family. In this work, we provide evidence that structurally important protein constituents of the Bacillus megaterium exosporium are different from those in the Bacillus cereus family. We also show that one of these proteins, when purified, can assemble to form sheets of exosporium-like material. This is significant, as it indicates that spore-forming bacteria employ different proteins and mechanisms of assembly to construct their external layers.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634872/

Identification of Novel Anthrax Toxin Countermeasures Using In Silico Methods

Anthrax is an acute infectious disease caused by the spore-forming, gram-positive, rod-shaped bacterium Bacillus anthracis. The anthrax toxin lethal factor (LF) is the primary anthrax toxin component responsible for cytotoxicity and host death and has been a heavily researched target for design of postexposure therapeutics in the event of a bioterror attack. Various computer-aided drug design methodologies have proven useful for pinpointing new antianthrax drug scaffolds, optimizing existing leads and probes, and elucidating key mechanisms of action. We present a selection of in silico virtual screening protocols incorporating docking and scoring, shape-based searching, and pharmacophore mapping techniques to identify and prioritize small molecules with potential biological activity against LF. We also recommend screening parameters that have been shown to increase the accuracy and reliability of these computational results.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795694/

The Quest for a Truly Universal Influenza Vaccine

There is an unmet public health need for a universal influenza vaccine (UIV) to provide broad and durable protection from influenza virus infections. The identification of broadly protective antibodies and cross-reactive T cells directed to influenza viral targets present a promising prospect for the development of a UIV. Multiple targets for cross-protection have been identified in the stalk and head of hemagglutinin (HA) to develop a UIV. Recently, neuraminidase (NA) has received significant attention as a critical component for increasing the breadth of protection. The HA stalk-based approaches have shown promising results of broader protection in animal studies, and their feasibility in humans are being evaluated in clinical trials. Mucosal immune responses and cross-reactive T cell immunity across influenza A and B viruses intrinsic to live attenuated influenza vaccine (LAIV) have emerged as essential features to be incorporated into a UIV. Complementing the weakness of the stand-alone approaches, prime-boost vaccination combining HA stalk, and LAIV is under clinical evaluation, with the aim to increase the efficacy and broaden the spectrum of protection. Preexisting immunity in humans established by prior exposure to influenza viruses may affect the hierarchy and magnitude of immune responses elicited by an influenza vaccine, limiting the interpretation of preclinical data based on naive animals, necessitating human challenge studies. A consensus is yet to be achieved on the spectrum of protection, efficacy, target population, and duration of protection to define a "universal" vaccine. This review discusses the recent advancements in the development of UIVs, rationales behind cross-protection and vaccine designs, and challenges faced in obtaining balanced protection potency, a wide spectrum of protection, and safety relevant to UIVs. Introduction Influenza viruses present a high level of antigenic diversity and variability due to their segmented RNA genome. These viruses are classified into four major types, A, B, C, and D, based on their nucleoprotein (NP) and matrix (M) genes. Human infecting type A and B viruses are further classified into multiple subtypes or lineages, respectively, depending on the antigenicity of viral surface proteins, hemagglutinin (HA), and neuraminidase (NA) genes (Paules and Subbarao, 2017 ). Influenza A and B viruses co-circulate in every season and, thus, c represents the primary targets of seasonal influenza vaccines (Sridhar et al., 2015 ). In addition to seasonal epidemics, influenza viruses have caused pandemics at the intervals of ~10–40 years since the 1918 Spanish flu H1N1, the 2009 pandemic H1N1 being the last outbreak (Saunders-Hastings and Krewski, 2016 ). While vaccination remains the most cost-effective measure to prevent influenza virus infections, antigenic drift in the surface antigens allows these viruses to escape antibody-mediated neutralization (Kim et al., 2018a ). In addition, the sudden occurrence of pandemics is often accompanied by zoonotic spillover of the surface genes into the human-infecting viruses, rendering preexisting vaccines ineffective to newly emerging viruses. The variation caused by genetic drift and shift is unpredictable, posing a serious challenge to the management of influenza outbreak. Based on the amino acid sequences of HAs, influenza A viruses (IAVs) are divided into two phylogenetic groups. The IAV HA group 1 viruses include H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18, while the group 2 viruses comprise H3, H4, H7, H10, H14, H15 ( Figure 1 ). The NAs of IAVs are also antigenically diverse, presenting two distinct groups. Influenza B viruses (IBVs) are not divided into subtypes but circulate as two distinct Yamagata-like and Victoria-like lineages. Influenza C viruses (ICVs) generally cause a mild respiratory disease in humans and do not cause epidemics (Dykes et al., 1980 ). Contemporary influenza epidemics are caused by the H1N1 and H3N2 of the IAVs and one or two lineages of the IBVs, dictating trivalent (TIV) or quadrivalent influenza vaccine (QIV) containing two IAV antigens and one or two IBV antigens, respectively (Ambrose and Levin, 2012 ). Figure 1 Phylogenetic trees representing HA and NA diversity among influenza viruses. The 18 subtypes of HAs of IAV are divided into two phylogenetic groups according to their amino acid sequences similarities. The HAs of IBVs are divided into Victoria-like and Yamagata-like lineages but they are closer to each other than any of two different subtypes of IAVs. The HAs of ICVs are antigenically distant from those of IAVs and IBVs. The NAs of IAVs also show high levels of antigenic variability and are divided into two groups. Phylogenetic trees were constructed based on amino acid sequence comparisons among influenza viruses. Multiple alignments were carried out using the representative sequence of each HA or NA subtype or lineage. The phylogenetic trees were constructed by the ClustalW algorithm using neighbor joining (N-J) method and visualized by FigTree v1.4.4. The scale bars represent amino acid change (%). Many strategies have been undertaken on the pursuit of developing a universal influenza vaccine (UIV) (Paules et al., 2017 ). The induction of cross-protective immune responses directed toward conserved B cell or T cell epitopes is a major principle underlying broad protection ( Figure 2 ). The direct binding of antibodies to the viral surface proteins interferes with their functions and results in virus neutralization before cell entry ( Figure 2A ). Alternatively, the antibodies may bind to viral antigens displayed on the surface of virus-infected cells and mediate effector functions to remove the infected cells. A cytotoxic T lymphocyte (CTL) can kill the virus-infected cells in an independent manner ( Figure 2B ). Several conserved viral antigens such as M2 extracellular domain (M2e), HA stalk and receptor binding site, NA, and T cell epitopes in the internal proteins such as polymerase basic protein 1 (PB1), NP, and M1 were defined as targets for eliciting cross-reactive immune response. A variety of vaccine platforms were tested for effective exposure of those antigens to the immune system (Wang et al., 2018a ; Estrada and Schultz-Cherry, 2019 ). While these vaccines showed broader cross-protection than the classical inactivated influenza vaccines (IIVs), their protection potency was weak. They provided partial protection against the antigenically distant viruses, and the protection breadth is usually limited to the same group (subtype-specific or HA group-specific) and not effective in another HA group. Thus, there exists a considerable gap between the current status, and the ultimate goal, of a truly universal vaccine. Figure 2 Major principles of developing a UIV. (A) Induction of broadly protective antibodies specific to conserved regions in the HA stalk or head, NA, or M2e has been extensively investigated. The antibodies bind to viral surface proteins either on virion or expressed on virus-infected cell membrane. (B) Cytotoxic T lymphocyte (CTL) recognizing the conserved epitopes of influenza internal proteins such as NP, M1, or PB1 has been shown to be critical for broad protection through the clearance of the virus-infected cells. Some studies have discovered rare antibodies specific to the conserved HA stalk in animals and humans with prior exposure to influenza viruses. Some of these antibodies show extremely broad specificity, encompassing both HA groups of IAVs (Ekiert et al., 2011 ) or even both IAVs and IBVs (Dreyfus et al., 2012 ), representing an optimistic prospect of developing a UIV. However, no studies so far have reported a successful vaccine approach that induces such antibodies to a protective level, reflecting considerable difficulty in selectively inducting particular antibodies in the context of vaccination. While antibody-dependent effector mechanisms and T cell immunity have emerged as potential correlates of broad protection against influenza infections (Krammer and Palese, 2015 ), the exact molecular mechanisms of their protective action are not completely understood, and immunopathology by T cell responses still remains a challenge (Peiris et al., 2010 ; Duan and Thomas, 2016 ; Erbelding et al., 2018 ). Moreover, understanding how preexisting immunity (immune imprinting or antigenic sin-like phenomenon) shaped by prior exposure to influenza viruses affects the magnitude, hierarchy, and sustainability of antibody response to vaccination is suggested as critical for designing a UIV in humans (Henry et al., 2018 ). The involvement of multiple factors in eliciting broad protection and the influence of pre-existing immunity on the subsequent vaccination pose a dilemma on establishing the correlate of cross-protection against heterologous or hetero-subtypic viruses, representing a critical obstacle for the licensure of a UIV by the regulatory authorities. This review updates the recent advances in UIV development and focuses on the critical issues to be addressed in designing a 'truly' UIV. Alternative vaccine antigens and vaccine strategies for durable and broader protection are also discussed in detail. This review is meant to help the readers to acquire general information on the cutting edge of UIV researches and to gain wide perspectives on rational design of a truly UIV with improved potency, breadth, and safety. Criteria for a UIV Definition of Universal Protection Although there is a clear consensus about the urgent need for a vaccine that provides durable and broad protection against multiple strains of influenza virus, the definition of the "universality" of UIV is still debated (Krammer et al., 2018b ). Recently, the National Institute of Allergy and Infectious Diseases (NIAID) held a meeting to identify and develop the criteria to define a UIV. The participants from multiple disciplines agreed that a reasonable UIV should be at least 75% effective against symptomatic influenza virus infection caused by group 1 and group 2 IAVs, with the protection lasting over 1 year for all age groups (Paules et al., 2017 ; Erbelding et al., 2018 ) ( Table 1 ). Similar criteria have also been suggested as preferred product characteristics (PPC) and target product profiles (TPP), describing the desired characteristics of a UIV, by the World Health Organization (WHO) and the Bill and Melinda Gates Foundation (BMGF), respectively ( Table 1 ). Despite the priority given to IAVs in WHO PPC ( Table 1 ), the IBVs represent a "low hanging fruit" because of their low antigenic diversity and the lack of animal reservoir, presenting credence to potential eradication from humans (Tan et al., 2018 ). The proposed consensus to define universal protection offers a valuable guideline to develop an effective and safe UIV. Ideally, the spectrum of protection may covers both IAVs and IBVs, considering the existence of extremely broadly protective antibodies and T cell epitopes across IAVs and IBVs from animals and humans (Corti et al., 2011 ; Koutsakos et al., 2019 ). However, as the antigenic difference between a vaccine and a target virus becomes larger, the number of conserved B cell or T cell epitopes between the two strains decreases. The limited availability of target epitopes may result in compromised protection robustness due to (1) decreased clonal diversity of cross-reactive antibodies and T cells, or (2) occurrence of escape mutants by even small amount of genetic mutations in the epitopes. Therefore, a balanced breadth and robustness of cross-protection should be considered when developing a reliable UIV ( Figure 3 ). Table 1 Consensus criteria for a UIV. NIAID WHO BMGF Consensus Breadth All influenza A viruses (influenza B protection would be the second target) All influenza A viruses All influenza A and B viruses All influenza A viruses Efficacy At least 75% effective against symptomatic influenza infection Better than that of current seasonal influenza vaccine At least 70% effective against symptomatic influenza infection At least 70% effective Target population All age groups >6 weeks, no upper age limit including high risk groups >6 weeks, no upper age limit including high risk groups >6 weeks, no upper age limit Duration of protection At least 1 year At least 5 years 3–5 years At least 1 year NIAID, National Institute of Allergy and Infectious Diseases ( https://www.niaid.nih.gov/diseases-conditions/universal-influenza-vaccine-research ); BMGF, Bill and Melinda Gates Foundation ( https://gcgh.grandchallenges.org/challenges ). WHO, World Health Organization ( https://apps.who.int/iris/bitstream/handle/10665/258767/9789241512466-eng.pdf ; https://jsessionid=37D9E056C3EA58A90EE237432AA7D65F?sequence=1 ) . Figure 3 Protection breadth of influenza vaccines ranging from strain-specific protection to pan-influenza universal protection. Currently licensed seasonal influenza vaccine provides only strain-specific protection against well-matched strains. Recently, many efforts have been put in order to improve the protection breadth of a vaccine from subtype-specific protection to, ideally, pan-influenza universal protection [adopted from Erbelding et al. ( 2018 )]. The NIAID and the researchers have proposed three research areas to address the knowledge gaps in developing a UIV; (1) understanding of influenza transmission, natural history, and viral pathogenesis, (2) characterization of correlates of protection, and (3) rational design of a UIV to improve potency and breadth of protection (Erbelding et al., 2018 ). In particular, the correlates of protection elicited by a UIV may vary considerably in quantity and quality, depending on the vaccine type used and the target influenza virus tested. For instance, while the protection potency of HA stalk-based vaccine may easily be evaluated by measuring the neutralizing activity or indirect effector mechanisms by the stalk-reactive antibodies (Jegaskanda et al., 2017b ), such correlates of protection cannot be used to evaluate the protective efficacy of a T cell epitope-based vaccine containing non-HA epitopes. Moreover, when using HA stalk-based vaccines, high levels of stalk-reactive antibodies represent a good protective efficacy against the same group IAVs ( Figure 1 ). However, the binding affinities of HA stalk antibodies are variable among different viruses, and therefore may require different antibody titers to exhibit sufficient protection against the viruses. This speculation is supported by the observations that broadly reactive HA stalk antibodies show considerably different neutralizing abilities and binding affinities to the viruses within the same HA group (Ekiert et al., 2011 , 2012 ). Furthermore, HA stalk-based vaccine and M2e-based vaccine may demonstrate a different balance of protective abilities to each other, between direct neutralization and indirect effector mechanisms (ADCC, for instance) by the respective antibodies. It has been shown that M2e antibodies provide protection via FcR-dependent effector functions rather than direct virus neutralization (Deng et al., 2015a ), whereas HA stalk antibodies exert both direct neutralization and indirect effector functions (Krammer and Palese, 2015 ). The antibody-mediated inhibition of virus attachment measured by hemagglutinin inhibition assay or microneutralization assay is the gold standard for seasonal influenza vaccines (Ohmit et al., 2011 ). However, the assay cannot be applied simply to reflect the cross-protection by a UIV against heterologous/heterosubtypic influenza viruses. Therefore, it is essential to develop mechanistically distinct in vitro or in vivo assays to measure potential correlates of protection in order to evaluate the protection potency and breadth of the vaccine. Mode of Protection by a UIV The cornerstone of developing a UIV is the determination of the precise protection mechanisms of immune response against influenza viruses. Influenza HA recognizes sialic acid on the cellular receptors and initiates infection by entering the cell via receptor-mediated endocytosis ( Figure 4 ). While HA inhibitory (HAI) antibodies have long been considered as the gold standard for strain-specific protection, very few of them were shown to elicit a broad protection by binding to the conserved receptor-binding site (RBS) of HA, thereby preventing viral entry to the cell (Krause et al., 2011 ; Ekiert et al., 2012 ). Recently, multifunctional protection mechanisms have been described for HA stalk-reactive antibodies. It has been shown that HA stalk antibodies may inhibit membrane fusion, the release of viral genome into the cytoplasm of the cell, and maturation of the HA precursor (Krammer and Palese, 2015 ). Furthermore, HA stalk antibodies can induce antibody-dependent effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytolysis (CDC), resulting in clearance of virus-infected cells by the immune cells or the complement system (Jegaskanda et al., 2017b ). During viral budding, NA cleaves the sialic acid from HA and supports multiple infection cycles by release of the newly assembled viral particles. NA inhibitory (NAI) antibodies specific to the conserved regions have shown an exceptional breadth, inhibiting divergent influenza viruses (Chen et al., 2018 ). In addition to the broadly protective antibodies, T cell immunity against conserved viral internal proteins also provides a broad protection. Cross-reactive cytotoxic T lymphocytes (CTLs) recognize the viral epitopes presented on MHC molecules and kill the infected cells. It is noteworthy that the cross-reactivity of T cell immunity has been recently shown to cover both IAVs and IBVs, and even the ICVs (Koutsakos et al., 2019 ), although its protective role in vivo has not been confirmed. Figure 4 Protection mode of action afforded by a UIV. Antibodies against the HA globular head domain inhibit viral attachment via HA-mediated receptor binding to the sialic acid on cellular receptors (a). HA stalk antibodies have multiple protective functions. As the virus enters the cell, pre-bound stalk antibodies prevent the fusion of viral and endosomal membranes and block the viral genome release into cytoplasm of the cell (b). Binding of stalk antibodies can also limit the access of cellular proteases to the cleavage site located in the stalk domain and inhibit the cleavage and subsequent conformational change of HA that is an essential step for acquiring viral infectivity (c). Different antibodies against HA stalk and also other viral proteins such as NA, M2, and NP are shown to mediate antibody-dependent effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), leading to the lysis of the virus-infected cells by immune cells or complement system (d–f). NA antibodies inhibit receptor destroying activity of NA and prevent the budding of newly formed viral particles from the cells (g). Cytotoxic T lymphocytes (CTLs) recognize the viral peptide presented on MHC-I molecule and kill the virus infected cell by the secretion of cytotoxic granules and cytokines (h). Definition of Universal Protection Although there is a clear consensus about the urgent need for a vaccine that provides durable and broad protection against multiple strains of influenza virus, the definition of the "universality" of UIV is still debated (Krammer et al., 2018b ). Recently, the National Institute of Allergy and Infectious Diseases (NIAID) held a meeting to identify and develop the criteria to define a UIV. The participants from multiple disciplines agreed that a reasonable UIV should be at least 75% effective against symptomatic influenza virus infection caused by group 1 and group 2 IAVs, with the protection lasting over 1 year for all age groups (Paules et al., 2017 ; Erbelding et al., 2018 ) ( Table 1 ). Similar criteria have also been suggested as preferred product characteristics (PPC) and target product profiles (TPP), describing the desired characteristics of a UIV, by the World Health Organization (WHO) and the Bill and Melinda Gates Foundation (BMGF), respectively ( Table 1 ). Despite the priority given to IAVs in WHO PPC ( Table 1 ), the IBVs represent a "low hanging fruit" because of their low antigenic diversity and the lack of animal reservoir, presenting credence to potential eradication from humans (Tan et al., 2018 ). The proposed consensus to define universal protection offers a valuable guideline to develop an effective and safe UIV. Ideally, the spectrum of protection may covers both IAVs and IBVs, considering the existence of extremely broadly protective antibodies and T cell epitopes across IAVs and IBVs from animals and humans (Corti et al., 2011 ; Koutsakos et al., 2019 ). However, as the antigenic difference between a vaccine and a target virus becomes larger, the number of conserved B cell or T cell epitopes between the two strains decreases. The limited availability of target epitopes may result in compromised protection robustness due to (1) decreased clonal diversity of cross-reactive antibodies and T cells, or (2) occurrence of escape mutants by even small amount of genetic mutations in the epitopes. Therefore, a balanced breadth and robustness of cross-protection should be considered when developing a reliable UIV ( Figure 3 ). Table 1 Consensus criteria for a UIV. NIAID WHO BMGF Consensus Breadth All influenza A viruses (influenza B protection would be the second target) All influenza A viruses All influenza A and B viruses All influenza A viruses Efficacy At least 75% effective against symptomatic influenza infection Better than that of current seasonal influenza vaccine At least 70% effective against symptomatic influenza infection At least 70% effective Target population All age groups >6 weeks, no upper age limit including high risk groups >6 weeks, no upper age limit including high risk groups >6 weeks, no upper age limit Duration of protection At least 1 year At least 5 years 3–5 years At least 1 year NIAID, National Institute of Allergy and Infectious Diseases ( https://www.niaid.nih.gov/diseases-conditions/universal-influenza-vaccine-research ); BMGF, Bill and Melinda Gates Foundation ( https://gcgh.grandchallenges.org/challenges ). WHO, World Health Organization ( https://apps.who.int/iris/bitstream/handle/10665/258767/9789241512466-eng.pdf ; https://jsessionid=37D9E056C3EA58A90EE237432AA7D65F?sequence=1 ) . Figure 3 Protection breadth of influenza vaccines ranging from strain-specific protection to pan-influenza universal protection. Currently licensed seasonal influenza vaccine provides only strain-specific protection against well-matched strains. Recently, many efforts have been put in order to improve the protection breadth of a vaccine from subtype-specific protection to, ideally, pan-influenza universal protection [adopted from Erbelding et al. ( 2018 )]. The NIAID and the researchers have proposed three research areas to address the knowledge gaps in developing a UIV; (1) understanding of influenza transmission, natural history, and viral pathogenesis, (2) characterization of correlates of protection, and (3) rational design of a UIV to improve potency and breadth of protection (Erbelding et al., 2018 ). In particular, the correlates of protection elicited by a UIV may vary considerably in quantity and quality, depending on the vaccine type used and the target influenza virus tested. For instance, while the protection potency of HA stalk-based vaccine may easily be evaluated by measuring the neutralizing activity or indirect effector mechanisms by the stalk-reactive antibodies (Jegaskanda et al., 2017b ), such correlates of protection cannot be used to evaluate the protective efficacy of a T cell epitope-based vaccine containing non-HA epitopes. Moreover, when using HA stalk-based vaccines, high levels of stalk-reactive antibodies represent a good protective efficacy against the same group IAVs ( Figure 1 ). However, the binding affinities of HA stalk antibodies are variable among different viruses, and therefore may require different antibody titers to exhibit sufficient protection against the viruses. This speculation is supported by the observations that broadly reactive HA stalk antibodies show considerably different neutralizing abilities and binding affinities to the viruses within the same HA group (Ekiert et al., 2011 , 2012 ). Furthermore, HA stalk-based vaccine and M2e-based vaccine may demonstrate a different balance of protective abilities to each other, between direct neutralization and indirect effector mechanisms (ADCC, for instance) by the respective antibodies. It has been shown that M2e antibodies provide protection via FcR-dependent effector functions rather than direct virus neutralization (Deng et al., 2015a ), whereas HA stalk antibodies exert both direct neutralization and indirect effector functions (Krammer and Palese, 2015 ). The antibody-mediated inhibition of virus attachment measured by hemagglutinin inhibition assay or microneutralization assay is the gold standard for seasonal influenza vaccines (Ohmit et al., 2011 ). However, the assay cannot be applied simply to reflect the cross-protection by a UIV against heterologous/heterosubtypic influenza viruses. Therefore, it is essential to develop mechanistically distinct in vitro or in vivo assays to measure potential correlates of protection in order to evaluate the protection potency and breadth of the vaccine. Mode of Protection by a UIV The cornerstone of developing a UIV is the determination of the precise protection mechanisms of immune response against influenza viruses. Influenza HA recognizes sialic acid on the cellular receptors and initiates infection by entering the cell via receptor-mediated endocytosis ( Figure 4 ). While HA inhibitory (HAI) antibodies have long been considered as the gold standard for strain-specific protection, very few of them were shown to elicit a broad protection by binding to the conserved receptor-binding site (RBS) of HA, thereby preventing viral entry to the cell (Krause et al., 2011 ; Ekiert et al., 2012 ). Recently, multifunctional protection mechanisms have been described for HA stalk-reactive antibodies. It has been shown that HA stalk antibodies may inhibit membrane fusion, the release of viral genome into the cytoplasm of the cell, and maturation of the HA precursor (Krammer and Palese, 2015 ). Furthermore, HA stalk antibodies can induce antibody-dependent effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytolysis (CDC), resulting in clearance of virus-infected cells by the immune cells or the complement system (Jegaskanda et al., 2017b ). During viral budding, NA cleaves the sialic acid from HA and supports multiple infection cycles by release of the newly assembled viral particles. NA inhibitory (NAI) antibodies specific to the conserved regions have shown an exceptional breadth, inhibiting divergent influenza viruses (Chen et al., 2018 ). In addition to the broadly protective antibodies, T cell immunity against conserved viral internal proteins also provides a broad protection. Cross-reactive cytotoxic T lymphocytes (CTLs) recognize the viral epitopes presented on MHC molecules and kill the infected cells. It is noteworthy that the cross-reactivity of T cell immunity has been recently shown to cover both IAVs and IBVs, and even the ICVs (Koutsakos et al., 2019 ), although its protective role in vivo has not been confirmed. Figure 4 Protection mode of action afforded by a UIV. Antibodies against the HA globular head domain inhibit viral attachment via HA-mediated receptor binding to the sialic acid on cellular receptors (a). HA stalk antibodies have multiple protective functions. As the virus enters the cell, pre-bound stalk antibodies prevent the fusion of viral and endosomal membranes and block the viral genome release into cytoplasm of the cell (b). Binding of stalk antibodies can also limit the access of cellular proteases to the cleavage site located in the stalk domain and inhibit the cleavage and subsequent conformational change of HA that is an essential step for acquiring viral infectivity (c). Different antibodies against HA stalk and also other viral proteins such as NA, M2, and NP are shown to mediate antibody-dependent effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), leading to the lysis of the virus-infected cells by immune cells or complement system (d–f). NA antibodies inhibit receptor destroying activity of NA and prevent the budding of newly formed viral particles from the cells (g). Cytotoxic T lymphocytes (CTLs) recognize the viral peptide presented on MHC-I molecule and kill the virus infected cell by the secretion of cytotoxic granules and cytokines (h). Current Status of M2e-Based UIV Approaches IAVs have two major surface proteins, HA and NA, and one minor surface protein, the M2 ion channel. During the infection cycle, the M2 ion channel is responsible for acidification of the viral interior, facilitating virus uncoating, and unloading of viral ribonucleoproteins (RNPs) into the host cytoplasm (Pinto et al., 1992 ). The extracellular domain of M2 protein (M2e) consists of 24 amino acids, among which 9 N-terminal amino acids are completely conserved among all IAVs (H1–H18) and minor mutations are observed in the distal portion (Deng et al., 2015a ; Tsybalova et al., 2018 ). Therefore, M2e is considered as a promising target for eliciting broadly reactive antibodies. However, due to poor immunogenicity of the small M2e region, UIV approaches targeting M2e required carriers, vectors, and adjuvants to enhance immune responses (Deng et al., 2015a ; Lee et al., 2015 ). One of the most efficient approaches is to make virus-like particles (VLPs) which display M2e on their surface. It has been shown that the hepatitis B virus core (HBc) protein fused with M2e self-assembled into VLPs that resemble wild type virus particles, expressing M2e on the surface (Neirynck et al., 1999 ). Following this work, substantial efforts were made to improve the immunogenicity and protective efficacy of M2e-HBc VLP vaccine constructs. Major strategies include the use of multiple copies of M2e by tandem repeats (Ravin et al., 2015 ; Sun et al., 2015 ; Tsybalova et al., 2015 ) and combination with adjuvants such as cholera toxin A1 (De Filette et al., 2006 ) or B subunit of Escherichia coli heat labile enterotoxin (LTB) (Zhang et al., 2009 ). The M2e-HBc VLP was further harnessed with T cell immunity by combining conserved T cell epitope from NP (Gao et al., 2013 ). The arginine-rich domain of HBc was shown to induce Th1-biased immune response of M2e-HBc VLP by binding to RNA, leading to improved protection (Ibanez et al., 2013 ). Besides the HBc, M2e was fused with various coat or capsid proteins derived from other viruses including Malva mosaic virus (Leclerc et al., 2013 ), tobacco mosaic virus (Petukhova et al., 2013 ), Papaya mosaic virus (Denis et al., 2008 ), T7 bacteriophage (Hashemi et al., 2012 ), and RNA phage Qβ (Bessa et al., 2008 ). Moreover, the enveloped VLPs displayed M2e in a membrane-anchored manner by combining influenza matrix protein and transmembrane (TM) domain of HA fused into M2e. The Sf9 insect cells infected with baculoviral vectors expressing influenza M1 and M2e-TM of influenza HA produced influenza VLPs displaying much higher levels of M2e than the wild type virions (Kim et al., 2015 , 2017 ). Other vaccine types, including DNA vaccines or recombinant protein vaccines expressing M2e with various carriers to enhance immune responses, have also been advanced (Deng et al., 2015a ). Many studies conducted on animals and humans have elucidated the mechanisms of cross-protection conferred by M2e-based vaccines. A common observation is that antibodies specific to M2e cannot directly neutralize the viruses but confer cross-protection by eliciting several mechanisms of antibody-mediated and cell-mediated immune responses. The most well-characterized protection mechanisms include ADCC, ADCP, and CDC ( Figure 4 ). Studies have shown that multiple types of immune cells including the natural killer (NK) cells, neutrophils, dendritic cells, or macrophages have the ability to sense the virus-infected cells by interaction with the Fc receptors (FcR) and the Fc region of M2e antibodies. This results in killing (ADCC) or phagocytosis (ADCP) of the infected cells (Huber et al., 2001 ; Jegerlehner et al., 2004 ; Hashimoto et al., 2007 ; Guilliams et al., 2014 ). The complement is also known to bind to the Fc of M2e antibodies and triggers the complement cascade, leading to formation of the membrane attack complex and target cell lysis (CDC) (Wang et al., 2008 ; Kim et al., 2018b ). These studies together suggest that M2e-specific non-neutralizing antibodies play a crucial role in broad protection against IAVs through the clearance of the virus-infected cells. Besides non-neutralizing effector functions, it has also been shown that M2e-displaying recombinant bacteriophages induce HAI antibodies in a mouse model (Deng et al., 2015b ). In addition, the T cell responses directed to M2e have been shown to contribute to cross-protection. Several studies have shown the presence of CD4 and CD8 T cell epitopes in M2e region in mice and humans (Jameson et al., 1998 ; Gianfrani et al., 2000 ; Mozdzanowska et al., 2003 ; Eliasson et al., 2008 ). In line with this, it has been demonstrated that CD4 T cell and CD8 T cell-mediated immunity are critical for cross-protection elicited by M2e-based UIVs including VLPs or recombinant peptide vaccines (Lee et al., 2014 ; Zhang et al., 2016 ; Arevalo et al., 2017 ; Pendzialek et al., 2017 ; Eliasson et al., 2018 ). It remains a challenge to overcome the low protective efficacy of M2e-based vaccines, due to intrinsic low immunogenicity of the vaccine antigen and the low abundance of M2 proteins on influenza virions and infected cells. From the clinical and practical standpoints, multiple immunizations with a high dose of vaccine antigens combined with potent adjuvants may pose safety concerns, rendering M2e-based vaccine approaches inadequate as a stand-alone UIV. Consequently, M2e antigen was combined with other influenza viral antigens (mostly HA) or supplemented with strain-specific vaccines, resulting in improved potency, and broader cross-protection. For instance, several studies combined M2e with influenza HA head or stalk domain to elicit broadly protective immunity against heterologous influenza infections with various vaccine platforms including nanoparticles, recombinant proteins, and recombinant influenza viruses (Guo et al., 2017 ; Bernasconi et al., 2018 ; Deng et al., 2018 ; Tsybalova et al., 2018 ). Supplemented vaccination with M2e was examined to overcome the strain-specific protection or poor immunogenicity of classical inactivated or split vaccines (Music et al., 2016 ; Song et al., 2016 ). Besides the approaches described above, a vast amount of research on M2e-based UIVs have accumulated in the last decade, which are described in specialized reviews (Deng et al., 2015a ). HA Stalk-Based UIV Approaches General Principles of HA Stalk-Based Approaches The HA stalk-based approach currently leads the mainstream of UIV development. Influenza HA is the primary target antigen of the currently licensed seasonal vaccine, and HAI antibodies serve as the "gold standard" when evaluating the protective efficacy of HA-based vaccines (Ohmit et al., 2011 ). However, HA-based vaccines provide only strain-specific protection to antigenically homologous viruses, necessitating annual update of the surface antigens of seasonal vaccines to match the circulating viral strains. HA comprises two distinct domains; globular head, and stalk domains. The head domain is highly variable and immunologically dominant and thus the majority of HI antibodies are directed to the head domain that harbors the RBS ( Figure 5 ). In contrast, the HA stalk domain is relatively conserved among the viruses, and therefore eliciting antibodies specific to the stalk is considered as a key to developing a UIV. However, the stalk is generally regarded as immunologically subdominant, due to masking effect by bulky head domain and close proximity to the viral membrane (Krammer and Palese, 2013 ; Crowe, 2018 ; Krammer, 2019 ), which necessitates rational design of a HA antigen and novel vaccination strategies to increase stalk-reactive antibodies. The major protective function of HA stalk antibodies is to lock the HA trimer in a prefusion state. This prevents pH-dependent conformational change of HA that triggers the membrane fusion and release of viral genome into cytoplasm of host cell (Ekiert et al., 2009 ). The membrane fusion inhibitory activity of the stalk antibodies may lead to neutralization of influenza viruses, although the neutralizing potency is weaker than head antibodies that directly prevent the receptor binding. Besides the neutralizing activity, stalk antibodies have multiple indirect mechanisms that contribute to broad protection, including antibody-dependent effector mechanisms (Jegaskanda et al., 2017b ) and the inhibition of the NA enzymatic activity (Wohlbold et al., 2016 ; Chen et al., 2019 ). To overcome the low immunogenicity of HA stalk, genetic modifications were required for efficient exposure of stalk to the host immune system. Prime-boost vaccination with chimeric HAs or "headless" HAs were designed to boost stalk-reactive antibodies ( Figure 5 ). Alternatively, hyperglycosylation in HA head resulted in the redirection of immune response from head to stalk (Krammer and Palese, 2015 ) ( Figure 5 ). These approaches have paved the way to increase the breadth of protection against influenza viruses. Figure 5 HA stalk-based approaches. Influenza HA comprises two functionally distinct domains; globular head and stalk. The globular head is variable among the viruses and immunologically dominant and, thus, the majority of neutralizing antibodies are directed to the head that harbors the receptor-binding site (RBS). The stalk is conserved and, therefore, stalk antibodies show broad protection against diverse strains within a group. However, the stalk is immunologically subdominant because of the physical shielding by the bulky head and the close proximity to the viral membrane. To preferably induce the broadly protective stalk-reactive antibodies, two vaccine approaches were designed; chimeric HA and headless HA. Chimeric HA consists of stalk domain derived from important target strains such as H1, H3, or B viruses fused with head domain from irrelevant avian strains such as H5, H6, or H8. Multiple immunizations with different chimeric HAs selectively increase stalk antibodies. Headless HA (stalk-only HA) lacking the head domain can induce only stalk antibodies through multiple vaccinations. Hyperglycosylation in antigenic sites in head domain results in the redirection of antibody responses toward stalk domain. The HA structure was downloaded from the Protein Data Bank (HA of A/swine/Iowa/15/1930, PDB ID 1RUY) and the final image was produced by PyMOL program). Protection Mechanisms of HA Stalk-Based Vaccine A primary function of stalk-reactive antibodies is membrane fusion inhibition. Following the influenza virus entry into the cells via endocytosis, a pH-dependent conformational change of HA proteins triggers the membrane fusion between the virus and endosome, leading to release of the viral genome into the cytoplasm. Binding of the antibody to the stalk interrupts the conformational change of HA and prevents subsequent membrane fusion. This leads to the trapping of the virus in the endosome, eventually aborting the infection (Ekiert et al., 2009 ; Sui et al., 2009 ). Moreover, binding of the stalk antibody blocks the access of proteases to the cleavage site located in stalk region and prevents protease-dependent cleavage of the HA precursor into HA1 and HA2 subunits, which is a prerequisite for the conformational change of HA (Ekiert et al., 2009 ; Brandenburg et al., 2013 ). These two overlapping functions of stalk-reactive antibodies result in inhibition of membrane fusion and direct neutralization of the virus. In addition to these mechanisms that operate at the early stage of infection, the stalk antibody may also bind to the newly expressed HA proteins on the cell surface, and interfere with the viral budding or release, at the later stage of infection (Tan et al., 2014 ). In addition to direct neutralization, stalk antibodies have indirect mechanisms that involve diverse innate immune cells to clear the virus-infected cells from the host. The NK cells sense virus-infected cells via interaction between FcR and Fc portion of antibody bound to HA stalk expressed on the cell surface, and kill the infected cells by releasing cytotoxic granules and antiviral cytokines, via ADCC mechanism (DiLillo et al., 2014 ) ( Figure 4 ). The FcR-Fc interaction is also required for ADCP, by which macrophages or neutrophils recognize and engulf antibody-bound influenza viral particles or infected cells (Ana-Sosa-Batiz et al., 2016 ). In addition, stalk antibodies bound to virus-infected cells are also able to activate the complement system, leading to the lysis of virus-infected cells (CDC) (Terajima et al., 2011 ). Animal challenge models have demonstrated broad protection by passive immunization with HA stalk monoclonal antibodies (DiLillo et al., 2014 , 2016 ). The stalk-reactive antibodies also inhibit NA enzymatic activity through steric hindrance, limiting NA access to the HA-bound sialic acid, thereby preventing viral egress (Wohlbold et al., 2016 ; Chen et al., 2019 ; Kosik et al., 2019 ). The failure to release newly assembled virions from the infected cells prevents the viruses from entering into multiple cycles of infection. Overall, the stalk antibodies confer a broad protection from influenza virus infection through multiple mechanisms affecting both HA and NA throughout the infection cycle of influenza virus. HA Stalk-Based Vaccine Constructs The first reported HA stalk-based UIV construct is the headless HA, a deletion mutant of HA lacking the globular head region (Graves et al., 1983 ; Sagawa et al., 1996 ; Steel et al., 2010 ) ( Figure 5 ). While the initial strategies demonstrated broadly protective potentials in animal models, the removal of the head region often led to misfolding of the stalk immunogen, rendering the critical neutralizing epitopes ineffective. Subsequent studies were conducted to enhance the structural integrity of the stalk immunogens by rational designs to mimic the native trimeric conformation (Lu et al., 2014 ; Mallajosyula et al., 2014 ; Impagliazzo et al., 2015 ). Other studies designed and evaluated nanoparticle structures consisting of the stalk antigens for broad protection in mice and ferret models (Yassine et al., 2015 ; Corbett et al., 2019 ). The second approach to preferably induce stalk-reactive antibodies is based on chimeric HA that consists of stalk domain derived from the major target strains such as H1, H3, or B viruses fused with head domain from irrelevant avian strains such as H5, H6, or H8 (Krammer and Palese, 2015 ) ( Figure 5 ). Multiple immunization was required to increase the stalk antibodies sufficient to provide protection from viruses within the same HA group (Hai et al., 2012 ; Krammer et al., 2013 , 2014 ). The chimeric HA approach involves a full-length functional HA protein and, therefore, it may be expressed in genetically engineered viruses of the wild type or live attenuated influenza viruses (Krammer et al., 2013 ; Isakova-Sivak et al., 2018 ). Third approach relies on hyperglycosylation in the HA head domain ( Figure 5 ). The introduction of additional N-glycosylation sites into the immunodominant head domain shields the major antigenic sites in the head and redirects the host immune responses toward the immune-subdominant stalk. The hyperglycosylated HA antigens induced higher stalk antibodies and provided better protection than the wild type HA in a mouse model (Eggink et al., 2014 ). Similar approaches were tested for avian H5N1 vaccines, in which hyperglycosylated HA antigens were delivered in various vaccine formats such as DNA, recombinant protein, VLP, and adenoviral vector (Lin et al., 2012 , 2014 ). A recent study has shown that glycan shielding of HA head resulted in immune focus on a conserved epitope occluded at the head interface, with Fc-dependent protection activity in mice (Bajic et al., 2019a ). These results together suggest that hyperglycosylation in the HA head is a promising strategy to find novel target epitopes hiding in the head and stalk in HA. Current Issues in HA Stalk-Based UIVs Vaccine Efficacy As discussed above, the protective efficacy of HA-stalk based vaccines is relatively weak, and multiple boost immunizations are required for efficient protection (Krammer et al., 2013 , 2014 ). In addition, the breadth of protection is within the same HA group rather than both the groups (Krammer et al., 2013 ). To complement the low efficacy, prime-boost approaches that entail sequential immunization by LAIV followed by inactivated virus containing chimeric HAs are being evaluated. As examined by a ferret challenge model, this approach provided potent cross protection (Nachbagauer et al., 2017 ). It was observed that there is a "disconnect" between stalk antibody titers in serum and the protection level (Nachbagauer et al., 2017 ). The results show that the broad protection observed in the ferret model was primarily mediated by multiple-level immune responses by LAIV and rather than the stalk antibodies (see section Live attenuated influenza vaccine as an alternative strategy below). This observation in preclinical model leads to the question of the relevance of HA stalk antibodies to cross-protection in humans. Studies with human infection model have led to a somewhat different interpretation on the protective role of the stalk antibodies, depending on the study design. A human challenge study showed that the naturally occurring stalk antibody titers were predictive of lowering viral shedding, but demonstrated poor correlation with the reduction of clinical symptoms upon the pandemic A/H1N1 challenge (Park et al., 2018 ). In contrast, a more recent study suggested the preexisting HA stalk antibodies as an independent correlate of protection against natural infection with the pandemic A/H1N1 virus (Ng et al., 2019 ). Thus, further studies are required for a conclusive demonstration of the breadth and efficacy of cross-protection offered by HA stalk antibodies in human challenge models. Potential Vaccine Safety Issues Although the HA stalk based strategies have shown promising broad protection in animal models, potential safety issues have been raised (Crowe, 2018 ; Khurana, 2018 ). It is suggested that the stalk antibodies are responsible for antibody-dependent enhancement of viral infectivity in a swine model. In a study, pigs vaccinated with inactivated H1N2 vaccine developed more severe respiratory diseases upon heterologous H1N1 challenge as compared to the non-vaccinated control, which was ascribed to infectivity-enhancing effects of stalk antibodies (Khurana et al., 2013 ). Similarly, the HA subunit vaccine resulted in vaccine-associated enhanced respiratory disease (VAERD) in pigs after heterologous challenge (Gauger et al., 2011 ; Rajao et al., 2014 ). This effect cannot be generalized as the enhancement was noticed only in the adjuvanted antigen in a swine model, but not seen in similar studies with non-adjuvanted vaccines in a ferret model (Krammer et al., 2014 ). In addition, it has been recently shown that head-reactive, non-neutralizing monoclonal antibodies increase the stalk flexibility and membrane fusion kinetics, resulting in enhanced respiratory disease in a mouse model (Winarski et al., 2019 ). These results indicate that head-reactive antibodies may also induce antibody-dependent enhancement. However, similar observations in humans are debatable. For instance, humans immunized with 2008–2009 inactivated TIV exhibited an increase in illness following infection with 2009 H1N1 pandemic virus (Janjua et al., 2010 ; Skowronski et al., 2010 ) due to the presence of cross-reactive antibodies (Monsalvo et al., 2011 ). Similar fatal cases were observed during 1957 H2N2 pandemic, although the relation of HA stalk antibodies to these observations is not known. It is noteworthy that NAI antibodies could reduce VAERD caused by mismatched heterologous HA, suggesting that vaccines which target HA protein alone may be prone to VAERD and cross-protective NAI antibodies may counteract VAERD (Rajao et al., 2016 ). Although there is no report on VAERD associated with the headless or chimeric HA vaccines, careful monitoring is advised as these approaches rely heavily on stalk antibodies. The inclusion of NA antigens into HA stalk-based vaccine merits further evaluation. Another issue raised by a recent study is the auto-reactivity of stalk antibodies to human proteins, which is significantly higher than the head antibodies, as confirmed by multiple in vitro assays (Bajic et al., 2019b ). Although biological implications of this observation have not been studied in vivo , it was speculated that vaccine strategies focused exclusively on the stalk, or any single conserved epitope may fail to induce adequate antibody titers due to negative selection of the auto-reactive B cell clones (Bajic et al., 2019b ). However, clinical observations showed that, despite under potential negative selection, preexisting stalk antibodies conferred protection against the 2009 pandemic A/H1N1 infection in humans (Ng et al., 2019 ). Stalk Variability Although conserved among the influenza viruses, the HA stalk is not antigenically identical, showing sequence variability even within the same HA subtype. In line with this, a study demonstrated that H1 HA stalk could undergo mutations in vitro by immune pressure, although the variability was less than the head region (Anderson et al., 2017 ). On the other hand, another study show that while the stalk domain does evolve over time, this evolution is slow and, historically, is not directed to aid in evading neutralizing antibody responses (Kirkpatrick et al., 2018 ). Several studies have isolated mutant influenza viruses showing resistance to stalk antibodies (Ekiert et al., 2011 ; Friesen et al., 2014 ; Chai et al., 2016 ). Notably, all isolated resistant viruses were seen to harbor three different mutations (Gln387Lys, Asp391Tyr, and Asp391Gly) in stalk region. While Gln387Lys mutation completely abolished the binding of the antibody to stalk region, the other two mutations rarely affected the antibody binding but enhanced the fusion ability of HA, representing two independent resistance mechanisms of the virus to escape stalk-reactive antibodies (Chai et al., 2016 ). These reports together show that the stalk may undergo natural or directed antigenic changes, asking important considerations in the context of developing stalk-based UIVs. Especially, the occurrence of mutations that enhance the membrane fusion of HA in presence of stalk antibodies presents a potential concern to vaccine safety issues. It should, however, be noted that the stalk antibody escape mutants tend to lose a viral fitness and are highly attenuated in vivo (Henry Dunand et al., 2015 ; Chai et al., 2016 ), alleviating the safety concerns. Rational Design of Stalk It is well-recognized that the stalk is less immunogenic than head and that the stalk antibodies are less potent in virus neutralization than the head antibodies. Therefore, multiple immunizations (three or four times) with stalk-based vaccines are required for inducing a sufficiently protective level of antibody response. More importantly, most humans have a diverse history of exposure to influenza antigens, by natural infection, or vaccination. The established immune memory influences the subsequent immune response to a UIV, posing a great challenge to rendering qualitatively uniform and protective immune responses in humans. As discussed earlier, the HA stalk-based vaccines elicit broad-spectrum protection within the same HA group, but usually fail to provide protection to strains belonging to different HA group (Krammer et al., 2013 ; Margine et al., 2013 ). These results apparently dissent from the finding that some of the stalk monoclonal antibodies isolated from humans recognize all IAV subtypes, neutralizing both group 1 and group 2 viruses, presenting a promising prospect of developing pan-influenza A therapeutic solution (Corti et al., 2011 ). Isolation of rare antibodies with extremely broad neutralization potency from humans with prior vaccinations or infections (Corti et al., 2011 ) indicates that our immune system is able to find the cryptic and conserved epitopes and generate specific antibodies to the regions. However, inducing such antibodies to a protective level by vaccination remains a big challenge. Some recent reports encourage other options such as the activation of the conserved "cryptic" epitopes via antigen processing mechanisms (Lee et al., 2018b ) or deliberately down-regulating surface antigens (Yang et al., 2013 ). Moreover, Fc-engineering technologies developed to enhance the therapeutic efficacy of antibodies may be harnessed to modulate FcR-Fc mediated effector functions (DiLillo et al., 2014 ) to achieve broad protection. A comprehensive understanding of immune response to broadly neutralizing epitopes and structure-based antigen design is required for the rational design of a pan-influenza A vaccine covering both group 1 and group 2 IAVs, as a consensus criterion of UIV ( Table 1 ). General Principles of HA Stalk-Based Approaches The HA stalk-based approach currently leads the mainstream of UIV development. Influenza HA is the primary target antigen of the currently licensed seasonal vaccine, and HAI antibodies serve as the "gold standard" when evaluating the protective efficacy of HA-based vaccines (Ohmit et al., 2011 ). However, HA-based vaccines provide only strain-specific protection to antigenically homologous viruses, necessitating annual update of the surface antigens of seasonal vaccines to match the circulating viral strains. HA comprises two distinct domains; globular head, and stalk domains. The head domain is highly variable and immunologically dominant and thus the majority of HI antibodies are directed to the head domain that harbors the RBS ( Figure 5 ). In contrast, the HA stalk domain is relatively conserved among the viruses, and therefore eliciting antibodies specific to the stalk is considered as a key to developing a UIV. However, the stalk is generally regarded as immunologically subdominant, due to masking effect by bulky head domain and close proximity to the viral membrane (Krammer and Palese, 2013 ; Crowe, 2018 ; Krammer, 2019 ), which necessitates rational design of a HA antigen and novel vaccination strategies to increase stalk-reactive antibodies. The major protective function of HA stalk antibodies is to lock the HA trimer in a prefusion state. This prevents pH-dependent conformational change of HA that triggers the membrane fusion and release of viral genome into cytoplasm of host cell (Ekiert et al., 2009 ). The membrane fusion inhibitory activity of the stalk antibodies may lead to neutralization of influenza viruses, although the neutralizing potency is weaker than head antibodies that directly prevent the receptor binding. Besides the neutralizing activity, stalk antibodies have multiple indirect mechanisms that contribute to broad protection, including antibody-dependent effector mechanisms (Jegaskanda et al., 2017b ) and the inhibition of the NA enzymatic activity (Wohlbold et al., 2016 ; Chen et al., 2019 ). To overcome the low immunogenicity of HA stalk, genetic modifications were required for efficient exposure of stalk to the host immune system. Prime-boost vaccination with chimeric HAs or "headless" HAs were designed to boost stalk-reactive antibodies ( Figure 5 ). Alternatively, hyperglycosylation in HA head resulted in the redirection of immune response from head to stalk (Krammer and Palese, 2015 ) ( Figure 5 ). These approaches have paved the way to increase the breadth of protection against influenza viruses. Figure 5 HA stalk-based approaches. Influenza HA comprises two functionally distinct domains; globular head and stalk. The globular head is variable among the viruses and immunologically dominant and, thus, the majority of neutralizing antibodies are directed to the head that harbors the receptor-binding site (RBS). The stalk is conserved and, therefore, stalk antibodies show broad protection against diverse strains within a group. However, the stalk is immunologically subdominant because of the physical shielding by the bulky head and the close proximity to the viral membrane. To preferably induce the broadly protective stalk-reactive antibodies, two vaccine approaches were designed; chimeric HA and headless HA. Chimeric HA consists of stalk domain derived from important target strains such as H1, H3, or B viruses fused with head domain from irrelevant avian strains such as H5, H6, or H8. Multiple immunizations with different chimeric HAs selectively increase stalk antibodies. Headless HA (stalk-only HA) lacking the head domain can induce only stalk antibodies through multiple vaccinations. Hyperglycosylation in antigenic sites in head domain results in the redirection of antibody responses toward stalk domain. The HA structure was downloaded from the Protein Data Bank (HA of A/swine/Iowa/15/1930, PDB ID 1RUY) and the final image was produced by PyMOL program). Protection Mechanisms of HA Stalk-Based Vaccine A primary function of stalk-reactive antibodies is membrane fusion inhibition. Following the influenza virus entry into the cells via endocytosis, a pH-dependent conformational change of HA proteins triggers the membrane fusion between the virus and endosome, leading to release of the viral genome into the cytoplasm. Binding of the antibody to the stalk interrupts the conformational change of HA and prevents subsequent membrane fusion. This leads to the trapping of the virus in the endosome, eventually aborting the infection (Ekiert et al., 2009 ; Sui et al., 2009 ). Moreover, binding of the stalk antibody blocks the access of proteases to the cleavage site located in stalk region and prevents protease-dependent cleavage of the HA precursor into HA1 and HA2 subunits, which is a prerequisite for the conformational change of HA (Ekiert et al., 2009 ; Brandenburg et al., 2013 ). These two overlapping functions of stalk-reactive antibodies result in inhibition of membrane fusion and direct neutralization of the virus. In addition to these mechanisms that operate at the early stage of infection, the stalk antibody may also bind to the newly expressed HA proteins on the cell surface, and interfere with the viral budding or release, at the later stage of infection (Tan et al., 2014 ). In addition to direct neutralization, stalk antibodies have indirect mechanisms that involve diverse innate immune cells to clear the virus-infected cells from the host. The NK cells sense virus-infected cells via interaction between FcR and Fc portion of antibody bound to HA stalk expressed on the cell surface, and kill the infected cells by releasing cytotoxic granules and antiviral cytokines, via ADCC mechanism (DiLillo et al., 2014 ) ( Figure 4 ). The FcR-Fc interaction is also required for ADCP, by which macrophages or neutrophils recognize and engulf antibody-bound influenza viral particles or infected cells (Ana-Sosa-Batiz et al., 2016 ). In addition, stalk antibodies bound to virus-infected cells are also able to activate the complement system, leading to the lysis of virus-infected cells (CDC) (Terajima et al., 2011 ). Animal challenge models have demonstrated broad protection by passive immunization with HA stalk monoclonal antibodies (DiLillo et al., 2014 , 2016 ). The stalk-reactive antibodies also inhibit NA enzymatic activity through steric hindrance, limiting NA access to the HA-bound sialic acid, thereby preventing viral egress (Wohlbold et al., 2016 ; Chen et al., 2019 ; Kosik et al., 2019 ). The failure to release newly assembled virions from the infected cells prevents the viruses from entering into multiple cycles of infection. Overall, the stalk antibodies confer a broad protection from influenza virus infection through multiple mechanisms affecting both HA and NA throughout the infection cycle of influenza virus. HA Stalk-Based Vaccine Constructs The first reported HA stalk-based UIV construct is the headless HA, a deletion mutant of HA lacking the globular head region (Graves et al., 1983 ; Sagawa et al., 1996 ; Steel et al., 2010 ) ( Figure 5 ). While the initial strategies demonstrated broadly protective potentials in animal models, the removal of the head region often led to misfolding of the stalk immunogen, rendering the critical neutralizing epitopes ineffective. Subsequent studies were conducted to enhance the structural integrity of the stalk immunogens by rational designs to mimic the native trimeric conformation (Lu et al., 2014 ; Mallajosyula et al., 2014 ; Impagliazzo et al., 2015 ). Other studies designed and evaluated nanoparticle structures consisting of the stalk antigens for broad protection in mice and ferret models (Yassine et al., 2015 ; Corbett et al., 2019 ). The second approach to preferably induce stalk-reactive antibodies is based on chimeric HA that consists of stalk domain derived from the major target strains such as H1, H3, or B viruses fused with head domain from irrelevant avian strains such as H5, H6, or H8 (Krammer and Palese, 2015 ) ( Figure 5 ). Multiple immunization was required to increase the stalk antibodies sufficient to provide protection from viruses within the same HA group (Hai et al., 2012 ; Krammer et al., 2013 , 2014 ). The chimeric HA approach involves a full-length functional HA protein and, therefore, it may be expressed in genetically engineered viruses of the wild type or live attenuated influenza viruses (Krammer et al., 2013 ; Isakova-Sivak et al., 2018 ). Third approach relies on hyperglycosylation in the HA head domain ( Figure 5 ). The introduction of additional N-glycosylation sites into the immunodominant head domain shields the major antigenic sites in the head and redirects the host immune responses toward the immune-subdominant stalk. The hyperglycosylated HA antigens induced higher stalk antibodies and provided better protection than the wild type HA in a mouse model (Eggink et al., 2014 ). Similar approaches were tested for avian H5N1 vaccines, in which hyperglycosylated HA antigens were delivered in various vaccine formats such as DNA, recombinant protein, VLP, and adenoviral vector (Lin et al., 2012 , 2014 ). A recent study has shown that glycan shielding of HA head resulted in immune focus on a conserved epitope occluded at the head interface, with Fc-dependent protection activity in mice (Bajic et al., 2019a ). These results together suggest that hyperglycosylation in the HA head is a promising strategy to find novel target epitopes hiding in the head and stalk in HA. Current Issues in HA Stalk-Based UIVs Vaccine Efficacy As discussed above, the protective efficacy of HA-stalk based vaccines is relatively weak, and multiple boost immunizations are required for efficient protection (Krammer et al., 2013 , 2014 ). In addition, the breadth of protection is within the same HA group rather than both the groups (Krammer et al., 2013 ). To complement the low efficacy, prime-boost approaches that entail sequential immunization by LAIV followed by inactivated virus containing chimeric HAs are being evaluated. As examined by a ferret challenge model, this approach provided potent cross protection (Nachbagauer et al., 2017 ). It was observed that there is a "disconnect" between stalk antibody titers in serum and the protection level (Nachbagauer et al., 2017 ). The results show that the broad protection observed in the ferret model was primarily mediated by multiple-level immune responses by LAIV and rather than the stalk antibodies (see section Live attenuated influenza vaccine as an alternative strategy below). This observation in preclinical model leads to the question of the relevance of HA stalk antibodies to cross-protection in humans. Studies with human infection model have led to a somewhat different interpretation on the protective role of the stalk antibodies, depending on the study design. A human challenge study showed that the naturally occurring stalk antibody titers were predictive of lowering viral shedding, but demonstrated poor correlation with the reduction of clinical symptoms upon the pandemic A/H1N1 challenge (Park et al., 2018 ). In contrast, a more recent study suggested the preexisting HA stalk antibodies as an independent correlate of protection against natural infection with the pandemic A/H1N1 virus (Ng et al., 2019 ). Thus, further studies are required for a conclusive demonstration of the breadth and efficacy of cross-protection offered by HA stalk antibodies in human challenge models. Potential Vaccine Safety Issues Although the HA stalk based strategies have shown promising broad protection in animal models, potential safety issues have been raised (Crowe, 2018 ; Khurana, 2018 ). It is suggested that the stalk antibodies are responsible for antibody-dependent enhancement of viral infectivity in a swine model. In a study, pigs vaccinated with inactivated H1N2 vaccine developed more severe respiratory diseases upon heterologous H1N1 challenge as compared to the non-vaccinated control, which was ascribed to infectivity-enhancing effects of stalk antibodies (Khurana et al., 2013 ). Similarly, the HA subunit vaccine resulted in vaccine-associated enhanced respiratory disease (VAERD) in pigs after heterologous challenge (Gauger et al., 2011 ; Rajao et al., 2014 ). This effect cannot be generalized as the enhancement was noticed only in the adjuvanted antigen in a swine model, but not seen in similar studies with non-adjuvanted vaccines in a ferret model (Krammer et al., 2014 ). In addition, it has been recently shown that head-reactive, non-neutralizing monoclonal antibodies increase the stalk flexibility and membrane fusion kinetics, resulting in enhanced respiratory disease in a mouse model (Winarski et al., 2019 ). These results indicate that head-reactive antibodies may also induce antibody-dependent enhancement. However, similar observations in humans are debatable. For instance, humans immunized with 2008–2009 inactivated TIV exhibited an increase in illness following infection with 2009 H1N1 pandemic virus (Janjua et al., 2010 ; Skowronski et al., 2010 ) due to the presence of cross-reactive antibodies (Monsalvo et al., 2011 ). Similar fatal cases were observed during 1957 H2N2 pandemic, although the relation of HA stalk antibodies to these observations is not known. It is noteworthy that NAI antibodies could reduce VAERD caused by mismatched heterologous HA, suggesting that vaccines which target HA protein alone may be prone to VAERD and cross-protective NAI antibodies may counteract VAERD (Rajao et al., 2016 ). Although there is no report on VAERD associated with the headless or chimeric HA vaccines, careful monitoring is advised as these approaches rely heavily on stalk antibodies. The inclusion of NA antigens into HA stalk-based vaccine merits further evaluation. Another issue raised by a recent study is the auto-reactivity of stalk antibodies to human proteins, which is significantly higher than the head antibodies, as confirmed by multiple in vitro assays (Bajic et al., 2019b ). Although biological implications of this observation have not been studied in vivo , it was speculated that vaccine strategies focused exclusively on the stalk, or any single conserved epitope may fail to induce adequate antibody titers due to negative selection of the auto-reactive B cell clones (Bajic et al., 2019b ). However, clinical observations showed that, despite under potential negative selection, preexisting stalk antibodies conferred protection against the 2009 pandemic A/H1N1 infection in humans (Ng et al., 2019 ). Stalk Variability Although conserved among the influenza viruses, the HA stalk is not antigenically identical, showing sequence variability even within the same HA subtype. In line with this, a study demonstrated that H1 HA stalk could undergo mutations in vitro by immune pressure, although the variability was less than the head region (Anderson et al., 2017 ). On the other hand, another study show that while the stalk domain does evolve over time, this evolution is slow and, historically, is not directed to aid in evading neutralizing antibody responses (Kirkpatrick et al., 2018 ). Several studies have isolated mutant influenza viruses showing resistance to stalk antibodies (Ekiert et al., 2011 ; Friesen et al., 2014 ; Chai et al., 2016 ). Notably, all isolated resistant viruses were seen to harbor three different mutations (Gln387Lys, Asp391Tyr, and Asp391Gly) in stalk region. While Gln387Lys mutation completely abolished the binding of the antibody to stalk region, the other two mutations rarely affected the antibody binding but enhanced the fusion ability of HA, representing two independent resistance mechanisms of the virus to escape stalk-reactive antibodies (Chai et al., 2016 ). These reports together show that the stalk may undergo natural or directed antigenic changes, asking important considerations in the context of developing stalk-based UIVs. Especially, the occurrence of mutations that enhance the membrane fusion of HA in presence of stalk antibodies presents a potential concern to vaccine safety issues. It should, however, be noted that the stalk antibody escape mutants tend to lose a viral fitness and are highly attenuated in vivo (Henry Dunand et al., 2015 ; Chai et al., 2016 ), alleviating the safety concerns. Rational Design of Stalk It is well-recognized that the stalk is less immunogenic than head and that the stalk antibodies are less potent in virus neutralization than the head antibodies. Therefore, multiple immunizations (three or four times) with stalk-based vaccines are required for inducing a sufficiently protective level of antibody response. More importantly, most humans have a diverse history of exposure to influenza antigens, by natural infection, or vaccination. The established immune memory influences the subsequent immune response to a UIV, posing a great challenge to rendering qualitatively uniform and protective immune responses in humans. As discussed earlier, the HA stalk-based vaccines elicit broad-spectrum protection within the same HA group, but usually fail to provide protection to strains belonging to different HA group (Krammer et al., 2013 ; Margine et al., 2013 ). These results apparently dissent from the finding that some of the stalk monoclonal antibodies isolated from humans recognize all IAV subtypes, neutralizing both group 1 and group 2 viruses, presenting a promising prospect of developing pan-influenza A therapeutic solution (Corti et al., 2011 ). Isolation of rare antibodies with extremely broad neutralization potency from humans with prior vaccinations or infections (Corti et al., 2011 ) indicates that our immune system is able to find the cryptic and conserved epitopes and generate specific antibodies to the regions. However, inducing such antibodies to a protective level by vaccination remains a big challenge. Some recent reports encourage other options such as the activation of the conserved "cryptic" epitopes via antigen processing mechanisms (Lee et al., 2018b ) or deliberately down-regulating surface antigens (Yang et al., 2013 ). Moreover, Fc-engineering technologies developed to enhance the therapeutic efficacy of antibodies may be harnessed to modulate FcR-Fc mediated effector functions (DiLillo et al., 2014 ) to achieve broad protection. A comprehensive understanding of immune response to broadly neutralizing epitopes and structure-based antigen design is required for the rational design of a pan-influenza A vaccine covering both group 1 and group 2 IAVs, as a consensus criterion of UIV ( Table 1 ). Vaccine Efficacy As discussed above, the protective efficacy of HA-stalk based vaccines is relatively weak, and multiple boost immunizations are required for efficient protection (Krammer et al., 2013 , 2014 ). In addition, the breadth of protection is within the same HA group rather than both the groups (Krammer et al., 2013 ). To complement the low efficacy, prime-boost approaches that entail sequential immunization by LAIV followed by inactivated virus containing chimeric HAs are being evaluated. As examined by a ferret challenge model, this approach provided potent cross protection (Nachbagauer et al., 2017 ). It was observed that there is a "disconnect" between stalk antibody titers in serum and the protection level (Nachbagauer et al., 2017 ). The results show that the broad protection observed in the ferret model was primarily mediated by multiple-level immune responses by LAIV and rather than the stalk antibodies (see section Live attenuated influenza vaccine as an alternative strategy below). This observation in preclinical model leads to the question of the relevance of HA stalk antibodies to cross-protection in humans. Studies with human infection model have led to a somewhat different interpretation on the protective role of the stalk antibodies, depending on the study design. A human challenge study showed that the naturally occurring stalk antibody titers were predictive of lowering viral shedding, but demonstrated poor correlation with the reduction of clinical symptoms upon the pandemic A/H1N1 challenge (Park et al., 2018 ). In contrast, a more recent study suggested the preexisting HA stalk antibodies as an independent correlate of protection against natural infection with the pandemic A/H1N1 virus (Ng et al., 2019 ). Thus, further studies are required for a conclusive demonstration of the breadth and efficacy of cross-protection offered by HA stalk antibodies in human challenge models. Potential Vaccine Safety Issues Although the HA stalk based strategies have shown promising broad protection in animal models, potential safety issues have been raised (Crowe, 2018 ; Khurana, 2018 ). It is suggested that the stalk antibodies are responsible for antibody-dependent enhancement of viral infectivity in a swine model. In a study, pigs vaccinated with inactivated H1N2 vaccine developed more severe respiratory diseases upon heterologous H1N1 challenge as compared to the non-vaccinated control, which was ascribed to infectivity-enhancing effects of stalk antibodies (Khurana et al., 2013 ). Similarly, the HA subunit vaccine resulted in vaccine-associated enhanced respiratory disease (VAERD) in pigs after heterologous challenge (Gauger et al., 2011 ; Rajao et al., 2014 ). This effect cannot be generalized as the enhancement was noticed only in the adjuvanted antigen in a swine model, but not seen in similar studies with non-adjuvanted vaccines in a ferret model (Krammer et al., 2014 ). In addition, it has been recently shown that head-reactive, non-neutralizing monoclonal antibodies increase the stalk flexibility and membrane fusion kinetics, resulting in enhanced respiratory disease in a mouse model (Winarski et al., 2019 ). These results indicate that head-reactive antibodies may also induce antibody-dependent enhancement. However, similar observations in humans are debatable. For instance, humans immunized with 2008–2009 inactivated TIV exhibited an increase in illness following infection with 2009 H1N1 pandemic virus (Janjua et al., 2010 ; Skowronski et al., 2010 ) due to the presence of cross-reactive antibodies (Monsalvo et al., 2011 ). Similar fatal cases were observed during 1957 H2N2 pandemic, although the relation of HA stalk antibodies to these observations is not known. It is noteworthy that NAI antibodies could reduce VAERD caused by mismatched heterologous HA, suggesting that vaccines which target HA protein alone may be prone to VAERD and cross-protective NAI antibodies may counteract VAERD (Rajao et al., 2016 ). Although there is no report on VAERD associated with the headless or chimeric HA vaccines, careful monitoring is advised as these approaches rely heavily on stalk antibodies. The inclusion of NA antigens into HA stalk-based vaccine merits further evaluation. Another issue raised by a recent study is the auto-reactivity of stalk antibodies to human proteins, which is significantly higher than the head antibodies, as confirmed by multiple in vitro assays (Bajic et al., 2019b ). Although biological implications of this observation have not been studied in vivo , it was speculated that vaccine strategies focused exclusively on the stalk, or any single conserved epitope may fail to induce adequate antibody titers due to negative selection of the auto-reactive B cell clones (Bajic et al., 2019b ). However, clinical observations showed that, despite under potential negative selection, preexisting stalk antibodies conferred protection against the 2009 pandemic A/H1N1 infection in humans (Ng et al., 2019 ). Stalk Variability Although conserved among the influenza viruses, the HA stalk is not antigenically identical, showing sequence variability even within the same HA subtype. In line with this, a study demonstrated that H1 HA stalk could undergo mutations in vitro by immune pressure, although the variability was less than the head region (Anderson et al., 2017 ). On the other hand, another study show that while the stalk domain does evolve over time, this evolution is slow and, historically, is not directed to aid in evading neutralizing antibody responses (Kirkpatrick et al., 2018 ). Several studies have isolated mutant influenza viruses showing resistance to stalk antibodies (Ekiert et al., 2011 ; Friesen et al., 2014 ; Chai et al., 2016 ). Notably, all isolated resistant viruses were seen to harbor three different mutations (Gln387Lys, Asp391Tyr, and Asp391Gly) in stalk region. While Gln387Lys mutation completely abolished the binding of the antibody to stalk region, the other two mutations rarely affected the antibody binding but enhanced the fusion ability of HA, representing two independent resistance mechanisms of the virus to escape stalk-reactive antibodies (Chai et al., 2016 ). These reports together show that the stalk may undergo natural or directed antigenic changes, asking important considerations in the context of developing stalk-based UIVs. Especially, the occurrence of mutations that enhance the membrane fusion of HA in presence of stalk antibodies presents a potential concern to vaccine safety issues. It should, however, be noted that the stalk antibody escape mutants tend to lose a viral fitness and are highly attenuated in vivo (Henry Dunand et al., 2015 ; Chai et al., 2016 ), alleviating the safety concerns. Rational Design of Stalk It is well-recognized that the stalk is less immunogenic than head and that the stalk antibodies are less potent in virus neutralization than the head antibodies. Therefore, multiple immunizations (three or four times) with stalk-based vaccines are required for inducing a sufficiently protective level of antibody response. More importantly, most humans have a diverse history of exposure to influenza antigens, by natural infection, or vaccination. The established immune memory influences the subsequent immune response to a UIV, posing a great challenge to rendering qualitatively uniform and protective immune responses in humans. As discussed earlier, the HA stalk-based vaccines elicit broad-spectrum protection within the same HA group, but usually fail to provide protection to strains belonging to different HA group (Krammer et al., 2013 ; Margine et al., 2013 ). These results apparently dissent from the finding that some of the stalk monoclonal antibodies isolated from humans recognize all IAV subtypes, neutralizing both group 1 and group 2 viruses, presenting a promising prospect of developing pan-influenza A therapeutic solution (Corti et al., 2011 ). Isolation of rare antibodies with extremely broad neutralization potency from humans with prior vaccinations or infections (Corti et al., 2011 ) indicates that our immune system is able to find the cryptic and conserved epitopes and generate specific antibodies to the regions. However, inducing such antibodies to a protective level by vaccination remains a big challenge. Some recent reports encourage other options such as the activation of the conserved "cryptic" epitopes via antigen processing mechanisms (Lee et al., 2018b ) or deliberately down-regulating surface antigens (Yang et al., 2013 ). Moreover, Fc-engineering technologies developed to enhance the therapeutic efficacy of antibodies may be harnessed to modulate FcR-Fc mediated effector functions (DiLillo et al., 2014 ) to achieve broad protection. A comprehensive understanding of immune response to broadly neutralizing epitopes and structure-based antigen design is required for the rational design of a pan-influenza A vaccine covering both group 1 and group 2 IAVs, as a consensus criterion of UIV ( Table 1 ). Conserved Targets in HA Other Than Stalk The inability to induce a complete protection by HA stalk-based approach led to a search for alternative targets for UIVs. Human monoclonal antibody CH65, mimicking the interaction with sialic acid, was shown to bind to the RBS in HA and neutralize multiple H1N1 influenza strains (Whittle et al., 2011 ). A caveat is that this antibody demonstrated stringent structural requirements for neutralization and mutation at the binding region led to generation of escape mutants. A panel of head-reactive monoclonal antibodies was also isolated and shown to recognize conserved region in the RBS and neutralize multiple influenza viruses (Krause et al., 2011 , 2012 ; Ekiert et al., 2012 ; Benjamin et al., 2014 ). The HA head domain was also reported to contain conserved epitopes outside the RBS. Different broadly neutralizing antibodies recognized the conserved regions located in HA head domain and neutralized multiple influenza viruses without detectable HI activity (Iba et al., 2014 ; Raymond et al., 2018 ). Generally, neutralization breadth of head antibodies was considerably variable to each other, ranging from pan-subtype (covering the same subtype) to pan-type (covering both group 1 and 2) coverage, indicating the presence of variably conserved regions. Additionally, a novel class of cross-reactive antibodies was discovered in humans vaccinated with seasonal TIV (Lee et al., 2016 ). These antibodies were shown to bind to a highly conserved region located on the HA RBS that was occluded in the HA trimer, conferring protective immunity against H1N1 and H3N2 strains in vivo , without neutralizing activity in vitro . Furthermore, a novel class of antibodies targeting vestigial esterase (VE) domain in HA has been characterized. The VE domain consists of two non-continuous sequences in HA head domain, which together forms a structurally distinct subdomain from the RBS and HA stalk domain (Zheng et al., 2018 ). The VE domain of the HEF protein of ICV is responsible for cleaving the host receptor to facilitate viral release, whereas in IAVs and IBVs the same receptor cleaving function is provided by a separate NA. The VE domains are found in both IAVs and IBVs, although their functions are not well-defined. The VE domains are highly conserved within a subtype of IAV but are variable among different subtypes (Ha et al., 2002 ). Monoclonal antibodies to the VE domain of H5N1 virus demonstrated broad neutralization against multiple clades of H5N1 subtype by preventing viral entry into cells (Oh et al., 2010 ). To date, different monoclonal antibodies have been isolated and they bind to different epitopes in the VE domain of H5N1 viruses, suggesting the presence of multiple neutralization epitopes in the VE domain (Paul et al., 2017 ). The VE-binding antibodies are reported to mediate ADCC for in vivo protection via FcR-Fc interaction (Wang et al., 2017 ). Besides H5N1, several monoclonal antibodies neutralizing H3 or H7 of IAVs or IBVs have also been recently characterized (Tan et al., 2016 ; Chai et al., 2017 ; Bangaru et al., 2018 ). Taken together, possibility remains to identify conserved neutralizing epitopes in the head domain in HA, in addition to extensively characterized HA stalk. Activation of these epitopes via antigen processing machineries (Lee et al., 2018b ) may offer an option for enhancing the potency of cross-protection. Despite the presence of conserved epitopes, the head domain is not a feasible vaccine antigen because of the immunodominance of the surrounding variable regions in the head that compete and prevent effective induction of antibodies toward the conserved regions. Therefore, several strategies were designed to enhance the breadth of protection by HA-based vaccines. The centralized HA was reconstructed such that whole HA contained consensus amino acids derived from diverse strains within a subtype (Weaver et al., 2011 ). This engineered HA antigen induced stronger immune response and provided better protection against heterologous influenza viruses, as compared to natural wild type HA antigen. Another approach, conceptually similar to the centralized HA, is based on a computationally optimized broadly reactive antigen (COBRA), in which the HA was designed to carry consensus sequences. The COBRA strategy was tested against H1N1, H3N2, and H5N1 influenza viruses, and demonstrated broad protection within a subtype (Giles and Ross, 2011 ; Crevar et al., 2015 ; Carter et al., 2016 ). A third strategy is the use of ancestral sequences as vaccine antigens to widen the window of cross-protection against diversified lineages or clades. Through phylogenetic tree analysis, putative ancestral HA and NA sequences have been determined and used as vaccine antigens, showing broadened cross-protection against multiple clades of H5N1 viruses in animal models (Ducatez et al., 2011 , 2013 ). Collectively, the UIV candidates using HA head or full-length antigen are based on reconstructed HA containing consensus sequences. Although the protection breadth of those strategies appears to be restricted to a subtype or a specific clade, similar concepts may be applied to other antigens such as HA stalk or NA to substantially improve the protection breadth. NA as a Novel Target For a UIV Multiple Function of NA in Infection Cycle NA is a tetrameric type II transmembrane glycoprotein and the second major surface protein of influenza viruses. The role of NA in influenza infection cycle is classically known as an expedited release of virus particle from infected cells by cleaving off the sialic acid residue present in host cell membrane, thus enabling multiple rounds of infection by the newly generated viral progeny. In addition to the canonical role that operates at later stage of infection, other functions of NA relevant to the infection cycle are being recognized. For instance, the sialidase activity of NA is critical for viral entry into a host cell at early stage of infection. At the entry site in the mucus, influenza virus meets mucosal defense proteins such as mucins that are highly glycosylated and capture viral HA. NA is able to release the captured viral particles via sialidase activity, allowing them to reach the host cells successfully (Cohen et al., 2013 ; Yang et al., 2014 ). Furthermore, with the same sialidase activity, NA facilitates HA-dependent membrane fusion and enhances the viral infectivity by removing the sialic acid residues from the virion-expressed HAs (Su et al., 2010 ). Additionally, NA cooperates with HA to enable crawling and gliding motions of influenza virus on cell surface to enhance viral entry into a cell (Sakai et al., 2017 ). More interestingly, some of the H3N2 viruses use their NA for receptor binding instead of HA, suggesting a novel role of NA other than receptor-destroying activity (Lin et al., 2010 ; Mogling et al., 2017 ). These observations show that NA performs multiple functions in the entire infection cycle, suggesting that NA antibodies may represent an important means of protection against influenza viruses. NA Antibodies as Important Correlate of Protection The 1968 H3N2 pandemic gave us important lessons pertaining to NA-mediated protection. The antigenic drift of NA is independent of HA; the pandemic involved a shift in HA, but NA remained close to the previous influenza A/H2N2 viruses (Schulman and Kilbourne, 1969 ). Notably, it has been shown that individuals with higher N2 antibody titers are less likely to be infected with the H3N2 pandemic, depicting the contribution of NA antibodies to broad protection (Schulman, 1969 ; Murphy et al., 1972 ; Monto and Kendal, 1973 ). However, NA has been largely ignored in the formulation of influenza vaccines due to the general beliefs that NA antibodies do not inhibit viral entry and that NA is less abundant than HA on a virion. Furthermore, the lack of a convenient assay to measure functional NA antibodies has rendered the NA forgotten antigens in influenza vaccines for decades (Eichelberger and Monto, 2019 ). Most of the current vaccine approaches focus on the induction of HA antibodies, both in trivalent/quadrivalent seasonal influenza vaccines and in the recent UIV candidates. However, it has been increasingly acknowledged that NA antibodies are important and independent correlates of protection and that NA immunity should be considered when evaluating vaccine potency. Clinical studies have shown a correlation between vaccination-induced or preexisting NAI antibody levels and decreased frequency of influenza infection and illness (Couch et al., 2013 ; Monto et al., 2015 ; Park et al., 2018 ). Further, a human challenge model depicted that NAI titers correlated more significantly with protection and disease severity than HAI titers (Memoli et al., 2016 ), or even HA stalk antibodies (Park et al., 2018 ). The observations in the human challenge models suggest that NA should be given full consideration as a vaccine antigen for better protection. Several animal studies have identified NAI monoclonal antibodies that show protective effects against heterologous influenza infection. The breadth of NAI antibodies varied from subtype-specific to pan-influenza, depending on the conserved epitopes (Doyle et al., 2013a , b ; Wan et al., 2013 ). Recently, it was reported that influenza infection in humans induces a variety of broadly reactive antibodies directed to the NA (Chen et al., 2018 ). In this study, it was shown that among the total influenza-specific antibodies induced by infection, the NA-reactive antibodies accounted for 23% and HA-reactive antibodies 35%. By contrast, the subunit or split vaccine resulted in antibody response directed predominantly to HA (87%), with only 1% for NA. The poor ability of the seasonal vaccine to induce NA antibodies was apparently due to insufficient content or structural integrity of NA antigen used in current vaccine formulation. This research suggests that correctly folded and immunologically relevant NA antigen is capable of inducing broadly protective antibody responses. NA-Based Vaccine as Low-Hanging Fruit for a UIV? Although the importance of NA-immunity in protection against homologous and heterologous influenza infections is clearly established, only a few literatures have demonstrated the cross-protection of NA-only vaccine constructs. One recent study in a mouse model has reported that computationally engineered recombinant NA proteins containing consensus sequences show broad protection within the H1N1 subtype (Job et al., 2018 ). Some other studies reported that VLPs expressing NA provided cross-protection against heterologous challenge in mice and ferrets (Easterbrook et al., 2012 ; Walz et al., 2018 ; Kim et al., 2019 ), and recombinant NA in a mouse model (Liu et al., 2015 ; Wohlbold et al., 2015 ). However, co-administration of H7 HA and N3 NA in modified vaccinia virus Ankara (MVA) vectors did not demonstrate enhanced protection efficacy as compared to the efficacy of MVA-HA or MVA-NA vaccine alone (Meseda et al., 2018 ). A predominant immune response in favor of HA over NA, when presented in an influenza virion, is already well-documented (Johansson et al., 1987 ), and the dissociation of HA and NA eliminates this antigenic competition (Johansson and Kilbourne, 1993 , 1996 ). These observations together suggest that NA-specific immunity may be dwarfed by competition with highly immunogenic HA in the final vaccine formulation. It could be argued that if the controlled ratio of HA and NA (dwarfing NA) is the strategy adopted by successful virus infection to minimize the host immune surveillance, then a deliberate perturbation of their ratio (increasing NA) in the vaccine formulation may be a promising strategy for effective protection. It was shown that the ratio of HA/NA could vary widely (up to 50 fold) without affecting viral fitness by a single mutation in the viral promoter (Lee and Seong, 1998 ). It remains to be seen if such a reverse-genetic approach could be harnessed to enhance the potency of NA-based vaccines. Currently, we have very limited knowledge about anti-NA immunity. To develop a broadly protective vaccine based on NA, there are several critical questions that need to be answered. Firstly, although the NAI antibodies have been determined as an independent correlate of protection in humans (Couch et al., 2013 ; Monto et al., 2015 ), this needs to be further validated by the NA-only vaccine constructs in animal and human models. Secondly, very little is known about the breadth of NA immunity. The literature discussed earlier has consistently demonstrated a subtype-specific protection (e.g., within N1 or N2) of NA-based vaccines in animal models. Considering the repertoire of influenza viruses infecting humans and animals (including N1, N2, N3, N7, and N9 encompassing both NA group 1 and 2) ( Figure 1 ), a successful NA-based vaccine should be designed to elicit broad protection covering both NA groups. Hence, the determination of conserved regions or epitopes hidden in NA is urgently needed. Thirdly, the mechanisms by which NA antibodies contribute to protection are not completely understood. Many NAI antibodies inhibit its enzymatic activity and thus prevent the release of newly formed viral particles. However, the extent to which NAI antibody titers may be considered protective has not been determined yet. Evaluation of cross-protection against mismatched or heterologous strains may be even more complicated. While ADCC was shown to be involved in protection by non-neutralizing NA antibodies (Jegaskanda et al., 2014 , 2017a ; Wohlbold et al., 2017 ), other protective mechanisms are yet to be further elucidated. Further isolation and characterization of broadly protective NA antibodies is required for better design of NA-based vaccines. Comprehensive reviews on NA-based immunity and the perspectives on current knowledge gaps to be addressed may be found in specialized reviews (Wohlbold and Krammer, 2014 ; Krammer et al., 2018a ). Multiple Function of NA in Infection Cycle NA is a tetrameric type II transmembrane glycoprotein and the second major surface protein of influenza viruses. The role of NA in influenza infection cycle is classically known as an expedited release of virus particle from infected cells by cleaving off the sialic acid residue present in host cell membrane, thus enabling multiple rounds of infection by the newly generated viral progeny. In addition to the canonical role that operates at later stage of infection, other functions of NA relevant to the infection cycle are being recognized. For instance, the sialidase activity of NA is critical for viral entry into a host cell at early stage of infection. At the entry site in the mucus, influenza virus meets mucosal defense proteins such as mucins that are highly glycosylated and capture viral HA. NA is able to release the captured viral particles via sialidase activity, allowing them to reach the host cells successfully (Cohen et al., 2013 ; Yang et al., 2014 ). Furthermore, with the same sialidase activity, NA facilitates HA-dependent membrane fusion and enhances the viral infectivity by removing the sialic acid residues from the virion-expressed HAs (Su et al., 2010 ). Additionally, NA cooperates with HA to enable crawling and gliding motions of influenza virus on cell surface to enhance viral entry into a cell (Sakai et al., 2017 ). More interestingly, some of the H3N2 viruses use their NA for receptor binding instead of HA, suggesting a novel role of NA other than receptor-destroying activity (Lin et al., 2010 ; Mogling et al., 2017 ). These observations show that NA performs multiple functions in the entire infection cycle, suggesting that NA antibodies may represent an important means of protection against influenza viruses. NA Antibodies as Important Correlate of Protection The 1968 H3N2 pandemic gave us important lessons pertaining to NA-mediated protection. The antigenic drift of NA is independent of HA; the pandemic involved a shift in HA, but NA remained close to the previous influenza A/H2N2 viruses (Schulman and Kilbourne, 1969 ). Notably, it has been shown that individuals with higher N2 antibody titers are less likely to be infected with the H3N2 pandemic, depicting the contribution of NA antibodies to broad protection (Schulman, 1969 ; Murphy et al., 1972 ; Monto and Kendal, 1973 ). However, NA has been largely ignored in the formulation of influenza vaccines due to the general beliefs that NA antibodies do not inhibit viral entry and that NA is less abundant than HA on a virion. Furthermore, the lack of a convenient assay to measure functional NA antibodies has rendered the NA forgotten antigens in influenza vaccines for decades (Eichelberger and Monto, 2019 ). Most of the current vaccine approaches focus on the induction of HA antibodies, both in trivalent/quadrivalent seasonal influenza vaccines and in the recent UIV candidates. However, it has been increasingly acknowledged that NA antibodies are important and independent correlates of protection and that NA immunity should be considered when evaluating vaccine potency. Clinical studies have shown a correlation between vaccination-induced or preexisting NAI antibody levels and decreased frequency of influenza infection and illness (Couch et al., 2013 ; Monto et al., 2015 ; Park et al., 2018 ). Further, a human challenge model depicted that NAI titers correlated more significantly with protection and disease severity than HAI titers (Memoli et al., 2016 ), or even HA stalk antibodies (Park et al., 2018 ). The observations in the human challenge models suggest that NA should be given full consideration as a vaccine antigen for better protection. Several animal studies have identified NAI monoclonal antibodies that show protective effects against heterologous influenza infection. The breadth of NAI antibodies varied from subtype-specific to pan-influenza, depending on the conserved epitopes (Doyle et al., 2013a , b ; Wan et al., 2013 ). Recently, it was reported that influenza infection in humans induces a variety of broadly reactive antibodies directed to the NA (Chen et al., 2018 ). In this study, it was shown that among the total influenza-specific antibodies induced by infection, the NA-reactive antibodies accounted for 23% and HA-reactive antibodies 35%. By contrast, the subunit or split vaccine resulted in antibody response directed predominantly to HA (87%), with only 1% for NA. The poor ability of the seasonal vaccine to induce NA antibodies was apparently due to insufficient content or structural integrity of NA antigen used in current vaccine formulation. This research suggests that correctly folded and immunologically relevant NA antigen is capable of inducing broadly protective antibody responses. NA-Based Vaccine as Low-Hanging Fruit for a UIV? Although the importance of NA-immunity in protection against homologous and heterologous influenza infections is clearly established, only a few literatures have demonstrated the cross-protection of NA-only vaccine constructs. One recent study in a mouse model has reported that computationally engineered recombinant NA proteins containing consensus sequences show broad protection within the H1N1 subtype (Job et al., 2018 ). Some other studies reported that VLPs expressing NA provided cross-protection against heterologous challenge in mice and ferrets (Easterbrook et al., 2012 ; Walz et al., 2018 ; Kim et al., 2019 ), and recombinant NA in a mouse model (Liu et al., 2015 ; Wohlbold et al., 2015 ). However, co-administration of H7 HA and N3 NA in modified vaccinia virus Ankara (MVA) vectors did not demonstrate enhanced protection efficacy as compared to the efficacy of MVA-HA or MVA-NA vaccine alone (Meseda et al., 2018 ). A predominant immune response in favor of HA over NA, when presented in an influenza virion, is already well-documented (Johansson et al., 1987 ), and the dissociation of HA and NA eliminates this antigenic competition (Johansson and Kilbourne, 1993 , 1996 ). These observations together suggest that NA-specific immunity may be dwarfed by competition with highly immunogenic HA in the final vaccine formulation. It could be argued that if the controlled ratio of HA and NA (dwarfing NA) is the strategy adopted by successful virus infection to minimize the host immune surveillance, then a deliberate perturbation of their ratio (increasing NA) in the vaccine formulation may be a promising strategy for effective protection. It was shown that the ratio of HA/NA could vary widely (up to 50 fold) without affecting viral fitness by a single mutation in the viral promoter (Lee and Seong, 1998 ). It remains to be seen if such a reverse-genetic approach could be harnessed to enhance the potency of NA-based vaccines. Currently, we have very limited knowledge about anti-NA immunity. To develop a broadly protective vaccine based on NA, there are several critical questions that need to be answered. Firstly, although the NAI antibodies have been determined as an independent correlate of protection in humans (Couch et al., 2013 ; Monto et al., 2015 ), this needs to be further validated by the NA-only vaccine constructs in animal and human models. Secondly, very little is known about the breadth of NA immunity. The literature discussed earlier has consistently demonstrated a subtype-specific protection (e.g., within N1 or N2) of NA-based vaccines in animal models. Considering the repertoire of influenza viruses infecting humans and animals (including N1, N2, N3, N7, and N9 encompassing both NA group 1 and 2) ( Figure 1 ), a successful NA-based vaccine should be designed to elicit broad protection covering both NA groups. Hence, the determination of conserved regions or epitopes hidden in NA is urgently needed. Thirdly, the mechanisms by which NA antibodies contribute to protection are not completely understood. Many NAI antibodies inhibit its enzymatic activity and thus prevent the release of newly formed viral particles. However, the extent to which NAI antibody titers may be considered protective has not been determined yet. Evaluation of cross-protection against mismatched or heterologous strains may be even more complicated. While ADCC was shown to be involved in protection by non-neutralizing NA antibodies (Jegaskanda et al., 2014 , 2017a ; Wohlbold et al., 2017 ), other protective mechanisms are yet to be further elucidated. Further isolation and characterization of broadly protective NA antibodies is required for better design of NA-based vaccines. Comprehensive reviews on NA-based immunity and the perspectives on current knowledge gaps to be addressed may be found in specialized reviews (Wohlbold and Krammer, 2014 ; Krammer et al., 2018a ). UIV Against Influenza B Viruses Besides IAVs, ~25% of all human influenza virus infections in each epidemic season is caused by IBVs that are classified into two distinct lineages, Victoria-like and Yamagata-like lineages (Ambrose and Levin, 2012 ). The current seasonal influenza vaccine is prepared in a trivalent or quadrivalent formulation, depending on the inclusion of one or two lineages of IBV antigens. Although priority is given to IAVs owing to a greater impact, IBVs may be more vulnerable targets against which to develop a UIV ( Table 1 ) because of their low antigenic diversity and lack of animal reservoir ( Figure 1 ) (Tan et al., 2018 ). Indirect evidence is being accumulated by the isolation of cross-protective antibodies against IBVs. Several broadly protective antibodies binding to head or stalk domain of influenza B HA have been isolated in humans. Overall, these monoclonal antibodies show lineage-specific neutralizing activity in vitro . Further, in vivo protection against both lineages was also demonstrated in mice by passive transfer, through non-neutralizing antibody-dependent effector functions (Shen et al., 2017 ; Hirano et al., 2018 ; Vigil et al., 2018 ; Asthagiri Arunkumar et al., 2019 ; Liu et al., 2019b ). Notably, some of B HA stalk antibodies demonstrated extremely broad binding ability (Hirano et al., 2018 ) or protection against both IAVs and IBVs (Dreyfus et al., 2012 ). Influenza B NA-reactive broadly neutralizing antibodies were also isolated in animals and humans. Seasonal QIV induced NA antibodies with broad and potent antiviral activity against both lineages in humans (Piepenbrink et al., 2019 ). Additionally, murine NA antibodies also showed broad protection against both lineages of IBV (Wohlbold et al., 2017 ). In line with these observations, chimeric HA strategy has also been tested for a UIV against IBVs. Chimeric HAs consisting of HA head domain from IAV and stalk domain from IBV, delivered as a DNA vaccine (prime), followed by two boosting immunizations with protein vaccines into mice, provided broad protection against both the lineages as well as an ancestral strain of IBV (Ermler et al., 2017 ). Mosaic HA is an advanced version, in which major antigenic sites of type B HA head domain were replaced by those of type A HA head so that antibodies directed to conserved regions both in the head and stalk domains of type B HA could be induced (Sun et al., 2019 ). The mosaic B HA provided broad protection against both lineages of IBV, through non-neutralizing ADCC-mediating antibody responses. There are only a few studies reporting B NA-based vaccine offering cross-lineage protection. A study showed that mice immunized with recombinant B NA protein of B/Yamagata/16/88 were protected from homologous Yamagata-like and Victoria-like lineages (Wohlbold et al., 2015 ). Another study demonstrated that a B NA-based vaccine inhibited the transmission of both homologous and heterologous IBVs in Guinea pig model (McMahon et al., 2019 ). As compared to IAVs, very little effort has been made so far to develop a UIV against IBVs. However, considering the limited diversity and variability ( Figure 1 ) compared to IAVs, further identifications of broadly protective B cell and T cell epitopes would make it possible to develop a pan-influenza B vaccine in the near future (Tan et al., 2018 ). T cell Immunity as an Essential Factor for Truly Universal Influenza Protection A vast majority of current efforts to develop a UIV are focused on inducing antibody response toward surface glycoproteins, M2e, HA, and NA. However, T cell immunity has been acknowledged as a potential immune correlate of broad protection against influenza infections (Sridhar, 2016 ; Clemens et al., 2018 ). T cell immunity may not provide sterilizing or neutralizing immunity against influenza viruses but improves the standard of care by reducing the disease severity and duration of infection, facilitating recovery from illness (Sridhar, 2016 ). It, therefore, seems that multiple immune arms including both antibodies and T cell immunity are critical to provide a truly universal protection against highly variable influenza viruses. The influenza-specific T cell immunity is known to be highly cross-reactive by recognition of conserved peptides between different subtypes of IAV (Assarsson et al., 2008 ; Kreijtz et al., 2008 ; Lee et al., 2008 ; van de Sandt et al., 2014 ). Extensive studies have proven the protective role of vaccination or infection-induced cross-reactive CD8+ T cells in various animal models (Kreijtz et al., 2007 , 2009 ; Bodewes et al., 2011 ; Hillaire et al., 2011 ). Additionally, in humans, CD8+ T cells offered cross-protection across seasonal, pandemic, avian IAVs, and both lineages of IBVs (Gras et al., 2010 ; Hayward et al., 2015 ; van de Sandt et al., 2015 ; Wang et al., 2018b ). The majority of cross-reactive T cell epitopes of IAVs are derived from internal proteins; among >70 T cell epitopes identified between H5N1 and H3N2 viruses, only one derived from HA and other from internal proteins (Lee et al., 2008 ). This is not surprising given that the conservation rate of internal proteins is >90%, whereas that of surface HA and NA is only 40–70% among IAVs (Lee et al., 2008 ), which shows that inducing T cell immunity directed to internal proteins of influenza virus may provide a basis of developing T cell-based UIVs. Despite poor sequence homology between the HAs of IAVs and IBVs, HA stalk harbors not only extremely broad B cell epitopes but also T cell epitopes encompassing both types of influenza viruses. The fusion peptide in HA stalk contains a cross-reactive CD4+ T cell epitope conserved in both IAVs and IBVs, although its protective role has not been examined in vivo (Babon et al., 2012 ). A number of CD4+ and CD8+ T cell epitopes are highly conserved in internal proteins (Terajima et al., 2013 ). A recent study has discovered a universal human CD8+ T cell epitope in PB1 (NMLSTVLGV PB1 413−412 ) that is identical across IAVs, IBVs, and ICVs (Koutsakos et al., 2019 ). The preexisting memory PB1 413−412 +CD8+ T cells were detected in the blood and lung tissues of healthy donors and clonally expanded upon infection with IAV or IBV. This report not only showed the existence of heterotypic memory CD8+ T cells in humans that could be induced by exposure to influenza viruses, but also presents the prospect of designing a T cell-based UIV. However, these cross-reactive T cells were not induced in HHD-A2 mouse model despite multiple influenza infections or vaccinations and the protective role of the T cells could not be assessed in the study. Nonetheless, the existence of a number of cross-reactive T cell epitopes between IAVs and IBVs provides an avenue to address to a UIV. Several T cell-based vaccine candidates are in different stages of clinical development, the major underlying strategy of which is to deliver multiple T cell epitopes derived from different viral antigens including internal as well as surface antigens (Sridhar, 2016 ). Delivery platforms include replicating or non-replicating viral vectors derived from vaccinia or adenovirus, recombinant VLPs, recombinant protein or peptide vaccines, and DNA vaccines (Sridhar, 2016 ; Clemens et al., 2018 ). Modified vaccinia Ankara (MVA) vector encoding NP and M1 was shown to induce robust T cell responses and provide cross-protection against multiple subtypes in animals and humans (Antrobus et al., 2012 ; Powell et al., 2013 ; Hessel et al., 2014 ; Folegatti et al., 2019 ). The baculovirus VLPs carrying influenza HA/NA and M1 offered cross-protection where T cells played a significant role in protection in mice (Hemann et al., 2013 ; Keshavarz et al., 2019 ). Synthetic peptides or fusion proteins harboring multiple conserved T cell epitopes have also been evaluated for immunogenicity and protective efficacy in animal models (Adar et al., 2009 ; Rosendahl Huber et al., 2015 ). While the vaccine approaches described above deliver exogenous antigens and induce CD4+ T cells as well as CD8+ T cells by cross-presentation, DNA vaccines are designed to predominantly activate cytotoxic CD8+ T cells to recognize endogenously expressed antigens presented on MHC class I molecules. In fact, the first report on DNA vaccines was targeted to influenza virus (Cohen, 1993 ; Ulmer et al., 1993 ), but initial success in a mouse model did not well-translate into higher animal models due to poor performance (Porter and Raviprakash, 2017 ). To date, much progress has been made to improve the efficacy of DNA vaccine against influenza virus, encompassing rational design of antigens and expression vectors and the development of novel adjuvants and delivery methods (Lee et al., 2018a ). Candidate universal DNA vaccines encoding NP, matrix proteins, or PB1 were shown to decrease viral load and cross-protect against heterologous challenges in diverse animal models including mice, pigs, ferrets, and macaques (Ulmer et al., 1993 ; Tompkins et al., 2007 ; Price et al., 2009 ; Bragstad et al., 2013 ; Wang et al., 2015 ; Koday et al., 2017 ). Further studies are required for to refine DNA vaccine approaches as a stand-alone UIV. Recent studies have indicated that DNA vaccines may serve an attractive component of prime-boost strategy, considering it as a very effective means to priming immune system when preexisting immunity is low (Ledgerwood et al., 2013 ; DeZure et al., 2017 ). Despite the potential for broad protection, the safety issues need to be monitored closely, especially because of the documented rise in pathological consequences associated with CTL responses (Peiris et al., 2010 ; Duan and Thomas, 2016 ). Live Attenuated Influenza Vaccine as an Alternative Strategy Cross-Protective Immunogenicity of LAIV LAIV has been approved for clinical use in humans since 2003 and is proven effective in preventing influenza infections (Mohn et al., 2018 ). The protection of LAIV is superior to IIVs due to multifaceted immune arms including antibody responses and cell-mediated responses that operate systemically and locally (Jang and Seong, 2013a , b ; Sridhar et al., 2015 ). Further, displaying a whole set of viral antigens in a native conformation into the host immune system presents a definite advantage of LAIV to generate better protective immunity than the other strategies relying on a limited set of antigens. As discussed above, T cell immunity directed to the conserved viral epitopes constitutes the cornerstone of cross-protection. A large number of researches have shown that T cell responses induced by LAIV are critical for broad protection against heterologous and heterosubtypic influenza infections in animal models (Cheng et al., 2013 ; Jang and Seong, 2013a ; Rekstin et al., 2017 ). In young children, only LAIVs were shown to induce durable and potent T cell responses, while developing similar levels of antibody response as compared to IIVs (Belshe et al., 2000 ; Hoft et al., 2011 ; Mohn et al., 2015 , 2017 ). Despite well-documented cross-protection, LAIV has received little attention to develop a UIV. This may be attributed to the general belief that LAIV is not effective at inducing broadly neutralizing antibodies against conserved domains in surface antigens (e.g., M2e or HA stalk). However, close attention is recently being given to LAIV as an alternative platform as a potent and cross-protective vehicle than previously thought, through inducing multifaceted immune correlates including T cell response and antibody-mediated effector functions (Jang and Seong, 2013a , 2014 ). UIV Approaches Using LAIV In the UIV approaches reported so far, LAIV was used either as a component in prime-boost regimens with other different vaccine formats such as IIV, DNA vaccine, or recombinant protein vaccine. Alternatively, LAIVs were also studies as a stand-alone vaccine given in single or multiple doses. A reassortant LAIV expressing a chimeric HA was constructed under the genetic background of Russian strain (A/Leningrad/134/17/57 cold-adapted virus) and used as a boosting vaccine in a ferret model (Nachbagauer et al., 2017 ). Notably, the LAIV-IIV regimen showed greater protective efficacy against the pandemic H1N1 challenge than the IIV-IIV regimen in terms of viral loads in the respiratory tissues, despite 32-fold lower stalk antibody titers in serum. Several factors were presumed to account for this disconnect between stalk antibody titers and protection efficacy, including anti-NA immunity, mucosal IgA antibodies, cell-mediated immunity, and non-specific innate immune responses offered by the LAIV. Similar results were obtained when using a different LAIV strain (A/Ann Arbor/6/60 cold-adapted strain) (Nachbagauer et al., 2018 ). In a subsequent study performed by the same group, chimeric HA-containing the LAIV-LAIV (A/Leningrad/134/17/57 cold-adapted strain) vaccine regimen was compared with the LAIV-IIV combination in terms of protection efficacy in a ferret model, in which the LAIV-LAIV vaccine regimen conferred superior protection against pandemic H1N1 and H6N1 challenges than the LAIV-IIV (Liu et al., 2019a ). Another group tested a vaccination regimen comprising only LAIV (A/Leningrad/134/17/57 cold-adapted strain) as prime-boost vaccination in a mouse model (Isakova-Sivak et al., 2018 ). To enhance the breadth of protection, an internal gene of cold-adapted virus was replaced with the wild type. This study compared the immunogenicity and cross-protection between chimeric HA-containing LAIVs and natural HA-containing LAIVs. The chimeric HA-containing LAIVs induced higher HA stalk antibody titers and showed better cross-protection against heterologous infection with various group 1 IAVs. Thus, a cooperative role of cell-mediated immunity and HA stalk antibodies was suggested, although their individual contribution to protection were not assessed directly. It would be interesting to investigate if the cross-protection could be extended to group 2 influenza viruses such as H3 or H7 strains. Besides the Leningrad and Ann Arbor strains, an independent cold-adapted vaccine strain (X-31ca) that was previously used for trivalent seasonal vaccine (Jang et al., 2014 ), H5N1 pre-pandemic vaccine (Jang et al., 2013c ), and 2009 pdmH1N1 vaccine (Jang et al., 2012 , 2013a ), was recently tested as a UIV in a mouse model (Jang et al., 2018 ). Mice vaccinated with single or two doses of X-31 ca LAIVs were completely protected against lethal challenge of heterosubtypic strains encompassing both HA group 1 and 2 IAVs. Interestingly, boosting with heterosubtypic LAIVs carrying different HA and NA surface antigens showed more potent cross-protection than homologous boosting. T cell immunity and NK cell-mediated ADCC activity was demonstrated to contribute significantly to the observed cross-protection. As the first report of pan-influenza A protection covering both HA groups, these results merit further studies in a ferret model for clinical relevance. Hence, the LAIVs appear to be a powerful tool to develop a UIV that confers broad and potent cross-protection as a stand-alone vaccine or in combination with other strategies. Future Prospects of LAIV-Based UIVs While the LAIV presents a remarkable prospect for a broadly protective influenza vaccine, several important issues need to be addressed for it to serve as a reliable vaccine modality. The protection efficacy of a LAIV substantially differs with age. The estimated protection efficacy of a LAIV is 80% in young children and only 40% in adults, to the matched strains (Jefferson et al., 2008 , 2010 ). As for T cell immune responses, clinical studies indicate that LAIVs induce better T cell response than IIVs in both children and adults (He et al., 2006 ; Subbramanian et al., 2010 ; Hoft et al., 2011 ). However, clinical studies have reported that LAIVs are not effective at inducing T cell responses in adults and the elderly, perhaps due to preexisting "vector" immunity, which limits the replication of LAIVs (He et al., 2006 ; Forrest et al., 2008 ; Hoft et al., 2011 ). Considering that both humoral and cell-mediated immunity contribute to broad protection, it will be important to elucidate how preexisting immunity or immunologic imprinting affects B cell and T cell immune response induced by LAIVs in humans (Gostic et al., 2016 ; Henry et al., 2018 ). For this purpose, animal models mimicking preexisting immunity and controlled human challenge studies will be needed. Considering that LAIVs mimic natural infection, a fundamental question remains to be answered: if infection (or vaccination with LAIV) is effective for conferring protection, why are humans vulnerable to repeated infections with homologous or heterologous strains? At the population level, currently used cold-adapted LAIVs provide a relatively low protection rates (~40% in adults) even against matched strains (Jefferson et al., 2010 ). However, little is known whether individuals who successfully acquired protective immunity by a cold-adapted LAIV were protected against other heterologous strain(s) in the next epidemic. This may be directly addressed by a well-controlled longitudinal cohort study using human challenge models. Some studies showed that LAIVs were able to generate cross-reactive T cell responses in children for up to 1 year after vaccination, as a basis of long-term cross-protection in humans (Mohn et al., 2015 , 2017 ). Most of the LAIV-based approaches are based on cold-adapted attenuated strains (Nachbagauer et al., 2017 , 2018 ; Jang et al., 2018 ; Liu et al., 2019a ). These strains acquire multiple attenuation mutations in the internal genes during the cold-adaptation process, which contribute to genetic stability to overall attenuation (Jang et al., 2016 ). A common concern for live vaccines is safety issues, especially those associated with potential reversion of attenuating mutation(s) into virulence during vaccination. LAIVs acquired multiple attenuation mutations among various internal genes and proven safe as seasonal influenza vaccines (especially for A/Ann Arbor strain as the master strain for FluMist). However, genetically engineered LAIVs with a limited set of attenuation mutations, e.g., NS1-deletion or elastase-dependent HA cleavage (Talon et al., 2000 ; Stech et al., 2005 ), may require additional monitoring on safety. Defining the precise correlates of protection represents the most challenging step in the development of a UIV (Erbelding et al., 2018 ). Significant efforts were made to identify the protection mechanisms of HA stalk-based vaccines, suggesting that direct neutralization in combination with Fc-dependent indirect effector mechanisms mediated by stalk antibodies were the primary correlates of protection. In contrast to HA stalk-based vaccines, LAIVs elicit multiple immunological factors including serum IgG antibodies and mucosal IgA antibodies to surface antigens (HA, NA, and M2) and cell-mediated immunity to internal antigens, synergistically contributing to protection. However, their quantitative relationship to protection has not been determined, not even for homologous protection (Sridhar et al., 2015 ; Mohn et al., 2018 ), let alone for cross-protection against heterologous infection. The development of standardized assays to quantitatively measure T cell-mediated protection is particularly challenging, as the magnitude and the subset of T cells critical for protection is likely to differ according to strains of LAIV and challenge viruses. Further, mucosal IgA antibodies are believed to correlate with cross-protection, but it is still challenging to measure the neutralizing activity or effector functions of mucosal IgA antibodies. The complicated nature of LAIV-induced immunity, including non-neutralizing antibodies and diverse subsets of T cells, present a bottleneck to identifying precise correlates of protection. Another important aspect of LAIV-based strategies lies on the LAIV strains. During the past century, H1N1 and H3N2 subtypes of influenza A viruses were the most prevalent strains in humans, causing annual epidemics as well as occasional pandemics, except for the temporal circulation of H2N2 during 1957–1968 (Kilbourne, 2006 ) ( Figure 6 ). Accordingly, seasonal influenza vaccines are recommended to include H1N1 and H3N2 vaccine antigens for more than 40 years since 1977. Therefore, it is likely that most contemporary population has preexisting T cell immunity to H1N1 and H3N2 strains through natural infections or vaccinations. It should be remembered that currently licensed LAIVs (A/Ann Arbor/6/60 ca and A/Leningrad/134/17/57 ca) are of H2N2 subtypes. Probably, the nature of strain itself does not really matter for seasonal influenza vaccine, for which strain-specific immunity is focused on the surface HA antigen. However, for eliciting cross-protection, the role of conserved region become important (see section HA stalk-based UIV approaches, Conserved targets in HA other than stalk, NA as a novel target for a UIV for HA/NA and section T cell immunity as an essential factor for truly universal influenza protection for internal proteins). It is likely that human populations under 50 years of age (born after 1968 when H2N2 became extinct) has little preexisting immunity against H2N2, but predominantly against H1N1 and H3N2 viruses. It will therefore be worthwhile to examine whether cold-adapted LAIVs of H1N1 (Jang et al., 2018 ) or H3N2 origin (non-existent, to our knowledge) offer a beneficial effects on boosting the preexisting cross-reactive T cell immunity and antibody effector functions (section Mode of protection by a UIV; Figure 3 ). Figure 6 Co-evolution of influenza viruses and influenza vaccines. Within the past century, there were four influenza pandemics; 1918 Spanish flu (H1N1), 1957 Asian flu (H2N2), 1968 Hong Kong flu (H3N2), and 2009 swine flu (H1N1) (Saunders-Hastings and Krewski, 2016 ). The 1918 Spanish flu (H1N1) evolved into seasonal influenza strain and had circulated for ~40 years until the next pandemic by 1957 Asian flu (H2N2), which after ~10 years of circulation was replaced by 1968 Hong Kong flu (H3N2). The 1968 Hong Kong flu (H3N2) has circulated until now as seasonal influenza strains. In 1977, H1N1 strain reemerged and was replaced by the 2009 swine flu (H1N1), which evolved into seasonal influenza strains circulating until now. Thus, H1N1 and H3N2 strains began to co-circulate from 1977. After the reemergence of H1N1 in 1977, World Health Organization has issued recommendations for trivalent vaccine composition containing A/H1N1, A/H3N2, and B strains (Hannoun, 2013 ). As for IBVs, two distinct lineages diverged and circulated after 1985, which necessitates the incorporation of Victoria-like and Yamagata-like lineages in seasonal influenza vaccines. This leads us to raise a possibility of using an ancestral influenza B strain (such as B/Lee/40) before divergence into two different lineages as a UIV candidate for IBVs. Establishing new cold-adapted LAIV strains is a time-consuming and laborious. However, recent advances in reverse genetics and rational approaches to attenuate viral virulence have enabled the rapid conversion of a wild type virus into a novel LAIV strain. These approaches include NS1 truncation, elastase-dependent HA cleavage, caspase-dependent NP and NS1 cleavage, microRNA-mediated silencing, and codon deoptimization (Talon et al., 2000 ; Stech et al., 2005 ; Coleman et al., 2008 ; Perez et al., 2009 ; Jang et al., 2013b ). The redirection of host immune responses from surface proteins toward internal proteins may be achieved by rational vaccination strategies with LAIVs. For example, the down regulation of expression levels of HA or NA in a LAIV (Yang et al., 2013 ) is likely to result in preferable induction of T cell immunity to internal proteins. Alternatively, vaccination with LAIV carrying HA from non-human influenza viruses such as H5 or H9 may be effective at boosting preexisting T cell immunity to internal proteins in humans. Given the cross-reactivity of T cell immunity between IAVs and IBVs, A type LAIVs and B type LAIVs may be administered as a bivalent formulation or by sequential vaccination to induce improved protection. Cross-Protective Immunogenicity of LAIV LAIV has been approved for clinical use in humans since 2003 and is proven effective in preventing influenza infections (Mohn et al., 2018 ). The protection of LAIV is superior to IIVs due to multifaceted immune arms including antibody responses and cell-mediated responses that operate systemically and locally (Jang and Seong, 2013a , b ; Sridhar et al., 2015 ). Further, displaying a whole set of viral antigens in a native conformation into the host immune system presents a definite advantage of LAIV to generate better protective immunity than the other strategies relying on a limited set of antigens. As discussed above, T cell immunity directed to the conserved viral epitopes constitutes the cornerstone of cross-protection. A large number of researches have shown that T cell responses induced by LAIV are critical for broad protection against heterologous and heterosubtypic influenza infections in animal models (Cheng et al., 2013 ; Jang and Seong, 2013a ; Rekstin et al., 2017 ). In young children, only LAIVs were shown to induce durable and potent T cell responses, while developing similar levels of antibody response as compared to IIVs (Belshe et al., 2000 ; Hoft et al., 2011 ; Mohn et al., 2015 , 2017 ). Despite well-documented cross-protection, LAIV has received little attention to develop a UIV. This may be attributed to the general belief that LAIV is not effective at inducing broadly neutralizing antibodies against conserved domains in surface antigens (e.g., M2e or HA stalk). However, close attention is recently being given to LAIV as an alternative platform as a potent and cross-protective vehicle than previously thought, through inducing multifaceted immune correlates including T cell response and antibody-mediated effector functions (Jang and Seong, 2013a , 2014 ). UIV Approaches Using LAIV In the UIV approaches reported so far, LAIV was used either as a component in prime-boost regimens with other different vaccine formats such as IIV, DNA vaccine, or recombinant protein vaccine. Alternatively, LAIVs were also studies as a stand-alone vaccine given in single or multiple doses. A reassortant LAIV expressing a chimeric HA was constructed under the genetic background of Russian strain (A/Leningrad/134/17/57 cold-adapted virus) and used as a boosting vaccine in a ferret model (Nachbagauer et al., 2017 ). Notably, the LAIV-IIV regimen showed greater protective efficacy against the pandemic H1N1 challenge than the IIV-IIV regimen in terms of viral loads in the respiratory tissues, despite 32-fold lower stalk antibody titers in serum. Several factors were presumed to account for this disconnect between stalk antibody titers and protection efficacy, including anti-NA immunity, mucosal IgA antibodies, cell-mediated immunity, and non-specific innate immune responses offered by the LAIV. Similar results were obtained when using a different LAIV strain (A/Ann Arbor/6/60 cold-adapted strain) (Nachbagauer et al., 2018 ). In a subsequent study performed by the same group, chimeric HA-containing the LAIV-LAIV (A/Leningrad/134/17/57 cold-adapted strain) vaccine regimen was compared with the LAIV-IIV combination in terms of protection efficacy in a ferret model, in which the LAIV-LAIV vaccine regimen conferred superior protection against pandemic H1N1 and H6N1 challenges than the LAIV-IIV (Liu et al., 2019a ). Another group tested a vaccination regimen comprising only LAIV (A/Leningrad/134/17/57 cold-adapted strain) as prime-boost vaccination in a mouse model (Isakova-Sivak et al., 2018 ). To enhance the breadth of protection, an internal gene of cold-adapted virus was replaced with the wild type. This study compared the immunogenicity and cross-protection between chimeric HA-containing LAIVs and natural HA-containing LAIVs. The chimeric HA-containing LAIVs induced higher HA stalk antibody titers and showed better cross-protection against heterologous infection with various group 1 IAVs. Thus, a cooperative role of cell-mediated immunity and HA stalk antibodies was suggested, although their individual contribution to protection were not assessed directly. It would be interesting to investigate if the cross-protection could be extended to group 2 influenza viruses such as H3 or H7 strains. Besides the Leningrad and Ann Arbor strains, an independent cold-adapted vaccine strain (X-31ca) that was previously used for trivalent seasonal vaccine (Jang et al., 2014 ), H5N1 pre-pandemic vaccine (Jang et al., 2013c ), and 2009 pdmH1N1 vaccine (Jang et al., 2012 , 2013a ), was recently tested as a UIV in a mouse model (Jang et al., 2018 ). Mice vaccinated with single or two doses of X-31 ca LAIVs were completely protected against lethal challenge of heterosubtypic strains encompassing both HA group 1 and 2 IAVs. Interestingly, boosting with heterosubtypic LAIVs carrying different HA and NA surface antigens showed more potent cross-protection than homologous boosting. T cell immunity and NK cell-mediated ADCC activity was demonstrated to contribute significantly to the observed cross-protection. As the first report of pan-influenza A protection covering both HA groups, these results merit further studies in a ferret model for clinical relevance. Hence, the LAIVs appear to be a powerful tool to develop a UIV that confers broad and potent cross-protection as a stand-alone vaccine or in combination with other strategies. Future Prospects of LAIV-Based UIVs While the LAIV presents a remarkable prospect for a broadly protective influenza vaccine, several important issues need to be addressed for it to serve as a reliable vaccine modality. The protection efficacy of a LAIV substantially differs with age. The estimated protection efficacy of a LAIV is 80% in young children and only 40% in adults, to the matched strains (Jefferson et al., 2008 , 2010 ). As for T cell immune responses, clinical studies indicate that LAIVs induce better T cell response than IIVs in both children and adults (He et al., 2006 ; Subbramanian et al., 2010 ; Hoft et al., 2011 ). However, clinical studies have reported that LAIVs are not effective at inducing T cell responses in adults and the elderly, perhaps due to preexisting "vector" immunity, which limits the replication of LAIVs (He et al., 2006 ; Forrest et al., 2008 ; Hoft et al., 2011 ). Considering that both humoral and cell-mediated immunity contribute to broad protection, it will be important to elucidate how preexisting immunity or immunologic imprinting affects B cell and T cell immune response induced by LAIVs in humans (Gostic et al., 2016 ; Henry et al., 2018 ). For this purpose, animal models mimicking preexisting immunity and controlled human challenge studies will be needed. Considering that LAIVs mimic natural infection, a fundamental question remains to be answered: if infection (or vaccination with LAIV) is effective for conferring protection, why are humans vulnerable to repeated infections with homologous or heterologous strains? At the population level, currently used cold-adapted LAIVs provide a relatively low protection rates (~40% in adults) even against matched strains (Jefferson et al., 2010 ). However, little is known whether individuals who successfully acquired protective immunity by a cold-adapted LAIV were protected against other heterologous strain(s) in the next epidemic. This may be directly addressed by a well-controlled longitudinal cohort study using human challenge models. Some studies showed that LAIVs were able to generate cross-reactive T cell responses in children for up to 1 year after vaccination, as a basis of long-term cross-protection in humans (Mohn et al., 2015 , 2017 ). Most of the LAIV-based approaches are based on cold-adapted attenuated strains (Nachbagauer et al., 2017 , 2018 ; Jang et al., 2018 ; Liu et al., 2019a ). These strains acquire multiple attenuation mutations in the internal genes during the cold-adaptation process, which contribute to genetic stability to overall attenuation (Jang et al., 2016 ). A common concern for live vaccines is safety issues, especially those associated with potential reversion of attenuating mutation(s) into virulence during vaccination. LAIVs acquired multiple attenuation mutations among various internal genes and proven safe as seasonal influenza vaccines (especially for A/Ann Arbor strain as the master strain for FluMist). However, genetically engineered LAIVs with a limited set of attenuation mutations, e.g., NS1-deletion or elastase-dependent HA cleavage (Talon et al., 2000 ; Stech et al., 2005 ), may require additional monitoring on safety. Defining the precise correlates of protection represents the most challenging step in the development of a UIV (Erbelding et al., 2018 ). Significant efforts were made to identify the protection mechanisms of HA stalk-based vaccines, suggesting that direct neutralization in combination with Fc-dependent indirect effector mechanisms mediated by stalk antibodies were the primary correlates of protection. In contrast to HA stalk-based vaccines, LAIVs elicit multiple immunological factors including serum IgG antibodies and mucosal IgA antibodies to surface antigens (HA, NA, and M2) and cell-mediated immunity to internal antigens, synergistically contributing to protection. However, their quantitative relationship to protection has not been determined, not even for homologous protection (Sridhar et al., 2015 ; Mohn et al., 2018 ), let alone for cross-protection against heterologous infection. The development of standardized assays to quantitatively measure T cell-mediated protection is particularly challenging, as the magnitude and the subset of T cells critical for protection is likely to differ according to strains of LAIV and challenge viruses. Further, mucosal IgA antibodies are believed to correlate with cross-protection, but it is still challenging to measure the neutralizing activity or effector functions of mucosal IgA antibodies. The complicated nature of LAIV-induced immunity, including non-neutralizing antibodies and diverse subsets of T cells, present a bottleneck to identifying precise correlates of protection. Another important aspect of LAIV-based strategies lies on the LAIV strains. During the past century, H1N1 and H3N2 subtypes of influenza A viruses were the most prevalent strains in humans, causing annual epidemics as well as occasional pandemics, except for the temporal circulation of H2N2 during 1957–1968 (Kilbourne, 2006 ) ( Figure 6 ). Accordingly, seasonal influenza vaccines are recommended to include H1N1 and H3N2 vaccine antigens for more than 40 years since 1977. Therefore, it is likely that most contemporary population has preexisting T cell immunity to H1N1 and H3N2 strains through natural infections or vaccinations. It should be remembered that currently licensed LAIVs (A/Ann Arbor/6/60 ca and A/Leningrad/134/17/57 ca) are of H2N2 subtypes. Probably, the nature of strain itself does not really matter for seasonal influenza vaccine, for which strain-specific immunity is focused on the surface HA antigen. However, for eliciting cross-protection, the role of conserved region become important (see section HA stalk-based UIV approaches, Conserved targets in HA other than stalk, NA as a novel target for a UIV for HA/NA and section T cell immunity as an essential factor for truly universal influenza protection for internal proteins). It is likely that human populations under 50 years of age (born after 1968 when H2N2 became extinct) has little preexisting immunity against H2N2, but predominantly against H1N1 and H3N2 viruses. It will therefore be worthwhile to examine whether cold-adapted LAIVs of H1N1 (Jang et al., 2018 ) or H3N2 origin (non-existent, to our knowledge) offer a beneficial effects on boosting the preexisting cross-reactive T cell immunity and antibody effector functions (section Mode of protection by a UIV; Figure 3 ). Figure 6 Co-evolution of influenza viruses and influenza vaccines. Within the past century, there were four influenza pandemics; 1918 Spanish flu (H1N1), 1957 Asian flu (H2N2), 1968 Hong Kong flu (H3N2), and 2009 swine flu (H1N1) (Saunders-Hastings and Krewski, 2016 ). The 1918 Spanish flu (H1N1) evolved into seasonal influenza strain and had circulated for ~40 years until the next pandemic by 1957 Asian flu (H2N2), which after ~10 years of circulation was replaced by 1968 Hong Kong flu (H3N2). The 1968 Hong Kong flu (H3N2) has circulated until now as seasonal influenza strains. In 1977, H1N1 strain reemerged and was replaced by the 2009 swine flu (H1N1), which evolved into seasonal influenza strains circulating until now. Thus, H1N1 and H3N2 strains began to co-circulate from 1977. After the reemergence of H1N1 in 1977, World Health Organization has issued recommendations for trivalent vaccine composition containing A/H1N1, A/H3N2, and B strains (Hannoun, 2013 ). As for IBVs, two distinct lineages diverged and circulated after 1985, which necessitates the incorporation of Victoria-like and Yamagata-like lineages in seasonal influenza vaccines. This leads us to raise a possibility of using an ancestral influenza B strain (such as B/Lee/40) before divergence into two different lineages as a UIV candidate for IBVs. Establishing new cold-adapted LAIV strains is a time-consuming and laborious. However, recent advances in reverse genetics and rational approaches to attenuate viral virulence have enabled the rapid conversion of a wild type virus into a novel LAIV strain. These approaches include NS1 truncation, elastase-dependent HA cleavage, caspase-dependent NP and NS1 cleavage, microRNA-mediated silencing, and codon deoptimization (Talon et al., 2000 ; Stech et al., 2005 ; Coleman et al., 2008 ; Perez et al., 2009 ; Jang et al., 2013b ). The redirection of host immune responses from surface proteins toward internal proteins may be achieved by rational vaccination strategies with LAIVs. For example, the down regulation of expression levels of HA or NA in a LAIV (Yang et al., 2013 ) is likely to result in preferable induction of T cell immunity to internal proteins. Alternatively, vaccination with LAIV carrying HA from non-human influenza viruses such as H5 or H9 may be effective at boosting preexisting T cell immunity to internal proteins in humans. Given the cross-reactivity of T cell immunity between IAVs and IBVs, A type LAIVs and B type LAIVs may be administered as a bivalent formulation or by sequential vaccination to induce improved protection. Other Strategies for UIVs A number of alternative strategies are being investigated for their potential to serve as a UIV platform. First approach is to use infection-competent but replication-defective viruses. For instance, the M2 knock-out influenza virus (M2SR) rescued from M2-producing cells is able to infect cells but does not produce progeny virus due to the lack of functional M2 protein. The M2SR was shown to induce strong cell-mediated and humoral immunity and to provide broad protection against both homologous and heterologous influenza virus challenge in mice and ferrets (Sarawar et al., 2016 ; Hatta et al., 2017 , 2018 ). A conceptually similar to M2SR, PB2 knock-out influenza virus could be produced in PB2-expressing cells as a vaccine. The protection efficacy of PB2-KO vaccine was tested against diverse influenza strains in mice (Victor et al., 2012 ; Uraki et al., 2013 ; Ui et al., 2017 ). Pseudotyped influenza A virus also presents a similar replication-incompetent virus approach. The pseudotyped influenza virus lacking HA produced in HA-expressing cells can infect cells and express all the viral proteins except for HA. Studies have shown that the vaccination of mice and ferrets with the pseudotyped influenza virus generated a vigorous T cell response and reduced lung viral loads and weight loss after challenge with homologous and heterologous influenza viruses (Powell et al., 2012 ; Baz et al., 2015 ). These approaches based on replication-defective viruses should be rigorously evaluated for safety, considering potential reversion into virulence by acquiring the wild type gene by reassortment with circulating viruses (Lowen, 2017 ). Secondly, mRNA vaccines have emerged as a promising alternative to conventional vaccine platforms against various infectious diseases including the influenza virus (Pardi et al., 2018a ). Initially, the mRNA vaccines successfully induced protective B cell and T cell immune response in mice, ferrets, and pigs (Petsch et al., 2012 ). In subsequent studies, mRNA vaccines encoding HA induced neutralizing antibodies and T cell immunity essential for heterologous protection in mice and ferrets (Brazzoli et al., 2016 ). Notably, mRNA vaccines encoding HA elicited HA stalk antibodies in mice, rabbits, and ferrets, and the stalk antibody responses were associated with protection against homologous and heterologous influenza virus infection in mice (Pardi et al., 2018b ), depicting promising potential as a UIV platform. Conclusions and Prospects During the past few years, several critically important issues have emerged in developing a UIV. While identification of a number of broadly protective antibodies presents an optimistic prospect for pan-influenza therapeutics, induction of such antibodies at a sufficiently protective level by vaccination has not been accomplished yet. A variety of viral targets have been identified to induce broad protection, including M2e and the conserved regions in the HA, such as stalk, RBS, and VE, and the long-neglected NA has emerged as an essential target for durable and broad protection. In virtually all cases, the rationale for broad protection is to redirect the immune responses from the variable immunodominant regions to conserved immunosubdominant regions. While effective in eliciting cross-protection, concerns were raised to non-neutralizing antibodies that potentially trigger the enhancement of disease. T-cell immunity has also been considered as an important correlate of cross-protection. LAIVs are able to induce multifaceted immune response and thus have increasingly received close attention as a promising vaccine platform, either alone, or in boosting format. Preexisting immunity or immunologic imprinting, established by prior exposure to influenza by infection or vaccination, may influence or thwart the desired breadth of immune response by UIVs. Therefore, disparities may exist between pre-clinical evaluation of a UIV without due recognition of immune imprinting and human challenge studies. Lastly, the correlates of protection and their precise molecular mechanisms have not been determined yet, which remains a significant gap between development and licensing of a UIV. Given the limited antigenic diversity of IBVs among humans and the lack of animal reservoir, pan-influenza B virus vaccine may be attainable in the near future. On the other hand, IAVs continually change their antigenicity via antigenic drift and shift and zoonotic spillover from animal reservoirs, requiring multi-faceted immune arms to increase the breadth of protection. Judicious choice of antigens and their efficient designing to confer a broad protection for a prolonged period are required to counterfeit the immune evasion and provide a truly universal protection against the ever-changing influenza viruses. Author Contributions BS designed and conceived the review. YJ and BS investigated the literature and wrote the manuscript. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7129771/

Killed in action: Microbiologists and clinicians as victims of their occupation Part 4: Tick-borne Relapsing Fever, Malta Fever, Glanders, SARS

Tick-borne Relapsing Fever A microbiologist and pathologist, who was engaged in research on African Tick-borne Relapsing Fever, Joseph Everett Dutton ( Fig. 1 ), became a victim of this disease. He was born on 9th of September 1874 in Higher Bebington, Cheshire, as the fifth son of the chemist John Dutton, was educated from 1888 to 1892 in the King's School of Chester, and entered the Liverpool Medical School and the Royal Infirmary in the same town. In 1897 he was graduated and was appointed to the George Holt Fellowship in Pathology. One year in the Departments of Surgery followed. He undertook his first scientific expedition in 1900 as a member of the third expedition of the Liverpool School of Tropical Medicine together with H.E. Annett and J.H. Elliott. They studied malaria and filariasis. At the beginning of 1901 Dutton travelled alone in the Gambia. It was the sixth expedition of the Liverpool School. On May 10, Dr. R.M. Forde, Colonial surgeon at the hospital in Bathurst, demonstrated a patient, where he (Forde) had found in the blood "very many actively moving worm-like bodies whose nature he was unable to ascertain" ( Dutton, 1902 ). The patient went back to England where Dutton examined him again, but he found no parasites in his blood. This patient, a 42-year-old government employee, came back to the Gambia several months later. Dutton, who was also back to the colony, examined him on December 15, 1901, and "found a flagellate protozoon evidently belonging to the genus Trypanosoma " and he suggested "that the name 'Trypanosoma gambiense'" [should] "be given to this trypanosoma" ( Dutton, 1902 ). We have told earlier the whole story in this journal ( Köhler, 2002 ). Because this patient was also ill with malaria Dutton did not realize the relation between sleeping sickness and trypanosomes. The tenth expedition of the Liverpool School led Dutton in 1902 together with J.L. Todd and C. Christy in 1903 to Belgian Congo. The King of Belgium placed a considerable amount of money at the disposal of this expedition. The researchers left Liverpool on September 23, and reached the Stanley Falls at the end of the year. Dutton's special interest was directed to tick-borne relapsing fever of the Central African type. He was able to infect monkeys by the bites of spirochaete-carrying soft ticks, Ornithodorus moubata . What he did not know, Philip Ross and Milne made the same experiment a few weeks before in Uganda. Dutton found also that Borrelia duttoni could pass into the eggs and larvae, and so confer infective potential on the tick of the succeeding generation ( Manson-Bahr, 1951 ). Robert Koch (1906) , who had no information about these results, found while he was in East Africa in 1905 that this transmission is possible up to the third generation. Christy and Dutton infected themselves with the disease, possibly at the dissection of a patient died of relapsing fever. Christy survived but Dutton died after the fourth relapse on 27th of February 1905 in Kosongo/Congo Free State. He was at the age of 31 years. The news about his death came relatively late to England because "The journey from Kosongo to the nearest telegraph station takes 2 months" ( Anonymus, 1905 ). Fig. 1 Joseph Everett Dutton (from Olpp, 1932 ). Malta Fever The "Malta Chronicle" of September 15, 1904, reported "Well known for his discovery of the microbe of Mediterranean or Malta Fever Prof. Tito Carbone died in Milan from a fever contracted during scientific experiments". The real story is another one, David Bruce discovered the microorganism, and Carbone is not so well-known in this respect. But, he was a victim of this disease. Tito Carbone was born on 16th of July 1863 in Carbonara Scrivia (Alessandria). He studied medicine in Turin, Pavia and Florence and was graduated in 1886 in Turin. Working with C. Lombroso (psychiatric clinic) he was engaged in the control of a cholera epidemic in Tortona, and later worked in the pathological laboratory of G. Bizzozero. In 1888 he was assistant at the Institute of Morbid Anatomy, headed by P. Foà , and changed in 1891 to the Laboratory of Pathological Physiology at the hospital Umberto I. at Turin. He went to Buenos Aires, and back to Italy he became a coworker of S. Belfanti at the Istituto Sieroterapia Milano. Later he followed an academic career and was appointed Professor of Morbid Anatomy in Cagliari (1897), Modena (1898) and Pisa (1903). His main field of research was the aetiopathogenesis of infectious diseases, he published on bacterial toxins and immunity. In 1904 he dissected a patient died of Malta Fever and published the pathological findings ( Carbone, 1904 ). Brucella melitensis was isolated by him and transferred to experimental animals. In order to get information about the toxin production he cultivated the microorganisms, and it seems that he infected himself by this way. Carbone died on 6th of September 1904, aged 41 years ( Dizionario Biografico degli Italiani, 1976 ). Glanders Glanders in humans was a rare disease, even when it was common in horses. But there have been some laboratory infections because "probably no organism, with the possible exception of tularaemia bacillus, is as dangerous to work with as the glanders bacillus. In one laboratory, several members of staff became ill a few days after the breaking of a centrifuge tube" ( Parker, 1984 ). Without giving details Bernstein and Carling (1909) mentioned: "glanders has been assigned as the most frequent case of accidental death amongst laboratory workers". The six patients, described by these authors, were horse keepers or horse-bus drivers. Howe and Miller (1947) reported on six cases of glanders occurring within 1 year (1944/45) in Camp Detrick, Frederick, MD. In the small laboratory a research program on glanders was performed. The first two cases occurred 2 weeks after an accident, when a technician dropped a flask containing a virulent suspension of Burkholderia mallei . The technician did not infect himself but two physicians staying in the laboratory fell ill. Two technicians became infected by routine laboratory work and the route of infection of two veterinarians was possibly aerogenic. The patients were cured by sulfadiazine. Little is known about the course of life and the death of Georg von Hofmann-Wellenhof . He was born on 14th of May 1860 in Vienna as the third child of Paul Ferdinand Hofmann von Wellenhof and his wife Klothilde, née Kraus (data from his thesis). He studied medicine in Vienna and Graz (where he was graduated), and was for some years First Assistant at the Institute of Pathology. Then he changed to the Institute of Hygiene, interrupted by some years of assistantship at the Institute of Zoology, all in Graz. As a result of his work in the Institute of Pathological Anatomy he reported on the detection of a "pseudodiphtheria bacillus" (later often cited as "Hofmann-Wellenhof'scher Bacillus"); first on 20th of September 1887 at the 66th Assembly of the "Gesellschaft Deutscher Ärzte und Naturforscher" in Wiesbaden, followed by the detailed publication ( von Hofmann-Wellenhof, 1888 ; Köhler, 2004 ). Before 25th of September 1889 (he was since 1888 assistant at the Institute of Hygiene at Vienna) he performed "infection experiments with the virus of glanders". Around October 8, his temperature raised and he had pain in his left chest (pleuritic rub). For some days he improved, then he complained about "rheumatoid discomfort". Eight days later purulent blisters and abscesses developed, and on 22nd of the month he was hospitalized. The clinical picture was that of acute glanders, and the dissection confirmed the diagnosis. The source of infection was unclear; an aerogenic infection was supposed. Georg von Hofmann-Wellenhof died on 23rd of October 1889, aged 30 years ( Anonymus, 1889 ). Severe acute respiratory syndrome (SARS) Deadly infections of physicians by microorganisms they were working with happened as shown in the four parts of this article mostly at the end of the 19th up to the first quarter of the 20th century. But also recently we had to deplore the loss of a colleague who died of SARS. It was Carlo Urbani , born on 19th of October 1956 in Castelplanio/Italy. As to Oranski (2003) in Hanoi as a representative of the WHO he examined Jonny Chen, a Chinese-American businessman on February 28, 2003, and he was the first one who identified this "new" disease. The patient was suspected for an avian influenza infection. Urbani himself realized that he became infected on 11th of March while taking care for the patient. About his career is known that he earned his medical degree in 1981 from the University of Ancona. For 3 years he was trained in infectious diseases at the University of Messina, and in 1990 he became deputy chief of the Department of Infectious Diseases at the General Hospital in Ancona. He worked for the WHO since 1993 and after moving to Médecins Sans Frontiérs in 1995 he became president of the Italian branch in 1999. His working places were in Vietnam, the Philippines and in Cambodia. On 29th of March 2003, 18 days after infection, Carlo Urbani died of SARS in Bangkok/Thailand. Some well-known bacteriologists and clinicians died of infections not connected with their professional duties ( Table 1 ; Vierordt, 1915 ; Köhler, 1997 ; Olpp, 1932 ; Vierhuff, 1907 ; Hetsch, 1935 ; Schloßberger, 1935 ). Table 1 Death of microbiologists and infectious disease specialists due to infections not connected with professional duties (except tuberculosis) Name Born Died Disease Semmelweis, Ignaz Philipp 1818, July 1 1865, August 13 Septicaemia Plehn, Friedrich 1862, November 15 1904, August 30 Malta Fever Schaudinn, Fritz 1871, September 19 1906, June 22 Septicaemia, rectal abscess Gabritschewsky, Georgiy Norbertovitch 1860 1907, March 23 Pneumonia Neisser, Albert 1855, January 22 1916, July 30 Septicaemia Gaffky, Georg 1850, February 17 1918, September 23 Influenza-pneumonia Kolle, Wilhelm 1868, November 2 1935, May 10 Infectious arthritis, pneumonia Many microbiologists and physicians lost their life in the battle against the disease they wanted to explore and to overcome for the welfare of mankind. A few of them were honoured by a monument of memory like Ricketts, Lazear, Noguchi, Thuillier or da Camara Pestana, others were mentioned only in a short obituary in scientific journals, or are remembered among microbiologists like E. Hailer of the Robert-Koch-Institute (Berlin) who died after a laboratory infection with anthrax bacilli in 1939 (Stefan Winkle, personal communication; Robert-Koch-Institut, 1991 ). Many are unnamed like those plague doctors in the Middle Ages who did not leave their patients, or the many army surgeons who died of typhus, variola or other infectious diseases. These men are shining examples and we should stand in awe of them. – This is the bright side, but there was (and is?) also a dark one. Microbiologists and so-called "physicians" used their knowledge and medical skills to kill people by application of virulent microorganisms in order to prove the efficacy of bacteriological weapons (e.g. see Geißler, 2003 ). We should treat them with scorn.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6165266/

Sleeping Beauties: Horizontal Transmission via Resting Spores of Species in the Entomophthoromycotina

Many of the almost 300 species of arthropod-pathogenic fungi in the Entomophthoromycotina (Zoopagomycota) are known for being quite host-specific and are able to cause epizootics. Most species produce two main types of spores, conidia and resting spores. Here, we present a review of the epizootiology of species of Entomophthoromycotina, focusing on their resting spores, and how this stage leads to horizontal transmission and persistence. Cadavers in which resting spores are produced can often be found in different locations than cadavers of the same host producing conidia. Resting spores generally are dormant directly after production and require specific conditions for germination. Fungal reproduction resulting from infections initiated by Entomophaga maimaiga resting spores can differ from reproduction resulting from conidial infections, although we do not know how commonly this occurs. Reservoirs of resting spores can germinate for variable lengths of time, including up to several months, providing primary infections to initiate secondary cycling based on conidial infections, and not all resting spores germinate every year. Molecular methods have been developed to improve environmental quantification of resting spores, which can exist at high titers after epizootics. Ecological studies of biological communities have demonstrated that this source of these spores providing primary inoculum in the environment can decrease not only because of germination, but also because of the activity of mycopathogens. 1. Introduction Diverse pathogens persist in the environment as long-lived forms in reservoirs, with perhaps the best documented example being the bacteria that causes anthrax ( Bacillus anthracis ) [ 1 ]. Long-lived forms allow persistence in the environment over periods when host stages that are present are not susceptible or prevailing environmental conditions are not ideal for successful pathogen transmission and development. It has been hypothesized that having such means for persistence allows these pathogen species to retain high virulence, even allowing all hosts in that location to be killed [ 2 , 3 , 4 ]. Of course, horizontal transmission is required for these persistent stages to infect hosts. Horizontal transmission, however, is more risky than vertical transmission and becoming active and successfully infecting hosts during the correct periods of time are critically important to pathogen survival and continued persistence. Most species in the fungal subphylum Entomophthoromycotina produce long-lived spores that persist in the environment to initiate infections in insects when susceptible host stages are present ( Figure 1 ). The subphylum Entomophthoromycotina (Zoopagomycota) includes highly virulent species that are capable of causing epizootics ( Figure 2 ) and have a specialized form for persistence. Among the >300 species of Entomophthoromycotina, most are obligate pathogens of arthropods with complex life cycles associated with their hosts. Most species in the Entomophthoromycotina produce at least two types of spores: conidia and either zygospores or azygospores ( Figure 3 ) or a mixture of these latter. While conidia are generally immediately able to germinate and infect hosts, azygospores or zygospores (commonly referred to as resting spores; Figure 3 ) are thought to enter dormancy after production, becoming environmentally persistent forms. It is not uncommon that resting spores are not included in species descriptions, as only conidia have been found by the describers (e.g., [ 5 ]). However, we will explain why it is possible or probable that most species in Entomophthoromycotina produce both types of spores. This is a review focusing on ecological aspects of resting spores made by species in the Entomophthoromycotina, with emphasis on horizontal transmission. We will not review all literature, but will especially explain research conducted since the review of entomophthoralean biology and ecology by Pell et al. [ 6 ], and the review of persistent forms of some Entomophthorales by Nielsen et al. [ 7 ]. 2. Systematics and Habits The systematics of the group Entomophthoromycotina has undergone major revisions, as new data from multigene analyses clarify phylogenetic relationships among genera traditionally identified in this group and with other basal fungi [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. Traditionally, this group was placed in the phylum Zygomycota, based on the shared characteristic of zygospore formation during the sexual cycle, after fusion of specialized hyphae called gametangia (but see below). Like most members of the Zygomycota, entomophthoralean fungi also have coenocytic hyphae (lacking cross walls or septa), in contrast to Ascomycota and Basidiomycota with septate mycelia [ 10 ]. Analysis of the rRNA, rpb1 and tef1 by James et al. [ 9 ], however, showed that the group Zygomycota was polyphyletic (i.e., with shared characteristics, but derived from two or more ancestral forms not common to all members). In a comprehensive review of the kingdom Fungi by Hibbett et al. [ 11 ] in 2007, based on available molecular phylogenetic analyses, a proposal was made to distribute taxa traditionally placed in Zygomycota among four subphyla incertae sedis (i.e., no phylum has been assigned, or the broader relationship is unknown), including the new subphylum Entomophthoromycotina for fungi in the order Entomophthorales. In 2012, Humber [ 8 ] proposed the creation of a new phylum, Entomophthoromycota, and discontinued use of Entomophthoromycotina, based on data from additional phylogenetic studies utilizing more loci and sampling from more representative taxa of entomophthoralean fungi that showed this group was monophyletic (i.e., with shared derived characters; descended from a common ancestor) [ 12 , 13 ]. Using a combination of molecular data and traditional taxonomic characters (i.e., nuclear characters, details of their mitoses, and morphology of primary and secondary conidia), a new classification was proposed, with the subphylum Entomophthoromycotina containing three classes, namely, Basidiobolomycetes, Neozygomycetes, and Entomophthoromycetes, and with each class consisting of only one order ([ 8 ]; see Table 1 ). The first two classes consisted of a single family each, Basiodiobolaceae and Neozygitaceae, respectively. The Entomophthoromycetes had four families, three of which contained arthropod pathogenic species ( Table 1 ). Also included in this proposal was the placement of the genera Eryniopsis and Tarichium as incertae sedis . Eryniopsis species identification had been based on morphological characters only, while Tarichium was a form genus, based on the absence of conidia. Further, multi-gene sequence data separated species of the former between two subfamilies, Entomophthoroideae and Erynioideae, in Entomophthoraceae [ 8 , 13 ]. Species in both genera need to be analyzed further by single- and multi-locus phylogenetic reconstructions to determine possible synonyms or placement of taxa in the correct genus (e.g., [ 15 ]). A more recent study by Spatafora et al. [ 14 ], utilizing genome-scale data to analyze zygomycete fungi, proposed the creation of a new phylum, Zoopagomycota, with subphyla Entomophthoromycotina, Kickxellomycotina, and Zoopagomycotina. The authors rejected use of the name Zygomycota for this phylum, because zygospore formation is not a synapomorphy, but a sympleisiomorphic trait (i.e., trait shared by common ancestors of Zoopagomycota and other phyla). The phylum name Zoopagomycota was selected over other possible names, including Entomophthoromycota, because of its broader and more inclusive meaning [ 14 ]. This new proposal retained the three classes in the subphylum Entomophthoromycotina proposed by Humber [ 8 ]. Studies on the molecular phylogeny of Entomophthoromycotina by Gryganski et al. [ 12 , 13 ], using multi-gene sequences (rRNA, rpb2 and mtssu) from 63 species representing 14 genera, showed that lineages inferred from molecular data were in agreement with groupings based on traditional taxonomic characters used to identify these fungi. These characters included morphological and ultrastructural features: type of conidiophores, type of primary conidia, type of secondary conidia, type of resting spores, and type of nuclear division [ 8 , 12 ]. Based on morphology, characteristics of the primary and secondary conidia were suggested as generally more useful than those of resting spores for species identification. However, resting spore characters such as shape, size, color, ornamentation, wall structure, and mode of germination are also key features in species identification in some genera (and see Section 3.2 ) [ 16 , 17 ]. For more information on traditional taxonomic characters and for identification keys to families, genera and species of Entomophthoromycotina, in particular arthropod pathogens in the order Entomophthorales, see Keller [ 18 , 19 ] and Keller and Petrini [ 20 ]. Most members of the Entomophthoromycotina are arthropod pathogens, with some plant pathogens, commensals, or soil saprobes. Among arthropod pathogens, another key character is the host, as many are host-specific, with only one known host or with a relatively narrow host range (i.e., hosts in the same family). The signs and symptoms of infection can also be quite useful in identifying the fungus or the etiological agent of disease. For example, flies in the families Anthomyiidae and Fanniidae infected with Strongwellsea spp. exhibit pathognomonic signs and symptoms (i.e., presence of a gaping hole in the ventral abdominal pleuron of adults, through which spores are dispersed from an internal hymenium, associated with the inability of infected female hosts to lay eggs [ 21 ]). Among the three classes of Entomophthoromycotina, most of the genera with insect pathogenic species are found in the class Entomophthorales, specifically in the families Ancylistaceae, Entomophthoraceae, and Meristacraceae ( Table 1 ). Identification of these fungi requires knowledge of host species in addition to descriptions of diagnostic structures that vary at different stages of infection [ 17 ]. Thus, morphology, in addition to molecular data, provides robust and stable classifications. Additional phylogenetic studies, however, are still needed to resolve several issues in the systematics of Entomophthoromycotina, including revising the Erynia / Furia / Pandora complex and sorting the identification of species in the genera Eryniopsis , Neozygites , and Tarichium [ 13 ]. In the past, morphological structures associated with conidiation were the sole means for species identification. However, cadavers of hosts killed by entomophthoralean pathogens can sometimes contain only resting spores and, in these situations, the pathogen could not be identified based on conidial morphology. In such cases, the species were included in the 'form genus' Tarichium , which is a temporary arrangement. At present, there are 40 species in the form genus Tarichium [ 15 ]. In recent years, however, the first species that was only known from the resting spores was described. Molecular evidence was used to place this species, Zoophthora independentia , in the correct genus [ 15 ]. In this case, resting spores are formed after death of adults of a native North American crane fly species, and cadavers are subsequently attached to the undersides of leaves by pseudorhizomorphs. The conidial stages have never been found and it is assumed that either adult hosts producing conidia are dying somewhere that collectors have not yet found them or conidial stages are produced from larvae or pupae in the leaf litter and soil and have not yet been found. At this time, questions remain for species in the genus Tarichium : are there actually no conidial stages, or they are limited, or have researchers not been looking at the correct times and/or locations to find them? 3. General Biology Fungi in the Entomophthoromycotina are described as having primary conidia that usually are formed externally on cadavers. Primary conidia may be uni- to multinucleate and are frequently forcibly discharged. Many species also form one or more types of secondary conidia (formed from the primary conidia), which may be forcibly discharged or passively dispersed. Secondary conidia can resemble primaries, although smaller, or can differ completely in appearance. One type of secondary conidia, called capilliconidia, are formed at the top of a thin stalk produced from the primary. Capilliconidia are sticky and adhere and subsequently infect when contacted by passing hosts. Resporulation can also proceed to tertiary conidia, but infectivity of this stage has not been proven. Primary and/or secondary conidia are infective, but this characteristic can vary by fungal species. Most species are also thought to form resting spores, either zygospores (after gametangial conjugation) or azygospores (no gametangial conjugation), with thick walls [ 8 ]. Germination studies suggest that resting spores themselves are not infective, but they germinate to produce infective germ conidia. Germ conidia can resemble primary conidia, but at least some studies have demonstrated differential reproduction after host infection by germ conidia [ 42 ]. Whether germ conidia should be considered a third type of spore for Entomophthoromycotina or a secondary spore produced by resting spores has not been discussed, as germ conidia are poorly known for most species. For many species, resting spores are produced within the body of the dead host, although for at least Pandora dipterigena , resting spores are produced externally on cadavers [ 5 ] and in Neozygites floridana , resting spores are produced within host mites when these hosts are still alive [ 43 , 44 ]. In the case of E. maimaiga , after host death the hyphal bodies round up on one end as they begin to become resting spores. Once round, the interiors of resting spores are granular and walls are thin, but this changes over the next 1–2 days while resting spores undergo maturation, until most resting spores have thick walls and contain large oil droplets [ 45 , 46 ]. There can be extreme diversity across species in the appearance of resting spores, but this has never been addressed as a taxonomic character, probably in part because resting spores are not known for many species. Among resting spores, some have no epispore while others have clear epispores that are ornamented or not, or some species have thick, opaque epispores that can be ornamented and colored. At least for the tipulid pathogen Z. independentia , the opaque epispore can be cracked and removed using pressure on a cover slip and the resting spore within has a thick wall and an appearance similar to some resting spores without epispores ( Figure 4 ; [ 15 ]). While resting spores of most species are spherical, those in the Neozygitaceae can be ovoid and dark brown to black [ 20 ]. 3.1. Resting Spores Versus Conidia and Species Identity Most entomophthoralean species can produce two types of spores (conidia and resting spores) from one host individual. Sometimes, both conidia and resting spores are produced from one cadaver (e.g., Entomophaga maimaiga ), but more commonly, only one type of spore is produced from the cadaver of one host individual. Usually, resting spores are formed under different conditions or host states than conidia (see Section 3.2 ). In many ways, entomophthoralean pathogens that produce conidia and resting spores can resemble two different species. The locations of the host cadavers, the manipulations of the host behavior before and after death, the fungal structures such as rhizoid holdfasts, as well as the fungal reproductive forms differ so much that if only one form (i.e., type of spore) is found, this may be described as a separate species from the other form (see Section 3.3 ). When resting spores are formed within host stages that are hidden and not easily found (e.g., within the soil), resting spores might never have been found and have not been described for that species. Conversely, sometimes resting spore-bearing hosts are found but individuals of the same host species in which conidia are produced are not found. In such a case, the species has been placed in the genus Tarichium . However, it is possible that the conidial stages have already been described as a different species. This sets up a situation similar to Ascomycota, where one species might have two different names, which has occurred when ascomycete anamorph and teleomorph forms are disconnected. The potential disjunct occurrence of different types of spores demonstrates the importance of detailed studies over time of a host/pathogen in order to understand all the stages. For instance, in the case of Erynia aquatica , the first researchers to study this pathogen of Aedes spp. snowpool mosquitoes found only conidial stages ( Figure 5 g). They correctly hypothesized that resting spores must exist because the hosts were univoltine [ 35 , 47 ]. Later, this hypothesis was proven to be true when Steinkraus and Kramer [ 48 ] found and described the resting spores ( Figure 5 h). 3.2. Resting Spore Production Empirical studies have repeatedly reported that resting spores are formed towards the end of a season, when few of the hosts will be present. Studies have demonstrated that numerous biotic and abiotic factors are associated with resting spore formation, but these can differ for different host/pathogen systems ( Table 2 ). For example, E. maimaiga shifted from making conidia to making resting spores at warmer temperatures, as later larval instars of its univoltine host pupate in summer. In contrast, Entomophthora muscae and Neozygites floridana produced resting spores at colder temperatures, when hosts would no longer be active. In the case of Eryniopsis lampyridarum in soldier beetles, resting spores are produced when the adult hosts are abundant. 3.3. Differential Locations of Cadavers Producing Resting Spores versus Conidia: Six Case Histories Because the conidial stage of entomophthoralean fungi functions in short term dissemination of the pathogen to new hosts and conidia are not durable structures that survive many months or years, hosts producing the conidial stage must be held in situations where hosts are active. For example, conidia-producing cadavers of rhagionids infected by Erynia ithacensis are underneath leaves [ 58 ]; Entomophthora scatophagae producing conidia from dead yellow dung flies ( Scatophaga stercoraria ) are found on flowering grasses where other dung flies are perching [ 59 ]; or, in the case of N. fresenii , infected conidial stage aphids are attached by their mouthpart stylets to the plant ( Figure 4 c), and spores are explosively discharged onto adjacent aphids in the colony, onto leaves, or into the air [ 60 ]. Many arthropod pathogenic Entomophthoromycotina are known only from their conidial stages [ 61 ]. There are two reasons for this. First, hosts killed by the conidial stage often occur in highly visible situations, such as on plants ( Figure 5 e), held by rhizoids, legs, or mouthparts ( Figure 5 i). From elevated positions conidia can be discharged onto potential hosts. In contrast, hosts producing resting spores are not as conspicuous. Second, many studies on arthropod hosts of Entomophthoromycotina have not been carried out in detail over a long enough period to detect hosts with resting spores that may occur in older hosts or towards the end of the season. With any entomophthoralean fungus that infects univoltine hosts, or multivoltine hosts that have an extended period when hosts are not available (such as during winter), there must be resting spores produced (or some other survival structure, e.g., N. floridana hyphal bodies within hibernating mites [ 44 ] or thick-walled conidia of the aphid pathogen Pandora neoaphidis [ 7 ]). Only careful studies over time with many specimens will result in finding the resting spore stage, as well as the conidial stage (or vice versa). Therefore, most species of Entomophthoromycotina must have a resting spore stage, but many have not yet have been discovered. In order to find hosts containing resting spores, scientists must examine host populations over time, often focusing on older hosts. If the prevalence of hosts with resting spores is low, one must collect many hosts, perhaps hundreds both alive and dead, keep them and examine them for resting spores that may develop over time (e.g., [ 43 , 44 ]). Careful, thorough searches should also be made of the soil surface or other areas near where hosts dying of conidial stages are found, where host eggs are oviposited, or where the next generation of hosts will be found. Following these suggestions could result in fruitful discovery of resting spore stages, greater understanding of entomophthoralean ecology and possibly utilization of these pathogens in biological control or integrated pest management. In this section, we will compare and contrast the conidial and resting spore stages of the following six species of fungi and their hosts: Massospora cicadina and Periodical Cicadas ( Magicicada spp.) Neozygites fresenii and Cotton Aphids ( Aphis gossypii ) Furia virescens and the True Armyworm ( Mythimna unipuncta ) Erynia aquatica and Aedes spp. Mosquitoes Eryniopsis lampyridarum and Soldier Beetles ( Chauliognathus spp.) Entomophaga maimaiga and Gypsy Moth ( Lymantria dispar ) 3.3.1. Massospora cicadina Infections in Periodical Cicadas This pathogen infects periodical cicadas in North America. Periodical cicadas ( Magicicada spp.) have the longest known life cycles of any insects [ 62 ]. These cicadas emerge in broods that occur either every 13 years (in the south) or 17 years (in the north). Therefore, it was assumed that the resting spores of M. cicadina must remain dormant in the soil either 13 or 17 years in order to infect new broods of cicadas [ 63 ] (but see Section 4.3.3 ). Both the conidial stage and the resting spore stage are produced in adult cicadas. Massospora cicadina is one of the most interesting of the fungal pathogens because it turns its living hosts into dispersers of both conidia and resting spores. The fungus forms its spores within the abdomen of the living cicadas but the insects remain able to fly and behave relatively normally and spread conidia to new victims or resting spores to the soil for the long wait for the next brood [ 64 ]. After spending either 13 or 17 years in the soil sucking on roots of plants, several weeks before the mass emergence cicada nymphs burrow near the surface of the soil. It is during this time and during the actual emergence of nymphs from the soil that they probably encounter infective spores produced by germinating resting spores and become infected. Research is needed to determine exactly when and where cicada nymphs become infected when resting spores germinate. Nymphs infected via germinating resting spores result in adult cicadas producing the conidial stage [ 65 ]. Infected cicadas with the conidial stage interact with and infect new hosts. Cicadas infected by conidia produce the resting spore stage. As in the conidial stage, the infected cicadas that produce resting spores remain alive, lose part of their terminal abdominal segments but in this case the abdomen becomes filled with a mass of loose dry resting spores. As the host flies about the emergence area, resting spores are scattered randomly onto the soil ( Figure 5 a,b). Cooley et al. [ 64 ] demonstrated that infected male periodical cicadas producing the conidial stage of the fungus exhibited "wing-flick" mating behaviors normally made only by female cicadas. This resulted in interactions where uninfected males attempted to mate with infected males, exposing more healthy males to infection. Somehow the fungal infection altered normal male behavior, resulting in increased spread of the pathogen. In contrast, male cicadas in which the fungus produced the resting spore stage did not exhibit this altered behavior. 3.3.2. Neozygites fresenii Infections in Aphis spp. Neozygites fresenii is an important pathogen of cotton aphids, Aphis gossypii , and other aphids in the USA and elsewhere [ 24 ]. It causes geographically and numerically vast epizootics in outbreak cotton aphid populations. Most infected individuals (both apterae and alatae) produce the conidial stage. Aphids producing conidia are held onto leaf surfaces by their mouthpart stylets alone ( Figure 5 c). The infection process is very complicated. Primary conidia are discharged from aphids, land on leaf surfaces, directly on aphids, or enter the air [ 66 , 67 ]. These aerial conidia can reach extremely high densities during epizootics and spread the pathogen widely and efficiently [ 66 ]. Primary conidia germinate to form capilliconidia on long slender stalks. Capilliconida are the infective stage and have a sticky apex called a mucoid hapteron that attaches them to new hosts. In contrast, when N. fresenii forms resting spores the cotton aphid cadaver is filled with black, ovoid resting spores and the body of the aphid liquefies, leaving a film of resting spores on the leaf surface ( Figure 5 d). Rain and leaf fall result in resting spores becoming deposited on the soil. Resting spores survive the winter and the next season germinate and infect new aphid populations. Ben-Ze'ev et al. [ 68 ] found that N. fresenii was an important pathogen of the citrus aphid, Aphis citricola , in Israel. They found that resting spores formed in aphids during the fall then germinated in the spring after a maturation/vernalization. Bitton et al. [ 69 ] found that N. fresenii overwintered as resting spores on the bark of citrus trees in Israel. These resting spores germinated in the spring in synchrony with the population increase of its host, Aphis spiraecola . 3.3.3. Furia virescens in the True Armyworm The true armyworm ( Mythimna unipuncta ) is a noctuid moth that is a serious pest of wheat, fescue, and other grasses [ 70 ]. It has many natural enemies including the fungus, Furia virescens . Furia virescens is a perfect example of how hosts producing the conidial stage of many Entomophthoromycotina are very obvious ( Figure 5 e), whereas the resting spore stages require greater effort to discover ( Figure 5 f). Infected larvae that produced the conidial stage died attached by their legs and prolegs to fescue plant stems a mean of 67 cm above the soil surface [ 70 ]. No rhizoids were formed and infected larvae were light grey and easily observed. Experiments demonstrated that larvae infected by conidia produced the resting spore stage [ 70 ]. Larvae with the resting spore stage were only discovered in the field by collecting live larvae beneath fescue plants and holding them for mortality. When M. unipuncta larvae collected from fescue were held in the lab, 21.6% produced resting spores when dying from F. virescens infections. The rest of the larvae either pupated or were killed by parasitoid wasps or unknown causes. Unlike the conidial stage, larvae producing resting spores died on the soil surface and large numbers of rhizoids attached the cadavers to the soil surface ( Figure 5 f). Without a diligent search of the soil surface or collection and rearing of large numbers of living larvae it would be impossible to find the cadavers containing the resting spore stage. Because resting spores are the source of inoculum for epizootics in subsequent years, it is clear that resting spore stages are a valuable resource in biological control. It is unknown how agronomic practices in wheat and fescue fields could affect survival of resting spores of F. virescens or similar management in cotton fields could affect survival of resting spores of N. fresenii . 3.3.4. Erynia aquatica Infections in Aedes spp. Mosquitoes Erynia aquatica is the only member of the Entomophthoromycotina known to infect immature stages of Culicidae [ 35 ]. This pathogen occurs in Aedes spp. that breed in snowpools, or ephemeral pools, and thus it must have a way of surviving periods when no hosts are available [ 35 ]. Mosquito larvae are infected in the spring by germ conidia produced from resting spores formed the previous year in adult mosquitoes. Infected immatures die either as larvae or pupae and are found floating on the water surface discharging conidia ( Figure 5 g). These conidia infect adult mosquitoes. Infected adults die attached by rhizoids to damp logs in or near the water pools ( Figure 5 h). Adult mosquitoes that produce the long-lived resting spores after death can disseminate the fungus to new mosquito breeding sites if they disperse before dying [ 48 ]. There are several striking differences between the conidial and resting spore stages of E. aquatica . First, host stage: conidia were produced only from late larval and pupal stages, while resting spores were produced only from adults. Second, rhizoids were not produced from conidial-stage mosquito immatures, whereas copious rhizoids were formed from adults producing resting spores. The rhizoids held the adult hosts firmly attached to moist wood in the snowpools, such as sticks and logs. Third, infected immatures were found floating on the surfaces of snowpools while infected adults with resting spores were found attached to moist logs associated with the snow pools. 3.3.5. Eryniopsis lampyridarum Infections in Soldier Beetles One of the most interesting of insect pathogens is Eryniopsis lampyridarum . It appears to have a host range restricted to two species of Coleoptera in the Cantharidae: Chauliognathus pensylvanicus and C. marginatus [ 71 ]. Adult beetles feed on pollen and also form mating leks on flowers. Adult beetles infected with the conidial stage of the fungus become attached to flowers before death by grasping a flower tightly with their mandibles ( Figure 5 i). There are no fungal attachment structures such as rhizoids and the legs are not involved in grasping the plant. The fungus causes its hosts to grasp plants with their mandibles, but the mechanism behind this is unknown. Similar behavior is exhibited by "zombie ants" infected with Ophiocordyceps spp. (Ascomycota) [ 72 ]. It is thought that the fungus produes chemicals that manipulate the ant brain. Infected beetles producing conidia are held in the midst of feeding and mating adult beetles, where transmission of conidia to new hosts can occur. Research by Steinkraus et al. [ 71 ] indicated that primary conidia were not explosively discharged (but see Carner [ 73 ] for a system where E. lampyridarum conidia apparently were actively discharged). By collecting large numbers of living adult C. pensylvanicus from flowers and holding them in the laboratory, it was shown that beetles producing resting spores stage fall to the ground, with no external growth of the fungus whatsoever (i.e., no cystidia, rhizoids, or conidiophores). Infected beetle abdomens were filled with dark brown resting spores ( Figure 5 j) and were friable and easily broken. Presumably the bodies break apart or are scavenged by other organisms and the resting spores are distributed on the soil under the flowering plants where the adults had fed and mated. There, they remain until the following spring or summer when they germinate and infect new soldier beetles. 3.3.6. Entomophaga maimaiga and Gypsy Moth, Lymantria dispar The locations of cadavers of gypsy moth, Lymantria dispar , larvae dying from E. maimaiga infections generally differ based on larval instar [ 74 ], which, in this host, also means that different types of spores are formed at different locations as conidia are produced from early instars and resting spores are usually produced from later instars [ 49 ]. Larvae of this species feed on leaves of many tree species and earlier instars are generally found in tree canopies. Conidia are actively ejected from cadavers of earlier instars killed by E. maimaiga , which are found with prolegs grasping the undersides of the wood of leaf-bearing twigs and the anterior portion of the body at a 90° angle to the twig, hanging vertically, with the head downward [ 75 ]. Later instar gypsy moth larvae have unusual behavior for lepidopteran larvae, as later instars climb trees every evening and descend in early morning to rest in darkened locations, often in the leaf litter, during the daylight hours; therefore, it is normal for later instars to walk up and down tree trunks each day [ 76 ]. Cadavers of later instar gypsy moth larvae that contain resting spores are usually found attached vertically to tree trunks, with prolegs extended horizontally and attached to the bark, and with heads downward ( Figure 2 ) [ 75 , 77 ]. Thus, some aspects of the differential distribution of conidia versus resting spore producing cadavers are consistent with the normal locations for gypsy moth larvae of early versus later instars [ 74 ]. However, some pre-death behaviors, for example, early instar larvae grasping twigs and not being on leaves and later instars having prolegs extended laterally, could be caused by pre-death manipulation of larval behavior by the fungus. 3.1. Resting Spores Versus Conidia and Species Identity Most entomophthoralean species can produce two types of spores (conidia and resting spores) from one host individual. Sometimes, both conidia and resting spores are produced from one cadaver (e.g., Entomophaga maimaiga ), but more commonly, only one type of spore is produced from the cadaver of one host individual. Usually, resting spores are formed under different conditions or host states than conidia (see Section 3.2 ). In many ways, entomophthoralean pathogens that produce conidia and resting spores can resemble two different species. The locations of the host cadavers, the manipulations of the host behavior before and after death, the fungal structures such as rhizoid holdfasts, as well as the fungal reproductive forms differ so much that if only one form (i.e., type of spore) is found, this may be described as a separate species from the other form (see Section 3.3 ). When resting spores are formed within host stages that are hidden and not easily found (e.g., within the soil), resting spores might never have been found and have not been described for that species. Conversely, sometimes resting spore-bearing hosts are found but individuals of the same host species in which conidia are produced are not found. In such a case, the species has been placed in the genus Tarichium . However, it is possible that the conidial stages have already been described as a different species. This sets up a situation similar to Ascomycota, where one species might have two different names, which has occurred when ascomycete anamorph and teleomorph forms are disconnected. The potential disjunct occurrence of different types of spores demonstrates the importance of detailed studies over time of a host/pathogen in order to understand all the stages. For instance, in the case of Erynia aquatica , the first researchers to study this pathogen of Aedes spp. snowpool mosquitoes found only conidial stages ( Figure 5 g). They correctly hypothesized that resting spores must exist because the hosts were univoltine [ 35 , 47 ]. Later, this hypothesis was proven to be true when Steinkraus and Kramer [ 48 ] found and described the resting spores ( Figure 5 h). 3.2. Resting Spore Production Empirical studies have repeatedly reported that resting spores are formed towards the end of a season, when few of the hosts will be present. Studies have demonstrated that numerous biotic and abiotic factors are associated with resting spore formation, but these can differ for different host/pathogen systems ( Table 2 ). For example, E. maimaiga shifted from making conidia to making resting spores at warmer temperatures, as later larval instars of its univoltine host pupate in summer. In contrast, Entomophthora muscae and Neozygites floridana produced resting spores at colder temperatures, when hosts would no longer be active. In the case of Eryniopsis lampyridarum in soldier beetles, resting spores are produced when the adult hosts are abundant. 3.3. Differential Locations of Cadavers Producing Resting Spores versus Conidia: Six Case Histories Because the conidial stage of entomophthoralean fungi functions in short term dissemination of the pathogen to new hosts and conidia are not durable structures that survive many months or years, hosts producing the conidial stage must be held in situations where hosts are active. For example, conidia-producing cadavers of rhagionids infected by Erynia ithacensis are underneath leaves [ 58 ]; Entomophthora scatophagae producing conidia from dead yellow dung flies ( Scatophaga stercoraria ) are found on flowering grasses where other dung flies are perching [ 59 ]; or, in the case of N. fresenii , infected conidial stage aphids are attached by their mouthpart stylets to the plant ( Figure 4 c), and spores are explosively discharged onto adjacent aphids in the colony, onto leaves, or into the air [ 60 ]. Many arthropod pathogenic Entomophthoromycotina are known only from their conidial stages [ 61 ]. There are two reasons for this. First, hosts killed by the conidial stage often occur in highly visible situations, such as on plants ( Figure 5 e), held by rhizoids, legs, or mouthparts ( Figure 5 i). From elevated positions conidia can be discharged onto potential hosts. In contrast, hosts producing resting spores are not as conspicuous. Second, many studies on arthropod hosts of Entomophthoromycotina have not been carried out in detail over a long enough period to detect hosts with resting spores that may occur in older hosts or towards the end of the season. With any entomophthoralean fungus that infects univoltine hosts, or multivoltine hosts that have an extended period when hosts are not available (such as during winter), there must be resting spores produced (or some other survival structure, e.g., N. floridana hyphal bodies within hibernating mites [ 44 ] or thick-walled conidia of the aphid pathogen Pandora neoaphidis [ 7 ]). Only careful studies over time with many specimens will result in finding the resting spore stage, as well as the conidial stage (or vice versa). Therefore, most species of Entomophthoromycotina must have a resting spore stage, but many have not yet have been discovered. In order to find hosts containing resting spores, scientists must examine host populations over time, often focusing on older hosts. If the prevalence of hosts with resting spores is low, one must collect many hosts, perhaps hundreds both alive and dead, keep them and examine them for resting spores that may develop over time (e.g., [ 43 , 44 ]). Careful, thorough searches should also be made of the soil surface or other areas near where hosts dying of conidial stages are found, where host eggs are oviposited, or where the next generation of hosts will be found. Following these suggestions could result in fruitful discovery of resting spore stages, greater understanding of entomophthoralean ecology and possibly utilization of these pathogens in biological control or integrated pest management. In this section, we will compare and contrast the conidial and resting spore stages of the following six species of fungi and their hosts: Massospora cicadina and Periodical Cicadas ( Magicicada spp.) Neozygites fresenii and Cotton Aphids ( Aphis gossypii ) Furia virescens and the True Armyworm ( Mythimna unipuncta ) Erynia aquatica and Aedes spp. Mosquitoes Eryniopsis lampyridarum and Soldier Beetles ( Chauliognathus spp.) Entomophaga maimaiga and Gypsy Moth ( Lymantria dispar ) 3.3.1. Massospora cicadina Infections in Periodical Cicadas This pathogen infects periodical cicadas in North America. Periodical cicadas ( Magicicada spp.) have the longest known life cycles of any insects [ 62 ]. These cicadas emerge in broods that occur either every 13 years (in the south) or 17 years (in the north). Therefore, it was assumed that the resting spores of M. cicadina must remain dormant in the soil either 13 or 17 years in order to infect new broods of cicadas [ 63 ] (but see Section 4.3.3 ). Both the conidial stage and the resting spore stage are produced in adult cicadas. Massospora cicadina is one of the most interesting of the fungal pathogens because it turns its living hosts into dispersers of both conidia and resting spores. The fungus forms its spores within the abdomen of the living cicadas but the insects remain able to fly and behave relatively normally and spread conidia to new victims or resting spores to the soil for the long wait for the next brood [ 64 ]. After spending either 13 or 17 years in the soil sucking on roots of plants, several weeks before the mass emergence cicada nymphs burrow near the surface of the soil. It is during this time and during the actual emergence of nymphs from the soil that they probably encounter infective spores produced by germinating resting spores and become infected. Research is needed to determine exactly when and where cicada nymphs become infected when resting spores germinate. Nymphs infected via germinating resting spores result in adult cicadas producing the conidial stage [ 65 ]. Infected cicadas with the conidial stage interact with and infect new hosts. Cicadas infected by conidia produce the resting spore stage. As in the conidial stage, the infected cicadas that produce resting spores remain alive, lose part of their terminal abdominal segments but in this case the abdomen becomes filled with a mass of loose dry resting spores. As the host flies about the emergence area, resting spores are scattered randomly onto the soil ( Figure 5 a,b). Cooley et al. [ 64 ] demonstrated that infected male periodical cicadas producing the conidial stage of the fungus exhibited "wing-flick" mating behaviors normally made only by female cicadas. This resulted in interactions where uninfected males attempted to mate with infected males, exposing more healthy males to infection. Somehow the fungal infection altered normal male behavior, resulting in increased spread of the pathogen. In contrast, male cicadas in which the fungus produced the resting spore stage did not exhibit this altered behavior. 3.3.2. Neozygites fresenii Infections in Aphis spp. Neozygites fresenii is an important pathogen of cotton aphids, Aphis gossypii , and other aphids in the USA and elsewhere [ 24 ]. It causes geographically and numerically vast epizootics in outbreak cotton aphid populations. Most infected individuals (both apterae and alatae) produce the conidial stage. Aphids producing conidia are held onto leaf surfaces by their mouthpart stylets alone ( Figure 5 c). The infection process is very complicated. Primary conidia are discharged from aphids, land on leaf surfaces, directly on aphids, or enter the air [ 66 , 67 ]. These aerial conidia can reach extremely high densities during epizootics and spread the pathogen widely and efficiently [ 66 ]. Primary conidia germinate to form capilliconidia on long slender stalks. Capilliconida are the infective stage and have a sticky apex called a mucoid hapteron that attaches them to new hosts. In contrast, when N. fresenii forms resting spores the cotton aphid cadaver is filled with black, ovoid resting spores and the body of the aphid liquefies, leaving a film of resting spores on the leaf surface ( Figure 5 d). Rain and leaf fall result in resting spores becoming deposited on the soil. Resting spores survive the winter and the next season germinate and infect new aphid populations. Ben-Ze'ev et al. [ 68 ] found that N. fresenii was an important pathogen of the citrus aphid, Aphis citricola , in Israel. They found that resting spores formed in aphids during the fall then germinated in the spring after a maturation/vernalization. Bitton et al. [ 69 ] found that N. fresenii overwintered as resting spores on the bark of citrus trees in Israel. These resting spores germinated in the spring in synchrony with the population increase of its host, Aphis spiraecola . 3.3.3. Furia virescens in the True Armyworm The true armyworm ( Mythimna unipuncta ) is a noctuid moth that is a serious pest of wheat, fescue, and other grasses [ 70 ]. It has many natural enemies including the fungus, Furia virescens . Furia virescens is a perfect example of how hosts producing the conidial stage of many Entomophthoromycotina are very obvious ( Figure 5 e), whereas the resting spore stages require greater effort to discover ( Figure 5 f). Infected larvae that produced the conidial stage died attached by their legs and prolegs to fescue plant stems a mean of 67 cm above the soil surface [ 70 ]. No rhizoids were formed and infected larvae were light grey and easily observed. Experiments demonstrated that larvae infected by conidia produced the resting spore stage [ 70 ]. Larvae with the resting spore stage were only discovered in the field by collecting live larvae beneath fescue plants and holding them for mortality. When M. unipuncta larvae collected from fescue were held in the lab, 21.6% produced resting spores when dying from F. virescens infections. The rest of the larvae either pupated or were killed by parasitoid wasps or unknown causes. Unlike the conidial stage, larvae producing resting spores died on the soil surface and large numbers of rhizoids attached the cadavers to the soil surface ( Figure 5 f). Without a diligent search of the soil surface or collection and rearing of large numbers of living larvae it would be impossible to find the cadavers containing the resting spore stage. Because resting spores are the source of inoculum for epizootics in subsequent years, it is clear that resting spore stages are a valuable resource in biological control. It is unknown how agronomic practices in wheat and fescue fields could affect survival of resting spores of F. virescens or similar management in cotton fields could affect survival of resting spores of N. fresenii . 3.3.4. Erynia aquatica Infections in Aedes spp. Mosquitoes Erynia aquatica is the only member of the Entomophthoromycotina known to infect immature stages of Culicidae [ 35 ]. This pathogen occurs in Aedes spp. that breed in snowpools, or ephemeral pools, and thus it must have a way of surviving periods when no hosts are available [ 35 ]. Mosquito larvae are infected in the spring by germ conidia produced from resting spores formed the previous year in adult mosquitoes. Infected immatures die either as larvae or pupae and are found floating on the water surface discharging conidia ( Figure 5 g). These conidia infect adult mosquitoes. Infected adults die attached by rhizoids to damp logs in or near the water pools ( Figure 5 h). Adult mosquitoes that produce the long-lived resting spores after death can disseminate the fungus to new mosquito breeding sites if they disperse before dying [ 48 ]. There are several striking differences between the conidial and resting spore stages of E. aquatica . First, host stage: conidia were produced only from late larval and pupal stages, while resting spores were produced only from adults. Second, rhizoids were not produced from conidial-stage mosquito immatures, whereas copious rhizoids were formed from adults producing resting spores. The rhizoids held the adult hosts firmly attached to moist wood in the snowpools, such as sticks and logs. Third, infected immatures were found floating on the surfaces of snowpools while infected adults with resting spores were found attached to moist logs associated with the snow pools. 3.3.5. Eryniopsis lampyridarum Infections in Soldier Beetles One of the most interesting of insect pathogens is Eryniopsis lampyridarum . It appears to have a host range restricted to two species of Coleoptera in the Cantharidae: Chauliognathus pensylvanicus and C. marginatus [ 71 ]. Adult beetles feed on pollen and also form mating leks on flowers. Adult beetles infected with the conidial stage of the fungus become attached to flowers before death by grasping a flower tightly with their mandibles ( Figure 5 i). There are no fungal attachment structures such as rhizoids and the legs are not involved in grasping the plant. The fungus causes its hosts to grasp plants with their mandibles, but the mechanism behind this is unknown. Similar behavior is exhibited by "zombie ants" infected with Ophiocordyceps spp. (Ascomycota) [ 72 ]. It is thought that the fungus produes chemicals that manipulate the ant brain. Infected beetles producing conidia are held in the midst of feeding and mating adult beetles, where transmission of conidia to new hosts can occur. Research by Steinkraus et al. [ 71 ] indicated that primary conidia were not explosively discharged (but see Carner [ 73 ] for a system where E. lampyridarum conidia apparently were actively discharged). By collecting large numbers of living adult C. pensylvanicus from flowers and holding them in the laboratory, it was shown that beetles producing resting spores stage fall to the ground, with no external growth of the fungus whatsoever (i.e., no cystidia, rhizoids, or conidiophores). Infected beetle abdomens were filled with dark brown resting spores ( Figure 5 j) and were friable and easily broken. Presumably the bodies break apart or are scavenged by other organisms and the resting spores are distributed on the soil under the flowering plants where the adults had fed and mated. There, they remain until the following spring or summer when they germinate and infect new soldier beetles. 3.3.6. Entomophaga maimaiga and Gypsy Moth, Lymantria dispar The locations of cadavers of gypsy moth, Lymantria dispar , larvae dying from E. maimaiga infections generally differ based on larval instar [ 74 ], which, in this host, also means that different types of spores are formed at different locations as conidia are produced from early instars and resting spores are usually produced from later instars [ 49 ]. Larvae of this species feed on leaves of many tree species and earlier instars are generally found in tree canopies. Conidia are actively ejected from cadavers of earlier instars killed by E. maimaiga , which are found with prolegs grasping the undersides of the wood of leaf-bearing twigs and the anterior portion of the body at a 90° angle to the twig, hanging vertically, with the head downward [ 75 ]. Later instar gypsy moth larvae have unusual behavior for lepidopteran larvae, as later instars climb trees every evening and descend in early morning to rest in darkened locations, often in the leaf litter, during the daylight hours; therefore, it is normal for later instars to walk up and down tree trunks each day [ 76 ]. Cadavers of later instar gypsy moth larvae that contain resting spores are usually found attached vertically to tree trunks, with prolegs extended horizontally and attached to the bark, and with heads downward ( Figure 2 ) [ 75 , 77 ]. Thus, some aspects of the differential distribution of conidia versus resting spore producing cadavers are consistent with the normal locations for gypsy moth larvae of early versus later instars [ 74 ]. However, some pre-death behaviors, for example, early instar larvae grasping twigs and not being on leaves and later instars having prolegs extended laterally, could be caused by pre-death manipulation of larval behavior by the fungus. 3.3.1. Massospora cicadina Infections in Periodical Cicadas This pathogen infects periodical cicadas in North America. Periodical cicadas ( Magicicada spp.) have the longest known life cycles of any insects [ 62 ]. These cicadas emerge in broods that occur either every 13 years (in the south) or 17 years (in the north). Therefore, it was assumed that the resting spores of M. cicadina must remain dormant in the soil either 13 or 17 years in order to infect new broods of cicadas [ 63 ] (but see Section 4.3.3 ). Both the conidial stage and the resting spore stage are produced in adult cicadas. Massospora cicadina is one of the most interesting of the fungal pathogens because it turns its living hosts into dispersers of both conidia and resting spores. The fungus forms its spores within the abdomen of the living cicadas but the insects remain able to fly and behave relatively normally and spread conidia to new victims or resting spores to the soil for the long wait for the next brood [ 64 ]. After spending either 13 or 17 years in the soil sucking on roots of plants, several weeks before the mass emergence cicada nymphs burrow near the surface of the soil. It is during this time and during the actual emergence of nymphs from the soil that they probably encounter infective spores produced by germinating resting spores and become infected. Research is needed to determine exactly when and where cicada nymphs become infected when resting spores germinate. Nymphs infected via germinating resting spores result in adult cicadas producing the conidial stage [ 65 ]. Infected cicadas with the conidial stage interact with and infect new hosts. Cicadas infected by conidia produce the resting spore stage. As in the conidial stage, the infected cicadas that produce resting spores remain alive, lose part of their terminal abdominal segments but in this case the abdomen becomes filled with a mass of loose dry resting spores. As the host flies about the emergence area, resting spores are scattered randomly onto the soil ( Figure 5 a,b). Cooley et al. [ 64 ] demonstrated that infected male periodical cicadas producing the conidial stage of the fungus exhibited "wing-flick" mating behaviors normally made only by female cicadas. This resulted in interactions where uninfected males attempted to mate with infected males, exposing more healthy males to infection. Somehow the fungal infection altered normal male behavior, resulting in increased spread of the pathogen. In contrast, male cicadas in which the fungus produced the resting spore stage did not exhibit this altered behavior. 3.3.2. Neozygites fresenii Infections in Aphis spp. Neozygites fresenii is an important pathogen of cotton aphids, Aphis gossypii , and other aphids in the USA and elsewhere [ 24 ]. It causes geographically and numerically vast epizootics in outbreak cotton aphid populations. Most infected individuals (both apterae and alatae) produce the conidial stage. Aphids producing conidia are held onto leaf surfaces by their mouthpart stylets alone ( Figure 5 c). The infection process is very complicated. Primary conidia are discharged from aphids, land on leaf surfaces, directly on aphids, or enter the air [ 66 , 67 ]. These aerial conidia can reach extremely high densities during epizootics and spread the pathogen widely and efficiently [ 66 ]. Primary conidia germinate to form capilliconidia on long slender stalks. Capilliconida are the infective stage and have a sticky apex called a mucoid hapteron that attaches them to new hosts. In contrast, when N. fresenii forms resting spores the cotton aphid cadaver is filled with black, ovoid resting spores and the body of the aphid liquefies, leaving a film of resting spores on the leaf surface ( Figure 5 d). Rain and leaf fall result in resting spores becoming deposited on the soil. Resting spores survive the winter and the next season germinate and infect new aphid populations. Ben-Ze'ev et al. [ 68 ] found that N. fresenii was an important pathogen of the citrus aphid, Aphis citricola , in Israel. They found that resting spores formed in aphids during the fall then germinated in the spring after a maturation/vernalization. Bitton et al. [ 69 ] found that N. fresenii overwintered as resting spores on the bark of citrus trees in Israel. These resting spores germinated in the spring in synchrony with the population increase of its host, Aphis spiraecola . 3.3.3. Furia virescens in the True Armyworm The true armyworm ( Mythimna unipuncta ) is a noctuid moth that is a serious pest of wheat, fescue, and other grasses [ 70 ]. It has many natural enemies including the fungus, Furia virescens . Furia virescens is a perfect example of how hosts producing the conidial stage of many Entomophthoromycotina are very obvious ( Figure 5 e), whereas the resting spore stages require greater effort to discover ( Figure 5 f). Infected larvae that produced the conidial stage died attached by their legs and prolegs to fescue plant stems a mean of 67 cm above the soil surface [ 70 ]. No rhizoids were formed and infected larvae were light grey and easily observed. Experiments demonstrated that larvae infected by conidia produced the resting spore stage [ 70 ]. Larvae with the resting spore stage were only discovered in the field by collecting live larvae beneath fescue plants and holding them for mortality. When M. unipuncta larvae collected from fescue were held in the lab, 21.6% produced resting spores when dying from F. virescens infections. The rest of the larvae either pupated or were killed by parasitoid wasps or unknown causes. Unlike the conidial stage, larvae producing resting spores died on the soil surface and large numbers of rhizoids attached the cadavers to the soil surface ( Figure 5 f). Without a diligent search of the soil surface or collection and rearing of large numbers of living larvae it would be impossible to find the cadavers containing the resting spore stage. Because resting spores are the source of inoculum for epizootics in subsequent years, it is clear that resting spore stages are a valuable resource in biological control. It is unknown how agronomic practices in wheat and fescue fields could affect survival of resting spores of F. virescens or similar management in cotton fields could affect survival of resting spores of N. fresenii . 3.3.4. Erynia aquatica Infections in Aedes spp. Mosquitoes Erynia aquatica is the only member of the Entomophthoromycotina known to infect immature stages of Culicidae [ 35 ]. This pathogen occurs in Aedes spp. that breed in snowpools, or ephemeral pools, and thus it must have a way of surviving periods when no hosts are available [ 35 ]. Mosquito larvae are infected in the spring by germ conidia produced from resting spores formed the previous year in adult mosquitoes. Infected immatures die either as larvae or pupae and are found floating on the water surface discharging conidia ( Figure 5 g). These conidia infect adult mosquitoes. Infected adults die attached by rhizoids to damp logs in or near the water pools ( Figure 5 h). Adult mosquitoes that produce the long-lived resting spores after death can disseminate the fungus to new mosquito breeding sites if they disperse before dying [ 48 ]. There are several striking differences between the conidial and resting spore stages of E. aquatica . First, host stage: conidia were produced only from late larval and pupal stages, while resting spores were produced only from adults. Second, rhizoids were not produced from conidial-stage mosquito immatures, whereas copious rhizoids were formed from adults producing resting spores. The rhizoids held the adult hosts firmly attached to moist wood in the snowpools, such as sticks and logs. Third, infected immatures were found floating on the surfaces of snowpools while infected adults with resting spores were found attached to moist logs associated with the snow pools. 3.3.5. Eryniopsis lampyridarum Infections in Soldier Beetles One of the most interesting of insect pathogens is Eryniopsis lampyridarum . It appears to have a host range restricted to two species of Coleoptera in the Cantharidae: Chauliognathus pensylvanicus and C. marginatus [ 71 ]. Adult beetles feed on pollen and also form mating leks on flowers. Adult beetles infected with the conidial stage of the fungus become attached to flowers before death by grasping a flower tightly with their mandibles ( Figure 5 i). There are no fungal attachment structures such as rhizoids and the legs are not involved in grasping the plant. The fungus causes its hosts to grasp plants with their mandibles, but the mechanism behind this is unknown. Similar behavior is exhibited by "zombie ants" infected with Ophiocordyceps spp. (Ascomycota) [ 72 ]. It is thought that the fungus produes chemicals that manipulate the ant brain. Infected beetles producing conidia are held in the midst of feeding and mating adult beetles, where transmission of conidia to new hosts can occur. Research by Steinkraus et al. [ 71 ] indicated that primary conidia were not explosively discharged (but see Carner [ 73 ] for a system where E. lampyridarum conidia apparently were actively discharged). By collecting large numbers of living adult C. pensylvanicus from flowers and holding them in the laboratory, it was shown that beetles producing resting spores stage fall to the ground, with no external growth of the fungus whatsoever (i.e., no cystidia, rhizoids, or conidiophores). Infected beetle abdomens were filled with dark brown resting spores ( Figure 5 j) and were friable and easily broken. Presumably the bodies break apart or are scavenged by other organisms and the resting spores are distributed on the soil under the flowering plants where the adults had fed and mated. There, they remain until the following spring or summer when they germinate and infect new soldier beetles. 3.3.6. Entomophaga maimaiga and Gypsy Moth, Lymantria dispar The locations of cadavers of gypsy moth, Lymantria dispar , larvae dying from E. maimaiga infections generally differ based on larval instar [ 74 ], which, in this host, also means that different types of spores are formed at different locations as conidia are produced from early instars and resting spores are usually produced from later instars [ 49 ]. Larvae of this species feed on leaves of many tree species and earlier instars are generally found in tree canopies. Conidia are actively ejected from cadavers of earlier instars killed by E. maimaiga , which are found with prolegs grasping the undersides of the wood of leaf-bearing twigs and the anterior portion of the body at a 90° angle to the twig, hanging vertically, with the head downward [ 75 ]. Later instar gypsy moth larvae have unusual behavior for lepidopteran larvae, as later instars climb trees every evening and descend in early morning to rest in darkened locations, often in the leaf litter, during the daylight hours; therefore, it is normal for later instars to walk up and down tree trunks each day [ 76 ]. Cadavers of later instar gypsy moth larvae that contain resting spores are usually found attached vertically to tree trunks, with prolegs extended horizontally and attached to the bark, and with heads downward ( Figure 2 ) [ 75 , 77 ]. Thus, some aspects of the differential distribution of conidia versus resting spore producing cadavers are consistent with the normal locations for gypsy moth larvae of early versus later instars [ 74 ]. However, some pre-death behaviors, for example, early instar larvae grasping twigs and not being on leaves and later instars having prolegs extended laterally, could be caused by pre-death manipulation of larval behavior by the fungus. 4. Resting Spores in the Environment 4.1. Quantification of Resting Spores in Soil A wet sieve method that may or may not be followed by density gradient separation has been routinely utilized for detection and quantification of resting spores of Entomophthoromycotina in the soil (e.g., [ 78 , 79 , 80 , 81 , 82 ]). Although this method is convenient to use, being adaptable to various species, it does not provide the specificity required when tracking an epizootic associated with a specific fungus of a given insect pest. The only way to track epizootics accurately and precisely is the application of molecular methods relying on species- or strain-specific primers. Thomsen and Jensen [ 83 ] designed species-specific primers that, in combination with a nested polymerase chain reaction (PCR) strategy, can be used to identify E. muscae resting spores. This two-step technique improved sensitivity and specificity of PCR assays when sampling DNA from in vivo samples (i.e., resting spores in host cadavers) [ 83 ]. In combination with real-time PCR, specific primers can also be used for quantitative detection of resting spores. So far, a species-specific real-time PCR assay has been developed for the resting spores of only one fungal species in the Entomophthoromycotina, E. maimaiga [ 84 ]. The primer pair and assay conditions developed by Castrillo et al. [ 84 ] were specific to E. maimaiga and were able to detect different strains of this species, but did not amplify the closely related E. aulicae . Entomophaga maimaiga is in the E. aulicae species complex [ 85 ]. A critical step in this method was breaking the thick walls of resting spores in soil samples to efficiently release fungal DNA. This was achieved through the use of high density beads such as silica and zirconia silica in combination with homogenization at high speeds (up to 5000 rpm) for 1 min. As the resting spores that were quantified were mixed in soil samples, the extracted fungal DNA samples were contaminated with DNA from other soil microorganisms and with soil organic matter. Humic acids present in the organic layer of the soil, where E. maimaiga resting spores are commonly found, can inhibit PCR reactions and affect assay sensitivity. Castrillo et al. [ 84 ] observed that at a high titer (10 4 resting spores/g of soil), estimates from mixed DNA samples were close to actual titers in different soil types tested. At lower titers, however, estimates obtained were significantly lower in soil samples with high percentages of organic matter present. 4.2. Spatial Distribution and Densities in the Environment The soil is generally considered the location where resting spores remain until receiving stimuli to germinate; therefore, resting spores must travel from infected hosts to the soil, but they have no independent means for transportation. Cadavers of cassava green mites, Mononychellus tanajoa , containing Neozygites tanajoae resting spores, are often attached to leaves by rhizoids. Cadavers then break apart while on top of leaves and resting spores are deposited on the leaf surface [ 43 ] (and see Figure 5 d). Less commonly, M. tanajoa cadavers containing resting spores fall to the ground intact. In either case, resting spores are washed into the soil. Later instar gypsy moth larvae that die from E. maimaiga infections predominantly contain resting spores within cadavers. Many cadavers are initially found on tree trunks (see Section 3.3.6 ), where they dry and subsequently fall to the ground. Once cadavers start to decompose, resting spores are released from cadavers into the soil; this process usually requires 1–2 weeks. Low titers of resting spores also overwinter and survive on tree bark near the bases of trees [ 75 ]. The distribution of resting spores in the environment has been quantified for E. maimaiga infecting gypsy moth larvae. Resting spores are found in highest concentrations (mean = 4751 resting spores/g dry soil) in the organic layer of the soil, 0–10 cm from the bases of oak trees, with numbers decreasing precipitously with increasing distance from the tree trunk as well as with increasing depth in the soil [ 86 ]. Resting spores extracted from the bark of trees reached a maximum mean of 277 resting spores/25 cm 2 , much lower than than densities in the soil at the bases of trees. Gypsy moth larvae do not enter the soil and when larvae were exposed to soil with E. maimaiga resting spores buried 1 cm below the soil surface, they did not become infected (although exposure to this same sample of spores on the surface of the soil resulted in infection); therefore, the fact that resting spores predominantly remain at or near the soil surface is appropriate for this host. Resting spores of E. maimaiga are also thought to be aggregated among trees; resting spore-bearing gypsy moth cadavers were found in larger numbers on tree species preferred by the host and not on non-preferred hosts. It is assumed that this distribution would result in greater densities of resting spores at the bases of preferred host trees [ 87 ]. 4.3. Resting Spore Activity 4.3.1. Dormancy and Germination Resting spores are thought to normally be constitutively dormant after production and maturation [ 6 , 88 ], although Conidiobolus thromboides does not appear to go into dormancy [ 42 ]. It has been hypothesized that dormancy acts to synchronize resting spore germination with developing insect hosts [ 89 ]. Once resting spores are dormant, the thick wall resists stains and viability assessments have never been successful. Therefore, to date, the only way to determine whether or not resting spores are alive is to conduct trials to assess germination. However, the conditions necessary to break dormancy so that germination is possible have been determined for only eight species [ 42 , 45 ]. For seven of the eight species, periods of storage under colder temperatures varying from two weeks to nine months, and varying within and between species, are required before germination will begin. As an exception, Zoophthora canadensis requires a photoperiod of longer than 12 h of light per day for germination to begin. Exposure to hosts has not been shown to activate or accelerate germination of E. maimaiga resting spores [ 90 ]. There is a trend of more resting spores being responsive and germinating if the resting spores are older or have been stored at colder temperatures [ 89 , 91 ]. Entomophaga maimaiga resting spores produced under laboratory conditions and not allowed to dry after production did not go into dormancy and germinated asynchronously for at least 200 days [ 46 ]. When resting spores that had not dried were then stored at 4 °C for from 1 to 8 months, they also demonstrated this prolonged asynchronous germination. To germinate, resting spores dissolve the large oil droplets and thick wall and one or two germ tubes are formed, which protrude through the epispore if one is present [ 45 , 89 ]. Germination does not require nutrients, generally has been demonstrated at temperatures of less than 20 °C, and is relatively slow and asynchronous. For E. maimaiga resting spores, more germination occurred with exposure to a photoperiod of 14:10 (light:dark) when compared with 13:11 or 12:12 [ 45 ]. 4.3.2. Horizontal Transmission Due to Resting Spores Resting spores do not directly infect hosts. They germinate to produce infective germ conidia that can be actively ejected. In the case of Entomophaga maimaiga , the germ conidia look exactly like primary conidia produced externally from cadavers, although they are slightly smaller. However, for Zoophthora radicans , germinating resting spores produce capilliconidia that are not actively ejected. One resting spore has been shown to produce from one to five germ conidia and this varies by species. In E. maimaiga , when germ conidia infect, the fungus always produces only conidia in the resulting cadaver and not resting spores [ 42 ]. However, this was not found for Furia gastropachae as infections initiated when Malacosoma disstria larvae were exposed to soil containing resting spores usually produced either resting spores or conidia, with both types of spores produced in the same cadavers for 500 resting spores/g dry soil were present four years after epizootics [ 92 ]. For M. cicadina , it has been thought that resting spores settled onto the ground and remained dormant for 13 or 17 years and were synchronized to germinate at the time of the mass emergence of cicada broods. However, Duke et al. [ 38 ] conducted an experimental study of M. cicadina resting spores from 17-year periodical cicadas captured in Iowa. The resting spores were sprayed on soil plots in an Arkansas forest that was due for an emergence of 13-year cicadas the next year. Massospora cicadina resting spores were capable of germinating and infecting Magicicada tredecassini after only one year of dormancy. This suggests that the resting spores of M. cicadina might be stimulated to germinate by proximity to mature periodical cicada nymphs and are not synchronized to germinate only after 13 or 17 years. We hypothesize that a chemical cue from periodical cicada nymphs could stimulate the resting spores to germinate and suggest that this should be tested experimentally. 4.4. Ecosystem Level Interactions Resting spores allow long term persistence of fungi in the Entomophthoromycotina in the field [ 6 ], but their longevity or viability can be affected by abiotic (e.g., [ 46 , 99 ]) and biotic factors [ 102 , 103 ]). Interspecific interactions with other microorganisms in the soil can result in antagonism following contact. These microorganisms include fungi and fungi-like organisms (Oomycetes), collectively termed mycoparasites, that derive most or all of their nutrients from other fungi. Mycoparasitic relationships can be biotrophic (narrow host ranges, with complex, controlled, and relatively non-destructive interactions) or necrotrophic (broad host ranges, with unspecialized parasitic mechanism, and kill their host) [ 104 ]. The relationship, however, can change during the course of parasitism, from being biotrophic to necrotrophic, and can vary between hosts for a given mycoparasite. Mycoparasites have been described from Oomycetes (phylum Heterokontophyta) and different fungal phyla, more commonly in Ascomycota and Chytridiomycota for invasive necrotrophs [ 104 ]. Information on mycoparasites attacking entomopthoromycotan fungi is sparse, but is critical in understanding factors that affect the persistence and prevalence of resting spores in the soil and their horizontal transmission. A sampling of E. maimaiga from L. dispar cadavers by Hajek et al. [ 102 ] revealed azygospores parasitized by the chytrid Gaertneriomyces semiglobifer . Laboratory bioassays where E. maimaiga azygospores were exposed to this chytrid resulted in up to 82% parasitism 4 day post exposure and 94% after 8 day [ 102 ]. Although parasitism observed was higher in immature azygospores, the results showed that even thick-walled mature azygospores were susceptible to this mycoparasite. Resting spores are nutrient rich reservoirs and can be valuable resources for mycoparasitic fungi capable of penetrating their thick walls [ 105 ]. In a follow up survey on mycoparasites associated with E. maimaiga conducted by Castrillo et al. [ 103 ], the results revealed a high percentage (up to 90% or more) of parasitism in E. maimaiga resting spores in multiple sites tested. Using a combination of soil baiting and molecular methods, prevalence of mycopasitism was determined microscopically and presumptive mycoparasites were identified by PCR and sequencing of the ITS locus with taxon-specific primers. Evidence of parasitism included misshapen resting spores that were filled with distinct fungal structures of various forms ( Figure 6 ). Microscopic examinations also suggested the possibility of multiple mycoparasites attacking a given resting spore. Identification of these presumptive mycoparasites revealed a Pochonia sp. (Ascomycota) and at least three Pythium species or strains (Oomycota). Given the various morphologically distinct fungal structures detected inside parasitized resting spores, it is likely that there were other mycoparasites not identified by the molecular method used. A few of the taxon-specific ITS primers tested had limited detection ranges [ 103 ]. Unlike the study by Hajek et al. [ 102 ], these presumptive mycoparasites were not cultured and details of their interaction with E. maimaga resting spores has not been studied in detail. 4.1. Quantification of Resting Spores in Soil A wet sieve method that may or may not be followed by density gradient separation has been routinely utilized for detection and quantification of resting spores of Entomophthoromycotina in the soil (e.g., [ 78 , 79 , 80 , 81 , 82 ]). Although this method is convenient to use, being adaptable to various species, it does not provide the specificity required when tracking an epizootic associated with a specific fungus of a given insect pest. The only way to track epizootics accurately and precisely is the application of molecular methods relying on species- or strain-specific primers. Thomsen and Jensen [ 83 ] designed species-specific primers that, in combination with a nested polymerase chain reaction (PCR) strategy, can be used to identify E. muscae resting spores. This two-step technique improved sensitivity and specificity of PCR assays when sampling DNA from in vivo samples (i.e., resting spores in host cadavers) [ 83 ]. In combination with real-time PCR, specific primers can also be used for quantitative detection of resting spores. So far, a species-specific real-time PCR assay has been developed for the resting spores of only one fungal species in the Entomophthoromycotina, E. maimaiga [ 84 ]. The primer pair and assay conditions developed by Castrillo et al. [ 84 ] were specific to E. maimaiga and were able to detect different strains of this species, but did not amplify the closely related E. aulicae . Entomophaga maimaiga is in the E. aulicae species complex [ 85 ]. A critical step in this method was breaking the thick walls of resting spores in soil samples to efficiently release fungal DNA. This was achieved through the use of high density beads such as silica and zirconia silica in combination with homogenization at high speeds (up to 5000 rpm) for 1 min. As the resting spores that were quantified were mixed in soil samples, the extracted fungal DNA samples were contaminated with DNA from other soil microorganisms and with soil organic matter. Humic acids present in the organic layer of the soil, where E. maimaiga resting spores are commonly found, can inhibit PCR reactions and affect assay sensitivity. Castrillo et al. [ 84 ] observed that at a high titer (10 4 resting spores/g of soil), estimates from mixed DNA samples were close to actual titers in different soil types tested. At lower titers, however, estimates obtained were significantly lower in soil samples with high percentages of organic matter present. 4.2. Spatial Distribution and Densities in the Environment The soil is generally considered the location where resting spores remain until receiving stimuli to germinate; therefore, resting spores must travel from infected hosts to the soil, but they have no independent means for transportation. Cadavers of cassava green mites, Mononychellus tanajoa , containing Neozygites tanajoae resting spores, are often attached to leaves by rhizoids. Cadavers then break apart while on top of leaves and resting spores are deposited on the leaf surface [ 43 ] (and see Figure 5 d). Less commonly, M. tanajoa cadavers containing resting spores fall to the ground intact. In either case, resting spores are washed into the soil. Later instar gypsy moth larvae that die from E. maimaiga infections predominantly contain resting spores within cadavers. Many cadavers are initially found on tree trunks (see Section 3.3.6 ), where they dry and subsequently fall to the ground. Once cadavers start to decompose, resting spores are released from cadavers into the soil; this process usually requires 1–2 weeks. Low titers of resting spores also overwinter and survive on tree bark near the bases of trees [ 75 ]. The distribution of resting spores in the environment has been quantified for E. maimaiga infecting gypsy moth larvae. Resting spores are found in highest concentrations (mean = 4751 resting spores/g dry soil) in the organic layer of the soil, 0–10 cm from the bases of oak trees, with numbers decreasing precipitously with increasing distance from the tree trunk as well as with increasing depth in the soil [ 86 ]. Resting spores extracted from the bark of trees reached a maximum mean of 277 resting spores/25 cm 2 , much lower than than densities in the soil at the bases of trees. Gypsy moth larvae do not enter the soil and when larvae were exposed to soil with E. maimaiga resting spores buried 1 cm below the soil surface, they did not become infected (although exposure to this same sample of spores on the surface of the soil resulted in infection); therefore, the fact that resting spores predominantly remain at or near the soil surface is appropriate for this host. Resting spores of E. maimaiga are also thought to be aggregated among trees; resting spore-bearing gypsy moth cadavers were found in larger numbers on tree species preferred by the host and not on non-preferred hosts. It is assumed that this distribution would result in greater densities of resting spores at the bases of preferred host trees [ 87 ]. 4.3. Resting Spore Activity 4.3.1. Dormancy and Germination Resting spores are thought to normally be constitutively dormant after production and maturation [ 6 , 88 ], although Conidiobolus thromboides does not appear to go into dormancy [ 42 ]. It has been hypothesized that dormancy acts to synchronize resting spore germination with developing insect hosts [ 89 ]. Once resting spores are dormant, the thick wall resists stains and viability assessments have never been successful. Therefore, to date, the only way to determine whether or not resting spores are alive is to conduct trials to assess germination. However, the conditions necessary to break dormancy so that germination is possible have been determined for only eight species [ 42 , 45 ]. For seven of the eight species, periods of storage under colder temperatures varying from two weeks to nine months, and varying within and between species, are required before germination will begin. As an exception, Zoophthora canadensis requires a photoperiod of longer than 12 h of light per day for germination to begin. Exposure to hosts has not been shown to activate or accelerate germination of E. maimaiga resting spores [ 90 ]. There is a trend of more resting spores being responsive and germinating if the resting spores are older or have been stored at colder temperatures [ 89 , 91 ]. Entomophaga maimaiga resting spores produced under laboratory conditions and not allowed to dry after production did not go into dormancy and germinated asynchronously for at least 200 days [ 46 ]. When resting spores that had not dried were then stored at 4 °C for from 1 to 8 months, they also demonstrated this prolonged asynchronous germination. To germinate, resting spores dissolve the large oil droplets and thick wall and one or two germ tubes are formed, which protrude through the epispore if one is present [ 45 , 89 ]. Germination does not require nutrients, generally has been demonstrated at temperatures of less than 20 °C, and is relatively slow and asynchronous. For E. maimaiga resting spores, more germination occurred with exposure to a photoperiod of 14:10 (light:dark) when compared with 13:11 or 12:12 [ 45 ]. 4.3.2. Horizontal Transmission Due to Resting Spores Resting spores do not directly infect hosts. They germinate to produce infective germ conidia that can be actively ejected. In the case of Entomophaga maimaiga , the germ conidia look exactly like primary conidia produced externally from cadavers, although they are slightly smaller. However, for Zoophthora radicans , germinating resting spores produce capilliconidia that are not actively ejected. One resting spore has been shown to produce from one to five germ conidia and this varies by species. In E. maimaiga , when germ conidia infect, the fungus always produces only conidia in the resulting cadaver and not resting spores [ 42 ]. However, this was not found for Furia gastropachae as infections initiated when Malacosoma disstria larvae were exposed to soil containing resting spores usually produced either resting spores or conidia, with both types of spores produced in the same cadavers for 500 resting spores/g dry soil were present four years after epizootics [ 92 ]. For M. cicadina , it has been thought that resting spores settled onto the ground and remained dormant for 13 or 17 years and were synchronized to germinate at the time of the mass emergence of cicada broods. However, Duke et al. [ 38 ] conducted an experimental study of M. cicadina resting spores from 17-year periodical cicadas captured in Iowa. The resting spores were sprayed on soil plots in an Arkansas forest that was due for an emergence of 13-year cicadas the next year. Massospora cicadina resting spores were capable of germinating and infecting Magicicada tredecassini after only one year of dormancy. This suggests that the resting spores of M. cicadina might be stimulated to germinate by proximity to mature periodical cicada nymphs and are not synchronized to germinate only after 13 or 17 years. We hypothesize that a chemical cue from periodical cicada nymphs could stimulate the resting spores to germinate and suggest that this should be tested experimentally. 4.3.1. Dormancy and Germination Resting spores are thought to normally be constitutively dormant after production and maturation [ 6 , 88 ], although Conidiobolus thromboides does not appear to go into dormancy [ 42 ]. It has been hypothesized that dormancy acts to synchronize resting spore germination with developing insect hosts [ 89 ]. Once resting spores are dormant, the thick wall resists stains and viability assessments have never been successful. Therefore, to date, the only way to determine whether or not resting spores are alive is to conduct trials to assess germination. However, the conditions necessary to break dormancy so that germination is possible have been determined for only eight species [ 42 , 45 ]. For seven of the eight species, periods of storage under colder temperatures varying from two weeks to nine months, and varying within and between species, are required before germination will begin. As an exception, Zoophthora canadensis requires a photoperiod of longer than 12 h of light per day for germination to begin. Exposure to hosts has not been shown to activate or accelerate germination of E. maimaiga resting spores [ 90 ]. There is a trend of more resting spores being responsive and germinating if the resting spores are older or have been stored at colder temperatures [ 89 , 91 ]. Entomophaga maimaiga resting spores produced under laboratory conditions and not allowed to dry after production did not go into dormancy and germinated asynchronously for at least 200 days [ 46 ]. When resting spores that had not dried were then stored at 4 °C for from 1 to 8 months, they also demonstrated this prolonged asynchronous germination. To germinate, resting spores dissolve the large oil droplets and thick wall and one or two germ tubes are formed, which protrude through the epispore if one is present [ 45 , 89 ]. Germination does not require nutrients, generally has been demonstrated at temperatures of less than 20 °C, and is relatively slow and asynchronous. For E. maimaiga resting spores, more germination occurred with exposure to a photoperiod of 14:10 (light:dark) when compared with 13:11 or 12:12 [ 45 ]. 4.3.2. Horizontal Transmission Due to Resting Spores Resting spores do not directly infect hosts. They germinate to produce infective germ conidia that can be actively ejected. In the case of Entomophaga maimaiga , the germ conidia look exactly like primary conidia produced externally from cadavers, although they are slightly smaller. However, for Zoophthora radicans , germinating resting spores produce capilliconidia that are not actively ejected. One resting spore has been shown to produce from one to five germ conidia and this varies by species. In E. maimaiga , when germ conidia infect, the fungus always produces only conidia in the resulting cadaver and not resting spores [ 42 ]. However, this was not found for Furia gastropachae as infections initiated when Malacosoma disstria larvae were exposed to soil containing resting spores usually produced either resting spores or conidia, with both types of spores produced in the same cadavers for 500 resting spores/g dry soil were present four years after epizootics [ 92 ]. For M. cicadina , it has been thought that resting spores settled onto the ground and remained dormant for 13 or 17 years and were synchronized to germinate at the time of the mass emergence of cicada broods. However, Duke et al. [ 38 ] conducted an experimental study of M. cicadina resting spores from 17-year periodical cicadas captured in Iowa. The resting spores were sprayed on soil plots in an Arkansas forest that was due for an emergence of 13-year cicadas the next year. Massospora cicadina resting spores were capable of germinating and infecting Magicicada tredecassini after only one year of dormancy. This suggests that the resting spores of M. cicadina might be stimulated to germinate by proximity to mature periodical cicada nymphs and are not synchronized to germinate only after 13 or 17 years. We hypothesize that a chemical cue from periodical cicada nymphs could stimulate the resting spores to germinate and suggest that this should be tested experimentally. 4.4. Ecosystem Level Interactions Resting spores allow long term persistence of fungi in the Entomophthoromycotina in the field [ 6 ], but their longevity or viability can be affected by abiotic (e.g., [ 46 , 99 ]) and biotic factors [ 102 , 103 ]). Interspecific interactions with other microorganisms in the soil can result in antagonism following contact. These microorganisms include fungi and fungi-like organisms (Oomycetes), collectively termed mycoparasites, that derive most or all of their nutrients from other fungi. Mycoparasitic relationships can be biotrophic (narrow host ranges, with complex, controlled, and relatively non-destructive interactions) or necrotrophic (broad host ranges, with unspecialized parasitic mechanism, and kill their host) [ 104 ]. The relationship, however, can change during the course of parasitism, from being biotrophic to necrotrophic, and can vary between hosts for a given mycoparasite. Mycoparasites have been described from Oomycetes (phylum Heterokontophyta) and different fungal phyla, more commonly in Ascomycota and Chytridiomycota for invasive necrotrophs [ 104 ]. Information on mycoparasites attacking entomopthoromycotan fungi is sparse, but is critical in understanding factors that affect the persistence and prevalence of resting spores in the soil and their horizontal transmission. A sampling of E. maimaiga from L. dispar cadavers by Hajek et al. [ 102 ] revealed azygospores parasitized by the chytrid Gaertneriomyces semiglobifer . Laboratory bioassays where E. maimaiga azygospores were exposed to this chytrid resulted in up to 82% parasitism 4 day post exposure and 94% after 8 day [ 102 ]. Although parasitism observed was higher in immature azygospores, the results showed that even thick-walled mature azygospores were susceptible to this mycoparasite. Resting spores are nutrient rich reservoirs and can be valuable resources for mycoparasitic fungi capable of penetrating their thick walls [ 105 ]. In a follow up survey on mycoparasites associated with E. maimaiga conducted by Castrillo et al. [ 103 ], the results revealed a high percentage (up to 90% or more) of parasitism in E. maimaiga resting spores in multiple sites tested. Using a combination of soil baiting and molecular methods, prevalence of mycopasitism was determined microscopically and presumptive mycoparasites were identified by PCR and sequencing of the ITS locus with taxon-specific primers. Evidence of parasitism included misshapen resting spores that were filled with distinct fungal structures of various forms ( Figure 6 ). Microscopic examinations also suggested the possibility of multiple mycoparasites attacking a given resting spore. Identification of these presumptive mycoparasites revealed a Pochonia sp. (Ascomycota) and at least three Pythium species or strains (Oomycota). Given the various morphologically distinct fungal structures detected inside parasitized resting spores, it is likely that there were other mycoparasites not identified by the molecular method used. A few of the taxon-specific ITS primers tested had limited detection ranges [ 103 ]. Unlike the study by Hajek et al. [ 102 ], these presumptive mycoparasites were not cultured and details of their interaction with E. maimaga resting spores has not been studied in detail. 5. Use of Resting Spores for Control Methods for in vitro production of resting spores have been developed for some entomophthoralean species (see table in [ 6 ]). Within E. maimaiga , there was extensive variability among different isolates regarding the numbers of resting spores that were formed in vitro [ 106 ]. Mass production methods were developed for a few aphid pathogenic species of Conidiobolus that readily make resting spores in vitro [ 6 ]. However, these methods did not result in marketable products. As an alternative, resting spores of E. maimaiga have been obtained for control purposes by collecting gypsy moth cadavers filled with resting spores and releasing them in new areas. This method was used to hasten the spread of E. maimaiga in the eastern United States [ 107 ] and later in the northern Midwestern United States (A. Diss personal communication). Releasing resting spore-filled cadavers was also used to augment naturally occurring E. maimaiga in urban forests [ 108 ] and studies have demonstrated higher levels of infection by E. maimaiga when water was applied to resting spore-bearing soil at the bases of trees (e.g., [ 99 ]). For classical biological control, cadavers containing resting spores have been successfully used to introduce E. maimaiga to Bulgaria in 1999 [ 109 ] and this method was then further used to spread E. maimaiga to more areas within Bulgaria (D. Pilarska pers. comm.). 6. Conclusions: Importance of Resting Spores to Epizootiology Most species in the Entomophthoromycotina produce more than one kind of spore. The persistent resting spore stages are important to epizootiology, but have been under-studied in the majority of host/pathogen systems. This is caused in part by the fact that cadavers in which resting spores are produced are often in different locations than cadavers from which conidia are produced; thus, for many species within Entomophthoromycotina, the resting spores have not been described and these species are only known from the conidial stages. Also, the reverse occurs when resting spores have been found but associated conidial stages have not. Among species for which resting spores are known, there are relatively few instances where the biology and ecology have been investigated, although in the past few decades, this information has increased for specific systems. We hope that this review of our present knowledge about the biology and ecology of resting spores of fungi in the Entomophthoromycotina will encourage others to gain more knowledge about these essential stages about which we know so little. In particular, we encourage additional studies on resting spore dormancy, environmental conditions leading to resting spore germination, spread due to airborne infective germ conidia produced by resting spores, altered behaviors of insects before death when resting spores versus conidia are produced, and interactions with biological communities that impact environmental reservoirs of resting spores. In addition, the other life stages used by some species in this group for survival, such as hyphal bodies, deserve investigation, for similar reasons.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8401346/

The Emergency Use Authorization of Pharmaceuticals: History and Utility During the COVID-19 Pandemic

The Emergency Use Authorization (EUA) originated in 2004 because of the need for emergency medical countermeasures (MCMs) against potential bioterrorist attacks. The EUA also proved useful in dealing with subsequent pandemics and has emerged as a critical regulatory pathway for therapeutics and vaccines throughout the Coronavirus Disease 2019 (COVID-19) pandemic. With the EUA process in the USA, we witnessed emergency authorizations, their expansions, as well as withdrawal of previously authorized products, which exemplifies the dynamic nature of scientific review of EUA products. EUAs proved vital for the first group of COVID-19 vaccines, including the temporary pause of one vaccine while emergency safety issues were evaluated. Although this review on the EUA is primarily focused on the USA, distinctions were made with other jurisdictions such as Europe and Canada with respect to the emergency authorizations of the vaccines. Finally, we discuss some important differences following EUA and formal new drug/vaccine application (NDA/BLA) approvals. Key Points The Emergency Use Authorization (EUA) acted as a critical regulatory pathway for therapeutics and vaccines throughout the Coronavirus Disease 2019 (COVID-19) pandemic. The EUA process demonstrates the dynamic nature of scientific review practices as characterized by emergency authorizations, expansion of the authorizations, and withdrawal of previously authorized products. EUAs were of critical utility in countering the pandemic and proved a remarkable regulatory framework for a monumental pharmaceutical achievement. Introduction Development of therapies and preventative vaccines to attenuate the often-devastating impact of the global COVID-19 pandemic have been at the daily forefront of both lay press and medical literature. While the basic public health hygienic measures were the foundation of efforts to ease the spread of infection and its burden, there was also great attention to innovative developments in pharmaceutical medicine. The pharmaceutical focus during the pandemic was on developing vaccines to protect individual patients and to foster herd immunity and therapies to treat the severe manifestations of disease. With respect to the latter, these developments included both novel therapeutics and, more often, the repurposing of drugs with known safety and efficacy in other indications (e.g., malaria) based on a working hypothesis of potential benefit against COVID-19. In both cases, the Emergency Use Authorization (EUA) served as a regulatory vehicle to allow patient use prior to formal New Drug Application (NDA) submissions. In the first year of the pandemic, we witnessed many approvals of EUAs; but also, amendments and withdrawals as the quickly evolving science drove decision making. We provide a historical summary of the EUA, including its conception and its application during the first year of the pandemic. We then describe how the EUA program operated to provide safe and effective emergency relief from a devastating pandemic with prescription therapeutics and the early availability of vaccines. While diagnostics were the most common medical product that received an EUA from the FDA, we focus exclusively on the use of EUAs for therapeutic interventions aimed at counteracting or preventing COVID-19 infections. The Purpose and Brief History of the EUA Bioterrorism Threats Prompt EUA After the 2001 anthrax bioterrorist attacks, the Project BioShield Act was enacted in 2004 to accelerate and bolster national security against potential bioterrorist attacks [ 1 ]. This legislation established the EUA under Section 564 of the Federal Food, Drug, and Cosmetic Act (FD&C Act), which permitted the emergency use of unapproved countermeasures against chemical, biological, radiological, or nuclear (CBRN) agent(s), among other authorities, when there are no adequate, approved, and available alternatives [ 2 ]. To enhance the flexibility of authorizations made under additional emergency circumstances, Section 564 of the FD&C Act was further amended by the Pandemic and All-Hazards Preparedness Reauthorization Act of 2013 (PAHPRA), the 21st Century Cures Act of 2016 , and Public Law 115-92 of 2017 [ 2 ]. The purpose of an EUA is to allow for the use of unapproved medical countermeasures (MCMs) to diagnose, treat, or prevent serious or life-threatening disease or condition in response to a public health, military, and domestic emergency [ 3 ]. If the determinations needed to support an EUA declaration in the USA is met, this will enable the Secretary of Homeland Security, Secretary of Defense, Secretary of Department of Health and Human Services (HHS) to declare that circumstances exist to support an authorization [ 3 ]. Given that a MCM is considered for EUA for treatment of serious or life-threatening consequences, the FDA will evaluate the evidence of potential effectiveness of the MCM, i.e., whether it may be effective, as well as the associated risk-benefit profile, allowing that there are no adequate, approved, and available alternative options [ 3 ]. There were several public statements and media broadcasts that brought a particular drug or agent into the spotlight, which have ultimately turned our attention to the FDA, and to its decision-making process that has led to an EUA issuance. Since the inception of the EUA, the first authorization for a vaccine was for the Anthrax Vaccine Adsorbed (AVA) used by the Armed Forced [ 4 ] (Table 1 ). Table 1 History of emergency use authorizations granted for vaccines in the USA [ 4 , 8 ] Date of first issuance Treatment Authorized use 14-Jan-05 Anthrax vaccine adsorbed For prevention of inhalation of Anthrax by individuals at heightened risk of exposure due to attack with Anthrax 11-Dec-20 Pfizer-BioNTech COVID-19 vaccine For the prevention of COVID-19 for individuals aged ≥ 16 years (Requested an EUA for the use in adolescents [ 53 ] and applied for full FDA approval [ 54 ]. Finally, on August 23, 2021, the FDA approved the vaccine for individuals 16 years or older [ 44 ].) 18-Dec-20 Moderna COVID-19 vaccine For the prevention of COVID-19 for individuals aged ≥ 18 years 27-Feb-21 Janssen COVID-19 vaccine For the prevention of Coronavirus Disease 2019 (COVID-19) for individuals aged ≥ 18 years COVID-19 Coronavirus Disease 2019, EUA emergency use authorization, FDA US Food and Drug Administration HIV Infection and Expanded Access The concept behind the EUA dates back to the decade after the first reported cases of human immunodeficiency virus and acquired immunodeficiency syndrome (HIV/AIDS) in 1981 [ 5 ]. Because of the urgent need for additional treatments, the United States Public Health Service (USPHS) created a "parallel track" expanded access program for an investigational drug known as dideoxyinosine (ddI) for AIDS patients with no other alternatives [ 5 ]. Notably, this pathway allowed eligible AIDS patients to access ddI while clinical trials of the drug were still ongoing. The fundamental principles of the parallel track/expanded access program formed the basis of the "accelerated approval (AA)" program, which was launched in 1992 [ 5 ]. Through the AA mechanism, the FDA was able to grant faster approval of new drugs that treat serious or life-threatening conditions based on a surrogate endpoint that is "reasonably likely" to predict clinical benefit [ 6 ], often well before clinical data supporting that benefit could be available. After granting an AA, confirmatory trials are required to verify its clinical effectiveness and benefit to support the AA [ 7 ]. The intentions of the EUA were reflected in the 1981 expanded access of ddI for AIDS patients. EUA in Previous Pandemics Apart from the significant role that EUAs played in authorizing the emergency use of treatments for 2009 H1N1 and COVID-19 vaccines [ 4 , 8 ] (Tables 1 , 2 ), other medical products (not shown here) including diagnostics, personal protective equipment (PPE), and medical devices have been authorized to combat the Zika virus, H7N9 virus, MERS-CoV, and Ebola virus [ 4 ]. Collectively, these events mark the historical successes that prove the utility of EUAs during times of public health emergencies, supporting its value as a regulatory tool. Table 2 Summary list of EUAs granted for therapeutics in the USA [ 4 ] Date of first issuance Treatment Authorized use 22-Jun-10 Antivirals: oseltamivir (Tamiflu), zanamivir (Relenza), and peramivir To treat and prevent 2009 H1N1 influenza 09-Jul-18 Pathogen-reduced leukocyte-depleted freeze-dried plasma For the treatment of hemorrhage or coagulopathy during an emergency involving agents of military combat (e.g., firearms, projectiles, and explosive devices) when plasma is not available for use or when the use of plasma is not practical 30-Apr-20 Fresenius Medical, multiFriltrate PRO System and multiBic/multiPlus solutions To provide CRRT to treat patients in an acute care environment during the COVID-19 pandemic 01-May-20 Remdesivir for certain hospitalized COVID-19 patients For the treatment of suspected or laboratory-confirmed COVID-19 in hospitalized pediatric patients weighing 3.5 kg to  16 with suspected or confirmed COVID-19 who require mechanical ventilation in an ICU setting 13-Aug-20 REGIOCIT replacement solution that contains citrate for RCA of the extracorporeal circuit To be used as a replacement solution only in adult patients treated with CRRT, and for whom regional citrate anticoagulation is appropriate, in a critical care setting 23-Aug-20 COVID-19 convalescent plasma For the treatment of hospitalized patients with COVID-19 09-Nov-20 Bamlanivimab For the treatment of mild-to-moderate COVID-19 in adult and pediatric patients with positive results of direct SARS-CoV-2 viral testing who are aged ≥ 12 years weighing at least 40 kg (about 88 pounds), and who are at high risk for progressing to severe COVID-19 and/or hospitalization 19-Nov-20 Baricitinib (Olumiant) in combination with remdesivir (Veklury) For the treatment of suspected or laboratory-confirmed COVID-19 in hospitalized adults and pediatric patients aged ≥ 2 years requiring supplemental oxygen, invasive mechanical ventilation, or ECMO 21-Nov-20 Casirivimab and imdevimab To be administered together for the treatment of mild-to-moderate COVID-19 in adults and pediatric patients (aged ≥ 12 years weighing at least 40 kg) with positive results of direct SARS-CoV-2 viral testing, and who are at high risk for progressing to severe COVID-19 and/or hospitalization 09-Feb-21 Bamlanivimab and estesevimab For the treatment of mild-to-moderate COVID-19 in adult and pediatric patients with positive results of direct SARS-CoV-2 viral testing who are aged ≥ 12 years weighing at least 40 kg (about 88 pounds), and who are at high risk for progressing to severe COVID-19 and/or hospitalization 12-Mar-21 Propofol-Lipuro 1% To maintain sedation via continuous infusion in patients greater than age 16 with suspected or confirmed COVID-19 who require mechanical ventilation in an ICU setting COVID-19 Coronavirus Disease 2019, CRRT continuous renal replacement therapy, ECMO extracorporeal membrane oxygenation , EUA Emergency Use Authorization, ICU intensive care unit, RCA regional citrate anticoagulation Bioterrorism Threats Prompt EUA After the 2001 anthrax bioterrorist attacks, the Project BioShield Act was enacted in 2004 to accelerate and bolster national security against potential bioterrorist attacks [ 1 ]. This legislation established the EUA under Section 564 of the Federal Food, Drug, and Cosmetic Act (FD&C Act), which permitted the emergency use of unapproved countermeasures against chemical, biological, radiological, or nuclear (CBRN) agent(s), among other authorities, when there are no adequate, approved, and available alternatives [ 2 ]. To enhance the flexibility of authorizations made under additional emergency circumstances, Section 564 of the FD&C Act was further amended by the Pandemic and All-Hazards Preparedness Reauthorization Act of 2013 (PAHPRA), the 21st Century Cures Act of 2016 , and Public Law 115-92 of 2017 [ 2 ]. The purpose of an EUA is to allow for the use of unapproved medical countermeasures (MCMs) to diagnose, treat, or prevent serious or life-threatening disease or condition in response to a public health, military, and domestic emergency [ 3 ]. If the determinations needed to support an EUA declaration in the USA is met, this will enable the Secretary of Homeland Security, Secretary of Defense, Secretary of Department of Health and Human Services (HHS) to declare that circumstances exist to support an authorization [ 3 ]. Given that a MCM is considered for EUA for treatment of serious or life-threatening consequences, the FDA will evaluate the evidence of potential effectiveness of the MCM, i.e., whether it may be effective, as well as the associated risk-benefit profile, allowing that there are no adequate, approved, and available alternative options [ 3 ]. There were several public statements and media broadcasts that brought a particular drug or agent into the spotlight, which have ultimately turned our attention to the FDA, and to its decision-making process that has led to an EUA issuance. Since the inception of the EUA, the first authorization for a vaccine was for the Anthrax Vaccine Adsorbed (AVA) used by the Armed Forced [ 4 ] (Table 1 ). Table 1 History of emergency use authorizations granted for vaccines in the USA [ 4 , 8 ] Date of first issuance Treatment Authorized use 14-Jan-05 Anthrax vaccine adsorbed For prevention of inhalation of Anthrax by individuals at heightened risk of exposure due to attack with Anthrax 11-Dec-20 Pfizer-BioNTech COVID-19 vaccine For the prevention of COVID-19 for individuals aged ≥ 16 years (Requested an EUA for the use in adolescents [ 53 ] and applied for full FDA approval [ 54 ]. Finally, on August 23, 2021, the FDA approved the vaccine for individuals 16 years or older [ 44 ].) 18-Dec-20 Moderna COVID-19 vaccine For the prevention of COVID-19 for individuals aged ≥ 18 years 27-Feb-21 Janssen COVID-19 vaccine For the prevention of Coronavirus Disease 2019 (COVID-19) for individuals aged ≥ 18 years COVID-19 Coronavirus Disease 2019, EUA emergency use authorization, FDA US Food and Drug Administration HIV Infection and Expanded Access The concept behind the EUA dates back to the decade after the first reported cases of human immunodeficiency virus and acquired immunodeficiency syndrome (HIV/AIDS) in 1981 [ 5 ]. Because of the urgent need for additional treatments, the United States Public Health Service (USPHS) created a "parallel track" expanded access program for an investigational drug known as dideoxyinosine (ddI) for AIDS patients with no other alternatives [ 5 ]. Notably, this pathway allowed eligible AIDS patients to access ddI while clinical trials of the drug were still ongoing. The fundamental principles of the parallel track/expanded access program formed the basis of the "accelerated approval (AA)" program, which was launched in 1992 [ 5 ]. Through the AA mechanism, the FDA was able to grant faster approval of new drugs that treat serious or life-threatening conditions based on a surrogate endpoint that is "reasonably likely" to predict clinical benefit [ 6 ], often well before clinical data supporting that benefit could be available. After granting an AA, confirmatory trials are required to verify its clinical effectiveness and benefit to support the AA [ 7 ]. The intentions of the EUA were reflected in the 1981 expanded access of ddI for AIDS patients. EUA in Previous Pandemics Apart from the significant role that EUAs played in authorizing the emergency use of treatments for 2009 H1N1 and COVID-19 vaccines [ 4 , 8 ] (Tables 1 , 2 ), other medical products (not shown here) including diagnostics, personal protective equipment (PPE), and medical devices have been authorized to combat the Zika virus, H7N9 virus, MERS-CoV, and Ebola virus [ 4 ]. Collectively, these events mark the historical successes that prove the utility of EUAs during times of public health emergencies, supporting its value as a regulatory tool. Table 2 Summary list of EUAs granted for therapeutics in the USA [ 4 ] Date of first issuance Treatment Authorized use 22-Jun-10 Antivirals: oseltamivir (Tamiflu), zanamivir (Relenza), and peramivir To treat and prevent 2009 H1N1 influenza 09-Jul-18 Pathogen-reduced leukocyte-depleted freeze-dried plasma For the treatment of hemorrhage or coagulopathy during an emergency involving agents of military combat (e.g., firearms, projectiles, and explosive devices) when plasma is not available for use or when the use of plasma is not practical 30-Apr-20 Fresenius Medical, multiFriltrate PRO System and multiBic/multiPlus solutions To provide CRRT to treat patients in an acute care environment during the COVID-19 pandemic 01-May-20 Remdesivir for certain hospitalized COVID-19 patients For the treatment of suspected or laboratory-confirmed COVID-19 in hospitalized pediatric patients weighing 3.5 kg to  16 with suspected or confirmed COVID-19 who require mechanical ventilation in an ICU setting 13-Aug-20 REGIOCIT replacement solution that contains citrate for RCA of the extracorporeal circuit To be used as a replacement solution only in adult patients treated with CRRT, and for whom regional citrate anticoagulation is appropriate, in a critical care setting 23-Aug-20 COVID-19 convalescent plasma For the treatment of hospitalized patients with COVID-19 09-Nov-20 Bamlanivimab For the treatment of mild-to-moderate COVID-19 in adult and pediatric patients with positive results of direct SARS-CoV-2 viral testing who are aged ≥ 12 years weighing at least 40 kg (about 88 pounds), and who are at high risk for progressing to severe COVID-19 and/or hospitalization 19-Nov-20 Baricitinib (Olumiant) in combination with remdesivir (Veklury) For the treatment of suspected or laboratory-confirmed COVID-19 in hospitalized adults and pediatric patients aged ≥ 2 years requiring supplemental oxygen, invasive mechanical ventilation, or ECMO 21-Nov-20 Casirivimab and imdevimab To be administered together for the treatment of mild-to-moderate COVID-19 in adults and pediatric patients (aged ≥ 12 years weighing at least 40 kg) with positive results of direct SARS-CoV-2 viral testing, and who are at high risk for progressing to severe COVID-19 and/or hospitalization 09-Feb-21 Bamlanivimab and estesevimab For the treatment of mild-to-moderate COVID-19 in adult and pediatric patients with positive results of direct SARS-CoV-2 viral testing who are aged ≥ 12 years weighing at least 40 kg (about 88 pounds), and who are at high risk for progressing to severe COVID-19 and/or hospitalization 12-Mar-21 Propofol-Lipuro 1% To maintain sedation via continuous infusion in patients greater than age 16 with suspected or confirmed COVID-19 who require mechanical ventilation in an ICU setting COVID-19 Coronavirus Disease 2019, CRRT continuous renal replacement therapy, ECMO extracorporeal membrane oxygenation , EUA Emergency Use Authorization, ICU intensive care unit, RCA regional citrate anticoagulation The EUA and Drugs Repurposed for COVID-19 Infection Repurposing Existing Drugs for COVID-19 A drug developed for one therapeutic indication can sometimes be found beneficial in selected situations for a different disease or condition. In fact, the first effective antiviral drug was being developed for cancer when insightful thinking led researchers to explore its antiviral activity [ 9 ]. This led to the development of acyclovir, an antiviral medication commonly used to treat human herpes infection, creating a new era in antiviral research, and leading to a Nobel Prize for Gertrude Elion and two other pharmaceutical industry colleagues [ 10 ]. Existing drugs repurposed for COVID-19 infection have been reviewed by Farne et al. [ 11 ] and classified into (1) antivirals, (2) promotors of innate antiviral response and (3) immunomodulatory/anti-inflammatory drugs. Drugs within these categories have reasonable likelihood of potential benefit, but the dose, dosing regimen, and precautions were not established for treatment of COVID-19 infections. The pathway to rapidly bridge these hypotheses to patient benefit is not straightforward. Early in the pandemic, two drugs in particular were hypothesized to have therapeutic potential and were frequently discussed in the media. Hydroxychloroquine and remdesivir were supported by reasonable therapeutic hypothesis and were seen as an immediate option while awaiting the discovery and development of a vaccine. While many took comfort in the fact that the drugs had a defined therapeutic window for the conditions they were originally developed to treat, we came to appreciate the intricacies of therapeutics in the often-overlooked efforts that go into defining a clear indication for the drug, its dose and dosing regimen, its safety and needed precautions and its contraindications—all the basic elements of a drug product label (i.e., package insert). The authority to use unapproved drugs for COVID-19 came to light as the pandemic was unfolding. On February 4, 2020, the U.S Department of Health and Human Services (HSS) declared a public health emergency that supported an EUA. This allowed the FDA to approve unapproved medical product(s) to be used in an emergency to diagnose, treat, or prevent COVID-19 infection, a life-threatening disease. Hydroxychloroquine On March 28, 2020, the FDA issued an EUA for oral formulations of chloroquine phosphate and hydroxychloroquine sulfate [ 12 ]. Hydroxychloroquine, despite demonstrating antiviral effects in laboratory models [ 13 ], ultimately did not show an acceptable benefit-risk relationship for the use to treat COVID-19 infection in humans [ 14 ]. As clinical trial data emerged, the agency concluded the drug may not be effective for COVID-19 infection and that any potential benefit did not outweigh its known and potential side effects and the EUA was revoked on June 15, 2020 [ 15 ] (Table 3 ). Chloroquine's EUA did not last long. In addition to chloroquine phosphate and hydroxychloroquine sulfate, there were a number of other therapies and diagnostics that had their EUA revoked during the COVID-19 pandemic (Table 4 ). Table 3 Repurposing of drugs for the treatment of COVID-19 [ 11 ] Description Antivirals Remdesivir Developed to treat Ebola; antiviral activity against respiratory infections including SARS-CoV-2 Acts as an adenosine analog to interfere with viral RdRp and induce premature or delayed RNA chain termination, while evading viral exoRNAase activity Recent study demonstrated accelerated recovery in patients during early disease Favipiravir Developed to treat influenza in Japan; effective in vitro against RNA viruses including SARS-CoV-2 Acts as purine analog to disrupt RdRp SARS-CoV-2 patients show shorter time to viral clearance and greater improvement in chest radiographic appearances (open-label RCT) Lopinavir/ritonavir ± ribavirin Developed to treat human HIV LPV inhibit proteases that cleave viral polyprotein to lead to the formation of RdRp; RTV inhibits cytochrome 450 to boost bioavailability of LPV One open-label RCT using combination of LPV/RTV with IFN-β showed clinical improvement in SARS-CoV-2 patients Chloroquine and hydroxychloroquine Chloroquine developed to treat malaria; hydroxychloroquine analogs introduced due to chloroquine-resistant strains of Plasmodium, treatment of autoimmune diseases including lupus erythematosus and rheumatoid arthritis Chloroquine and analogs undergo protonation and subsequent accumulation in lysosomes to increase lysosomal pH, and interferes with viral trafficking; impairs autophagosome fusion with lysosomes; inhibits glycosylation of angiotensin-converting enzyme 2 on cell surface required for viral entry; interferes with TLR signaling and cyclic guanosine monophosphate synthase to diminish downstream production of type-I IFNs and other cytokines Majority of clinical studies reported no benefit; one study showed improvement in symptom burden and reduction in C-reactive protein at 28 days at the cost of increased risks of adverse events Promoter of the innate antiviral response Exogenous interferon therapy Used for the treatment of chronic hepatitis B and C High-dose of type-I and -III IFN shown to be effective against SARS-CoV and MERS-CoV both in vitro and in vivo Single pilot clinical trial in SARS revealed that addition of IFN-α to corticosteroids was associated with clinical improvement. Limitations: IFN-α group received higher doses of corticosteroids, small sample size, IFNα given at a median 8 days after symptom onset Azithromycin Drug screen preprint demonstrated antiviral activity against SARS-CoV-2 A macrolide antibiotic that induces antiviral IFN; shown to double levels of antiviral type-I and type-III IFN released from virus-infected bronchial epithelial cells; possesses anti-inflammatory properties Azithromycin with oseltamivir showed clinical benefit in patients with influenza; MERS patients treated with macrolides found no improvement in 90-day mortality or viral clearance (only included critically ill, not treated with macrolide until admission to intensive care, subset did not receive azithromycin) Exogenous anti-SARS-CoV-2 antibody Transfer of antibodies from recovered patient; passive immunity; received interest during the Ebola epidemic An RCT showed no benefit in the primary outcome of time to clinical improvement but significant reduction in patients with severe but non-life-threatening COVID-19 Serious adverse events were infrequent but not absent Immunomodulatory and anti-inflammatory drugs Anti-IL-6 (tocilizumab, siltuximab) Humanized monoclonal antibody acting to block IL-6 Approved treatment for cytokine release syndrome, rheumatoid arthritis, systemic juvenile idiopathic arthritis Clinical studies show mixed results with tocilizumab, and siltuximab, individually IL-1 receptor antagonist (anakinra) Developed to treat rheumatoid arthritis Binding of SARS-CoV-2 activates the inflammasome to mediate the cleavage of pro-IL-1β into biologically active, and mature IL-1β; results in influx of inflammatory cells; lung inflammation and fibrosis Anti-IL-1 therapy with the recombinant IL-1RA (IL-1 receptor antagonist) protein anakinra shown to reduce mortality in patients with sepsis-related hyperinflammation Corticosteroids Synthetic analogues of steroid hormones; primarily used to treat immune-mediated diseases Systemic corticosteroids to suppress SARS-COV-2-induced lung inflammation to prevent and/or treat ARDS; risk of inhibiting immune responses and impair pathogen clearance Twenty-eight-day mortality was reduced with dexamethasone; reduced mortality in patients on mechanical ventilation, treated with oxygen but worsened survival in mild cases not requiring oxygen Treatments that were granted an EUA are given in italics ARDS acute respiratory distress syndrome, COVID-19 Coronavirus Disease 2019, HIV immunodeficiency virus, IFN interferon, IL interleukin, LPV Lopinavir, MERS Middle East respiratory syndrome, RCT randomized control trial, RdRp RNA-dependent RNA-polymerase, RNA ribonucleic acid, RTV Ritonavir, TLR toll-like receptor Table 4 Summary list of revoked EUAs during the COVID-19 pandemic in the USA [ 4 ] Date of first issuance Medical product Authorized use Reason for revocation 28-Mar-20 Chloroquine phosphate and hydroxychloroquine sulfate for treatment of COVID-19 To only treat adults and adolescents who weigh 50 kg or more and are hospitalized with COVID-19 for whom a clinical trial is not available, or participation is not feasible New information including clinical trial data results that have concluded this drug may not be effective to treat COVID-19. In addition, the drug's potential benefits for such use do not outweigh its known and potential risks 24-Apr-20 DPP COVID-19 IgM/IgG System (Chembio Diagnostic System, Inc.) Qualitative detection and differentiation of IgM and IgG antibodies against SARS-CoV-2 in serum, plasma (EDTA or lithium heparin), venous whole blood, or fingerstick whole blood from individuals suspected of COVID-19 by their healthcare provider Poor device performance and may not be effective in detecting antibodies against SARS-CoV-2 28-Apr-20 Umbrella EUA for independently validated serology tests for SARS-CoV-2 Intended for use as an aid in identifying individuals with an adaptive immune response to SARS-CoV-2, indicating recent or prior infection, by detecting antibodies (IgG, or IgG and IgM, or total), as specified in each authorized device's instructions for use, to SARS-CoV-2 in human plasma and/or serum FDA has determined that circumstances support revocation of the umbrella EUA to protect the public health or safety 24-Apr-20 Anti-SARS-CoV-2 Rapid Test (Autobio Diagnostics Co. Ltd.) Anti-SARS-CoV-2 rapid test for the qualitative detection and differentiation of IgM and IgG antibodies to SARS-CoV-2 in human plasma from anticoagulated blood (heparin/ EDTA/ sodium citrate) or serum Anti-SARS-CoV-2 rapid test does not meet current clinical performance estimates for serology tests that are generally necessary to satisfy the effectiveness and risk/benefit standards for issuance of an EUA 01-May-20 Protective barrier enclosures Protective barrier enclosures by HCP when caring for or performing medical procedures on patients who are known or suspected to have COVID-19 in healthcare settings to prevent HCP exposure to pathogenic biological airborne particulates by providing an extra layer of barrier protection in addition to PPE FDA believes it is no longer reasonable to believe that the authorized protective barrier enclosures may be effective at preventing HCP exposure to pathogenic biological airborne particulates by providing an extra layer of barrier protection in addition to PPE when caring for or performing medical procedures on patients who are known or suspected to have COVID-19 in healthcare settings 13-May-20 Infusion pumps and infusion pump accessories To treat conditions caused by the COVID-19 with the controlled infusion of medications, TPN, and/or other fluids FDA has determined that circumstances support revocation of the umbrella EUA to protect the public health or safety 12-Feb-21 Nova2200 for Decontaminating Compatible N95 Respirators (NovaSterillis, Inc.) For use in decontaminating compatible N95 respirators that are contaminated or potentially contaminated with SARS-CoV-2 or other pathogenic microorganisms, for a maximum of one decontamination cycle per respirator, for single-user reuse by HCP to prevent exposure to pathogenic biological airborne particulates during the COVID-19 pandemic FDA has become aware of new data and evidence suggesting that 3M Model 1860 and Halyard FLUIDSHIELD N95 respirators, the only compatible N95 respirators identified in this EUA, may not maintain adequate fit and filtration efficiency following one decontamination cycle using the Nova2200 17-Mar-21 BioFire Respiratory Panel 2.1 (RP2.1) (BioFire Diagnostics, LLC) Device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test FDA has determined that the criteria for issuance of such authorization under section 564(c) of the Act are no longer met. Under section 564(c)(3) of the Act, an EUA may be issued only if FDA concludes there is no adequate, approved, and available alternative to the product for diagnosing, preventing, or treating the disease or condition 16-Apr-21 Bamlanivimab For the treatment of mild-to-moderate COVID-19 in adult and pediatric patients with positive results of direct SARS-CoV-2 viral testing who are 12 years of age and older weighing at least 40 kg (about 88 pounds), and who are at high risk for progressing to severe COVID-19 and/or hospitalization The known and potential benefits of bamlanivimab alone no longer outweigh the known and potential risks for the product 30-Apr-21 Battelle Decontamination System For use in decontaminating compatible N95 respirators for multiple-user reuse by HCP to prevent exposure to pathogenic biological airborne particulates when there are insufficient supplies of FFRs resulting from the COVID-19 pandemic Battelle has notified FDA that it has ceased operations and associated activities and requests withdrawal of the authorization, FDA has determined that it is appropriate to protect the public health or safety to revoke this authorization COVID-19 Coronavirus Disease 2019, Ig immunoglobulin, EDTA ethylenediaminetetraacetic acid, EUA Emergency Use Authorization, FDA US Food and Drug Administration , FFRs filtering facepiece respirators , HCP healthcare providers, PPE personal protective equipment, TPN total parenteral nutrition Hydroxychloroquine's failure to show meaningful benefit became more critical when serious safety issues emerged. The FDA announced reports of serious arrythmias in patients with COVID-19 treated with hydroxychloroquine or chloroquine, often in combination with azithromycin and other QT prolonging medicines [ 16 ]. The FDA also noted their concern for public health as they observed increase outpatient prescriptions, possibly indicating increased requests for prescriptions to physicians by patients. The public was able to witness firsthand the two ends of a therapeutic index (benefit and risk) and the importance of each in therapeutic decisions. The hope of repurposing hydroxychloroquine had faded despite misguided enthusiasm by some influencers including country presidents and even a physician who used sermons as a minister to foster misinformation [ 17 ]. Remdesivir The FDA issued an EUA for remdesivir in hospitalized patients with severe disease from COVID-19 infection on May 1, 2020 [ 18 ] (Tables 2 , 3 ). Use for the treatment of suspected or laboratory-confirmed COVID-19 in adults and children hospitalized with severe disease was expanded on August 28, 2020 [ 19 ], and was FDA approved on October 22, 2020 [ 20 ]. Although remdesivir was being developed primarily against Ebola, like hydroxychloroquine, it showed an effect against coronaviruses in laboratory models [ 21 , 22 ]. A clinical study demonstrated that remdesivir use was not associated with an overall difference in time to clinical improvement. In patients with symptoms for 10 days or less, faster time to clinical improvement, compared to placebo, was observed but the difference was not statistically significant [ 23 ]. In addition to conducting the trial noted above, the remdesivir manufacturer began accepting physician requests for compassionate use. With safety data for some 500 patients in the Ebola program [ 24 , 25 ], a reasonable hypothesis for benefit, and no other therapy available, a compassionate use program was initiated. The investigators were able to observe clinical improvement in 68% of patients and nobody experienced side effects [ 26 ]. Compassionate use was followed by reporting of the ACTT-1 Trial where remdesivir demonstrated superiority to placebo based on a shortened time to recovery in hospitalized adults with COVID-19 and evidence of lower respiratory tract infection [ 27 ]. These data led to the drug's authorization in the USA, EU, and UK. However, some uncertainty concerning the drug's ultimate utility arose from the WHO 12,000 Patient Solidarity Trial [ 28 ], where no effect on survival was observed. The lack of any alternative therapy at this phase of the pandemic reinforced the need to continue remdesivir clinical development and allow compassionate use in the interim. Ultimately, enough evidence became available for FDA NDA approval [ 29 ]. Antibody Therapies and Convalescent Plasma Subsequent to the frequent public discussion on hydroxychloroquine and remdesivir, this was followed by EUAs for antibody therapy with convalescent plasma for the treatment of hospitalized patients with COVID-19 [ 30 ] (Tables 2 , 3 ). This was proceeded by EUAs for antibody therapy with bamlanivimab [ 31 ] as well as casirivimab and imdevimab [ 32 ] co-administration (Table 2 ). An important public health component is accurate and truthful communication of drug effects to the public in order to build trust in the regulatory processes. In the case of plasma administration, the FDA commissioner overstated the efficacy of therapy to the public, misinformation that was criticized by other health experts [ 33 ]. The commissioner's prompt admission that the data could have been better represented helped reinforce trust, an element that subsequently came to the forefront in optimizing vaccine roll out. Repurposing Existing Drugs for COVID-19 A drug developed for one therapeutic indication can sometimes be found beneficial in selected situations for a different disease or condition. In fact, the first effective antiviral drug was being developed for cancer when insightful thinking led researchers to explore its antiviral activity [ 9 ]. This led to the development of acyclovir, an antiviral medication commonly used to treat human herpes infection, creating a new era in antiviral research, and leading to a Nobel Prize for Gertrude Elion and two other pharmaceutical industry colleagues [ 10 ]. Existing drugs repurposed for COVID-19 infection have been reviewed by Farne et al. [ 11 ] and classified into (1) antivirals, (2) promotors of innate antiviral response and (3) immunomodulatory/anti-inflammatory drugs. Drugs within these categories have reasonable likelihood of potential benefit, but the dose, dosing regimen, and precautions were not established for treatment of COVID-19 infections. The pathway to rapidly bridge these hypotheses to patient benefit is not straightforward. Early in the pandemic, two drugs in particular were hypothesized to have therapeutic potential and were frequently discussed in the media. Hydroxychloroquine and remdesivir were supported by reasonable therapeutic hypothesis and were seen as an immediate option while awaiting the discovery and development of a vaccine. While many took comfort in the fact that the drugs had a defined therapeutic window for the conditions they were originally developed to treat, we came to appreciate the intricacies of therapeutics in the often-overlooked efforts that go into defining a clear indication for the drug, its dose and dosing regimen, its safety and needed precautions and its contraindications—all the basic elements of a drug product label (i.e., package insert). The authority to use unapproved drugs for COVID-19 came to light as the pandemic was unfolding. On February 4, 2020, the U.S Department of Health and Human Services (HSS) declared a public health emergency that supported an EUA. This allowed the FDA to approve unapproved medical product(s) to be used in an emergency to diagnose, treat, or prevent COVID-19 infection, a life-threatening disease. Hydroxychloroquine On March 28, 2020, the FDA issued an EUA for oral formulations of chloroquine phosphate and hydroxychloroquine sulfate [ 12 ]. Hydroxychloroquine, despite demonstrating antiviral effects in laboratory models [ 13 ], ultimately did not show an acceptable benefit-risk relationship for the use to treat COVID-19 infection in humans [ 14 ]. As clinical trial data emerged, the agency concluded the drug may not be effective for COVID-19 infection and that any potential benefit did not outweigh its known and potential side effects and the EUA was revoked on June 15, 2020 [ 15 ] (Table 3 ). Chloroquine's EUA did not last long. In addition to chloroquine phosphate and hydroxychloroquine sulfate, there were a number of other therapies and diagnostics that had their EUA revoked during the COVID-19 pandemic (Table 4 ). Table 3 Repurposing of drugs for the treatment of COVID-19 [ 11 ] Description Antivirals Remdesivir Developed to treat Ebola; antiviral activity against respiratory infections including SARS-CoV-2 Acts as an adenosine analog to interfere with viral RdRp and induce premature or delayed RNA chain termination, while evading viral exoRNAase activity Recent study demonstrated accelerated recovery in patients during early disease Favipiravir Developed to treat influenza in Japan; effective in vitro against RNA viruses including SARS-CoV-2 Acts as purine analog to disrupt RdRp SARS-CoV-2 patients show shorter time to viral clearance and greater improvement in chest radiographic appearances (open-label RCT) Lopinavir/ritonavir ± ribavirin Developed to treat human HIV LPV inhibit proteases that cleave viral polyprotein to lead to the formation of RdRp; RTV inhibits cytochrome 450 to boost bioavailability of LPV One open-label RCT using combination of LPV/RTV with IFN-β showed clinical improvement in SARS-CoV-2 patients Chloroquine and hydroxychloroquine Chloroquine developed to treat malaria; hydroxychloroquine analogs introduced due to chloroquine-resistant strains of Plasmodium, treatment of autoimmune diseases including lupus erythematosus and rheumatoid arthritis Chloroquine and analogs undergo protonation and subsequent accumulation in lysosomes to increase lysosomal pH, and interferes with viral trafficking; impairs autophagosome fusion with lysosomes; inhibits glycosylation of angiotensin-converting enzyme 2 on cell surface required for viral entry; interferes with TLR signaling and cyclic guanosine monophosphate synthase to diminish downstream production of type-I IFNs and other cytokines Majority of clinical studies reported no benefit; one study showed improvement in symptom burden and reduction in C-reactive protein at 28 days at the cost of increased risks of adverse events Promoter of the innate antiviral response Exogenous interferon therapy Used for the treatment of chronic hepatitis B and C High-dose of type-I and -III IFN shown to be effective against SARS-CoV and MERS-CoV both in vitro and in vivo Single pilot clinical trial in SARS revealed that addition of IFN-α to corticosteroids was associated with clinical improvement. Limitations: IFN-α group received higher doses of corticosteroids, small sample size, IFNα given at a median 8 days after symptom onset Azithromycin Drug screen preprint demonstrated antiviral activity against SARS-CoV-2 A macrolide antibiotic that induces antiviral IFN; shown to double levels of antiviral type-I and type-III IFN released from virus-infected bronchial epithelial cells; possesses anti-inflammatory properties Azithromycin with oseltamivir showed clinical benefit in patients with influenza; MERS patients treated with macrolides found no improvement in 90-day mortality or viral clearance (only included critically ill, not treated with macrolide until admission to intensive care, subset did not receive azithromycin) Exogenous anti-SARS-CoV-2 antibody Transfer of antibodies from recovered patient; passive immunity; received interest during the Ebola epidemic An RCT showed no benefit in the primary outcome of time to clinical improvement but significant reduction in patients with severe but non-life-threatening COVID-19 Serious adverse events were infrequent but not absent Immunomodulatory and anti-inflammatory drugs Anti-IL-6 (tocilizumab, siltuximab) Humanized monoclonal antibody acting to block IL-6 Approved treatment for cytokine release syndrome, rheumatoid arthritis, systemic juvenile idiopathic arthritis Clinical studies show mixed results with tocilizumab, and siltuximab, individually IL-1 receptor antagonist (anakinra) Developed to treat rheumatoid arthritis Binding of SARS-CoV-2 activates the inflammasome to mediate the cleavage of pro-IL-1β into biologically active, and mature IL-1β; results in influx of inflammatory cells; lung inflammation and fibrosis Anti-IL-1 therapy with the recombinant IL-1RA (IL-1 receptor antagonist) protein anakinra shown to reduce mortality in patients with sepsis-related hyperinflammation Corticosteroids Synthetic analogues of steroid hormones; primarily used to treat immune-mediated diseases Systemic corticosteroids to suppress SARS-COV-2-induced lung inflammation to prevent and/or treat ARDS; risk of inhibiting immune responses and impair pathogen clearance Twenty-eight-day mortality was reduced with dexamethasone; reduced mortality in patients on mechanical ventilation, treated with oxygen but worsened survival in mild cases not requiring oxygen Treatments that were granted an EUA are given in italics ARDS acute respiratory distress syndrome, COVID-19 Coronavirus Disease 2019, HIV immunodeficiency virus, IFN interferon, IL interleukin, LPV Lopinavir, MERS Middle East respiratory syndrome, RCT randomized control trial, RdRp RNA-dependent RNA-polymerase, RNA ribonucleic acid, RTV Ritonavir, TLR toll-like receptor Table 4 Summary list of revoked EUAs during the COVID-19 pandemic in the USA [ 4 ] Date of first issuance Medical product Authorized use Reason for revocation 28-Mar-20 Chloroquine phosphate and hydroxychloroquine sulfate for treatment of COVID-19 To only treat adults and adolescents who weigh 50 kg or more and are hospitalized with COVID-19 for whom a clinical trial is not available, or participation is not feasible New information including clinical trial data results that have concluded this drug may not be effective to treat COVID-19. In addition, the drug's potential benefits for such use do not outweigh its known and potential risks 24-Apr-20 DPP COVID-19 IgM/IgG System (Chembio Diagnostic System, Inc.) Qualitative detection and differentiation of IgM and IgG antibodies against SARS-CoV-2 in serum, plasma (EDTA or lithium heparin), venous whole blood, or fingerstick whole blood from individuals suspected of COVID-19 by their healthcare provider Poor device performance and may not be effective in detecting antibodies against SARS-CoV-2 28-Apr-20 Umbrella EUA for independently validated serology tests for SARS-CoV-2 Intended for use as an aid in identifying individuals with an adaptive immune response to SARS-CoV-2, indicating recent or prior infection, by detecting antibodies (IgG, or IgG and IgM, or total), as specified in each authorized device's instructions for use, to SARS-CoV-2 in human plasma and/or serum FDA has determined that circumstances support revocation of the umbrella EUA to protect the public health or safety 24-Apr-20 Anti-SARS-CoV-2 Rapid Test (Autobio Diagnostics Co. Ltd.) Anti-SARS-CoV-2 rapid test for the qualitative detection and differentiation of IgM and IgG antibodies to SARS-CoV-2 in human plasma from anticoagulated blood (heparin/ EDTA/ sodium citrate) or serum Anti-SARS-CoV-2 rapid test does not meet current clinical performance estimates for serology tests that are generally necessary to satisfy the effectiveness and risk/benefit standards for issuance of an EUA 01-May-20 Protective barrier enclosures Protective barrier enclosures by HCP when caring for or performing medical procedures on patients who are known or suspected to have COVID-19 in healthcare settings to prevent HCP exposure to pathogenic biological airborne particulates by providing an extra layer of barrier protection in addition to PPE FDA believes it is no longer reasonable to believe that the authorized protective barrier enclosures may be effective at preventing HCP exposure to pathogenic biological airborne particulates by providing an extra layer of barrier protection in addition to PPE when caring for or performing medical procedures on patients who are known or suspected to have COVID-19 in healthcare settings 13-May-20 Infusion pumps and infusion pump accessories To treat conditions caused by the COVID-19 with the controlled infusion of medications, TPN, and/or other fluids FDA has determined that circumstances support revocation of the umbrella EUA to protect the public health or safety 12-Feb-21 Nova2200 for Decontaminating Compatible N95 Respirators (NovaSterillis, Inc.) For use in decontaminating compatible N95 respirators that are contaminated or potentially contaminated with SARS-CoV-2 or other pathogenic microorganisms, for a maximum of one decontamination cycle per respirator, for single-user reuse by HCP to prevent exposure to pathogenic biological airborne particulates during the COVID-19 pandemic FDA has become aware of new data and evidence suggesting that 3M Model 1860 and Halyard FLUIDSHIELD N95 respirators, the only compatible N95 respirators identified in this EUA, may not maintain adequate fit and filtration efficiency following one decontamination cycle using the Nova2200 17-Mar-21 BioFire Respiratory Panel 2.1 (RP2.1) (BioFire Diagnostics, LLC) Device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test FDA has determined that the criteria for issuance of such authorization under section 564(c) of the Act are no longer met. Under section 564(c)(3) of the Act, an EUA may be issued only if FDA concludes there is no adequate, approved, and available alternative to the product for diagnosing, preventing, or treating the disease or condition 16-Apr-21 Bamlanivimab For the treatment of mild-to-moderate COVID-19 in adult and pediatric patients with positive results of direct SARS-CoV-2 viral testing who are 12 years of age and older weighing at least 40 kg (about 88 pounds), and who are at high risk for progressing to severe COVID-19 and/or hospitalization The known and potential benefits of bamlanivimab alone no longer outweigh the known and potential risks for the product 30-Apr-21 Battelle Decontamination System For use in decontaminating compatible N95 respirators for multiple-user reuse by HCP to prevent exposure to pathogenic biological airborne particulates when there are insufficient supplies of FFRs resulting from the COVID-19 pandemic Battelle has notified FDA that it has ceased operations and associated activities and requests withdrawal of the authorization, FDA has determined that it is appropriate to protect the public health or safety to revoke this authorization COVID-19 Coronavirus Disease 2019, Ig immunoglobulin, EDTA ethylenediaminetetraacetic acid, EUA Emergency Use Authorization, FDA US Food and Drug Administration , FFRs filtering facepiece respirators , HCP healthcare providers, PPE personal protective equipment, TPN total parenteral nutrition Hydroxychloroquine's failure to show meaningful benefit became more critical when serious safety issues emerged. The FDA announced reports of serious arrythmias in patients with COVID-19 treated with hydroxychloroquine or chloroquine, often in combination with azithromycin and other QT prolonging medicines [ 16 ]. The FDA also noted their concern for public health as they observed increase outpatient prescriptions, possibly indicating increased requests for prescriptions to physicians by patients. The public was able to witness firsthand the two ends of a therapeutic index (benefit and risk) and the importance of each in therapeutic decisions. The hope of repurposing hydroxychloroquine had faded despite misguided enthusiasm by some influencers including country presidents and even a physician who used sermons as a minister to foster misinformation [ 17 ]. Remdesivir The FDA issued an EUA for remdesivir in hospitalized patients with severe disease from COVID-19 infection on May 1, 2020 [ 18 ] (Tables 2 , 3 ). Use for the treatment of suspected or laboratory-confirmed COVID-19 in adults and children hospitalized with severe disease was expanded on August 28, 2020 [ 19 ], and was FDA approved on October 22, 2020 [ 20 ]. Although remdesivir was being developed primarily against Ebola, like hydroxychloroquine, it showed an effect against coronaviruses in laboratory models [ 21 , 22 ]. A clinical study demonstrated that remdesivir use was not associated with an overall difference in time to clinical improvement. In patients with symptoms for 10 days or less, faster time to clinical improvement, compared to placebo, was observed but the difference was not statistically significant [ 23 ]. In addition to conducting the trial noted above, the remdesivir manufacturer began accepting physician requests for compassionate use. With safety data for some 500 patients in the Ebola program [ 24 , 25 ], a reasonable hypothesis for benefit, and no other therapy available, a compassionate use program was initiated. The investigators were able to observe clinical improvement in 68% of patients and nobody experienced side effects [ 26 ]. Compassionate use was followed by reporting of the ACTT-1 Trial where remdesivir demonstrated superiority to placebo based on a shortened time to recovery in hospitalized adults with COVID-19 and evidence of lower respiratory tract infection [ 27 ]. These data led to the drug's authorization in the USA, EU, and UK. However, some uncertainty concerning the drug's ultimate utility arose from the WHO 12,000 Patient Solidarity Trial [ 28 ], where no effect on survival was observed. The lack of any alternative therapy at this phase of the pandemic reinforced the need to continue remdesivir clinical development and allow compassionate use in the interim. Ultimately, enough evidence became available for FDA NDA approval [ 29 ]. Antibody Therapies and Convalescent Plasma Subsequent to the frequent public discussion on hydroxychloroquine and remdesivir, this was followed by EUAs for antibody therapy with convalescent plasma for the treatment of hospitalized patients with COVID-19 [ 30 ] (Tables 2 , 3 ). This was proceeded by EUAs for antibody therapy with bamlanivimab [ 31 ] as well as casirivimab and imdevimab [ 32 ] co-administration (Table 2 ). An important public health component is accurate and truthful communication of drug effects to the public in order to build trust in the regulatory processes. In the case of plasma administration, the FDA commissioner overstated the efficacy of therapy to the public, misinformation that was criticized by other health experts [ 33 ]. The commissioner's prompt admission that the data could have been better represented helped reinforce trust, an element that subsequently came to the forefront in optimizing vaccine roll out. The EUA and the Urgency for Vaccine Development and Deployment The most celebrated use of EUAs came in mid-December 2020, with approvals for the Pfizer-BioNTech vaccine [ 34 ] followed a week later by ModernaTX's application [ 35 ], with the first administration of both occurring days later. Nearing the end of February 2021, the third EUA was issued for the Janssen's (Johnson & Johnson [J&J]) single-dose vaccine [ 36 ] (Table 1 ). Thus, within a year of declaring a global pandemic, three vaccines using two platforms (mRNA and viral vector-based) were authorized for emergency use in the USA. Basic pharmacovigilance led to interrupted authorization for the Janssen's viral vector vaccine. On April 13, the FDA and the Centers for Disease Control and Prevention (CDC) recommended a pause on the use of J&J's vaccine after 6 reported cases of blood clots following vaccination [ 37 – 39 ]. After several days of data evaluation, the authorization was reinstituted with a warning on the low-risk association of vaccine-induced thrombotic thrombocytopenia (VITT) with the vaccine [ 40 ]. During this pause period, physicians became alerted to the type of clotting and appropriate therapy. AstraZeneca's viral vector vaccine was authorized in Europe and elsewhere in early 2021 but has not yet been granted an EUA by the FDA. Concerns about robustness of Phase 3 trial data are probably related to lagging US approval and the need for additional data [ 41 ]. During the early stages of the pandemic, the FDA has guided the pharmaceutical industry to facilitate the satisfaction of scientific and regulatory requirements for the development and licensure, and EUA issuance of COVID-19 vaccines [ 42 , 43 ]. Once vaccine manufacturers complete a primary efficacy endpoint analysis in Phase 3 clinical trials that achieved the pre-specified success criteria of efficacy, an EUA application can be submitted to the FDA for review [ 43 ]. The FDA reviews data independent from the sponsor and can utilize an advisory committee of expert scientists and physicians from the Vaccines and Related Biological Products Advisory Committee (VRBPAC) [ 43 ]. This Committee deliberates on data in a public forum and votes on answers to questions provided by the FDA. While FDA is not bound by their recommendations, they usually follow them. Robust pharmacovigilance follows EUA authorization, as demonstrated by the J&J case [ 37 ]. Full review of an NDA/BLA will generally follow an EUA, which, in the case of vaccines, will include follow-up observations as defined by the Phase 3 protocol and evaluated in 'real world' studies of various designs. It is worthy to note that some COVID-19 vaccines are now progressing to full NDAs as their EUA will expire once the declared emergency is over. More recently, on August 23, 2021, the FDA approved Pfizer's COVID-19 vaccine for individuals 16 years or older [ 44 ]. Perspective on EUA The approval of ddI for the treatment of AIDS was the first accelerated mechanism to bring lifesaving drugs to patients with no available alternatives [ 5 ]. Over a decade later, the origin of the EUA introduced the emergency use of the AVA to facilitate anthrax vaccine immunization of US military personnel with an increased risk of anthrax attacks [ 45 ]. These events along with the earlier pandemics, led to the use of EUAs, which have further supported and confirmed its utility during early public health emergencies. In the COVID-19 pandemic, EUA vaccines were a remarkable accomplishment that was critical to mitigating the pandemic's negative effects. The temporary pause in one authorization reflected a system that was active and effective [ 37 ]. EUAs of therapeutics were also critical, as was the dynamic nature of their intention where we witnessed expansions of use, withdrawal of use, and progression to approved NDAs. While our review focused on the USA, other jurisdictions used similar processes to expedite safe and effective authorization of vaccinations given the unmet needs posed by the COVID-19 pandemic. There are, however, noteworthy differences. In the USA, three vaccines are currently authorized for emergency use, which does not include the AstraZeneca product. This product was granted conditional marketing authorization by the European Medicines Agency (EMA), also a fast-track authorization procedure to speed up approval of treatments and vaccines during public health emergencies [ 46 ]. Similarly, a Marketing Authorization with Conditions was granted by Health Canada. Information on what additional data may have been requested by FDA and whether AstraZeneca will submit for an EUA, or full BLA, or no authorization or approval in the USA remains unclear [ 47 ]. Another distinction of the EUA review process in the USA is the independent advisory committee proceedings being open to the public [ 48 ]. While EMA and Health Canada strive for transparency in data access [ 49 ], the ability to witness the debates and rationale of opinions of independent experts is most transparent and informative, a model that should be considered by other authorities. Finally, the process in the USA resulted in authorization based on the product label, which was informed by findings of robust clinical trial. In Canada, for example, vaccine procurement issues early on, forced authorities to make recommendations away from the product monograph. Specifically, the dosing intervals in the two-dose vaccines were allowed to be broadened [ 50 ]. This was followed by non-regulator recommendations allowing two-dose vaccine products to be mixed. Advisories then suggested limited use of the AstraZeneca vaccine [ 51 ]. Both of these practices were not studied in the pivotal trials driving the authorization, thus 'uncoupling' the data from these controlled studies, including long-term safety. While the ultimate benefit-risk of these debatable practices is unknown [ 52 ], it is noteworthy in the context of EUAs granted in the USA that ultimate dosing and administration did not stray from the data submitted by the sponsor and assessed by the FDA. Conclusion The EUA, originally designed to provide rapid antidotes for potential bioterrorism, acted as a critical regulatory pathway for vaccines and therapeutics throughout the COVID-19 pandemic. Our experiences demonstrated the dynamic nature of the scientific review process where some products had their emergency use expanded, some withdrawn after evaluation of new data, and some moving on to full NDA approval. The EUA was a critical component in counteracting the pandemic through pharmaceutical medicine and proved a remarkable framework for the monumental drug development achievements. This experience will inform regulatory processes in future pandemics and public health crises.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992447/

In vitro biosynthesis and substrate tolerance of the plantazolicin family of natural products

Plantazolicin (PZN) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that exhibits extraordinarily narrow-spectrum antibacterial activity towards the causative agent of anthrax, Bacillus anthracis . During PZN biosynthesis, a cyclodehydratase catalyzes cyclization of cysteine, serine, and threonine residues in the PZN precursor peptide (BamA) to azolines. Subsequently, a dehydrogenase then oxidizes most of these azolines to thiazoles and (methyl)oxazoles. The final biosynthetic steps consist of leader peptide removal and dimethylation of the nascent N -terminus. Using heterologously expressed and purified heterocycle synthetase, the BamA peptide was processed in vitro concordant with the pattern of post-translational modification found in the naturally occurring compound. Using a suite of BamA-derived peptides, including amino acid substitutions as well as contracted and expanded substrate variants, the substrate tolerance of the heterocycle synthetase was elucidated in vitro , and the residues crucial for leader peptide binding were identified. Despite increased promiscuity compared to what was previously observed during heterologous production in E. coli , the synthetase retained exquisite selectivity in cyclization of unnatural peptides only at positions which correspond to those cyclized in the natural product. A cleavage site was subsequently introduced to facilitate leader peptide removal, yielding mature PZN variants after enzymatic or chemical dimethylation. In addition, we report the isolation and characterization of two novel PZN-like natural products was predicted from genome sequences but whose production had not yet been observed.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5407842/

Evaluation of immunogenicity and protective efficacy of recombinant outer membrane proteins of Haemophilus parasuis serovar 5 in a murine model

Glässer's disease is an economically important infectious disease of pigs caused by Haemophilus parasuis . Few vaccines are currently available that could provide effective cross-protection against various serovars of H . parasuis . In this study, five OMPs (OppA, TolC, HxuC, LppC, and HAPS_0926) identified by bioinformatic approaches, were cloned and expressed as recombinant proteins. Antigenicity of the purified proteins was verified through Western blotting, and primary screening for protective potential was evaluated in vivo . Recombinant TolC (rTolC), rLppC, and rHAPS_0926 proteins showing marked protection of mice against H . parasuis infection, and were further evaluated individually or in combination. Mice treated with these three OMPs produced humoral and host cell-mediated responses, with a significant rise in antigen-specific IgG titer and lymphoproliferative response in contrast with the mock-immunized group. Significant increases were noted in CD4 + , CD8 + T cells, and three cytokines (IL-2, IL-4, and IFN-γ) in vaccinated animals. The antisera against candidate antigens could efficiently impede bacterial survival in whole blood bactericidal assay against H . parasuis infection. The multi-protein vaccine induced more pronounced immune responses and offered better protection than individual vaccines. Our findings indicate that these three OMPs are promising antigens for the development of multi-component subunit vaccines against Glässer's disease. Introduction Haemophilus parasuis is an early colonizer of the upper respiratory tract of pigs, and the etiological agent of Glässer's disease, which is characterized by polyserositis, polyarthritis, meningitis, and arthritis [ 1 – 2 ]. Glässer's disease has been reported sporadically and is usually associated with precipitating stress factors. With the effects of immunosuppressive viruses and increasingly intensive swine production, there is an apparent increase in the prevalence of the disease [ 3 ]. Nowadays, H . parasuis is a widespread epidemic pathogenic bacteria and leads to huge economic losses to the world swine industry, while prevention and control of Glässer's disease remain a big challenge [ 4 ]. Fifteen distinct serovars of H . parasuis have been described, while approximately 26% of the isolates were reported as non-typeable using traditional serotyping [ 5 – 6 ], and this percentage was lower when detected by molecular serotyping methods [ 7 – 8 ]. Commercial vaccines mainly comprise inactivated whole-cell vaccines and could not confer cross-protection against different serovars [ 9 ]. Currently, the development of subunit vaccines has attracted more attention, and they mainly concentrate upon outer membrane proteins (OMPs) as vaccine candidate antigens [ 10 ]. OMPs are unique to Gram-negative bacteria and have been shown to be potential candidates for vaccine development against infections in recent years [ 11 ]. Several OMPs of H . parasuis , such as PalA, D15, OmpP2, HPS-06257, OapA, HPS-0675, GAPDH, native outer membrane proteins with affinity to porcine transferrin (NPAPT) and many more have been confirmed to exhibit a strong potential as vaccine candidates [ 12 – 16 ]. However, a combination of protective antigens may be able to provide effective protection against multiple H . parasuis serovars. In our previous study, six secreted proteins and seven OMPs were predicted using bioinformatic analysis and were evaluated as potential vaccine candidates of H . parasuis serovar 5 [ 17 – 18 ]. In the present study, we adopted the same approach to identify protective antigens. Five OMPs, including OppA (oligopeptide permease ABC transporter membrane protein), TolC (RND efflux system outer membrane lipoprotein), LppC (lipoprotein C), HAPS_0926 (DNA uptake lipoprotein), and HxuC (haem-haemopexin utilization protein C/outer membrane receptor protein) were cloned, expressed, and purified, and initially screening for protective potential was performed in a murine model. Then rTolC, rLppC, and rHAPS_0926, showing a good protective potential, were administered individually or in combination to evaluate the protective immunity against H . parasuis . Materials and methods Ethics statement All animal procedures were approved by the Ethics Committee of Institute of Animal Health, Guangdong Academy of Agricultural Sciences according to Guangdong Province Laboratory Animal Management Regulations—2010. The license number was SYXK(Yue) 2011–0116. All efforts were made to minimize suffering. Humane endpoints used during the animal survival study were: rapid weight loss of >20% of body weight, poor physical appearance (reduced mobility, rough coat and depression), rapid breathing, swollen eyes, and joint tumefaction. Following infection, the healthy status of animals was evaluated every 8 h and there were not unexpected deaths. Animals that reached humane endpoints were euthanized through complete exsanguination via cardiac puncture under general anesthesia with inhaled 2% isoflurane. Bacterial strains and growth conditions H . parasuis was maintained in tryptic soy broth (TSB) (Difco, Detroit, MI, USA) with 10% inactivated newborn calf serum and 0.01% nicotinamide adenine dinucleotide (NAD) (Sigma, St. Louis, MO, USA) or plated on tryptic soy agar (TSA) (Difco, USA) plus 10% serum, and 0.01% NAD at 37°C. Escherichia coli strains were cultured in Luria-Bertani (LB) medium. If necessary, 100 μg/mL ampicillin or 50 μg/mL kanamycin was complemented. E . coli DH5α (Invitrogen, Carlsbad, CA, USA) and BL21(DE3) (Invitrogen, USA) were used for the cloning of plasmids and expression of recombinant proteins. The H . parasuis serovar 5 H46 was isolated from a pig farm in Guangdong Province, China [ 18 ]. For challenge test, H46 was cultured on TSA agar for 16 h at 37°C. Then a single clone was randomly picked, inoculated into 5 mL TSB (plus 10% serum and 0.01% NAD) and shaken at 37°C overnight. The overnight culture was reinoculated into 500 mL of TSB medium to bacteria counting. Screening, cloning, expression, and purification of the recombinant proteins To identify the OMPs of H . parasuis serovar 5 as candidate vaccines, we used a strategy combining bioinformatic analysis with an experimental approach as described previously [ 17 ]. The gene sequences of five selected antigens (OppA, TolC, LppC, HAPS_0926, and HxuC) were collated from H . parasuis SH0165 complete genome sequence [ 19 ]. Total genomic DNA was prepared from an H . parasuis serovar 5 H46 strain. Briefly, H46 was cultured in TSB overnight and 5 mL of culture was collected and lysed with the Bacterial DNA extraction kit (Sangon, Shanghai, China). The DNA region encoding each protein without the putative secreted signal was amplified with the primer pairs ( Table 1 ) from H46 genomic DNA. The amplified PCR products digested with restriction enzymes ( BamH I/ Xho I) were used to transform into the pET-30a(+) vector (Novagen, Billerica, MA, USA), and then were transformed into E . coli DH5α. The plasmid constructs were verified by restriction digestion, PCR, and sequencing. The constructed expression vectors were transformed to E . coli BL21(DE3) for expression with a C-terminal 6 × His-tag. Recombinant proteins were induced upon treatment with 0.6 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h. These expressed OMPs were purified by Ni 2+ -NTA affinity chromatography (Qiagen, Dusseldorf, Germany) in accordance with the instructions. Protein quantifications were detected with the bicinchoninic acid (BCA) protein assay kit (Tiangen, Beijing, China) and stored at –80°C. 10.1371/journal.pone.0176537.t001 Table 1 Experimental design for grouping and antigen dose of immunization. Group Vaccine Dose 1 rTolC 60 μg/200 μL 2 rLppC 60 μg/200 μL 3 rHAPS_0926 60 μg/200 μL 4 rTolC + rLppC 30μg each/200 μL 5 rTolC + rHAPS_0926 30μg each/200 μL 6 rLppC + rHAPS_0926 30μg each/200 μL 7 rTolC + rLppC+ rHAPS_0926 20 μg each/200 μL 8 PBS 200 μL SDS-PAGE and Western blotting analysis Five purified recombinant OMPs were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the transfer of proteins to a polyvinylidene fluoride (PVDF) membrane. Western blotting analysis was carried out using our previous study [ 17 – 18 ]. Convalescent swine sera (1:500 diluted with 5% skim milk in PBST) were added and incubated for 1 h at room temperature (RT) as primary antibody, and goat anti-porcine IgG (H + L)-HRP antibody (1:5,000) (Sigma, USA) was added and incubated at RT for 1 h as secondary antibody. After washing three times with PBS, the specific antigen-bound antibody was visualized with ECL (Biovision, Milpitas, CA, USA) following the manufacturer's instructions. Immunization-challenge test of the selected five OMPs in mice All animals used in this study were maintained under optimal conditions of temperature, hygiene, humidity, and light with a 12-h dark/light cycle. Sixty female BALB/c mice (seven-week-old) were randomly divided into six groups of 10 mice each. Five groups were vaccinated subcutaneously with 50 μg each of recombinant OMP emulsified in 200 μL of complete Freund's adjuvant (CFA) (Sigma, USA). The mice were boosted at 21 days post-immunization (dpi) using the identical dosage of antigens, but with incomplete Freund's adjuvant (IFA) (Sigma, USA). The sixth group, serving as a negative control (NC), was immunized with phosphate-buffered saline (PBS) emulsified in the corresponding Freund's adjuvant. At 42 days following the first immunization, all of the mice were intraperitoneally infected with 2 × 10 9 colony forming unit (CFU) log-phase H . parasuis H46 strain. After the challenge, clinical signs were monitored for two weeks, and mice judged to be in a moribund state were euthanized. Evaluation of the efficacy of the three screened OMPs in mice One hundred and twenty mice were randomly assigned to eight groups of 15 mice each. Three screened OMPs rTolC, rLppC, and rHAPS_0926 were placed in eight groups ( Table 2 ). Groups 1 to 3 were immunized with 60 μg of each recombinant protein emulsified in 200 μL of CFA via subcutaneous injection. Groups 4 to 6 received a mixture of two kinds of the recombinant OMPs (containing 30 μg of each antigen) with an equal volume of CFA. Group 7 was immunized with mixed rTolC, rLppC, and rHAPS_0926 (20 μg each). Mice in Group 8 were immunized with 200 μL of PBS emulsified in CFA serving as a NC. A booster immunization was given at 21 dpi using the same antigen and IFA. 10.1371/journal.pone.0176537.t002 Table 2 Primers for amplifying the genes encoding the five outer membrane proteins. Genes Primer sequences a OppA GC GGATCC TTATTAGCCAGTGCGATT GCC CTCGAG TTACTGCTTAATGATATA TolC GC GGATCC TTACTTTCTGCACTGGTA CC CTCGAG CTATTTGCGATATTTCCC LppC GC GGATCC ACCGCCACGATATTGTAG GG CTCGAG GTTTGCATCAACAATAGA HxuC GC GGATCC ATTTAATATTGCGCCCAG GCC CTCGAG ATGAGACTATCAAAAATT HAPS_0926 GCC GGATCC ATATTTGTAAAGGTTTTA GG CTCGAG AAATTGCTCGCCATATTT a Introduced restriction sites were highlighted in italics and underlined. At 42 days following the primary immunization, the immunized mice (ten mice in each group) were intraperitoneally inoculated with 5.0 × 10 9 CFU log-phase H . parasuis H46 strain. The animals were intensively monitored daily after the challenge for the presence and severity of clinical symptoms of illness or mortality. At 14 days post-challenge (dpc), all surviving mice were euthanized, and different tissues (heart, liver, spleen, lung, and kidney) were collected and subjected to pathological and immunohistochemistry (IHC) examinations and PCR. Indirect ELISA An indirect enzyme-linked immunosorbent assay (ELISA) was made in accordance with the method described in previous study [ 13 ]. The sera collected from pre-immune mice by tail vein bleeding were used as controls. Briefly, ELISA plates were coated with 0.2 μg/well of purified recombinant protein antigen diluted in coating buffer (0.02 M carbonate-bicarbonate buffer, pH 9.6), incubated overnight at 4°C. Following washing, 1% (w/v) bovine serum albumin in PBST (PBS containing 0.05% Tween-20, pH 7.4) was added to wells for blocking. Then the plates were incubated with serially diluted serum samples (initially in 1:100) at 37°C for 1 h. Horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma, USA), at a dilution of 1:5,000, was subsequently added to incubate at 37°C for 1 h. The tetramethylbenzidine was added for 10 min in the dark and then 2 M H 2 SO 4 was added to stop the reaction. The absorbance was read at optical density of 450 nm (OD 450 nm ) with an ELISA plate reader (Bio-Rad, Hercules, CA, USA). Antibody titer was calculated as the reciprocal of the maximum serum dilution that gave an OD yielding the cutoff value of ELISA (OD 450 nm = 0.35, which is the mean values of negative sera plus three-fold standard errors). Flow cytometry Fourteen days after the booster immunization, mice were killed and aseptically collected spleens were washed in PBS (pH 7.4). The splenocytes were harvested from the immunized mice as described previously [ 17 , 20 ]. Lymphocyte subtype analysis was performed as described previously [ 21 ]. Briefly, splenocytes were labeled with fluorescein isothiocyanate (FITC)-anti-mouse CD4 + , allophycocyanin-cyanine 7 (APC-Cy7)-anti-mouse CD3 + , and phycoerythrin (PE)-anti-mouse CD8 + . FITC-, APC-Cy7-, or PE-conjugated antibodies were used as isotype controls (eBioscience, San Diego, CA, USA). Then the percentage of CD3 + , CD4 + , and CD8 + T cells was quantified. Lymphocyte proliferation assay Spleen lymphocytes resuspended in RPMI 1640 complete medium (Gibco, USA) supplemented with 10% inactivated fetal bovine serum were adjusted to 10 6 cells/mL and 100 μL of the suspension was added per well in 96-well plates, and incubated with 5 μg/well recombinant proteins in 5% CO 2 for 72 h at 37°C. Splenocytes stimulated with 5 μg/well of concanavalin A (ConA) (Sigma, USA) served as a positive control, while negative control cells received medium only. Lymphoproliferation assay was carried out with a MTS cell proliferation detection kit (Promega, Madison, WI, USA). The lymphocytes were incubated with MTS reagent for 4 h. The proliferation of cells was measured at OD 490 nm using a plate reader (Bio-Rad, USA). Cytokine assay Cytokine assay was performed as described previously [ 17 ]. Supernatants obtained in Section of lymphocyte proliferation assay were harvested and stored at ÿ80°C. Interleukin 2 (IL-2), IL-4, and interferon gamma (IFN-γ) levels were measured using cytokine detection kits following the manufacturer's instructions (Research & Diagnostics Systems, Minneapolis, MN, USA). In vitro whole blood bactericidal assay The whole blood bactericidal assay was carried out as described previously [ 18 ]. The H46 cultures were washed three times and diluted in sterilized PBS to 10 8 CFU. Subsequently, a mixture of 10 μL of H46 suspension and 190 μL of each serum was incubated for 30 min at 37°C. Then 100 μL of nonimmune heparinized blood was added and incubated for 1 h at 37°C with shaking. The samples were plated on TSA plates and colonies were determined after 24 h. The resulting expression was performed as described previously [ 18 ]. Bacterium re-isolation from the immunized mice following challenge At 1, 2, and 7 dpc, the presence of H . parasuis in the liver, spleen, and lung was detected. The tissue sections were prepared as described previously [ 18 ]. Then serial 10-fold dilution samples were determined by plating on TSA plates and incubated for 16 h at 37°C. Statistical analysis The immunized groups were compared with the negative group. Descriptive statistics (mean, standard error), normality (Shapiro-Wilk test), and homoscedasticity (Bartlett' s test) were determined. Data were analyzed by ANOVA test using the software Statistics Package for Social Science19.0 (SPSS 19.0). Ethics statement All animal procedures were approved by the Ethics Committee of Institute of Animal Health, Guangdong Academy of Agricultural Sciences according to Guangdong Province Laboratory Animal Management Regulations—2010. The license number was SYXK(Yue) 2011–0116. All efforts were made to minimize suffering. Humane endpoints used during the animal survival study were: rapid weight loss of >20% of body weight, poor physical appearance (reduced mobility, rough coat and depression), rapid breathing, swollen eyes, and joint tumefaction. Following infection, the healthy status of animals was evaluated every 8 h and there were not unexpected deaths. Animals that reached humane endpoints were euthanized through complete exsanguination via cardiac puncture under general anesthesia with inhaled 2% isoflurane. Bacterial strains and growth conditions H . parasuis was maintained in tryptic soy broth (TSB) (Difco, Detroit, MI, USA) with 10% inactivated newborn calf serum and 0.01% nicotinamide adenine dinucleotide (NAD) (Sigma, St. Louis, MO, USA) or plated on tryptic soy agar (TSA) (Difco, USA) plus 10% serum, and 0.01% NAD at 37°C. Escherichia coli strains were cultured in Luria-Bertani (LB) medium. If necessary, 100 μg/mL ampicillin or 50 μg/mL kanamycin was complemented. E . coli DH5α (Invitrogen, Carlsbad, CA, USA) and BL21(DE3) (Invitrogen, USA) were used for the cloning of plasmids and expression of recombinant proteins. The H . parasuis serovar 5 H46 was isolated from a pig farm in Guangdong Province, China [ 18 ]. For challenge test, H46 was cultured on TSA agar for 16 h at 37°C. Then a single clone was randomly picked, inoculated into 5 mL TSB (plus 10% serum and 0.01% NAD) and shaken at 37°C overnight. The overnight culture was reinoculated into 500 mL of TSB medium to bacteria counting. Screening, cloning, expression, and purification of the recombinant proteins To identify the OMPs of H . parasuis serovar 5 as candidate vaccines, we used a strategy combining bioinformatic analysis with an experimental approach as described previously [ 17 ]. The gene sequences of five selected antigens (OppA, TolC, LppC, HAPS_0926, and HxuC) were collated from H . parasuis SH0165 complete genome sequence [ 19 ]. Total genomic DNA was prepared from an H . parasuis serovar 5 H46 strain. Briefly, H46 was cultured in TSB overnight and 5 mL of culture was collected and lysed with the Bacterial DNA extraction kit (Sangon, Shanghai, China). The DNA region encoding each protein without the putative secreted signal was amplified with the primer pairs ( Table 1 ) from H46 genomic DNA. The amplified PCR products digested with restriction enzymes ( BamH I/ Xho I) were used to transform into the pET-30a(+) vector (Novagen, Billerica, MA, USA), and then were transformed into E . coli DH5α. The plasmid constructs were verified by restriction digestion, PCR, and sequencing. The constructed expression vectors were transformed to E . coli BL21(DE3) for expression with a C-terminal 6 × His-tag. Recombinant proteins were induced upon treatment with 0.6 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h. These expressed OMPs were purified by Ni 2+ -NTA affinity chromatography (Qiagen, Dusseldorf, Germany) in accordance with the instructions. Protein quantifications were detected with the bicinchoninic acid (BCA) protein assay kit (Tiangen, Beijing, China) and stored at –80°C. 10.1371/journal.pone.0176537.t001 Table 1 Experimental design for grouping and antigen dose of immunization. Group Vaccine Dose 1 rTolC 60 μg/200 μL 2 rLppC 60 μg/200 μL 3 rHAPS_0926 60 μg/200 μL 4 rTolC + rLppC 30μg each/200 μL 5 rTolC + rHAPS_0926 30μg each/200 μL 6 rLppC + rHAPS_0926 30μg each/200 μL 7 rTolC + rLppC+ rHAPS_0926 20 μg each/200 μL 8 PBS 200 μL SDS-PAGE and Western blotting analysis Five purified recombinant OMPs were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the transfer of proteins to a polyvinylidene fluoride (PVDF) membrane. Western blotting analysis was carried out using our previous study [ 17 – 18 ]. Convalescent swine sera (1:500 diluted with 5% skim milk in PBST) were added and incubated for 1 h at room temperature (RT) as primary antibody, and goat anti-porcine IgG (H + L)-HRP antibody (1:5,000) (Sigma, USA) was added and incubated at RT for 1 h as secondary antibody. After washing three times with PBS, the specific antigen-bound antibody was visualized with ECL (Biovision, Milpitas, CA, USA) following the manufacturer's instructions. Immunization-challenge test of the selected five OMPs in mice All animals used in this study were maintained under optimal conditions of temperature, hygiene, humidity, and light with a 12-h dark/light cycle. Sixty female BALB/c mice (seven-week-old) were randomly divided into six groups of 10 mice each. Five groups were vaccinated subcutaneously with 50 μg each of recombinant OMP emulsified in 200 μL of complete Freund's adjuvant (CFA) (Sigma, USA). The mice were boosted at 21 days post-immunization (dpi) using the identical dosage of antigens, but with incomplete Freund's adjuvant (IFA) (Sigma, USA). The sixth group, serving as a negative control (NC), was immunized with phosphate-buffered saline (PBS) emulsified in the corresponding Freund's adjuvant. At 42 days following the first immunization, all of the mice were intraperitoneally infected with 2 × 10 9 colony forming unit (CFU) log-phase H . parasuis H46 strain. After the challenge, clinical signs were monitored for two weeks, and mice judged to be in a moribund state were euthanized. Evaluation of the efficacy of the three screened OMPs in mice One hundred and twenty mice were randomly assigned to eight groups of 15 mice each. Three screened OMPs rTolC, rLppC, and rHAPS_0926 were placed in eight groups ( Table 2 ). Groups 1 to 3 were immunized with 60 μg of each recombinant protein emulsified in 200 μL of CFA via subcutaneous injection. Groups 4 to 6 received a mixture of two kinds of the recombinant OMPs (containing 30 μg of each antigen) with an equal volume of CFA. Group 7 was immunized with mixed rTolC, rLppC, and rHAPS_0926 (20 μg each). Mice in Group 8 were immunized with 200 μL of PBS emulsified in CFA serving as a NC. A booster immunization was given at 21 dpi using the same antigen and IFA. 10.1371/journal.pone.0176537.t002 Table 2 Primers for amplifying the genes encoding the five outer membrane proteins. Genes Primer sequences a OppA GC GGATCC TTATTAGCCAGTGCGATT GCC CTCGAG TTACTGCTTAATGATATA TolC GC GGATCC TTACTTTCTGCACTGGTA CC CTCGAG CTATTTGCGATATTTCCC LppC GC GGATCC ACCGCCACGATATTGTAG GG CTCGAG GTTTGCATCAACAATAGA HxuC GC GGATCC ATTTAATATTGCGCCCAG GCC CTCGAG ATGAGACTATCAAAAATT HAPS_0926 GCC GGATCC ATATTTGTAAAGGTTTTA GG CTCGAG AAATTGCTCGCCATATTT a Introduced restriction sites were highlighted in italics and underlined. At 42 days following the primary immunization, the immunized mice (ten mice in each group) were intraperitoneally inoculated with 5.0 × 10 9 CFU log-phase H . parasuis H46 strain. The animals were intensively monitored daily after the challenge for the presence and severity of clinical symptoms of illness or mortality. At 14 days post-challenge (dpc), all surviving mice were euthanized, and different tissues (heart, liver, spleen, lung, and kidney) were collected and subjected to pathological and immunohistochemistry (IHC) examinations and PCR. Indirect ELISA An indirect enzyme-linked immunosorbent assay (ELISA) was made in accordance with the method described in previous study [ 13 ]. The sera collected from pre-immune mice by tail vein bleeding were used as controls. Briefly, ELISA plates were coated with 0.2 μg/well of purified recombinant protein antigen diluted in coating buffer (0.02 M carbonate-bicarbonate buffer, pH 9.6), incubated overnight at 4°C. Following washing, 1% (w/v) bovine serum albumin in PBST (PBS containing 0.05% Tween-20, pH 7.4) was added to wells for blocking. Then the plates were incubated with serially diluted serum samples (initially in 1:100) at 37°C for 1 h. Horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma, USA), at a dilution of 1:5,000, was subsequently added to incubate at 37°C for 1 h. The tetramethylbenzidine was added for 10 min in the dark and then 2 M H 2 SO 4 was added to stop the reaction. The absorbance was read at optical density of 450 nm (OD 450 nm ) with an ELISA plate reader (Bio-Rad, Hercules, CA, USA). Antibody titer was calculated as the reciprocal of the maximum serum dilution that gave an OD yielding the cutoff value of ELISA (OD 450 nm = 0.35, which is the mean values of negative sera plus three-fold standard errors). Flow cytometry Fourteen days after the booster immunization, mice were killed and aseptically collected spleens were washed in PBS (pH 7.4). The splenocytes were harvested from the immunized mice as described previously [ 17 , 20 ]. Lymphocyte subtype analysis was performed as described previously [ 21 ]. Briefly, splenocytes were labeled with fluorescein isothiocyanate (FITC)-anti-mouse CD4 + , allophycocyanin-cyanine 7 (APC-Cy7)-anti-mouse CD3 + , and phycoerythrin (PE)-anti-mouse CD8 + . FITC-, APC-Cy7-, or PE-conjugated antibodies were used as isotype controls (eBioscience, San Diego, CA, USA). Then the percentage of CD3 + , CD4 + , and CD8 + T cells was quantified. Lymphocyte proliferation assay Spleen lymphocytes resuspended in RPMI 1640 complete medium (Gibco, USA) supplemented with 10% inactivated fetal bovine serum were adjusted to 10 6 cells/mL and 100 μL of the suspension was added per well in 96-well plates, and incubated with 5 μg/well recombinant proteins in 5% CO 2 for 72 h at 37°C. Splenocytes stimulated with 5 μg/well of concanavalin A (ConA) (Sigma, USA) served as a positive control, while negative control cells received medium only. Lymphoproliferation assay was carried out with a MTS cell proliferation detection kit (Promega, Madison, WI, USA). The lymphocytes were incubated with MTS reagent for 4 h. The proliferation of cells was measured at OD 490 nm using a plate reader (Bio-Rad, USA). Cytokine assay Cytokine assay was performed as described previously [ 17 ]. Supernatants obtained in Section of lymphocyte proliferation assay were harvested and stored at ÿ80°C. Interleukin 2 (IL-2), IL-4, and interferon gamma (IFN-γ) levels were measured using cytokine detection kits following the manufacturer's instructions (Research & Diagnostics Systems, Minneapolis, MN, USA). In vitro whole blood bactericidal assay The whole blood bactericidal assay was carried out as described previously [ 18 ]. The H46 cultures were washed three times and diluted in sterilized PBS to 10 8 CFU. Subsequently, a mixture of 10 μL of H46 suspension and 190 μL of each serum was incubated for 30 min at 37°C. Then 100 μL of nonimmune heparinized blood was added and incubated for 1 h at 37°C with shaking. The samples were plated on TSA plates and colonies were determined after 24 h. The resulting expression was performed as described previously [ 18 ]. Bacterium re-isolation from the immunized mice following challenge At 1, 2, and 7 dpc, the presence of H . parasuis in the liver, spleen, and lung was detected. The tissue sections were prepared as described previously [ 18 ]. Then serial 10-fold dilution samples were determined by plating on TSA plates and incubated for 16 h at 37°C. Statistical analysis The immunized groups were compared with the negative group. Descriptive statistics (mean, standard error), normality (Shapiro-Wilk test), and homoscedasticity (Bartlett' s test) were determined. Data were analyzed by ANOVA test using the software Statistics Package for Social Science19.0 (SPSS 19.0). Results Expression and purification of five recombinant OMPs The DNA fragments encoding the protein of OppA, TolC, LppC, HAPS_0926, and HxuC were successfully cloned into pET-30a(+), confirmed by sequencing (data not shown) and the digestion of Bam H I/ Xho I (data not shown). The recombinant plasmids harboring the foreign genes were used to transform into E . coli BL21(DE3). The SDS-PAGE results indicated that all of the recombinant proteins were expressed in E . coli as His-tagged fusion proteins with an expected size ( Fig 1 ). 10.1371/journal.pone.0176537.g001 Fig 1 SDS-PAGE analysis of the E . coli -expressed five recombinant proteins purified by Ni 2+ -NTA affinity chromatography. Lane M: protein marker; Lanes 1–6: TolC (approx. 51 kDa), HAPS_0926 (approx. 25 kDa), OppA (approx. 60 kDa), LppC (approx. 62 kDa), and HxuC (approx.78 kDa). Antigenicity of five recombinant OMPs The expression of the five recombinant OMPs was further confirmed by Western blotting using the porcine convalescent sera to H . parasuis [ 18 ] ( Fig 2 ). The results showed that the H . parasuis convalescent sera reacted with five recombinant proteins, indicating that the obtained recombinant proteins can be used for further evaluation of immunoprotection. 10.1371/journal.pone.0176537.g002 Fig 2 Western blotting analysis of the purified five recombinant proteins. Lanes 1–6: purified HxuC (approx.78 kDa), LppC (approx. 62 kDa), OppA (approx. 60 kDa), TolC (approx. 51 kDa), and HAPS_0926 (approx. 25 kDa); Lane 7: sonicated whole cells of E . coli serving as a NC; Lane M: protein molecular weight marker. Protective potential of the five antigens in mice The protective potential of the five OMPs against a lethal challenge with H46 was evaluated in mice. The results indicated that rTolC, rLppC, and rHAPS_0926 provided 80%, 70%, and 60% protection, respectively. rOppA and rHxuC did not afford significant protection (survival of ≤ 50%). Therefore, in the following experiment, the immune responses and efficacy of rTolC, rLppC, and rHAPS_0926 were further evaluated separately and in combination. Antibody responses in the vaccinated mice To determine whether the mice immunized with the recombinant proteins can induce specific humoral immune responses, an indirect ELISA was carried out. Serum samples from the immunized mice were collected two weeks after the booster immunization. The individual or mixed recombinant proteins were used as coating antigens. As shown in Fig 3 , specific IgG antibody titers against all of the recombinant protein-immunized groups were significantly higher than that of the negative group ( p < 0.01). The mice immunized with two antigens in combination displayed slightly higher levels of IgG titers than that of the mice immunized with single antigens. Compared to the other groups, the rTolC + rLppC + rHAPS_0926 (triple-rOMP)-immunized group developed the highest antigen-specific response. 10.1371/journal.pone.0176537.g003 Fig 3 Serum IgG antibody titers against the recombinant proteins in mice. Serum samples were collected two weeks after the booster immunization and tested for the antibody titers by indirect ELISA. Standard deviations were shown as error bars. **, p < 0.01; ***, p < 0.001. Cell-mediated immune responses in the immunized mice To detect the cell-mediated immune responses, the immunized mice were sacrificed. The splenocytes isolated at two weeks following the booster immunization were analyzed using flow cytometry ( Fig 4A ). Compared to the NC, the proportion of proliferated CD4 + ( p < 0.01) and CD8 + ( p < 0.05) T cells was significantly higher for the recombinant protein-immunized groups, and the percentage of proliferated CD4 + T cells was much higher than that of CD8 + T cells. In contrast with the mice immunized with individual antigens or two antigens combination, the mice immunized with triple-rOMP displayed a very significant increase in the percentage of CD4 + and CD8 + T cells ( p < 0.01) ( Fig 4B ). 10.1371/journal.pone.0176537.g004 Fig 4 (A) Dot plot analysis of CD4 + and CD8 + T cell proliferation. (B) Expression of T lymphocyte subsets in the spleens collected from the mice immunized with recombinant proteins or PBS (NC). Splenocytes were stained with FITC-labeled anti-mouse CD4 + and PE-labeled anti-mouse CD8 + antibodies. Significant increase in CD4 + ( p < 0.01) and CD8 + ( p < 0.05) subsets was observed for the vaccinated group compared to the NC group. (C) Lymphocyte proliferation assay. Splenocytes from the mice immunized with the recombinant proteins or PBS (NC) were stimulated in vitro with the corresponding recombinant proteins for 72 h and the lymphoproliferative responses were measured by MTS assay. Stimulation with ConA serving as a positive control. (D) Expression of IFN-γ, IL-2, and IL-4 in the spleens isolated from the mice immunized with the recombinant proteins or PBS (NC). Standard deviations were shown as error bars. ***, p < 0.001; **, p < 0.01; *, p < 0.05. As shown in Fig 4C , the seven groups of vaccinated animals showed significant antigen-specific proliferative cell immune responses ( p < 0.01). Similarly, a strong proliferative response was determined to ConA as a positive control. However, no antigen-specific lymphoproliferation was found in the NC group. The cytokine response of splenic lymphocytes was detected by ELISA. Compared to the NC, splenocytes from the recombinant protein-immunized mice induced a significant cytotoxic response of IFN-γ ( p < 0.001), IL-2 ( p < 0.001), and IL-4 ( p < 0.01) ( Fig 4D ). Higher levels of IL-2 and IFN-γ were secreted than that of IL-4. These results suggested that the immunization of mice with individual antigens and multi-proteins could induce the Th1 type response. Bactericidal activities of the antisera from the immunized mice Antisera against the recombinant proteins significantly inhibited ( p < 0.001) the growth of H . parasuis compared to the negative NC ( Fig 5 ). Compared to the individual recombinant proteins, the bactericidal activities of antiserum of triple-rOMP-immunized mice were much higher. The results suggest that the immune responses induced by the recombinant proteins are able to provide partial protection against H . parasuis infection. 10.1371/journal.pone.0176537.g005 Fig 5 Bactericidal activities of the whole blood from the mice immunized with the recombinant proteins or PBS (NC). The results were expressed in pg/mL. Error bars represent the standard errors of the means from three replicates. ***, p < 0.001. Protection of the immunized mice from lethal H . parasuis challenge To evaluate the protective effectiveness of the three vaccine candidates, groups 1–8 were each challenged with 5.0 × 10 9 CFU H46. The mortality and clinical signs of the mice were recorded daily for 14 dpc. As shown in Fig 6 , the mice of the NC group died within 2 dpc and showed severe pathological changes, including pulmonary consolidation with massive proliferation of fibroblasts, pleura edematous, fibrin in the abdomen, and fibrin in the thorax/hydrothorax. At 14 dpc, all surviving mice were euthanized and subjected to pathological examination. None of the mice immunized with the recombinant proteins showed pathological changes (data not shown). Examination of the heart, liver, spleen, lung, and kidney by PCR[ 22 ]showed that no H . parasuis was detectable in the tissues of any animal of the vaccination groups (data not shown). 10.1371/journal.pone.0176537.g006 Fig 6 The immunized mice' survival following challenge with H . parasuis serovar 5. The post-challenge survival data showed that 80% of the animals survived in the triple-rOMP-immunized group. In contrast with the individual antigens, immunization with the combined three proteins provided better protection against the H . parasuis serovar 5 challenge. Bacterial loads in various tissues of immunized mice following challenge As shown in Fig 7 , from 1 to 7 dpc, the bacterial loads in the liver and spleen were higher than those in the lung. Analyzing the viable counts in tissues, we observed a significant reduction in the recombinant protein-immunized groups at 7 dpc, and in Groups 4, 5, and 7, there were no bacteria in the spleen, liver, or lung. Compared with others, the animals vaccinated with triple-rOMP showed lowest counts in tested tissues. All of the isolated bacteria were confirmed to be H . parasuis serovar 5 by PCR (data not shown) [ 22 ]. 10.1371/journal.pone.0176537.g007 Fig 7 Bacterial counts in different tissues from the vaccinated animals at 1, 2, and 7 dpc following challenge with H . parasuis serovar 5. The bacterial loads in the tissues of the animals were expressed as log 10 CFU/50 μg of tissues. Expression and purification of five recombinant OMPs The DNA fragments encoding the protein of OppA, TolC, LppC, HAPS_0926, and HxuC were successfully cloned into pET-30a(+), confirmed by sequencing (data not shown) and the digestion of Bam H I/ Xho I (data not shown). The recombinant plasmids harboring the foreign genes were used to transform into E . coli BL21(DE3). The SDS-PAGE results indicated that all of the recombinant proteins were expressed in E . coli as His-tagged fusion proteins with an expected size ( Fig 1 ). 10.1371/journal.pone.0176537.g001 Fig 1 SDS-PAGE analysis of the E . coli -expressed five recombinant proteins purified by Ni 2+ -NTA affinity chromatography. Lane M: protein marker; Lanes 1–6: TolC (approx. 51 kDa), HAPS_0926 (approx. 25 kDa), OppA (approx. 60 kDa), LppC (approx. 62 kDa), and HxuC (approx.78 kDa). Antigenicity of five recombinant OMPs The expression of the five recombinant OMPs was further confirmed by Western blotting using the porcine convalescent sera to H . parasuis [ 18 ] ( Fig 2 ). The results showed that the H . parasuis convalescent sera reacted with five recombinant proteins, indicating that the obtained recombinant proteins can be used for further evaluation of immunoprotection. 10.1371/journal.pone.0176537.g002 Fig 2 Western blotting analysis of the purified five recombinant proteins. Lanes 1–6: purified HxuC (approx.78 kDa), LppC (approx. 62 kDa), OppA (approx. 60 kDa), TolC (approx. 51 kDa), and HAPS_0926 (approx. 25 kDa); Lane 7: sonicated whole cells of E . coli serving as a NC; Lane M: protein molecular weight marker. Protective potential of the five antigens in mice The protective potential of the five OMPs against a lethal challenge with H46 was evaluated in mice. The results indicated that rTolC, rLppC, and rHAPS_0926 provided 80%, 70%, and 60% protection, respectively. rOppA and rHxuC did not afford significant protection (survival of ≤ 50%). Therefore, in the following experiment, the immune responses and efficacy of rTolC, rLppC, and rHAPS_0926 were further evaluated separately and in combination. Antibody responses in the vaccinated mice To determine whether the mice immunized with the recombinant proteins can induce specific humoral immune responses, an indirect ELISA was carried out. Serum samples from the immunized mice were collected two weeks after the booster immunization. The individual or mixed recombinant proteins were used as coating antigens. As shown in Fig 3 , specific IgG antibody titers against all of the recombinant protein-immunized groups were significantly higher than that of the negative group ( p < 0.01). The mice immunized with two antigens in combination displayed slightly higher levels of IgG titers than that of the mice immunized with single antigens. Compared to the other groups, the rTolC + rLppC + rHAPS_0926 (triple-rOMP)-immunized group developed the highest antigen-specific response. 10.1371/journal.pone.0176537.g003 Fig 3 Serum IgG antibody titers against the recombinant proteins in mice. Serum samples were collected two weeks after the booster immunization and tested for the antibody titers by indirect ELISA. Standard deviations were shown as error bars. **, p < 0.01; ***, p < 0.001. Cell-mediated immune responses in the immunized mice To detect the cell-mediated immune responses, the immunized mice were sacrificed. The splenocytes isolated at two weeks following the booster immunization were analyzed using flow cytometry ( Fig 4A ). Compared to the NC, the proportion of proliferated CD4 + ( p < 0.01) and CD8 + ( p < 0.05) T cells was significantly higher for the recombinant protein-immunized groups, and the percentage of proliferated CD4 + T cells was much higher than that of CD8 + T cells. In contrast with the mice immunized with individual antigens or two antigens combination, the mice immunized with triple-rOMP displayed a very significant increase in the percentage of CD4 + and CD8 + T cells ( p < 0.01) ( Fig 4B ). 10.1371/journal.pone.0176537.g004 Fig 4 (A) Dot plot analysis of CD4 + and CD8 + T cell proliferation. (B) Expression of T lymphocyte subsets in the spleens collected from the mice immunized with recombinant proteins or PBS (NC). Splenocytes were stained with FITC-labeled anti-mouse CD4 + and PE-labeled anti-mouse CD8 + antibodies. Significant increase in CD4 + ( p < 0.01) and CD8 + ( p < 0.05) subsets was observed for the vaccinated group compared to the NC group. (C) Lymphocyte proliferation assay. Splenocytes from the mice immunized with the recombinant proteins or PBS (NC) were stimulated in vitro with the corresponding recombinant proteins for 72 h and the lymphoproliferative responses were measured by MTS assay. Stimulation with ConA serving as a positive control. (D) Expression of IFN-γ, IL-2, and IL-4 in the spleens isolated from the mice immunized with the recombinant proteins or PBS (NC). Standard deviations were shown as error bars. ***, p < 0.001; **, p < 0.01; *, p < 0.05. As shown in Fig 4C , the seven groups of vaccinated animals showed significant antigen-specific proliferative cell immune responses ( p < 0.01). Similarly, a strong proliferative response was determined to ConA as a positive control. However, no antigen-specific lymphoproliferation was found in the NC group. The cytokine response of splenic lymphocytes was detected by ELISA. Compared to the NC, splenocytes from the recombinant protein-immunized mice induced a significant cytotoxic response of IFN-γ ( p < 0.001), IL-2 ( p < 0.001), and IL-4 ( p < 0.01) ( Fig 4D ). Higher levels of IL-2 and IFN-γ were secreted than that of IL-4. These results suggested that the immunization of mice with individual antigens and multi-proteins could induce the Th1 type response. Bactericidal activities of the antisera from the immunized mice Antisera against the recombinant proteins significantly inhibited ( p < 0.001) the growth of H . parasuis compared to the negative NC ( Fig 5 ). Compared to the individual recombinant proteins, the bactericidal activities of antiserum of triple-rOMP-immunized mice were much higher. The results suggest that the immune responses induced by the recombinant proteins are able to provide partial protection against H . parasuis infection. 10.1371/journal.pone.0176537.g005 Fig 5 Bactericidal activities of the whole blood from the mice immunized with the recombinant proteins or PBS (NC). The results were expressed in pg/mL. Error bars represent the standard errors of the means from three replicates. ***, p < 0.001. Protection of the immunized mice from lethal H . parasuis challenge To evaluate the protective effectiveness of the three vaccine candidates, groups 1–8 were each challenged with 5.0 × 10 9 CFU H46. The mortality and clinical signs of the mice were recorded daily for 14 dpc. As shown in Fig 6 , the mice of the NC group died within 2 dpc and showed severe pathological changes, including pulmonary consolidation with massive proliferation of fibroblasts, pleura edematous, fibrin in the abdomen, and fibrin in the thorax/hydrothorax. At 14 dpc, all surviving mice were euthanized and subjected to pathological examination. None of the mice immunized with the recombinant proteins showed pathological changes (data not shown). Examination of the heart, liver, spleen, lung, and kidney by PCR[ 22 ]showed that no H . parasuis was detectable in the tissues of any animal of the vaccination groups (data not shown). 10.1371/journal.pone.0176537.g006 Fig 6 The immunized mice' survival following challenge with H . parasuis serovar 5. The post-challenge survival data showed that 80% of the animals survived in the triple-rOMP-immunized group. In contrast with the individual antigens, immunization with the combined three proteins provided better protection against the H . parasuis serovar 5 challenge. Bacterial loads in various tissues of immunized mice following challenge As shown in Fig 7 , from 1 to 7 dpc, the bacterial loads in the liver and spleen were higher than those in the lung. Analyzing the viable counts in tissues, we observed a significant reduction in the recombinant protein-immunized groups at 7 dpc, and in Groups 4, 5, and 7, there were no bacteria in the spleen, liver, or lung. Compared with others, the animals vaccinated with triple-rOMP showed lowest counts in tested tissues. All of the isolated bacteria were confirmed to be H . parasuis serovar 5 by PCR (data not shown) [ 22 ]. 10.1371/journal.pone.0176537.g007 Fig 7 Bacterial counts in different tissues from the vaccinated animals at 1, 2, and 7 dpc following challenge with H . parasuis serovar 5. The bacterial loads in the tissues of the animals were expressed as log 10 CFU/50 μg of tissues. Discussion H . parasuis is an important respiratory tract pathogen causing severe infections in pigs. It is difficult to control the systemic infection of this organism because of the limitation of knowledge regarding its pathogenesis and immunogenicity [ 23 – 24 ]. Conventional vaccines for H . parasuis are inefficient for inducing cross-protective immunity [ 25 ]. OMPs of H . parasuis have been confirmed to have the potential for developing effective subunit vaccines [ 12 – 16 , 18 ]. The advances in whole-genome sequencing and bioinformatics techniques made it possible to search protective antigens for bacterial pathogens [ 13 ]. Animal models have widely been used to screen bacterial proteins as subunit candidate vaccines. For instance, mice are good models to evaluate the immunological parameters of H . parasuis [ 12 – 13 , 17 ]. In the present study, screening for the vaccine candidates was primarily performed in in vivo protection assay, and immunization with rTolC, rLppC, and rHAPS_0926 markedly protected mice from H . parasuis infection. The abilities of these three antigens to induce humoral and cell-mediated immunity and protection were further evaluated separately or in cocktails. The TolC protein is used by both the type I protein secretion pathway and multidrug efflux pumps as a prototypical outer membrane channel component [ 26 ]. TolC participates in the formation of type I secretion systems of bacterial virulence factors, for instance, adenylate cyclase toxin ( Bordetella pertussis ), alkaline protease ( Pseudomonas aeruginosa ), and α-hemolysin ( E . coli ) [ 27 ]. A previous study demonstrated that efflux protein TolC plays an important role in biofilm formation in E . coli [ 28 ]. It is one of the most potent antigens of Salmonella paratyphi A and a promising candidate target for the development of new vaccines [ 29 ]. Both of LppC and HAPS_0926 are outer membrane lipoproteins. Lipoproteins are widely distributed in Gram-negative bacteria, which are involved in diverse mechanisms of physiology/pathogenesis and are considered potential target antigens for vaccine development against many infectious diseases [ 30 – 31 ]. Our previous study demonstrated that the VacJ lipoprotein was a good vaccine candidate against Glässer's disease [ 18 ]. We chose these three OMPs for further analysis of immunogenicity and immunoprotection. The results revealed that the mice immunized with rTolC, rLppC, or rHAPS_0926 alone provided partial protection against H . parasuis infection. The advantage of a multivalent subunit vaccine is that it would provide better protection than the univalent component because of its ability to generate abundant immunogens. In previous studies, researchers have used a 1:1 or 1:1:1 ratio of single proteins to evaluate the efficacy of subunit vaccine candidates [ 24 , 32 ]. Hence, we chose the same ratio of single OMPs to compose the multi-protein vaccines. In the current study, these subunit vaccines were shown to be capable of inducing a high titer of antibodies and cell-mediated immunity in mice, providing strong protective potential. Similar to the previous reports, our results showed that the combination of recombinant antigens induced higher antibody titers, confirming that the antigen cocktails are more immunogenic than the individual ones [ 33 – 35 ]. The induction of cell-mediated immune responses characterized by antigen-specific T cell proliferation revealed the immunogenicity of multi-proteins with a higher proliferation index. The splenocytes from the multi-proteins-immunized groups secreted higher levels of cytokines in response to corresponding antigens than those of the single-components vaccinated groups, which is consistent with the lymphocyte proliferation. It was possible to verify that the multi-protein vaccines induced specific Th1-mediated immune protection in the immunized animals, as demonstrated by the specific production of IL-2 and IFN-γ in vitro , along with a low expression of IL-4. Our results of immunogenicity proved that the multi-protein vaccine was better than individual proteins. After the challenge, bacterial loads in all evaluated tissues were decreased significantly in the triple-rOMP-immunized mice, which were related to bactericidal activities of the antisera. Besides the three vaccine candidates, two other proteins, OppA and HxuC, should not be ignored. OppA is an oligopeptide permease ABC transporter membrane protein, which has been identified as a vaccine development target against several pathogenic bacteria, such as Yersinia pestis and Moraxella catarrhalis [ 36 – 37 ]. Recently, the researchers used recombinant OppA as the antigen of an indirect hemagglutination assay for detecting antibodies against H . parasuis [ 38 ]. The TonB-dependent haem receptor HxuC belongs to the hxuCBA gene cluster, which is a virulence factor of H . Influenzae [ 39 ]. The results revealed that these two proteins could react with convalescent sera, but did not induce effective protection against H . parasuis infection in the mouse model. It is important to optimize the dose and route of administration for evaluating new antigens. Development of cross-protective vaccines is needed urgently for the control and prevention against H . parasuis infection, because single antigens are insufficient to offer complete protection. Additional studies to evaluate the triple-rOMP vaccine against other serovars of H . parasuis , such as serovars 1, 4, 12, and 13, should also be performed in future studies. Indeed, subunit vaccines should be tested for the protection efficacy conferred in practice in target animals. Piglets are quite suitable for evaluating the protective potency against the heterologous challenge of H . parasuis . In previous studies, our group had already screened four secreted proteins (RnfC, Ndk, Gcp, and HsdS) and three OMPs (Omp26, VacJ, and HAPS_0742) as vaccine candidates against Glässer's disease[ 18 , 22 ]. Therefore, we will further evaluate the screened bacterial proteins in cocktails in pigs, which might contribute in the development of an efficacious multi-protein vaccine against Glässer's disease. In summary, humoral and cellular immunity and protection efficacy of rTolC, rLppC, and rHAPS_0926 identified by a selective bioinformatic approach were evaluated in this study. Our results showed that three recombinant proteins elicited high-titer antibodies, T cell-mediated immunity, and substantial protection (survival rate ≥ 50%) against H . parasuis serovar 5 infection. The combination of three antigens elicited a much stronger immune response and effective protection. Therefore, an antigen cocktail containing triple-rOMP would be a valuable candidate for developing an efficient and improved multivalent subunit vaccine against H . parasuis serovar 5 infection. Supporting information S1 File The excel data file used to generate the Fig 4C and 4D in this manuscript. (XLS) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2850155/

Practically Useful: What the R osetta Protein Modeling Suite Can Do for You

The objective of this review is to enable researchers to use the software package R osetta for biochemical and biomedicinal studies. We provide a brief review of the six most frequent research problems tackled with R osetta . For each of these six tasks, we provide a tutorial that illustrates a basic R osetta protocol. The R osetta method was originally developed for de novo protein structure prediction and is regularly one of the best performers in the community-wide biennial Critical Assessment of Structure Prediction. Predictions for protein domains with fewer than 125 amino acids regularly have a backbone root-mean-square deviation of better than 5.0 à . More impressively, there are several cases in which R osetta has been used to predict structures with atomic level accuracy better than 2.5 à . In addition to de novo structure prediction, R osetta also has methods for molecular docking, homology modeling, determining protein structures from sparse experimental NMR or EPR data, and protein design. R osetta has been used to accurately design a novel protein structure, predict the structure of protein−protein complexes, design altered specificity protein−protein and protein−DNA interactions, and stabilize proteins and protein complexes. Most recently, R osetta has been used to solve the X-ray crystallographic phase problem. Rosetta Conformational Sampling Strategies The majority of conformational sampling protocols in R osetta use the Metropolis Monte Carlo algorithm to guide sampling. Gradient-based minimization is often employed for the last step of refinement of initial models. Since each R osetta protocol allows degrees of freedom specific for the task, R osetta can perform a diverse set of protein modeling tasks ( 3 ). Sampling Strategies for Backbone Degrees of Freedom R osetta separates large backbone conformational sampling from local backbone refinement. Large backbone conformational changes are modeled by exchanging the backbone conformations of nine or three amino acid peptide fragments. Peptide conformations are collected from the PDB for homologous stretches of sequence ( 4 ) that capture the structural bias for the local sequence ( 5 ). For local refinement of protein models, R osetta utilizes Metropolis Monte Carlo sampling of ϕ and ψ angles that are calculated not to disturb the global fold of the protein. Rohl ( 6 ) provides a review of the fragment selection and backbone refinement algorithms in R osetta . Sampling Strategies for Side Chain Degrees of Freedom Systematic sampling of side chain degrees of freedom of even short peptides quickly becomes intractable ( 7 ). R osetta drastically reduces the number of conformations sampled through the use of discrete conformations of side chains observed in the PDB ( 8 , 9 ). These "rotamers" capture allowed combinations between side chain torsion angles, as well as the backbone ϕ and ψ angles, and thereby reduce the amount of conformational space ( 9 ). A Metropolis Monte Carlo simulated annealing run is used to search for the combination of rotamers occupying the global minimum in the energy function ( 8 , 10 ). This general approach is extended to protein design via replacement of a rotamer of amino acid A with a rotamer of amino acid B in the Monte Carlo step. Sampling Strategies for Backbone Degrees of Freedom R osetta separates large backbone conformational sampling from local backbone refinement. Large backbone conformational changes are modeled by exchanging the backbone conformations of nine or three amino acid peptide fragments. Peptide conformations are collected from the PDB for homologous stretches of sequence ( 4 ) that capture the structural bias for the local sequence ( 5 ). For local refinement of protein models, R osetta utilizes Metropolis Monte Carlo sampling of ϕ and ψ angles that are calculated not to disturb the global fold of the protein. Rohl ( 6 ) provides a review of the fragment selection and backbone refinement algorithms in R osetta . Sampling Strategies for Side Chain Degrees of Freedom Systematic sampling of side chain degrees of freedom of even short peptides quickly becomes intractable ( 7 ). R osetta drastically reduces the number of conformations sampled through the use of discrete conformations of side chains observed in the PDB ( 8 , 9 ). These "rotamers" capture allowed combinations between side chain torsion angles, as well as the backbone ϕ and ψ angles, and thereby reduce the amount of conformational space ( 9 ). A Metropolis Monte Carlo simulated annealing run is used to search for the combination of rotamers occupying the global minimum in the energy function ( 8 , 10 ). This general approach is extended to protein design via replacement of a rotamer of amino acid A with a rotamer of amino acid B in the Monte Carlo step. R osetta Energy Function Simulations with R osetta can be classified on the basis of whether amino acid side chains are represented by super atoms or centroids in the low-resolution mode or at atomic detail in the high-resolution mode. Both modes come with tailored energy functions that have been reviewed previously by Rohl ( 6 ). R osetta Knowledge-Based Centroid Energy Function The low-resolution energy function treats the side chains as centroids ( 4 , 11 ). The energy function models solvation, electrostatics, hydrogen bonding between β strands, and steric clashes. Solvation effects are modeled as the probability of seeing a particular amino acid with a given number of α carbons within an amino acid-dependent cutoff distance. Electrostatic interactions are modeled as the probability of observing a given distance between centroids of amino acids. Hydrogen bonding between β strands is evaluated on the basis of the relative geometric arrangement of strand fragments. Backbone atom and side chain centroid overlap is penalized and thus provides the repulsive component of a van der Waals force. A radius of gyration term is used to model the effect of van der Waals attraction. All probability profiles have been derived using Bayesian statistics on crystal structures from the PDB. The lower resolution of this centroid-based energy function smoothes the energy landscape at the expense of its accuracy. The smoother energy landscape allows structures that are close to the true global minima to maintain a low energy even with structural defects that a full atom energy function would stiffly penalize. Knowledge-Based All Atom Energy Function The all atom high-resolution energy function used by R osetta was originally developed for protein design ( 8 , 12 ). It combines the 6-12 Lennard-Jones potential for van der Waals forces, a solvation approximation ( 13 ), an orientation-dependent hydrogen bonding potential ( 14 ), a knowledge-based electrostatics term, and a knowledge-based conformation-dependent amino acid internal free energy term ( 9 ). An important consideration in the construction of this potential was that all energy terms are pairwise decomposable. The pairwise decomposition of each of the terms limits the total number of energy contributions to 1 / 2 N ( N − 1) when N is the number of atoms within the system. This limitation allows precomputation and storage of many of these energy contributions in the computer memory, which is necessary for rapid execution of the Metropolis Monte Carlo sampling strategies employed by R osetta during protein design and atomic-detail protein structure prediction. R osetta Knowledge-Based Centroid Energy Function The low-resolution energy function treats the side chains as centroids ( 4 , 11 ). The energy function models solvation, electrostatics, hydrogen bonding between β strands, and steric clashes. Solvation effects are modeled as the probability of seeing a particular amino acid with a given number of α carbons within an amino acid-dependent cutoff distance. Electrostatic interactions are modeled as the probability of observing a given distance between centroids of amino acids. Hydrogen bonding between β strands is evaluated on the basis of the relative geometric arrangement of strand fragments. Backbone atom and side chain centroid overlap is penalized and thus provides the repulsive component of a van der Waals force. A radius of gyration term is used to model the effect of van der Waals attraction. All probability profiles have been derived using Bayesian statistics on crystal structures from the PDB. The lower resolution of this centroid-based energy function smoothes the energy landscape at the expense of its accuracy. The smoother energy landscape allows structures that are close to the true global minima to maintain a low energy even with structural defects that a full atom energy function would stiffly penalize. Knowledge-Based All Atom Energy Function The all atom high-resolution energy function used by R osetta was originally developed for protein design ( 8 , 12 ). It combines the 6-12 Lennard-Jones potential for van der Waals forces, a solvation approximation ( 13 ), an orientation-dependent hydrogen bonding potential ( 14 ), a knowledge-based electrostatics term, and a knowledge-based conformation-dependent amino acid internal free energy term ( 9 ). An important consideration in the construction of this potential was that all energy terms are pairwise decomposable. The pairwise decomposition of each of the terms limits the total number of energy contributions to 1 / 2 N ( N − 1) when N is the number of atoms within the system. This limitation allows precomputation and storage of many of these energy contributions in the computer memory, which is necessary for rapid execution of the Metropolis Monte Carlo sampling strategies employed by R osetta during protein design and atomic-detail protein structure prediction. Protein Structure Prediction The central tenet of structural biology is that structure determines function. Thus, the structure of a protein is critical for a full understanding of its function. Experimental structure determination techniques such as X-ray crystallography, nuclear magnetic resonance, electron paramagnetic resonance, and electron microscopy require significant investments of effort to produce structures of a molecule. Conversely, the advent of the genomic revolution created an explosion in the number of known sequences for biopolymers. For example, from October 2008 to March 2009, approximately 8 million (!) new, nonredundant sequences were added to the BLAST database. R osetta remedies the shortfall in structural information by predicting high-probability structures for a given amino acid sequence. De Novo Folding Simulation The "protein folding problem" (given an amino acid sequence, predict the tertiary structure into which it folds) is considered the greatest challenge in computational structural biology. The R osetta de novo structure prediction algorithm has been reviewed and described in detail elsewhere ( 4 , 6 , 11 , 15 ). Briefly, R osetta begins with an extended peptide chain. Insertion of backbone fragments rapidly "folds" the protein using the low-resolution energy function and sampling approaches (Figure 1 ). R osetta attempts approximately 30000 nine-residue fragment insertions followed by another 10000 three-residue fragment insertions to generate a protein model ( 6 ). Usually, 20000−50000 models are folded for each individual protein ( 15 ). The resulting models can undergo atomic-detail refinement, or if computational expense is an issue, clustering based on the C α root-mean-square deviation (rmsd) ( 16 , 17 ) can reduce the number of models before performing refinement. The clustering parameters are chosen by the user to generate clusters that maintain the same overall fold (i.e., C α rmsd 50% identical sequence), the protein backbone is only rebuilt in regions surrounding insertions and deletions in the sequence alignment ( 19 , 22 ). Consequently, R osetta starts with the template structure and builds in missing loops using fragment insertion or randomization of ϕ and ψ angles followed by one of the loop closure algorithms such as cyclic coordinate descent or kinematic closure ( 23 − 25 ). In the case of a medium to low degree of sequence identity between the template and target, Raman et al. applied a more aggressive iterative stochastic rebuild and refine protocol that allowed the complete rebuilding of large regions of the protein, which in some cases included entire secondary structure elements ( 19 ). Mandell et al. ( 24 ) recently developed a Loop Closure algorithm in R osetta that achieves rmsd values of better than 1 à . Their adaptation of Kinematic closure (KIC) first selects three C α atoms as pivots. Next, nonpivot (φ and ψ) torsion angles are sampled, leading to a chain break at the middle pivot. Finally, KIC is used to determine φ and ψ torsion angles for the pivot atoms that close the loop. For a data set of 25 loops containing 12 residues each, R osetta achieved a median accuracy of 0.8 à rmsd (see Figure 2 ). This demonstrates an improvement over both the standard R osetta cyclic coordinate descent protocol and a state-of-the-art molecular dynamics protocol (median accuracies of 2.0 and 1.2 à rmsd, respectively). The "Loop Modeling" tutorial demonstrates the kinematic loop closure protocol. Figure 2 Kinematic loop closure. (a) The kinematic loop closure algorithm samples φ and ψ angles at the cyan C α spheres from a residue specific Ramachandran map. The φ and ψ angles at green C α spheres are determined analytically to close the loop. (b) The energy vs rmsd plot shows accuracies for the prediction of loop conformation better than 1 à achieved through the improved sampling offered by the kinematic closure protocol. (c) The kinematic closure prediction (blue) closely resembles the crystallographic conformation (cyan). From ( 24 ) reprinted with permission from Nature Methods . Model Relaxation and Refinement After construction of a protein backbone via de novo protein folding or comparative modeling, the model enters atom-detail refinement ( 15 , 26 , 27 ). During the iterative relaxation protocol, ϕ and ψ angles of the backbone are perturbed slightly while the overall global conformation of the protein is maintained. The side chains of the protein are adjusted using a simulated annealing Metropolis Monte Carlo search of the rotamer space. Finally, gradient minimization is applied to all torsional degrees of freedom (ϕ, ψ, ω, and χ). The repulsive portion of van der Waals potential is increased incrementally, moving the structure to the nearest energy minimum. Extensive use of the all atom model refinement has proven integral to the success of R osetta in the recent Critical Assessment of Structure Prediction (CASP) experiments. A basic refinement protocol is introduced in the "Refinement" tutorial. Recently, Qian et al. applied the refinement protocol to protein structures determined de novo , via comparative modeling, or using NMR-derived restraints ( 26 ). In this protocol, protein models derived from NMR constraints or comparative modeling were used as a basis for solving the crystallographic phase problem. The model was initially minimized using R osetta 's all atom Monte Carlo Refinement protocol. The results of this refinement were used to identify regions likely to deviate from the native structure. In this context, it was demonstrated that regions of high variability between refined models often correlate to areas of deviation from the native structure. The conformational space in these areas was sampled extensively using the fragment replacement approach used by R osetta 's de novo structure prediction protocol. The resulting models are then subjected to another round of all atom refinement. This cycle of refinement and conformational sampling is performed iteratively, each time using only the lowest-energy models from the previous round of refinement, until the system converges. The final models were then used in molecular replacement to solve the crystallographic phase problem. In a blind test, this ab initio phase solution method resulted in significant improvement in structural resolution compared to that of the original unrefined models. The protocol can also be applied for the refinement of models derived from medium-resolution NMR data. R osetta 's Performance in the CASP Experiment R osetta has displayed remarkable success in de novo structure prediction in the last several blind CASP experiments; this is evidenced by the method's ranking among the top structure prediction groups ( 16 , 19 , 22 , 28 − 31 ). During CASP, sequences of proteins not yet reported in the PDB are distributed among participating laboratories. Within a given time limit, predictions are collected and assessed on the basis of the experimental structure that is typically available shortly after the CASP experiment ( http://www.predictioncenter.org ). Generally, R osetta has superseded competing approaches at predicting the structure of small to moderately sized protein domains with fewer than 200 amino acids de novo . Shortly after the CASP6 (held in 2004), Bradley et al. showed that for a benchmark of small proteins R osetta de novo structure prediction found models at atomic detail accuracy in an encouraging eight of 16 cases ( 15 , 29 ). In that same year, Misura et al. found that homology models built with R osetta can be more accurate than their templates ( 15 ). During CASP 7, with the assistance of the R osetta @Home distributed computer network, several moderately sized domains were predicted to atomic-detail accuracy within 2 à of the experimental structure for the first time ( 16 ). On the basis of the performance of R osetta in improving models over the best template structures available (see Figure 3 ) ( 19 ), Raman et al. suggest that the limitation of the R osetta structure prediction protocol remains in the sampling algorithms rather than the energy function. For this reason, prediction of larger domains becomes possible upon introduction of experimental data which restricts the conformational space. Figure 3 Comparative modeling CASP performance. Raman and colleagues demonstrated that comparative models refined with R osetta improved upon the best template structure available for several CASP targets, for example, (a) T0492 and (b) T0464. The native structure is colored blue, the best submitted R osetta model red, and the best template structure green. The R osetta models for T0492 resulted in atomic-level accuracy for side chains in the core of the protein. For T0464, a 25-residue insertion was predicted which resulted in models that were significantly improved over the best templates available. One of the models was further improved in the model refinement category. From ( 19 ) reprinted with permission from Proteins . R osetta Leverages Sparse Experimental Data from NMR and EPR Experiments R osetta allows incorporation of many types of experimental restraints. R osetta 's ability to deal with restraints derived from nuclear magnetic resonance (NMR) spectroscopy is the most developed ( 32 ). NMR restraints have two entry points into the R osetta protein structure prediction routine. Chemical shift assignments for backbone atoms can be converted to ϕ and ψ backbone angle restraints ( 33 ) and are used during the selection of the fragment libraries. Distance and orientation restraints [e.g., nuclear Overhauser effect (NOE) restraints and residual dipolar couplings (RDCs), respectively] are incorporated into the scoring function used during folding. Bowers et al. showed that a sparse mixture of short- and long-range NOE restraints (approximately one restraint per residue) in addition to the backbone chemical shifts enables R osetta to build models at atomic-detail accuracy ( 34 ). Rohl and Baker ( 35 ) likewise demonstrated that limited RDC measurements (approximately one per residue) in combination with backbone chemical shifts identify the correct fold. Meiler and Baker presented a protocol that uses unassigned NMR restraints for rapid protein fold determination ( 36 ). More recently, Shen et al. showed the use of a modified fragment selection protocol in R osetta to generate structures of a quality comparable to those from traditional NMR structure determination methods ( 37 ). Furthermore, Shen found that R osetta sampling can compensate for the incomplete and incorrect NMR restraints ( 38 ). A major point to note is that in each of these examples R osetta is used to complement structure restraints obtained early in the structure determination process. Consistently, R osetta models are accurate at the atomic level of detail that would only be apparent from either significantly more or higher-resolution experimental information. For example, Rohl and Baker found that R osetta produced ubiquitin models with an rmsd of 20 à ( 35 ). Beyond NMR restraints, any experimental data suitable to represent the distance between atoms can be used in the simulation. Through site-directed spin labeling (SDSL), such distance restraints can be obtained from electron paramagnetic resonance (EPR) spectroscopy ( 39 ). Alexander et al. generated accurate atomic-detail models of T4 lysozyme (see Figure 4 ) and the heat shock protein α-crystallin using SDSL-EPR data using as few as one distance restraint for every four residues. Similar approaches can be used to model multimeric complexes from monomers, as Hanson et al. showed for the Arrestin tetramer in solution ( 40 ). Both the "Protein Folding" and "Loop Modeling" tutorials demonstrate the use of distance restraints. Figure 4 De novo protein structure prediction from sparse EPR data. Alexander et al. demonstrated that approximately one low-resolution distance restraint for every four residues is sufficient to determine a model of T4 lysozyme that is accurate at an atomic level of detail. The distance restraints had been determined using SDSL-EPR experiments. The native T4 lysozyme structure is colored gray, while the model with an rmsd of 1.0 à is shown with a rainbow coloring scheme. Side chain conformations in the core of the protein are accurately represented in the model. From ( 39 ) reprinted with permission from Structure . R osetta Models Assist in the Determination of Molecular Structures from Electron Diffraction Data De novo -predicted models have also been used to assist in phasing of X-ray diffraction data (see Figure 5 ) ( 26 , 41 , 42 ). Das and Baker found that for 15 of 30 benchmark cases, R osetta de novo models successfully solved the phase problem by molecular replacement ( 43 ). Das and Baker suggest that approximately one in six X-ray diffraction data sets for proteins with 100 or fewer residues can be solved via molecular replacement using R osetta -generated de novo models. In a subsequent study, Ramelot et al. showed that refinement of NMR ensembles using R osetta results in higher-quality molecular replacement solutions to X-ray diffraction data than direct use of the NMR ensembles ( 42 ). DiMaio et al. extended R osetta to directly build models from electron density ( 44 ). Both Lindert and DiMaio have obtained atomic accuracy models via cryo-electron microscopy density maps at resolutions of 4−7 à using R osetta 44 − 46 . In both cases, resulting models have a higher resolution than the density from which they were built. Figure 5 Crysallographic phase problem. Qian et al. demonstrated that R osetta -predicted protein models can be used in conjunction with automated molecular replacement algorithms to determine phases for electron density maps. The coordinates of BH3980 from Bacillus halodurans [PDB entry 2hh6 (unpublished), colored red] fit well into the isosurface of the electron density determined by molecular replacement using a R osetta prediction for T0283 at CASP 7. From ( 26 ) reprinted with permission from Nature . Protein Structure Prediction Servers Large parts of the R osetta protein structure prediction protocol, including generation of fragments, de novo folding, and comparative modeling, have been replicated in an automated server R obetta 30 , 47 , 48 . Chivian found that comparative models built with early versions of R obetta generally did not improve upon templates from close homologues; however, recently, R obetta performed well in fold recognition and produced models that serve as good starting points for further refinement ( 48 ). In the most recent CASP, however, R obetta produced several models with accuracy comparable to that of the best human predictions ( 19 ). For instance, R obetta 's top model for the server only target, T0513 domain 2, had an rmsd of 0.84 à . In general, the performance of R obetta compared to that of other servers increases as the quality of the template structures decreases ( 19 ). R obetta is publicly accessible at http://robetta.bakerlab.org . De Novo Folding Simulation The "protein folding problem" (given an amino acid sequence, predict the tertiary structure into which it folds) is considered the greatest challenge in computational structural biology. The R osetta de novo structure prediction algorithm has been reviewed and described in detail elsewhere ( 4 , 6 , 11 , 15 ). Briefly, R osetta begins with an extended peptide chain. Insertion of backbone fragments rapidly "folds" the protein using the low-resolution energy function and sampling approaches (Figure 1 ). R osetta attempts approximately 30000 nine-residue fragment insertions followed by another 10000 three-residue fragment insertions to generate a protein model ( 6 ). Usually, 20000−50000 models are folded for each individual protein ( 15 ). The resulting models can undergo atomic-detail refinement, or if computational expense is an issue, clustering based on the C α root-mean-square deviation (rmsd) ( 16 , 17 ) can reduce the number of models before performing refinement. The clustering parameters are chosen by the user to generate clusters that maintain the same overall fold (i.e., C α rmsd 50% identical sequence), the protein backbone is only rebuilt in regions surrounding insertions and deletions in the sequence alignment ( 19 , 22 ). Consequently, R osetta starts with the template structure and builds in missing loops using fragment insertion or randomization of ϕ and ψ angles followed by one of the loop closure algorithms such as cyclic coordinate descent or kinematic closure ( 23 − 25 ). In the case of a medium to low degree of sequence identity between the template and target, Raman et al. applied a more aggressive iterative stochastic rebuild and refine protocol that allowed the complete rebuilding of large regions of the protein, which in some cases included entire secondary structure elements ( 19 ). Mandell et al. ( 24 ) recently developed a Loop Closure algorithm in R osetta that achieves rmsd values of better than 1 à . Their adaptation of Kinematic closure (KIC) first selects three C α atoms as pivots. Next, nonpivot (φ and ψ) torsion angles are sampled, leading to a chain break at the middle pivot. Finally, KIC is used to determine φ and ψ torsion angles for the pivot atoms that close the loop. For a data set of 25 loops containing 12 residues each, R osetta achieved a median accuracy of 0.8 à rmsd (see Figure 2 ). This demonstrates an improvement over both the standard R osetta cyclic coordinate descent protocol and a state-of-the-art molecular dynamics protocol (median accuracies of 2.0 and 1.2 à rmsd, respectively). The "Loop Modeling" tutorial demonstrates the kinematic loop closure protocol. Figure 2 Kinematic loop closure. (a) The kinematic loop closure algorithm samples φ and ψ angles at the cyan C α spheres from a residue specific Ramachandran map. The φ and ψ angles at green C α spheres are determined analytically to close the loop. (b) The energy vs rmsd plot shows accuracies for the prediction of loop conformation better than 1 à achieved through the improved sampling offered by the kinematic closure protocol. (c) The kinematic closure prediction (blue) closely resembles the crystallographic conformation (cyan). From ( 24 ) reprinted with permission from Nature Methods . Model Relaxation and Refinement After construction of a protein backbone via de novo protein folding or comparative modeling, the model enters atom-detail refinement ( 15 , 26 , 27 ). During the iterative relaxation protocol, ϕ and ψ angles of the backbone are perturbed slightly while the overall global conformation of the protein is maintained. The side chains of the protein are adjusted using a simulated annealing Metropolis Monte Carlo search of the rotamer space. Finally, gradient minimization is applied to all torsional degrees of freedom (ϕ, ψ, ω, and χ). The repulsive portion of van der Waals potential is increased incrementally, moving the structure to the nearest energy minimum. Extensive use of the all atom model refinement has proven integral to the success of R osetta in the recent Critical Assessment of Structure Prediction (CASP) experiments. A basic refinement protocol is introduced in the "Refinement" tutorial. Recently, Qian et al. applied the refinement protocol to protein structures determined de novo , via comparative modeling, or using NMR-derived restraints ( 26 ). In this protocol, protein models derived from NMR constraints or comparative modeling were used as a basis for solving the crystallographic phase problem. The model was initially minimized using R osetta 's all atom Monte Carlo Refinement protocol. The results of this refinement were used to identify regions likely to deviate from the native structure. In this context, it was demonstrated that regions of high variability between refined models often correlate to areas of deviation from the native structure. The conformational space in these areas was sampled extensively using the fragment replacement approach used by R osetta 's de novo structure prediction protocol. The resulting models are then subjected to another round of all atom refinement. This cycle of refinement and conformational sampling is performed iteratively, each time using only the lowest-energy models from the previous round of refinement, until the system converges. The final models were then used in molecular replacement to solve the crystallographic phase problem. In a blind test, this ab initio phase solution method resulted in significant improvement in structural resolution compared to that of the original unrefined models. The protocol can also be applied for the refinement of models derived from medium-resolution NMR data. R osetta 's Performance in the CASP Experiment R osetta has displayed remarkable success in de novo structure prediction in the last several blind CASP experiments; this is evidenced by the method's ranking among the top structure prediction groups ( 16 , 19 , 22 , 28 − 31 ). During CASP, sequences of proteins not yet reported in the PDB are distributed among participating laboratories. Within a given time limit, predictions are collected and assessed on the basis of the experimental structure that is typically available shortly after the CASP experiment ( http://www.predictioncenter.org ). Generally, R osetta has superseded competing approaches at predicting the structure of small to moderately sized protein domains with fewer than 200 amino acids de novo . Shortly after the CASP6 (held in 2004), Bradley et al. showed that for a benchmark of small proteins R osetta de novo structure prediction found models at atomic detail accuracy in an encouraging eight of 16 cases ( 15 , 29 ). In that same year, Misura et al. found that homology models built with R osetta can be more accurate than their templates ( 15 ). During CASP 7, with the assistance of the R osetta @Home distributed computer network, several moderately sized domains were predicted to atomic-detail accuracy within 2 à of the experimental structure for the first time ( 16 ). On the basis of the performance of R osetta in improving models over the best template structures available (see Figure 3 ) ( 19 ), Raman et al. suggest that the limitation of the R osetta structure prediction protocol remains in the sampling algorithms rather than the energy function. For this reason, prediction of larger domains becomes possible upon introduction of experimental data which restricts the conformational space. Figure 3 Comparative modeling CASP performance. Raman and colleagues demonstrated that comparative models refined with R osetta improved upon the best template structure available for several CASP targets, for example, (a) T0492 and (b) T0464. The native structure is colored blue, the best submitted R osetta model red, and the best template structure green. The R osetta models for T0492 resulted in atomic-level accuracy for side chains in the core of the protein. For T0464, a 25-residue insertion was predicted which resulted in models that were significantly improved over the best templates available. One of the models was further improved in the model refinement category. From ( 19 ) reprinted with permission from Proteins . R osetta Leverages Sparse Experimental Data from NMR and EPR Experiments R osetta allows incorporation of many types of experimental restraints. R osetta 's ability to deal with restraints derived from nuclear magnetic resonance (NMR) spectroscopy is the most developed ( 32 ). NMR restraints have two entry points into the R osetta protein structure prediction routine. Chemical shift assignments for backbone atoms can be converted to ϕ and ψ backbone angle restraints ( 33 ) and are used during the selection of the fragment libraries. Distance and orientation restraints [e.g., nuclear Overhauser effect (NOE) restraints and residual dipolar couplings (RDCs), respectively] are incorporated into the scoring function used during folding. Bowers et al. showed that a sparse mixture of short- and long-range NOE restraints (approximately one restraint per residue) in addition to the backbone chemical shifts enables R osetta to build models at atomic-detail accuracy ( 34 ). Rohl and Baker ( 35 ) likewise demonstrated that limited RDC measurements (approximately one per residue) in combination with backbone chemical shifts identify the correct fold. Meiler and Baker presented a protocol that uses unassigned NMR restraints for rapid protein fold determination ( 36 ). More recently, Shen et al. showed the use of a modified fragment selection protocol in R osetta to generate structures of a quality comparable to those from traditional NMR structure determination methods ( 37 ). Furthermore, Shen found that R osetta sampling can compensate for the incomplete and incorrect NMR restraints ( 38 ). A major point to note is that in each of these examples R osetta is used to complement structure restraints obtained early in the structure determination process. Consistently, R osetta models are accurate at the atomic level of detail that would only be apparent from either significantly more or higher-resolution experimental information. For example, Rohl and Baker found that R osetta produced ubiquitin models with an rmsd of 20 à ( 35 ). Beyond NMR restraints, any experimental data suitable to represent the distance between atoms can be used in the simulation. Through site-directed spin labeling (SDSL), such distance restraints can be obtained from electron paramagnetic resonance (EPR) spectroscopy ( 39 ). Alexander et al. generated accurate atomic-detail models of T4 lysozyme (see Figure 4 ) and the heat shock protein α-crystallin using SDSL-EPR data using as few as one distance restraint for every four residues. Similar approaches can be used to model multimeric complexes from monomers, as Hanson et al. showed for the Arrestin tetramer in solution ( 40 ). Both the "Protein Folding" and "Loop Modeling" tutorials demonstrate the use of distance restraints. Figure 4 De novo protein structure prediction from sparse EPR data. Alexander et al. demonstrated that approximately one low-resolution distance restraint for every four residues is sufficient to determine a model of T4 lysozyme that is accurate at an atomic level of detail. The distance restraints had been determined using SDSL-EPR experiments. The native T4 lysozyme structure is colored gray, while the model with an rmsd of 1.0 à is shown with a rainbow coloring scheme. Side chain conformations in the core of the protein are accurately represented in the model. From ( 39 ) reprinted with permission from Structure . R osetta Models Assist in the Determination of Molecular Structures from Electron Diffraction Data De novo -predicted models have also been used to assist in phasing of X-ray diffraction data (see Figure 5 ) ( 26 , 41 , 42 ). Das and Baker found that for 15 of 30 benchmark cases, R osetta de novo models successfully solved the phase problem by molecular replacement ( 43 ). Das and Baker suggest that approximately one in six X-ray diffraction data sets for proteins with 100 or fewer residues can be solved via molecular replacement using R osetta -generated de novo models. In a subsequent study, Ramelot et al. showed that refinement of NMR ensembles using R osetta results in higher-quality molecular replacement solutions to X-ray diffraction data than direct use of the NMR ensembles ( 42 ). DiMaio et al. extended R osetta to directly build models from electron density ( 44 ). Both Lindert and DiMaio have obtained atomic accuracy models via cryo-electron microscopy density maps at resolutions of 4−7 à using R osetta 44 − 46 . In both cases, resulting models have a higher resolution than the density from which they were built. Figure 5 Crysallographic phase problem. Qian et al. demonstrated that R osetta -predicted protein models can be used in conjunction with automated molecular replacement algorithms to determine phases for electron density maps. The coordinates of BH3980 from Bacillus halodurans [PDB entry 2hh6 (unpublished), colored red] fit well into the isosurface of the electron density determined by molecular replacement using a R osetta prediction for T0283 at CASP 7. From ( 26 ) reprinted with permission from Nature . Protein Structure Prediction Servers Large parts of the R osetta protein structure prediction protocol, including generation of fragments, de novo folding, and comparative modeling, have been replicated in an automated server R obetta 30 , 47 , 48 . Chivian found that comparative models built with early versions of R obetta generally did not improve upon templates from close homologues; however, recently, R obetta performed well in fold recognition and produced models that serve as good starting points for further refinement ( 48 ). In the most recent CASP, however, R obetta produced several models with accuracy comparable to that of the best human predictions ( 19 ). For instance, R obetta 's top model for the server only target, T0513 domain 2, had an rmsd of 0.84 à . In general, the performance of R obetta compared to that of other servers increases as the quality of the template structures decreases ( 19 ). R obetta is publicly accessible at http://robetta.bakerlab.org . Protein−Protein Docking While protein function is often determined by interactions with other proteins, most structures found in the PDB contain single chains. Because of the difficulties in determining structures of protein complexes, computational methods for predicting protein−protein interactions are important. R osetta D ock provides tools for predicting the interaction of two proteins ( 49 ). R osetta D ock employs first a low-resolution rigid-body docking. The second high-resolution refinement stage provides for side chain conformational sampling and backbone relaxation. The R osetta D ock algorithm begins with random reorientation of both proteins ( 49 ). Next, one protein slides into contact with the other. The following low-resolution docking conformational search involves 500 Monte Carlo rigid-body movements. These moves rotate and translate one protein around the surface of the other with movements chosen from a Gaussian distribution centered at 5° and 0.7 à . Each conformation is scored using the low-resolution energy function based on residue pair interaction statistics, residue environment statistics, and van der Waals attractive and repulsive terms. In this low-resolution step, side chains are represented by their centroids. Next, 50 cycles of high-resolution refinement at the atomic level of detail are performed. Each cycle consists of a 0.1 à random rigid-body translation, Monte Carlo-based side chain rotamer sampling (packing), and gradient-based rigid-body minimization to find a local energy minimum. Finally, backbone flexibility is introduced around the protein interface. The "Protein−Protein Docking" tutorial demonstrates the entire protocol. R osetta D ock is available as a web server ( http://rosettadock.graylab.jhu.edu ) but is limited to complexes for which the approximate binding orientation is known. The server produces 1000 structures and returns details for the 10 lowest-energy models ( 50 ). R osetta D ock successfully recovered the native structures of 42 of 54 benchmark targets from which side chains had been removed ( 49 ). Starting with randomly placed proteins, R osetta D ock predicts more than 50% of the interface contacts for 23 of 32 benchmark targets ( 49 ). These results have improved with the addition of backbone flexibility ( 3 ) and conformational sampling ( 51 ). R osetta D ock has been used to predict the structures of anthrax protective antigen ( 52 ) and epidermal growth factor ( 53 ) bound to monoclonal antibodies. Both docking studies led to predicted interface structures consistent with known mutant binding properties and were used to select residues for site-directed mutagenesis. The antibody modeling protocol has been made accessible through a web server ( http://antibody.graylab.jhu.edu ). R osetta D ock was benchmarked in the Critical Assessment of PRediction of Interactions (CAPRI) experiment (Figure 6 ), where it achieved full-atom rmsds of better than 1.6 à for most targets ( 54 ). Its success has been attributed to advances such as the inclusion of gradient-based energy minimization of side chain torsion angles ( 54 ), incorporation of biochemical data ( 55 ), and coupling of docking with loop modeling ( 55 ). Figure 6 Protein interface prediction. High-resolution CAPRI prediction of the colicin D−immunity protein D interface. Both rigid-body orientation and side chain conformation were modeled. The crystal structure is colored red and orange, and the R osetta model is colored blue. (a) Whole protein complex. (b) The interface shows the side chains of catalytic residue H611 and additional positively charged residues that are thought to bind to the RNA, as well as their matching negatively charged residues in the immunity protein. From ( 54 ) reprinted with permission from Proteins: Structure, Function, and Bioinformatics . Sircar and Gray recently reported on an extension of the R osetta D ock algorithm that allows for accurate modeling of antibody−antigen complexes in the absence of an antibody crystal structure ( 56 ). S nug D ock simultaneously samples the rigid-body antibody−antigen positions, the orientation of antibody light and heavy chains, and the conformations of the six complementary determining loops. Additionally, antibody conformational ensembles can be provided to mimic conformational selection. As in R osetta D ock , side chain rotamers are sampled during high-resolution refinement. S nug D ock was compared with R osetta D ock in a blind prediction of human MCP-1 binding 11k2 antibody (PDB entry 2bdn ) ( 57 ). While the lowest-energy structure produced by R osetta D ock is incorrect, the model produced by S nug D ock meets the CAPRI acceptable criterion of having more than 30% of the residue−residue contacts predicted correctly. When combined with ensemble sampling, five of the 10 lowest-energy models meet the CAPRI medium-quality criterion of correctly predicting more than 50% residue−residue contacts. Similar results were seen in a benchmark of 15 antibody−antigen complexes. Protein−Ligand Docking Ligand docking seeks to predict the interaction between a protein and a small molecule. Most ligand docking applications struggle to correctly predict conformational selection or induced-fit effects ( 58 ) resulting from ligand and protein flexibility. As applications were originally designed for protein−ligand docking, flexibility is often a feature added as an afterthought. On the other hand, R osetta was originally developed for de novo structure prediction. As such, it was designed from its inception to efficiently model flexibility. While protein flexibility is well-defined by side chain rotamers and backbone ϕ and ψ angle changes, small molecule flexibility was newly introduced into R osetta ( 59 ). Modeling ligand flexibility using knowledge-based score functions is especially challenging since the available small molecule crystal structures pale in comparison to the possible chemical diversity available to small molecules. R osetta L igand is an application for docking a small molecule in the binding pocket of a protein that considers both ligand and protein flexibility ( 60 ). The R osetta L igand algorithm is a modification of the R osetta D ock algorithm. First, a ligand conformer is chosen randomly from a user-provided ligand conformational ensemble. Second, the ligand is moved to a user-defined putative binding site. A low-resolution shape complementarity search translates and rotates the ligand optimizing attractive and repulsive score terms. In the high-resolution phase, cycles of Monte Carlo minimization perturb the ligand pose and optimize amino acid side chain rotamers and ligand conformers. Lastly, all torsion degrees of freedom in the ligand and protein undergo gradient minimization, and the model is output. The "Small Molecule Docking" tutorial demonstrates this protocol. In a benchmark, R osetta L igand successfully recovered the native structure of 80 of 100 protein−ligand complexes with an rmsd better than 2.0 à . When docking ligands into experimental protein structures determined with different binding partners (cross-docking), R osetta L igand recovered the native structure in 14 of 20 cases. Comparing binding energy predictions with 229 experimentally determined binding energies from the Ligand-Protein Database ( http://lpdb.chem.lsa.umich.edu ) ( 61 ), R osetta L igand achieved an overall correlation coefficient of 0.63, which is comparable to the best scoring functions available for protein−ligand interfaces ( 62 ). Recently, backbone flexibility was added to the docking algorithm which led to improved predictions, including lower rmsds among top scoring ligands ( 63 ). Backbone flexibility allows prediction of induced-fit effects that occur upon ligand binding. When R osetta L igand was tested in a blind study on a set of lead-like compounds, its performance was comparable to those of other commercially available docking programs ( 64 ). The authors caution, however, that current docking programs fail 70% of the time on at least one of the receptors in the test set. Often researchers seek to understand the interaction of a small molecule with a target protein whose structure has not yet been determined. In such cases, docking studies utilize comparative models. R osetta L igand was recently used by Kaufmann et al. ( 65 ) to dock serotonin into comparative models of human and Drosophila serotonin transporters (hSERT and dSERT, respectively). The models were based on the leucine transporter (LeuTAa) structure reported by Yamashita et al. ( 66 ) which has an overall sequence similarity of 17% and a binding site similarity of 50% with SERT. Using these models alone, R osetta L igand predicted a binding mode that places serotonin deep in the binding pocket of SERT (see Figure 7 ). This binding mode is consistent with site-directed mutagenesis studies and substituted cysteine accessibility method (SCAM) data and retains the amine placement seen in the LeuTAa structure. Additionally, binding energy predictions of serotonin analogues agree with experimental data ( R = 0.72). Figure 7 Complex of the human serotonin transporter with its substrate. The color scheme of serotonin displays the differential sensitivity of human and Drosophila serotonin transporter (SERT) for serotonin derivatives as dervied from a QSAR study. Blue indicates a higher sensitivity in dSERT, while red indicates a higher sensitivity in hSERT. The QSAR data indicate that the docking pose predicted by R osetta L igand is plausible. From ( 65 ) reprinted with permission from Proteins . Kaufmann and Meiler find that R osetta L igand successfully docks a variety of small molecules into comparative models (unpublished results). R osetta L igand identified the binding mode within 2 à rmsd for six of nine protein−ligand complexes in which models had been submitted in the eighth CASP competition. In seven additional examples, Kaufmann and Meiler observe that R osetta L igand samples the correct binding mode in at least one template for most ligands, yielding an overall success rate of better than 70%. This success rate is comparable to R osetta L igand 's performance with an experimental structure for the protein partner and can be attributed to R osetta L igand 's ability to sample protein conformational changes. Protein Design All protocols discussed up to this point relate to protein structure prediction and seek to determine the position of amino acid atoms in space. Protein design, on the other hand, seeks to determine an amino acid sequence that folds into a given protein structure or performs a given function. In this context, the protein design problem (to find a sequence that folds into a given tertiary structure) is also known as the "inverse protein folding problem". The R osetta D esign algorithm ( 12 ) is an iterative process that energetically optimizes both the structure and sequence of a protein. R osetta D esign alternates between rounds of fixed backbone sequence optimization and flexible backbone energy minimization ( 12 ). During the sequence optimization step, a Monte Carlo simulated annealing search is used to sample the sequence space. Every amino acid is considered at each position in the sequence, and rotamers are constrained to the Dunbrack Library ( 67 ). After each round of Monte Carlo sequence optimization, the backbone is relaxed to accommodate the designed amino acids ( 12 ). The practical uses of R osetta D esign can be divided into five basic categories: design of novel folds ( 12 ), redesign of existing proteins ( 68 ), protein interface design ( 69 ), enzyme design ( 70 ), and prediction of fibril-forming regions in proteins ( 71 ). The " De Novo Protein Design" tutorial demonstrates the complete redesign of the protein ubiquitin. De Novo Protein Design The R osetta D esign method has been used for the de novo design of a fold that was not (yet) represented in the PDB. A starting backbone model consisting of a five-strand β sheet and two packed α helices was constructed with the R osetta de novo protocol using distance constraints derived from a two-dimensional sketch ( 12 ). The sequence was iteratively designed with five simulation trials of 15 cycles each. The final sequence was expressed, and the structure was determined using X-ray crystallography. The experimental structure has an rmsd with respect to the computational design of <1.1 à (see Figure 8 ) ( 12 ). Figure 8 Design of a novel protein fold. (a) The experimentally determined structure of the Top7 (red) fold displays an rmsd of 1.17 à with respect to the model that had been previously designed for this protein (blue). (b) In the core of the protein, side chain conformations have been designed to atomic-detail accuracy. From ( 12 ) reprinted with permission from AAAS . Similarly, a molecular switch that folded into a trimeric coiled coil in the absence of zinc, and a monomeric zinc finger in the presence of zinc, was designed by extending R osetta D esign to simultaneously optimize a sequence in two different folds. The sequence of an existing zinc finger domain was aligned with a coiled-coil hemaglutinin domain. During the design protocol, the sequence was optimized to fold into both tertiary structures ( 72 ). Redesign of Existing Proteins When nine globular proteins were stripped of all side chains and then redesigned using R osetta D esign , the average sequence recovery was 35% for all residues ( 73 ). In four of nine cases, the protein stability improved as measured by chemical denaturation. The structure of a redesigned human procarboxypeptidase (PDB entry 1aye ) ( 74 ) was determined experimentally. R osetta D esign was then used to systematically identify mutations of procarboxypeptidase that would improve the stability of the protein. All of the tested mutants were more stable than the wild-type protein, with the top-scoring mutant having a reduction of free energy of 5.2 kcal/mol ( 75 ). The R osetta D esign server ( http://rosettadesign.med.unc.edu ) ( 76 ) is a Web-based interface to the fixed backbone design module of R osetta that allows design of proteins with up to 200 residues. The average design takes 5−30 min to complete. Interface Design Computational design techniques have been used to engineer an endonuclease with altered specificity. A 1400 à 2 interface was designed between individual domains of two homodimeric endonucleases (I-DomI and I-CreI). The design retained specificity and catalytic activity and crystallized with an rmsd of 0.8 à with respect to the model ( 77 ). Similarly, a highly effective specificity switch was designed into the colicin E7 DNase−Im7 immunity complex through the design of a novel hydrogen bond network (Figure 9 ). This designed network exhibited a 300-fold increase in specificity ( 78 ). R osetta 's alanine scanning application simulates experimental alanine scanning in silico . Each residue in the protein complex is iteratively mutated to an alanine, and the change in binding free energy is calculated. In silico alanine scanning has been implemented in the current version of R osetta and is available through a Web-based interface ( http://robetta.bakerlab.org ) ( 69 ). More recently, multispecific designs have been generated in which a single protein interface sequence is simultaneously optimized to bind to multiple targets, producing a so-called "hub" protein ( 79 ). Figure 9 Design of a novel protein interface. Comparison of the designed specificity switch in the colicin E7 DNase−Im7 immunity complex with the experimentally determined structure. (a) Experimentally determined coordinates, including a density map for computationally designed residues. (b) The computational design (yellow) is superimposed on an experimental structure (orange). (c and d) Side-by-side comparison of the designed and experimentally determined hydrogen bond networks. (e) Hydrogen bonding connectivity in the context of the interface region. From ( 78 ) reprinted with permission from Journal of Molecular Biology . Protein design approaches have enhanced our knowledge of how protein sequence relates to protein structure. For instance, the finding that designed protein sequences are highly similar to the native sequence suggests that native protein sequences are optimal for their structure ( 8 ). Recently, Babor and Kortemme investigated the antibody sequence−structure relationship using R osetta protein design. They demonstrated that native sequences of antibody H3 loops are optimal for conformational flexibility ( 80 ). The authors collected pairs of unbound and antigen-bound antibody structures. They used multiconstraint design to find low-scoring sequences that were consistent with both unbound and bound structures. The sequences predicted by multiconstraint design were more similar to the native sequences than the sequences predicted to preferentially bind either the unbound or bound conformations. Next, they collected pairs of antibody structures differing only in their degree of maturation. They used protein design in R osetta to show that mature antibody sequences are optimized for the bound conformation. A current major challenge in protein interface design is the de novo design of a novel protein−protein interface. So far, the most successful attempts at de novo interface design have been relatively modest, focusing on small proteins and yielding micromolar affinity ( 20 , 81 ). Enzyme Design The R osetta M atch algorithm ( 82 ) starts from the protein backbone and attempts to build toward the specified transition state geometry. In this method, all possible active site positions are defined for the protein scaffold, and rotamers from the Dunbrack library ( 67 ) are placed at each sequence position in the catalytic site. The sequence of the area surrounding the catalytic site is then designed using the R osetta D esign method ( 82 ). Recently, the R osetta M atch algorithm was used to design enzymes that catalyze the retro-aldol reaction ( 70 ). The degrees of freedom in the transition state, the orientation of the active site side chains, and the conformations of the active site side chains were simultaneously optimized. Of 72 models tested, a total of 32 were found to have catalytic activity as much as four orders of magnitude greater than that of an uncatalyzed reaction. Two of the active enzymes were crystallized. The experimental structures share a high degree of similarity with the computational design (rmsd better than 1.1 à ), although the loop regions surrounding the catalytic site show significant variance from the model ( 70 ). Röthlisberger et al. have computationally designed functional Kemp elimination catalysts using R osetta M atch . Quantum chemical predictions were used to generate an idealized transition state model, and R osetta M atch was used to search for backbone configurations that would support the predicted transition sate. The resulting designs were expressed and found to have k cat / K m values between 6 and 160 M −1 s −1 . Directed evolution was then performed on the designed enzymes to produce an optimized enzyme with a k cat / K m of 2600 M −1 s −1 ( 83 ). The R osetta D esign Algorithm Can Be Used To Identify Structurally Similar Peptide Fragments A method for predicting peptides capable of forming amyloid fibrils was recently developed using the R osetta D esign protocol ( 71 ). The most well understood fibril-forming fragment, the NNQQNY peptide, was used as a template, and the sequence space was searched for alternative fibril-forming sequences. This method was then used to predict fibril-forming regions in proteins known to form amyloids. De Novo Protein Design The R osetta D esign method has been used for the de novo design of a fold that was not (yet) represented in the PDB. A starting backbone model consisting of a five-strand β sheet and two packed α helices was constructed with the R osetta de novo protocol using distance constraints derived from a two-dimensional sketch ( 12 ). The sequence was iteratively designed with five simulation trials of 15 cycles each. The final sequence was expressed, and the structure was determined using X-ray crystallography. The experimental structure has an rmsd with respect to the computational design of <1.1 à (see Figure 8 ) ( 12 ). Figure 8 Design of a novel protein fold. (a) The experimentally determined structure of the Top7 (red) fold displays an rmsd of 1.17 à with respect to the model that had been previously designed for this protein (blue). (b) In the core of the protein, side chain conformations have been designed to atomic-detail accuracy. From ( 12 ) reprinted with permission from AAAS . Similarly, a molecular switch that folded into a trimeric coiled coil in the absence of zinc, and a monomeric zinc finger in the presence of zinc, was designed by extending R osetta D esign to simultaneously optimize a sequence in two different folds. The sequence of an existing zinc finger domain was aligned with a coiled-coil hemaglutinin domain. During the design protocol, the sequence was optimized to fold into both tertiary structures ( 72 ). Redesign of Existing Proteins When nine globular proteins were stripped of all side chains and then redesigned using R osetta D esign , the average sequence recovery was 35% for all residues ( 73 ). In four of nine cases, the protein stability improved as measured by chemical denaturation. The structure of a redesigned human procarboxypeptidase (PDB entry 1aye ) ( 74 ) was determined experimentally. R osetta D esign was then used to systematically identify mutations of procarboxypeptidase that would improve the stability of the protein. All of the tested mutants were more stable than the wild-type protein, with the top-scoring mutant having a reduction of free energy of 5.2 kcal/mol ( 75 ). The R osetta D esign server ( http://rosettadesign.med.unc.edu ) ( 76 ) is a Web-based interface to the fixed backbone design module of R osetta that allows design of proteins with up to 200 residues. The average design takes 5−30 min to complete. Interface Design Computational design techniques have been used to engineer an endonuclease with altered specificity. A 1400 à 2 interface was designed between individual domains of two homodimeric endonucleases (I-DomI and I-CreI). The design retained specificity and catalytic activity and crystallized with an rmsd of 0.8 à with respect to the model ( 77 ). Similarly, a highly effective specificity switch was designed into the colicin E7 DNase−Im7 immunity complex through the design of a novel hydrogen bond network (Figure 9 ). This designed network exhibited a 300-fold increase in specificity ( 78 ). R osetta 's alanine scanning application simulates experimental alanine scanning in silico . Each residue in the protein complex is iteratively mutated to an alanine, and the change in binding free energy is calculated. In silico alanine scanning has been implemented in the current version of R osetta and is available through a Web-based interface ( http://robetta.bakerlab.org ) ( 69 ). More recently, multispecific designs have been generated in which a single protein interface sequence is simultaneously optimized to bind to multiple targets, producing a so-called "hub" protein ( 79 ). Figure 9 Design of a novel protein interface. Comparison of the designed specificity switch in the colicin E7 DNase−Im7 immunity complex with the experimentally determined structure. (a) Experimentally determined coordinates, including a density map for computationally designed residues. (b) The computational design (yellow) is superimposed on an experimental structure (orange). (c and d) Side-by-side comparison of the designed and experimentally determined hydrogen bond networks. (e) Hydrogen bonding connectivity in the context of the interface region. From ( 78 ) reprinted with permission from Journal of Molecular Biology . Protein design approaches have enhanced our knowledge of how protein sequence relates to protein structure. For instance, the finding that designed protein sequences are highly similar to the native sequence suggests that native protein sequences are optimal for their structure ( 8 ). Recently, Babor and Kortemme investigated the antibody sequence−structure relationship using R osetta protein design. They demonstrated that native sequences of antibody H3 loops are optimal for conformational flexibility ( 80 ). The authors collected pairs of unbound and antigen-bound antibody structures. They used multiconstraint design to find low-scoring sequences that were consistent with both unbound and bound structures. The sequences predicted by multiconstraint design were more similar to the native sequences than the sequences predicted to preferentially bind either the unbound or bound conformations. Next, they collected pairs of antibody structures differing only in their degree of maturation. They used protein design in R osetta to show that mature antibody sequences are optimized for the bound conformation. A current major challenge in protein interface design is the de novo design of a novel protein−protein interface. So far, the most successful attempts at de novo interface design have been relatively modest, focusing on small proteins and yielding micromolar affinity ( 20 , 81 ). Enzyme Design The R osetta M atch algorithm ( 82 ) starts from the protein backbone and attempts to build toward the specified transition state geometry. In this method, all possible active site positions are defined for the protein scaffold, and rotamers from the Dunbrack library ( 67 ) are placed at each sequence position in the catalytic site. The sequence of the area surrounding the catalytic site is then designed using the R osetta D esign method ( 82 ). Recently, the R osetta M atch algorithm was used to design enzymes that catalyze the retro-aldol reaction ( 70 ). The degrees of freedom in the transition state, the orientation of the active site side chains, and the conformations of the active site side chains were simultaneously optimized. Of 72 models tested, a total of 32 were found to have catalytic activity as much as four orders of magnitude greater than that of an uncatalyzed reaction. Two of the active enzymes were crystallized. The experimental structures share a high degree of similarity with the computational design (rmsd better than 1.1 à ), although the loop regions surrounding the catalytic site show significant variance from the model ( 70 ). Röthlisberger et al. have computationally designed functional Kemp elimination catalysts using R osetta M atch . Quantum chemical predictions were used to generate an idealized transition state model, and R osetta M atch was used to search for backbone configurations that would support the predicted transition sate. The resulting designs were expressed and found to have k cat / K m values between 6 and 160 M −1 s −1 . Directed evolution was then performed on the designed enzymes to produce an optimized enzyme with a k cat / K m of 2600 M −1 s −1 ( 83 ). The R osetta D esign Algorithm Can Be Used To Identify Structurally Similar Peptide Fragments A method for predicting peptides capable of forming amyloid fibrils was recently developed using the R osetta D esign protocol ( 71 ). The most well understood fibril-forming fragment, the NNQQNY peptide, was used as a template, and the sequence space was searched for alternative fibril-forming sequences. This method was then used to predict fibril-forming regions in proteins known to form amyloids. Caveats of Modeling Despite R osetta 's success in producing accurate and precise models, some of its predictions will necessarily be incorrect, whether due to imperfections in the statistically derived energy function or to practical limits on exhaustive sampling. The following four strategies must be employed by the skeptical researcher to reject incorrect models and validate the low-energy predictions. (1) Model precision is a necessary prerequisite for model accuracy. Hence, it is an important strategy to ensure precision by insisting upon convergence of multiple independent trials toward a single low-energy solution. This strategy is employed, for example, during the analysis of pairwise rmsd values of low-energy models in an "energy funnel". (2) A modification of the precision analysis is clustering. If more than one low-energy solution is found, clustering assesses whether independent trajectories converged to a limited number of low-energy solutions. For example, clustering of models and ranking by cluster size is commonly used in de novo structure prediction, based on the observation that the deepest energy well is frequently also the widest ( 84 ). This is important because even using Monte Carlo search, adequate sampling is expensive to achieve due to the extreme roughness of the energy landscape ( 15 ). (3) Every mode of R osetta described in this review has been benchmarked on a set of test cases. Before these protocols are applied to a system that falls outside the scope of the test cases, it is necessary to apply the protocol to a closely related system of known structure. This experiment serves as a positive control for the method. Even if the application falls within the scope of the original benchmark, it is advisible to reproduce the benchmark results to ensure the operator-independent performance of the respective version of the software and accurate application of the protocol. (4) It is insufficient to rely solely on the R osetta energy function to discriminate good models from bad. The reliability of the result can be improved by incorporating the scores from disparate structure evaluators such as PROCHECK ( 85 ) and the DOPE scoring function implemented in Modeler ( 86 ). All of the preceding avenues are available without a departure from purely computational methodology. However, the most powerful and only conclusive method to ensure the reliability of computational models is the incorporation of experimental data. There are three strategies to incorporate experimental data into a modeling project: (a) Experimental restraints can be applied during the simulation (compare protein structure determination from NMR/EPR restraints); (b) Experimental restraints can discriminate inaccurate models in a post-simulation filtering; (c) Experimental restraints can be recorded to verify a computational model or hypothesis. More broadly, R osetta is most valuable as an integrated component of a research program in which initial structural models are used to guide hypothesis generation, and then data from experimental testing of these predictions are used to select and refine supported models in an iterative process. Conclusion The R osetta protein modeling suite provides a variety of tools for protein structure prediction and functional design. These techniques have been used in conjunction with traditional molecular and biochemical techniques to make predictions that would be prohibitively expensive or time-consuming via noncomputational methods. The quality of predictions has reached atomic-detail accuracy in many examples and is a practical tool for biochemical and biomedical research. R osetta 's de novo folding protocol is applicable if the protein of interest has no detectable homologues in the PDB and is fewer than 100 residues in length. For comparative models based on medium to distant homologues (25 and 50% identical sequence), R osetta 's comparative modeling protocols offer the ability of remodeling variable regions and regions of poor alignment. R osetta 's knowledge-based energy function and large-scale sampling strategies allow for construction of models from incomplete or limited experimental data sets. R osetta shows the capability of supplying structural detail in regions of the models underdetermined by the experimental data. R osetta 's protein−protein and protein−ligand docking protocols have proven to be particularly helpful if induced fit and conformational selection play a critical role in the interaction. Specialized protocols make R osetta an attractive option for antibody modeling. While de novo protein design remains a challenging problem, R osetta can routinely be applied when searching for thermo-stabilizing mutations, when redesigning protein−protein interfaces, and when performing in silico mutagenesis studies such as alanine scanning. Installation and Licensing The R osetta licenses are available at http://www.rosettacommons.org/software free of charge for noncommercial use. R osetta is compatible with most Unix-based operating systems and is distributed as source code. A user manual describing compilation, installation, and usage for the current release can be found at http://www.rosettacommons.org/manuals/rosetta3_user_guide . Interested developers can join the R osetta C ommons setup to contribute to the R osetta software package. Installation and Licensing The R osetta licenses are available at http://www.rosettacommons.org/software free of charge for noncommercial use. R osetta is compatible with most Unix-based operating systems and is distributed as source code. A user manual describing compilation, installation, and usage for the current release can be found at http://www.rosettacommons.org/manuals/rosetta3_user_guide . Interested developers can join the R osetta C ommons setup to contribute to the R osetta software package. Additional Features Several R osetta Methods under Development Have Been Excluded from This Review In addition to the protocols described above, several additional methods are currently in development. These methods have been excluded from this review as they are not yet fully implemented in the release version of the software. R osetta M embrane is a transmembrane helix scoring potential that allows R osetta to predict and design membrane bound proteins at atomic detail. In 2007, Barth et al. used this potential to predict the structure of small transmembrane helices at up to 2.5 à rmsd ( 87 ). The R osetta D esign protocol has also been adapted to model DNA−protein interactions. In 2002, Chevalier et al. used a DNA−protein interaction scoring function in combination with R osetta D esign to produce a novel endonuclease with high specificity ( 77 ). In addition to DNA−protein interactions, scoring potentials have been developed to score RNA−RNA interactions and allow for de novo prediction of RNA tertiary structure. This method was developed by Das et al. and uses the R osetta fragment-based design approach in conjunction with a knowledge-based potential for modeling RNA interactions. Through the use of this method, RNA structures have been predicted with a 4.0 à rmsd with respect to the backbone ( 16 ). Sheffler et al. implemented a space filling VDW model called R osetta H oles that detects voids and packing errors in protein structures ( 88 ). Extensions to the experimental modes available for docking small molecule ligands are also under development. These extensions will allow users to simultaneously dock multiple ligands and perform fragment-based design based on a scaffold and a library of small chemical fragments. R osetta Interfaces R osetta provides several optional user interfaces for interacting with the R osetta library. In addition to the standard command line interface, pyR osetta ( http://pyrosetta.org ) has been developed. It contains a set of Python bindings to the R osetta libraries which integrates many aspects of R osetta into Python scripts. A simple XML-based scripting language is available which allows users without programming experience to quickly generate custom protocols consisting of existing R osetta movers and filters. In addition to these conventional interfaces, the "FoldIt" game has been developed in which the player manually alters the protein conformation to identify energy minima using the R osetta scoring function ( http://www.fold.it ). Several R osetta Methods under Development Have Been Excluded from This Review In addition to the protocols described above, several additional methods are currently in development. These methods have been excluded from this review as they are not yet fully implemented in the release version of the software. R osetta M embrane is a transmembrane helix scoring potential that allows R osetta to predict and design membrane bound proteins at atomic detail. In 2007, Barth et al. used this potential to predict the structure of small transmembrane helices at up to 2.5 à rmsd ( 87 ). The R osetta D esign protocol has also been adapted to model DNA−protein interactions. In 2002, Chevalier et al. used a DNA−protein interaction scoring function in combination with R osetta D esign to produce a novel endonuclease with high specificity ( 77 ). In addition to DNA−protein interactions, scoring potentials have been developed to score RNA−RNA interactions and allow for de novo prediction of RNA tertiary structure. This method was developed by Das et al. and uses the R osetta fragment-based design approach in conjunction with a knowledge-based potential for modeling RNA interactions. Through the use of this method, RNA structures have been predicted with a 4.0 à rmsd with respect to the backbone ( 16 ). Sheffler et al. implemented a space filling VDW model called R osetta H oles that detects voids and packing errors in protein structures ( 88 ). Extensions to the experimental modes available for docking small molecule ligands are also under development. These extensions will allow users to simultaneously dock multiple ligands and perform fragment-based design based on a scaffold and a library of small chemical fragments. R osetta Interfaces R osetta provides several optional user interfaces for interacting with the R osetta library. In addition to the standard command line interface, pyR osetta ( http://pyrosetta.org ) has been developed. It contains a set of Python bindings to the R osetta libraries which integrates many aspects of R osetta into Python scripts. A simple XML-based scripting language is available which allows users without programming experience to quickly generate custom protocols consisting of existing R osetta movers and filters. In addition to these conventional interfaces, the "FoldIt" game has been developed in which the player manually alters the protein conformation to identify energy minima using the R osetta scoring function ( http://www.fold.it ). Supplementary Material bi902153g_si_001.pdf bi902153g_si_002.zip bi902153g_si_003.zip

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2583027/

β-Sheet Pore-Forming Peptides Selected from a Rational Combinatorial Library: Mechanism of Pore Formation in Lipid Vesicles and Activity in Biological Membranes †

In a previous report we described the selection of potent, β-sheet pore-forming peptides from a combinatorial library designed to mimic membrane-spanning β-hairpins (Rausch JM, Marks JR and Wimley WC, (2005) PNAS, 102:10511-5). Here, we characterize their mechanism of action and compare the structure-function relationships in lipid vesicles to their activity in biological membranes. The pore-forming peptides bind to membrane interfaces and self-assemble into β-sheets that cause a transient burst of graded leakage across the bilayers. Despite the continued presence of the structured peptides in the bilayer, at most peptide concentrations leakage is incomplete and ceases quickly after peptide addition with a deactivation half-time of several minutes. Molecules up to 3,000 Da escape from the transient pores, but much larger molecules do not. Fluorescence spectroscopy and quenching showed that the peptides reside mainly on the bilayer surface and are partially exposed to water, rather than in a membrane-spanning state. The "carpet" or "sinking raft" model of peptide pore formation offers a viable explanation for our observations and suggests that the selected pore formers function with a mechanism that is similar to the natural pore-forming antimicrobial peptides. We therefore also characterized the antimicrobial and cytotoxic activity of these peptides. All peptides studied, including non pore-formers, had sterilizing antimicrobial activity against at least some microbes, and most have low activity against mammalian cell membranes. Thus, the structure-function relationships that were apparent in the vesicle systems are similar to, but do not correlate completely with the activity of the same peptides in biological membranes. However, of the peptides tested, only the pore-formers selected in the high throughput screen have potent, broad-spectrum sterilizing activity against Gram-positive and Gram-negative bacteria as well as against fungi, while having only small lytic effects on human cells.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9018666/

Catalogue of the Diptera ( Insecta ) of Morocco— an annotated checklist, with distributions and a bibliography

Abstract The faunistic knowledge of the Diptera of Morocco recorded from 1787 to 2021 is summarized and updated in this first catalogue of Moroccan Diptera species. A total of 3057 species, classified into 948 genera and 93 families (21 Nematocera and 72 Brachycera ), are listed. Taxa (superfamily, family, genus and species) have been updated according to current interpretations, based on reviews in the literature, the expertise of authors and contributors, and recently conducted fieldwork. Data to compile this catalogue were primarily gathered from the literature. In total, 1225 references were consulted and some information was also obtained from online databases. Each family was reviewed and the checklist updated by the respective taxon expert(s), including the number of species that can be expected for that family in Morocco. For each valid species, synonyms known to have been used for published records from Morocco are listed under the currently accepted name. Where available, distribution within Morocco is also included. One new combination is proposed: Assuania melanoleuca (Séguy, 1941), comb. nov. ( Chloropidae ). Citation Kettani K, Ebejer MJ, Ackland DM, Bächli G, Barraclough D, Barták M, Carles-Tolrá M, Černý M, Cerretti P, Chandler P, Dakki M, Daugeron C, De Jong H, Dils J, Disney H, Droz B, Evenhuis N, Gatt P, Graciolli G, Grichanov IY, Haenni J-P, Hauser M, Himmi O, Macgowan I, Mathieu B, Mouna M, Munari L, Nartshuk EP, Negrobov OP, Oosterbroek P, Pape T, Pont AC, Popov GV, Rognes K, Skuhravá M, Skuhravý V, Speight M, Tomasovic G, Trari B, Tschorsnig H-P, Vala J-C, von Tschirnhaus M, Wagner R, Whitmore D, Woźnica AJ, Zatwarnicki T, Zwick P (2022) Catalogue of the Diptera (Insecta) of Morocco—an annotated checklist, with distributions and a bibliography. ZooKeys 1094: 1–466. https://doi.org/10.3897/zookeys.1094.62644 Foreword Almost everyone is now aware that there is a biodiversity crisis, and even the popular press regularly reports on general declines in the diversity of species as well as more specific threats to the distribution and abundance of species. Despite this growing awareness of the importance of biodiversity to the health of our planet and thus to the welfare of humankind, relatively little attention has been paid to the glaringly obvious point that it is impossible to objectively assess changes such as declines in diversity in the absence of baseline data and a good taxonomic infrastructure. In other words, if you don't know what you have to start with it is impossible to know what you are losing (or gaining). Recognition of invasions, extirpations, extinctions, range changes and changes in phenology all pivot on the documentation of what species occur in an area to begin with. Regional catalogues like this one provide both a baseline and a startling wake-up call; in this case with the announcements of 156 previously undocumented species, records of many hitherto unrecorded genera, and the first recognition of half a dozen families new to the country. Documentation of the dipteran diversity of Morocco is of special significance both in terms of taxon and area. The taxon, Diptera or true flies, is one of the two or three largest orders of living things, with hundreds of thousands of undescribed species worldwide in addition to the approximately 160,000 currently named species. The Moroccan Diptera fauna is relatively well known compared to other parts of Africa, but nonetheless the current catalogue more than doubles the number of species known from the country as of the last inventories a couple of decades ago. This impressive advance in the documentation of Morocco's significant dipteran fauna is of course in part due to the great dedication and Herculean effort on the part of the lead author, Prof. Kettani. But, it is also a great credit to the community of dipterists who rallied to the cause by meticulously cataloguing their chosen families. Approximately 33 of the 60 contributors are retired entomologists or amateur dipterists who volunteered their time to contribute to this work. Sadly, a few have passed away since they made their contribution. It is both impressive and worrisome that more than half of those 60 top experts on the taxonomy of the region's Diptera are retired or amateur, since there is a pressing and ongoing need for specialized expertise on all of the families of Diptera . Hopefully the framework, and the challenge, provided by this thorough description of our current state of knowledge of Moroccan flies will help and encourage new students of dipterology while at the same time providing a foundation for ongoing assessments of the dipteran diversity of North Africa. I congratulate all involved for the production of such an important contribution to our understanding of the biodiversity of the region, and for the successful completion of the first full Diptera catalogue for any African country. Stephen A. Marshall Professor, University of Guelph, Canada October 2020 Contributions This catalogue was conceived by K. Kettani (KK) in 2011 and then further developed and planned together with Martin J. Ebejer (MJE). These plans included more fieldwork, predominantly by KK and MJE, with some contribution by students, carried out in 2012–2019, which resulted in about 1000 new species records for Morocco and 23 species (in 12 families) new to science. These records were published prior to the end of 2020 and the papers are included in the references and cited under the appropriate families. KK compiled the data and the preliminary list of species for each family. The taxonomic specialists (co-authors and contributors) revised any taxonomic issues, added data available to them and updated nomenclature. Contributors are thanked in the Acknowledgements, as well as mentioned in their sections of the checklist. The distributions of species in Morocco, with the appropriate citations, were added by KK. The introduction was written by KK and supplemented with additional information by MJE, who also checked the English language. Introduction Aims of the catalogue Although the national study on biodiversity, Etude nationale sur la biodiversité ( Dakki 1997 ; Mouna 1998 ), was the only important synthesis of entomological studies carried out in Morocco until now, important data on the Diptera are lacking. For example, no taxonomic rank such as subfamily and tribe, and no synonyms or revised generic combinations, names of museums or private collections that house species described from Morocco were included. No information was provided on the habitats and distribution of species in Morocco. Furthermore, the information resulting from the national study on biodiversity is now outdated. A large number of important recent publications, cited appropriately under each family, provide much additional data to the lists published in 1997 and 1998, almost doubling the number of known species. The main aims of this catalogue are to bring together all this information by providing an up-to-date checklist according to the current nomenclature and systematics, to provide synonyms used in older literature that dealt with the Moroccan fauna and to give regional distribution data. Finally, an essential comprehensive bibliography is provided. This catalogue is the first for a North African country and it may prove useful also for neighbouring countries. It is hoped that it will provide a sound foundation for anyone choosing to study a group of Moroccan Diptera . It could indicate where the greatest need for investigation lies, both with regard to areas of the country that have been poorly studied and taxonomic groups (or families) that may benefit from more attention. It is undoubtedly not a complete inventory and many more discoveries are expected. The faunistic knowledge of the Diptera of Morocco from 1787 to 2021 is summarized and updated, providing the first catalogue of the Moroccan species of Diptera . In total, 3057 species (975 Nematocera and 2082 Brachycera ) in 93 families, classified into 949 genera (250 in Nematocera and 699 in Brachycera ) are listed (Tables 1 , 2 ). Table 1. Families of Nematocera with contributing specialists and numbers of taxa. Family Specialist(s) Subfamilies Genera Species Anisopodidae Haenni 1 1 3 Bibionidae Haenni 1 2 10 Blephariceridae Zwick 1 1 4 Cecidomyiidae Skuhravá & Skuhravý 3 31 57 Ceratopogonidae Mathieu 3 6 62 Chaoboridae Wagner 1 2 2 Chironomidae - 7 100 412 Culicidae Trari, Himmi & Dakki 2 7 43 Dixidae Wagner - 2 12 Keroplatidae Chandler 1 2 2 Limoniidae Oosterbroek 4 22 67 Mycetophilidae Chandler 5 25 64 Pediciidae Oosterbroek 1 2 6 Psychodidae Wagner 2 12 51 Ptychopteridae Wagner 1 1 5 Scatopsidae Haenni 3 11 13 Sciaridae Heller - 12 70 Simuliidae - 1 6 43 Thaumaleidae Wagner - 1 2 Tipulidae Oosterbroek & de Jong 2 3 39 Trichoceridae Krzemińska 1 1 8 Total: 250 Total: 975 Table 2. Families of Brachycera with contributing specialists and numbers of taxa. Family Specialist(s) Subfamilies Genera Species Acroceridae Nartshuk 2 6 13 Agromyzidae Černý 2 12 62 Anthomyiidae Ackland 2 14 36 Anthomyzidae Ebejer - 2 2 Asilidae Tomasovic 9 42 131 Asteiidae Ebejer 1 2 5 Atelestidae Gatt - 1 1 Athericidae Mouna 1 1 2 Aulacigastridae Ebejer - 1 1 Bombyliidae Ebejer & Dils 11 48 248 Braulidae - 1 1 1 Calliphoridae Rognes 5 5 8 Camillidae Ebejer - 1 4 Canacidae Munari 2 3 15 Carnidae Ebejer - 1 3 Chamaemyiidae Ebejer 2 5 18 Chloropidae von Tschirnhaus & Ebejer 3 34 74 Chyromyidae Ebejer 2 3 22 Clusiidae Ebejer - 1 1 Coelopidae - 1 1 1 Conopidae - 5 10 34 Cryptochetidae Nartshuk - 1 1 Diastatidae Ebejer 2 2 2 Dolichopodidae Grichanov & Negrobov 12 36 112 Drosophilidae Bächli 2 9 26 Dryomyzidae - 1 1 1 Empididae Daugeron 3 8 40 Ephydridae Zatwarnicki 5 43 117 Fanniidae Pont - 1 10 Helcomyzidae - 1 1 1 Heleomyzidae Woźnica 3 5 19 Hippoboscidae Droz 3 10 17 Hybotidae Gatt 2 10 44 Lauxaniidae Ebejer 2 8 27 Lonchaeidae MacGowan 1 4 5 Lonchopteridae Barták 1 1 4 Micropezidae Ebejer - 1 1 Milichiidae - 3 6 8 Muscidae Pont 6 25 115 Mydidae Dikow 3 4 9 Mythicomyiidae Evenhuis 4 6 8 Nemestrinidae Barraclough 2 4 13 Nycteribiidae Graciolli 1 4 8 Odiniidae Ebejer 1 1 2 Oestridae Pape 2 5 10 Opomyzidae - - 2 5 Pallopteridae Ebejer - 1 1 Phoridae Disney - 2 3 Piophilidae Ebejer 1 3 3 Pipunculidae Ebejer 2 7 16 Platypezidae Ebejer 1 3 3 Platystomatidae Popov 1 2 4 Polleniidae Rognes - 1 12 Psilidae Ebejer - 1 1 Rhagionidae Ebejer 1 1 4 Rhiniidae Rognes 1 5 17 Rhinophoridae Pape 1 6 8 Sarcophagidae Whitmore & Pape 3 16 66 Scathophagidae - 1 2 3 Scenopinidae Carles-Tolrá 1 2 11 Sciomyzidae Vala 1 12 25 Sepsidae Haenni 1 4 12 Sphaeroceridae Gatt 3 29 67 Stratiomyidae Woodley 5 11 40 Streblidae Graciolli 1 2 2 Syrphidae Speight 2 50 166 Tabanidae - 3 11 69 Tachinidae Cerretti & Tschorsnig 4 88 147 Tephritidae Norrbom 3 29 69 Therevidae Hauser 2 9 27 Ulidiidae Ebejer 2 8 13 Vermileonidae Ebejer 1 2 6 Total: 698 Total: 2082 Brief history of Moroccan dipterology The study of Diptera in Morocco is still far from being extensive despite the numerous investigations that have taken place so far. Until now, a synthesis of Moroccan Diptera had never been produced. The studies that have been devoted to this group of insects have been limited and sporadic. They were mainly concerned with either taxonomy and descriptions of new species, or faunal studies from specific areas of Morocco with little reference to the habitats of species. This was particularly so for early works such as Becker and Stein (1913) , Séguy (1930a , 1934b , 1935a , 1941a , 1941d , 1949a , 1953a ), Timon-David (1951) , Vaillant (1956b) and, recently, Pârvu et al. (2006) , Popescu-Mirceni (2011) , and Ebejer et al. (2019) . Very few recent works have provided relevant ecological data ( Cassar et al. 2005 , 2008 ). Becker and Stein (1913) were the first to publish a list of Moroccan Diptera , at that time comprising only 204 species. Séguy followed with a long series of valuable publications. In 1930 he produced his first list of Diptera , which included 471 species in 230 genera grouped into 37 families (Séguy 1930). Over the subsequent years, he contributed greatly to the knowledge of the Diptera of Morocco ( Séguy 1926 , 1934b , 1935a , 1941a , 1941d , 1949a , 1953a , 1957 , 1961 ), where he also referenced an extensive bibliography. Later, Bailly-Choumara (1977) compiled a bibliography of Moroccan Diptera . It took until 1997–1998, with a national study on the biodiversity of Morocco co-ordinated by what was then the Ministry of the Environment, to update the list of the Moroccan insect fauna. This national study was dedicated to the compilation of the results of entomological research conducted in Morocco in order to highlight Moroccan biodiversity. It included a comprehensive inventory of published entomological research conducted in Morocco and was the last work to include a broad overview of Moroccan Diptera . It listed 623 species of aquatic Diptera in 210 genera and 25 families ( Dakki 1997 ) and 928 species of terrestrial Diptera in 350 genera and 57 families ( Mouna 1998 ). Terrain and bioclimatic regions Morocco, located in the westernmost corner of North Africa, constitutes a biogeographical crossroads between the Afrotropical and the western Palaearctic faunas, which may have allowed some exchange of genetic material. The country has a remarkable variety of bioclimates, ranging from humid in the Rif and the Middle and High Atlas to very arid in the Sahara in the south and to the sub-humid and semi-arid plains and foothills (Fig. 1 ). As a result of this diversity of landscapes and climates, there exists a great biological and ecological diversity in the country. 10.3897/zookeys.1094.62644.figure1 Figure 1. The major bioclimatic regions of the northern part of Morocco. https://binary.pensoft.net/fig/671550 The geomorphological, orographic and bioclimatic diversity of the country is reflected in its seven distinct biogeographical areas: the Rif, the Atlantic Plain, Eastern Morocco, the Middle Atlas, the High Atlas, the Anti-Atlas and the Sahara. In the checklist, an abbreviated distribution (in bold) is given for each species according to these seven biogeographical areas (Fig. 2 ). 10.3897/zookeys.1094.62644.figure2 Figure 2. The seven biogeographical regions covering the studied area in the northern part of Morocco. https://binary.pensoft.net/fig/671551 Rif : The Rif consists of a mountainous domain overlooking the Mediterranean Sea in the north of Morocco. It belongs to the Gibraltar Arc or Alborán Sea geological region, part of the Alpine orogenic belt, where the maximum altitude is 2,452 m in the Jbel Tidirrhine mountain range. The climate is mainly of the Mediterranean type and is characterised by high levels of precipitation. This domain is considered very rich and diverse in vegetation cover ( Valdés et al. 2002 ), including nearly all of the Moroccan forest plant species ( Médail and Quézel 1999 ) and integrating five bioclimatic stages. Atlantic Plain ( AP ) : Also known as the Moroccan Central Plateau, the Atlantic Plain is an ancient massif located in the northwest of Morocco, between the Atlantic coast and the Middle Atlas Mountains. It consists of a plateau surrounded by medium-altitude mountain massifs and covers an area of 8500 km². With an asymmetrical shape, the plateau culminates in the south-east at Jebel Mtourzgene (1627 m), located near the town of Oulmès. Its complex geomorphology presents tectonic troughs that have favoured the formation of lakes and rivers. The annual rainfall of this region, concentrated in winter and spring, totals an average of 500 mm, allowing the establishment of Holm Oak and Cork Oak forests in a semi-arid bioclimate ( Piqué 1994 ). Towards the coast, the vegetation is mainly a steppe with Jujube shrubs and Dwarf Palms ( Chamaerops humilis ). The maritime fringe is underlined by cliffs and ancient consolidated dunes. Eastern Morocco ( EM ) : This is an arid region covering the Hauts Plateaux to the northeast of the country. It is the only region of Morocco subject to a true Mediterranean bioclimate, with some influence from the Sahara. The annual rainfall is less than 300 mm, encouraging the dominance of esparto steppe ( Stipa tenacissima ) ( Piqué 1994 ). Its geomorphology is represented by a plateau at an altitude of 1000 m, which gradually decreases from 1500 to 500 m from the city of Midelt to the province of Guercif and is criss-crossed by the Moulouya wadi. In the northwestern part of Eastern Morocco is the Béni Snassen massif, beyond the Guercif depression. Middle Atlas ( MA ) : A mountainous massif located in the centre of Morocco, oriented from south-west to north-east and extending over 350 km ( Piqué 1994 ). This mass of highlands is essentially composed of limestone layers, tabular and of medium altitude in the southwest (tabular Middle Atlas) and undulating in the northeast (folded Middle Atlas) to culminate at 3340 m at Jebel Bou Naceur, which overlooks the high plateaux of Eastern Morocco. The climate, generally subhumid to humid, is cold in winter, giving rise to cedar and Holm Oak forests covering the western slope, which is wetter than the eastern slope and where lakes are abundant and many rivers and streams have their source. High Atlas ( HA ) : The main mountainous massif of Morocco, oriented from west-south-west to east-north-east, is the High Atlas, stretching over more than 700 km from the Atlantic to the east of Morocco, with a north-south width of 50 to 100 km and reaching at Jebel Toubkal an altitude of 4165 m. The whole massif is high, incised by deep valleys that lead to passes through which the mountainous barrier is crossed: the Tizi-n'Test (2100 m) towards the Souss plain and the Tizi-n'Tichka (2200 m) towards the depression of Ouarzazate. The chain is formed by a massif of Palaeozoic rocks surrounded by sedimentary deposits. The relief of the High Atlas is divided into three different entities from west to east: the Western High Atlas, which is the oldest massif and is made up mainly of Jurassic or Cretaceous formations with deep valleys and culminates at Jebel Toubkal; the central High Atlas, which is an essentially limestone massif morphologically dominated by tabular zones culminating at an altitude of 2500 m, stretches between the towns of Azilal and Ouarzazate and is sheltered by the Jebel M'Goum (4071 m); and the Eastern High Atlas, which is formed by the vast high plateaux of the Upper Moulouya, stretches between the cities of Midelt and Imilchil and contains the Jebel Ayachi (3747 m) ( Piqué 1994 ). The bioclimate of this area includes subhumid, humid and semi-arid zones, but with more than 800 mm of annual precipitation on the highest peaks and a significant snow cover from September to May, with temperatures dropping below -20 °C (AEFCS 1996) and bright sunshine even in winter. These climatic conditions have favoured large pine, Holm Oak, Thuja and thuriferous juniper forests (HCEFLCD 2017). The aridity of the southern slope of the High Atlas allows only steppe vegetation of sagebrush and esparto grass to grow. Anti-Atlas ( AA ) : This is the pre-Saharan area, which extends north of the Wadi Draa and rises to an average altitude of 2000 m. It is a semi-arid region with Jebel Bani and Jebel Ouarkziz as its main elevations, and is home to numerous oases. The western part of the Anti-Atlas is characterised by the Saharan plateau, with a temperate winter except in the high mountains. On the other hand, in the eastern part of the Anti-Atlas, the greatest arid area is marked by the northward extension of the Saharan landscape and a bioclimate of cool and temperate winters. These bioclimatic stages are characterised by plant communities such as wooded steppes with Acacia raddiana , Artemisia herba alba and Stipa tenuissima , interspersed with thuriferous juniper ( Juniperus thurifera ) at the top of the slopes ( Piqué 1994 ). The vegetation is also represented by two formations that partially interpenetrate each other: groves of the Argan tree ( Argania spinosa ) and the Euphorbia steppe ( Euphorbia echinus ). Sahara ( SA ) : This area in the south of the country consists of a wide arid zone with a typical Saharan bioclimate of high daytime temperatures and very low precipitation. From a geological point of view it is part of the West African Craton and its margins. It is mainly ancient terrain with Precambrian basement rock and Palaeozoic cover ( Piqué 1994 ). This area is represented by a peneplain of very modest altitudes from 200 to 500 m, established on ancient crystalline terrain. Precipitation from July to September is very low, of the order of 100 mm. The dipteran species richness of each biogeographical region of Morocco is summarised in Table 3 , which highlights the great disparity between the different regions and reflects the lack of knowledge about this insect order in some areas of Morocco. The Rif region in the north of the country has the greatest recorded diversity, but this undoubtedly reflects a greater sampling effort in this area, with large parts of the country still very poorly explored for Diptera . The catalogue does not include any data from the southern part of the Moroccan Sahara. Table 3. Species richness of Diptera families each biogeographical region in Morocco. Rif ; AP – Atlantic Plain; EM – Eastern Morocco; MA – Middle Atlas; HA – High Atlas; AA – Anti-Atlas; SA – Sahara. * No precise localities known. Rif AP EM MA HA AA SA Acroceridae 7 1 0 1 3 0 0 Agromyzidae 7 15 0 10 22 10 0 Anisopodidae * Anthomyiidae 4 12 0 13 2 4 1 Anthomyzidae 1 0 0 0 1 0 0 Asilidae 34 27 4 35 25 24 5 Asteiidae 0 3 0 0 1 1 0 Atelestidae 1 0 0 0 0 0 0 Athericidae 0 0 0 1 2 0 0 Aulacigastridae 1 0 0 0 0 0 0 Bibionidae 4 2 0 3 1 2 2 Blephariceridae 1 0 0 0 3 0 0 Bombyliidae 35 80 12 59 48 56 19 Braulidae * Calliphoridae 3 8 2 4 4 0 0 Camillidae 1 2 0 0 1 0 0 Canacidae 6 10 0 0 0 6 0 Carnidae 1 0 0 0 0 0 0 Cecidomyiidae 8 27 21 25 20 7 4 Ceratopogonidae 15 43 14 35 33 24 36 Chamaemyiidae 5 5 0 4 8 6 0 Chaoboridae * Chironomidae 230 30 7 105 120 14 1 Chloropidae 35 22 0 7 6 25 1 Chyromyidae 6 6 0 0 1 12 1 Clusiidae 1 0 0 0 0 0 0 Coelopidae 1 0 0 0 0 0 0 Conopidae 7 13 0 16 7 9 0 Cryptochetidae 0 0 0 1 0 0 0 Culicidae 32 41 24 30 29 22 11 Diastatidae 0 1 0 0 1 0 0 Dixidae 2 2 0 2 5 0 0 Dolichopodidae 72 28 3 6 18 26 0 Drosophilidae 11 14 0 8 12 5 1 Dryomyzidae 1 0 0 0 0 0 0 Empididae 4 3 0 13 20 1 0 Ephydridae 47 44 1 60 9 20 1 Fanniidae 4 2 0 2 5 0 0 Helcomyzidae 1 0 0 0 0 0 0 Heleomyzidae 11 3 0 2 2 3 0 Hippoboscidae 0 2 3 4 4 4 1 Hybotidae 20 7 3 8 7 8 0 Keroplatidae 1 0 0 0 0 0 0 Lauxaniidae 23 3 2 1 8 2 0 Limoniidae 43 9 4 5 34 5 0 Lonchaeidae 3 3 0 1 1 0 0 Lonchopteridae 0 0 0 1 1 0 0 Micropezidae 1 0 0 0 0 0 0 Milichiidae 2 1 0 0 4 3 1 Muscidae 25 47 13 38 68 20 6 Mycetophilidae 60 3 7 3 0 0 0 Mydidae 0 3 2 1 0 3 1 Mythicomyiidae 2 0 0 0 1 2 1 Nemestrinidae 1 1 1 5 3 0 1 Nycteribiidae 6 5 0 6 0 2 1 Odiniidae 1 0 0 0 0 0 0 Oestridae 0 2 1 1 1 0 1 Opomyzidae 1 1 0 1 2 0 0 Pallopteridae 0 0 0 1 0 0 0 Pediciidae 3 1 0 0 0 0 0 Phoridae 0 2 0 0 0 0 0 Piophilidae 2 1 0 1 1 0 0 Pipunculidae 13 1 0 0 0 0 0 Platypezidae 2 1 0 0 0 0 0 Platystomatidae 0 2 1 1 0 0 0 Polleniidae 1 4 2 5 5 1 1 Psilidae 1 0 0 1 0 0 0 Psychodidae 25 12 12 12 34 9 2 Ptychopteridae * Rhagionidae 2 0 0 0 2 0 0 Rhiniidae 2 5 3 5 0 5 4 Rhinophoridae 6 2 0 1 0 0 0 Sarcophagidae 11 19 1 13 8 9 2 Scathophagidae 3 1 0 1 0 0 0 Scatopsidae 10 2 0 0 1 3 0 Scenopinidae 3 4 1 1 0 0 1 Sciaridae 29 12 2 8 45 9 3 Sciomyzidae 8 4 2 11 18 3 0 Sepsidae 3 6 0 4 7 3 0 Simuliidae 32 8 0 19 32 6 0 Sphaeroceridae 42 3 2 10 1 3 3 Stratiomyidae 20 13 5 13 6 1 0 Streblidae 1 1 0 0 0 1 0 Syrphidae 70 49 11 82 64 25 4 Tabanidae 26 27 11 35 21 7 1 Tachinidae 18 27 4 46 43 29 3 Tephritidae 34 13 4 16 13 23 3 Thaumaleidae 0 0 0 0 2 0 0 Therevidae 9 7 2 9 4 5 2 Tipulidae 28 7 0 16 18 2 0 Trichoceridae 7 1 3 1 0 0 0 Ulidiidae 6 2 0 1 4 4 1 Vermileonidae 1 2 1 1 4 0 0 Total species 1126 770 191 831 876 474 126 Sources of data Data for the present study were gathered from the literature, supplemented by information taken from a number of websites. The cut-off date for inclusion of species published in the literature is 30 June 2021. Taxonomic, nomenclatural, and distributional data for the present catalogue were obtained from: studies by various foreign and Moroccan researchers published between 1787 and 2021, including theses, books, catalogues, checklists and notes from the 1997–1998 synthesis studies on Morocco's biodiversity. data from websites and organisations such as Global Species, the Global Biodiversity Information Facility, the BioSystematic Database of World Diptera, Systema Dipterorum, Catalogue of Life, Systema Naturae 2000 and others, or from relevant homepages specific to particular families. the collection of Diptera in the National Museum of Morocco (Muséum National d'Histoire Naturelle à l'Institut Scientifique, Université Mohammed V, Rabat). collaboration with specialists who communicated reliable identifications from their personal collections or provided data for species deposited in foreign museums. published records of specimens in the personal collection of the lead author (PCKK), deposited at Abdelmalek Essaadi University, Tetouan, identified with the assistance of specialists. Classification The arrangement of families in this catalogue (Table 4 ) is mainly based on Pape et al. (2011) and Wiegmann et al. (2011) . All families are listed alphabetically within each superfamily. Nomenclature follows the most recent version of Systema Dipterorum ( Evenhuis and Pape 2021 ) and the Polleniidae are listed as a separate family ( Gisondi et al. 2020 ). Table 4. Diptera classification used in the catalogue. Suborder NEMATOCERA Tipuloidea Limoniidae Pediciidae Tipulidae Trichoceridoidea Trichoceridae Psychodoidea Psychodidae Scatopsoidea Scatopsidae Ptychopteridae Culicoidea Chaoboridae Culicidae Dixidae Chironomoidea Ceratopogonidae Chironomidae Simuliidae Thaumaleidae Blephariceridae Bibionoidea Anisopodidae Bibionidae Sciaroidea Cecidomyiidae Keroplatidae Mycetophilidae Sciaridae Suborder BRACHYCERA Stratiomyoidea Stratiomyidae Tabanoidea Athericidae Rhagionidae Tabanidae Vermileonidae Nemestrinoidea Acroceridae Nemestrinidae Asiloidea Asilidae Bombyliidae Mydidae Mythicomyiidae Scenopinidae Therevidae Empidoidea Atelestidae Empididae Dolichopodidae Hybotidae Platypezoidea Phoridae Platypezidae Lonchopteridae Syrphoidea Pipunculidae Syrphidae Conopoidea Conopidae Nerioidea Micropezidae Tanypezoidea Psilidae Tephritoidea Lonchaeidae Pallopteridae Piophilidae Platystomatidae Tephritidae Ulidiidae Lauxanioidea Chamaemyiidae Lauxaniidae Sciomyzoidea Coelopidae Dryomyzidae Helcomyzidae Sciomyzidae Sepsidae Opomyzoidea Agromyzidae Anthomyzidae Asteiidae Aulacigastridae Clusiidae Odiniidae Opomyzidae Carnoidea Canacidae Carnidae Chloropidae Milichiidae Sphaeroceroidea Chyromyidae Heleomyzidae Sphaeroceridae Ephydroidea Braulidae Camillidae Cryptochetidae Diastatidae Drosophilidae Ephydridae Hippoboscoidea Hippoboscidae Nycteribiidae Streblidae Muscoidea Anthomyiidae Fanniidae Muscoidea Muscidae Scathophagidae Oestroidea Calliphoridae Oestridae Polleniidae Rhiniidae Rhinophoridae Sarcophagidae Tachinidae Introduction Aims of the catalogue Although the national study on biodiversity, Etude nationale sur la biodiversité ( Dakki 1997 ; Mouna 1998 ), was the only important synthesis of entomological studies carried out in Morocco until now, important data on the Diptera are lacking. For example, no taxonomic rank such as subfamily and tribe, and no synonyms or revised generic combinations, names of museums or private collections that house species described from Morocco were included. No information was provided on the habitats and distribution of species in Morocco. Furthermore, the information resulting from the national study on biodiversity is now outdated. A large number of important recent publications, cited appropriately under each family, provide much additional data to the lists published in 1997 and 1998, almost doubling the number of known species. The main aims of this catalogue are to bring together all this information by providing an up-to-date checklist according to the current nomenclature and systematics, to provide synonyms used in older literature that dealt with the Moroccan fauna and to give regional distribution data. Finally, an essential comprehensive bibliography is provided. This catalogue is the first for a North African country and it may prove useful also for neighbouring countries. It is hoped that it will provide a sound foundation for anyone choosing to study a group of Moroccan Diptera . It could indicate where the greatest need for investigation lies, both with regard to areas of the country that have been poorly studied and taxonomic groups (or families) that may benefit from more attention. It is undoubtedly not a complete inventory and many more discoveries are expected. The faunistic knowledge of the Diptera of Morocco from 1787 to 2021 is summarized and updated, providing the first catalogue of the Moroccan species of Diptera . In total, 3057 species (975 Nematocera and 2082 Brachycera ) in 93 families, classified into 949 genera (250 in Nematocera and 699 in Brachycera ) are listed (Tables 1 , 2 ). Table 1. Families of Nematocera with contributing specialists and numbers of taxa. Family Specialist(s) Subfamilies Genera Species Anisopodidae Haenni 1 1 3 Bibionidae Haenni 1 2 10 Blephariceridae Zwick 1 1 4 Cecidomyiidae Skuhravá & Skuhravý 3 31 57 Ceratopogonidae Mathieu 3 6 62 Chaoboridae Wagner 1 2 2 Chironomidae - 7 100 412 Culicidae Trari, Himmi & Dakki 2 7 43 Dixidae Wagner - 2 12 Keroplatidae Chandler 1 2 2 Limoniidae Oosterbroek 4 22 67 Mycetophilidae Chandler 5 25 64 Pediciidae Oosterbroek 1 2 6 Psychodidae Wagner 2 12 51 Ptychopteridae Wagner 1 1 5 Scatopsidae Haenni 3 11 13 Sciaridae Heller - 12 70 Simuliidae - 1 6 43 Thaumaleidae Wagner - 1 2 Tipulidae Oosterbroek & de Jong 2 3 39 Trichoceridae Krzemińska 1 1 8 Total: 250 Total: 975 Table 2. Families of Brachycera with contributing specialists and numbers of taxa. Family Specialist(s) Subfamilies Genera Species Acroceridae Nartshuk 2 6 13 Agromyzidae Černý 2 12 62 Anthomyiidae Ackland 2 14 36 Anthomyzidae Ebejer - 2 2 Asilidae Tomasovic 9 42 131 Asteiidae Ebejer 1 2 5 Atelestidae Gatt - 1 1 Athericidae Mouna 1 1 2 Aulacigastridae Ebejer - 1 1 Bombyliidae Ebejer & Dils 11 48 248 Braulidae - 1 1 1 Calliphoridae Rognes 5 5 8 Camillidae Ebejer - 1 4 Canacidae Munari 2 3 15 Carnidae Ebejer - 1 3 Chamaemyiidae Ebejer 2 5 18 Chloropidae von Tschirnhaus & Ebejer 3 34 74 Chyromyidae Ebejer 2 3 22 Clusiidae Ebejer - 1 1 Coelopidae - 1 1 1 Conopidae - 5 10 34 Cryptochetidae Nartshuk - 1 1 Diastatidae Ebejer 2 2 2 Dolichopodidae Grichanov & Negrobov 12 36 112 Drosophilidae Bächli 2 9 26 Dryomyzidae - 1 1 1 Empididae Daugeron 3 8 40 Ephydridae Zatwarnicki 5 43 117 Fanniidae Pont - 1 10 Helcomyzidae - 1 1 1 Heleomyzidae Woźnica 3 5 19 Hippoboscidae Droz 3 10 17 Hybotidae Gatt 2 10 44 Lauxaniidae Ebejer 2 8 27 Lonchaeidae MacGowan 1 4 5 Lonchopteridae Barták 1 1 4 Micropezidae Ebejer - 1 1 Milichiidae - 3 6 8 Muscidae Pont 6 25 115 Mydidae Dikow 3 4 9 Mythicomyiidae Evenhuis 4 6 8 Nemestrinidae Barraclough 2 4 13 Nycteribiidae Graciolli 1 4 8 Odiniidae Ebejer 1 1 2 Oestridae Pape 2 5 10 Opomyzidae - - 2 5 Pallopteridae Ebejer - 1 1 Phoridae Disney - 2 3 Piophilidae Ebejer 1 3 3 Pipunculidae Ebejer 2 7 16 Platypezidae Ebejer 1 3 3 Platystomatidae Popov 1 2 4 Polleniidae Rognes - 1 12 Psilidae Ebejer - 1 1 Rhagionidae Ebejer 1 1 4 Rhiniidae Rognes 1 5 17 Rhinophoridae Pape 1 6 8 Sarcophagidae Whitmore & Pape 3 16 66 Scathophagidae - 1 2 3 Scenopinidae Carles-Tolrá 1 2 11 Sciomyzidae Vala 1 12 25 Sepsidae Haenni 1 4 12 Sphaeroceridae Gatt 3 29 67 Stratiomyidae Woodley 5 11 40 Streblidae Graciolli 1 2 2 Syrphidae Speight 2 50 166 Tabanidae - 3 11 69 Tachinidae Cerretti & Tschorsnig 4 88 147 Tephritidae Norrbom 3 29 69 Therevidae Hauser 2 9 27 Ulidiidae Ebejer 2 8 13 Vermileonidae Ebejer 1 2 6 Total: 698 Total: 2082 Brief history of Moroccan dipterology The study of Diptera in Morocco is still far from being extensive despite the numerous investigations that have taken place so far. Until now, a synthesis of Moroccan Diptera had never been produced. The studies that have been devoted to this group of insects have been limited and sporadic. They were mainly concerned with either taxonomy and descriptions of new species, or faunal studies from specific areas of Morocco with little reference to the habitats of species. This was particularly so for early works such as Becker and Stein (1913) , Séguy (1930a , 1934b , 1935a , 1941a , 1941d , 1949a , 1953a ), Timon-David (1951) , Vaillant (1956b) and, recently, Pârvu et al. (2006) , Popescu-Mirceni (2011) , and Ebejer et al. (2019) . Very few recent works have provided relevant ecological data ( Cassar et al. 2005 , 2008 ). Becker and Stein (1913) were the first to publish a list of Moroccan Diptera , at that time comprising only 204 species. Séguy followed with a long series of valuable publications. In 1930 he produced his first list of Diptera , which included 471 species in 230 genera grouped into 37 families (Séguy 1930). Over the subsequent years, he contributed greatly to the knowledge of the Diptera of Morocco ( Séguy 1926 , 1934b , 1935a , 1941a , 1941d , 1949a , 1953a , 1957 , 1961 ), where he also referenced an extensive bibliography. Later, Bailly-Choumara (1977) compiled a bibliography of Moroccan Diptera . It took until 1997–1998, with a national study on the biodiversity of Morocco co-ordinated by what was then the Ministry of the Environment, to update the list of the Moroccan insect fauna. This national study was dedicated to the compilation of the results of entomological research conducted in Morocco in order to highlight Moroccan biodiversity. It included a comprehensive inventory of published entomological research conducted in Morocco and was the last work to include a broad overview of Moroccan Diptera . It listed 623 species of aquatic Diptera in 210 genera and 25 families ( Dakki 1997 ) and 928 species of terrestrial Diptera in 350 genera and 57 families ( Mouna 1998 ). Terrain and bioclimatic regions Morocco, located in the westernmost corner of North Africa, constitutes a biogeographical crossroads between the Afrotropical and the western Palaearctic faunas, which may have allowed some exchange of genetic material. The country has a remarkable variety of bioclimates, ranging from humid in the Rif and the Middle and High Atlas to very arid in the Sahara in the south and to the sub-humid and semi-arid plains and foothills (Fig. 1 ). As a result of this diversity of landscapes and climates, there exists a great biological and ecological diversity in the country. 10.3897/zookeys.1094.62644.figure1 Figure 1. The major bioclimatic regions of the northern part of Morocco. https://binary.pensoft.net/fig/671550 The geomorphological, orographic and bioclimatic diversity of the country is reflected in its seven distinct biogeographical areas: the Rif, the Atlantic Plain, Eastern Morocco, the Middle Atlas, the High Atlas, the Anti-Atlas and the Sahara. In the checklist, an abbreviated distribution (in bold) is given for each species according to these seven biogeographical areas (Fig. 2 ). 10.3897/zookeys.1094.62644.figure2 Figure 2. The seven biogeographical regions covering the studied area in the northern part of Morocco. https://binary.pensoft.net/fig/671551 Rif : The Rif consists of a mountainous domain overlooking the Mediterranean Sea in the north of Morocco. It belongs to the Gibraltar Arc or Alborán Sea geological region, part of the Alpine orogenic belt, where the maximum altitude is 2,452 m in the Jbel Tidirrhine mountain range. The climate is mainly of the Mediterranean type and is characterised by high levels of precipitation. This domain is considered very rich and diverse in vegetation cover ( Valdés et al. 2002 ), including nearly all of the Moroccan forest plant species ( Médail and Quézel 1999 ) and integrating five bioclimatic stages. Atlantic Plain ( AP ) : Also known as the Moroccan Central Plateau, the Atlantic Plain is an ancient massif located in the northwest of Morocco, between the Atlantic coast and the Middle Atlas Mountains. It consists of a plateau surrounded by medium-altitude mountain massifs and covers an area of 8500 km². With an asymmetrical shape, the plateau culminates in the south-east at Jebel Mtourzgene (1627 m), located near the town of Oulmès. Its complex geomorphology presents tectonic troughs that have favoured the formation of lakes and rivers. The annual rainfall of this region, concentrated in winter and spring, totals an average of 500 mm, allowing the establishment of Holm Oak and Cork Oak forests in a semi-arid bioclimate ( Piqué 1994 ). Towards the coast, the vegetation is mainly a steppe with Jujube shrubs and Dwarf Palms ( Chamaerops humilis ). The maritime fringe is underlined by cliffs and ancient consolidated dunes. Eastern Morocco ( EM ) : This is an arid region covering the Hauts Plateaux to the northeast of the country. It is the only region of Morocco subject to a true Mediterranean bioclimate, with some influence from the Sahara. The annual rainfall is less than 300 mm, encouraging the dominance of esparto steppe ( Stipa tenacissima ) ( Piqué 1994 ). Its geomorphology is represented by a plateau at an altitude of 1000 m, which gradually decreases from 1500 to 500 m from the city of Midelt to the province of Guercif and is criss-crossed by the Moulouya wadi. In the northwestern part of Eastern Morocco is the Béni Snassen massif, beyond the Guercif depression. Middle Atlas ( MA ) : A mountainous massif located in the centre of Morocco, oriented from south-west to north-east and extending over 350 km ( Piqué 1994 ). This mass of highlands is essentially composed of limestone layers, tabular and of medium altitude in the southwest (tabular Middle Atlas) and undulating in the northeast (folded Middle Atlas) to culminate at 3340 m at Jebel Bou Naceur, which overlooks the high plateaux of Eastern Morocco. The climate, generally subhumid to humid, is cold in winter, giving rise to cedar and Holm Oak forests covering the western slope, which is wetter than the eastern slope and where lakes are abundant and many rivers and streams have their source. High Atlas ( HA ) : The main mountainous massif of Morocco, oriented from west-south-west to east-north-east, is the High Atlas, stretching over more than 700 km from the Atlantic to the east of Morocco, with a north-south width of 50 to 100 km and reaching at Jebel Toubkal an altitude of 4165 m. The whole massif is high, incised by deep valleys that lead to passes through which the mountainous barrier is crossed: the Tizi-n'Test (2100 m) towards the Souss plain and the Tizi-n'Tichka (2200 m) towards the depression of Ouarzazate. The chain is formed by a massif of Palaeozoic rocks surrounded by sedimentary deposits. The relief of the High Atlas is divided into three different entities from west to east: the Western High Atlas, which is the oldest massif and is made up mainly of Jurassic or Cretaceous formations with deep valleys and culminates at Jebel Toubkal; the central High Atlas, which is an essentially limestone massif morphologically dominated by tabular zones culminating at an altitude of 2500 m, stretches between the towns of Azilal and Ouarzazate and is sheltered by the Jebel M'Goum (4071 m); and the Eastern High Atlas, which is formed by the vast high plateaux of the Upper Moulouya, stretches between the cities of Midelt and Imilchil and contains the Jebel Ayachi (3747 m) ( Piqué 1994 ). The bioclimate of this area includes subhumid, humid and semi-arid zones, but with more than 800 mm of annual precipitation on the highest peaks and a significant snow cover from September to May, with temperatures dropping below -20 °C (AEFCS 1996) and bright sunshine even in winter. These climatic conditions have favoured large pine, Holm Oak, Thuja and thuriferous juniper forests (HCEFLCD 2017). The aridity of the southern slope of the High Atlas allows only steppe vegetation of sagebrush and esparto grass to grow. Anti-Atlas ( AA ) : This is the pre-Saharan area, which extends north of the Wadi Draa and rises to an average altitude of 2000 m. It is a semi-arid region with Jebel Bani and Jebel Ouarkziz as its main elevations, and is home to numerous oases. The western part of the Anti-Atlas is characterised by the Saharan plateau, with a temperate winter except in the high mountains. On the other hand, in the eastern part of the Anti-Atlas, the greatest arid area is marked by the northward extension of the Saharan landscape and a bioclimate of cool and temperate winters. These bioclimatic stages are characterised by plant communities such as wooded steppes with Acacia raddiana , Artemisia herba alba and Stipa tenuissima , interspersed with thuriferous juniper ( Juniperus thurifera ) at the top of the slopes ( Piqué 1994 ). The vegetation is also represented by two formations that partially interpenetrate each other: groves of the Argan tree ( Argania spinosa ) and the Euphorbia steppe ( Euphorbia echinus ). Sahara ( SA ) : This area in the south of the country consists of a wide arid zone with a typical Saharan bioclimate of high daytime temperatures and very low precipitation. From a geological point of view it is part of the West African Craton and its margins. It is mainly ancient terrain with Precambrian basement rock and Palaeozoic cover ( Piqué 1994 ). This area is represented by a peneplain of very modest altitudes from 200 to 500 m, established on ancient crystalline terrain. Precipitation from July to September is very low, of the order of 100 mm. The dipteran species richness of each biogeographical region of Morocco is summarised in Table 3 , which highlights the great disparity between the different regions and reflects the lack of knowledge about this insect order in some areas of Morocco. The Rif region in the north of the country has the greatest recorded diversity, but this undoubtedly reflects a greater sampling effort in this area, with large parts of the country still very poorly explored for Diptera . The catalogue does not include any data from the southern part of the Moroccan Sahara. Table 3. Species richness of Diptera families each biogeographical region in Morocco. Rif ; AP – Atlantic Plain; EM – Eastern Morocco; MA – Middle Atlas; HA – High Atlas; AA – Anti-Atlas; SA – Sahara. * No precise localities known. Rif AP EM MA HA AA SA Acroceridae 7 1 0 1 3 0 0 Agromyzidae 7 15 0 10 22 10 0 Anisopodidae * Anthomyiidae 4 12 0 13 2 4 1 Anthomyzidae 1 0 0 0 1 0 0 Asilidae 34 27 4 35 25 24 5 Asteiidae 0 3 0 0 1 1 0 Atelestidae 1 0 0 0 0 0 0 Athericidae 0 0 0 1 2 0 0 Aulacigastridae 1 0 0 0 0 0 0 Bibionidae 4 2 0 3 1 2 2 Blephariceridae 1 0 0 0 3 0 0 Bombyliidae 35 80 12 59 48 56 19 Braulidae * Calliphoridae 3 8 2 4 4 0 0 Camillidae 1 2 0 0 1 0 0 Canacidae 6 10 0 0 0 6 0 Carnidae 1 0 0 0 0 0 0 Cecidomyiidae 8 27 21 25 20 7 4 Ceratopogonidae 15 43 14 35 33 24 36 Chamaemyiidae 5 5 0 4 8 6 0 Chaoboridae * Chironomidae 230 30 7 105 120 14 1 Chloropidae 35 22 0 7 6 25 1 Chyromyidae 6 6 0 0 1 12 1 Clusiidae 1 0 0 0 0 0 0 Coelopidae 1 0 0 0 0 0 0 Conopidae 7 13 0 16 7 9 0 Cryptochetidae 0 0 0 1 0 0 0 Culicidae 32 41 24 30 29 22 11 Diastatidae 0 1 0 0 1 0 0 Dixidae 2 2 0 2 5 0 0 Dolichopodidae 72 28 3 6 18 26 0 Drosophilidae 11 14 0 8 12 5 1 Dryomyzidae 1 0 0 0 0 0 0 Empididae 4 3 0 13 20 1 0 Ephydridae 47 44 1 60 9 20 1 Fanniidae 4 2 0 2 5 0 0 Helcomyzidae 1 0 0 0 0 0 0 Heleomyzidae 11 3 0 2 2 3 0 Hippoboscidae 0 2 3 4 4 4 1 Hybotidae 20 7 3 8 7 8 0 Keroplatidae 1 0 0 0 0 0 0 Lauxaniidae 23 3 2 1 8 2 0 Limoniidae 43 9 4 5 34 5 0 Lonchaeidae 3 3 0 1 1 0 0 Lonchopteridae 0 0 0 1 1 0 0 Micropezidae 1 0 0 0 0 0 0 Milichiidae 2 1 0 0 4 3 1 Muscidae 25 47 13 38 68 20 6 Mycetophilidae 60 3 7 3 0 0 0 Mydidae 0 3 2 1 0 3 1 Mythicomyiidae 2 0 0 0 1 2 1 Nemestrinidae 1 1 1 5 3 0 1 Nycteribiidae 6 5 0 6 0 2 1 Odiniidae 1 0 0 0 0 0 0 Oestridae 0 2 1 1 1 0 1 Opomyzidae 1 1 0 1 2 0 0 Pallopteridae 0 0 0 1 0 0 0 Pediciidae 3 1 0 0 0 0 0 Phoridae 0 2 0 0 0 0 0 Piophilidae 2 1 0 1 1 0 0 Pipunculidae 13 1 0 0 0 0 0 Platypezidae 2 1 0 0 0 0 0 Platystomatidae 0 2 1 1 0 0 0 Polleniidae 1 4 2 5 5 1 1 Psilidae 1 0 0 1 0 0 0 Psychodidae 25 12 12 12 34 9 2 Ptychopteridae * Rhagionidae 2 0 0 0 2 0 0 Rhiniidae 2 5 3 5 0 5 4 Rhinophoridae 6 2 0 1 0 0 0 Sarcophagidae 11 19 1 13 8 9 2 Scathophagidae 3 1 0 1 0 0 0 Scatopsidae 10 2 0 0 1 3 0 Scenopinidae 3 4 1 1 0 0 1 Sciaridae 29 12 2 8 45 9 3 Sciomyzidae 8 4 2 11 18 3 0 Sepsidae 3 6 0 4 7 3 0 Simuliidae 32 8 0 19 32 6 0 Sphaeroceridae 42 3 2 10 1 3 3 Stratiomyidae 20 13 5 13 6 1 0 Streblidae 1 1 0 0 0 1 0 Syrphidae 70 49 11 82 64 25 4 Tabanidae 26 27 11 35 21 7 1 Tachinidae 18 27 4 46 43 29 3 Tephritidae 34 13 4 16 13 23 3 Thaumaleidae 0 0 0 0 2 0 0 Therevidae 9 7 2 9 4 5 2 Tipulidae 28 7 0 16 18 2 0 Trichoceridae 7 1 3 1 0 0 0 Ulidiidae 6 2 0 1 4 4 1 Vermileonidae 1 2 1 1 4 0 0 Total species 1126 770 191 831 876 474 126 Sources of data Data for the present study were gathered from the literature, supplemented by information taken from a number of websites. The cut-off date for inclusion of species published in the literature is 30 June 2021. Taxonomic, nomenclatural, and distributional data for the present catalogue were obtained from: studies by various foreign and Moroccan researchers published between 1787 and 2021, including theses, books, catalogues, checklists and notes from the 1997–1998 synthesis studies on Morocco's biodiversity. data from websites and organisations such as Global Species, the Global Biodiversity Information Facility, the BioSystematic Database of World Diptera, Systema Dipterorum, Catalogue of Life, Systema Naturae 2000 and others, or from relevant homepages specific to particular families. the collection of Diptera in the National Museum of Morocco (Muséum National d'Histoire Naturelle à l'Institut Scientifique, Université Mohammed V, Rabat). collaboration with specialists who communicated reliable identifications from their personal collections or provided data for species deposited in foreign museums. published records of specimens in the personal collection of the lead author (PCKK), deposited at Abdelmalek Essaadi University, Tetouan, identified with the assistance of specialists. Classification The arrangement of families in this catalogue (Table 4 ) is mainly based on Pape et al. (2011) and Wiegmann et al. (2011) . All families are listed alphabetically within each superfamily. Nomenclature follows the most recent version of Systema Dipterorum ( Evenhuis and Pape 2021 ) and the Polleniidae are listed as a separate family ( Gisondi et al. 2020 ). Table 4. Diptera classification used in the catalogue. Suborder NEMATOCERA Tipuloidea Limoniidae Pediciidae Tipulidae Trichoceridoidea Trichoceridae Psychodoidea Psychodidae Scatopsoidea Scatopsidae Ptychopteridae Culicoidea Chaoboridae Culicidae Dixidae Chironomoidea Ceratopogonidae Chironomidae Simuliidae Thaumaleidae Blephariceridae Bibionoidea Anisopodidae Bibionidae Sciaroidea Cecidomyiidae Keroplatidae Mycetophilidae Sciaridae Suborder BRACHYCERA Stratiomyoidea Stratiomyidae Tabanoidea Athericidae Rhagionidae Tabanidae Vermileonidae Nemestrinoidea Acroceridae Nemestrinidae Asiloidea Asilidae Bombyliidae Mydidae Mythicomyiidae Scenopinidae Therevidae Empidoidea Atelestidae Empididae Dolichopodidae Hybotidae Platypezoidea Phoridae Platypezidae Lonchopteridae Syrphoidea Pipunculidae Syrphidae Conopoidea Conopidae Nerioidea Micropezidae Tanypezoidea Psilidae Tephritoidea Lonchaeidae Pallopteridae Piophilidae Platystomatidae Tephritidae Ulidiidae Lauxanioidea Chamaemyiidae Lauxaniidae Sciomyzoidea Coelopidae Dryomyzidae Helcomyzidae Sciomyzidae Sepsidae Opomyzoidea Agromyzidae Anthomyzidae Asteiidae Aulacigastridae Clusiidae Odiniidae Opomyzidae Carnoidea Canacidae Carnidae Chloropidae Milichiidae Sphaeroceroidea Chyromyidae Heleomyzidae Sphaeroceridae Ephydroidea Braulidae Camillidae Cryptochetidae Diastatidae Drosophilidae Ephydridae Hippoboscoidea Hippoboscidae Nycteribiidae Streblidae Muscoidea Anthomyiidae Fanniidae Muscoidea Muscidae Scathophagidae Oestroidea Calliphoridae Oestridae Polleniidae Rhiniidae Rhinophoridae Sarcophagidae Tachinidae Arrangement of the catalogue The catalogue includes all names of taxonomically valid species of the Order Diptera so far known to occur in Morocco. For each family, synonyms and alternative combinations that have been published as records of Moroccan provenance are listed under the currently accepted name. The transliteration of Moroccan place names in the literature, maps, and sometimes on data labels presented a small problem. Although it would have been preferable to use standard names, this would have meant changing some place names from their original spellings in the literature. Most transliteration used the French language for spelling and Roman letters, but not all. English was sometimes used, and occasionally also Spanish. Readers are therefore advised to take this into account when searching for place names given in the catalogue and in the cited references. Some common examples are "Jebel", "Jbel", "Djebel" or "Dj.", used for "mountain", or "Kbir" and "Kebir", for "large" or "great". The article "the" in Arabic is El or L; it is sometimes separated by a hyphen from its associated noun or joined to it, sometimes capitalised while the noun is not, both are capitalised, or only the noun is. When the article "el" is followed by a noun beginning with a certain consonant, like n, r, s and t, the l is changed to match the noun's initial, for example "Er-Rifiyine". In transliteration, Arabic speakers would make this change, whereas non-Arabic speakers may not. In cases where the article is joined to the noun, the resulting versions may appear as being different place names; for example, El Rachidia, Er Rachidia and Errachidia are alternative transliterations of the same place name. In some entries, the French terms "affluent" and "aval affluent" are left as in the original citation, because "tributary" may not be an accurate translation in all instances. For some of the sites, the French term refers to a downstream overflow of water into a side tributary or waterfall, whereas for others it refers to an actual tributary feeding into a main river or stream. Older authors were imprecise with localities, and many references did not give the coordinates of localities from which species were collected. We could not reliably provide accurate coordinates for each species. Where known, species depositories are included. Individual family checklists may include, at the end, taxonomic notes and comments on species doubtfully present in the country. New records are indicated with an asterisk (*) and the relevant data listed at the end of the family section. Limitations One of the major difficulties encountered while compiling the catalogue was the limited ability to check old identifications. In some cases, identifications were accepted as given in the original sources, even when doubtful, as it was usually not possible to re-examine specimens. The inaccessibility of some references was also an obstacle to updating the checklists for some families. Collections cited The following museums and collections house some of the specimens mentioned in this catalogue. The National Museum of Natural History of Morocco is located in the Institut Scientifique, Université Mohammed V, Rabat. However, the Diptera collection housed in that museum is not yet included in any world database such as the GBIF Registry of Scientific Collections, https://www.gbif.org/grscicoll , or the Insect and Spider collections of the World Website, http://hbs.bishopmuseum.org/codens/ . HNHM Hungarian Natural History Museum, Budapest, Hungary ; INHS Illinois Natural History Survey, Urbana, USA ; IRSNB Institut Royal des Sciences Naturelles de Belgique, Brussels, Belgium ; MfN Museum für Naturkunde, Berlin, Germany ; MHNN Muséum d'Histoire Naturelle de Neuchâtel, Neuchâtel, Switzerland ; MISR Museum National d'Histoire Naturelle à l'Institut Scientifique, Université Mohammed V, Rabat, Morocco; MNCN Museo Nacional de Ciencias Naturales, Madrid, Spain ; MNHN Muséum National d'Histoire Naturelle, Paris, France ; NHMD Natural History Museum of Denmark, Copenhagen, Denmark; NHMUK Natural History Museum, London, United Kingdom ; NMWC National Museum of Wales, Cardiff, United Kingdom ; OUMNH Oxford University Museum of Natural History, Oxford, United Kingdom ; RMNH Rijksmuseum voor Natuurlijke Historie, Leiden, The Netherlands; SMNS Staatliches Museum für Naturkunde Stuttgart, Stuttgart, Germany ; ZMUM Zoological Museum of Moscow University, Moscow, Russia; ZSM Zoologische Staatssammlung, Munich, Germany ; ZSM Zoologische Staatssammlung, Munich, Germany. The personal collection of Dr Michael von Tschirnhaus has been donated to the ZSM , but is currently on loan to Dr von Tschirnhaus. Other abbreviations HT Holotype; NPH National Park of Al Hoceima; NPHAO National Park of Haut Atlas Oriental; NPI National Park of Ifrane; NPT National Park of Talassemtane; PCJD Private Collection of Jos Dils, Belgium; PCKK Private Collection of Kawtar Kettani, Morocco; PNPB Project of Natural Park of Bouhachem. Limitations One of the major difficulties encountered while compiling the catalogue was the limited ability to check old identifications. In some cases, identifications were accepted as given in the original sources, even when doubtful, as it was usually not possible to re-examine specimens. The inaccessibility of some references was also an obstacle to updating the checklists for some families. Collections cited The following museums and collections house some of the specimens mentioned in this catalogue. The National Museum of Natural History of Morocco is located in the Institut Scientifique, Université Mohammed V, Rabat. However, the Diptera collection housed in that museum is not yet included in any world database such as the GBIF Registry of Scientific Collections, https://www.gbif.org/grscicoll , or the Insect and Spider collections of the World Website, http://hbs.bishopmuseum.org/codens/ . HNHM Hungarian Natural History Museum, Budapest, Hungary ; INHS Illinois Natural History Survey, Urbana, USA ; IRSNB Institut Royal des Sciences Naturelles de Belgique, Brussels, Belgium ; MfN Museum für Naturkunde, Berlin, Germany ; MHNN Muséum d'Histoire Naturelle de Neuchâtel, Neuchâtel, Switzerland ; MISR Museum National d'Histoire Naturelle à l'Institut Scientifique, Université Mohammed V, Rabat, Morocco; MNCN Museo Nacional de Ciencias Naturales, Madrid, Spain ; MNHN Muséum National d'Histoire Naturelle, Paris, France ; NHMD Natural History Museum of Denmark, Copenhagen, Denmark; NHMUK Natural History Museum, London, United Kingdom ; NMWC National Museum of Wales, Cardiff, United Kingdom ; OUMNH Oxford University Museum of Natural History, Oxford, United Kingdom ; RMNH Rijksmuseum voor Natuurlijke Historie, Leiden, The Netherlands; SMNS Staatliches Museum für Naturkunde Stuttgart, Stuttgart, Germany ; ZMUM Zoological Museum of Moscow University, Moscow, Russia; ZSM Zoologische Staatssammlung, Munich, Germany ; ZSM Zoologische Staatssammlung, Munich, Germany. The personal collection of Dr Michael von Tschirnhaus has been donated to the ZSM , but is currently on loan to Dr von Tschirnhaus. Other abbreviations HT Holotype; NPH National Park of Al Hoceima; NPHAO National Park of Haut Atlas Oriental; NPI National Park of Ifrane; NPT National Park of Talassemtane; PCJD Private Collection of Jos Dils, Belgium; PCKK Private Collection of Kawtar Kettani, Morocco; PNPB Project of Natural Park of Bouhachem. Other abbreviations HT Holotype; NPH National Park of Al Hoceima; NPHAO National Park of Haut Atlas Oriental; NPI National Park of Ifrane; NPT National Park of Talassemtane; PCJD Private Collection of Jos Dils, Belgium; PCKK Private Collection of Kawtar Kettani, Morocco; PNPB Project of Natural Park of Bouhachem. Acknowledgements The authors are deeply grateful to the reviewers for their constructive comments, which helped to improve the manuscript. Our sincere thanks are expressed to the subject editor, Torsten Dikow, for his significant contribution and support as editor. We are most grateful to the following specialists who generously contributed in various ways to this catalogue by sharing information and knowledge of their group or by reviewing a family section: Patrick Ashe (Dublin, Ireland)— Chironomidae ; Torsten Dikow (Washington, D.C., USA)— Mydidae ; the late Amnon Freidberg (Tel Aviv, Israel)— Tephritidae ; Kai Heller (Heikendorf, Germany)— Sciaridae ; Gail Kampmeier (Champaign, USA)— Therevidae ; Valery Korneyev (Kiev, Ukraine)— Tephritidae ; Ewa Krzemińska (Kraków, Poland)— Trichoceridae ; Allen Norrbom (Washington, D.C., USA)— Tephritidae ; Adrian R. Plant (Mahasarakham, Thailand)— Empididae , Hemerodrominae ; Bradley J. Sinclair (Ottawa, Canada)— Empididae , Clinocerinae ; Jens-Hermann Stuke (Leer, Germany)— Conopidae ; Norman Woodley (Hereford, AZ, USA)— Stratiomyidae . Mohamed Mouna and Mohamed Dakki are acknowledged and thanked for their encouragement and inspiration that led the first author to commence this project, as well as for generously donating to her a large bibliography on the subject. Mohamed Mouna was instrumental in ensuring that she had full access to the collections and library and full cooperation from the staff of these departments. He is immensely thanked by the first author for donating to her his library, consisting of many books and publications. Mohammed Fekhaoui, Abdellatif Bayed, Mohamed-Aziz El Agbani, Mohamed Arahou from the administration of Museum National d'Histoire Naturelle à l'Institut Scientifique, Université Mohammed V, Rabat are warmly thanked for their valuable assistance in providing access to the library and Diptera collections of the museum. Thanks also to Amina Zouida, the librarian at the said institute, for her tireless and precious help in searching for obscure publications, and to Abderrahmane Mataame (responsible for the zoological collections of the museum) for his kind and patient assistance during the first author's frequent visits to the museum. Yassine Fekrani is acknowledged for his kind help producing the maps. Suborder NEMATOCERA Tipuloidea LIMONIIDAE K. Kettani, P. Oosterbroek Number of species: 67 . Expected: 85 Faunistic knowledge of the family in Morocco: moderate Chioneinae Baeoura Alexander, 1924 Baeoura ebenina Starý, 1981 Driauach and Belqat 2015 , Rif , Oued Tazarine (Mezine village); Dayat near Aïn Afersiw; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: Rif ) Baeoura staryi Driauach & Belqat, 2015 Driauach and Belqat 2015 , Rif , Jnane Niche; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: Rif ) Cheilotrichia Rossi, 1848 Cheilotrichia ( Empeda ) cinerascens (Meigen, 1804) Driauach et al. 2013 , Rif , Ikadjiouen; Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Amsa, Oued Maggou, Aïn Quanquben (Jebel Bou Bessoui), EM , Grotte du Chameau (Zegzel, Béni Snassen); Oosterbroek 2020 (CCW: Rif ; EM ) Cheilotrichia ( Empeda ) fuscohalterata (Strobl, 1906) Driauach and Belqat 2016 , Rif , Dayat Fifi, tributary of Oued El Fondak, Barrage Ajras, Dayat Mghara, Oued Zarka, Oued Tizekhte; Oosterbroek 2020 (CCW: Rif ) Cheilotrichia ( Empeda ) minima (Strobl, 1898) Driauach and Belqat 2016 , Rif , Oued Zendoula, Oued Jnane Niche; Oosterbroek 2020 (CCW: Rif ) Ellipteroides Becker, 1907 Ellipteroides ( Ellipteroides ) lateralis (Macquart, 1835) = Gonomyia lateralis Macquart, in Pierre 1922a : 22, Dakki 1997 : 62 = Gonomyia cincta Egger, in Séguy 1941a : 26 Pierre 1922a , AP , Dradek (near Rabat); Lackschewitz 1940a , HA , Tachdirt (2200–2900 m); Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 ; Dakki 1997 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Dayat Afrate; Oosterbroek 2020 (CCW: Rif ) Ellipteroides ( Protogonomyia ) alboscutellatus (von Roser, 1840) Lachschewitz 1940a, HA ; Savchenko et al. 1992 ; Starý and Oosterbroek 2008 ; Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Ellipteroides ( Protogonomyia ) hutsoni (Starý, 1971) Starý 1971 , HA , Jebel Ayachi; Savchenko et al. 1992 , HA , Jebel Ayachi; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Erioconopa Starý, 1976 Erioconopa diuturna (Walker, 1848) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Dayat Jebel Zemzem, Oued Mezine, Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ) Erioconopa symplectoides (Kuntze, 1914) = Dactylolabis symplectoides Egger, in Pierre 1922a : 23 Pierre 1922a , HA , Marrakech; Savchenko et al. 1992 ; Starý and Oosterbroek 2008 , HA ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Erioptera Meigen, 1803 Erioptera ( Erioptera ) fuscipennis Meigen, 1818 Pierre 1922a , AP , Casablanca (Oued Guerera); Pierre 1922b , HA , Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 , Rif , Sidi Brahim Ben Arrif (Bab Hachef Aissa); Driauach and Belqat 2016 , Rif , Aïn El Ma Bared, Oued Amsemlil, Oued Maggou (Bridge), Oued Tkarae, Dayat near Aïn Afersiw, Aïn Afersiw, Oued Abou Bnar, Oued Maggou (Zaouiet El Habtiyne), Dayat Amsemlil, Dayat Aïn Jdi­oui; Dayat Afrate, Oued Mezine; Oosterbroek 2020 (CCW: Rif , HA ) Erioptera ( Erioptera ) lutea lutea Meigen, 1804 Driauach and Belqat 2016 , Rif , Aïn Boughaba; Oosterbroek 2020 (CCW: Rif ) Erioptera ( Erioptera ) transmarina Bergroth, 1889 = Mesocyphona transmarina Bergroth, in Pierre 1922a : 22, Pierre 1922b : 148, Dakki 1997 : 62 Pierre 1922a , HA , Marrakech; Pierre 1922b , HA , Tannaout (1000 m), Marrakech; Dakki 1997 ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Gonomyia Meigen, 1818 Gonomyia ( Gonomyia ) abscondita Lackschewitz, 1935 Driauach and Belqat 2016 , Rif , maison forestière; Oosterbroek 2020 (CCW: Rif ) Gonomyia ( Gonomyia ) sicula Lackschewitz, 1940 Driauach and Belqat 2016 , Rif , Oued Kbir, Dayat Jebel Zemzem, Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Aïn El Maounzil; Oosterbroek 2020 (CCW: Rif ) Gonomyia ( Gonomyia ) subtenella Savchenko, 1972 Starý and Oosterbroek 2008 , HA , Massif Toubkal, Aghbalou, 43 km S Marrakech (1000 m); Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Jnane Niche, Oued Sidi Yahia Aârab, EM , Oued Béni Ouachekradi; Oosterbroek 2020 (CCW: Rif , EM ) Gonomyia ( Gonomyia ) tenella (Meigen, 1818) Pierre 1924a , HA , Asni (1200 m); Séguy 1930a , HA , Asni; Savchenko et al. 1992 ; Dakki 1997 ; Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis Osten-Sacken, 1869 Hoplolabis ( Parilisia ) obtusiapex (Savchenko, 1982) Starý 2006 , HA , Oasis Meski, AA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis ( Parilisia ) punctigera (Lackschewitz, 1940) Starý 2006 , HA , Oasis Meski, AA ; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis ( Parilisia ) sororcula (Lackschewitz, 1940) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Barrage Ajras, Oued Ouringa, Oued El Kanar, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Idiocera Dale, 1842 Idiocera ( Euptilostena ) jucunda (Loew, 1873) = Gonomyia jucunda Loew, in Pierre 1924a : 201, Séguy 1930a : 22 Pierre 1924a , HA , Asni (1200 m); Séguy 1930a , HA , Asni; Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Idiocera ( Idiocera ) ampullifera (Starý, 1979) Driauach and Belqat 2016 , AA , Oued Zag; Oosterbroek 2020 (CCW: AA ) Idiocera ( Idiocera ) pulchripennis (Loew, 1856) = Gonomyia sexpunctata Dale, in Pierre 1922b : 148 = Gonomyia pulchripennis (Loew), in Dakki 1997 : 62 Pierre 1922b , AP , Atlantic coast; Ramdani 1981 , AP , Merja Sidi Boughaba; Savchenko et al. 1992 ; Dakki 1997 ; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Jnane Niche, Oued Aârkoub, Aïn Jdioui; Oosterbroek 2020 (CCW) Idiocera ( Idiocera ) sziladyi (Lackschewitz, 1940) Driauach and Belqat 2016 , Rif , Oued Zarka; Oosterbroek 2020 (CCW: Rif ) Ilisia Rondani, 1856 Ilisia maculata (Meigen, 1804) Driauach and Belqat 2016 , Rif , Oued Maggou, EM , Oued Tafoughalt; Oosterbroek 2020 (CCW: Rif ; EM ) Molophilus Curtis, 1833 Molophilus ( Molophilus ) ibericus Starý, 2011 Starý 2011 , HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Molophilus ( Molophilus ) obscurus (Meigen, 1818) Starý and Oosterbroek 2008 , HA , Massif Toubkal, Oukaimeden, 2500–2800 m; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , tributary of Oued Ouara, tributary of Oued Maggou, Dayat El Ânassar, Dayat Jebel Zemzem, Aïn El Ma Bared, Dayat Rmali, Oued Amsemlil, Aïn Mâaze, Dayat Afrate, Dayat Lemtahane; Oosterbroek 2020 (CCW: Rif ) Molophilus ( Molophilus ) propinquus propinquus (Egger, 1863) Savchenko et al. 1992 ; Starý and Oosterbroek 2008 , HA , Oukaimeden (2500–2800 m); Starý 2011 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Farda, tributary of Oued Taida, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Molophilus ( Molophilus ) testaceus Lackschewitz, 1940 Driauach and Belqat 2016 , Rif , Dayat Amsemlil, Dayat Lemtahane, Marj El kheyl, Oued Tkarae, Dayat near Aïn Afersiw, Dayat Mezine, Dayat Tazia, Dayat Rmali, Dayat Amsemlil, Dayat near Aïn Afersiw; Oosterbroek 2020 (CCW: Rif ) Symplecta Meigen, 1830 Symplecta ( Symplecta ) grata Loew, 1873 Ebejer et al. 2020, Rif , Aïn Jdioui (Tahaddart, 8 m) Symplecta ( Symplecta ) hybrida (Meigen, 1804) Lackshewitz 1940a, HA , Goundafa (1200 m); Séguy 1941a , HA , Taroudant; Savchenko et al. 1992 ; Oosterbroek et al. 2007; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Barrage Ajras, Oued El Kanar, Oued Aârkoub, Oued Jnane Niche, MA , Barrage Allal El Fassi; Oosterbroek 2020 (CCW: Rif ; MA ) Symplecta ( Trimicra ) pilipes (Fabricius, 1787) = Trimicra pilipes Fabricius, in Pierre 1922a : 23 = Trimicra andalusiaca Strobl, in Pierre 1922b : 149 = Trimicra hirsutipes Macquart, in Séguy 1930a : 22 Pierre 1922a , AP , Dradek (near Rabat), HA , Marrakech; Pierre 1922b , MA , Volubilis, HA , Oued Tensift; Séguy 1930a ; Séguy 1941d , AA , Taroudant; Savchenko et al. 1992 ; Dakki 1997 ; Pârvu and Zaharia 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , tributary of Oued Hachef, Oued Aârk­ob, Oued El Kanar, Oued Mezine, AP , Aïn Chouk (Larache); Oosterbroek 2020 (CCW: Rif ) Tasiocera ( Dasymolophilus ) murina (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued Farda, Oued Amsemlil; Oosterbroek 2020 (CCW: Rif ) Dactylolabinae Dactylolabis Osten-Sacken, 1860 Dactylolabis ( Dactylolabis ) symplectoidea Egger, 1863 Pierre 1922a , AP , Around Casablanca (coastal meseta); Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limnophilinae Austrolimnophila Alexander, 1920 Austrolimnophila ( Austrolimnophila ) latistyla Starý, 1977 Driauach and Belqat 2016 , Rif , Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Dicranophragma Osten-Sacken, 1860 Dicranophragma ( Brachylimnophila ) adjunctum (Walker, 1848) = Neolimnomyia adjuncta (Walker, 1848), in Pârvu et al. 2006 : 273 Pârvu et al. 2006 , AP , Merja Zerga; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranophragma ( Brachylimnophila ) nemorale (Meigen, 1818) Lackschewitz 1940b , HA , Tachdirt (2200–2700 m); Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Aïn Sidi Brahim Ben Arrif, tributary Oued Ouara, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Eloeophila Rondani , 1856 Eloeophila maroccana Starý, 2009 Starý 2009b , HA , Okaïmeden (2500–2800 m); Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Euphylidorea Alexander, 1972 Euphylidorea ( Euphylidorea ) crocotula (Séguy, 1941) = Phylidorea crocotula (Séguy), in Séguy 1941a : 28 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 , HA , Tachdirt (Toubkal); Starý and Oosterbroek 2008 , HA , Oukaimeden (2500 m–2800 m), Imlil (1400 m); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ; HA ) Euphylidorea ( Euphylidorea ) dispar (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued at 15 km from Fifi; Oosterbroek 2020 (CCW: Rif ) Euphylidorea ( Euphylidorea ) lineola (Meigen, 1804) Lackschewitz 1940b , HA ; Savchenko et al. 1992 ; Starý and Freidberg 2007 ; Driauch et al. 2013; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hexatoma Latreille, 1809 Hexatoma ( Hexatoma ) bicolor (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued at 15 km from Fifi, tributary of Oued Ouara, Oued Madissouka, Oued Maggou, Aïn Quanquben (Jebel Bou Bessoui), Oued Tkarae, Oued Tamerte; Oosterbroek 2020 (CCW: Rif ) Hexatoma ( Hexatoma ) gaedii (Meigen, 1830) Lackschewitz 1940b , HA , Tachdirt (2200 m–2700 m); Savchenko et al. 1992 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Pseudolimnophila Alexander, 1919 Pseudolimnophila ( Pseudolimnophila ) sepium (Verrall, 1886) Driauach et al. 2013 , Rif , Guelta Tazia; Driauach and Belqat 2016 , Rif , Dayat Tazia, Aïn Sidi Brahim Ben Ar­rif, Aïn Afersiw, Oued Maggou, Dayat Tazia, Oued Taida, Aïn Sidi Brahim Ben Arrif; Oosterbroek 2020 (CCW: Rif ) Limoniinae Dicranomyia Stephens, 1829 Dicranomyia ( Dicranomyia ) affinis (Schummel, 1829) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Amsemlil, Dayat Lemtahane, Oued Tkarae, Marj El Kheyl, Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Aïn El Maounzil, Aïn El Malâab, Dayat near Aïn Afersiw, Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) chorea (Meigen, 1818) Pierre 1922b , HA , Haute Réghaya, Tannaout, Asni (1000 m–1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Farda, Oued Kelâa, Aïn Ras el Ma, Oued Jnane Niche, Cascade Chrafate, maison forestière, Oued El Kanar, tributary of Oued Zarka, HA , Oued Sidi Fares (National Park of Toubkal); Oosterbroek 2020 (CCW: Rif ; HA ) Dicranomyia ( Dicranomyia ) didyma (Meigen, 1804) Pierre 1922b , HA , Haute Réghaya (2000 m); Dakki 1997 ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Dicranomyia ( Dicranomyia ) goritiensis (Mik, 1864) Lackschewitz 1940b ; Vaillant 1956b ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Maggou, Cascade Chrafate, Oued El Koub, Cascade Zarka, Âounsar Aheramen, maison forestière; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) longicollis (Macquart, 1846) = Telecephala longicollis (Macquart), in Pierre 1922a : 21, Pierre 1922b : 148, Séguy 1930a : 22 Séguy 1930a , AP , Dradek (near Rabat); Dakki 1997 ; Pierre 1922a , AP , Dradek, HA , Marrakech; Pierre 1922b , AP , Rabat; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Aârate, Barrage Moulay Bouchta, Oued Kbir, MA , Barrage Allal El Fassi; Oosterbroek 2020 (CCW) Dicranomyia ( Dicranomyia ) mitis (Meigen, 1830) = Dicranomyia hygropetrica Vaillant, in Vaillant 1956b : 42 Vaillant 1956b , HA , Asif Tessaout (M'Goum), Izourar, Tahanaout, Tamesrit, Imi-N'Ifri, Aguelmous, Sidi Chamarouch, Lac Tamhda (Anremer), Oukaimeden; Savchenko et al. 1992 ; Pârvu et al. 2006 , AA , near Agadir, Souss plain to High Atlas occidental; Pârvu and Zaharia 2007 ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 , Rif , Tazia, Tisgris; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia ( Dicranomyia ) modesta (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued Farda, Oued El Kanar, Oued Jnane Niche, Oued Sidi Yahia Aârab; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) novemmaculata (Strobl, 1906) Driauach and Belqat 2016 , Rif , tributary of Oued el Fondak, Oued Aârate; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) ventralis (Schummel, 1829) Driauach and Belqat 2016 , Rif , Lake Badriouen; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Glochina ) sericata (Meigen, 1830) Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia ( Melanolimonia ) hamata Becker, 1908 Driauach and Belqat 2016 , Rif , tributary of Oued Kbir, Oued Aârate, Aïn Sidi Brahim Ben Arrif, Dayat Tazia; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Melanolimonia ) morio (Fabricius, 1787) = Limonia pauliani Séguy, in Séguy 1941a : 26 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 , HA , Tachdirt (Toubkal); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Seguia Lemtahane, Dayat near Aïn Afersiw, Dayat Jebel Zemzem; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia majuscula Pierre, 1924 1 Pierre 1924a , HA , Haut Imminen (2400 m); Séguy 1930a , HA , Haut Imminen; Dakki 1997 ; Savchenko et al. 1992 , HA , Haut Imminen; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranoptycha Osten-Sacken, 1860 Dicranoptycha fuscescens (Schummel, 1829) Eiroa 2000 , Rif ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Mlilah, Oued Zendoula, AP , Oued Loukous; Oosterbroek 2020 (CCW) Geranomyia Haliday, 1833 Geranomyia caloptera (Mik, 1867) Driauach et al. 2013 , HA , Setti Fatma; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Geranomyia obscura Strobl, 1900 Vaillant 1956b , HA , Lac Tamhda (Anremer), Oukaimeden, Izourar, Sidi Chamarouch, Tamesrit; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Helius Lepeletier & Serville, 1828 Helius ( Helius ) hispanicus Lackschewitz, 1928 Starý and Oosterbroek 2008 , HA , Massif Toubkal, Imlil (17 km S Asni, 1700–1900 m); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Amsemlil; Oosterbroek 2020 (CCW: Rif ) Helius ( Helius ) pallirostris Edwards, 1921 Driauach and Belqat 2016 , AP , Aïn Chouk (Larache); Oosterbroek 2020 (CCW: Rif ) Limonia Meigen, 1803 Limonia flavipes (Fabricius, 1787) Pierre 1922b , HA , Haute Réghaya, Asni (1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limonia hercegovinae (Strobl, 1898) Starý and Oosterbroek 2008 , MA , Ifrane (1700 m), HA ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Limonia macrostigma (Schummel, 1829) Starý and Oosterbroek 2008 , HA , 5 km from Oukaimeden (2350 m); Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limonia nubeculosa Meigen, 1804 Pierre 1922b , HA , Haute Réghaya, Asni (1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Gavryushin pers. comm. 2012, HA ; Driauach and Belqat 2016 , Rif , tributary of Oued El Fondak, Aïn Ras el Ma, Aïn Boughaba, Âounsar Aheramen, Aïn Takhninjoute, maison forestière, Oued Madissouka, Aïn Quan­quben (Jebel Bou Bessoui), EM , Oued Azila; Oosterbroek 2020 (CCW: Rif ; HA ) Limonia phragmitidis (Schrank, 1781) Starý and Oosterbroek 2008 , MA , Ifrane (1700 m); Driauach and Belqat 2016 , Rif , Aïn Quanquben (Jebel Bou Bessoui); Oosterbroek 2020 (CCW) PEDICIIDAE K. Kettani, P. Oosterbroek Number of species: 6 . Expected: 10 Faunistic knowledge of the family in Morocco: moderate Pediciinae Dicranota Zetterstedt, 1838 Dicranota ( Dicranota ) bimaculata (Schummel, 1829) Pierre 1922b , HA , Haute Réghaya (1800 m); Savchenko et al. 1992 (? Morocco); Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Dicranota ) irregularis Pierre, 1921 Pierre 1922b , HA , Haute Réghaya (1800 m); Savchenko et al. 1992 , HA , Cirque d'Arround (Haute Réghaya); Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Ludicia ) claripennis (Verrall, 1888) Driauach and Belqat 2016 , Rif , Oued Amsemlil, maison forestière; Oosterbroek 2020 (CCW) Dicranota ( Paradicranota ) candelisequa Starý, 1981 Pârvu et al. 2006 , AP , Merja Zerga; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Paradicranota ) landrocki Czižek, 1931 Driauach et al. 2013 , Rif , Fifi (1252 m); Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Taida, Aïn Sidi Brahim Ben Arrif, Âounsar Aheramen, Oued Tizekhte, Oued Mezine, maison forestière, Aïn Bab Tariouant, HA , Imlil (Assif Haouz); Oosterbroek 2020 (CCW) Tricyphona Zetterstedt, 1838 Tricyphona ( Tricyphona ) immaculata (Meigen, 1804) Driauach and Belqat 2016 , Rif , maison forestière, Dayat Lemtahane; Oosterbroek 2020 (CCW) TIPULIDAE K. Kettani, P. Oosterbroek, H. de Jong Number of species: 39 . Expected: 42 Faunistic knowledge of the family in Morocco: moderate Dolichopezinae Dolichopeza Curtis, 1825 Dolichopeza ( Dolichopeza ) hispanica Mannheims, 1951 Theowald and Oosterbroek 1980 , HA , Aghbalou, Oukaimeden, Imlil, Tadmant; Oosterbroek and Theowald 1992 ; Oosterbroek and Lantsov 2011 , HA , Oukaimeden (2300 m), Aghbalou (Massif Toubkal), 43 km S Marrakech (1000 m), Imlil (17 km S Asni, 1700–1900 m), Tadmant (17 km E Asni); Adghir et al. 2018 , Rif , Kitane, Aîn Ras el Ma (Chefchaouen); Oosterbroek 2020 (CCW) Tipulinae Nephrotoma Meigen, 1803 Nephrotoma alluaudi (Pierre, 1922) = Pachyrhina lunulicornis Schummel, in Pierre 1922a : 24 = Pachyrhina alluaudi Pierre, in Pierre 1922b : 150, Dakki 1997 : 62 = Pales alluaudi (Pierre), in Mannheims 1951 : 47 Pierre 1922b , HA , Tannaout (1000 m); Mannheims 1951 , Rif , Beni Seddat, AP , Lagune Guedira; HA , Taddert north of Marrakech, Goundafa (1200 m); AA , Llano Amarillo, Tlata Reisana; Oosterbroek 1979b , Rif , HA , Imlil (1400 m), Tizi-n'Tichka (2200 m); Theowald and Oosterbroek 1980 , AP , Rabat, Guedira lagoon, MA , Immouzer, Ifrane, Timahdit, Aghbalou, Tizi-n'Zou, HA , Marrakech, Goundafa, Taddert, Dayat, Tizi-n'Test, Tizi-n'Tichka, Asni, Imlil, Oukaimeden, Setti Fatma, Tinmel, Acif Tifni, AA , Taroudant, Mikdana, Sidi Said bou Merdoul, Tlata Reisana, Llano Amarillo; Eiroa 1990 , MA , Azrou, Ajabo; Oosterbroek and Theowald 1992 , HA , Tannaout; Dakki 1997 ; Mouna 1998 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Oued Laou, Dardara, Douar Mouarâa, Oued Zandoula, Laghmari-Rmal, Douar Louamera, Douar Laheyayda, Oued Jnane Niche, Cabo Negro, Oued Beni Said, Oued El Kanar, Oued Amsemlil; Oosterbroek 2020 (CCW) – MISR ( HA ), MHNV, MNCNM, MAKB Nephrotoma appendiculata pertenua Oosterbroek, 1978 Oosterbroek 1978 , Rif , 9 km SW Chefchaouen; Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Fès, Immouzer, Khemisset; Oosterbroek and Theowald 1992 ; de Jong 1993 , 1998 , Rif ; Adghir et al. 2018 , Rif , Oued Ametrasse, Dayat Aïn Jdioui, Barrage Moulay Bouchta, Oued Sahil, Dayat Jebel Zemzem, Aïn Sidi Brahim Ben Arrif, Oued Nakhla, Oued Tizekhte, Lot Hemmadi, Douar Ayacha, Douar Louamera, Dayat Mezine, Aïn El Malâab, Aïn Takhninjoute, Dayat Tazia, Oued Taida, maison forestière Tazia, Tourbière Amesmlil, Oued El Hamma, Oued Kbir, Jebel Lakraâ; Oosterbroek 2020 (CCW) Nephrotoma astigma Pierre, 1925 Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Taza; Adghir et al. 2018 , Rif , Oued Tabandout, Etang Maggou, Aïn El Malâab, Douar Remla; Oosterbroek 2020 (CCW) Nephrotoma fontana Oosterbroek, 1978 de Jong 1998 , Rif ; Adghir et al. 2018 , Rif , Ketama, maison forestière Tazia, Dayat Tazia; Oosterbroek 2020 (CCW) Nephrotoma guestfalica vaillanti de Jong, Adghir & Bosch, 2021 de Jong et al. 2021 , Rif , Ras el Ma (Chefchaouen, 500 m), Fomento, Oued Laou (6 km north-west of Chefchaouen, 200 m), Jebel Tissouka (3 km south of Chefchaouen, 500 m; 3 km south of Chefchaouen, 700 m; 4 km south-east of Chefchaouen, 700–800 m; 5 km southeast of Chefchaouen, 700–900 m; 5 km south of Chefchaouen, 900–1000 m), Bab Taza (25 km south-east of Chefchaouen, 750–800 m), Dardara (10 km south of Chefchaouen, 300 m), Oued Martil, Nakhla, Âounsar Aheramen, Boumerouil, Oued Maggou, Oued Maggou (Aïn Ras el Ma), Douar Kitane, Oued Sidi Yahia Aârab, Oued Tamerte, Oued Zandoula, Oued El Hamma, Lot Hemmadi, Oued Tamerte, Oued Sidi Mohamed Saâda, Oued El Koub, Douar Iholebatine, Dayat Tazia, Belouazen, Oued Lemtahane, Oued Siflaou, Oued Amsemlil, AP , Oued Loukous, Aïn el-Aouda, MA , Ifrane (road to Mischliffen, 1680 m), N.S. and W of Ifrane (1400–1800 m), Khemisset, Azrou, Oum-er-Rbia, HA , M'semrir (bord de l'Oued), Rich, Haute Rhégaya (identified by Mannheims in 1951 as surcoufi ) Nephrotoma luteata (Meigen, 1818) Oosterbroek 1979a , Rif , Targuist, Chefchaouen, HA , Kasba Taguendaft, near Oukaimeden; Theowald and Oosterbroek 1980 , Rif , Targuist, Chefchaouen, HA , Kasba Taguendaft, near Oukaimeden; Oosterbroek and Theowald 1992 ; Pârvu et al. 2006 , AP , Merja Zerga; Adghir et al. 2018 , Rif , Oued Laou, Dardera, Oued Nakhla; Oosterbroek 2020 (CCW) Nephrotoma subanalis (Mannheims, 1951) = ? Pachyrhina analis Schummel, in Pierre 1922a : 24, Pierre 1922b : 150 = Pales subanalis Mannheims, in Mannheims 1951 : 56 Mannheims 1951 , HA , Tachdirt (2200–2900 m); Vaillant 1956b , HA , Oukaimeden (2250 m); Oosterbroek 1979c, HA , Tachdirt (2200–2900 m); Theowald and Oosterbroek 1980 , HA , Tachdirt, Oukaimeden, M'Goum, Tadmant, Imlil, Tizi-N'Tichka; Oosterbroek and Theowald 1992 , HA , Tachdirt; Oosterbroek 2020 (CCW) Nephrotoma submaculosa Edwards, 1928 Oosterbroek 1982 , Rif , Ketama, Dardara, MA , Azrou; Oosterbroek and Theowald 1992 ; de Jong 1998 , Rif , Atlas ; Oosterbroek 2011 ; Adghir et al. 2018 , Rif , Jebel Dahedouh, Ketama, Aïn Sidi Brahim Ben Arrif, Oued Taida, tributary of Oued Ouara, Sidi Chouiref, Dayat Tazia; Oosterbroek 2020 (CCW) Nephrotoma sullingtonensis Edwards, 1938 Oosterbroek 1978 ; Theowald and Oosterbroek 1980 , Rif , Bab Berred; Oosterbroek 1982 Rif , Bab Berred; de Jong 1993 , 1998 , Rif ; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 , Rif , 4 km SE Ketama, Barrage Moulay Bouchta, Oued Aârate, Aïn Takhninjoute, Oued Jbara, Aïn El Malâab, Aïn El Ma Bared, Oued Lemtahane, maison forestière Tazia, Oued Taida, Dayat Tazia; Oosterbroek 2020 (CCW) Tipula Linnaeus, 1758 Tipula ( Acutipula ) anormalipennis Pierre, 1924 Pierre 1924b , HA , Haut Imminen; Séguy 1930a , HA , Haut Imminen; Séguy 1941a , HA , Tachdirt (2500 m), Haut Imminen; Mannheims 1952 , HA , Tachdirt (2500 m), Imminen (2400–2500 m), Arround (1950 m); Vaillant 1956b , HA , Lake of Tamhda (Anremer, 2900 m); Theowald and Oosterbroek 1980 , HA , Anremer, Haut-Immenen, Tachdirt, Arround, Oukaimeden; Vermoolen 1983 , HA , Haut Imminen, Oukaimeden (2500–2800 m), Arround (1950 m), Tachdirt (2500 m); Oosterbroek and Theowald 1992 ; de Jong 1994a , HA ; de Jong 1998 , HA ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Acutipula ) repentina Mannheims, 1952 = Tipula maxima Poda, in Séguy 1941a : 26 Séguy 1941a , HA , Tachdirt (2500 m); Mannheims 1952 , HA , Tachdirt (2200–2700 m); Vaillant 1956b , HA , Lac Tamhda (Anremer, 2900), M'Goum (2500 m); Theowald and Oosterbroek 1980 , HA , Anremer, M'Goum, Tachdirt, Tizi-N'Tichka, Asni, Oukaimeden, Setti Fatma, Imlil, Tadmant; Vermoolen 1983 , MA , Ifrane, HA , Androment, M'Goum, Tadmant, Tizi-N'Test, Setti Fatma, Oukaimeden, Tizi-N'Tichka; Oosterbroek and Theowald 1992 , HA , Tachdirt; de Jong 1994a , MA , HA ; de Jong 1998 , HA ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Acutipula ) rifensis Theowald & Oosterbroek, 1980 Theowald and Oosterbroek 1980 , Rif , Targuist; Vermoolen 1983 , Rif , Targuist, Tidiguin (90 km E. of Ouezzane, 2350 m); Oosterbroek and Theowald 1992 , Rif ; de Jong 1994a , 1998 , Rif ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Emodotipula ) leo Dufour, 1991 = Tipula ( Emodotipula ) obscuriventris Strobl 2 , in Oosterbroek and Theowald 1992 : 99 Oosterbroek and Theowald 1992 (?); Dufour 2003 , Rif ; Adghir et al. 2018 , Rif , Jebel Tissouka; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) bivittata Pierre, 1922 Pierre 1922a , AP , Maâmora between Kénitra and Oued Beth, Dradek (near Rabat); Pierre 1922b , AP , Rabat; Mannheims 1968 ; Theowald and Oosterbroek 1980 , AP , forest of Maâmora, Dradek; Oosterbroek and Theowald 1992 , AP , forest of Maâmora, Dradek; Dakki 1997 ; Oosterbroek 2020 (CCW) – MISR ( AP , Kénitra, Dradek) Tipula ( Lunatipula ) cinereicolor Pierre, 1924 Pierre 1924b , HA , Haut Imminen; Séguy 1930a , HA , Tachdirt (3100–3200 m); Theowald 1973 , HA , Haut Imminen (2400 m); Theowald and Oosterbroek 1980 , MA , Ifrane, HA , Oukaimeden, Tachdirt, Haut-Imminen; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Imminen; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) cornicula Pierre, 1922 Pierre 1922b , HA , Arround (2000 m); Séguy 1930a , HA , Tachdirt (3100–3200 m); Theowald 1973 , HA , Cirque d'Arround (2000 m), Tachdirt (2200–2700 m), Goundafa (1200 m); Theowald and Oosterbroek 1980 , HA , Oukaimeden, Tachdirt, Goundafa; Oosterbroek and Theowald 1992 , HA , Tachdirt; Dakki 1997 ; Oosterbroek 2020 (CCW) – MISR ( HA ) Tipula ( Lunatipula ) fabiola Mannheims, 1968 Theowald and Oosterbroek 1980 , Rif , Bab Berred, Ras El Ma (Chefchaouen), MA , Jebel Abad, Ifrane; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) hermes Theischinger, 1977 Theischinger 1977 , Rif , north of Ouezzane; Theowald 1980 ; Theowald and Oosterbroek 1980 , Rif , north of Ouezzane; Oosterbroek and Theowald 1992 , Rif , Ouezzane; Adghir et al. 2018 , Rif , 4 km SE Ketama, Oued Ametrasse, Aïn El Ma Bared; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) iberica iberica Mannheims, 1963 = Tipula lunata Linnaeus, in Pierre 1922b : 149, Séguy 1930a : 23 Pierre 1922b , HA , Haute Réghaya, Tannaout (1000 m); Séguy 1930a , HA , Haut Imminen; Mannheims 1963 , MA ; Theowald and Oosterbroek 1980 , Rif , Bab Berred, MA , Ifrane, Taounate; Eiroa 1990 , MA , Ifrane; Oosterbroek and Theowald 1992 ; Oosterbroek 2009 ; Adghir et al. 2018 , Rif , Ketama; Oosterbroek 2020 (CCW) – MISR ( HA ) Tipula ( Lunatipula ) iberica spinula Theischinger, 1980 Theischinger 1980 , HA , Oukaimeden (1300–2800 m); Theowald and Oosterbroek 1980 , HA , Oukaimeden; Oosterbroek and Theowald 1992 , HA , Oukaimeden; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) peliostigma peliostigma Schummel, 1833 Eiroa 1990 , MA , Azrou; Mouna 1997; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) pjotri de Jong & Adghir, 2018 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m) Tipula ( Lunatipula ) pseudocinerascens Strobl, 1906 Adghir et al. 2018 , Rif , Stehat, Oued Taida, Perdicaris Park, Dayat Tazia Tipula ( Lunatipula ) rocina Theischinger, 1979 Theowald and Oosterbroek 1980 , Rif , Tétouan; Oosterbroek and Theowald 1992 ; Oosterbroek 2009 ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) selenaria Mannheims, 1967 Mannheims 1967 , MA , Jebel Tazzeka (1500–1989 m), HA , Goundafa (1200 m); Theowald and Oosterbroek 1980 , MA , Tazzeka, HA , Foum Keneg, Goundafa, Oukaimeden; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Goundafa; de Jong 1995 , MA , HA , Oukaimeden; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) stimulosa Mannheims, 1973 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m) Tipula ( Lunatipula ) subfalcata Mannheims, 1967 de Jong 1995 , 1998 , Rif ; Adghir et al. 2018 , Rif , Jebel Tissouka, Oued Tamerte, Oued El Koub; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) subpustulata Mannheims, 1963 = Tipula pustulata Pierre, in Pierre 1922b : 150 Pierre 1922b , AP , Mogador; Vaillant 1956b , HA , M'Goun (2500 m); Mannheims 1963 , AP , Aïn el Aouda, MA , Jebel Tazzeka (1600–1989 m), HA , Goundafa (1200 m), Tachdirt (2200–2900 m), AA , Lac Goulmima; Theowald 1972 ; Theowald and Oosterbroek 1980 , AP , Aïn el Aouda, MA , Iebel Tazzeka, HA , M'Goum, Goundafa, Tachdirt, Oukaimeden, Imlil; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Goundafa; Dakki 1997 ; Adghir et al. 2018 , Rif , 4 km SE Ketama, Aïn El Ma Bared; Oosterbroek 2020 (CCW) – MISR ( AP , Mogador) Tipula ( Lunatipula ) tazzekai Theowald, 1973 Theowald 1973 , MA , Jebel Tazzeka (1600–1989 m); Theowald and Oosterbroek 1980 , MA , Jebel Tazzeka; Eiroa 1990 , MA , Jebel Hebri; Oosterbroek and Theowald 1992 , MA , Jebel Tazzeka; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) atlas Pierre, 1924 Pierre 1924b , HA , Tachdirt (3100–3250 m); Séguy 1930a , HA , Tachdirt (3100–3200 m); Vaillant 1956b , HA , Cascade Siroua (3000 m), M'Goun (2500 m), Toubkal (3350 m), Lake of Tamhda (Anremer, 2900 m); Mannheims 1964 , HA ; Theowald 1973 , HA ; Theowald 1980 , HA ; Theowald and Oosterbroek 1980 , HA , Toubkal, Sources de Tessaouts, M'Goum, Siroua, Anremer, Tachdirt, Oukaimeden, Tizi-N'Tichka, Tadmant; Eiroa 1990 , MA , Oum-Er-Rbia; Oosterbroek and Theowald 1992 , HA , Tachdirt; de Jong 1994b ; de Jong 1998 , Atlas ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) breviantennata Lackschewitz, 1933 de Jong 1998 , Rif ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Oued Maggou, Douar Aouzighen; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) confusa van der Wulp, 1883 Adghir et al. 2018 , Rif , maison forestière (Talassemtane) Tipula ( Savtshenkia ) rufina rufina Meigen, 1818 Theowald and Oosterbroek 1980 , HA , Oukaimeden; Theowald and Oosterbroek 1983 , Rif , Atlas ; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen); Oosterbroek 2020 (CCW) Tipula ( Tipula ) mediterranea Lackschewitz, 1930 Vaillant 1956b , HA , Oukaimeden (2250 m), M'Goun (2500 m); Mannheims 1952 ; Theowald and Oosterbroek 1980 , MA , Ifrane, HA , Oukaimeden, M'Goum, Asni, M'Semrir, Ifni, Bab-Rou-Idie, Tizi-N'Tichka, Setti Fatma; Theowald 1984 , Rif ; Eiroa 1990 , MA , Azrou, Oum-Er-Rbia; Oosterbroek and Theowald 1992 ; Pârvu et al. 2006 AP , Merja Zerga; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Jebel Tissouka, Bab Taza, Dardara, 4 km SE Ketama, Aïn Afersiw, Douar Kitane, Dayat Jebel Zemzem, Wilaya (Tétouan), Aïn El Ma Bared, Oued Tamerte, Douar Louamera, Tourbière Amesmlil, Dayat Mezine, Hejar Nehal, tributary of Oued Ouara, Oued Tkarae, Oued Jnane Niche, Dayat Afrate, Oued Ametrasse; Oosterbroek 2020 (CCW) Tipula ( Tipula ) oleracea Linnaeus, 1758 Mannheims 1952 , Rif , Tlata Ketama; Theowald and Oosterbroek 1980 , Rif , Tlata Ketama; Theowald 1984 , Rif ; Oosterbroek and Theowald 1992 ; Dakki 1997 ; Pârvu and Zaharia 2007 ; Oosterbroek 2011 ; Adghir et al. 2018 , Rif , Oued Laou, Dardara, Jebel Tissouka, Ksar Rimal, 15 km from Fifi, Oued Aârate, Oued El Hamma, Douar Louamera, Oued Zaouya, Près de Beni Said, Wilaya (Tétouan), Tourbière Amesmlil, Oued Smir, Aïn Jdida; Oosterbroek 2020 (CCW) – MISR Tipula ( Vestiplex ) vaillanti vaillanti Theowald, 1977 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m), Douar Kitane Tipula ( Yamatotipula ) afriberia afriberia Theowald & Oosterbroek, 1980 Theowald and Oosterbroek 1980 , HA , Oukaimeden; Oosterbroek and Theowald 1992 , HA , Oukaimeden; Oosterbroek 1994a ; Adghir et al. 2018 , Rif , Jebel Tissouka, Dardara, Douar Mokedassen, Oued Zarka; Oosterbroek 2020 (CCW) Tipula ( Yamatotipula ) barbarensis Theowald & Oosterbroek, 1980 = Tipula lateralis Meigen, in Pierre 1922a : 23, Pierre 1922b : 150, Séguy 1930a : 23, Séguy 1941a : 26, Mannheims 1952 : 98 (in part), Vaillant 1956b : 238 Pierre 1922a , AP , Dradek (Rabat), MA , Azrou (riverside of Oued Tigrigra); Pierre 1922b , AP , Mogador, MA , Beni Méllal, HA , Asni; Séguy 1930a ; Séguy 1941a , HA , Imi n'Ouaka (1500 m); Mannheims 1952 ; Vaillant 1956b , HA , Oukaimeden; Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Ifrane, Aghbalou, HA , Asni, Oukaimeden, Setti Fatma, Imlil, Tizi-N'Tichka, Tadmant, Tachdirt; Eiroa 1990 , MA , Azrou, Oum-er-Rbia, Ifrane; Oosterbroek and Theowald 1992 , HA , Setti Fatma; Oosterbroek 1994a , Rif , Dardara, MA , Ifrane, Aghbalou, HA , Oukaimeden, Setti Fatma, Imlil, Tizi-N'Tichka, Tadmant, Asni, Tachdirt; Dakki 1997 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Jebel Tissouka, Bab Taza, Dardara, 4 km SE Ketama, Aïn El Manzela, Aïn Bab Tariouante, Dayat Aïn Afersiw, Oued El Kanar, Dayat Afrate, Douar Kitane, Wilaya (Tétouan), 15 km from Fifi, Aïn Sidi Brahim Ben Arrif, Oued Nakhla, Âounsar Aheramen, Oued Boumerouil, Douar Zaouya, Oued Tizekhte, Oued Samsa, Dayat Aïn Jdioui, Douar Ouled Laghmari-Rmal, maison forestière Tazia, Hejar Nehal, Lot Hemmadi, tributary of Oued Ouara, Oued Sidi Mohamed Saâda, Oued Amsemlil, Tourbière Amesmlil, Beni Salah, Oued Jnane Niche, Oued Maggou, Souk Lhed Beni Darkoul, Oued Imassouden, Aïn Helouma, Source Zarka, Aïn Kchour; Oosterbroek 2020 (CCW) – MISR Trichoceridoidea TRICHOCERIDAE K. Kettani, E. Krzemińska Number of species: 8 . Expected: 10 Faunistic knowledge of the family in Morocco: good Trichocerinae Trichocera Meigen, 1803 Trichocera ( Trichocera ) hiemalis (DeGeer, 1776) Pierre 1922b ( AP , Rabat); Dakki 1997 ; Mouna 1998 ; AP (Rabat) – MISR Trichocera ( Trichocera ) marocana Driauach, Krzemińska & Belqat, 2015 Driauach et al. 2015 , Rif , Oued Akrir Trichocera ( Saltrichocera ) annulata Meigen, 1818 Driauach et al. 2015 , Rif , affluent Oued Akrir, Dayat Fifi, Oued Taria, Oued Kelâa, Oued Ouara, Oued à 15 km de Fifi, Aïn Mâaze, Oued Maggou, Aïn Quanquben, Oued Tizekhte, Forêt Jebel Bouhachem, EM , Grotte du Chameau, Oued Zegzel, Aïn Sidi Yahia, Cascade Grotte des pigeons, Oued Tafoughalt Trichocera ( Saltrichocera ) pappi Krzemińska, 2003 Driauach et al. 2015 , Rif , Dayat Fifi, affluent Oued Akrir, Oued Amsemlil, Ruisseau Agoummir, maison forestière, EM , Cascade Grotte des Pigeons, MA , Seguia El Hajeb Trichocera ( Saltrichocera ) saltator (Harris, 1776) Driauach et al. 2015 , Rif , Dayat Fifi, Oued à 15 km de Fifi, Forêt Jebel Bouhachem, maison forestière, EM , Cascade Grotte des pigeons Trichocera ( Saltrichocera ) sardiniensis Petrašiūnas, 2009 Driauach et al. 2015 , Rif , Oued à 15 km de Fifi, affluent Oued Akrir, Oued Amsemlil, maison forestière Trichocera ( Saltrichocera ) regelationis (Linnaeus, 1758) Driauach et al. 2015 , Rif , Oued Ouara, Aïn el Ma Bared, Oued à 15 km de Fifi, Cascade Chrafate, Aïn Quanquben Trichocera ( Saltrichocera ) rufescens Edwards, 1921 Driauach et al. 2015 , Rif , affluent Oued Akrir, Dayat Fifi, Oued Tassikeste, Oued Farda, Aïn Mâaze Psychodoidea PSYCHODIDAE K. Kettani, R. Wagner Number of species: 51 . Expected: 70 Faunistic knowledge of the family in Morocco: moderate Phlebotominae Phlebotomus Loew, 1845 Phlebotomus ( Larroussius ) ariasi Tonnoir, 1921 Gaud 1947a ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , AP , MA , HA ; Rioux et al. 1974 ; Mouna 1998 ; Guernaoui et al. 2005 , MA , HA ; Bounamous 2010 ; Boussaa et al. 2005 ; Boussaa et al. 2010 Phlebotomus ( Larroussius ) chadlii Rioux, Juminer & Gibily, 1966 Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Croset et al. 1978 ; Mouna 1998 ; Bounamous 2010 Phlebotomus ( Larroussius ) langeroni Nitzulescu, 1930 Rislorcelli 1941, HA ; Bailly-Choumara et al. 1971 , AP , EM ; Croset et al. 1978 ; Mouna 1998 Phlebotomus ( Larroussius ) longicuspis Nitzulescu, 1930 Rislorcelli 1941, HA ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , Rif , EM , AP , MA , HA , AA ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Guernaoui et al. 2005 , Rif , Chefchaouen, HA , AA , Agadir; Boussaa et al. 2005 ; Boussaa 2008 ; Boussaa et al. 2008 ; Boussaa et al. 2010 ; Bounamous 2010 ; Zarrouk et al. 2016 Phlebotomus ( Larroussius ) mariae Rioux, Croset, Léger and Bailly-Choumara, 1974 Hervy et al. 1994 ; Rioux et al. 1974 , HA ; Mouna 1998 ; Guernaoui et al. 2005 , MA , HA Phlebotomus ( Larroussius ) perfiliewi Parrot, 1930 Rioux et al. 1977 ; Croset et al. 1978 ; Mouna 1998 Phlebotomus ( Larroussius ) perniciosus Newstead, 1911 Séguy 1930a ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , AP , EM , MA , HA , AA ; Mouna 1998 ; Guernaoui et al. 2005 , Rif , Chefchaouen, HA ; Boussaa et al. 2008 ; Bounamous 2010 ; Boussaa et al. 2010 ; Zarrouk et al. 2016 Phlebotomus ( Paraphlebotomus ) alexandri Sinton, 1928 Bailly-Choumara et al. 1971 , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Colacicco-Mayhugh et al. 2010 , EM ; Boussaa et al. 2010 ; Bounamous 2010 Phlebotomus ( Paraphlebotomus ) chabaudii Croset, Abonnenc & Rioux, 1970 Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Mouna 1998 Phlebotmus ( Paraphlebotomus ) kazeruni Theodor & Mesghali, 1964 Mouna 1998 Phlebotomus ( Paraphlebotomus ) riouxi Depaquit, Killick-Kendrick & Léger, 1998 Bounamous 2010 Phlebotomus ( Paraphlebotomus ) sergenti Parrot, 1917 Séguy 1930a ; Rislorcelli 1941; Rislorcelli 1947; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , Rif , AP , EM , MA , HA , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Mouna 1998 ; Boussaa et al. 2009 ; Bounamous 2010 Phlebotomus ( Phlebotomus ) bergeroti Parrot, 1934 Rioux et al. 1975 , HA ; Mouna 1998 ; Bounamous 2010 Phlebotomus ( Phlebotomus ) papatasi (Scopoli, 1786) Séguy 1930a ; Rislorcelli 1941, Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , AP , EM , MA , HA , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Boussaa et al. 2005 ; Boussaa 2008 ; Colacicco-Mayhugh et al. 2010 , Mediterranean region; Bounamous 2010 ; Boussaa et al. 2010 ; Prudhomme et al. 2012 Phlebotomus clydei Sinton, 1928 Bailly-Choumara et al. 1971 , EM ; Mouna 1998 Phlebotomus lewisi Parrot, 1948 Bailly-Choumara et al. 1971 , South AP ; Mouna 1998 Sergentomyia França & Parrot, 1920 Sergentomyia ( Grassomyia ) dreyfussi (Parrot, 1933) Rislorcelli 1941, Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , AP , EM , MA , HA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 Sergentomyia ( Parrotomyia ) africana (Newstead, 1912) = Phlebotomus ( Parrotomyia ) africana (Newstead), in Rislorcelli 1941: 522, Bailly-Choumara et al. 1971 : 454; Rioux et al. 1974 : 99 Séguy 1930a (as subspecies of minutus Rondani: 43), HA , Marrakech; Rislorcelli 1941 (as subspecies of minutus Rondani: 528), AA , Ksar es Souk; Gaud and Laurent 1952 (as subspecies of minutus Rondani: 75), AP , Rabat; Bailly-Choumara et al. 1971 (as subspecies of minutus Rondani: 438), AP , AA ; Rioux et al. 1974 , HA ; Croset et al. 1978 (as subspecies of minutus Rondani: 722); Mouna 1998 ; Boussaa et al. 2005 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 ; Boussaa et al. 2010 Sergentomyia ( Sergentomyia ) antennata (Newstead, 1912) = Phlebotomus cinctus Parrot & Martin, 1944, in Mouna 1998 : 86 = Phlebotomus signatipennis Newstead, 1920, in Mouna 1998 : 86 Bailly-Choumara et al. 1971 , EM ; Rioux et al. 1974 , HA ; Léger et al. 1974 , AA (south of Morocco); Mouna 1998 Sergentomyia ( Sergentomyia ) fallax (Parrot, 1921) Rislorcelli 1947; Gaud 1954 ; Bailly-Choumara et al. 1971 , AP , EM , MA , AA ; Rioux et al. 1974 , HA ; Mouna 1998 ; Guernaoui et al. 2005 ; Boussaa et al. 2005 ; Boussaa et al. 2007 ; Boumezzough et al. 2009 , HA , Marrakech; Boussaa et al. 2010 ; Bounamous 2010 Sergentomyia ( Sergentomyia ) minuta (Rondani, 1843) = Phlebotomus minutus Rondani, in Gaud and Laurent 1952: 75, Mouna 1998 : 86 = Phlebotomus ( Sergentomyia ) parroti (Adler and Theodor), in Rislorcelli 1941: 526, Rislorcelli 1947: 487, Bailly-Choumara et al. 1971 : 450, Rioux et al. 1974 , 99 Séguy 1930a , HA , Marrakech; Gaud and Laurent 1952, AP , Rabat; Rislorcelli 1941; Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , Rif , AP , EM , MA , HA ; Rioux et al. 1974 , HA ; Croset et al. 1978 (from the mediterranean region to the Sahara); Mouna 1998 ; Boussaa et al. 2005 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 ; Boussaa et al. 2010 ; Depaquit et al. 2015 , Rif , Chefchaouen Sergentomyia ( Sergentomyia ) schwetzi Adier, Theodor & Parrot, 1929 Bailly-Choumara and Léger 1976, SA , Aouinet-Torkoz; Mouna 1998 Sergentomyia ( Sintonius ) christophersi (Sinton, 1927) Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Croset et al. 1978 ; Mouna 1998 Psychodinae Maruinini Tonnoiriella Vaillant, 1982 Tonnoiriella paveli Ježek, 1999 Ježek 1999 , HA , AA ; Afzan and Belqat 2016 Tonnoiriella pulchra (Eaton, 1893) (?) [probably mis-identification] Wagner 1990 ; Ježek and Hájek 2007 ; Afzan and Belqat 2016 Mormiini Mormia Enderlein, 1935 Mormia tenebricosa Vaillant, 1954 = Telmatoscopus ( Mormia ) tenebricosus Vaillant, in Vaillant 1956b : 244 Vaillant 1956b , HA , Imi-N'Ifri; Afzan and Belqat 2016 , Rif , Oued Achekrade Paramormiini Clogmia Enderlein, 1937 Clogmia albipunctata (Williston, 1893) Afzan and Belqat 2016 , Rif , Douar Kitane, Douar Moukhlata, Oued Mhannech, AP , Douar Aoulad Ali (Central Plateau (Coastal region)) Panimerus Eaton, 1913 Panimerus thienemanni (Vaillant, 1954) = Panimerus maynei (Tonnoir, 1919), in Dakki 1997 : 62 Boumezzough and Vaillant 1986b , HA , Assif Réghaya; Afzan and Belqat 2016 Paramormia Enderlein, 1935 Paramormia ustulata (Walker, 1856) Vaillant 1956b , HA ; Mouna 1998 ; Ježek and Yağcı 2005; Omelková and Ježek 2012a ; Afzan and Belqat 2016 , Rif , Seguia Barrage Dar Chaoui, Douar Kitane, Oued Jnane Niche Pericomini Bazarella Vaillant, 1964 Bazarella atra (Vaillant, 1955) = Pericoma atra Vaillant, in Vaillant 1956b : 234, 238, 242 Vaillant 1956b , HA , Assif Tasouat (M'Goum), Siroua, Imi-N'Ifri, Aguelmous, Sidi Chamarouch, Lac Tamhda (Anremer), Oukaimeden; Boumezzough and Thomas 1987 , HA , Oued Réghaya (Imlil, 1750 m); Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Inesmane, Oued Madissouka, Aïn Quanquben Pericoma Walker, 1856 Pericoma ( Pachypericoma ) blandula Eaton, 1893 Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Ježek 2004 , Rif ; Ježek and Hájek 2007 ; Dakki and Himmi 2008 , MA , Oued Sebou; Omelková and Ježek 2012a ; Afzan and Belqat 2016 , Rif , Oued Taida, Âounsar Aherman, Oued Beni Ouachekradi, Oued Aâyaden, Cascade Ras el Ma Pericoma ( Pericoma ) barbarica Vaillant, 1955 Vaillant 1956b , HA , M'Goum; Afzan and Belqat 2016 , Rif , Oued Taida, Douar Taria, Cascade Grotte des pigeons Pericoma ( Pericoma ) granadica Vaillant, 1978 Boumezzough and Vaillant 1986b , HA ; Vaillant and Moubayed 1987; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou; Afzan and Belqat 2016 , Rif , Oued Taida, Ametrasse, Oued Farda, Oued Aâyaden, Oued Ras el Ma, MA , Aïn Vittel, HA , Cascade sur sol cuivreux, Oued Réghaya Pericoma ( Pericoma ) diversa Tonnoir, 1920 Vaillant 1978 , HA ; Afzan and Belqat 2016 , Rif , Cascade Chrafate Pericoma ( Pericoma ) exquisita Eaton, 1893 Ježek 2004 , Rif , HA ; Afzan and Belqat 2016 Pericoma ( Pericoma ) latina Sarà , 1954 Vaillant 1955, HA ; Afzan and Belqat 2016 , Rif , Cascade Chrafate, Oued Maggou, Nord Village Maggou, Oued Kelâa, Oued Talembote, Oued associé à Dayat Fifi, Oued Tiffert, Oued à 20 km de Fifi, Oued El Kanar, Beni Fenzar Pericoma ( Pericoma ) maroccana Vaillant, 1955 = Pericoma numidica var. marocana Vaillant, 1955 Boumezzough and Vaillant 1986b , HA , Tissaout; Dakki 1997 ; Afzan and Belqat 2016 , Rif , Cascade Chrafate, ruisseau maison forestière; Dakki and Himmi 2008 , MA , Oued Sebou Pericoma ( Pericoma ) modesta Tonnoir, 1922 = Pericoma numidica Vaillant, in Vaillant 1956b : 236, 237, 240 Vaillant 1956b , HA , Assif Tassouat (M'Goum), Lac Tamhda (L'Anremer); Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou; Afzan and Belqat 2016 Pericoma pseudexquisita Tonnoir, 1940 Afzan and Belqat 2016 , Rif , Oued Azila Pneumia Enderlein, 1935 Pneumia nubila (Meigen, 1818) Afzan and Belqat 2016 , Rif , Aïn Mâaze Pneumia pilularia (Tonnoir, 1940) Ježek 2004 ; Ježek and Hájek 2007 ; Omelková and Ježek 2012a Pneumia propinqua (Satchell, 1955) Afzan and Belqat 2016 , Rif , Chrafate, Oued Zarka Pneumia reghayana (Boumezzough & Vaillant, 1986) = Satchelliella reghayana Boumezzough & Vaillant, 1986, in Boumezzough and Vaillant 1986b : 238 Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 Pneumia toubkalensis Omelková & Ježek, 2012 Omelková and Ježek 2012b , HA , Toubkal; Afzan and Belqat 2016 , Rif , Oued Aâyaden, Aïn Ras el Ma Psychodini Philosepedon Eaton, 1904 Philosepedon ( Philosepedon ) humeralis (Meigen, 1818) Afzan and Belqat 2016 , Rif , Oued Hachef, Cascade Ras el Ma, Oued El Kanar, 2 km de Douar Assoul, Oued Aâyaden Psychoda Latreille, 1796 Psychoda ( Logima ) albipennis (Zettersdedt, 1850) = Logima albipennis (Zettersdedt), in Mouna 1998 : 86 Mouna 1998 Psychoda ( Psycha ) grisescens Tonnoir, 1922 Ježek 2004 , Rif ; Afzan and Belqat 2016 , Rif , Douar Kitane, MA , Gîte Aït Ayoub Psychoda ( Psychoda ) uniformata Haseman, 1907 Ježek 2004 , Rif ; Afzan and Belqat 2016 Psychoda ( Psychodocha ) cinerea Banks, 1894 Boumezzough and Thomas 1987 , HA , Azib Oukaimeden (2730 m); Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Tazzarine, Douar Taria, Douar Kitane, Oued Chrafate, Oued Aâyaden, EM , Cascade Grotte des Pigeons (Béni Snassen) Psychoda ( Psychodocha ) gemina (Eaton, 1904) Afzan and Belqat 2016 , Rif , Dayat Fifi, Oued Zarka, Douar Kitane, Oued Aâyaden Psychoda ( Tinearia ) alternata Say, 1824 Tonnoir 1920, HA , La Maire; Boumezzough and Thomas 1987 , HA , Oued Réghaya (1740 m), Imlil; Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Nakhla, Oued Farda, Oued Ouara, Oued Ametrasse, Oued Chrafate, Douar Derâa, Douar Ihermochene, Douar Ikhlafene, Douar Taria, Douar Idrene, Douar Kitane, Oued 2 km de Douar Assoul, Oued Aâyaden, ruisseau maison forestière, Oued Mhannech, Aïn Sidi Yahia, MA , Gîte Aït Ayoub Scatopsoidea SCATOPSIDAE 3 K. Kettani, J.P. Haenni Number of species: 13 . Expected: 30–40 Faunistic knowledge of the family in Morocco: poor Ectaetiinae Ectaetia Enderlein, 1912 Ectaetia clavipes (Loew, 1846) = Scatopse clavipes Loew, in Mouna 1998 : 84 Mouna 1998 ; Haenni and Kettani 2016 , Rif , Amsa – MISR Psectrosciarinae Anapausis Enderlein, 1912 Anapausis sp.* Rif , Source Aïn Tissemlal Psectrosciara Kieffer, 1911 Psectrosciara sp. 1* Rif , Adrou Psectrosciara sp. 2* Rif , Adrou Scatopsinae Coboldia Melander, 1916 Coboldia fuscipes (Meigen, 1830) Haenni and Kettani 2011 , Rif , Kitane, AP , El Jadida, Rabat, AA , Souss; Haenni and Kettani 2016 , Rif , Maggou, Aârkob, Jnane Niche; Rif (M'Diq farm) – MISR Parascatopse Cook, 1955 Parascatopse sp. Haenni and Kettani 2011 , AA , Ouarzazate ­– MHNN Quateiella Cook, 1975 Quateiella inexpectata Haenni, 1988 Haenni and Kettani 2016 , Rif , Afertane Reichertella Enderlein, 1912 Reichertella geniculata (Zetterstedt, 1850) Haenni and Kettani 2016 , Rif , Jnane Niche Reichertella maroccana Haenni, 2011 Haenni and Kettani 2011 , HA , Oukaimeden – RMNH Rhegmoclemina Enderlein, 1936 Rhegmoclemina lunensis Haenni & Godfrey, 2009 Haenni and Kettani 2011 , Rif , Boujdad Rhexoza Enderlein, 1936 Rhexoza freyi (Duda, 1936) Haenni and Kettani 2011 , AA , Agadir – MHNN Scatopse Geoffroy, 1762 Scatopse notata (Linnaeus, 1758) Mouna 1998 ; Haenni and Kettani 2016 , Rif , Onsar Lile – MISR Swammerdamella Enderlein, 1912 Swammerdamella brevicornis (Meigen, 1830) Haenni and Kettani 2011 , Rif , Kitane, Oued Laou, Ketama, AP , El Jadida, Essaouira, HA , Ouarzazate; Haenni and Kettani 2016 , Rif , Amsa, Jnane Niche, Ametrasse – MISR New records for Morocco An undescribed species of Anapausis has been collected in the Rif mountains in 2014 by K. Kettani and will be described elsewhere. Anapausis sp. Rif: Forêt Azilane ( NPT ), Source Aïn Tissemlal, 1255 m, 35°11.67N, 5°15.20W , 7.vi.2014, Sapinière à Abies maroccana et Pinus nigra , 1♂. Two undescribed species of Psectrosciara have been collected in the Rif mountains in 2013 by K. Kettani and will be described elsewhere. Psectrosciara sp. 1 Rif: Taghzout ( PNPB ), Adrou, 556 m, 35°22.39N, 05°32.28W , chênes-liège, 14.vii–15.viii.2013, 6♂♂. Psectrosciara sp. 2 Rif: Taghzout ( PNPB ), Adrou, 556m, 35°22.39N, 05°32.28W , chênes-liège, 14.vii–15.viii.2013, 3♂♂. PTYCHOPTERIDAE K. Kettani, R. Wagner Number of species: 5 . Expected: 8 Faunistic knowledge of the family in Morocco: moderate Ptychopterinae Ptychoptera Meigen, 1803 Ptychoptera albimana (Fabricius, 1787) MISR (No locality given) Ptychoptera contaminata (Linnaeus, 1758) MISR (No locality given) Ptychoptera lacustris Meigen, 1830 MISR (No locality given) Ptychoptera paludosa Meigen, 1804 MISR (No locality given) Ptychoptera scutellaris Meigen, 1818 MISR (No locality given) Culicoidea CHAOBORIDAE K. Kettani, R. Wagner Number of species: 2 . Expected: 4 Faunistic knowledge of the family in Morocco: poor Chaoborinae Chaoborus Lichtenstein, 1800 Chaoborus crystallinus (De Geer, 1776) Dakki 1997 : 60 Mochlonyx Loew, 1844 Mochlonyx culiciformis (De Geer, 1776) Dakki 1997 : 60 CULICIDAE K. Kettani, B. Trari, O. Himmi, M. Dakki Number of species: 43 . Expected: 60 Faunistic knowledge of the family in Morocco: good Anophelinae Anopheles Meigen, 1818 Anopheles ( Anopheles ) algeriensis Theobald, 1903 Viallate 1922, AP , Kénitra; Séguy 1930a ; Bonjean 1947 , EM , MA ; Gaud 1957a , HA , north of High Atlas; Guy 1959a , MA , Béni Mellal, HA ; Guy 1959b , HA ; Guy 1959a , MA , Béni Mellal; Bailly-Choumara 1967a , MA , Ghorm El Alem; Benmansour et al. 1972 , MA , Barrage Bin El Ouidane; Bailly-Choumara 1973b , AP , Sidi Yahia du Gharb; Metge 1986 , AP , Casablanca; Trari and Himmi 1987 , AP ; Himmi et al. 1995 ; Louah 1995 , Rif , Tahaddart, Schroda; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Casablanca; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 , Rif , Chefchaouen; El Ouali Lalami et al. 2010 a,b, MA , Fès, Boulmane; El Ouali Lalami 2012 , MA , Fès; Trari 2017 , Rif , Chefchaouen, AA , Tiznit; Trari and Dakki 2017a , Rif , Chefchaouen, AA , Tiznit; Trari and Dakki 2017b , AA , Tiznit; Trari et al. 2017 Anopheles ( Anopheles ) claviger (Meigen, 1804) Vialatte 1923 , AP , Kénitra; Séguy 1930a , AP , Rabat; Langeron 1938 , HA , Tounfit, Massou, Anefgou, Tirghist, Tighermine, Louggouargh; Callot 1940 , HA , Anefgou, Tirghist; Bonjean 1947 , EM , MA ; Gaud 1947c , MA , Sefrou, Meknès; Gaud 1948 , AP , Rabat, MA , El Hajeb, AA , Errachidia, Tadla; Gaud et al. 1948 , AP , Skhirat; Guy 1963 , Rif , Taounate, MA , Meknès, Ifrane; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, piste Ksiba-Naour, piste Naour-Arbala, Zaouia Cheikh, Oued Sarif (environs El Ksiba), Dayet Aoua (environs Ifrane), Boulemane; Bailly-Choumara 1967b , EM , 8 km N Itzer; Bailly-Choumara 1967c , Rif , piste Ketama-Mt Tiguidin; Guy 1967 , HA , Marrakech, AA , Tafilalt; Guy and Holstein 1968 , SA ; Metge 1986 , AP , Casablanca; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris drains, Tamaris merja; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 , Rif , Bab Berred, Tanakoub; Faraj et al. 2008b , Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Boulmane; Larhbali et al. 2010 , MA , Zhiliga, Boukachmir, Aït Ichou; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) labranchiae Falleroni, 1926 d'Anfreville 1916 , AP , Salé; Delanoe 1917, AP , El Jadida; Viallate 1922, AP , Rabat, Boulhaut, Bouznika, Gharb, MA , Sidi Kacem, Tiflet, Fès, Taza; Charrier 1924 , Rif , Tanger; Séguy 1930a , MA ; Roubaud 1935, AP , Rabat; Sicault et al. 1935 , AP , Merja Ras Eddaoura, Merja Zerga, dayas entre Sebou et Maâmora, Dar bel Hamri (entre barrage El Kansera et Sidi Slimane), à proximité de Merja Zerga, Douar Anabsa; Langeron 1938 , AA ; Callot 1940 , AA ; Ristorcelli 1946a , b , HA , Oued Tensift, Oued Issil; Bonjean 1947 , AP , Gharb; Gaud 1947c , MA , Oulmès, HA , Marrakech; Gaud et al. 1948 , AP , Merja Ras Eddaoura; Gaud et al. 1949 , AP , Salé, SA , Foum Zguid, Tagounit; Gaud 1953a , AP , from Tanger to El Jadida, MA , Tissa, Timhadit, Bekrit, Meknès, Ifrane (1700 m), HA , Sidi Aissa, Tizi-n'Tichka; Guy 1958 , HA , Oued N'fis; Sacca and Guy 1960b, Rif , Tétouan, AP , Skhirat, Sidi Yahia, Sidi Bettache, Mazagan, Braila (près Sidi Allal Tazi), Aït Lahsen, MA , Meknès, HA , Marrakech; Guy 1962 , Rif , Taounate, AP , Gharb, HA , Marrakech (ville et banlieu); Guy 1963 , Rif , Tanger, Tétouan, EM , Berkane, Debdou, Oujda, AP , Kénitra, Souk Larba, Settat, Chamaîa, Safi, Casablanca, Rabat, Essaouira, MA , Meknès, Fès, Azrou, Oued Zem, Béni Mellal, HA , Kelaâ of Sraghna; Bailly-Choumara 1967a , MA , piste Tafechna-Znan Imes, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Aguelmane Azigza, piste Aguelmane Azigza-Aïn Leuh, maison forestière Ouiouane, route Khénifra-Tafechna, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Bords Moulouya, piste Itzer-Boumia, RN P.33 Boumia-El Kbab, Ouaoumana, piste Ksiba-Naour, piste Naour-Arbala, piste Arbala-El Kbab, Kafensour, Oued Sarif (environs El Ksiba), Dayet Aoua, Ifrane, Imouzer Marmoucha; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Douar Sherba, Douar Aïn Shebbak, Douar Aïn Zabia, Douar Madarh, Douar Sidi Hashas, Mechraa safsaf; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, piste Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Al Hoceima Club Med, Béni Bouayache, Targuist et environs; Guy 1967 , HA , Marrakech, AA , Tafilalt; Bailly-Choumara 1968b , AP , Larache; Guy and Holstein 1968 , AA , Ouarzazate; Bailly-Choumara 1970 , Rif , Tétouan, EM , Berkane, AP , Larache, Sidi Yahia du Gharb, HA , Marrakech; Benmansour et al. 1972 , AA , Agadir; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1972b , AP , Merja Sheishat; Bailly-Choumara 1973a , AP , Merja de l'Oued Smir; Bailly-Choumara 1973b , Rif , Tétouan, EM , Berkane, AP , Merja Sheishat, HA , Souk des Oudaias; de Zulueta et al. 1983 , Rif ; Ibn Jilali 1984, AP , Maâmora; Metge 1986 , AP , Entre Oulad Dlim et Al Ara'ra; Trari and Himmi 1987 , AP , Gharb; Himmi 1991 , AP , Kénitra, Maâmora; Metge 1991 , AP , Sidi Bettache; Trari 1991 , Rif , Chefchaouen, Tanger, Taounate, EM , Oujda, AP , Sidi Amira, Sidi Boughaba, Merja Zerga, Sidi Allal Tazi, Aïn Chouk, Oued Loukous, Merja Oulad Skhar, MA , Khémissat, Béni Mellal, Khouribga, HA , El Kelaâ des Sraghrna, AA , Ouarzazate; El Bermaki 1993, AP , entre l'aeroport Anfa et l'aménagement d'El Oulfa, Sidi Maârouf, El Oulfa; Chlaida and Bouzidi 1995 , AP , Aïn Blal, Oued Sidi Messoud; Louah 1995 , Rif , Tres piedras, Marina Smir, Bouzerlal, Oued Maleh, Zekri, Lajour, Azla, Tahaddart, Skroda, Stehat, Moulay Bouchta, Kebbache, Talembote, Loubart, Chefchaouen, Oued Maggou, Bab Berred, Sidi Kankoch, Oued kbir, Oued Jebel Lehbib; Louah et al. 1995 ; Chlaida 1997 , AP , Aïn Blal, Oued Sidi Messoud, Douar Chlihat, Barrage Al Massira; Moussalim 1997 , AP , Sidi Allal Tazi, Maâmora; Ramdani 1997 , AP , Tamaris, Skhirat; Faraj et al. 1997 , AP , Ouled Moussa; Handaq 1998 , HA , Oukaimeden, Amizmiz, Tiguenziouine; Himmi et al. 1998 , AP , Dayat d'El Menzeh (north-east of Kénitra), Sidi Boughaba; Alaoui Slimani et al. 1999 , AP , Bou-Regreg Salé, Sidi Bouguettaya, Quartier industriel Takaddoum, Marjane; Alaoui Slimani 2002 , AP , Rabat-Salé; Trari et al. 2002 ; Faraj et al. 2003 , Rif , Azib Bouflou, Azib Jrou, Imzouren, Amezzaourou, Tizi-Tamalout, MA , Aït Abdelsalam, Aït Lamfadel; Faraj et al. 2004 , Rif , Azib Jrou, Tanghaya Akarkar, Amezzaourou, Ouled Nsar, AP , Fedalate, MA , Talaa Chougaga, Aïn Smen (Fès), Aït Lamfadel; Trari et al. 2004a , Rif , Larache; Trari et al. 2004b , Rif , Ketama, Gzenaya, Taounate, Bouzaghlal, Oued Laou, Smir (Merja), Béni Hassane, Azib Jrou, Azib Bouflou, AP , Laaouamra, Aarabat Sidi Abdelaziz, Moukaouama, Dar Belamri, Laksibia, Mgadid, Beggara, Rabat (Chellah), MA , Adouz, Rommani (Khémisset), Aït Abderrahmane, Aït Ishaq, Oulad Messoud, Ouled Fennane, El karma, Oulad Zguida, Oulad Abbou; Aouinty et al. 2006 , AP , Mohammedia; Himmi 2007 , Rif , Bab Berred, Bab Taza, Tanakoub, AP , Skhirate, Maâmora, Oulja, Bouknadel, Sidi Azzouz, Tamaris, MA , Khémisset; Faraj et al. 2008a , AP , Laouamra, Boucharen, MA , Béni Khlef, Talaa Chougaga; Faraj et al. 2008b , Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010b , MA , Fès; Faraj et al. 2010 , AP , Begara, Boucharen, Ben Slimane, Skhirat, Rabat, Sehoul, MA , Sidi Allal Msader, Aïn Aghbal, Aïn Elouali, Sidi Kacem; Larhbali et al. 2010 , MA , Aït Haddou Said; Adlaoui et al. 2011 , AP , Larache; Larhbali et al. 2011 , MA , Oulmès, Aït Yadin, Sfassif, Mâaziz, Rommani, Laghoualem, Ezzhiliga, Sidi Allal Bahraoui, Boukachmine, Aït Malek, Sidi Boukhalkhal, Bni Ounzar, Ganzra, Aït Siberne, Sidi Allal Msader, El Ghandour; El Ouali Lalami 2012 , MA , Pont Diamant vert, Sidi Harazem, Oued El Himmer, Moulay Yakoub, Oued Sebou, Aïn Kansara, Oued Aïn Chkef, Sefrou, Boulemane; Laboudi et al. 2012 , AP , Larache; Hadji et al. 2013 , AP , Sidi Slimane; El Joubari et al. 2014 , Rif , Smir lagoon; Laboudi et al. 2014 , Rif , Tétouan, Tanger, Chefchaouen, Al Hoceima, AP , Larache, Salé, MA , Taza, Khémissat; El Joubari et al. 2015a , Rif , Smir lagoon; El Joubari et al. 2015b , Rif , Smir lagoon; Marc et al. 2016 , AP , Kénitra; Trari 2017 , Rif , Chefchaouen, Tétouan, AP , Larache, Rabat, Settat, EM , Oujda, MA , Khémisset, Meknès, Khouribga, HA , Marrakech, AA , Tiznit, Ouarzazate; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, AP , Larache, Rabat, Settat, EM , Oujda, MA , Khémisset, Meknès, Khouribga, HA , Marrakech, AA , Tiznit, Ouarzazate; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) marteri Senevet & Prunnelle, 1927 Gaud 1945b , MA , El Hajeb, Khénifra, HA , Tizi-n'test, Tillougite; Gaud et al. 1949 , HA , Tizi-n'test; Gaud 1953b , HA , Tizi-n'test; Bailly-Choumara 1967b , EM , Grotte du Zegzel; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Taza-Asifane, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Boured; Benmansour et al. 1972 , MA , Taza; Trari 1991 , Rif , Taounate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; Trari 2017 , Rif , Chefchaouen, AP , Settat; Trari and Dakki 2017a , Rif , Chefchaouen, AP , Settat; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) ziemanni Grünberg, 1902 Senevet 1935 , MA ; Gaud et al. 1949 , HA , plain of south and north of the Occidental Atlas; Gaud et al. 1950 , HA , plain of south and north of the Occidental Atlas; Guy 1958 , HA , Marrakech; Guy et al. 1958, HA , Oued N'fis; Guy 1967 , EM , Oujda, AP , Rabat, HA , Marrakech, AA , Tafilelt; Bailly-Choumara 1970 , HA , Marrakech; Benmansour et al. 1972 , MA , Taza, HA , Haouz, Tadla Azilal; Bailly-Choumara 1973b , HA , Souk des Oudaias (Souk Tnine des Oudaias, 470 m); Trari 1991 , MA , Tissa; Moussalim 1997 , AP , 7.5 km de Sidi Allal Tazi; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 ; Trari 2017 , Rif , Tétouan, AA , Tiznit; Trari and Dakki 2017a , Rif , Tétouan, AA , Tiznit; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) cinereus Theobald, 1901 Viallate 1922, MA , Sefrou, Sidi Lamine, HA , Mtougui; Sicault et al. 1935 , AP , Souk Larba of Gharb; Senevet 1935 , AP , Souk Larba of Zemmour; Langeron 1938 , HA , Anefgou, Tirghist, Valley of Sidi Yahia Ouyoussef, Tighermine, Louggouargh, Massou; Callot 1940 , HA , Anefgou, Tirghist; Gaud 1945a , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud and Duthu 1945, HA , Marrakech; Viamonte and Ramirez 1945 , Rif ; Viamonte and Ramirez 1946 , Rif ; Ristorcelli 1946a , HA , Oued Tensift, Oued Issil; Ristorcelli 1946b , HA , Oued Tensift, Oued Issil; Gaud 1945b , AA , Tansikht; Gaud et al. 1949 , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud et al. 1950 , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud 1953a ; Gaud 1958, HA , Marrakech; Guy 1962 , Rif , Taounate, HA , Marrakech; Guy 1963 , MA , Midelt, AA , Hamada of Draa; Bailly-Choumara 1965, EM , Aman d'Aït Oussa, Tiglit, El Megrinat, Taskala, Aïn Aït delouine, Oued mesdourt, Talmesdourt, Assa, AA , Aït Melloul, Oued Teima, Issen, Taroudant, Talaint, Tiznit, Oued Assaka, Anezi, Pont de la route Agadir-Tiznit, valley of low Draa, Tafraoute, Tacharicht, Bou Izakarn, Jemâa N'tirhirte, Aït Erkha,Tazert, Barrage Taourirt, AA , Goulmima, SA , Aouinet Torkoz, Tirh Mzoun; Bailly-Choumara 1966 , AA , piste Foum Zguid-Lac Iriqui, Agadir-Tissint, Akka-Iguiren, Tirhem, Taoujgelt, Souk El Khémis Dades, Akka, Aït Ouabelli, Foum-el-Hassan, Tarhjicht, Aït melloul, Taliouine, Tazenakht (Rocade of Draa); Bailly-Choumara 1967a , MA , route Azrou-Khénifra, Jnane Imasse, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Sources Oum-er-Rbia, Itzer, piste Itzer-Boumia, Ouaoumana, piste Ghorm El Alem-El Ksiba, piste Naour-Arbala, Zaouia Cheich, kafensour, Ifrane, Imouzzer Marmoucha; Bailly-Choumara 1967b , EM , 7 km N Itzer, 8 km N Itzer, route Itzer-Midelt, Aïn Srouna, Gouttitir, Cascade Oued Za, Grotte du Zegzel, Douar Aïn Soultane, Mechraa Safsaf, Oujda, Berguent (valley of Moulouya), Figuig; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Assifane, Anasar, piste Bab berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, route Al Hoceima-Arba Taourirt, Arba Taourirt, Targuist et environs, route Targuist Al Hoceima, Jebha, Oued Ouergha, route Aknoul-Al Hoceima; Guy 1967 , AP , Rabat, EM , Oujda, HA , Marrakech, AA , Tafilalet; Bailly-Choumara 1970 , HA , Marrakech; Bailly-Choumara 1973b , HA , Souk des Oudaias; Trari 1991 , Rif , Al Hoceima, Chefchaouen, Taounate, EM , Nador, Oujda, Figuig, AP , Larache, Settat, Ben Slimane, MA , Khénifra, Taza, Khouribga, HA , Kelaâ of Sraghna, AA , Ouarzazate, Goulmima; Bouallam 1992, HA , Oued N'fis; Louah 1995 , Rif , Marina Smir, Bouzerlal, Tahaddart, Stehat, Moulay Bouchta, Kebbache, Loubart, 0ued Maggou; Louah et al. 1995 ; Bouallam et al. 1997b , HA , Marrakech; Handaq 1998 , HA , Amizmiz, Tiguenziouine; Bouallam 2001, HA , Oued N'fis; Trari et al. 2002 ; Trari et al. 2004b , Rif , Ketama, Azib Jrou, Sidi Mokhfi, MA , Taghzirt, Aghbala, Aït Shak, Smaala, Mlalih, Ouled Fennane, Béni Khlef, Tachrafte; Faraj et al. 2007 , Rif , Assoul, Mizgane; Himmi 2007 , Rif , Bab Berred, Bab Taza, Stehat, Tanakoub; Faraj et al. 2008, Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Fès; El Ouali Lalami et al. 2010b , MA , Oued El Himmer; Larhbali et al. 2010 , MA , Roumani Aïn Sbite, Jamâa M. B., Ghoualem, Zhiliga, Oulmès, Tarmilate, Boukachmir, Mrirte, Aït Ichou, Mâaziz, Tiddas, Bni Ounzar, Ganzra; El Ouali Lalami 2012 , MA , Oued El Himmer; Trari 2017 , Rif , Chefchaouen, Tétouan, MA , Khouribga, AA , Tiznit; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, MA , Khouribga, AA , Tiznit; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Anopheles ( Cellia ) dthali Patton, 1905 Saccà 1960 , AA , Aoufous, Meski, Erfoud, Agdz, Zogora, Tagounit, Tamsrruth; Guy 1961 , HA , Sud de Zagora (en bordure Hamada du Draa); Guy 1963 , AA , Zagora and south of the High Atlas (at edge of Hamada of Draa), Oued Ziz; Bailly-Choumara 1965c , AA , Tiznit, EM , Aïn Aït Delouine, Aouinet Torkoz, Rich Tamlougout (eastern borders of Jebel Bani); Bailly-Choumara 1966 , AA , piste Foum Zguid au Lac Iriqui, Agadir-Tissint, Akka-Iguiren, Souk El Khémis Dades, Akka et Environs, Aït Ouabelli, Tarhjicht, piste Tazenakhte à Foum Zguid (Rocade du Draa); Bailly-Choumara 1967b , EM , valley of Moulouya; Guy 1967 , HA , Marrakech, AA , Tafilalet; Guy and Holstein 1968 , EM , N Outat El Haj (valley of Moulouya), Gouttitir (environs de Taourirt); Bailly-Choumara 1970 , AA , Foum Zquid; Bailly-Choumara 1973b , AA , Foum Zquid; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari et al. 2004b ; Faraj et al. 2007 , Rif , Assoul, Mizgane (SE of Bab Berred); Faraj et al. 2008, Rif , Assoul, Mizgane (SE Bab Berred); Himmi 2007 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) multicolor Cambouliu, 1902 Messerlin and Treillard 1938 , HA , Marrakech; Viamonte and Ramirez 1945 , Rif ; Guy 1963 , SA ; Bailly-Choumara 1965c , EM , Aït Oussa, Aman d'Aït Oussa, Aïn Aït Delouine, Aouinet Torkoz, Rich tamlougout (Confins orientaux du Jebel Bani), AA , Tafnidilt, Guelta Zerga, Aïn Temda (valley of low Draa), Tirhmert (Goulmima), SA , Vallée et embouchure de l'Oued Assaka, Tantan ville, Tirh Mzoun; Bailly-Choumara 1966 , AA , Rocade of Draa; Bailly-Choumara 1967b , EM , 40 km N de Outat El Haj, Gouttitir, Cascade Oued Za, Douar Aïn Shebbak (valley of Moulouya); Guy 1967 , HA , Marrakech, AA , Tafilelt; Guy and Holstein 1968 , HA , south of Atlas, AP , plain located between Marrakech and the Atlantic from Tanger along the length of the Mediterranean; Bailly-Choumara 1970 , AA , Foum Zquid; Bailly-Choumara 1973b , AA , Foum Zquid; Metge 1986 , AP , Casablanca; Trari 1991 , Rif , Al Hoceima, Taounate, Oued Maleh, Bab Berred, AP , Tamaris Merja, AA , Ouarzazate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari et al. 2004b ; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) sergentii (Theobald, 1907) Séguy 1930a ; Messerlin and Treillard 1938 , HA , Tamelelt; Langeron 1938 , Rif , Targhist; Callot 1940 , AA , Taghjicht; Gaud 1947c , AA , Wadi Draa; Gaud et al. 1949 , Rif , Zoumi, HA , Zaouia Sidi Hamza, Tizi-n'test (1700 m), Tillougit (1800 m); Gaud et al. 1950 , Rif , Zoumi, MA ; Guy et al. 1958, HA , Oued N'fis; Guy 1961 , Rif , AP , south of Casablanca, AA , Sud de Zagora; Guy 1962 , Rif , AP , south of Casablanca, HA , Marrakech; Guy 1963 , Rif , Tanger, EM , Berkane, AP , south of Casablanca, MA , Béni Mellal, HA , Oued Tensift, Oued Ziz, Marrakech, Chichaoua, AA , Oued Draâ, Oued Dades, Goulmima, sud de Zagora, SA , Foum Zguid; Bailly-Choumara 1965c , EM , iglit, Aman d'Aït Oussa, Oued Isker, El Megrinat, Aïn Aït Delouine, Oued Mesdourt, almesdourt, Aouinet Torkoz, Bouanama, Rich Tamlougout, Assa, AA , Tiznit, Tafraoute, Oued Izi, Bou Izakarn, Abeino (region of Goulmima), SA , Aouzeroual, Tirhmert, Tacharicht, Jebel Bani, Tantan; Bailly-Choumara 1966 , AA , Rocade of Draa, Taliouine; Bailly-Choumara 1967a , Rif , AP , south of Casablanca, MA , Itzer, Ghorm El Alem, Zaouia Cheikh, Kafensour, HA , north of High Atlas; Bailly-Choumara 1967b , EM , Aïn Srouna, Grotte du Zegzel, Douar Mardarh, Douar Aïn Soultane, Merja Boubker, Selouane, Driouch (valley of Moulouya); Bailly-Choumara 1967c , Rif , route nationale Jebha, Targuist-Béni Boufrah, Al Hoceima, Béni Bouayache, Marchica, Had El Rouadi, Pont du Srah; Guy 1967 , HA , Marrakech, AA , Tafilelt; Guy and Holstein 1968 , AP , Casablanca; Bailly-Choumara 1970 , EM , Berkane, HA , Marrakech; Bailly-Choumara 1973b , EM , Berkane, HA , Marrakech; Metge 1986 , AP , Casablanca; Trari 1991 , Rif , Al Hoceima, Taounate, AP , Larache, AA , Ouarzazate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Faraj et al. 2003 , Rif , Al Hoceima, Chefchaouen, Taounate, HA , Khouribga; Trari et al. 2004b , Rif , Ketama, Sidi Mokhfi; Faraj et al. 2007 , Rif , Assoul, Mizgane; Faraj et al. 2008, Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Fès; Larhbaliet al. 2010 , MA , Oulmès, Ganzra; Trari 2017 , Rif , Chefchaouen, Tétouan, EM , Oujda, MA , Khouribga; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, EM , Oujda, MA , Khouribga; Trari and Dakki 2017b ; Trari et al. 2017 ; Benabdelkrim Filali et al. 2018 ; Mouatassem et al. 2019, MA , Fès Culicinae Aedini Aedes Meigen, 1818 Aedes ( Acartomyia ) mariae (Sergent & Sergent, 1903) Séguy 1930b ; Messerlin 1938 , AP , Rabat; Séguy 1930a , Rif , littoral méditerranéen; Bailly-Choumara 1967b , Rif , Al Hoceima; Bailly-Choumara 1967c , Rif , Al Hoceima; Bailly-Choumara 1968a , Rif , Al Hoceima, AP , Larache, Sidi Yahia, Sidi Allal Tazi, Rabat, HA , Marrakech, AA , Tiznit; Himmi et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Aedimorphus ) vexans (Meigen, 1830) Gaud 1947c , AP , Sidi Allal Tazi, MA , Khémisset; Metge 1986 , AP , Littoral Casablanca; Himmi et al. 1995 ; Handaq 1998 , AP , Gharbia; Dakki 1997 ; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Dahliana ) echinus (Edwards, 1920) Séguy 1924 ; Séguy 1930a ; Gaud 1953a , AP , Rabat, Sidi Yahia, MA , Moulay Bouazza, Taza, Fès, Meknès, Ifrane; Bailly-Choumara 1965b , AP , Maâmora; Bailly-Choumara 1967c , Rif , piste Bab Taza-Talassemtane; Himmi et al. 1995 ; Trari et al. 2002 ; Nikookar et al. 2010 ; El Joubari et al. 2014 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Dahliana ) geniculatus (Olivier, 1791) Séguy 1924 , MA ; Séguy 1930a ; Metge and El Alaoui 1987 , AP , Subéraies de Béni Abid-Benslimane (Casablanca); Metge and Belakoul 1989 , AP , Sidi Bettache; Himmi et al. 1995 ; El Ouali Lalami et al. 2010a , MA , Fès Boulmane; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) berlandi (Séguy, 1921) Séguy 1930a , AP , Rabat; Gaud 1953a , MA , Fès; Bailly-Choumara 1967a , MA , Jnane Imasse, piste Tafechna-Taoujgelt; Bailly-Choumara 1967c , Rif , piste Bab Taza-Béni Ahmed; Belakoul 1985 , AP , Benslimane, Sidi Bettache; Metge and El Alaoui 1987 , AP , Casablanca; Metge and Belakoul 1989 , AP , Benslimane, Sidi Bettache; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) caspius (Pallas, 1771) Séguy 1930b ; Viamonte et Ramirez 1946, AP , Larache; Gaud 1952 , AP , Rabat, Casablanca; Gaud 1953a , AP , Rabat, Casablanca; Senevet and Andarelli 1954 , EM , Embouchure de la Moulouya, Figuig, AP , Jorf Lasfar, Mohammedia, Rabat, MA , Meknès, Fès, Taza, HA , Marrakech, Midelt, AA , Tiznit; Bailly-Choumara 1966 , AA , environs de Tiznit; Bailly-Choumara 1967a , EM , Bords Moulouya (près Itzer), Cherarba; Bailly-Choumara 1967b , EM , Cherarba, Aïn Shebbak, Saidia, Berguent; Bailly-Choumara 1967c , Rif , piste Al Hoceima-Arba Taourirt; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , Rif , Merja de l'Oued Smir; El Kaim 1972 , AP , Bou-Regreg; Rioux et al. 1975 , AP , Rabat-Salé; Metge 1986 , AP , Littoral casablancais; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba, Merja Zerga, Oued Loukous; Himmi et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Kénitra; Ramdani 1997 , AP , Tamaris Merja; Handaq 1998 , AP , Zemamra, B. Iffou (Entre El Oualidia et Youssoufia), MA , Béni Mellal, HA , Marrakech, Zaouiet Ben Sassi, Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; Alaoui Slimani 2002 , AP , Rabat; Aouinty et al. 2006 , AP , Mohammedia; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) coluzzii Rioux, Guilvard & Pasteur, 1998 et Aedes ( Ochlerotatus ) detritus (Haliday, 1833) [Complexe detritus] Charrier 1924 , Rif , Tanger; Séguy 1930a ; Gaud 1953a , EM , Saidia, AP , Kénitra, Rabat, Bouznika, El jadida, Oualidia, HA , Marrakech, AA , Agadir, Tafnidilt; Bailly-Choumara 1965c , EM , Aïn Aït delouine, SA , Tirhmert; Bailly-Choumara 1970 , Rif , Tétouan; Knight 1971 , AP , Kénitra; El Kaim 1972 , AP , Bou-Regreg; Bailly-Choumara 1973b , Rif , Merja de l'Oued Smir; Rioux et al. 1975 , AP , Rabat; Pasteur et al. 1978 , AP , Bou-Regreg; Metge 1986 , AP , Littoral casablancais; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba, Merja Zerga, Oued Loukous; Louah 1995 , Rif , Tres piedras, Cabo Negro, Lajour, Azla, Tahaddart; Himmi et al. 1995 ; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Rabat; Ramdani 1997 , AP , Tamaris Merja; Handaq 1998 , AP , Essaouira, Zima-Chemaîa; Himmi et al. 1998 , AP , Sidi Boughaba; Himmi 2007 , AP , Sidi Boughaba; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) pulchritarsis (Rondani, 1872) Gaud 1953a , AP , Benslimane, Sidi Yahia, Rabat, MA , Oued Zem, Khénifra, Fès; Metge and El Alaoui 1987 , AP , Benslimane; Himmi et al. 1995 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Rusticoidus ) rusticus (Rossi, 1790) Viamonte and Ramirez 1946 , Rif , Tétouan, AP , Larache; Gaud 1953a , MA , Taza; Himmi et al. 1995 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Stegomyia ) aegypti (Linnaeus in Hasselquist, 1762) d'Anfreville 1916 , AP , Salé; Vialatte 1923 , AP , Rabat, Casablanca, HA , Marrakech; Charrier 1924 , Rif , Tanger; Gaud 1953a , AP , Salé, HA , Marrakech; Himmi et al. 1995 ; Dakki 1997 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Stegomyia ) albopictus (Skuse, 1895) Bennouna et al. 2016 , AP , Agdal (Rabat); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Faraj et al. 2018 ; Amraoui et al. 2019 Culicini Culex Linnaeus, 1758 Culex ( Barraudius ) modestus Ficalbi, 1889 Séguy 1930a ; Bailly-Choumara 1968a , AP , Larache; Trari 1991 , AP , Gharb; Himmi et al. 1995 ; Dakki 1997 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, AP , Maâmora; Hadji et al. 2013 , AP , Sidi yahia du Gharb, Kcebia, Sidi Hagouch (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) brumpti Galliard, 1931 Bailly-Choumara 1968a , AP , Merja Bokka, Larache, HA , Marrakech; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi et al. 1995 ; Dakki 1997 ; Himmi 2007 ; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) laticintus Edwards, 1913 Charrier 1924 , Rif , Tanger; Callot 1940 , AA , Goulmima (mares); Gaud 1953a , HA , Marrakech, AA , Agadir; Gaud 1957a , EM , Nador; Bailly-Choumara 1965c , EM , Oued Isker, Aïn Aït delouine, Talmesdourt, AA , Ouled Teima, ounaamane, Bou Izakarn, Agunil Khnufa, Akka-guiren; Bailly-Choumara 1966 , AA , Akka-Iguiren, Tirherm, Taoujgelt, Aït Ouabelli, Anamere-Smougue, Aït melloul, Tiznit (Rocade de Draa); Bailly-Choumara 1967b , Rif , Béni Bouayache, Targuist et environs, EM , Grotte du Zegzel; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 , AP , Skhirat; Faraj et al. 2008b , AP , Louamra; Hadji et al. 2013 , AP , Sidi Hagouch (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) mimeticus Noè, 1899 Séguy 1930a ; Viamonte and Ramirez 1946 , Rif , Béni Ider, Fnideq, Khemis Anjra, Ketama, Oued Amsa, Oued Krikra, Oued Martil, Oued Laou; Gaud 1953a , Rif , Ouezzane, Ghafsai, EM , Berkane, Martinpray (près Berkane), El Aïoun Sidi Mellouk, AP , Tamri, MA , Meknès, Fès, Ifrane, Taza, Béni Mellal, HA , Midelt, Marrakech, Azilal, AA , Tinghir, Tichka; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , AA , Oued Noun, Anezi, Tafraoute; Bailly-Choumara 1966 , AA , Agadir; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Taoujgelt, Source Oumerrbia, Ghorm El Alem, piste Ksiba-Naour, piste Naour-Arbala, Zaouia Cheikh, Oued Sarif; Bailly-Choumara 1967b , EM , 7 km N d'Itzer, 8 km d'Itzer; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Taza-Asifane, piste Bab Berred-Tamorote, Ketama, piste Ketama-Jebel Tidighine, route Ketama-Targuist, route nationale Jebha, Al Hoceima, Béni Bouayache, Marchica, Targuist et environs, Jebha, Boured; Trari 1991 , AP , Sidi Yahia du Gharb; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, Tres piedras, Marina Smir, Bouzaghlal, M'diq, Oued Maleh, Azla, Tahaddart, Moulay Bouchta, Schroda, Kebbache, Talambote, Oued Maggou; Louah et al. 1995 ; Chlaida 1997 , AP , Oued Sidi Messoud, Aïn Blal, Douar Chlihat, Barrage Al Massira; Chlaida and Bouzidi 1995 , AP , Barrage El Massira; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris Merja; Handaq 1998 , HA , Oukaimeden, Amizmiz, Tiguenziouine (près Oued N'fis); Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, AP , Bouknadel, Douar jdid (Skhirat); El Ouali Lalami et al. 2010a , MA , Fès; Larhbali et al. 2010 , MA , Oulmès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) perexiguus Theobald, 1903 Séguy 1930a ; Callot 1940 , AA , Assa; Senevet and Andarelli 1959a, EM , Oujda, Taourirt, AP , Aïn el Aouda, Arbaoua, Had Kourt, Oued Beht, Rabat, Allal Tazi, Oued Sahli, Zaouia Ech cheikh, Taghzirt, MA , Béni Mellal, Foum Zabel, Ifrane, Meknès, Aït Atta du Rteb, Fès, Sidi Mokhfi, Tahala, HA , Tazert, Midelt, AA , akka, Tamri; Bailly-Choumara 1965c , EM , Assa; Bailly-Choumara 1966 , AA , Souk El Khémis Dades, Aït Ouabelli, Tarhjicht, Tirherm, Taoujgelt, Aït melloul; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Ajdir; Bailly-Choumara 1967b , EM , Madagh, Merja Boubker, Aïn Béni Mathar; Bailly-Choumara 1967c , Rif , piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Béni Bouayache, Targusit et environs; Bailly-Choumara 1972a , AP , Merja Sheishat; Louah 1995 , Rif , Riffien, Tres piedras, Marina Smir, Bouzeghlal, Oued Maleh, Tahaddart, Talembote, Schroda, Tanger; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, El Oulja, Fouarate; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , HA , Marrakech, entre Oued N'fis et Chichaoua, Kelaâ Sraghna; Alaoui Slimani 2002 , AP , Rabat, Salé; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Bab Taza; Faraj et al. 2008c, AP , Larache Louamra; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Culex ) pipiens Linnaeus, 1758 d'Anfreville 1916 , AP , Salé; Charrier 1924 , Rif , Tanger; Séguy 1930a ; Callot 1940 , SA , Goulimine; Viamonte and Ramirez 1946 , Rif , Boudinar, Dar Benkarrich, Tétouan, Tanger, Asilah, Ksar El Kébir, Chefchaouen, Ketama, Nador; Gaud 1952 , AP , Gharb; Séguy 1953a , SA , Tindouf; Guy 1958 , HA , Oued N'fis; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , EM , Aouinet Aït Oussa, Aïn Isker, Aïn Aït Delouine, Oued Mesdourt, Talmesdourt, Toudi, AA , Aït Onmar, Oulad Teima, Taroudant, Tiznit ville, Talaint, Hassi Tafnidilt, Zaouiat Cheikh, Aïn Guerzim, Tafraout ville, SA , Goulimine ville, Vallée de l'Oued Assaka, Ouaroun, Zriouila, Labyar, Tighmert, Abeino, Tantan ville, Zag; Bailly-Choumara 1966 , AA , piste d'Akka au Draa, Akka et environs, Aït Ouabelli, Anamere-Smougue, Tarhjicht, Aït Melloul, Tiznit et environs; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, piste Itzer-Boumia, Ghorm El Alem, Ghorm El Alem-El Ksiba, El Ksiba, piste Ksiba-Naour, Zaouia Cheikh, Oued Sarif, Dayet Aoua, Pont Tarmilate, Ifrane, Imilchil; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Gaada de Debdou, Guercif ville, Cascade Oued Za, Grotte du Zegzel, Environs Saidia, Douar Aïn Shebbak, Douar Aïn Zabia, Douar Mardarh, Douar Sidi Hashas, Saidia, Merja Boubker, Berguent, Tendrara, Figuig; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Al Hoceima, Al Hoceima Club Med, Béni Bouayache, Marchica, Targuist et environs, Jebha, Ghafsai; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , AP , Merja Bokka, Merja Qodiya; Metge and Belakoul 1989 , AP , Benslimane, Sidi Bettache; Himmi 1991 , AP , Sidi Boughaba, Sidi Amira; Trari 1991 , AP , Maâmora, El Menzeh, Sidi Boughaba, Chkaïfien, Sidi Yahia du Gharb, Bokka, Merja Zerga, Sidi Allal Tazi, Oued Loukous, Aïn Chouk, Merja Bargha, Merja Oulad Skhar; Bouallam and Ramdani 1992 , HA , Marrakech; El Bermaki 1993, AP , Sidi Maârouf; Himmi et al. 1995 ; Louah 1995 , Rif , Fnideq, Riffien, Tres piedras, Marina Smir, M'diq, Cabo Negro, Zekri, Azla, Tahaddart, Schroda, Kebbache, Ouadras, Punta cirres, Sidi Kankoch, Oued Kbir; Louah et al. 1995 ; Bouallam et al. 1997b , HA , Marrakech; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Himmi et al. 1998 , AP , Sidi Boughaba, Puits de Sidi Amira (forest of Maâmora); Moussalim 1997 , AP , El Oulja, Maâmora, Fouarate, Sidi Boughaba; Ramdani 1997 , AP , meseta côtière (Témara-Casablanca); Faraj et al. 1997 , AP , Kénitra; Handaq 1998 , AP , Zemamra, MA , Oued Zem, HA , Marrakech, Zaouiet Ben Sassi, Sidi Bou Othmane, Kelaâ Sraghna, Bengrir; Bouallam 2001, HA , Marrakech; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Aouinty et al. 2006 , AP , Mohammedia; Faraj et al. 2006 , AP , Salé; Himmi 2007 , Rif , Bab Berred, Bab Taza, Stehat, AP , Skhirate, forest of Maâmora, forest of Hilton, Sidi Boughaba, El Oulja, Ouled Salem, Ouled dlim, Douar Ould Yahia Ben Ali, Larouaza, Douar Elarja, Douar Jdid, Douar Jnaja; Faraj et al. 2008b , AP , Louamra; El Ouali Lalami et al. 2010a , MA , Fès; Larhbali et al. 2010 , MA , Oulmès, Tarmilate, Bni Ounzar, Ganzra;Louali Lalami et al. 2010b, MA , Oued El Himmer; Amraoui 2012 , HA , Marrakech; Amraoui et al. 2012 , Rif , Tanger, AP , Mohammedia, Casablanca, HA , Marrakech; Amraoui et al. 2012 , AP , Mohammedia, Casablanca; El Ouali lalami 2012 , MA , route de Sidi Harazem; Hadji et al. 2013 , AP , Sidi Slimane; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Marc et al. 2016 , AP , Kénitra; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Bkhache et al. 2018 , Rif , Tanger, AP , Rabat, Mohammedia, HA , Marrakech; Tmimi et al. 2018 , AP , Mohammedia; Mouatassem et al. 2019, MA , Fès Culex ( Culex ) simpsoni Theobald, 1905 Callot 1940 , AA , Taghicht; Senevet et al. 1949 , AA , Oued Noun, Tafnidelt, Akka; Gaud 1953a , AA , Imsouane, Aït Melloul, Akka, O'Noun, Taghjicht, Assa, Tafnidilt; Bailly-Choumara 1965c , EM , Aman d'Aït Oussa, El Megrinat, AA , Oued Izi, Oued Massa-Pont de la route Agadir-Tiznit, Guelta Zerga, Tafnidilt, SA , Poste militaire de Boujrif, Tirhmert, Taourirt-Barrage, Aouinet Torkoz; Bailly-Choumara 1966 , AA , Tirherm, Taoujgelt, Taghjicht, Aït melloul, Tiznit; Chlaida and Bouzidi 1995 , AP , Sidi M'barek, Oued Sidi Messoud, Mechrâa, Sidi Boulâarais, Douar Chlihat (south of Settat); Chlaida 1997 , AP , Sidi M'barek, Mechrâa Settir, Sidi Boulâarais, Douar Chlihat (south of Settat); Dakki 1997 ; Handaq 1998 , HA , Sidi Bou Othmane (Marrakech); Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, MA , Khémisset, AA , sud Anti Atlas; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) theileri Theobald, 1903 Callot 1940 , AA , Taghjicht; Viamonte and Ramirez 1946 , Rif , Dar Benkarrich, Boudinar, Ketama, Tanger, Tétouan, Chefchaouen, AP , Larache; Guy 1958 , HA , Oued N'fis; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , EM , Aouinet Aït Oussa, Aïn Oumesdour, Aouinet Torkoz, SA , Tirh Mzoun; Bailly-Choumara 1966 , AA , Souk El Khémis Dades, Akka et environs, Aït Melloul, Tazenakhte, piste Tiznit-Tafraout, SA , Goulimine; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Aguelmane Azigza, piste Aguelmane Azigza-Aïn Leuh, maison forestière Ouiouane, Khénifra, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Col du Zad, piste Itzer-Boumia, Ghorm El Alem, piste Ghorm El Alem-El Ksiba, El Ksiba, piste Ksiba-Naour, Aguelmane Moulay Yakoub, Zaouia Cheich, Kafensour, Dayet Aoua, Pont Tarmilate; Bailly-Choumara 1967b , EM , route Itzer-Midelt, 40 km N Outat El Haj, Taourirt, Driouch, Figuig; Bailly-Choumara 1967c , Rif , piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, ArbaaTaourirt, Al Hoceima, Targuist et environs, Ghafsai; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , AP , Merja Sheishat, Merja Bokka; Himmi 1991 , AP , Sidi Boughaba, El menzeh, Sidi Amira; Trari 1991 , AP , Maâmora, Oued Sebou, Bordure Oued Loukous, Sidi Yahia du Gharb, Bokka, Entre Moulay Bousselham et Larache, Merja Zerga, Larache; El Bermaki 1993, AP , Sidi Maârouf, El Oulfa; Louah 1995 , Rif , Bouzeghlal, M'Diq, Oued Maleh, Zekri, Lajour, Tahaddart, Loubart, Chefchaouen, Tanger; Himmi et al. 1995 ; Louah et al. 1995 ; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Maâmora, Rabat, Kénitra; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , AP , Zemamra, MA , Béni Mellal, HA , Oukaimeden, Amizmiz, Tiguenziouine, Marrakech, Sidi Bou Othmane, Seguarta-Kelaâ, Had Mhara, Aîn Äounate, Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra), Gharb; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Tanakoub, AP , Skhirate, forest of Maâmora, Chiahna, Sidi Amira, Sidi Boughaba, Bouknadel, Sidi Azzouz, Ehssaïne, Bettana, Sidi Yahia, Sebbah, Larouaza, Douar Elarja, Douar Jdid, Douar Jnaja; Faraj et al. 2008b , AP , Larache Louamra; El Ouali Lalami et al. 2010a , AP , Boucharen; Larhbali et al. 2010 , MA , Roumani, Aïn Sbite, Ezzhiliga, Oulmès, Tarmilate, M'rirt, Bni Ounzar, Ganzra (Khémisset); Hadji et al. 2013 , AP , Sidi yahia du Gharb, Kcebia, Sidi Hagouch, Dar Belamri (Sidi Slimane); El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Maillotia ) deserticola Kirkpatrick, 1925 Gaud 1947c , AA , Tansikht; Gaud 1953a , EM , Figuig, Berguent, Anoual, Boudnib, Aïn Chair, Aoufous, Tarhit, HA , Tazenakht, Tichka, Tizi-n'Telghemt, AA , Ouarzazate, Zagora; Bailly-Choumara 1965c , EM , Oued Isker, El Megrinat, Taskala, Aïn Aït Delouine, Talmesdourt, Aouinet Torkoz, Bouanama, Rich Tamlougout, Assa, AA , Tafraout ville, Oued Jemâa Idaousmaal, Aït abdallah, Issedrim Igmur Igues, SA , Vallée de l'Oued Assaka, Tacharicht, Bou Izakarn, Aïn Erkha; Chlaida and Bouzidi 1995 , AP , Sud de Settat; Himmi et al. 1995 ; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Maillotia ) hortensis Ficalbi, 1889 Charrier 1924 , Rif , Tanger; Séguy 1930a ; Langeron 1938 , HA , Tounfite; Callot 1940 , HA , Anefgou (2500 m), Tirghist (2500 m), Tighermine (2500 m); Gaud 1953a , HA , Tizi-n'Telghemt, Tizi-n'Tichka; Bailly-Choumara 1967a , MA , piste tafechna-Znan Imes, Khénifra, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Ghorm El Alem, El Ksiba, piste Ksiba-Naour, Piste Naour-Arbala, piste Arbala-El Kbab, Zouia Cheikh, Oued Sarif, Ifrane, Boulmane, Imilchil; Bailly-Choumara 1967b , EM , Tafraout, Grotte du Zegzel; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Tamorote, route Bab Berred-Ketama, Ketama, piste Ketama-Mt Tiguidin, route nationale Jebha, route Targuist-Béni Boufrah, Trari 1991 , AP , Gharb; El Bermaki 1993, AP , Casablanca; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, Marina Smir, M'diq, Oued Maleh, Lajour, Tahaddart, Schroda, Talembote, Bab Berred, Punta Cirres, Sidi Kankoch, Oued kbir, Oued Jebel Lehbib; Louah et al. 1995 ; Chlaida and Bouzidi 1995 , AP , Barrage Al Massira; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , HA , Amizmiz, Sidi Bou Othmane, Had Mhara, Bengrir; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Bab Taza, AP , Skhirate; El Ouali Lalami et al. 2010b , MA , Oued Fès, Oued El Himmer, Camping Sidi Harazem; Larhbali et al. 2010 , MA , Merchouch, Ghoualem, Ezzhiliga, Oulmès, Tarmilate, M'rirt, Bni Ounzar, Ganzra (Khémisset); El Ouali Lalami 2012 , MA , Oued Fès; Oued El Himmer, route de Sidi Harazem, Camping Sidi Harazem; Hadji et al. 2013 , AP , Sidi Yahia du Gharb, Kcebia, Sidi Hagouch, Dar Belamri, Lalla Itto, Soualem (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Neoculex ) impudicus Ficalbi, 1890 Charrier 1924 , Rif , Tanger; Séguy 1930a ; Bailly-Choumara 1966 , AA , Taliouine; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Senoual-Itzer, Itzer, piste Naour-Arbala, Zaouia Cheikh, Pont Tarmilate; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Cascade Oued Za, Grotte du Zegzel; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Asifane, piste Bab Berred-Tamorote, Ketama, route nationale Jebha, Al Hoceima Club Med, Targuist et environs; Trari 1991 , AP , Sidi Amira, El Menzeh, Oued Sebou, Sidi Yahia du Gharb, Moulay Bousselham, Larache, Bordure Oued Loukous; Dakki 1997 ; Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra), Puits de Sidi Amira (forest of Maâmora); Trari et al. 2002 ; Himmi 1991 , AP , Sidi Boughaba, El Menzeh, Sidi Amira; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir, Tahaddart, Tanger; Louah et al. 1995 ; Ramdani 1997 , AP , Tamaris; Alaoui Slimani 2002 , AP , Rabat; Himmi 2007 , Rif , Bab Berred, Bab Taza, AP , forest Maâmora; El Ouali Lalami et al. 2010a , MA , Fès; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Neoculex ) martinii Medschid, 1930 Bailly-Choumara 1968a , Rif , Al Hoceima, AP , Sidi Yahia du Gharb, Sidi Allal Tazi, Larache, Rabat, HA , Marrakech, AA , Tiznit; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir, Cabo Negro, Oued Maleh; Louah et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culisetini Culiseta Felt, 1904 Culiseta ( Allotheobaldia ) longiareolata (Macquart, 1838) d'Anfreville 1916 , AP , Salé; Séguy 1930a ; Gaud 1947c , AA , Tansikht; Gaud 1953a , HA , Marrakech; Bailly-Choumara 1965c , EM , El Aïoun du Draa, Aïn Aït Delouine, Talmesdourt, Aouinet Torkoz, Rich Tamlougout, AA , Aït Onmar, Ouled Teima, Tiznit ville, Bounaamane, Id Baha, Tafnidilt, Guelta Zerga, Aïn Kerma, Tafraout ville, Oued Jemâa Idaousmaal, Toudi, Aït Abdallah, Igherm, Issedrim Igmur Igues, SA , Goulimine ville, Poste militaire de Boujrif, Embouchure de l'Oued Assaka, Ouaroun, Labyar, Asrir, Tacharicht, Bou Izakarn ville, Jemâa N'Tirhirte, Aït Erkha, Tantan ville; Bailly-Choumara 1966 , AA , Aït melloul, Tiznit; Bailly-Choumara 1967a , MA , Jnane Imasse, Khénifra, piste Tafechna-Senoual-Itzer, Itzer, Ghorm El Alem, El ksiba, piste Naour-Arbala, Ifrane, Pont Tarmilate, Imouzzer Marmoucha; Bailly-Choumara 1967b , EM , Tafraout, Guercif ville, Grotte du Zegzel, Berguent, Tendrara; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, route Bab Taza-Bab Berred, route Ketama-Targuist, route nationale Jebha, Targuist et environs, Aïn Hamra; Bailly-Choumara 1968a , AP , Meja Bokka, AA , Tiznit; Himmi 1991 , AP , Sidi Boughaba, El Menzeh; Trari 1991 , AP , El Menzeh, Gharb; El Bermaki 1993, AP , Sidi Maârouf; Himmi et al. 1995 ; Louah 1995 , Rif , Fnideq, Riffien, Marina Smir, M'diq, Cabo Negro, Lajour, Ouadras, Punta Cirres, Ksar Sghir, Tanger; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Maâmora, Fouarate; Ramdani 1997 , AP , Témara, Skhirat, Tamaris; Dakki 1997 ; Handaq 1998 , HA , Marrakech, Kelaâ Sraghna, Bengrir Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra); Bouallam 2001, HA , Bordure Oued N'fis; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Aouinty et al. 2006 , AP , Mohammedia; Himmi 2007 , Rif , Bab Berred, AP , Skhirate, Maâmora, forest of Hilton; Koçak and Kemal 2013; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culiseta ( Culicella ) fumipennis (Stephens, 1825) Gaud 1947c , AP , Rabat, Sidi Allal Tazi, MA , Khémisset; Senevet and Andarelli 1959, AP , Rabat, Casablanca, Bouznika, MA , Fès; Bailly-Choumara 1967a , MA , Jnane Imasse; Bailly-Choumara 1967c , Rif , route Bab Taza-Bab Berred; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culicella ) litorea (Shute, 1928) Metge 1986 , AP , Casablanca; Dakki 1997 ; Carles-Tolrá 2002 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culiseta ) annulata (Schrank, 1776) d'Anfreville 1916 , AP , Salé; Charrier 1924 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Viamonte and Ramirez 1946 , Rif , Ben Karrich, Asilah, Ketama, Malaliene, Tétouan, AP , Larache; Gaud 1952 , AP , Souk El Hadd (Gharb); Gaud 1953a , Rif , Tanger, Tétouan, EM , Saidia, Talsint, AP , Casablanca, Rabat, Kénitra, MA , Meknès, Fès, Ifrane, Taza, HA , Marrakech, Aït Bouguemez, Midelt; Gaud 1957b , Rif , Tétouan, Tanger, AP , Sidi Allal Tazi, Rabat, Casablanca, MA , Meknès, Ifrane; Bailly-Choumara 1967a , MA , Jnane Imasse, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, Ghorm El Alem, Zaouia Cheikh, Pont Tarmilate, Ifrane; Bailly-Choumara 1967b , EM , Tafraout, route Itzer-Midelt; Bailly-Choumara 1967c , Rif , piste Bab Taza-Talassemtane; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, M'diq, Tanger; Louah et al. 1995 ; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Rabat; Ramdani 1997 , AP , meseta côtière (Casablanca-Rabat); Himmi et al. 1998 , AP , Sidi Boughaba; Himmi 1991 , AP , Sidi Boughaba, El Menzeh; Trari 1991 , AP , Sidi Boughaba, El Menzeh; Trari et al. 2002 ; Himmi 2007 , AP , Skhirate, Maâmora, forest of Hilton, Oulja; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culiseta ) subochrea (Edwards, 1921) Gaud 1952 , AP , Gharb; Gaud 1957b , Rif , Tanger, Chefchaouen, EM , Oujda, AP , Sidi Allal Tazi, Casablanca; Bailly-Choumara 1967a , MA , Imilchil; Bailly-Choumara 1967c , Rif , Ketama; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba; Louah 1995 , Rif , Riffien, Tres piedras, M'diq, Cabo Negro, Oued Maleh, Tanger; Louah et al. 1995 ; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Kénitra; Ramdani 1997 , AP , Témara, Skhirat, Casablanca; Handaq 1998 , AP , El Jadida, HA , Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Skhirat, Dayet Aïn Chems, Maâmora, forest of Hilton; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Mansoniini Coquillettidia Dyar, 1905 Coquillettidia ( Coquillettidia ) buxtoni (Edwards, 1923) Bailly-Choumara 1965a , AP , Merja Bokka; Bailly-Choumara 1970 , AP , Merja Bokka; Bailly-Choumara 1972a , AP , Merja Sheishat (Larache); Bailly-Choumara 1973b , AP , Merja Bokka, Larache; Himmi et al. 1995 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Coquillettidia ( Coquillettidia ) richiardii (Ficalbi, 1889) Bailly-Choumara 1965a , AP , Merja Bokka; Bailly-Choumara 1967a , MA , Zaouia Cheikh; Bailly-Choumara 1970 , AP , Merja Bokka; Bailly-Choumara 1972a , AP , Merja Sheishat (Larache); Bailly-Choumara 1973b , AP , Merja Bokka; Trari 1991 , AP , Sidi Yahia du Gharb, Aïn Chouk, Merja Bargha; Himmi et al. 1995 ; Dakki 1997 ; Moussalim 1997 , AP , Fouarate sur les bordures de la Merja; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Orthopodomyiini Orthopodomyia Theobald, 1904 Orthopodomyia pulcripalpis (Rondani, 1872) Bailly-Choumara 1965b , AP , Maâmora; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Uranotaeniini Uranotaenia Lynch Arribálzaga, 1891 Uranotaenia ( Pseudoficalbia ) unguiculata Edwards, 1913 Séguy 1930a ; Gaud 1953a , HA , Marrakech; Senevet and Andarelli 1959a, EM , Oujda, Berguent, Guercif, AP , Sidi Allal Tazi, Bouznika, Casablanca, Béni Moussa, MA , Oulad Massine, Meknès, Moulay Yacoub, Béni Mellal, AA , Ksar Mzizel, Aït Melloul; Bailly-Choumara 1965c , EM , Aïn Aït Delouine, Talmesdourt, Rich Tamlougout; Bailly-Choumara 1966 , AA , Aït Ouabelli; Bailly-Choumara 1967a , MA , Pont Tarmilate; Bailly-Choumara 1967b , EM , environs de Taourirt, Madagh, Merja Boubker; Bailly-Choumara 1967c , Rif , route Bab Taza-Bab Berred, piste Ketama-Jebel Tidighine; Bailly-Choumara 1968a , AP , Oued Loukous, Merja Bokka, Kénitra, Oued Bou-Regreg; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Oued Sebou; El Bermaki 1993, AP , El Oulfa (Casablanca); Himmi et al. 1995 ; Dakki 1997 ; Ramdani 1997 , AP , Tamaris; Handaq 1998 , AP , Sidi Bennour, HA , Zima Chemaîa (Bengrir); Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Uranotaenia ( Uranotaenia ) balfouri Theobald, 1904 Bailly-Choumara 1968a , AP , Merja Bokka, Oued Loukous, Sidi Yahia, Oued Sebou (Kénitra), Oued Bou-Regreg; Himmi et al. 1995 ; Dakki 1997 ; Moussalim 1997 , AP , Sidi Allal Tazi, Fouarate, Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; El Ouali Lalami et al. 2010a , MA , Fès Boulmane; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès DIXIDAE K. Kettani, R. Wagner Number of species: 12 . Expected: 15 Faunistic knowledge of the family in Morocco: moderate Dixa Meigen, 1818 Dixa caudatula Séguy, 1928 Séguy 1930a , HA , Arround, Skoutana (2400 m); Vaillant 1959 ; Dakki 1997 ; Mouna 1998 Dixa dilatata Strobl, 1900 = Dixa riparia Vaillant, in Vaillant 1959 : 180 Vaillant 1959 , HA , Source de M'Goum (2500 m), Gorges d'Imi-N'Ifri (1050 m); Vaillant 1965 ; Dakki 1997 Dixa maculata Meigen, 1818 Séguy 1930a ; Dakki 1997 ; Mouna 1998 Dixa mera Séguy, 1930 Séguy 1930a , MA , forest of Timelilt (1900 m); Vaillant 1959 ; Dakki 1997 ; Mouna 1998 Dixa nebulosa Meigen, 1830 Séguy 1930a , HA ; Dakki 1997 ; Mouna 1998 Dixa perexilis Séguy, 1928 Séguy 1930a , HA , riverside of Oued Imminen (Tachdirt, 2400 m); Vaillant 1959 ; Dakki 1997 ; Mouna 1998 Dixa puberula Loew, 1849 Vaillant 1959 , HA , headwaters of Asif M'Goum (2500 m); Vaillant 1965 ; Dakki 1997 ; Mouna 1998 Dixa submaculata Edwards, 1920 Séguy 1930a , MA , Sidi Yahia, Talzent (1800 m); Dakki 1997 ; Mouna 1998 Dixella Dyar & Shannon, 1924 Dixella aestivalis (Meigen, 1818) Séguy 1930a , AP , Merja Boughaba; Vaillant 1965 ; Ramdani 1981 ; Ramdani and Tourenq 1982 ; Mouna 1998 Dixella attica (Pandazis, 1933) = Dixella numidica (Sicart, 1955) Ebejer et al. 2019 , Rif , Cabo Negro (indoors: 10 m) Dixella martinii (Peus, 1934) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m) Dixella serotina (Meigen, 1818) = Dixa serotina Wied, in Mouna 1998 : 85 Séguy 1930a , AP , Casablanca (between Kénitra and Oued Beth); Dakki 1997 ; Mouna 1998 Chironomoidea CERATOPOGONIDAE K. Kettani, B. Mathieu Number of species: 62 . Expected: 80 Faunistic knowledge of the family in Morocco: moderate Ceratopogoninae Culicoidini Culicoides Latreille, 1809 Culicoides ( Avaritia ) imicola Kieffer, 1913 Kremer et al. 1971 , MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Fès, Rhafsai, AA , Torkoz, Tarhjisht; Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, EM , Oriental, AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Draa-Tafilalet, Souss-Massa, SA , Guelmim-Oued Noun; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Aïn Leuh, Ait Siberne, Meknès, AA , Errachidia, Sidi Dahmane, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) montanus Shakirzjanova, 1962 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Safi, HA , Marrakech; Chaker et al. 1980 , Rif , Al Hoceima, AP , Oued Cherrat, HA , Souk Tnine de Oudaias (Haouz), Marrakech, AA , Torkoz; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) obsoletus (Meigen, 1818) Callot et al. 1968 , Rif , Al Hoceima, AP , Merja Bokka, Sidi Yahia du Gharb, Sidi-Bettache (Zaeir), Rabat-Salé-Kénitra, HA , El Harcha (plateau central); Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Béni Mellal-Khénifra, HA , Marrakech; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, HA , Marrakech; Chaker et al. 1980 , Rif , Al Hoceima, AP , Zaers, Sidi Bettache, HA , El Harcha, Talet Inaouane (Haouz); Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Souss-Massa, SA , Guelmim-Oued Noun; Cêtre-Sossah and Baldet 2004 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Ait Siberne; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) scoticus Downes & Kettle, 1952 Kremer et al. 1971 , AP , Safi, MA , Béni Mellal-Khénifra, HA , El Harcha, Talet Inaouane (Haouz), Marrakech; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat, Safi, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Beltranmyia ) circumscriptus Kieffer, 1918 Callot et al. 1968 ; Bailly-Choumara and Kremer 1970 , Rif , Smir lagoon, Oued Negro, EM , Merja Boubker (Berkane), Gouttitir (NE Guercif), AP , Merja Sheishat (Larache), Aïn Muelha (near Oued Sidi Allal Tazi, estuaire Oued Bou-Regreg, Dayat Qoudiya (Sidi Yahia Gharb); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, AA , Souss Massa; Chaker et al. 1980 , Rif , Al Hoceima, AP , Merja Qoudiya, Merja Bokka, Sidi Yahia du Gharb, MA , Aïn Karma (Saiss), Oulmès, HA , Setti Fatma, AA , Aït Melloul (Souss); Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Meknès, AA , Errachidia, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) fagineus Edwards, in Edwards et al. 1939 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Béni Mellal-Khénifra, HA , Marrakech; Kremer et al. 1975 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Kremer et al. 1979 ; Chaker et al. 1980 , AP , Rabat, Sidi Bettache, MA , Khemisset, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) newsteadi Austen, 1921 = Culicoides ( Culicoides ) halophilus Kieffer, in Callot et al. 1968 : 886, Bailly-Choumara and Kremer 1970 : 386, Dakki 1997 : 60 Callot et al. 1968 , Rif , Cabo Negro (Ferma), Tétouan, Talerhza, Tanger-Tétouan-Al Hoceima, AP , Larache, Merja Bokka (Gharb), Aïn Chok, HA , Talet-Inaouan (Haouz); Bailly-Choumara and Kremer 1970 , Rif , Smir lagoon, Oued Negro, AP , Merja Sheishat (Larache), Aïn Muelha (near Oued Sidi Allal Tazi, estuaire Oued Bou-Regreg, Dayat Qoudiya (Sidi Yahia du Gharb), EM , Merja Boubker (Berkane), Ksabi (NE Midelt), HA , Souk Tnine des Oudaias (bordure Oued N'fis), AA , Aïn Sefra (south Foum Zquid); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Casablanca, Settat, MA , Fès-Meknès, Béni Mellal-Khénifra, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, Casablanca, Settat, MA , Béni Mellal- Khénifra, HA , Marrakech; Kremer et al. 1979 ; Baylis et al. 1997 , Rif , Tanger, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , MA , Aïn Leuh, Ait Siberne, Meknès, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) pulicaris (Linnaeus, 1758) Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, Béni Mellal-Khénifra, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Lalla Outka, Khénifra, Oulmès, HA , Talet Inaouan (Haouz), AA , Aouinet-Torkoz, Tarhjicht; Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, EM , Oriental, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Drâa-Tafilalet, Souss-Massa; Cêtre-Sossah and Baldet 2004 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Aïn Leuh, Aït Siberne, Meknès; Bourquia et al. 2019 Culicoides ( Culicoides ) punctatus (Meigen, 1804) Callot et al. 1968 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1970 , Rif , Merja Smir, Oued Negro, AP , Merja Sheishat (Larache), estuaire Bou-Regreg, EM , Merja Boubker (Berkane); Kremer et al. 1971 , Rif , Cabo Negro (Ferma), Tétouan, EM , Berkane, AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, AA , Foum Zguid; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat; Dakki 1997 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Aïn Leuh, Meknès; Bourquia et al. 2019 Culicoides ( Culicoides ) subfagineus Delécolle & Ortega, 1998 Bourquia et al. 2019 , AP , Rabat Culicoides ( Monoculicoides ) parroti Kieffer, 1922 Bailly-Choumara and Kremer 1970 , HA , Dar Saâda (Haouz); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Safi, HA , Marrakech; Chakeret al. 1980 , AP , Rabat, HA , Marrakech, Souk Tnine des Oudaias (Haouz); Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Monoculicoides ) puncticollis (Becker, 1903) Callot et al. 1968 , AP , Merja Qoudiya, Sidi Yahia du Gharb, Romani (Zaers), Rabat-Salé-Kénitra, MA , Aïn Karma (Saiss), HA , Souk Tnine des Oudaias (Haouz); Bailly-Choumara and Kremer 1970 (reported as C. riethi and corrected by Kremer et al. 1971 ), Rif , Merja Smir, AP , Merja Sheishat (Larache), estuaire Bou-Regreg, Dayat Qoudiya (Sidi Yahia du Gharb), HA , Souk Tnine des Oudaias (bordure de l'Oued N'fis), Dar Saâda (Haouz), Talet Inouane (bordure marécageuse du lac du Barrage Lalla Taguergoust); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, HA , Marrakech; Chaker et al. 1980 , AP , Aïn Karma, Zaers, Roumani, HA , Souk-Tnine des Oudaias; Remm 1988a ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) azerbajdzhanicus Dhzafarov, 1962 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Safi, MA , Fès-Meknès, Beni Mellal-Khénifra, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 , AA , Souss-Massa SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Kkénifra, Rhafsai, HA , Marrakech, AA , Torkoz, Tarhjisht; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) longipennis Khalaf, 1957 Kremer et al. 1971 , EM , Berkane AP , Safi, MA , Fès-Meknès, HA , Marrakech; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) marcleti Callot, Kremer & Basset, 1968 Kremer et al. 1971 , MA , Rhafsai, Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) pallidus Khalaf, 1957 = Culicoides stackelbergi Dhzafarov, in Kremer et al. 1971 : 662, Dakki 1997 : 61 Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1971 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) ravus De Meillon, 1936 = Culicoides ( Synhelea ) subravus Cornet and Château, in Kremer et al. 1971 : 664, Chaker et al. 1980 : 85, Dakki 1997 : 61 Kremer et al. 1971 , AP , Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AA , Souss-Massa, SA , Guelmim-Oued Noun; Chaker et al. 1980 , HA , Marrakech, AA , Torkoz, Tarhjicht, Aït Ouaballi (Draa); Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) sahariensis Kieffer, 1923 = Culicoides colluzzii Callot, Kremer and Bailly-Choumara, in Bailly-Choumara and Kremer 1970 : 386, Chaker et al. 1980 : 83, Dakki 1997 : 61 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), EM , Merja Boubker (Berkane), HA , Souk Tnine des Oudaias (bordure Oued N'fis); Callot et al. 1970 , AP , Larache, HA , Marrakech; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Merja Bokka (Gharb), Rabat (Zaers), EM , Berkane, MA , Fès, Khémisset, AA , Tarhjisht; Baylis et al. 1997 ; Dakki 1997 ; Bouayoune et al. 1998; Cêtre-Sossah and Baldet 2004 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) santonicus Callot, Kremer, Rault & Bach, 1966 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Bailly-Choumara et al. 1980, AP , Larache, Sidi Bettache, MA , Oulmès, EM , El-Harcha; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) semimaculatus Clastrier, 1958 Kremer et al. 1975 , Rif , Tanger-Tétouan-Al Hoceima, AP , Larache, Casablanca-Settat, MA , Plateau Central (Khatouate); Chaker et al. 1979 ; Remm 1988a ; Dakki 1997 ; Szadziewski and Dominiak 2006 ; Bourquia et al. 2019 Culicoides ( Oecacta ) sergenti Kieffer, 1921 = Culicoides ( Oecacta ) mosulensis Khalaf, in Chaker et al. 1980 : 84, Dakki 1997 : 60 Kremer et al. 1979 , EM , Oriental, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AA , Tarhjicht, SA , Bou-Arfa; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) similis Carter, Ingram & Macfie, 1920 Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) truncorum Edwards, 1939 = Culicoides ( Oecacta ) sylvarum Callot and Kremer, in Kremer et al. 1971 : 662, Remm 1988a : 65, Dakki 1997 : 61 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Béni Mellal- Khénifra; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Pontoculicoides ) saevus Kieffer, 1922 Callot et al. 1968 , AP , Sidi Yahia du Gharb, Rabat-Salé-Kénitra, Safi, MA , Aïn Karma (Saiss), HA , Talet Inouane, Marrakech, Souk Tnine des Oudaias (Haouz), AA , Aït Melloul (Souss), Ksar er Souk (Tafilalt), Tarhjicht, SA , Bou-Arfa; Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), HA , Souk Tnine des Oudaias (bordure de l'Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AP , Safi, MA , Fès-Meknès, HA , Marrakech, AA , Drâa-Tafilalet, Souss-Massa, SA , Guelmim-Oued Noun; Bailly-Choumara and Kremer 1980; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Pontoculicoides ) sejfadinei Dzhafarov, 1958 Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AA , Drâa-Tafilalet; Bourquia et al. 2019 Culicoides ( Remmia ) kingi Austen, 1912 Bailly-Choumara and Kremer 1970 , AA , Mrimima (Oued de Foum Zquid), Souss-Massa; Chaker et al. 1980 , MA , Meknès, AA , Tarhjisht; Cornet and Brunhes 1994 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Remmia ) schultzei (Enderlein, 1908) Callot et al. 1968 , AP , Safi, HA , Talet Inouane, Marrakech; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) badooshensis Khalaf, 1961 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), HA , Marrakech, Souk Tnine des Oudaias; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech; Dakki 1997 ; Chaker et al. 1980 , AP , Oued Cherrat, Bousselham, Sidi Yahia, MA , Fès, HA , Marrakech; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) cataneii Clastrier, 1957 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra; Chaker et al. 1980 , AP , Oued Cherrat, Rabat; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) clastrieri Callot, Kremer & Deduit, 1962 Bourquia et al. 2019 , MA , Fès-Meknès Culicoides ( Sensiculicoides ) derisor Callot & Kremer, 1965 Chaker et al. 1980 , AP , Rabat; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès; Kremer et al. 1975 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Kremer et al. 1979 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) duddingstoni Kettle & Lawson, 1955 Bourquia et al. 2019 , MA , Fès-Meknès Culicoides ( Sensiculicoides ) dzhafarovi Remm, 1967 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Oued Cherrat, AA , Tarhjisht; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) festivipennis Kieffer, 1914 = Culicoides ( Oecacta ) odibilis Austen, in Bailly-Choumara and Kremer 1970 : 387, Chaker et al. 1980 : 84, Dakki 1997 : 61 Callot et al. 1968 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1970 , Rif , Merja Smir, HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, Casablanca-Settat, HA , Marrakech; Chaker et al. 1980 , Rif , Tétouan, EM , El-Harcha, AP , Aïn Chok, Rabat, Larache, MA , Oulmès, HA , Marrakech, Talet-Inaouan (Haouz); Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) heteroclitus Kremer and Callot, in Callot & Kremer, 1965 Kremer et al. 1975 , AP , Safi, HA , Marrakech, AA , Tafraout, Tiznit, Souss-Massa; Chaker et al. 1979 ; HA , Haouz; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) jumineri Callot & Kremer, 1969 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, AA , Souss-Massa, SA , Guelmim-Oued Noun; Chaket et al. 1980, AP , Oued Cherrat, Merja Bokka, Rabat, MA , Fès, HA , Talet-Inaouan (Haouz), AA , Torkoz, Tarhjisht, Aït Oubelli, SA , Bou-Arfa; Remm 1988a ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) kibunensis Tokunaga, 1937 = Culicoides ( Oecata ) cubitalis Edwards, in Kremer et al. 1975 : 205, Dakki 1997 : 60 Kremer et al. 1975 , AP , Safi, MA , Ifrane, Imouzzer-du-Kander, Fès-Meknès, HA , Haouz, Marrakech; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) kurensis Dzhafarov in Gutsevich, 1960 Remm 1988a ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) landauae Kremer, Rebholtz-Hirtzel & Bailly-Choumara, 1975 Kremer et al. 1975 , MA , Imouzzer-du-Kander, Sefrou, Fès-Meknès; Chaker et al. 1979 ; Hervy et al. 1994 ; Borkent and Wirth 1997 ; Remm 1988a ; Dakki 1997 ; Chilasse and Dakki 2004, MA ; Borkent 2012 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) langeroni Kieffer, 1921 Bailly-Choumara and Kremer 1970 , HA , Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Bailly-Choumara and Kremer 1980, MA , Khénifra, AA , Tarhjicht, Torkoz (Draa); Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) maritimus Kieffer, 1924 Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Bailly-Choumara and Kremer 1980, Rif , Tétouan, AP , Larache, Rabat, Sidi Bettache, HA , Talet-Inaouan (Haouz), Souk Tnine des Oudaias (Haouz); Remm 1988; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) odiatus Austen, 1921 = Culicoides lailae Khalaf, in Bailly-Choumara and Kremer 1970 : 387, Dakki 1997 : 60 = Culicoides indistinctus Khalaf, in Kremer et al. 1975 : 206, Dakki 1997 : 60 Bailly-Choumara and Kremer 1970 , HA , Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1975 , AA , Tafraout, Souss-Massa; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Kénitra, Merja Bokka (Gharb), HA , Souk Tnine des Oudaias (Haouz), AA , Tarhjicht; Remm 1988a ; Dakki 1997 ; Baylis et al. 1997 ; Bouayoune et al. 1998; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Sarvašová et al. 2014 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) paolae Boorman, 1996 Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) pictipennis (Staeger, 1839) Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 ; Remm 1988a ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) pseudopallidus Khalaf, 1961 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Safi, MA , Rhafsai, Fès-Meknès, HA , Marrakech; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) shaklawensis Khalaf, 1957 Kremer et al. 1975 , AP , Safi, MA , Sefrou, Fès-Meknès, HA , Setti Fatma, Marrakech; Chaker et al. 1979 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) simulator Edwards, 1939 Kremer et al. 1975 , MA , Ifrane, Fès-Meknès, HA , Setti Fatma; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) univittatus Vimmer, 1932 = Culicoides agathensis Callot, Kremer and Rioux, in Bailly-Choumara and Kremer 1970 : 386, Kremer et al. 1971 : 663, Chaker et al. 1980 : 82, Dakki 1997 : 60 Bailly-Choumara and Kremer 1970 , AP , estuaire Bou-Regreg; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Rif , Tanger-Tétouan-Al Hoceima; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, HA , Marrakech; Chaker et al. 1980 , Rif , Tétouan, AP , Larache, Sidi Bettache MA , Oulmès; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) vidourlensis Callot, Kremer, Molet & Bach, 1968 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), estuaire de Oued Bou-Regreg, HA , Souk Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 ; Remm 1988a ; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) pallidicornis Kieffer, 1919 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Dakki 1997 ; Balenghien et al. 2014 , SA , Guelmim-Oued Noun; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) picturatus Kremer & Deduit, 1961 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache); Kremer et al. 1971 , Rif , Talerhza, EM , El-Harcha, AP , Bousselham, Rabat-Salé-Kénitra, MA , Oulmès; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat, MA , Béni Mellal-Khénifra; Remm 1988a ; Dakki 1997 ; Sarvašová et al. 2014 ; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) subfasciipennis Kieffer, 1919 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, Béni Mellal-Khénifra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1980, AP , Larache, Zaers, Rabat, Aïn Chok, MA , Sefrou; Remm 1988a ; Hervy et al. 1994 ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Wirthomyia ) faghihi Navai, 1971 Kremer et al. 1975 , AA , Tafraout, Souss-Massa; Chaker et al. 1979 ; Remm 1988a ; Hervy et al. 1994 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Wirthomyia ) minutissimus (Zetterstedt, 1855) Referred as C. pumilus Culicoides ( Wirthomyia ) pumilus (Winnertz, 1852) Kremer et al. 1975 , AP , Safi, MA , Ifrane, Imouzzer-du-Kander, Fès-Meknès, HA , Setti Fatma, Marrakech; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides calloti Kremer, Delécolle, Bailly-Choumara & Chaker, 1979 Chaker et al. 1979 , AA , Souss Massa, SA , Guelmim-Oued Noun; Kremer et al. 1979 , AA , Tarhjigt, Aït Ouaballi, Souss Massa, SA , Guelmim-Oued Noun; Chaker et al. 1980 ; Remm 1988a ; Hervy et al. 1994 ; Borkent and Wirth 1997 ; Baylis et al. 1997 ; Dakki 1997 ; Koçak and Kemal 2010 ; Borkent 2012 ; Bourquia et al. 2019 Ceratopogonini Alluaudomyia Kieffer, 1913 Alluaudomyia hygropetrica Vaillant, 1954 Vaillant 1956b , HA , Sidi Chamarouch Palpomyiini Bezzia Kieffer, 1899 Bezzia ( Bezzia ) nigritula (Zetterstedt, 1838) = Palpomyia tenebricosa Goetghebuer, 1912, in Vaillant 1956b : 241 Vaillant 1956b , HA , Tamesrit Palpomyia Meigen, 1818 Palpomyia helviscutellata Borkent, in Borkent & Wirth 1997 = Dasyhelea flavoscutellata (Zetterstedt, 1850), in Vaillant 1956b : 244 Vaillant 1956b , HA , Tahanaout Forcipomyiinae Dasyheleini Dasyhelea Kieffer, 1911 Dasyhelea ( Prokempia ) flaviventris (Goetghebuer, 1910) Dominiak 2012 Dasyhelea ( Pseudoculicoides ) turficola Kieffer, 1925 Dominiak 2012 Leptoconopinae Leptoconops Skuse, 1889 Leptoconops ( Holoconops ) laurae (Weiss, 1912) Remm 1988b CHIRONOMIDAE K. Kettani Number of species: 412 . Expected: 600 Faunistic knowledge of the family in Morocco: good Buchonomyiinae Buchonomyia Fittkau, 1955 Buchonomyia thienemanni Fittkau, 1955 Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe et al. 2015 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Moubayed-Breil 2018 , Rif Podonominae Paraboreochlus Thienemann, 1939 Paraboreochlus minutissimus (Strobl, 1895) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Tanypodinae Macropelopiini Apsectrotanypus Fittkau, 1962 Apsectrotanypus trifascipennis (Zetterstedt, 1838) Kettani et al. 2010 , Rif , Aïn Abou Hayane (Tiouertiouane, 880 m), Oued Maggou (Maggou village, 777 m), Oued Kanar (Gorges Kanar, 280 m); Kettani and Langton 2012 Macropelopia Thienemann, 1916 Macropelopia adaucta Kieffer, 1916 Kettani and Langton 2011 , Rif , Fifi, Issaguen; Kettani and Langton 2012 Macropelopia nebulosa (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrotanypus Kieffer, 1909 Psectrotanypus varius (Fabricius, 1787) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Pentaneurini Ablabesmyia Johannsen, 1905 Ablabesmyia ( Ablabesmyia ) ebbae Lehmann, 1981 Lehmann 1981 ; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Ablabesmyia ( Ablabesmyia ) longistyla Fittkau, 1962 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , source Maggou (Maggou, 1300 m), Oued Talembote; Kettani and Langton 2012 Ablabesmyia ( Ablabesmyia ) monilis (Linnaeus, 1758) Reiss 1977 , Rif , Tétouan, HA , kranichsee (Dra-Tal); Azzouzi and Laville 1987 , Rif , retenue El Makhazine; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Conchapelopia Fittkau, 1957 Conchapelopia ( Conchapelopia ) melanops (Meigen, 1818) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Conchapelopia ( Conchapelopia ) pallidula (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Conchapelopia ( Conchapelopia ) viator (Kieffer, 1911) = Conchapelopia Pe 1 Langton 1991 in Kettani et al. 1994 : 28, Kettani et al. 1995 : 256 Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 ; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Larsia Fittkau, 1962 Larsia atrocincta (Goetghebuer, 1942) Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , Oued Moulay Bouchta; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Larsia curticalcar (Kieffer, 1918) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m); Kettani and Langton 2012 Nilotanypus Kieffer, 1923 Nilotanypus dubius (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kanar (Gorges Kanar, 280 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paramerina Fittkau, 1962 Paramerina cingulata (Walker, 1856) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paramerina divisa (Walker, 1856) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Paramerina mauretanica Fittkau, 1962 Fittkau 1962 , Atlas (850 m), SA ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 , EM , Figuig; Kettani et al. 2001 ; Ashe and O'Connor 2009 , EM , Figuig; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Paramerina spec. Greichenland (Fittkau, 1962) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani et al. 2010 , Rif , Oued Chrafat (Armotah, 900 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Moubayed-Breil 2018 , Rif Pentaneurella Fittkau & Murray, 1983 Pentaneurella sp. Ourika Azzouzi et al. 1992 , HA ; Kettani et al. 2001 Rheopelopia Fittkau, 1962 Rheopelopia maculipennis (Zetterstedt, 1838) Naya 1988 , MA , Haut et Moyen Sebou; Azzouzi and Laville 1987 , MA , Oum-er-Rbia, HA , Tensift; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Ruisselet maison forestière (Talassemtane, 1683 m), Source Maggou (Maggou, 1300 m), Oued Talembote (avant village Talembote, 320 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheopelopia murrayi Dowling, 1983 Dowling 1983 , AA , Tata (Moyen Draa); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; 2012 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheopelopia ornata (Meigen, 1838) Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Telopelopia Roback, 1971 Telopelopia fascigera (Verneaux, 1970) = Telopelopia maroccana Murray, 1980, in Reiss 1977 : 91, Murray 1980 : 151, Azzouzi and Laville 1987 : 218, Ashe and Cranston 1990 : 133 Reiss 1977 , AP , Larache, HA , Dra-Tal; Murray 1980 , AP , Larache, HA , Dra-Tal; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Telmatopelopia Fittkau, 1962 Telmatopelopia nemorum (Goetghebuer, 1921) Kettani et al. 1996 , Rif , Oued Khizana (Oued Laou); Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Thienemannimyia Fittkau, 1957 Thienemannimyia ( Thienemannimyia ) berkanea Dowling, 1987 Dowling 1987 , EM , Berkane; Azzouzi et al. 1992 , EM , Environs de Berkane, HA , Ouarzazate (1160 m), Oasis Meski (1160 m), Aït Saoun; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) carnea (Fabricius, 1805) Kettani and Langton 2012 , Rif Thienemannimyia ( Thienemannimyia ) choumara Dowling, 1983 Dowling 1983 , EM , Environ de Berkane (Monts de Bni Snassen), HA , Souk des Judais (Marrakech); Azzouzi et al. 1987, HA , Dra-Tal; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Thienemannimyia ( Thienemannimyia ) geijskesi (Goetghebuer, 1934) Kettani and Langton 2012 , Rif , Oued Zarka Thienemannimyia ( Thienemannimyia ) laeta (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) lentiginosa (Fries, 1823) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) northumbrica (Edwards, 1929) Fittkau 1962 ; Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Trissopelopia Kieffer, 1923 Trissopelopia longimana (Staeger, 1839) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Xenopelopia Fittkau, 1962 Xenopelopia falcigera (Kieffer, 1911) Kettani and Langton 2011 , Rif , Anasser, Fifi, AP , marais de Loukous; Kettani and Langton 2012 Xenopelopia nigricans (Goetghebuer, 1927) Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia Fittkau, 1962 Zavrelimyia ( Zavrelimyia ) barbatipes (Kieffer, 1911) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 (?); Kettani et al. 2010 , Rif , Oued Tiffert (Tiffert Talassemtane, 1230 m), Aïn Abou Hayane (Tiouertiouane, 880 m), Oued Abiyati (Ifansa, 140 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) berberi Fittkau, 1962 Azzouzi and Laville 1987 ; Ashe and Cranston 1990 , HA , Tamhda; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) hirtimana (Kieffer, 1918) Kettani and Langton 2012 Zavrelimyia ( Zavrelimyia ) melanura (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) nubila (Meigen, 1830) Kettani and Langton 2011 , Rif , marais de Lemtahane ( PNPB ), Dayat Aïn Rami, Dayat Amlay; Kettani and Langton 2012 Procladiini Procladius Skuse, 1889 Procladius ( Holotanypus ) brevipetiolatus (Goetghebuer, 1935) Azzouzi et al. 1992 , HA , Oued Meski (1160 m), Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Procladius ( Holotanypus ) choreus (Meigen, 1804) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , AP , Merja Sidi Boughaba; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2010 , Rif , Aïn Talassemtane (Talassemtane, 1700 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Procladius ( Holotanypus ) culiciformis (Linnaeus, 1767) Kettani and Moubayed-Breil 2018 , Rif Procladius ( Holotanypus ) noctivagus (Kieffer, 1910) Azzouzi et al. 1992 , HA , Ouarzazate (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 Procladius ( Holotanypus ) sagittalis (Kieffer, 1909) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Procladius ( Psilotanypus ) anomalus Kieffer, 1906 Nomen dubium in Ashe and O'Connor 2009 : 213 Naya 1988 , MA ; Kettani et al. 2001 ; Kettani and Langton 2012 Procladius Pe 3 Langton 1991 Kettani et al. 1994 , Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 Tanypodini Tanypus Meigen, 1803 Tanypus ( Tanypus ) brevipalpis (Kieffer, 1923) Reiss 1977 , EM , Berkane; Ashe and O'Connor 2009 (?); Kettani and Langton 2012 Tanypus ( Tanypus ) kraatzi (Kieffer, 1912) Azzouzi et al. 1992 , HA , Oasis Meski; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Tanypus ( Tanypus ) punctipennis Meigen, 1818 Reiss 1977 , EM , Berkane; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesinae Boreoheptagyiini Boreoheptagyia Brundin, 1966 Boreoheptagyia legeri (Goetghebuer, 1933) = Boreoheptagyia punctulata (Goetghebuer, 1934), in Kettani et al. 2001 : 327 Ashe and Cranston 1990 ; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesini Diamesa Meigen, 1835 Diamesa aberrata Lundbeck, 1898 Saether 1968 ; Serra-Tosio 1973 ; Fittkau and Reiss 1987 ; Serra-Tosio 1983 ; Azzouzi and Laville 1987 , HA (2500–3350 m); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa bertrami Edwards, 1935 Serra-Tosio 1983 , HA , Gorges de Todra (2500 m); Azzouzi and Laville 1987 , HA , Gorges Todra; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa hamaticornis Kieffer, 1924 Reiss 1977 ; Serra-Tosio 1983 , HA , M'Goum; Azzouzi and Laville 1987 , HA , M'Goum; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa insignipes Kieffer, 1908 Serra-Tosio 1983 , HA (2500 m); Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut and Moyen Sebou; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Diamesa latitarsis (Goetghebuer, 1921) Vaillant 1955b ; Vaillant 1956b , HA , Asif Tessaout (M'Goum), Lac Tamhda (Anremer); Serra-Tosio 1967 ; Serra-Tosio 1967 ; Saether 1968 ; Serra-Tosio 1973 ; Azzouzi and Laville 1987 , HA ; Ashe and Cranston 1990 ; Kettani et al. 2001 , Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa steinboecki Goetghebuer, 1933 Vaillant 1956b , HA , Cascade Siroua, Oukaimeden, Sidi Chamarouch Diamesa tonsa (Haliday in Walker, 1856) = Diamesa thienemanni Kieffer, 1909 Naya 1988 , MA , Haut Sebou (Arhbalou Yahya, Oued Arbi, Pont Aït hamza); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 Diamesa vaillanti Serra-Tosio, 1972 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa veletensis Serra-Tosio, 1971 Serra-Tosio 1983 , HA (2500 m); Azzouzi and Laville 1987 , HA ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa zernyi Edwards, 1933 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Potthastia Kieffer, 1922 Potthastia gaedii (Meigen, 1838) Azzouzi and Laville 1987 , MA , oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Laou, Oued Afertane; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Potthastia pastoris (Edwards, 1933) Kettani and Moubayed-Breil 2018 , Rif Pseudodiamesa Goetghebuer, 1939 Pseudodiamesa ( Pseudodiamesa ) branickii (Nowicki, 1873) Naya 1988 , MA , Haut Sebou; Ashe and Cranston 1990 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Pseudodiamesa ( Pseudodiamesa ) nivosa (Goetghebuer, 1928) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Sympothastia Pagast, 1947 Sympothastia zavreli Pagast, 1947 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , aval Oued Krikra; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Syndiamesa Kieffer, 1918 Syndiamesa hygropterica (Kieffer, 1909) Naya 1988 , MA , Moyen Sebou (Sidi Abdellah, Dar El Arsa, Pont Oulad Slimane, Pont Portugais); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Protanypini Protanypus Kieffer, 1906 Protanypus morio (Zetterstedt, 1838) Naya 1988 , MA , Moyen Sebou; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Prodiamesinae Odontomesa Pagast, 1947 Odontomesa fulva (Kieffer, 1919) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Prodiamesa Kieffer, 1906 Prodiamesa olivacea (Meigen, 1818) Naya 1988 , MA , Haut Sebou (Haut Guigou); Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Maggou village, Ifansa; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladiinae Orthocladiini Acricotopus Kieffer, 1921 Acricotopus lucens (Zetterstedt, 1850) Kettani and Moubayed-Breil 2018 , Rif Brilla Kieffer, 1913 Brillia bifida (Kieffer, 1909) = Brilla modesta (Meigen, 1830) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Brillia flavifrons (Johannsen, 1905) Kettani and Langton 2012 Brilla longifurca Kieffer, 1921 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1995 , Rif , amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Talembote (Usine électrique, 120 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Bryophaenocladius Thienemann, 1934 Bryophaenocladius aestivus (Brundin, 1947) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius flexidens (Brundin, 1947) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius cf. furcatus Thienemann & Strenzke, 1940 Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius illimbatus (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius muscicola (Kieffer, 1906) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius nidorum (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius subvernalis (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius tuberculatus (Edwards 1929) Kettani and Moubayed-Breil 2018 , Rif Camptocladius Wulp, 1874 Camptocladius stercorarius (De Geer, 1976) Kettani and Moubayed-Breil 2018 , Rif Cardiocladius Kieffer, 1912 Cardiocladius capucinus (Zetterstedt, 1850) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cardiocladius fuscus Kieffer, 1924 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou (Amont de Aïn Tadout, Skhounate, amont confluence avec Oued Atchane, Pont Aït Hamza); Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius Kieffer, 1911 Chaetocladius ( Chaetocladius ) acuticornis (Kieffer in Potthast, 1914) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius dentiforceps (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Chaetocladius dissipatus (Edwards, 1929) Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Chaetocladius ( Chaetocladius ) melaleucus (Meigen, 1818) Kettani and Langton 2011 , Rif , Oued Sgara, Bab Tariouant, Bouztata; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius piger (Goetghebuer, 1913) Kettani and Moubayed-Breil 2018 , Rif Chaetocladius ( Chaetocladius ) perennis (Meigen, 1830) Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 Chaetocladius ( Chaetocladius ) vitellinus (Kieffer in Kieffer & Thienemann, 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Corynoneura Winnertz, 1846 Corynoneura carriana Edwards, 1924 Naya 1988 , MA , Haut Sebou (Haut Guigou, Aïn Nokra); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura celtica Edwards, 1924 Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura coronata Edwards, 1924 Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Corynoneura edwardsi Brundin, 1949 Kettani and Langton 2012 Corynoneura lacustris Edwards, 1924 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura lobata Edwards, 1924 Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Azzouzi et al. 1992 , HA ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura scutellata Winnertz, 1846 Kettani and Moubayed-Breil 2018 , Rif Corynoneura Pe 2 Langton 1991 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Corynoneurella Brundin, 1949 Corynoneurella paludosa Brundin, 1949 Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus van der Wulp, 1874 Cricotopus ( Cricotopus ) albiforceps (Kieffer in Thienemann and Kieffer 1916) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) annulator Goetghebuer, 1927 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2011 , Rif , Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) beckeri Hirvenoja, 1973 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Langton and Laville 2002; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) bicinctus (Meigen, 1818) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Naya 1988 , MA , Haut et Moyen Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) caducus Hirvenoja, 1973 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) ephippium (Zetterstedt, 1838) Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) levantinus Moubayed & Hirvenoja, 1986 Kettani et al. 1996 , Rif , Haut Maggou; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Inesmane (Adeldal, 1173 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Cricotopus ) pallidipes Edwards, 1929 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) pulchripes Verrall, 1912 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) similis Goetgnebuer, 1921 Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Afertane, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) tremulus (Linnaeus, 1758) Kettani et al. 2010 , Rif , Oued Maggou (Maggou village, 777 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) triannulatus (Macquart, 1826) Kettani et al. 1994 , Rif , Haut Laou, Oued Moulay Bouchta, Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) trifascia Edwards, 1929 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Maggou (Maggou village, 777 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) vierriensis Goetghebuer, 1935 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Isocladius ) brevipalpis Kieffer, 1909 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) laetus Hirvenoja, 1973 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) ornatus (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) sylvestris (Fabricius, 1794) Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Isocladius ) tricinctus (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) micans (Kieffer, 1918) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Talembote, Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) osellai Rossaro, 1990 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) rufiventris (Meigen, 1830) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; (Dar El Arsa, Pont oulad Slimane, Pont portugais); Naya 1988 , MA , Haut et Moyen Sebou; Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) skirwithensis (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukieferiella Thienemann, 1926 Eukieferiella ancyla Svensson, 1986 Kettani and Langton 2011 , Rif , Oued Tkarae; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella bedmari Vilchez-Quero & Laville, 1988 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Laville and Langton 2002 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella brehmi Gowin, 1943 Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (Usine électrique, 120 m) Eukiefferiella brevicalcar (Kieffer, 1911) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m), Oued Ametrasse (Ametrasse, 820 m); Kettani and Langton 2011 , Rif , Oued Issaguen, Oued Ketama, Oued Sgara; Bab Tariouant, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella claripennis (Lundbeck, 1898) Fittkau and Reiss 1978 ; Naya 1988 , MA , Moyen Sebou (Dar Cheik Harazem); Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukieffeiella clypeata (Thienemann, 1919) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella coerulescens (Kieffer in Zavřel, 1926) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou (Skhounate, Arhbalou Aberchane); Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Nord Maggou village (Maggou, 905 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella cyanea Thienemann, 1936 Vaillant 1955b , HA ; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , HA ; Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella devonica (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella dittmari Lehmann, 1972 Kettani and Langton 2011 , Rif , Oued Boujdad, Fifi; Kettani and Langton 2012 , Rif , Oued Zarka; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella fittkaui Lehmann, 1972 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella fuldensis Lehmann, 1972 Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella gracei (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Tassikeste; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella ilkleyensis (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella lobifera Goetghebuer, 1934 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella minor (Edwards, 1929) Vaillant 1955b , HA (1050 m); Vaillant 1956b , HA , Imi-N'Ifri; Azzouzi and Laville 1987 , HA ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella pseudomontana Goetghebuer, 1935 Kettani et al. 2010 , Rif , Oued Madissouka (Talassemtane, 1530 m), Oued Dchar d'Amran (Béni M'Hamed, 1180 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella similis Goetghebuer, 1939 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella tirolensis Goetghebuer, 1938 Kettani et al. 1994 , Rif , Oued Afertane; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Azzouzi et al. 1992 , HA , Oued Tensift; Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella Pe 2 Langton 1991 Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Halocladius Hirvenoja, 1973 Halocladius ( Halocladius ) varians (Staeger, 1839) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m); Ashe and Cranston 1990 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heleniella Gowin, 1943 Heleniella dorieri Serra-Tosio, 1967 Kettani and Langton 2012 Heleniella ornaticollis (Edwards, 1929) Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heleniella serratosioi Ringe, 1976 Kettani and Langton 2011 , Rif , Oued Hamma, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heterotrissocladius Spärck, 1923 Heterotrissocladius marcidus (Walker, 1856) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2012 Hydrobaenus Fries, 1830 Hydrobaenus conformis (Holmgren, 1869) Kettani and Moubayed-Breil 2018 , Rif Hydrosmittia Ferrington & Sæther, 2011 Hydrosmittia oxoniana (Edwards, 1929) = Pseudosmittia recta (Edwards, 1929), in Azzouzi and Laville 1987 : 218, Kettani et al. 2001 : 330, Kettani and Langton 2012 : 422 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Hydrosmittia ruttneri (Strenzke & Thienemann, 1942) Kettani and Moubayed-Breil 2018 , Rif Krenosmittia Thienemann & Krüger, 1939 Krenosmittia boreoalpina (Goetghebuer, 1944) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Krenosmittia camptophleps (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Krenosmittia halvorseni (Cranston & Sæther, 1986) Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Krenosmittia hispanica Wülker, 1957 Kettani et al. 2001 ; Laville and Langton 2002 ; Ashe and O'Connor 2012 Limnophyes Eaton, 1875 Limnophyes difficilis Brunidin, 1947 Kettani and Moubayed-Breil 2018 , Rif Limnophyes gelasinus Saether, 1990 Kettani and Moubayed-Breil 2018 , Rif Limnophyes habilis (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Limnophyes madeirae Sæther, 1985 Kettani and Moubayed-Breil 2018 , Rif Limnophyes minimus (Meigen, 1818) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Kettani et al. 2001 ; Kettani and Langton 2011 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Limnophyes natalensis (Kieffer, 1914) Kettani and Moubayed-Breil 2018 , Rif Limnophyes ninae Sæther, 1975 Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Limnophyes pentaplastus (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Limnophyes pumilio (Holmgren, 1869) Kettani and Moubayed-Breil 2018 , Rif Limnophyes punctipennis (Goetghebuer, 1919) Kettani and Langton 2012 Limnophyes Pe 1a Langton 1991 Kettani et al. 2010 , Rif , Oued Talembote (Usine électrique, 120 m); Kettani and Langton 2012 Metriocnemus van der Wulp, 1874 Metriocnemus ( Metriocnemus ) albolineatus Meigen, 1818 Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) eurynotus (Holmgren, 1883) = Metriocnemus hygropetricus Kieffer, 1912, in Ashe and O'Connor 2012 : 372 = Metriocnemus ( Metriocnemus ) obscuripes (Holmgren, 1869), in Azzouzi et al. 1992 : 229, Kettani et al. 2001 : 329, Kettani and Langton 2012 : 421 Boumezzough and Thomas 1987 , HA , Oued Réghaya (1740 m), Imlil; Azzouzi and Laville 1987 , HA , Oued Tensift; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Metriocnemus ( Metriocnemus ) fuscipes (Meigen, 1818) Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) hirticollis (Staeger, 1839) Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) ursinus Holmgren, 1869 Kettani and Moubayed-Breil 2018 , Rif Nanocladius Kieffer, 1913 Nanocladius ( Nanocladius ) balticus (Palmén, 1959) Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) dichromus (Kieffer 1906) Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) parvulus (Kieffer 1909) Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) rectinervis (Kieffer, 1911) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Inesmane (Adeldal, 1173 m), Oued Talembote (aval Barrage Talembote, 245 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius van der Wulp, 1874 Orthocladius ( Eudactylocladius ) fuscimanus (Kieffer, 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued Krikra, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , source Maggou (Maggou, 1300 m), Oued Chrafat (Armotah, 900 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) ashei Soponis, 1990 = Orthocladius luteipes Goetghebuer, in Azzouzi and Laville 1987 : 218 = Orthocladius rivicola Kieffer, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Fès, Oued boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique), aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) rivulorum Kieffer, 1909 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor; 2012 Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) thienemanni Kieffer, 1906 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Mesorthocladius ) frigidus (Zetterstedt, 1838) Vaillant 1955, HA (2900 m); Vaillant 1956b , HA , Lac Tamhda (Anremer); Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut Sebou (Haut Guigou); Fekhaoui et al. 1993 ; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Dchar d'Amran (Béni M'Hamed, 1180 m), Nord Maggou village (Maggou, 905 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Orthocladius ( Orthocladius ) oblidens (Walker, 1856) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) obumbratus Johannsen, 1905 = Orthocladius excavatus Brundin, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Orthocladius ( Orthocladius ) pedestris Kieffer, 1909 Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) rubicundus (Meigen, 1818) = Orthocladius saxicola Kieffer, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) vaillanti Langton & Cranston, 1991 Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Symposiocladius ) lignicola Kieffer in Potthast, 1914 = Symposiocladius lignicola Kieffer, in Kettani et al. 2010 : 70 Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Orthocladius ( Symposiocladius ) ruffoi Rossaro & Prato, 1991 = Orthocladius Pe 1 Langton 1991, in Azzouzi and Laville 1987 : 218 = Rheortocladius sp A Langton 1991, in Kettani et al. 1995 : 257 = Rheorthocladius ruffoi Rossaro, in Kettani et al. 1997 : 184 Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Ifansa, 105 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Paracricotopus Brundin, 1956 Paracricotopus niger (Kieffer, 1913) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès; Kettani et al. 1994 , Rif , Oued Afertane; Kettani et al. 1995 , Rif , amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou, Oued Tamaridine (Zaouit et Habtyiène, 819 m), Oued Kelaâ (Akoumi, 400 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (155 m), Oued Tassikeste (240 m), Oued Laou (Ifansa, 105 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parakiefferiella Thienemann, 1936 Parakiefferiella coronata (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parakiefferiella wuelkeri Moubayed, 1994 = Parakiefferiella sp. d Wülker, in Azzouzi et al. 1992 : 230 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parametriocnemus Goetghebuer, 1932 Parametriocnemus boreoalpinus Gowin & Thienemann, 1942 Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parametriocnemus stylatus (Spärck, 1923) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Béni M'Hamed (1330 m), Haut Maggou (1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (320 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Parametriocnemus valescurensis Moubayed & Langton, 1999 Kettani and Langton 2011 , Rif , Oued Issaguen; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parametriocnemus Pe 1 Langton 1991 Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Talembote (320 m); Kettani and Langton 2012 Paraphaenocladius Thienemann, 1924 Paraphaenocladius exagitans ssp. 1 Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius impensus impensus (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius irritus Walker, 1856 Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius pseudirritus Strenzke, 1950 Kettani and Moubayed-Breil 2018 , Rif Paratrissocladius Zavřel, 1937 Paratrissocladius excerptus (Walker, 1856) Kettani et al. 1996 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parorthocladius Thienemann, 1935 Parorthocladius nudipennis (Kieffer in Kieffer & Thienemann 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psecrocladius Kieffer, 1906 Psectrocladius ( Allopsectrocladius ) obvius (Walker, 1856) = Psectrocladius dilatatus (van der Wulp, 1859), in Naya 1988 : 48 Naya 1988 , MA , Moyen Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2011 , Rif , sources de Issaguen; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Allopsectrocladius ) platypus (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Mesopsectrocladius ) barbatipes Kieffer, 1923 Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Laou (Afertane, 56 m); Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psecrocladius ( Psectrocladius ) brehmi Kieffer, 1923 Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psectrocladius ( Psectrocladius ) fennicus Storå, 1939 Kettani and Langton 2012 Psectrocladius ( Psectrocladius ) limbatellus (Holmgren, 1869) Wülker 1959 ; Azzouzi and Laville 1987 , HA , Lac Tamhda (2800 m); Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Psectrocladius ) octomoculatus Wülker, 1956 Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psectrocladius ( Psectrocladius ) sordidellus (Zetterstedt, 1838) Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous (NE Boucharene); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Psectrocladius ) ventricosus Kieffer, 1925 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Pseudosmittia Edwards, 1932 Pseudosmittia albipennis (Goetghebuer, 1921) Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia baueri Strenzke, 1960 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia danconai (Marcuzzi, 1947) Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia holsata Thienemann & Stenzke, 1940 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia obtusa Strenzke, 1960 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia trilobata Edwards, 1929 Kettani and Moubayed-Breil 2018 , Rif Pseudorthocladius Goetghebuer, 1943 Pseudorthocladius ( Pseudorthocladius ) berthelemyi Moubayed, 1990 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Pseudorthocladius ( Pseudorthocladius ) curtistylus (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oasis Meski (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Pseudorthocladius near Pe 3 Langton 1991 Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 Rheocricotopus Brundin, 1956 Rheocricotopus ( Psilocricotopus ) atripes (Kieffer, 1913) = Rheocricotopus ( Psilocricotopus ) foveatus foveatus (Edwards, 1929), in Naya 1988 : 40 Naya 1988 , MA , Haut Sebou (Haut Guigou); Azzouzi et al. 1992 , HA , Oued Tensift, Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , Haut Laou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote, Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 56 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) chalybeatus subsp. chalybeatus (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) gallicus Lehamnn 1969 Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) glabricollis (Meigen, 1830) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Ametrasse (Ametrasse, 820 m), Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) meridionalis Moubayed-Breil, 2016 Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) tirolus Lehmann, 1969 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) effusus (Walker, 1856) Reiss 1977 ; Naya 1988 , MA , Haut et Moyen Sebou; Fekhaoui et al. 1993 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) fuscipes (Kieffer, 1909) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Maggou (905 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) rifensis Moubayed & Kettani, 2019 Moubayed-Breil and Kettani, Rif , Chrafate, Challal Sghir (Akchour) Synorthocladius Thienemann, 1935 Synorthocladius semivirens (Kieffer, 1909) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Smittia Holmgren, 1869 Smittia alpicola Goetghebuer, 1941 Kettani and Moubayed-Breil 2018 , Rif Smittia aterrima Meigen, 1818 Kettani and Moubayed-Breil 2018 , Rif Smittia contingens Walker, 1856 Kettani and Moubayed-Breil 2018 , Rif Smittia foliacea (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Smittia pratorum Goetghebuer, 1927 Kettani and Moubayed-Breil 2018 , Rif Thienemannia Kieffer, 1909 Thienemannia cf. fulvofasciata (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Thienemannia gracilis Kieffer, 1909 Kettani and Moubayed-Breil 2018 , Rif Thienemanniella Kieffer, 1911 Thienemanniella acuticornis (Kieffer, 1912) Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (320 m); Kettani and Langton 2011 , Rif , Oued Hamma, Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Thienemanniella clavicornis (Kieffer, 1911) Kettani and Moubayed-Breil 2018 , Rif Thienemanniella majuscula (Edwards, 1924) Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemanniella vittata (Edwards, 1924) Kettani et al. 1996 , Rif , Haut Maggou; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou (1300 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemanniella Pe 2a Langton 1991 Kettani et al. 2010 , Rif , Oued Maggou (905 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote; Kettani and Langton 2012 Thienemanniella Pe 2b Langton 1991 Kettani et al. 2010 , Rif , Oued Maggou (905 m), Oued Talembote; Kettani and Langton 2012 Trissocladius Kieffer, 1908 Trissocladius brevipalpis Kieffer in Kieffer & Thienemann 1908 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia Kieffer, 1922 Tvetenia bavarica (Goetghebuer, 1934) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia calvescens (Edwards, 1929) Naya 1988 , MA , Moyen Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m), Oued Talembote (245 m), Oued Laou (Afertane, 56 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tvetenia discoloripes (Goetghebuer & Thienemann in Thienemann, 1936) Kettani and Langton 2011 , Rif , Oued Nakhla, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia verralli (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, amont Oued Nakhla; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , ruisselet maison forestière Talassemtane (1683 m), Oued Tamaridine (Zaouiet et Habtiyiène, 819 m), Oued Talembote (245 m), Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zalutschia Lipina, 1939 Zalutschia humphriesiae Dowling & Murray, 1980 Kettani and Langton 2011 , Rif , marais de Lemtahane ( PNPB ), Dayat Fifi; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Chironominae Chironomini Chironomus Meigen, 1803 Chironomus ( Baeotendipes ) noctivagus (Kieffer, 1911) Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) annularius Meigen, 1818 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) aprilinus sensu Meigen, 1818 = Chironomus halophilus Kieffer, in Ramdani and Tourenq 1982 : 180, in Naya 1988 : 50 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut Sebou; Fekhaoui et al. 1993 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) bernensis Klötzli, 1973 = Chironomus sp 1 Kettani 1994 Kettani et al. 1994 ; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) calipterus Kieffer, 1908 Reiss 1977 , AP , Larache; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) longistylus Goetghebuer, 1921 Kettani et al. 2011, Rif , Oued Ketama; Kettani and Langton 2012 Chironomus ( Chironomus ) luridus Strenzke, 1959 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) nuditarsis Keyl, 1961 Kettani et al. 2011, Rif , Oued Boujdad (Kitane, 42 m), Oued El Hatba (SIBE Jebel Moussa, 165 m); Kettani and Langton 2012 , Rif , SIBE Jebel Moussa Chironomus ( Chironomus ) piger (Strenzke, 1956) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) plumosus (Linnaeus, 1758) Reiss 1977 , AP , Larache, AA , Dra-Tal; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 ; Naya 1988 , MA , Sidi Abdellah, Dar Cheih Harazem, Dar El Arsa; Fekhaoui et al. 1993 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Aïn Tissmelal (Tissmelal, 1046 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) prasinus sensu Pinder, 1978 Kettani et al. 2011, Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 Chironomus ( Chironomus ) riparius Meigen, 1804 = Chironomus thummi Kieffer, in Naya 1988 : 51, Fekhaoui et al. 1993 : 26 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou; Naya 1988 , MA , Moyen Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) salinarius Kieffer, 1915 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) tentans Fabricius, 1805 = Camptochironomus tentans Fabricius, 1805, in Naya 1988 : 50 Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Cladopelma Kieffer, 1921 Cladopelma virescens (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus Kieffer, 1918 Cryptochironomus ( Cryptochironomus ) albofasciatus (Staeger, 1839) = Cryptochironomus obreptans Walker, 1856, in Kettani 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 Cryptochironomus ( Cryptochironomus ) psittacinus (Meigen, 1830) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Cryptochironomus ( Cryptochironomus ) rostratus Kieffer, 1921 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou, oued Oum-er-Rbia, Oued Boufekrane, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus ( Cryptochironomus ) supplicans (Meigen, 1830) Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus Pe 5 Langton 1991 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 Demicryptochironomus Lenz, 1941 Demicryptochironomus ( Demicryptochironomus ) vulneratus (Zetterstedt, 1838) Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m); Kettani and Langton 2012 Demicryptochironomus ( Irmakia ) neglectus Reiss, 1988 Kettani and Moubayed-Breil 2018 , Rif Demicryptochironomus ( Irmakia ) Pe 1 Langton 1991 Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, aval Oued Khemis; Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes Kieffer, 1913 Dicrotendipes collarti (Goetghebuer, 1936) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes cordatus Kieffer, 1922 Kettani et al. 1996 , Rif , Oued Khizana (Oued Laou); Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes fusconotatus (Kieffer, 1922) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes modestus (Say, 1823) Kettani et al. 2011, Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 Dicrotendipes nervosus (Staeger, 1839) = Limnochirononomus nervosus Staeger, in Naya 1988 : 53 Naya 1988 , MA , Moyen Sebou (Sidi Abdellah); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes notatus (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes pallidicornis (Goetghebuer, 1934) Azzouzi and Laville 1987 , Rif , Retenue El Makhazine, MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes peringueyanus Kieffer, 1924 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 Dicrotendipes septemmaculatus (Becker, 1908) = Dicrotendipes pilosimanus Kieffer, in Reiss 1977 : 91, Azzouzi and Laville 1987 : 219 Reiss 1977 , AP , Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , AP , Larache; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 Endochironomus Kieffer, 1918 Endochironomus albipennis (Meigen, 1830) Naya 1988 , MA , Haut Sebou (Skhounata); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Endochironomus tendens (Fabricius, 1775) Naya 1988 , MA , Moyen Sebou (Gantra Mdez, Azzaba); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes Kieffer, 1913 Glyptotendipes ( Caulochironomus ) viridis (Macquart, 1834) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes ( Glyptotendipes ) cauliginellus (Kieffer, 1913) = Glyptotendipes gripekoveni (Kieffer) Naya 1988 , MA , Haut Sebout (Guigou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes ( Glyptotendipes ) pallens (Meigen, 1804) Azzouzi and Laville 1987 , Rif , Retenue El Makhazine; Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes sp A Langton 1991 Naya 1988 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Glyptotendipes sp B Langton 1991 Naya 1988 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Harnischia Kieffer, 1921 Harnischia curtilamellata (Malloch, 1915) Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Harnischia fuscimanus Kieffer, 1921 Azzouzi and Laville 1987 , Rif , Retenue El Makhazine, MA , Oued Boufekrane; Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Kiefferulus Goetghebuer, 1922 Kiefferulus ( Kiefferulus ) tendipediformis (Goetghebuer, 1921) Reiss 1977 , Rif , Tétouan; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2010 , Rif , Guelta 1 km après Amariguen (Jebel Setsou, 1280 m); Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Dalia (SIBE Jebel Moussa, 169 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Kloosia Kruseman, 1933 Kloosia pusilla (Linnaeus, 1767) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Lauterborniella Thienemann & Bause, 1913 Lauterborniella agrayloides (Kieffer, 1911) Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus Kieffer, 1918 Microchironomus deribae (Freeman, 1957) = Leptochirononomus deribae Freeman, in Reiss 1977 : 91, Ramdani and Tourenq 1982 : 180 Reiss 1977 , AP , Rabat; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus lendli (Kieffer, 1918) Reiss 1986 , AA , Oasis Meski; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus tener (Kieffer, 1918) Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Azzouzi et al. 1992 , HA , Oued Tensift, Barrage Lalla Takerkoust; Kettani and Langton 2012 Microtendipes Kieffer, 1915 Microtendipes britteni (Edwards, 1929) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Microtendipes chloris (Meigen, 1818) Kettani et al. 2011, Rif , Dayat En-Nâsser (Khandek En-Nâsser, 1177 m), source Bab Karn (Fifi, 1216 m), Dayat Fifi (1179 m); Kettani and Langton 2012 Microtendipes confinis (Meigen, 1830) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 Microtendipes diffinis (Edwards, 1929) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani et al. 2011, Rif , Dayat En-Nâsser (Khandek En-Nâsser, 1177 m), Dayat Aïn Rami, source Bab Karn (Fifi, 1216 m); Kettani and Langton 2012 Microtendipes pedellus (De Geer, 1776) Reiss 1977 , Rif , Environ de Tétouan; Fittkau and Reiss 1978 ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , Rif , Tétouan, HA ; Naya 1988 , MA , Haut Sebou (amont Aîn Tadout, Skhounate, Arhbalou Aberchane); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Nubensia Spies, 2015 Nubensia nubens (Edwards, 1929) = Polypedilum nubens (Edwards, 1929), in Azzouzi and Laville 1987 : 219; Kettani et al. 1994 : 28, 1995 : 257, 1996 : 137, 1997 : 184, 2001 : 331, 2010 : 70; Dakki 1997 : 65; Dakki et al. 2008: 32, Kettani and Langton 2012 : 423 Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Laou (Ifansa, 105 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parachironomus Lenz, 1921 Parachironomus frequens (Johannsen, 1905) Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Parachironomus parilis (Walker, 1856) Reiss 1977 , AP , Environ de Larache; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Ashe and Cranston 1990 ; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Paracladopelma Harnisch, 1923 Paracladopelma camptolabis (Kieffer, 1913) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paracladopelma galaptera (Townes, 1945) Azzouzi et al. 1992 , HA , Ouarzazate (1140 m), Gorges de Todra (1400 m); Kettani et al. 2001 ; Kettani and Langton 2012 Paracladopelma graminicolor (Kieffer, 1925) = Cryptotendipes graminicolor (Kieffer), in Azzouzi et al. 1992 : 230 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Paracladopelma laminatum (Kieffer, 1921) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paracladopelma mikianum (Goetghebuer, 1937) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paralauterborniella Lenz, 1941 Paralauterborniella nigrohalteralis (Malloch, 1915) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Paratendipes Kieffer, 1911 Paratendipes albimanus (Meigen, 1818) Naya 1988 , MA , Moyen Sebou (Mdez); Kettani et al. 1994 , Rif , aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued Mhajrat, aval Oued Khemis, Oued Laou (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratendipes nudisquama (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Paratendipes striatus (Kieffer, 1925) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Phaenopsectra Kieffer, 1921 Phaenopsectra flavipes (Meigen, 1818) Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum Kieffer, 1912 Polypedilum ( Pentapedilum ) ruandae Freeman, 1955 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Pentapedilum ) sordens (van der Wulp, 1875) = Polypedilum sp 1, in Kettani et al. 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Pentapedilum ) uncinatum (Goetghebuer, 1921) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Polypedilum ) albicorne (Meigen, 1838) Naya 1988 , MA , Haut Sebou; Kettani et al. 1995 , Rif , aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) arundineti (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 Polypedilum ( Polypedilum ) laetum (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) nubeculosum (Meigen, 1804) Reiss 1977 , Rif , Environ de Tétouan; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , Rif , Environ Tétouan, MA , Oued Sebou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) nubifer (Skuse, 1889) = Polypedilum pharao Kieffer, in Reiss 1977 : 91, Naya 1998: 55, Ramdani and Tourenq 1982 : 180 Kügler and Wool 1968 ; Reiss 1977 , AP , Larache, Rabat; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 , AP , Environ de Larache, Rabat, Merja Sidi Boughaba; Naya 1988 , MA , Haut Sebou; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) pedestre (Meigen, 1830) Reiss 1977 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 1994 , Rif , aval Barrage Talembote; Kettani et al. 1995 , Rif , Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Tripodura ) acifer Townes, 1945 Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 , MA , Oued Boufekroune, Oued Fès, Oued Sebou; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Polypedilum ( Tripodura ) aegyptium Kieffer, 1925 = Polypedilum pruina Freeman, in Reiss 1977 : 91 Reiss 1977 , AP , Larache, HA , Marrakech, AA , Dra-Tal; Reiss 1985 ; Azzouzi and Laville 1987 , AP , Larache, HA , Marrakech, AA , Gorges de Todra; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Polypedilum ( Tripodura ) bicrenatum Kieffer, 1921 Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) pullum (Zetterstedt, 1838) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) quadriguttatum Kieffer, 1921 Naya 1988 , MA , Moyen Sebou; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Polypedilum ( Tripodura ) scalaenum (Schrank, 1803) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Kettani et al. 1996 , Rif , Ras el Ma (Chefchaouen); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) tetracrenatum Hirvenoja, 1962 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) tridens Freeman, 1955 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Uresipedilum ) convictum (Walker, 1856) Reiss 1977 , AP , Environ de Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane (Gantra Mdez), Naya 1988 , MA , Haut Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued pont Béni M'Hamed (Béni M'Hamed, 1330 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Uresipedilum ) cultellatum Goetghebuer, 1931 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Polypedilum ontario -group sp. 1 Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Rheomus Laville & Reiss, 1988 Rheomus alatus Laville & Reiss, 1988 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Rheomus yahiae Laville & Reiss, 1988 Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Stenochironomus Kieffer, 1919 Stenochironomus gibbus Fabricius, 1794 Kettani and Moubayed-Breil 2018 , Rif Stictochironomus Kieffer, 1919 Stictochironomus caffrarius (Kieffer, 1921) Reiss 1977 ; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 Stictochironomus maculipennis (Meigen, 1818) Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Afertane; Kettani et al. 1995 , Rif , aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Stictochironomus pictulus (Meigen, 1830) Reiss 1977 , AP , Environ de Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1995 , Rif , aval Oued Kbir, aval Oued Krikra, Oued El Kbir; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Stictochironomus rosenschoeldi Zetterstedt, 1838 Kettani and Moubayed-Breil 2018 , Rif Stictochironomus reissi Cranston, 1989 = Stictochironomus sp. nov. Reiss, in Reiss 1977 : 91 Reiss 1977 ; Azzouzi and Laville 1987 , AA , M'Hamid, Dra-Tal; Kettani et al. 2001 ; Kettani and Langton 2012 Stictochironomus sticticus (Fabricius, 1781) = Stictochironomus histrio (Fabricius, 1794), in Kettani et al. 1996 : 138 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Berranda (Bouztate, 1259 m), Dayat Dalia (SIBE Jebel Moussa); Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Stictochironomus Pe 2 Langton 1991 Kettani et al. 2001 Xenochironomus Kieffer, 1921 Xenochironomus xenolabis (Kieffer, 1916) Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsini Cladotanytarsus Kieffer, 1921 Cladotanytarsus ( Cladotanytarsus ) atridorsum Kieffer, 1924 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Azzouzi et al. 1992 , HA , Aït Saoun, Gorges de Dadès (1900 m), vallée de Drâa, Marrakech; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cladotanytarsus ( Cladotanytarsus ) capensis (Freeman, 1954) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) ecristatus Reiss, 1991 = Tanytarsus sp. nov. (Morokko) Reiss, in Azzouzi and Laville 1987 : 219 Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 , EM , Berkane; Reiss 1991 ; Azzouzi et al. 1992 , HA ; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) mancus (Walker, 1856) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cladotanytarsus ( Cladotanytarsus ) pallidus Kieffer, 1922 = Cladotanytarsus Pe 5 Langton 1984 Azzouzi and Laville 1987 , MA , Oued Sebou, Oum Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) vanderwulpi (Edwards, 1929) Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Lithotanytarsus Thienemann, 1933 Lithotanytarsus dadesi Reiss, 1991 Reiss 1991 ; Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Lithotanytarsus emarginatus (Goetghebuer, 1933) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani and Langton 2012 Micropsectra Kieffer, 1909 Micropsectra andalusiaca Marcuzzi, 1950 Kettani and Moubayed-Breil 2018 , Rif Micropsectra apposita (Walker, 1856) = Micropsectra contracta Reiss, 1965 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Chrafat (Armotah, 900 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra aristata Pinder, 1976 Kettani and Langton 2012 , Rif , Oued Zarka Micropsectra atrofasciata (Kieffer, 1911) = Micropsectra bidentata (Goetghebuer, 1921), in Azzouzi et al. 1992 : 230; Kettani et al. 2001 : 332; Kettani and Langton 2011 : 590, 2012 : 424 Fittkau and Reiss 1978 ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Sebou (Arhbalou Aberchane), Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Madissouka (Talassemtane, 1530 m), Oued Chrafat (Armotah, 900 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval affluent Talembote, 155 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam, 650 m), cascade Zarka, Dayat En-Nâsser (Khandek En-Nâsser, 1177 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra junci (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra lacustris Säwedal, 1975 Kettani and Langton 2012 , Rif , Oued Zarka Micropsectra lindrothi Goetghebuer, 1931 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra notescens (Walker, 1856) Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara, ruisselet Bab Tariouant, Oued Berranda (Bouztate, 1259 m), source Bab Karn (Fifi, 1220 m), Dayat Fifi (Fifi, 1179); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra pallidula (Meigen, 1830) Kettani and Moubayed-Breil 2018 , Rif Micropsectra schrankelae Stur & Ekrem, 2006 Kettani and Moubayed-Breil 2018 , Rif Micropsectra zernyi Marcuzzi, 1950 Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus Thienemann & Bause, 1913 Paratanytarsus bituberculatus (Edwards, 1929) Azzouzi et al. 1992 , MA , Lac Aguelmane Azigza (1510 m); Kettani et al. 1995 , Rif , Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Paratanytarsus dissimilis (Johannsen, 1905) = Paratanytarsus confusus Palmén, 1960, in Naya 1988 : 40; Dakki et al. 2008: 32; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 423 Naya 1988 , MA , Haut Sebou; Dakki et al. 2008, MA , Oued Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus grimmii (Schneider, 1885) Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Paratanytarsus inopertus (Walker, 1856) Reiss 1977 , Rif , Environ Tétouan; Fittkau and Reiss 1978 ; Reiss and Säwedal 1981 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus mediterraneus Reiss & Säwedal, 1981 Reiss and Säwedal 1981 , Rif , Estuaire Oued Mharka (Tanger), AP , Oued Loukous; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous; Kettani and Langton 2012 Paratanytarsus tenellulus (Goetghebuer, 1921) = Microspsectra tenellula Reiss 1977 : 91; Azzouzi and Laville 1987 : 219 Reiss 1977 , MA , Lac Kranichsee; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Paratanytarsus tenuis (Meigen, 1830) = Tanytarsus tenuis Meigen, in Naya 1988 : 57 Naya 1988 , MA , Moyen Sebou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Rheotanytarsus Thienemann & Bause, 1913 Rheotanytarsus ceratophylli Dejoux, 1973 Naya 1988 , MA , Moyen et Bas Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Rheotanytarsus curtistylus (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oasis Meski (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus langtoni Moubayed & Kettani, 2018 Moubayed-Breil and Kettani 2018 , Rif , Oued Farda; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Rheotanytarsus muscicola Thienemann, 1929 Reiss 1977 , AP , Environ de Larache, AA , Dra-Tal (Tissint Moyen Dra); Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus nigricauda Fittkau, 1960 Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus pellucidus (Walker, 1818) = Rheotanytarsus distinctissimus (Brundin, 1947), in Kettani et al. 1995 : 258; Kettani et al. 1996 : 138, 1997 : 185; Kettani and Langton 2012 : 424 Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus pentapoda (Kieffer, 1909) = Rheotanytarsus sp 1, in Kettani et al. 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Rheotanytarsus photophilus (Goetghebuer, 1921) Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Rheotanytarsus procerus Reiss, 1991 Reiss 1991 , HA ; Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus reissi Lehmann, 1970 Lehmann, 1970; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus rhenanus Klink, 1983 Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus ringei Lehmann, 1970 Lehmann, 1970; Reiss 1977 , Rif , Environ Tétouan; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , Rif , Tétouan, MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus Pe 3 Langton 1991 Kettani et al. 2010 ; Kettani and Langton 2011 , Rif , Oued Sgara (Ketama, 1300 m); Kettani and Langton 2012 Stempellina Thienemann & Bause, 1913 Stempellina almi Brundin, 1947 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2012 Stempellina bausei (Kieffer, 1911) Kettani and Langton 2012 , Rif , Ketama; Kettani and Moubayed-Breil 2018 , Rif Stempellinella Brundin, 1947 Stempellinella brevis (Edwards, 1929) Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Tanytarsus van der Wulp, 1874 Tanytarsus brundini Lindeberg, 1963 Kettani et al. 1994 , Rif , Oued Moulay Bouchta, aval Oued Laou; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus chinyensis Goetghebuer, 1934 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Fifi (Fifi, 1179 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus cretensis Reiss, 1987 = Tanytarsus sp. nov. ( creticus ), in Reiss 1977 : 91; Azzouzi and Laville 1987 : 219 = Cladotanytarsus sp 1, in Kettani et al. 1995 : 258 Reiss and Fittkau 1971 ; Reiss 1977 , EM , Environ de Berkane; Reiss 1987 ; Azzouzi and Laville 1987 , Rif , Tétouan, AP , Larache, Kénitra; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsus dibranchius Kieffer, 1926 = Tanytarsus separabilis Brundin, 1947, in Kettani et al. 1994 : 29; Kettani et al. 1995 : 258, 1996 : 138, 2001 : 332; Dakki 1997 : 63; Kettani and Langton 2012 : 424 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsus ejuncidus (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Tanytarsus eminulus (Walker, 1856) Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus formosanus Kieffer, 1912 = Tanytarsus horni Goetghebuer, 1934, in Reiss and Fittkau 1971 : 122; Reiss 1977 : 91; Fittkau and Reiss 1978 : 439; Ramdani and Tourenq 1982 : 180; El Mezdi and Giudicelli 1985 : 292; Azzouzi and Laville 1987 : 219; Ashe and Cranston 1990 : 341; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 424 Reiss and Fittkau 1971 , Rif , M'Diq; Reiss 1977 , Rif , Environ Tétouan, AP , Larache, Rabat, Kénitra; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , HA , Oued Tensift; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus gregarius Kieffer, 1909 Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Tanytarsus heusdensis Goetghebuer, 1923 Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Ifansa, 105 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus mendax Kieffer, 1925 Kettani and Moubayed-Breil 2018 , Rif Tanytarsus medius Reiss & Fittkau, 1971 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus palettaris Verneaux, 1969 Kettani et al. 1994 , Rif , aval Oued Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus pallidicornis (Walker, 1856) Kettani and Langton 2011 , Rif , Dayat Fifi (Fifi, 1179 m), Oued El Hatba (SIBE Jebel Moussa, 165 m); Kettani and Langton 2012 Tanytarsus recurvatus Brundin, 1947 Kettani and Langton 2011 , Rif , Oued El Hamma (El Hamma, 240 m); Kettani and Langton 2012 Tanytarsus signatus (van der Wulp, 1859) = Tanytarsus Pe 5 Langton 1991, in Azzouzi and Laville 1987 : 219 Kügler and Reiss 1973 ; Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Aïn Rami (373 m), Dayat Amlay (258 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus verralli Goetghebuer, 1928 Kettani and Langton 2011 , Rif , Oued Taida (650 m); Kettani and Langton 2012 Tanytarsus volgensis Miseiko, 1967 = Tanytarsus fimbriatus Reiss & Fittkau, 1971, in Fittkau and Reiss 1978 : 439; Azzouzi and Laville 1987 : 219; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 424 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou, HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus Pe 14 Langton 1991 Kettani and Langton 2011 , Rif , source Issaguen (Ketama, 1600 m); Kettani and Langton 2012 Tanytarsus Pe 23 Langton 1991 Kettani and Langton 2011 , Rif , Oued El Hamma (El Hamma, 240 m); Kettani and Langton 2012 Virgatanytarsus Pinder, 1982 Virgatanytarsus albisutus (Santos-Abreu, 1918) = Virgatanytarsus maroccanus Kügler and Reiss, in Azzouzi and Laville 1987 : 219 Fittkau and Reiss 1978 ; Reiss and Schurch 1984 , AA , Dra-Tal; Reiss 1986 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia, AA , Dra-Tal; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Virgatanytarsus ansatus Reiss & Schürch, 1984 Reiss and Schurch 1984 , HA ; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Virgatanytarsus arduennensis (Goetghebuer, 1922) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Virgatanytarsus triangularis (Goetghebuer, 1928) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Virgatanytarsus Pe 1 Langton 1991 Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 Zavrelia Kieffer, Thienemann & Bause, 1913 Zavrelia pentatoma Kieffer & Bause, 1913 Kettani and Langton 2012 Zavrelia Pe 1 Langton, 1991 Kettani and Langton 2011 , Rif , Oued Berranda (Bouztate, 1259 m); Kettani and Langton 2012 Acknowledgment We gratefully acknowledge the invaluable assistance and cooperation of Patrick Ashe (Dublin, Ireland) who contributed greatly to the revision of this family. SIMULIIDAE K. Kettani Number of species: 43 . Faunistic knowledge of the family in Morocco: good Simulinae Prosimuliini Helodon Enderlein, 1921 Helodon laamii (Beaucournu-Saguez and Bailly-Choumara, 1981) Beaucournu-Saguez and Bailly-Choumara 1981 , Rif ; Clergue-Gazeau et al. 1991 ; Hervy et al. 1994 ; Belqat et al. 2001a ; Belqat 2002 ; Belqat and Dakki 2004 ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium Roubaud, 1906 Prosimulium hirtipes species group Bailly-Choumara and Beaucournu-Saguez 1981 : 53–54: groupe latimucro (species nova ?); Beaucournu-Saguez and Bailly-Choumara 1981 : 119: groupe latimucro , groupe tomosvaryi and groupe rufipes - hirtipes ; Clergue-Gazeau et al. 1991 : 54 as «sp. gr. Hirtipes » Prosimulium latimucro (Enderlein, 1925) 4 Bailly-Choumara and Beaucournu-Saguez 1981 ; Beaucournu-Saguez and Bailly-Choumara 1981 ; Giudicelli and Thiery 1985 , HA ; Giudicelli and Bouzidi 1989 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'Goum en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Adler and Belqat 2001 , Rif , Oued Iouchirene, Oued Ketama (Al Hoceima); Belqat et al. 2001a , Rif , HA ; Belqat and Adler 2001 , Rif , Aïn Khandek En Nâsser, Oued Iouchirene, Oued Ketama; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Koçak and Kemal 2010 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium rachiliense Djafarov, 1954 (complex) 5 Beaucournu-Saguez and Bailly-Choumara 1981 ; Adler and Belqat 2001 ; Belqat and Adler 2001 ; Belqat 2002 ; Belqat and Dakki 2004 ; Belqat et al. 2005 ; Belqat et al. 2008 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium tomosvaryi (Enderlein, 1921) Beaucournu-Saguez and Bailly-Choumara 1981 ; Giudicelli and Thiery 1985 , HA ; Giudicelli and Bouzidi 1989 , Giudicelli et al. 2000 ; Adler and Belqat 2001 , Rif , Oued Iouchirene (Al Hoceima); Belqat and Adler 2001 , Rif , Oued Ouringa Tamdâ, oued Iouchirene, Oued Mrinet, Oued Ketama, Aîn Ksour, Oued Tisgris, Aîn Sidi Brahim Ben Arrif, Oued Hannacha; Belqat et al. 2001a , Rif ; Belqat et al. 2001b ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Koçak and Kemal 2010 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Urosimulium Contini, 1963 Urosimulium faurei (Bernard, Grenier & Bailly-Choumara, 1972) Grenier et al. 1957 , MA ; Bernard et al. 1972 : 63–68 (original description), MA , Plateau de Talerhza (environ de Meknès); Clergue-Gazeau et al. 1991 , MA ; Hervy et al. 1994 ; Belqat and Adler 2001 , Rif , Oued Iouchirene, Oued Mrinet, Oued Biyada, Oued Hannacha, Oued Ankouda; Belqat et al. 2001a , Rif , MA ; Belqat 2002 , Rif , MA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simuliini Greniera Doby & David, 1959 Greniera fabri Doby & David, 1959 Clergue-Gazeau et al. 1991 , MA ; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Metacnephia Crosskey, 1969 Metacnephia blanci (Grenier & Théodoridès, 1953) = Cnephia sp. in Grenier 1953 : 157 = Cnephia blanci Grenier and Théodoridès, in Grenier and Théodoridès 1953 : 430–435 = Eusimulium latinum Rubzov, in Benhoussa et al. 1988 : 160–164 Grenier 1953 , HA ; Grenier and Théodoridès 1953 , HA ; Grenier et al. 1957 , MA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Clergue-Gazeau et al. 1991 , AA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane: 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Metacnephia nuragica Rivosecchi, Raastad & Contini, 1975 6 = Cnephia tredecimatum (Edwards), in Grenier et al. 1957 : 226 Grenier et al. 1957 , AP , Coastal meseta (region of Rabat); Belqat et al. 2001a , AP , Rabat; Belqat 2002 , AP , Rabat; Belqat and Dakki 2004 , AP , Rabat; Belqat et al. 2011 , AP ; Belqat et al. 2018 Simulium Latreille, 1802 Simulium ( Crosskeyellum ) gracilipes Edwards, 1921 Edwards 1921 : 143 (original description), MA ; Séguy 1925 : 233, MA ; Séguy 1930a , MA ; Grenier 1953 , MA ; Crosskey 1964 , MA , Fès; Grenier and Bailly-Choumara 1970 : 96–102 (original description of subgenus Crosskeyellum , description of gracilipes ), MA ; Clergue-Gazeau et al. 1991 , MA ; Hervy et al. 1994 ; Dakki 1997 ; Belqat et al. 2001a , MA ; Belqat 2002 , MA ; Belqat and Dakki 2004 , MA ; Belqat et al. 2011 , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) angustipes Edwards, 1915 Clergue-Gazeau et al. 1991 , MA , HA ; Dakki 1997 ; Belqat et al. 2001a , MA , HA ; Belqat 2002 , MA , HA ; Belqat and Dakki 2004 , MA , HA ; Dakki et al. 2008, MA , O. Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) mellah Giudicelli & Bouzidi, 2000 [in Giudicelli, Bouzidi and Abdelaali 2000] Giudicelli et al. 2000 : 63 (original description), HA , Oued Mellah (Bassin Draa); Belqat et al. 2001a , HA ; Belqat 2002 , MA , HA ; Belqat and Dakki 2004 , MA , HA ; Dakki et al. 2008, MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , MA , HA ; Adler et al. 2015 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) petricolum (Rivosecchi, 1963) = Simulium latizonum Bailly-Choumara and Beaucournu-Saguez, in Bailly-Choumara and Beaucournu-Saguez 1978 : 143–144 (misidentified); Bailly-Choumara and Beaucournu-Saguez 1981 : 53–54 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, Rif , MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , HA ; Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) rubzovianum (Sherban, 1961) Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) velutinum sensu stricto (Santos Abreu, 1922) = Eusimilium latinum Rubzov, in El Mezdi and Giudicelli 1985 : 292–295; Benhoussa et al. 1988 : 160–164 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; El Mezdi and Giudicelli 1985 , HA , Khettaras of Marrakech; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Clergue-Gazeau et al. 1991 , AA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) velutinum cytospecies '5' Adler et al. 2015 , Rif , Tanger-Anjra, HA , Marrakech; Belqat et al. 2018 Simulium ( Nevermannia ) ruficorne species group Simulium ( Nevermannia ) angustitarse (Lundström, 1911) Belqat et al. 2001a , Rif ; Belqat et al. 2001b , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) ibleum (Rivosecchi, 1966) Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) lundstromi (Enderlein, 1921) Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , Rif , Kanar (280 m), Majjo (905 m), 10 km before the Issaguen source (1200 m), HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) ruficorne Macquart, 1838 = Eusimulium ruficorne Macquart, in El Mezdi and Giudicelli 1985 : 292, 294–295 Grenier et al. 1957 , AA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; El Mezdi and Giudicelli 1985 , HA , Khettaras of Marrakech; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA , AA ; Belqat 2002 , Rif , HA , AA ; Crosskey et al. 2002; Belqat and Dakki 2004 , Rif , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , AP , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) vernum species group Simulium ( Nevermannia ) brevidens (Rubtsov, 1956) Clergue-Gazeau et al. 1991 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) carthusiense (Grenier & Dorier, 1959) Giudicelli and Dakki 1984, Rif ; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) costatum Friederichs, 1920 Grenier et al. 1957 , Rif , Pré-Rif, MA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) cryophilum (Rubtsov, 1959) (complex) = Simulium pusillum Fries, in Séguy 1930a : 52 (misidentification); Grenier 1953 : 159 (after Séguy) Séguy 1930a , HA ; Grenier 1953 , Rif , HA , Lac Ifni; Bouzidi and Giudicelli 1986 , HA ; Bouzidi and Giudicelli 1989, HA ; Clergue-Gazeau et al. 1991 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Giudicelli Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) toubkal (Bouzidi & Giudicelli, 1986) Bouzidi and Giudicelli 1986 : 41–52 (original description), HA , assif n'Ouarzane (Oued Nfis); Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) vernum Macquart, 1826 (complex) [ latipes authors pre-1972, not Meigen] Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Rubzovia ) knidirii (Giudicelli & Thiery, 1985) Giudicelli and Thiery 1985 : 109–123 (original description in new subgenus Simulium ( Crenosimulium ) , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Rubzovia ) lamachi (Doby & David, 1960) Giudicelli and Dakki 1984, Rif ; Giudicelli and Thiery 1985 , Rif ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane: 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) bezzii species group Simulium ( Simulium ) bezzii (Corti, 1914) (complex) = Simulium atlas Séguy, 1930, in Séguy 1930a : 50 (original description); Grenier 1953 : 158 (synonymy of atlas Séguy with bezzii suggested) Séguy 1930a , MA ; Grenier 1953 ; Grenier 1953 , MA , HA ; Grenier and Théodoridès 1953 ; Grenier et al. 1957 , AA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Bouzidi and Giudicelli 1986 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) ornatum species group Simulium ( Simulium ) egregium Séguy, 1930 Grenier 1930, HA ; Séguy 1930a : 51 (original description), HA ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) intermedium Roubaud, 1906 = Simulium reptans var. fasciatum Séguy, in Séguy 1930a : 52 (misidentification) = Simulium ornatum var. nitidifrons Edwards, in Grenier 1953 : 159, Grenier and Théodoridès 1953 : 441, Grenier and Faure 1957 [1956]: 840, Grenier and Bailly-Choumara 1970 : 102, Bailly-Choumara and Beaucournu-Saguez 1978 : 143–144 = Odagmia nitidifrons Edwards, in Giudicelli and Dakki 1984: 95, Benhoussa et al. 1988 : 160–164 = Simulium nitidifrons Edwards, in El Mezdi and Giudicelli 1985 : 292, 294–295 Séguy 1930a , HA ; Grenier 1953 , MA , HA ; Grenier and Théodoridès 1953 , MA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif, AP , S Rabat; MA , Plain of Meknès; Grenier et al. 1957 , Rif , Pré-Rif, HA ; Grenier and Bailly-Choumara 1970 , MA ; Bernard et al. 1972 , MA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Giudicelli and Dakki 1984, Rif , MA ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , MA , HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'oun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) ornatum Meigen, 1818 (complex) = reptans var fasciatum , in Séguy 1930: 52 [ subornatum : Séguy 1925 /1930, not Edwards] Séguy 1930a : 52 ( ornatum and subornatum records), HA ; Grenier 1953 , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Clergue-Gazeau et al. 1991 , MA , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) trifasciatum Curtis, 1839 Belqat et al. 2001a , Rif ; 2001b, Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) variegatum species group Bailly-Choumara and Beaucournu-Saguez (1981 : 52–54): groupe monticola ("sp. nova A" and "sp. nova B") Simulium ( Simulium ) atlasicum Giudicelli & Bouzidi, 1989 Giudicelli and Bouzid 1989: 146–151 (original description), HA , near village Aguelmous; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) berberum Giudicelli & Bouzidi, 1989 Giudicelli and Bouzidi 1989 : 151–156 (original description), HA , assif n'Ouarzane; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) variegatum Meigen, 1818 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Giudicelli and Bouzidi 1989 ; Clergue-Gazeau et al. 1991 ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif , HA ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) xanthinum Edwards, 1933 = Simulium gaudi Grenier and Faure, in Grenier and Faure 1957 [1956]: 838–840 Grenier and Faure 1957 [1956], Rif , Pré-Rif, HA ; Grenier et al. 1957 ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Clergue-Gazeau et al. 1991 , MA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Carles-Tolrá 2002 ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) albellum species group Simulium ( Trichodagmia ) auricoma Meigen, 1818 = Simulium ( Obuchovia ) auricoma Meigen, 1818, in Belqat et al. 2011 : 52 Belqat 2000 , Rif ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) galloprovinciale Giudicelli, 1963 [1962] = Simulium ( Obuchovia ) galloprovinciale Giudicelli, 1963, in Belqat et al. 2011 : 52 Belqat 2000 , Rif ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) marocanum Bouzidi & Giudicelli, 1988 [1987] = Simulium ( Obuchovia ) marocanum Bouzidi & Giudicelli, 1987, in Belqat et al. 2011 : 52 Bouzidi and Giudicelli 1987: 185–195 (original description), Rif , near village Bou Adel, HA , Oued Rdat (affluent de l'Oued Tensift); Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) equinum species group Simulium ( Wilhelmia ) equinum (Linnaeus, 1758) = Simulium equinum Linnaeus, in Grenier et al. 1957 : 231–232 Grenier et al. 1957 , MA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Dakki 1997 ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) pseudequinum Séguy, 1921 = Simulium barbaricum Séguy, in Séguy 1930a : 51 = Simulium equinum var. mediterraneum Puri, in Grenier 1953 : 145–148; Grenier and Théodoridès 1953 : 436 = Simulium equinum mediterraneum Puri, in Grenier and Faure 1957 [1956]: 840; Grenier et al. 1957 : 232–234 = Wilhelmia pseudequinum Séguy, in Benhoussa et al. 1988 : 160–164 Séguy 1930a , HA ; Grenier 1953 , HA ; Grenier and Théodoridès 1953 , HA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif, AP , HA , AA ; Meknès; Grenier et al. 1957 , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Clergue-Gazeau et al. 1991 , HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 Simulium ( Wilhelmia ) quadrifila Grenier, Faure & Laurent, 1957 [1956] Grenier et al. 1957 : 238–239 (original description as form of sergenti ), Rif , Pré-Rif, AP , S Casablanca, MA , Meknès, HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , MA , HA ; Clergue-Gazeau et al. 1991 , Rif , Pré-Rif; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif , AP , S Casablanca, MA , HA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) sergenti (Edwards, 1923) = Simulium ariasi Séguy, in Séguy 1925 : 231–238; Séguy 1930a : 50; Grenier 1953 : 144 = Simulium equinum mediterraneum Puri, in Grenier and Faure 1957 [1956]: 840; Grenier et al. 1957 : 238–240 = Wilhelmia sergenti Edwards, in Benhoussa et al. 1993 : 249 Séguy 1930a , MA ; Grenier 1953 , MA ; Grenier and Théodoridès 1953 , HA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif; Grenier et al. 1957 , Rif , Pré-Rif, AP , S Casablanca, MA , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , AP , MA , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Clergue-Gazeau et al. 1991 , Rif , Pré-Rif, HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 THAUMALEIDAE K. Kettani, R. Wagner Number of species: 2 . Expected: 10 Faunistic knowledge of the family in Morocco: poor Thaumalea Ruthe, 1831 Thaumalea bernardi Vaillant, 1956 Vaillant 1956b , HA , Toubkal, Siroua, Lac Tamhda (Anremer), Sidi Chamarouch, Izourar, M'Goum, Oukaimeden; Dakki 1997 Thaumalea spinata Vaillant, 1954 7 Vaillant 1954a , HA , M'Goum, springs powering d'Ameskeur el Fougani, springs powering Izourar lagoon (Azourki), springs powering the lake Tamhda (Anremer), torrent at the bottom of Jebel Siroua, Oukaimeden (Toubkal), Jebel Toubkal, Atend (Sidi Chamarouch) BLEPHARICERIDAE K. Kettani, P. Zwick Number of species: 4 Blepharicerinae Liponeura Loew, 1844 Liponeura alticola Giudicelli & Bouzidi, 1987 Giudicelli and Bouzidi 1987 , HA , Oued Réghaya; Dakki 1997 Liponeura megalatlantica (Vaillant, 1956) Vaillant 1956c , HA , Izourar, Imi-N'Ifri; Giudicelli and Lavandier 1974 ; Dakki 1997 Liponeura rifincola Zwick, 2013 Zwick 2013 , Rif , Issaguen (Ketama, 1800 m) Liponeura sirouana (Vaillant, 1956) = Cardiocrepsis sirouana Vaillant, in Vaillant 1956b : 234 Vaillant 1956b , HA , Siroua (3000 m); Giudicelli and Lavandier 1974 ; Dakki 1997 Bibionoidea ANISOPODIDAE 8 K. Kettani, J.-P. Haenni Number of species: 3 . Expected: 7–8 Faunistic knowledge of the family in Morocco: poor Anisopodinae Sylvicola Harris, 1780 Sylvicola fenestralis (Scopoli, 1763) 9 = Rhyphus fenestralis (Scopoli, 1763), in Mouna 1998 : 86 Mouna 1998 (no locality given) – MISR Sylvicola fuscatus (Fabricius, 1775) 1 = Rhyphus fuscatus (Fabricius, 1775), in Mouna 1998 : 86 Mouna 1998 (no locality given) – MISR Sylvicola punctatus (Fabricius, 1787) 1 = Rhyphus punctatus (Fabricius, 1787), in Mouna 1998 : 86 Mouna 1998 (no locality given) – MISR BIBIONIDAE 10 K. Kettani, J.-P. Haenni Number of species: 10 . Expected: 20 Faunistic knowledge of the family in Morocco: poor Bibioninae Bibio Geoffroy, 1762 Bibio hortulanus (Linnaeus, 1758) = Bibio hortularum Linnaeus, 1758, in Becker and Stein 1913 : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1949a , SA , Foum Zguid Bibio lanigerus Meigen, 1818 Ebejer et al. 2019 , Rif , Amsemlil ( PNPB , 1067 m) Bibio laufferi Strobl, 1906 Ebejer et al. 2019 , MA , 3.5 km S of Azrou (Ifrane, 1450 m), 20 km S of Azrou (Ifrane, 1720 m) Bibio leucopterus (Meigen, 1804) Ebejer et al. 2019 , MA , 6 km S of Azrou (Ifrane, 1610 m) Bibio marci (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Haenni 2009 , Rif , Tanger, MA , Ifrane Dilophus Meigen, 1803 Dilophus antipedalis Wiedemann in Meigen, 1818 Ebejer et al. 2019 , Rif , Oued Azla (Nwawel, 57 m), Oued Azla (Hallila, 95 m) Dilophus (cf. bispinosus Lundström, 1914) 11 Séguy 1953a , AA , Aïn Chaib (Souss) Dilophus febrilis (Linnaeus, 1758) = Dilophus vulgaris Meigen, 1818, in Séguy 1949a : 153 Séguy 1949a , AA , Goulimine, Foum-el-Hassan Dilophus femoratus Meigen, 1804 = Philia femorata Meigen, 1804, in Séguy 1941: 2 Séguy 1941d , HA , Tizi-n'Test (2000 m); Pârvu et al. 2006 , AP , Merja Zerga Dilophus tridentatus Walker, 1848 Ebejer et al. 2019 , AP , Sidi Mokhtar (Essaouira) – MHNN (J.-P. Haenni leg.), MNHN ( SA , Foum-el-Hassan) Sciaroidea CECIDOMYIIDAE K. Kettani, M. Skuhravá, V. Skuhravý Number of species: 57 . Expected: 100 Faunistic knowledge of the family in Morocco: moderate Lestremiinae Lestremia Macquart, 1826 Lestremia parvostylia Jaschhof, 1994 Jaschhof 1994 , SA , Abeino, 15 km N Goulimine; Papp 2007 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Micromyinae Campylomyza Meigen, 1818 Campylomyza flavipes Meigen, 1818 Jaschhof 1998 , HA , Telouet; Skuhravá et al. 2017 Campylomyza fusca Winnertz, 1853 Jaschhof 1998 , HA , Telouet; Skuhravá et al. 2017 Campylomyza mohrigi Jaschhof, 2009 Jaschhof 2009 (south Morocco); Gagné 2010 ; Skuhravá et al. 2017 Monardia Kieffer, 1895 Monardia ( Xylopriona ) toxicodendri (Felt, 1907) Jaschhof 1998 (South Morocco); Jaschhof 2009; Gagné 2010 ; Skuhravá et al. 2017 Cecidomyiinae Asphondylia Loew, 1850 Asphondylia capparis Rübsaamen, 1893 Houard 1921 , MA , Fès; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Asphondylia cytisi Frauenfeld, 1873 Mimeur 1949 , HA , Tanzat (1800 m), AA , Jebel Sargho, Amalou Bou Mansour (2000 m); Skuhravá et al. 2017 Asphondylia punica Marchal, 1897 = Asphondylia conglomerata De Stefani, 1900 Houard 1922 , EM , Zousfana (Jebel Tagla); Mimeur 1949 , AA , Agdz; Mouna 1998 ; Skuhravá et al. 2017 Asphondylia scrophulariae Schiner, 1856 Mimeur 1949 , AP , Rabat, Arcilla; Skuhravá et al. 2017 Asphondylia verbasci (Vallot, 1827) Mimeur 1949 , AP , Maâmora, Rabat, Zaërs; Skuhravá et al. 2017 Baldratia Kieffer, 1897 Baldratia salicorniae Kieffer, 1897 Mimeur 1949 , AP , Rabat, Salé, Bou-Regreg; Möhn 1966 , EM , Melilla, Bocona, AP , Rabat; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Bayeriola Gagné, 1991 Bayeriola thymicola (Kieffer, 1888) Houard 1923 , HA , Réghaya; Mimeur 1949 , EM , Sidi Ali Oujda, Jebel Hamra, Itzer, MA , Ifrane, Azrou, Bordj-Doumergue, Timhadite, Aguelmane; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 1993 ; Gagné 2010 (south of Morocco); Bruun et al. 2012 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Blastomyia Kieffer, 1913 Blastomyia origani (Tavares, 1901) Houard 1922 , MA , Col de Bouchtata, Zalagh (Mouret); Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 (south of Morocco); Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Braueriella Kieffer, 1896 Braueriella phillyreae (Löw, 1877) Houard 1922 , Rif , Jebel Kébir; Houard 1923 , AP , Cap Ghir (south of Morocco); Mimeur 1949 , Rif , Zoumi, Ouezzane, EM , Béni Snassen, AP , Larache, Zaërs, Mehdia, Sehoul, MA , Jebel Said, Taza, Tahala, Tadla; Mouna 1998 ; Skuhravá et al. 2017 – MNHN ( AP , Mehdia) Contarinia Rondani, 1860 Contarinia ilicis Kieffer, 1898 Houard 1919 , MA , Immouzer; Mimeur 1949 , EM , Béni Snassen, Ras Foughal, El-Harcha, MA , Ifrane, Aït Bou-Mzil, Monts Zaian, Agoumi-n´Aït Mguild; Skuhravá et al. 2017 Contarinia luteola Tavares, 1902 Mimeur 1949 , EM , El Harcha, MA , Ifrane, Djaba, Imouzzer-du-Kandar, Tafechna; Mouna 1998 ; Skuhravá et al. 2017 Contarinia nasturtii (Kieffer, 1888) Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 Contarinia pyrivora (Riley, 1886) = Diplosis pirivora Riley, in Mouna 1998 : 85 Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 Dasineura Rondani, 1840 Dasineura affinis (Kieffer, 1886) = Perrisia affinis (Kieffer), in Mouna 1998 : 85 Mimeur 1949 , Rif , Tanger, EM , Oujda, AP , Gharb, Port-Lyautey, Rabat, Fedala, Casablanca, Mazagan, Settat, Oued-Zem, Mogador, MA , Fès, Tahala, Marchand; Mouna 1998 ; Skuhravá et al. 2017 ; AP (Rabat) – MISR Dasineura asparagi (Tavares, 1902) Mimeur 1949 , AP , Rabat, Zaërs, HA , Chaouia; Skuhravá et al. 2017 Dasineura crataegi (Winnertz, 1853) Mimeur 1949 , EM , Chaouia des Béni Snassen, AP , Rabat, Zaërs, MA , Ifrane, Tahala Aguelmane de Sidi Ali, Fès, Agoumi-n´Aït Mguild, Tarhzirt, AA , Argana, Imi-n-Tanoute; Skuhravá et al. 2017 Dasineura ericaescopariae (Dufour, 1837) Houard 1912 , Rif , Cap Spartel; Rübsaamen 1899 ; Skuhravá et al. 2017 Dasineura helianthemi (Hardy, 1850) = Contarinia helianthemi (Hardy, 1850) Mimeur 1949 , AP , Gharb, Maâmora, Zaërs; Skuhravá et al. 2017 Dasineura napi (Loew, 1850) = Dasineura brassicae (Winnertz, 1853) in Mouna 1998 : 85 Mouna 1998 (no accurate locality); Skuhravá et al. 2017 Dasineura periclymeni (Rübsaamen, 1889) Mimeur 1949 , AP , Rabat, Chellah, Yquem, Grou, Korifla, EM , Berkane; Skuhravá et al. 2017 Dasineura plicatrix (Loew, 1850) Mimeur 1949 , EM , Massif des Béni Snassen, El-Harcha, AP , Larache, Korifla, Grou, Yquem, Malah, Rabat, MA , Azrou, Ifrane, Monts Zaian, Oulmés, Khénifra, HA , Tizi-Machou, AA , Bigoudine; Skuhravá et al. 2017 Dasineura rosae (Loew, 1850) = Wachtliella rosarum (Hardy, 1850) Mimeur 1949 , MA , Ifrane, Aguelmane de Sidi Ali, Aguelmane Azigza, Zaad, Bordj-Doumergue, Bekrite, HA , Tizi-Machou; Skuhravá et al. 2017 Dicrodiplosis Kieffer, 1895 Dicrodiplosis pseudococci (Felt, 1914) Harris 1968 , AP , Rabat, Salé, HA , Asni; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Dryomyia Kieffer, 1898 Dryomyia lichtensteinii (Löw, 1878) Mimeur 1949 , EM , Debdou, Ras Foughal, El-Harcha, MA , Taza, Oulmès, Azrou, Aguelmane Azigza, Ifrane, Imouzzer-du-Kandar, Tizi-n´Tretten, Ida-ou Tanane, Azilal, HA , Ayachi; Skuhravá et al. 1984 ; Skuhravá 1986 ; Oosterbroek 2007 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; MA (Oulmès) – MISR Etsuhoa Inouye, 1959 Etsuhoa thuriferae Skuhravá, 1996 Mimeur 1949 , HA , Sidi-Chamharouch, Aït Bou-Jafar; Skuhravá 1995 ; Skuhravá et al. 2017 Feltiella Rübsaamen, 1910 Feltiella acarisuga (Vallot, 1827) Gagné 1995 , AP , Rabat; Osborne et al. 2002 (productive areas of cereals); Gagné 2004 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Gephyraulus Rübsaamen, 1916 Gephyraulus diplotaxis (Solinas, 1982) Houard 1922 , EM , Oasis de Figuig, Jebel Ouazzani; Mimeur 1949 , AP , Vallée de l´Oued Korifla, Sidi Bouknadel; Skuhravá et al. 2017 Gephyraulus raphanistri (Kieffer, 1886) Houard 1923 , AP , Mogador; Mimeur 1949 , AP , Gharb, Skhirat, Bouznika; Skuhravá et al. 2017 Houardiella Kieffer, 1912 Houardiella salicorniae Kieffer, 1912 Trotter 1904 , Rif , Tingis; Houard 1912 , Rif , Tanger; Mimeur 1949 , AP , Loukous, Larache, Ksob, Ameur, Mogador, Rabat, Salé, Bou-Regreg, EM , Moulouya, AA , Agadir; Skuhravá et al. 2017 Iteomyia Kieffer, 1913 Iteomyia major (Kieffer, 1889) Mimeur 1949 , AP , Korifla, Grou, MA , Leghzel; Skuhravá et al. 2017 Jaapiella Rübsaamen, 1916 Jaapiella bryoniae (Bouché, 1847) Mimeur 1949 , Rif , Ouezzane, AP , Sidi Bouknadel, Mehdia, Temara, Skhirat, Pont-Blondin, Larache, Monod, Boulhaut, Safi, MA , Tahala, Fès, Khemisset, Marchand, AA , Agadir; Skuhravá et al. 2017 Lasioptera Meigen, 1818 Lasioptera berlesiana Paoli, 1907 Mimeur 1949 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Lasioptera rubi (Schrank, 1803) Mimeur 1949 , AP , Tinkert, MA , Ifrane, Taza, Tarhzirt, HA , N´Fis, Oumer-Rbia, Jebel Tardema; Skuhravá et al. 2017 Lasioptera thapsiae Kieffer, 1898 = Lasioptera carophila F. Löw, in Houard 1923 : 699 Houard 1921 ; Houard 1923 , Rif , Tanger; Mimeur 1949 , EM , Béni Snassen, Guercif, AP , Settat, MA , Tiddas; Mouna 1998 ; Skuhravá et al. 2017 Lestodiplosis Kieffer, 1894 Lestodiplosis aonidiellae Harris, 1968 Harris 1968 , EM , Oujda, AP , Rabat, MA , Fès; Skuhravá et al. 2017 Mayetiola Kieffer, 1896 Mayetiola avenae (Marchal, 1895) Mouna 1998 ; Lahloui et al. 2005, AP , Settat, Safi, El Jadida, MA , Béni Mellal, Khouribga, HA , Marrakech, El Kelaâ; Skuhravá et al. 2017 Mayetiola destructor (Say, 1817) Vayssière 1920 , EM , Oujda; Jourdan 1937 ; Hudault and Zelensky 1939 ; Mimeur 1949 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné et al. 1991 ; Amri et al. 1992 (productive areas of cereals); El Bouhssini et al. 1992a , 1992b ; Lhaloui et al. 1992 ; El Bouhssini et al. 1996a , 1996b ; Khalifi et al. 1996 ; Azzam et al. 1997 ; El Bouhssini et al. 1997 , 1999 ; El Bouhssini et al. 1998 ; Mouna 1998 ; Naber et al. 2000 , 2003 ; Lhaloui et al. 2001 ; Lhaloui et al. 2005 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; AP (Rabat), MA – MISR Mayetiola hordei Kieffer, 1909 Mouna 1998 ; Gagné et al. 1991 ; Lhaloui et al. 2005 , AP , Settat, Safi, El Jadida, MA , Béni Mellal, Khouribga, HA , Marrakech, El Kelaâ; Skuhravá et al. 2005 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Oligotrophus Latreille, 1805 Oligotrophus panteli Kieffer, 1898 Mimeur 1949 , EM , Béni Snassen, MA , Aguelmane Azigza, Azrou, HA , Ayachi, Aït Bou-Jafar; Skuhravá et al. 2017 Oligotrophus valerii (Tavares, 1904) = Arceuthomyia valerii (Tavares, 1904) Mimeur 1949 , HA , Ayachi, Aït Bou-Jafar; Skuhravá et al. 2017 Orseolia Kieffer & Massalongo, 1902 Orseolia cynodontis Kieffer & Massalongo, 1902 Houard 1922 , Rif , Tanger, Aïn Dalia ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Phyllodiplosis Kieffer, 1912 Phyllodiplosis cocciferae (Tavares, 1902) = Blastodiplosis cocciferae (Tavares, 1902) Trotter 1904 , Rif , Cap Spartel; Houard 1912 ; Mimeur 1949 , Rif , Cap Spartel, Ouezzane, EM , Béni Snassen, El-Harcha, AP , Larache, Maâmora, Boulhaut, Zaërs, MA , Taza, Djaba, Ifrane, Imouzzer-du-Kandar, Dayet Achlaf, Dayet Ifrah, Michlifen, Bordj-Doumergue, Zaad, Bekrite, Aguelmane Azigza, Agoumi-n´Aït Mguild, Bou-Mzil, HA , Bou-Jafar, Jebel Tardema; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 Psectrosema Kieffer, 1904 Psectrosema tamaricum (Kieffer, 1912) = Amblardiella tamaricum Kieffer, in Mouna 1998 : 85 Houard 1922 , EM , Zousfana, near Sidi Youssef; Mimeur 1949 , Morocco Mediterranean, continental and sub-saharan, EM , Berkane, Moulouya, AP , MA , Tadla, HA , Haouz, AA , Tafilalet, SA , Agad; Skuhravá et al. 1984 ; Skuhravá 1986 ; Mouna 1998 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Resseliella Seitner, 1906 Resseliella oleisuga (Targioni-Tozzetti, 1887) = Clinodiplosis oleisuga Targioni-Tozzetti, in Mouna 1998 : 85 Hafraoui 1966 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Mouna 1998 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Rhopalomyia Rübsaamen, 1892 Rhopalomyia navasi Tavares, 1904 Houard 1922 , EM , Jebel Mais, Djahifa, HA , Aït Ameli; Houard 1923 ; Mimeur 1949 , EM , Figuig, Jebel Nokra, Ifkern, Haute Moulouya, MA , Tadla; Skuhravá et al. 1993 ; Skuhravá et al. 2014b ; Skuhravá et al. 2017 Schizomyia Kieffer, 1889 Schizomyia buboniae (Frauenfeld, 1859) Houard 1917 , EM , Djorf de Taouriet; Houard 1922 , EM , Jebel Tagla, Aïn Yalon; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Stefaniella Kieffer, 1898 Stefaniella trinacriae De Stefani, 1900 Mimeur 1949 , AP , Oualidia, Zima, Casablanca; Skuhravá et al. 2017 Stefaniola Kieffer, 1913 Stefaniola africana Möhn, 1971 Mimeur 1949 , AP , Rabat, Salé, Bou-Regreg; Skuhravá et al. 2017 Stefaniola bilobata (Kieffer, 1913) Houard 1922 – 1923 ; Mimeur 1949 , MA , Tadla, HA , Ksar-es-Souk, AA , Haouz; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 1993 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Stefaniola opulenta Möhn, 1971 Möhn 1971 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; Pape and Thompson 2019 Stefaniola ventriosa Möhn, 1971 Möhn 1971 , MA , Oued Gheris; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Thecodiplosis Kieffer, 1895 Thecodiplosis brachyntera (Schwägrichen, 1835) Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 KEROPLATIDAE K. Kettani, P.J. Chandler Number of species: 2 . Expected: 50 Faunistic knowledge of the family in Morocco: poor Keroplatinae Keroplatus Bosc, 1792 Keroplatus reaumurii (Dufour, 1839) Matile 1986 ; Chandler et al. 2005 ; Evenhuis 2006 Macrocera Meigen, 1803 Macrocera fasciata Meigen, 1804 Becker and Stein 1913 , Rif , Tanger; Chandler and Ribeiro 1995 ; Evenhuis 2006 MYCETOPHILIDAE K. Kettani, P.J. Chandler Number of species: 64 . Expected: 250 Faunistic knowledge of the family in Morocco: poor Mycetophilinae Exechiini Allodiopsis Tuomikoski, 1966 Allodiopsis rustica Edwards, 1941 Banamar et al. 2020 , Rif , Dayat Tazia Anatella Winnertz, 1864 Anatella concava Plassmann, 1990 Banamar et al. 2020 , Rif , Oued Aârate Brevicornu Marshall, 1896 Brevicornu intermedium (Santos Abréu, 1920) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Tisgris, Oued Aârate, Dayat Tazia, Oued Maggou (Maggou Village), Douar Tizga, maison forestière de Talassemtane, Dayat Jebel Zemzem, Aïn Takhninjoute, Forêt Adrou, Dayat Amsemlil Brevicornu griseicolle Staeger, 1840 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Aârate, Dayat Tazia, Oued Maggou (Maggou Village), Aïn El Malaâb, maison forestière de Talassemtane, Douar Tizga, Aïn Takhninjoute, Dayat Amsemlil, Dayat Lemtahane, Dayat avant Taida Brevicornu sericoma (Meigen, 1830) Banamar et al. 2020 , Rif , Chefchaouen, Dayat Fifi, Forêt Jebel Lakraâ, Dayat Fifi, Oued Maggou (Maggou Village), maison forestière de Talassemtane, Douar Tizga, Oued Amsemlil, Grotte d'Hercule, Forêt Adrou, Cascade Chrafate, Oued Aârate, Bab el Karn, Dayat Amsemlil – MNHN (coll. J. Beaucournu) Brevicornu verralli (Edwards, 1925) Banamar et al. 2020 , Rif , Dayat Afersiw, Forêt Jebel Lakraâ Cordyla Meigen, 1803 Cordyla crassicornis Meigen, 1818 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Douar Abou Boubnar, maison forestière de Talassemtane, Forêt Adrou, Oued Sidi Yahia Aârab, EM , Oued Tafoughalt; Rif (Chefchaouen) – MNHN Cordyla insons Laštovka & Matile, 1974 Banamar et al. 2020 , Rif , Oued Maggou Cordyla murina Winnertz, 1864 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba Cordyla styliforceps (Bukowski, 1934) Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Oued Tkaraâ, Oued Sidi Yahia Aârab Exechia Winnertz, 1863 Exechia bicincta (Staeger, 1840) Banamar et al. 2020 , Rif , Oued Kelaâ, Oued Aârate, EM , Grotte des Pigeons, Oued Tafoughalt Exechia dorsalis (Staeger, 1840) Banamar et al. 2020 , Rif , Bab el Karn Exechia fulva Santos Abreu, 1920 = Rymosia exornata Séguy, in Séguy 1941a : 26 Séguy 1941a , HA , Toubkal; Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Oued Kelaâ, Forêt Aïn Boughaba, Forêt Jebel Lakraâ, Dayat Fifi, Aïn Sidi Brahim Ben Arrif, Oued Maggou (Maggou Village), Aïn Takhninjoute, maison forestière de Talassemtane, Oued Amsemlil, Dayat Jebel Zemzem, Aïn Takhninjoute, Bab el Karn, Dayat Fifi, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN ; Rif (20 km west of Targuist, coll. A.M. Hutson) – NHMUK Exechia fusca (Meigen, 1804) Banamar et al. 2020 , Rif , Oued Kelaâ, Forêt Jebel Lakraâ, Douar Kitane, Dayat Amsemlil Exechiopsis Tuomikoski, 1966 Exechiopsis coremura (Edwards, 1928) Banamar et al. 2020 , Rif , Cascade Chrafate Pseudexechia Tuomikoski, 1966 Pseudexechia tuomikoskii (Kjærandsen, 2009) Banamar et al. 2020 , Rif , Source Aheramen Rymosia Winnertz, 1864 Rymosia affinis Winnertz, 1864 Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Amsemlil, Aïn Tiouila, Dayat Fifi Rymosia beaucournui Matile, 1963 Chandler 1994 ; Chandler and Ribeiro 1995 ; Chandler et al. 2005 ; Banamar et al. 2020 , EM , Grotte des Pigeons; AP (Oued y Kern, coll. H. Choumara) – MNHN Rymosia pseudocretensis Burghele-Balacesco, 1966 Chandler 1994 ; Chandler et al. 2005 ; Banamar et al. 2020 ; AP (Oued y Kern, coll. H. Choumara) – MNHN Stigmatomeria Tuomikoski, 1966 Stigmatomeria crassicornis (Stannius, 1831) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Dayat Fifi, Aïn Takhninjoute, maison forestière de Talassemtane, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Tarnania Tuomikoski, 1966 Tarnania dziedzickii (Edwards, 1941) Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Amsemlil, Cascade Chrafate, Dayat Amsemlil, Grotte Aïn El-Aouda – MNHN Mycetophilini Mycetophila Meigen, 1804 Mycetophila alea Laffoon, 1965 Banamar et al. 2020 , Rif , Aïn el Ma Bared, maison forestière de Talassemtane, Oued Aârate, Bab el Karn; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila britannica Laštovka & Kidd, 1975 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, Forêt Jebel Lakraâ, Douar Kitane, Aïn El Malaâb, oued à 15 km de Fifi, Aïn El Ma Bared, maison forestière de Talassemtane, Oued Maggou (Maggou Village), Aïn Takhninjoute, Grotte d'Hercule, Oued Aârate, Bab el Karn, Dayat Amsemlil, Dayat Lemtahane, Dayat avant Taida, Forêt Taghzout – MNHN Mycetophila deflexa Chandler, 2001 Banamar et al. 2020 , Rif , Forêt Taghzout, route Ksar el Kebir–Chefchaouen Mycetophila edwardsi Lundström, 1913 Banamar et al. 2020 , Rif , Dayat Fifi, Dayat Tazia, Oued Tkarae, Forêt Jebel Lakraâ, Forêt Taghzout, Grotte d'Hercule, EM , Béni Snassen Mycetophila formosa Lundström, 1911 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Amsemlil, Dayat Amsemlil, Dayat avant Taida Mycetophila marginata Winnertz, 1864 Banamar et al. 2020 , Rif , Aïn Ras el Ma, Oued Maggou (Maggou Village), maison forestière de Talassemtane, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila perpallida Chandler, 1993 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, Aïn El Malaâb, maison forestière de Talassemtane, Bab el Karn, Dayat Amsemlil, Douar Kitane Mycetophila pictula Meigen, 1830 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Maggou (Maggou Village); Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila sordida van der Wulp, 1874 Chandler 1994 ; Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ; Oued Maggou (Maggou Village); MA (Khenolap-el-Ouaer, 1580 m, coll. J. Beaucournu) – MNHN Mycetophila spectabilis Winnertz, 1864 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Forêt Aïn Boughaba, oued à 15 km de Fifi, Aïn El Malaâb Mycetophila strigatoides (Landrock, 1927) Banamar et al. 2020 , Rif , Forêt Taghzout, Oued Aârate Mycetophila unicolor Stannius, 1831 Banamar et al. 2020 , Rif , Oued Kelaâ Mycetophila vittipes Zetterstedt, 1852 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Dayat Amsemlil Phronia Winnertz, 1863 Phronia biarcuata (Becker, 1908) Chandler 1994 ; Chandler and Ribeiro 1995 ; Chandler et al. 2005 ; Banamar et al. 2020 , Rif , Dayat Tazia, Aïn el Ma Bared, Aïn El Malaâb, Aïn Takhninjoute, maison forestière de Talassemtane, Grotte d'Hercule, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Phronia cinerascens Winnertz, 1864 Banamar et al. 2020 , Rif , Dayat Amsemlil Phronia nitidiventris (van der Wulp, 1858) Banamar et al. 2020 , Rif , Dayat Afersiw, Oued Aârate Phronia tenuis Winnertz, 1864 Banamar et al. 2020 , Rif , Oued Kelaâ, Oued Aârate, Oued Maggou (Maggou Village), Aïn Takhninjoute, Grotte d'Hercule Phronia tyrrhenica Edwards, 1928 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Dayat Amsemlil, Aïn Takhninjoute Phronia willistoni Dziedzicki, 1889 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Aârate, maison forestière de Talassemtane, Dayat Amsemlil, Cascade Chrafate, Bab el Karn Sceptonia Winnertz, 1864 Sceptonia intestata Plassmann & Schacht, 1990 Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Aïn El Malaâb Sceptonia membranacea Edwards, 1925 Banamar et al. 2020 , Rif , oued à 15 km de Fifi, Oued Aârate Trichonta Winnertz, 1864 Trichonta foeda Loew, 1869 Banamar et al. 2020 , Rif , Aïn Takhninjoute, Bab el Karn, Dayat Amsemlil, Oued Tkarae Trichonta icenica Edwards, 1925 Banamar et al. 2020 , EM , Grotte du Chameau Trichonta vitta (Meigen, 1830) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Bab el Karn Trichonta vulcani Dziedzicki, 1889 Banamar et al. 2020 , EM , Grotte du Chameau Zygomyia Winnertz, 1864 Zygomyia humeralis (Wiedemann, 1817) Banamar et al. 2020 , Rif , maison forestière de Talassemtane Zygomyia valida Winnertz, 1864 Banamar et al. 2020 , Rif , Aïn Ras el Ma, Oued Aârate Leiinae Docosia Winnertz, 1864 Docosia gilvipes (Walker, 1856) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Aïn Takhninjoute, Dayat Amsemlil Leia Meigen, 1818 Leia arsona Hutson, 1978 Banamar et al. 2020 , Rif , Oued Maggou (Maggou village), Oued Sidi Yahia Aârab, MA , Aïn Walili Leia beckeri Landrock, 1940 Banamar et al. 2020 , Rif , Aïn Ras el Ma Leia bimaculata (Meigen, 1804) Chandler 1994 ; Chandler et al. 2005 ; Banamar et al. 2020 , Rif , Dayat Tazia, maison forestière de Talassemtane, Aïn el Ma Bared, MA , Forêt 3.5 km S Azrou; MA (Forêt Ifrane, coll. P.N. Lawrence) – NHMUK Novakia Strobl, 1893 Novakia scatopsiformis Strobl, 1893 Banamar et al. 2020 , Rif , maison forestière de Talassemtane Novakia simillima Strobl, 1910 Banamar et al. 2020 , Rif , Oued Aârate, maison forestière de Talassemtane Gnoristinae Boletina Staeger, 1840 Boletina gripha Dziedzicki, 1885 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Aârate, Aïn Sidi Brahim Ben Arrif, Dayat Amsemlil, Grotte d'Hercule, Cascade Chrafate, Oued Sidi Yahia Aârab, Aïn el Ma Bared, Bab el Karn, MA , forêt 3.5 km S Azrou Coelosia Winnertz, 1864 Coelosia fusca Bezzi, 1892 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Kelaâ, Oued Amsemlil, Dayat Amsemlil, Aïn Takhninjoute, Cascade Chrafate, Bab el Karn, Dayat avant Taida Synapha Meigen, 1818 Synapha fasciata Meigen, 1818 Banamar et al. 2020 , Rif , Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Forêt Adrou, Dayat Amsemlil, Dayat avant Taida Synapha vitripennis (Meigen, 1818) Banamar et al. 2020 , Rif , Dayat Amsemlil Tetragoneura Winnertz, 1846 Tetragoneura ambigua Grzegorzek, 1885 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, EM , Oued Tafoughalt Mycomyinae Mycomya Rondani, 1856 Mycomya flavicollis (Zetterstedt, 1852) Banamar et al. 2020 , Rif , Aïn El Malaâb, maison forestière de Talassemtane Mycomya pygmalion Väisänen, 1984 Banamar et al. 2020 , Rif , Oued Amsemlil, Aïn Sidi Brahim Ben Arrif Mycomya tumida (Winnertz, 1864) Banamar et al. 2020 , Rif , Dayat Fifi Sciophilinae Azana Walker, 1856 Azana anomala Staeger, 1840 Banamar et al. 2020 , Rif , Oued Maggou (Maggou village), maison forestière de Talassemtane Sciophila Meigen, 1818 Sciophila iberolutea Chandler & Blasco-Zumeta, 2001 Chandler and Gatt 2000 , AP , Oued y Kern; Chandler and Blasco-Zumeta 2001 , AP , Oued y Kern; Bechev and Koç 2006 ; Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Jebel Zemzem, Oued Sidi Yahia Aârab, Bab el Karn, Marabout el Khaloua; AP (Oued y Kern, coll. H. Choumara) – MNHN SCIARIDAE K. Kettani, K. Heller Number of species: 70 . Expected: 200–500 Faunistic knowledge of the family in Morocco: poor Austrosciara Schmitz & Mjöberg, 1924 Austrosciara hyalipennis (Meigen, 1804) El Ouazzani et al. 2019 , Rif , Douar El Hamma, MA , Lac Ouiouane Bradysia Winnertz, 1867 Bradysia alpicola (Winnertz, 1867) El Ouazzani et al. 2019 , MA , Lac Ouiouane Bradysia bulbigera Mohrig & Kauschke, 1994 El Ouazzani et al. 2019 , Rif , Oued Ouara, Oued Ametrasse, Merzouk Bni Salah, Dayat Bayn widane, HA , Ouirgane Bradysia cavernicola Mohrig & Eckert, 1999 Menzel and Heller 2004 , HA , Ouirgane Bradysia cinerascens (Grzegorzek, 1884) El Ouazzani et al. 2019 , Rif , Issaguen, Anissar ( PNPB ) Bradysia crinita Mohrig, 1992 El Ouazzani et al. 2019 , Rif , Issaguen, Douar El Hamma Bradysia fenestralis (Zetterstedt, 1838) El Ouazzani et al. 2019 , Rif , Forêt R'milat Bradysia fenestrata (Meigen, 1818) El Ouazzani et al. 2019 , Rif , Jebel Zemzem, Oued Tkarâa, Ben Karrich, Dayat Tazia, Perdicaris Park, Tourbière Amsemlil, Dayat Tazia Bradysia flavipila Tuomikoski, 1960 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia iberiana Rudzinski & Baumjohann, 2009 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia lembkei Mohrig & Menzel, 1990 El Ouazzani et al. 2019 , Rif , Oued Maggou, Dayat Tazia, AP , Forêt Maâmora, HA , Ouirgane, Gerifodene Bradysia lucichaeta Mohrig & Krivosheina, 1989 Mohrig et al. 1997 , AA , Sidi Rbat (40 km S Agadir) Bradysia mediterranea Mohrig & Menzel, 1992 Mohrig and Menzel 1992 , HA Bradysia nigrispina Menzel, 2006 El Ouazzani et al. 2019 , HA , Gerifodene Bradysia pectoralis (Staeger, 1840) El Ouazzani et al. 2019 , MA , Lac Ouiouane, HA , Ouirgane Bradysia placida (Winnertz, 1867) El Ouazzani et al. 2019 , HA , Ouirgane Bradysia promissa Mohrig & Röschmann, 1999 El Ouazzani et al. 2019 , Rif , Beni Barou, Anissar ( PNPB ), Oued Tkarâa ( PNPB ), Taida, Marabout Moulay Abdelsalam Bradysia reflexa Tuomikoski, 1960 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia regularis (Lengersdorf, 1934) El Ouazzani et al. 2019 , Rif , Talassemtane (maison forestière), HA , Ouirgane Bradysia ruginosa Mohrig, 1994 Mohrig et al. 1997 , SA , Ablino (15 km N Goulimine); El Ouazzani et al. 2019 , Rif , Jebel Lakraâ, Aïn El Fakir, HA , Amizmiz Bradysia santorina Mohrig & Menzel, 1992 Mohrig et al. 1997 , AA , Sidi Rbat (40 km S Agadir) Bradysia scabricornis Tuomikoski, 1960 El Ouazzani et al. 2019 , Rif , Oued Ouara, Maggou, Oued Azla, Douar El Hamma, HA , Ouirgane, Setti Fatma, Oued Imlil Bradysia subrufescens Mohrig & Krivosheina, 1989 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia subsantorina Mohrig & Kauschke, 1997 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia tilicola (Loew, 1850) El Ouazzani et al. 2019 , Rif , Douar Tissouka, MA , Lac Ouiouane, HA , Ouirgane Bradysia transitata Rudzinski & Baumjohann, 2013 El Ouazzani et al. 2019 , Rif , Oued Ez-Zarka, Oued Tkarâa ( PNPB ), Oued Laou, AP , Larache (Strawberry farm), Forêt Maâmora, AA , Barrage Aoulouz, Assif Tifnout Bradysia trivittata (Staeger, 1840) Mohrig and Röschmann 1993, HA , AA , Sidi Rbat (40 km S Agadir); El Ouazzani et al. 2019 , Rif , Aïn Tayattine, Oued Ez-Zarka, Oued Bayine, Beni Barou, Douar El Hamma, Douar Mouarâa, Tétouan, MA , Lac Ouiouane, HA , Télouet, Ouirgane Bradysia vagans (Winnertz, 1868) misidentified as Bradysia rufescens (Zetterstedt, 1852) in Röschmann and Mohrig 1993 : 111 Röschmann and Mohrig 1993 , HA , Talouete; El Ouazzani et al. 2019 , Rif , Aïn Fouara, HA , Ouirgane Bradysia xenoreflexa Mohrig & Menzel, 1993 El Ouazzani et al. 2019 , AP , Forêt Maâmora Bradysiopsis Tuomikoski, 1960 Bradysiopsis vittata (Meigen, 1830) El Ouazzani et al. 2019 , HA , Setti Fatma Camptochaeta Hippa & Vilkamaa, 1994 Camptochaeta jeskei (Mohrig & Röschmann, 1993) = Corynoptera jeskei Mohrig and Röschmann, in Röschmann and Mohrig 1993 : 109 Röschmann and Mohrig 1993 , HA , Talouete (1800 m) Corynoptera Winnertz, 1867 Corynoptera andalusica Hippa, Vilkamaa & Heller, 2010 El Ouazzani et al. 2019 , MA , Lac Ouiouane, HA , Ouirgane Corynoptera bicuspidata (Lengersdorf, 1926) Hippa et al. 2010 , HA , Ouirgane, Lac Ouiouane Corynoptera bispinulosa Mohrig & Dimitrova, 1992 El Ouazzani et al. 2019 , EM , Tafoughalt Corynoptera caesula Hippa & Menzel, 2004 El Ouazzani et al. 2019 , Rif , Aïn Kchour, AP , Forêt Maâmora Corynoptera cincinnata Mohrig & Blasco-Zumeta, 1996 Hippa et al. 2010 , HA , Ouirgane Corynoptera dentiforceps (Bukowski & Lengersdorf, 1936) El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera deserta Heller & Menzel, 2006 El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera fatigans (Johannsen, 1912) = Corynoptera perpusilla Winnertz, 1867, in Mohrig et al. 1997 : 384 Mohrig et al. 1997 , HA , Anezal; Hippa et al. 2010 [for nomenclature see Mohrig et al. 2013 ] Corynoptera gemina (Hippa & Vilkamaa, 1994) El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera globiformis (Frey, 1945) El Ouazzani et al. 2019 , Rif , Talassemtane (maison forestière) Corynoptera hemiacantha Mohrig & Mamaev, 1992 El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera iberica Hippa, Vilkamaa & Heller, 2010 El Ouazzani et al. 2019 , AP , Forêt Maâmora, Sidi Boughaba Corynoptera inclinata Hippa, Vilkamaa & Heller, 2010 Hippa et al. 2010 , HA , Ouirgane Corynoptera irmgardis (Lengersdorf, 1930) Hippa et al. 2010 , HA , Ouirgane Corynoptera postglobiformis Mohrig, 1993 El Ouazzani et al. 2019 , Rif , Talassemtane (maison forestière), HA , Ouirgane Corynoptera praeparvula Mohrig & Krivosheina, 1983 El Ouazzani et al. 2019 , HA , Ouirgane, Amizmiz, Gerifodene Corynoptera saccata Tuomikoski, 1960 Hippa et al. 2010 , SA , Goulimine; Mohrig et al. 2012 Corynoptera semipedestris Mohrig & Blasco-Zumeta, 1996 Mohrig et al. 1997 , SA , Ablino (15 km N Goulimine) Corynoptera spiciceps Hippa, Vilkamaa & Heller, 2010 Hippa et al. 2010 , HA , Ouirgane Corynoptera stipidaria Mohrig, 1994 Hippa et al. 2010 , HA , Ouirgane Corynoptera subcavipes Menzel & Smith, 2007 El Ouazzani et al. 2019 , Rif , Douar El Hamma, Talassemtane (maison forestière) Corynoptera subparvula Tuomikoski, 1960 El Ouazzani et al. 2019 , HA , Ouirgane Epidapus Haliday, 1851 Epidapus atomarius (De Geer, 1778) El Ouazzani et al. 2019 , Rif , Aïn el Ma Bared (Fifi) Leptosciarella Tuomikoski, 1960 Leptosciarella dives (Johannsen, 1912) Mohrig et al. 2012 , HA , Ouirgane Leptosciarella parcepilosa (Strobl, 1900) El Ouazzani et al. 2019 , AP , Sidi Boughaba Leptosciarella subviatica Mohrig & Menzel, 1997 El Ouazzani et al. 2019 , HA , Ouirgane Leptosciarella tomentosa (Mohrig & Kauschke, 1994) El Ouazzani et al. 2019 , AP , Forêt Maâmora Lycoriella Frey, 1942 Lycoriella agraria (Felt, 1898) El Ouazzani et al. 2019 , Rif , Douar Tissouka Lycoriella sativae (Johannsen, 1912) El Ouazzani et al. 2019 , Rif , M'Diq, Oued Zaouya, AP , Larache (strawberry farm), HA , Ouirgane Pseudolycoriella Menzel & Mohrig, 1998 Pseudolycoriella morenae (Strobl, 1900) El Ouazzani et al. 2019 , Rif , Perdicaris Park, Bab Tariouant, Oued Maggou, EM , Zegzel, HA , Ouirgane Scatopsciara Edwards, 1927 Scatopsciara ( Scatopsciara ) atomaria (Zetterstedt, 1851) = Scatopsciara vivida (Winnertz, 1867), in Röschmann and Mohrig 1993 : 111 Röschmann and Mohrig 1993 , HA , Talouete; El Ouazzani et al. 2019 , Rif , Oued Ez-Zarka, Oued Tkarâa ( PNPB ), Oued Guallet, Marécage Lemtahane ( PNPB ), Oued Ametrasse, Issaguen, Talassemtane (maison forestière), Douar El Hamma, Aïn Fouara, Oued Souk El Had, Merzouk Bni Salah, Oued Maggou, AP , Sidi Boughaba, HA , Assif Tifnout, Gerifodene, Armed, Amzmiz, Lac Tislit, AA , Barrage Mokhtar Soussi Scatopsciara ( Scatopsciara ) maroccoensis Mohrig & Jaschhof, 1997 Mohrig et al. 1997 , AA , Sidi Rbat (40 km S Agadir) Scatopsiara ( Scatopsciara ) nana (Winnertz, 1871) El Ouazzani et al. 2019 , Rif , Oued Ez-Zarka, Oued Maâmala, Oued Aârkob, Ben Karrich, Merja Sidi Lhaj Merzouk, Tétouan, HA , Ouirgane, Anafgou Scatopsciara ( Scatopsciara ) vitripennis (Meigen, 1818) El Ouazzani et al. 2019 , Rif , Oued Ouarra, Oued Tkarâa ( PNPB ), Oued Aoudour, Oued Ametrasse, Oued Aârkob, Oued Boumarouil, Ben Karrich, Dayat Tazia, Aïn El Fakir, Azib de Khmis Mdik, Merzouk Bni Salah, Oued Souk El Had, Oued Maggou, El Malâab (Talassemtane), AP , Sidi Boughaba, MA , Lac Ouiouane, HA , Ouirgane, AA , Barrage Aoulouz, Assif Tifnout, Barrage Mokhtar Soussi Scatopsciara ( Xenopygina ) curvilinea (Lengersdorf, 1934) El Ouazzani et al. 2019 , AP , Sidi Boughaba, HA , Aïn Taferaout, Amzmiz, AA , Assif Tifnout, Barrage Mokhtar Soussi Scatopsciara ( Xenopygina ) subarmata Mohrig & Mamaev, 1983 El Ouazzani et al. 2019 , Rif , Oued Amsa, AP , Larache, MA , Mont Habri, HA , Ouirgane, AA , Id Aissa, Tissint Schwenckfeldina Frey, 1942 Schwenckfeldina carbonaria (Meigen, 1830) = Sciara carbonaria Meigen, in Séguy 1941d : 2 Séguy 1941d , HA , Tizi-n'Test (2000 m) Sciara Meigen, 1803 Sciara flavimana Zetterstedt, 1851 El Ouazzani et al. 2019 , Rif , Douar El Hamma Sciara hemerobioides (Scopoli, 1763) = Lycoria ( Sciara ) thomae Linnaeus, in Becker and Stein 1913 : 85 Becker and Stein 1913 , Rif , Tanger Tipuloidea LIMONIIDAE K. Kettani, P. Oosterbroek Number of species: 67 . Expected: 85 Faunistic knowledge of the family in Morocco: moderate Chioneinae Baeoura Alexander, 1924 Baeoura ebenina Starý, 1981 Driauach and Belqat 2015 , Rif , Oued Tazarine (Mezine village); Dayat near Aïn Afersiw; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: Rif ) Baeoura staryi Driauach & Belqat, 2015 Driauach and Belqat 2015 , Rif , Jnane Niche; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: Rif ) Cheilotrichia Rossi, 1848 Cheilotrichia ( Empeda ) cinerascens (Meigen, 1804) Driauach et al. 2013 , Rif , Ikadjiouen; Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Amsa, Oued Maggou, Aïn Quanquben (Jebel Bou Bessoui), EM , Grotte du Chameau (Zegzel, Béni Snassen); Oosterbroek 2020 (CCW: Rif ; EM ) Cheilotrichia ( Empeda ) fuscohalterata (Strobl, 1906) Driauach and Belqat 2016 , Rif , Dayat Fifi, tributary of Oued El Fondak, Barrage Ajras, Dayat Mghara, Oued Zarka, Oued Tizekhte; Oosterbroek 2020 (CCW: Rif ) Cheilotrichia ( Empeda ) minima (Strobl, 1898) Driauach and Belqat 2016 , Rif , Oued Zendoula, Oued Jnane Niche; Oosterbroek 2020 (CCW: Rif ) Ellipteroides Becker, 1907 Ellipteroides ( Ellipteroides ) lateralis (Macquart, 1835) = Gonomyia lateralis Macquart, in Pierre 1922a : 22, Dakki 1997 : 62 = Gonomyia cincta Egger, in Séguy 1941a : 26 Pierre 1922a , AP , Dradek (near Rabat); Lackschewitz 1940a , HA , Tachdirt (2200–2900 m); Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 ; Dakki 1997 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Dayat Afrate; Oosterbroek 2020 (CCW: Rif ) Ellipteroides ( Protogonomyia ) alboscutellatus (von Roser, 1840) Lachschewitz 1940a, HA ; Savchenko et al. 1992 ; Starý and Oosterbroek 2008 ; Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Ellipteroides ( Protogonomyia ) hutsoni (Starý, 1971) Starý 1971 , HA , Jebel Ayachi; Savchenko et al. 1992 , HA , Jebel Ayachi; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Erioconopa Starý, 1976 Erioconopa diuturna (Walker, 1848) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Dayat Jebel Zemzem, Oued Mezine, Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ) Erioconopa symplectoides (Kuntze, 1914) = Dactylolabis symplectoides Egger, in Pierre 1922a : 23 Pierre 1922a , HA , Marrakech; Savchenko et al. 1992 ; Starý and Oosterbroek 2008 , HA ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Erioptera Meigen, 1803 Erioptera ( Erioptera ) fuscipennis Meigen, 1818 Pierre 1922a , AP , Casablanca (Oued Guerera); Pierre 1922b , HA , Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 , Rif , Sidi Brahim Ben Arrif (Bab Hachef Aissa); Driauach and Belqat 2016 , Rif , Aïn El Ma Bared, Oued Amsemlil, Oued Maggou (Bridge), Oued Tkarae, Dayat near Aïn Afersiw, Aïn Afersiw, Oued Abou Bnar, Oued Maggou (Zaouiet El Habtiyne), Dayat Amsemlil, Dayat Aïn Jdi­oui; Dayat Afrate, Oued Mezine; Oosterbroek 2020 (CCW: Rif , HA ) Erioptera ( Erioptera ) lutea lutea Meigen, 1804 Driauach and Belqat 2016 , Rif , Aïn Boughaba; Oosterbroek 2020 (CCW: Rif ) Erioptera ( Erioptera ) transmarina Bergroth, 1889 = Mesocyphona transmarina Bergroth, in Pierre 1922a : 22, Pierre 1922b : 148, Dakki 1997 : 62 Pierre 1922a , HA , Marrakech; Pierre 1922b , HA , Tannaout (1000 m), Marrakech; Dakki 1997 ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Gonomyia Meigen, 1818 Gonomyia ( Gonomyia ) abscondita Lackschewitz, 1935 Driauach and Belqat 2016 , Rif , maison forestière; Oosterbroek 2020 (CCW: Rif ) Gonomyia ( Gonomyia ) sicula Lackschewitz, 1940 Driauach and Belqat 2016 , Rif , Oued Kbir, Dayat Jebel Zemzem, Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Aïn El Maounzil; Oosterbroek 2020 (CCW: Rif ) Gonomyia ( Gonomyia ) subtenella Savchenko, 1972 Starý and Oosterbroek 2008 , HA , Massif Toubkal, Aghbalou, 43 km S Marrakech (1000 m); Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Jnane Niche, Oued Sidi Yahia Aârab, EM , Oued Béni Ouachekradi; Oosterbroek 2020 (CCW: Rif , EM ) Gonomyia ( Gonomyia ) tenella (Meigen, 1818) Pierre 1924a , HA , Asni (1200 m); Séguy 1930a , HA , Asni; Savchenko et al. 1992 ; Dakki 1997 ; Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis Osten-Sacken, 1869 Hoplolabis ( Parilisia ) obtusiapex (Savchenko, 1982) Starý 2006 , HA , Oasis Meski, AA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis ( Parilisia ) punctigera (Lackschewitz, 1940) Starý 2006 , HA , Oasis Meski, AA ; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis ( Parilisia ) sororcula (Lackschewitz, 1940) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Barrage Ajras, Oued Ouringa, Oued El Kanar, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Idiocera Dale, 1842 Idiocera ( Euptilostena ) jucunda (Loew, 1873) = Gonomyia jucunda Loew, in Pierre 1924a : 201, Séguy 1930a : 22 Pierre 1924a , HA , Asni (1200 m); Séguy 1930a , HA , Asni; Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Idiocera ( Idiocera ) ampullifera (Starý, 1979) Driauach and Belqat 2016 , AA , Oued Zag; Oosterbroek 2020 (CCW: AA ) Idiocera ( Idiocera ) pulchripennis (Loew, 1856) = Gonomyia sexpunctata Dale, in Pierre 1922b : 148 = Gonomyia pulchripennis (Loew), in Dakki 1997 : 62 Pierre 1922b , AP , Atlantic coast; Ramdani 1981 , AP , Merja Sidi Boughaba; Savchenko et al. 1992 ; Dakki 1997 ; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Jnane Niche, Oued Aârkoub, Aïn Jdioui; Oosterbroek 2020 (CCW) Idiocera ( Idiocera ) sziladyi (Lackschewitz, 1940) Driauach and Belqat 2016 , Rif , Oued Zarka; Oosterbroek 2020 (CCW: Rif ) Ilisia Rondani, 1856 Ilisia maculata (Meigen, 1804) Driauach and Belqat 2016 , Rif , Oued Maggou, EM , Oued Tafoughalt; Oosterbroek 2020 (CCW: Rif ; EM ) Molophilus Curtis, 1833 Molophilus ( Molophilus ) ibericus Starý, 2011 Starý 2011 , HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Molophilus ( Molophilus ) obscurus (Meigen, 1818) Starý and Oosterbroek 2008 , HA , Massif Toubkal, Oukaimeden, 2500–2800 m; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , tributary of Oued Ouara, tributary of Oued Maggou, Dayat El Ânassar, Dayat Jebel Zemzem, Aïn El Ma Bared, Dayat Rmali, Oued Amsemlil, Aïn Mâaze, Dayat Afrate, Dayat Lemtahane; Oosterbroek 2020 (CCW: Rif ) Molophilus ( Molophilus ) propinquus propinquus (Egger, 1863) Savchenko et al. 1992 ; Starý and Oosterbroek 2008 , HA , Oukaimeden (2500–2800 m); Starý 2011 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Farda, tributary of Oued Taida, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Molophilus ( Molophilus ) testaceus Lackschewitz, 1940 Driauach and Belqat 2016 , Rif , Dayat Amsemlil, Dayat Lemtahane, Marj El kheyl, Oued Tkarae, Dayat near Aïn Afersiw, Dayat Mezine, Dayat Tazia, Dayat Rmali, Dayat Amsemlil, Dayat near Aïn Afersiw; Oosterbroek 2020 (CCW: Rif ) Symplecta Meigen, 1830 Symplecta ( Symplecta ) grata Loew, 1873 Ebejer et al. 2020, Rif , Aïn Jdioui (Tahaddart, 8 m) Symplecta ( Symplecta ) hybrida (Meigen, 1804) Lackshewitz 1940a, HA , Goundafa (1200 m); Séguy 1941a , HA , Taroudant; Savchenko et al. 1992 ; Oosterbroek et al. 2007; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Barrage Ajras, Oued El Kanar, Oued Aârkoub, Oued Jnane Niche, MA , Barrage Allal El Fassi; Oosterbroek 2020 (CCW: Rif ; MA ) Symplecta ( Trimicra ) pilipes (Fabricius, 1787) = Trimicra pilipes Fabricius, in Pierre 1922a : 23 = Trimicra andalusiaca Strobl, in Pierre 1922b : 149 = Trimicra hirsutipes Macquart, in Séguy 1930a : 22 Pierre 1922a , AP , Dradek (near Rabat), HA , Marrakech; Pierre 1922b , MA , Volubilis, HA , Oued Tensift; Séguy 1930a ; Séguy 1941d , AA , Taroudant; Savchenko et al. 1992 ; Dakki 1997 ; Pârvu and Zaharia 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , tributary of Oued Hachef, Oued Aârk­ob, Oued El Kanar, Oued Mezine, AP , Aïn Chouk (Larache); Oosterbroek 2020 (CCW: Rif ) Tasiocera ( Dasymolophilus ) murina (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued Farda, Oued Amsemlil; Oosterbroek 2020 (CCW: Rif ) Dactylolabinae Dactylolabis Osten-Sacken, 1860 Dactylolabis ( Dactylolabis ) symplectoidea Egger, 1863 Pierre 1922a , AP , Around Casablanca (coastal meseta); Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limnophilinae Austrolimnophila Alexander, 1920 Austrolimnophila ( Austrolimnophila ) latistyla Starý, 1977 Driauach and Belqat 2016 , Rif , Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Dicranophragma Osten-Sacken, 1860 Dicranophragma ( Brachylimnophila ) adjunctum (Walker, 1848) = Neolimnomyia adjuncta (Walker, 1848), in Pârvu et al. 2006 : 273 Pârvu et al. 2006 , AP , Merja Zerga; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranophragma ( Brachylimnophila ) nemorale (Meigen, 1818) Lackschewitz 1940b , HA , Tachdirt (2200–2700 m); Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Aïn Sidi Brahim Ben Arrif, tributary Oued Ouara, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Eloeophila Rondani , 1856 Eloeophila maroccana Starý, 2009 Starý 2009b , HA , Okaïmeden (2500–2800 m); Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Euphylidorea Alexander, 1972 Euphylidorea ( Euphylidorea ) crocotula (Séguy, 1941) = Phylidorea crocotula (Séguy), in Séguy 1941a : 28 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 , HA , Tachdirt (Toubkal); Starý and Oosterbroek 2008 , HA , Oukaimeden (2500 m–2800 m), Imlil (1400 m); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ; HA ) Euphylidorea ( Euphylidorea ) dispar (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued at 15 km from Fifi; Oosterbroek 2020 (CCW: Rif ) Euphylidorea ( Euphylidorea ) lineola (Meigen, 1804) Lackschewitz 1940b , HA ; Savchenko et al. 1992 ; Starý and Freidberg 2007 ; Driauch et al. 2013; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hexatoma Latreille, 1809 Hexatoma ( Hexatoma ) bicolor (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued at 15 km from Fifi, tributary of Oued Ouara, Oued Madissouka, Oued Maggou, Aïn Quanquben (Jebel Bou Bessoui), Oued Tkarae, Oued Tamerte; Oosterbroek 2020 (CCW: Rif ) Hexatoma ( Hexatoma ) gaedii (Meigen, 1830) Lackschewitz 1940b , HA , Tachdirt (2200 m–2700 m); Savchenko et al. 1992 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Pseudolimnophila Alexander, 1919 Pseudolimnophila ( Pseudolimnophila ) sepium (Verrall, 1886) Driauach et al. 2013 , Rif , Guelta Tazia; Driauach and Belqat 2016 , Rif , Dayat Tazia, Aïn Sidi Brahim Ben Ar­rif, Aïn Afersiw, Oued Maggou, Dayat Tazia, Oued Taida, Aïn Sidi Brahim Ben Arrif; Oosterbroek 2020 (CCW: Rif ) Limoniinae Dicranomyia Stephens, 1829 Dicranomyia ( Dicranomyia ) affinis (Schummel, 1829) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Amsemlil, Dayat Lemtahane, Oued Tkarae, Marj El Kheyl, Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Aïn El Maounzil, Aïn El Malâab, Dayat near Aïn Afersiw, Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) chorea (Meigen, 1818) Pierre 1922b , HA , Haute Réghaya, Tannaout, Asni (1000 m–1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Farda, Oued Kelâa, Aïn Ras el Ma, Oued Jnane Niche, Cascade Chrafate, maison forestière, Oued El Kanar, tributary of Oued Zarka, HA , Oued Sidi Fares (National Park of Toubkal); Oosterbroek 2020 (CCW: Rif ; HA ) Dicranomyia ( Dicranomyia ) didyma (Meigen, 1804) Pierre 1922b , HA , Haute Réghaya (2000 m); Dakki 1997 ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Dicranomyia ( Dicranomyia ) goritiensis (Mik, 1864) Lackschewitz 1940b ; Vaillant 1956b ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Maggou, Cascade Chrafate, Oued El Koub, Cascade Zarka, Âounsar Aheramen, maison forestière; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) longicollis (Macquart, 1846) = Telecephala longicollis (Macquart), in Pierre 1922a : 21, Pierre 1922b : 148, Séguy 1930a : 22 Séguy 1930a , AP , Dradek (near Rabat); Dakki 1997 ; Pierre 1922a , AP , Dradek, HA , Marrakech; Pierre 1922b , AP , Rabat; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Aârate, Barrage Moulay Bouchta, Oued Kbir, MA , Barrage Allal El Fassi; Oosterbroek 2020 (CCW) Dicranomyia ( Dicranomyia ) mitis (Meigen, 1830) = Dicranomyia hygropetrica Vaillant, in Vaillant 1956b : 42 Vaillant 1956b , HA , Asif Tessaout (M'Goum), Izourar, Tahanaout, Tamesrit, Imi-N'Ifri, Aguelmous, Sidi Chamarouch, Lac Tamhda (Anremer), Oukaimeden; Savchenko et al. 1992 ; Pârvu et al. 2006 , AA , near Agadir, Souss plain to High Atlas occidental; Pârvu and Zaharia 2007 ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 , Rif , Tazia, Tisgris; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia ( Dicranomyia ) modesta (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued Farda, Oued El Kanar, Oued Jnane Niche, Oued Sidi Yahia Aârab; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) novemmaculata (Strobl, 1906) Driauach and Belqat 2016 , Rif , tributary of Oued el Fondak, Oued Aârate; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) ventralis (Schummel, 1829) Driauach and Belqat 2016 , Rif , Lake Badriouen; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Glochina ) sericata (Meigen, 1830) Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia ( Melanolimonia ) hamata Becker, 1908 Driauach and Belqat 2016 , Rif , tributary of Oued Kbir, Oued Aârate, Aïn Sidi Brahim Ben Arrif, Dayat Tazia; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Melanolimonia ) morio (Fabricius, 1787) = Limonia pauliani Séguy, in Séguy 1941a : 26 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 , HA , Tachdirt (Toubkal); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Seguia Lemtahane, Dayat near Aïn Afersiw, Dayat Jebel Zemzem; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia majuscula Pierre, 1924 1 Pierre 1924a , HA , Haut Imminen (2400 m); Séguy 1930a , HA , Haut Imminen; Dakki 1997 ; Savchenko et al. 1992 , HA , Haut Imminen; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranoptycha Osten-Sacken, 1860 Dicranoptycha fuscescens (Schummel, 1829) Eiroa 2000 , Rif ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Mlilah, Oued Zendoula, AP , Oued Loukous; Oosterbroek 2020 (CCW) Geranomyia Haliday, 1833 Geranomyia caloptera (Mik, 1867) Driauach et al. 2013 , HA , Setti Fatma; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Geranomyia obscura Strobl, 1900 Vaillant 1956b , HA , Lac Tamhda (Anremer), Oukaimeden, Izourar, Sidi Chamarouch, Tamesrit; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Helius Lepeletier & Serville, 1828 Helius ( Helius ) hispanicus Lackschewitz, 1928 Starý and Oosterbroek 2008 , HA , Massif Toubkal, Imlil (17 km S Asni, 1700–1900 m); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Amsemlil; Oosterbroek 2020 (CCW: Rif ) Helius ( Helius ) pallirostris Edwards, 1921 Driauach and Belqat 2016 , AP , Aïn Chouk (Larache); Oosterbroek 2020 (CCW: Rif ) Limonia Meigen, 1803 Limonia flavipes (Fabricius, 1787) Pierre 1922b , HA , Haute Réghaya, Asni (1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limonia hercegovinae (Strobl, 1898) Starý and Oosterbroek 2008 , MA , Ifrane (1700 m), HA ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Limonia macrostigma (Schummel, 1829) Starý and Oosterbroek 2008 , HA , 5 km from Oukaimeden (2350 m); Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limonia nubeculosa Meigen, 1804 Pierre 1922b , HA , Haute Réghaya, Asni (1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Gavryushin pers. comm. 2012, HA ; Driauach and Belqat 2016 , Rif , tributary of Oued El Fondak, Aïn Ras el Ma, Aïn Boughaba, Âounsar Aheramen, Aïn Takhninjoute, maison forestière, Oued Madissouka, Aïn Quan­quben (Jebel Bou Bessoui), EM , Oued Azila; Oosterbroek 2020 (CCW: Rif ; HA ) Limonia phragmitidis (Schrank, 1781) Starý and Oosterbroek 2008 , MA , Ifrane (1700 m); Driauach and Belqat 2016 , Rif , Aïn Quanquben (Jebel Bou Bessoui); Oosterbroek 2020 (CCW) PEDICIIDAE K. Kettani, P. Oosterbroek Number of species: 6 . Expected: 10 Faunistic knowledge of the family in Morocco: moderate Pediciinae Dicranota Zetterstedt, 1838 Dicranota ( Dicranota ) bimaculata (Schummel, 1829) Pierre 1922b , HA , Haute Réghaya (1800 m); Savchenko et al. 1992 (? Morocco); Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Dicranota ) irregularis Pierre, 1921 Pierre 1922b , HA , Haute Réghaya (1800 m); Savchenko et al. 1992 , HA , Cirque d'Arround (Haute Réghaya); Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Ludicia ) claripennis (Verrall, 1888) Driauach and Belqat 2016 , Rif , Oued Amsemlil, maison forestière; Oosterbroek 2020 (CCW) Dicranota ( Paradicranota ) candelisequa Starý, 1981 Pârvu et al. 2006 , AP , Merja Zerga; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Paradicranota ) landrocki Czižek, 1931 Driauach et al. 2013 , Rif , Fifi (1252 m); Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Taida, Aïn Sidi Brahim Ben Arrif, Âounsar Aheramen, Oued Tizekhte, Oued Mezine, maison forestière, Aïn Bab Tariouant, HA , Imlil (Assif Haouz); Oosterbroek 2020 (CCW) Tricyphona Zetterstedt, 1838 Tricyphona ( Tricyphona ) immaculata (Meigen, 1804) Driauach and Belqat 2016 , Rif , maison forestière, Dayat Lemtahane; Oosterbroek 2020 (CCW) TIPULIDAE K. Kettani, P. Oosterbroek, H. de Jong Number of species: 39 . Expected: 42 Faunistic knowledge of the family in Morocco: moderate Dolichopezinae Dolichopeza Curtis, 1825 Dolichopeza ( Dolichopeza ) hispanica Mannheims, 1951 Theowald and Oosterbroek 1980 , HA , Aghbalou, Oukaimeden, Imlil, Tadmant; Oosterbroek and Theowald 1992 ; Oosterbroek and Lantsov 2011 , HA , Oukaimeden (2300 m), Aghbalou (Massif Toubkal), 43 km S Marrakech (1000 m), Imlil (17 km S Asni, 1700–1900 m), Tadmant (17 km E Asni); Adghir et al. 2018 , Rif , Kitane, Aîn Ras el Ma (Chefchaouen); Oosterbroek 2020 (CCW) Tipulinae Nephrotoma Meigen, 1803 Nephrotoma alluaudi (Pierre, 1922) = Pachyrhina lunulicornis Schummel, in Pierre 1922a : 24 = Pachyrhina alluaudi Pierre, in Pierre 1922b : 150, Dakki 1997 : 62 = Pales alluaudi (Pierre), in Mannheims 1951 : 47 Pierre 1922b , HA , Tannaout (1000 m); Mannheims 1951 , Rif , Beni Seddat, AP , Lagune Guedira; HA , Taddert north of Marrakech, Goundafa (1200 m); AA , Llano Amarillo, Tlata Reisana; Oosterbroek 1979b , Rif , HA , Imlil (1400 m), Tizi-n'Tichka (2200 m); Theowald and Oosterbroek 1980 , AP , Rabat, Guedira lagoon, MA , Immouzer, Ifrane, Timahdit, Aghbalou, Tizi-n'Zou, HA , Marrakech, Goundafa, Taddert, Dayat, Tizi-n'Test, Tizi-n'Tichka, Asni, Imlil, Oukaimeden, Setti Fatma, Tinmel, Acif Tifni, AA , Taroudant, Mikdana, Sidi Said bou Merdoul, Tlata Reisana, Llano Amarillo; Eiroa 1990 , MA , Azrou, Ajabo; Oosterbroek and Theowald 1992 , HA , Tannaout; Dakki 1997 ; Mouna 1998 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Oued Laou, Dardara, Douar Mouarâa, Oued Zandoula, Laghmari-Rmal, Douar Louamera, Douar Laheyayda, Oued Jnane Niche, Cabo Negro, Oued Beni Said, Oued El Kanar, Oued Amsemlil; Oosterbroek 2020 (CCW) – MISR ( HA ), MHNV, MNCNM, MAKB Nephrotoma appendiculata pertenua Oosterbroek, 1978 Oosterbroek 1978 , Rif , 9 km SW Chefchaouen; Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Fès, Immouzer, Khemisset; Oosterbroek and Theowald 1992 ; de Jong 1993 , 1998 , Rif ; Adghir et al. 2018 , Rif , Oued Ametrasse, Dayat Aïn Jdioui, Barrage Moulay Bouchta, Oued Sahil, Dayat Jebel Zemzem, Aïn Sidi Brahim Ben Arrif, Oued Nakhla, Oued Tizekhte, Lot Hemmadi, Douar Ayacha, Douar Louamera, Dayat Mezine, Aïn El Malâab, Aïn Takhninjoute, Dayat Tazia, Oued Taida, maison forestière Tazia, Tourbière Amesmlil, Oued El Hamma, Oued Kbir, Jebel Lakraâ; Oosterbroek 2020 (CCW) Nephrotoma astigma Pierre, 1925 Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Taza; Adghir et al. 2018 , Rif , Oued Tabandout, Etang Maggou, Aïn El Malâab, Douar Remla; Oosterbroek 2020 (CCW) Nephrotoma fontana Oosterbroek, 1978 de Jong 1998 , Rif ; Adghir et al. 2018 , Rif , Ketama, maison forestière Tazia, Dayat Tazia; Oosterbroek 2020 (CCW) Nephrotoma guestfalica vaillanti de Jong, Adghir & Bosch, 2021 de Jong et al. 2021 , Rif , Ras el Ma (Chefchaouen, 500 m), Fomento, Oued Laou (6 km north-west of Chefchaouen, 200 m), Jebel Tissouka (3 km south of Chefchaouen, 500 m; 3 km south of Chefchaouen, 700 m; 4 km south-east of Chefchaouen, 700–800 m; 5 km southeast of Chefchaouen, 700–900 m; 5 km south of Chefchaouen, 900–1000 m), Bab Taza (25 km south-east of Chefchaouen, 750–800 m), Dardara (10 km south of Chefchaouen, 300 m), Oued Martil, Nakhla, Âounsar Aheramen, Boumerouil, Oued Maggou, Oued Maggou (Aïn Ras el Ma), Douar Kitane, Oued Sidi Yahia Aârab, Oued Tamerte, Oued Zandoula, Oued El Hamma, Lot Hemmadi, Oued Tamerte, Oued Sidi Mohamed Saâda, Oued El Koub, Douar Iholebatine, Dayat Tazia, Belouazen, Oued Lemtahane, Oued Siflaou, Oued Amsemlil, AP , Oued Loukous, Aïn el-Aouda, MA , Ifrane (road to Mischliffen, 1680 m), N.S. and W of Ifrane (1400–1800 m), Khemisset, Azrou, Oum-er-Rbia, HA , M'semrir (bord de l'Oued), Rich, Haute Rhégaya (identified by Mannheims in 1951 as surcoufi ) Nephrotoma luteata (Meigen, 1818) Oosterbroek 1979a , Rif , Targuist, Chefchaouen, HA , Kasba Taguendaft, near Oukaimeden; Theowald and Oosterbroek 1980 , Rif , Targuist, Chefchaouen, HA , Kasba Taguendaft, near Oukaimeden; Oosterbroek and Theowald 1992 ; Pârvu et al. 2006 , AP , Merja Zerga; Adghir et al. 2018 , Rif , Oued Laou, Dardera, Oued Nakhla; Oosterbroek 2020 (CCW) Nephrotoma subanalis (Mannheims, 1951) = ? Pachyrhina analis Schummel, in Pierre 1922a : 24, Pierre 1922b : 150 = Pales subanalis Mannheims, in Mannheims 1951 : 56 Mannheims 1951 , HA , Tachdirt (2200–2900 m); Vaillant 1956b , HA , Oukaimeden (2250 m); Oosterbroek 1979c, HA , Tachdirt (2200–2900 m); Theowald and Oosterbroek 1980 , HA , Tachdirt, Oukaimeden, M'Goum, Tadmant, Imlil, Tizi-N'Tichka; Oosterbroek and Theowald 1992 , HA , Tachdirt; Oosterbroek 2020 (CCW) Nephrotoma submaculosa Edwards, 1928 Oosterbroek 1982 , Rif , Ketama, Dardara, MA , Azrou; Oosterbroek and Theowald 1992 ; de Jong 1998 , Rif , Atlas ; Oosterbroek 2011 ; Adghir et al. 2018 , Rif , Jebel Dahedouh, Ketama, Aïn Sidi Brahim Ben Arrif, Oued Taida, tributary of Oued Ouara, Sidi Chouiref, Dayat Tazia; Oosterbroek 2020 (CCW) Nephrotoma sullingtonensis Edwards, 1938 Oosterbroek 1978 ; Theowald and Oosterbroek 1980 , Rif , Bab Berred; Oosterbroek 1982 Rif , Bab Berred; de Jong 1993 , 1998 , Rif ; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 , Rif , 4 km SE Ketama, Barrage Moulay Bouchta, Oued Aârate, Aïn Takhninjoute, Oued Jbara, Aïn El Malâab, Aïn El Ma Bared, Oued Lemtahane, maison forestière Tazia, Oued Taida, Dayat Tazia; Oosterbroek 2020 (CCW) Tipula Linnaeus, 1758 Tipula ( Acutipula ) anormalipennis Pierre, 1924 Pierre 1924b , HA , Haut Imminen; Séguy 1930a , HA , Haut Imminen; Séguy 1941a , HA , Tachdirt (2500 m), Haut Imminen; Mannheims 1952 , HA , Tachdirt (2500 m), Imminen (2400–2500 m), Arround (1950 m); Vaillant 1956b , HA , Lake of Tamhda (Anremer, 2900 m); Theowald and Oosterbroek 1980 , HA , Anremer, Haut-Immenen, Tachdirt, Arround, Oukaimeden; Vermoolen 1983 , HA , Haut Imminen, Oukaimeden (2500–2800 m), Arround (1950 m), Tachdirt (2500 m); Oosterbroek and Theowald 1992 ; de Jong 1994a , HA ; de Jong 1998 , HA ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Acutipula ) repentina Mannheims, 1952 = Tipula maxima Poda, in Séguy 1941a : 26 Séguy 1941a , HA , Tachdirt (2500 m); Mannheims 1952 , HA , Tachdirt (2200–2700 m); Vaillant 1956b , HA , Lac Tamhda (Anremer, 2900), M'Goum (2500 m); Theowald and Oosterbroek 1980 , HA , Anremer, M'Goum, Tachdirt, Tizi-N'Tichka, Asni, Oukaimeden, Setti Fatma, Imlil, Tadmant; Vermoolen 1983 , MA , Ifrane, HA , Androment, M'Goum, Tadmant, Tizi-N'Test, Setti Fatma, Oukaimeden, Tizi-N'Tichka; Oosterbroek and Theowald 1992 , HA , Tachdirt; de Jong 1994a , MA , HA ; de Jong 1998 , HA ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Acutipula ) rifensis Theowald & Oosterbroek, 1980 Theowald and Oosterbroek 1980 , Rif , Targuist; Vermoolen 1983 , Rif , Targuist, Tidiguin (90 km E. of Ouezzane, 2350 m); Oosterbroek and Theowald 1992 , Rif ; de Jong 1994a , 1998 , Rif ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Emodotipula ) leo Dufour, 1991 = Tipula ( Emodotipula ) obscuriventris Strobl 2 , in Oosterbroek and Theowald 1992 : 99 Oosterbroek and Theowald 1992 (?); Dufour 2003 , Rif ; Adghir et al. 2018 , Rif , Jebel Tissouka; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) bivittata Pierre, 1922 Pierre 1922a , AP , Maâmora between Kénitra and Oued Beth, Dradek (near Rabat); Pierre 1922b , AP , Rabat; Mannheims 1968 ; Theowald and Oosterbroek 1980 , AP , forest of Maâmora, Dradek; Oosterbroek and Theowald 1992 , AP , forest of Maâmora, Dradek; Dakki 1997 ; Oosterbroek 2020 (CCW) – MISR ( AP , Kénitra, Dradek) Tipula ( Lunatipula ) cinereicolor Pierre, 1924 Pierre 1924b , HA , Haut Imminen; Séguy 1930a , HA , Tachdirt (3100–3200 m); Theowald 1973 , HA , Haut Imminen (2400 m); Theowald and Oosterbroek 1980 , MA , Ifrane, HA , Oukaimeden, Tachdirt, Haut-Imminen; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Imminen; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) cornicula Pierre, 1922 Pierre 1922b , HA , Arround (2000 m); Séguy 1930a , HA , Tachdirt (3100–3200 m); Theowald 1973 , HA , Cirque d'Arround (2000 m), Tachdirt (2200–2700 m), Goundafa (1200 m); Theowald and Oosterbroek 1980 , HA , Oukaimeden, Tachdirt, Goundafa; Oosterbroek and Theowald 1992 , HA , Tachdirt; Dakki 1997 ; Oosterbroek 2020 (CCW) – MISR ( HA ) Tipula ( Lunatipula ) fabiola Mannheims, 1968 Theowald and Oosterbroek 1980 , Rif , Bab Berred, Ras El Ma (Chefchaouen), MA , Jebel Abad, Ifrane; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) hermes Theischinger, 1977 Theischinger 1977 , Rif , north of Ouezzane; Theowald 1980 ; Theowald and Oosterbroek 1980 , Rif , north of Ouezzane; Oosterbroek and Theowald 1992 , Rif , Ouezzane; Adghir et al. 2018 , Rif , 4 km SE Ketama, Oued Ametrasse, Aïn El Ma Bared; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) iberica iberica Mannheims, 1963 = Tipula lunata Linnaeus, in Pierre 1922b : 149, Séguy 1930a : 23 Pierre 1922b , HA , Haute Réghaya, Tannaout (1000 m); Séguy 1930a , HA , Haut Imminen; Mannheims 1963 , MA ; Theowald and Oosterbroek 1980 , Rif , Bab Berred, MA , Ifrane, Taounate; Eiroa 1990 , MA , Ifrane; Oosterbroek and Theowald 1992 ; Oosterbroek 2009 ; Adghir et al. 2018 , Rif , Ketama; Oosterbroek 2020 (CCW) – MISR ( HA ) Tipula ( Lunatipula ) iberica spinula Theischinger, 1980 Theischinger 1980 , HA , Oukaimeden (1300–2800 m); Theowald and Oosterbroek 1980 , HA , Oukaimeden; Oosterbroek and Theowald 1992 , HA , Oukaimeden; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) peliostigma peliostigma Schummel, 1833 Eiroa 1990 , MA , Azrou; Mouna 1997; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) pjotri de Jong & Adghir, 2018 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m) Tipula ( Lunatipula ) pseudocinerascens Strobl, 1906 Adghir et al. 2018 , Rif , Stehat, Oued Taida, Perdicaris Park, Dayat Tazia Tipula ( Lunatipula ) rocina Theischinger, 1979 Theowald and Oosterbroek 1980 , Rif , Tétouan; Oosterbroek and Theowald 1992 ; Oosterbroek 2009 ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) selenaria Mannheims, 1967 Mannheims 1967 , MA , Jebel Tazzeka (1500–1989 m), HA , Goundafa (1200 m); Theowald and Oosterbroek 1980 , MA , Tazzeka, HA , Foum Keneg, Goundafa, Oukaimeden; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Goundafa; de Jong 1995 , MA , HA , Oukaimeden; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) stimulosa Mannheims, 1973 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m) Tipula ( Lunatipula ) subfalcata Mannheims, 1967 de Jong 1995 , 1998 , Rif ; Adghir et al. 2018 , Rif , Jebel Tissouka, Oued Tamerte, Oued El Koub; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) subpustulata Mannheims, 1963 = Tipula pustulata Pierre, in Pierre 1922b : 150 Pierre 1922b , AP , Mogador; Vaillant 1956b , HA , M'Goun (2500 m); Mannheims 1963 , AP , Aïn el Aouda, MA , Jebel Tazzeka (1600–1989 m), HA , Goundafa (1200 m), Tachdirt (2200–2900 m), AA , Lac Goulmima; Theowald 1972 ; Theowald and Oosterbroek 1980 , AP , Aïn el Aouda, MA , Iebel Tazzeka, HA , M'Goum, Goundafa, Tachdirt, Oukaimeden, Imlil; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Goundafa; Dakki 1997 ; Adghir et al. 2018 , Rif , 4 km SE Ketama, Aïn El Ma Bared; Oosterbroek 2020 (CCW) – MISR ( AP , Mogador) Tipula ( Lunatipula ) tazzekai Theowald, 1973 Theowald 1973 , MA , Jebel Tazzeka (1600–1989 m); Theowald and Oosterbroek 1980 , MA , Jebel Tazzeka; Eiroa 1990 , MA , Jebel Hebri; Oosterbroek and Theowald 1992 , MA , Jebel Tazzeka; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) atlas Pierre, 1924 Pierre 1924b , HA , Tachdirt (3100–3250 m); Séguy 1930a , HA , Tachdirt (3100–3200 m); Vaillant 1956b , HA , Cascade Siroua (3000 m), M'Goun (2500 m), Toubkal (3350 m), Lake of Tamhda (Anremer, 2900 m); Mannheims 1964 , HA ; Theowald 1973 , HA ; Theowald 1980 , HA ; Theowald and Oosterbroek 1980 , HA , Toubkal, Sources de Tessaouts, M'Goum, Siroua, Anremer, Tachdirt, Oukaimeden, Tizi-N'Tichka, Tadmant; Eiroa 1990 , MA , Oum-Er-Rbia; Oosterbroek and Theowald 1992 , HA , Tachdirt; de Jong 1994b ; de Jong 1998 , Atlas ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) breviantennata Lackschewitz, 1933 de Jong 1998 , Rif ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Oued Maggou, Douar Aouzighen; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) confusa van der Wulp, 1883 Adghir et al. 2018 , Rif , maison forestière (Talassemtane) Tipula ( Savtshenkia ) rufina rufina Meigen, 1818 Theowald and Oosterbroek 1980 , HA , Oukaimeden; Theowald and Oosterbroek 1983 , Rif , Atlas ; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen); Oosterbroek 2020 (CCW) Tipula ( Tipula ) mediterranea Lackschewitz, 1930 Vaillant 1956b , HA , Oukaimeden (2250 m), M'Goun (2500 m); Mannheims 1952 ; Theowald and Oosterbroek 1980 , MA , Ifrane, HA , Oukaimeden, M'Goum, Asni, M'Semrir, Ifni, Bab-Rou-Idie, Tizi-N'Tichka, Setti Fatma; Theowald 1984 , Rif ; Eiroa 1990 , MA , Azrou, Oum-Er-Rbia; Oosterbroek and Theowald 1992 ; Pârvu et al. 2006 AP , Merja Zerga; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Jebel Tissouka, Bab Taza, Dardara, 4 km SE Ketama, Aïn Afersiw, Douar Kitane, Dayat Jebel Zemzem, Wilaya (Tétouan), Aïn El Ma Bared, Oued Tamerte, Douar Louamera, Tourbière Amesmlil, Dayat Mezine, Hejar Nehal, tributary of Oued Ouara, Oued Tkarae, Oued Jnane Niche, Dayat Afrate, Oued Ametrasse; Oosterbroek 2020 (CCW) Tipula ( Tipula ) oleracea Linnaeus, 1758 Mannheims 1952 , Rif , Tlata Ketama; Theowald and Oosterbroek 1980 , Rif , Tlata Ketama; Theowald 1984 , Rif ; Oosterbroek and Theowald 1992 ; Dakki 1997 ; Pârvu and Zaharia 2007 ; Oosterbroek 2011 ; Adghir et al. 2018 , Rif , Oued Laou, Dardara, Jebel Tissouka, Ksar Rimal, 15 km from Fifi, Oued Aârate, Oued El Hamma, Douar Louamera, Oued Zaouya, Près de Beni Said, Wilaya (Tétouan), Tourbière Amesmlil, Oued Smir, Aïn Jdida; Oosterbroek 2020 (CCW) – MISR Tipula ( Vestiplex ) vaillanti vaillanti Theowald, 1977 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m), Douar Kitane Tipula ( Yamatotipula ) afriberia afriberia Theowald & Oosterbroek, 1980 Theowald and Oosterbroek 1980 , HA , Oukaimeden; Oosterbroek and Theowald 1992 , HA , Oukaimeden; Oosterbroek 1994a ; Adghir et al. 2018 , Rif , Jebel Tissouka, Dardara, Douar Mokedassen, Oued Zarka; Oosterbroek 2020 (CCW) Tipula ( Yamatotipula ) barbarensis Theowald & Oosterbroek, 1980 = Tipula lateralis Meigen, in Pierre 1922a : 23, Pierre 1922b : 150, Séguy 1930a : 23, Séguy 1941a : 26, Mannheims 1952 : 98 (in part), Vaillant 1956b : 238 Pierre 1922a , AP , Dradek (Rabat), MA , Azrou (riverside of Oued Tigrigra); Pierre 1922b , AP , Mogador, MA , Beni Méllal, HA , Asni; Séguy 1930a ; Séguy 1941a , HA , Imi n'Ouaka (1500 m); Mannheims 1952 ; Vaillant 1956b , HA , Oukaimeden; Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Ifrane, Aghbalou, HA , Asni, Oukaimeden, Setti Fatma, Imlil, Tizi-N'Tichka, Tadmant, Tachdirt; Eiroa 1990 , MA , Azrou, Oum-er-Rbia, Ifrane; Oosterbroek and Theowald 1992 , HA , Setti Fatma; Oosterbroek 1994a , Rif , Dardara, MA , Ifrane, Aghbalou, HA , Oukaimeden, Setti Fatma, Imlil, Tizi-N'Tichka, Tadmant, Asni, Tachdirt; Dakki 1997 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Jebel Tissouka, Bab Taza, Dardara, 4 km SE Ketama, Aïn El Manzela, Aïn Bab Tariouante, Dayat Aïn Afersiw, Oued El Kanar, Dayat Afrate, Douar Kitane, Wilaya (Tétouan), 15 km from Fifi, Aïn Sidi Brahim Ben Arrif, Oued Nakhla, Âounsar Aheramen, Oued Boumerouil, Douar Zaouya, Oued Tizekhte, Oued Samsa, Dayat Aïn Jdioui, Douar Ouled Laghmari-Rmal, maison forestière Tazia, Hejar Nehal, Lot Hemmadi, tributary of Oued Ouara, Oued Sidi Mohamed Saâda, Oued Amsemlil, Tourbière Amesmlil, Beni Salah, Oued Jnane Niche, Oued Maggou, Souk Lhed Beni Darkoul, Oued Imassouden, Aïn Helouma, Source Zarka, Aïn Kchour; Oosterbroek 2020 (CCW) – MISR LIMONIIDAE K. Kettani, P. Oosterbroek Number of species: 67 . Expected: 85 Faunistic knowledge of the family in Morocco: moderate Chioneinae Baeoura Alexander, 1924 Baeoura ebenina Starý, 1981 Driauach and Belqat 2015 , Rif , Oued Tazarine (Mezine village); Dayat near Aïn Afersiw; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: Rif ) Baeoura staryi Driauach & Belqat, 2015 Driauach and Belqat 2015 , Rif , Jnane Niche; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: Rif ) Cheilotrichia Rossi, 1848 Cheilotrichia ( Empeda ) cinerascens (Meigen, 1804) Driauach et al. 2013 , Rif , Ikadjiouen; Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Amsa, Oued Maggou, Aïn Quanquben (Jebel Bou Bessoui), EM , Grotte du Chameau (Zegzel, Béni Snassen); Oosterbroek 2020 (CCW: Rif ; EM ) Cheilotrichia ( Empeda ) fuscohalterata (Strobl, 1906) Driauach and Belqat 2016 , Rif , Dayat Fifi, tributary of Oued El Fondak, Barrage Ajras, Dayat Mghara, Oued Zarka, Oued Tizekhte; Oosterbroek 2020 (CCW: Rif ) Cheilotrichia ( Empeda ) minima (Strobl, 1898) Driauach and Belqat 2016 , Rif , Oued Zendoula, Oued Jnane Niche; Oosterbroek 2020 (CCW: Rif ) Ellipteroides Becker, 1907 Ellipteroides ( Ellipteroides ) lateralis (Macquart, 1835) = Gonomyia lateralis Macquart, in Pierre 1922a : 22, Dakki 1997 : 62 = Gonomyia cincta Egger, in Séguy 1941a : 26 Pierre 1922a , AP , Dradek (near Rabat); Lackschewitz 1940a , HA , Tachdirt (2200–2900 m); Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 ; Dakki 1997 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Dayat Afrate; Oosterbroek 2020 (CCW: Rif ) Ellipteroides ( Protogonomyia ) alboscutellatus (von Roser, 1840) Lachschewitz 1940a, HA ; Savchenko et al. 1992 ; Starý and Oosterbroek 2008 ; Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Ellipteroides ( Protogonomyia ) hutsoni (Starý, 1971) Starý 1971 , HA , Jebel Ayachi; Savchenko et al. 1992 , HA , Jebel Ayachi; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Erioconopa Starý, 1976 Erioconopa diuturna (Walker, 1848) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Dayat Jebel Zemzem, Oued Mezine, Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ) Erioconopa symplectoides (Kuntze, 1914) = Dactylolabis symplectoides Egger, in Pierre 1922a : 23 Pierre 1922a , HA , Marrakech; Savchenko et al. 1992 ; Starý and Oosterbroek 2008 , HA ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Erioptera Meigen, 1803 Erioptera ( Erioptera ) fuscipennis Meigen, 1818 Pierre 1922a , AP , Casablanca (Oued Guerera); Pierre 1922b , HA , Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 , Rif , Sidi Brahim Ben Arrif (Bab Hachef Aissa); Driauach and Belqat 2016 , Rif , Aïn El Ma Bared, Oued Amsemlil, Oued Maggou (Bridge), Oued Tkarae, Dayat near Aïn Afersiw, Aïn Afersiw, Oued Abou Bnar, Oued Maggou (Zaouiet El Habtiyne), Dayat Amsemlil, Dayat Aïn Jdi­oui; Dayat Afrate, Oued Mezine; Oosterbroek 2020 (CCW: Rif , HA ) Erioptera ( Erioptera ) lutea lutea Meigen, 1804 Driauach and Belqat 2016 , Rif , Aïn Boughaba; Oosterbroek 2020 (CCW: Rif ) Erioptera ( Erioptera ) transmarina Bergroth, 1889 = Mesocyphona transmarina Bergroth, in Pierre 1922a : 22, Pierre 1922b : 148, Dakki 1997 : 62 Pierre 1922a , HA , Marrakech; Pierre 1922b , HA , Tannaout (1000 m), Marrakech; Dakki 1997 ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Gonomyia Meigen, 1818 Gonomyia ( Gonomyia ) abscondita Lackschewitz, 1935 Driauach and Belqat 2016 , Rif , maison forestière; Oosterbroek 2020 (CCW: Rif ) Gonomyia ( Gonomyia ) sicula Lackschewitz, 1940 Driauach and Belqat 2016 , Rif , Oued Kbir, Dayat Jebel Zemzem, Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Aïn El Maounzil; Oosterbroek 2020 (CCW: Rif ) Gonomyia ( Gonomyia ) subtenella Savchenko, 1972 Starý and Oosterbroek 2008 , HA , Massif Toubkal, Aghbalou, 43 km S Marrakech (1000 m); Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Jnane Niche, Oued Sidi Yahia Aârab, EM , Oued Béni Ouachekradi; Oosterbroek 2020 (CCW: Rif , EM ) Gonomyia ( Gonomyia ) tenella (Meigen, 1818) Pierre 1924a , HA , Asni (1200 m); Séguy 1930a , HA , Asni; Savchenko et al. 1992 ; Dakki 1997 ; Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis Osten-Sacken, 1869 Hoplolabis ( Parilisia ) obtusiapex (Savchenko, 1982) Starý 2006 , HA , Oasis Meski, AA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis ( Parilisia ) punctigera (Lackschewitz, 1940) Starý 2006 , HA , Oasis Meski, AA ; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis ( Parilisia ) sororcula (Lackschewitz, 1940) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Barrage Ajras, Oued Ouringa, Oued El Kanar, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Idiocera Dale, 1842 Idiocera ( Euptilostena ) jucunda (Loew, 1873) = Gonomyia jucunda Loew, in Pierre 1924a : 201, Séguy 1930a : 22 Pierre 1924a , HA , Asni (1200 m); Séguy 1930a , HA , Asni; Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Idiocera ( Idiocera ) ampullifera (Starý, 1979) Driauach and Belqat 2016 , AA , Oued Zag; Oosterbroek 2020 (CCW: AA ) Idiocera ( Idiocera ) pulchripennis (Loew, 1856) = Gonomyia sexpunctata Dale, in Pierre 1922b : 148 = Gonomyia pulchripennis (Loew), in Dakki 1997 : 62 Pierre 1922b , AP , Atlantic coast; Ramdani 1981 , AP , Merja Sidi Boughaba; Savchenko et al. 1992 ; Dakki 1997 ; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Jnane Niche, Oued Aârkoub, Aïn Jdioui; Oosterbroek 2020 (CCW) Idiocera ( Idiocera ) sziladyi (Lackschewitz, 1940) Driauach and Belqat 2016 , Rif , Oued Zarka; Oosterbroek 2020 (CCW: Rif ) Ilisia Rondani, 1856 Ilisia maculata (Meigen, 1804) Driauach and Belqat 2016 , Rif , Oued Maggou, EM , Oued Tafoughalt; Oosterbroek 2020 (CCW: Rif ; EM ) Molophilus Curtis, 1833 Molophilus ( Molophilus ) ibericus Starý, 2011 Starý 2011 , HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Molophilus ( Molophilus ) obscurus (Meigen, 1818) Starý and Oosterbroek 2008 , HA , Massif Toubkal, Oukaimeden, 2500–2800 m; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , tributary of Oued Ouara, tributary of Oued Maggou, Dayat El Ânassar, Dayat Jebel Zemzem, Aïn El Ma Bared, Dayat Rmali, Oued Amsemlil, Aïn Mâaze, Dayat Afrate, Dayat Lemtahane; Oosterbroek 2020 (CCW: Rif ) Molophilus ( Molophilus ) propinquus propinquus (Egger, 1863) Savchenko et al. 1992 ; Starý and Oosterbroek 2008 , HA , Oukaimeden (2500–2800 m); Starý 2011 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Farda, tributary of Oued Taida, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Molophilus ( Molophilus ) testaceus Lackschewitz, 1940 Driauach and Belqat 2016 , Rif , Dayat Amsemlil, Dayat Lemtahane, Marj El kheyl, Oued Tkarae, Dayat near Aïn Afersiw, Dayat Mezine, Dayat Tazia, Dayat Rmali, Dayat Amsemlil, Dayat near Aïn Afersiw; Oosterbroek 2020 (CCW: Rif ) Symplecta Meigen, 1830 Symplecta ( Symplecta ) grata Loew, 1873 Ebejer et al. 2020, Rif , Aïn Jdioui (Tahaddart, 8 m) Symplecta ( Symplecta ) hybrida (Meigen, 1804) Lackshewitz 1940a, HA , Goundafa (1200 m); Séguy 1941a , HA , Taroudant; Savchenko et al. 1992 ; Oosterbroek et al. 2007; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Barrage Ajras, Oued El Kanar, Oued Aârkoub, Oued Jnane Niche, MA , Barrage Allal El Fassi; Oosterbroek 2020 (CCW: Rif ; MA ) Symplecta ( Trimicra ) pilipes (Fabricius, 1787) = Trimicra pilipes Fabricius, in Pierre 1922a : 23 = Trimicra andalusiaca Strobl, in Pierre 1922b : 149 = Trimicra hirsutipes Macquart, in Séguy 1930a : 22 Pierre 1922a , AP , Dradek (near Rabat), HA , Marrakech; Pierre 1922b , MA , Volubilis, HA , Oued Tensift; Séguy 1930a ; Séguy 1941d , AA , Taroudant; Savchenko et al. 1992 ; Dakki 1997 ; Pârvu and Zaharia 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , tributary of Oued Hachef, Oued Aârk­ob, Oued El Kanar, Oued Mezine, AP , Aïn Chouk (Larache); Oosterbroek 2020 (CCW: Rif ) Tasiocera ( Dasymolophilus ) murina (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued Farda, Oued Amsemlil; Oosterbroek 2020 (CCW: Rif ) Dactylolabinae Dactylolabis Osten-Sacken, 1860 Dactylolabis ( Dactylolabis ) symplectoidea Egger, 1863 Pierre 1922a , AP , Around Casablanca (coastal meseta); Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limnophilinae Austrolimnophila Alexander, 1920 Austrolimnophila ( Austrolimnophila ) latistyla Starý, 1977 Driauach and Belqat 2016 , Rif , Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Dicranophragma Osten-Sacken, 1860 Dicranophragma ( Brachylimnophila ) adjunctum (Walker, 1848) = Neolimnomyia adjuncta (Walker, 1848), in Pârvu et al. 2006 : 273 Pârvu et al. 2006 , AP , Merja Zerga; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranophragma ( Brachylimnophila ) nemorale (Meigen, 1818) Lackschewitz 1940b , HA , Tachdirt (2200–2700 m); Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Aïn Sidi Brahim Ben Arrif, tributary Oued Ouara, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Eloeophila Rondani , 1856 Eloeophila maroccana Starý, 2009 Starý 2009b , HA , Okaïmeden (2500–2800 m); Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Euphylidorea Alexander, 1972 Euphylidorea ( Euphylidorea ) crocotula (Séguy, 1941) = Phylidorea crocotula (Séguy), in Séguy 1941a : 28 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 , HA , Tachdirt (Toubkal); Starý and Oosterbroek 2008 , HA , Oukaimeden (2500 m–2800 m), Imlil (1400 m); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ; HA ) Euphylidorea ( Euphylidorea ) dispar (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued at 15 km from Fifi; Oosterbroek 2020 (CCW: Rif ) Euphylidorea ( Euphylidorea ) lineola (Meigen, 1804) Lackschewitz 1940b , HA ; Savchenko et al. 1992 ; Starý and Freidberg 2007 ; Driauch et al. 2013; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hexatoma Latreille, 1809 Hexatoma ( Hexatoma ) bicolor (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued at 15 km from Fifi, tributary of Oued Ouara, Oued Madissouka, Oued Maggou, Aïn Quanquben (Jebel Bou Bessoui), Oued Tkarae, Oued Tamerte; Oosterbroek 2020 (CCW: Rif ) Hexatoma ( Hexatoma ) gaedii (Meigen, 1830) Lackschewitz 1940b , HA , Tachdirt (2200 m–2700 m); Savchenko et al. 1992 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Pseudolimnophila Alexander, 1919 Pseudolimnophila ( Pseudolimnophila ) sepium (Verrall, 1886) Driauach et al. 2013 , Rif , Guelta Tazia; Driauach and Belqat 2016 , Rif , Dayat Tazia, Aïn Sidi Brahim Ben Ar­rif, Aïn Afersiw, Oued Maggou, Dayat Tazia, Oued Taida, Aïn Sidi Brahim Ben Arrif; Oosterbroek 2020 (CCW: Rif ) Limoniinae Dicranomyia Stephens, 1829 Dicranomyia ( Dicranomyia ) affinis (Schummel, 1829) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Amsemlil, Dayat Lemtahane, Oued Tkarae, Marj El Kheyl, Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Aïn El Maounzil, Aïn El Malâab, Dayat near Aïn Afersiw, Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) chorea (Meigen, 1818) Pierre 1922b , HA , Haute Réghaya, Tannaout, Asni (1000 m–1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Farda, Oued Kelâa, Aïn Ras el Ma, Oued Jnane Niche, Cascade Chrafate, maison forestière, Oued El Kanar, tributary of Oued Zarka, HA , Oued Sidi Fares (National Park of Toubkal); Oosterbroek 2020 (CCW: Rif ; HA ) Dicranomyia ( Dicranomyia ) didyma (Meigen, 1804) Pierre 1922b , HA , Haute Réghaya (2000 m); Dakki 1997 ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Dicranomyia ( Dicranomyia ) goritiensis (Mik, 1864) Lackschewitz 1940b ; Vaillant 1956b ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Maggou, Cascade Chrafate, Oued El Koub, Cascade Zarka, Âounsar Aheramen, maison forestière; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) longicollis (Macquart, 1846) = Telecephala longicollis (Macquart), in Pierre 1922a : 21, Pierre 1922b : 148, Séguy 1930a : 22 Séguy 1930a , AP , Dradek (near Rabat); Dakki 1997 ; Pierre 1922a , AP , Dradek, HA , Marrakech; Pierre 1922b , AP , Rabat; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Aârate, Barrage Moulay Bouchta, Oued Kbir, MA , Barrage Allal El Fassi; Oosterbroek 2020 (CCW) Dicranomyia ( Dicranomyia ) mitis (Meigen, 1830) = Dicranomyia hygropetrica Vaillant, in Vaillant 1956b : 42 Vaillant 1956b , HA , Asif Tessaout (M'Goum), Izourar, Tahanaout, Tamesrit, Imi-N'Ifri, Aguelmous, Sidi Chamarouch, Lac Tamhda (Anremer), Oukaimeden; Savchenko et al. 1992 ; Pârvu et al. 2006 , AA , near Agadir, Souss plain to High Atlas occidental; Pârvu and Zaharia 2007 ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 , Rif , Tazia, Tisgris; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia ( Dicranomyia ) modesta (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued Farda, Oued El Kanar, Oued Jnane Niche, Oued Sidi Yahia Aârab; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) novemmaculata (Strobl, 1906) Driauach and Belqat 2016 , Rif , tributary of Oued el Fondak, Oued Aârate; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) ventralis (Schummel, 1829) Driauach and Belqat 2016 , Rif , Lake Badriouen; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Glochina ) sericata (Meigen, 1830) Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia ( Melanolimonia ) hamata Becker, 1908 Driauach and Belqat 2016 , Rif , tributary of Oued Kbir, Oued Aârate, Aïn Sidi Brahim Ben Arrif, Dayat Tazia; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Melanolimonia ) morio (Fabricius, 1787) = Limonia pauliani Séguy, in Séguy 1941a : 26 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 , HA , Tachdirt (Toubkal); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Seguia Lemtahane, Dayat near Aïn Afersiw, Dayat Jebel Zemzem; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia majuscula Pierre, 1924 1 Pierre 1924a , HA , Haut Imminen (2400 m); Séguy 1930a , HA , Haut Imminen; Dakki 1997 ; Savchenko et al. 1992 , HA , Haut Imminen; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranoptycha Osten-Sacken, 1860 Dicranoptycha fuscescens (Schummel, 1829) Eiroa 2000 , Rif ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Mlilah, Oued Zendoula, AP , Oued Loukous; Oosterbroek 2020 (CCW) Geranomyia Haliday, 1833 Geranomyia caloptera (Mik, 1867) Driauach et al. 2013 , HA , Setti Fatma; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Geranomyia obscura Strobl, 1900 Vaillant 1956b , HA , Lac Tamhda (Anremer), Oukaimeden, Izourar, Sidi Chamarouch, Tamesrit; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Helius Lepeletier & Serville, 1828 Helius ( Helius ) hispanicus Lackschewitz, 1928 Starý and Oosterbroek 2008 , HA , Massif Toubkal, Imlil (17 km S Asni, 1700–1900 m); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Amsemlil; Oosterbroek 2020 (CCW: Rif ) Helius ( Helius ) pallirostris Edwards, 1921 Driauach and Belqat 2016 , AP , Aïn Chouk (Larache); Oosterbroek 2020 (CCW: Rif ) Limonia Meigen, 1803 Limonia flavipes (Fabricius, 1787) Pierre 1922b , HA , Haute Réghaya, Asni (1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limonia hercegovinae (Strobl, 1898) Starý and Oosterbroek 2008 , MA , Ifrane (1700 m), HA ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Limonia macrostigma (Schummel, 1829) Starý and Oosterbroek 2008 , HA , 5 km from Oukaimeden (2350 m); Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limonia nubeculosa Meigen, 1804 Pierre 1922b , HA , Haute Réghaya, Asni (1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Gavryushin pers. comm. 2012, HA ; Driauach and Belqat 2016 , Rif , tributary of Oued El Fondak, Aïn Ras el Ma, Aïn Boughaba, Âounsar Aheramen, Aïn Takhninjoute, maison forestière, Oued Madissouka, Aïn Quan­quben (Jebel Bou Bessoui), EM , Oued Azila; Oosterbroek 2020 (CCW: Rif ; HA ) Limonia phragmitidis (Schrank, 1781) Starý and Oosterbroek 2008 , MA , Ifrane (1700 m); Driauach and Belqat 2016 , Rif , Aïn Quanquben (Jebel Bou Bessoui); Oosterbroek 2020 (CCW) Chioneinae Baeoura Alexander, 1924 Baeoura ebenina Starý, 1981 Driauach and Belqat 2015 , Rif , Oued Tazarine (Mezine village); Dayat near Aïn Afersiw; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: Rif ) Baeoura staryi Driauach & Belqat, 2015 Driauach and Belqat 2015 , Rif , Jnane Niche; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: Rif ) Cheilotrichia Rossi, 1848 Cheilotrichia ( Empeda ) cinerascens (Meigen, 1804) Driauach et al. 2013 , Rif , Ikadjiouen; Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Amsa, Oued Maggou, Aïn Quanquben (Jebel Bou Bessoui), EM , Grotte du Chameau (Zegzel, Béni Snassen); Oosterbroek 2020 (CCW: Rif ; EM ) Cheilotrichia ( Empeda ) fuscohalterata (Strobl, 1906) Driauach and Belqat 2016 , Rif , Dayat Fifi, tributary of Oued El Fondak, Barrage Ajras, Dayat Mghara, Oued Zarka, Oued Tizekhte; Oosterbroek 2020 (CCW: Rif ) Cheilotrichia ( Empeda ) minima (Strobl, 1898) Driauach and Belqat 2016 , Rif , Oued Zendoula, Oued Jnane Niche; Oosterbroek 2020 (CCW: Rif ) Ellipteroides Becker, 1907 Ellipteroides ( Ellipteroides ) lateralis (Macquart, 1835) = Gonomyia lateralis Macquart, in Pierre 1922a : 22, Dakki 1997 : 62 = Gonomyia cincta Egger, in Séguy 1941a : 26 Pierre 1922a , AP , Dradek (near Rabat); Lackschewitz 1940a , HA , Tachdirt (2200–2900 m); Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 ; Dakki 1997 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Dayat Afrate; Oosterbroek 2020 (CCW: Rif ) Ellipteroides ( Protogonomyia ) alboscutellatus (von Roser, 1840) Lachschewitz 1940a, HA ; Savchenko et al. 1992 ; Starý and Oosterbroek 2008 ; Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Ellipteroides ( Protogonomyia ) hutsoni (Starý, 1971) Starý 1971 , HA , Jebel Ayachi; Savchenko et al. 1992 , HA , Jebel Ayachi; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Erioconopa Starý, 1976 Erioconopa diuturna (Walker, 1848) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Dayat Jebel Zemzem, Oued Mezine, Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ) Erioconopa symplectoides (Kuntze, 1914) = Dactylolabis symplectoides Egger, in Pierre 1922a : 23 Pierre 1922a , HA , Marrakech; Savchenko et al. 1992 ; Starý and Oosterbroek 2008 , HA ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Erioptera Meigen, 1803 Erioptera ( Erioptera ) fuscipennis Meigen, 1818 Pierre 1922a , AP , Casablanca (Oued Guerera); Pierre 1922b , HA , Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 , Rif , Sidi Brahim Ben Arrif (Bab Hachef Aissa); Driauach and Belqat 2016 , Rif , Aïn El Ma Bared, Oued Amsemlil, Oued Maggou (Bridge), Oued Tkarae, Dayat near Aïn Afersiw, Aïn Afersiw, Oued Abou Bnar, Oued Maggou (Zaouiet El Habtiyne), Dayat Amsemlil, Dayat Aïn Jdi­oui; Dayat Afrate, Oued Mezine; Oosterbroek 2020 (CCW: Rif , HA ) Erioptera ( Erioptera ) lutea lutea Meigen, 1804 Driauach and Belqat 2016 , Rif , Aïn Boughaba; Oosterbroek 2020 (CCW: Rif ) Erioptera ( Erioptera ) transmarina Bergroth, 1889 = Mesocyphona transmarina Bergroth, in Pierre 1922a : 22, Pierre 1922b : 148, Dakki 1997 : 62 Pierre 1922a , HA , Marrakech; Pierre 1922b , HA , Tannaout (1000 m), Marrakech; Dakki 1997 ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Gonomyia Meigen, 1818 Gonomyia ( Gonomyia ) abscondita Lackschewitz, 1935 Driauach and Belqat 2016 , Rif , maison forestière; Oosterbroek 2020 (CCW: Rif ) Gonomyia ( Gonomyia ) sicula Lackschewitz, 1940 Driauach and Belqat 2016 , Rif , Oued Kbir, Dayat Jebel Zemzem, Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Aïn El Maounzil; Oosterbroek 2020 (CCW: Rif ) Gonomyia ( Gonomyia ) subtenella Savchenko, 1972 Starý and Oosterbroek 2008 , HA , Massif Toubkal, Aghbalou, 43 km S Marrakech (1000 m); Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Jnane Niche, Oued Sidi Yahia Aârab, EM , Oued Béni Ouachekradi; Oosterbroek 2020 (CCW: Rif , EM ) Gonomyia ( Gonomyia ) tenella (Meigen, 1818) Pierre 1924a , HA , Asni (1200 m); Séguy 1930a , HA , Asni; Savchenko et al. 1992 ; Dakki 1997 ; Starý 2009a ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis Osten-Sacken, 1869 Hoplolabis ( Parilisia ) obtusiapex (Savchenko, 1982) Starý 2006 , HA , Oasis Meski, AA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis ( Parilisia ) punctigera (Lackschewitz, 1940) Starý 2006 , HA , Oasis Meski, AA ; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hoplolabis ( Parilisia ) sororcula (Lackschewitz, 1940) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Barrage Ajras, Oued Ouringa, Oued El Kanar, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Idiocera Dale, 1842 Idiocera ( Euptilostena ) jucunda (Loew, 1873) = Gonomyia jucunda Loew, in Pierre 1924a : 201, Séguy 1930a : 22 Pierre 1924a , HA , Asni (1200 m); Séguy 1930a , HA , Asni; Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Idiocera ( Idiocera ) ampullifera (Starý, 1979) Driauach and Belqat 2016 , AA , Oued Zag; Oosterbroek 2020 (CCW: AA ) Idiocera ( Idiocera ) pulchripennis (Loew, 1856) = Gonomyia sexpunctata Dale, in Pierre 1922b : 148 = Gonomyia pulchripennis (Loew), in Dakki 1997 : 62 Pierre 1922b , AP , Atlantic coast; Ramdani 1981 , AP , Merja Sidi Boughaba; Savchenko et al. 1992 ; Dakki 1997 ; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Jnane Niche, Oued Aârkoub, Aïn Jdioui; Oosterbroek 2020 (CCW) Idiocera ( Idiocera ) sziladyi (Lackschewitz, 1940) Driauach and Belqat 2016 , Rif , Oued Zarka; Oosterbroek 2020 (CCW: Rif ) Ilisia Rondani, 1856 Ilisia maculata (Meigen, 1804) Driauach and Belqat 2016 , Rif , Oued Maggou, EM , Oued Tafoughalt; Oosterbroek 2020 (CCW: Rif ; EM ) Molophilus Curtis, 1833 Molophilus ( Molophilus ) ibericus Starý, 2011 Starý 2011 , HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Molophilus ( Molophilus ) obscurus (Meigen, 1818) Starý and Oosterbroek 2008 , HA , Massif Toubkal, Oukaimeden, 2500–2800 m; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , tributary of Oued Ouara, tributary of Oued Maggou, Dayat El Ânassar, Dayat Jebel Zemzem, Aïn El Ma Bared, Dayat Rmali, Oued Amsemlil, Aïn Mâaze, Dayat Afrate, Dayat Lemtahane; Oosterbroek 2020 (CCW: Rif ) Molophilus ( Molophilus ) propinquus propinquus (Egger, 1863) Savchenko et al. 1992 ; Starý and Oosterbroek 2008 , HA , Oukaimeden (2500–2800 m); Starý 2011 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Farda, tributary of Oued Taida, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Molophilus ( Molophilus ) testaceus Lackschewitz, 1940 Driauach and Belqat 2016 , Rif , Dayat Amsemlil, Dayat Lemtahane, Marj El kheyl, Oued Tkarae, Dayat near Aïn Afersiw, Dayat Mezine, Dayat Tazia, Dayat Rmali, Dayat Amsemlil, Dayat near Aïn Afersiw; Oosterbroek 2020 (CCW: Rif ) Symplecta Meigen, 1830 Symplecta ( Symplecta ) grata Loew, 1873 Ebejer et al. 2020, Rif , Aïn Jdioui (Tahaddart, 8 m) Symplecta ( Symplecta ) hybrida (Meigen, 1804) Lackshewitz 1940a, HA , Goundafa (1200 m); Séguy 1941a , HA , Taroudant; Savchenko et al. 1992 ; Oosterbroek et al. 2007; Koçak and Kemal 2010 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Barrage Ajras, Oued El Kanar, Oued Aârkoub, Oued Jnane Niche, MA , Barrage Allal El Fassi; Oosterbroek 2020 (CCW: Rif ; MA ) Symplecta ( Trimicra ) pilipes (Fabricius, 1787) = Trimicra pilipes Fabricius, in Pierre 1922a : 23 = Trimicra andalusiaca Strobl, in Pierre 1922b : 149 = Trimicra hirsutipes Macquart, in Séguy 1930a : 22 Pierre 1922a , AP , Dradek (near Rabat), HA , Marrakech; Pierre 1922b , MA , Volubilis, HA , Oued Tensift; Séguy 1930a ; Séguy 1941d , AA , Taroudant; Savchenko et al. 1992 ; Dakki 1997 ; Pârvu and Zaharia 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , tributary of Oued Hachef, Oued Aârk­ob, Oued El Kanar, Oued Mezine, AP , Aïn Chouk (Larache); Oosterbroek 2020 (CCW: Rif ) Tasiocera ( Dasymolophilus ) murina (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued Farda, Oued Amsemlil; Oosterbroek 2020 (CCW: Rif ) Dactylolabinae Dactylolabis Osten-Sacken, 1860 Dactylolabis ( Dactylolabis ) symplectoidea Egger, 1863 Pierre 1922a , AP , Around Casablanca (coastal meseta); Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limnophilinae Austrolimnophila Alexander, 1920 Austrolimnophila ( Austrolimnophila ) latistyla Starý, 1977 Driauach and Belqat 2016 , Rif , Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Dicranophragma Osten-Sacken, 1860 Dicranophragma ( Brachylimnophila ) adjunctum (Walker, 1848) = Neolimnomyia adjuncta (Walker, 1848), in Pârvu et al. 2006 : 273 Pârvu et al. 2006 , AP , Merja Zerga; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranophragma ( Brachylimnophila ) nemorale (Meigen, 1818) Lackschewitz 1940b , HA , Tachdirt (2200–2700 m); Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Aïn Sidi Brahim Ben Arrif, tributary Oued Ouara, Oued Maggou; Oosterbroek 2020 (CCW: Rif ) Eloeophila Rondani , 1856 Eloeophila maroccana Starý, 2009 Starý 2009b , HA , Okaïmeden (2500–2800 m); Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Euphylidorea Alexander, 1972 Euphylidorea ( Euphylidorea ) crocotula (Séguy, 1941) = Phylidorea crocotula (Séguy), in Séguy 1941a : 28 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 , HA , Tachdirt (Toubkal); Starý and Oosterbroek 2008 , HA , Oukaimeden (2500 m–2800 m), Imlil (1400 m); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ; HA ) Euphylidorea ( Euphylidorea ) dispar (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued at 15 km from Fifi; Oosterbroek 2020 (CCW: Rif ) Euphylidorea ( Euphylidorea ) lineola (Meigen, 1804) Lackschewitz 1940b , HA ; Savchenko et al. 1992 ; Starý and Freidberg 2007 ; Driauch et al. 2013; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Hexatoma Latreille, 1809 Hexatoma ( Hexatoma ) bicolor (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued at 15 km from Fifi, tributary of Oued Ouara, Oued Madissouka, Oued Maggou, Aïn Quanquben (Jebel Bou Bessoui), Oued Tkarae, Oued Tamerte; Oosterbroek 2020 (CCW: Rif ) Hexatoma ( Hexatoma ) gaedii (Meigen, 1830) Lackschewitz 1940b , HA , Tachdirt (2200 m–2700 m); Savchenko et al. 1992 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Pseudolimnophila Alexander, 1919 Pseudolimnophila ( Pseudolimnophila ) sepium (Verrall, 1886) Driauach et al. 2013 , Rif , Guelta Tazia; Driauach and Belqat 2016 , Rif , Dayat Tazia, Aïn Sidi Brahim Ben Ar­rif, Aïn Afersiw, Oued Maggou, Dayat Tazia, Oued Taida, Aïn Sidi Brahim Ben Arrif; Oosterbroek 2020 (CCW: Rif ) Limoniinae Dicranomyia Stephens, 1829 Dicranomyia ( Dicranomyia ) affinis (Schummel, 1829) Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Amsemlil, Dayat Lemtahane, Oued Tkarae, Marj El Kheyl, Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Aïn El Maounzil, Aïn El Malâab, Dayat near Aïn Afersiw, Aïn Bab Tariouant; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) chorea (Meigen, 1818) Pierre 1922b , HA , Haute Réghaya, Tannaout, Asni (1000 m–1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Starý and Freidberg 2007 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Farda, Oued Kelâa, Aïn Ras el Ma, Oued Jnane Niche, Cascade Chrafate, maison forestière, Oued El Kanar, tributary of Oued Zarka, HA , Oued Sidi Fares (National Park of Toubkal); Oosterbroek 2020 (CCW: Rif ; HA ) Dicranomyia ( Dicranomyia ) didyma (Meigen, 1804) Pierre 1922b , HA , Haute Réghaya (2000 m); Dakki 1997 ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Dicranomyia ( Dicranomyia ) goritiensis (Mik, 1864) Lackschewitz 1940b ; Vaillant 1956b ; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Maggou, Cascade Chrafate, Oued El Koub, Cascade Zarka, Âounsar Aheramen, maison forestière; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) longicollis (Macquart, 1846) = Telecephala longicollis (Macquart), in Pierre 1922a : 21, Pierre 1922b : 148, Séguy 1930a : 22 Séguy 1930a , AP , Dradek (near Rabat); Dakki 1997 ; Pierre 1922a , AP , Dradek, HA , Marrakech; Pierre 1922b , AP , Rabat; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Aârate, Barrage Moulay Bouchta, Oued Kbir, MA , Barrage Allal El Fassi; Oosterbroek 2020 (CCW) Dicranomyia ( Dicranomyia ) mitis (Meigen, 1830) = Dicranomyia hygropetrica Vaillant, in Vaillant 1956b : 42 Vaillant 1956b , HA , Asif Tessaout (M'Goum), Izourar, Tahanaout, Tamesrit, Imi-N'Ifri, Aguelmous, Sidi Chamarouch, Lac Tamhda (Anremer), Oukaimeden; Savchenko et al. 1992 ; Pârvu et al. 2006 , AA , near Agadir, Souss plain to High Atlas occidental; Pârvu and Zaharia 2007 ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 , Rif , Tazia, Tisgris; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia ( Dicranomyia ) modesta (Meigen, 1818) Driauach and Belqat 2016 , Rif , Oued Farda, Oued El Kanar, Oued Jnane Niche, Oued Sidi Yahia Aârab; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) novemmaculata (Strobl, 1906) Driauach and Belqat 2016 , Rif , tributary of Oued el Fondak, Oued Aârate; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Dicranomyia ) ventralis (Schummel, 1829) Driauach and Belqat 2016 , Rif , Lake Badriouen; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Glochina ) sericata (Meigen, 1830) Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia ( Melanolimonia ) hamata Becker, 1908 Driauach and Belqat 2016 , Rif , tributary of Oued Kbir, Oued Aârate, Aïn Sidi Brahim Ben Arrif, Dayat Tazia; Oosterbroek 2020 (CCW: Rif ) Dicranomyia ( Melanolimonia ) morio (Fabricius, 1787) = Limonia pauliani Séguy, in Séguy 1941a : 26 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Savchenko et al. 1992 , HA , Tachdirt (Toubkal); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Seguia Lemtahane, Dayat near Aïn Afersiw, Dayat Jebel Zemzem; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranomyia majuscula Pierre, 1924 1 Pierre 1924a , HA , Haut Imminen (2400 m); Séguy 1930a , HA , Haut Imminen; Dakki 1997 ; Savchenko et al. 1992 , HA , Haut Imminen; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranoptycha Osten-Sacken, 1860 Dicranoptycha fuscescens (Schummel, 1829) Eiroa 2000 , Rif ; Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Mlilah, Oued Zendoula, AP , Oued Loukous; Oosterbroek 2020 (CCW) Geranomyia Haliday, 1833 Geranomyia caloptera (Mik, 1867) Driauach et al. 2013 , HA , Setti Fatma; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Geranomyia obscura Strobl, 1900 Vaillant 1956b , HA , Lac Tamhda (Anremer), Oukaimeden, Izourar, Sidi Chamarouch, Tamesrit; Savchenko et al. 1992 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Helius Lepeletier & Serville, 1828 Helius ( Helius ) hispanicus Lackschewitz, 1928 Starý and Oosterbroek 2008 , HA , Massif Toubkal, Imlil (17 km S Asni, 1700–1900 m); Driauach et al. 2013 ; Driauach and Belqat 2016 , Rif , Oued Amsemlil; Oosterbroek 2020 (CCW: Rif ) Helius ( Helius ) pallirostris Edwards, 1921 Driauach and Belqat 2016 , AP , Aïn Chouk (Larache); Oosterbroek 2020 (CCW: Rif ) Limonia Meigen, 1803 Limonia flavipes (Fabricius, 1787) Pierre 1922b , HA , Haute Réghaya, Asni (1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limonia hercegovinae (Strobl, 1898) Starý and Oosterbroek 2008 , MA , Ifrane (1700 m), HA ; Gavryushin pers. comm. 2012, HA ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW: HA ) Limonia macrostigma (Schummel, 1829) Starý and Oosterbroek 2008 , HA , 5 km from Oukaimeden (2350 m); Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Limonia nubeculosa Meigen, 1804 Pierre 1922b , HA , Haute Réghaya, Asni (1250 m); Savchenko et al. 1992 ; Dakki 1997 ; Gavryushin pers. comm. 2012, HA ; Driauach and Belqat 2016 , Rif , tributary of Oued El Fondak, Aïn Ras el Ma, Aïn Boughaba, Âounsar Aheramen, Aïn Takhninjoute, maison forestière, Oued Madissouka, Aïn Quan­quben (Jebel Bou Bessoui), EM , Oued Azila; Oosterbroek 2020 (CCW: Rif ; HA ) Limonia phragmitidis (Schrank, 1781) Starý and Oosterbroek 2008 , MA , Ifrane (1700 m); Driauach and Belqat 2016 , Rif , Aïn Quanquben (Jebel Bou Bessoui); Oosterbroek 2020 (CCW) PEDICIIDAE K. Kettani, P. Oosterbroek Number of species: 6 . Expected: 10 Faunistic knowledge of the family in Morocco: moderate Pediciinae Dicranota Zetterstedt, 1838 Dicranota ( Dicranota ) bimaculata (Schummel, 1829) Pierre 1922b , HA , Haute Réghaya (1800 m); Savchenko et al. 1992 (? Morocco); Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Dicranota ) irregularis Pierre, 1921 Pierre 1922b , HA , Haute Réghaya (1800 m); Savchenko et al. 1992 , HA , Cirque d'Arround (Haute Réghaya); Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Ludicia ) claripennis (Verrall, 1888) Driauach and Belqat 2016 , Rif , Oued Amsemlil, maison forestière; Oosterbroek 2020 (CCW) Dicranota ( Paradicranota ) candelisequa Starý, 1981 Pârvu et al. 2006 , AP , Merja Zerga; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Paradicranota ) landrocki Czižek, 1931 Driauach et al. 2013 , Rif , Fifi (1252 m); Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Taida, Aïn Sidi Brahim Ben Arrif, Âounsar Aheramen, Oued Tizekhte, Oued Mezine, maison forestière, Aïn Bab Tariouant, HA , Imlil (Assif Haouz); Oosterbroek 2020 (CCW) Tricyphona Zetterstedt, 1838 Tricyphona ( Tricyphona ) immaculata (Meigen, 1804) Driauach and Belqat 2016 , Rif , maison forestière, Dayat Lemtahane; Oosterbroek 2020 (CCW) Pediciinae Dicranota Zetterstedt, 1838 Dicranota ( Dicranota ) bimaculata (Schummel, 1829) Pierre 1922b , HA , Haute Réghaya (1800 m); Savchenko et al. 1992 (? Morocco); Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Dicranota ) irregularis Pierre, 1921 Pierre 1922b , HA , Haute Réghaya (1800 m); Savchenko et al. 1992 , HA , Cirque d'Arround (Haute Réghaya); Dakki 1997 ; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Ludicia ) claripennis (Verrall, 1888) Driauach and Belqat 2016 , Rif , Oued Amsemlil, maison forestière; Oosterbroek 2020 (CCW) Dicranota ( Paradicranota ) candelisequa Starý, 1981 Pârvu et al. 2006 , AP , Merja Zerga; Driauach et al. 2013 ; Driauach and Belqat 2016 ; Oosterbroek 2020 (CCW) Dicranota ( Paradicranota ) landrocki Czižek, 1931 Driauach et al. 2013 , Rif , Fifi (1252 m); Driauach and Belqat 2016 , Rif , Oued Ouara, Oued Taida, Aïn Sidi Brahim Ben Arrif, Âounsar Aheramen, Oued Tizekhte, Oued Mezine, maison forestière, Aïn Bab Tariouant, HA , Imlil (Assif Haouz); Oosterbroek 2020 (CCW) Tricyphona Zetterstedt, 1838 Tricyphona ( Tricyphona ) immaculata (Meigen, 1804) Driauach and Belqat 2016 , Rif , maison forestière, Dayat Lemtahane; Oosterbroek 2020 (CCW) TIPULIDAE K. Kettani, P. Oosterbroek, H. de Jong Number of species: 39 . Expected: 42 Faunistic knowledge of the family in Morocco: moderate Dolichopezinae Dolichopeza Curtis, 1825 Dolichopeza ( Dolichopeza ) hispanica Mannheims, 1951 Theowald and Oosterbroek 1980 , HA , Aghbalou, Oukaimeden, Imlil, Tadmant; Oosterbroek and Theowald 1992 ; Oosterbroek and Lantsov 2011 , HA , Oukaimeden (2300 m), Aghbalou (Massif Toubkal), 43 km S Marrakech (1000 m), Imlil (17 km S Asni, 1700–1900 m), Tadmant (17 km E Asni); Adghir et al. 2018 , Rif , Kitane, Aîn Ras el Ma (Chefchaouen); Oosterbroek 2020 (CCW) Tipulinae Nephrotoma Meigen, 1803 Nephrotoma alluaudi (Pierre, 1922) = Pachyrhina lunulicornis Schummel, in Pierre 1922a : 24 = Pachyrhina alluaudi Pierre, in Pierre 1922b : 150, Dakki 1997 : 62 = Pales alluaudi (Pierre), in Mannheims 1951 : 47 Pierre 1922b , HA , Tannaout (1000 m); Mannheims 1951 , Rif , Beni Seddat, AP , Lagune Guedira; HA , Taddert north of Marrakech, Goundafa (1200 m); AA , Llano Amarillo, Tlata Reisana; Oosterbroek 1979b , Rif , HA , Imlil (1400 m), Tizi-n'Tichka (2200 m); Theowald and Oosterbroek 1980 , AP , Rabat, Guedira lagoon, MA , Immouzer, Ifrane, Timahdit, Aghbalou, Tizi-n'Zou, HA , Marrakech, Goundafa, Taddert, Dayat, Tizi-n'Test, Tizi-n'Tichka, Asni, Imlil, Oukaimeden, Setti Fatma, Tinmel, Acif Tifni, AA , Taroudant, Mikdana, Sidi Said bou Merdoul, Tlata Reisana, Llano Amarillo; Eiroa 1990 , MA , Azrou, Ajabo; Oosterbroek and Theowald 1992 , HA , Tannaout; Dakki 1997 ; Mouna 1998 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Oued Laou, Dardara, Douar Mouarâa, Oued Zandoula, Laghmari-Rmal, Douar Louamera, Douar Laheyayda, Oued Jnane Niche, Cabo Negro, Oued Beni Said, Oued El Kanar, Oued Amsemlil; Oosterbroek 2020 (CCW) – MISR ( HA ), MHNV, MNCNM, MAKB Nephrotoma appendiculata pertenua Oosterbroek, 1978 Oosterbroek 1978 , Rif , 9 km SW Chefchaouen; Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Fès, Immouzer, Khemisset; Oosterbroek and Theowald 1992 ; de Jong 1993 , 1998 , Rif ; Adghir et al. 2018 , Rif , Oued Ametrasse, Dayat Aïn Jdioui, Barrage Moulay Bouchta, Oued Sahil, Dayat Jebel Zemzem, Aïn Sidi Brahim Ben Arrif, Oued Nakhla, Oued Tizekhte, Lot Hemmadi, Douar Ayacha, Douar Louamera, Dayat Mezine, Aïn El Malâab, Aïn Takhninjoute, Dayat Tazia, Oued Taida, maison forestière Tazia, Tourbière Amesmlil, Oued El Hamma, Oued Kbir, Jebel Lakraâ; Oosterbroek 2020 (CCW) Nephrotoma astigma Pierre, 1925 Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Taza; Adghir et al. 2018 , Rif , Oued Tabandout, Etang Maggou, Aïn El Malâab, Douar Remla; Oosterbroek 2020 (CCW) Nephrotoma fontana Oosterbroek, 1978 de Jong 1998 , Rif ; Adghir et al. 2018 , Rif , Ketama, maison forestière Tazia, Dayat Tazia; Oosterbroek 2020 (CCW) Nephrotoma guestfalica vaillanti de Jong, Adghir & Bosch, 2021 de Jong et al. 2021 , Rif , Ras el Ma (Chefchaouen, 500 m), Fomento, Oued Laou (6 km north-west of Chefchaouen, 200 m), Jebel Tissouka (3 km south of Chefchaouen, 500 m; 3 km south of Chefchaouen, 700 m; 4 km south-east of Chefchaouen, 700–800 m; 5 km southeast of Chefchaouen, 700–900 m; 5 km south of Chefchaouen, 900–1000 m), Bab Taza (25 km south-east of Chefchaouen, 750–800 m), Dardara (10 km south of Chefchaouen, 300 m), Oued Martil, Nakhla, Âounsar Aheramen, Boumerouil, Oued Maggou, Oued Maggou (Aïn Ras el Ma), Douar Kitane, Oued Sidi Yahia Aârab, Oued Tamerte, Oued Zandoula, Oued El Hamma, Lot Hemmadi, Oued Tamerte, Oued Sidi Mohamed Saâda, Oued El Koub, Douar Iholebatine, Dayat Tazia, Belouazen, Oued Lemtahane, Oued Siflaou, Oued Amsemlil, AP , Oued Loukous, Aïn el-Aouda, MA , Ifrane (road to Mischliffen, 1680 m), N.S. and W of Ifrane (1400–1800 m), Khemisset, Azrou, Oum-er-Rbia, HA , M'semrir (bord de l'Oued), Rich, Haute Rhégaya (identified by Mannheims in 1951 as surcoufi ) Nephrotoma luteata (Meigen, 1818) Oosterbroek 1979a , Rif , Targuist, Chefchaouen, HA , Kasba Taguendaft, near Oukaimeden; Theowald and Oosterbroek 1980 , Rif , Targuist, Chefchaouen, HA , Kasba Taguendaft, near Oukaimeden; Oosterbroek and Theowald 1992 ; Pârvu et al. 2006 , AP , Merja Zerga; Adghir et al. 2018 , Rif , Oued Laou, Dardera, Oued Nakhla; Oosterbroek 2020 (CCW) Nephrotoma subanalis (Mannheims, 1951) = ? Pachyrhina analis Schummel, in Pierre 1922a : 24, Pierre 1922b : 150 = Pales subanalis Mannheims, in Mannheims 1951 : 56 Mannheims 1951 , HA , Tachdirt (2200–2900 m); Vaillant 1956b , HA , Oukaimeden (2250 m); Oosterbroek 1979c, HA , Tachdirt (2200–2900 m); Theowald and Oosterbroek 1980 , HA , Tachdirt, Oukaimeden, M'Goum, Tadmant, Imlil, Tizi-N'Tichka; Oosterbroek and Theowald 1992 , HA , Tachdirt; Oosterbroek 2020 (CCW) Nephrotoma submaculosa Edwards, 1928 Oosterbroek 1982 , Rif , Ketama, Dardara, MA , Azrou; Oosterbroek and Theowald 1992 ; de Jong 1998 , Rif , Atlas ; Oosterbroek 2011 ; Adghir et al. 2018 , Rif , Jebel Dahedouh, Ketama, Aïn Sidi Brahim Ben Arrif, Oued Taida, tributary of Oued Ouara, Sidi Chouiref, Dayat Tazia; Oosterbroek 2020 (CCW) Nephrotoma sullingtonensis Edwards, 1938 Oosterbroek 1978 ; Theowald and Oosterbroek 1980 , Rif , Bab Berred; Oosterbroek 1982 Rif , Bab Berred; de Jong 1993 , 1998 , Rif ; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 , Rif , 4 km SE Ketama, Barrage Moulay Bouchta, Oued Aârate, Aïn Takhninjoute, Oued Jbara, Aïn El Malâab, Aïn El Ma Bared, Oued Lemtahane, maison forestière Tazia, Oued Taida, Dayat Tazia; Oosterbroek 2020 (CCW) Tipula Linnaeus, 1758 Tipula ( Acutipula ) anormalipennis Pierre, 1924 Pierre 1924b , HA , Haut Imminen; Séguy 1930a , HA , Haut Imminen; Séguy 1941a , HA , Tachdirt (2500 m), Haut Imminen; Mannheims 1952 , HA , Tachdirt (2500 m), Imminen (2400–2500 m), Arround (1950 m); Vaillant 1956b , HA , Lake of Tamhda (Anremer, 2900 m); Theowald and Oosterbroek 1980 , HA , Anremer, Haut-Immenen, Tachdirt, Arround, Oukaimeden; Vermoolen 1983 , HA , Haut Imminen, Oukaimeden (2500–2800 m), Arround (1950 m), Tachdirt (2500 m); Oosterbroek and Theowald 1992 ; de Jong 1994a , HA ; de Jong 1998 , HA ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Acutipula ) repentina Mannheims, 1952 = Tipula maxima Poda, in Séguy 1941a : 26 Séguy 1941a , HA , Tachdirt (2500 m); Mannheims 1952 , HA , Tachdirt (2200–2700 m); Vaillant 1956b , HA , Lac Tamhda (Anremer, 2900), M'Goum (2500 m); Theowald and Oosterbroek 1980 , HA , Anremer, M'Goum, Tachdirt, Tizi-N'Tichka, Asni, Oukaimeden, Setti Fatma, Imlil, Tadmant; Vermoolen 1983 , MA , Ifrane, HA , Androment, M'Goum, Tadmant, Tizi-N'Test, Setti Fatma, Oukaimeden, Tizi-N'Tichka; Oosterbroek and Theowald 1992 , HA , Tachdirt; de Jong 1994a , MA , HA ; de Jong 1998 , HA ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Acutipula ) rifensis Theowald & Oosterbroek, 1980 Theowald and Oosterbroek 1980 , Rif , Targuist; Vermoolen 1983 , Rif , Targuist, Tidiguin (90 km E. of Ouezzane, 2350 m); Oosterbroek and Theowald 1992 , Rif ; de Jong 1994a , 1998 , Rif ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Emodotipula ) leo Dufour, 1991 = Tipula ( Emodotipula ) obscuriventris Strobl 2 , in Oosterbroek and Theowald 1992 : 99 Oosterbroek and Theowald 1992 (?); Dufour 2003 , Rif ; Adghir et al. 2018 , Rif , Jebel Tissouka; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) bivittata Pierre, 1922 Pierre 1922a , AP , Maâmora between Kénitra and Oued Beth, Dradek (near Rabat); Pierre 1922b , AP , Rabat; Mannheims 1968 ; Theowald and Oosterbroek 1980 , AP , forest of Maâmora, Dradek; Oosterbroek and Theowald 1992 , AP , forest of Maâmora, Dradek; Dakki 1997 ; Oosterbroek 2020 (CCW) – MISR ( AP , Kénitra, Dradek) Tipula ( Lunatipula ) cinereicolor Pierre, 1924 Pierre 1924b , HA , Haut Imminen; Séguy 1930a , HA , Tachdirt (3100–3200 m); Theowald 1973 , HA , Haut Imminen (2400 m); Theowald and Oosterbroek 1980 , MA , Ifrane, HA , Oukaimeden, Tachdirt, Haut-Imminen; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Imminen; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) cornicula Pierre, 1922 Pierre 1922b , HA , Arround (2000 m); Séguy 1930a , HA , Tachdirt (3100–3200 m); Theowald 1973 , HA , Cirque d'Arround (2000 m), Tachdirt (2200–2700 m), Goundafa (1200 m); Theowald and Oosterbroek 1980 , HA , Oukaimeden, Tachdirt, Goundafa; Oosterbroek and Theowald 1992 , HA , Tachdirt; Dakki 1997 ; Oosterbroek 2020 (CCW) – MISR ( HA ) Tipula ( Lunatipula ) fabiola Mannheims, 1968 Theowald and Oosterbroek 1980 , Rif , Bab Berred, Ras El Ma (Chefchaouen), MA , Jebel Abad, Ifrane; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) hermes Theischinger, 1977 Theischinger 1977 , Rif , north of Ouezzane; Theowald 1980 ; Theowald and Oosterbroek 1980 , Rif , north of Ouezzane; Oosterbroek and Theowald 1992 , Rif , Ouezzane; Adghir et al. 2018 , Rif , 4 km SE Ketama, Oued Ametrasse, Aïn El Ma Bared; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) iberica iberica Mannheims, 1963 = Tipula lunata Linnaeus, in Pierre 1922b : 149, Séguy 1930a : 23 Pierre 1922b , HA , Haute Réghaya, Tannaout (1000 m); Séguy 1930a , HA , Haut Imminen; Mannheims 1963 , MA ; Theowald and Oosterbroek 1980 , Rif , Bab Berred, MA , Ifrane, Taounate; Eiroa 1990 , MA , Ifrane; Oosterbroek and Theowald 1992 ; Oosterbroek 2009 ; Adghir et al. 2018 , Rif , Ketama; Oosterbroek 2020 (CCW) – MISR ( HA ) Tipula ( Lunatipula ) iberica spinula Theischinger, 1980 Theischinger 1980 , HA , Oukaimeden (1300–2800 m); Theowald and Oosterbroek 1980 , HA , Oukaimeden; Oosterbroek and Theowald 1992 , HA , Oukaimeden; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) peliostigma peliostigma Schummel, 1833 Eiroa 1990 , MA , Azrou; Mouna 1997; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) pjotri de Jong & Adghir, 2018 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m) Tipula ( Lunatipula ) pseudocinerascens Strobl, 1906 Adghir et al. 2018 , Rif , Stehat, Oued Taida, Perdicaris Park, Dayat Tazia Tipula ( Lunatipula ) rocina Theischinger, 1979 Theowald and Oosterbroek 1980 , Rif , Tétouan; Oosterbroek and Theowald 1992 ; Oosterbroek 2009 ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) selenaria Mannheims, 1967 Mannheims 1967 , MA , Jebel Tazzeka (1500–1989 m), HA , Goundafa (1200 m); Theowald and Oosterbroek 1980 , MA , Tazzeka, HA , Foum Keneg, Goundafa, Oukaimeden; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Goundafa; de Jong 1995 , MA , HA , Oukaimeden; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) stimulosa Mannheims, 1973 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m) Tipula ( Lunatipula ) subfalcata Mannheims, 1967 de Jong 1995 , 1998 , Rif ; Adghir et al. 2018 , Rif , Jebel Tissouka, Oued Tamerte, Oued El Koub; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) subpustulata Mannheims, 1963 = Tipula pustulata Pierre, in Pierre 1922b : 150 Pierre 1922b , AP , Mogador; Vaillant 1956b , HA , M'Goun (2500 m); Mannheims 1963 , AP , Aïn el Aouda, MA , Jebel Tazzeka (1600–1989 m), HA , Goundafa (1200 m), Tachdirt (2200–2900 m), AA , Lac Goulmima; Theowald 1972 ; Theowald and Oosterbroek 1980 , AP , Aïn el Aouda, MA , Iebel Tazzeka, HA , M'Goum, Goundafa, Tachdirt, Oukaimeden, Imlil; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Goundafa; Dakki 1997 ; Adghir et al. 2018 , Rif , 4 km SE Ketama, Aïn El Ma Bared; Oosterbroek 2020 (CCW) – MISR ( AP , Mogador) Tipula ( Lunatipula ) tazzekai Theowald, 1973 Theowald 1973 , MA , Jebel Tazzeka (1600–1989 m); Theowald and Oosterbroek 1980 , MA , Jebel Tazzeka; Eiroa 1990 , MA , Jebel Hebri; Oosterbroek and Theowald 1992 , MA , Jebel Tazzeka; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) atlas Pierre, 1924 Pierre 1924b , HA , Tachdirt (3100–3250 m); Séguy 1930a , HA , Tachdirt (3100–3200 m); Vaillant 1956b , HA , Cascade Siroua (3000 m), M'Goun (2500 m), Toubkal (3350 m), Lake of Tamhda (Anremer, 2900 m); Mannheims 1964 , HA ; Theowald 1973 , HA ; Theowald 1980 , HA ; Theowald and Oosterbroek 1980 , HA , Toubkal, Sources de Tessaouts, M'Goum, Siroua, Anremer, Tachdirt, Oukaimeden, Tizi-N'Tichka, Tadmant; Eiroa 1990 , MA , Oum-Er-Rbia; Oosterbroek and Theowald 1992 , HA , Tachdirt; de Jong 1994b ; de Jong 1998 , Atlas ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) breviantennata Lackschewitz, 1933 de Jong 1998 , Rif ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Oued Maggou, Douar Aouzighen; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) confusa van der Wulp, 1883 Adghir et al. 2018 , Rif , maison forestière (Talassemtane) Tipula ( Savtshenkia ) rufina rufina Meigen, 1818 Theowald and Oosterbroek 1980 , HA , Oukaimeden; Theowald and Oosterbroek 1983 , Rif , Atlas ; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen); Oosterbroek 2020 (CCW) Tipula ( Tipula ) mediterranea Lackschewitz, 1930 Vaillant 1956b , HA , Oukaimeden (2250 m), M'Goun (2500 m); Mannheims 1952 ; Theowald and Oosterbroek 1980 , MA , Ifrane, HA , Oukaimeden, M'Goum, Asni, M'Semrir, Ifni, Bab-Rou-Idie, Tizi-N'Tichka, Setti Fatma; Theowald 1984 , Rif ; Eiroa 1990 , MA , Azrou, Oum-Er-Rbia; Oosterbroek and Theowald 1992 ; Pârvu et al. 2006 AP , Merja Zerga; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Jebel Tissouka, Bab Taza, Dardara, 4 km SE Ketama, Aïn Afersiw, Douar Kitane, Dayat Jebel Zemzem, Wilaya (Tétouan), Aïn El Ma Bared, Oued Tamerte, Douar Louamera, Tourbière Amesmlil, Dayat Mezine, Hejar Nehal, tributary of Oued Ouara, Oued Tkarae, Oued Jnane Niche, Dayat Afrate, Oued Ametrasse; Oosterbroek 2020 (CCW) Tipula ( Tipula ) oleracea Linnaeus, 1758 Mannheims 1952 , Rif , Tlata Ketama; Theowald and Oosterbroek 1980 , Rif , Tlata Ketama; Theowald 1984 , Rif ; Oosterbroek and Theowald 1992 ; Dakki 1997 ; Pârvu and Zaharia 2007 ; Oosterbroek 2011 ; Adghir et al. 2018 , Rif , Oued Laou, Dardara, Jebel Tissouka, Ksar Rimal, 15 km from Fifi, Oued Aârate, Oued El Hamma, Douar Louamera, Oued Zaouya, Près de Beni Said, Wilaya (Tétouan), Tourbière Amesmlil, Oued Smir, Aïn Jdida; Oosterbroek 2020 (CCW) – MISR Tipula ( Vestiplex ) vaillanti vaillanti Theowald, 1977 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m), Douar Kitane Tipula ( Yamatotipula ) afriberia afriberia Theowald & Oosterbroek, 1980 Theowald and Oosterbroek 1980 , HA , Oukaimeden; Oosterbroek and Theowald 1992 , HA , Oukaimeden; Oosterbroek 1994a ; Adghir et al. 2018 , Rif , Jebel Tissouka, Dardara, Douar Mokedassen, Oued Zarka; Oosterbroek 2020 (CCW) Tipula ( Yamatotipula ) barbarensis Theowald & Oosterbroek, 1980 = Tipula lateralis Meigen, in Pierre 1922a : 23, Pierre 1922b : 150, Séguy 1930a : 23, Séguy 1941a : 26, Mannheims 1952 : 98 (in part), Vaillant 1956b : 238 Pierre 1922a , AP , Dradek (Rabat), MA , Azrou (riverside of Oued Tigrigra); Pierre 1922b , AP , Mogador, MA , Beni Méllal, HA , Asni; Séguy 1930a ; Séguy 1941a , HA , Imi n'Ouaka (1500 m); Mannheims 1952 ; Vaillant 1956b , HA , Oukaimeden; Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Ifrane, Aghbalou, HA , Asni, Oukaimeden, Setti Fatma, Imlil, Tizi-N'Tichka, Tadmant, Tachdirt; Eiroa 1990 , MA , Azrou, Oum-er-Rbia, Ifrane; Oosterbroek and Theowald 1992 , HA , Setti Fatma; Oosterbroek 1994a , Rif , Dardara, MA , Ifrane, Aghbalou, HA , Oukaimeden, Setti Fatma, Imlil, Tizi-N'Tichka, Tadmant, Asni, Tachdirt; Dakki 1997 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Jebel Tissouka, Bab Taza, Dardara, 4 km SE Ketama, Aïn El Manzela, Aïn Bab Tariouante, Dayat Aïn Afersiw, Oued El Kanar, Dayat Afrate, Douar Kitane, Wilaya (Tétouan), 15 km from Fifi, Aïn Sidi Brahim Ben Arrif, Oued Nakhla, Âounsar Aheramen, Oued Boumerouil, Douar Zaouya, Oued Tizekhte, Oued Samsa, Dayat Aïn Jdioui, Douar Ouled Laghmari-Rmal, maison forestière Tazia, Hejar Nehal, Lot Hemmadi, tributary of Oued Ouara, Oued Sidi Mohamed Saâda, Oued Amsemlil, Tourbière Amesmlil, Beni Salah, Oued Jnane Niche, Oued Maggou, Souk Lhed Beni Darkoul, Oued Imassouden, Aïn Helouma, Source Zarka, Aïn Kchour; Oosterbroek 2020 (CCW) – MISR Dolichopezinae Dolichopeza Curtis, 1825 Dolichopeza ( Dolichopeza ) hispanica Mannheims, 1951 Theowald and Oosterbroek 1980 , HA , Aghbalou, Oukaimeden, Imlil, Tadmant; Oosterbroek and Theowald 1992 ; Oosterbroek and Lantsov 2011 , HA , Oukaimeden (2300 m), Aghbalou (Massif Toubkal), 43 km S Marrakech (1000 m), Imlil (17 km S Asni, 1700–1900 m), Tadmant (17 km E Asni); Adghir et al. 2018 , Rif , Kitane, Aîn Ras el Ma (Chefchaouen); Oosterbroek 2020 (CCW) Tipulinae Nephrotoma Meigen, 1803 Nephrotoma alluaudi (Pierre, 1922) = Pachyrhina lunulicornis Schummel, in Pierre 1922a : 24 = Pachyrhina alluaudi Pierre, in Pierre 1922b : 150, Dakki 1997 : 62 = Pales alluaudi (Pierre), in Mannheims 1951 : 47 Pierre 1922b , HA , Tannaout (1000 m); Mannheims 1951 , Rif , Beni Seddat, AP , Lagune Guedira; HA , Taddert north of Marrakech, Goundafa (1200 m); AA , Llano Amarillo, Tlata Reisana; Oosterbroek 1979b , Rif , HA , Imlil (1400 m), Tizi-n'Tichka (2200 m); Theowald and Oosterbroek 1980 , AP , Rabat, Guedira lagoon, MA , Immouzer, Ifrane, Timahdit, Aghbalou, Tizi-n'Zou, HA , Marrakech, Goundafa, Taddert, Dayat, Tizi-n'Test, Tizi-n'Tichka, Asni, Imlil, Oukaimeden, Setti Fatma, Tinmel, Acif Tifni, AA , Taroudant, Mikdana, Sidi Said bou Merdoul, Tlata Reisana, Llano Amarillo; Eiroa 1990 , MA , Azrou, Ajabo; Oosterbroek and Theowald 1992 , HA , Tannaout; Dakki 1997 ; Mouna 1998 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Oued Laou, Dardara, Douar Mouarâa, Oued Zandoula, Laghmari-Rmal, Douar Louamera, Douar Laheyayda, Oued Jnane Niche, Cabo Negro, Oued Beni Said, Oued El Kanar, Oued Amsemlil; Oosterbroek 2020 (CCW) – MISR ( HA ), MHNV, MNCNM, MAKB Nephrotoma appendiculata pertenua Oosterbroek, 1978 Oosterbroek 1978 , Rif , 9 km SW Chefchaouen; Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Fès, Immouzer, Khemisset; Oosterbroek and Theowald 1992 ; de Jong 1993 , 1998 , Rif ; Adghir et al. 2018 , Rif , Oued Ametrasse, Dayat Aïn Jdioui, Barrage Moulay Bouchta, Oued Sahil, Dayat Jebel Zemzem, Aïn Sidi Brahim Ben Arrif, Oued Nakhla, Oued Tizekhte, Lot Hemmadi, Douar Ayacha, Douar Louamera, Dayat Mezine, Aïn El Malâab, Aïn Takhninjoute, Dayat Tazia, Oued Taida, maison forestière Tazia, Tourbière Amesmlil, Oued El Hamma, Oued Kbir, Jebel Lakraâ; Oosterbroek 2020 (CCW) Nephrotoma astigma Pierre, 1925 Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Taza; Adghir et al. 2018 , Rif , Oued Tabandout, Etang Maggou, Aïn El Malâab, Douar Remla; Oosterbroek 2020 (CCW) Nephrotoma fontana Oosterbroek, 1978 de Jong 1998 , Rif ; Adghir et al. 2018 , Rif , Ketama, maison forestière Tazia, Dayat Tazia; Oosterbroek 2020 (CCW) Nephrotoma guestfalica vaillanti de Jong, Adghir & Bosch, 2021 de Jong et al. 2021 , Rif , Ras el Ma (Chefchaouen, 500 m), Fomento, Oued Laou (6 km north-west of Chefchaouen, 200 m), Jebel Tissouka (3 km south of Chefchaouen, 500 m; 3 km south of Chefchaouen, 700 m; 4 km south-east of Chefchaouen, 700–800 m; 5 km southeast of Chefchaouen, 700–900 m; 5 km south of Chefchaouen, 900–1000 m), Bab Taza (25 km south-east of Chefchaouen, 750–800 m), Dardara (10 km south of Chefchaouen, 300 m), Oued Martil, Nakhla, Âounsar Aheramen, Boumerouil, Oued Maggou, Oued Maggou (Aïn Ras el Ma), Douar Kitane, Oued Sidi Yahia Aârab, Oued Tamerte, Oued Zandoula, Oued El Hamma, Lot Hemmadi, Oued Tamerte, Oued Sidi Mohamed Saâda, Oued El Koub, Douar Iholebatine, Dayat Tazia, Belouazen, Oued Lemtahane, Oued Siflaou, Oued Amsemlil, AP , Oued Loukous, Aïn el-Aouda, MA , Ifrane (road to Mischliffen, 1680 m), N.S. and W of Ifrane (1400–1800 m), Khemisset, Azrou, Oum-er-Rbia, HA , M'semrir (bord de l'Oued), Rich, Haute Rhégaya (identified by Mannheims in 1951 as surcoufi ) Nephrotoma luteata (Meigen, 1818) Oosterbroek 1979a , Rif , Targuist, Chefchaouen, HA , Kasba Taguendaft, near Oukaimeden; Theowald and Oosterbroek 1980 , Rif , Targuist, Chefchaouen, HA , Kasba Taguendaft, near Oukaimeden; Oosterbroek and Theowald 1992 ; Pârvu et al. 2006 , AP , Merja Zerga; Adghir et al. 2018 , Rif , Oued Laou, Dardera, Oued Nakhla; Oosterbroek 2020 (CCW) Nephrotoma subanalis (Mannheims, 1951) = ? Pachyrhina analis Schummel, in Pierre 1922a : 24, Pierre 1922b : 150 = Pales subanalis Mannheims, in Mannheims 1951 : 56 Mannheims 1951 , HA , Tachdirt (2200–2900 m); Vaillant 1956b , HA , Oukaimeden (2250 m); Oosterbroek 1979c, HA , Tachdirt (2200–2900 m); Theowald and Oosterbroek 1980 , HA , Tachdirt, Oukaimeden, M'Goum, Tadmant, Imlil, Tizi-N'Tichka; Oosterbroek and Theowald 1992 , HA , Tachdirt; Oosterbroek 2020 (CCW) Nephrotoma submaculosa Edwards, 1928 Oosterbroek 1982 , Rif , Ketama, Dardara, MA , Azrou; Oosterbroek and Theowald 1992 ; de Jong 1998 , Rif , Atlas ; Oosterbroek 2011 ; Adghir et al. 2018 , Rif , Jebel Dahedouh, Ketama, Aïn Sidi Brahim Ben Arrif, Oued Taida, tributary of Oued Ouara, Sidi Chouiref, Dayat Tazia; Oosterbroek 2020 (CCW) Nephrotoma sullingtonensis Edwards, 1938 Oosterbroek 1978 ; Theowald and Oosterbroek 1980 , Rif , Bab Berred; Oosterbroek 1982 Rif , Bab Berred; de Jong 1993 , 1998 , Rif ; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 , Rif , 4 km SE Ketama, Barrage Moulay Bouchta, Oued Aârate, Aïn Takhninjoute, Oued Jbara, Aïn El Malâab, Aïn El Ma Bared, Oued Lemtahane, maison forestière Tazia, Oued Taida, Dayat Tazia; Oosterbroek 2020 (CCW) Tipula Linnaeus, 1758 Tipula ( Acutipula ) anormalipennis Pierre, 1924 Pierre 1924b , HA , Haut Imminen; Séguy 1930a , HA , Haut Imminen; Séguy 1941a , HA , Tachdirt (2500 m), Haut Imminen; Mannheims 1952 , HA , Tachdirt (2500 m), Imminen (2400–2500 m), Arround (1950 m); Vaillant 1956b , HA , Lake of Tamhda (Anremer, 2900 m); Theowald and Oosterbroek 1980 , HA , Anremer, Haut-Immenen, Tachdirt, Arround, Oukaimeden; Vermoolen 1983 , HA , Haut Imminen, Oukaimeden (2500–2800 m), Arround (1950 m), Tachdirt (2500 m); Oosterbroek and Theowald 1992 ; de Jong 1994a , HA ; de Jong 1998 , HA ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Acutipula ) repentina Mannheims, 1952 = Tipula maxima Poda, in Séguy 1941a : 26 Séguy 1941a , HA , Tachdirt (2500 m); Mannheims 1952 , HA , Tachdirt (2200–2700 m); Vaillant 1956b , HA , Lac Tamhda (Anremer, 2900), M'Goum (2500 m); Theowald and Oosterbroek 1980 , HA , Anremer, M'Goum, Tachdirt, Tizi-N'Tichka, Asni, Oukaimeden, Setti Fatma, Imlil, Tadmant; Vermoolen 1983 , MA , Ifrane, HA , Androment, M'Goum, Tadmant, Tizi-N'Test, Setti Fatma, Oukaimeden, Tizi-N'Tichka; Oosterbroek and Theowald 1992 , HA , Tachdirt; de Jong 1994a , MA , HA ; de Jong 1998 , HA ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Acutipula ) rifensis Theowald & Oosterbroek, 1980 Theowald and Oosterbroek 1980 , Rif , Targuist; Vermoolen 1983 , Rif , Targuist, Tidiguin (90 km E. of Ouezzane, 2350 m); Oosterbroek and Theowald 1992 , Rif ; de Jong 1994a , 1998 , Rif ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Emodotipula ) leo Dufour, 1991 = Tipula ( Emodotipula ) obscuriventris Strobl 2 , in Oosterbroek and Theowald 1992 : 99 Oosterbroek and Theowald 1992 (?); Dufour 2003 , Rif ; Adghir et al. 2018 , Rif , Jebel Tissouka; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) bivittata Pierre, 1922 Pierre 1922a , AP , Maâmora between Kénitra and Oued Beth, Dradek (near Rabat); Pierre 1922b , AP , Rabat; Mannheims 1968 ; Theowald and Oosterbroek 1980 , AP , forest of Maâmora, Dradek; Oosterbroek and Theowald 1992 , AP , forest of Maâmora, Dradek; Dakki 1997 ; Oosterbroek 2020 (CCW) – MISR ( AP , Kénitra, Dradek) Tipula ( Lunatipula ) cinereicolor Pierre, 1924 Pierre 1924b , HA , Haut Imminen; Séguy 1930a , HA , Tachdirt (3100–3200 m); Theowald 1973 , HA , Haut Imminen (2400 m); Theowald and Oosterbroek 1980 , MA , Ifrane, HA , Oukaimeden, Tachdirt, Haut-Imminen; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Imminen; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) cornicula Pierre, 1922 Pierre 1922b , HA , Arround (2000 m); Séguy 1930a , HA , Tachdirt (3100–3200 m); Theowald 1973 , HA , Cirque d'Arround (2000 m), Tachdirt (2200–2700 m), Goundafa (1200 m); Theowald and Oosterbroek 1980 , HA , Oukaimeden, Tachdirt, Goundafa; Oosterbroek and Theowald 1992 , HA , Tachdirt; Dakki 1997 ; Oosterbroek 2020 (CCW) – MISR ( HA ) Tipula ( Lunatipula ) fabiola Mannheims, 1968 Theowald and Oosterbroek 1980 , Rif , Bab Berred, Ras El Ma (Chefchaouen), MA , Jebel Abad, Ifrane; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) hermes Theischinger, 1977 Theischinger 1977 , Rif , north of Ouezzane; Theowald 1980 ; Theowald and Oosterbroek 1980 , Rif , north of Ouezzane; Oosterbroek and Theowald 1992 , Rif , Ouezzane; Adghir et al. 2018 , Rif , 4 km SE Ketama, Oued Ametrasse, Aïn El Ma Bared; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) iberica iberica Mannheims, 1963 = Tipula lunata Linnaeus, in Pierre 1922b : 149, Séguy 1930a : 23 Pierre 1922b , HA , Haute Réghaya, Tannaout (1000 m); Séguy 1930a , HA , Haut Imminen; Mannheims 1963 , MA ; Theowald and Oosterbroek 1980 , Rif , Bab Berred, MA , Ifrane, Taounate; Eiroa 1990 , MA , Ifrane; Oosterbroek and Theowald 1992 ; Oosterbroek 2009 ; Adghir et al. 2018 , Rif , Ketama; Oosterbroek 2020 (CCW) – MISR ( HA ) Tipula ( Lunatipula ) iberica spinula Theischinger, 1980 Theischinger 1980 , HA , Oukaimeden (1300–2800 m); Theowald and Oosterbroek 1980 , HA , Oukaimeden; Oosterbroek and Theowald 1992 , HA , Oukaimeden; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) peliostigma peliostigma Schummel, 1833 Eiroa 1990 , MA , Azrou; Mouna 1997; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) pjotri de Jong & Adghir, 2018 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m) Tipula ( Lunatipula ) pseudocinerascens Strobl, 1906 Adghir et al. 2018 , Rif , Stehat, Oued Taida, Perdicaris Park, Dayat Tazia Tipula ( Lunatipula ) rocina Theischinger, 1979 Theowald and Oosterbroek 1980 , Rif , Tétouan; Oosterbroek and Theowald 1992 ; Oosterbroek 2009 ; Adghir et al. 2018 ; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) selenaria Mannheims, 1967 Mannheims 1967 , MA , Jebel Tazzeka (1500–1989 m), HA , Goundafa (1200 m); Theowald and Oosterbroek 1980 , MA , Tazzeka, HA , Foum Keneg, Goundafa, Oukaimeden; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Goundafa; de Jong 1995 , MA , HA , Oukaimeden; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) stimulosa Mannheims, 1973 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m) Tipula ( Lunatipula ) subfalcata Mannheims, 1967 de Jong 1995 , 1998 , Rif ; Adghir et al. 2018 , Rif , Jebel Tissouka, Oued Tamerte, Oued El Koub; Oosterbroek 2020 (CCW) Tipula ( Lunatipula ) subpustulata Mannheims, 1963 = Tipula pustulata Pierre, in Pierre 1922b : 150 Pierre 1922b , AP , Mogador; Vaillant 1956b , HA , M'Goun (2500 m); Mannheims 1963 , AP , Aïn el Aouda, MA , Jebel Tazzeka (1600–1989 m), HA , Goundafa (1200 m), Tachdirt (2200–2900 m), AA , Lac Goulmima; Theowald 1972 ; Theowald and Oosterbroek 1980 , AP , Aïn el Aouda, MA , Iebel Tazzeka, HA , M'Goum, Goundafa, Tachdirt, Oukaimeden, Imlil; Eiroa 1990 , MA , Ajabo; Oosterbroek and Theowald 1992 , HA , Goundafa; Dakki 1997 ; Adghir et al. 2018 , Rif , 4 km SE Ketama, Aïn El Ma Bared; Oosterbroek 2020 (CCW) – MISR ( AP , Mogador) Tipula ( Lunatipula ) tazzekai Theowald, 1973 Theowald 1973 , MA , Jebel Tazzeka (1600–1989 m); Theowald and Oosterbroek 1980 , MA , Jebel Tazzeka; Eiroa 1990 , MA , Jebel Hebri; Oosterbroek and Theowald 1992 , MA , Jebel Tazzeka; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) atlas Pierre, 1924 Pierre 1924b , HA , Tachdirt (3100–3250 m); Séguy 1930a , HA , Tachdirt (3100–3200 m); Vaillant 1956b , HA , Cascade Siroua (3000 m), M'Goun (2500 m), Toubkal (3350 m), Lake of Tamhda (Anremer, 2900 m); Mannheims 1964 , HA ; Theowald 1973 , HA ; Theowald 1980 , HA ; Theowald and Oosterbroek 1980 , HA , Toubkal, Sources de Tessaouts, M'Goum, Siroua, Anremer, Tachdirt, Oukaimeden, Tizi-N'Tichka, Tadmant; Eiroa 1990 , MA , Oum-Er-Rbia; Oosterbroek and Theowald 1992 , HA , Tachdirt; de Jong 1994b ; de Jong 1998 , Atlas ; Dakki 1997 ; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) breviantennata Lackschewitz, 1933 de Jong 1998 , Rif ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Oued Maggou, Douar Aouzighen; Oosterbroek 2020 (CCW) Tipula ( Savtshenkia ) confusa van der Wulp, 1883 Adghir et al. 2018 , Rif , maison forestière (Talassemtane) Tipula ( Savtshenkia ) rufina rufina Meigen, 1818 Theowald and Oosterbroek 1980 , HA , Oukaimeden; Theowald and Oosterbroek 1983 , Rif , Atlas ; Oosterbroek and Theowald 1992 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen); Oosterbroek 2020 (CCW) Tipula ( Tipula ) mediterranea Lackschewitz, 1930 Vaillant 1956b , HA , Oukaimeden (2250 m), M'Goun (2500 m); Mannheims 1952 ; Theowald and Oosterbroek 1980 , MA , Ifrane, HA , Oukaimeden, M'Goum, Asni, M'Semrir, Ifni, Bab-Rou-Idie, Tizi-N'Tichka, Setti Fatma; Theowald 1984 , Rif ; Eiroa 1990 , MA , Azrou, Oum-Er-Rbia; Oosterbroek and Theowald 1992 ; Pârvu et al. 2006 AP , Merja Zerga; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Jebel Tissouka, Bab Taza, Dardara, 4 km SE Ketama, Aïn Afersiw, Douar Kitane, Dayat Jebel Zemzem, Wilaya (Tétouan), Aïn El Ma Bared, Oued Tamerte, Douar Louamera, Tourbière Amesmlil, Dayat Mezine, Hejar Nehal, tributary of Oued Ouara, Oued Tkarae, Oued Jnane Niche, Dayat Afrate, Oued Ametrasse; Oosterbroek 2020 (CCW) Tipula ( Tipula ) oleracea Linnaeus, 1758 Mannheims 1952 , Rif , Tlata Ketama; Theowald and Oosterbroek 1980 , Rif , Tlata Ketama; Theowald 1984 , Rif ; Oosterbroek and Theowald 1992 ; Dakki 1997 ; Pârvu and Zaharia 2007 ; Oosterbroek 2011 ; Adghir et al. 2018 , Rif , Oued Laou, Dardara, Jebel Tissouka, Ksar Rimal, 15 km from Fifi, Oued Aârate, Oued El Hamma, Douar Louamera, Oued Zaouya, Près de Beni Said, Wilaya (Tétouan), Tourbière Amesmlil, Oued Smir, Aïn Jdida; Oosterbroek 2020 (CCW) – MISR Tipula ( Vestiplex ) vaillanti vaillanti Theowald, 1977 Adghir et al. 2018 , Rif , Jebel El Kelâa (Talassemtane, 1340 m), Douar Kitane Tipula ( Yamatotipula ) afriberia afriberia Theowald & Oosterbroek, 1980 Theowald and Oosterbroek 1980 , HA , Oukaimeden; Oosterbroek and Theowald 1992 , HA , Oukaimeden; Oosterbroek 1994a ; Adghir et al. 2018 , Rif , Jebel Tissouka, Dardara, Douar Mokedassen, Oued Zarka; Oosterbroek 2020 (CCW) Tipula ( Yamatotipula ) barbarensis Theowald & Oosterbroek, 1980 = Tipula lateralis Meigen, in Pierre 1922a : 23, Pierre 1922b : 150, Séguy 1930a : 23, Séguy 1941a : 26, Mannheims 1952 : 98 (in part), Vaillant 1956b : 238 Pierre 1922a , AP , Dradek (Rabat), MA , Azrou (riverside of Oued Tigrigra); Pierre 1922b , AP , Mogador, MA , Beni Méllal, HA , Asni; Séguy 1930a ; Séguy 1941a , HA , Imi n'Ouaka (1500 m); Mannheims 1952 ; Vaillant 1956b , HA , Oukaimeden; Theowald and Oosterbroek 1980 , Rif , Dardara, MA , Ifrane, Aghbalou, HA , Asni, Oukaimeden, Setti Fatma, Imlil, Tizi-N'Tichka, Tadmant, Tachdirt; Eiroa 1990 , MA , Azrou, Oum-er-Rbia, Ifrane; Oosterbroek and Theowald 1992 , HA , Setti Fatma; Oosterbroek 1994a , Rif , Dardara, MA , Ifrane, Aghbalou, HA , Oukaimeden, Setti Fatma, Imlil, Tizi-N'Tichka, Tadmant, Asni, Tachdirt; Dakki 1997 ; Adghir et al. 2018 , Rif , Ras el Ma (Chefchaouen), Jebel Tissouka, Bab Taza, Dardara, 4 km SE Ketama, Aïn El Manzela, Aïn Bab Tariouante, Dayat Aïn Afersiw, Oued El Kanar, Dayat Afrate, Douar Kitane, Wilaya (Tétouan), 15 km from Fifi, Aïn Sidi Brahim Ben Arrif, Oued Nakhla, Âounsar Aheramen, Oued Boumerouil, Douar Zaouya, Oued Tizekhte, Oued Samsa, Dayat Aïn Jdioui, Douar Ouled Laghmari-Rmal, maison forestière Tazia, Hejar Nehal, Lot Hemmadi, tributary of Oued Ouara, Oued Sidi Mohamed Saâda, Oued Amsemlil, Tourbière Amesmlil, Beni Salah, Oued Jnane Niche, Oued Maggou, Souk Lhed Beni Darkoul, Oued Imassouden, Aïn Helouma, Source Zarka, Aïn Kchour; Oosterbroek 2020 (CCW) – MISR Trichoceridoidea TRICHOCERIDAE K. Kettani, E. Krzemińska Number of species: 8 . Expected: 10 Faunistic knowledge of the family in Morocco: good Trichocerinae Trichocera Meigen, 1803 Trichocera ( Trichocera ) hiemalis (DeGeer, 1776) Pierre 1922b ( AP , Rabat); Dakki 1997 ; Mouna 1998 ; AP (Rabat) – MISR Trichocera ( Trichocera ) marocana Driauach, Krzemińska & Belqat, 2015 Driauach et al. 2015 , Rif , Oued Akrir Trichocera ( Saltrichocera ) annulata Meigen, 1818 Driauach et al. 2015 , Rif , affluent Oued Akrir, Dayat Fifi, Oued Taria, Oued Kelâa, Oued Ouara, Oued à 15 km de Fifi, Aïn Mâaze, Oued Maggou, Aïn Quanquben, Oued Tizekhte, Forêt Jebel Bouhachem, EM , Grotte du Chameau, Oued Zegzel, Aïn Sidi Yahia, Cascade Grotte des pigeons, Oued Tafoughalt Trichocera ( Saltrichocera ) pappi Krzemińska, 2003 Driauach et al. 2015 , Rif , Dayat Fifi, affluent Oued Akrir, Oued Amsemlil, Ruisseau Agoummir, maison forestière, EM , Cascade Grotte des Pigeons, MA , Seguia El Hajeb Trichocera ( Saltrichocera ) saltator (Harris, 1776) Driauach et al. 2015 , Rif , Dayat Fifi, Oued à 15 km de Fifi, Forêt Jebel Bouhachem, maison forestière, EM , Cascade Grotte des pigeons Trichocera ( Saltrichocera ) sardiniensis Petrašiūnas, 2009 Driauach et al. 2015 , Rif , Oued à 15 km de Fifi, affluent Oued Akrir, Oued Amsemlil, maison forestière Trichocera ( Saltrichocera ) regelationis (Linnaeus, 1758) Driauach et al. 2015 , Rif , Oued Ouara, Aïn el Ma Bared, Oued à 15 km de Fifi, Cascade Chrafate, Aïn Quanquben Trichocera ( Saltrichocera ) rufescens Edwards, 1921 Driauach et al. 2015 , Rif , affluent Oued Akrir, Dayat Fifi, Oued Tassikeste, Oued Farda, Aïn Mâaze TRICHOCERIDAE K. Kettani, E. Krzemińska Number of species: 8 . Expected: 10 Faunistic knowledge of the family in Morocco: good Trichocerinae Trichocera Meigen, 1803 Trichocera ( Trichocera ) hiemalis (DeGeer, 1776) Pierre 1922b ( AP , Rabat); Dakki 1997 ; Mouna 1998 ; AP (Rabat) – MISR Trichocera ( Trichocera ) marocana Driauach, Krzemińska & Belqat, 2015 Driauach et al. 2015 , Rif , Oued Akrir Trichocera ( Saltrichocera ) annulata Meigen, 1818 Driauach et al. 2015 , Rif , affluent Oued Akrir, Dayat Fifi, Oued Taria, Oued Kelâa, Oued Ouara, Oued à 15 km de Fifi, Aïn Mâaze, Oued Maggou, Aïn Quanquben, Oued Tizekhte, Forêt Jebel Bouhachem, EM , Grotte du Chameau, Oued Zegzel, Aïn Sidi Yahia, Cascade Grotte des pigeons, Oued Tafoughalt Trichocera ( Saltrichocera ) pappi Krzemińska, 2003 Driauach et al. 2015 , Rif , Dayat Fifi, affluent Oued Akrir, Oued Amsemlil, Ruisseau Agoummir, maison forestière, EM , Cascade Grotte des Pigeons, MA , Seguia El Hajeb Trichocera ( Saltrichocera ) saltator (Harris, 1776) Driauach et al. 2015 , Rif , Dayat Fifi, Oued à 15 km de Fifi, Forêt Jebel Bouhachem, maison forestière, EM , Cascade Grotte des pigeons Trichocera ( Saltrichocera ) sardiniensis Petrašiūnas, 2009 Driauach et al. 2015 , Rif , Oued à 15 km de Fifi, affluent Oued Akrir, Oued Amsemlil, maison forestière Trichocera ( Saltrichocera ) regelationis (Linnaeus, 1758) Driauach et al. 2015 , Rif , Oued Ouara, Aïn el Ma Bared, Oued à 15 km de Fifi, Cascade Chrafate, Aïn Quanquben Trichocera ( Saltrichocera ) rufescens Edwards, 1921 Driauach et al. 2015 , Rif , affluent Oued Akrir, Dayat Fifi, Oued Tassikeste, Oued Farda, Aïn Mâaze Trichocerinae Trichocera Meigen, 1803 Trichocera ( Trichocera ) hiemalis (DeGeer, 1776) Pierre 1922b ( AP , Rabat); Dakki 1997 ; Mouna 1998 ; AP (Rabat) – MISR Trichocera ( Trichocera ) marocana Driauach, Krzemińska & Belqat, 2015 Driauach et al. 2015 , Rif , Oued Akrir Trichocera ( Saltrichocera ) annulata Meigen, 1818 Driauach et al. 2015 , Rif , affluent Oued Akrir, Dayat Fifi, Oued Taria, Oued Kelâa, Oued Ouara, Oued à 15 km de Fifi, Aïn Mâaze, Oued Maggou, Aïn Quanquben, Oued Tizekhte, Forêt Jebel Bouhachem, EM , Grotte du Chameau, Oued Zegzel, Aïn Sidi Yahia, Cascade Grotte des pigeons, Oued Tafoughalt Trichocera ( Saltrichocera ) pappi Krzemińska, 2003 Driauach et al. 2015 , Rif , Dayat Fifi, affluent Oued Akrir, Oued Amsemlil, Ruisseau Agoummir, maison forestière, EM , Cascade Grotte des Pigeons, MA , Seguia El Hajeb Trichocera ( Saltrichocera ) saltator (Harris, 1776) Driauach et al. 2015 , Rif , Dayat Fifi, Oued à 15 km de Fifi, Forêt Jebel Bouhachem, maison forestière, EM , Cascade Grotte des pigeons Trichocera ( Saltrichocera ) sardiniensis Petrašiūnas, 2009 Driauach et al. 2015 , Rif , Oued à 15 km de Fifi, affluent Oued Akrir, Oued Amsemlil, maison forestière Trichocera ( Saltrichocera ) regelationis (Linnaeus, 1758) Driauach et al. 2015 , Rif , Oued Ouara, Aïn el Ma Bared, Oued à 15 km de Fifi, Cascade Chrafate, Aïn Quanquben Trichocera ( Saltrichocera ) rufescens Edwards, 1921 Driauach et al. 2015 , Rif , affluent Oued Akrir, Dayat Fifi, Oued Tassikeste, Oued Farda, Aïn Mâaze Psychodoidea PSYCHODIDAE K. Kettani, R. Wagner Number of species: 51 . Expected: 70 Faunistic knowledge of the family in Morocco: moderate Phlebotominae Phlebotomus Loew, 1845 Phlebotomus ( Larroussius ) ariasi Tonnoir, 1921 Gaud 1947a ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , AP , MA , HA ; Rioux et al. 1974 ; Mouna 1998 ; Guernaoui et al. 2005 , MA , HA ; Bounamous 2010 ; Boussaa et al. 2005 ; Boussaa et al. 2010 Phlebotomus ( Larroussius ) chadlii Rioux, Juminer & Gibily, 1966 Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Croset et al. 1978 ; Mouna 1998 ; Bounamous 2010 Phlebotomus ( Larroussius ) langeroni Nitzulescu, 1930 Rislorcelli 1941, HA ; Bailly-Choumara et al. 1971 , AP , EM ; Croset et al. 1978 ; Mouna 1998 Phlebotomus ( Larroussius ) longicuspis Nitzulescu, 1930 Rislorcelli 1941, HA ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , Rif , EM , AP , MA , HA , AA ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Guernaoui et al. 2005 , Rif , Chefchaouen, HA , AA , Agadir; Boussaa et al. 2005 ; Boussaa 2008 ; Boussaa et al. 2008 ; Boussaa et al. 2010 ; Bounamous 2010 ; Zarrouk et al. 2016 Phlebotomus ( Larroussius ) mariae Rioux, Croset, Léger and Bailly-Choumara, 1974 Hervy et al. 1994 ; Rioux et al. 1974 , HA ; Mouna 1998 ; Guernaoui et al. 2005 , MA , HA Phlebotomus ( Larroussius ) perfiliewi Parrot, 1930 Rioux et al. 1977 ; Croset et al. 1978 ; Mouna 1998 Phlebotomus ( Larroussius ) perniciosus Newstead, 1911 Séguy 1930a ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , AP , EM , MA , HA , AA ; Mouna 1998 ; Guernaoui et al. 2005 , Rif , Chefchaouen, HA ; Boussaa et al. 2008 ; Bounamous 2010 ; Boussaa et al. 2010 ; Zarrouk et al. 2016 Phlebotomus ( Paraphlebotomus ) alexandri Sinton, 1928 Bailly-Choumara et al. 1971 , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Colacicco-Mayhugh et al. 2010 , EM ; Boussaa et al. 2010 ; Bounamous 2010 Phlebotomus ( Paraphlebotomus ) chabaudii Croset, Abonnenc & Rioux, 1970 Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Mouna 1998 Phlebotmus ( Paraphlebotomus ) kazeruni Theodor & Mesghali, 1964 Mouna 1998 Phlebotomus ( Paraphlebotomus ) riouxi Depaquit, Killick-Kendrick & Léger, 1998 Bounamous 2010 Phlebotomus ( Paraphlebotomus ) sergenti Parrot, 1917 Séguy 1930a ; Rislorcelli 1941; Rislorcelli 1947; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , Rif , AP , EM , MA , HA , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Mouna 1998 ; Boussaa et al. 2009 ; Bounamous 2010 Phlebotomus ( Phlebotomus ) bergeroti Parrot, 1934 Rioux et al. 1975 , HA ; Mouna 1998 ; Bounamous 2010 Phlebotomus ( Phlebotomus ) papatasi (Scopoli, 1786) Séguy 1930a ; Rislorcelli 1941, Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , AP , EM , MA , HA , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Boussaa et al. 2005 ; Boussaa 2008 ; Colacicco-Mayhugh et al. 2010 , Mediterranean region; Bounamous 2010 ; Boussaa et al. 2010 ; Prudhomme et al. 2012 Phlebotomus clydei Sinton, 1928 Bailly-Choumara et al. 1971 , EM ; Mouna 1998 Phlebotomus lewisi Parrot, 1948 Bailly-Choumara et al. 1971 , South AP ; Mouna 1998 Sergentomyia França & Parrot, 1920 Sergentomyia ( Grassomyia ) dreyfussi (Parrot, 1933) Rislorcelli 1941, Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , AP , EM , MA , HA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 Sergentomyia ( Parrotomyia ) africana (Newstead, 1912) = Phlebotomus ( Parrotomyia ) africana (Newstead), in Rislorcelli 1941: 522, Bailly-Choumara et al. 1971 : 454; Rioux et al. 1974 : 99 Séguy 1930a (as subspecies of minutus Rondani: 43), HA , Marrakech; Rislorcelli 1941 (as subspecies of minutus Rondani: 528), AA , Ksar es Souk; Gaud and Laurent 1952 (as subspecies of minutus Rondani: 75), AP , Rabat; Bailly-Choumara et al. 1971 (as subspecies of minutus Rondani: 438), AP , AA ; Rioux et al. 1974 , HA ; Croset et al. 1978 (as subspecies of minutus Rondani: 722); Mouna 1998 ; Boussaa et al. 2005 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 ; Boussaa et al. 2010 Sergentomyia ( Sergentomyia ) antennata (Newstead, 1912) = Phlebotomus cinctus Parrot & Martin, 1944, in Mouna 1998 : 86 = Phlebotomus signatipennis Newstead, 1920, in Mouna 1998 : 86 Bailly-Choumara et al. 1971 , EM ; Rioux et al. 1974 , HA ; Léger et al. 1974 , AA (south of Morocco); Mouna 1998 Sergentomyia ( Sergentomyia ) fallax (Parrot, 1921) Rislorcelli 1947; Gaud 1954 ; Bailly-Choumara et al. 1971 , AP , EM , MA , AA ; Rioux et al. 1974 , HA ; Mouna 1998 ; Guernaoui et al. 2005 ; Boussaa et al. 2005 ; Boussaa et al. 2007 ; Boumezzough et al. 2009 , HA , Marrakech; Boussaa et al. 2010 ; Bounamous 2010 Sergentomyia ( Sergentomyia ) minuta (Rondani, 1843) = Phlebotomus minutus Rondani, in Gaud and Laurent 1952: 75, Mouna 1998 : 86 = Phlebotomus ( Sergentomyia ) parroti (Adler and Theodor), in Rislorcelli 1941: 526, Rislorcelli 1947: 487, Bailly-Choumara et al. 1971 : 450, Rioux et al. 1974 , 99 Séguy 1930a , HA , Marrakech; Gaud and Laurent 1952, AP , Rabat; Rislorcelli 1941; Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , Rif , AP , EM , MA , HA ; Rioux et al. 1974 , HA ; Croset et al. 1978 (from the mediterranean region to the Sahara); Mouna 1998 ; Boussaa et al. 2005 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 ; Boussaa et al. 2010 ; Depaquit et al. 2015 , Rif , Chefchaouen Sergentomyia ( Sergentomyia ) schwetzi Adier, Theodor & Parrot, 1929 Bailly-Choumara and Léger 1976, SA , Aouinet-Torkoz; Mouna 1998 Sergentomyia ( Sintonius ) christophersi (Sinton, 1927) Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Croset et al. 1978 ; Mouna 1998 Psychodinae Maruinini Tonnoiriella Vaillant, 1982 Tonnoiriella paveli Ježek, 1999 Ježek 1999 , HA , AA ; Afzan and Belqat 2016 Tonnoiriella pulchra (Eaton, 1893) (?) [probably mis-identification] Wagner 1990 ; Ježek and Hájek 2007 ; Afzan and Belqat 2016 Mormiini Mormia Enderlein, 1935 Mormia tenebricosa Vaillant, 1954 = Telmatoscopus ( Mormia ) tenebricosus Vaillant, in Vaillant 1956b : 244 Vaillant 1956b , HA , Imi-N'Ifri; Afzan and Belqat 2016 , Rif , Oued Achekrade Paramormiini Clogmia Enderlein, 1937 Clogmia albipunctata (Williston, 1893) Afzan and Belqat 2016 , Rif , Douar Kitane, Douar Moukhlata, Oued Mhannech, AP , Douar Aoulad Ali (Central Plateau (Coastal region)) Panimerus Eaton, 1913 Panimerus thienemanni (Vaillant, 1954) = Panimerus maynei (Tonnoir, 1919), in Dakki 1997 : 62 Boumezzough and Vaillant 1986b , HA , Assif Réghaya; Afzan and Belqat 2016 Paramormia Enderlein, 1935 Paramormia ustulata (Walker, 1856) Vaillant 1956b , HA ; Mouna 1998 ; Ježek and Yağcı 2005; Omelková and Ježek 2012a ; Afzan and Belqat 2016 , Rif , Seguia Barrage Dar Chaoui, Douar Kitane, Oued Jnane Niche Pericomini Bazarella Vaillant, 1964 Bazarella atra (Vaillant, 1955) = Pericoma atra Vaillant, in Vaillant 1956b : 234, 238, 242 Vaillant 1956b , HA , Assif Tasouat (M'Goum), Siroua, Imi-N'Ifri, Aguelmous, Sidi Chamarouch, Lac Tamhda (Anremer), Oukaimeden; Boumezzough and Thomas 1987 , HA , Oued Réghaya (Imlil, 1750 m); Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Inesmane, Oued Madissouka, Aïn Quanquben Pericoma Walker, 1856 Pericoma ( Pachypericoma ) blandula Eaton, 1893 Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Ježek 2004 , Rif ; Ježek and Hájek 2007 ; Dakki and Himmi 2008 , MA , Oued Sebou; Omelková and Ježek 2012a ; Afzan and Belqat 2016 , Rif , Oued Taida, Âounsar Aherman, Oued Beni Ouachekradi, Oued Aâyaden, Cascade Ras el Ma Pericoma ( Pericoma ) barbarica Vaillant, 1955 Vaillant 1956b , HA , M'Goum; Afzan and Belqat 2016 , Rif , Oued Taida, Douar Taria, Cascade Grotte des pigeons Pericoma ( Pericoma ) granadica Vaillant, 1978 Boumezzough and Vaillant 1986b , HA ; Vaillant and Moubayed 1987; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou; Afzan and Belqat 2016 , Rif , Oued Taida, Ametrasse, Oued Farda, Oued Aâyaden, Oued Ras el Ma, MA , Aïn Vittel, HA , Cascade sur sol cuivreux, Oued Réghaya Pericoma ( Pericoma ) diversa Tonnoir, 1920 Vaillant 1978 , HA ; Afzan and Belqat 2016 , Rif , Cascade Chrafate Pericoma ( Pericoma ) exquisita Eaton, 1893 Ježek 2004 , Rif , HA ; Afzan and Belqat 2016 Pericoma ( Pericoma ) latina Sarà , 1954 Vaillant 1955, HA ; Afzan and Belqat 2016 , Rif , Cascade Chrafate, Oued Maggou, Nord Village Maggou, Oued Kelâa, Oued Talembote, Oued associé à Dayat Fifi, Oued Tiffert, Oued à 20 km de Fifi, Oued El Kanar, Beni Fenzar Pericoma ( Pericoma ) maroccana Vaillant, 1955 = Pericoma numidica var. marocana Vaillant, 1955 Boumezzough and Vaillant 1986b , HA , Tissaout; Dakki 1997 ; Afzan and Belqat 2016 , Rif , Cascade Chrafate, ruisseau maison forestière; Dakki and Himmi 2008 , MA , Oued Sebou Pericoma ( Pericoma ) modesta Tonnoir, 1922 = Pericoma numidica Vaillant, in Vaillant 1956b : 236, 237, 240 Vaillant 1956b , HA , Assif Tassouat (M'Goum), Lac Tamhda (L'Anremer); Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou; Afzan and Belqat 2016 Pericoma pseudexquisita Tonnoir, 1940 Afzan and Belqat 2016 , Rif , Oued Azila Pneumia Enderlein, 1935 Pneumia nubila (Meigen, 1818) Afzan and Belqat 2016 , Rif , Aïn Mâaze Pneumia pilularia (Tonnoir, 1940) Ježek 2004 ; Ježek and Hájek 2007 ; Omelková and Ježek 2012a Pneumia propinqua (Satchell, 1955) Afzan and Belqat 2016 , Rif , Chrafate, Oued Zarka Pneumia reghayana (Boumezzough & Vaillant, 1986) = Satchelliella reghayana Boumezzough & Vaillant, 1986, in Boumezzough and Vaillant 1986b : 238 Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 Pneumia toubkalensis Omelková & Ježek, 2012 Omelková and Ježek 2012b , HA , Toubkal; Afzan and Belqat 2016 , Rif , Oued Aâyaden, Aïn Ras el Ma Psychodini Philosepedon Eaton, 1904 Philosepedon ( Philosepedon ) humeralis (Meigen, 1818) Afzan and Belqat 2016 , Rif , Oued Hachef, Cascade Ras el Ma, Oued El Kanar, 2 km de Douar Assoul, Oued Aâyaden Psychoda Latreille, 1796 Psychoda ( Logima ) albipennis (Zettersdedt, 1850) = Logima albipennis (Zettersdedt), in Mouna 1998 : 86 Mouna 1998 Psychoda ( Psycha ) grisescens Tonnoir, 1922 Ježek 2004 , Rif ; Afzan and Belqat 2016 , Rif , Douar Kitane, MA , Gîte Aït Ayoub Psychoda ( Psychoda ) uniformata Haseman, 1907 Ježek 2004 , Rif ; Afzan and Belqat 2016 Psychoda ( Psychodocha ) cinerea Banks, 1894 Boumezzough and Thomas 1987 , HA , Azib Oukaimeden (2730 m); Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Tazzarine, Douar Taria, Douar Kitane, Oued Chrafate, Oued Aâyaden, EM , Cascade Grotte des Pigeons (Béni Snassen) Psychoda ( Psychodocha ) gemina (Eaton, 1904) Afzan and Belqat 2016 , Rif , Dayat Fifi, Oued Zarka, Douar Kitane, Oued Aâyaden Psychoda ( Tinearia ) alternata Say, 1824 Tonnoir 1920, HA , La Maire; Boumezzough and Thomas 1987 , HA , Oued Réghaya (1740 m), Imlil; Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Nakhla, Oued Farda, Oued Ouara, Oued Ametrasse, Oued Chrafate, Douar Derâa, Douar Ihermochene, Douar Ikhlafene, Douar Taria, Douar Idrene, Douar Kitane, Oued 2 km de Douar Assoul, Oued Aâyaden, ruisseau maison forestière, Oued Mhannech, Aïn Sidi Yahia, MA , Gîte Aït Ayoub Scatopsoidea SCATOPSIDAE 3 K. Kettani, J.P. Haenni Number of species: 13 . Expected: 30–40 Faunistic knowledge of the family in Morocco: poor Ectaetiinae Ectaetia Enderlein, 1912 Ectaetia clavipes (Loew, 1846) = Scatopse clavipes Loew, in Mouna 1998 : 84 Mouna 1998 ; Haenni and Kettani 2016 , Rif , Amsa – MISR Psectrosciarinae Anapausis Enderlein, 1912 Anapausis sp.* Rif , Source Aïn Tissemlal Psectrosciara Kieffer, 1911 Psectrosciara sp. 1* Rif , Adrou Psectrosciara sp. 2* Rif , Adrou Scatopsinae Coboldia Melander, 1916 Coboldia fuscipes (Meigen, 1830) Haenni and Kettani 2011 , Rif , Kitane, AP , El Jadida, Rabat, AA , Souss; Haenni and Kettani 2016 , Rif , Maggou, Aârkob, Jnane Niche; Rif (M'Diq farm) – MISR Parascatopse Cook, 1955 Parascatopse sp. Haenni and Kettani 2011 , AA , Ouarzazate ­– MHNN Quateiella Cook, 1975 Quateiella inexpectata Haenni, 1988 Haenni and Kettani 2016 , Rif , Afertane Reichertella Enderlein, 1912 Reichertella geniculata (Zetterstedt, 1850) Haenni and Kettani 2016 , Rif , Jnane Niche Reichertella maroccana Haenni, 2011 Haenni and Kettani 2011 , HA , Oukaimeden – RMNH Rhegmoclemina Enderlein, 1936 Rhegmoclemina lunensis Haenni & Godfrey, 2009 Haenni and Kettani 2011 , Rif , Boujdad Rhexoza Enderlein, 1936 Rhexoza freyi (Duda, 1936) Haenni and Kettani 2011 , AA , Agadir – MHNN Scatopse Geoffroy, 1762 Scatopse notata (Linnaeus, 1758) Mouna 1998 ; Haenni and Kettani 2016 , Rif , Onsar Lile – MISR Swammerdamella Enderlein, 1912 Swammerdamella brevicornis (Meigen, 1830) Haenni and Kettani 2011 , Rif , Kitane, Oued Laou, Ketama, AP , El Jadida, Essaouira, HA , Ouarzazate; Haenni and Kettani 2016 , Rif , Amsa, Jnane Niche, Ametrasse – MISR New records for Morocco An undescribed species of Anapausis has been collected in the Rif mountains in 2014 by K. Kettani and will be described elsewhere. Anapausis sp. Rif: Forêt Azilane ( NPT ), Source Aïn Tissemlal, 1255 m, 35°11.67N, 5°15.20W , 7.vi.2014, Sapinière à Abies maroccana et Pinus nigra , 1♂. Two undescribed species of Psectrosciara have been collected in the Rif mountains in 2013 by K. Kettani and will be described elsewhere. Psectrosciara sp. 1 Rif: Taghzout ( PNPB ), Adrou, 556 m, 35°22.39N, 05°32.28W , chênes-liège, 14.vii–15.viii.2013, 6♂♂. Psectrosciara sp. 2 Rif: Taghzout ( PNPB ), Adrou, 556m, 35°22.39N, 05°32.28W , chênes-liège, 14.vii–15.viii.2013, 3♂♂. PTYCHOPTERIDAE K. Kettani, R. Wagner Number of species: 5 . Expected: 8 Faunistic knowledge of the family in Morocco: moderate Ptychopterinae Ptychoptera Meigen, 1803 Ptychoptera albimana (Fabricius, 1787) MISR (No locality given) Ptychoptera contaminata (Linnaeus, 1758) MISR (No locality given) Ptychoptera lacustris Meigen, 1830 MISR (No locality given) Ptychoptera paludosa Meigen, 1804 MISR (No locality given) Ptychoptera scutellaris Meigen, 1818 MISR (No locality given) PSYCHODIDAE K. Kettani, R. Wagner Number of species: 51 . Expected: 70 Faunistic knowledge of the family in Morocco: moderate Phlebotominae Phlebotomus Loew, 1845 Phlebotomus ( Larroussius ) ariasi Tonnoir, 1921 Gaud 1947a ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , AP , MA , HA ; Rioux et al. 1974 ; Mouna 1998 ; Guernaoui et al. 2005 , MA , HA ; Bounamous 2010 ; Boussaa et al. 2005 ; Boussaa et al. 2010 Phlebotomus ( Larroussius ) chadlii Rioux, Juminer & Gibily, 1966 Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Croset et al. 1978 ; Mouna 1998 ; Bounamous 2010 Phlebotomus ( Larroussius ) langeroni Nitzulescu, 1930 Rislorcelli 1941, HA ; Bailly-Choumara et al. 1971 , AP , EM ; Croset et al. 1978 ; Mouna 1998 Phlebotomus ( Larroussius ) longicuspis Nitzulescu, 1930 Rislorcelli 1941, HA ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , Rif , EM , AP , MA , HA , AA ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Guernaoui et al. 2005 , Rif , Chefchaouen, HA , AA , Agadir; Boussaa et al. 2005 ; Boussaa 2008 ; Boussaa et al. 2008 ; Boussaa et al. 2010 ; Bounamous 2010 ; Zarrouk et al. 2016 Phlebotomus ( Larroussius ) mariae Rioux, Croset, Léger and Bailly-Choumara, 1974 Hervy et al. 1994 ; Rioux et al. 1974 , HA ; Mouna 1998 ; Guernaoui et al. 2005 , MA , HA Phlebotomus ( Larroussius ) perfiliewi Parrot, 1930 Rioux et al. 1977 ; Croset et al. 1978 ; Mouna 1998 Phlebotomus ( Larroussius ) perniciosus Newstead, 1911 Séguy 1930a ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , AP , EM , MA , HA , AA ; Mouna 1998 ; Guernaoui et al. 2005 , Rif , Chefchaouen, HA ; Boussaa et al. 2008 ; Bounamous 2010 ; Boussaa et al. 2010 ; Zarrouk et al. 2016 Phlebotomus ( Paraphlebotomus ) alexandri Sinton, 1928 Bailly-Choumara et al. 1971 , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Colacicco-Mayhugh et al. 2010 , EM ; Boussaa et al. 2010 ; Bounamous 2010 Phlebotomus ( Paraphlebotomus ) chabaudii Croset, Abonnenc & Rioux, 1970 Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Mouna 1998 Phlebotmus ( Paraphlebotomus ) kazeruni Theodor & Mesghali, 1964 Mouna 1998 Phlebotomus ( Paraphlebotomus ) riouxi Depaquit, Killick-Kendrick & Léger, 1998 Bounamous 2010 Phlebotomus ( Paraphlebotomus ) sergenti Parrot, 1917 Séguy 1930a ; Rislorcelli 1941; Rislorcelli 1947; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , Rif , AP , EM , MA , HA , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Mouna 1998 ; Boussaa et al. 2009 ; Bounamous 2010 Phlebotomus ( Phlebotomus ) bergeroti Parrot, 1934 Rioux et al. 1975 , HA ; Mouna 1998 ; Bounamous 2010 Phlebotomus ( Phlebotomus ) papatasi (Scopoli, 1786) Séguy 1930a ; Rislorcelli 1941, Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , AP , EM , MA , HA , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Boussaa et al. 2005 ; Boussaa 2008 ; Colacicco-Mayhugh et al. 2010 , Mediterranean region; Bounamous 2010 ; Boussaa et al. 2010 ; Prudhomme et al. 2012 Phlebotomus clydei Sinton, 1928 Bailly-Choumara et al. 1971 , EM ; Mouna 1998 Phlebotomus lewisi Parrot, 1948 Bailly-Choumara et al. 1971 , South AP ; Mouna 1998 Sergentomyia França & Parrot, 1920 Sergentomyia ( Grassomyia ) dreyfussi (Parrot, 1933) Rislorcelli 1941, Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , AP , EM , MA , HA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 Sergentomyia ( Parrotomyia ) africana (Newstead, 1912) = Phlebotomus ( Parrotomyia ) africana (Newstead), in Rislorcelli 1941: 522, Bailly-Choumara et al. 1971 : 454; Rioux et al. 1974 : 99 Séguy 1930a (as subspecies of minutus Rondani: 43), HA , Marrakech; Rislorcelli 1941 (as subspecies of minutus Rondani: 528), AA , Ksar es Souk; Gaud and Laurent 1952 (as subspecies of minutus Rondani: 75), AP , Rabat; Bailly-Choumara et al. 1971 (as subspecies of minutus Rondani: 438), AP , AA ; Rioux et al. 1974 , HA ; Croset et al. 1978 (as subspecies of minutus Rondani: 722); Mouna 1998 ; Boussaa et al. 2005 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 ; Boussaa et al. 2010 Sergentomyia ( Sergentomyia ) antennata (Newstead, 1912) = Phlebotomus cinctus Parrot & Martin, 1944, in Mouna 1998 : 86 = Phlebotomus signatipennis Newstead, 1920, in Mouna 1998 : 86 Bailly-Choumara et al. 1971 , EM ; Rioux et al. 1974 , HA ; Léger et al. 1974 , AA (south of Morocco); Mouna 1998 Sergentomyia ( Sergentomyia ) fallax (Parrot, 1921) Rislorcelli 1947; Gaud 1954 ; Bailly-Choumara et al. 1971 , AP , EM , MA , AA ; Rioux et al. 1974 , HA ; Mouna 1998 ; Guernaoui et al. 2005 ; Boussaa et al. 2005 ; Boussaa et al. 2007 ; Boumezzough et al. 2009 , HA , Marrakech; Boussaa et al. 2010 ; Bounamous 2010 Sergentomyia ( Sergentomyia ) minuta (Rondani, 1843) = Phlebotomus minutus Rondani, in Gaud and Laurent 1952: 75, Mouna 1998 : 86 = Phlebotomus ( Sergentomyia ) parroti (Adler and Theodor), in Rislorcelli 1941: 526, Rislorcelli 1947: 487, Bailly-Choumara et al. 1971 : 450, Rioux et al. 1974 , 99 Séguy 1930a , HA , Marrakech; Gaud and Laurent 1952, AP , Rabat; Rislorcelli 1941; Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , Rif , AP , EM , MA , HA ; Rioux et al. 1974 , HA ; Croset et al. 1978 (from the mediterranean region to the Sahara); Mouna 1998 ; Boussaa et al. 2005 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 ; Boussaa et al. 2010 ; Depaquit et al. 2015 , Rif , Chefchaouen Sergentomyia ( Sergentomyia ) schwetzi Adier, Theodor & Parrot, 1929 Bailly-Choumara and Léger 1976, SA , Aouinet-Torkoz; Mouna 1998 Sergentomyia ( Sintonius ) christophersi (Sinton, 1927) Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Croset et al. 1978 ; Mouna 1998 Psychodinae Maruinini Tonnoiriella Vaillant, 1982 Tonnoiriella paveli Ježek, 1999 Ježek 1999 , HA , AA ; Afzan and Belqat 2016 Tonnoiriella pulchra (Eaton, 1893) (?) [probably mis-identification] Wagner 1990 ; Ježek and Hájek 2007 ; Afzan and Belqat 2016 Mormiini Mormia Enderlein, 1935 Mormia tenebricosa Vaillant, 1954 = Telmatoscopus ( Mormia ) tenebricosus Vaillant, in Vaillant 1956b : 244 Vaillant 1956b , HA , Imi-N'Ifri; Afzan and Belqat 2016 , Rif , Oued Achekrade Paramormiini Clogmia Enderlein, 1937 Clogmia albipunctata (Williston, 1893) Afzan and Belqat 2016 , Rif , Douar Kitane, Douar Moukhlata, Oued Mhannech, AP , Douar Aoulad Ali (Central Plateau (Coastal region)) Panimerus Eaton, 1913 Panimerus thienemanni (Vaillant, 1954) = Panimerus maynei (Tonnoir, 1919), in Dakki 1997 : 62 Boumezzough and Vaillant 1986b , HA , Assif Réghaya; Afzan and Belqat 2016 Paramormia Enderlein, 1935 Paramormia ustulata (Walker, 1856) Vaillant 1956b , HA ; Mouna 1998 ; Ježek and Yağcı 2005; Omelková and Ježek 2012a ; Afzan and Belqat 2016 , Rif , Seguia Barrage Dar Chaoui, Douar Kitane, Oued Jnane Niche Pericomini Bazarella Vaillant, 1964 Bazarella atra (Vaillant, 1955) = Pericoma atra Vaillant, in Vaillant 1956b : 234, 238, 242 Vaillant 1956b , HA , Assif Tasouat (M'Goum), Siroua, Imi-N'Ifri, Aguelmous, Sidi Chamarouch, Lac Tamhda (Anremer), Oukaimeden; Boumezzough and Thomas 1987 , HA , Oued Réghaya (Imlil, 1750 m); Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Inesmane, Oued Madissouka, Aïn Quanquben Pericoma Walker, 1856 Pericoma ( Pachypericoma ) blandula Eaton, 1893 Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Ježek 2004 , Rif ; Ježek and Hájek 2007 ; Dakki and Himmi 2008 , MA , Oued Sebou; Omelková and Ježek 2012a ; Afzan and Belqat 2016 , Rif , Oued Taida, Âounsar Aherman, Oued Beni Ouachekradi, Oued Aâyaden, Cascade Ras el Ma Pericoma ( Pericoma ) barbarica Vaillant, 1955 Vaillant 1956b , HA , M'Goum; Afzan and Belqat 2016 , Rif , Oued Taida, Douar Taria, Cascade Grotte des pigeons Pericoma ( Pericoma ) granadica Vaillant, 1978 Boumezzough and Vaillant 1986b , HA ; Vaillant and Moubayed 1987; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou; Afzan and Belqat 2016 , Rif , Oued Taida, Ametrasse, Oued Farda, Oued Aâyaden, Oued Ras el Ma, MA , Aïn Vittel, HA , Cascade sur sol cuivreux, Oued Réghaya Pericoma ( Pericoma ) diversa Tonnoir, 1920 Vaillant 1978 , HA ; Afzan and Belqat 2016 , Rif , Cascade Chrafate Pericoma ( Pericoma ) exquisita Eaton, 1893 Ježek 2004 , Rif , HA ; Afzan and Belqat 2016 Pericoma ( Pericoma ) latina Sarà , 1954 Vaillant 1955, HA ; Afzan and Belqat 2016 , Rif , Cascade Chrafate, Oued Maggou, Nord Village Maggou, Oued Kelâa, Oued Talembote, Oued associé à Dayat Fifi, Oued Tiffert, Oued à 20 km de Fifi, Oued El Kanar, Beni Fenzar Pericoma ( Pericoma ) maroccana Vaillant, 1955 = Pericoma numidica var. marocana Vaillant, 1955 Boumezzough and Vaillant 1986b , HA , Tissaout; Dakki 1997 ; Afzan and Belqat 2016 , Rif , Cascade Chrafate, ruisseau maison forestière; Dakki and Himmi 2008 , MA , Oued Sebou Pericoma ( Pericoma ) modesta Tonnoir, 1922 = Pericoma numidica Vaillant, in Vaillant 1956b : 236, 237, 240 Vaillant 1956b , HA , Assif Tassouat (M'Goum), Lac Tamhda (L'Anremer); Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou; Afzan and Belqat 2016 Pericoma pseudexquisita Tonnoir, 1940 Afzan and Belqat 2016 , Rif , Oued Azila Pneumia Enderlein, 1935 Pneumia nubila (Meigen, 1818) Afzan and Belqat 2016 , Rif , Aïn Mâaze Pneumia pilularia (Tonnoir, 1940) Ježek 2004 ; Ježek and Hájek 2007 ; Omelková and Ježek 2012a Pneumia propinqua (Satchell, 1955) Afzan and Belqat 2016 , Rif , Chrafate, Oued Zarka Pneumia reghayana (Boumezzough & Vaillant, 1986) = Satchelliella reghayana Boumezzough & Vaillant, 1986, in Boumezzough and Vaillant 1986b : 238 Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 Pneumia toubkalensis Omelková & Ježek, 2012 Omelková and Ježek 2012b , HA , Toubkal; Afzan and Belqat 2016 , Rif , Oued Aâyaden, Aïn Ras el Ma Psychodini Philosepedon Eaton, 1904 Philosepedon ( Philosepedon ) humeralis (Meigen, 1818) Afzan and Belqat 2016 , Rif , Oued Hachef, Cascade Ras el Ma, Oued El Kanar, 2 km de Douar Assoul, Oued Aâyaden Psychoda Latreille, 1796 Psychoda ( Logima ) albipennis (Zettersdedt, 1850) = Logima albipennis (Zettersdedt), in Mouna 1998 : 86 Mouna 1998 Psychoda ( Psycha ) grisescens Tonnoir, 1922 Ježek 2004 , Rif ; Afzan and Belqat 2016 , Rif , Douar Kitane, MA , Gîte Aït Ayoub Psychoda ( Psychoda ) uniformata Haseman, 1907 Ježek 2004 , Rif ; Afzan and Belqat 2016 Psychoda ( Psychodocha ) cinerea Banks, 1894 Boumezzough and Thomas 1987 , HA , Azib Oukaimeden (2730 m); Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Tazzarine, Douar Taria, Douar Kitane, Oued Chrafate, Oued Aâyaden, EM , Cascade Grotte des Pigeons (Béni Snassen) Psychoda ( Psychodocha ) gemina (Eaton, 1904) Afzan and Belqat 2016 , Rif , Dayat Fifi, Oued Zarka, Douar Kitane, Oued Aâyaden Psychoda ( Tinearia ) alternata Say, 1824 Tonnoir 1920, HA , La Maire; Boumezzough and Thomas 1987 , HA , Oued Réghaya (1740 m), Imlil; Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Nakhla, Oued Farda, Oued Ouara, Oued Ametrasse, Oued Chrafate, Douar Derâa, Douar Ihermochene, Douar Ikhlafene, Douar Taria, Douar Idrene, Douar Kitane, Oued 2 km de Douar Assoul, Oued Aâyaden, ruisseau maison forestière, Oued Mhannech, Aïn Sidi Yahia, MA , Gîte Aït Ayoub Phlebotominae Phlebotomus Loew, 1845 Phlebotomus ( Larroussius ) ariasi Tonnoir, 1921 Gaud 1947a ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , AP , MA , HA ; Rioux et al. 1974 ; Mouna 1998 ; Guernaoui et al. 2005 , MA , HA ; Bounamous 2010 ; Boussaa et al. 2005 ; Boussaa et al. 2010 Phlebotomus ( Larroussius ) chadlii Rioux, Juminer & Gibily, 1966 Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Croset et al. 1978 ; Mouna 1998 ; Bounamous 2010 Phlebotomus ( Larroussius ) langeroni Nitzulescu, 1930 Rislorcelli 1941, HA ; Bailly-Choumara et al. 1971 , AP , EM ; Croset et al. 1978 ; Mouna 1998 Phlebotomus ( Larroussius ) longicuspis Nitzulescu, 1930 Rislorcelli 1941, HA ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , Rif , EM , AP , MA , HA , AA ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Guernaoui et al. 2005 , Rif , Chefchaouen, HA , AA , Agadir; Boussaa et al. 2005 ; Boussaa 2008 ; Boussaa et al. 2008 ; Boussaa et al. 2010 ; Bounamous 2010 ; Zarrouk et al. 2016 Phlebotomus ( Larroussius ) mariae Rioux, Croset, Léger and Bailly-Choumara, 1974 Hervy et al. 1994 ; Rioux et al. 1974 , HA ; Mouna 1998 ; Guernaoui et al. 2005 , MA , HA Phlebotomus ( Larroussius ) perfiliewi Parrot, 1930 Rioux et al. 1977 ; Croset et al. 1978 ; Mouna 1998 Phlebotomus ( Larroussius ) perniciosus Newstead, 1911 Séguy 1930a ; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , AP , EM , MA , HA , AA ; Mouna 1998 ; Guernaoui et al. 2005 , Rif , Chefchaouen, HA ; Boussaa et al. 2008 ; Bounamous 2010 ; Boussaa et al. 2010 ; Zarrouk et al. 2016 Phlebotomus ( Paraphlebotomus ) alexandri Sinton, 1928 Bailly-Choumara et al. 1971 , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Colacicco-Mayhugh et al. 2010 , EM ; Boussaa et al. 2010 ; Bounamous 2010 Phlebotomus ( Paraphlebotomus ) chabaudii Croset, Abonnenc & Rioux, 1970 Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Mouna 1998 Phlebotmus ( Paraphlebotomus ) kazeruni Theodor & Mesghali, 1964 Mouna 1998 Phlebotomus ( Paraphlebotomus ) riouxi Depaquit, Killick-Kendrick & Léger, 1998 Bounamous 2010 Phlebotomus ( Paraphlebotomus ) sergenti Parrot, 1917 Séguy 1930a ; Rislorcelli 1941; Rislorcelli 1947; Gaud and Laurent 1952, AP , Rabat; Bailly-Choumara et al. 1971 , Rif , AP , EM , MA , HA , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Mouna 1998 ; Boussaa et al. 2009 ; Bounamous 2010 Phlebotomus ( Phlebotomus ) bergeroti Parrot, 1934 Rioux et al. 1975 , HA ; Mouna 1998 ; Bounamous 2010 Phlebotomus ( Phlebotomus ) papatasi (Scopoli, 1786) Séguy 1930a ; Rislorcelli 1941, Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , AP , EM , MA , HA , AA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Boussaa et al. 2005 ; Boussaa 2008 ; Colacicco-Mayhugh et al. 2010 , Mediterranean region; Bounamous 2010 ; Boussaa et al. 2010 ; Prudhomme et al. 2012 Phlebotomus clydei Sinton, 1928 Bailly-Choumara et al. 1971 , EM ; Mouna 1998 Phlebotomus lewisi Parrot, 1948 Bailly-Choumara et al. 1971 , South AP ; Mouna 1998 Sergentomyia França & Parrot, 1920 Sergentomyia ( Grassomyia ) dreyfussi (Parrot, 1933) Rislorcelli 1941, Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , AP , EM , MA , HA ; Abonnenc 1972 ; Rioux et al. 1974 , HA ; Croset et al. 1978 ; Mouna 1998 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 Sergentomyia ( Parrotomyia ) africana (Newstead, 1912) = Phlebotomus ( Parrotomyia ) africana (Newstead), in Rislorcelli 1941: 522, Bailly-Choumara et al. 1971 : 454; Rioux et al. 1974 : 99 Séguy 1930a (as subspecies of minutus Rondani: 43), HA , Marrakech; Rislorcelli 1941 (as subspecies of minutus Rondani: 528), AA , Ksar es Souk; Gaud and Laurent 1952 (as subspecies of minutus Rondani: 75), AP , Rabat; Bailly-Choumara et al. 1971 (as subspecies of minutus Rondani: 438), AP , AA ; Rioux et al. 1974 , HA ; Croset et al. 1978 (as subspecies of minutus Rondani: 722); Mouna 1998 ; Boussaa et al. 2005 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 ; Boussaa et al. 2010 Sergentomyia ( Sergentomyia ) antennata (Newstead, 1912) = Phlebotomus cinctus Parrot & Martin, 1944, in Mouna 1998 : 86 = Phlebotomus signatipennis Newstead, 1920, in Mouna 1998 : 86 Bailly-Choumara et al. 1971 , EM ; Rioux et al. 1974 , HA ; Léger et al. 1974 , AA (south of Morocco); Mouna 1998 Sergentomyia ( Sergentomyia ) fallax (Parrot, 1921) Rislorcelli 1947; Gaud 1954 ; Bailly-Choumara et al. 1971 , AP , EM , MA , AA ; Rioux et al. 1974 , HA ; Mouna 1998 ; Guernaoui et al. 2005 ; Boussaa et al. 2005 ; Boussaa et al. 2007 ; Boumezzough et al. 2009 , HA , Marrakech; Boussaa et al. 2010 ; Bounamous 2010 Sergentomyia ( Sergentomyia ) minuta (Rondani, 1843) = Phlebotomus minutus Rondani, in Gaud and Laurent 1952: 75, Mouna 1998 : 86 = Phlebotomus ( Sergentomyia ) parroti (Adler and Theodor), in Rislorcelli 1941: 526, Rislorcelli 1947: 487, Bailly-Choumara et al. 1971 : 450, Rioux et al. 1974 , 99 Séguy 1930a , HA , Marrakech; Gaud and Laurent 1952, AP , Rabat; Rislorcelli 1941; Rislorcelli 1947, HA ; Bailly-Choumara et al. 1971 , Rif , AP , EM , MA , HA ; Rioux et al. 1974 , HA ; Croset et al. 1978 (from the mediterranean region to the Sahara); Mouna 1998 ; Boussaa et al. 2005 ; Boumezzough et al. 2009 , HA , Marrakech; Bounamous 2010 ; Boussaa et al. 2010 ; Depaquit et al. 2015 , Rif , Chefchaouen Sergentomyia ( Sergentomyia ) schwetzi Adier, Theodor & Parrot, 1929 Bailly-Choumara and Léger 1976, SA , Aouinet-Torkoz; Mouna 1998 Sergentomyia ( Sintonius ) christophersi (Sinton, 1927) Rioux et al. 1974 , HA ; Rioux et al. 1975 ; Croset et al. 1978 ; Mouna 1998 Psychodinae Maruinini Tonnoiriella Vaillant, 1982 Tonnoiriella paveli Ježek, 1999 Ježek 1999 , HA , AA ; Afzan and Belqat 2016 Tonnoiriella pulchra (Eaton, 1893) (?) [probably mis-identification] Wagner 1990 ; Ježek and Hájek 2007 ; Afzan and Belqat 2016 Mormiini Mormia Enderlein, 1935 Mormia tenebricosa Vaillant, 1954 = Telmatoscopus ( Mormia ) tenebricosus Vaillant, in Vaillant 1956b : 244 Vaillant 1956b , HA , Imi-N'Ifri; Afzan and Belqat 2016 , Rif , Oued Achekrade Paramormiini Clogmia Enderlein, 1937 Clogmia albipunctata (Williston, 1893) Afzan and Belqat 2016 , Rif , Douar Kitane, Douar Moukhlata, Oued Mhannech, AP , Douar Aoulad Ali (Central Plateau (Coastal region)) Panimerus Eaton, 1913 Panimerus thienemanni (Vaillant, 1954) = Panimerus maynei (Tonnoir, 1919), in Dakki 1997 : 62 Boumezzough and Vaillant 1986b , HA , Assif Réghaya; Afzan and Belqat 2016 Paramormia Enderlein, 1935 Paramormia ustulata (Walker, 1856) Vaillant 1956b , HA ; Mouna 1998 ; Ježek and Yağcı 2005; Omelková and Ježek 2012a ; Afzan and Belqat 2016 , Rif , Seguia Barrage Dar Chaoui, Douar Kitane, Oued Jnane Niche Pericomini Bazarella Vaillant, 1964 Bazarella atra (Vaillant, 1955) = Pericoma atra Vaillant, in Vaillant 1956b : 234, 238, 242 Vaillant 1956b , HA , Assif Tasouat (M'Goum), Siroua, Imi-N'Ifri, Aguelmous, Sidi Chamarouch, Lac Tamhda (Anremer), Oukaimeden; Boumezzough and Thomas 1987 , HA , Oued Réghaya (Imlil, 1750 m); Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Inesmane, Oued Madissouka, Aïn Quanquben Pericoma Walker, 1856 Pericoma ( Pachypericoma ) blandula Eaton, 1893 Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Ježek 2004 , Rif ; Ježek and Hájek 2007 ; Dakki and Himmi 2008 , MA , Oued Sebou; Omelková and Ježek 2012a ; Afzan and Belqat 2016 , Rif , Oued Taida, Âounsar Aherman, Oued Beni Ouachekradi, Oued Aâyaden, Cascade Ras el Ma Pericoma ( Pericoma ) barbarica Vaillant, 1955 Vaillant 1956b , HA , M'Goum; Afzan and Belqat 2016 , Rif , Oued Taida, Douar Taria, Cascade Grotte des pigeons Pericoma ( Pericoma ) granadica Vaillant, 1978 Boumezzough and Vaillant 1986b , HA ; Vaillant and Moubayed 1987; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou; Afzan and Belqat 2016 , Rif , Oued Taida, Ametrasse, Oued Farda, Oued Aâyaden, Oued Ras el Ma, MA , Aïn Vittel, HA , Cascade sur sol cuivreux, Oued Réghaya Pericoma ( Pericoma ) diversa Tonnoir, 1920 Vaillant 1978 , HA ; Afzan and Belqat 2016 , Rif , Cascade Chrafate Pericoma ( Pericoma ) exquisita Eaton, 1893 Ježek 2004 , Rif , HA ; Afzan and Belqat 2016 Pericoma ( Pericoma ) latina Sarà , 1954 Vaillant 1955, HA ; Afzan and Belqat 2016 , Rif , Cascade Chrafate, Oued Maggou, Nord Village Maggou, Oued Kelâa, Oued Talembote, Oued associé à Dayat Fifi, Oued Tiffert, Oued à 20 km de Fifi, Oued El Kanar, Beni Fenzar Pericoma ( Pericoma ) maroccana Vaillant, 1955 = Pericoma numidica var. marocana Vaillant, 1955 Boumezzough and Vaillant 1986b , HA , Tissaout; Dakki 1997 ; Afzan and Belqat 2016 , Rif , Cascade Chrafate, ruisseau maison forestière; Dakki and Himmi 2008 , MA , Oued Sebou Pericoma ( Pericoma ) modesta Tonnoir, 1922 = Pericoma numidica Vaillant, in Vaillant 1956b : 236, 237, 240 Vaillant 1956b , HA , Assif Tassouat (M'Goum), Lac Tamhda (L'Anremer); Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou; Afzan and Belqat 2016 Pericoma pseudexquisita Tonnoir, 1940 Afzan and Belqat 2016 , Rif , Oued Azila Pneumia Enderlein, 1935 Pneumia nubila (Meigen, 1818) Afzan and Belqat 2016 , Rif , Aïn Mâaze Pneumia pilularia (Tonnoir, 1940) Ježek 2004 ; Ježek and Hájek 2007 ; Omelková and Ježek 2012a Pneumia propinqua (Satchell, 1955) Afzan and Belqat 2016 , Rif , Chrafate, Oued Zarka Pneumia reghayana (Boumezzough & Vaillant, 1986) = Satchelliella reghayana Boumezzough & Vaillant, 1986, in Boumezzough and Vaillant 1986b : 238 Boumezzough and Vaillant 1986b , HA ; Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 Pneumia toubkalensis Omelková & Ježek, 2012 Omelková and Ježek 2012b , HA , Toubkal; Afzan and Belqat 2016 , Rif , Oued Aâyaden, Aïn Ras el Ma Psychodini Philosepedon Eaton, 1904 Philosepedon ( Philosepedon ) humeralis (Meigen, 1818) Afzan and Belqat 2016 , Rif , Oued Hachef, Cascade Ras el Ma, Oued El Kanar, 2 km de Douar Assoul, Oued Aâyaden Psychoda Latreille, 1796 Psychoda ( Logima ) albipennis (Zettersdedt, 1850) = Logima albipennis (Zettersdedt), in Mouna 1998 : 86 Mouna 1998 Psychoda ( Psycha ) grisescens Tonnoir, 1922 Ježek 2004 , Rif ; Afzan and Belqat 2016 , Rif , Douar Kitane, MA , Gîte Aït Ayoub Psychoda ( Psychoda ) uniformata Haseman, 1907 Ježek 2004 , Rif ; Afzan and Belqat 2016 Psychoda ( Psychodocha ) cinerea Banks, 1894 Boumezzough and Thomas 1987 , HA , Azib Oukaimeden (2730 m); Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Tazzarine, Douar Taria, Douar Kitane, Oued Chrafate, Oued Aâyaden, EM , Cascade Grotte des Pigeons (Béni Snassen) Psychoda ( Psychodocha ) gemina (Eaton, 1904) Afzan and Belqat 2016 , Rif , Dayat Fifi, Oued Zarka, Douar Kitane, Oued Aâyaden Psychoda ( Tinearia ) alternata Say, 1824 Tonnoir 1920, HA , La Maire; Boumezzough and Thomas 1987 , HA , Oued Réghaya (1740 m), Imlil; Dakki 1997 ; Dakki and Himmi 2008 ; Afzan and Belqat 2016 , Rif , Oued Nakhla, Oued Farda, Oued Ouara, Oued Ametrasse, Oued Chrafate, Douar Derâa, Douar Ihermochene, Douar Ikhlafene, Douar Taria, Douar Idrene, Douar Kitane, Oued 2 km de Douar Assoul, Oued Aâyaden, ruisseau maison forestière, Oued Mhannech, Aïn Sidi Yahia, MA , Gîte Aït Ayoub Scatopsoidea SCATOPSIDAE 3 K. Kettani, J.P. Haenni Number of species: 13 . Expected: 30–40 Faunistic knowledge of the family in Morocco: poor Ectaetiinae Ectaetia Enderlein, 1912 Ectaetia clavipes (Loew, 1846) = Scatopse clavipes Loew, in Mouna 1998 : 84 Mouna 1998 ; Haenni and Kettani 2016 , Rif , Amsa – MISR Psectrosciarinae Anapausis Enderlein, 1912 Anapausis sp.* Rif , Source Aïn Tissemlal Psectrosciara Kieffer, 1911 Psectrosciara sp. 1* Rif , Adrou Psectrosciara sp. 2* Rif , Adrou Scatopsinae Coboldia Melander, 1916 Coboldia fuscipes (Meigen, 1830) Haenni and Kettani 2011 , Rif , Kitane, AP , El Jadida, Rabat, AA , Souss; Haenni and Kettani 2016 , Rif , Maggou, Aârkob, Jnane Niche; Rif (M'Diq farm) – MISR Parascatopse Cook, 1955 Parascatopse sp. Haenni and Kettani 2011 , AA , Ouarzazate ­– MHNN Quateiella Cook, 1975 Quateiella inexpectata Haenni, 1988 Haenni and Kettani 2016 , Rif , Afertane Reichertella Enderlein, 1912 Reichertella geniculata (Zetterstedt, 1850) Haenni and Kettani 2016 , Rif , Jnane Niche Reichertella maroccana Haenni, 2011 Haenni and Kettani 2011 , HA , Oukaimeden – RMNH Rhegmoclemina Enderlein, 1936 Rhegmoclemina lunensis Haenni & Godfrey, 2009 Haenni and Kettani 2011 , Rif , Boujdad Rhexoza Enderlein, 1936 Rhexoza freyi (Duda, 1936) Haenni and Kettani 2011 , AA , Agadir – MHNN Scatopse Geoffroy, 1762 Scatopse notata (Linnaeus, 1758) Mouna 1998 ; Haenni and Kettani 2016 , Rif , Onsar Lile – MISR Swammerdamella Enderlein, 1912 Swammerdamella brevicornis (Meigen, 1830) Haenni and Kettani 2011 , Rif , Kitane, Oued Laou, Ketama, AP , El Jadida, Essaouira, HA , Ouarzazate; Haenni and Kettani 2016 , Rif , Amsa, Jnane Niche, Ametrasse – MISR New records for Morocco An undescribed species of Anapausis has been collected in the Rif mountains in 2014 by K. Kettani and will be described elsewhere. Anapausis sp. Rif: Forêt Azilane ( NPT ), Source Aïn Tissemlal, 1255 m, 35°11.67N, 5°15.20W , 7.vi.2014, Sapinière à Abies maroccana et Pinus nigra , 1♂. Two undescribed species of Psectrosciara have been collected in the Rif mountains in 2013 by K. Kettani and will be described elsewhere. Psectrosciara sp. 1 Rif: Taghzout ( PNPB ), Adrou, 556 m, 35°22.39N, 05°32.28W , chênes-liège, 14.vii–15.viii.2013, 6♂♂. Psectrosciara sp. 2 Rif: Taghzout ( PNPB ), Adrou, 556m, 35°22.39N, 05°32.28W , chênes-liège, 14.vii–15.viii.2013, 3♂♂. Ectaetiinae Ectaetia Enderlein, 1912 Ectaetia clavipes (Loew, 1846) = Scatopse clavipes Loew, in Mouna 1998 : 84 Mouna 1998 ; Haenni and Kettani 2016 , Rif , Amsa – MISR Psectrosciarinae Anapausis Enderlein, 1912 Anapausis sp.* Rif , Source Aïn Tissemlal Psectrosciara Kieffer, 1911 Psectrosciara sp. 1* Rif , Adrou Psectrosciara sp. 2* Rif , Adrou Scatopsinae Coboldia Melander, 1916 Coboldia fuscipes (Meigen, 1830) Haenni and Kettani 2011 , Rif , Kitane, AP , El Jadida, Rabat, AA , Souss; Haenni and Kettani 2016 , Rif , Maggou, Aârkob, Jnane Niche; Rif (M'Diq farm) – MISR Parascatopse Cook, 1955 Parascatopse sp. Haenni and Kettani 2011 , AA , Ouarzazate ­– MHNN Quateiella Cook, 1975 Quateiella inexpectata Haenni, 1988 Haenni and Kettani 2016 , Rif , Afertane Reichertella Enderlein, 1912 Reichertella geniculata (Zetterstedt, 1850) Haenni and Kettani 2016 , Rif , Jnane Niche Reichertella maroccana Haenni, 2011 Haenni and Kettani 2011 , HA , Oukaimeden – RMNH Rhegmoclemina Enderlein, 1936 Rhegmoclemina lunensis Haenni & Godfrey, 2009 Haenni and Kettani 2011 , Rif , Boujdad Rhexoza Enderlein, 1936 Rhexoza freyi (Duda, 1936) Haenni and Kettani 2011 , AA , Agadir – MHNN Scatopse Geoffroy, 1762 Scatopse notata (Linnaeus, 1758) Mouna 1998 ; Haenni and Kettani 2016 , Rif , Onsar Lile – MISR Swammerdamella Enderlein, 1912 Swammerdamella brevicornis (Meigen, 1830) Haenni and Kettani 2011 , Rif , Kitane, Oued Laou, Ketama, AP , El Jadida, Essaouira, HA , Ouarzazate; Haenni and Kettani 2016 , Rif , Amsa, Jnane Niche, Ametrasse – MISR New records for Morocco An undescribed species of Anapausis has been collected in the Rif mountains in 2014 by K. Kettani and will be described elsewhere. Anapausis sp. Rif: Forêt Azilane ( NPT ), Source Aïn Tissemlal, 1255 m, 35°11.67N, 5°15.20W , 7.vi.2014, Sapinière à Abies maroccana et Pinus nigra , 1♂. Two undescribed species of Psectrosciara have been collected in the Rif mountains in 2013 by K. Kettani and will be described elsewhere. Psectrosciara sp. 1 Rif: Taghzout ( PNPB ), Adrou, 556 m, 35°22.39N, 05°32.28W , chênes-liège, 14.vii–15.viii.2013, 6♂♂. Psectrosciara sp. 2 Rif: Taghzout ( PNPB ), Adrou, 556m, 35°22.39N, 05°32.28W , chênes-liège, 14.vii–15.viii.2013, 3♂♂. PTYCHOPTERIDAE K. Kettani, R. Wagner Number of species: 5 . Expected: 8 Faunistic knowledge of the family in Morocco: moderate Ptychopterinae Ptychoptera Meigen, 1803 Ptychoptera albimana (Fabricius, 1787) MISR (No locality given) Ptychoptera contaminata (Linnaeus, 1758) MISR (No locality given) Ptychoptera lacustris Meigen, 1830 MISR (No locality given) Ptychoptera paludosa Meigen, 1804 MISR (No locality given) Ptychoptera scutellaris Meigen, 1818 MISR (No locality given) Ptychopterinae Ptychoptera Meigen, 1803 Ptychoptera albimana (Fabricius, 1787) MISR (No locality given) Ptychoptera contaminata (Linnaeus, 1758) MISR (No locality given) Ptychoptera lacustris Meigen, 1830 MISR (No locality given) Ptychoptera paludosa Meigen, 1804 MISR (No locality given) Ptychoptera scutellaris Meigen, 1818 MISR (No locality given) Culicoidea CHAOBORIDAE K. Kettani, R. Wagner Number of species: 2 . Expected: 4 Faunistic knowledge of the family in Morocco: poor Chaoborinae Chaoborus Lichtenstein, 1800 Chaoborus crystallinus (De Geer, 1776) Dakki 1997 : 60 Mochlonyx Loew, 1844 Mochlonyx culiciformis (De Geer, 1776) Dakki 1997 : 60 CULICIDAE K. Kettani, B. Trari, O. Himmi, M. Dakki Number of species: 43 . Expected: 60 Faunistic knowledge of the family in Morocco: good Anophelinae Anopheles Meigen, 1818 Anopheles ( Anopheles ) algeriensis Theobald, 1903 Viallate 1922, AP , Kénitra; Séguy 1930a ; Bonjean 1947 , EM , MA ; Gaud 1957a , HA , north of High Atlas; Guy 1959a , MA , Béni Mellal, HA ; Guy 1959b , HA ; Guy 1959a , MA , Béni Mellal; Bailly-Choumara 1967a , MA , Ghorm El Alem; Benmansour et al. 1972 , MA , Barrage Bin El Ouidane; Bailly-Choumara 1973b , AP , Sidi Yahia du Gharb; Metge 1986 , AP , Casablanca; Trari and Himmi 1987 , AP ; Himmi et al. 1995 ; Louah 1995 , Rif , Tahaddart, Schroda; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Casablanca; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 , Rif , Chefchaouen; El Ouali Lalami et al. 2010 a,b, MA , Fès, Boulmane; El Ouali Lalami 2012 , MA , Fès; Trari 2017 , Rif , Chefchaouen, AA , Tiznit; Trari and Dakki 2017a , Rif , Chefchaouen, AA , Tiznit; Trari and Dakki 2017b , AA , Tiznit; Trari et al. 2017 Anopheles ( Anopheles ) claviger (Meigen, 1804) Vialatte 1923 , AP , Kénitra; Séguy 1930a , AP , Rabat; Langeron 1938 , HA , Tounfit, Massou, Anefgou, Tirghist, Tighermine, Louggouargh; Callot 1940 , HA , Anefgou, Tirghist; Bonjean 1947 , EM , MA ; Gaud 1947c , MA , Sefrou, Meknès; Gaud 1948 , AP , Rabat, MA , El Hajeb, AA , Errachidia, Tadla; Gaud et al. 1948 , AP , Skhirat; Guy 1963 , Rif , Taounate, MA , Meknès, Ifrane; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, piste Ksiba-Naour, piste Naour-Arbala, Zaouia Cheikh, Oued Sarif (environs El Ksiba), Dayet Aoua (environs Ifrane), Boulemane; Bailly-Choumara 1967b , EM , 8 km N Itzer; Bailly-Choumara 1967c , Rif , piste Ketama-Mt Tiguidin; Guy 1967 , HA , Marrakech, AA , Tafilalt; Guy and Holstein 1968 , SA ; Metge 1986 , AP , Casablanca; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris drains, Tamaris merja; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 , Rif , Bab Berred, Tanakoub; Faraj et al. 2008b , Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Boulmane; Larhbali et al. 2010 , MA , Zhiliga, Boukachmir, Aït Ichou; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) labranchiae Falleroni, 1926 d'Anfreville 1916 , AP , Salé; Delanoe 1917, AP , El Jadida; Viallate 1922, AP , Rabat, Boulhaut, Bouznika, Gharb, MA , Sidi Kacem, Tiflet, Fès, Taza; Charrier 1924 , Rif , Tanger; Séguy 1930a , MA ; Roubaud 1935, AP , Rabat; Sicault et al. 1935 , AP , Merja Ras Eddaoura, Merja Zerga, dayas entre Sebou et Maâmora, Dar bel Hamri (entre barrage El Kansera et Sidi Slimane), à proximité de Merja Zerga, Douar Anabsa; Langeron 1938 , AA ; Callot 1940 , AA ; Ristorcelli 1946a , b , HA , Oued Tensift, Oued Issil; Bonjean 1947 , AP , Gharb; Gaud 1947c , MA , Oulmès, HA , Marrakech; Gaud et al. 1948 , AP , Merja Ras Eddaoura; Gaud et al. 1949 , AP , Salé, SA , Foum Zguid, Tagounit; Gaud 1953a , AP , from Tanger to El Jadida, MA , Tissa, Timhadit, Bekrit, Meknès, Ifrane (1700 m), HA , Sidi Aissa, Tizi-n'Tichka; Guy 1958 , HA , Oued N'fis; Sacca and Guy 1960b, Rif , Tétouan, AP , Skhirat, Sidi Yahia, Sidi Bettache, Mazagan, Braila (près Sidi Allal Tazi), Aït Lahsen, MA , Meknès, HA , Marrakech; Guy 1962 , Rif , Taounate, AP , Gharb, HA , Marrakech (ville et banlieu); Guy 1963 , Rif , Tanger, Tétouan, EM , Berkane, Debdou, Oujda, AP , Kénitra, Souk Larba, Settat, Chamaîa, Safi, Casablanca, Rabat, Essaouira, MA , Meknès, Fès, Azrou, Oued Zem, Béni Mellal, HA , Kelaâ of Sraghna; Bailly-Choumara 1967a , MA , piste Tafechna-Znan Imes, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Aguelmane Azigza, piste Aguelmane Azigza-Aïn Leuh, maison forestière Ouiouane, route Khénifra-Tafechna, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Bords Moulouya, piste Itzer-Boumia, RN P.33 Boumia-El Kbab, Ouaoumana, piste Ksiba-Naour, piste Naour-Arbala, piste Arbala-El Kbab, Kafensour, Oued Sarif (environs El Ksiba), Dayet Aoua, Ifrane, Imouzer Marmoucha; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Douar Sherba, Douar Aïn Shebbak, Douar Aïn Zabia, Douar Madarh, Douar Sidi Hashas, Mechraa safsaf; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, piste Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Al Hoceima Club Med, Béni Bouayache, Targuist et environs; Guy 1967 , HA , Marrakech, AA , Tafilalt; Bailly-Choumara 1968b , AP , Larache; Guy and Holstein 1968 , AA , Ouarzazate; Bailly-Choumara 1970 , Rif , Tétouan, EM , Berkane, AP , Larache, Sidi Yahia du Gharb, HA , Marrakech; Benmansour et al. 1972 , AA , Agadir; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1972b , AP , Merja Sheishat; Bailly-Choumara 1973a , AP , Merja de l'Oued Smir; Bailly-Choumara 1973b , Rif , Tétouan, EM , Berkane, AP , Merja Sheishat, HA , Souk des Oudaias; de Zulueta et al. 1983 , Rif ; Ibn Jilali 1984, AP , Maâmora; Metge 1986 , AP , Entre Oulad Dlim et Al Ara'ra; Trari and Himmi 1987 , AP , Gharb; Himmi 1991 , AP , Kénitra, Maâmora; Metge 1991 , AP , Sidi Bettache; Trari 1991 , Rif , Chefchaouen, Tanger, Taounate, EM , Oujda, AP , Sidi Amira, Sidi Boughaba, Merja Zerga, Sidi Allal Tazi, Aïn Chouk, Oued Loukous, Merja Oulad Skhar, MA , Khémissat, Béni Mellal, Khouribga, HA , El Kelaâ des Sraghrna, AA , Ouarzazate; El Bermaki 1993, AP , entre l'aeroport Anfa et l'aménagement d'El Oulfa, Sidi Maârouf, El Oulfa; Chlaida and Bouzidi 1995 , AP , Aïn Blal, Oued Sidi Messoud; Louah 1995 , Rif , Tres piedras, Marina Smir, Bouzerlal, Oued Maleh, Zekri, Lajour, Azla, Tahaddart, Skroda, Stehat, Moulay Bouchta, Kebbache, Talembote, Loubart, Chefchaouen, Oued Maggou, Bab Berred, Sidi Kankoch, Oued kbir, Oued Jebel Lehbib; Louah et al. 1995 ; Chlaida 1997 , AP , Aïn Blal, Oued Sidi Messoud, Douar Chlihat, Barrage Al Massira; Moussalim 1997 , AP , Sidi Allal Tazi, Maâmora; Ramdani 1997 , AP , Tamaris, Skhirat; Faraj et al. 1997 , AP , Ouled Moussa; Handaq 1998 , HA , Oukaimeden, Amizmiz, Tiguenziouine; Himmi et al. 1998 , AP , Dayat d'El Menzeh (north-east of Kénitra), Sidi Boughaba; Alaoui Slimani et al. 1999 , AP , Bou-Regreg Salé, Sidi Bouguettaya, Quartier industriel Takaddoum, Marjane; Alaoui Slimani 2002 , AP , Rabat-Salé; Trari et al. 2002 ; Faraj et al. 2003 , Rif , Azib Bouflou, Azib Jrou, Imzouren, Amezzaourou, Tizi-Tamalout, MA , Aït Abdelsalam, Aït Lamfadel; Faraj et al. 2004 , Rif , Azib Jrou, Tanghaya Akarkar, Amezzaourou, Ouled Nsar, AP , Fedalate, MA , Talaa Chougaga, Aïn Smen (Fès), Aït Lamfadel; Trari et al. 2004a , Rif , Larache; Trari et al. 2004b , Rif , Ketama, Gzenaya, Taounate, Bouzaghlal, Oued Laou, Smir (Merja), Béni Hassane, Azib Jrou, Azib Bouflou, AP , Laaouamra, Aarabat Sidi Abdelaziz, Moukaouama, Dar Belamri, Laksibia, Mgadid, Beggara, Rabat (Chellah), MA , Adouz, Rommani (Khémisset), Aït Abderrahmane, Aït Ishaq, Oulad Messoud, Ouled Fennane, El karma, Oulad Zguida, Oulad Abbou; Aouinty et al. 2006 , AP , Mohammedia; Himmi 2007 , Rif , Bab Berred, Bab Taza, Tanakoub, AP , Skhirate, Maâmora, Oulja, Bouknadel, Sidi Azzouz, Tamaris, MA , Khémisset; Faraj et al. 2008a , AP , Laouamra, Boucharen, MA , Béni Khlef, Talaa Chougaga; Faraj et al. 2008b , Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010b , MA , Fès; Faraj et al. 2010 , AP , Begara, Boucharen, Ben Slimane, Skhirat, Rabat, Sehoul, MA , Sidi Allal Msader, Aïn Aghbal, Aïn Elouali, Sidi Kacem; Larhbali et al. 2010 , MA , Aït Haddou Said; Adlaoui et al. 2011 , AP , Larache; Larhbali et al. 2011 , MA , Oulmès, Aït Yadin, Sfassif, Mâaziz, Rommani, Laghoualem, Ezzhiliga, Sidi Allal Bahraoui, Boukachmine, Aït Malek, Sidi Boukhalkhal, Bni Ounzar, Ganzra, Aït Siberne, Sidi Allal Msader, El Ghandour; El Ouali Lalami 2012 , MA , Pont Diamant vert, Sidi Harazem, Oued El Himmer, Moulay Yakoub, Oued Sebou, Aïn Kansara, Oued Aïn Chkef, Sefrou, Boulemane; Laboudi et al. 2012 , AP , Larache; Hadji et al. 2013 , AP , Sidi Slimane; El Joubari et al. 2014 , Rif , Smir lagoon; Laboudi et al. 2014 , Rif , Tétouan, Tanger, Chefchaouen, Al Hoceima, AP , Larache, Salé, MA , Taza, Khémissat; El Joubari et al. 2015a , Rif , Smir lagoon; El Joubari et al. 2015b , Rif , Smir lagoon; Marc et al. 2016 , AP , Kénitra; Trari 2017 , Rif , Chefchaouen, Tétouan, AP , Larache, Rabat, Settat, EM , Oujda, MA , Khémisset, Meknès, Khouribga, HA , Marrakech, AA , Tiznit, Ouarzazate; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, AP , Larache, Rabat, Settat, EM , Oujda, MA , Khémisset, Meknès, Khouribga, HA , Marrakech, AA , Tiznit, Ouarzazate; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) marteri Senevet & Prunnelle, 1927 Gaud 1945b , MA , El Hajeb, Khénifra, HA , Tizi-n'test, Tillougite; Gaud et al. 1949 , HA , Tizi-n'test; Gaud 1953b , HA , Tizi-n'test; Bailly-Choumara 1967b , EM , Grotte du Zegzel; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Taza-Asifane, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Boured; Benmansour et al. 1972 , MA , Taza; Trari 1991 , Rif , Taounate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; Trari 2017 , Rif , Chefchaouen, AP , Settat; Trari and Dakki 2017a , Rif , Chefchaouen, AP , Settat; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) ziemanni Grünberg, 1902 Senevet 1935 , MA ; Gaud et al. 1949 , HA , plain of south and north of the Occidental Atlas; Gaud et al. 1950 , HA , plain of south and north of the Occidental Atlas; Guy 1958 , HA , Marrakech; Guy et al. 1958, HA , Oued N'fis; Guy 1967 , EM , Oujda, AP , Rabat, HA , Marrakech, AA , Tafilelt; Bailly-Choumara 1970 , HA , Marrakech; Benmansour et al. 1972 , MA , Taza, HA , Haouz, Tadla Azilal; Bailly-Choumara 1973b , HA , Souk des Oudaias (Souk Tnine des Oudaias, 470 m); Trari 1991 , MA , Tissa; Moussalim 1997 , AP , 7.5 km de Sidi Allal Tazi; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 ; Trari 2017 , Rif , Tétouan, AA , Tiznit; Trari and Dakki 2017a , Rif , Tétouan, AA , Tiznit; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) cinereus Theobald, 1901 Viallate 1922, MA , Sefrou, Sidi Lamine, HA , Mtougui; Sicault et al. 1935 , AP , Souk Larba of Gharb; Senevet 1935 , AP , Souk Larba of Zemmour; Langeron 1938 , HA , Anefgou, Tirghist, Valley of Sidi Yahia Ouyoussef, Tighermine, Louggouargh, Massou; Callot 1940 , HA , Anefgou, Tirghist; Gaud 1945a , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud and Duthu 1945, HA , Marrakech; Viamonte and Ramirez 1945 , Rif ; Viamonte and Ramirez 1946 , Rif ; Ristorcelli 1946a , HA , Oued Tensift, Oued Issil; Ristorcelli 1946b , HA , Oued Tensift, Oued Issil; Gaud 1945b , AA , Tansikht; Gaud et al. 1949 , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud et al. 1950 , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud 1953a ; Gaud 1958, HA , Marrakech; Guy 1962 , Rif , Taounate, HA , Marrakech; Guy 1963 , MA , Midelt, AA , Hamada of Draa; Bailly-Choumara 1965, EM , Aman d'Aït Oussa, Tiglit, El Megrinat, Taskala, Aïn Aït delouine, Oued mesdourt, Talmesdourt, Assa, AA , Aït Melloul, Oued Teima, Issen, Taroudant, Talaint, Tiznit, Oued Assaka, Anezi, Pont de la route Agadir-Tiznit, valley of low Draa, Tafraoute, Tacharicht, Bou Izakarn, Jemâa N'tirhirte, Aït Erkha,Tazert, Barrage Taourirt, AA , Goulmima, SA , Aouinet Torkoz, Tirh Mzoun; Bailly-Choumara 1966 , AA , piste Foum Zguid-Lac Iriqui, Agadir-Tissint, Akka-Iguiren, Tirhem, Taoujgelt, Souk El Khémis Dades, Akka, Aït Ouabelli, Foum-el-Hassan, Tarhjicht, Aït melloul, Taliouine, Tazenakht (Rocade of Draa); Bailly-Choumara 1967a , MA , route Azrou-Khénifra, Jnane Imasse, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Sources Oum-er-Rbia, Itzer, piste Itzer-Boumia, Ouaoumana, piste Ghorm El Alem-El Ksiba, piste Naour-Arbala, Zaouia Cheich, kafensour, Ifrane, Imouzzer Marmoucha; Bailly-Choumara 1967b , EM , 7 km N Itzer, 8 km N Itzer, route Itzer-Midelt, Aïn Srouna, Gouttitir, Cascade Oued Za, Grotte du Zegzel, Douar Aïn Soultane, Mechraa Safsaf, Oujda, Berguent (valley of Moulouya), Figuig; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Assifane, Anasar, piste Bab berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, route Al Hoceima-Arba Taourirt, Arba Taourirt, Targuist et environs, route Targuist Al Hoceima, Jebha, Oued Ouergha, route Aknoul-Al Hoceima; Guy 1967 , AP , Rabat, EM , Oujda, HA , Marrakech, AA , Tafilalet; Bailly-Choumara 1970 , HA , Marrakech; Bailly-Choumara 1973b , HA , Souk des Oudaias; Trari 1991 , Rif , Al Hoceima, Chefchaouen, Taounate, EM , Nador, Oujda, Figuig, AP , Larache, Settat, Ben Slimane, MA , Khénifra, Taza, Khouribga, HA , Kelaâ of Sraghna, AA , Ouarzazate, Goulmima; Bouallam 1992, HA , Oued N'fis; Louah 1995 , Rif , Marina Smir, Bouzerlal, Tahaddart, Stehat, Moulay Bouchta, Kebbache, Loubart, 0ued Maggou; Louah et al. 1995 ; Bouallam et al. 1997b , HA , Marrakech; Handaq 1998 , HA , Amizmiz, Tiguenziouine; Bouallam 2001, HA , Oued N'fis; Trari et al. 2002 ; Trari et al. 2004b , Rif , Ketama, Azib Jrou, Sidi Mokhfi, MA , Taghzirt, Aghbala, Aït Shak, Smaala, Mlalih, Ouled Fennane, Béni Khlef, Tachrafte; Faraj et al. 2007 , Rif , Assoul, Mizgane; Himmi 2007 , Rif , Bab Berred, Bab Taza, Stehat, Tanakoub; Faraj et al. 2008, Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Fès; El Ouali Lalami et al. 2010b , MA , Oued El Himmer; Larhbali et al. 2010 , MA , Roumani Aïn Sbite, Jamâa M. B., Ghoualem, Zhiliga, Oulmès, Tarmilate, Boukachmir, Mrirte, Aït Ichou, Mâaziz, Tiddas, Bni Ounzar, Ganzra; El Ouali Lalami 2012 , MA , Oued El Himmer; Trari 2017 , Rif , Chefchaouen, Tétouan, MA , Khouribga, AA , Tiznit; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, MA , Khouribga, AA , Tiznit; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Anopheles ( Cellia ) dthali Patton, 1905 Saccà 1960 , AA , Aoufous, Meski, Erfoud, Agdz, Zogora, Tagounit, Tamsrruth; Guy 1961 , HA , Sud de Zagora (en bordure Hamada du Draa); Guy 1963 , AA , Zagora and south of the High Atlas (at edge of Hamada of Draa), Oued Ziz; Bailly-Choumara 1965c , AA , Tiznit, EM , Aïn Aït Delouine, Aouinet Torkoz, Rich Tamlougout (eastern borders of Jebel Bani); Bailly-Choumara 1966 , AA , piste Foum Zguid au Lac Iriqui, Agadir-Tissint, Akka-Iguiren, Souk El Khémis Dades, Akka et Environs, Aït Ouabelli, Tarhjicht, piste Tazenakhte à Foum Zguid (Rocade du Draa); Bailly-Choumara 1967b , EM , valley of Moulouya; Guy 1967 , HA , Marrakech, AA , Tafilalet; Guy and Holstein 1968 , EM , N Outat El Haj (valley of Moulouya), Gouttitir (environs de Taourirt); Bailly-Choumara 1970 , AA , Foum Zquid; Bailly-Choumara 1973b , AA , Foum Zquid; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari et al. 2004b ; Faraj et al. 2007 , Rif , Assoul, Mizgane (SE of Bab Berred); Faraj et al. 2008, Rif , Assoul, Mizgane (SE Bab Berred); Himmi 2007 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) multicolor Cambouliu, 1902 Messerlin and Treillard 1938 , HA , Marrakech; Viamonte and Ramirez 1945 , Rif ; Guy 1963 , SA ; Bailly-Choumara 1965c , EM , Aït Oussa, Aman d'Aït Oussa, Aïn Aït Delouine, Aouinet Torkoz, Rich tamlougout (Confins orientaux du Jebel Bani), AA , Tafnidilt, Guelta Zerga, Aïn Temda (valley of low Draa), Tirhmert (Goulmima), SA , Vallée et embouchure de l'Oued Assaka, Tantan ville, Tirh Mzoun; Bailly-Choumara 1966 , AA , Rocade of Draa; Bailly-Choumara 1967b , EM , 40 km N de Outat El Haj, Gouttitir, Cascade Oued Za, Douar Aïn Shebbak (valley of Moulouya); Guy 1967 , HA , Marrakech, AA , Tafilelt; Guy and Holstein 1968 , HA , south of Atlas, AP , plain located between Marrakech and the Atlantic from Tanger along the length of the Mediterranean; Bailly-Choumara 1970 , AA , Foum Zquid; Bailly-Choumara 1973b , AA , Foum Zquid; Metge 1986 , AP , Casablanca; Trari 1991 , Rif , Al Hoceima, Taounate, Oued Maleh, Bab Berred, AP , Tamaris Merja, AA , Ouarzazate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari et al. 2004b ; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) sergentii (Theobald, 1907) Séguy 1930a ; Messerlin and Treillard 1938 , HA , Tamelelt; Langeron 1938 , Rif , Targhist; Callot 1940 , AA , Taghjicht; Gaud 1947c , AA , Wadi Draa; Gaud et al. 1949 , Rif , Zoumi, HA , Zaouia Sidi Hamza, Tizi-n'test (1700 m), Tillougit (1800 m); Gaud et al. 1950 , Rif , Zoumi, MA ; Guy et al. 1958, HA , Oued N'fis; Guy 1961 , Rif , AP , south of Casablanca, AA , Sud de Zagora; Guy 1962 , Rif , AP , south of Casablanca, HA , Marrakech; Guy 1963 , Rif , Tanger, EM , Berkane, AP , south of Casablanca, MA , Béni Mellal, HA , Oued Tensift, Oued Ziz, Marrakech, Chichaoua, AA , Oued Draâ, Oued Dades, Goulmima, sud de Zagora, SA , Foum Zguid; Bailly-Choumara 1965c , EM , iglit, Aman d'Aït Oussa, Oued Isker, El Megrinat, Aïn Aït Delouine, Oued Mesdourt, almesdourt, Aouinet Torkoz, Bouanama, Rich Tamlougout, Assa, AA , Tiznit, Tafraoute, Oued Izi, Bou Izakarn, Abeino (region of Goulmima), SA , Aouzeroual, Tirhmert, Tacharicht, Jebel Bani, Tantan; Bailly-Choumara 1966 , AA , Rocade of Draa, Taliouine; Bailly-Choumara 1967a , Rif , AP , south of Casablanca, MA , Itzer, Ghorm El Alem, Zaouia Cheikh, Kafensour, HA , north of High Atlas; Bailly-Choumara 1967b , EM , Aïn Srouna, Grotte du Zegzel, Douar Mardarh, Douar Aïn Soultane, Merja Boubker, Selouane, Driouch (valley of Moulouya); Bailly-Choumara 1967c , Rif , route nationale Jebha, Targuist-Béni Boufrah, Al Hoceima, Béni Bouayache, Marchica, Had El Rouadi, Pont du Srah; Guy 1967 , HA , Marrakech, AA , Tafilelt; Guy and Holstein 1968 , AP , Casablanca; Bailly-Choumara 1970 , EM , Berkane, HA , Marrakech; Bailly-Choumara 1973b , EM , Berkane, HA , Marrakech; Metge 1986 , AP , Casablanca; Trari 1991 , Rif , Al Hoceima, Taounate, AP , Larache, AA , Ouarzazate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Faraj et al. 2003 , Rif , Al Hoceima, Chefchaouen, Taounate, HA , Khouribga; Trari et al. 2004b , Rif , Ketama, Sidi Mokhfi; Faraj et al. 2007 , Rif , Assoul, Mizgane; Faraj et al. 2008, Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Fès; Larhbaliet al. 2010 , MA , Oulmès, Ganzra; Trari 2017 , Rif , Chefchaouen, Tétouan, EM , Oujda, MA , Khouribga; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, EM , Oujda, MA , Khouribga; Trari and Dakki 2017b ; Trari et al. 2017 ; Benabdelkrim Filali et al. 2018 ; Mouatassem et al. 2019, MA , Fès Culicinae Aedini Aedes Meigen, 1818 Aedes ( Acartomyia ) mariae (Sergent & Sergent, 1903) Séguy 1930b ; Messerlin 1938 , AP , Rabat; Séguy 1930a , Rif , littoral méditerranéen; Bailly-Choumara 1967b , Rif , Al Hoceima; Bailly-Choumara 1967c , Rif , Al Hoceima; Bailly-Choumara 1968a , Rif , Al Hoceima, AP , Larache, Sidi Yahia, Sidi Allal Tazi, Rabat, HA , Marrakech, AA , Tiznit; Himmi et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Aedimorphus ) vexans (Meigen, 1830) Gaud 1947c , AP , Sidi Allal Tazi, MA , Khémisset; Metge 1986 , AP , Littoral Casablanca; Himmi et al. 1995 ; Handaq 1998 , AP , Gharbia; Dakki 1997 ; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Dahliana ) echinus (Edwards, 1920) Séguy 1924 ; Séguy 1930a ; Gaud 1953a , AP , Rabat, Sidi Yahia, MA , Moulay Bouazza, Taza, Fès, Meknès, Ifrane; Bailly-Choumara 1965b , AP , Maâmora; Bailly-Choumara 1967c , Rif , piste Bab Taza-Talassemtane; Himmi et al. 1995 ; Trari et al. 2002 ; Nikookar et al. 2010 ; El Joubari et al. 2014 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Dahliana ) geniculatus (Olivier, 1791) Séguy 1924 , MA ; Séguy 1930a ; Metge and El Alaoui 1987 , AP , Subéraies de Béni Abid-Benslimane (Casablanca); Metge and Belakoul 1989 , AP , Sidi Bettache; Himmi et al. 1995 ; El Ouali Lalami et al. 2010a , MA , Fès Boulmane; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) berlandi (Séguy, 1921) Séguy 1930a , AP , Rabat; Gaud 1953a , MA , Fès; Bailly-Choumara 1967a , MA , Jnane Imasse, piste Tafechna-Taoujgelt; Bailly-Choumara 1967c , Rif , piste Bab Taza-Béni Ahmed; Belakoul 1985 , AP , Benslimane, Sidi Bettache; Metge and El Alaoui 1987 , AP , Casablanca; Metge and Belakoul 1989 , AP , Benslimane, Sidi Bettache; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) caspius (Pallas, 1771) Séguy 1930b ; Viamonte et Ramirez 1946, AP , Larache; Gaud 1952 , AP , Rabat, Casablanca; Gaud 1953a , AP , Rabat, Casablanca; Senevet and Andarelli 1954 , EM , Embouchure de la Moulouya, Figuig, AP , Jorf Lasfar, Mohammedia, Rabat, MA , Meknès, Fès, Taza, HA , Marrakech, Midelt, AA , Tiznit; Bailly-Choumara 1966 , AA , environs de Tiznit; Bailly-Choumara 1967a , EM , Bords Moulouya (près Itzer), Cherarba; Bailly-Choumara 1967b , EM , Cherarba, Aïn Shebbak, Saidia, Berguent; Bailly-Choumara 1967c , Rif , piste Al Hoceima-Arba Taourirt; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , Rif , Merja de l'Oued Smir; El Kaim 1972 , AP , Bou-Regreg; Rioux et al. 1975 , AP , Rabat-Salé; Metge 1986 , AP , Littoral casablancais; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba, Merja Zerga, Oued Loukous; Himmi et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Kénitra; Ramdani 1997 , AP , Tamaris Merja; Handaq 1998 , AP , Zemamra, B. Iffou (Entre El Oualidia et Youssoufia), MA , Béni Mellal, HA , Marrakech, Zaouiet Ben Sassi, Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; Alaoui Slimani 2002 , AP , Rabat; Aouinty et al. 2006 , AP , Mohammedia; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) coluzzii Rioux, Guilvard & Pasteur, 1998 et Aedes ( Ochlerotatus ) detritus (Haliday, 1833) [Complexe detritus] Charrier 1924 , Rif , Tanger; Séguy 1930a ; Gaud 1953a , EM , Saidia, AP , Kénitra, Rabat, Bouznika, El jadida, Oualidia, HA , Marrakech, AA , Agadir, Tafnidilt; Bailly-Choumara 1965c , EM , Aïn Aït delouine, SA , Tirhmert; Bailly-Choumara 1970 , Rif , Tétouan; Knight 1971 , AP , Kénitra; El Kaim 1972 , AP , Bou-Regreg; Bailly-Choumara 1973b , Rif , Merja de l'Oued Smir; Rioux et al. 1975 , AP , Rabat; Pasteur et al. 1978 , AP , Bou-Regreg; Metge 1986 , AP , Littoral casablancais; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba, Merja Zerga, Oued Loukous; Louah 1995 , Rif , Tres piedras, Cabo Negro, Lajour, Azla, Tahaddart; Himmi et al. 1995 ; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Rabat; Ramdani 1997 , AP , Tamaris Merja; Handaq 1998 , AP , Essaouira, Zima-Chemaîa; Himmi et al. 1998 , AP , Sidi Boughaba; Himmi 2007 , AP , Sidi Boughaba; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) pulchritarsis (Rondani, 1872) Gaud 1953a , AP , Benslimane, Sidi Yahia, Rabat, MA , Oued Zem, Khénifra, Fès; Metge and El Alaoui 1987 , AP , Benslimane; Himmi et al. 1995 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Rusticoidus ) rusticus (Rossi, 1790) Viamonte and Ramirez 1946 , Rif , Tétouan, AP , Larache; Gaud 1953a , MA , Taza; Himmi et al. 1995 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Stegomyia ) aegypti (Linnaeus in Hasselquist, 1762) d'Anfreville 1916 , AP , Salé; Vialatte 1923 , AP , Rabat, Casablanca, HA , Marrakech; Charrier 1924 , Rif , Tanger; Gaud 1953a , AP , Salé, HA , Marrakech; Himmi et al. 1995 ; Dakki 1997 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Stegomyia ) albopictus (Skuse, 1895) Bennouna et al. 2016 , AP , Agdal (Rabat); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Faraj et al. 2018 ; Amraoui et al. 2019 Culicini Culex Linnaeus, 1758 Culex ( Barraudius ) modestus Ficalbi, 1889 Séguy 1930a ; Bailly-Choumara 1968a , AP , Larache; Trari 1991 , AP , Gharb; Himmi et al. 1995 ; Dakki 1997 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, AP , Maâmora; Hadji et al. 2013 , AP , Sidi yahia du Gharb, Kcebia, Sidi Hagouch (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) brumpti Galliard, 1931 Bailly-Choumara 1968a , AP , Merja Bokka, Larache, HA , Marrakech; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi et al. 1995 ; Dakki 1997 ; Himmi 2007 ; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) laticintus Edwards, 1913 Charrier 1924 , Rif , Tanger; Callot 1940 , AA , Goulmima (mares); Gaud 1953a , HA , Marrakech, AA , Agadir; Gaud 1957a , EM , Nador; Bailly-Choumara 1965c , EM , Oued Isker, Aïn Aït delouine, Talmesdourt, AA , Ouled Teima, ounaamane, Bou Izakarn, Agunil Khnufa, Akka-guiren; Bailly-Choumara 1966 , AA , Akka-Iguiren, Tirherm, Taoujgelt, Aït Ouabelli, Anamere-Smougue, Aït melloul, Tiznit (Rocade de Draa); Bailly-Choumara 1967b , Rif , Béni Bouayache, Targuist et environs, EM , Grotte du Zegzel; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 , AP , Skhirat; Faraj et al. 2008b , AP , Louamra; Hadji et al. 2013 , AP , Sidi Hagouch (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) mimeticus Noè, 1899 Séguy 1930a ; Viamonte and Ramirez 1946 , Rif , Béni Ider, Fnideq, Khemis Anjra, Ketama, Oued Amsa, Oued Krikra, Oued Martil, Oued Laou; Gaud 1953a , Rif , Ouezzane, Ghafsai, EM , Berkane, Martinpray (près Berkane), El Aïoun Sidi Mellouk, AP , Tamri, MA , Meknès, Fès, Ifrane, Taza, Béni Mellal, HA , Midelt, Marrakech, Azilal, AA , Tinghir, Tichka; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , AA , Oued Noun, Anezi, Tafraoute; Bailly-Choumara 1966 , AA , Agadir; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Taoujgelt, Source Oumerrbia, Ghorm El Alem, piste Ksiba-Naour, piste Naour-Arbala, Zaouia Cheikh, Oued Sarif; Bailly-Choumara 1967b , EM , 7 km N d'Itzer, 8 km d'Itzer; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Taza-Asifane, piste Bab Berred-Tamorote, Ketama, piste Ketama-Jebel Tidighine, route Ketama-Targuist, route nationale Jebha, Al Hoceima, Béni Bouayache, Marchica, Targuist et environs, Jebha, Boured; Trari 1991 , AP , Sidi Yahia du Gharb; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, Tres piedras, Marina Smir, Bouzaghlal, M'diq, Oued Maleh, Azla, Tahaddart, Moulay Bouchta, Schroda, Kebbache, Talambote, Oued Maggou; Louah et al. 1995 ; Chlaida 1997 , AP , Oued Sidi Messoud, Aïn Blal, Douar Chlihat, Barrage Al Massira; Chlaida and Bouzidi 1995 , AP , Barrage El Massira; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris Merja; Handaq 1998 , HA , Oukaimeden, Amizmiz, Tiguenziouine (près Oued N'fis); Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, AP , Bouknadel, Douar jdid (Skhirat); El Ouali Lalami et al. 2010a , MA , Fès; Larhbali et al. 2010 , MA , Oulmès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) perexiguus Theobald, 1903 Séguy 1930a ; Callot 1940 , AA , Assa; Senevet and Andarelli 1959a, EM , Oujda, Taourirt, AP , Aïn el Aouda, Arbaoua, Had Kourt, Oued Beht, Rabat, Allal Tazi, Oued Sahli, Zaouia Ech cheikh, Taghzirt, MA , Béni Mellal, Foum Zabel, Ifrane, Meknès, Aït Atta du Rteb, Fès, Sidi Mokhfi, Tahala, HA , Tazert, Midelt, AA , akka, Tamri; Bailly-Choumara 1965c , EM , Assa; Bailly-Choumara 1966 , AA , Souk El Khémis Dades, Aït Ouabelli, Tarhjicht, Tirherm, Taoujgelt, Aït melloul; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Ajdir; Bailly-Choumara 1967b , EM , Madagh, Merja Boubker, Aïn Béni Mathar; Bailly-Choumara 1967c , Rif , piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Béni Bouayache, Targusit et environs; Bailly-Choumara 1972a , AP , Merja Sheishat; Louah 1995 , Rif , Riffien, Tres piedras, Marina Smir, Bouzeghlal, Oued Maleh, Tahaddart, Talembote, Schroda, Tanger; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, El Oulja, Fouarate; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , HA , Marrakech, entre Oued N'fis et Chichaoua, Kelaâ Sraghna; Alaoui Slimani 2002 , AP , Rabat, Salé; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Bab Taza; Faraj et al. 2008c, AP , Larache Louamra; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Culex ) pipiens Linnaeus, 1758 d'Anfreville 1916 , AP , Salé; Charrier 1924 , Rif , Tanger; Séguy 1930a ; Callot 1940 , SA , Goulimine; Viamonte and Ramirez 1946 , Rif , Boudinar, Dar Benkarrich, Tétouan, Tanger, Asilah, Ksar El Kébir, Chefchaouen, Ketama, Nador; Gaud 1952 , AP , Gharb; Séguy 1953a , SA , Tindouf; Guy 1958 , HA , Oued N'fis; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , EM , Aouinet Aït Oussa, Aïn Isker, Aïn Aït Delouine, Oued Mesdourt, Talmesdourt, Toudi, AA , Aït Onmar, Oulad Teima, Taroudant, Tiznit ville, Talaint, Hassi Tafnidilt, Zaouiat Cheikh, Aïn Guerzim, Tafraout ville, SA , Goulimine ville, Vallée de l'Oued Assaka, Ouaroun, Zriouila, Labyar, Tighmert, Abeino, Tantan ville, Zag; Bailly-Choumara 1966 , AA , piste d'Akka au Draa, Akka et environs, Aït Ouabelli, Anamere-Smougue, Tarhjicht, Aït Melloul, Tiznit et environs; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, piste Itzer-Boumia, Ghorm El Alem, Ghorm El Alem-El Ksiba, El Ksiba, piste Ksiba-Naour, Zaouia Cheikh, Oued Sarif, Dayet Aoua, Pont Tarmilate, Ifrane, Imilchil; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Gaada de Debdou, Guercif ville, Cascade Oued Za, Grotte du Zegzel, Environs Saidia, Douar Aïn Shebbak, Douar Aïn Zabia, Douar Mardarh, Douar Sidi Hashas, Saidia, Merja Boubker, Berguent, Tendrara, Figuig; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Al Hoceima, Al Hoceima Club Med, Béni Bouayache, Marchica, Targuist et environs, Jebha, Ghafsai; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , AP , Merja Bokka, Merja Qodiya; Metge and Belakoul 1989 , AP , Benslimane, Sidi Bettache; Himmi 1991 , AP , Sidi Boughaba, Sidi Amira; Trari 1991 , AP , Maâmora, El Menzeh, Sidi Boughaba, Chkaïfien, Sidi Yahia du Gharb, Bokka, Merja Zerga, Sidi Allal Tazi, Oued Loukous, Aïn Chouk, Merja Bargha, Merja Oulad Skhar; Bouallam and Ramdani 1992 , HA , Marrakech; El Bermaki 1993, AP , Sidi Maârouf; Himmi et al. 1995 ; Louah 1995 , Rif , Fnideq, Riffien, Tres piedras, Marina Smir, M'diq, Cabo Negro, Zekri, Azla, Tahaddart, Schroda, Kebbache, Ouadras, Punta cirres, Sidi Kankoch, Oued Kbir; Louah et al. 1995 ; Bouallam et al. 1997b , HA , Marrakech; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Himmi et al. 1998 , AP , Sidi Boughaba, Puits de Sidi Amira (forest of Maâmora); Moussalim 1997 , AP , El Oulja, Maâmora, Fouarate, Sidi Boughaba; Ramdani 1997 , AP , meseta côtière (Témara-Casablanca); Faraj et al. 1997 , AP , Kénitra; Handaq 1998 , AP , Zemamra, MA , Oued Zem, HA , Marrakech, Zaouiet Ben Sassi, Sidi Bou Othmane, Kelaâ Sraghna, Bengrir; Bouallam 2001, HA , Marrakech; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Aouinty et al. 2006 , AP , Mohammedia; Faraj et al. 2006 , AP , Salé; Himmi 2007 , Rif , Bab Berred, Bab Taza, Stehat, AP , Skhirate, forest of Maâmora, forest of Hilton, Sidi Boughaba, El Oulja, Ouled Salem, Ouled dlim, Douar Ould Yahia Ben Ali, Larouaza, Douar Elarja, Douar Jdid, Douar Jnaja; Faraj et al. 2008b , AP , Louamra; El Ouali Lalami et al. 2010a , MA , Fès; Larhbali et al. 2010 , MA , Oulmès, Tarmilate, Bni Ounzar, Ganzra;Louali Lalami et al. 2010b, MA , Oued El Himmer; Amraoui 2012 , HA , Marrakech; Amraoui et al. 2012 , Rif , Tanger, AP , Mohammedia, Casablanca, HA , Marrakech; Amraoui et al. 2012 , AP , Mohammedia, Casablanca; El Ouali lalami 2012 , MA , route de Sidi Harazem; Hadji et al. 2013 , AP , Sidi Slimane; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Marc et al. 2016 , AP , Kénitra; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Bkhache et al. 2018 , Rif , Tanger, AP , Rabat, Mohammedia, HA , Marrakech; Tmimi et al. 2018 , AP , Mohammedia; Mouatassem et al. 2019, MA , Fès Culex ( Culex ) simpsoni Theobald, 1905 Callot 1940 , AA , Taghicht; Senevet et al. 1949 , AA , Oued Noun, Tafnidelt, Akka; Gaud 1953a , AA , Imsouane, Aït Melloul, Akka, O'Noun, Taghjicht, Assa, Tafnidilt; Bailly-Choumara 1965c , EM , Aman d'Aït Oussa, El Megrinat, AA , Oued Izi, Oued Massa-Pont de la route Agadir-Tiznit, Guelta Zerga, Tafnidilt, SA , Poste militaire de Boujrif, Tirhmert, Taourirt-Barrage, Aouinet Torkoz; Bailly-Choumara 1966 , AA , Tirherm, Taoujgelt, Taghjicht, Aït melloul, Tiznit; Chlaida and Bouzidi 1995 , AP , Sidi M'barek, Oued Sidi Messoud, Mechrâa, Sidi Boulâarais, Douar Chlihat (south of Settat); Chlaida 1997 , AP , Sidi M'barek, Mechrâa Settir, Sidi Boulâarais, Douar Chlihat (south of Settat); Dakki 1997 ; Handaq 1998 , HA , Sidi Bou Othmane (Marrakech); Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, MA , Khémisset, AA , sud Anti Atlas; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) theileri Theobald, 1903 Callot 1940 , AA , Taghjicht; Viamonte and Ramirez 1946 , Rif , Dar Benkarrich, Boudinar, Ketama, Tanger, Tétouan, Chefchaouen, AP , Larache; Guy 1958 , HA , Oued N'fis; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , EM , Aouinet Aït Oussa, Aïn Oumesdour, Aouinet Torkoz, SA , Tirh Mzoun; Bailly-Choumara 1966 , AA , Souk El Khémis Dades, Akka et environs, Aït Melloul, Tazenakhte, piste Tiznit-Tafraout, SA , Goulimine; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Aguelmane Azigza, piste Aguelmane Azigza-Aïn Leuh, maison forestière Ouiouane, Khénifra, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Col du Zad, piste Itzer-Boumia, Ghorm El Alem, piste Ghorm El Alem-El Ksiba, El Ksiba, piste Ksiba-Naour, Aguelmane Moulay Yakoub, Zaouia Cheich, Kafensour, Dayet Aoua, Pont Tarmilate; Bailly-Choumara 1967b , EM , route Itzer-Midelt, 40 km N Outat El Haj, Taourirt, Driouch, Figuig; Bailly-Choumara 1967c , Rif , piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, ArbaaTaourirt, Al Hoceima, Targuist et environs, Ghafsai; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , AP , Merja Sheishat, Merja Bokka; Himmi 1991 , AP , Sidi Boughaba, El menzeh, Sidi Amira; Trari 1991 , AP , Maâmora, Oued Sebou, Bordure Oued Loukous, Sidi Yahia du Gharb, Bokka, Entre Moulay Bousselham et Larache, Merja Zerga, Larache; El Bermaki 1993, AP , Sidi Maârouf, El Oulfa; Louah 1995 , Rif , Bouzeghlal, M'Diq, Oued Maleh, Zekri, Lajour, Tahaddart, Loubart, Chefchaouen, Tanger; Himmi et al. 1995 ; Louah et al. 1995 ; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Maâmora, Rabat, Kénitra; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , AP , Zemamra, MA , Béni Mellal, HA , Oukaimeden, Amizmiz, Tiguenziouine, Marrakech, Sidi Bou Othmane, Seguarta-Kelaâ, Had Mhara, Aîn Äounate, Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra), Gharb; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Tanakoub, AP , Skhirate, forest of Maâmora, Chiahna, Sidi Amira, Sidi Boughaba, Bouknadel, Sidi Azzouz, Ehssaïne, Bettana, Sidi Yahia, Sebbah, Larouaza, Douar Elarja, Douar Jdid, Douar Jnaja; Faraj et al. 2008b , AP , Larache Louamra; El Ouali Lalami et al. 2010a , AP , Boucharen; Larhbali et al. 2010 , MA , Roumani, Aïn Sbite, Ezzhiliga, Oulmès, Tarmilate, M'rirt, Bni Ounzar, Ganzra (Khémisset); Hadji et al. 2013 , AP , Sidi yahia du Gharb, Kcebia, Sidi Hagouch, Dar Belamri (Sidi Slimane); El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Maillotia ) deserticola Kirkpatrick, 1925 Gaud 1947c , AA , Tansikht; Gaud 1953a , EM , Figuig, Berguent, Anoual, Boudnib, Aïn Chair, Aoufous, Tarhit, HA , Tazenakht, Tichka, Tizi-n'Telghemt, AA , Ouarzazate, Zagora; Bailly-Choumara 1965c , EM , Oued Isker, El Megrinat, Taskala, Aïn Aït Delouine, Talmesdourt, Aouinet Torkoz, Bouanama, Rich Tamlougout, Assa, AA , Tafraout ville, Oued Jemâa Idaousmaal, Aït abdallah, Issedrim Igmur Igues, SA , Vallée de l'Oued Assaka, Tacharicht, Bou Izakarn, Aïn Erkha; Chlaida and Bouzidi 1995 , AP , Sud de Settat; Himmi et al. 1995 ; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Maillotia ) hortensis Ficalbi, 1889 Charrier 1924 , Rif , Tanger; Séguy 1930a ; Langeron 1938 , HA , Tounfite; Callot 1940 , HA , Anefgou (2500 m), Tirghist (2500 m), Tighermine (2500 m); Gaud 1953a , HA , Tizi-n'Telghemt, Tizi-n'Tichka; Bailly-Choumara 1967a , MA , piste tafechna-Znan Imes, Khénifra, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Ghorm El Alem, El Ksiba, piste Ksiba-Naour, Piste Naour-Arbala, piste Arbala-El Kbab, Zouia Cheikh, Oued Sarif, Ifrane, Boulmane, Imilchil; Bailly-Choumara 1967b , EM , Tafraout, Grotte du Zegzel; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Tamorote, route Bab Berred-Ketama, Ketama, piste Ketama-Mt Tiguidin, route nationale Jebha, route Targuist-Béni Boufrah, Trari 1991 , AP , Gharb; El Bermaki 1993, AP , Casablanca; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, Marina Smir, M'diq, Oued Maleh, Lajour, Tahaddart, Schroda, Talembote, Bab Berred, Punta Cirres, Sidi Kankoch, Oued kbir, Oued Jebel Lehbib; Louah et al. 1995 ; Chlaida and Bouzidi 1995 , AP , Barrage Al Massira; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , HA , Amizmiz, Sidi Bou Othmane, Had Mhara, Bengrir; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Bab Taza, AP , Skhirate; El Ouali Lalami et al. 2010b , MA , Oued Fès, Oued El Himmer, Camping Sidi Harazem; Larhbali et al. 2010 , MA , Merchouch, Ghoualem, Ezzhiliga, Oulmès, Tarmilate, M'rirt, Bni Ounzar, Ganzra (Khémisset); El Ouali Lalami 2012 , MA , Oued Fès; Oued El Himmer, route de Sidi Harazem, Camping Sidi Harazem; Hadji et al. 2013 , AP , Sidi Yahia du Gharb, Kcebia, Sidi Hagouch, Dar Belamri, Lalla Itto, Soualem (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Neoculex ) impudicus Ficalbi, 1890 Charrier 1924 , Rif , Tanger; Séguy 1930a ; Bailly-Choumara 1966 , AA , Taliouine; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Senoual-Itzer, Itzer, piste Naour-Arbala, Zaouia Cheikh, Pont Tarmilate; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Cascade Oued Za, Grotte du Zegzel; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Asifane, piste Bab Berred-Tamorote, Ketama, route nationale Jebha, Al Hoceima Club Med, Targuist et environs; Trari 1991 , AP , Sidi Amira, El Menzeh, Oued Sebou, Sidi Yahia du Gharb, Moulay Bousselham, Larache, Bordure Oued Loukous; Dakki 1997 ; Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra), Puits de Sidi Amira (forest of Maâmora); Trari et al. 2002 ; Himmi 1991 , AP , Sidi Boughaba, El Menzeh, Sidi Amira; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir, Tahaddart, Tanger; Louah et al. 1995 ; Ramdani 1997 , AP , Tamaris; Alaoui Slimani 2002 , AP , Rabat; Himmi 2007 , Rif , Bab Berred, Bab Taza, AP , forest Maâmora; El Ouali Lalami et al. 2010a , MA , Fès; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Neoculex ) martinii Medschid, 1930 Bailly-Choumara 1968a , Rif , Al Hoceima, AP , Sidi Yahia du Gharb, Sidi Allal Tazi, Larache, Rabat, HA , Marrakech, AA , Tiznit; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir, Cabo Negro, Oued Maleh; Louah et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culisetini Culiseta Felt, 1904 Culiseta ( Allotheobaldia ) longiareolata (Macquart, 1838) d'Anfreville 1916 , AP , Salé; Séguy 1930a ; Gaud 1947c , AA , Tansikht; Gaud 1953a , HA , Marrakech; Bailly-Choumara 1965c , EM , El Aïoun du Draa, Aïn Aït Delouine, Talmesdourt, Aouinet Torkoz, Rich Tamlougout, AA , Aït Onmar, Ouled Teima, Tiznit ville, Bounaamane, Id Baha, Tafnidilt, Guelta Zerga, Aïn Kerma, Tafraout ville, Oued Jemâa Idaousmaal, Toudi, Aït Abdallah, Igherm, Issedrim Igmur Igues, SA , Goulimine ville, Poste militaire de Boujrif, Embouchure de l'Oued Assaka, Ouaroun, Labyar, Asrir, Tacharicht, Bou Izakarn ville, Jemâa N'Tirhirte, Aït Erkha, Tantan ville; Bailly-Choumara 1966 , AA , Aït melloul, Tiznit; Bailly-Choumara 1967a , MA , Jnane Imasse, Khénifra, piste Tafechna-Senoual-Itzer, Itzer, Ghorm El Alem, El ksiba, piste Naour-Arbala, Ifrane, Pont Tarmilate, Imouzzer Marmoucha; Bailly-Choumara 1967b , EM , Tafraout, Guercif ville, Grotte du Zegzel, Berguent, Tendrara; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, route Bab Taza-Bab Berred, route Ketama-Targuist, route nationale Jebha, Targuist et environs, Aïn Hamra; Bailly-Choumara 1968a , AP , Meja Bokka, AA , Tiznit; Himmi 1991 , AP , Sidi Boughaba, El Menzeh; Trari 1991 , AP , El Menzeh, Gharb; El Bermaki 1993, AP , Sidi Maârouf; Himmi et al. 1995 ; Louah 1995 , Rif , Fnideq, Riffien, Marina Smir, M'diq, Cabo Negro, Lajour, Ouadras, Punta Cirres, Ksar Sghir, Tanger; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Maâmora, Fouarate; Ramdani 1997 , AP , Témara, Skhirat, Tamaris; Dakki 1997 ; Handaq 1998 , HA , Marrakech, Kelaâ Sraghna, Bengrir Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra); Bouallam 2001, HA , Bordure Oued N'fis; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Aouinty et al. 2006 , AP , Mohammedia; Himmi 2007 , Rif , Bab Berred, AP , Skhirate, Maâmora, forest of Hilton; Koçak and Kemal 2013; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culiseta ( Culicella ) fumipennis (Stephens, 1825) Gaud 1947c , AP , Rabat, Sidi Allal Tazi, MA , Khémisset; Senevet and Andarelli 1959, AP , Rabat, Casablanca, Bouznika, MA , Fès; Bailly-Choumara 1967a , MA , Jnane Imasse; Bailly-Choumara 1967c , Rif , route Bab Taza-Bab Berred; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culicella ) litorea (Shute, 1928) Metge 1986 , AP , Casablanca; Dakki 1997 ; Carles-Tolrá 2002 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culiseta ) annulata (Schrank, 1776) d'Anfreville 1916 , AP , Salé; Charrier 1924 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Viamonte and Ramirez 1946 , Rif , Ben Karrich, Asilah, Ketama, Malaliene, Tétouan, AP , Larache; Gaud 1952 , AP , Souk El Hadd (Gharb); Gaud 1953a , Rif , Tanger, Tétouan, EM , Saidia, Talsint, AP , Casablanca, Rabat, Kénitra, MA , Meknès, Fès, Ifrane, Taza, HA , Marrakech, Aït Bouguemez, Midelt; Gaud 1957b , Rif , Tétouan, Tanger, AP , Sidi Allal Tazi, Rabat, Casablanca, MA , Meknès, Ifrane; Bailly-Choumara 1967a , MA , Jnane Imasse, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, Ghorm El Alem, Zaouia Cheikh, Pont Tarmilate, Ifrane; Bailly-Choumara 1967b , EM , Tafraout, route Itzer-Midelt; Bailly-Choumara 1967c , Rif , piste Bab Taza-Talassemtane; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, M'diq, Tanger; Louah et al. 1995 ; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Rabat; Ramdani 1997 , AP , meseta côtière (Casablanca-Rabat); Himmi et al. 1998 , AP , Sidi Boughaba; Himmi 1991 , AP , Sidi Boughaba, El Menzeh; Trari 1991 , AP , Sidi Boughaba, El Menzeh; Trari et al. 2002 ; Himmi 2007 , AP , Skhirate, Maâmora, forest of Hilton, Oulja; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culiseta ) subochrea (Edwards, 1921) Gaud 1952 , AP , Gharb; Gaud 1957b , Rif , Tanger, Chefchaouen, EM , Oujda, AP , Sidi Allal Tazi, Casablanca; Bailly-Choumara 1967a , MA , Imilchil; Bailly-Choumara 1967c , Rif , Ketama; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba; Louah 1995 , Rif , Riffien, Tres piedras, M'diq, Cabo Negro, Oued Maleh, Tanger; Louah et al. 1995 ; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Kénitra; Ramdani 1997 , AP , Témara, Skhirat, Casablanca; Handaq 1998 , AP , El Jadida, HA , Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Skhirat, Dayet Aïn Chems, Maâmora, forest of Hilton; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Mansoniini Coquillettidia Dyar, 1905 Coquillettidia ( Coquillettidia ) buxtoni (Edwards, 1923) Bailly-Choumara 1965a , AP , Merja Bokka; Bailly-Choumara 1970 , AP , Merja Bokka; Bailly-Choumara 1972a , AP , Merja Sheishat (Larache); Bailly-Choumara 1973b , AP , Merja Bokka, Larache; Himmi et al. 1995 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Coquillettidia ( Coquillettidia ) richiardii (Ficalbi, 1889) Bailly-Choumara 1965a , AP , Merja Bokka; Bailly-Choumara 1967a , MA , Zaouia Cheikh; Bailly-Choumara 1970 , AP , Merja Bokka; Bailly-Choumara 1972a , AP , Merja Sheishat (Larache); Bailly-Choumara 1973b , AP , Merja Bokka; Trari 1991 , AP , Sidi Yahia du Gharb, Aïn Chouk, Merja Bargha; Himmi et al. 1995 ; Dakki 1997 ; Moussalim 1997 , AP , Fouarate sur les bordures de la Merja; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Orthopodomyiini Orthopodomyia Theobald, 1904 Orthopodomyia pulcripalpis (Rondani, 1872) Bailly-Choumara 1965b , AP , Maâmora; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Uranotaeniini Uranotaenia Lynch Arribálzaga, 1891 Uranotaenia ( Pseudoficalbia ) unguiculata Edwards, 1913 Séguy 1930a ; Gaud 1953a , HA , Marrakech; Senevet and Andarelli 1959a, EM , Oujda, Berguent, Guercif, AP , Sidi Allal Tazi, Bouznika, Casablanca, Béni Moussa, MA , Oulad Massine, Meknès, Moulay Yacoub, Béni Mellal, AA , Ksar Mzizel, Aït Melloul; Bailly-Choumara 1965c , EM , Aïn Aït Delouine, Talmesdourt, Rich Tamlougout; Bailly-Choumara 1966 , AA , Aït Ouabelli; Bailly-Choumara 1967a , MA , Pont Tarmilate; Bailly-Choumara 1967b , EM , environs de Taourirt, Madagh, Merja Boubker; Bailly-Choumara 1967c , Rif , route Bab Taza-Bab Berred, piste Ketama-Jebel Tidighine; Bailly-Choumara 1968a , AP , Oued Loukous, Merja Bokka, Kénitra, Oued Bou-Regreg; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Oued Sebou; El Bermaki 1993, AP , El Oulfa (Casablanca); Himmi et al. 1995 ; Dakki 1997 ; Ramdani 1997 , AP , Tamaris; Handaq 1998 , AP , Sidi Bennour, HA , Zima Chemaîa (Bengrir); Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Uranotaenia ( Uranotaenia ) balfouri Theobald, 1904 Bailly-Choumara 1968a , AP , Merja Bokka, Oued Loukous, Sidi Yahia, Oued Sebou (Kénitra), Oued Bou-Regreg; Himmi et al. 1995 ; Dakki 1997 ; Moussalim 1997 , AP , Sidi Allal Tazi, Fouarate, Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; El Ouali Lalami et al. 2010a , MA , Fès Boulmane; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès DIXIDAE K. Kettani, R. Wagner Number of species: 12 . Expected: 15 Faunistic knowledge of the family in Morocco: moderate Dixa Meigen, 1818 Dixa caudatula Séguy, 1928 Séguy 1930a , HA , Arround, Skoutana (2400 m); Vaillant 1959 ; Dakki 1997 ; Mouna 1998 Dixa dilatata Strobl, 1900 = Dixa riparia Vaillant, in Vaillant 1959 : 180 Vaillant 1959 , HA , Source de M'Goum (2500 m), Gorges d'Imi-N'Ifri (1050 m); Vaillant 1965 ; Dakki 1997 Dixa maculata Meigen, 1818 Séguy 1930a ; Dakki 1997 ; Mouna 1998 Dixa mera Séguy, 1930 Séguy 1930a , MA , forest of Timelilt (1900 m); Vaillant 1959 ; Dakki 1997 ; Mouna 1998 Dixa nebulosa Meigen, 1830 Séguy 1930a , HA ; Dakki 1997 ; Mouna 1998 Dixa perexilis Séguy, 1928 Séguy 1930a , HA , riverside of Oued Imminen (Tachdirt, 2400 m); Vaillant 1959 ; Dakki 1997 ; Mouna 1998 Dixa puberula Loew, 1849 Vaillant 1959 , HA , headwaters of Asif M'Goum (2500 m); Vaillant 1965 ; Dakki 1997 ; Mouna 1998 Dixa submaculata Edwards, 1920 Séguy 1930a , MA , Sidi Yahia, Talzent (1800 m); Dakki 1997 ; Mouna 1998 Dixella Dyar & Shannon, 1924 Dixella aestivalis (Meigen, 1818) Séguy 1930a , AP , Merja Boughaba; Vaillant 1965 ; Ramdani 1981 ; Ramdani and Tourenq 1982 ; Mouna 1998 Dixella attica (Pandazis, 1933) = Dixella numidica (Sicart, 1955) Ebejer et al. 2019 , Rif , Cabo Negro (indoors: 10 m) Dixella martinii (Peus, 1934) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m) Dixella serotina (Meigen, 1818) = Dixa serotina Wied, in Mouna 1998 : 85 Séguy 1930a , AP , Casablanca (between Kénitra and Oued Beth); Dakki 1997 ; Mouna 1998 CHAOBORIDAE K. Kettani, R. Wagner Number of species: 2 . Expected: 4 Faunistic knowledge of the family in Morocco: poor Chaoborinae Chaoborus Lichtenstein, 1800 Chaoborus crystallinus (De Geer, 1776) Dakki 1997 : 60 Mochlonyx Loew, 1844 Mochlonyx culiciformis (De Geer, 1776) Dakki 1997 : 60 Chaoborinae Chaoborus Lichtenstein, 1800 Chaoborus crystallinus (De Geer, 1776) Dakki 1997 : 60 Mochlonyx Loew, 1844 Mochlonyx culiciformis (De Geer, 1776) Dakki 1997 : 60 CULICIDAE K. Kettani, B. Trari, O. Himmi, M. Dakki Number of species: 43 . Expected: 60 Faunistic knowledge of the family in Morocco: good Anophelinae Anopheles Meigen, 1818 Anopheles ( Anopheles ) algeriensis Theobald, 1903 Viallate 1922, AP , Kénitra; Séguy 1930a ; Bonjean 1947 , EM , MA ; Gaud 1957a , HA , north of High Atlas; Guy 1959a , MA , Béni Mellal, HA ; Guy 1959b , HA ; Guy 1959a , MA , Béni Mellal; Bailly-Choumara 1967a , MA , Ghorm El Alem; Benmansour et al. 1972 , MA , Barrage Bin El Ouidane; Bailly-Choumara 1973b , AP , Sidi Yahia du Gharb; Metge 1986 , AP , Casablanca; Trari and Himmi 1987 , AP ; Himmi et al. 1995 ; Louah 1995 , Rif , Tahaddart, Schroda; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Casablanca; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 , Rif , Chefchaouen; El Ouali Lalami et al. 2010 a,b, MA , Fès, Boulmane; El Ouali Lalami 2012 , MA , Fès; Trari 2017 , Rif , Chefchaouen, AA , Tiznit; Trari and Dakki 2017a , Rif , Chefchaouen, AA , Tiznit; Trari and Dakki 2017b , AA , Tiznit; Trari et al. 2017 Anopheles ( Anopheles ) claviger (Meigen, 1804) Vialatte 1923 , AP , Kénitra; Séguy 1930a , AP , Rabat; Langeron 1938 , HA , Tounfit, Massou, Anefgou, Tirghist, Tighermine, Louggouargh; Callot 1940 , HA , Anefgou, Tirghist; Bonjean 1947 , EM , MA ; Gaud 1947c , MA , Sefrou, Meknès; Gaud 1948 , AP , Rabat, MA , El Hajeb, AA , Errachidia, Tadla; Gaud et al. 1948 , AP , Skhirat; Guy 1963 , Rif , Taounate, MA , Meknès, Ifrane; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, piste Ksiba-Naour, piste Naour-Arbala, Zaouia Cheikh, Oued Sarif (environs El Ksiba), Dayet Aoua (environs Ifrane), Boulemane; Bailly-Choumara 1967b , EM , 8 km N Itzer; Bailly-Choumara 1967c , Rif , piste Ketama-Mt Tiguidin; Guy 1967 , HA , Marrakech, AA , Tafilalt; Guy and Holstein 1968 , SA ; Metge 1986 , AP , Casablanca; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris drains, Tamaris merja; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 , Rif , Bab Berred, Tanakoub; Faraj et al. 2008b , Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Boulmane; Larhbali et al. 2010 , MA , Zhiliga, Boukachmir, Aït Ichou; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) labranchiae Falleroni, 1926 d'Anfreville 1916 , AP , Salé; Delanoe 1917, AP , El Jadida; Viallate 1922, AP , Rabat, Boulhaut, Bouznika, Gharb, MA , Sidi Kacem, Tiflet, Fès, Taza; Charrier 1924 , Rif , Tanger; Séguy 1930a , MA ; Roubaud 1935, AP , Rabat; Sicault et al. 1935 , AP , Merja Ras Eddaoura, Merja Zerga, dayas entre Sebou et Maâmora, Dar bel Hamri (entre barrage El Kansera et Sidi Slimane), à proximité de Merja Zerga, Douar Anabsa; Langeron 1938 , AA ; Callot 1940 , AA ; Ristorcelli 1946a , b , HA , Oued Tensift, Oued Issil; Bonjean 1947 , AP , Gharb; Gaud 1947c , MA , Oulmès, HA , Marrakech; Gaud et al. 1948 , AP , Merja Ras Eddaoura; Gaud et al. 1949 , AP , Salé, SA , Foum Zguid, Tagounit; Gaud 1953a , AP , from Tanger to El Jadida, MA , Tissa, Timhadit, Bekrit, Meknès, Ifrane (1700 m), HA , Sidi Aissa, Tizi-n'Tichka; Guy 1958 , HA , Oued N'fis; Sacca and Guy 1960b, Rif , Tétouan, AP , Skhirat, Sidi Yahia, Sidi Bettache, Mazagan, Braila (près Sidi Allal Tazi), Aït Lahsen, MA , Meknès, HA , Marrakech; Guy 1962 , Rif , Taounate, AP , Gharb, HA , Marrakech (ville et banlieu); Guy 1963 , Rif , Tanger, Tétouan, EM , Berkane, Debdou, Oujda, AP , Kénitra, Souk Larba, Settat, Chamaîa, Safi, Casablanca, Rabat, Essaouira, MA , Meknès, Fès, Azrou, Oued Zem, Béni Mellal, HA , Kelaâ of Sraghna; Bailly-Choumara 1967a , MA , piste Tafechna-Znan Imes, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Aguelmane Azigza, piste Aguelmane Azigza-Aïn Leuh, maison forestière Ouiouane, route Khénifra-Tafechna, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Bords Moulouya, piste Itzer-Boumia, RN P.33 Boumia-El Kbab, Ouaoumana, piste Ksiba-Naour, piste Naour-Arbala, piste Arbala-El Kbab, Kafensour, Oued Sarif (environs El Ksiba), Dayet Aoua, Ifrane, Imouzer Marmoucha; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Douar Sherba, Douar Aïn Shebbak, Douar Aïn Zabia, Douar Madarh, Douar Sidi Hashas, Mechraa safsaf; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, piste Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Al Hoceima Club Med, Béni Bouayache, Targuist et environs; Guy 1967 , HA , Marrakech, AA , Tafilalt; Bailly-Choumara 1968b , AP , Larache; Guy and Holstein 1968 , AA , Ouarzazate; Bailly-Choumara 1970 , Rif , Tétouan, EM , Berkane, AP , Larache, Sidi Yahia du Gharb, HA , Marrakech; Benmansour et al. 1972 , AA , Agadir; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1972b , AP , Merja Sheishat; Bailly-Choumara 1973a , AP , Merja de l'Oued Smir; Bailly-Choumara 1973b , Rif , Tétouan, EM , Berkane, AP , Merja Sheishat, HA , Souk des Oudaias; de Zulueta et al. 1983 , Rif ; Ibn Jilali 1984, AP , Maâmora; Metge 1986 , AP , Entre Oulad Dlim et Al Ara'ra; Trari and Himmi 1987 , AP , Gharb; Himmi 1991 , AP , Kénitra, Maâmora; Metge 1991 , AP , Sidi Bettache; Trari 1991 , Rif , Chefchaouen, Tanger, Taounate, EM , Oujda, AP , Sidi Amira, Sidi Boughaba, Merja Zerga, Sidi Allal Tazi, Aïn Chouk, Oued Loukous, Merja Oulad Skhar, MA , Khémissat, Béni Mellal, Khouribga, HA , El Kelaâ des Sraghrna, AA , Ouarzazate; El Bermaki 1993, AP , entre l'aeroport Anfa et l'aménagement d'El Oulfa, Sidi Maârouf, El Oulfa; Chlaida and Bouzidi 1995 , AP , Aïn Blal, Oued Sidi Messoud; Louah 1995 , Rif , Tres piedras, Marina Smir, Bouzerlal, Oued Maleh, Zekri, Lajour, Azla, Tahaddart, Skroda, Stehat, Moulay Bouchta, Kebbache, Talembote, Loubart, Chefchaouen, Oued Maggou, Bab Berred, Sidi Kankoch, Oued kbir, Oued Jebel Lehbib; Louah et al. 1995 ; Chlaida 1997 , AP , Aïn Blal, Oued Sidi Messoud, Douar Chlihat, Barrage Al Massira; Moussalim 1997 , AP , Sidi Allal Tazi, Maâmora; Ramdani 1997 , AP , Tamaris, Skhirat; Faraj et al. 1997 , AP , Ouled Moussa; Handaq 1998 , HA , Oukaimeden, Amizmiz, Tiguenziouine; Himmi et al. 1998 , AP , Dayat d'El Menzeh (north-east of Kénitra), Sidi Boughaba; Alaoui Slimani et al. 1999 , AP , Bou-Regreg Salé, Sidi Bouguettaya, Quartier industriel Takaddoum, Marjane; Alaoui Slimani 2002 , AP , Rabat-Salé; Trari et al. 2002 ; Faraj et al. 2003 , Rif , Azib Bouflou, Azib Jrou, Imzouren, Amezzaourou, Tizi-Tamalout, MA , Aït Abdelsalam, Aït Lamfadel; Faraj et al. 2004 , Rif , Azib Jrou, Tanghaya Akarkar, Amezzaourou, Ouled Nsar, AP , Fedalate, MA , Talaa Chougaga, Aïn Smen (Fès), Aït Lamfadel; Trari et al. 2004a , Rif , Larache; Trari et al. 2004b , Rif , Ketama, Gzenaya, Taounate, Bouzaghlal, Oued Laou, Smir (Merja), Béni Hassane, Azib Jrou, Azib Bouflou, AP , Laaouamra, Aarabat Sidi Abdelaziz, Moukaouama, Dar Belamri, Laksibia, Mgadid, Beggara, Rabat (Chellah), MA , Adouz, Rommani (Khémisset), Aït Abderrahmane, Aït Ishaq, Oulad Messoud, Ouled Fennane, El karma, Oulad Zguida, Oulad Abbou; Aouinty et al. 2006 , AP , Mohammedia; Himmi 2007 , Rif , Bab Berred, Bab Taza, Tanakoub, AP , Skhirate, Maâmora, Oulja, Bouknadel, Sidi Azzouz, Tamaris, MA , Khémisset; Faraj et al. 2008a , AP , Laouamra, Boucharen, MA , Béni Khlef, Talaa Chougaga; Faraj et al. 2008b , Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010b , MA , Fès; Faraj et al. 2010 , AP , Begara, Boucharen, Ben Slimane, Skhirat, Rabat, Sehoul, MA , Sidi Allal Msader, Aïn Aghbal, Aïn Elouali, Sidi Kacem; Larhbali et al. 2010 , MA , Aït Haddou Said; Adlaoui et al. 2011 , AP , Larache; Larhbali et al. 2011 , MA , Oulmès, Aït Yadin, Sfassif, Mâaziz, Rommani, Laghoualem, Ezzhiliga, Sidi Allal Bahraoui, Boukachmine, Aït Malek, Sidi Boukhalkhal, Bni Ounzar, Ganzra, Aït Siberne, Sidi Allal Msader, El Ghandour; El Ouali Lalami 2012 , MA , Pont Diamant vert, Sidi Harazem, Oued El Himmer, Moulay Yakoub, Oued Sebou, Aïn Kansara, Oued Aïn Chkef, Sefrou, Boulemane; Laboudi et al. 2012 , AP , Larache; Hadji et al. 2013 , AP , Sidi Slimane; El Joubari et al. 2014 , Rif , Smir lagoon; Laboudi et al. 2014 , Rif , Tétouan, Tanger, Chefchaouen, Al Hoceima, AP , Larache, Salé, MA , Taza, Khémissat; El Joubari et al. 2015a , Rif , Smir lagoon; El Joubari et al. 2015b , Rif , Smir lagoon; Marc et al. 2016 , AP , Kénitra; Trari 2017 , Rif , Chefchaouen, Tétouan, AP , Larache, Rabat, Settat, EM , Oujda, MA , Khémisset, Meknès, Khouribga, HA , Marrakech, AA , Tiznit, Ouarzazate; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, AP , Larache, Rabat, Settat, EM , Oujda, MA , Khémisset, Meknès, Khouribga, HA , Marrakech, AA , Tiznit, Ouarzazate; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) marteri Senevet & Prunnelle, 1927 Gaud 1945b , MA , El Hajeb, Khénifra, HA , Tizi-n'test, Tillougite; Gaud et al. 1949 , HA , Tizi-n'test; Gaud 1953b , HA , Tizi-n'test; Bailly-Choumara 1967b , EM , Grotte du Zegzel; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Taza-Asifane, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Boured; Benmansour et al. 1972 , MA , Taza; Trari 1991 , Rif , Taounate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; Trari 2017 , Rif , Chefchaouen, AP , Settat; Trari and Dakki 2017a , Rif , Chefchaouen, AP , Settat; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) ziemanni Grünberg, 1902 Senevet 1935 , MA ; Gaud et al. 1949 , HA , plain of south and north of the Occidental Atlas; Gaud et al. 1950 , HA , plain of south and north of the Occidental Atlas; Guy 1958 , HA , Marrakech; Guy et al. 1958, HA , Oued N'fis; Guy 1967 , EM , Oujda, AP , Rabat, HA , Marrakech, AA , Tafilelt; Bailly-Choumara 1970 , HA , Marrakech; Benmansour et al. 1972 , MA , Taza, HA , Haouz, Tadla Azilal; Bailly-Choumara 1973b , HA , Souk des Oudaias (Souk Tnine des Oudaias, 470 m); Trari 1991 , MA , Tissa; Moussalim 1997 , AP , 7.5 km de Sidi Allal Tazi; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 ; Trari 2017 , Rif , Tétouan, AA , Tiznit; Trari and Dakki 2017a , Rif , Tétouan, AA , Tiznit; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) cinereus Theobald, 1901 Viallate 1922, MA , Sefrou, Sidi Lamine, HA , Mtougui; Sicault et al. 1935 , AP , Souk Larba of Gharb; Senevet 1935 , AP , Souk Larba of Zemmour; Langeron 1938 , HA , Anefgou, Tirghist, Valley of Sidi Yahia Ouyoussef, Tighermine, Louggouargh, Massou; Callot 1940 , HA , Anefgou, Tirghist; Gaud 1945a , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud and Duthu 1945, HA , Marrakech; Viamonte and Ramirez 1945 , Rif ; Viamonte and Ramirez 1946 , Rif ; Ristorcelli 1946a , HA , Oued Tensift, Oued Issil; Ristorcelli 1946b , HA , Oued Tensift, Oued Issil; Gaud 1945b , AA , Tansikht; Gaud et al. 1949 , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud et al. 1950 , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud 1953a ; Gaud 1958, HA , Marrakech; Guy 1962 , Rif , Taounate, HA , Marrakech; Guy 1963 , MA , Midelt, AA , Hamada of Draa; Bailly-Choumara 1965, EM , Aman d'Aït Oussa, Tiglit, El Megrinat, Taskala, Aïn Aït delouine, Oued mesdourt, Talmesdourt, Assa, AA , Aït Melloul, Oued Teima, Issen, Taroudant, Talaint, Tiznit, Oued Assaka, Anezi, Pont de la route Agadir-Tiznit, valley of low Draa, Tafraoute, Tacharicht, Bou Izakarn, Jemâa N'tirhirte, Aït Erkha,Tazert, Barrage Taourirt, AA , Goulmima, SA , Aouinet Torkoz, Tirh Mzoun; Bailly-Choumara 1966 , AA , piste Foum Zguid-Lac Iriqui, Agadir-Tissint, Akka-Iguiren, Tirhem, Taoujgelt, Souk El Khémis Dades, Akka, Aït Ouabelli, Foum-el-Hassan, Tarhjicht, Aït melloul, Taliouine, Tazenakht (Rocade of Draa); Bailly-Choumara 1967a , MA , route Azrou-Khénifra, Jnane Imasse, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Sources Oum-er-Rbia, Itzer, piste Itzer-Boumia, Ouaoumana, piste Ghorm El Alem-El Ksiba, piste Naour-Arbala, Zaouia Cheich, kafensour, Ifrane, Imouzzer Marmoucha; Bailly-Choumara 1967b , EM , 7 km N Itzer, 8 km N Itzer, route Itzer-Midelt, Aïn Srouna, Gouttitir, Cascade Oued Za, Grotte du Zegzel, Douar Aïn Soultane, Mechraa Safsaf, Oujda, Berguent (valley of Moulouya), Figuig; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Assifane, Anasar, piste Bab berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, route Al Hoceima-Arba Taourirt, Arba Taourirt, Targuist et environs, route Targuist Al Hoceima, Jebha, Oued Ouergha, route Aknoul-Al Hoceima; Guy 1967 , AP , Rabat, EM , Oujda, HA , Marrakech, AA , Tafilalet; Bailly-Choumara 1970 , HA , Marrakech; Bailly-Choumara 1973b , HA , Souk des Oudaias; Trari 1991 , Rif , Al Hoceima, Chefchaouen, Taounate, EM , Nador, Oujda, Figuig, AP , Larache, Settat, Ben Slimane, MA , Khénifra, Taza, Khouribga, HA , Kelaâ of Sraghna, AA , Ouarzazate, Goulmima; Bouallam 1992, HA , Oued N'fis; Louah 1995 , Rif , Marina Smir, Bouzerlal, Tahaddart, Stehat, Moulay Bouchta, Kebbache, Loubart, 0ued Maggou; Louah et al. 1995 ; Bouallam et al. 1997b , HA , Marrakech; Handaq 1998 , HA , Amizmiz, Tiguenziouine; Bouallam 2001, HA , Oued N'fis; Trari et al. 2002 ; Trari et al. 2004b , Rif , Ketama, Azib Jrou, Sidi Mokhfi, MA , Taghzirt, Aghbala, Aït Shak, Smaala, Mlalih, Ouled Fennane, Béni Khlef, Tachrafte; Faraj et al. 2007 , Rif , Assoul, Mizgane; Himmi 2007 , Rif , Bab Berred, Bab Taza, Stehat, Tanakoub; Faraj et al. 2008, Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Fès; El Ouali Lalami et al. 2010b , MA , Oued El Himmer; Larhbali et al. 2010 , MA , Roumani Aïn Sbite, Jamâa M. B., Ghoualem, Zhiliga, Oulmès, Tarmilate, Boukachmir, Mrirte, Aït Ichou, Mâaziz, Tiddas, Bni Ounzar, Ganzra; El Ouali Lalami 2012 , MA , Oued El Himmer; Trari 2017 , Rif , Chefchaouen, Tétouan, MA , Khouribga, AA , Tiznit; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, MA , Khouribga, AA , Tiznit; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Anopheles ( Cellia ) dthali Patton, 1905 Saccà 1960 , AA , Aoufous, Meski, Erfoud, Agdz, Zogora, Tagounit, Tamsrruth; Guy 1961 , HA , Sud de Zagora (en bordure Hamada du Draa); Guy 1963 , AA , Zagora and south of the High Atlas (at edge of Hamada of Draa), Oued Ziz; Bailly-Choumara 1965c , AA , Tiznit, EM , Aïn Aït Delouine, Aouinet Torkoz, Rich Tamlougout (eastern borders of Jebel Bani); Bailly-Choumara 1966 , AA , piste Foum Zguid au Lac Iriqui, Agadir-Tissint, Akka-Iguiren, Souk El Khémis Dades, Akka et Environs, Aït Ouabelli, Tarhjicht, piste Tazenakhte à Foum Zguid (Rocade du Draa); Bailly-Choumara 1967b , EM , valley of Moulouya; Guy 1967 , HA , Marrakech, AA , Tafilalet; Guy and Holstein 1968 , EM , N Outat El Haj (valley of Moulouya), Gouttitir (environs de Taourirt); Bailly-Choumara 1970 , AA , Foum Zquid; Bailly-Choumara 1973b , AA , Foum Zquid; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari et al. 2004b ; Faraj et al. 2007 , Rif , Assoul, Mizgane (SE of Bab Berred); Faraj et al. 2008, Rif , Assoul, Mizgane (SE Bab Berred); Himmi 2007 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) multicolor Cambouliu, 1902 Messerlin and Treillard 1938 , HA , Marrakech; Viamonte and Ramirez 1945 , Rif ; Guy 1963 , SA ; Bailly-Choumara 1965c , EM , Aït Oussa, Aman d'Aït Oussa, Aïn Aït Delouine, Aouinet Torkoz, Rich tamlougout (Confins orientaux du Jebel Bani), AA , Tafnidilt, Guelta Zerga, Aïn Temda (valley of low Draa), Tirhmert (Goulmima), SA , Vallée et embouchure de l'Oued Assaka, Tantan ville, Tirh Mzoun; Bailly-Choumara 1966 , AA , Rocade of Draa; Bailly-Choumara 1967b , EM , 40 km N de Outat El Haj, Gouttitir, Cascade Oued Za, Douar Aïn Shebbak (valley of Moulouya); Guy 1967 , HA , Marrakech, AA , Tafilelt; Guy and Holstein 1968 , HA , south of Atlas, AP , plain located between Marrakech and the Atlantic from Tanger along the length of the Mediterranean; Bailly-Choumara 1970 , AA , Foum Zquid; Bailly-Choumara 1973b , AA , Foum Zquid; Metge 1986 , AP , Casablanca; Trari 1991 , Rif , Al Hoceima, Taounate, Oued Maleh, Bab Berred, AP , Tamaris Merja, AA , Ouarzazate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari et al. 2004b ; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) sergentii (Theobald, 1907) Séguy 1930a ; Messerlin and Treillard 1938 , HA , Tamelelt; Langeron 1938 , Rif , Targhist; Callot 1940 , AA , Taghjicht; Gaud 1947c , AA , Wadi Draa; Gaud et al. 1949 , Rif , Zoumi, HA , Zaouia Sidi Hamza, Tizi-n'test (1700 m), Tillougit (1800 m); Gaud et al. 1950 , Rif , Zoumi, MA ; Guy et al. 1958, HA , Oued N'fis; Guy 1961 , Rif , AP , south of Casablanca, AA , Sud de Zagora; Guy 1962 , Rif , AP , south of Casablanca, HA , Marrakech; Guy 1963 , Rif , Tanger, EM , Berkane, AP , south of Casablanca, MA , Béni Mellal, HA , Oued Tensift, Oued Ziz, Marrakech, Chichaoua, AA , Oued Draâ, Oued Dades, Goulmima, sud de Zagora, SA , Foum Zguid; Bailly-Choumara 1965c , EM , iglit, Aman d'Aït Oussa, Oued Isker, El Megrinat, Aïn Aït Delouine, Oued Mesdourt, almesdourt, Aouinet Torkoz, Bouanama, Rich Tamlougout, Assa, AA , Tiznit, Tafraoute, Oued Izi, Bou Izakarn, Abeino (region of Goulmima), SA , Aouzeroual, Tirhmert, Tacharicht, Jebel Bani, Tantan; Bailly-Choumara 1966 , AA , Rocade of Draa, Taliouine; Bailly-Choumara 1967a , Rif , AP , south of Casablanca, MA , Itzer, Ghorm El Alem, Zaouia Cheikh, Kafensour, HA , north of High Atlas; Bailly-Choumara 1967b , EM , Aïn Srouna, Grotte du Zegzel, Douar Mardarh, Douar Aïn Soultane, Merja Boubker, Selouane, Driouch (valley of Moulouya); Bailly-Choumara 1967c , Rif , route nationale Jebha, Targuist-Béni Boufrah, Al Hoceima, Béni Bouayache, Marchica, Had El Rouadi, Pont du Srah; Guy 1967 , HA , Marrakech, AA , Tafilelt; Guy and Holstein 1968 , AP , Casablanca; Bailly-Choumara 1970 , EM , Berkane, HA , Marrakech; Bailly-Choumara 1973b , EM , Berkane, HA , Marrakech; Metge 1986 , AP , Casablanca; Trari 1991 , Rif , Al Hoceima, Taounate, AP , Larache, AA , Ouarzazate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Faraj et al. 2003 , Rif , Al Hoceima, Chefchaouen, Taounate, HA , Khouribga; Trari et al. 2004b , Rif , Ketama, Sidi Mokhfi; Faraj et al. 2007 , Rif , Assoul, Mizgane; Faraj et al. 2008, Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Fès; Larhbaliet al. 2010 , MA , Oulmès, Ganzra; Trari 2017 , Rif , Chefchaouen, Tétouan, EM , Oujda, MA , Khouribga; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, EM , Oujda, MA , Khouribga; Trari and Dakki 2017b ; Trari et al. 2017 ; Benabdelkrim Filali et al. 2018 ; Mouatassem et al. 2019, MA , Fès Culicinae Aedini Aedes Meigen, 1818 Aedes ( Acartomyia ) mariae (Sergent & Sergent, 1903) Séguy 1930b ; Messerlin 1938 , AP , Rabat; Séguy 1930a , Rif , littoral méditerranéen; Bailly-Choumara 1967b , Rif , Al Hoceima; Bailly-Choumara 1967c , Rif , Al Hoceima; Bailly-Choumara 1968a , Rif , Al Hoceima, AP , Larache, Sidi Yahia, Sidi Allal Tazi, Rabat, HA , Marrakech, AA , Tiznit; Himmi et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Aedimorphus ) vexans (Meigen, 1830) Gaud 1947c , AP , Sidi Allal Tazi, MA , Khémisset; Metge 1986 , AP , Littoral Casablanca; Himmi et al. 1995 ; Handaq 1998 , AP , Gharbia; Dakki 1997 ; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Dahliana ) echinus (Edwards, 1920) Séguy 1924 ; Séguy 1930a ; Gaud 1953a , AP , Rabat, Sidi Yahia, MA , Moulay Bouazza, Taza, Fès, Meknès, Ifrane; Bailly-Choumara 1965b , AP , Maâmora; Bailly-Choumara 1967c , Rif , piste Bab Taza-Talassemtane; Himmi et al. 1995 ; Trari et al. 2002 ; Nikookar et al. 2010 ; El Joubari et al. 2014 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Dahliana ) geniculatus (Olivier, 1791) Séguy 1924 , MA ; Séguy 1930a ; Metge and El Alaoui 1987 , AP , Subéraies de Béni Abid-Benslimane (Casablanca); Metge and Belakoul 1989 , AP , Sidi Bettache; Himmi et al. 1995 ; El Ouali Lalami et al. 2010a , MA , Fès Boulmane; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) berlandi (Séguy, 1921) Séguy 1930a , AP , Rabat; Gaud 1953a , MA , Fès; Bailly-Choumara 1967a , MA , Jnane Imasse, piste Tafechna-Taoujgelt; Bailly-Choumara 1967c , Rif , piste Bab Taza-Béni Ahmed; Belakoul 1985 , AP , Benslimane, Sidi Bettache; Metge and El Alaoui 1987 , AP , Casablanca; Metge and Belakoul 1989 , AP , Benslimane, Sidi Bettache; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) caspius (Pallas, 1771) Séguy 1930b ; Viamonte et Ramirez 1946, AP , Larache; Gaud 1952 , AP , Rabat, Casablanca; Gaud 1953a , AP , Rabat, Casablanca; Senevet and Andarelli 1954 , EM , Embouchure de la Moulouya, Figuig, AP , Jorf Lasfar, Mohammedia, Rabat, MA , Meknès, Fès, Taza, HA , Marrakech, Midelt, AA , Tiznit; Bailly-Choumara 1966 , AA , environs de Tiznit; Bailly-Choumara 1967a , EM , Bords Moulouya (près Itzer), Cherarba; Bailly-Choumara 1967b , EM , Cherarba, Aïn Shebbak, Saidia, Berguent; Bailly-Choumara 1967c , Rif , piste Al Hoceima-Arba Taourirt; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , Rif , Merja de l'Oued Smir; El Kaim 1972 , AP , Bou-Regreg; Rioux et al. 1975 , AP , Rabat-Salé; Metge 1986 , AP , Littoral casablancais; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba, Merja Zerga, Oued Loukous; Himmi et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Kénitra; Ramdani 1997 , AP , Tamaris Merja; Handaq 1998 , AP , Zemamra, B. Iffou (Entre El Oualidia et Youssoufia), MA , Béni Mellal, HA , Marrakech, Zaouiet Ben Sassi, Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; Alaoui Slimani 2002 , AP , Rabat; Aouinty et al. 2006 , AP , Mohammedia; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) coluzzii Rioux, Guilvard & Pasteur, 1998 et Aedes ( Ochlerotatus ) detritus (Haliday, 1833) [Complexe detritus] Charrier 1924 , Rif , Tanger; Séguy 1930a ; Gaud 1953a , EM , Saidia, AP , Kénitra, Rabat, Bouznika, El jadida, Oualidia, HA , Marrakech, AA , Agadir, Tafnidilt; Bailly-Choumara 1965c , EM , Aïn Aït delouine, SA , Tirhmert; Bailly-Choumara 1970 , Rif , Tétouan; Knight 1971 , AP , Kénitra; El Kaim 1972 , AP , Bou-Regreg; Bailly-Choumara 1973b , Rif , Merja de l'Oued Smir; Rioux et al. 1975 , AP , Rabat; Pasteur et al. 1978 , AP , Bou-Regreg; Metge 1986 , AP , Littoral casablancais; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba, Merja Zerga, Oued Loukous; Louah 1995 , Rif , Tres piedras, Cabo Negro, Lajour, Azla, Tahaddart; Himmi et al. 1995 ; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Rabat; Ramdani 1997 , AP , Tamaris Merja; Handaq 1998 , AP , Essaouira, Zima-Chemaîa; Himmi et al. 1998 , AP , Sidi Boughaba; Himmi 2007 , AP , Sidi Boughaba; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) pulchritarsis (Rondani, 1872) Gaud 1953a , AP , Benslimane, Sidi Yahia, Rabat, MA , Oued Zem, Khénifra, Fès; Metge and El Alaoui 1987 , AP , Benslimane; Himmi et al. 1995 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Rusticoidus ) rusticus (Rossi, 1790) Viamonte and Ramirez 1946 , Rif , Tétouan, AP , Larache; Gaud 1953a , MA , Taza; Himmi et al. 1995 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Stegomyia ) aegypti (Linnaeus in Hasselquist, 1762) d'Anfreville 1916 , AP , Salé; Vialatte 1923 , AP , Rabat, Casablanca, HA , Marrakech; Charrier 1924 , Rif , Tanger; Gaud 1953a , AP , Salé, HA , Marrakech; Himmi et al. 1995 ; Dakki 1997 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Stegomyia ) albopictus (Skuse, 1895) Bennouna et al. 2016 , AP , Agdal (Rabat); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Faraj et al. 2018 ; Amraoui et al. 2019 Culicini Culex Linnaeus, 1758 Culex ( Barraudius ) modestus Ficalbi, 1889 Séguy 1930a ; Bailly-Choumara 1968a , AP , Larache; Trari 1991 , AP , Gharb; Himmi et al. 1995 ; Dakki 1997 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, AP , Maâmora; Hadji et al. 2013 , AP , Sidi yahia du Gharb, Kcebia, Sidi Hagouch (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) brumpti Galliard, 1931 Bailly-Choumara 1968a , AP , Merja Bokka, Larache, HA , Marrakech; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi et al. 1995 ; Dakki 1997 ; Himmi 2007 ; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) laticintus Edwards, 1913 Charrier 1924 , Rif , Tanger; Callot 1940 , AA , Goulmima (mares); Gaud 1953a , HA , Marrakech, AA , Agadir; Gaud 1957a , EM , Nador; Bailly-Choumara 1965c , EM , Oued Isker, Aïn Aït delouine, Talmesdourt, AA , Ouled Teima, ounaamane, Bou Izakarn, Agunil Khnufa, Akka-guiren; Bailly-Choumara 1966 , AA , Akka-Iguiren, Tirherm, Taoujgelt, Aït Ouabelli, Anamere-Smougue, Aït melloul, Tiznit (Rocade de Draa); Bailly-Choumara 1967b , Rif , Béni Bouayache, Targuist et environs, EM , Grotte du Zegzel; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 , AP , Skhirat; Faraj et al. 2008b , AP , Louamra; Hadji et al. 2013 , AP , Sidi Hagouch (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) mimeticus Noè, 1899 Séguy 1930a ; Viamonte and Ramirez 1946 , Rif , Béni Ider, Fnideq, Khemis Anjra, Ketama, Oued Amsa, Oued Krikra, Oued Martil, Oued Laou; Gaud 1953a , Rif , Ouezzane, Ghafsai, EM , Berkane, Martinpray (près Berkane), El Aïoun Sidi Mellouk, AP , Tamri, MA , Meknès, Fès, Ifrane, Taza, Béni Mellal, HA , Midelt, Marrakech, Azilal, AA , Tinghir, Tichka; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , AA , Oued Noun, Anezi, Tafraoute; Bailly-Choumara 1966 , AA , Agadir; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Taoujgelt, Source Oumerrbia, Ghorm El Alem, piste Ksiba-Naour, piste Naour-Arbala, Zaouia Cheikh, Oued Sarif; Bailly-Choumara 1967b , EM , 7 km N d'Itzer, 8 km d'Itzer; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Taza-Asifane, piste Bab Berred-Tamorote, Ketama, piste Ketama-Jebel Tidighine, route Ketama-Targuist, route nationale Jebha, Al Hoceima, Béni Bouayache, Marchica, Targuist et environs, Jebha, Boured; Trari 1991 , AP , Sidi Yahia du Gharb; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, Tres piedras, Marina Smir, Bouzaghlal, M'diq, Oued Maleh, Azla, Tahaddart, Moulay Bouchta, Schroda, Kebbache, Talambote, Oued Maggou; Louah et al. 1995 ; Chlaida 1997 , AP , Oued Sidi Messoud, Aïn Blal, Douar Chlihat, Barrage Al Massira; Chlaida and Bouzidi 1995 , AP , Barrage El Massira; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris Merja; Handaq 1998 , HA , Oukaimeden, Amizmiz, Tiguenziouine (près Oued N'fis); Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, AP , Bouknadel, Douar jdid (Skhirat); El Ouali Lalami et al. 2010a , MA , Fès; Larhbali et al. 2010 , MA , Oulmès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) perexiguus Theobald, 1903 Séguy 1930a ; Callot 1940 , AA , Assa; Senevet and Andarelli 1959a, EM , Oujda, Taourirt, AP , Aïn el Aouda, Arbaoua, Had Kourt, Oued Beht, Rabat, Allal Tazi, Oued Sahli, Zaouia Ech cheikh, Taghzirt, MA , Béni Mellal, Foum Zabel, Ifrane, Meknès, Aït Atta du Rteb, Fès, Sidi Mokhfi, Tahala, HA , Tazert, Midelt, AA , akka, Tamri; Bailly-Choumara 1965c , EM , Assa; Bailly-Choumara 1966 , AA , Souk El Khémis Dades, Aït Ouabelli, Tarhjicht, Tirherm, Taoujgelt, Aït melloul; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Ajdir; Bailly-Choumara 1967b , EM , Madagh, Merja Boubker, Aïn Béni Mathar; Bailly-Choumara 1967c , Rif , piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Béni Bouayache, Targusit et environs; Bailly-Choumara 1972a , AP , Merja Sheishat; Louah 1995 , Rif , Riffien, Tres piedras, Marina Smir, Bouzeghlal, Oued Maleh, Tahaddart, Talembote, Schroda, Tanger; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, El Oulja, Fouarate; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , HA , Marrakech, entre Oued N'fis et Chichaoua, Kelaâ Sraghna; Alaoui Slimani 2002 , AP , Rabat, Salé; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Bab Taza; Faraj et al. 2008c, AP , Larache Louamra; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Culex ) pipiens Linnaeus, 1758 d'Anfreville 1916 , AP , Salé; Charrier 1924 , Rif , Tanger; Séguy 1930a ; Callot 1940 , SA , Goulimine; Viamonte and Ramirez 1946 , Rif , Boudinar, Dar Benkarrich, Tétouan, Tanger, Asilah, Ksar El Kébir, Chefchaouen, Ketama, Nador; Gaud 1952 , AP , Gharb; Séguy 1953a , SA , Tindouf; Guy 1958 , HA , Oued N'fis; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , EM , Aouinet Aït Oussa, Aïn Isker, Aïn Aït Delouine, Oued Mesdourt, Talmesdourt, Toudi, AA , Aït Onmar, Oulad Teima, Taroudant, Tiznit ville, Talaint, Hassi Tafnidilt, Zaouiat Cheikh, Aïn Guerzim, Tafraout ville, SA , Goulimine ville, Vallée de l'Oued Assaka, Ouaroun, Zriouila, Labyar, Tighmert, Abeino, Tantan ville, Zag; Bailly-Choumara 1966 , AA , piste d'Akka au Draa, Akka et environs, Aït Ouabelli, Anamere-Smougue, Tarhjicht, Aït Melloul, Tiznit et environs; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, piste Itzer-Boumia, Ghorm El Alem, Ghorm El Alem-El Ksiba, El Ksiba, piste Ksiba-Naour, Zaouia Cheikh, Oued Sarif, Dayet Aoua, Pont Tarmilate, Ifrane, Imilchil; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Gaada de Debdou, Guercif ville, Cascade Oued Za, Grotte du Zegzel, Environs Saidia, Douar Aïn Shebbak, Douar Aïn Zabia, Douar Mardarh, Douar Sidi Hashas, Saidia, Merja Boubker, Berguent, Tendrara, Figuig; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Al Hoceima, Al Hoceima Club Med, Béni Bouayache, Marchica, Targuist et environs, Jebha, Ghafsai; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , AP , Merja Bokka, Merja Qodiya; Metge and Belakoul 1989 , AP , Benslimane, Sidi Bettache; Himmi 1991 , AP , Sidi Boughaba, Sidi Amira; Trari 1991 , AP , Maâmora, El Menzeh, Sidi Boughaba, Chkaïfien, Sidi Yahia du Gharb, Bokka, Merja Zerga, Sidi Allal Tazi, Oued Loukous, Aïn Chouk, Merja Bargha, Merja Oulad Skhar; Bouallam and Ramdani 1992 , HA , Marrakech; El Bermaki 1993, AP , Sidi Maârouf; Himmi et al. 1995 ; Louah 1995 , Rif , Fnideq, Riffien, Tres piedras, Marina Smir, M'diq, Cabo Negro, Zekri, Azla, Tahaddart, Schroda, Kebbache, Ouadras, Punta cirres, Sidi Kankoch, Oued Kbir; Louah et al. 1995 ; Bouallam et al. 1997b , HA , Marrakech; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Himmi et al. 1998 , AP , Sidi Boughaba, Puits de Sidi Amira (forest of Maâmora); Moussalim 1997 , AP , El Oulja, Maâmora, Fouarate, Sidi Boughaba; Ramdani 1997 , AP , meseta côtière (Témara-Casablanca); Faraj et al. 1997 , AP , Kénitra; Handaq 1998 , AP , Zemamra, MA , Oued Zem, HA , Marrakech, Zaouiet Ben Sassi, Sidi Bou Othmane, Kelaâ Sraghna, Bengrir; Bouallam 2001, HA , Marrakech; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Aouinty et al. 2006 , AP , Mohammedia; Faraj et al. 2006 , AP , Salé; Himmi 2007 , Rif , Bab Berred, Bab Taza, Stehat, AP , Skhirate, forest of Maâmora, forest of Hilton, Sidi Boughaba, El Oulja, Ouled Salem, Ouled dlim, Douar Ould Yahia Ben Ali, Larouaza, Douar Elarja, Douar Jdid, Douar Jnaja; Faraj et al. 2008b , AP , Louamra; El Ouali Lalami et al. 2010a , MA , Fès; Larhbali et al. 2010 , MA , Oulmès, Tarmilate, Bni Ounzar, Ganzra;Louali Lalami et al. 2010b, MA , Oued El Himmer; Amraoui 2012 , HA , Marrakech; Amraoui et al. 2012 , Rif , Tanger, AP , Mohammedia, Casablanca, HA , Marrakech; Amraoui et al. 2012 , AP , Mohammedia, Casablanca; El Ouali lalami 2012 , MA , route de Sidi Harazem; Hadji et al. 2013 , AP , Sidi Slimane; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Marc et al. 2016 , AP , Kénitra; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Bkhache et al. 2018 , Rif , Tanger, AP , Rabat, Mohammedia, HA , Marrakech; Tmimi et al. 2018 , AP , Mohammedia; Mouatassem et al. 2019, MA , Fès Culex ( Culex ) simpsoni Theobald, 1905 Callot 1940 , AA , Taghicht; Senevet et al. 1949 , AA , Oued Noun, Tafnidelt, Akka; Gaud 1953a , AA , Imsouane, Aït Melloul, Akka, O'Noun, Taghjicht, Assa, Tafnidilt; Bailly-Choumara 1965c , EM , Aman d'Aït Oussa, El Megrinat, AA , Oued Izi, Oued Massa-Pont de la route Agadir-Tiznit, Guelta Zerga, Tafnidilt, SA , Poste militaire de Boujrif, Tirhmert, Taourirt-Barrage, Aouinet Torkoz; Bailly-Choumara 1966 , AA , Tirherm, Taoujgelt, Taghjicht, Aït melloul, Tiznit; Chlaida and Bouzidi 1995 , AP , Sidi M'barek, Oued Sidi Messoud, Mechrâa, Sidi Boulâarais, Douar Chlihat (south of Settat); Chlaida 1997 , AP , Sidi M'barek, Mechrâa Settir, Sidi Boulâarais, Douar Chlihat (south of Settat); Dakki 1997 ; Handaq 1998 , HA , Sidi Bou Othmane (Marrakech); Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, MA , Khémisset, AA , sud Anti Atlas; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) theileri Theobald, 1903 Callot 1940 , AA , Taghjicht; Viamonte and Ramirez 1946 , Rif , Dar Benkarrich, Boudinar, Ketama, Tanger, Tétouan, Chefchaouen, AP , Larache; Guy 1958 , HA , Oued N'fis; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , EM , Aouinet Aït Oussa, Aïn Oumesdour, Aouinet Torkoz, SA , Tirh Mzoun; Bailly-Choumara 1966 , AA , Souk El Khémis Dades, Akka et environs, Aït Melloul, Tazenakhte, piste Tiznit-Tafraout, SA , Goulimine; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Aguelmane Azigza, piste Aguelmane Azigza-Aïn Leuh, maison forestière Ouiouane, Khénifra, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Col du Zad, piste Itzer-Boumia, Ghorm El Alem, piste Ghorm El Alem-El Ksiba, El Ksiba, piste Ksiba-Naour, Aguelmane Moulay Yakoub, Zaouia Cheich, Kafensour, Dayet Aoua, Pont Tarmilate; Bailly-Choumara 1967b , EM , route Itzer-Midelt, 40 km N Outat El Haj, Taourirt, Driouch, Figuig; Bailly-Choumara 1967c , Rif , piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, ArbaaTaourirt, Al Hoceima, Targuist et environs, Ghafsai; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , AP , Merja Sheishat, Merja Bokka; Himmi 1991 , AP , Sidi Boughaba, El menzeh, Sidi Amira; Trari 1991 , AP , Maâmora, Oued Sebou, Bordure Oued Loukous, Sidi Yahia du Gharb, Bokka, Entre Moulay Bousselham et Larache, Merja Zerga, Larache; El Bermaki 1993, AP , Sidi Maârouf, El Oulfa; Louah 1995 , Rif , Bouzeghlal, M'Diq, Oued Maleh, Zekri, Lajour, Tahaddart, Loubart, Chefchaouen, Tanger; Himmi et al. 1995 ; Louah et al. 1995 ; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Maâmora, Rabat, Kénitra; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , AP , Zemamra, MA , Béni Mellal, HA , Oukaimeden, Amizmiz, Tiguenziouine, Marrakech, Sidi Bou Othmane, Seguarta-Kelaâ, Had Mhara, Aîn Äounate, Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra), Gharb; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Tanakoub, AP , Skhirate, forest of Maâmora, Chiahna, Sidi Amira, Sidi Boughaba, Bouknadel, Sidi Azzouz, Ehssaïne, Bettana, Sidi Yahia, Sebbah, Larouaza, Douar Elarja, Douar Jdid, Douar Jnaja; Faraj et al. 2008b , AP , Larache Louamra; El Ouali Lalami et al. 2010a , AP , Boucharen; Larhbali et al. 2010 , MA , Roumani, Aïn Sbite, Ezzhiliga, Oulmès, Tarmilate, M'rirt, Bni Ounzar, Ganzra (Khémisset); Hadji et al. 2013 , AP , Sidi yahia du Gharb, Kcebia, Sidi Hagouch, Dar Belamri (Sidi Slimane); El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Maillotia ) deserticola Kirkpatrick, 1925 Gaud 1947c , AA , Tansikht; Gaud 1953a , EM , Figuig, Berguent, Anoual, Boudnib, Aïn Chair, Aoufous, Tarhit, HA , Tazenakht, Tichka, Tizi-n'Telghemt, AA , Ouarzazate, Zagora; Bailly-Choumara 1965c , EM , Oued Isker, El Megrinat, Taskala, Aïn Aït Delouine, Talmesdourt, Aouinet Torkoz, Bouanama, Rich Tamlougout, Assa, AA , Tafraout ville, Oued Jemâa Idaousmaal, Aït abdallah, Issedrim Igmur Igues, SA , Vallée de l'Oued Assaka, Tacharicht, Bou Izakarn, Aïn Erkha; Chlaida and Bouzidi 1995 , AP , Sud de Settat; Himmi et al. 1995 ; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Maillotia ) hortensis Ficalbi, 1889 Charrier 1924 , Rif , Tanger; Séguy 1930a ; Langeron 1938 , HA , Tounfite; Callot 1940 , HA , Anefgou (2500 m), Tirghist (2500 m), Tighermine (2500 m); Gaud 1953a , HA , Tizi-n'Telghemt, Tizi-n'Tichka; Bailly-Choumara 1967a , MA , piste tafechna-Znan Imes, Khénifra, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Ghorm El Alem, El Ksiba, piste Ksiba-Naour, Piste Naour-Arbala, piste Arbala-El Kbab, Zouia Cheikh, Oued Sarif, Ifrane, Boulmane, Imilchil; Bailly-Choumara 1967b , EM , Tafraout, Grotte du Zegzel; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Tamorote, route Bab Berred-Ketama, Ketama, piste Ketama-Mt Tiguidin, route nationale Jebha, route Targuist-Béni Boufrah, Trari 1991 , AP , Gharb; El Bermaki 1993, AP , Casablanca; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, Marina Smir, M'diq, Oued Maleh, Lajour, Tahaddart, Schroda, Talembote, Bab Berred, Punta Cirres, Sidi Kankoch, Oued kbir, Oued Jebel Lehbib; Louah et al. 1995 ; Chlaida and Bouzidi 1995 , AP , Barrage Al Massira; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , HA , Amizmiz, Sidi Bou Othmane, Had Mhara, Bengrir; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Bab Taza, AP , Skhirate; El Ouali Lalami et al. 2010b , MA , Oued Fès, Oued El Himmer, Camping Sidi Harazem; Larhbali et al. 2010 , MA , Merchouch, Ghoualem, Ezzhiliga, Oulmès, Tarmilate, M'rirt, Bni Ounzar, Ganzra (Khémisset); El Ouali Lalami 2012 , MA , Oued Fès; Oued El Himmer, route de Sidi Harazem, Camping Sidi Harazem; Hadji et al. 2013 , AP , Sidi Yahia du Gharb, Kcebia, Sidi Hagouch, Dar Belamri, Lalla Itto, Soualem (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Neoculex ) impudicus Ficalbi, 1890 Charrier 1924 , Rif , Tanger; Séguy 1930a ; Bailly-Choumara 1966 , AA , Taliouine; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Senoual-Itzer, Itzer, piste Naour-Arbala, Zaouia Cheikh, Pont Tarmilate; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Cascade Oued Za, Grotte du Zegzel; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Asifane, piste Bab Berred-Tamorote, Ketama, route nationale Jebha, Al Hoceima Club Med, Targuist et environs; Trari 1991 , AP , Sidi Amira, El Menzeh, Oued Sebou, Sidi Yahia du Gharb, Moulay Bousselham, Larache, Bordure Oued Loukous; Dakki 1997 ; Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra), Puits de Sidi Amira (forest of Maâmora); Trari et al. 2002 ; Himmi 1991 , AP , Sidi Boughaba, El Menzeh, Sidi Amira; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir, Tahaddart, Tanger; Louah et al. 1995 ; Ramdani 1997 , AP , Tamaris; Alaoui Slimani 2002 , AP , Rabat; Himmi 2007 , Rif , Bab Berred, Bab Taza, AP , forest Maâmora; El Ouali Lalami et al. 2010a , MA , Fès; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Neoculex ) martinii Medschid, 1930 Bailly-Choumara 1968a , Rif , Al Hoceima, AP , Sidi Yahia du Gharb, Sidi Allal Tazi, Larache, Rabat, HA , Marrakech, AA , Tiznit; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir, Cabo Negro, Oued Maleh; Louah et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culisetini Culiseta Felt, 1904 Culiseta ( Allotheobaldia ) longiareolata (Macquart, 1838) d'Anfreville 1916 , AP , Salé; Séguy 1930a ; Gaud 1947c , AA , Tansikht; Gaud 1953a , HA , Marrakech; Bailly-Choumara 1965c , EM , El Aïoun du Draa, Aïn Aït Delouine, Talmesdourt, Aouinet Torkoz, Rich Tamlougout, AA , Aït Onmar, Ouled Teima, Tiznit ville, Bounaamane, Id Baha, Tafnidilt, Guelta Zerga, Aïn Kerma, Tafraout ville, Oued Jemâa Idaousmaal, Toudi, Aït Abdallah, Igherm, Issedrim Igmur Igues, SA , Goulimine ville, Poste militaire de Boujrif, Embouchure de l'Oued Assaka, Ouaroun, Labyar, Asrir, Tacharicht, Bou Izakarn ville, Jemâa N'Tirhirte, Aït Erkha, Tantan ville; Bailly-Choumara 1966 , AA , Aït melloul, Tiznit; Bailly-Choumara 1967a , MA , Jnane Imasse, Khénifra, piste Tafechna-Senoual-Itzer, Itzer, Ghorm El Alem, El ksiba, piste Naour-Arbala, Ifrane, Pont Tarmilate, Imouzzer Marmoucha; Bailly-Choumara 1967b , EM , Tafraout, Guercif ville, Grotte du Zegzel, Berguent, Tendrara; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, route Bab Taza-Bab Berred, route Ketama-Targuist, route nationale Jebha, Targuist et environs, Aïn Hamra; Bailly-Choumara 1968a , AP , Meja Bokka, AA , Tiznit; Himmi 1991 , AP , Sidi Boughaba, El Menzeh; Trari 1991 , AP , El Menzeh, Gharb; El Bermaki 1993, AP , Sidi Maârouf; Himmi et al. 1995 ; Louah 1995 , Rif , Fnideq, Riffien, Marina Smir, M'diq, Cabo Negro, Lajour, Ouadras, Punta Cirres, Ksar Sghir, Tanger; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Maâmora, Fouarate; Ramdani 1997 , AP , Témara, Skhirat, Tamaris; Dakki 1997 ; Handaq 1998 , HA , Marrakech, Kelaâ Sraghna, Bengrir Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra); Bouallam 2001, HA , Bordure Oued N'fis; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Aouinty et al. 2006 , AP , Mohammedia; Himmi 2007 , Rif , Bab Berred, AP , Skhirate, Maâmora, forest of Hilton; Koçak and Kemal 2013; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culiseta ( Culicella ) fumipennis (Stephens, 1825) Gaud 1947c , AP , Rabat, Sidi Allal Tazi, MA , Khémisset; Senevet and Andarelli 1959, AP , Rabat, Casablanca, Bouznika, MA , Fès; Bailly-Choumara 1967a , MA , Jnane Imasse; Bailly-Choumara 1967c , Rif , route Bab Taza-Bab Berred; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culicella ) litorea (Shute, 1928) Metge 1986 , AP , Casablanca; Dakki 1997 ; Carles-Tolrá 2002 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culiseta ) annulata (Schrank, 1776) d'Anfreville 1916 , AP , Salé; Charrier 1924 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Viamonte and Ramirez 1946 , Rif , Ben Karrich, Asilah, Ketama, Malaliene, Tétouan, AP , Larache; Gaud 1952 , AP , Souk El Hadd (Gharb); Gaud 1953a , Rif , Tanger, Tétouan, EM , Saidia, Talsint, AP , Casablanca, Rabat, Kénitra, MA , Meknès, Fès, Ifrane, Taza, HA , Marrakech, Aït Bouguemez, Midelt; Gaud 1957b , Rif , Tétouan, Tanger, AP , Sidi Allal Tazi, Rabat, Casablanca, MA , Meknès, Ifrane; Bailly-Choumara 1967a , MA , Jnane Imasse, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, Ghorm El Alem, Zaouia Cheikh, Pont Tarmilate, Ifrane; Bailly-Choumara 1967b , EM , Tafraout, route Itzer-Midelt; Bailly-Choumara 1967c , Rif , piste Bab Taza-Talassemtane; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, M'diq, Tanger; Louah et al. 1995 ; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Rabat; Ramdani 1997 , AP , meseta côtière (Casablanca-Rabat); Himmi et al. 1998 , AP , Sidi Boughaba; Himmi 1991 , AP , Sidi Boughaba, El Menzeh; Trari 1991 , AP , Sidi Boughaba, El Menzeh; Trari et al. 2002 ; Himmi 2007 , AP , Skhirate, Maâmora, forest of Hilton, Oulja; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culiseta ) subochrea (Edwards, 1921) Gaud 1952 , AP , Gharb; Gaud 1957b , Rif , Tanger, Chefchaouen, EM , Oujda, AP , Sidi Allal Tazi, Casablanca; Bailly-Choumara 1967a , MA , Imilchil; Bailly-Choumara 1967c , Rif , Ketama; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba; Louah 1995 , Rif , Riffien, Tres piedras, M'diq, Cabo Negro, Oued Maleh, Tanger; Louah et al. 1995 ; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Kénitra; Ramdani 1997 , AP , Témara, Skhirat, Casablanca; Handaq 1998 , AP , El Jadida, HA , Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Skhirat, Dayet Aïn Chems, Maâmora, forest of Hilton; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Mansoniini Coquillettidia Dyar, 1905 Coquillettidia ( Coquillettidia ) buxtoni (Edwards, 1923) Bailly-Choumara 1965a , AP , Merja Bokka; Bailly-Choumara 1970 , AP , Merja Bokka; Bailly-Choumara 1972a , AP , Merja Sheishat (Larache); Bailly-Choumara 1973b , AP , Merja Bokka, Larache; Himmi et al. 1995 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Coquillettidia ( Coquillettidia ) richiardii (Ficalbi, 1889) Bailly-Choumara 1965a , AP , Merja Bokka; Bailly-Choumara 1967a , MA , Zaouia Cheikh; Bailly-Choumara 1970 , AP , Merja Bokka; Bailly-Choumara 1972a , AP , Merja Sheishat (Larache); Bailly-Choumara 1973b , AP , Merja Bokka; Trari 1991 , AP , Sidi Yahia du Gharb, Aïn Chouk, Merja Bargha; Himmi et al. 1995 ; Dakki 1997 ; Moussalim 1997 , AP , Fouarate sur les bordures de la Merja; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Orthopodomyiini Orthopodomyia Theobald, 1904 Orthopodomyia pulcripalpis (Rondani, 1872) Bailly-Choumara 1965b , AP , Maâmora; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Uranotaeniini Uranotaenia Lynch Arribálzaga, 1891 Uranotaenia ( Pseudoficalbia ) unguiculata Edwards, 1913 Séguy 1930a ; Gaud 1953a , HA , Marrakech; Senevet and Andarelli 1959a, EM , Oujda, Berguent, Guercif, AP , Sidi Allal Tazi, Bouznika, Casablanca, Béni Moussa, MA , Oulad Massine, Meknès, Moulay Yacoub, Béni Mellal, AA , Ksar Mzizel, Aït Melloul; Bailly-Choumara 1965c , EM , Aïn Aït Delouine, Talmesdourt, Rich Tamlougout; Bailly-Choumara 1966 , AA , Aït Ouabelli; Bailly-Choumara 1967a , MA , Pont Tarmilate; Bailly-Choumara 1967b , EM , environs de Taourirt, Madagh, Merja Boubker; Bailly-Choumara 1967c , Rif , route Bab Taza-Bab Berred, piste Ketama-Jebel Tidighine; Bailly-Choumara 1968a , AP , Oued Loukous, Merja Bokka, Kénitra, Oued Bou-Regreg; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Oued Sebou; El Bermaki 1993, AP , El Oulfa (Casablanca); Himmi et al. 1995 ; Dakki 1997 ; Ramdani 1997 , AP , Tamaris; Handaq 1998 , AP , Sidi Bennour, HA , Zima Chemaîa (Bengrir); Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Uranotaenia ( Uranotaenia ) balfouri Theobald, 1904 Bailly-Choumara 1968a , AP , Merja Bokka, Oued Loukous, Sidi Yahia, Oued Sebou (Kénitra), Oued Bou-Regreg; Himmi et al. 1995 ; Dakki 1997 ; Moussalim 1997 , AP , Sidi Allal Tazi, Fouarate, Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; El Ouali Lalami et al. 2010a , MA , Fès Boulmane; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Anophelinae Anopheles Meigen, 1818 Anopheles ( Anopheles ) algeriensis Theobald, 1903 Viallate 1922, AP , Kénitra; Séguy 1930a ; Bonjean 1947 , EM , MA ; Gaud 1957a , HA , north of High Atlas; Guy 1959a , MA , Béni Mellal, HA ; Guy 1959b , HA ; Guy 1959a , MA , Béni Mellal; Bailly-Choumara 1967a , MA , Ghorm El Alem; Benmansour et al. 1972 , MA , Barrage Bin El Ouidane; Bailly-Choumara 1973b , AP , Sidi Yahia du Gharb; Metge 1986 , AP , Casablanca; Trari and Himmi 1987 , AP ; Himmi et al. 1995 ; Louah 1995 , Rif , Tahaddart, Schroda; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Casablanca; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 , Rif , Chefchaouen; El Ouali Lalami et al. 2010 a,b, MA , Fès, Boulmane; El Ouali Lalami 2012 , MA , Fès; Trari 2017 , Rif , Chefchaouen, AA , Tiznit; Trari and Dakki 2017a , Rif , Chefchaouen, AA , Tiznit; Trari and Dakki 2017b , AA , Tiznit; Trari et al. 2017 Anopheles ( Anopheles ) claviger (Meigen, 1804) Vialatte 1923 , AP , Kénitra; Séguy 1930a , AP , Rabat; Langeron 1938 , HA , Tounfit, Massou, Anefgou, Tirghist, Tighermine, Louggouargh; Callot 1940 , HA , Anefgou, Tirghist; Bonjean 1947 , EM , MA ; Gaud 1947c , MA , Sefrou, Meknès; Gaud 1948 , AP , Rabat, MA , El Hajeb, AA , Errachidia, Tadla; Gaud et al. 1948 , AP , Skhirat; Guy 1963 , Rif , Taounate, MA , Meknès, Ifrane; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, piste Ksiba-Naour, piste Naour-Arbala, Zaouia Cheikh, Oued Sarif (environs El Ksiba), Dayet Aoua (environs Ifrane), Boulemane; Bailly-Choumara 1967b , EM , 8 km N Itzer; Bailly-Choumara 1967c , Rif , piste Ketama-Mt Tiguidin; Guy 1967 , HA , Marrakech, AA , Tafilalt; Guy and Holstein 1968 , SA ; Metge 1986 , AP , Casablanca; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris drains, Tamaris merja; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 , Rif , Bab Berred, Tanakoub; Faraj et al. 2008b , Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Boulmane; Larhbali et al. 2010 , MA , Zhiliga, Boukachmir, Aït Ichou; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) labranchiae Falleroni, 1926 d'Anfreville 1916 , AP , Salé; Delanoe 1917, AP , El Jadida; Viallate 1922, AP , Rabat, Boulhaut, Bouznika, Gharb, MA , Sidi Kacem, Tiflet, Fès, Taza; Charrier 1924 , Rif , Tanger; Séguy 1930a , MA ; Roubaud 1935, AP , Rabat; Sicault et al. 1935 , AP , Merja Ras Eddaoura, Merja Zerga, dayas entre Sebou et Maâmora, Dar bel Hamri (entre barrage El Kansera et Sidi Slimane), à proximité de Merja Zerga, Douar Anabsa; Langeron 1938 , AA ; Callot 1940 , AA ; Ristorcelli 1946a , b , HA , Oued Tensift, Oued Issil; Bonjean 1947 , AP , Gharb; Gaud 1947c , MA , Oulmès, HA , Marrakech; Gaud et al. 1948 , AP , Merja Ras Eddaoura; Gaud et al. 1949 , AP , Salé, SA , Foum Zguid, Tagounit; Gaud 1953a , AP , from Tanger to El Jadida, MA , Tissa, Timhadit, Bekrit, Meknès, Ifrane (1700 m), HA , Sidi Aissa, Tizi-n'Tichka; Guy 1958 , HA , Oued N'fis; Sacca and Guy 1960b, Rif , Tétouan, AP , Skhirat, Sidi Yahia, Sidi Bettache, Mazagan, Braila (près Sidi Allal Tazi), Aït Lahsen, MA , Meknès, HA , Marrakech; Guy 1962 , Rif , Taounate, AP , Gharb, HA , Marrakech (ville et banlieu); Guy 1963 , Rif , Tanger, Tétouan, EM , Berkane, Debdou, Oujda, AP , Kénitra, Souk Larba, Settat, Chamaîa, Safi, Casablanca, Rabat, Essaouira, MA , Meknès, Fès, Azrou, Oued Zem, Béni Mellal, HA , Kelaâ of Sraghna; Bailly-Choumara 1967a , MA , piste Tafechna-Znan Imes, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Aguelmane Azigza, piste Aguelmane Azigza-Aïn Leuh, maison forestière Ouiouane, route Khénifra-Tafechna, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Bords Moulouya, piste Itzer-Boumia, RN P.33 Boumia-El Kbab, Ouaoumana, piste Ksiba-Naour, piste Naour-Arbala, piste Arbala-El Kbab, Kafensour, Oued Sarif (environs El Ksiba), Dayet Aoua, Ifrane, Imouzer Marmoucha; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Douar Sherba, Douar Aïn Shebbak, Douar Aïn Zabia, Douar Madarh, Douar Sidi Hashas, Mechraa safsaf; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, piste Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Al Hoceima Club Med, Béni Bouayache, Targuist et environs; Guy 1967 , HA , Marrakech, AA , Tafilalt; Bailly-Choumara 1968b , AP , Larache; Guy and Holstein 1968 , AA , Ouarzazate; Bailly-Choumara 1970 , Rif , Tétouan, EM , Berkane, AP , Larache, Sidi Yahia du Gharb, HA , Marrakech; Benmansour et al. 1972 , AA , Agadir; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1972b , AP , Merja Sheishat; Bailly-Choumara 1973a , AP , Merja de l'Oued Smir; Bailly-Choumara 1973b , Rif , Tétouan, EM , Berkane, AP , Merja Sheishat, HA , Souk des Oudaias; de Zulueta et al. 1983 , Rif ; Ibn Jilali 1984, AP , Maâmora; Metge 1986 , AP , Entre Oulad Dlim et Al Ara'ra; Trari and Himmi 1987 , AP , Gharb; Himmi 1991 , AP , Kénitra, Maâmora; Metge 1991 , AP , Sidi Bettache; Trari 1991 , Rif , Chefchaouen, Tanger, Taounate, EM , Oujda, AP , Sidi Amira, Sidi Boughaba, Merja Zerga, Sidi Allal Tazi, Aïn Chouk, Oued Loukous, Merja Oulad Skhar, MA , Khémissat, Béni Mellal, Khouribga, HA , El Kelaâ des Sraghrna, AA , Ouarzazate; El Bermaki 1993, AP , entre l'aeroport Anfa et l'aménagement d'El Oulfa, Sidi Maârouf, El Oulfa; Chlaida and Bouzidi 1995 , AP , Aïn Blal, Oued Sidi Messoud; Louah 1995 , Rif , Tres piedras, Marina Smir, Bouzerlal, Oued Maleh, Zekri, Lajour, Azla, Tahaddart, Skroda, Stehat, Moulay Bouchta, Kebbache, Talembote, Loubart, Chefchaouen, Oued Maggou, Bab Berred, Sidi Kankoch, Oued kbir, Oued Jebel Lehbib; Louah et al. 1995 ; Chlaida 1997 , AP , Aïn Blal, Oued Sidi Messoud, Douar Chlihat, Barrage Al Massira; Moussalim 1997 , AP , Sidi Allal Tazi, Maâmora; Ramdani 1997 , AP , Tamaris, Skhirat; Faraj et al. 1997 , AP , Ouled Moussa; Handaq 1998 , HA , Oukaimeden, Amizmiz, Tiguenziouine; Himmi et al. 1998 , AP , Dayat d'El Menzeh (north-east of Kénitra), Sidi Boughaba; Alaoui Slimani et al. 1999 , AP , Bou-Regreg Salé, Sidi Bouguettaya, Quartier industriel Takaddoum, Marjane; Alaoui Slimani 2002 , AP , Rabat-Salé; Trari et al. 2002 ; Faraj et al. 2003 , Rif , Azib Bouflou, Azib Jrou, Imzouren, Amezzaourou, Tizi-Tamalout, MA , Aït Abdelsalam, Aït Lamfadel; Faraj et al. 2004 , Rif , Azib Jrou, Tanghaya Akarkar, Amezzaourou, Ouled Nsar, AP , Fedalate, MA , Talaa Chougaga, Aïn Smen (Fès), Aït Lamfadel; Trari et al. 2004a , Rif , Larache; Trari et al. 2004b , Rif , Ketama, Gzenaya, Taounate, Bouzaghlal, Oued Laou, Smir (Merja), Béni Hassane, Azib Jrou, Azib Bouflou, AP , Laaouamra, Aarabat Sidi Abdelaziz, Moukaouama, Dar Belamri, Laksibia, Mgadid, Beggara, Rabat (Chellah), MA , Adouz, Rommani (Khémisset), Aït Abderrahmane, Aït Ishaq, Oulad Messoud, Ouled Fennane, El karma, Oulad Zguida, Oulad Abbou; Aouinty et al. 2006 , AP , Mohammedia; Himmi 2007 , Rif , Bab Berred, Bab Taza, Tanakoub, AP , Skhirate, Maâmora, Oulja, Bouknadel, Sidi Azzouz, Tamaris, MA , Khémisset; Faraj et al. 2008a , AP , Laouamra, Boucharen, MA , Béni Khlef, Talaa Chougaga; Faraj et al. 2008b , Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010b , MA , Fès; Faraj et al. 2010 , AP , Begara, Boucharen, Ben Slimane, Skhirat, Rabat, Sehoul, MA , Sidi Allal Msader, Aïn Aghbal, Aïn Elouali, Sidi Kacem; Larhbali et al. 2010 , MA , Aït Haddou Said; Adlaoui et al. 2011 , AP , Larache; Larhbali et al. 2011 , MA , Oulmès, Aït Yadin, Sfassif, Mâaziz, Rommani, Laghoualem, Ezzhiliga, Sidi Allal Bahraoui, Boukachmine, Aït Malek, Sidi Boukhalkhal, Bni Ounzar, Ganzra, Aït Siberne, Sidi Allal Msader, El Ghandour; El Ouali Lalami 2012 , MA , Pont Diamant vert, Sidi Harazem, Oued El Himmer, Moulay Yakoub, Oued Sebou, Aïn Kansara, Oued Aïn Chkef, Sefrou, Boulemane; Laboudi et al. 2012 , AP , Larache; Hadji et al. 2013 , AP , Sidi Slimane; El Joubari et al. 2014 , Rif , Smir lagoon; Laboudi et al. 2014 , Rif , Tétouan, Tanger, Chefchaouen, Al Hoceima, AP , Larache, Salé, MA , Taza, Khémissat; El Joubari et al. 2015a , Rif , Smir lagoon; El Joubari et al. 2015b , Rif , Smir lagoon; Marc et al. 2016 , AP , Kénitra; Trari 2017 , Rif , Chefchaouen, Tétouan, AP , Larache, Rabat, Settat, EM , Oujda, MA , Khémisset, Meknès, Khouribga, HA , Marrakech, AA , Tiznit, Ouarzazate; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, AP , Larache, Rabat, Settat, EM , Oujda, MA , Khémisset, Meknès, Khouribga, HA , Marrakech, AA , Tiznit, Ouarzazate; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) marteri Senevet & Prunnelle, 1927 Gaud 1945b , MA , El Hajeb, Khénifra, HA , Tizi-n'test, Tillougite; Gaud et al. 1949 , HA , Tizi-n'test; Gaud 1953b , HA , Tizi-n'test; Bailly-Choumara 1967b , EM , Grotte du Zegzel; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Taza-Asifane, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Boured; Benmansour et al. 1972 , MA , Taza; Trari 1991 , Rif , Taounate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; Trari 2017 , Rif , Chefchaouen, AP , Settat; Trari and Dakki 2017a , Rif , Chefchaouen, AP , Settat; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Anopheles ) ziemanni Grünberg, 1902 Senevet 1935 , MA ; Gaud et al. 1949 , HA , plain of south and north of the Occidental Atlas; Gaud et al. 1950 , HA , plain of south and north of the Occidental Atlas; Guy 1958 , HA , Marrakech; Guy et al. 1958, HA , Oued N'fis; Guy 1967 , EM , Oujda, AP , Rabat, HA , Marrakech, AA , Tafilelt; Bailly-Choumara 1970 , HA , Marrakech; Benmansour et al. 1972 , MA , Taza, HA , Haouz, Tadla Azilal; Bailly-Choumara 1973b , HA , Souk des Oudaias (Souk Tnine des Oudaias, 470 m); Trari 1991 , MA , Tissa; Moussalim 1997 , AP , 7.5 km de Sidi Allal Tazi; Trari et al. 2002 ; Trari et al. 2004b ; Himmi 2007 ; Trari 2017 , Rif , Tétouan, AA , Tiznit; Trari and Dakki 2017a , Rif , Tétouan, AA , Tiznit; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) cinereus Theobald, 1901 Viallate 1922, MA , Sefrou, Sidi Lamine, HA , Mtougui; Sicault et al. 1935 , AP , Souk Larba of Gharb; Senevet 1935 , AP , Souk Larba of Zemmour; Langeron 1938 , HA , Anefgou, Tirghist, Valley of Sidi Yahia Ouyoussef, Tighermine, Louggouargh, Massou; Callot 1940 , HA , Anefgou, Tirghist; Gaud 1945a , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud and Duthu 1945, HA , Marrakech; Viamonte and Ramirez 1945 , Rif ; Viamonte and Ramirez 1946 , Rif ; Ristorcelli 1946a , HA , Oued Tensift, Oued Issil; Ristorcelli 1946b , HA , Oued Tensift, Oued Issil; Gaud 1945b , AA , Tansikht; Gaud et al. 1949 , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud et al. 1950 , Rif , Meridional Rif, EM , Moulouya, HA , Marrakech, AA , Tansikht, valley de Sous; Gaud 1953a ; Gaud 1958, HA , Marrakech; Guy 1962 , Rif , Taounate, HA , Marrakech; Guy 1963 , MA , Midelt, AA , Hamada of Draa; Bailly-Choumara 1965, EM , Aman d'Aït Oussa, Tiglit, El Megrinat, Taskala, Aïn Aït delouine, Oued mesdourt, Talmesdourt, Assa, AA , Aït Melloul, Oued Teima, Issen, Taroudant, Talaint, Tiznit, Oued Assaka, Anezi, Pont de la route Agadir-Tiznit, valley of low Draa, Tafraoute, Tacharicht, Bou Izakarn, Jemâa N'tirhirte, Aït Erkha,Tazert, Barrage Taourirt, AA , Goulmima, SA , Aouinet Torkoz, Tirh Mzoun; Bailly-Choumara 1966 , AA , piste Foum Zguid-Lac Iriqui, Agadir-Tissint, Akka-Iguiren, Tirhem, Taoujgelt, Souk El Khémis Dades, Akka, Aït Ouabelli, Foum-el-Hassan, Tarhjicht, Aït melloul, Taliouine, Tazenakht (Rocade of Draa); Bailly-Choumara 1967a , MA , route Azrou-Khénifra, Jnane Imasse, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Sources Oum-er-Rbia, Itzer, piste Itzer-Boumia, Ouaoumana, piste Ghorm El Alem-El Ksiba, piste Naour-Arbala, Zaouia Cheich, kafensour, Ifrane, Imouzzer Marmoucha; Bailly-Choumara 1967b , EM , 7 km N Itzer, 8 km N Itzer, route Itzer-Midelt, Aïn Srouna, Gouttitir, Cascade Oued Za, Grotte du Zegzel, Douar Aïn Soultane, Mechraa Safsaf, Oujda, Berguent (valley of Moulouya), Figuig; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Assifane, Anasar, piste Bab berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, route Al Hoceima-Arba Taourirt, Arba Taourirt, Targuist et environs, route Targuist Al Hoceima, Jebha, Oued Ouergha, route Aknoul-Al Hoceima; Guy 1967 , AP , Rabat, EM , Oujda, HA , Marrakech, AA , Tafilalet; Bailly-Choumara 1970 , HA , Marrakech; Bailly-Choumara 1973b , HA , Souk des Oudaias; Trari 1991 , Rif , Al Hoceima, Chefchaouen, Taounate, EM , Nador, Oujda, Figuig, AP , Larache, Settat, Ben Slimane, MA , Khénifra, Taza, Khouribga, HA , Kelaâ of Sraghna, AA , Ouarzazate, Goulmima; Bouallam 1992, HA , Oued N'fis; Louah 1995 , Rif , Marina Smir, Bouzerlal, Tahaddart, Stehat, Moulay Bouchta, Kebbache, Loubart, 0ued Maggou; Louah et al. 1995 ; Bouallam et al. 1997b , HA , Marrakech; Handaq 1998 , HA , Amizmiz, Tiguenziouine; Bouallam 2001, HA , Oued N'fis; Trari et al. 2002 ; Trari et al. 2004b , Rif , Ketama, Azib Jrou, Sidi Mokhfi, MA , Taghzirt, Aghbala, Aït Shak, Smaala, Mlalih, Ouled Fennane, Béni Khlef, Tachrafte; Faraj et al. 2007 , Rif , Assoul, Mizgane; Himmi 2007 , Rif , Bab Berred, Bab Taza, Stehat, Tanakoub; Faraj et al. 2008, Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Fès; El Ouali Lalami et al. 2010b , MA , Oued El Himmer; Larhbali et al. 2010 , MA , Roumani Aïn Sbite, Jamâa M. B., Ghoualem, Zhiliga, Oulmès, Tarmilate, Boukachmir, Mrirte, Aït Ichou, Mâaziz, Tiddas, Bni Ounzar, Ganzra; El Ouali Lalami 2012 , MA , Oued El Himmer; Trari 2017 , Rif , Chefchaouen, Tétouan, MA , Khouribga, AA , Tiznit; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, MA , Khouribga, AA , Tiznit; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Anopheles ( Cellia ) dthali Patton, 1905 Saccà 1960 , AA , Aoufous, Meski, Erfoud, Agdz, Zogora, Tagounit, Tamsrruth; Guy 1961 , HA , Sud de Zagora (en bordure Hamada du Draa); Guy 1963 , AA , Zagora and south of the High Atlas (at edge of Hamada of Draa), Oued Ziz; Bailly-Choumara 1965c , AA , Tiznit, EM , Aïn Aït Delouine, Aouinet Torkoz, Rich Tamlougout (eastern borders of Jebel Bani); Bailly-Choumara 1966 , AA , piste Foum Zguid au Lac Iriqui, Agadir-Tissint, Akka-Iguiren, Souk El Khémis Dades, Akka et Environs, Aït Ouabelli, Tarhjicht, piste Tazenakhte à Foum Zguid (Rocade du Draa); Bailly-Choumara 1967b , EM , valley of Moulouya; Guy 1967 , HA , Marrakech, AA , Tafilalet; Guy and Holstein 1968 , EM , N Outat El Haj (valley of Moulouya), Gouttitir (environs de Taourirt); Bailly-Choumara 1970 , AA , Foum Zquid; Bailly-Choumara 1973b , AA , Foum Zquid; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari et al. 2004b ; Faraj et al. 2007 , Rif , Assoul, Mizgane (SE of Bab Berred); Faraj et al. 2008, Rif , Assoul, Mizgane (SE Bab Berred); Himmi 2007 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) multicolor Cambouliu, 1902 Messerlin and Treillard 1938 , HA , Marrakech; Viamonte and Ramirez 1945 , Rif ; Guy 1963 , SA ; Bailly-Choumara 1965c , EM , Aït Oussa, Aman d'Aït Oussa, Aïn Aït Delouine, Aouinet Torkoz, Rich tamlougout (Confins orientaux du Jebel Bani), AA , Tafnidilt, Guelta Zerga, Aïn Temda (valley of low Draa), Tirhmert (Goulmima), SA , Vallée et embouchure de l'Oued Assaka, Tantan ville, Tirh Mzoun; Bailly-Choumara 1966 , AA , Rocade of Draa; Bailly-Choumara 1967b , EM , 40 km N de Outat El Haj, Gouttitir, Cascade Oued Za, Douar Aïn Shebbak (valley of Moulouya); Guy 1967 , HA , Marrakech, AA , Tafilelt; Guy and Holstein 1968 , HA , south of Atlas, AP , plain located between Marrakech and the Atlantic from Tanger along the length of the Mediterranean; Bailly-Choumara 1970 , AA , Foum Zquid; Bailly-Choumara 1973b , AA , Foum Zquid; Metge 1986 , AP , Casablanca; Trari 1991 , Rif , Al Hoceima, Taounate, Oued Maleh, Bab Berred, AP , Tamaris Merja, AA , Ouarzazate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari et al. 2004b ; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Anopheles ( Cellia ) sergentii (Theobald, 1907) Séguy 1930a ; Messerlin and Treillard 1938 , HA , Tamelelt; Langeron 1938 , Rif , Targhist; Callot 1940 , AA , Taghjicht; Gaud 1947c , AA , Wadi Draa; Gaud et al. 1949 , Rif , Zoumi, HA , Zaouia Sidi Hamza, Tizi-n'test (1700 m), Tillougit (1800 m); Gaud et al. 1950 , Rif , Zoumi, MA ; Guy et al. 1958, HA , Oued N'fis; Guy 1961 , Rif , AP , south of Casablanca, AA , Sud de Zagora; Guy 1962 , Rif , AP , south of Casablanca, HA , Marrakech; Guy 1963 , Rif , Tanger, EM , Berkane, AP , south of Casablanca, MA , Béni Mellal, HA , Oued Tensift, Oued Ziz, Marrakech, Chichaoua, AA , Oued Draâ, Oued Dades, Goulmima, sud de Zagora, SA , Foum Zguid; Bailly-Choumara 1965c , EM , iglit, Aman d'Aït Oussa, Oued Isker, El Megrinat, Aïn Aït Delouine, Oued Mesdourt, almesdourt, Aouinet Torkoz, Bouanama, Rich Tamlougout, Assa, AA , Tiznit, Tafraoute, Oued Izi, Bou Izakarn, Abeino (region of Goulmima), SA , Aouzeroual, Tirhmert, Tacharicht, Jebel Bani, Tantan; Bailly-Choumara 1966 , AA , Rocade of Draa, Taliouine; Bailly-Choumara 1967a , Rif , AP , south of Casablanca, MA , Itzer, Ghorm El Alem, Zaouia Cheikh, Kafensour, HA , north of High Atlas; Bailly-Choumara 1967b , EM , Aïn Srouna, Grotte du Zegzel, Douar Mardarh, Douar Aïn Soultane, Merja Boubker, Selouane, Driouch (valley of Moulouya); Bailly-Choumara 1967c , Rif , route nationale Jebha, Targuist-Béni Boufrah, Al Hoceima, Béni Bouayache, Marchica, Had El Rouadi, Pont du Srah; Guy 1967 , HA , Marrakech, AA , Tafilelt; Guy and Holstein 1968 , AP , Casablanca; Bailly-Choumara 1970 , EM , Berkane, HA , Marrakech; Bailly-Choumara 1973b , EM , Berkane, HA , Marrakech; Metge 1986 , AP , Casablanca; Trari 1991 , Rif , Al Hoceima, Taounate, AP , Larache, AA , Ouarzazate; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Faraj et al. 2003 , Rif , Al Hoceima, Chefchaouen, Taounate, HA , Khouribga; Trari et al. 2004b , Rif , Ketama, Sidi Mokhfi; Faraj et al. 2007 , Rif , Assoul, Mizgane; Faraj et al. 2008, Rif , Assoul, Mizgane; El Ouali Lalami et al. 2010a , MA , Fès; Larhbaliet al. 2010 , MA , Oulmès, Ganzra; Trari 2017 , Rif , Chefchaouen, Tétouan, EM , Oujda, MA , Khouribga; Trari and Dakki 2017a , Rif , Chefchaouen, Tétouan, EM , Oujda, MA , Khouribga; Trari and Dakki 2017b ; Trari et al. 2017 ; Benabdelkrim Filali et al. 2018 ; Mouatassem et al. 2019, MA , Fès Culicinae Aedini Aedes Meigen, 1818 Aedes ( Acartomyia ) mariae (Sergent & Sergent, 1903) Séguy 1930b ; Messerlin 1938 , AP , Rabat; Séguy 1930a , Rif , littoral méditerranéen; Bailly-Choumara 1967b , Rif , Al Hoceima; Bailly-Choumara 1967c , Rif , Al Hoceima; Bailly-Choumara 1968a , Rif , Al Hoceima, AP , Larache, Sidi Yahia, Sidi Allal Tazi, Rabat, HA , Marrakech, AA , Tiznit; Himmi et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Aedimorphus ) vexans (Meigen, 1830) Gaud 1947c , AP , Sidi Allal Tazi, MA , Khémisset; Metge 1986 , AP , Littoral Casablanca; Himmi et al. 1995 ; Handaq 1998 , AP , Gharbia; Dakki 1997 ; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Dahliana ) echinus (Edwards, 1920) Séguy 1924 ; Séguy 1930a ; Gaud 1953a , AP , Rabat, Sidi Yahia, MA , Moulay Bouazza, Taza, Fès, Meknès, Ifrane; Bailly-Choumara 1965b , AP , Maâmora; Bailly-Choumara 1967c , Rif , piste Bab Taza-Talassemtane; Himmi et al. 1995 ; Trari et al. 2002 ; Nikookar et al. 2010 ; El Joubari et al. 2014 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Dahliana ) geniculatus (Olivier, 1791) Séguy 1924 , MA ; Séguy 1930a ; Metge and El Alaoui 1987 , AP , Subéraies de Béni Abid-Benslimane (Casablanca); Metge and Belakoul 1989 , AP , Sidi Bettache; Himmi et al. 1995 ; El Ouali Lalami et al. 2010a , MA , Fès Boulmane; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) berlandi (Séguy, 1921) Séguy 1930a , AP , Rabat; Gaud 1953a , MA , Fès; Bailly-Choumara 1967a , MA , Jnane Imasse, piste Tafechna-Taoujgelt; Bailly-Choumara 1967c , Rif , piste Bab Taza-Béni Ahmed; Belakoul 1985 , AP , Benslimane, Sidi Bettache; Metge and El Alaoui 1987 , AP , Casablanca; Metge and Belakoul 1989 , AP , Benslimane, Sidi Bettache; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) caspius (Pallas, 1771) Séguy 1930b ; Viamonte et Ramirez 1946, AP , Larache; Gaud 1952 , AP , Rabat, Casablanca; Gaud 1953a , AP , Rabat, Casablanca; Senevet and Andarelli 1954 , EM , Embouchure de la Moulouya, Figuig, AP , Jorf Lasfar, Mohammedia, Rabat, MA , Meknès, Fès, Taza, HA , Marrakech, Midelt, AA , Tiznit; Bailly-Choumara 1966 , AA , environs de Tiznit; Bailly-Choumara 1967a , EM , Bords Moulouya (près Itzer), Cherarba; Bailly-Choumara 1967b , EM , Cherarba, Aïn Shebbak, Saidia, Berguent; Bailly-Choumara 1967c , Rif , piste Al Hoceima-Arba Taourirt; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , Rif , Merja de l'Oued Smir; El Kaim 1972 , AP , Bou-Regreg; Rioux et al. 1975 , AP , Rabat-Salé; Metge 1986 , AP , Littoral casablancais; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba, Merja Zerga, Oued Loukous; Himmi et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Kénitra; Ramdani 1997 , AP , Tamaris Merja; Handaq 1998 , AP , Zemamra, B. Iffou (Entre El Oualidia et Youssoufia), MA , Béni Mellal, HA , Marrakech, Zaouiet Ben Sassi, Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; Alaoui Slimani 2002 , AP , Rabat; Aouinty et al. 2006 , AP , Mohammedia; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) coluzzii Rioux, Guilvard & Pasteur, 1998 et Aedes ( Ochlerotatus ) detritus (Haliday, 1833) [Complexe detritus] Charrier 1924 , Rif , Tanger; Séguy 1930a ; Gaud 1953a , EM , Saidia, AP , Kénitra, Rabat, Bouznika, El jadida, Oualidia, HA , Marrakech, AA , Agadir, Tafnidilt; Bailly-Choumara 1965c , EM , Aïn Aït delouine, SA , Tirhmert; Bailly-Choumara 1970 , Rif , Tétouan; Knight 1971 , AP , Kénitra; El Kaim 1972 , AP , Bou-Regreg; Bailly-Choumara 1973b , Rif , Merja de l'Oued Smir; Rioux et al. 1975 , AP , Rabat; Pasteur et al. 1978 , AP , Bou-Regreg; Metge 1986 , AP , Littoral casablancais; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba, Merja Zerga, Oued Loukous; Louah 1995 , Rif , Tres piedras, Cabo Negro, Lajour, Azla, Tahaddart; Himmi et al. 1995 ; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Rabat; Ramdani 1997 , AP , Tamaris Merja; Handaq 1998 , AP , Essaouira, Zima-Chemaîa; Himmi et al. 1998 , AP , Sidi Boughaba; Himmi 2007 , AP , Sidi Boughaba; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Ochlerotatus ) pulchritarsis (Rondani, 1872) Gaud 1953a , AP , Benslimane, Sidi Yahia, Rabat, MA , Oued Zem, Khénifra, Fès; Metge and El Alaoui 1987 , AP , Benslimane; Himmi et al. 1995 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Rusticoidus ) rusticus (Rossi, 1790) Viamonte and Ramirez 1946 , Rif , Tétouan, AP , Larache; Gaud 1953a , MA , Taza; Himmi et al. 1995 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Stegomyia ) aegypti (Linnaeus in Hasselquist, 1762) d'Anfreville 1916 , AP , Salé; Vialatte 1923 , AP , Rabat, Casablanca, HA , Marrakech; Charrier 1924 , Rif , Tanger; Gaud 1953a , AP , Salé, HA , Marrakech; Himmi et al. 1995 ; Dakki 1997 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Aedes ( Stegomyia ) albopictus (Skuse, 1895) Bennouna et al. 2016 , AP , Agdal (Rabat); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Faraj et al. 2018 ; Amraoui et al. 2019 Culicini Culex Linnaeus, 1758 Culex ( Barraudius ) modestus Ficalbi, 1889 Séguy 1930a ; Bailly-Choumara 1968a , AP , Larache; Trari 1991 , AP , Gharb; Himmi et al. 1995 ; Dakki 1997 ; Handaq 1998 , HA , Bengrir; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, AP , Maâmora; Hadji et al. 2013 , AP , Sidi yahia du Gharb, Kcebia, Sidi Hagouch (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) brumpti Galliard, 1931 Bailly-Choumara 1968a , AP , Merja Bokka, Larache, HA , Marrakech; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi et al. 1995 ; Dakki 1997 ; Himmi 2007 ; Trari et al. 2002 ; El Ouali Lalami et al. 2010a , MA , Fès; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) laticintus Edwards, 1913 Charrier 1924 , Rif , Tanger; Callot 1940 , AA , Goulmima (mares); Gaud 1953a , HA , Marrakech, AA , Agadir; Gaud 1957a , EM , Nador; Bailly-Choumara 1965c , EM , Oued Isker, Aïn Aït delouine, Talmesdourt, AA , Ouled Teima, ounaamane, Bou Izakarn, Agunil Khnufa, Akka-guiren; Bailly-Choumara 1966 , AA , Akka-Iguiren, Tirherm, Taoujgelt, Aït Ouabelli, Anamere-Smougue, Aït melloul, Tiznit (Rocade de Draa); Bailly-Choumara 1967b , Rif , Béni Bouayache, Targuist et environs, EM , Grotte du Zegzel; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 , AP , Skhirat; Faraj et al. 2008b , AP , Louamra; Hadji et al. 2013 , AP , Sidi Hagouch (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) mimeticus Noè, 1899 Séguy 1930a ; Viamonte and Ramirez 1946 , Rif , Béni Ider, Fnideq, Khemis Anjra, Ketama, Oued Amsa, Oued Krikra, Oued Martil, Oued Laou; Gaud 1953a , Rif , Ouezzane, Ghafsai, EM , Berkane, Martinpray (près Berkane), El Aïoun Sidi Mellouk, AP , Tamri, MA , Meknès, Fès, Ifrane, Taza, Béni Mellal, HA , Midelt, Marrakech, Azilal, AA , Tinghir, Tichka; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , AA , Oued Noun, Anezi, Tafraoute; Bailly-Choumara 1966 , AA , Agadir; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Taoujgelt, Source Oumerrbia, Ghorm El Alem, piste Ksiba-Naour, piste Naour-Arbala, Zaouia Cheikh, Oued Sarif; Bailly-Choumara 1967b , EM , 7 km N d'Itzer, 8 km d'Itzer; Bailly-Choumara 1967c , Rif , route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Taza-Asifane, piste Bab Berred-Tamorote, Ketama, piste Ketama-Jebel Tidighine, route Ketama-Targuist, route nationale Jebha, Al Hoceima, Béni Bouayache, Marchica, Targuist et environs, Jebha, Boured; Trari 1991 , AP , Sidi Yahia du Gharb; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, Tres piedras, Marina Smir, Bouzaghlal, M'diq, Oued Maleh, Azla, Tahaddart, Moulay Bouchta, Schroda, Kebbache, Talambote, Oued Maggou; Louah et al. 1995 ; Chlaida 1997 , AP , Oued Sidi Messoud, Aïn Blal, Douar Chlihat, Barrage Al Massira; Chlaida and Bouzidi 1995 , AP , Barrage El Massira; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris Merja; Handaq 1998 , HA , Oukaimeden, Amizmiz, Tiguenziouine (près Oued N'fis); Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, AP , Bouknadel, Douar jdid (Skhirat); El Ouali Lalami et al. 2010a , MA , Fès; Larhbali et al. 2010 , MA , Oulmès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) perexiguus Theobald, 1903 Séguy 1930a ; Callot 1940 , AA , Assa; Senevet and Andarelli 1959a, EM , Oujda, Taourirt, AP , Aïn el Aouda, Arbaoua, Had Kourt, Oued Beht, Rabat, Allal Tazi, Oued Sahli, Zaouia Ech cheikh, Taghzirt, MA , Béni Mellal, Foum Zabel, Ifrane, Meknès, Aït Atta du Rteb, Fès, Sidi Mokhfi, Tahala, HA , Tazert, Midelt, AA , akka, Tamri; Bailly-Choumara 1965c , EM , Assa; Bailly-Choumara 1966 , AA , Souk El Khémis Dades, Aït Ouabelli, Tarhjicht, Tirherm, Taoujgelt, Aït melloul; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Ajdir; Bailly-Choumara 1967b , EM , Madagh, Merja Boubker, Aïn Béni Mathar; Bailly-Choumara 1967c , Rif , piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Béni Bouayache, Targusit et environs; Bailly-Choumara 1972a , AP , Merja Sheishat; Louah 1995 , Rif , Riffien, Tres piedras, Marina Smir, Bouzeghlal, Oued Maleh, Tahaddart, Talembote, Schroda, Tanger; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, El Oulja, Fouarate; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , HA , Marrakech, entre Oued N'fis et Chichaoua, Kelaâ Sraghna; Alaoui Slimani 2002 , AP , Rabat, Salé; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Bab Taza; Faraj et al. 2008c, AP , Larache Louamra; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Culex ) pipiens Linnaeus, 1758 d'Anfreville 1916 , AP , Salé; Charrier 1924 , Rif , Tanger; Séguy 1930a ; Callot 1940 , SA , Goulimine; Viamonte and Ramirez 1946 , Rif , Boudinar, Dar Benkarrich, Tétouan, Tanger, Asilah, Ksar El Kébir, Chefchaouen, Ketama, Nador; Gaud 1952 , AP , Gharb; Séguy 1953a , SA , Tindouf; Guy 1958 , HA , Oued N'fis; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , EM , Aouinet Aït Oussa, Aïn Isker, Aïn Aït Delouine, Oued Mesdourt, Talmesdourt, Toudi, AA , Aït Onmar, Oulad Teima, Taroudant, Tiznit ville, Talaint, Hassi Tafnidilt, Zaouiat Cheikh, Aïn Guerzim, Tafraout ville, SA , Goulimine ville, Vallée de l'Oued Assaka, Ouaroun, Zriouila, Labyar, Tighmert, Abeino, Tantan ville, Zag; Bailly-Choumara 1966 , AA , piste d'Akka au Draa, Akka et environs, Aït Ouabelli, Anamere-Smougue, Tarhjicht, Aït Melloul, Tiznit et environs; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Taoujgelt, piste Tafechna-Assoul, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, piste Itzer-Boumia, Ghorm El Alem, Ghorm El Alem-El Ksiba, El Ksiba, piste Ksiba-Naour, Zaouia Cheikh, Oued Sarif, Dayet Aoua, Pont Tarmilate, Ifrane, Imilchil; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Gaada de Debdou, Guercif ville, Cascade Oued Za, Grotte du Zegzel, Environs Saidia, Douar Aïn Shebbak, Douar Aïn Zabia, Douar Mardarh, Douar Sidi Hashas, Saidia, Merja Boubker, Berguent, Tendrara, Figuig; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, route nationale Jebha, Al Hoceima, Al Hoceima Club Med, Béni Bouayache, Marchica, Targuist et environs, Jebha, Ghafsai; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , AP , Merja Bokka, Merja Qodiya; Metge and Belakoul 1989 , AP , Benslimane, Sidi Bettache; Himmi 1991 , AP , Sidi Boughaba, Sidi Amira; Trari 1991 , AP , Maâmora, El Menzeh, Sidi Boughaba, Chkaïfien, Sidi Yahia du Gharb, Bokka, Merja Zerga, Sidi Allal Tazi, Oued Loukous, Aïn Chouk, Merja Bargha, Merja Oulad Skhar; Bouallam and Ramdani 1992 , HA , Marrakech; El Bermaki 1993, AP , Sidi Maârouf; Himmi et al. 1995 ; Louah 1995 , Rif , Fnideq, Riffien, Tres piedras, Marina Smir, M'diq, Cabo Negro, Zekri, Azla, Tahaddart, Schroda, Kebbache, Ouadras, Punta cirres, Sidi Kankoch, Oued Kbir; Louah et al. 1995 ; Bouallam et al. 1997b , HA , Marrakech; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Himmi et al. 1998 , AP , Sidi Boughaba, Puits de Sidi Amira (forest of Maâmora); Moussalim 1997 , AP , El Oulja, Maâmora, Fouarate, Sidi Boughaba; Ramdani 1997 , AP , meseta côtière (Témara-Casablanca); Faraj et al. 1997 , AP , Kénitra; Handaq 1998 , AP , Zemamra, MA , Oued Zem, HA , Marrakech, Zaouiet Ben Sassi, Sidi Bou Othmane, Kelaâ Sraghna, Bengrir; Bouallam 2001, HA , Marrakech; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Aouinty et al. 2006 , AP , Mohammedia; Faraj et al. 2006 , AP , Salé; Himmi 2007 , Rif , Bab Berred, Bab Taza, Stehat, AP , Skhirate, forest of Maâmora, forest of Hilton, Sidi Boughaba, El Oulja, Ouled Salem, Ouled dlim, Douar Ould Yahia Ben Ali, Larouaza, Douar Elarja, Douar Jdid, Douar Jnaja; Faraj et al. 2008b , AP , Louamra; El Ouali Lalami et al. 2010a , MA , Fès; Larhbali et al. 2010 , MA , Oulmès, Tarmilate, Bni Ounzar, Ganzra;Louali Lalami et al. 2010b, MA , Oued El Himmer; Amraoui 2012 , HA , Marrakech; Amraoui et al. 2012 , Rif , Tanger, AP , Mohammedia, Casablanca, HA , Marrakech; Amraoui et al. 2012 , AP , Mohammedia, Casablanca; El Ouali lalami 2012 , MA , route de Sidi Harazem; Hadji et al. 2013 , AP , Sidi Slimane; El Joubari et al. 2014 , Rif , Smir lagoon; El Joubari et al. 2015a , Rif , Smir lagoon; Marc et al. 2016 , AP , Kénitra; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Bkhache et al. 2018 , Rif , Tanger, AP , Rabat, Mohammedia, HA , Marrakech; Tmimi et al. 2018 , AP , Mohammedia; Mouatassem et al. 2019, MA , Fès Culex ( Culex ) simpsoni Theobald, 1905 Callot 1940 , AA , Taghicht; Senevet et al. 1949 , AA , Oued Noun, Tafnidelt, Akka; Gaud 1953a , AA , Imsouane, Aït Melloul, Akka, O'Noun, Taghjicht, Assa, Tafnidilt; Bailly-Choumara 1965c , EM , Aman d'Aït Oussa, El Megrinat, AA , Oued Izi, Oued Massa-Pont de la route Agadir-Tiznit, Guelta Zerga, Tafnidilt, SA , Poste militaire de Boujrif, Tirhmert, Taourirt-Barrage, Aouinet Torkoz; Bailly-Choumara 1966 , AA , Tirherm, Taoujgelt, Taghjicht, Aït melloul, Tiznit; Chlaida and Bouzidi 1995 , AP , Sidi M'barek, Oued Sidi Messoud, Mechrâa, Sidi Boulâarais, Douar Chlihat (south of Settat); Chlaida 1997 , AP , Sidi M'barek, Mechrâa Settir, Sidi Boulâarais, Douar Chlihat (south of Settat); Dakki 1997 ; Handaq 1998 , HA , Sidi Bou Othmane (Marrakech); Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, MA , Khémisset, AA , sud Anti Atlas; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Culex ) theileri Theobald, 1903 Callot 1940 , AA , Taghjicht; Viamonte and Ramirez 1946 , Rif , Dar Benkarrich, Boudinar, Ketama, Tanger, Tétouan, Chefchaouen, AP , Larache; Guy 1958 , HA , Oued N'fis; Guy et al. 1958, HA , Oued N'fis; Bailly-Choumara 1965c , EM , Aouinet Aït Oussa, Aïn Oumesdour, Aouinet Torkoz, SA , Tirh Mzoun; Bailly-Choumara 1966 , AA , Souk El Khémis Dades, Akka et environs, Aït Melloul, Tazenakhte, piste Tiznit-Tafraout, SA , Goulimine; Bailly-Choumara 1967a , MA , piste Tafechna-Taoujgelt, piste Tafechna-Assoul, Aguelmane Azigza, piste Aguelmane Azigza-Aïn Leuh, maison forestière Ouiouane, Khénifra, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Col du Zad, piste Itzer-Boumia, Ghorm El Alem, piste Ghorm El Alem-El Ksiba, El Ksiba, piste Ksiba-Naour, Aguelmane Moulay Yakoub, Zaouia Cheich, Kafensour, Dayet Aoua, Pont Tarmilate; Bailly-Choumara 1967b , EM , route Itzer-Midelt, 40 km N Outat El Haj, Taourirt, Driouch, Figuig; Bailly-Choumara 1967c , Rif , piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, route Bab Taza-Bab Berred, Anasar, piste Bab Berred-Tamorote, Ketama, route Ketama-Targuist, ArbaaTaourirt, Al Hoceima, Targuist et environs, Ghafsai; Bailly-Choumara 1972a , AP , Merja Sheishat; Bailly-Choumara 1973b , AP , Merja Sheishat, Merja Bokka; Himmi 1991 , AP , Sidi Boughaba, El menzeh, Sidi Amira; Trari 1991 , AP , Maâmora, Oued Sebou, Bordure Oued Loukous, Sidi Yahia du Gharb, Bokka, Entre Moulay Bousselham et Larache, Merja Zerga, Larache; El Bermaki 1993, AP , Sidi Maârouf, El Oulfa; Louah 1995 , Rif , Bouzeghlal, M'Diq, Oued Maleh, Zekri, Lajour, Tahaddart, Loubart, Chefchaouen, Tanger; Himmi et al. 1995 ; Louah et al. 1995 ; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Sidi Allal Tazi, Maâmora, Rabat, Kénitra; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , AP , Zemamra, MA , Béni Mellal, HA , Oukaimeden, Amizmiz, Tiguenziouine, Marrakech, Sidi Bou Othmane, Seguarta-Kelaâ, Had Mhara, Aîn Äounate, Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra), Gharb; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Tanakoub, AP , Skhirate, forest of Maâmora, Chiahna, Sidi Amira, Sidi Boughaba, Bouknadel, Sidi Azzouz, Ehssaïne, Bettana, Sidi Yahia, Sebbah, Larouaza, Douar Elarja, Douar Jdid, Douar Jnaja; Faraj et al. 2008b , AP , Larache Louamra; El Ouali Lalami et al. 2010a , AP , Boucharen; Larhbali et al. 2010 , MA , Roumani, Aïn Sbite, Ezzhiliga, Oulmès, Tarmilate, M'rirt, Bni Ounzar, Ganzra (Khémisset); Hadji et al. 2013 , AP , Sidi yahia du Gharb, Kcebia, Sidi Hagouch, Dar Belamri (Sidi Slimane); El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Maillotia ) deserticola Kirkpatrick, 1925 Gaud 1947c , AA , Tansikht; Gaud 1953a , EM , Figuig, Berguent, Anoual, Boudnib, Aïn Chair, Aoufous, Tarhit, HA , Tazenakht, Tichka, Tizi-n'Telghemt, AA , Ouarzazate, Zagora; Bailly-Choumara 1965c , EM , Oued Isker, El Megrinat, Taskala, Aïn Aït Delouine, Talmesdourt, Aouinet Torkoz, Bouanama, Rich Tamlougout, Assa, AA , Tafraout ville, Oued Jemâa Idaousmaal, Aït abdallah, Issedrim Igmur Igues, SA , Vallée de l'Oued Assaka, Tacharicht, Bou Izakarn, Aïn Erkha; Chlaida and Bouzidi 1995 , AP , Sud de Settat; Himmi et al. 1995 ; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; El Ouali Lalami et al. 2010a , MA , Fès; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Maillotia ) hortensis Ficalbi, 1889 Charrier 1924 , Rif , Tanger; Séguy 1930a ; Langeron 1938 , HA , Tounfite; Callot 1940 , HA , Anefgou (2500 m), Tirghist (2500 m), Tighermine (2500 m); Gaud 1953a , HA , Tizi-n'Telghemt, Tizi-n'Tichka; Bailly-Choumara 1967a , MA , piste tafechna-Znan Imes, Khénifra, piste Tafechna-Senoual-Itzer, Ajdir, Itzer, Ghorm El Alem, El Ksiba, piste Ksiba-Naour, Piste Naour-Arbala, piste Arbala-El Kbab, Zouia Cheikh, Oued Sarif, Ifrane, Boulmane, Imilchil; Bailly-Choumara 1967b , EM , Tafraout, Grotte du Zegzel; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Tamorote, route Bab Berred-Ketama, Ketama, piste Ketama-Mt Tiguidin, route nationale Jebha, route Targuist-Béni Boufrah, Trari 1991 , AP , Gharb; El Bermaki 1993, AP , Casablanca; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, Marina Smir, M'diq, Oued Maleh, Lajour, Tahaddart, Schroda, Talembote, Bab Berred, Punta Cirres, Sidi Kankoch, Oued kbir, Oued Jebel Lehbib; Louah et al. 1995 ; Chlaida and Bouzidi 1995 , AP , Barrage Al Massira; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Ramdani 1997 , AP , Skhirat, Tamaris; Handaq 1998 , HA , Amizmiz, Sidi Bou Othmane, Had Mhara, Bengrir; Trari et al. 2002 ; Himmi 2007 , Rif , Bab Berred, Bab Taza, AP , Skhirate; El Ouali Lalami et al. 2010b , MA , Oued Fès, Oued El Himmer, Camping Sidi Harazem; Larhbali et al. 2010 , MA , Merchouch, Ghoualem, Ezzhiliga, Oulmès, Tarmilate, M'rirt, Bni Ounzar, Ganzra (Khémisset); El Ouali Lalami 2012 , MA , Oued Fès; Oued El Himmer, route de Sidi Harazem, Camping Sidi Harazem; Hadji et al. 2013 , AP , Sidi Yahia du Gharb, Kcebia, Sidi Hagouch, Dar Belamri, Lalla Itto, Soualem (Sidi Slimane); Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culex ( Neoculex ) impudicus Ficalbi, 1890 Charrier 1924 , Rif , Tanger; Séguy 1930a ; Bailly-Choumara 1966 , AA , Taliouine; Bailly-Choumara 1967a , MA , route Azrou-Khénifra, piste Tafechna-Senoual-Itzer, Itzer, piste Naour-Arbala, Zaouia Cheikh, Pont Tarmilate; Bailly-Choumara 1967b , EM , route Itzer-Midelt, Cascade Oued Za, Grotte du Zegzel; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, piste Bab Taza-Fifi, piste Bab Taza-Talassemtane, piste Bab Taza-Béni Ahmed, route Bab Taza-Bab Berred, piste Bab Berred-Asifane, piste Bab Berred-Tamorote, Ketama, route nationale Jebha, Al Hoceima Club Med, Targuist et environs; Trari 1991 , AP , Sidi Amira, El Menzeh, Oued Sebou, Sidi Yahia du Gharb, Moulay Bousselham, Larache, Bordure Oued Loukous; Dakki 1997 ; Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra), Puits de Sidi Amira (forest of Maâmora); Trari et al. 2002 ; Himmi 1991 , AP , Sidi Boughaba, El Menzeh, Sidi Amira; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir, Tahaddart, Tanger; Louah et al. 1995 ; Ramdani 1997 , AP , Tamaris; Alaoui Slimani 2002 , AP , Rabat; Himmi 2007 , Rif , Bab Berred, Bab Taza, AP , forest Maâmora; El Ouali Lalami et al. 2010a , MA , Fès; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culex ( Neoculex ) martinii Medschid, 1930 Bailly-Choumara 1968a , Rif , Al Hoceima, AP , Sidi Yahia du Gharb, Sidi Allal Tazi, Larache, Rabat, HA , Marrakech, AA , Tiznit; Himmi et al. 1995 ; Louah 1995 , Rif , Haidra, Marina Smir, Cabo Negro, Oued Maleh; Louah et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Himmi 2007 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culisetini Culiseta Felt, 1904 Culiseta ( Allotheobaldia ) longiareolata (Macquart, 1838) d'Anfreville 1916 , AP , Salé; Séguy 1930a ; Gaud 1947c , AA , Tansikht; Gaud 1953a , HA , Marrakech; Bailly-Choumara 1965c , EM , El Aïoun du Draa, Aïn Aït Delouine, Talmesdourt, Aouinet Torkoz, Rich Tamlougout, AA , Aït Onmar, Ouled Teima, Tiznit ville, Bounaamane, Id Baha, Tafnidilt, Guelta Zerga, Aïn Kerma, Tafraout ville, Oued Jemâa Idaousmaal, Toudi, Aït Abdallah, Igherm, Issedrim Igmur Igues, SA , Goulimine ville, Poste militaire de Boujrif, Embouchure de l'Oued Assaka, Ouaroun, Labyar, Asrir, Tacharicht, Bou Izakarn ville, Jemâa N'Tirhirte, Aït Erkha, Tantan ville; Bailly-Choumara 1966 , AA , Aït melloul, Tiznit; Bailly-Choumara 1967a , MA , Jnane Imasse, Khénifra, piste Tafechna-Senoual-Itzer, Itzer, Ghorm El Alem, El ksiba, piste Naour-Arbala, Ifrane, Pont Tarmilate, Imouzzer Marmoucha; Bailly-Choumara 1967b , EM , Tafraout, Guercif ville, Grotte du Zegzel, Berguent, Tendrara; Bailly-Choumara 1967c , Rif , Chaouen ville, route Chaouen-Bab Taza, Bab Taza, route Bab Taza-Bab Berred, route Ketama-Targuist, route nationale Jebha, Targuist et environs, Aïn Hamra; Bailly-Choumara 1968a , AP , Meja Bokka, AA , Tiznit; Himmi 1991 , AP , Sidi Boughaba, El Menzeh; Trari 1991 , AP , El Menzeh, Gharb; El Bermaki 1993, AP , Sidi Maârouf; Himmi et al. 1995 ; Louah 1995 , Rif , Fnideq, Riffien, Marina Smir, M'diq, Cabo Negro, Lajour, Ouadras, Punta Cirres, Ksar Sghir, Tanger; Louah et al. 1995 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Maâmora, Fouarate; Ramdani 1997 , AP , Témara, Skhirat, Tamaris; Dakki 1997 ; Handaq 1998 , HA , Marrakech, Kelaâ Sraghna, Bengrir Himmi et al. 1998 , AP , Sidi Boughaba, Dayat d'El Menzeh (north east of Kénitra); Bouallam 2001, HA , Bordure Oued N'fis; Alaoui Slimani 2002 , AP , Rabat; Trari et al. 2002 ; Aouinty et al. 2006 , AP , Mohammedia; Himmi 2007 , Rif , Bab Berred, AP , Skhirate, Maâmora, forest of Hilton; Koçak and Kemal 2013; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Culiseta ( Culicella ) fumipennis (Stephens, 1825) Gaud 1947c , AP , Rabat, Sidi Allal Tazi, MA , Khémisset; Senevet and Andarelli 1959, AP , Rabat, Casablanca, Bouznika, MA , Fès; Bailly-Choumara 1967a , MA , Jnane Imasse; Bailly-Choumara 1967c , Rif , route Bab Taza-Bab Berred; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culicella ) litorea (Shute, 1928) Metge 1986 , AP , Casablanca; Dakki 1997 ; Carles-Tolrá 2002 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culiseta ) annulata (Schrank, 1776) d'Anfreville 1916 , AP , Salé; Charrier 1924 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Viamonte and Ramirez 1946 , Rif , Ben Karrich, Asilah, Ketama, Malaliene, Tétouan, AP , Larache; Gaud 1952 , AP , Souk El Hadd (Gharb); Gaud 1953a , Rif , Tanger, Tétouan, EM , Saidia, Talsint, AP , Casablanca, Rabat, Kénitra, MA , Meknès, Fès, Ifrane, Taza, HA , Marrakech, Aït Bouguemez, Midelt; Gaud 1957b , Rif , Tétouan, Tanger, AP , Sidi Allal Tazi, Rabat, Casablanca, MA , Meknès, Ifrane; Bailly-Choumara 1967a , MA , Jnane Imasse, maison forestière Ouiouane, piste Tafechna-Senoual-Itzer, Ghorm El Alem, Zaouia Cheikh, Pont Tarmilate, Ifrane; Bailly-Choumara 1967b , EM , Tafraout, route Itzer-Midelt; Bailly-Choumara 1967c , Rif , piste Bab Taza-Talassemtane; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi et al. 1995 ; Louah 1995 , Rif , Riffien, M'diq, Tanger; Louah et al. 1995 ; Chlaida 1997 , AP , Barrage Al Massira; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Rabat; Ramdani 1997 , AP , meseta côtière (Casablanca-Rabat); Himmi et al. 1998 , AP , Sidi Boughaba; Himmi 1991 , AP , Sidi Boughaba, El Menzeh; Trari 1991 , AP , Sidi Boughaba, El Menzeh; Trari et al. 2002 ; Himmi 2007 , AP , Skhirate, Maâmora, forest of Hilton, Oulja; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Culiseta ( Culiseta ) subochrea (Edwards, 1921) Gaud 1952 , AP , Gharb; Gaud 1957b , Rif , Tanger, Chefchaouen, EM , Oujda, AP , Sidi Allal Tazi, Casablanca; Bailly-Choumara 1967a , MA , Imilchil; Bailly-Choumara 1967c , Rif , Ketama; Bailly-Choumara 1972a , AP , Merja Sheishat; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Sidi Boughaba; Louah 1995 , Rif , Riffien, Tres piedras, M'diq, Cabo Negro, Oued Maleh, Tanger; Louah et al. 1995 ; Dakki 1997 ; Mestari 1997 , AP , Mohammedia; Moussalim 1997 , AP , Kénitra; Ramdani 1997 , AP , Témara, Skhirat, Casablanca; Handaq 1998 , AP , El Jadida, HA , Bengrir; Himmi et al. 1998 , AP , Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Skhirat, Dayet Aïn Chems, Maâmora, forest of Hilton; El Joubari et al. 2014 , Rif , Smir lagoon; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Mansoniini Coquillettidia Dyar, 1905 Coquillettidia ( Coquillettidia ) buxtoni (Edwards, 1923) Bailly-Choumara 1965a , AP , Merja Bokka; Bailly-Choumara 1970 , AP , Merja Bokka; Bailly-Choumara 1972a , AP , Merja Sheishat (Larache); Bailly-Choumara 1973b , AP , Merja Bokka, Larache; Himmi et al. 1995 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Coquillettidia ( Coquillettidia ) richiardii (Ficalbi, 1889) Bailly-Choumara 1965a , AP , Merja Bokka; Bailly-Choumara 1967a , MA , Zaouia Cheikh; Bailly-Choumara 1970 , AP , Merja Bokka; Bailly-Choumara 1972a , AP , Merja Sheishat (Larache); Bailly-Choumara 1973b , AP , Merja Bokka; Trari 1991 , AP , Sidi Yahia du Gharb, Aïn Chouk, Merja Bargha; Himmi et al. 1995 ; Dakki 1997 ; Moussalim 1997 , AP , Fouarate sur les bordures de la Merja; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Orthopodomyiini Orthopodomyia Theobald, 1904 Orthopodomyia pulcripalpis (Rondani, 1872) Bailly-Choumara 1965b , AP , Maâmora; Himmi et al. 1995 ; Dakki 1997 ; Trari et al. 2002 ; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 Uranotaeniini Uranotaenia Lynch Arribálzaga, 1891 Uranotaenia ( Pseudoficalbia ) unguiculata Edwards, 1913 Séguy 1930a ; Gaud 1953a , HA , Marrakech; Senevet and Andarelli 1959a, EM , Oujda, Berguent, Guercif, AP , Sidi Allal Tazi, Bouznika, Casablanca, Béni Moussa, MA , Oulad Massine, Meknès, Moulay Yacoub, Béni Mellal, AA , Ksar Mzizel, Aït Melloul; Bailly-Choumara 1965c , EM , Aïn Aït Delouine, Talmesdourt, Rich Tamlougout; Bailly-Choumara 1966 , AA , Aït Ouabelli; Bailly-Choumara 1967a , MA , Pont Tarmilate; Bailly-Choumara 1967b , EM , environs de Taourirt, Madagh, Merja Boubker; Bailly-Choumara 1967c , Rif , route Bab Taza-Bab Berred, piste Ketama-Jebel Tidighine; Bailly-Choumara 1968a , AP , Oued Loukous, Merja Bokka, Kénitra, Oued Bou-Regreg; Himmi 1991 , AP , Sidi Boughaba; Trari 1991 , AP , Oued Sebou; El Bermaki 1993, AP , El Oulfa (Casablanca); Himmi et al. 1995 ; Dakki 1997 ; Ramdani 1997 , AP , Tamaris; Handaq 1998 , AP , Sidi Bennour, HA , Zima Chemaîa (Bengrir); Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès Uranotaenia ( Uranotaenia ) balfouri Theobald, 1904 Bailly-Choumara 1968a , AP , Merja Bokka, Oued Loukous, Sidi Yahia, Oued Sebou (Kénitra), Oued Bou-Regreg; Himmi et al. 1995 ; Dakki 1997 ; Moussalim 1997 , AP , Sidi Allal Tazi, Fouarate, Sidi Boughaba; Trari et al. 2002 ; Himmi 2007 , AP , Sidi Boughaba; El Ouali Lalami et al. 2010a , MA , Fès Boulmane; Trari 2017 ; Trari and Dakki 2017b ; Trari et al. 2017 ; Mouatassem et al. 2019, MA , Fès DIXIDAE K. Kettani, R. Wagner Number of species: 12 . Expected: 15 Faunistic knowledge of the family in Morocco: moderate Dixa Meigen, 1818 Dixa caudatula Séguy, 1928 Séguy 1930a , HA , Arround, Skoutana (2400 m); Vaillant 1959 ; Dakki 1997 ; Mouna 1998 Dixa dilatata Strobl, 1900 = Dixa riparia Vaillant, in Vaillant 1959 : 180 Vaillant 1959 , HA , Source de M'Goum (2500 m), Gorges d'Imi-N'Ifri (1050 m); Vaillant 1965 ; Dakki 1997 Dixa maculata Meigen, 1818 Séguy 1930a ; Dakki 1997 ; Mouna 1998 Dixa mera Séguy, 1930 Séguy 1930a , MA , forest of Timelilt (1900 m); Vaillant 1959 ; Dakki 1997 ; Mouna 1998 Dixa nebulosa Meigen, 1830 Séguy 1930a , HA ; Dakki 1997 ; Mouna 1998 Dixa perexilis Séguy, 1928 Séguy 1930a , HA , riverside of Oued Imminen (Tachdirt, 2400 m); Vaillant 1959 ; Dakki 1997 ; Mouna 1998 Dixa puberula Loew, 1849 Vaillant 1959 , HA , headwaters of Asif M'Goum (2500 m); Vaillant 1965 ; Dakki 1997 ; Mouna 1998 Dixa submaculata Edwards, 1920 Séguy 1930a , MA , Sidi Yahia, Talzent (1800 m); Dakki 1997 ; Mouna 1998 Dixella Dyar & Shannon, 1924 Dixella aestivalis (Meigen, 1818) Séguy 1930a , AP , Merja Boughaba; Vaillant 1965 ; Ramdani 1981 ; Ramdani and Tourenq 1982 ; Mouna 1998 Dixella attica (Pandazis, 1933) = Dixella numidica (Sicart, 1955) Ebejer et al. 2019 , Rif , Cabo Negro (indoors: 10 m) Dixella martinii (Peus, 1934) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m) Dixella serotina (Meigen, 1818) = Dixa serotina Wied, in Mouna 1998 : 85 Séguy 1930a , AP , Casablanca (between Kénitra and Oued Beth); Dakki 1997 ; Mouna 1998 Chironomoidea CERATOPOGONIDAE K. Kettani, B. Mathieu Number of species: 62 . Expected: 80 Faunistic knowledge of the family in Morocco: moderate Ceratopogoninae Culicoidini Culicoides Latreille, 1809 Culicoides ( Avaritia ) imicola Kieffer, 1913 Kremer et al. 1971 , MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Fès, Rhafsai, AA , Torkoz, Tarhjisht; Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, EM , Oriental, AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Draa-Tafilalet, Souss-Massa, SA , Guelmim-Oued Noun; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Aïn Leuh, Ait Siberne, Meknès, AA , Errachidia, Sidi Dahmane, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) montanus Shakirzjanova, 1962 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Safi, HA , Marrakech; Chaker et al. 1980 , Rif , Al Hoceima, AP , Oued Cherrat, HA , Souk Tnine de Oudaias (Haouz), Marrakech, AA , Torkoz; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) obsoletus (Meigen, 1818) Callot et al. 1968 , Rif , Al Hoceima, AP , Merja Bokka, Sidi Yahia du Gharb, Sidi-Bettache (Zaeir), Rabat-Salé-Kénitra, HA , El Harcha (plateau central); Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Béni Mellal-Khénifra, HA , Marrakech; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, HA , Marrakech; Chaker et al. 1980 , Rif , Al Hoceima, AP , Zaers, Sidi Bettache, HA , El Harcha, Talet Inaouane (Haouz); Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Souss-Massa, SA , Guelmim-Oued Noun; Cêtre-Sossah and Baldet 2004 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Ait Siberne; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) scoticus Downes & Kettle, 1952 Kremer et al. 1971 , AP , Safi, MA , Béni Mellal-Khénifra, HA , El Harcha, Talet Inaouane (Haouz), Marrakech; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat, Safi, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Beltranmyia ) circumscriptus Kieffer, 1918 Callot et al. 1968 ; Bailly-Choumara and Kremer 1970 , Rif , Smir lagoon, Oued Negro, EM , Merja Boubker (Berkane), Gouttitir (NE Guercif), AP , Merja Sheishat (Larache), Aïn Muelha (near Oued Sidi Allal Tazi, estuaire Oued Bou-Regreg, Dayat Qoudiya (Sidi Yahia Gharb); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, AA , Souss Massa; Chaker et al. 1980 , Rif , Al Hoceima, AP , Merja Qoudiya, Merja Bokka, Sidi Yahia du Gharb, MA , Aïn Karma (Saiss), Oulmès, HA , Setti Fatma, AA , Aït Melloul (Souss); Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Meknès, AA , Errachidia, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) fagineus Edwards, in Edwards et al. 1939 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Béni Mellal-Khénifra, HA , Marrakech; Kremer et al. 1975 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Kremer et al. 1979 ; Chaker et al. 1980 , AP , Rabat, Sidi Bettache, MA , Khemisset, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) newsteadi Austen, 1921 = Culicoides ( Culicoides ) halophilus Kieffer, in Callot et al. 1968 : 886, Bailly-Choumara and Kremer 1970 : 386, Dakki 1997 : 60 Callot et al. 1968 , Rif , Cabo Negro (Ferma), Tétouan, Talerhza, Tanger-Tétouan-Al Hoceima, AP , Larache, Merja Bokka (Gharb), Aïn Chok, HA , Talet-Inaouan (Haouz); Bailly-Choumara and Kremer 1970 , Rif , Smir lagoon, Oued Negro, AP , Merja Sheishat (Larache), Aïn Muelha (near Oued Sidi Allal Tazi, estuaire Oued Bou-Regreg, Dayat Qoudiya (Sidi Yahia du Gharb), EM , Merja Boubker (Berkane), Ksabi (NE Midelt), HA , Souk Tnine des Oudaias (bordure Oued N'fis), AA , Aïn Sefra (south Foum Zquid); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Casablanca, Settat, MA , Fès-Meknès, Béni Mellal-Khénifra, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, Casablanca, Settat, MA , Béni Mellal- Khénifra, HA , Marrakech; Kremer et al. 1979 ; Baylis et al. 1997 , Rif , Tanger, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , MA , Aïn Leuh, Ait Siberne, Meknès, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) pulicaris (Linnaeus, 1758) Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, Béni Mellal-Khénifra, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Lalla Outka, Khénifra, Oulmès, HA , Talet Inaouan (Haouz), AA , Aouinet-Torkoz, Tarhjicht; Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, EM , Oriental, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Drâa-Tafilalet, Souss-Massa; Cêtre-Sossah and Baldet 2004 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Aïn Leuh, Aït Siberne, Meknès; Bourquia et al. 2019 Culicoides ( Culicoides ) punctatus (Meigen, 1804) Callot et al. 1968 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1970 , Rif , Merja Smir, Oued Negro, AP , Merja Sheishat (Larache), estuaire Bou-Regreg, EM , Merja Boubker (Berkane); Kremer et al. 1971 , Rif , Cabo Negro (Ferma), Tétouan, EM , Berkane, AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, AA , Foum Zguid; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat; Dakki 1997 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Aïn Leuh, Meknès; Bourquia et al. 2019 Culicoides ( Culicoides ) subfagineus Delécolle & Ortega, 1998 Bourquia et al. 2019 , AP , Rabat Culicoides ( Monoculicoides ) parroti Kieffer, 1922 Bailly-Choumara and Kremer 1970 , HA , Dar Saâda (Haouz); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Safi, HA , Marrakech; Chakeret al. 1980 , AP , Rabat, HA , Marrakech, Souk Tnine des Oudaias (Haouz); Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Monoculicoides ) puncticollis (Becker, 1903) Callot et al. 1968 , AP , Merja Qoudiya, Sidi Yahia du Gharb, Romani (Zaers), Rabat-Salé-Kénitra, MA , Aïn Karma (Saiss), HA , Souk Tnine des Oudaias (Haouz); Bailly-Choumara and Kremer 1970 (reported as C. riethi and corrected by Kremer et al. 1971 ), Rif , Merja Smir, AP , Merja Sheishat (Larache), estuaire Bou-Regreg, Dayat Qoudiya (Sidi Yahia du Gharb), HA , Souk Tnine des Oudaias (bordure de l'Oued N'fis), Dar Saâda (Haouz), Talet Inouane (bordure marécageuse du lac du Barrage Lalla Taguergoust); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, HA , Marrakech; Chaker et al. 1980 , AP , Aïn Karma, Zaers, Roumani, HA , Souk-Tnine des Oudaias; Remm 1988a ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) azerbajdzhanicus Dhzafarov, 1962 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Safi, MA , Fès-Meknès, Beni Mellal-Khénifra, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 , AA , Souss-Massa SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Kkénifra, Rhafsai, HA , Marrakech, AA , Torkoz, Tarhjisht; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) longipennis Khalaf, 1957 Kremer et al. 1971 , EM , Berkane AP , Safi, MA , Fès-Meknès, HA , Marrakech; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) marcleti Callot, Kremer & Basset, 1968 Kremer et al. 1971 , MA , Rhafsai, Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) pallidus Khalaf, 1957 = Culicoides stackelbergi Dhzafarov, in Kremer et al. 1971 : 662, Dakki 1997 : 61 Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1971 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) ravus De Meillon, 1936 = Culicoides ( Synhelea ) subravus Cornet and Château, in Kremer et al. 1971 : 664, Chaker et al. 1980 : 85, Dakki 1997 : 61 Kremer et al. 1971 , AP , Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AA , Souss-Massa, SA , Guelmim-Oued Noun; Chaker et al. 1980 , HA , Marrakech, AA , Torkoz, Tarhjicht, Aït Ouaballi (Draa); Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) sahariensis Kieffer, 1923 = Culicoides colluzzii Callot, Kremer and Bailly-Choumara, in Bailly-Choumara and Kremer 1970 : 386, Chaker et al. 1980 : 83, Dakki 1997 : 61 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), EM , Merja Boubker (Berkane), HA , Souk Tnine des Oudaias (bordure Oued N'fis); Callot et al. 1970 , AP , Larache, HA , Marrakech; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Merja Bokka (Gharb), Rabat (Zaers), EM , Berkane, MA , Fès, Khémisset, AA , Tarhjisht; Baylis et al. 1997 ; Dakki 1997 ; Bouayoune et al. 1998; Cêtre-Sossah and Baldet 2004 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) santonicus Callot, Kremer, Rault & Bach, 1966 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Bailly-Choumara et al. 1980, AP , Larache, Sidi Bettache, MA , Oulmès, EM , El-Harcha; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) semimaculatus Clastrier, 1958 Kremer et al. 1975 , Rif , Tanger-Tétouan-Al Hoceima, AP , Larache, Casablanca-Settat, MA , Plateau Central (Khatouate); Chaker et al. 1979 ; Remm 1988a ; Dakki 1997 ; Szadziewski and Dominiak 2006 ; Bourquia et al. 2019 Culicoides ( Oecacta ) sergenti Kieffer, 1921 = Culicoides ( Oecacta ) mosulensis Khalaf, in Chaker et al. 1980 : 84, Dakki 1997 : 60 Kremer et al. 1979 , EM , Oriental, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AA , Tarhjicht, SA , Bou-Arfa; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) similis Carter, Ingram & Macfie, 1920 Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) truncorum Edwards, 1939 = Culicoides ( Oecacta ) sylvarum Callot and Kremer, in Kremer et al. 1971 : 662, Remm 1988a : 65, Dakki 1997 : 61 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Béni Mellal- Khénifra; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Pontoculicoides ) saevus Kieffer, 1922 Callot et al. 1968 , AP , Sidi Yahia du Gharb, Rabat-Salé-Kénitra, Safi, MA , Aïn Karma (Saiss), HA , Talet Inouane, Marrakech, Souk Tnine des Oudaias (Haouz), AA , Aït Melloul (Souss), Ksar er Souk (Tafilalt), Tarhjicht, SA , Bou-Arfa; Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), HA , Souk Tnine des Oudaias (bordure de l'Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AP , Safi, MA , Fès-Meknès, HA , Marrakech, AA , Drâa-Tafilalet, Souss-Massa, SA , Guelmim-Oued Noun; Bailly-Choumara and Kremer 1980; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Pontoculicoides ) sejfadinei Dzhafarov, 1958 Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AA , Drâa-Tafilalet; Bourquia et al. 2019 Culicoides ( Remmia ) kingi Austen, 1912 Bailly-Choumara and Kremer 1970 , AA , Mrimima (Oued de Foum Zquid), Souss-Massa; Chaker et al. 1980 , MA , Meknès, AA , Tarhjisht; Cornet and Brunhes 1994 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Remmia ) schultzei (Enderlein, 1908) Callot et al. 1968 , AP , Safi, HA , Talet Inouane, Marrakech; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) badooshensis Khalaf, 1961 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), HA , Marrakech, Souk Tnine des Oudaias; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech; Dakki 1997 ; Chaker et al. 1980 , AP , Oued Cherrat, Bousselham, Sidi Yahia, MA , Fès, HA , Marrakech; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) cataneii Clastrier, 1957 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra; Chaker et al. 1980 , AP , Oued Cherrat, Rabat; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) clastrieri Callot, Kremer & Deduit, 1962 Bourquia et al. 2019 , MA , Fès-Meknès Culicoides ( Sensiculicoides ) derisor Callot & Kremer, 1965 Chaker et al. 1980 , AP , Rabat; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès; Kremer et al. 1975 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Kremer et al. 1979 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) duddingstoni Kettle & Lawson, 1955 Bourquia et al. 2019 , MA , Fès-Meknès Culicoides ( Sensiculicoides ) dzhafarovi Remm, 1967 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Oued Cherrat, AA , Tarhjisht; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) festivipennis Kieffer, 1914 = Culicoides ( Oecacta ) odibilis Austen, in Bailly-Choumara and Kremer 1970 : 387, Chaker et al. 1980 : 84, Dakki 1997 : 61 Callot et al. 1968 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1970 , Rif , Merja Smir, HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, Casablanca-Settat, HA , Marrakech; Chaker et al. 1980 , Rif , Tétouan, EM , El-Harcha, AP , Aïn Chok, Rabat, Larache, MA , Oulmès, HA , Marrakech, Talet-Inaouan (Haouz); Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) heteroclitus Kremer and Callot, in Callot & Kremer, 1965 Kremer et al. 1975 , AP , Safi, HA , Marrakech, AA , Tafraout, Tiznit, Souss-Massa; Chaker et al. 1979 ; HA , Haouz; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) jumineri Callot & Kremer, 1969 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, AA , Souss-Massa, SA , Guelmim-Oued Noun; Chaket et al. 1980, AP , Oued Cherrat, Merja Bokka, Rabat, MA , Fès, HA , Talet-Inaouan (Haouz), AA , Torkoz, Tarhjisht, Aït Oubelli, SA , Bou-Arfa; Remm 1988a ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) kibunensis Tokunaga, 1937 = Culicoides ( Oecata ) cubitalis Edwards, in Kremer et al. 1975 : 205, Dakki 1997 : 60 Kremer et al. 1975 , AP , Safi, MA , Ifrane, Imouzzer-du-Kander, Fès-Meknès, HA , Haouz, Marrakech; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) kurensis Dzhafarov in Gutsevich, 1960 Remm 1988a ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) landauae Kremer, Rebholtz-Hirtzel & Bailly-Choumara, 1975 Kremer et al. 1975 , MA , Imouzzer-du-Kander, Sefrou, Fès-Meknès; Chaker et al. 1979 ; Hervy et al. 1994 ; Borkent and Wirth 1997 ; Remm 1988a ; Dakki 1997 ; Chilasse and Dakki 2004, MA ; Borkent 2012 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) langeroni Kieffer, 1921 Bailly-Choumara and Kremer 1970 , HA , Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Bailly-Choumara and Kremer 1980, MA , Khénifra, AA , Tarhjicht, Torkoz (Draa); Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) maritimus Kieffer, 1924 Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Bailly-Choumara and Kremer 1980, Rif , Tétouan, AP , Larache, Rabat, Sidi Bettache, HA , Talet-Inaouan (Haouz), Souk Tnine des Oudaias (Haouz); Remm 1988; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) odiatus Austen, 1921 = Culicoides lailae Khalaf, in Bailly-Choumara and Kremer 1970 : 387, Dakki 1997 : 60 = Culicoides indistinctus Khalaf, in Kremer et al. 1975 : 206, Dakki 1997 : 60 Bailly-Choumara and Kremer 1970 , HA , Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1975 , AA , Tafraout, Souss-Massa; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Kénitra, Merja Bokka (Gharb), HA , Souk Tnine des Oudaias (Haouz), AA , Tarhjicht; Remm 1988a ; Dakki 1997 ; Baylis et al. 1997 ; Bouayoune et al. 1998; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Sarvašová et al. 2014 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) paolae Boorman, 1996 Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) pictipennis (Staeger, 1839) Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 ; Remm 1988a ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) pseudopallidus Khalaf, 1961 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Safi, MA , Rhafsai, Fès-Meknès, HA , Marrakech; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) shaklawensis Khalaf, 1957 Kremer et al. 1975 , AP , Safi, MA , Sefrou, Fès-Meknès, HA , Setti Fatma, Marrakech; Chaker et al. 1979 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) simulator Edwards, 1939 Kremer et al. 1975 , MA , Ifrane, Fès-Meknès, HA , Setti Fatma; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) univittatus Vimmer, 1932 = Culicoides agathensis Callot, Kremer and Rioux, in Bailly-Choumara and Kremer 1970 : 386, Kremer et al. 1971 : 663, Chaker et al. 1980 : 82, Dakki 1997 : 60 Bailly-Choumara and Kremer 1970 , AP , estuaire Bou-Regreg; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Rif , Tanger-Tétouan-Al Hoceima; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, HA , Marrakech; Chaker et al. 1980 , Rif , Tétouan, AP , Larache, Sidi Bettache MA , Oulmès; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) vidourlensis Callot, Kremer, Molet & Bach, 1968 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), estuaire de Oued Bou-Regreg, HA , Souk Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 ; Remm 1988a ; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) pallidicornis Kieffer, 1919 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Dakki 1997 ; Balenghien et al. 2014 , SA , Guelmim-Oued Noun; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) picturatus Kremer & Deduit, 1961 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache); Kremer et al. 1971 , Rif , Talerhza, EM , El-Harcha, AP , Bousselham, Rabat-Salé-Kénitra, MA , Oulmès; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat, MA , Béni Mellal-Khénifra; Remm 1988a ; Dakki 1997 ; Sarvašová et al. 2014 ; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) subfasciipennis Kieffer, 1919 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, Béni Mellal-Khénifra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1980, AP , Larache, Zaers, Rabat, Aïn Chok, MA , Sefrou; Remm 1988a ; Hervy et al. 1994 ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Wirthomyia ) faghihi Navai, 1971 Kremer et al. 1975 , AA , Tafraout, Souss-Massa; Chaker et al. 1979 ; Remm 1988a ; Hervy et al. 1994 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Wirthomyia ) minutissimus (Zetterstedt, 1855) Referred as C. pumilus Culicoides ( Wirthomyia ) pumilus (Winnertz, 1852) Kremer et al. 1975 , AP , Safi, MA , Ifrane, Imouzzer-du-Kander, Fès-Meknès, HA , Setti Fatma, Marrakech; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides calloti Kremer, Delécolle, Bailly-Choumara & Chaker, 1979 Chaker et al. 1979 , AA , Souss Massa, SA , Guelmim-Oued Noun; Kremer et al. 1979 , AA , Tarhjigt, Aït Ouaballi, Souss Massa, SA , Guelmim-Oued Noun; Chaker et al. 1980 ; Remm 1988a ; Hervy et al. 1994 ; Borkent and Wirth 1997 ; Baylis et al. 1997 ; Dakki 1997 ; Koçak and Kemal 2010 ; Borkent 2012 ; Bourquia et al. 2019 Ceratopogonini Alluaudomyia Kieffer, 1913 Alluaudomyia hygropetrica Vaillant, 1954 Vaillant 1956b , HA , Sidi Chamarouch Palpomyiini Bezzia Kieffer, 1899 Bezzia ( Bezzia ) nigritula (Zetterstedt, 1838) = Palpomyia tenebricosa Goetghebuer, 1912, in Vaillant 1956b : 241 Vaillant 1956b , HA , Tamesrit Palpomyia Meigen, 1818 Palpomyia helviscutellata Borkent, in Borkent & Wirth 1997 = Dasyhelea flavoscutellata (Zetterstedt, 1850), in Vaillant 1956b : 244 Vaillant 1956b , HA , Tahanaout Forcipomyiinae Dasyheleini Dasyhelea Kieffer, 1911 Dasyhelea ( Prokempia ) flaviventris (Goetghebuer, 1910) Dominiak 2012 Dasyhelea ( Pseudoculicoides ) turficola Kieffer, 1925 Dominiak 2012 Leptoconopinae Leptoconops Skuse, 1889 Leptoconops ( Holoconops ) laurae (Weiss, 1912) Remm 1988b CHIRONOMIDAE K. Kettani Number of species: 412 . Expected: 600 Faunistic knowledge of the family in Morocco: good Buchonomyiinae Buchonomyia Fittkau, 1955 Buchonomyia thienemanni Fittkau, 1955 Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe et al. 2015 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Moubayed-Breil 2018 , Rif Podonominae Paraboreochlus Thienemann, 1939 Paraboreochlus minutissimus (Strobl, 1895) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Tanypodinae Macropelopiini Apsectrotanypus Fittkau, 1962 Apsectrotanypus trifascipennis (Zetterstedt, 1838) Kettani et al. 2010 , Rif , Aïn Abou Hayane (Tiouertiouane, 880 m), Oued Maggou (Maggou village, 777 m), Oued Kanar (Gorges Kanar, 280 m); Kettani and Langton 2012 Macropelopia Thienemann, 1916 Macropelopia adaucta Kieffer, 1916 Kettani and Langton 2011 , Rif , Fifi, Issaguen; Kettani and Langton 2012 Macropelopia nebulosa (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrotanypus Kieffer, 1909 Psectrotanypus varius (Fabricius, 1787) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Pentaneurini Ablabesmyia Johannsen, 1905 Ablabesmyia ( Ablabesmyia ) ebbae Lehmann, 1981 Lehmann 1981 ; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Ablabesmyia ( Ablabesmyia ) longistyla Fittkau, 1962 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , source Maggou (Maggou, 1300 m), Oued Talembote; Kettani and Langton 2012 Ablabesmyia ( Ablabesmyia ) monilis (Linnaeus, 1758) Reiss 1977 , Rif , Tétouan, HA , kranichsee (Dra-Tal); Azzouzi and Laville 1987 , Rif , retenue El Makhazine; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Conchapelopia Fittkau, 1957 Conchapelopia ( Conchapelopia ) melanops (Meigen, 1818) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Conchapelopia ( Conchapelopia ) pallidula (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Conchapelopia ( Conchapelopia ) viator (Kieffer, 1911) = Conchapelopia Pe 1 Langton 1991 in Kettani et al. 1994 : 28, Kettani et al. 1995 : 256 Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 ; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Larsia Fittkau, 1962 Larsia atrocincta (Goetghebuer, 1942) Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , Oued Moulay Bouchta; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Larsia curticalcar (Kieffer, 1918) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m); Kettani and Langton 2012 Nilotanypus Kieffer, 1923 Nilotanypus dubius (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kanar (Gorges Kanar, 280 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paramerina Fittkau, 1962 Paramerina cingulata (Walker, 1856) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paramerina divisa (Walker, 1856) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Paramerina mauretanica Fittkau, 1962 Fittkau 1962 , Atlas (850 m), SA ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 , EM , Figuig; Kettani et al. 2001 ; Ashe and O'Connor 2009 , EM , Figuig; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Paramerina spec. Greichenland (Fittkau, 1962) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani et al. 2010 , Rif , Oued Chrafat (Armotah, 900 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Moubayed-Breil 2018 , Rif Pentaneurella Fittkau & Murray, 1983 Pentaneurella sp. Ourika Azzouzi et al. 1992 , HA ; Kettani et al. 2001 Rheopelopia Fittkau, 1962 Rheopelopia maculipennis (Zetterstedt, 1838) Naya 1988 , MA , Haut et Moyen Sebou; Azzouzi and Laville 1987 , MA , Oum-er-Rbia, HA , Tensift; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Ruisselet maison forestière (Talassemtane, 1683 m), Source Maggou (Maggou, 1300 m), Oued Talembote (avant village Talembote, 320 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheopelopia murrayi Dowling, 1983 Dowling 1983 , AA , Tata (Moyen Draa); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; 2012 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheopelopia ornata (Meigen, 1838) Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Telopelopia Roback, 1971 Telopelopia fascigera (Verneaux, 1970) = Telopelopia maroccana Murray, 1980, in Reiss 1977 : 91, Murray 1980 : 151, Azzouzi and Laville 1987 : 218, Ashe and Cranston 1990 : 133 Reiss 1977 , AP , Larache, HA , Dra-Tal; Murray 1980 , AP , Larache, HA , Dra-Tal; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Telmatopelopia Fittkau, 1962 Telmatopelopia nemorum (Goetghebuer, 1921) Kettani et al. 1996 , Rif , Oued Khizana (Oued Laou); Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Thienemannimyia Fittkau, 1957 Thienemannimyia ( Thienemannimyia ) berkanea Dowling, 1987 Dowling 1987 , EM , Berkane; Azzouzi et al. 1992 , EM , Environs de Berkane, HA , Ouarzazate (1160 m), Oasis Meski (1160 m), Aït Saoun; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) carnea (Fabricius, 1805) Kettani and Langton 2012 , Rif Thienemannimyia ( Thienemannimyia ) choumara Dowling, 1983 Dowling 1983 , EM , Environ de Berkane (Monts de Bni Snassen), HA , Souk des Judais (Marrakech); Azzouzi et al. 1987, HA , Dra-Tal; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Thienemannimyia ( Thienemannimyia ) geijskesi (Goetghebuer, 1934) Kettani and Langton 2012 , Rif , Oued Zarka Thienemannimyia ( Thienemannimyia ) laeta (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) lentiginosa (Fries, 1823) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) northumbrica (Edwards, 1929) Fittkau 1962 ; Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Trissopelopia Kieffer, 1923 Trissopelopia longimana (Staeger, 1839) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Xenopelopia Fittkau, 1962 Xenopelopia falcigera (Kieffer, 1911) Kettani and Langton 2011 , Rif , Anasser, Fifi, AP , marais de Loukous; Kettani and Langton 2012 Xenopelopia nigricans (Goetghebuer, 1927) Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia Fittkau, 1962 Zavrelimyia ( Zavrelimyia ) barbatipes (Kieffer, 1911) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 (?); Kettani et al. 2010 , Rif , Oued Tiffert (Tiffert Talassemtane, 1230 m), Aïn Abou Hayane (Tiouertiouane, 880 m), Oued Abiyati (Ifansa, 140 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) berberi Fittkau, 1962 Azzouzi and Laville 1987 ; Ashe and Cranston 1990 , HA , Tamhda; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) hirtimana (Kieffer, 1918) Kettani and Langton 2012 Zavrelimyia ( Zavrelimyia ) melanura (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) nubila (Meigen, 1830) Kettani and Langton 2011 , Rif , marais de Lemtahane ( PNPB ), Dayat Aïn Rami, Dayat Amlay; Kettani and Langton 2012 Procladiini Procladius Skuse, 1889 Procladius ( Holotanypus ) brevipetiolatus (Goetghebuer, 1935) Azzouzi et al. 1992 , HA , Oued Meski (1160 m), Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Procladius ( Holotanypus ) choreus (Meigen, 1804) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , AP , Merja Sidi Boughaba; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2010 , Rif , Aïn Talassemtane (Talassemtane, 1700 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Procladius ( Holotanypus ) culiciformis (Linnaeus, 1767) Kettani and Moubayed-Breil 2018 , Rif Procladius ( Holotanypus ) noctivagus (Kieffer, 1910) Azzouzi et al. 1992 , HA , Ouarzazate (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 Procladius ( Holotanypus ) sagittalis (Kieffer, 1909) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Procladius ( Psilotanypus ) anomalus Kieffer, 1906 Nomen dubium in Ashe and O'Connor 2009 : 213 Naya 1988 , MA ; Kettani et al. 2001 ; Kettani and Langton 2012 Procladius Pe 3 Langton 1991 Kettani et al. 1994 , Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 Tanypodini Tanypus Meigen, 1803 Tanypus ( Tanypus ) brevipalpis (Kieffer, 1923) Reiss 1977 , EM , Berkane; Ashe and O'Connor 2009 (?); Kettani and Langton 2012 Tanypus ( Tanypus ) kraatzi (Kieffer, 1912) Azzouzi et al. 1992 , HA , Oasis Meski; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Tanypus ( Tanypus ) punctipennis Meigen, 1818 Reiss 1977 , EM , Berkane; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesinae Boreoheptagyiini Boreoheptagyia Brundin, 1966 Boreoheptagyia legeri (Goetghebuer, 1933) = Boreoheptagyia punctulata (Goetghebuer, 1934), in Kettani et al. 2001 : 327 Ashe and Cranston 1990 ; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesini Diamesa Meigen, 1835 Diamesa aberrata Lundbeck, 1898 Saether 1968 ; Serra-Tosio 1973 ; Fittkau and Reiss 1987 ; Serra-Tosio 1983 ; Azzouzi and Laville 1987 , HA (2500–3350 m); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa bertrami Edwards, 1935 Serra-Tosio 1983 , HA , Gorges de Todra (2500 m); Azzouzi and Laville 1987 , HA , Gorges Todra; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa hamaticornis Kieffer, 1924 Reiss 1977 ; Serra-Tosio 1983 , HA , M'Goum; Azzouzi and Laville 1987 , HA , M'Goum; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa insignipes Kieffer, 1908 Serra-Tosio 1983 , HA (2500 m); Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut and Moyen Sebou; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Diamesa latitarsis (Goetghebuer, 1921) Vaillant 1955b ; Vaillant 1956b , HA , Asif Tessaout (M'Goum), Lac Tamhda (Anremer); Serra-Tosio 1967 ; Serra-Tosio 1967 ; Saether 1968 ; Serra-Tosio 1973 ; Azzouzi and Laville 1987 , HA ; Ashe and Cranston 1990 ; Kettani et al. 2001 , Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa steinboecki Goetghebuer, 1933 Vaillant 1956b , HA , Cascade Siroua, Oukaimeden, Sidi Chamarouch Diamesa tonsa (Haliday in Walker, 1856) = Diamesa thienemanni Kieffer, 1909 Naya 1988 , MA , Haut Sebou (Arhbalou Yahya, Oued Arbi, Pont Aït hamza); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 Diamesa vaillanti Serra-Tosio, 1972 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa veletensis Serra-Tosio, 1971 Serra-Tosio 1983 , HA (2500 m); Azzouzi and Laville 1987 , HA ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa zernyi Edwards, 1933 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Potthastia Kieffer, 1922 Potthastia gaedii (Meigen, 1838) Azzouzi and Laville 1987 , MA , oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Laou, Oued Afertane; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Potthastia pastoris (Edwards, 1933) Kettani and Moubayed-Breil 2018 , Rif Pseudodiamesa Goetghebuer, 1939 Pseudodiamesa ( Pseudodiamesa ) branickii (Nowicki, 1873) Naya 1988 , MA , Haut Sebou; Ashe and Cranston 1990 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Pseudodiamesa ( Pseudodiamesa ) nivosa (Goetghebuer, 1928) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Sympothastia Pagast, 1947 Sympothastia zavreli Pagast, 1947 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , aval Oued Krikra; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Syndiamesa Kieffer, 1918 Syndiamesa hygropterica (Kieffer, 1909) Naya 1988 , MA , Moyen Sebou (Sidi Abdellah, Dar El Arsa, Pont Oulad Slimane, Pont Portugais); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Protanypini Protanypus Kieffer, 1906 Protanypus morio (Zetterstedt, 1838) Naya 1988 , MA , Moyen Sebou; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Prodiamesinae Odontomesa Pagast, 1947 Odontomesa fulva (Kieffer, 1919) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Prodiamesa Kieffer, 1906 Prodiamesa olivacea (Meigen, 1818) Naya 1988 , MA , Haut Sebou (Haut Guigou); Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Maggou village, Ifansa; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladiinae Orthocladiini Acricotopus Kieffer, 1921 Acricotopus lucens (Zetterstedt, 1850) Kettani and Moubayed-Breil 2018 , Rif Brilla Kieffer, 1913 Brillia bifida (Kieffer, 1909) = Brilla modesta (Meigen, 1830) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Brillia flavifrons (Johannsen, 1905) Kettani and Langton 2012 Brilla longifurca Kieffer, 1921 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1995 , Rif , amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Talembote (Usine électrique, 120 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Bryophaenocladius Thienemann, 1934 Bryophaenocladius aestivus (Brundin, 1947) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius flexidens (Brundin, 1947) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius cf. furcatus Thienemann & Strenzke, 1940 Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius illimbatus (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius muscicola (Kieffer, 1906) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius nidorum (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius subvernalis (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius tuberculatus (Edwards 1929) Kettani and Moubayed-Breil 2018 , Rif Camptocladius Wulp, 1874 Camptocladius stercorarius (De Geer, 1976) Kettani and Moubayed-Breil 2018 , Rif Cardiocladius Kieffer, 1912 Cardiocladius capucinus (Zetterstedt, 1850) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cardiocladius fuscus Kieffer, 1924 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou (Amont de Aïn Tadout, Skhounate, amont confluence avec Oued Atchane, Pont Aït Hamza); Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius Kieffer, 1911 Chaetocladius ( Chaetocladius ) acuticornis (Kieffer in Potthast, 1914) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius dentiforceps (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Chaetocladius dissipatus (Edwards, 1929) Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Chaetocladius ( Chaetocladius ) melaleucus (Meigen, 1818) Kettani and Langton 2011 , Rif , Oued Sgara, Bab Tariouant, Bouztata; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius piger (Goetghebuer, 1913) Kettani and Moubayed-Breil 2018 , Rif Chaetocladius ( Chaetocladius ) perennis (Meigen, 1830) Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 Chaetocladius ( Chaetocladius ) vitellinus (Kieffer in Kieffer & Thienemann, 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Corynoneura Winnertz, 1846 Corynoneura carriana Edwards, 1924 Naya 1988 , MA , Haut Sebou (Haut Guigou, Aïn Nokra); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura celtica Edwards, 1924 Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura coronata Edwards, 1924 Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Corynoneura edwardsi Brundin, 1949 Kettani and Langton 2012 Corynoneura lacustris Edwards, 1924 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura lobata Edwards, 1924 Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Azzouzi et al. 1992 , HA ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura scutellata Winnertz, 1846 Kettani and Moubayed-Breil 2018 , Rif Corynoneura Pe 2 Langton 1991 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Corynoneurella Brundin, 1949 Corynoneurella paludosa Brundin, 1949 Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus van der Wulp, 1874 Cricotopus ( Cricotopus ) albiforceps (Kieffer in Thienemann and Kieffer 1916) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) annulator Goetghebuer, 1927 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2011 , Rif , Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) beckeri Hirvenoja, 1973 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Langton and Laville 2002; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) bicinctus (Meigen, 1818) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Naya 1988 , MA , Haut et Moyen Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) caducus Hirvenoja, 1973 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) ephippium (Zetterstedt, 1838) Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) levantinus Moubayed & Hirvenoja, 1986 Kettani et al. 1996 , Rif , Haut Maggou; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Inesmane (Adeldal, 1173 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Cricotopus ) pallidipes Edwards, 1929 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) pulchripes Verrall, 1912 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) similis Goetgnebuer, 1921 Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Afertane, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) tremulus (Linnaeus, 1758) Kettani et al. 2010 , Rif , Oued Maggou (Maggou village, 777 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) triannulatus (Macquart, 1826) Kettani et al. 1994 , Rif , Haut Laou, Oued Moulay Bouchta, Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) trifascia Edwards, 1929 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Maggou (Maggou village, 777 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) vierriensis Goetghebuer, 1935 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Isocladius ) brevipalpis Kieffer, 1909 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) laetus Hirvenoja, 1973 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) ornatus (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) sylvestris (Fabricius, 1794) Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Isocladius ) tricinctus (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) micans (Kieffer, 1918) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Talembote, Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) osellai Rossaro, 1990 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) rufiventris (Meigen, 1830) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; (Dar El Arsa, Pont oulad Slimane, Pont portugais); Naya 1988 , MA , Haut et Moyen Sebou; Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) skirwithensis (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukieferiella Thienemann, 1926 Eukieferiella ancyla Svensson, 1986 Kettani and Langton 2011 , Rif , Oued Tkarae; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella bedmari Vilchez-Quero & Laville, 1988 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Laville and Langton 2002 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella brehmi Gowin, 1943 Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (Usine électrique, 120 m) Eukiefferiella brevicalcar (Kieffer, 1911) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m), Oued Ametrasse (Ametrasse, 820 m); Kettani and Langton 2011 , Rif , Oued Issaguen, Oued Ketama, Oued Sgara; Bab Tariouant, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella claripennis (Lundbeck, 1898) Fittkau and Reiss 1978 ; Naya 1988 , MA , Moyen Sebou (Dar Cheik Harazem); Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukieffeiella clypeata (Thienemann, 1919) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella coerulescens (Kieffer in Zavřel, 1926) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou (Skhounate, Arhbalou Aberchane); Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Nord Maggou village (Maggou, 905 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella cyanea Thienemann, 1936 Vaillant 1955b , HA ; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , HA ; Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella devonica (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella dittmari Lehmann, 1972 Kettani and Langton 2011 , Rif , Oued Boujdad, Fifi; Kettani and Langton 2012 , Rif , Oued Zarka; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella fittkaui Lehmann, 1972 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella fuldensis Lehmann, 1972 Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella gracei (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Tassikeste; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella ilkleyensis (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella lobifera Goetghebuer, 1934 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella minor (Edwards, 1929) Vaillant 1955b , HA (1050 m); Vaillant 1956b , HA , Imi-N'Ifri; Azzouzi and Laville 1987 , HA ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella pseudomontana Goetghebuer, 1935 Kettani et al. 2010 , Rif , Oued Madissouka (Talassemtane, 1530 m), Oued Dchar d'Amran (Béni M'Hamed, 1180 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella similis Goetghebuer, 1939 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella tirolensis Goetghebuer, 1938 Kettani et al. 1994 , Rif , Oued Afertane; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Azzouzi et al. 1992 , HA , Oued Tensift; Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella Pe 2 Langton 1991 Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Halocladius Hirvenoja, 1973 Halocladius ( Halocladius ) varians (Staeger, 1839) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m); Ashe and Cranston 1990 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heleniella Gowin, 1943 Heleniella dorieri Serra-Tosio, 1967 Kettani and Langton 2012 Heleniella ornaticollis (Edwards, 1929) Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heleniella serratosioi Ringe, 1976 Kettani and Langton 2011 , Rif , Oued Hamma, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heterotrissocladius Spärck, 1923 Heterotrissocladius marcidus (Walker, 1856) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2012 Hydrobaenus Fries, 1830 Hydrobaenus conformis (Holmgren, 1869) Kettani and Moubayed-Breil 2018 , Rif Hydrosmittia Ferrington & Sæther, 2011 Hydrosmittia oxoniana (Edwards, 1929) = Pseudosmittia recta (Edwards, 1929), in Azzouzi and Laville 1987 : 218, Kettani et al. 2001 : 330, Kettani and Langton 2012 : 422 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Hydrosmittia ruttneri (Strenzke & Thienemann, 1942) Kettani and Moubayed-Breil 2018 , Rif Krenosmittia Thienemann & Krüger, 1939 Krenosmittia boreoalpina (Goetghebuer, 1944) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Krenosmittia camptophleps (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Krenosmittia halvorseni (Cranston & Sæther, 1986) Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Krenosmittia hispanica Wülker, 1957 Kettani et al. 2001 ; Laville and Langton 2002 ; Ashe and O'Connor 2012 Limnophyes Eaton, 1875 Limnophyes difficilis Brunidin, 1947 Kettani and Moubayed-Breil 2018 , Rif Limnophyes gelasinus Saether, 1990 Kettani and Moubayed-Breil 2018 , Rif Limnophyes habilis (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Limnophyes madeirae Sæther, 1985 Kettani and Moubayed-Breil 2018 , Rif Limnophyes minimus (Meigen, 1818) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Kettani et al. 2001 ; Kettani and Langton 2011 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Limnophyes natalensis (Kieffer, 1914) Kettani and Moubayed-Breil 2018 , Rif Limnophyes ninae Sæther, 1975 Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Limnophyes pentaplastus (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Limnophyes pumilio (Holmgren, 1869) Kettani and Moubayed-Breil 2018 , Rif Limnophyes punctipennis (Goetghebuer, 1919) Kettani and Langton 2012 Limnophyes Pe 1a Langton 1991 Kettani et al. 2010 , Rif , Oued Talembote (Usine électrique, 120 m); Kettani and Langton 2012 Metriocnemus van der Wulp, 1874 Metriocnemus ( Metriocnemus ) albolineatus Meigen, 1818 Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) eurynotus (Holmgren, 1883) = Metriocnemus hygropetricus Kieffer, 1912, in Ashe and O'Connor 2012 : 372 = Metriocnemus ( Metriocnemus ) obscuripes (Holmgren, 1869), in Azzouzi et al. 1992 : 229, Kettani et al. 2001 : 329, Kettani and Langton 2012 : 421 Boumezzough and Thomas 1987 , HA , Oued Réghaya (1740 m), Imlil; Azzouzi and Laville 1987 , HA , Oued Tensift; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Metriocnemus ( Metriocnemus ) fuscipes (Meigen, 1818) Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) hirticollis (Staeger, 1839) Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) ursinus Holmgren, 1869 Kettani and Moubayed-Breil 2018 , Rif Nanocladius Kieffer, 1913 Nanocladius ( Nanocladius ) balticus (Palmén, 1959) Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) dichromus (Kieffer 1906) Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) parvulus (Kieffer 1909) Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) rectinervis (Kieffer, 1911) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Inesmane (Adeldal, 1173 m), Oued Talembote (aval Barrage Talembote, 245 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius van der Wulp, 1874 Orthocladius ( Eudactylocladius ) fuscimanus (Kieffer, 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued Krikra, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , source Maggou (Maggou, 1300 m), Oued Chrafat (Armotah, 900 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) ashei Soponis, 1990 = Orthocladius luteipes Goetghebuer, in Azzouzi and Laville 1987 : 218 = Orthocladius rivicola Kieffer, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Fès, Oued boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique), aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) rivulorum Kieffer, 1909 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor; 2012 Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) thienemanni Kieffer, 1906 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Mesorthocladius ) frigidus (Zetterstedt, 1838) Vaillant 1955, HA (2900 m); Vaillant 1956b , HA , Lac Tamhda (Anremer); Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut Sebou (Haut Guigou); Fekhaoui et al. 1993 ; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Dchar d'Amran (Béni M'Hamed, 1180 m), Nord Maggou village (Maggou, 905 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Orthocladius ( Orthocladius ) oblidens (Walker, 1856) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) obumbratus Johannsen, 1905 = Orthocladius excavatus Brundin, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Orthocladius ( Orthocladius ) pedestris Kieffer, 1909 Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) rubicundus (Meigen, 1818) = Orthocladius saxicola Kieffer, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) vaillanti Langton & Cranston, 1991 Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Symposiocladius ) lignicola Kieffer in Potthast, 1914 = Symposiocladius lignicola Kieffer, in Kettani et al. 2010 : 70 Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Orthocladius ( Symposiocladius ) ruffoi Rossaro & Prato, 1991 = Orthocladius Pe 1 Langton 1991, in Azzouzi and Laville 1987 : 218 = Rheortocladius sp A Langton 1991, in Kettani et al. 1995 : 257 = Rheorthocladius ruffoi Rossaro, in Kettani et al. 1997 : 184 Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Ifansa, 105 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Paracricotopus Brundin, 1956 Paracricotopus niger (Kieffer, 1913) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès; Kettani et al. 1994 , Rif , Oued Afertane; Kettani et al. 1995 , Rif , amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou, Oued Tamaridine (Zaouit et Habtyiène, 819 m), Oued Kelaâ (Akoumi, 400 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (155 m), Oued Tassikeste (240 m), Oued Laou (Ifansa, 105 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parakiefferiella Thienemann, 1936 Parakiefferiella coronata (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parakiefferiella wuelkeri Moubayed, 1994 = Parakiefferiella sp. d Wülker, in Azzouzi et al. 1992 : 230 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parametriocnemus Goetghebuer, 1932 Parametriocnemus boreoalpinus Gowin & Thienemann, 1942 Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parametriocnemus stylatus (Spärck, 1923) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Béni M'Hamed (1330 m), Haut Maggou (1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (320 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Parametriocnemus valescurensis Moubayed & Langton, 1999 Kettani and Langton 2011 , Rif , Oued Issaguen; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parametriocnemus Pe 1 Langton 1991 Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Talembote (320 m); Kettani and Langton 2012 Paraphaenocladius Thienemann, 1924 Paraphaenocladius exagitans ssp. 1 Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius impensus impensus (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius irritus Walker, 1856 Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius pseudirritus Strenzke, 1950 Kettani and Moubayed-Breil 2018 , Rif Paratrissocladius Zavřel, 1937 Paratrissocladius excerptus (Walker, 1856) Kettani et al. 1996 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parorthocladius Thienemann, 1935 Parorthocladius nudipennis (Kieffer in Kieffer & Thienemann 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psecrocladius Kieffer, 1906 Psectrocladius ( Allopsectrocladius ) obvius (Walker, 1856) = Psectrocladius dilatatus (van der Wulp, 1859), in Naya 1988 : 48 Naya 1988 , MA , Moyen Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2011 , Rif , sources de Issaguen; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Allopsectrocladius ) platypus (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Mesopsectrocladius ) barbatipes Kieffer, 1923 Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Laou (Afertane, 56 m); Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psecrocladius ( Psectrocladius ) brehmi Kieffer, 1923 Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psectrocladius ( Psectrocladius ) fennicus Storå, 1939 Kettani and Langton 2012 Psectrocladius ( Psectrocladius ) limbatellus (Holmgren, 1869) Wülker 1959 ; Azzouzi and Laville 1987 , HA , Lac Tamhda (2800 m); Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Psectrocladius ) octomoculatus Wülker, 1956 Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psectrocladius ( Psectrocladius ) sordidellus (Zetterstedt, 1838) Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous (NE Boucharene); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Psectrocladius ) ventricosus Kieffer, 1925 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Pseudosmittia Edwards, 1932 Pseudosmittia albipennis (Goetghebuer, 1921) Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia baueri Strenzke, 1960 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia danconai (Marcuzzi, 1947) Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia holsata Thienemann & Stenzke, 1940 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia obtusa Strenzke, 1960 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia trilobata Edwards, 1929 Kettani and Moubayed-Breil 2018 , Rif Pseudorthocladius Goetghebuer, 1943 Pseudorthocladius ( Pseudorthocladius ) berthelemyi Moubayed, 1990 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Pseudorthocladius ( Pseudorthocladius ) curtistylus (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oasis Meski (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Pseudorthocladius near Pe 3 Langton 1991 Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 Rheocricotopus Brundin, 1956 Rheocricotopus ( Psilocricotopus ) atripes (Kieffer, 1913) = Rheocricotopus ( Psilocricotopus ) foveatus foveatus (Edwards, 1929), in Naya 1988 : 40 Naya 1988 , MA , Haut Sebou (Haut Guigou); Azzouzi et al. 1992 , HA , Oued Tensift, Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , Haut Laou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote, Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 56 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) chalybeatus subsp. chalybeatus (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) gallicus Lehamnn 1969 Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) glabricollis (Meigen, 1830) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Ametrasse (Ametrasse, 820 m), Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) meridionalis Moubayed-Breil, 2016 Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) tirolus Lehmann, 1969 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) effusus (Walker, 1856) Reiss 1977 ; Naya 1988 , MA , Haut et Moyen Sebou; Fekhaoui et al. 1993 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) fuscipes (Kieffer, 1909) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Maggou (905 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) rifensis Moubayed & Kettani, 2019 Moubayed-Breil and Kettani, Rif , Chrafate, Challal Sghir (Akchour) Synorthocladius Thienemann, 1935 Synorthocladius semivirens (Kieffer, 1909) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Smittia Holmgren, 1869 Smittia alpicola Goetghebuer, 1941 Kettani and Moubayed-Breil 2018 , Rif Smittia aterrima Meigen, 1818 Kettani and Moubayed-Breil 2018 , Rif Smittia contingens Walker, 1856 Kettani and Moubayed-Breil 2018 , Rif Smittia foliacea (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Smittia pratorum Goetghebuer, 1927 Kettani and Moubayed-Breil 2018 , Rif Thienemannia Kieffer, 1909 Thienemannia cf. fulvofasciata (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Thienemannia gracilis Kieffer, 1909 Kettani and Moubayed-Breil 2018 , Rif Thienemanniella Kieffer, 1911 Thienemanniella acuticornis (Kieffer, 1912) Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (320 m); Kettani and Langton 2011 , Rif , Oued Hamma, Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Thienemanniella clavicornis (Kieffer, 1911) Kettani and Moubayed-Breil 2018 , Rif Thienemanniella majuscula (Edwards, 1924) Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemanniella vittata (Edwards, 1924) Kettani et al. 1996 , Rif , Haut Maggou; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou (1300 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemanniella Pe 2a Langton 1991 Kettani et al. 2010 , Rif , Oued Maggou (905 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote; Kettani and Langton 2012 Thienemanniella Pe 2b Langton 1991 Kettani et al. 2010 , Rif , Oued Maggou (905 m), Oued Talembote; Kettani and Langton 2012 Trissocladius Kieffer, 1908 Trissocladius brevipalpis Kieffer in Kieffer & Thienemann 1908 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia Kieffer, 1922 Tvetenia bavarica (Goetghebuer, 1934) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia calvescens (Edwards, 1929) Naya 1988 , MA , Moyen Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m), Oued Talembote (245 m), Oued Laou (Afertane, 56 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tvetenia discoloripes (Goetghebuer & Thienemann in Thienemann, 1936) Kettani and Langton 2011 , Rif , Oued Nakhla, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia verralli (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, amont Oued Nakhla; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , ruisselet maison forestière Talassemtane (1683 m), Oued Tamaridine (Zaouiet et Habtiyiène, 819 m), Oued Talembote (245 m), Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zalutschia Lipina, 1939 Zalutschia humphriesiae Dowling & Murray, 1980 Kettani and Langton 2011 , Rif , marais de Lemtahane ( PNPB ), Dayat Fifi; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Chironominae Chironomini Chironomus Meigen, 1803 Chironomus ( Baeotendipes ) noctivagus (Kieffer, 1911) Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) annularius Meigen, 1818 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) aprilinus sensu Meigen, 1818 = Chironomus halophilus Kieffer, in Ramdani and Tourenq 1982 : 180, in Naya 1988 : 50 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut Sebou; Fekhaoui et al. 1993 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) bernensis Klötzli, 1973 = Chironomus sp 1 Kettani 1994 Kettani et al. 1994 ; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) calipterus Kieffer, 1908 Reiss 1977 , AP , Larache; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) longistylus Goetghebuer, 1921 Kettani et al. 2011, Rif , Oued Ketama; Kettani and Langton 2012 Chironomus ( Chironomus ) luridus Strenzke, 1959 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) nuditarsis Keyl, 1961 Kettani et al. 2011, Rif , Oued Boujdad (Kitane, 42 m), Oued El Hatba (SIBE Jebel Moussa, 165 m); Kettani and Langton 2012 , Rif , SIBE Jebel Moussa Chironomus ( Chironomus ) piger (Strenzke, 1956) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) plumosus (Linnaeus, 1758) Reiss 1977 , AP , Larache, AA , Dra-Tal; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 ; Naya 1988 , MA , Sidi Abdellah, Dar Cheih Harazem, Dar El Arsa; Fekhaoui et al. 1993 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Aïn Tissmelal (Tissmelal, 1046 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) prasinus sensu Pinder, 1978 Kettani et al. 2011, Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 Chironomus ( Chironomus ) riparius Meigen, 1804 = Chironomus thummi Kieffer, in Naya 1988 : 51, Fekhaoui et al. 1993 : 26 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou; Naya 1988 , MA , Moyen Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) salinarius Kieffer, 1915 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) tentans Fabricius, 1805 = Camptochironomus tentans Fabricius, 1805, in Naya 1988 : 50 Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Cladopelma Kieffer, 1921 Cladopelma virescens (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus Kieffer, 1918 Cryptochironomus ( Cryptochironomus ) albofasciatus (Staeger, 1839) = Cryptochironomus obreptans Walker, 1856, in Kettani 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 Cryptochironomus ( Cryptochironomus ) psittacinus (Meigen, 1830) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Cryptochironomus ( Cryptochironomus ) rostratus Kieffer, 1921 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou, oued Oum-er-Rbia, Oued Boufekrane, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus ( Cryptochironomus ) supplicans (Meigen, 1830) Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus Pe 5 Langton 1991 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 Demicryptochironomus Lenz, 1941 Demicryptochironomus ( Demicryptochironomus ) vulneratus (Zetterstedt, 1838) Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m); Kettani and Langton 2012 Demicryptochironomus ( Irmakia ) neglectus Reiss, 1988 Kettani and Moubayed-Breil 2018 , Rif Demicryptochironomus ( Irmakia ) Pe 1 Langton 1991 Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, aval Oued Khemis; Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes Kieffer, 1913 Dicrotendipes collarti (Goetghebuer, 1936) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes cordatus Kieffer, 1922 Kettani et al. 1996 , Rif , Oued Khizana (Oued Laou); Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes fusconotatus (Kieffer, 1922) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes modestus (Say, 1823) Kettani et al. 2011, Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 Dicrotendipes nervosus (Staeger, 1839) = Limnochirononomus nervosus Staeger, in Naya 1988 : 53 Naya 1988 , MA , Moyen Sebou (Sidi Abdellah); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes notatus (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes pallidicornis (Goetghebuer, 1934) Azzouzi and Laville 1987 , Rif , Retenue El Makhazine, MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes peringueyanus Kieffer, 1924 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 Dicrotendipes septemmaculatus (Becker, 1908) = Dicrotendipes pilosimanus Kieffer, in Reiss 1977 : 91, Azzouzi and Laville 1987 : 219 Reiss 1977 , AP , Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , AP , Larache; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 Endochironomus Kieffer, 1918 Endochironomus albipennis (Meigen, 1830) Naya 1988 , MA , Haut Sebou (Skhounata); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Endochironomus tendens (Fabricius, 1775) Naya 1988 , MA , Moyen Sebou (Gantra Mdez, Azzaba); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes Kieffer, 1913 Glyptotendipes ( Caulochironomus ) viridis (Macquart, 1834) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes ( Glyptotendipes ) cauliginellus (Kieffer, 1913) = Glyptotendipes gripekoveni (Kieffer) Naya 1988 , MA , Haut Sebout (Guigou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes ( Glyptotendipes ) pallens (Meigen, 1804) Azzouzi and Laville 1987 , Rif , Retenue El Makhazine; Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes sp A Langton 1991 Naya 1988 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Glyptotendipes sp B Langton 1991 Naya 1988 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Harnischia Kieffer, 1921 Harnischia curtilamellata (Malloch, 1915) Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Harnischia fuscimanus Kieffer, 1921 Azzouzi and Laville 1987 , Rif , Retenue El Makhazine, MA , Oued Boufekrane; Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Kiefferulus Goetghebuer, 1922 Kiefferulus ( Kiefferulus ) tendipediformis (Goetghebuer, 1921) Reiss 1977 , Rif , Tétouan; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2010 , Rif , Guelta 1 km après Amariguen (Jebel Setsou, 1280 m); Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Dalia (SIBE Jebel Moussa, 169 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Kloosia Kruseman, 1933 Kloosia pusilla (Linnaeus, 1767) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Lauterborniella Thienemann & Bause, 1913 Lauterborniella agrayloides (Kieffer, 1911) Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus Kieffer, 1918 Microchironomus deribae (Freeman, 1957) = Leptochirononomus deribae Freeman, in Reiss 1977 : 91, Ramdani and Tourenq 1982 : 180 Reiss 1977 , AP , Rabat; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus lendli (Kieffer, 1918) Reiss 1986 , AA , Oasis Meski; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus tener (Kieffer, 1918) Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Azzouzi et al. 1992 , HA , Oued Tensift, Barrage Lalla Takerkoust; Kettani and Langton 2012 Microtendipes Kieffer, 1915 Microtendipes britteni (Edwards, 1929) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Microtendipes chloris (Meigen, 1818) Kettani et al. 2011, Rif , Dayat En-Nâsser (Khandek En-Nâsser, 1177 m), source Bab Karn (Fifi, 1216 m), Dayat Fifi (1179 m); Kettani and Langton 2012 Microtendipes confinis (Meigen, 1830) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 Microtendipes diffinis (Edwards, 1929) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani et al. 2011, Rif , Dayat En-Nâsser (Khandek En-Nâsser, 1177 m), Dayat Aïn Rami, source Bab Karn (Fifi, 1216 m); Kettani and Langton 2012 Microtendipes pedellus (De Geer, 1776) Reiss 1977 , Rif , Environ de Tétouan; Fittkau and Reiss 1978 ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , Rif , Tétouan, HA ; Naya 1988 , MA , Haut Sebou (amont Aîn Tadout, Skhounate, Arhbalou Aberchane); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Nubensia Spies, 2015 Nubensia nubens (Edwards, 1929) = Polypedilum nubens (Edwards, 1929), in Azzouzi and Laville 1987 : 219; Kettani et al. 1994 : 28, 1995 : 257, 1996 : 137, 1997 : 184, 2001 : 331, 2010 : 70; Dakki 1997 : 65; Dakki et al. 2008: 32, Kettani and Langton 2012 : 423 Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Laou (Ifansa, 105 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parachironomus Lenz, 1921 Parachironomus frequens (Johannsen, 1905) Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Parachironomus parilis (Walker, 1856) Reiss 1977 , AP , Environ de Larache; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Ashe and Cranston 1990 ; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Paracladopelma Harnisch, 1923 Paracladopelma camptolabis (Kieffer, 1913) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paracladopelma galaptera (Townes, 1945) Azzouzi et al. 1992 , HA , Ouarzazate (1140 m), Gorges de Todra (1400 m); Kettani et al. 2001 ; Kettani and Langton 2012 Paracladopelma graminicolor (Kieffer, 1925) = Cryptotendipes graminicolor (Kieffer), in Azzouzi et al. 1992 : 230 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Paracladopelma laminatum (Kieffer, 1921) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paracladopelma mikianum (Goetghebuer, 1937) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paralauterborniella Lenz, 1941 Paralauterborniella nigrohalteralis (Malloch, 1915) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Paratendipes Kieffer, 1911 Paratendipes albimanus (Meigen, 1818) Naya 1988 , MA , Moyen Sebou (Mdez); Kettani et al. 1994 , Rif , aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued Mhajrat, aval Oued Khemis, Oued Laou (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratendipes nudisquama (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Paratendipes striatus (Kieffer, 1925) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Phaenopsectra Kieffer, 1921 Phaenopsectra flavipes (Meigen, 1818) Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum Kieffer, 1912 Polypedilum ( Pentapedilum ) ruandae Freeman, 1955 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Pentapedilum ) sordens (van der Wulp, 1875) = Polypedilum sp 1, in Kettani et al. 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Pentapedilum ) uncinatum (Goetghebuer, 1921) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Polypedilum ) albicorne (Meigen, 1838) Naya 1988 , MA , Haut Sebou; Kettani et al. 1995 , Rif , aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) arundineti (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 Polypedilum ( Polypedilum ) laetum (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) nubeculosum (Meigen, 1804) Reiss 1977 , Rif , Environ de Tétouan; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , Rif , Environ Tétouan, MA , Oued Sebou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) nubifer (Skuse, 1889) = Polypedilum pharao Kieffer, in Reiss 1977 : 91, Naya 1998: 55, Ramdani and Tourenq 1982 : 180 Kügler and Wool 1968 ; Reiss 1977 , AP , Larache, Rabat; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 , AP , Environ de Larache, Rabat, Merja Sidi Boughaba; Naya 1988 , MA , Haut Sebou; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) pedestre (Meigen, 1830) Reiss 1977 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 1994 , Rif , aval Barrage Talembote; Kettani et al. 1995 , Rif , Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Tripodura ) acifer Townes, 1945 Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 , MA , Oued Boufekroune, Oued Fès, Oued Sebou; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Polypedilum ( Tripodura ) aegyptium Kieffer, 1925 = Polypedilum pruina Freeman, in Reiss 1977 : 91 Reiss 1977 , AP , Larache, HA , Marrakech, AA , Dra-Tal; Reiss 1985 ; Azzouzi and Laville 1987 , AP , Larache, HA , Marrakech, AA , Gorges de Todra; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Polypedilum ( Tripodura ) bicrenatum Kieffer, 1921 Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) pullum (Zetterstedt, 1838) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) quadriguttatum Kieffer, 1921 Naya 1988 , MA , Moyen Sebou; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Polypedilum ( Tripodura ) scalaenum (Schrank, 1803) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Kettani et al. 1996 , Rif , Ras el Ma (Chefchaouen); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) tetracrenatum Hirvenoja, 1962 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) tridens Freeman, 1955 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Uresipedilum ) convictum (Walker, 1856) Reiss 1977 , AP , Environ de Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane (Gantra Mdez), Naya 1988 , MA , Haut Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued pont Béni M'Hamed (Béni M'Hamed, 1330 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Uresipedilum ) cultellatum Goetghebuer, 1931 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Polypedilum ontario -group sp. 1 Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Rheomus Laville & Reiss, 1988 Rheomus alatus Laville & Reiss, 1988 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Rheomus yahiae Laville & Reiss, 1988 Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Stenochironomus Kieffer, 1919 Stenochironomus gibbus Fabricius, 1794 Kettani and Moubayed-Breil 2018 , Rif Stictochironomus Kieffer, 1919 Stictochironomus caffrarius (Kieffer, 1921) Reiss 1977 ; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 Stictochironomus maculipennis (Meigen, 1818) Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Afertane; Kettani et al. 1995 , Rif , aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Stictochironomus pictulus (Meigen, 1830) Reiss 1977 , AP , Environ de Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1995 , Rif , aval Oued Kbir, aval Oued Krikra, Oued El Kbir; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Stictochironomus rosenschoeldi Zetterstedt, 1838 Kettani and Moubayed-Breil 2018 , Rif Stictochironomus reissi Cranston, 1989 = Stictochironomus sp. nov. Reiss, in Reiss 1977 : 91 Reiss 1977 ; Azzouzi and Laville 1987 , AA , M'Hamid, Dra-Tal; Kettani et al. 2001 ; Kettani and Langton 2012 Stictochironomus sticticus (Fabricius, 1781) = Stictochironomus histrio (Fabricius, 1794), in Kettani et al. 1996 : 138 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Berranda (Bouztate, 1259 m), Dayat Dalia (SIBE Jebel Moussa); Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Stictochironomus Pe 2 Langton 1991 Kettani et al. 2001 Xenochironomus Kieffer, 1921 Xenochironomus xenolabis (Kieffer, 1916) Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsini Cladotanytarsus Kieffer, 1921 Cladotanytarsus ( Cladotanytarsus ) atridorsum Kieffer, 1924 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Azzouzi et al. 1992 , HA , Aït Saoun, Gorges de Dadès (1900 m), vallée de Drâa, Marrakech; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cladotanytarsus ( Cladotanytarsus ) capensis (Freeman, 1954) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) ecristatus Reiss, 1991 = Tanytarsus sp. nov. (Morokko) Reiss, in Azzouzi and Laville 1987 : 219 Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 , EM , Berkane; Reiss 1991 ; Azzouzi et al. 1992 , HA ; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) mancus (Walker, 1856) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cladotanytarsus ( Cladotanytarsus ) pallidus Kieffer, 1922 = Cladotanytarsus Pe 5 Langton 1984 Azzouzi and Laville 1987 , MA , Oued Sebou, Oum Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) vanderwulpi (Edwards, 1929) Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Lithotanytarsus Thienemann, 1933 Lithotanytarsus dadesi Reiss, 1991 Reiss 1991 ; Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Lithotanytarsus emarginatus (Goetghebuer, 1933) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani and Langton 2012 Micropsectra Kieffer, 1909 Micropsectra andalusiaca Marcuzzi, 1950 Kettani and Moubayed-Breil 2018 , Rif Micropsectra apposita (Walker, 1856) = Micropsectra contracta Reiss, 1965 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Chrafat (Armotah, 900 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra aristata Pinder, 1976 Kettani and Langton 2012 , Rif , Oued Zarka Micropsectra atrofasciata (Kieffer, 1911) = Micropsectra bidentata (Goetghebuer, 1921), in Azzouzi et al. 1992 : 230; Kettani et al. 2001 : 332; Kettani and Langton 2011 : 590, 2012 : 424 Fittkau and Reiss 1978 ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Sebou (Arhbalou Aberchane), Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Madissouka (Talassemtane, 1530 m), Oued Chrafat (Armotah, 900 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval affluent Talembote, 155 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam, 650 m), cascade Zarka, Dayat En-Nâsser (Khandek En-Nâsser, 1177 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra junci (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra lacustris Säwedal, 1975 Kettani and Langton 2012 , Rif , Oued Zarka Micropsectra lindrothi Goetghebuer, 1931 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra notescens (Walker, 1856) Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara, ruisselet Bab Tariouant, Oued Berranda (Bouztate, 1259 m), source Bab Karn (Fifi, 1220 m), Dayat Fifi (Fifi, 1179); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra pallidula (Meigen, 1830) Kettani and Moubayed-Breil 2018 , Rif Micropsectra schrankelae Stur & Ekrem, 2006 Kettani and Moubayed-Breil 2018 , Rif Micropsectra zernyi Marcuzzi, 1950 Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus Thienemann & Bause, 1913 Paratanytarsus bituberculatus (Edwards, 1929) Azzouzi et al. 1992 , MA , Lac Aguelmane Azigza (1510 m); Kettani et al. 1995 , Rif , Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Paratanytarsus dissimilis (Johannsen, 1905) = Paratanytarsus confusus Palmén, 1960, in Naya 1988 : 40; Dakki et al. 2008: 32; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 423 Naya 1988 , MA , Haut Sebou; Dakki et al. 2008, MA , Oued Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus grimmii (Schneider, 1885) Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Paratanytarsus inopertus (Walker, 1856) Reiss 1977 , Rif , Environ Tétouan; Fittkau and Reiss 1978 ; Reiss and Säwedal 1981 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus mediterraneus Reiss & Säwedal, 1981 Reiss and Säwedal 1981 , Rif , Estuaire Oued Mharka (Tanger), AP , Oued Loukous; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous; Kettani and Langton 2012 Paratanytarsus tenellulus (Goetghebuer, 1921) = Microspsectra tenellula Reiss 1977 : 91; Azzouzi and Laville 1987 : 219 Reiss 1977 , MA , Lac Kranichsee; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Paratanytarsus tenuis (Meigen, 1830) = Tanytarsus tenuis Meigen, in Naya 1988 : 57 Naya 1988 , MA , Moyen Sebou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Rheotanytarsus Thienemann & Bause, 1913 Rheotanytarsus ceratophylli Dejoux, 1973 Naya 1988 , MA , Moyen et Bas Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Rheotanytarsus curtistylus (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oasis Meski (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus langtoni Moubayed & Kettani, 2018 Moubayed-Breil and Kettani 2018 , Rif , Oued Farda; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Rheotanytarsus muscicola Thienemann, 1929 Reiss 1977 , AP , Environ de Larache, AA , Dra-Tal (Tissint Moyen Dra); Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus nigricauda Fittkau, 1960 Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus pellucidus (Walker, 1818) = Rheotanytarsus distinctissimus (Brundin, 1947), in Kettani et al. 1995 : 258; Kettani et al. 1996 : 138, 1997 : 185; Kettani and Langton 2012 : 424 Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus pentapoda (Kieffer, 1909) = Rheotanytarsus sp 1, in Kettani et al. 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Rheotanytarsus photophilus (Goetghebuer, 1921) Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Rheotanytarsus procerus Reiss, 1991 Reiss 1991 , HA ; Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus reissi Lehmann, 1970 Lehmann, 1970; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus rhenanus Klink, 1983 Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus ringei Lehmann, 1970 Lehmann, 1970; Reiss 1977 , Rif , Environ Tétouan; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , Rif , Tétouan, MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus Pe 3 Langton 1991 Kettani et al. 2010 ; Kettani and Langton 2011 , Rif , Oued Sgara (Ketama, 1300 m); Kettani and Langton 2012 Stempellina Thienemann & Bause, 1913 Stempellina almi Brundin, 1947 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2012 Stempellina bausei (Kieffer, 1911) Kettani and Langton 2012 , Rif , Ketama; Kettani and Moubayed-Breil 2018 , Rif Stempellinella Brundin, 1947 Stempellinella brevis (Edwards, 1929) Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Tanytarsus van der Wulp, 1874 Tanytarsus brundini Lindeberg, 1963 Kettani et al. 1994 , Rif , Oued Moulay Bouchta, aval Oued Laou; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus chinyensis Goetghebuer, 1934 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Fifi (Fifi, 1179 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus cretensis Reiss, 1987 = Tanytarsus sp. nov. ( creticus ), in Reiss 1977 : 91; Azzouzi and Laville 1987 : 219 = Cladotanytarsus sp 1, in Kettani et al. 1995 : 258 Reiss and Fittkau 1971 ; Reiss 1977 , EM , Environ de Berkane; Reiss 1987 ; Azzouzi and Laville 1987 , Rif , Tétouan, AP , Larache, Kénitra; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsus dibranchius Kieffer, 1926 = Tanytarsus separabilis Brundin, 1947, in Kettani et al. 1994 : 29; Kettani et al. 1995 : 258, 1996 : 138, 2001 : 332; Dakki 1997 : 63; Kettani and Langton 2012 : 424 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsus ejuncidus (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Tanytarsus eminulus (Walker, 1856) Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus formosanus Kieffer, 1912 = Tanytarsus horni Goetghebuer, 1934, in Reiss and Fittkau 1971 : 122; Reiss 1977 : 91; Fittkau and Reiss 1978 : 439; Ramdani and Tourenq 1982 : 180; El Mezdi and Giudicelli 1985 : 292; Azzouzi and Laville 1987 : 219; Ashe and Cranston 1990 : 341; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 424 Reiss and Fittkau 1971 , Rif , M'Diq; Reiss 1977 , Rif , Environ Tétouan, AP , Larache, Rabat, Kénitra; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , HA , Oued Tensift; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus gregarius Kieffer, 1909 Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Tanytarsus heusdensis Goetghebuer, 1923 Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Ifansa, 105 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus mendax Kieffer, 1925 Kettani and Moubayed-Breil 2018 , Rif Tanytarsus medius Reiss & Fittkau, 1971 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus palettaris Verneaux, 1969 Kettani et al. 1994 , Rif , aval Oued Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus pallidicornis (Walker, 1856) Kettani and Langton 2011 , Rif , Dayat Fifi (Fifi, 1179 m), Oued El Hatba (SIBE Jebel Moussa, 165 m); Kettani and Langton 2012 Tanytarsus recurvatus Brundin, 1947 Kettani and Langton 2011 , Rif , Oued El Hamma (El Hamma, 240 m); Kettani and Langton 2012 Tanytarsus signatus (van der Wulp, 1859) = Tanytarsus Pe 5 Langton 1991, in Azzouzi and Laville 1987 : 219 Kügler and Reiss 1973 ; Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Aïn Rami (373 m), Dayat Amlay (258 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus verralli Goetghebuer, 1928 Kettani and Langton 2011 , Rif , Oued Taida (650 m); Kettani and Langton 2012 Tanytarsus volgensis Miseiko, 1967 = Tanytarsus fimbriatus Reiss & Fittkau, 1971, in Fittkau and Reiss 1978 : 439; Azzouzi and Laville 1987 : 219; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 424 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou, HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus Pe 14 Langton 1991 Kettani and Langton 2011 , Rif , source Issaguen (Ketama, 1600 m); Kettani and Langton 2012 Tanytarsus Pe 23 Langton 1991 Kettani and Langton 2011 , Rif , Oued El Hamma (El Hamma, 240 m); Kettani and Langton 2012 Virgatanytarsus Pinder, 1982 Virgatanytarsus albisutus (Santos-Abreu, 1918) = Virgatanytarsus maroccanus Kügler and Reiss, in Azzouzi and Laville 1987 : 219 Fittkau and Reiss 1978 ; Reiss and Schurch 1984 , AA , Dra-Tal; Reiss 1986 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia, AA , Dra-Tal; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Virgatanytarsus ansatus Reiss & Schürch, 1984 Reiss and Schurch 1984 , HA ; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Virgatanytarsus arduennensis (Goetghebuer, 1922) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Virgatanytarsus triangularis (Goetghebuer, 1928) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Virgatanytarsus Pe 1 Langton 1991 Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 Zavrelia Kieffer, Thienemann & Bause, 1913 Zavrelia pentatoma Kieffer & Bause, 1913 Kettani and Langton 2012 Zavrelia Pe 1 Langton, 1991 Kettani and Langton 2011 , Rif , Oued Berranda (Bouztate, 1259 m); Kettani and Langton 2012 Acknowledgment We gratefully acknowledge the invaluable assistance and cooperation of Patrick Ashe (Dublin, Ireland) who contributed greatly to the revision of this family. SIMULIIDAE K. Kettani Number of species: 43 . Faunistic knowledge of the family in Morocco: good Simulinae Prosimuliini Helodon Enderlein, 1921 Helodon laamii (Beaucournu-Saguez and Bailly-Choumara, 1981) Beaucournu-Saguez and Bailly-Choumara 1981 , Rif ; Clergue-Gazeau et al. 1991 ; Hervy et al. 1994 ; Belqat et al. 2001a ; Belqat 2002 ; Belqat and Dakki 2004 ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium Roubaud, 1906 Prosimulium hirtipes species group Bailly-Choumara and Beaucournu-Saguez 1981 : 53–54: groupe latimucro (species nova ?); Beaucournu-Saguez and Bailly-Choumara 1981 : 119: groupe latimucro , groupe tomosvaryi and groupe rufipes - hirtipes ; Clergue-Gazeau et al. 1991 : 54 as «sp. gr. Hirtipes » Prosimulium latimucro (Enderlein, 1925) 4 Bailly-Choumara and Beaucournu-Saguez 1981 ; Beaucournu-Saguez and Bailly-Choumara 1981 ; Giudicelli and Thiery 1985 , HA ; Giudicelli and Bouzidi 1989 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'Goum en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Adler and Belqat 2001 , Rif , Oued Iouchirene, Oued Ketama (Al Hoceima); Belqat et al. 2001a , Rif , HA ; Belqat and Adler 2001 , Rif , Aïn Khandek En Nâsser, Oued Iouchirene, Oued Ketama; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Koçak and Kemal 2010 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium rachiliense Djafarov, 1954 (complex) 5 Beaucournu-Saguez and Bailly-Choumara 1981 ; Adler and Belqat 2001 ; Belqat and Adler 2001 ; Belqat 2002 ; Belqat and Dakki 2004 ; Belqat et al. 2005 ; Belqat et al. 2008 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium tomosvaryi (Enderlein, 1921) Beaucournu-Saguez and Bailly-Choumara 1981 ; Giudicelli and Thiery 1985 , HA ; Giudicelli and Bouzidi 1989 , Giudicelli et al. 2000 ; Adler and Belqat 2001 , Rif , Oued Iouchirene (Al Hoceima); Belqat and Adler 2001 , Rif , Oued Ouringa Tamdâ, oued Iouchirene, Oued Mrinet, Oued Ketama, Aîn Ksour, Oued Tisgris, Aîn Sidi Brahim Ben Arrif, Oued Hannacha; Belqat et al. 2001a , Rif ; Belqat et al. 2001b ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Koçak and Kemal 2010 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Urosimulium Contini, 1963 Urosimulium faurei (Bernard, Grenier & Bailly-Choumara, 1972) Grenier et al. 1957 , MA ; Bernard et al. 1972 : 63–68 (original description), MA , Plateau de Talerhza (environ de Meknès); Clergue-Gazeau et al. 1991 , MA ; Hervy et al. 1994 ; Belqat and Adler 2001 , Rif , Oued Iouchirene, Oued Mrinet, Oued Biyada, Oued Hannacha, Oued Ankouda; Belqat et al. 2001a , Rif , MA ; Belqat 2002 , Rif , MA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simuliini Greniera Doby & David, 1959 Greniera fabri Doby & David, 1959 Clergue-Gazeau et al. 1991 , MA ; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Metacnephia Crosskey, 1969 Metacnephia blanci (Grenier & Théodoridès, 1953) = Cnephia sp. in Grenier 1953 : 157 = Cnephia blanci Grenier and Théodoridès, in Grenier and Théodoridès 1953 : 430–435 = Eusimulium latinum Rubzov, in Benhoussa et al. 1988 : 160–164 Grenier 1953 , HA ; Grenier and Théodoridès 1953 , HA ; Grenier et al. 1957 , MA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Clergue-Gazeau et al. 1991 , AA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane: 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Metacnephia nuragica Rivosecchi, Raastad & Contini, 1975 6 = Cnephia tredecimatum (Edwards), in Grenier et al. 1957 : 226 Grenier et al. 1957 , AP , Coastal meseta (region of Rabat); Belqat et al. 2001a , AP , Rabat; Belqat 2002 , AP , Rabat; Belqat and Dakki 2004 , AP , Rabat; Belqat et al. 2011 , AP ; Belqat et al. 2018 Simulium Latreille, 1802 Simulium ( Crosskeyellum ) gracilipes Edwards, 1921 Edwards 1921 : 143 (original description), MA ; Séguy 1925 : 233, MA ; Séguy 1930a , MA ; Grenier 1953 , MA ; Crosskey 1964 , MA , Fès; Grenier and Bailly-Choumara 1970 : 96–102 (original description of subgenus Crosskeyellum , description of gracilipes ), MA ; Clergue-Gazeau et al. 1991 , MA ; Hervy et al. 1994 ; Dakki 1997 ; Belqat et al. 2001a , MA ; Belqat 2002 , MA ; Belqat and Dakki 2004 , MA ; Belqat et al. 2011 , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) angustipes Edwards, 1915 Clergue-Gazeau et al. 1991 , MA , HA ; Dakki 1997 ; Belqat et al. 2001a , MA , HA ; Belqat 2002 , MA , HA ; Belqat and Dakki 2004 , MA , HA ; Dakki et al. 2008, MA , O. Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) mellah Giudicelli & Bouzidi, 2000 [in Giudicelli, Bouzidi and Abdelaali 2000] Giudicelli et al. 2000 : 63 (original description), HA , Oued Mellah (Bassin Draa); Belqat et al. 2001a , HA ; Belqat 2002 , MA , HA ; Belqat and Dakki 2004 , MA , HA ; Dakki et al. 2008, MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , MA , HA ; Adler et al. 2015 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) petricolum (Rivosecchi, 1963) = Simulium latizonum Bailly-Choumara and Beaucournu-Saguez, in Bailly-Choumara and Beaucournu-Saguez 1978 : 143–144 (misidentified); Bailly-Choumara and Beaucournu-Saguez 1981 : 53–54 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, Rif , MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , HA ; Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) rubzovianum (Sherban, 1961) Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) velutinum sensu stricto (Santos Abreu, 1922) = Eusimilium latinum Rubzov, in El Mezdi and Giudicelli 1985 : 292–295; Benhoussa et al. 1988 : 160–164 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; El Mezdi and Giudicelli 1985 , HA , Khettaras of Marrakech; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Clergue-Gazeau et al. 1991 , AA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) velutinum cytospecies '5' Adler et al. 2015 , Rif , Tanger-Anjra, HA , Marrakech; Belqat et al. 2018 Simulium ( Nevermannia ) ruficorne species group Simulium ( Nevermannia ) angustitarse (Lundström, 1911) Belqat et al. 2001a , Rif ; Belqat et al. 2001b , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) ibleum (Rivosecchi, 1966) Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) lundstromi (Enderlein, 1921) Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , Rif , Kanar (280 m), Majjo (905 m), 10 km before the Issaguen source (1200 m), HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) ruficorne Macquart, 1838 = Eusimulium ruficorne Macquart, in El Mezdi and Giudicelli 1985 : 292, 294–295 Grenier et al. 1957 , AA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; El Mezdi and Giudicelli 1985 , HA , Khettaras of Marrakech; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA , AA ; Belqat 2002 , Rif , HA , AA ; Crosskey et al. 2002; Belqat and Dakki 2004 , Rif , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , AP , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) vernum species group Simulium ( Nevermannia ) brevidens (Rubtsov, 1956) Clergue-Gazeau et al. 1991 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) carthusiense (Grenier & Dorier, 1959) Giudicelli and Dakki 1984, Rif ; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) costatum Friederichs, 1920 Grenier et al. 1957 , Rif , Pré-Rif, MA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) cryophilum (Rubtsov, 1959) (complex) = Simulium pusillum Fries, in Séguy 1930a : 52 (misidentification); Grenier 1953 : 159 (after Séguy) Séguy 1930a , HA ; Grenier 1953 , Rif , HA , Lac Ifni; Bouzidi and Giudicelli 1986 , HA ; Bouzidi and Giudicelli 1989, HA ; Clergue-Gazeau et al. 1991 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Giudicelli Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) toubkal (Bouzidi & Giudicelli, 1986) Bouzidi and Giudicelli 1986 : 41–52 (original description), HA , assif n'Ouarzane (Oued Nfis); Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) vernum Macquart, 1826 (complex) [ latipes authors pre-1972, not Meigen] Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Rubzovia ) knidirii (Giudicelli & Thiery, 1985) Giudicelli and Thiery 1985 : 109–123 (original description in new subgenus Simulium ( Crenosimulium ) , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Rubzovia ) lamachi (Doby & David, 1960) Giudicelli and Dakki 1984, Rif ; Giudicelli and Thiery 1985 , Rif ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane: 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) bezzii species group Simulium ( Simulium ) bezzii (Corti, 1914) (complex) = Simulium atlas Séguy, 1930, in Séguy 1930a : 50 (original description); Grenier 1953 : 158 (synonymy of atlas Séguy with bezzii suggested) Séguy 1930a , MA ; Grenier 1953 ; Grenier 1953 , MA , HA ; Grenier and Théodoridès 1953 ; Grenier et al. 1957 , AA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Bouzidi and Giudicelli 1986 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) ornatum species group Simulium ( Simulium ) egregium Séguy, 1930 Grenier 1930, HA ; Séguy 1930a : 51 (original description), HA ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) intermedium Roubaud, 1906 = Simulium reptans var. fasciatum Séguy, in Séguy 1930a : 52 (misidentification) = Simulium ornatum var. nitidifrons Edwards, in Grenier 1953 : 159, Grenier and Théodoridès 1953 : 441, Grenier and Faure 1957 [1956]: 840, Grenier and Bailly-Choumara 1970 : 102, Bailly-Choumara and Beaucournu-Saguez 1978 : 143–144 = Odagmia nitidifrons Edwards, in Giudicelli and Dakki 1984: 95, Benhoussa et al. 1988 : 160–164 = Simulium nitidifrons Edwards, in El Mezdi and Giudicelli 1985 : 292, 294–295 Séguy 1930a , HA ; Grenier 1953 , MA , HA ; Grenier and Théodoridès 1953 , MA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif, AP , S Rabat; MA , Plain of Meknès; Grenier et al. 1957 , Rif , Pré-Rif, HA ; Grenier and Bailly-Choumara 1970 , MA ; Bernard et al. 1972 , MA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Giudicelli and Dakki 1984, Rif , MA ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , MA , HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'oun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) ornatum Meigen, 1818 (complex) = reptans var fasciatum , in Séguy 1930: 52 [ subornatum : Séguy 1925 /1930, not Edwards] Séguy 1930a : 52 ( ornatum and subornatum records), HA ; Grenier 1953 , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Clergue-Gazeau et al. 1991 , MA , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) trifasciatum Curtis, 1839 Belqat et al. 2001a , Rif ; 2001b, Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) variegatum species group Bailly-Choumara and Beaucournu-Saguez (1981 : 52–54): groupe monticola ("sp. nova A" and "sp. nova B") Simulium ( Simulium ) atlasicum Giudicelli & Bouzidi, 1989 Giudicelli and Bouzid 1989: 146–151 (original description), HA , near village Aguelmous; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) berberum Giudicelli & Bouzidi, 1989 Giudicelli and Bouzidi 1989 : 151–156 (original description), HA , assif n'Ouarzane; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) variegatum Meigen, 1818 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Giudicelli and Bouzidi 1989 ; Clergue-Gazeau et al. 1991 ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif , HA ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) xanthinum Edwards, 1933 = Simulium gaudi Grenier and Faure, in Grenier and Faure 1957 [1956]: 838–840 Grenier and Faure 1957 [1956], Rif , Pré-Rif, HA ; Grenier et al. 1957 ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Clergue-Gazeau et al. 1991 , MA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Carles-Tolrá 2002 ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) albellum species group Simulium ( Trichodagmia ) auricoma Meigen, 1818 = Simulium ( Obuchovia ) auricoma Meigen, 1818, in Belqat et al. 2011 : 52 Belqat 2000 , Rif ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) galloprovinciale Giudicelli, 1963 [1962] = Simulium ( Obuchovia ) galloprovinciale Giudicelli, 1963, in Belqat et al. 2011 : 52 Belqat 2000 , Rif ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) marocanum Bouzidi & Giudicelli, 1988 [1987] = Simulium ( Obuchovia ) marocanum Bouzidi & Giudicelli, 1987, in Belqat et al. 2011 : 52 Bouzidi and Giudicelli 1987: 185–195 (original description), Rif , near village Bou Adel, HA , Oued Rdat (affluent de l'Oued Tensift); Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) equinum species group Simulium ( Wilhelmia ) equinum (Linnaeus, 1758) = Simulium equinum Linnaeus, in Grenier et al. 1957 : 231–232 Grenier et al. 1957 , MA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Dakki 1997 ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) pseudequinum Séguy, 1921 = Simulium barbaricum Séguy, in Séguy 1930a : 51 = Simulium equinum var. mediterraneum Puri, in Grenier 1953 : 145–148; Grenier and Théodoridès 1953 : 436 = Simulium equinum mediterraneum Puri, in Grenier and Faure 1957 [1956]: 840; Grenier et al. 1957 : 232–234 = Wilhelmia pseudequinum Séguy, in Benhoussa et al. 1988 : 160–164 Séguy 1930a , HA ; Grenier 1953 , HA ; Grenier and Théodoridès 1953 , HA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif, AP , HA , AA ; Meknès; Grenier et al. 1957 , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Clergue-Gazeau et al. 1991 , HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 Simulium ( Wilhelmia ) quadrifila Grenier, Faure & Laurent, 1957 [1956] Grenier et al. 1957 : 238–239 (original description as form of sergenti ), Rif , Pré-Rif, AP , S Casablanca, MA , Meknès, HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , MA , HA ; Clergue-Gazeau et al. 1991 , Rif , Pré-Rif; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif , AP , S Casablanca, MA , HA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) sergenti (Edwards, 1923) = Simulium ariasi Séguy, in Séguy 1925 : 231–238; Séguy 1930a : 50; Grenier 1953 : 144 = Simulium equinum mediterraneum Puri, in Grenier and Faure 1957 [1956]: 840; Grenier et al. 1957 : 238–240 = Wilhelmia sergenti Edwards, in Benhoussa et al. 1993 : 249 Séguy 1930a , MA ; Grenier 1953 , MA ; Grenier and Théodoridès 1953 , HA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif; Grenier et al. 1957 , Rif , Pré-Rif, AP , S Casablanca, MA , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , AP , MA , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Clergue-Gazeau et al. 1991 , Rif , Pré-Rif, HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 THAUMALEIDAE K. Kettani, R. Wagner Number of species: 2 . Expected: 10 Faunistic knowledge of the family in Morocco: poor Thaumalea Ruthe, 1831 Thaumalea bernardi Vaillant, 1956 Vaillant 1956b , HA , Toubkal, Siroua, Lac Tamhda (Anremer), Sidi Chamarouch, Izourar, M'Goum, Oukaimeden; Dakki 1997 Thaumalea spinata Vaillant, 1954 7 Vaillant 1954a , HA , M'Goum, springs powering d'Ameskeur el Fougani, springs powering Izourar lagoon (Azourki), springs powering the lake Tamhda (Anremer), torrent at the bottom of Jebel Siroua, Oukaimeden (Toubkal), Jebel Toubkal, Atend (Sidi Chamarouch) BLEPHARICERIDAE K. Kettani, P. Zwick Number of species: 4 Blepharicerinae Liponeura Loew, 1844 Liponeura alticola Giudicelli & Bouzidi, 1987 Giudicelli and Bouzidi 1987 , HA , Oued Réghaya; Dakki 1997 Liponeura megalatlantica (Vaillant, 1956) Vaillant 1956c , HA , Izourar, Imi-N'Ifri; Giudicelli and Lavandier 1974 ; Dakki 1997 Liponeura rifincola Zwick, 2013 Zwick 2013 , Rif , Issaguen (Ketama, 1800 m) Liponeura sirouana (Vaillant, 1956) = Cardiocrepsis sirouana Vaillant, in Vaillant 1956b : 234 Vaillant 1956b , HA , Siroua (3000 m); Giudicelli and Lavandier 1974 ; Dakki 1997 CERATOPOGONIDAE K. Kettani, B. Mathieu Number of species: 62 . Expected: 80 Faunistic knowledge of the family in Morocco: moderate Ceratopogoninae Culicoidini Culicoides Latreille, 1809 Culicoides ( Avaritia ) imicola Kieffer, 1913 Kremer et al. 1971 , MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Fès, Rhafsai, AA , Torkoz, Tarhjisht; Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, EM , Oriental, AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Draa-Tafilalet, Souss-Massa, SA , Guelmim-Oued Noun; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Aïn Leuh, Ait Siberne, Meknès, AA , Errachidia, Sidi Dahmane, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) montanus Shakirzjanova, 1962 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Safi, HA , Marrakech; Chaker et al. 1980 , Rif , Al Hoceima, AP , Oued Cherrat, HA , Souk Tnine de Oudaias (Haouz), Marrakech, AA , Torkoz; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) obsoletus (Meigen, 1818) Callot et al. 1968 , Rif , Al Hoceima, AP , Merja Bokka, Sidi Yahia du Gharb, Sidi-Bettache (Zaeir), Rabat-Salé-Kénitra, HA , El Harcha (plateau central); Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Béni Mellal-Khénifra, HA , Marrakech; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, HA , Marrakech; Chaker et al. 1980 , Rif , Al Hoceima, AP , Zaers, Sidi Bettache, HA , El Harcha, Talet Inaouane (Haouz); Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Souss-Massa, SA , Guelmim-Oued Noun; Cêtre-Sossah and Baldet 2004 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Ait Siberne; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) scoticus Downes & Kettle, 1952 Kremer et al. 1971 , AP , Safi, MA , Béni Mellal-Khénifra, HA , El Harcha, Talet Inaouane (Haouz), Marrakech; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat, Safi, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Beltranmyia ) circumscriptus Kieffer, 1918 Callot et al. 1968 ; Bailly-Choumara and Kremer 1970 , Rif , Smir lagoon, Oued Negro, EM , Merja Boubker (Berkane), Gouttitir (NE Guercif), AP , Merja Sheishat (Larache), Aïn Muelha (near Oued Sidi Allal Tazi, estuaire Oued Bou-Regreg, Dayat Qoudiya (Sidi Yahia Gharb); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, AA , Souss Massa; Chaker et al. 1980 , Rif , Al Hoceima, AP , Merja Qoudiya, Merja Bokka, Sidi Yahia du Gharb, MA , Aïn Karma (Saiss), Oulmès, HA , Setti Fatma, AA , Aït Melloul (Souss); Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Meknès, AA , Errachidia, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) fagineus Edwards, in Edwards et al. 1939 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Béni Mellal-Khénifra, HA , Marrakech; Kremer et al. 1975 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Kremer et al. 1979 ; Chaker et al. 1980 , AP , Rabat, Sidi Bettache, MA , Khemisset, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) newsteadi Austen, 1921 = Culicoides ( Culicoides ) halophilus Kieffer, in Callot et al. 1968 : 886, Bailly-Choumara and Kremer 1970 : 386, Dakki 1997 : 60 Callot et al. 1968 , Rif , Cabo Negro (Ferma), Tétouan, Talerhza, Tanger-Tétouan-Al Hoceima, AP , Larache, Merja Bokka (Gharb), Aïn Chok, HA , Talet-Inaouan (Haouz); Bailly-Choumara and Kremer 1970 , Rif , Smir lagoon, Oued Negro, AP , Merja Sheishat (Larache), Aïn Muelha (near Oued Sidi Allal Tazi, estuaire Oued Bou-Regreg, Dayat Qoudiya (Sidi Yahia du Gharb), EM , Merja Boubker (Berkane), Ksabi (NE Midelt), HA , Souk Tnine des Oudaias (bordure Oued N'fis), AA , Aïn Sefra (south Foum Zquid); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Casablanca, Settat, MA , Fès-Meknès, Béni Mellal-Khénifra, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, Casablanca, Settat, MA , Béni Mellal- Khénifra, HA , Marrakech; Kremer et al. 1979 ; Baylis et al. 1997 , Rif , Tanger, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , MA , Aïn Leuh, Ait Siberne, Meknès, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) pulicaris (Linnaeus, 1758) Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, Béni Mellal-Khénifra, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Lalla Outka, Khénifra, Oulmès, HA , Talet Inaouan (Haouz), AA , Aouinet-Torkoz, Tarhjicht; Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, EM , Oriental, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Drâa-Tafilalet, Souss-Massa; Cêtre-Sossah and Baldet 2004 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Aïn Leuh, Aït Siberne, Meknès; Bourquia et al. 2019 Culicoides ( Culicoides ) punctatus (Meigen, 1804) Callot et al. 1968 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1970 , Rif , Merja Smir, Oued Negro, AP , Merja Sheishat (Larache), estuaire Bou-Regreg, EM , Merja Boubker (Berkane); Kremer et al. 1971 , Rif , Cabo Negro (Ferma), Tétouan, EM , Berkane, AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, AA , Foum Zguid; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat; Dakki 1997 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Aïn Leuh, Meknès; Bourquia et al. 2019 Culicoides ( Culicoides ) subfagineus Delécolle & Ortega, 1998 Bourquia et al. 2019 , AP , Rabat Culicoides ( Monoculicoides ) parroti Kieffer, 1922 Bailly-Choumara and Kremer 1970 , HA , Dar Saâda (Haouz); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Safi, HA , Marrakech; Chakeret al. 1980 , AP , Rabat, HA , Marrakech, Souk Tnine des Oudaias (Haouz); Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Monoculicoides ) puncticollis (Becker, 1903) Callot et al. 1968 , AP , Merja Qoudiya, Sidi Yahia du Gharb, Romani (Zaers), Rabat-Salé-Kénitra, MA , Aïn Karma (Saiss), HA , Souk Tnine des Oudaias (Haouz); Bailly-Choumara and Kremer 1970 (reported as C. riethi and corrected by Kremer et al. 1971 ), Rif , Merja Smir, AP , Merja Sheishat (Larache), estuaire Bou-Regreg, Dayat Qoudiya (Sidi Yahia du Gharb), HA , Souk Tnine des Oudaias (bordure de l'Oued N'fis), Dar Saâda (Haouz), Talet Inouane (bordure marécageuse du lac du Barrage Lalla Taguergoust); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, HA , Marrakech; Chaker et al. 1980 , AP , Aïn Karma, Zaers, Roumani, HA , Souk-Tnine des Oudaias; Remm 1988a ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) azerbajdzhanicus Dhzafarov, 1962 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Safi, MA , Fès-Meknès, Beni Mellal-Khénifra, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 , AA , Souss-Massa SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Kkénifra, Rhafsai, HA , Marrakech, AA , Torkoz, Tarhjisht; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) longipennis Khalaf, 1957 Kremer et al. 1971 , EM , Berkane AP , Safi, MA , Fès-Meknès, HA , Marrakech; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) marcleti Callot, Kremer & Basset, 1968 Kremer et al. 1971 , MA , Rhafsai, Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) pallidus Khalaf, 1957 = Culicoides stackelbergi Dhzafarov, in Kremer et al. 1971 : 662, Dakki 1997 : 61 Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1971 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) ravus De Meillon, 1936 = Culicoides ( Synhelea ) subravus Cornet and Château, in Kremer et al. 1971 : 664, Chaker et al. 1980 : 85, Dakki 1997 : 61 Kremer et al. 1971 , AP , Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AA , Souss-Massa, SA , Guelmim-Oued Noun; Chaker et al. 1980 , HA , Marrakech, AA , Torkoz, Tarhjicht, Aït Ouaballi (Draa); Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) sahariensis Kieffer, 1923 = Culicoides colluzzii Callot, Kremer and Bailly-Choumara, in Bailly-Choumara and Kremer 1970 : 386, Chaker et al. 1980 : 83, Dakki 1997 : 61 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), EM , Merja Boubker (Berkane), HA , Souk Tnine des Oudaias (bordure Oued N'fis); Callot et al. 1970 , AP , Larache, HA , Marrakech; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Merja Bokka (Gharb), Rabat (Zaers), EM , Berkane, MA , Fès, Khémisset, AA , Tarhjisht; Baylis et al. 1997 ; Dakki 1997 ; Bouayoune et al. 1998; Cêtre-Sossah and Baldet 2004 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) santonicus Callot, Kremer, Rault & Bach, 1966 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Bailly-Choumara et al. 1980, AP , Larache, Sidi Bettache, MA , Oulmès, EM , El-Harcha; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) semimaculatus Clastrier, 1958 Kremer et al. 1975 , Rif , Tanger-Tétouan-Al Hoceima, AP , Larache, Casablanca-Settat, MA , Plateau Central (Khatouate); Chaker et al. 1979 ; Remm 1988a ; Dakki 1997 ; Szadziewski and Dominiak 2006 ; Bourquia et al. 2019 Culicoides ( Oecacta ) sergenti Kieffer, 1921 = Culicoides ( Oecacta ) mosulensis Khalaf, in Chaker et al. 1980 : 84, Dakki 1997 : 60 Kremer et al. 1979 , EM , Oriental, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AA , Tarhjicht, SA , Bou-Arfa; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) similis Carter, Ingram & Macfie, 1920 Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) truncorum Edwards, 1939 = Culicoides ( Oecacta ) sylvarum Callot and Kremer, in Kremer et al. 1971 : 662, Remm 1988a : 65, Dakki 1997 : 61 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Béni Mellal- Khénifra; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Pontoculicoides ) saevus Kieffer, 1922 Callot et al. 1968 , AP , Sidi Yahia du Gharb, Rabat-Salé-Kénitra, Safi, MA , Aïn Karma (Saiss), HA , Talet Inouane, Marrakech, Souk Tnine des Oudaias (Haouz), AA , Aït Melloul (Souss), Ksar er Souk (Tafilalt), Tarhjicht, SA , Bou-Arfa; Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), HA , Souk Tnine des Oudaias (bordure de l'Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AP , Safi, MA , Fès-Meknès, HA , Marrakech, AA , Drâa-Tafilalet, Souss-Massa, SA , Guelmim-Oued Noun; Bailly-Choumara and Kremer 1980; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Pontoculicoides ) sejfadinei Dzhafarov, 1958 Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AA , Drâa-Tafilalet; Bourquia et al. 2019 Culicoides ( Remmia ) kingi Austen, 1912 Bailly-Choumara and Kremer 1970 , AA , Mrimima (Oued de Foum Zquid), Souss-Massa; Chaker et al. 1980 , MA , Meknès, AA , Tarhjisht; Cornet and Brunhes 1994 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Remmia ) schultzei (Enderlein, 1908) Callot et al. 1968 , AP , Safi, HA , Talet Inouane, Marrakech; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) badooshensis Khalaf, 1961 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), HA , Marrakech, Souk Tnine des Oudaias; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech; Dakki 1997 ; Chaker et al. 1980 , AP , Oued Cherrat, Bousselham, Sidi Yahia, MA , Fès, HA , Marrakech; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) cataneii Clastrier, 1957 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra; Chaker et al. 1980 , AP , Oued Cherrat, Rabat; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) clastrieri Callot, Kremer & Deduit, 1962 Bourquia et al. 2019 , MA , Fès-Meknès Culicoides ( Sensiculicoides ) derisor Callot & Kremer, 1965 Chaker et al. 1980 , AP , Rabat; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès; Kremer et al. 1975 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Kremer et al. 1979 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) duddingstoni Kettle & Lawson, 1955 Bourquia et al. 2019 , MA , Fès-Meknès Culicoides ( Sensiculicoides ) dzhafarovi Remm, 1967 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Oued Cherrat, AA , Tarhjisht; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) festivipennis Kieffer, 1914 = Culicoides ( Oecacta ) odibilis Austen, in Bailly-Choumara and Kremer 1970 : 387, Chaker et al. 1980 : 84, Dakki 1997 : 61 Callot et al. 1968 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1970 , Rif , Merja Smir, HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, Casablanca-Settat, HA , Marrakech; Chaker et al. 1980 , Rif , Tétouan, EM , El-Harcha, AP , Aïn Chok, Rabat, Larache, MA , Oulmès, HA , Marrakech, Talet-Inaouan (Haouz); Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) heteroclitus Kremer and Callot, in Callot & Kremer, 1965 Kremer et al. 1975 , AP , Safi, HA , Marrakech, AA , Tafraout, Tiznit, Souss-Massa; Chaker et al. 1979 ; HA , Haouz; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) jumineri Callot & Kremer, 1969 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, AA , Souss-Massa, SA , Guelmim-Oued Noun; Chaket et al. 1980, AP , Oued Cherrat, Merja Bokka, Rabat, MA , Fès, HA , Talet-Inaouan (Haouz), AA , Torkoz, Tarhjisht, Aït Oubelli, SA , Bou-Arfa; Remm 1988a ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) kibunensis Tokunaga, 1937 = Culicoides ( Oecata ) cubitalis Edwards, in Kremer et al. 1975 : 205, Dakki 1997 : 60 Kremer et al. 1975 , AP , Safi, MA , Ifrane, Imouzzer-du-Kander, Fès-Meknès, HA , Haouz, Marrakech; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) kurensis Dzhafarov in Gutsevich, 1960 Remm 1988a ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) landauae Kremer, Rebholtz-Hirtzel & Bailly-Choumara, 1975 Kremer et al. 1975 , MA , Imouzzer-du-Kander, Sefrou, Fès-Meknès; Chaker et al. 1979 ; Hervy et al. 1994 ; Borkent and Wirth 1997 ; Remm 1988a ; Dakki 1997 ; Chilasse and Dakki 2004, MA ; Borkent 2012 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) langeroni Kieffer, 1921 Bailly-Choumara and Kremer 1970 , HA , Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Bailly-Choumara and Kremer 1980, MA , Khénifra, AA , Tarhjicht, Torkoz (Draa); Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) maritimus Kieffer, 1924 Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Bailly-Choumara and Kremer 1980, Rif , Tétouan, AP , Larache, Rabat, Sidi Bettache, HA , Talet-Inaouan (Haouz), Souk Tnine des Oudaias (Haouz); Remm 1988; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) odiatus Austen, 1921 = Culicoides lailae Khalaf, in Bailly-Choumara and Kremer 1970 : 387, Dakki 1997 : 60 = Culicoides indistinctus Khalaf, in Kremer et al. 1975 : 206, Dakki 1997 : 60 Bailly-Choumara and Kremer 1970 , HA , Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1975 , AA , Tafraout, Souss-Massa; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Kénitra, Merja Bokka (Gharb), HA , Souk Tnine des Oudaias (Haouz), AA , Tarhjicht; Remm 1988a ; Dakki 1997 ; Baylis et al. 1997 ; Bouayoune et al. 1998; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Sarvašová et al. 2014 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) paolae Boorman, 1996 Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) pictipennis (Staeger, 1839) Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 ; Remm 1988a ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) pseudopallidus Khalaf, 1961 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Safi, MA , Rhafsai, Fès-Meknès, HA , Marrakech; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) shaklawensis Khalaf, 1957 Kremer et al. 1975 , AP , Safi, MA , Sefrou, Fès-Meknès, HA , Setti Fatma, Marrakech; Chaker et al. 1979 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) simulator Edwards, 1939 Kremer et al. 1975 , MA , Ifrane, Fès-Meknès, HA , Setti Fatma; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) univittatus Vimmer, 1932 = Culicoides agathensis Callot, Kremer and Rioux, in Bailly-Choumara and Kremer 1970 : 386, Kremer et al. 1971 : 663, Chaker et al. 1980 : 82, Dakki 1997 : 60 Bailly-Choumara and Kremer 1970 , AP , estuaire Bou-Regreg; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Rif , Tanger-Tétouan-Al Hoceima; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, HA , Marrakech; Chaker et al. 1980 , Rif , Tétouan, AP , Larache, Sidi Bettache MA , Oulmès; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) vidourlensis Callot, Kremer, Molet & Bach, 1968 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), estuaire de Oued Bou-Regreg, HA , Souk Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 ; Remm 1988a ; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) pallidicornis Kieffer, 1919 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Dakki 1997 ; Balenghien et al. 2014 , SA , Guelmim-Oued Noun; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) picturatus Kremer & Deduit, 1961 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache); Kremer et al. 1971 , Rif , Talerhza, EM , El-Harcha, AP , Bousselham, Rabat-Salé-Kénitra, MA , Oulmès; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat, MA , Béni Mellal-Khénifra; Remm 1988a ; Dakki 1997 ; Sarvašová et al. 2014 ; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) subfasciipennis Kieffer, 1919 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, Béni Mellal-Khénifra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1980, AP , Larache, Zaers, Rabat, Aïn Chok, MA , Sefrou; Remm 1988a ; Hervy et al. 1994 ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Wirthomyia ) faghihi Navai, 1971 Kremer et al. 1975 , AA , Tafraout, Souss-Massa; Chaker et al. 1979 ; Remm 1988a ; Hervy et al. 1994 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Wirthomyia ) minutissimus (Zetterstedt, 1855) Referred as C. pumilus Culicoides ( Wirthomyia ) pumilus (Winnertz, 1852) Kremer et al. 1975 , AP , Safi, MA , Ifrane, Imouzzer-du-Kander, Fès-Meknès, HA , Setti Fatma, Marrakech; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides calloti Kremer, Delécolle, Bailly-Choumara & Chaker, 1979 Chaker et al. 1979 , AA , Souss Massa, SA , Guelmim-Oued Noun; Kremer et al. 1979 , AA , Tarhjigt, Aït Ouaballi, Souss Massa, SA , Guelmim-Oued Noun; Chaker et al. 1980 ; Remm 1988a ; Hervy et al. 1994 ; Borkent and Wirth 1997 ; Baylis et al. 1997 ; Dakki 1997 ; Koçak and Kemal 2010 ; Borkent 2012 ; Bourquia et al. 2019 Ceratopogonini Alluaudomyia Kieffer, 1913 Alluaudomyia hygropetrica Vaillant, 1954 Vaillant 1956b , HA , Sidi Chamarouch Palpomyiini Bezzia Kieffer, 1899 Bezzia ( Bezzia ) nigritula (Zetterstedt, 1838) = Palpomyia tenebricosa Goetghebuer, 1912, in Vaillant 1956b : 241 Vaillant 1956b , HA , Tamesrit Palpomyia Meigen, 1818 Palpomyia helviscutellata Borkent, in Borkent & Wirth 1997 = Dasyhelea flavoscutellata (Zetterstedt, 1850), in Vaillant 1956b : 244 Vaillant 1956b , HA , Tahanaout Forcipomyiinae Dasyheleini Dasyhelea Kieffer, 1911 Dasyhelea ( Prokempia ) flaviventris (Goetghebuer, 1910) Dominiak 2012 Dasyhelea ( Pseudoculicoides ) turficola Kieffer, 1925 Dominiak 2012 Leptoconopinae Leptoconops Skuse, 1889 Leptoconops ( Holoconops ) laurae (Weiss, 1912) Remm 1988b Ceratopogoninae Culicoidini Culicoides Latreille, 1809 Culicoides ( Avaritia ) imicola Kieffer, 1913 Kremer et al. 1971 , MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Fès, Rhafsai, AA , Torkoz, Tarhjisht; Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, EM , Oriental, AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Draa-Tafilalet, Souss-Massa, SA , Guelmim-Oued Noun; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Aïn Leuh, Ait Siberne, Meknès, AA , Errachidia, Sidi Dahmane, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) montanus Shakirzjanova, 1962 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Safi, HA , Marrakech; Chaker et al. 1980 , Rif , Al Hoceima, AP , Oued Cherrat, HA , Souk Tnine de Oudaias (Haouz), Marrakech, AA , Torkoz; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) obsoletus (Meigen, 1818) Callot et al. 1968 , Rif , Al Hoceima, AP , Merja Bokka, Sidi Yahia du Gharb, Sidi-Bettache (Zaeir), Rabat-Salé-Kénitra, HA , El Harcha (plateau central); Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Béni Mellal-Khénifra, HA , Marrakech; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, HA , Marrakech; Chaker et al. 1980 , Rif , Al Hoceima, AP , Zaers, Sidi Bettache, HA , El Harcha, Talet Inaouane (Haouz); Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Souss-Massa, SA , Guelmim-Oued Noun; Cêtre-Sossah and Baldet 2004 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Ait Siberne; Bourquia et al. 2019 , AP , Rabat Culicoides ( Avaritia ) scoticus Downes & Kettle, 1952 Kremer et al. 1971 , AP , Safi, MA , Béni Mellal-Khénifra, HA , El Harcha, Talet Inaouane (Haouz), Marrakech; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat, Safi, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Beltranmyia ) circumscriptus Kieffer, 1918 Callot et al. 1968 ; Bailly-Choumara and Kremer 1970 , Rif , Smir lagoon, Oued Negro, EM , Merja Boubker (Berkane), Gouttitir (NE Guercif), AP , Merja Sheishat (Larache), Aïn Muelha (near Oued Sidi Allal Tazi, estuaire Oued Bou-Regreg, Dayat Qoudiya (Sidi Yahia Gharb); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, AA , Souss Massa; Chaker et al. 1980 , Rif , Al Hoceima, AP , Merja Qoudiya, Merja Bokka, Sidi Yahia du Gharb, MA , Aïn Karma (Saiss), Oulmès, HA , Setti Fatma, AA , Aït Melloul (Souss); Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Meknès, AA , Errachidia, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) fagineus Edwards, in Edwards et al. 1939 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Béni Mellal-Khénifra, HA , Marrakech; Kremer et al. 1975 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Kremer et al. 1979 ; Chaker et al. 1980 , AP , Rabat, Sidi Bettache, MA , Khemisset, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) newsteadi Austen, 1921 = Culicoides ( Culicoides ) halophilus Kieffer, in Callot et al. 1968 : 886, Bailly-Choumara and Kremer 1970 : 386, Dakki 1997 : 60 Callot et al. 1968 , Rif , Cabo Negro (Ferma), Tétouan, Talerhza, Tanger-Tétouan-Al Hoceima, AP , Larache, Merja Bokka (Gharb), Aïn Chok, HA , Talet-Inaouan (Haouz); Bailly-Choumara and Kremer 1970 , Rif , Smir lagoon, Oued Negro, AP , Merja Sheishat (Larache), Aïn Muelha (near Oued Sidi Allal Tazi, estuaire Oued Bou-Regreg, Dayat Qoudiya (Sidi Yahia du Gharb), EM , Merja Boubker (Berkane), Ksabi (NE Midelt), HA , Souk Tnine des Oudaias (bordure Oued N'fis), AA , Aïn Sefra (south Foum Zquid); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Casablanca, Settat, MA , Fès-Meknès, Béni Mellal-Khénifra, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, Casablanca, Settat, MA , Béni Mellal- Khénifra, HA , Marrakech; Kremer et al. 1979 ; Baylis et al. 1997 , Rif , Tanger, HA , Marrakech; Remm 1988a ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Lhor et al. 2015 , MA , Aïn Leuh, Ait Siberne, Meknès, SA , Foum El Oued; Bourquia et al. 2019 , AP , Rabat Culicoides ( Culicoides ) pulicaris (Linnaeus, 1758) Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, Béni Mellal-Khénifra, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Lalla Outka, Khénifra, Oulmès, HA , Talet Inaouan (Haouz), AA , Aouinet-Torkoz, Tarhjicht; Remm 1988a ; Dakki 1997 ; Bouayoune et al. 1998, Rif , Tanger-Tétouan-Al Hoceima, EM , Oriental, AP , Rabat-Salé-Kénitra, Casablanca, Settat, Safi, MA , Fès-Meknès, Béni Mellal-Khénifra, HA , Marrakech, AA , Drâa-Tafilalet, Souss-Massa; Cêtre-Sossah and Baldet 2004 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Aïn Leuh, Aït Siberne, Meknès; Bourquia et al. 2019 Culicoides ( Culicoides ) punctatus (Meigen, 1804) Callot et al. 1968 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1970 , Rif , Merja Smir, Oued Negro, AP , Merja Sheishat (Larache), estuaire Bou-Regreg, EM , Merja Boubker (Berkane); Kremer et al. 1971 , Rif , Cabo Negro (Ferma), Tétouan, EM , Berkane, AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, AA , Foum Zguid; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat; Dakki 1997 ; Lhor et al. 2015 , Rif , Sahel Chamali, MA , Sidi Hammadi, Benioukil, Aïn Leuh, Meknès; Bourquia et al. 2019 Culicoides ( Culicoides ) subfagineus Delécolle & Ortega, 1998 Bourquia et al. 2019 , AP , Rabat Culicoides ( Monoculicoides ) parroti Kieffer, 1922 Bailly-Choumara and Kremer 1970 , HA , Dar Saâda (Haouz); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Safi, HA , Marrakech; Chakeret al. 1980 , AP , Rabat, HA , Marrakech, Souk Tnine des Oudaias (Haouz); Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Monoculicoides ) puncticollis (Becker, 1903) Callot et al. 1968 , AP , Merja Qoudiya, Sidi Yahia du Gharb, Romani (Zaers), Rabat-Salé-Kénitra, MA , Aïn Karma (Saiss), HA , Souk Tnine des Oudaias (Haouz); Bailly-Choumara and Kremer 1970 (reported as C. riethi and corrected by Kremer et al. 1971 ), Rif , Merja Smir, AP , Merja Sheishat (Larache), estuaire Bou-Regreg, Dayat Qoudiya (Sidi Yahia du Gharb), HA , Souk Tnine des Oudaias (bordure de l'Oued N'fis), Dar Saâda (Haouz), Talet Inouane (bordure marécageuse du lac du Barrage Lalla Taguergoust); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, HA , Marrakech; Chaker et al. 1980 , AP , Aïn Karma, Zaers, Roumani, HA , Souk-Tnine des Oudaias; Remm 1988a ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) azerbajdzhanicus Dhzafarov, 1962 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Safi, MA , Fès-Meknès, Beni Mellal-Khénifra, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 , AA , Souss-Massa SA , Guelmim-Oued Noun; Chaker et al. 1980 , MA , Kkénifra, Rhafsai, HA , Marrakech, AA , Torkoz, Tarhjisht; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) longipennis Khalaf, 1957 Kremer et al. 1971 , EM , Berkane AP , Safi, MA , Fès-Meknès, HA , Marrakech; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) marcleti Callot, Kremer & Basset, 1968 Kremer et al. 1971 , MA , Rhafsai, Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) pallidus Khalaf, 1957 = Culicoides stackelbergi Dhzafarov, in Kremer et al. 1971 : 662, Dakki 1997 : 61 Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1971 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) ravus De Meillon, 1936 = Culicoides ( Synhelea ) subravus Cornet and Château, in Kremer et al. 1971 : 664, Chaker et al. 1980 : 85, Dakki 1997 : 61 Kremer et al. 1971 , AP , Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AA , Souss-Massa, SA , Guelmim-Oued Noun; Chaker et al. 1980 , HA , Marrakech, AA , Torkoz, Tarhjicht, Aït Ouaballi (Draa); Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) sahariensis Kieffer, 1923 = Culicoides colluzzii Callot, Kremer and Bailly-Choumara, in Bailly-Choumara and Kremer 1970 : 386, Chaker et al. 1980 : 83, Dakki 1997 : 61 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), EM , Merja Boubker (Berkane), HA , Souk Tnine des Oudaias (bordure Oued N'fis); Callot et al. 1970 , AP , Larache, HA , Marrakech; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Merja Bokka (Gharb), Rabat (Zaers), EM , Berkane, MA , Fès, Khémisset, AA , Tarhjisht; Baylis et al. 1997 ; Dakki 1997 ; Bouayoune et al. 1998; Cêtre-Sossah and Baldet 2004 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Oecacta ) santonicus Callot, Kremer, Rault & Bach, 1966 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Bailly-Choumara et al. 1980, AP , Larache, Sidi Bettache, MA , Oulmès, EM , El-Harcha; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) semimaculatus Clastrier, 1958 Kremer et al. 1975 , Rif , Tanger-Tétouan-Al Hoceima, AP , Larache, Casablanca-Settat, MA , Plateau Central (Khatouate); Chaker et al. 1979 ; Remm 1988a ; Dakki 1997 ; Szadziewski and Dominiak 2006 ; Bourquia et al. 2019 Culicoides ( Oecacta ) sergenti Kieffer, 1921 = Culicoides ( Oecacta ) mosulensis Khalaf, in Chaker et al. 1980 : 84, Dakki 1997 : 60 Kremer et al. 1979 , EM , Oriental, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AA , Tarhjicht, SA , Bou-Arfa; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) similis Carter, Ingram & Macfie, 1920 Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Oecacta ) truncorum Edwards, 1939 = Culicoides ( Oecacta ) sylvarum Callot and Kremer, in Kremer et al. 1971 : 662, Remm 1988a : 65, Dakki 1997 : 61 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Béni Mellal- Khénifra; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Pontoculicoides ) saevus Kieffer, 1922 Callot et al. 1968 , AP , Sidi Yahia du Gharb, Rabat-Salé-Kénitra, Safi, MA , Aïn Karma (Saiss), HA , Talet Inouane, Marrakech, Souk Tnine des Oudaias (Haouz), AA , Aït Melloul (Souss), Ksar er Souk (Tafilalt), Tarhjicht, SA , Bou-Arfa; Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), HA , Souk Tnine des Oudaias (bordure de l'Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AP , Safi, MA , Fès-Meknès, HA , Marrakech, AA , Drâa-Tafilalet, Souss-Massa, SA , Guelmim-Oued Noun; Bailly-Choumara and Kremer 1980; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Pontoculicoides ) sejfadinei Dzhafarov, 1958 Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AA , Drâa-Tafilalet; Bourquia et al. 2019 Culicoides ( Remmia ) kingi Austen, 1912 Bailly-Choumara and Kremer 1970 , AA , Mrimima (Oued de Foum Zquid), Souss-Massa; Chaker et al. 1980 , MA , Meknès, AA , Tarhjisht; Cornet and Brunhes 1994 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Remmia ) schultzei (Enderlein, 1908) Callot et al. 1968 , AP , Safi, HA , Talet Inouane, Marrakech; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) badooshensis Khalaf, 1961 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), HA , Marrakech, Souk Tnine des Oudaias; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès; Kremer et al. 1975 ; Kremer et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech; Dakki 1997 ; Chaker et al. 1980 , AP , Oued Cherrat, Bousselham, Sidi Yahia, MA , Fès, HA , Marrakech; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) cataneii Clastrier, 1957 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra; Chaker et al. 1980 , AP , Oued Cherrat, Rabat; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) clastrieri Callot, Kremer & Deduit, 1962 Bourquia et al. 2019 , MA , Fès-Meknès Culicoides ( Sensiculicoides ) derisor Callot & Kremer, 1965 Chaker et al. 1980 , AP , Rabat; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès; Kremer et al. 1975 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra; Kremer et al. 1979 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) duddingstoni Kettle & Lawson, 1955 Bourquia et al. 2019 , MA , Fès-Meknès Culicoides ( Sensiculicoides ) dzhafarovi Remm, 1967 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Oued Cherrat, AA , Tarhjisht; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) festivipennis Kieffer, 1914 = Culicoides ( Oecacta ) odibilis Austen, in Bailly-Choumara and Kremer 1970 : 387, Chaker et al. 1980 : 84, Dakki 1997 : 61 Callot et al. 1968 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1970 , Rif , Merja Smir, HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, Casablanca-Settat, HA , Marrakech; Chaker et al. 1980 , Rif , Tétouan, EM , El-Harcha, AP , Aïn Chok, Rabat, Larache, MA , Oulmès, HA , Marrakech, Talet-Inaouan (Haouz); Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) heteroclitus Kremer and Callot, in Callot & Kremer, 1965 Kremer et al. 1975 , AP , Safi, HA , Marrakech, AA , Tafraout, Tiznit, Souss-Massa; Chaker et al. 1979 ; HA , Haouz; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) jumineri Callot & Kremer, 1969 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, SA , Guelmim-Oued Noun; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , EM , Oriental, AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, AA , Souss-Massa, SA , Guelmim-Oued Noun; Chaket et al. 1980, AP , Oued Cherrat, Merja Bokka, Rabat, MA , Fès, HA , Talet-Inaouan (Haouz), AA , Torkoz, Tarhjisht, Aït Oubelli, SA , Bou-Arfa; Remm 1988a ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) kibunensis Tokunaga, 1937 = Culicoides ( Oecata ) cubitalis Edwards, in Kremer et al. 1975 : 205, Dakki 1997 : 60 Kremer et al. 1975 , AP , Safi, MA , Ifrane, Imouzzer-du-Kander, Fès-Meknès, HA , Haouz, Marrakech; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) kurensis Dzhafarov in Gutsevich, 1960 Remm 1988a ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) landauae Kremer, Rebholtz-Hirtzel & Bailly-Choumara, 1975 Kremer et al. 1975 , MA , Imouzzer-du-Kander, Sefrou, Fès-Meknès; Chaker et al. 1979 ; Hervy et al. 1994 ; Borkent and Wirth 1997 ; Remm 1988a ; Dakki 1997 ; Chilasse and Dakki 2004, MA ; Borkent 2012 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) langeroni Kieffer, 1921 Bailly-Choumara and Kremer 1970 , HA , Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Bailly-Choumara and Kremer 1980, MA , Khénifra, AA , Tarhjicht, Torkoz (Draa); Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) maritimus Kieffer, 1924 Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra; Kremer et al. 1975 ; Kremer et al. 1979 ; Bailly-Choumara and Kremer 1980, Rif , Tétouan, AP , Larache, Rabat, Sidi Bettache, HA , Talet-Inaouan (Haouz), Souk Tnine des Oudaias (Haouz); Remm 1988; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) odiatus Austen, 1921 = Culicoides lailae Khalaf, in Bailly-Choumara and Kremer 1970 : 387, Dakki 1997 : 60 = Culicoides indistinctus Khalaf, in Kremer et al. 1975 : 206, Dakki 1997 : 60 Bailly-Choumara and Kremer 1970 , HA , Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, AA , Torkoz, SA , Guelmim-Oued Noun; Kremer et al. 1975 , AA , Tafraout, Souss-Massa; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Rabat-Salé-Kénitra, Safi, HA , Marrakech, SA , Guelmim-Oued Noun; Chaker et al. 1980 , AP , Kénitra, Merja Bokka (Gharb), HA , Souk Tnine des Oudaias (Haouz), AA , Tarhjicht; Remm 1988a ; Dakki 1997 ; Baylis et al. 1997 ; Bouayoune et al. 1998; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Sarvašová et al. 2014 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) paolae Boorman, 1996 Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) pictipennis (Staeger, 1839) Bailly-Choumara and Kremer 1970 , AP , estuaire de Bou-Regreg; Kremer et al. 1971 ; Remm 1988a ; Bourquia et al. 2019 , AP , Rabat Culicoides ( Sensiculicoides ) pseudopallidus Khalaf, 1961 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AP , Safi, MA , Rhafsai, Fès-Meknès, HA , Marrakech; Kremer et al. 1975 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) shaklawensis Khalaf, 1957 Kremer et al. 1975 , AP , Safi, MA , Sefrou, Fès-Meknès, HA , Setti Fatma, Marrakech; Chaker et al. 1979 ; Remm 1988a ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) simulator Edwards, 1939 Kremer et al. 1975 , MA , Ifrane, Fès-Meknès, HA , Setti Fatma; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) univittatus Vimmer, 1932 = Culicoides agathensis Callot, Kremer and Rioux, in Bailly-Choumara and Kremer 1970 : 386, Kremer et al. 1971 : 663, Chaker et al. 1980 : 82, Dakki 1997 : 60 Bailly-Choumara and Kremer 1970 , AP , estuaire Bou-Regreg; Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, Rif , Tanger-Tétouan-Al Hoceima; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima, AP , Rabat-Salé-Kénitra, Safi, MA , Fès-Meknès, HA , Marrakech; Chaker et al. 1980 , Rif , Tétouan, AP , Larache, Sidi Bettache MA , Oulmès; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Sensiculicoides ) vidourlensis Callot, Kremer, Molet & Bach, 1968 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache), estuaire de Oued Bou-Regreg, HA , Souk Tnine des Oudaias (bordure de Oued N'fis); Kremer et al. 1971 ; Remm 1988a ; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) pallidicornis Kieffer, 1919 Bailly-Choumara and Kremer 1970 , HA , Souk Tnine des Oudaias (bordure Oued N'fis); Kremer et al. 1971 , AA , Torkoz, SA , Guelmim-Oued Noun; Dakki 1997 ; Balenghien et al. 2014 , SA , Guelmim-Oued Noun; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) picturatus Kremer & Deduit, 1961 Bailly-Choumara and Kremer 1970 , AP , Merja Sheishat (Larache); Kremer et al. 1971 , Rif , Talerhza, EM , El-Harcha, AP , Bousselham, Rabat-Salé-Kénitra, MA , Oulmès; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , AP , Casablanca-Settat, MA , Béni Mellal-Khénifra; Remm 1988a ; Dakki 1997 ; Sarvašová et al. 2014 ; Bourquia et al. 2019 Culicoides ( Silvaticulicoides ) subfasciipennis Kieffer, 1919 Kremer et al. 1971 , AP , Rabat-Salé-Kénitra, MA , Fès-Meknès, Béni Mellal-Khénifra; Kremer et al. 1975 ; Kremer et al. 1979 ; Chaker et al. 1979 , Rif , Tanger-Tétouan-Al Hoceima; Bailly-Choumara and Kremer 1980, AP , Larache, Zaers, Rabat, Aïn Chok, MA , Sefrou; Remm 1988a ; Hervy et al. 1994 ; Dakki 1997 ; Cêtre-Sossah and Baldet 2004 , AP , Rabat-Salé-Kénitra; Bourquia et al. 2019 Culicoides ( Wirthomyia ) faghihi Navai, 1971 Kremer et al. 1975 , AA , Tafraout, Souss-Massa; Chaker et al. 1979 ; Remm 1988a ; Hervy et al. 1994 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides ( Wirthomyia ) minutissimus (Zetterstedt, 1855) Referred as C. pumilus Culicoides ( Wirthomyia ) pumilus (Winnertz, 1852) Kremer et al. 1975 , AP , Safi, MA , Ifrane, Imouzzer-du-Kander, Fès-Meknès, HA , Setti Fatma, Marrakech; Chaker et al. 1979 ; Dakki 1997 ; Bourquia et al. 2019 Culicoides calloti Kremer, Delécolle, Bailly-Choumara & Chaker, 1979 Chaker et al. 1979 , AA , Souss Massa, SA , Guelmim-Oued Noun; Kremer et al. 1979 , AA , Tarhjigt, Aït Ouaballi, Souss Massa, SA , Guelmim-Oued Noun; Chaker et al. 1980 ; Remm 1988a ; Hervy et al. 1994 ; Borkent and Wirth 1997 ; Baylis et al. 1997 ; Dakki 1997 ; Koçak and Kemal 2010 ; Borkent 2012 ; Bourquia et al. 2019 Ceratopogonini Alluaudomyia Kieffer, 1913 Alluaudomyia hygropetrica Vaillant, 1954 Vaillant 1956b , HA , Sidi Chamarouch Palpomyiini Bezzia Kieffer, 1899 Bezzia ( Bezzia ) nigritula (Zetterstedt, 1838) = Palpomyia tenebricosa Goetghebuer, 1912, in Vaillant 1956b : 241 Vaillant 1956b , HA , Tamesrit Palpomyia Meigen, 1818 Palpomyia helviscutellata Borkent, in Borkent & Wirth 1997 = Dasyhelea flavoscutellata (Zetterstedt, 1850), in Vaillant 1956b : 244 Vaillant 1956b , HA , Tahanaout Forcipomyiinae Dasyheleini Dasyhelea Kieffer, 1911 Dasyhelea ( Prokempia ) flaviventris (Goetghebuer, 1910) Dominiak 2012 Dasyhelea ( Pseudoculicoides ) turficola Kieffer, 1925 Dominiak 2012 Leptoconopinae Leptoconops Skuse, 1889 Leptoconops ( Holoconops ) laurae (Weiss, 1912) Remm 1988b CHIRONOMIDAE K. Kettani Number of species: 412 . Expected: 600 Faunistic knowledge of the family in Morocco: good Buchonomyiinae Buchonomyia Fittkau, 1955 Buchonomyia thienemanni Fittkau, 1955 Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe et al. 2015 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Moubayed-Breil 2018 , Rif Podonominae Paraboreochlus Thienemann, 1939 Paraboreochlus minutissimus (Strobl, 1895) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Tanypodinae Macropelopiini Apsectrotanypus Fittkau, 1962 Apsectrotanypus trifascipennis (Zetterstedt, 1838) Kettani et al. 2010 , Rif , Aïn Abou Hayane (Tiouertiouane, 880 m), Oued Maggou (Maggou village, 777 m), Oued Kanar (Gorges Kanar, 280 m); Kettani and Langton 2012 Macropelopia Thienemann, 1916 Macropelopia adaucta Kieffer, 1916 Kettani and Langton 2011 , Rif , Fifi, Issaguen; Kettani and Langton 2012 Macropelopia nebulosa (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrotanypus Kieffer, 1909 Psectrotanypus varius (Fabricius, 1787) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Pentaneurini Ablabesmyia Johannsen, 1905 Ablabesmyia ( Ablabesmyia ) ebbae Lehmann, 1981 Lehmann 1981 ; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Ablabesmyia ( Ablabesmyia ) longistyla Fittkau, 1962 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , source Maggou (Maggou, 1300 m), Oued Talembote; Kettani and Langton 2012 Ablabesmyia ( Ablabesmyia ) monilis (Linnaeus, 1758) Reiss 1977 , Rif , Tétouan, HA , kranichsee (Dra-Tal); Azzouzi and Laville 1987 , Rif , retenue El Makhazine; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Conchapelopia Fittkau, 1957 Conchapelopia ( Conchapelopia ) melanops (Meigen, 1818) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Conchapelopia ( Conchapelopia ) pallidula (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Conchapelopia ( Conchapelopia ) viator (Kieffer, 1911) = Conchapelopia Pe 1 Langton 1991 in Kettani et al. 1994 : 28, Kettani et al. 1995 : 256 Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 ; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Larsia Fittkau, 1962 Larsia atrocincta (Goetghebuer, 1942) Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , Oued Moulay Bouchta; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Larsia curticalcar (Kieffer, 1918) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m); Kettani and Langton 2012 Nilotanypus Kieffer, 1923 Nilotanypus dubius (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kanar (Gorges Kanar, 280 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paramerina Fittkau, 1962 Paramerina cingulata (Walker, 1856) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paramerina divisa (Walker, 1856) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Paramerina mauretanica Fittkau, 1962 Fittkau 1962 , Atlas (850 m), SA ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 , EM , Figuig; Kettani et al. 2001 ; Ashe and O'Connor 2009 , EM , Figuig; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Paramerina spec. Greichenland (Fittkau, 1962) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani et al. 2010 , Rif , Oued Chrafat (Armotah, 900 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Moubayed-Breil 2018 , Rif Pentaneurella Fittkau & Murray, 1983 Pentaneurella sp. Ourika Azzouzi et al. 1992 , HA ; Kettani et al. 2001 Rheopelopia Fittkau, 1962 Rheopelopia maculipennis (Zetterstedt, 1838) Naya 1988 , MA , Haut et Moyen Sebou; Azzouzi and Laville 1987 , MA , Oum-er-Rbia, HA , Tensift; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Ruisselet maison forestière (Talassemtane, 1683 m), Source Maggou (Maggou, 1300 m), Oued Talembote (avant village Talembote, 320 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheopelopia murrayi Dowling, 1983 Dowling 1983 , AA , Tata (Moyen Draa); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; 2012 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheopelopia ornata (Meigen, 1838) Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Telopelopia Roback, 1971 Telopelopia fascigera (Verneaux, 1970) = Telopelopia maroccana Murray, 1980, in Reiss 1977 : 91, Murray 1980 : 151, Azzouzi and Laville 1987 : 218, Ashe and Cranston 1990 : 133 Reiss 1977 , AP , Larache, HA , Dra-Tal; Murray 1980 , AP , Larache, HA , Dra-Tal; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Telmatopelopia Fittkau, 1962 Telmatopelopia nemorum (Goetghebuer, 1921) Kettani et al. 1996 , Rif , Oued Khizana (Oued Laou); Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Thienemannimyia Fittkau, 1957 Thienemannimyia ( Thienemannimyia ) berkanea Dowling, 1987 Dowling 1987 , EM , Berkane; Azzouzi et al. 1992 , EM , Environs de Berkane, HA , Ouarzazate (1160 m), Oasis Meski (1160 m), Aït Saoun; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) carnea (Fabricius, 1805) Kettani and Langton 2012 , Rif Thienemannimyia ( Thienemannimyia ) choumara Dowling, 1983 Dowling 1983 , EM , Environ de Berkane (Monts de Bni Snassen), HA , Souk des Judais (Marrakech); Azzouzi et al. 1987, HA , Dra-Tal; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Thienemannimyia ( Thienemannimyia ) geijskesi (Goetghebuer, 1934) Kettani and Langton 2012 , Rif , Oued Zarka Thienemannimyia ( Thienemannimyia ) laeta (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) lentiginosa (Fries, 1823) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) northumbrica (Edwards, 1929) Fittkau 1962 ; Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Trissopelopia Kieffer, 1923 Trissopelopia longimana (Staeger, 1839) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Xenopelopia Fittkau, 1962 Xenopelopia falcigera (Kieffer, 1911) Kettani and Langton 2011 , Rif , Anasser, Fifi, AP , marais de Loukous; Kettani and Langton 2012 Xenopelopia nigricans (Goetghebuer, 1927) Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia Fittkau, 1962 Zavrelimyia ( Zavrelimyia ) barbatipes (Kieffer, 1911) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 (?); Kettani et al. 2010 , Rif , Oued Tiffert (Tiffert Talassemtane, 1230 m), Aïn Abou Hayane (Tiouertiouane, 880 m), Oued Abiyati (Ifansa, 140 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) berberi Fittkau, 1962 Azzouzi and Laville 1987 ; Ashe and Cranston 1990 , HA , Tamhda; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) hirtimana (Kieffer, 1918) Kettani and Langton 2012 Zavrelimyia ( Zavrelimyia ) melanura (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) nubila (Meigen, 1830) Kettani and Langton 2011 , Rif , marais de Lemtahane ( PNPB ), Dayat Aïn Rami, Dayat Amlay; Kettani and Langton 2012 Procladiini Procladius Skuse, 1889 Procladius ( Holotanypus ) brevipetiolatus (Goetghebuer, 1935) Azzouzi et al. 1992 , HA , Oued Meski (1160 m), Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Procladius ( Holotanypus ) choreus (Meigen, 1804) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , AP , Merja Sidi Boughaba; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2010 , Rif , Aïn Talassemtane (Talassemtane, 1700 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Procladius ( Holotanypus ) culiciformis (Linnaeus, 1767) Kettani and Moubayed-Breil 2018 , Rif Procladius ( Holotanypus ) noctivagus (Kieffer, 1910) Azzouzi et al. 1992 , HA , Ouarzazate (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 Procladius ( Holotanypus ) sagittalis (Kieffer, 1909) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Procladius ( Psilotanypus ) anomalus Kieffer, 1906 Nomen dubium in Ashe and O'Connor 2009 : 213 Naya 1988 , MA ; Kettani et al. 2001 ; Kettani and Langton 2012 Procladius Pe 3 Langton 1991 Kettani et al. 1994 , Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 Tanypodini Tanypus Meigen, 1803 Tanypus ( Tanypus ) brevipalpis (Kieffer, 1923) Reiss 1977 , EM , Berkane; Ashe and O'Connor 2009 (?); Kettani and Langton 2012 Tanypus ( Tanypus ) kraatzi (Kieffer, 1912) Azzouzi et al. 1992 , HA , Oasis Meski; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Tanypus ( Tanypus ) punctipennis Meigen, 1818 Reiss 1977 , EM , Berkane; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesinae Boreoheptagyiini Boreoheptagyia Brundin, 1966 Boreoheptagyia legeri (Goetghebuer, 1933) = Boreoheptagyia punctulata (Goetghebuer, 1934), in Kettani et al. 2001 : 327 Ashe and Cranston 1990 ; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesini Diamesa Meigen, 1835 Diamesa aberrata Lundbeck, 1898 Saether 1968 ; Serra-Tosio 1973 ; Fittkau and Reiss 1987 ; Serra-Tosio 1983 ; Azzouzi and Laville 1987 , HA (2500–3350 m); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa bertrami Edwards, 1935 Serra-Tosio 1983 , HA , Gorges de Todra (2500 m); Azzouzi and Laville 1987 , HA , Gorges Todra; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa hamaticornis Kieffer, 1924 Reiss 1977 ; Serra-Tosio 1983 , HA , M'Goum; Azzouzi and Laville 1987 , HA , M'Goum; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa insignipes Kieffer, 1908 Serra-Tosio 1983 , HA (2500 m); Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut and Moyen Sebou; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Diamesa latitarsis (Goetghebuer, 1921) Vaillant 1955b ; Vaillant 1956b , HA , Asif Tessaout (M'Goum), Lac Tamhda (Anremer); Serra-Tosio 1967 ; Serra-Tosio 1967 ; Saether 1968 ; Serra-Tosio 1973 ; Azzouzi and Laville 1987 , HA ; Ashe and Cranston 1990 ; Kettani et al. 2001 , Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa steinboecki Goetghebuer, 1933 Vaillant 1956b , HA , Cascade Siroua, Oukaimeden, Sidi Chamarouch Diamesa tonsa (Haliday in Walker, 1856) = Diamesa thienemanni Kieffer, 1909 Naya 1988 , MA , Haut Sebou (Arhbalou Yahya, Oued Arbi, Pont Aït hamza); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 Diamesa vaillanti Serra-Tosio, 1972 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa veletensis Serra-Tosio, 1971 Serra-Tosio 1983 , HA (2500 m); Azzouzi and Laville 1987 , HA ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa zernyi Edwards, 1933 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Potthastia Kieffer, 1922 Potthastia gaedii (Meigen, 1838) Azzouzi and Laville 1987 , MA , oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Laou, Oued Afertane; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Potthastia pastoris (Edwards, 1933) Kettani and Moubayed-Breil 2018 , Rif Pseudodiamesa Goetghebuer, 1939 Pseudodiamesa ( Pseudodiamesa ) branickii (Nowicki, 1873) Naya 1988 , MA , Haut Sebou; Ashe and Cranston 1990 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Pseudodiamesa ( Pseudodiamesa ) nivosa (Goetghebuer, 1928) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Sympothastia Pagast, 1947 Sympothastia zavreli Pagast, 1947 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , aval Oued Krikra; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Syndiamesa Kieffer, 1918 Syndiamesa hygropterica (Kieffer, 1909) Naya 1988 , MA , Moyen Sebou (Sidi Abdellah, Dar El Arsa, Pont Oulad Slimane, Pont Portugais); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Protanypini Protanypus Kieffer, 1906 Protanypus morio (Zetterstedt, 1838) Naya 1988 , MA , Moyen Sebou; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Prodiamesinae Odontomesa Pagast, 1947 Odontomesa fulva (Kieffer, 1919) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Prodiamesa Kieffer, 1906 Prodiamesa olivacea (Meigen, 1818) Naya 1988 , MA , Haut Sebou (Haut Guigou); Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Maggou village, Ifansa; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladiinae Orthocladiini Acricotopus Kieffer, 1921 Acricotopus lucens (Zetterstedt, 1850) Kettani and Moubayed-Breil 2018 , Rif Brilla Kieffer, 1913 Brillia bifida (Kieffer, 1909) = Brilla modesta (Meigen, 1830) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Brillia flavifrons (Johannsen, 1905) Kettani and Langton 2012 Brilla longifurca Kieffer, 1921 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1995 , Rif , amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Talembote (Usine électrique, 120 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Bryophaenocladius Thienemann, 1934 Bryophaenocladius aestivus (Brundin, 1947) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius flexidens (Brundin, 1947) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius cf. furcatus Thienemann & Strenzke, 1940 Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius illimbatus (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius muscicola (Kieffer, 1906) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius nidorum (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius subvernalis (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius tuberculatus (Edwards 1929) Kettani and Moubayed-Breil 2018 , Rif Camptocladius Wulp, 1874 Camptocladius stercorarius (De Geer, 1976) Kettani and Moubayed-Breil 2018 , Rif Cardiocladius Kieffer, 1912 Cardiocladius capucinus (Zetterstedt, 1850) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cardiocladius fuscus Kieffer, 1924 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou (Amont de Aïn Tadout, Skhounate, amont confluence avec Oued Atchane, Pont Aït Hamza); Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius Kieffer, 1911 Chaetocladius ( Chaetocladius ) acuticornis (Kieffer in Potthast, 1914) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius dentiforceps (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Chaetocladius dissipatus (Edwards, 1929) Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Chaetocladius ( Chaetocladius ) melaleucus (Meigen, 1818) Kettani and Langton 2011 , Rif , Oued Sgara, Bab Tariouant, Bouztata; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius piger (Goetghebuer, 1913) Kettani and Moubayed-Breil 2018 , Rif Chaetocladius ( Chaetocladius ) perennis (Meigen, 1830) Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 Chaetocladius ( Chaetocladius ) vitellinus (Kieffer in Kieffer & Thienemann, 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Corynoneura Winnertz, 1846 Corynoneura carriana Edwards, 1924 Naya 1988 , MA , Haut Sebou (Haut Guigou, Aïn Nokra); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura celtica Edwards, 1924 Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura coronata Edwards, 1924 Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Corynoneura edwardsi Brundin, 1949 Kettani and Langton 2012 Corynoneura lacustris Edwards, 1924 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura lobata Edwards, 1924 Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Azzouzi et al. 1992 , HA ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura scutellata Winnertz, 1846 Kettani and Moubayed-Breil 2018 , Rif Corynoneura Pe 2 Langton 1991 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Corynoneurella Brundin, 1949 Corynoneurella paludosa Brundin, 1949 Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus van der Wulp, 1874 Cricotopus ( Cricotopus ) albiforceps (Kieffer in Thienemann and Kieffer 1916) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) annulator Goetghebuer, 1927 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2011 , Rif , Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) beckeri Hirvenoja, 1973 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Langton and Laville 2002; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) bicinctus (Meigen, 1818) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Naya 1988 , MA , Haut et Moyen Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) caducus Hirvenoja, 1973 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) ephippium (Zetterstedt, 1838) Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) levantinus Moubayed & Hirvenoja, 1986 Kettani et al. 1996 , Rif , Haut Maggou; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Inesmane (Adeldal, 1173 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Cricotopus ) pallidipes Edwards, 1929 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) pulchripes Verrall, 1912 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) similis Goetgnebuer, 1921 Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Afertane, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) tremulus (Linnaeus, 1758) Kettani et al. 2010 , Rif , Oued Maggou (Maggou village, 777 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) triannulatus (Macquart, 1826) Kettani et al. 1994 , Rif , Haut Laou, Oued Moulay Bouchta, Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) trifascia Edwards, 1929 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Maggou (Maggou village, 777 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) vierriensis Goetghebuer, 1935 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Isocladius ) brevipalpis Kieffer, 1909 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) laetus Hirvenoja, 1973 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) ornatus (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) sylvestris (Fabricius, 1794) Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Isocladius ) tricinctus (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) micans (Kieffer, 1918) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Talembote, Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) osellai Rossaro, 1990 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) rufiventris (Meigen, 1830) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; (Dar El Arsa, Pont oulad Slimane, Pont portugais); Naya 1988 , MA , Haut et Moyen Sebou; Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) skirwithensis (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukieferiella Thienemann, 1926 Eukieferiella ancyla Svensson, 1986 Kettani and Langton 2011 , Rif , Oued Tkarae; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella bedmari Vilchez-Quero & Laville, 1988 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Laville and Langton 2002 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella brehmi Gowin, 1943 Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (Usine électrique, 120 m) Eukiefferiella brevicalcar (Kieffer, 1911) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m), Oued Ametrasse (Ametrasse, 820 m); Kettani and Langton 2011 , Rif , Oued Issaguen, Oued Ketama, Oued Sgara; Bab Tariouant, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella claripennis (Lundbeck, 1898) Fittkau and Reiss 1978 ; Naya 1988 , MA , Moyen Sebou (Dar Cheik Harazem); Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukieffeiella clypeata (Thienemann, 1919) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella coerulescens (Kieffer in Zavřel, 1926) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou (Skhounate, Arhbalou Aberchane); Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Nord Maggou village (Maggou, 905 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella cyanea Thienemann, 1936 Vaillant 1955b , HA ; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , HA ; Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella devonica (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella dittmari Lehmann, 1972 Kettani and Langton 2011 , Rif , Oued Boujdad, Fifi; Kettani and Langton 2012 , Rif , Oued Zarka; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella fittkaui Lehmann, 1972 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella fuldensis Lehmann, 1972 Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella gracei (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Tassikeste; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella ilkleyensis (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella lobifera Goetghebuer, 1934 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella minor (Edwards, 1929) Vaillant 1955b , HA (1050 m); Vaillant 1956b , HA , Imi-N'Ifri; Azzouzi and Laville 1987 , HA ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella pseudomontana Goetghebuer, 1935 Kettani et al. 2010 , Rif , Oued Madissouka (Talassemtane, 1530 m), Oued Dchar d'Amran (Béni M'Hamed, 1180 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella similis Goetghebuer, 1939 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella tirolensis Goetghebuer, 1938 Kettani et al. 1994 , Rif , Oued Afertane; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Azzouzi et al. 1992 , HA , Oued Tensift; Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella Pe 2 Langton 1991 Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Halocladius Hirvenoja, 1973 Halocladius ( Halocladius ) varians (Staeger, 1839) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m); Ashe and Cranston 1990 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heleniella Gowin, 1943 Heleniella dorieri Serra-Tosio, 1967 Kettani and Langton 2012 Heleniella ornaticollis (Edwards, 1929) Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heleniella serratosioi Ringe, 1976 Kettani and Langton 2011 , Rif , Oued Hamma, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heterotrissocladius Spärck, 1923 Heterotrissocladius marcidus (Walker, 1856) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2012 Hydrobaenus Fries, 1830 Hydrobaenus conformis (Holmgren, 1869) Kettani and Moubayed-Breil 2018 , Rif Hydrosmittia Ferrington & Sæther, 2011 Hydrosmittia oxoniana (Edwards, 1929) = Pseudosmittia recta (Edwards, 1929), in Azzouzi and Laville 1987 : 218, Kettani et al. 2001 : 330, Kettani and Langton 2012 : 422 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Hydrosmittia ruttneri (Strenzke & Thienemann, 1942) Kettani and Moubayed-Breil 2018 , Rif Krenosmittia Thienemann & Krüger, 1939 Krenosmittia boreoalpina (Goetghebuer, 1944) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Krenosmittia camptophleps (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Krenosmittia halvorseni (Cranston & Sæther, 1986) Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Krenosmittia hispanica Wülker, 1957 Kettani et al. 2001 ; Laville and Langton 2002 ; Ashe and O'Connor 2012 Limnophyes Eaton, 1875 Limnophyes difficilis Brunidin, 1947 Kettani and Moubayed-Breil 2018 , Rif Limnophyes gelasinus Saether, 1990 Kettani and Moubayed-Breil 2018 , Rif Limnophyes habilis (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Limnophyes madeirae Sæther, 1985 Kettani and Moubayed-Breil 2018 , Rif Limnophyes minimus (Meigen, 1818) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Kettani et al. 2001 ; Kettani and Langton 2011 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Limnophyes natalensis (Kieffer, 1914) Kettani and Moubayed-Breil 2018 , Rif Limnophyes ninae Sæther, 1975 Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Limnophyes pentaplastus (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Limnophyes pumilio (Holmgren, 1869) Kettani and Moubayed-Breil 2018 , Rif Limnophyes punctipennis (Goetghebuer, 1919) Kettani and Langton 2012 Limnophyes Pe 1a Langton 1991 Kettani et al. 2010 , Rif , Oued Talembote (Usine électrique, 120 m); Kettani and Langton 2012 Metriocnemus van der Wulp, 1874 Metriocnemus ( Metriocnemus ) albolineatus Meigen, 1818 Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) eurynotus (Holmgren, 1883) = Metriocnemus hygropetricus Kieffer, 1912, in Ashe and O'Connor 2012 : 372 = Metriocnemus ( Metriocnemus ) obscuripes (Holmgren, 1869), in Azzouzi et al. 1992 : 229, Kettani et al. 2001 : 329, Kettani and Langton 2012 : 421 Boumezzough and Thomas 1987 , HA , Oued Réghaya (1740 m), Imlil; Azzouzi and Laville 1987 , HA , Oued Tensift; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Metriocnemus ( Metriocnemus ) fuscipes (Meigen, 1818) Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) hirticollis (Staeger, 1839) Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) ursinus Holmgren, 1869 Kettani and Moubayed-Breil 2018 , Rif Nanocladius Kieffer, 1913 Nanocladius ( Nanocladius ) balticus (Palmén, 1959) Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) dichromus (Kieffer 1906) Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) parvulus (Kieffer 1909) Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) rectinervis (Kieffer, 1911) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Inesmane (Adeldal, 1173 m), Oued Talembote (aval Barrage Talembote, 245 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius van der Wulp, 1874 Orthocladius ( Eudactylocladius ) fuscimanus (Kieffer, 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued Krikra, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , source Maggou (Maggou, 1300 m), Oued Chrafat (Armotah, 900 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) ashei Soponis, 1990 = Orthocladius luteipes Goetghebuer, in Azzouzi and Laville 1987 : 218 = Orthocladius rivicola Kieffer, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Fès, Oued boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique), aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) rivulorum Kieffer, 1909 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor; 2012 Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) thienemanni Kieffer, 1906 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Mesorthocladius ) frigidus (Zetterstedt, 1838) Vaillant 1955, HA (2900 m); Vaillant 1956b , HA , Lac Tamhda (Anremer); Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut Sebou (Haut Guigou); Fekhaoui et al. 1993 ; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Dchar d'Amran (Béni M'Hamed, 1180 m), Nord Maggou village (Maggou, 905 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Orthocladius ( Orthocladius ) oblidens (Walker, 1856) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) obumbratus Johannsen, 1905 = Orthocladius excavatus Brundin, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Orthocladius ( Orthocladius ) pedestris Kieffer, 1909 Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) rubicundus (Meigen, 1818) = Orthocladius saxicola Kieffer, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) vaillanti Langton & Cranston, 1991 Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Symposiocladius ) lignicola Kieffer in Potthast, 1914 = Symposiocladius lignicola Kieffer, in Kettani et al. 2010 : 70 Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Orthocladius ( Symposiocladius ) ruffoi Rossaro & Prato, 1991 = Orthocladius Pe 1 Langton 1991, in Azzouzi and Laville 1987 : 218 = Rheortocladius sp A Langton 1991, in Kettani et al. 1995 : 257 = Rheorthocladius ruffoi Rossaro, in Kettani et al. 1997 : 184 Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Ifansa, 105 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Paracricotopus Brundin, 1956 Paracricotopus niger (Kieffer, 1913) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès; Kettani et al. 1994 , Rif , Oued Afertane; Kettani et al. 1995 , Rif , amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou, Oued Tamaridine (Zaouit et Habtyiène, 819 m), Oued Kelaâ (Akoumi, 400 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (155 m), Oued Tassikeste (240 m), Oued Laou (Ifansa, 105 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parakiefferiella Thienemann, 1936 Parakiefferiella coronata (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parakiefferiella wuelkeri Moubayed, 1994 = Parakiefferiella sp. d Wülker, in Azzouzi et al. 1992 : 230 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parametriocnemus Goetghebuer, 1932 Parametriocnemus boreoalpinus Gowin & Thienemann, 1942 Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parametriocnemus stylatus (Spärck, 1923) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Béni M'Hamed (1330 m), Haut Maggou (1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (320 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Parametriocnemus valescurensis Moubayed & Langton, 1999 Kettani and Langton 2011 , Rif , Oued Issaguen; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parametriocnemus Pe 1 Langton 1991 Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Talembote (320 m); Kettani and Langton 2012 Paraphaenocladius Thienemann, 1924 Paraphaenocladius exagitans ssp. 1 Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius impensus impensus (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius irritus Walker, 1856 Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius pseudirritus Strenzke, 1950 Kettani and Moubayed-Breil 2018 , Rif Paratrissocladius Zavřel, 1937 Paratrissocladius excerptus (Walker, 1856) Kettani et al. 1996 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parorthocladius Thienemann, 1935 Parorthocladius nudipennis (Kieffer in Kieffer & Thienemann 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psecrocladius Kieffer, 1906 Psectrocladius ( Allopsectrocladius ) obvius (Walker, 1856) = Psectrocladius dilatatus (van der Wulp, 1859), in Naya 1988 : 48 Naya 1988 , MA , Moyen Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2011 , Rif , sources de Issaguen; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Allopsectrocladius ) platypus (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Mesopsectrocladius ) barbatipes Kieffer, 1923 Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Laou (Afertane, 56 m); Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psecrocladius ( Psectrocladius ) brehmi Kieffer, 1923 Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psectrocladius ( Psectrocladius ) fennicus Storå, 1939 Kettani and Langton 2012 Psectrocladius ( Psectrocladius ) limbatellus (Holmgren, 1869) Wülker 1959 ; Azzouzi and Laville 1987 , HA , Lac Tamhda (2800 m); Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Psectrocladius ) octomoculatus Wülker, 1956 Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psectrocladius ( Psectrocladius ) sordidellus (Zetterstedt, 1838) Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous (NE Boucharene); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Psectrocladius ) ventricosus Kieffer, 1925 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Pseudosmittia Edwards, 1932 Pseudosmittia albipennis (Goetghebuer, 1921) Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia baueri Strenzke, 1960 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia danconai (Marcuzzi, 1947) Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia holsata Thienemann & Stenzke, 1940 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia obtusa Strenzke, 1960 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia trilobata Edwards, 1929 Kettani and Moubayed-Breil 2018 , Rif Pseudorthocladius Goetghebuer, 1943 Pseudorthocladius ( Pseudorthocladius ) berthelemyi Moubayed, 1990 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Pseudorthocladius ( Pseudorthocladius ) curtistylus (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oasis Meski (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Pseudorthocladius near Pe 3 Langton 1991 Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 Rheocricotopus Brundin, 1956 Rheocricotopus ( Psilocricotopus ) atripes (Kieffer, 1913) = Rheocricotopus ( Psilocricotopus ) foveatus foveatus (Edwards, 1929), in Naya 1988 : 40 Naya 1988 , MA , Haut Sebou (Haut Guigou); Azzouzi et al. 1992 , HA , Oued Tensift, Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , Haut Laou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote, Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 56 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) chalybeatus subsp. chalybeatus (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) gallicus Lehamnn 1969 Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) glabricollis (Meigen, 1830) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Ametrasse (Ametrasse, 820 m), Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) meridionalis Moubayed-Breil, 2016 Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) tirolus Lehmann, 1969 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) effusus (Walker, 1856) Reiss 1977 ; Naya 1988 , MA , Haut et Moyen Sebou; Fekhaoui et al. 1993 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) fuscipes (Kieffer, 1909) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Maggou (905 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) rifensis Moubayed & Kettani, 2019 Moubayed-Breil and Kettani, Rif , Chrafate, Challal Sghir (Akchour) Synorthocladius Thienemann, 1935 Synorthocladius semivirens (Kieffer, 1909) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Smittia Holmgren, 1869 Smittia alpicola Goetghebuer, 1941 Kettani and Moubayed-Breil 2018 , Rif Smittia aterrima Meigen, 1818 Kettani and Moubayed-Breil 2018 , Rif Smittia contingens Walker, 1856 Kettani and Moubayed-Breil 2018 , Rif Smittia foliacea (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Smittia pratorum Goetghebuer, 1927 Kettani and Moubayed-Breil 2018 , Rif Thienemannia Kieffer, 1909 Thienemannia cf. fulvofasciata (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Thienemannia gracilis Kieffer, 1909 Kettani and Moubayed-Breil 2018 , Rif Thienemanniella Kieffer, 1911 Thienemanniella acuticornis (Kieffer, 1912) Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (320 m); Kettani and Langton 2011 , Rif , Oued Hamma, Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Thienemanniella clavicornis (Kieffer, 1911) Kettani and Moubayed-Breil 2018 , Rif Thienemanniella majuscula (Edwards, 1924) Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemanniella vittata (Edwards, 1924) Kettani et al. 1996 , Rif , Haut Maggou; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou (1300 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemanniella Pe 2a Langton 1991 Kettani et al. 2010 , Rif , Oued Maggou (905 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote; Kettani and Langton 2012 Thienemanniella Pe 2b Langton 1991 Kettani et al. 2010 , Rif , Oued Maggou (905 m), Oued Talembote; Kettani and Langton 2012 Trissocladius Kieffer, 1908 Trissocladius brevipalpis Kieffer in Kieffer & Thienemann 1908 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia Kieffer, 1922 Tvetenia bavarica (Goetghebuer, 1934) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia calvescens (Edwards, 1929) Naya 1988 , MA , Moyen Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m), Oued Talembote (245 m), Oued Laou (Afertane, 56 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tvetenia discoloripes (Goetghebuer & Thienemann in Thienemann, 1936) Kettani and Langton 2011 , Rif , Oued Nakhla, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia verralli (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, amont Oued Nakhla; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , ruisselet maison forestière Talassemtane (1683 m), Oued Tamaridine (Zaouiet et Habtiyiène, 819 m), Oued Talembote (245 m), Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zalutschia Lipina, 1939 Zalutschia humphriesiae Dowling & Murray, 1980 Kettani and Langton 2011 , Rif , marais de Lemtahane ( PNPB ), Dayat Fifi; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Chironominae Chironomini Chironomus Meigen, 1803 Chironomus ( Baeotendipes ) noctivagus (Kieffer, 1911) Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) annularius Meigen, 1818 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) aprilinus sensu Meigen, 1818 = Chironomus halophilus Kieffer, in Ramdani and Tourenq 1982 : 180, in Naya 1988 : 50 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut Sebou; Fekhaoui et al. 1993 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) bernensis Klötzli, 1973 = Chironomus sp 1 Kettani 1994 Kettani et al. 1994 ; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) calipterus Kieffer, 1908 Reiss 1977 , AP , Larache; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) longistylus Goetghebuer, 1921 Kettani et al. 2011, Rif , Oued Ketama; Kettani and Langton 2012 Chironomus ( Chironomus ) luridus Strenzke, 1959 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) nuditarsis Keyl, 1961 Kettani et al. 2011, Rif , Oued Boujdad (Kitane, 42 m), Oued El Hatba (SIBE Jebel Moussa, 165 m); Kettani and Langton 2012 , Rif , SIBE Jebel Moussa Chironomus ( Chironomus ) piger (Strenzke, 1956) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) plumosus (Linnaeus, 1758) Reiss 1977 , AP , Larache, AA , Dra-Tal; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 ; Naya 1988 , MA , Sidi Abdellah, Dar Cheih Harazem, Dar El Arsa; Fekhaoui et al. 1993 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Aïn Tissmelal (Tissmelal, 1046 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) prasinus sensu Pinder, 1978 Kettani et al. 2011, Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 Chironomus ( Chironomus ) riparius Meigen, 1804 = Chironomus thummi Kieffer, in Naya 1988 : 51, Fekhaoui et al. 1993 : 26 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou; Naya 1988 , MA , Moyen Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) salinarius Kieffer, 1915 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) tentans Fabricius, 1805 = Camptochironomus tentans Fabricius, 1805, in Naya 1988 : 50 Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Cladopelma Kieffer, 1921 Cladopelma virescens (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus Kieffer, 1918 Cryptochironomus ( Cryptochironomus ) albofasciatus (Staeger, 1839) = Cryptochironomus obreptans Walker, 1856, in Kettani 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 Cryptochironomus ( Cryptochironomus ) psittacinus (Meigen, 1830) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Cryptochironomus ( Cryptochironomus ) rostratus Kieffer, 1921 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou, oued Oum-er-Rbia, Oued Boufekrane, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus ( Cryptochironomus ) supplicans (Meigen, 1830) Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus Pe 5 Langton 1991 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 Demicryptochironomus Lenz, 1941 Demicryptochironomus ( Demicryptochironomus ) vulneratus (Zetterstedt, 1838) Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m); Kettani and Langton 2012 Demicryptochironomus ( Irmakia ) neglectus Reiss, 1988 Kettani and Moubayed-Breil 2018 , Rif Demicryptochironomus ( Irmakia ) Pe 1 Langton 1991 Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, aval Oued Khemis; Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes Kieffer, 1913 Dicrotendipes collarti (Goetghebuer, 1936) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes cordatus Kieffer, 1922 Kettani et al. 1996 , Rif , Oued Khizana (Oued Laou); Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes fusconotatus (Kieffer, 1922) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes modestus (Say, 1823) Kettani et al. 2011, Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 Dicrotendipes nervosus (Staeger, 1839) = Limnochirononomus nervosus Staeger, in Naya 1988 : 53 Naya 1988 , MA , Moyen Sebou (Sidi Abdellah); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes notatus (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes pallidicornis (Goetghebuer, 1934) Azzouzi and Laville 1987 , Rif , Retenue El Makhazine, MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes peringueyanus Kieffer, 1924 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 Dicrotendipes septemmaculatus (Becker, 1908) = Dicrotendipes pilosimanus Kieffer, in Reiss 1977 : 91, Azzouzi and Laville 1987 : 219 Reiss 1977 , AP , Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , AP , Larache; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 Endochironomus Kieffer, 1918 Endochironomus albipennis (Meigen, 1830) Naya 1988 , MA , Haut Sebou (Skhounata); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Endochironomus tendens (Fabricius, 1775) Naya 1988 , MA , Moyen Sebou (Gantra Mdez, Azzaba); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes Kieffer, 1913 Glyptotendipes ( Caulochironomus ) viridis (Macquart, 1834) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes ( Glyptotendipes ) cauliginellus (Kieffer, 1913) = Glyptotendipes gripekoveni (Kieffer) Naya 1988 , MA , Haut Sebout (Guigou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes ( Glyptotendipes ) pallens (Meigen, 1804) Azzouzi and Laville 1987 , Rif , Retenue El Makhazine; Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes sp A Langton 1991 Naya 1988 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Glyptotendipes sp B Langton 1991 Naya 1988 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Harnischia Kieffer, 1921 Harnischia curtilamellata (Malloch, 1915) Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Harnischia fuscimanus Kieffer, 1921 Azzouzi and Laville 1987 , Rif , Retenue El Makhazine, MA , Oued Boufekrane; Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Kiefferulus Goetghebuer, 1922 Kiefferulus ( Kiefferulus ) tendipediformis (Goetghebuer, 1921) Reiss 1977 , Rif , Tétouan; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2010 , Rif , Guelta 1 km après Amariguen (Jebel Setsou, 1280 m); Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Dalia (SIBE Jebel Moussa, 169 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Kloosia Kruseman, 1933 Kloosia pusilla (Linnaeus, 1767) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Lauterborniella Thienemann & Bause, 1913 Lauterborniella agrayloides (Kieffer, 1911) Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus Kieffer, 1918 Microchironomus deribae (Freeman, 1957) = Leptochirononomus deribae Freeman, in Reiss 1977 : 91, Ramdani and Tourenq 1982 : 180 Reiss 1977 , AP , Rabat; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus lendli (Kieffer, 1918) Reiss 1986 , AA , Oasis Meski; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus tener (Kieffer, 1918) Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Azzouzi et al. 1992 , HA , Oued Tensift, Barrage Lalla Takerkoust; Kettani and Langton 2012 Microtendipes Kieffer, 1915 Microtendipes britteni (Edwards, 1929) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Microtendipes chloris (Meigen, 1818) Kettani et al. 2011, Rif , Dayat En-Nâsser (Khandek En-Nâsser, 1177 m), source Bab Karn (Fifi, 1216 m), Dayat Fifi (1179 m); Kettani and Langton 2012 Microtendipes confinis (Meigen, 1830) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 Microtendipes diffinis (Edwards, 1929) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani et al. 2011, Rif , Dayat En-Nâsser (Khandek En-Nâsser, 1177 m), Dayat Aïn Rami, source Bab Karn (Fifi, 1216 m); Kettani and Langton 2012 Microtendipes pedellus (De Geer, 1776) Reiss 1977 , Rif , Environ de Tétouan; Fittkau and Reiss 1978 ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , Rif , Tétouan, HA ; Naya 1988 , MA , Haut Sebou (amont Aîn Tadout, Skhounate, Arhbalou Aberchane); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Nubensia Spies, 2015 Nubensia nubens (Edwards, 1929) = Polypedilum nubens (Edwards, 1929), in Azzouzi and Laville 1987 : 219; Kettani et al. 1994 : 28, 1995 : 257, 1996 : 137, 1997 : 184, 2001 : 331, 2010 : 70; Dakki 1997 : 65; Dakki et al. 2008: 32, Kettani and Langton 2012 : 423 Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Laou (Ifansa, 105 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parachironomus Lenz, 1921 Parachironomus frequens (Johannsen, 1905) Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Parachironomus parilis (Walker, 1856) Reiss 1977 , AP , Environ de Larache; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Ashe and Cranston 1990 ; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Paracladopelma Harnisch, 1923 Paracladopelma camptolabis (Kieffer, 1913) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paracladopelma galaptera (Townes, 1945) Azzouzi et al. 1992 , HA , Ouarzazate (1140 m), Gorges de Todra (1400 m); Kettani et al. 2001 ; Kettani and Langton 2012 Paracladopelma graminicolor (Kieffer, 1925) = Cryptotendipes graminicolor (Kieffer), in Azzouzi et al. 1992 : 230 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Paracladopelma laminatum (Kieffer, 1921) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paracladopelma mikianum (Goetghebuer, 1937) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paralauterborniella Lenz, 1941 Paralauterborniella nigrohalteralis (Malloch, 1915) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Paratendipes Kieffer, 1911 Paratendipes albimanus (Meigen, 1818) Naya 1988 , MA , Moyen Sebou (Mdez); Kettani et al. 1994 , Rif , aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued Mhajrat, aval Oued Khemis, Oued Laou (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratendipes nudisquama (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Paratendipes striatus (Kieffer, 1925) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Phaenopsectra Kieffer, 1921 Phaenopsectra flavipes (Meigen, 1818) Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum Kieffer, 1912 Polypedilum ( Pentapedilum ) ruandae Freeman, 1955 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Pentapedilum ) sordens (van der Wulp, 1875) = Polypedilum sp 1, in Kettani et al. 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Pentapedilum ) uncinatum (Goetghebuer, 1921) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Polypedilum ) albicorne (Meigen, 1838) Naya 1988 , MA , Haut Sebou; Kettani et al. 1995 , Rif , aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) arundineti (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 Polypedilum ( Polypedilum ) laetum (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) nubeculosum (Meigen, 1804) Reiss 1977 , Rif , Environ de Tétouan; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , Rif , Environ Tétouan, MA , Oued Sebou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) nubifer (Skuse, 1889) = Polypedilum pharao Kieffer, in Reiss 1977 : 91, Naya 1998: 55, Ramdani and Tourenq 1982 : 180 Kügler and Wool 1968 ; Reiss 1977 , AP , Larache, Rabat; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 , AP , Environ de Larache, Rabat, Merja Sidi Boughaba; Naya 1988 , MA , Haut Sebou; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) pedestre (Meigen, 1830) Reiss 1977 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 1994 , Rif , aval Barrage Talembote; Kettani et al. 1995 , Rif , Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Tripodura ) acifer Townes, 1945 Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 , MA , Oued Boufekroune, Oued Fès, Oued Sebou; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Polypedilum ( Tripodura ) aegyptium Kieffer, 1925 = Polypedilum pruina Freeman, in Reiss 1977 : 91 Reiss 1977 , AP , Larache, HA , Marrakech, AA , Dra-Tal; Reiss 1985 ; Azzouzi and Laville 1987 , AP , Larache, HA , Marrakech, AA , Gorges de Todra; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Polypedilum ( Tripodura ) bicrenatum Kieffer, 1921 Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) pullum (Zetterstedt, 1838) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) quadriguttatum Kieffer, 1921 Naya 1988 , MA , Moyen Sebou; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Polypedilum ( Tripodura ) scalaenum (Schrank, 1803) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Kettani et al. 1996 , Rif , Ras el Ma (Chefchaouen); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) tetracrenatum Hirvenoja, 1962 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) tridens Freeman, 1955 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Uresipedilum ) convictum (Walker, 1856) Reiss 1977 , AP , Environ de Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane (Gantra Mdez), Naya 1988 , MA , Haut Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued pont Béni M'Hamed (Béni M'Hamed, 1330 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Uresipedilum ) cultellatum Goetghebuer, 1931 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Polypedilum ontario -group sp. 1 Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Rheomus Laville & Reiss, 1988 Rheomus alatus Laville & Reiss, 1988 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Rheomus yahiae Laville & Reiss, 1988 Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Stenochironomus Kieffer, 1919 Stenochironomus gibbus Fabricius, 1794 Kettani and Moubayed-Breil 2018 , Rif Stictochironomus Kieffer, 1919 Stictochironomus caffrarius (Kieffer, 1921) Reiss 1977 ; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 Stictochironomus maculipennis (Meigen, 1818) Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Afertane; Kettani et al. 1995 , Rif , aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Stictochironomus pictulus (Meigen, 1830) Reiss 1977 , AP , Environ de Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1995 , Rif , aval Oued Kbir, aval Oued Krikra, Oued El Kbir; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Stictochironomus rosenschoeldi Zetterstedt, 1838 Kettani and Moubayed-Breil 2018 , Rif Stictochironomus reissi Cranston, 1989 = Stictochironomus sp. nov. Reiss, in Reiss 1977 : 91 Reiss 1977 ; Azzouzi and Laville 1987 , AA , M'Hamid, Dra-Tal; Kettani et al. 2001 ; Kettani and Langton 2012 Stictochironomus sticticus (Fabricius, 1781) = Stictochironomus histrio (Fabricius, 1794), in Kettani et al. 1996 : 138 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Berranda (Bouztate, 1259 m), Dayat Dalia (SIBE Jebel Moussa); Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Stictochironomus Pe 2 Langton 1991 Kettani et al. 2001 Xenochironomus Kieffer, 1921 Xenochironomus xenolabis (Kieffer, 1916) Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsini Cladotanytarsus Kieffer, 1921 Cladotanytarsus ( Cladotanytarsus ) atridorsum Kieffer, 1924 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Azzouzi et al. 1992 , HA , Aït Saoun, Gorges de Dadès (1900 m), vallée de Drâa, Marrakech; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cladotanytarsus ( Cladotanytarsus ) capensis (Freeman, 1954) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) ecristatus Reiss, 1991 = Tanytarsus sp. nov. (Morokko) Reiss, in Azzouzi and Laville 1987 : 219 Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 , EM , Berkane; Reiss 1991 ; Azzouzi et al. 1992 , HA ; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) mancus (Walker, 1856) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cladotanytarsus ( Cladotanytarsus ) pallidus Kieffer, 1922 = Cladotanytarsus Pe 5 Langton 1984 Azzouzi and Laville 1987 , MA , Oued Sebou, Oum Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) vanderwulpi (Edwards, 1929) Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Lithotanytarsus Thienemann, 1933 Lithotanytarsus dadesi Reiss, 1991 Reiss 1991 ; Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Lithotanytarsus emarginatus (Goetghebuer, 1933) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani and Langton 2012 Micropsectra Kieffer, 1909 Micropsectra andalusiaca Marcuzzi, 1950 Kettani and Moubayed-Breil 2018 , Rif Micropsectra apposita (Walker, 1856) = Micropsectra contracta Reiss, 1965 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Chrafat (Armotah, 900 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra aristata Pinder, 1976 Kettani and Langton 2012 , Rif , Oued Zarka Micropsectra atrofasciata (Kieffer, 1911) = Micropsectra bidentata (Goetghebuer, 1921), in Azzouzi et al. 1992 : 230; Kettani et al. 2001 : 332; Kettani and Langton 2011 : 590, 2012 : 424 Fittkau and Reiss 1978 ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Sebou (Arhbalou Aberchane), Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Madissouka (Talassemtane, 1530 m), Oued Chrafat (Armotah, 900 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval affluent Talembote, 155 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam, 650 m), cascade Zarka, Dayat En-Nâsser (Khandek En-Nâsser, 1177 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra junci (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra lacustris Säwedal, 1975 Kettani and Langton 2012 , Rif , Oued Zarka Micropsectra lindrothi Goetghebuer, 1931 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra notescens (Walker, 1856) Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara, ruisselet Bab Tariouant, Oued Berranda (Bouztate, 1259 m), source Bab Karn (Fifi, 1220 m), Dayat Fifi (Fifi, 1179); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra pallidula (Meigen, 1830) Kettani and Moubayed-Breil 2018 , Rif Micropsectra schrankelae Stur & Ekrem, 2006 Kettani and Moubayed-Breil 2018 , Rif Micropsectra zernyi Marcuzzi, 1950 Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus Thienemann & Bause, 1913 Paratanytarsus bituberculatus (Edwards, 1929) Azzouzi et al. 1992 , MA , Lac Aguelmane Azigza (1510 m); Kettani et al. 1995 , Rif , Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Paratanytarsus dissimilis (Johannsen, 1905) = Paratanytarsus confusus Palmén, 1960, in Naya 1988 : 40; Dakki et al. 2008: 32; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 423 Naya 1988 , MA , Haut Sebou; Dakki et al. 2008, MA , Oued Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus grimmii (Schneider, 1885) Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Paratanytarsus inopertus (Walker, 1856) Reiss 1977 , Rif , Environ Tétouan; Fittkau and Reiss 1978 ; Reiss and Säwedal 1981 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus mediterraneus Reiss & Säwedal, 1981 Reiss and Säwedal 1981 , Rif , Estuaire Oued Mharka (Tanger), AP , Oued Loukous; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous; Kettani and Langton 2012 Paratanytarsus tenellulus (Goetghebuer, 1921) = Microspsectra tenellula Reiss 1977 : 91; Azzouzi and Laville 1987 : 219 Reiss 1977 , MA , Lac Kranichsee; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Paratanytarsus tenuis (Meigen, 1830) = Tanytarsus tenuis Meigen, in Naya 1988 : 57 Naya 1988 , MA , Moyen Sebou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Rheotanytarsus Thienemann & Bause, 1913 Rheotanytarsus ceratophylli Dejoux, 1973 Naya 1988 , MA , Moyen et Bas Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Rheotanytarsus curtistylus (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oasis Meski (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus langtoni Moubayed & Kettani, 2018 Moubayed-Breil and Kettani 2018 , Rif , Oued Farda; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Rheotanytarsus muscicola Thienemann, 1929 Reiss 1977 , AP , Environ de Larache, AA , Dra-Tal (Tissint Moyen Dra); Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus nigricauda Fittkau, 1960 Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus pellucidus (Walker, 1818) = Rheotanytarsus distinctissimus (Brundin, 1947), in Kettani et al. 1995 : 258; Kettani et al. 1996 : 138, 1997 : 185; Kettani and Langton 2012 : 424 Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus pentapoda (Kieffer, 1909) = Rheotanytarsus sp 1, in Kettani et al. 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Rheotanytarsus photophilus (Goetghebuer, 1921) Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Rheotanytarsus procerus Reiss, 1991 Reiss 1991 , HA ; Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus reissi Lehmann, 1970 Lehmann, 1970; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus rhenanus Klink, 1983 Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus ringei Lehmann, 1970 Lehmann, 1970; Reiss 1977 , Rif , Environ Tétouan; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , Rif , Tétouan, MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus Pe 3 Langton 1991 Kettani et al. 2010 ; Kettani and Langton 2011 , Rif , Oued Sgara (Ketama, 1300 m); Kettani and Langton 2012 Stempellina Thienemann & Bause, 1913 Stempellina almi Brundin, 1947 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2012 Stempellina bausei (Kieffer, 1911) Kettani and Langton 2012 , Rif , Ketama; Kettani and Moubayed-Breil 2018 , Rif Stempellinella Brundin, 1947 Stempellinella brevis (Edwards, 1929) Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Tanytarsus van der Wulp, 1874 Tanytarsus brundini Lindeberg, 1963 Kettani et al. 1994 , Rif , Oued Moulay Bouchta, aval Oued Laou; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus chinyensis Goetghebuer, 1934 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Fifi (Fifi, 1179 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus cretensis Reiss, 1987 = Tanytarsus sp. nov. ( creticus ), in Reiss 1977 : 91; Azzouzi and Laville 1987 : 219 = Cladotanytarsus sp 1, in Kettani et al. 1995 : 258 Reiss and Fittkau 1971 ; Reiss 1977 , EM , Environ de Berkane; Reiss 1987 ; Azzouzi and Laville 1987 , Rif , Tétouan, AP , Larache, Kénitra; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsus dibranchius Kieffer, 1926 = Tanytarsus separabilis Brundin, 1947, in Kettani et al. 1994 : 29; Kettani et al. 1995 : 258, 1996 : 138, 2001 : 332; Dakki 1997 : 63; Kettani and Langton 2012 : 424 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsus ejuncidus (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Tanytarsus eminulus (Walker, 1856) Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus formosanus Kieffer, 1912 = Tanytarsus horni Goetghebuer, 1934, in Reiss and Fittkau 1971 : 122; Reiss 1977 : 91; Fittkau and Reiss 1978 : 439; Ramdani and Tourenq 1982 : 180; El Mezdi and Giudicelli 1985 : 292; Azzouzi and Laville 1987 : 219; Ashe and Cranston 1990 : 341; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 424 Reiss and Fittkau 1971 , Rif , M'Diq; Reiss 1977 , Rif , Environ Tétouan, AP , Larache, Rabat, Kénitra; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , HA , Oued Tensift; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus gregarius Kieffer, 1909 Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Tanytarsus heusdensis Goetghebuer, 1923 Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Ifansa, 105 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus mendax Kieffer, 1925 Kettani and Moubayed-Breil 2018 , Rif Tanytarsus medius Reiss & Fittkau, 1971 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus palettaris Verneaux, 1969 Kettani et al. 1994 , Rif , aval Oued Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus pallidicornis (Walker, 1856) Kettani and Langton 2011 , Rif , Dayat Fifi (Fifi, 1179 m), Oued El Hatba (SIBE Jebel Moussa, 165 m); Kettani and Langton 2012 Tanytarsus recurvatus Brundin, 1947 Kettani and Langton 2011 , Rif , Oued El Hamma (El Hamma, 240 m); Kettani and Langton 2012 Tanytarsus signatus (van der Wulp, 1859) = Tanytarsus Pe 5 Langton 1991, in Azzouzi and Laville 1987 : 219 Kügler and Reiss 1973 ; Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Aïn Rami (373 m), Dayat Amlay (258 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus verralli Goetghebuer, 1928 Kettani and Langton 2011 , Rif , Oued Taida (650 m); Kettani and Langton 2012 Tanytarsus volgensis Miseiko, 1967 = Tanytarsus fimbriatus Reiss & Fittkau, 1971, in Fittkau and Reiss 1978 : 439; Azzouzi and Laville 1987 : 219; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 424 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou, HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus Pe 14 Langton 1991 Kettani and Langton 2011 , Rif , source Issaguen (Ketama, 1600 m); Kettani and Langton 2012 Tanytarsus Pe 23 Langton 1991 Kettani and Langton 2011 , Rif , Oued El Hamma (El Hamma, 240 m); Kettani and Langton 2012 Virgatanytarsus Pinder, 1982 Virgatanytarsus albisutus (Santos-Abreu, 1918) = Virgatanytarsus maroccanus Kügler and Reiss, in Azzouzi and Laville 1987 : 219 Fittkau and Reiss 1978 ; Reiss and Schurch 1984 , AA , Dra-Tal; Reiss 1986 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia, AA , Dra-Tal; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Virgatanytarsus ansatus Reiss & Schürch, 1984 Reiss and Schurch 1984 , HA ; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Virgatanytarsus arduennensis (Goetghebuer, 1922) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Virgatanytarsus triangularis (Goetghebuer, 1928) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Virgatanytarsus Pe 1 Langton 1991 Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 Zavrelia Kieffer, Thienemann & Bause, 1913 Zavrelia pentatoma Kieffer & Bause, 1913 Kettani and Langton 2012 Zavrelia Pe 1 Langton, 1991 Kettani and Langton 2011 , Rif , Oued Berranda (Bouztate, 1259 m); Kettani and Langton 2012 Acknowledgment We gratefully acknowledge the invaluable assistance and cooperation of Patrick Ashe (Dublin, Ireland) who contributed greatly to the revision of this family. Buchonomyiinae Buchonomyia Fittkau, 1955 Buchonomyia thienemanni Fittkau, 1955 Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe et al. 2015 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Moubayed-Breil 2018 , Rif Podonominae Paraboreochlus Thienemann, 1939 Paraboreochlus minutissimus (Strobl, 1895) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Tanypodinae Macropelopiini Apsectrotanypus Fittkau, 1962 Apsectrotanypus trifascipennis (Zetterstedt, 1838) Kettani et al. 2010 , Rif , Aïn Abou Hayane (Tiouertiouane, 880 m), Oued Maggou (Maggou village, 777 m), Oued Kanar (Gorges Kanar, 280 m); Kettani and Langton 2012 Macropelopia Thienemann, 1916 Macropelopia adaucta Kieffer, 1916 Kettani and Langton 2011 , Rif , Fifi, Issaguen; Kettani and Langton 2012 Macropelopia nebulosa (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrotanypus Kieffer, 1909 Psectrotanypus varius (Fabricius, 1787) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Pentaneurini Ablabesmyia Johannsen, 1905 Ablabesmyia ( Ablabesmyia ) ebbae Lehmann, 1981 Lehmann 1981 ; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Ablabesmyia ( Ablabesmyia ) longistyla Fittkau, 1962 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , source Maggou (Maggou, 1300 m), Oued Talembote; Kettani and Langton 2012 Ablabesmyia ( Ablabesmyia ) monilis (Linnaeus, 1758) Reiss 1977 , Rif , Tétouan, HA , kranichsee (Dra-Tal); Azzouzi and Laville 1987 , Rif , retenue El Makhazine; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Conchapelopia Fittkau, 1957 Conchapelopia ( Conchapelopia ) melanops (Meigen, 1818) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Conchapelopia ( Conchapelopia ) pallidula (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Conchapelopia ( Conchapelopia ) viator (Kieffer, 1911) = Conchapelopia Pe 1 Langton 1991 in Kettani et al. 1994 : 28, Kettani et al. 1995 : 256 Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 ; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Larsia Fittkau, 1962 Larsia atrocincta (Goetghebuer, 1942) Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , Oued Moulay Bouchta; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Larsia curticalcar (Kieffer, 1918) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m); Kettani and Langton 2012 Nilotanypus Kieffer, 1923 Nilotanypus dubius (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kanar (Gorges Kanar, 280 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paramerina Fittkau, 1962 Paramerina cingulata (Walker, 1856) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paramerina divisa (Walker, 1856) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Paramerina mauretanica Fittkau, 1962 Fittkau 1962 , Atlas (850 m), SA ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 , EM , Figuig; Kettani et al. 2001 ; Ashe and O'Connor 2009 , EM , Figuig; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Paramerina spec. Greichenland (Fittkau, 1962) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani et al. 2010 , Rif , Oued Chrafat (Armotah, 900 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Moubayed-Breil 2018 , Rif Pentaneurella Fittkau & Murray, 1983 Pentaneurella sp. Ourika Azzouzi et al. 1992 , HA ; Kettani et al. 2001 Rheopelopia Fittkau, 1962 Rheopelopia maculipennis (Zetterstedt, 1838) Naya 1988 , MA , Haut et Moyen Sebou; Azzouzi and Laville 1987 , MA , Oum-er-Rbia, HA , Tensift; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Ruisselet maison forestière (Talassemtane, 1683 m), Source Maggou (Maggou, 1300 m), Oued Talembote (avant village Talembote, 320 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheopelopia murrayi Dowling, 1983 Dowling 1983 , AA , Tata (Moyen Draa); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; 2012 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheopelopia ornata (Meigen, 1838) Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Telopelopia Roback, 1971 Telopelopia fascigera (Verneaux, 1970) = Telopelopia maroccana Murray, 1980, in Reiss 1977 : 91, Murray 1980 : 151, Azzouzi and Laville 1987 : 218, Ashe and Cranston 1990 : 133 Reiss 1977 , AP , Larache, HA , Dra-Tal; Murray 1980 , AP , Larache, HA , Dra-Tal; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Telmatopelopia Fittkau, 1962 Telmatopelopia nemorum (Goetghebuer, 1921) Kettani et al. 1996 , Rif , Oued Khizana (Oued Laou); Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Thienemannimyia Fittkau, 1957 Thienemannimyia ( Thienemannimyia ) berkanea Dowling, 1987 Dowling 1987 , EM , Berkane; Azzouzi et al. 1992 , EM , Environs de Berkane, HA , Ouarzazate (1160 m), Oasis Meski (1160 m), Aït Saoun; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) carnea (Fabricius, 1805) Kettani and Langton 2012 , Rif Thienemannimyia ( Thienemannimyia ) choumara Dowling, 1983 Dowling 1983 , EM , Environ de Berkane (Monts de Bni Snassen), HA , Souk des Judais (Marrakech); Azzouzi et al. 1987, HA , Dra-Tal; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Thienemannimyia ( Thienemannimyia ) geijskesi (Goetghebuer, 1934) Kettani and Langton 2012 , Rif , Oued Zarka Thienemannimyia ( Thienemannimyia ) laeta (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) lentiginosa (Fries, 1823) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemannimyia ( Thienemannimyia ) northumbrica (Edwards, 1929) Fittkau 1962 ; Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Trissopelopia Kieffer, 1923 Trissopelopia longimana (Staeger, 1839) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Xenopelopia Fittkau, 1962 Xenopelopia falcigera (Kieffer, 1911) Kettani and Langton 2011 , Rif , Anasser, Fifi, AP , marais de Loukous; Kettani and Langton 2012 Xenopelopia nigricans (Goetghebuer, 1927) Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia Fittkau, 1962 Zavrelimyia ( Zavrelimyia ) barbatipes (Kieffer, 1911) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 (?); Kettani et al. 2010 , Rif , Oued Tiffert (Tiffert Talassemtane, 1230 m), Aïn Abou Hayane (Tiouertiouane, 880 m), Oued Abiyati (Ifansa, 140 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) berberi Fittkau, 1962 Azzouzi and Laville 1987 ; Ashe and Cranston 1990 , HA , Tamhda; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) hirtimana (Kieffer, 1918) Kettani and Langton 2012 Zavrelimyia ( Zavrelimyia ) melanura (Meigen, 1804) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zavrelimyia ( Zavrelimyia ) nubila (Meigen, 1830) Kettani and Langton 2011 , Rif , marais de Lemtahane ( PNPB ), Dayat Aïn Rami, Dayat Amlay; Kettani and Langton 2012 Procladiini Procladius Skuse, 1889 Procladius ( Holotanypus ) brevipetiolatus (Goetghebuer, 1935) Azzouzi et al. 1992 , HA , Oued Meski (1160 m), Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Procladius ( Holotanypus ) choreus (Meigen, 1804) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , AP , Merja Sidi Boughaba; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2010 , Rif , Aïn Talassemtane (Talassemtane, 1700 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Procladius ( Holotanypus ) culiciformis (Linnaeus, 1767) Kettani and Moubayed-Breil 2018 , Rif Procladius ( Holotanypus ) noctivagus (Kieffer, 1910) Azzouzi et al. 1992 , HA , Ouarzazate (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 Procladius ( Holotanypus ) sagittalis (Kieffer, 1909) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Procladius ( Psilotanypus ) anomalus Kieffer, 1906 Nomen dubium in Ashe and O'Connor 2009 : 213 Naya 1988 , MA ; Kettani et al. 2001 ; Kettani and Langton 2012 Procladius Pe 3 Langton 1991 Kettani et al. 1994 , Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 Tanypodini Tanypus Meigen, 1803 Tanypus ( Tanypus ) brevipalpis (Kieffer, 1923) Reiss 1977 , EM , Berkane; Ashe and O'Connor 2009 (?); Kettani and Langton 2012 Tanypus ( Tanypus ) kraatzi (Kieffer, 1912) Azzouzi et al. 1992 , HA , Oasis Meski; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Tanypus ( Tanypus ) punctipennis Meigen, 1818 Reiss 1977 , EM , Berkane; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesinae Boreoheptagyiini Boreoheptagyia Brundin, 1966 Boreoheptagyia legeri (Goetghebuer, 1933) = Boreoheptagyia punctulata (Goetghebuer, 1934), in Kettani et al. 2001 : 327 Ashe and Cranston 1990 ; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesini Diamesa Meigen, 1835 Diamesa aberrata Lundbeck, 1898 Saether 1968 ; Serra-Tosio 1973 ; Fittkau and Reiss 1987 ; Serra-Tosio 1983 ; Azzouzi and Laville 1987 , HA (2500–3350 m); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa bertrami Edwards, 1935 Serra-Tosio 1983 , HA , Gorges de Todra (2500 m); Azzouzi and Laville 1987 , HA , Gorges Todra; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa hamaticornis Kieffer, 1924 Reiss 1977 ; Serra-Tosio 1983 , HA , M'Goum; Azzouzi and Laville 1987 , HA , M'Goum; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa insignipes Kieffer, 1908 Serra-Tosio 1983 , HA (2500 m); Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut and Moyen Sebou; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Diamesa latitarsis (Goetghebuer, 1921) Vaillant 1955b ; Vaillant 1956b , HA , Asif Tessaout (M'Goum), Lac Tamhda (Anremer); Serra-Tosio 1967 ; Serra-Tosio 1967 ; Saether 1968 ; Serra-Tosio 1973 ; Azzouzi and Laville 1987 , HA ; Ashe and Cranston 1990 ; Kettani et al. 2001 , Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa steinboecki Goetghebuer, 1933 Vaillant 1956b , HA , Cascade Siroua, Oukaimeden, Sidi Chamarouch Diamesa tonsa (Haliday in Walker, 1856) = Diamesa thienemanni Kieffer, 1909 Naya 1988 , MA , Haut Sebou (Arhbalou Yahya, Oued Arbi, Pont Aït hamza); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 Diamesa vaillanti Serra-Tosio, 1972 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Diamesa veletensis Serra-Tosio, 1971 Serra-Tosio 1983 , HA (2500 m); Azzouzi and Laville 1987 , HA ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Diamesa zernyi Edwards, 1933 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Potthastia Kieffer, 1922 Potthastia gaedii (Meigen, 1838) Azzouzi and Laville 1987 , MA , oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Oued Laou, Oued Afertane; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Potthastia pastoris (Edwards, 1933) Kettani and Moubayed-Breil 2018 , Rif Pseudodiamesa Goetghebuer, 1939 Pseudodiamesa ( Pseudodiamesa ) branickii (Nowicki, 1873) Naya 1988 , MA , Haut Sebou; Ashe and Cranston 1990 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Pseudodiamesa ( Pseudodiamesa ) nivosa (Goetghebuer, 1928) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Sympothastia Pagast, 1947 Sympothastia zavreli Pagast, 1947 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , aval Oued Krikra; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Syndiamesa Kieffer, 1918 Syndiamesa hygropterica (Kieffer, 1909) Naya 1988 , MA , Moyen Sebou (Sidi Abdellah, Dar El Arsa, Pont Oulad Slimane, Pont Portugais); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Protanypini Protanypus Kieffer, 1906 Protanypus morio (Zetterstedt, 1838) Naya 1988 , MA , Moyen Sebou; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Prodiamesinae Odontomesa Pagast, 1947 Odontomesa fulva (Kieffer, 1919) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani and Langton 2012 Prodiamesa Kieffer, 1906 Prodiamesa olivacea (Meigen, 1818) Naya 1988 , MA , Haut Sebou (Haut Guigou); Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Ashe and O'Connor 2009 ; Kettani et al. 2010 , Rif , Maggou village, Ifansa; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladiinae Orthocladiini Acricotopus Kieffer, 1921 Acricotopus lucens (Zetterstedt, 1850) Kettani and Moubayed-Breil 2018 , Rif Brilla Kieffer, 1913 Brillia bifida (Kieffer, 1909) = Brilla modesta (Meigen, 1830) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Brillia flavifrons (Johannsen, 1905) Kettani and Langton 2012 Brilla longifurca Kieffer, 1921 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1995 , Rif , amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Talembote (Usine électrique, 120 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Bryophaenocladius Thienemann, 1934 Bryophaenocladius aestivus (Brundin, 1947) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius flexidens (Brundin, 1947) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius cf. furcatus Thienemann & Strenzke, 1940 Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius illimbatus (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius muscicola (Kieffer, 1906) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius nidorum (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Bryophaenocladius subvernalis (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Bryophaenocladius tuberculatus (Edwards 1929) Kettani and Moubayed-Breil 2018 , Rif Camptocladius Wulp, 1874 Camptocladius stercorarius (De Geer, 1976) Kettani and Moubayed-Breil 2018 , Rif Cardiocladius Kieffer, 1912 Cardiocladius capucinus (Zetterstedt, 1850) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cardiocladius fuscus Kieffer, 1924 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou (Amont de Aïn Tadout, Skhounate, amont confluence avec Oued Atchane, Pont Aït Hamza); Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius Kieffer, 1911 Chaetocladius ( Chaetocladius ) acuticornis (Kieffer in Potthast, 1914) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius dentiforceps (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Chaetocladius dissipatus (Edwards, 1929) Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Chaetocladius ( Chaetocladius ) melaleucus (Meigen, 1818) Kettani and Langton 2011 , Rif , Oued Sgara, Bab Tariouant, Bouztata; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chaetocladius piger (Goetghebuer, 1913) Kettani and Moubayed-Breil 2018 , Rif Chaetocladius ( Chaetocladius ) perennis (Meigen, 1830) Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 Chaetocladius ( Chaetocladius ) vitellinus (Kieffer in Kieffer & Thienemann, 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Corynoneura Winnertz, 1846 Corynoneura carriana Edwards, 1924 Naya 1988 , MA , Haut Sebou (Haut Guigou, Aïn Nokra); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura celtica Edwards, 1924 Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura coronata Edwards, 1924 Kettani and Langton 2011 , Rif , Oued Hamma; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Corynoneura edwardsi Brundin, 1949 Kettani and Langton 2012 Corynoneura lacustris Edwards, 1924 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura lobata Edwards, 1924 Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Azzouzi et al. 1992 , HA ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Corynoneura scutellata Winnertz, 1846 Kettani and Moubayed-Breil 2018 , Rif Corynoneura Pe 2 Langton 1991 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Corynoneurella Brundin, 1949 Corynoneurella paludosa Brundin, 1949 Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus van der Wulp, 1874 Cricotopus ( Cricotopus ) albiforceps (Kieffer in Thienemann and Kieffer 1916) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) annulator Goetghebuer, 1927 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2011 , Rif , Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) beckeri Hirvenoja, 1973 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Langton and Laville 2002; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) bicinctus (Meigen, 1818) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Naya 1988 , MA , Haut et Moyen Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) caducus Hirvenoja, 1973 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) ephippium (Zetterstedt, 1838) Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) levantinus Moubayed & Hirvenoja, 1986 Kettani et al. 1996 , Rif , Haut Maggou; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Inesmane (Adeldal, 1173 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Cricotopus ) pallidipes Edwards, 1929 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) pulchripes Verrall, 1912 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) similis Goetgnebuer, 1921 Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Afertane, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) tremulus (Linnaeus, 1758) Kettani et al. 2010 , Rif , Oued Maggou (Maggou village, 777 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) triannulatus (Macquart, 1826) Kettani et al. 1994 , Rif , Haut Laou, Oued Moulay Bouchta, Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) trifascia Edwards, 1929 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Maggou (Maggou village, 777 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Cricotopus ) vierriensis Goetghebuer, 1935 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Isocladius ) brevipalpis Kieffer, 1909 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) laetus Hirvenoja, 1973 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) ornatus (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Cricotopus ( Isocladius ) sylvestris (Fabricius, 1794) Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Isocladius ) tricinctus (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) micans (Kieffer, 1918) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Talembote, Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) osellai Rossaro, 1990 Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) rufiventris (Meigen, 1830) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; (Dar El Arsa, Pont oulad Slimane, Pont portugais); Naya 1988 , MA , Haut et Moyen Sebou; Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cricotopus ( Paratrichocladius ) skirwithensis (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukieferiella Thienemann, 1926 Eukieferiella ancyla Svensson, 1986 Kettani and Langton 2011 , Rif , Oued Tkarae; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella bedmari Vilchez-Quero & Laville, 1988 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Laville and Langton 2002 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella brehmi Gowin, 1943 Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (Usine électrique, 120 m) Eukiefferiella brevicalcar (Kieffer, 1911) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m), Oued Ametrasse (Ametrasse, 820 m); Kettani and Langton 2011 , Rif , Oued Issaguen, Oued Ketama, Oued Sgara; Bab Tariouant, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella claripennis (Lundbeck, 1898) Fittkau and Reiss 1978 ; Naya 1988 , MA , Moyen Sebou (Dar Cheik Harazem); Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukieffeiella clypeata (Thienemann, 1919) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella coerulescens (Kieffer in Zavřel, 1926) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou (Skhounate, Arhbalou Aberchane); Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Nord Maggou village (Maggou, 905 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella cyanea Thienemann, 1936 Vaillant 1955b , HA ; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , HA ; Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella devonica (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella dittmari Lehmann, 1972 Kettani and Langton 2011 , Rif , Oued Boujdad, Fifi; Kettani and Langton 2012 , Rif , Oued Zarka; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella fittkaui Lehmann, 1972 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella fuldensis Lehmann, 1972 Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella gracei (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Tassikeste; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Eukiefferiella ilkleyensis (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella lobifera Goetghebuer, 1934 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella minor (Edwards, 1929) Vaillant 1955b , HA (1050 m); Vaillant 1956b , HA , Imi-N'Ifri; Azzouzi and Laville 1987 , HA ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Eukiefferiella pseudomontana Goetghebuer, 1935 Kettani et al. 2010 , Rif , Oued Madissouka (Talassemtane, 1530 m), Oued Dchar d'Amran (Béni M'Hamed, 1180 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella similis Goetghebuer, 1939 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella tirolensis Goetghebuer, 1938 Kettani et al. 1994 , Rif , Oued Afertane; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Azzouzi et al. 1992 , HA , Oued Tensift; Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Eukiefferiella Pe 2 Langton 1991 Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Halocladius Hirvenoja, 1973 Halocladius ( Halocladius ) varians (Staeger, 1839) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m); Ashe and Cranston 1990 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heleniella Gowin, 1943 Heleniella dorieri Serra-Tosio, 1967 Kettani and Langton 2012 Heleniella ornaticollis (Edwards, 1929) Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heleniella serratosioi Ringe, 1976 Kettani and Langton 2011 , Rif , Oued Hamma, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Heterotrissocladius Spärck, 1923 Heterotrissocladius marcidus (Walker, 1856) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 ; Dakki et al. 2008, MA , Oued Sebou; Ashe and O'Connor 2012 Hydrobaenus Fries, 1830 Hydrobaenus conformis (Holmgren, 1869) Kettani and Moubayed-Breil 2018 , Rif Hydrosmittia Ferrington & Sæther, 2011 Hydrosmittia oxoniana (Edwards, 1929) = Pseudosmittia recta (Edwards, 1929), in Azzouzi and Laville 1987 : 218, Kettani et al. 2001 : 330, Kettani and Langton 2012 : 422 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Hydrosmittia ruttneri (Strenzke & Thienemann, 1942) Kettani and Moubayed-Breil 2018 , Rif Krenosmittia Thienemann & Krüger, 1939 Krenosmittia boreoalpina (Goetghebuer, 1944) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Krenosmittia camptophleps (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Krenosmittia halvorseni (Cranston & Sæther, 1986) Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Krenosmittia hispanica Wülker, 1957 Kettani et al. 2001 ; Laville and Langton 2002 ; Ashe and O'Connor 2012 Limnophyes Eaton, 1875 Limnophyes difficilis Brunidin, 1947 Kettani and Moubayed-Breil 2018 , Rif Limnophyes gelasinus Saether, 1990 Kettani and Moubayed-Breil 2018 , Rif Limnophyes habilis (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Limnophyes madeirae Sæther, 1985 Kettani and Moubayed-Breil 2018 , Rif Limnophyes minimus (Meigen, 1818) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Kettani et al. 2001 ; Kettani and Langton 2011 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Limnophyes natalensis (Kieffer, 1914) Kettani and Moubayed-Breil 2018 , Rif Limnophyes ninae Sæther, 1975 Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Limnophyes pentaplastus (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Limnophyes pumilio (Holmgren, 1869) Kettani and Moubayed-Breil 2018 , Rif Limnophyes punctipennis (Goetghebuer, 1919) Kettani and Langton 2012 Limnophyes Pe 1a Langton 1991 Kettani et al. 2010 , Rif , Oued Talembote (Usine électrique, 120 m); Kettani and Langton 2012 Metriocnemus van der Wulp, 1874 Metriocnemus ( Metriocnemus ) albolineatus Meigen, 1818 Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) eurynotus (Holmgren, 1883) = Metriocnemus hygropetricus Kieffer, 1912, in Ashe and O'Connor 2012 : 372 = Metriocnemus ( Metriocnemus ) obscuripes (Holmgren, 1869), in Azzouzi et al. 1992 : 229, Kettani et al. 2001 : 329, Kettani and Langton 2012 : 421 Boumezzough and Thomas 1987 , HA , Oued Réghaya (1740 m), Imlil; Azzouzi and Laville 1987 , HA , Oued Tensift; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Metriocnemus ( Metriocnemus ) fuscipes (Meigen, 1818) Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) hirticollis (Staeger, 1839) Kettani and Moubayed-Breil 2018 , Rif Metriocnemus ( Metriocnemus ) ursinus Holmgren, 1869 Kettani and Moubayed-Breil 2018 , Rif Nanocladius Kieffer, 1913 Nanocladius ( Nanocladius ) balticus (Palmén, 1959) Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) dichromus (Kieffer 1906) Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) parvulus (Kieffer 1909) Kettani and Moubayed-Breil 2018 , Rif Nanocladius ( Nanocladius ) rectinervis (Kieffer, 1911) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Inesmane (Adeldal, 1173 m), Oued Talembote (aval Barrage Talembote, 245 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius van der Wulp, 1874 Orthocladius ( Eudactylocladius ) fuscimanus (Kieffer, 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued Krikra, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , source Maggou (Maggou, 1300 m), Oued Chrafat (Armotah, 900 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) ashei Soponis, 1990 = Orthocladius luteipes Goetghebuer, in Azzouzi and Laville 1987 : 218 = Orthocladius rivicola Kieffer, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Fès, Oued boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique), aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) rivulorum Kieffer, 1909 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor; 2012 Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Euorthocladius ) thienemanni Kieffer, 1906 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Barrage Talembote, aval Oued Talembote (usine éléctrique), aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Mesorthocladius ) frigidus (Zetterstedt, 1838) Vaillant 1955, HA (2900 m); Vaillant 1956b , HA , Lac Tamhda (Anremer); Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut Sebou (Haut Guigou); Fekhaoui et al. 1993 ; Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Dchar d'Amran (Béni M'Hamed, 1180 m), Nord Maggou village (Maggou, 905 m), Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Orthocladius ( Orthocladius ) oblidens (Walker, 1856) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) obumbratus Johannsen, 1905 = Orthocladius excavatus Brundin, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Orthocladius ( Orthocladius ) pedestris Kieffer, 1909 Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) rubicundus (Meigen, 1818) = Orthocladius saxicola Kieffer, in Azzouzi and Laville 1987 : 218 Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (Usine électrique, 120 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Orthocladius ) vaillanti Langton & Cranston, 1991 Kettani and Moubayed-Breil 2018 , Rif Orthocladius ( Symposiocladius ) lignicola Kieffer in Potthast, 1914 = Symposiocladius lignicola Kieffer, in Kettani et al. 2010 : 70 Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Orthocladius ( Symposiocladius ) ruffoi Rossaro & Prato, 1991 = Orthocladius Pe 1 Langton 1991, in Azzouzi and Laville 1987 : 218 = Rheortocladius sp A Langton 1991, in Kettani et al. 1995 : 257 = Rheorthocladius ruffoi Rossaro, in Kettani et al. 1997 : 184 Azzouzi and Laville 1987 , MA , Oum-er-Rbia; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet El Habtiyine, 819 m), Oued Maggou (Maggou village, 777 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Ifansa, 105 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Paracricotopus Brundin, 1956 Paracricotopus niger (Kieffer, 1913) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès; Kettani et al. 1994 , Rif , Oued Afertane; Kettani et al. 1995 , Rif , amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou, Oued Tamaridine (Zaouit et Habtyiène, 819 m), Oued Kelaâ (Akoumi, 400 m), Oued Kanar (Gorges Kanar, 280 m), Oued Talembote (155 m), Oued Tassikeste (240 m), Oued Laou (Ifansa, 105 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parakiefferiella Thienemann, 1936 Parakiefferiella coronata (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parakiefferiella wuelkeri Moubayed, 1994 = Parakiefferiella sp. d Wülker, in Azzouzi et al. 1992 : 230 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parametriocnemus Goetghebuer, 1932 Parametriocnemus boreoalpinus Gowin & Thienemann, 1942 Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Parametriocnemus stylatus (Spärck, 1923) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Béni M'Hamed (1330 m), Haut Maggou (1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (320 m), Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Parametriocnemus valescurensis Moubayed & Langton, 1999 Kettani and Langton 2011 , Rif , Oued Issaguen; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parametriocnemus Pe 1 Langton 1991 Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Talembote (320 m); Kettani and Langton 2012 Paraphaenocladius Thienemann, 1924 Paraphaenocladius exagitans ssp. 1 Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius impensus impensus (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius irritus Walker, 1856 Kettani and Moubayed-Breil 2018 , Rif Paraphaenocladius pseudirritus Strenzke, 1950 Kettani and Moubayed-Breil 2018 , Rif Paratrissocladius Zavřel, 1937 Paratrissocladius excerptus (Walker, 1856) Kettani et al. 1996 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parorthocladius Thienemann, 1935 Parorthocladius nudipennis (Kieffer in Kieffer & Thienemann 1908) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psecrocladius Kieffer, 1906 Psectrocladius ( Allopsectrocladius ) obvius (Walker, 1856) = Psectrocladius dilatatus (van der Wulp, 1859), in Naya 1988 : 48 Naya 1988 , MA , Moyen Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2011 , Rif , sources de Issaguen; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Allopsectrocladius ) platypus (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Mesopsectrocladius ) barbatipes Kieffer, 1923 Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou (1300 m), Oued Laou (Afertane, 56 m); Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psecrocladius ( Psectrocladius ) brehmi Kieffer, 1923 Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psectrocladius ( Psectrocladius ) fennicus Storå, 1939 Kettani and Langton 2012 Psectrocladius ( Psectrocladius ) limbatellus (Holmgren, 1869) Wülker 1959 ; Azzouzi and Laville 1987 , HA , Lac Tamhda (2800 m); Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Psectrocladius ) octomoculatus Wülker, 1956 Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Psectrocladius ( Psectrocladius ) sordidellus (Zetterstedt, 1838) Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous (NE Boucharene); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Psectrocladius ( Psectrocladius ) ventricosus Kieffer, 1925 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Pseudosmittia Edwards, 1932 Pseudosmittia albipennis (Goetghebuer, 1921) Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia baueri Strenzke, 1960 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia danconai (Marcuzzi, 1947) Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia holsata Thienemann & Stenzke, 1940 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia obtusa Strenzke, 1960 Kettani and Moubayed-Breil 2018 , Rif Pseudosmittia trilobata Edwards, 1929 Kettani and Moubayed-Breil 2018 , Rif Pseudorthocladius Goetghebuer, 1943 Pseudorthocladius ( Pseudorthocladius ) berthelemyi Moubayed, 1990 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Laville and Langton 2002 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Pseudorthocladius ( Pseudorthocladius ) curtistylus (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oasis Meski (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Pseudorthocladius near Pe 3 Langton 1991 Kettani and Langton 2011 , Rif , Bouztate (Fifi); Kettani and Langton 2012 Rheocricotopus Brundin, 1956 Rheocricotopus ( Psilocricotopus ) atripes (Kieffer, 1913) = Rheocricotopus ( Psilocricotopus ) foveatus foveatus (Edwards, 1929), in Naya 1988 : 40 Naya 1988 , MA , Haut Sebou (Haut Guigou); Azzouzi et al. 1992 , HA , Oued Tensift, Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , Haut Laou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote, Oued Tassikeste (Afechtal, 240 m), Oued Laou (Afertane, 56 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) chalybeatus subsp. chalybeatus (Edwards, 1929) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) gallicus Lehamnn 1969 Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) glabricollis (Meigen, 1830) Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Ametrasse (Ametrasse, 820 m), Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) meridionalis Moubayed-Breil, 2016 Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Psilocricotopus ) tirolus Lehmann, 1969 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Dakki 1997 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) effusus (Walker, 1856) Reiss 1977 ; Naya 1988 , MA , Haut et Moyen Sebou; Fekhaoui et al. 1993 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) fuscipes (Kieffer, 1909) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Maggou (905 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheocricotopus ( Rheocricotopus ) rifensis Moubayed & Kettani, 2019 Moubayed-Breil and Kettani, Rif , Chrafate, Challal Sghir (Akchour) Synorthocladius Thienemann, 1935 Synorthocladius semivirens (Kieffer, 1909) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Smittia Holmgren, 1869 Smittia alpicola Goetghebuer, 1941 Kettani and Moubayed-Breil 2018 , Rif Smittia aterrima Meigen, 1818 Kettani and Moubayed-Breil 2018 , Rif Smittia contingens Walker, 1856 Kettani and Moubayed-Breil 2018 , Rif Smittia foliacea (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Smittia pratorum Goetghebuer, 1927 Kettani and Moubayed-Breil 2018 , Rif Thienemannia Kieffer, 1909 Thienemannia cf. fulvofasciata (Kieffer, 1921) Kettani and Moubayed-Breil 2018 , Rif Thienemannia gracilis Kieffer, 1909 Kettani and Moubayed-Breil 2018 , Rif Thienemanniella Kieffer, 1911 Thienemanniella acuticornis (Kieffer, 1912) Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (320 m); Kettani and Langton 2011 , Rif , Oued Hamma, Oued Ketama, Oued Sgara; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Thienemanniella clavicornis (Kieffer, 1911) Kettani and Moubayed-Breil 2018 , Rif Thienemanniella majuscula (Edwards, 1924) Kettani et al. 1995 , Rif , aval Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemanniella vittata (Edwards, 1924) Kettani et al. 1996 , Rif , Haut Maggou; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Haut Maggou (1300 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Thienemanniella Pe 2a Langton 1991 Kettani et al. 2010 , Rif , Oued Maggou (905 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote; Kettani and Langton 2012 Thienemanniella Pe 2b Langton 1991 Kettani et al. 2010 , Rif , Oued Maggou (905 m), Oued Talembote; Kettani and Langton 2012 Trissocladius Kieffer, 1908 Trissocladius brevipalpis Kieffer in Kieffer & Thienemann 1908 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia Kieffer, 1922 Tvetenia bavarica (Goetghebuer, 1934) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia calvescens (Edwards, 1929) Naya 1988 , MA , Moyen Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tamaridine (Zaouiet et Habtiyiène, 819 m), Oued Talembote (245 m), Oued Laou (Afertane, 56 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tvetenia discoloripes (Goetghebuer & Thienemann in Thienemann, 1936) Kettani and Langton 2011 , Rif , Oued Nakhla, Bouztate (Fifi); Kettani and Langton 2012 ; Ashe and O'Connor 2012 Tvetenia verralli (Edwards, 1929) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta; Kettani et al. 1995 , Rif , aval Oued El Kbir, amont Oued Nakhla; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , ruisselet maison forestière Talassemtane (1683 m), Oued Tamaridine (Zaouiet et Habtiyiène, 819 m), Oued Talembote (245 m), Oued Laou (Afertane, 56 m); Kettani and Langton 2012 ; Ashe and O'Connor 2012 ; Kettani and Moubayed-Breil 2018 , Rif Zalutschia Lipina, 1939 Zalutschia humphriesiae Dowling & Murray, 1980 Kettani and Langton 2011 , Rif , marais de Lemtahane ( PNPB ), Dayat Fifi; Kettani and Langton 2012 ; Ashe and O'Connor 2012 Chironominae Chironomini Chironomus Meigen, 1803 Chironomus ( Baeotendipes ) noctivagus (Kieffer, 1911) Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) annularius Meigen, 1818 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) aprilinus sensu Meigen, 1818 = Chironomus halophilus Kieffer, in Ramdani and Tourenq 1982 : 180, in Naya 1988 : 50 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Naya 1988 , MA , Haut Sebou; Fekhaoui et al. 1993 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) bernensis Klötzli, 1973 = Chironomus sp 1 Kettani 1994 Kettani et al. 1994 ; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) calipterus Kieffer, 1908 Reiss 1977 , AP , Larache; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) longistylus Goetghebuer, 1921 Kettani et al. 2011, Rif , Oued Ketama; Kettani and Langton 2012 Chironomus ( Chironomus ) luridus Strenzke, 1959 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) nuditarsis Keyl, 1961 Kettani et al. 2011, Rif , Oued Boujdad (Kitane, 42 m), Oued El Hatba (SIBE Jebel Moussa, 165 m); Kettani and Langton 2012 , Rif , SIBE Jebel Moussa Chironomus ( Chironomus ) piger (Strenzke, 1956) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) plumosus (Linnaeus, 1758) Reiss 1977 , AP , Larache, AA , Dra-Tal; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 ; Naya 1988 , MA , Sidi Abdellah, Dar Cheih Harazem, Dar El Arsa; Fekhaoui et al. 1993 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Aïn Tissmelal (Tissmelal, 1046 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) prasinus sensu Pinder, 1978 Kettani et al. 2011, Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 Chironomus ( Chironomus ) riparius Meigen, 1804 = Chironomus thummi Kieffer, in Naya 1988 : 51, Fekhaoui et al. 1993 : 26 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou; Naya 1988 , MA , Moyen Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) salinarius Kieffer, 1915 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Chironomus ( Chironomus ) tentans Fabricius, 1805 = Camptochironomus tentans Fabricius, 1805, in Naya 1988 : 50 Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Cladopelma Kieffer, 1921 Cladopelma virescens (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus Kieffer, 1918 Cryptochironomus ( Cryptochironomus ) albofasciatus (Staeger, 1839) = Cryptochironomus obreptans Walker, 1856, in Kettani 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 Cryptochironomus ( Cryptochironomus ) psittacinus (Meigen, 1830) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Cryptochironomus ( Cryptochironomus ) rostratus Kieffer, 1921 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou, oued Oum-er-Rbia, Oued Boufekrane, HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus ( Cryptochironomus ) supplicans (Meigen, 1830) Kettani and Moubayed-Breil 2018 , Rif Cryptochironomus Pe 5 Langton 1991 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 Demicryptochironomus Lenz, 1941 Demicryptochironomus ( Demicryptochironomus ) vulneratus (Zetterstedt, 1838) Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Nord Maggou village (Maggou, 905 m); Kettani and Langton 2012 Demicryptochironomus ( Irmakia ) neglectus Reiss, 1988 Kettani and Moubayed-Breil 2018 , Rif Demicryptochironomus ( Irmakia ) Pe 1 Langton 1991 Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, aval Oued Khemis; Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes Kieffer, 1913 Dicrotendipes collarti (Goetghebuer, 1936) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes cordatus Kieffer, 1922 Kettani et al. 1996 , Rif , Oued Khizana (Oued Laou); Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Dicrotendipes fusconotatus (Kieffer, 1922) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes modestus (Say, 1823) Kettani et al. 2011, Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 Dicrotendipes nervosus (Staeger, 1839) = Limnochirononomus nervosus Staeger, in Naya 1988 : 53 Naya 1988 , MA , Moyen Sebou (Sidi Abdellah); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes notatus (Meigen, 1818) Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes pallidicornis (Goetghebuer, 1934) Azzouzi and Laville 1987 , Rif , Retenue El Makhazine, MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Dicrotendipes peringueyanus Kieffer, 1924 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 Dicrotendipes septemmaculatus (Becker, 1908) = Dicrotendipes pilosimanus Kieffer, in Reiss 1977 : 91, Azzouzi and Laville 1987 : 219 Reiss 1977 , AP , Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , AP , Larache; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued Krikra, amont Oued Nakhla, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 Endochironomus Kieffer, 1918 Endochironomus albipennis (Meigen, 1830) Naya 1988 , MA , Haut Sebou (Skhounata); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Endochironomus tendens (Fabricius, 1775) Naya 1988 , MA , Moyen Sebou (Gantra Mdez, Azzaba); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes Kieffer, 1913 Glyptotendipes ( Caulochironomus ) viridis (Macquart, 1834) Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes ( Glyptotendipes ) cauliginellus (Kieffer, 1913) = Glyptotendipes gripekoveni (Kieffer) Naya 1988 , MA , Haut Sebout (Guigou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes ( Glyptotendipes ) pallens (Meigen, 1804) Azzouzi and Laville 1987 , Rif , Retenue El Makhazine; Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Glyptotendipes sp A Langton 1991 Naya 1988 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Glyptotendipes sp B Langton 1991 Naya 1988 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Harnischia Kieffer, 1921 Harnischia curtilamellata (Malloch, 1915) Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou; Kettani et al. 1994 , Rif , Oued Siflaou, Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Harnischia fuscimanus Kieffer, 1921 Azzouzi and Laville 1987 , Rif , Retenue El Makhazine, MA , Oued Boufekrane; Kettani et al. 1995 , Rif , Oued El Kbir, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Kiefferulus Goetghebuer, 1922 Kiefferulus ( Kiefferulus ) tendipediformis (Goetghebuer, 1921) Reiss 1977 , Rif , Tétouan; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2010 , Rif , Guelta 1 km après Amariguen (Jebel Setsou, 1280 m); Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Dalia (SIBE Jebel Moussa, 169 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Kloosia Kruseman, 1933 Kloosia pusilla (Linnaeus, 1767) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Lauterborniella Thienemann & Bause, 1913 Lauterborniella agrayloides (Kieffer, 1911) Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus Kieffer, 1918 Microchironomus deribae (Freeman, 1957) = Leptochirononomus deribae Freeman, in Reiss 1977 : 91, Ramdani and Tourenq 1982 : 180 Reiss 1977 , AP , Rabat; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus lendli (Kieffer, 1918) Reiss 1986 , AA , Oasis Meski; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 Microchironomus tener (Kieffer, 1918) Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Azzouzi et al. 1992 , HA , Oued Tensift, Barrage Lalla Takerkoust; Kettani and Langton 2012 Microtendipes Kieffer, 1915 Microtendipes britteni (Edwards, 1929) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Microtendipes chloris (Meigen, 1818) Kettani et al. 2011, Rif , Dayat En-Nâsser (Khandek En-Nâsser, 1177 m), source Bab Karn (Fifi, 1216 m), Dayat Fifi (1179 m); Kettani and Langton 2012 Microtendipes confinis (Meigen, 1830) Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 Microtendipes diffinis (Edwards, 1929) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani et al. 2011, Rif , Dayat En-Nâsser (Khandek En-Nâsser, 1177 m), Dayat Aïn Rami, source Bab Karn (Fifi, 1216 m); Kettani and Langton 2012 Microtendipes pedellus (De Geer, 1776) Reiss 1977 , Rif , Environ de Tétouan; Fittkau and Reiss 1978 ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , Rif , Tétouan, HA ; Naya 1988 , MA , Haut Sebou (amont Aîn Tadout, Skhounate, Arhbalou Aberchane); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Nubensia Spies, 2015 Nubensia nubens (Edwards, 1929) = Polypedilum nubens (Edwards, 1929), in Azzouzi and Laville 1987 : 219; Kettani et al. 1994 : 28, 1995 : 257, 1996 : 137, 1997 : 184, 2001 : 331, 2010 : 70; Dakki 1997 : 65; Dakki et al. 2008: 32, Kettani and Langton 2012 : 423 Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Laou (Ifansa, 105 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Parachironomus Lenz, 1921 Parachironomus frequens (Johannsen, 1905) Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Parachironomus parilis (Walker, 1856) Reiss 1977 , AP , Environ de Larache; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Ashe and Cranston 1990 ; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Paracladopelma Harnisch, 1923 Paracladopelma camptolabis (Kieffer, 1913) Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paracladopelma galaptera (Townes, 1945) Azzouzi et al. 1992 , HA , Ouarzazate (1140 m), Gorges de Todra (1400 m); Kettani et al. 2001 ; Kettani and Langton 2012 Paracladopelma graminicolor (Kieffer, 1925) = Cryptotendipes graminicolor (Kieffer), in Azzouzi et al. 1992 : 230 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Paracladopelma laminatum (Kieffer, 1921) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paracladopelma mikianum (Goetghebuer, 1937) Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paralauterborniella Lenz, 1941 Paralauterborniella nigrohalteralis (Malloch, 1915) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès, Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Paratendipes Kieffer, 1911 Paratendipes albimanus (Meigen, 1818) Naya 1988 , MA , Moyen Sebou (Mdez); Kettani et al. 1994 , Rif , aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued Mhajrat, aval Oued Khemis, Oued Laou (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratendipes nudisquama (Edwards, 1929) Kettani and Moubayed-Breil 2018 , Rif Paratendipes striatus (Kieffer, 1925) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Phaenopsectra Kieffer, 1921 Phaenopsectra flavipes (Meigen, 1818) Kettani et al. 1994 , Rif , Haut Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum Kieffer, 1912 Polypedilum ( Pentapedilum ) ruandae Freeman, 1955 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Pentapedilum ) sordens (van der Wulp, 1875) = Polypedilum sp 1, in Kettani et al. 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Pentapedilum ) uncinatum (Goetghebuer, 1921) Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Polypedilum ) albicorne (Meigen, 1838) Naya 1988 , MA , Haut Sebou; Kettani et al. 1995 , Rif , aval Oued Krikra, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) arundineti (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 Polypedilum ( Polypedilum ) laetum (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) nubeculosum (Meigen, 1804) Reiss 1977 , Rif , Environ de Tétouan; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , Rif , Environ Tétouan, MA , Oued Sebou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) nubifer (Skuse, 1889) = Polypedilum pharao Kieffer, in Reiss 1977 : 91, Naya 1998: 55, Ramdani and Tourenq 1982 : 180 Kügler and Wool 1968 ; Reiss 1977 , AP , Larache, Rabat; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 , AP , Environ de Larache, Rabat, Merja Sidi Boughaba; Naya 1988 , MA , Haut Sebou; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Polypedilum ) pedestre (Meigen, 1830) Reiss 1977 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 1994 , Rif , aval Barrage Talembote; Kettani et al. 1995 , Rif , Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani et al. 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Tripodura ) acifer Townes, 1945 Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 , MA , Oued Boufekroune, Oued Fès, Oued Sebou; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Polypedilum ( Tripodura ) aegyptium Kieffer, 1925 = Polypedilum pruina Freeman, in Reiss 1977 : 91 Reiss 1977 , AP , Larache, HA , Marrakech, AA , Dra-Tal; Reiss 1985 ; Azzouzi and Laville 1987 , AP , Larache, HA , Marrakech, AA , Gorges de Todra; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 Polypedilum ( Tripodura ) bicrenatum Kieffer, 1921 Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) pullum (Zetterstedt, 1838) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia, HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) quadriguttatum Kieffer, 1921 Naya 1988 , MA , Moyen Sebou; Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Polypedilum ( Tripodura ) scalaenum (Schrank, 1803) Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Kettani et al. 1996 , Rif , Ras el Ma (Chefchaouen); Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) tetracrenatum Hirvenoja, 1962 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Tripodura ) tridens Freeman, 1955 El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Polypedilum ( Uresipedilum ) convictum (Walker, 1856) Reiss 1977 , AP , Environ de Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane (Gantra Mdez), Naya 1988 , MA , Haut Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Tassikeste; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Dakki et al. 2008, MA , Oued Sebou; Kettani et al. 2010 , Rif , Oued pont Béni M'Hamed (Béni M'Hamed, 1330 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Polypedilum ( Uresipedilum ) cultellatum Goetghebuer, 1931 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani et al. 1996 , Rif , Oued Nakhla; Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Polypedilum ontario -group sp. 1 Kettani et al. 1995 , Rif , aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Rheomus Laville & Reiss, 1988 Rheomus alatus Laville & Reiss, 1988 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Rheomus yahiae Laville & Reiss, 1988 Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Stenochironomus Kieffer, 1919 Stenochironomus gibbus Fabricius, 1794 Kettani and Moubayed-Breil 2018 , Rif Stictochironomus Kieffer, 1919 Stictochironomus caffrarius (Kieffer, 1921) Reiss 1977 ; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 Stictochironomus maculipennis (Meigen, 1818) Azzouzi and Laville 1987 , MA , Oued Sebou; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Afertane; Kettani et al. 1995 , Rif , aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Stictochironomus pictulus (Meigen, 1830) Reiss 1977 , AP , Environ de Larache; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou; Kettani et al. 1995 , Rif , aval Oued Kbir, aval Oued Krikra, Oued El Kbir; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , Oued Nakhla; Kettani and Langton 2012 Stictochironomus rosenschoeldi Zetterstedt, 1838 Kettani and Moubayed-Breil 2018 , Rif Stictochironomus reissi Cranston, 1989 = Stictochironomus sp. nov. Reiss, in Reiss 1977 : 91 Reiss 1977 ; Azzouzi and Laville 1987 , AA , M'Hamid, Dra-Tal; Kettani et al. 2001 ; Kettani and Langton 2012 Stictochironomus sticticus (Fabricius, 1781) = Stictochironomus histrio (Fabricius, 1794), in Kettani et al. 1996 : 138 Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Oued Berranda (Bouztate, 1259 m), Dayat Dalia (SIBE Jebel Moussa); Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Stictochironomus Pe 2 Langton 1991 Kettani et al. 2001 Xenochironomus Kieffer, 1921 Xenochironomus xenolabis (Kieffer, 1916) Azzouzi and Laville 1987 , MA , Oued Fès; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsini Cladotanytarsus Kieffer, 1921 Cladotanytarsus ( Cladotanytarsus ) atridorsum Kieffer, 1924 Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Azzouzi et al. 1992 , HA , Aït Saoun, Gorges de Dadès (1900 m), vallée de Drâa, Marrakech; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cladotanytarsus ( Cladotanytarsus ) capensis (Freeman, 1954) El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) ecristatus Reiss, 1991 = Tanytarsus sp. nov. (Morokko) Reiss, in Azzouzi and Laville 1987 : 219 Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 , EM , Berkane; Reiss 1991 ; Azzouzi et al. 1992 , HA ; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) mancus (Walker, 1856) Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Cladotanytarsus ( Cladotanytarsus ) pallidus Kieffer, 1922 = Cladotanytarsus Pe 5 Langton 1984 Azzouzi and Laville 1987 , MA , Oued Sebou, Oum Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 Cladotanytarsus ( Cladotanytarsus ) vanderwulpi (Edwards, 1929) Azzouzi and Laville 1987 , HA , Oued Tensift; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued Mhajrat, Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Lithotanytarsus Thienemann, 1933 Lithotanytarsus dadesi Reiss, 1991 Reiss 1991 ; Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique), Oued Afertane; Kettani et al. 1995 , Rif , Oued Mhajrat; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Lithotanytarsus emarginatus (Goetghebuer, 1933) Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia; Kettani and Langton 2012 Micropsectra Kieffer, 1909 Micropsectra andalusiaca Marcuzzi, 1950 Kettani and Moubayed-Breil 2018 , Rif Micropsectra apposita (Walker, 1856) = Micropsectra contracta Reiss, 1965 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Chrafat (Armotah, 900 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra aristata Pinder, 1976 Kettani and Langton 2012 , Rif , Oued Zarka Micropsectra atrofasciata (Kieffer, 1911) = Micropsectra bidentata (Goetghebuer, 1921), in Azzouzi et al. 1992 : 230; Kettani et al. 2001 : 332; Kettani and Langton 2011 : 590, 2012 : 424 Fittkau and Reiss 1978 ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , MA , Oued Sebou (Arhbalou Aberchane), Oued Oum-er-Rbia; Naya 1988 , MA , Haut Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Madissouka (Talassemtane, 1530 m), Oued Chrafat (Armotah, 900 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval affluent Talembote, 155 m); Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2011 , Rif , Oued Taida (Moulay Abdelsalam, 650 m), cascade Zarka, Dayat En-Nâsser (Khandek En-Nâsser, 1177 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra junci (Meigen, 1818) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra lacustris Säwedal, 1975 Kettani and Langton 2012 , Rif , Oued Zarka Micropsectra lindrothi Goetghebuer, 1931 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra notescens (Walker, 1856) Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2011 , Rif , Oued Ketama, Oued Sgara, ruisselet Bab Tariouant, Oued Berranda (Bouztate, 1259 m), source Bab Karn (Fifi, 1220 m), Dayat Fifi (Fifi, 1179); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Micropsectra pallidula (Meigen, 1830) Kettani and Moubayed-Breil 2018 , Rif Micropsectra schrankelae Stur & Ekrem, 2006 Kettani and Moubayed-Breil 2018 , Rif Micropsectra zernyi Marcuzzi, 1950 Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus Thienemann & Bause, 1913 Paratanytarsus bituberculatus (Edwards, 1929) Azzouzi et al. 1992 , MA , Lac Aguelmane Azigza (1510 m); Kettani et al. 1995 , Rif , Oued Martil (Tamuda); Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 Paratanytarsus dissimilis (Johannsen, 1905) = Paratanytarsus confusus Palmén, 1960, in Naya 1988 : 40; Dakki et al. 2008: 32; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 423 Naya 1988 , MA , Haut Sebou; Dakki et al. 2008, MA , Oued Sebou; Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus grimmii (Schneider, 1885) Kettani et al. 2010 , Rif , Oued Laou (Afertane, 55 m); Kettani and Langton 2012 Paratanytarsus inopertus (Walker, 1856) Reiss 1977 , Rif , Environ Tétouan; Fittkau and Reiss 1978 ; Reiss and Säwedal 1981 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , merja Mtalssi (Tamuda, 31 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Paratanytarsus mediterraneus Reiss & Säwedal, 1981 Reiss and Säwedal 1981 , Rif , Estuaire Oued Mharka (Tanger), AP , Oued Loukous; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2011 , AP , marais de Loukous; Kettani and Langton 2012 Paratanytarsus tenellulus (Goetghebuer, 1921) = Microspsectra tenellula Reiss 1977 : 91; Azzouzi and Laville 1987 : 219 Reiss 1977 , MA , Lac Kranichsee; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Paratanytarsus tenuis (Meigen, 1830) = Tanytarsus tenuis Meigen, in Naya 1988 : 57 Naya 1988 , MA , Moyen Sebou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Oued Khizana (Oued Laou); Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Rheotanytarsus Thienemann & Bause, 1913 Rheotanytarsus ceratophylli Dejoux, 1973 Naya 1988 , MA , Moyen et Bas Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Rheotanytarsus curtistylus (Goetghebuer, 1921) Azzouzi et al. 1992 , HA , Oasis Meski (1160 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus langtoni Moubayed & Kettani, 2018 Moubayed-Breil and Kettani 2018 , Rif , Oued Farda; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Rheotanytarsus muscicola Thienemann, 1929 Reiss 1977 , AP , Environ de Larache, AA , Dra-Tal (Tissint Moyen Dra); Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus nigricauda Fittkau, 1960 Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus pellucidus (Walker, 1818) = Rheotanytarsus distinctissimus (Brundin, 1947), in Kettani et al. 1995 : 258; Kettani et al. 1996 : 138, 1997 : 185; Kettani and Langton 2012 : 424 Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra; Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus pentapoda (Kieffer, 1909) = Rheotanytarsus sp 1, in Kettani et al. 1994 : 28 Kettani et al. 1994 , Rif , Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote; Kettani et al. 1995 , Rif , aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval Barrage Talembote, 245 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif ; Moubayed-Breil and Kettani 2019 , Rif , Chrafate, Challal Sghir (Akchour) Rheotanytarsus photophilus (Goetghebuer, 1921) Naya 1988 , MA , Haut Sebou; Kettani et al. 2001 ; Kettani and Langton 2012 Rheotanytarsus procerus Reiss, 1991 Reiss 1991 , HA ; Azzouzi et al. 1992 , HA , Gorges de Dadès (Imdiazen, 1900 m); Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus reissi Lehmann, 1970 Lehmann, 1970; Azzouzi and Laville 1987 , MA , Oued Boufekrane, Oued Oum-er-Rbia; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Source Maggou (Maggou, 1300 m), Oued Kelaâ (Akoumi, 400 m), Oued Talembote (aval Barrage Talembote, 245 m), Oued Tassikeste (Afechtal, 240 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Afertane, 55 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus rhenanus Klink, 1983 Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus ringei Lehmann, 1970 Lehmann, 1970; Reiss 1977 , Rif , Environ Tétouan; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , Rif , Tétouan, MA , Oued Boufekrane, Oued Fès, Oued Sebou, Oued Oum-er-Rbia; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Rheotanytarsus Pe 3 Langton 1991 Kettani et al. 2010 ; Kettani and Langton 2011 , Rif , Oued Sgara (Ketama, 1300 m); Kettani and Langton 2012 Stempellina Thienemann & Bause, 1913 Stempellina almi Brundin, 1947 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Boufekrane; Kettani et al. 2001 ; Kettani and Langton 2012 Stempellina bausei (Kieffer, 1911) Kettani and Langton 2012 , Rif , Ketama; Kettani and Moubayed-Breil 2018 , Rif Stempellinella Brundin, 1947 Stempellinella brevis (Edwards, 1929) Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 Tanytarsus van der Wulp, 1874 Tanytarsus brundini Lindeberg, 1963 Kettani et al. 1994 , Rif , Oued Moulay Bouchta, aval Oued Laou; Kettani et al. 1995 , Rif , amont Oued Nakhla, aval Oued Khemis; Kettani et al. 1996 ; Kettani et al. 2001 ; Dakki 1997 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus chinyensis Goetghebuer, 1934 Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Fifi (Fifi, 1179 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus cretensis Reiss, 1987 = Tanytarsus sp. nov. ( creticus ), in Reiss 1977 : 91; Azzouzi and Laville 1987 : 219 = Cladotanytarsus sp 1, in Kettani et al. 1995 : 258 Reiss and Fittkau 1971 ; Reiss 1977 , EM , Environ de Berkane; Reiss 1987 ; Azzouzi and Laville 1987 , Rif , Tétouan, AP , Larache, Kénitra; Kettani et al. 1996 ; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsus dibranchius Kieffer, 1926 = Tanytarsus separabilis Brundin, 1947, in Kettani et al. 1994 : 29; Kettani et al. 1995 : 258, 1996 : 138, 2001 : 332; Dakki 1997 : 63; Kettani and Langton 2012 : 424 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, aval Barrage Talembote, aval Oued Talembote (usine éléctrique); Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 Tanytarsus ejuncidus (Walker, 1856) Kettani and Moubayed-Breil 2018 , Rif Tanytarsus eminulus (Walker, 1856) Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus formosanus Kieffer, 1912 = Tanytarsus horni Goetghebuer, 1934, in Reiss and Fittkau 1971 : 122; Reiss 1977 : 91; Fittkau and Reiss 1978 : 439; Ramdani and Tourenq 1982 : 180; El Mezdi and Giudicelli 1985 : 292; Azzouzi and Laville 1987 : 219; Ashe and Cranston 1990 : 341; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 424 Reiss and Fittkau 1971 , Rif , M'Diq; Reiss 1977 , Rif , Environ Tétouan, AP , Larache, Rabat, Kénitra; Fittkau and Reiss 1978 ; Ramdani and Tourenq 1982 , AP , Merja Sidi Boughaba; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Azzouzi and Laville 1987 , HA , Oued Tensift; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus gregarius Kieffer, 1909 Naya 1988 , MA , Moyen Sebou; Kettani et al. 2001 ; Dakki et al. 2008, MA , Oued Sebou; Kettani and Langton 2012 Tanytarsus heusdensis Goetghebuer, 1923 Reiss 1977 , AA , Dra-Tal; Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 ; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Kelaâ (Akoumi, 400 m), Oued Talembote (avant village Talembote, 320 m), Oued Talembote (aval affluent Talembote, 155 m), Oued Laou (Ifansa, 105 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus mendax Kieffer, 1925 Kettani and Moubayed-Breil 2018 , Rif Tanytarsus medius Reiss & Fittkau, 1971 Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, aval Oued Krikra, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus palettaris Verneaux, 1969 Kettani et al. 1994 , Rif , aval Oued Laou; Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus pallidicornis (Walker, 1856) Kettani and Langton 2011 , Rif , Dayat Fifi (Fifi, 1179 m), Oued El Hatba (SIBE Jebel Moussa, 165 m); Kettani and Langton 2012 Tanytarsus recurvatus Brundin, 1947 Kettani and Langton 2011 , Rif , Oued El Hamma (El Hamma, 240 m); Kettani and Langton 2012 Tanytarsus signatus (van der Wulp, 1859) = Tanytarsus Pe 5 Langton 1991, in Azzouzi and Laville 1987 : 219 Kügler and Reiss 1973 ; Reiss 1977 , AA , Dra-Tal; Azzouzi and Laville 1987 ; Kettani et al. 2001 ; Kettani and Langton 2011 , Rif , Dayat Aïn Rami (373 m), Dayat Amlay (258 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus verralli Goetghebuer, 1928 Kettani and Langton 2011 , Rif , Oued Taida (650 m); Kettani and Langton 2012 Tanytarsus volgensis Miseiko, 1967 = Tanytarsus fimbriatus Reiss & Fittkau, 1971, in Fittkau and Reiss 1978 : 439; Azzouzi and Laville 1987 : 219; Kettani et al. 2001 : 332; Kettani and Langton 2012 : 424 Fittkau and Reiss 1978 ; Azzouzi and Laville 1987 , MA , Oued Fès, Oued Sebou, HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Tanytarsus Pe 14 Langton 1991 Kettani and Langton 2011 , Rif , source Issaguen (Ketama, 1600 m); Kettani and Langton 2012 Tanytarsus Pe 23 Langton 1991 Kettani and Langton 2011 , Rif , Oued El Hamma (El Hamma, 240 m); Kettani and Langton 2012 Virgatanytarsus Pinder, 1982 Virgatanytarsus albisutus (Santos-Abreu, 1918) = Virgatanytarsus maroccanus Kügler and Reiss, in Azzouzi and Laville 1987 : 219 Fittkau and Reiss 1978 ; Reiss and Schurch 1984 , AA , Dra-Tal; Reiss 1986 ; Azzouzi and Laville 1987 , MA , Oued Oum-er-Rbia, AA , Dra-Tal; Ashe and Cranston 1990 ; Kettani et al. 1994 , Rif , Haut Laou, Oued Siflaou, Oued Moulay Bouchta, aval Barrage Talembote, aval Oued Talembote (usine éléctrique), Oued Afertane, aval Oued Tassikeste, aval Oued Laou; Kettani et al. 1995 , Rif , aval Oued El Kbir, Oued El Kbir, amont Oued Nakhla, Oued Mhajrat, aval Oued Khemis, Oued Martil (Tamuda); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou), Ras el Ma (Chefchaouen); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani et al. 2010 , Rif , Oued Talembote (aval Barrage Talembote, 245 m), Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Virgatanytarsus ansatus Reiss & Schürch, 1984 Reiss and Schurch 1984 , HA ; Azzouzi and Laville 1987 , MA , Lac Aguelmane Azigza; Ashe and Cranston 1990 ; Kettani et al. 2001 ; Kettani and Langton 2012 Virgatanytarsus arduennensis (Goetghebuer, 1922) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 1994 , Rif , aval Oued Talembote (usine éléctrique); Kettani et al. 1996 ; Dakki 1997 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou); Kettani et al. 2001 ; Kettani et al. 2010 , Rif , Oued Talembote (aval affluent Talembote, 155 m); Kettani and Langton 2012 ; Kettani and Moubayed-Breil 2018 , Rif Virgatanytarsus triangularis (Goetghebuer, 1928) Azzouzi et al. 1992 , HA , Oued Tensift; Kettani et al. 2001 ; Kettani and Langton 2012 Virgatanytarsus Pe 1 Langton 1991 Kettani et al. 1996 ; Kettani et al. 1997 , Rif , Maggou (Oued Laou), Oued Khizana (Oued Laou); Kettani et al. 2001 ; Kettani and El Ouazzani 2005, Rif , amont Oued Nakhla; Kettani and Langton 2012 Zavrelia Kieffer, Thienemann & Bause, 1913 Zavrelia pentatoma Kieffer & Bause, 1913 Kettani and Langton 2012 Zavrelia Pe 1 Langton, 1991 Kettani and Langton 2011 , Rif , Oued Berranda (Bouztate, 1259 m); Kettani and Langton 2012 Acknowledgment We gratefully acknowledge the invaluable assistance and cooperation of Patrick Ashe (Dublin, Ireland) who contributed greatly to the revision of this family. SIMULIIDAE K. Kettani Number of species: 43 . Faunistic knowledge of the family in Morocco: good Simulinae Prosimuliini Helodon Enderlein, 1921 Helodon laamii (Beaucournu-Saguez and Bailly-Choumara, 1981) Beaucournu-Saguez and Bailly-Choumara 1981 , Rif ; Clergue-Gazeau et al. 1991 ; Hervy et al. 1994 ; Belqat et al. 2001a ; Belqat 2002 ; Belqat and Dakki 2004 ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium Roubaud, 1906 Prosimulium hirtipes species group Bailly-Choumara and Beaucournu-Saguez 1981 : 53–54: groupe latimucro (species nova ?); Beaucournu-Saguez and Bailly-Choumara 1981 : 119: groupe latimucro , groupe tomosvaryi and groupe rufipes - hirtipes ; Clergue-Gazeau et al. 1991 : 54 as «sp. gr. Hirtipes » Prosimulium latimucro (Enderlein, 1925) 4 Bailly-Choumara and Beaucournu-Saguez 1981 ; Beaucournu-Saguez and Bailly-Choumara 1981 ; Giudicelli and Thiery 1985 , HA ; Giudicelli and Bouzidi 1989 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'Goum en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Adler and Belqat 2001 , Rif , Oued Iouchirene, Oued Ketama (Al Hoceima); Belqat et al. 2001a , Rif , HA ; Belqat and Adler 2001 , Rif , Aïn Khandek En Nâsser, Oued Iouchirene, Oued Ketama; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Koçak and Kemal 2010 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium rachiliense Djafarov, 1954 (complex) 5 Beaucournu-Saguez and Bailly-Choumara 1981 ; Adler and Belqat 2001 ; Belqat and Adler 2001 ; Belqat 2002 ; Belqat and Dakki 2004 ; Belqat et al. 2005 ; Belqat et al. 2008 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium tomosvaryi (Enderlein, 1921) Beaucournu-Saguez and Bailly-Choumara 1981 ; Giudicelli and Thiery 1985 , HA ; Giudicelli and Bouzidi 1989 , Giudicelli et al. 2000 ; Adler and Belqat 2001 , Rif , Oued Iouchirene (Al Hoceima); Belqat and Adler 2001 , Rif , Oued Ouringa Tamdâ, oued Iouchirene, Oued Mrinet, Oued Ketama, Aîn Ksour, Oued Tisgris, Aîn Sidi Brahim Ben Arrif, Oued Hannacha; Belqat et al. 2001a , Rif ; Belqat et al. 2001b ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Koçak and Kemal 2010 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Urosimulium Contini, 1963 Urosimulium faurei (Bernard, Grenier & Bailly-Choumara, 1972) Grenier et al. 1957 , MA ; Bernard et al. 1972 : 63–68 (original description), MA , Plateau de Talerhza (environ de Meknès); Clergue-Gazeau et al. 1991 , MA ; Hervy et al. 1994 ; Belqat and Adler 2001 , Rif , Oued Iouchirene, Oued Mrinet, Oued Biyada, Oued Hannacha, Oued Ankouda; Belqat et al. 2001a , Rif , MA ; Belqat 2002 , Rif , MA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simuliini Greniera Doby & David, 1959 Greniera fabri Doby & David, 1959 Clergue-Gazeau et al. 1991 , MA ; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Metacnephia Crosskey, 1969 Metacnephia blanci (Grenier & Théodoridès, 1953) = Cnephia sp. in Grenier 1953 : 157 = Cnephia blanci Grenier and Théodoridès, in Grenier and Théodoridès 1953 : 430–435 = Eusimulium latinum Rubzov, in Benhoussa et al. 1988 : 160–164 Grenier 1953 , HA ; Grenier and Théodoridès 1953 , HA ; Grenier et al. 1957 , MA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Clergue-Gazeau et al. 1991 , AA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane: 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Metacnephia nuragica Rivosecchi, Raastad & Contini, 1975 6 = Cnephia tredecimatum (Edwards), in Grenier et al. 1957 : 226 Grenier et al. 1957 , AP , Coastal meseta (region of Rabat); Belqat et al. 2001a , AP , Rabat; Belqat 2002 , AP , Rabat; Belqat and Dakki 2004 , AP , Rabat; Belqat et al. 2011 , AP ; Belqat et al. 2018 Simulium Latreille, 1802 Simulium ( Crosskeyellum ) gracilipes Edwards, 1921 Edwards 1921 : 143 (original description), MA ; Séguy 1925 : 233, MA ; Séguy 1930a , MA ; Grenier 1953 , MA ; Crosskey 1964 , MA , Fès; Grenier and Bailly-Choumara 1970 : 96–102 (original description of subgenus Crosskeyellum , description of gracilipes ), MA ; Clergue-Gazeau et al. 1991 , MA ; Hervy et al. 1994 ; Dakki 1997 ; Belqat et al. 2001a , MA ; Belqat 2002 , MA ; Belqat and Dakki 2004 , MA ; Belqat et al. 2011 , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) angustipes Edwards, 1915 Clergue-Gazeau et al. 1991 , MA , HA ; Dakki 1997 ; Belqat et al. 2001a , MA , HA ; Belqat 2002 , MA , HA ; Belqat and Dakki 2004 , MA , HA ; Dakki et al. 2008, MA , O. Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) mellah Giudicelli & Bouzidi, 2000 [in Giudicelli, Bouzidi and Abdelaali 2000] Giudicelli et al. 2000 : 63 (original description), HA , Oued Mellah (Bassin Draa); Belqat et al. 2001a , HA ; Belqat 2002 , MA , HA ; Belqat and Dakki 2004 , MA , HA ; Dakki et al. 2008, MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , MA , HA ; Adler et al. 2015 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) petricolum (Rivosecchi, 1963) = Simulium latizonum Bailly-Choumara and Beaucournu-Saguez, in Bailly-Choumara and Beaucournu-Saguez 1978 : 143–144 (misidentified); Bailly-Choumara and Beaucournu-Saguez 1981 : 53–54 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, Rif , MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , HA ; Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) rubzovianum (Sherban, 1961) Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) velutinum sensu stricto (Santos Abreu, 1922) = Eusimilium latinum Rubzov, in El Mezdi and Giudicelli 1985 : 292–295; Benhoussa et al. 1988 : 160–164 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; El Mezdi and Giudicelli 1985 , HA , Khettaras of Marrakech; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Clergue-Gazeau et al. 1991 , AA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) velutinum cytospecies '5' Adler et al. 2015 , Rif , Tanger-Anjra, HA , Marrakech; Belqat et al. 2018 Simulium ( Nevermannia ) ruficorne species group Simulium ( Nevermannia ) angustitarse (Lundström, 1911) Belqat et al. 2001a , Rif ; Belqat et al. 2001b , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) ibleum (Rivosecchi, 1966) Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) lundstromi (Enderlein, 1921) Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , Rif , Kanar (280 m), Majjo (905 m), 10 km before the Issaguen source (1200 m), HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) ruficorne Macquart, 1838 = Eusimulium ruficorne Macquart, in El Mezdi and Giudicelli 1985 : 292, 294–295 Grenier et al. 1957 , AA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; El Mezdi and Giudicelli 1985 , HA , Khettaras of Marrakech; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA , AA ; Belqat 2002 , Rif , HA , AA ; Crosskey et al. 2002; Belqat and Dakki 2004 , Rif , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , AP , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) vernum species group Simulium ( Nevermannia ) brevidens (Rubtsov, 1956) Clergue-Gazeau et al. 1991 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) carthusiense (Grenier & Dorier, 1959) Giudicelli and Dakki 1984, Rif ; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) costatum Friederichs, 1920 Grenier et al. 1957 , Rif , Pré-Rif, MA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) cryophilum (Rubtsov, 1959) (complex) = Simulium pusillum Fries, in Séguy 1930a : 52 (misidentification); Grenier 1953 : 159 (after Séguy) Séguy 1930a , HA ; Grenier 1953 , Rif , HA , Lac Ifni; Bouzidi and Giudicelli 1986 , HA ; Bouzidi and Giudicelli 1989, HA ; Clergue-Gazeau et al. 1991 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Giudicelli Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) toubkal (Bouzidi & Giudicelli, 1986) Bouzidi and Giudicelli 1986 : 41–52 (original description), HA , assif n'Ouarzane (Oued Nfis); Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) vernum Macquart, 1826 (complex) [ latipes authors pre-1972, not Meigen] Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Rubzovia ) knidirii (Giudicelli & Thiery, 1985) Giudicelli and Thiery 1985 : 109–123 (original description in new subgenus Simulium ( Crenosimulium ) , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Rubzovia ) lamachi (Doby & David, 1960) Giudicelli and Dakki 1984, Rif ; Giudicelli and Thiery 1985 , Rif ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane: 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) bezzii species group Simulium ( Simulium ) bezzii (Corti, 1914) (complex) = Simulium atlas Séguy, 1930, in Séguy 1930a : 50 (original description); Grenier 1953 : 158 (synonymy of atlas Séguy with bezzii suggested) Séguy 1930a , MA ; Grenier 1953 ; Grenier 1953 , MA , HA ; Grenier and Théodoridès 1953 ; Grenier et al. 1957 , AA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Bouzidi and Giudicelli 1986 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) ornatum species group Simulium ( Simulium ) egregium Séguy, 1930 Grenier 1930, HA ; Séguy 1930a : 51 (original description), HA ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) intermedium Roubaud, 1906 = Simulium reptans var. fasciatum Séguy, in Séguy 1930a : 52 (misidentification) = Simulium ornatum var. nitidifrons Edwards, in Grenier 1953 : 159, Grenier and Théodoridès 1953 : 441, Grenier and Faure 1957 [1956]: 840, Grenier and Bailly-Choumara 1970 : 102, Bailly-Choumara and Beaucournu-Saguez 1978 : 143–144 = Odagmia nitidifrons Edwards, in Giudicelli and Dakki 1984: 95, Benhoussa et al. 1988 : 160–164 = Simulium nitidifrons Edwards, in El Mezdi and Giudicelli 1985 : 292, 294–295 Séguy 1930a , HA ; Grenier 1953 , MA , HA ; Grenier and Théodoridès 1953 , MA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif, AP , S Rabat; MA , Plain of Meknès; Grenier et al. 1957 , Rif , Pré-Rif, HA ; Grenier and Bailly-Choumara 1970 , MA ; Bernard et al. 1972 , MA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Giudicelli and Dakki 1984, Rif , MA ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , MA , HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'oun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) ornatum Meigen, 1818 (complex) = reptans var fasciatum , in Séguy 1930: 52 [ subornatum : Séguy 1925 /1930, not Edwards] Séguy 1930a : 52 ( ornatum and subornatum records), HA ; Grenier 1953 , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Clergue-Gazeau et al. 1991 , MA , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) trifasciatum Curtis, 1839 Belqat et al. 2001a , Rif ; 2001b, Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) variegatum species group Bailly-Choumara and Beaucournu-Saguez (1981 : 52–54): groupe monticola ("sp. nova A" and "sp. nova B") Simulium ( Simulium ) atlasicum Giudicelli & Bouzidi, 1989 Giudicelli and Bouzid 1989: 146–151 (original description), HA , near village Aguelmous; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) berberum Giudicelli & Bouzidi, 1989 Giudicelli and Bouzidi 1989 : 151–156 (original description), HA , assif n'Ouarzane; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) variegatum Meigen, 1818 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Giudicelli and Bouzidi 1989 ; Clergue-Gazeau et al. 1991 ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif , HA ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) xanthinum Edwards, 1933 = Simulium gaudi Grenier and Faure, in Grenier and Faure 1957 [1956]: 838–840 Grenier and Faure 1957 [1956], Rif , Pré-Rif, HA ; Grenier et al. 1957 ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Clergue-Gazeau et al. 1991 , MA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Carles-Tolrá 2002 ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) albellum species group Simulium ( Trichodagmia ) auricoma Meigen, 1818 = Simulium ( Obuchovia ) auricoma Meigen, 1818, in Belqat et al. 2011 : 52 Belqat 2000 , Rif ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) galloprovinciale Giudicelli, 1963 [1962] = Simulium ( Obuchovia ) galloprovinciale Giudicelli, 1963, in Belqat et al. 2011 : 52 Belqat 2000 , Rif ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) marocanum Bouzidi & Giudicelli, 1988 [1987] = Simulium ( Obuchovia ) marocanum Bouzidi & Giudicelli, 1987, in Belqat et al. 2011 : 52 Bouzidi and Giudicelli 1987: 185–195 (original description), Rif , near village Bou Adel, HA , Oued Rdat (affluent de l'Oued Tensift); Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) equinum species group Simulium ( Wilhelmia ) equinum (Linnaeus, 1758) = Simulium equinum Linnaeus, in Grenier et al. 1957 : 231–232 Grenier et al. 1957 , MA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Dakki 1997 ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) pseudequinum Séguy, 1921 = Simulium barbaricum Séguy, in Séguy 1930a : 51 = Simulium equinum var. mediterraneum Puri, in Grenier 1953 : 145–148; Grenier and Théodoridès 1953 : 436 = Simulium equinum mediterraneum Puri, in Grenier and Faure 1957 [1956]: 840; Grenier et al. 1957 : 232–234 = Wilhelmia pseudequinum Séguy, in Benhoussa et al. 1988 : 160–164 Séguy 1930a , HA ; Grenier 1953 , HA ; Grenier and Théodoridès 1953 , HA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif, AP , HA , AA ; Meknès; Grenier et al. 1957 , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Clergue-Gazeau et al. 1991 , HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 Simulium ( Wilhelmia ) quadrifila Grenier, Faure & Laurent, 1957 [1956] Grenier et al. 1957 : 238–239 (original description as form of sergenti ), Rif , Pré-Rif, AP , S Casablanca, MA , Meknès, HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , MA , HA ; Clergue-Gazeau et al. 1991 , Rif , Pré-Rif; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif , AP , S Casablanca, MA , HA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) sergenti (Edwards, 1923) = Simulium ariasi Séguy, in Séguy 1925 : 231–238; Séguy 1930a : 50; Grenier 1953 : 144 = Simulium equinum mediterraneum Puri, in Grenier and Faure 1957 [1956]: 840; Grenier et al. 1957 : 238–240 = Wilhelmia sergenti Edwards, in Benhoussa et al. 1993 : 249 Séguy 1930a , MA ; Grenier 1953 , MA ; Grenier and Théodoridès 1953 , HA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif; Grenier et al. 1957 , Rif , Pré-Rif, AP , S Casablanca, MA , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , AP , MA , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Clergue-Gazeau et al. 1991 , Rif , Pré-Rif, HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulinae Prosimuliini Helodon Enderlein, 1921 Helodon laamii (Beaucournu-Saguez and Bailly-Choumara, 1981) Beaucournu-Saguez and Bailly-Choumara 1981 , Rif ; Clergue-Gazeau et al. 1991 ; Hervy et al. 1994 ; Belqat et al. 2001a ; Belqat 2002 ; Belqat and Dakki 2004 ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium Roubaud, 1906 Prosimulium hirtipes species group Bailly-Choumara and Beaucournu-Saguez 1981 : 53–54: groupe latimucro (species nova ?); Beaucournu-Saguez and Bailly-Choumara 1981 : 119: groupe latimucro , groupe tomosvaryi and groupe rufipes - hirtipes ; Clergue-Gazeau et al. 1991 : 54 as «sp. gr. Hirtipes » Prosimulium latimucro (Enderlein, 1925) 4 Bailly-Choumara and Beaucournu-Saguez 1981 ; Beaucournu-Saguez and Bailly-Choumara 1981 ; Giudicelli and Thiery 1985 , HA ; Giudicelli and Bouzidi 1989 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'Goum en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Adler and Belqat 2001 , Rif , Oued Iouchirene, Oued Ketama (Al Hoceima); Belqat et al. 2001a , Rif , HA ; Belqat and Adler 2001 , Rif , Aïn Khandek En Nâsser, Oued Iouchirene, Oued Ketama; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Koçak and Kemal 2010 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium rachiliense Djafarov, 1954 (complex) 5 Beaucournu-Saguez and Bailly-Choumara 1981 ; Adler and Belqat 2001 ; Belqat and Adler 2001 ; Belqat 2002 ; Belqat and Dakki 2004 ; Belqat et al. 2005 ; Belqat et al. 2008 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Prosimulium tomosvaryi (Enderlein, 1921) Beaucournu-Saguez and Bailly-Choumara 1981 ; Giudicelli and Thiery 1985 , HA ; Giudicelli and Bouzidi 1989 , Giudicelli et al. 2000 ; Adler and Belqat 2001 , Rif , Oued Iouchirene (Al Hoceima); Belqat and Adler 2001 , Rif , Oued Ouringa Tamdâ, oued Iouchirene, Oued Mrinet, Oued Ketama, Aîn Ksour, Oued Tisgris, Aîn Sidi Brahim Ben Arrif, Oued Hannacha; Belqat et al. 2001a , Rif ; Belqat et al. 2001b ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Koçak and Kemal 2010 ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Urosimulium Contini, 1963 Urosimulium faurei (Bernard, Grenier & Bailly-Choumara, 1972) Grenier et al. 1957 , MA ; Bernard et al. 1972 : 63–68 (original description), MA , Plateau de Talerhza (environ de Meknès); Clergue-Gazeau et al. 1991 , MA ; Hervy et al. 1994 ; Belqat and Adler 2001 , Rif , Oued Iouchirene, Oued Mrinet, Oued Biyada, Oued Hannacha, Oued Ankouda; Belqat et al. 2001a , Rif , MA ; Belqat 2002 , Rif , MA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simuliini Greniera Doby & David, 1959 Greniera fabri Doby & David, 1959 Clergue-Gazeau et al. 1991 , MA ; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Metacnephia Crosskey, 1969 Metacnephia blanci (Grenier & Théodoridès, 1953) = Cnephia sp. in Grenier 1953 : 157 = Cnephia blanci Grenier and Théodoridès, in Grenier and Théodoridès 1953 : 430–435 = Eusimulium latinum Rubzov, in Benhoussa et al. 1988 : 160–164 Grenier 1953 , HA ; Grenier and Théodoridès 1953 , HA ; Grenier et al. 1957 , MA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Clergue-Gazeau et al. 1991 , AA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane: 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Metacnephia nuragica Rivosecchi, Raastad & Contini, 1975 6 = Cnephia tredecimatum (Edwards), in Grenier et al. 1957 : 226 Grenier et al. 1957 , AP , Coastal meseta (region of Rabat); Belqat et al. 2001a , AP , Rabat; Belqat 2002 , AP , Rabat; Belqat and Dakki 2004 , AP , Rabat; Belqat et al. 2011 , AP ; Belqat et al. 2018 Simulium Latreille, 1802 Simulium ( Crosskeyellum ) gracilipes Edwards, 1921 Edwards 1921 : 143 (original description), MA ; Séguy 1925 : 233, MA ; Séguy 1930a , MA ; Grenier 1953 , MA ; Crosskey 1964 , MA , Fès; Grenier and Bailly-Choumara 1970 : 96–102 (original description of subgenus Crosskeyellum , description of gracilipes ), MA ; Clergue-Gazeau et al. 1991 , MA ; Hervy et al. 1994 ; Dakki 1997 ; Belqat et al. 2001a , MA ; Belqat 2002 , MA ; Belqat and Dakki 2004 , MA ; Belqat et al. 2011 , MA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) angustipes Edwards, 1915 Clergue-Gazeau et al. 1991 , MA , HA ; Dakki 1997 ; Belqat et al. 2001a , MA , HA ; Belqat 2002 , MA , HA ; Belqat and Dakki 2004 , MA , HA ; Dakki et al. 2008, MA , O. Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) mellah Giudicelli & Bouzidi, 2000 [in Giudicelli, Bouzidi and Abdelaali 2000] Giudicelli et al. 2000 : 63 (original description), HA , Oued Mellah (Bassin Draa); Belqat et al. 2001a , HA ; Belqat 2002 , MA , HA ; Belqat and Dakki 2004 , MA , HA ; Dakki et al. 2008, MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , MA , HA ; Adler et al. 2015 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) petricolum (Rivosecchi, 1963) = Simulium latizonum Bailly-Choumara and Beaucournu-Saguez, in Bailly-Choumara and Beaucournu-Saguez 1978 : 143–144 (misidentified); Bailly-Choumara and Beaucournu-Saguez 1981 : 53–54 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, Rif , MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , HA ; Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) rubzovianum (Sherban, 1961) Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) velutinum sensu stricto (Santos Abreu, 1922) = Eusimilium latinum Rubzov, in El Mezdi and Giudicelli 1985 : 292–295; Benhoussa et al. 1988 : 160–164 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; El Mezdi and Giudicelli 1985 , HA , Khettaras of Marrakech; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Clergue-Gazeau et al. 1991 , AA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Koçak and Kemal 2010 ; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler et al. 2015 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Eusimulium ) velutinum cytospecies '5' Adler et al. 2015 , Rif , Tanger-Anjra, HA , Marrakech; Belqat et al. 2018 Simulium ( Nevermannia ) ruficorne species group Simulium ( Nevermannia ) angustitarse (Lundström, 1911) Belqat et al. 2001a , Rif ; Belqat et al. 2001b , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) ibleum (Rivosecchi, 1966) Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) lundstromi (Enderlein, 1921) Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , Rif , Kanar (280 m), Majjo (905 m), 10 km before the Issaguen source (1200 m), HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) ruficorne Macquart, 1838 = Eusimulium ruficorne Macquart, in El Mezdi and Giudicelli 1985 : 292, 294–295 Grenier et al. 1957 , AA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; El Mezdi and Giudicelli 1985 , HA , Khettaras of Marrakech; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA , AA ; Belqat 2002 , Rif , HA , AA ; Crosskey et al. 2002; Belqat and Dakki 2004 , Rif , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , AP , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) vernum species group Simulium ( Nevermannia ) brevidens (Rubtsov, 1956) Clergue-Gazeau et al. 1991 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) carthusiense (Grenier & Dorier, 1959) Giudicelli and Dakki 1984, Rif ; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) costatum Friederichs, 1920 Grenier et al. 1957 , Rif , Pré-Rif, MA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) cryophilum (Rubtsov, 1959) (complex) = Simulium pusillum Fries, in Séguy 1930a : 52 (misidentification); Grenier 1953 : 159 (after Séguy) Séguy 1930a , HA ; Grenier 1953 , Rif , HA , Lac Ifni; Bouzidi and Giudicelli 1986 , HA ; Bouzidi and Giudicelli 1989, HA ; Clergue-Gazeau et al. 1991 , HA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Giudicelli Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) toubkal (Bouzidi & Giudicelli, 1986) Bouzidi and Giudicelli 1986 : 41–52 (original description), HA , assif n'Ouarzane (Oued Nfis); Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Nevermannia ) vernum Macquart, 1826 (complex) [ latipes authors pre-1972, not Meigen] Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Rubzovia ) knidirii (Giudicelli & Thiery, 1985) Giudicelli and Thiery 1985 : 109–123 (original description in new subgenus Simulium ( Crenosimulium ) , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Rubzovia ) lamachi (Doby & David, 1960) Giudicelli and Dakki 1984, Rif ; Giudicelli and Thiery 1985 , Rif ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane: 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) bezzii species group Simulium ( Simulium ) bezzii (Corti, 1914) (complex) = Simulium atlas Séguy, 1930, in Séguy 1930a : 50 (original description); Grenier 1953 : 158 (synonymy of atlas Séguy with bezzii suggested) Séguy 1930a , MA ; Grenier 1953 ; Grenier 1953 , MA , HA ; Grenier and Théodoridès 1953 ; Grenier et al. 1957 , AA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Bouzidi and Giudicelli 1986 , HA ; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) ornatum species group Simulium ( Simulium ) egregium Séguy, 1930 Grenier 1930, HA ; Séguy 1930a : 51 (original description), HA ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) intermedium Roubaud, 1906 = Simulium reptans var. fasciatum Séguy, in Séguy 1930a : 52 (misidentification) = Simulium ornatum var. nitidifrons Edwards, in Grenier 1953 : 159, Grenier and Théodoridès 1953 : 441, Grenier and Faure 1957 [1956]: 840, Grenier and Bailly-Choumara 1970 : 102, Bailly-Choumara and Beaucournu-Saguez 1978 : 143–144 = Odagmia nitidifrons Edwards, in Giudicelli and Dakki 1984: 95, Benhoussa et al. 1988 : 160–164 = Simulium nitidifrons Edwards, in El Mezdi and Giudicelli 1985 : 292, 294–295 Séguy 1930a , HA ; Grenier 1953 , MA , HA ; Grenier and Théodoridès 1953 , MA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif, AP , S Rabat; MA , Plain of Meknès; Grenier et al. 1957 , Rif , Pré-Rif, HA ; Grenier and Bailly-Choumara 1970 , MA ; Bernard et al. 1972 , MA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Giudicelli and Dakki 1984, Rif , MA ; El Mezdi and Giudicelli 1985 , HA , Khettaras de Marrakech; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Giudicelli and Bouzidi 1989 , HA ; Clergue-Gazeau et al. 1991 , MA , HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'oun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) ornatum Meigen, 1818 (complex) = reptans var fasciatum , in Séguy 1930: 52 [ subornatum : Séguy 1925 /1930, not Edwards] Séguy 1930a : 52 ( ornatum and subornatum records), HA ; Grenier 1953 , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Clergue-Gazeau et al. 1991 , MA , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) trifasciatum Curtis, 1839 Belqat et al. 2001a , Rif ; 2001b, Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) variegatum species group Bailly-Choumara and Beaucournu-Saguez (1981 : 52–54): groupe monticola ("sp. nova A" and "sp. nova B") Simulium ( Simulium ) atlasicum Giudicelli & Bouzidi, 1989 Giudicelli and Bouzid 1989: 146–151 (original description), HA , near village Aguelmous; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) berberum Giudicelli & Bouzidi, 1989 Giudicelli and Bouzidi 1989 : 151–156 (original description), HA , assif n'Ouarzane; Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) variegatum Meigen, 1818 Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Giudicelli and Bouzidi 1989 ; Clergue-Gazeau et al. 1991 ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , HA ; Belqat 2002 , Rif , HA ; Belqat and Dakki 2004 , Rif , HA ; Belqat et al. 2005 , Rif , HA ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Simulium ) xanthinum Edwards, 1933 = Simulium gaudi Grenier and Faure, in Grenier and Faure 1957 [1956]: 838–840 Grenier and Faure 1957 [1956], Rif , Pré-Rif, HA ; Grenier et al. 1957 ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Clergue-Gazeau et al. 1991 , MA ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Carles-Tolrá 2002 ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Belqat et al. 2011 , Rif , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) albellum species group Simulium ( Trichodagmia ) auricoma Meigen, 1818 = Simulium ( Obuchovia ) auricoma Meigen, 1818, in Belqat et al. 2011 : 52 Belqat 2000 , Rif ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) galloprovinciale Giudicelli, 1963 [1962] = Simulium ( Obuchovia ) galloprovinciale Giudicelli, 1963, in Belqat et al. 2011 : 52 Belqat 2000 , Rif ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Belqat et al. 2011 , Rif ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Trichodagmia ) marocanum Bouzidi & Giudicelli, 1988 [1987] = Simulium ( Obuchovia ) marocanum Bouzidi & Giudicelli, 1987, in Belqat et al. 2011 : 52 Bouzidi and Giudicelli 1987: 185–195 (original description), Rif , near village Bou Adel, HA , Oued Rdat (affluent de l'Oued Tensift); Clergue-Gazeau et al. 1991 , HA ; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Belqat et al. 2011 , Rif , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) equinum species group Simulium ( Wilhelmia ) equinum (Linnaeus, 1758) = Simulium equinum Linnaeus, in Grenier et al. 1957 : 231–232 Grenier et al. 1957 , MA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Dakki 1997 ; Belqat et al. 2001a , HA ; Belqat 2002 , HA ; Belqat and Dakki 2004 , HA ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) pseudequinum Séguy, 1921 = Simulium barbaricum Séguy, in Séguy 1930a : 51 = Simulium equinum var. mediterraneum Puri, in Grenier 1953 : 145–148; Grenier and Théodoridès 1953 : 436 = Simulium equinum mediterraneum Puri, in Grenier and Faure 1957 [1956]: 840; Grenier et al. 1957 : 232–234 = Wilhelmia pseudequinum Séguy, in Benhoussa et al. 1988 : 160–164 Séguy 1930a , HA ; Grenier 1953 , HA ; Grenier and Théodoridès 1953 , HA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif, AP , HA , AA ; Meknès; Grenier et al. 1957 , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Benhoussa et al. 1988 , AP , Oued Bou-Regreg; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Clergue-Gazeau et al. 1991 , HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA , AA ; Belqat 2002 , Rif , MA , HA , AA ; Belqat and Dakki 2004 , Rif , MA , HA , AA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA , AA ; Adler and Crosskey 2017 ; Belqat et al. 2018 Simulium ( Wilhelmia ) quadrifila Grenier, Faure & Laurent, 1957 [1956] Grenier et al. 1957 : 238–239 (original description as form of sergenti ), Rif , Pré-Rif, AP , S Casablanca, MA , Meknès, HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , MA , HA ; Clergue-Gazeau et al. 1991 , Rif , Pré-Rif; Dakki 1997 ; Belqat et al. 2001a , Rif ; Belqat 2002 , Rif , AP , S Casablanca, MA , HA ; Belqat and Dakki 2004 , Rif ; Belqat et al. 2005 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 Simulium ( Wilhelmia ) sergenti (Edwards, 1923) = Simulium ariasi Séguy, in Séguy 1925 : 231–238; Séguy 1930a : 50; Grenier 1953 : 144 = Simulium equinum mediterraneum Puri, in Grenier and Faure 1957 [1956]: 840; Grenier et al. 1957 : 238–240 = Wilhelmia sergenti Edwards, in Benhoussa et al. 1993 : 249 Séguy 1930a , MA ; Grenier 1953 , MA ; Grenier and Théodoridès 1953 , HA ; Grenier and Faure 1957 [1956], Rif , Pré-Rif; Grenier et al. 1957 , Rif , Pré-Rif, AP , S Casablanca, MA , HA ; Bailly-Choumara and Beaucournu-Saguez 1978 , Rif , AP , MA , HA ; Bailly-Choumara and Beaucournu-Saguez 1981 , HA ; Clergue-Gazeau et al. 1991 , Rif , Pré-Rif, HA ; Benhoussa et al. 1993 , AP , Oued Bou-Regreg; Dakki 1997 ; Giudicelli et al. 2000 , HA , Oued Réghaya (Neltner, 3800 m), Oued Réghaya (Sidi Chamharouch, 2300 m), Oued Réghaya (lmlil, 1740 m), Oued Réghaya (Aguersioual, 1550 m), Oued Réghaya (Moulay Brahim, 1200 m), Oued Réghaya (Tahanaout, 890 m), ruisselet émissaire de source débouchant dans Oued Réghaya en amont d'lmlil (1750 m), ruisselet émissaire de source débouchant dans l'assif M'zik (1850 m), ruisselet émissaire de source débouchant dans l'assif N'Ouarzane (3000 m), ruisseau émissaire de source (assif N'Ouarzane, 3000 m), assif N'Ouarzane (Irhoulidene, 2800 m), ruisseau affluent en rive droite de l'assif N'Ouarzane (2400 m), Oued N'fis (amont Ijoukak, 1600 m), Oued N'fis (amont Wirgan, 1200 m), Oued N'fis (980 m), Oued N'fis (amont retenue Lalla Takerkoust, 660 m), ruisseau de Tinzart (émissaire de source: 2850 m), ruisseau de Tifni (émissaire de source: 2780 m), ruisseau de Likemt (émissaire de source: 2670 m), ruisseau de Tougroudadene (émissaire de source: 2660 m), assif Oukaimeden (2600 m), source hélocrène au niveau du cirque d'Oukaimeden (2660 m), assif Tiferguine (2500 m), assif Oukaimeden (2450 m), ruisseau émissaire de source débouchant dans l'assif Oukaimeden (1740 m), complexe rhéocrène formé par des émissaires de source débouchant dans l'assif Oukaimeden (1730 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1630 m), affluent temporaire en rive gauche de l'assif Oukaimeden (1360 m), affluent temporaire en rive droite de l'assif Oukaimeden (1260 m), affluent en rive droite de l'assif Oukaimeden (1300 m), assif Tarzaza (1200 m), assif Tarzaza (1000 m), cours inférieur de l'oued Ourika (850 m), Oued Rdat en amont de Taddert (1850 m), affluent temporaire en rive gauche de Oued Rdat (1400 m), Oued Rdat (1600 m), Oued Rdat (1230 m), Oued Tensift (600–700 m), khetarras (450–600 m), Oued Tessaout au niveau d'Aït Tamli (1620 m), Oued Lakdar en aval de la retenue de Sidi Driss (1030 m), ruisseau émissaire de source formant le début de l'assif Imini (2090 m), assif Imini (1560 m), Oued Ounila (1820 m), ruisseau affluent en rive gauche de l'oued Ounila (1820 m), Oued Ounila (Timhlt, 1600 m), Oued Mellah (Anghessa, 1400 m), Oued Dadès en amont des gorges (1630 m), Oued Dadès (Boumalne, 1530 m), Oued Dadès (Sidi Flah, 1100 m), Oued M'Goun (1530 m), Oued M'Goun en aval de Kelaâ (1370 m), ruisseau émissaire de source débouchant dans un affluent de l'Oued Souss (2350 m); Belqat et al. 2001a , Rif , MA , HA ; Belqat 2002 , Rif , MA , HA ; Belqat and Dakki 2004 , Rif , MA , HA ; Belqat et al. 2005 , Rif ; Belqat et al. 2008 , Rif ; Dakki et al. 2008, MA , Oued Sebou; Belqat et al. 2011 , Rif , AP , MA , HA ; Adler and Crosskey 2017 ; Belqat et al. 2018 ; Adler 2019 THAUMALEIDAE K. Kettani, R. Wagner Number of species: 2 . Expected: 10 Faunistic knowledge of the family in Morocco: poor Thaumalea Ruthe, 1831 Thaumalea bernardi Vaillant, 1956 Vaillant 1956b , HA , Toubkal, Siroua, Lac Tamhda (Anremer), Sidi Chamarouch, Izourar, M'Goum, Oukaimeden; Dakki 1997 Thaumalea spinata Vaillant, 1954 7 Vaillant 1954a , HA , M'Goum, springs powering d'Ameskeur el Fougani, springs powering Izourar lagoon (Azourki), springs powering the lake Tamhda (Anremer), torrent at the bottom of Jebel Siroua, Oukaimeden (Toubkal), Jebel Toubkal, Atend (Sidi Chamarouch) BLEPHARICERIDAE K. Kettani, P. Zwick Number of species: 4 Blepharicerinae Liponeura Loew, 1844 Liponeura alticola Giudicelli & Bouzidi, 1987 Giudicelli and Bouzidi 1987 , HA , Oued Réghaya; Dakki 1997 Liponeura megalatlantica (Vaillant, 1956) Vaillant 1956c , HA , Izourar, Imi-N'Ifri; Giudicelli and Lavandier 1974 ; Dakki 1997 Liponeura rifincola Zwick, 2013 Zwick 2013 , Rif , Issaguen (Ketama, 1800 m) Liponeura sirouana (Vaillant, 1956) = Cardiocrepsis sirouana Vaillant, in Vaillant 1956b : 234 Vaillant 1956b , HA , Siroua (3000 m); Giudicelli and Lavandier 1974 ; Dakki 1997 Bibionoidea ANISOPODIDAE 8 K. Kettani, J.-P. Haenni Number of species: 3 . Expected: 7–8 Faunistic knowledge of the family in Morocco: poor Anisopodinae Sylvicola Harris, 1780 Sylvicola fenestralis (Scopoli, 1763) 9 = Rhyphus fenestralis (Scopoli, 1763), in Mouna 1998 : 86 Mouna 1998 (no locality given) – MISR Sylvicola fuscatus (Fabricius, 1775) 1 = Rhyphus fuscatus (Fabricius, 1775), in Mouna 1998 : 86 Mouna 1998 (no locality given) – MISR Sylvicola punctatus (Fabricius, 1787) 1 = Rhyphus punctatus (Fabricius, 1787), in Mouna 1998 : 86 Mouna 1998 (no locality given) – MISR BIBIONIDAE 10 K. Kettani, J.-P. Haenni Number of species: 10 . Expected: 20 Faunistic knowledge of the family in Morocco: poor Bibioninae Bibio Geoffroy, 1762 Bibio hortulanus (Linnaeus, 1758) = Bibio hortularum Linnaeus, 1758, in Becker and Stein 1913 : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1949a , SA , Foum Zguid Bibio lanigerus Meigen, 1818 Ebejer et al. 2019 , Rif , Amsemlil ( PNPB , 1067 m) Bibio laufferi Strobl, 1906 Ebejer et al. 2019 , MA , 3.5 km S of Azrou (Ifrane, 1450 m), 20 km S of Azrou (Ifrane, 1720 m) Bibio leucopterus (Meigen, 1804) Ebejer et al. 2019 , MA , 6 km S of Azrou (Ifrane, 1610 m) Bibio marci (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Haenni 2009 , Rif , Tanger, MA , Ifrane Dilophus Meigen, 1803 Dilophus antipedalis Wiedemann in Meigen, 1818 Ebejer et al. 2019 , Rif , Oued Azla (Nwawel, 57 m), Oued Azla (Hallila, 95 m) Dilophus (cf. bispinosus Lundström, 1914) 11 Séguy 1953a , AA , Aïn Chaib (Souss) Dilophus febrilis (Linnaeus, 1758) = Dilophus vulgaris Meigen, 1818, in Séguy 1949a : 153 Séguy 1949a , AA , Goulimine, Foum-el-Hassan Dilophus femoratus Meigen, 1804 = Philia femorata Meigen, 1804, in Séguy 1941: 2 Séguy 1941d , HA , Tizi-n'Test (2000 m); Pârvu et al. 2006 , AP , Merja Zerga Dilophus tridentatus Walker, 1848 Ebejer et al. 2019 , AP , Sidi Mokhtar (Essaouira) – MHNN (J.-P. Haenni leg.), MNHN ( SA , Foum-el-Hassan) ANISOPODIDAE 8 K. Kettani, J.-P. Haenni Number of species: 3 . Expected: 7–8 Faunistic knowledge of the family in Morocco: poor Anisopodinae Sylvicola Harris, 1780 Sylvicola fenestralis (Scopoli, 1763) 9 = Rhyphus fenestralis (Scopoli, 1763), in Mouna 1998 : 86 Mouna 1998 (no locality given) – MISR Sylvicola fuscatus (Fabricius, 1775) 1 = Rhyphus fuscatus (Fabricius, 1775), in Mouna 1998 : 86 Mouna 1998 (no locality given) – MISR Sylvicola punctatus (Fabricius, 1787) 1 = Rhyphus punctatus (Fabricius, 1787), in Mouna 1998 : 86 Mouna 1998 (no locality given) – MISR BIBIONIDAE 10 K. Kettani, J.-P. Haenni Number of species: 10 . Expected: 20 Faunistic knowledge of the family in Morocco: poor Bibioninae Bibio Geoffroy, 1762 Bibio hortulanus (Linnaeus, 1758) = Bibio hortularum Linnaeus, 1758, in Becker and Stein 1913 : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1949a , SA , Foum Zguid Bibio lanigerus Meigen, 1818 Ebejer et al. 2019 , Rif , Amsemlil ( PNPB , 1067 m) Bibio laufferi Strobl, 1906 Ebejer et al. 2019 , MA , 3.5 km S of Azrou (Ifrane, 1450 m), 20 km S of Azrou (Ifrane, 1720 m) Bibio leucopterus (Meigen, 1804) Ebejer et al. 2019 , MA , 6 km S of Azrou (Ifrane, 1610 m) Bibio marci (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Haenni 2009 , Rif , Tanger, MA , Ifrane Dilophus Meigen, 1803 Dilophus antipedalis Wiedemann in Meigen, 1818 Ebejer et al. 2019 , Rif , Oued Azla (Nwawel, 57 m), Oued Azla (Hallila, 95 m) Dilophus (cf. bispinosus Lundström, 1914) 11 Séguy 1953a , AA , Aïn Chaib (Souss) Dilophus febrilis (Linnaeus, 1758) = Dilophus vulgaris Meigen, 1818, in Séguy 1949a : 153 Séguy 1949a , AA , Goulimine, Foum-el-Hassan Dilophus femoratus Meigen, 1804 = Philia femorata Meigen, 1804, in Séguy 1941: 2 Séguy 1941d , HA , Tizi-n'Test (2000 m); Pârvu et al. 2006 , AP , Merja Zerga Dilophus tridentatus Walker, 1848 Ebejer et al. 2019 , AP , Sidi Mokhtar (Essaouira) – MHNN (J.-P. Haenni leg.), MNHN ( SA , Foum-el-Hassan) Bibioninae Bibio Geoffroy, 1762 Bibio hortulanus (Linnaeus, 1758) = Bibio hortularum Linnaeus, 1758, in Becker and Stein 1913 : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1949a , SA , Foum Zguid Bibio lanigerus Meigen, 1818 Ebejer et al. 2019 , Rif , Amsemlil ( PNPB , 1067 m) Bibio laufferi Strobl, 1906 Ebejer et al. 2019 , MA , 3.5 km S of Azrou (Ifrane, 1450 m), 20 km S of Azrou (Ifrane, 1720 m) Bibio leucopterus (Meigen, 1804) Ebejer et al. 2019 , MA , 6 km S of Azrou (Ifrane, 1610 m) Bibio marci (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Haenni 2009 , Rif , Tanger, MA , Ifrane Dilophus Meigen, 1803 Dilophus antipedalis Wiedemann in Meigen, 1818 Ebejer et al. 2019 , Rif , Oued Azla (Nwawel, 57 m), Oued Azla (Hallila, 95 m) Dilophus (cf. bispinosus Lundström, 1914) 11 Séguy 1953a , AA , Aïn Chaib (Souss) Dilophus febrilis (Linnaeus, 1758) = Dilophus vulgaris Meigen, 1818, in Séguy 1949a : 153 Séguy 1949a , AA , Goulimine, Foum-el-Hassan Dilophus femoratus Meigen, 1804 = Philia femorata Meigen, 1804, in Séguy 1941: 2 Séguy 1941d , HA , Tizi-n'Test (2000 m); Pârvu et al. 2006 , AP , Merja Zerga Dilophus tridentatus Walker, 1848 Ebejer et al. 2019 , AP , Sidi Mokhtar (Essaouira) – MHNN (J.-P. Haenni leg.), MNHN ( SA , Foum-el-Hassan) Sciaroidea CECIDOMYIIDAE K. Kettani, M. Skuhravá, V. Skuhravý Number of species: 57 . Expected: 100 Faunistic knowledge of the family in Morocco: moderate Lestremiinae Lestremia Macquart, 1826 Lestremia parvostylia Jaschhof, 1994 Jaschhof 1994 , SA , Abeino, 15 km N Goulimine; Papp 2007 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Micromyinae Campylomyza Meigen, 1818 Campylomyza flavipes Meigen, 1818 Jaschhof 1998 , HA , Telouet; Skuhravá et al. 2017 Campylomyza fusca Winnertz, 1853 Jaschhof 1998 , HA , Telouet; Skuhravá et al. 2017 Campylomyza mohrigi Jaschhof, 2009 Jaschhof 2009 (south Morocco); Gagné 2010 ; Skuhravá et al. 2017 Monardia Kieffer, 1895 Monardia ( Xylopriona ) toxicodendri (Felt, 1907) Jaschhof 1998 (South Morocco); Jaschhof 2009; Gagné 2010 ; Skuhravá et al. 2017 Cecidomyiinae Asphondylia Loew, 1850 Asphondylia capparis Rübsaamen, 1893 Houard 1921 , MA , Fès; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Asphondylia cytisi Frauenfeld, 1873 Mimeur 1949 , HA , Tanzat (1800 m), AA , Jebel Sargho, Amalou Bou Mansour (2000 m); Skuhravá et al. 2017 Asphondylia punica Marchal, 1897 = Asphondylia conglomerata De Stefani, 1900 Houard 1922 , EM , Zousfana (Jebel Tagla); Mimeur 1949 , AA , Agdz; Mouna 1998 ; Skuhravá et al. 2017 Asphondylia scrophulariae Schiner, 1856 Mimeur 1949 , AP , Rabat, Arcilla; Skuhravá et al. 2017 Asphondylia verbasci (Vallot, 1827) Mimeur 1949 , AP , Maâmora, Rabat, Zaërs; Skuhravá et al. 2017 Baldratia Kieffer, 1897 Baldratia salicorniae Kieffer, 1897 Mimeur 1949 , AP , Rabat, Salé, Bou-Regreg; Möhn 1966 , EM , Melilla, Bocona, AP , Rabat; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Bayeriola Gagné, 1991 Bayeriola thymicola (Kieffer, 1888) Houard 1923 , HA , Réghaya; Mimeur 1949 , EM , Sidi Ali Oujda, Jebel Hamra, Itzer, MA , Ifrane, Azrou, Bordj-Doumergue, Timhadite, Aguelmane; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 1993 ; Gagné 2010 (south of Morocco); Bruun et al. 2012 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Blastomyia Kieffer, 1913 Blastomyia origani (Tavares, 1901) Houard 1922 , MA , Col de Bouchtata, Zalagh (Mouret); Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 (south of Morocco); Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Braueriella Kieffer, 1896 Braueriella phillyreae (Löw, 1877) Houard 1922 , Rif , Jebel Kébir; Houard 1923 , AP , Cap Ghir (south of Morocco); Mimeur 1949 , Rif , Zoumi, Ouezzane, EM , Béni Snassen, AP , Larache, Zaërs, Mehdia, Sehoul, MA , Jebel Said, Taza, Tahala, Tadla; Mouna 1998 ; Skuhravá et al. 2017 – MNHN ( AP , Mehdia) Contarinia Rondani, 1860 Contarinia ilicis Kieffer, 1898 Houard 1919 , MA , Immouzer; Mimeur 1949 , EM , Béni Snassen, Ras Foughal, El-Harcha, MA , Ifrane, Aït Bou-Mzil, Monts Zaian, Agoumi-n´Aït Mguild; Skuhravá et al. 2017 Contarinia luteola Tavares, 1902 Mimeur 1949 , EM , El Harcha, MA , Ifrane, Djaba, Imouzzer-du-Kandar, Tafechna; Mouna 1998 ; Skuhravá et al. 2017 Contarinia nasturtii (Kieffer, 1888) Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 Contarinia pyrivora (Riley, 1886) = Diplosis pirivora Riley, in Mouna 1998 : 85 Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 Dasineura Rondani, 1840 Dasineura affinis (Kieffer, 1886) = Perrisia affinis (Kieffer), in Mouna 1998 : 85 Mimeur 1949 , Rif , Tanger, EM , Oujda, AP , Gharb, Port-Lyautey, Rabat, Fedala, Casablanca, Mazagan, Settat, Oued-Zem, Mogador, MA , Fès, Tahala, Marchand; Mouna 1998 ; Skuhravá et al. 2017 ; AP (Rabat) – MISR Dasineura asparagi (Tavares, 1902) Mimeur 1949 , AP , Rabat, Zaërs, HA , Chaouia; Skuhravá et al. 2017 Dasineura crataegi (Winnertz, 1853) Mimeur 1949 , EM , Chaouia des Béni Snassen, AP , Rabat, Zaërs, MA , Ifrane, Tahala Aguelmane de Sidi Ali, Fès, Agoumi-n´Aït Mguild, Tarhzirt, AA , Argana, Imi-n-Tanoute; Skuhravá et al. 2017 Dasineura ericaescopariae (Dufour, 1837) Houard 1912 , Rif , Cap Spartel; Rübsaamen 1899 ; Skuhravá et al. 2017 Dasineura helianthemi (Hardy, 1850) = Contarinia helianthemi (Hardy, 1850) Mimeur 1949 , AP , Gharb, Maâmora, Zaërs; Skuhravá et al. 2017 Dasineura napi (Loew, 1850) = Dasineura brassicae (Winnertz, 1853) in Mouna 1998 : 85 Mouna 1998 (no accurate locality); Skuhravá et al. 2017 Dasineura periclymeni (Rübsaamen, 1889) Mimeur 1949 , AP , Rabat, Chellah, Yquem, Grou, Korifla, EM , Berkane; Skuhravá et al. 2017 Dasineura plicatrix (Loew, 1850) Mimeur 1949 , EM , Massif des Béni Snassen, El-Harcha, AP , Larache, Korifla, Grou, Yquem, Malah, Rabat, MA , Azrou, Ifrane, Monts Zaian, Oulmés, Khénifra, HA , Tizi-Machou, AA , Bigoudine; Skuhravá et al. 2017 Dasineura rosae (Loew, 1850) = Wachtliella rosarum (Hardy, 1850) Mimeur 1949 , MA , Ifrane, Aguelmane de Sidi Ali, Aguelmane Azigza, Zaad, Bordj-Doumergue, Bekrite, HA , Tizi-Machou; Skuhravá et al. 2017 Dicrodiplosis Kieffer, 1895 Dicrodiplosis pseudococci (Felt, 1914) Harris 1968 , AP , Rabat, Salé, HA , Asni; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Dryomyia Kieffer, 1898 Dryomyia lichtensteinii (Löw, 1878) Mimeur 1949 , EM , Debdou, Ras Foughal, El-Harcha, MA , Taza, Oulmès, Azrou, Aguelmane Azigza, Ifrane, Imouzzer-du-Kandar, Tizi-n´Tretten, Ida-ou Tanane, Azilal, HA , Ayachi; Skuhravá et al. 1984 ; Skuhravá 1986 ; Oosterbroek 2007 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; MA (Oulmès) – MISR Etsuhoa Inouye, 1959 Etsuhoa thuriferae Skuhravá, 1996 Mimeur 1949 , HA , Sidi-Chamharouch, Aït Bou-Jafar; Skuhravá 1995 ; Skuhravá et al. 2017 Feltiella Rübsaamen, 1910 Feltiella acarisuga (Vallot, 1827) Gagné 1995 , AP , Rabat; Osborne et al. 2002 (productive areas of cereals); Gagné 2004 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Gephyraulus Rübsaamen, 1916 Gephyraulus diplotaxis (Solinas, 1982) Houard 1922 , EM , Oasis de Figuig, Jebel Ouazzani; Mimeur 1949 , AP , Vallée de l´Oued Korifla, Sidi Bouknadel; Skuhravá et al. 2017 Gephyraulus raphanistri (Kieffer, 1886) Houard 1923 , AP , Mogador; Mimeur 1949 , AP , Gharb, Skhirat, Bouznika; Skuhravá et al. 2017 Houardiella Kieffer, 1912 Houardiella salicorniae Kieffer, 1912 Trotter 1904 , Rif , Tingis; Houard 1912 , Rif , Tanger; Mimeur 1949 , AP , Loukous, Larache, Ksob, Ameur, Mogador, Rabat, Salé, Bou-Regreg, EM , Moulouya, AA , Agadir; Skuhravá et al. 2017 Iteomyia Kieffer, 1913 Iteomyia major (Kieffer, 1889) Mimeur 1949 , AP , Korifla, Grou, MA , Leghzel; Skuhravá et al. 2017 Jaapiella Rübsaamen, 1916 Jaapiella bryoniae (Bouché, 1847) Mimeur 1949 , Rif , Ouezzane, AP , Sidi Bouknadel, Mehdia, Temara, Skhirat, Pont-Blondin, Larache, Monod, Boulhaut, Safi, MA , Tahala, Fès, Khemisset, Marchand, AA , Agadir; Skuhravá et al. 2017 Lasioptera Meigen, 1818 Lasioptera berlesiana Paoli, 1907 Mimeur 1949 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Lasioptera rubi (Schrank, 1803) Mimeur 1949 , AP , Tinkert, MA , Ifrane, Taza, Tarhzirt, HA , N´Fis, Oumer-Rbia, Jebel Tardema; Skuhravá et al. 2017 Lasioptera thapsiae Kieffer, 1898 = Lasioptera carophila F. Löw, in Houard 1923 : 699 Houard 1921 ; Houard 1923 , Rif , Tanger; Mimeur 1949 , EM , Béni Snassen, Guercif, AP , Settat, MA , Tiddas; Mouna 1998 ; Skuhravá et al. 2017 Lestodiplosis Kieffer, 1894 Lestodiplosis aonidiellae Harris, 1968 Harris 1968 , EM , Oujda, AP , Rabat, MA , Fès; Skuhravá et al. 2017 Mayetiola Kieffer, 1896 Mayetiola avenae (Marchal, 1895) Mouna 1998 ; Lahloui et al. 2005, AP , Settat, Safi, El Jadida, MA , Béni Mellal, Khouribga, HA , Marrakech, El Kelaâ; Skuhravá et al. 2017 Mayetiola destructor (Say, 1817) Vayssière 1920 , EM , Oujda; Jourdan 1937 ; Hudault and Zelensky 1939 ; Mimeur 1949 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné et al. 1991 ; Amri et al. 1992 (productive areas of cereals); El Bouhssini et al. 1992a , 1992b ; Lhaloui et al. 1992 ; El Bouhssini et al. 1996a , 1996b ; Khalifi et al. 1996 ; Azzam et al. 1997 ; El Bouhssini et al. 1997 , 1999 ; El Bouhssini et al. 1998 ; Mouna 1998 ; Naber et al. 2000 , 2003 ; Lhaloui et al. 2001 ; Lhaloui et al. 2005 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; AP (Rabat), MA – MISR Mayetiola hordei Kieffer, 1909 Mouna 1998 ; Gagné et al. 1991 ; Lhaloui et al. 2005 , AP , Settat, Safi, El Jadida, MA , Béni Mellal, Khouribga, HA , Marrakech, El Kelaâ; Skuhravá et al. 2005 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Oligotrophus Latreille, 1805 Oligotrophus panteli Kieffer, 1898 Mimeur 1949 , EM , Béni Snassen, MA , Aguelmane Azigza, Azrou, HA , Ayachi, Aït Bou-Jafar; Skuhravá et al. 2017 Oligotrophus valerii (Tavares, 1904) = Arceuthomyia valerii (Tavares, 1904) Mimeur 1949 , HA , Ayachi, Aït Bou-Jafar; Skuhravá et al. 2017 Orseolia Kieffer & Massalongo, 1902 Orseolia cynodontis Kieffer & Massalongo, 1902 Houard 1922 , Rif , Tanger, Aïn Dalia ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Phyllodiplosis Kieffer, 1912 Phyllodiplosis cocciferae (Tavares, 1902) = Blastodiplosis cocciferae (Tavares, 1902) Trotter 1904 , Rif , Cap Spartel; Houard 1912 ; Mimeur 1949 , Rif , Cap Spartel, Ouezzane, EM , Béni Snassen, El-Harcha, AP , Larache, Maâmora, Boulhaut, Zaërs, MA , Taza, Djaba, Ifrane, Imouzzer-du-Kandar, Dayet Achlaf, Dayet Ifrah, Michlifen, Bordj-Doumergue, Zaad, Bekrite, Aguelmane Azigza, Agoumi-n´Aït Mguild, Bou-Mzil, HA , Bou-Jafar, Jebel Tardema; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 Psectrosema Kieffer, 1904 Psectrosema tamaricum (Kieffer, 1912) = Amblardiella tamaricum Kieffer, in Mouna 1998 : 85 Houard 1922 , EM , Zousfana, near Sidi Youssef; Mimeur 1949 , Morocco Mediterranean, continental and sub-saharan, EM , Berkane, Moulouya, AP , MA , Tadla, HA , Haouz, AA , Tafilalet, SA , Agad; Skuhravá et al. 1984 ; Skuhravá 1986 ; Mouna 1998 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Resseliella Seitner, 1906 Resseliella oleisuga (Targioni-Tozzetti, 1887) = Clinodiplosis oleisuga Targioni-Tozzetti, in Mouna 1998 : 85 Hafraoui 1966 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Mouna 1998 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Rhopalomyia Rübsaamen, 1892 Rhopalomyia navasi Tavares, 1904 Houard 1922 , EM , Jebel Mais, Djahifa, HA , Aït Ameli; Houard 1923 ; Mimeur 1949 , EM , Figuig, Jebel Nokra, Ifkern, Haute Moulouya, MA , Tadla; Skuhravá et al. 1993 ; Skuhravá et al. 2014b ; Skuhravá et al. 2017 Schizomyia Kieffer, 1889 Schizomyia buboniae (Frauenfeld, 1859) Houard 1917 , EM , Djorf de Taouriet; Houard 1922 , EM , Jebel Tagla, Aïn Yalon; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Stefaniella Kieffer, 1898 Stefaniella trinacriae De Stefani, 1900 Mimeur 1949 , AP , Oualidia, Zima, Casablanca; Skuhravá et al. 2017 Stefaniola Kieffer, 1913 Stefaniola africana Möhn, 1971 Mimeur 1949 , AP , Rabat, Salé, Bou-Regreg; Skuhravá et al. 2017 Stefaniola bilobata (Kieffer, 1913) Houard 1922 – 1923 ; Mimeur 1949 , MA , Tadla, HA , Ksar-es-Souk, AA , Haouz; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 1993 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Stefaniola opulenta Möhn, 1971 Möhn 1971 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; Pape and Thompson 2019 Stefaniola ventriosa Möhn, 1971 Möhn 1971 , MA , Oued Gheris; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Thecodiplosis Kieffer, 1895 Thecodiplosis brachyntera (Schwägrichen, 1835) Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 KEROPLATIDAE K. Kettani, P.J. Chandler Number of species: 2 . Expected: 50 Faunistic knowledge of the family in Morocco: poor Keroplatinae Keroplatus Bosc, 1792 Keroplatus reaumurii (Dufour, 1839) Matile 1986 ; Chandler et al. 2005 ; Evenhuis 2006 Macrocera Meigen, 1803 Macrocera fasciata Meigen, 1804 Becker and Stein 1913 , Rif , Tanger; Chandler and Ribeiro 1995 ; Evenhuis 2006 MYCETOPHILIDAE K. Kettani, P.J. Chandler Number of species: 64 . Expected: 250 Faunistic knowledge of the family in Morocco: poor Mycetophilinae Exechiini Allodiopsis Tuomikoski, 1966 Allodiopsis rustica Edwards, 1941 Banamar et al. 2020 , Rif , Dayat Tazia Anatella Winnertz, 1864 Anatella concava Plassmann, 1990 Banamar et al. 2020 , Rif , Oued Aârate Brevicornu Marshall, 1896 Brevicornu intermedium (Santos Abréu, 1920) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Tisgris, Oued Aârate, Dayat Tazia, Oued Maggou (Maggou Village), Douar Tizga, maison forestière de Talassemtane, Dayat Jebel Zemzem, Aïn Takhninjoute, Forêt Adrou, Dayat Amsemlil Brevicornu griseicolle Staeger, 1840 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Aârate, Dayat Tazia, Oued Maggou (Maggou Village), Aïn El Malaâb, maison forestière de Talassemtane, Douar Tizga, Aïn Takhninjoute, Dayat Amsemlil, Dayat Lemtahane, Dayat avant Taida Brevicornu sericoma (Meigen, 1830) Banamar et al. 2020 , Rif , Chefchaouen, Dayat Fifi, Forêt Jebel Lakraâ, Dayat Fifi, Oued Maggou (Maggou Village), maison forestière de Talassemtane, Douar Tizga, Oued Amsemlil, Grotte d'Hercule, Forêt Adrou, Cascade Chrafate, Oued Aârate, Bab el Karn, Dayat Amsemlil – MNHN (coll. J. Beaucournu) Brevicornu verralli (Edwards, 1925) Banamar et al. 2020 , Rif , Dayat Afersiw, Forêt Jebel Lakraâ Cordyla Meigen, 1803 Cordyla crassicornis Meigen, 1818 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Douar Abou Boubnar, maison forestière de Talassemtane, Forêt Adrou, Oued Sidi Yahia Aârab, EM , Oued Tafoughalt; Rif (Chefchaouen) – MNHN Cordyla insons Laštovka & Matile, 1974 Banamar et al. 2020 , Rif , Oued Maggou Cordyla murina Winnertz, 1864 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba Cordyla styliforceps (Bukowski, 1934) Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Oued Tkaraâ, Oued Sidi Yahia Aârab Exechia Winnertz, 1863 Exechia bicincta (Staeger, 1840) Banamar et al. 2020 , Rif , Oued Kelaâ, Oued Aârate, EM , Grotte des Pigeons, Oued Tafoughalt Exechia dorsalis (Staeger, 1840) Banamar et al. 2020 , Rif , Bab el Karn Exechia fulva Santos Abreu, 1920 = Rymosia exornata Séguy, in Séguy 1941a : 26 Séguy 1941a , HA , Toubkal; Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Oued Kelaâ, Forêt Aïn Boughaba, Forêt Jebel Lakraâ, Dayat Fifi, Aïn Sidi Brahim Ben Arrif, Oued Maggou (Maggou Village), Aïn Takhninjoute, maison forestière de Talassemtane, Oued Amsemlil, Dayat Jebel Zemzem, Aïn Takhninjoute, Bab el Karn, Dayat Fifi, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN ; Rif (20 km west of Targuist, coll. A.M. Hutson) – NHMUK Exechia fusca (Meigen, 1804) Banamar et al. 2020 , Rif , Oued Kelaâ, Forêt Jebel Lakraâ, Douar Kitane, Dayat Amsemlil Exechiopsis Tuomikoski, 1966 Exechiopsis coremura (Edwards, 1928) Banamar et al. 2020 , Rif , Cascade Chrafate Pseudexechia Tuomikoski, 1966 Pseudexechia tuomikoskii (Kjærandsen, 2009) Banamar et al. 2020 , Rif , Source Aheramen Rymosia Winnertz, 1864 Rymosia affinis Winnertz, 1864 Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Amsemlil, Aïn Tiouila, Dayat Fifi Rymosia beaucournui Matile, 1963 Chandler 1994 ; Chandler and Ribeiro 1995 ; Chandler et al. 2005 ; Banamar et al. 2020 , EM , Grotte des Pigeons; AP (Oued y Kern, coll. H. Choumara) – MNHN Rymosia pseudocretensis Burghele-Balacesco, 1966 Chandler 1994 ; Chandler et al. 2005 ; Banamar et al. 2020 ; AP (Oued y Kern, coll. H. Choumara) – MNHN Stigmatomeria Tuomikoski, 1966 Stigmatomeria crassicornis (Stannius, 1831) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Dayat Fifi, Aïn Takhninjoute, maison forestière de Talassemtane, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Tarnania Tuomikoski, 1966 Tarnania dziedzickii (Edwards, 1941) Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Amsemlil, Cascade Chrafate, Dayat Amsemlil, Grotte Aïn El-Aouda – MNHN Mycetophilini Mycetophila Meigen, 1804 Mycetophila alea Laffoon, 1965 Banamar et al. 2020 , Rif , Aïn el Ma Bared, maison forestière de Talassemtane, Oued Aârate, Bab el Karn; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila britannica Laštovka & Kidd, 1975 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, Forêt Jebel Lakraâ, Douar Kitane, Aïn El Malaâb, oued à 15 km de Fifi, Aïn El Ma Bared, maison forestière de Talassemtane, Oued Maggou (Maggou Village), Aïn Takhninjoute, Grotte d'Hercule, Oued Aârate, Bab el Karn, Dayat Amsemlil, Dayat Lemtahane, Dayat avant Taida, Forêt Taghzout – MNHN Mycetophila deflexa Chandler, 2001 Banamar et al. 2020 , Rif , Forêt Taghzout, route Ksar el Kebir–Chefchaouen Mycetophila edwardsi Lundström, 1913 Banamar et al. 2020 , Rif , Dayat Fifi, Dayat Tazia, Oued Tkarae, Forêt Jebel Lakraâ, Forêt Taghzout, Grotte d'Hercule, EM , Béni Snassen Mycetophila formosa Lundström, 1911 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Amsemlil, Dayat Amsemlil, Dayat avant Taida Mycetophila marginata Winnertz, 1864 Banamar et al. 2020 , Rif , Aïn Ras el Ma, Oued Maggou (Maggou Village), maison forestière de Talassemtane, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila perpallida Chandler, 1993 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, Aïn El Malaâb, maison forestière de Talassemtane, Bab el Karn, Dayat Amsemlil, Douar Kitane Mycetophila pictula Meigen, 1830 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Maggou (Maggou Village); Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila sordida van der Wulp, 1874 Chandler 1994 ; Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ; Oued Maggou (Maggou Village); MA (Khenolap-el-Ouaer, 1580 m, coll. J. Beaucournu) – MNHN Mycetophila spectabilis Winnertz, 1864 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Forêt Aïn Boughaba, oued à 15 km de Fifi, Aïn El Malaâb Mycetophila strigatoides (Landrock, 1927) Banamar et al. 2020 , Rif , Forêt Taghzout, Oued Aârate Mycetophila unicolor Stannius, 1831 Banamar et al. 2020 , Rif , Oued Kelaâ Mycetophila vittipes Zetterstedt, 1852 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Dayat Amsemlil Phronia Winnertz, 1863 Phronia biarcuata (Becker, 1908) Chandler 1994 ; Chandler and Ribeiro 1995 ; Chandler et al. 2005 ; Banamar et al. 2020 , Rif , Dayat Tazia, Aïn el Ma Bared, Aïn El Malaâb, Aïn Takhninjoute, maison forestière de Talassemtane, Grotte d'Hercule, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Phronia cinerascens Winnertz, 1864 Banamar et al. 2020 , Rif , Dayat Amsemlil Phronia nitidiventris (van der Wulp, 1858) Banamar et al. 2020 , Rif , Dayat Afersiw, Oued Aârate Phronia tenuis Winnertz, 1864 Banamar et al. 2020 , Rif , Oued Kelaâ, Oued Aârate, Oued Maggou (Maggou Village), Aïn Takhninjoute, Grotte d'Hercule Phronia tyrrhenica Edwards, 1928 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Dayat Amsemlil, Aïn Takhninjoute Phronia willistoni Dziedzicki, 1889 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Aârate, maison forestière de Talassemtane, Dayat Amsemlil, Cascade Chrafate, Bab el Karn Sceptonia Winnertz, 1864 Sceptonia intestata Plassmann & Schacht, 1990 Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Aïn El Malaâb Sceptonia membranacea Edwards, 1925 Banamar et al. 2020 , Rif , oued à 15 km de Fifi, Oued Aârate Trichonta Winnertz, 1864 Trichonta foeda Loew, 1869 Banamar et al. 2020 , Rif , Aïn Takhninjoute, Bab el Karn, Dayat Amsemlil, Oued Tkarae Trichonta icenica Edwards, 1925 Banamar et al. 2020 , EM , Grotte du Chameau Trichonta vitta (Meigen, 1830) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Bab el Karn Trichonta vulcani Dziedzicki, 1889 Banamar et al. 2020 , EM , Grotte du Chameau Zygomyia Winnertz, 1864 Zygomyia humeralis (Wiedemann, 1817) Banamar et al. 2020 , Rif , maison forestière de Talassemtane Zygomyia valida Winnertz, 1864 Banamar et al. 2020 , Rif , Aïn Ras el Ma, Oued Aârate Leiinae Docosia Winnertz, 1864 Docosia gilvipes (Walker, 1856) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Aïn Takhninjoute, Dayat Amsemlil Leia Meigen, 1818 Leia arsona Hutson, 1978 Banamar et al. 2020 , Rif , Oued Maggou (Maggou village), Oued Sidi Yahia Aârab, MA , Aïn Walili Leia beckeri Landrock, 1940 Banamar et al. 2020 , Rif , Aïn Ras el Ma Leia bimaculata (Meigen, 1804) Chandler 1994 ; Chandler et al. 2005 ; Banamar et al. 2020 , Rif , Dayat Tazia, maison forestière de Talassemtane, Aïn el Ma Bared, MA , Forêt 3.5 km S Azrou; MA (Forêt Ifrane, coll. P.N. Lawrence) – NHMUK Novakia Strobl, 1893 Novakia scatopsiformis Strobl, 1893 Banamar et al. 2020 , Rif , maison forestière de Talassemtane Novakia simillima Strobl, 1910 Banamar et al. 2020 , Rif , Oued Aârate, maison forestière de Talassemtane Gnoristinae Boletina Staeger, 1840 Boletina gripha Dziedzicki, 1885 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Aârate, Aïn Sidi Brahim Ben Arrif, Dayat Amsemlil, Grotte d'Hercule, Cascade Chrafate, Oued Sidi Yahia Aârab, Aïn el Ma Bared, Bab el Karn, MA , forêt 3.5 km S Azrou Coelosia Winnertz, 1864 Coelosia fusca Bezzi, 1892 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Kelaâ, Oued Amsemlil, Dayat Amsemlil, Aïn Takhninjoute, Cascade Chrafate, Bab el Karn, Dayat avant Taida Synapha Meigen, 1818 Synapha fasciata Meigen, 1818 Banamar et al. 2020 , Rif , Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Forêt Adrou, Dayat Amsemlil, Dayat avant Taida Synapha vitripennis (Meigen, 1818) Banamar et al. 2020 , Rif , Dayat Amsemlil Tetragoneura Winnertz, 1846 Tetragoneura ambigua Grzegorzek, 1885 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, EM , Oued Tafoughalt Mycomyinae Mycomya Rondani, 1856 Mycomya flavicollis (Zetterstedt, 1852) Banamar et al. 2020 , Rif , Aïn El Malaâb, maison forestière de Talassemtane Mycomya pygmalion Väisänen, 1984 Banamar et al. 2020 , Rif , Oued Amsemlil, Aïn Sidi Brahim Ben Arrif Mycomya tumida (Winnertz, 1864) Banamar et al. 2020 , Rif , Dayat Fifi Sciophilinae Azana Walker, 1856 Azana anomala Staeger, 1840 Banamar et al. 2020 , Rif , Oued Maggou (Maggou village), maison forestière de Talassemtane Sciophila Meigen, 1818 Sciophila iberolutea Chandler & Blasco-Zumeta, 2001 Chandler and Gatt 2000 , AP , Oued y Kern; Chandler and Blasco-Zumeta 2001 , AP , Oued y Kern; Bechev and Koç 2006 ; Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Jebel Zemzem, Oued Sidi Yahia Aârab, Bab el Karn, Marabout el Khaloua; AP (Oued y Kern, coll. H. Choumara) – MNHN SCIARIDAE K. Kettani, K. Heller Number of species: 70 . Expected: 200–500 Faunistic knowledge of the family in Morocco: poor Austrosciara Schmitz & Mjöberg, 1924 Austrosciara hyalipennis (Meigen, 1804) El Ouazzani et al. 2019 , Rif , Douar El Hamma, MA , Lac Ouiouane Bradysia Winnertz, 1867 Bradysia alpicola (Winnertz, 1867) El Ouazzani et al. 2019 , MA , Lac Ouiouane Bradysia bulbigera Mohrig & Kauschke, 1994 El Ouazzani et al. 2019 , Rif , Oued Ouara, Oued Ametrasse, Merzouk Bni Salah, Dayat Bayn widane, HA , Ouirgane Bradysia cavernicola Mohrig & Eckert, 1999 Menzel and Heller 2004 , HA , Ouirgane Bradysia cinerascens (Grzegorzek, 1884) El Ouazzani et al. 2019 , Rif , Issaguen, Anissar ( PNPB ) Bradysia crinita Mohrig, 1992 El Ouazzani et al. 2019 , Rif , Issaguen, Douar El Hamma Bradysia fenestralis (Zetterstedt, 1838) El Ouazzani et al. 2019 , Rif , Forêt R'milat Bradysia fenestrata (Meigen, 1818) El Ouazzani et al. 2019 , Rif , Jebel Zemzem, Oued Tkarâa, Ben Karrich, Dayat Tazia, Perdicaris Park, Tourbière Amsemlil, Dayat Tazia Bradysia flavipila Tuomikoski, 1960 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia iberiana Rudzinski & Baumjohann, 2009 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia lembkei Mohrig & Menzel, 1990 El Ouazzani et al. 2019 , Rif , Oued Maggou, Dayat Tazia, AP , Forêt Maâmora, HA , Ouirgane, Gerifodene Bradysia lucichaeta Mohrig & Krivosheina, 1989 Mohrig et al. 1997 , AA , Sidi Rbat (40 km S Agadir) Bradysia mediterranea Mohrig & Menzel, 1992 Mohrig and Menzel 1992 , HA Bradysia nigrispina Menzel, 2006 El Ouazzani et al. 2019 , HA , Gerifodene Bradysia pectoralis (Staeger, 1840) El Ouazzani et al. 2019 , MA , Lac Ouiouane, HA , Ouirgane Bradysia placida (Winnertz, 1867) El Ouazzani et al. 2019 , HA , Ouirgane Bradysia promissa Mohrig & Röschmann, 1999 El Ouazzani et al. 2019 , Rif , Beni Barou, Anissar ( PNPB ), Oued Tkarâa ( PNPB ), Taida, Marabout Moulay Abdelsalam Bradysia reflexa Tuomikoski, 1960 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia regularis (Lengersdorf, 1934) El Ouazzani et al. 2019 , Rif , Talassemtane (maison forestière), HA , Ouirgane Bradysia ruginosa Mohrig, 1994 Mohrig et al. 1997 , SA , Ablino (15 km N Goulimine); El Ouazzani et al. 2019 , Rif , Jebel Lakraâ, Aïn El Fakir, HA , Amizmiz Bradysia santorina Mohrig & Menzel, 1992 Mohrig et al. 1997 , AA , Sidi Rbat (40 km S Agadir) Bradysia scabricornis Tuomikoski, 1960 El Ouazzani et al. 2019 , Rif , Oued Ouara, Maggou, Oued Azla, Douar El Hamma, HA , Ouirgane, Setti Fatma, Oued Imlil Bradysia subrufescens Mohrig & Krivosheina, 1989 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia subsantorina Mohrig & Kauschke, 1997 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia tilicola (Loew, 1850) El Ouazzani et al. 2019 , Rif , Douar Tissouka, MA , Lac Ouiouane, HA , Ouirgane Bradysia transitata Rudzinski & Baumjohann, 2013 El Ouazzani et al. 2019 , Rif , Oued Ez-Zarka, Oued Tkarâa ( PNPB ), Oued Laou, AP , Larache (Strawberry farm), Forêt Maâmora, AA , Barrage Aoulouz, Assif Tifnout Bradysia trivittata (Staeger, 1840) Mohrig and Röschmann 1993, HA , AA , Sidi Rbat (40 km S Agadir); El Ouazzani et al. 2019 , Rif , Aïn Tayattine, Oued Ez-Zarka, Oued Bayine, Beni Barou, Douar El Hamma, Douar Mouarâa, Tétouan, MA , Lac Ouiouane, HA , Télouet, Ouirgane Bradysia vagans (Winnertz, 1868) misidentified as Bradysia rufescens (Zetterstedt, 1852) in Röschmann and Mohrig 1993 : 111 Röschmann and Mohrig 1993 , HA , Talouete; El Ouazzani et al. 2019 , Rif , Aïn Fouara, HA , Ouirgane Bradysia xenoreflexa Mohrig & Menzel, 1993 El Ouazzani et al. 2019 , AP , Forêt Maâmora Bradysiopsis Tuomikoski, 1960 Bradysiopsis vittata (Meigen, 1830) El Ouazzani et al. 2019 , HA , Setti Fatma Camptochaeta Hippa & Vilkamaa, 1994 Camptochaeta jeskei (Mohrig & Röschmann, 1993) = Corynoptera jeskei Mohrig and Röschmann, in Röschmann and Mohrig 1993 : 109 Röschmann and Mohrig 1993 , HA , Talouete (1800 m) Corynoptera Winnertz, 1867 Corynoptera andalusica Hippa, Vilkamaa & Heller, 2010 El Ouazzani et al. 2019 , MA , Lac Ouiouane, HA , Ouirgane Corynoptera bicuspidata (Lengersdorf, 1926) Hippa et al. 2010 , HA , Ouirgane, Lac Ouiouane Corynoptera bispinulosa Mohrig & Dimitrova, 1992 El Ouazzani et al. 2019 , EM , Tafoughalt Corynoptera caesula Hippa & Menzel, 2004 El Ouazzani et al. 2019 , Rif , Aïn Kchour, AP , Forêt Maâmora Corynoptera cincinnata Mohrig & Blasco-Zumeta, 1996 Hippa et al. 2010 , HA , Ouirgane Corynoptera dentiforceps (Bukowski & Lengersdorf, 1936) El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera deserta Heller & Menzel, 2006 El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera fatigans (Johannsen, 1912) = Corynoptera perpusilla Winnertz, 1867, in Mohrig et al. 1997 : 384 Mohrig et al. 1997 , HA , Anezal; Hippa et al. 2010 [for nomenclature see Mohrig et al. 2013 ] Corynoptera gemina (Hippa & Vilkamaa, 1994) El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera globiformis (Frey, 1945) El Ouazzani et al. 2019 , Rif , Talassemtane (maison forestière) Corynoptera hemiacantha Mohrig & Mamaev, 1992 El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera iberica Hippa, Vilkamaa & Heller, 2010 El Ouazzani et al. 2019 , AP , Forêt Maâmora, Sidi Boughaba Corynoptera inclinata Hippa, Vilkamaa & Heller, 2010 Hippa et al. 2010 , HA , Ouirgane Corynoptera irmgardis (Lengersdorf, 1930) Hippa et al. 2010 , HA , Ouirgane Corynoptera postglobiformis Mohrig, 1993 El Ouazzani et al. 2019 , Rif , Talassemtane (maison forestière), HA , Ouirgane Corynoptera praeparvula Mohrig & Krivosheina, 1983 El Ouazzani et al. 2019 , HA , Ouirgane, Amizmiz, Gerifodene Corynoptera saccata Tuomikoski, 1960 Hippa et al. 2010 , SA , Goulimine; Mohrig et al. 2012 Corynoptera semipedestris Mohrig & Blasco-Zumeta, 1996 Mohrig et al. 1997 , SA , Ablino (15 km N Goulimine) Corynoptera spiciceps Hippa, Vilkamaa & Heller, 2010 Hippa et al. 2010 , HA , Ouirgane Corynoptera stipidaria Mohrig, 1994 Hippa et al. 2010 , HA , Ouirgane Corynoptera subcavipes Menzel & Smith, 2007 El Ouazzani et al. 2019 , Rif , Douar El Hamma, Talassemtane (maison forestière) Corynoptera subparvula Tuomikoski, 1960 El Ouazzani et al. 2019 , HA , Ouirgane Epidapus Haliday, 1851 Epidapus atomarius (De Geer, 1778) El Ouazzani et al. 2019 , Rif , Aïn el Ma Bared (Fifi) Leptosciarella Tuomikoski, 1960 Leptosciarella dives (Johannsen, 1912) Mohrig et al. 2012 , HA , Ouirgane Leptosciarella parcepilosa (Strobl, 1900) El Ouazzani et al. 2019 , AP , Sidi Boughaba Leptosciarella subviatica Mohrig & Menzel, 1997 El Ouazzani et al. 2019 , HA , Ouirgane Leptosciarella tomentosa (Mohrig & Kauschke, 1994) El Ouazzani et al. 2019 , AP , Forêt Maâmora Lycoriella Frey, 1942 Lycoriella agraria (Felt, 1898) El Ouazzani et al. 2019 , Rif , Douar Tissouka Lycoriella sativae (Johannsen, 1912) El Ouazzani et al. 2019 , Rif , M'Diq, Oued Zaouya, AP , Larache (strawberry farm), HA , Ouirgane Pseudolycoriella Menzel & Mohrig, 1998 Pseudolycoriella morenae (Strobl, 1900) El Ouazzani et al. 2019 , Rif , Perdicaris Park, Bab Tariouant, Oued Maggou, EM , Zegzel, HA , Ouirgane Scatopsciara Edwards, 1927 Scatopsciara ( Scatopsciara ) atomaria (Zetterstedt, 1851) = Scatopsciara vivida (Winnertz, 1867), in Röschmann and Mohrig 1993 : 111 Röschmann and Mohrig 1993 , HA , Talouete; El Ouazzani et al. 2019 , Rif , Oued Ez-Zarka, Oued Tkarâa ( PNPB ), Oued Guallet, Marécage Lemtahane ( PNPB ), Oued Ametrasse, Issaguen, Talassemtane (maison forestière), Douar El Hamma, Aïn Fouara, Oued Souk El Had, Merzouk Bni Salah, Oued Maggou, AP , Sidi Boughaba, HA , Assif Tifnout, Gerifodene, Armed, Amzmiz, Lac Tislit, AA , Barrage Mokhtar Soussi Scatopsciara ( Scatopsciara ) maroccoensis Mohrig & Jaschhof, 1997 Mohrig et al. 1997 , AA , Sidi Rbat (40 km S Agadir) Scatopsiara ( Scatopsciara ) nana (Winnertz, 1871) El Ouazzani et al. 2019 , Rif , Oued Ez-Zarka, Oued Maâmala, Oued Aârkob, Ben Karrich, Merja Sidi Lhaj Merzouk, Tétouan, HA , Ouirgane, Anafgou Scatopsciara ( Scatopsciara ) vitripennis (Meigen, 1818) El Ouazzani et al. 2019 , Rif , Oued Ouarra, Oued Tkarâa ( PNPB ), Oued Aoudour, Oued Ametrasse, Oued Aârkob, Oued Boumarouil, Ben Karrich, Dayat Tazia, Aïn El Fakir, Azib de Khmis Mdik, Merzouk Bni Salah, Oued Souk El Had, Oued Maggou, El Malâab (Talassemtane), AP , Sidi Boughaba, MA , Lac Ouiouane, HA , Ouirgane, AA , Barrage Aoulouz, Assif Tifnout, Barrage Mokhtar Soussi Scatopsciara ( Xenopygina ) curvilinea (Lengersdorf, 1934) El Ouazzani et al. 2019 , AP , Sidi Boughaba, HA , Aïn Taferaout, Amzmiz, AA , Assif Tifnout, Barrage Mokhtar Soussi Scatopsciara ( Xenopygina ) subarmata Mohrig & Mamaev, 1983 El Ouazzani et al. 2019 , Rif , Oued Amsa, AP , Larache, MA , Mont Habri, HA , Ouirgane, AA , Id Aissa, Tissint Schwenckfeldina Frey, 1942 Schwenckfeldina carbonaria (Meigen, 1830) = Sciara carbonaria Meigen, in Séguy 1941d : 2 Séguy 1941d , HA , Tizi-n'Test (2000 m) Sciara Meigen, 1803 Sciara flavimana Zetterstedt, 1851 El Ouazzani et al. 2019 , Rif , Douar El Hamma Sciara hemerobioides (Scopoli, 1763) = Lycoria ( Sciara ) thomae Linnaeus, in Becker and Stein 1913 : 85 Becker and Stein 1913 , Rif , Tanger CECIDOMYIIDAE K. Kettani, M. Skuhravá, V. Skuhravý Number of species: 57 . Expected: 100 Faunistic knowledge of the family in Morocco: moderate Lestremiinae Lestremia Macquart, 1826 Lestremia parvostylia Jaschhof, 1994 Jaschhof 1994 , SA , Abeino, 15 km N Goulimine; Papp 2007 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Micromyinae Campylomyza Meigen, 1818 Campylomyza flavipes Meigen, 1818 Jaschhof 1998 , HA , Telouet; Skuhravá et al. 2017 Campylomyza fusca Winnertz, 1853 Jaschhof 1998 , HA , Telouet; Skuhravá et al. 2017 Campylomyza mohrigi Jaschhof, 2009 Jaschhof 2009 (south Morocco); Gagné 2010 ; Skuhravá et al. 2017 Monardia Kieffer, 1895 Monardia ( Xylopriona ) toxicodendri (Felt, 1907) Jaschhof 1998 (South Morocco); Jaschhof 2009; Gagné 2010 ; Skuhravá et al. 2017 Cecidomyiinae Asphondylia Loew, 1850 Asphondylia capparis Rübsaamen, 1893 Houard 1921 , MA , Fès; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Asphondylia cytisi Frauenfeld, 1873 Mimeur 1949 , HA , Tanzat (1800 m), AA , Jebel Sargho, Amalou Bou Mansour (2000 m); Skuhravá et al. 2017 Asphondylia punica Marchal, 1897 = Asphondylia conglomerata De Stefani, 1900 Houard 1922 , EM , Zousfana (Jebel Tagla); Mimeur 1949 , AA , Agdz; Mouna 1998 ; Skuhravá et al. 2017 Asphondylia scrophulariae Schiner, 1856 Mimeur 1949 , AP , Rabat, Arcilla; Skuhravá et al. 2017 Asphondylia verbasci (Vallot, 1827) Mimeur 1949 , AP , Maâmora, Rabat, Zaërs; Skuhravá et al. 2017 Baldratia Kieffer, 1897 Baldratia salicorniae Kieffer, 1897 Mimeur 1949 , AP , Rabat, Salé, Bou-Regreg; Möhn 1966 , EM , Melilla, Bocona, AP , Rabat; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Bayeriola Gagné, 1991 Bayeriola thymicola (Kieffer, 1888) Houard 1923 , HA , Réghaya; Mimeur 1949 , EM , Sidi Ali Oujda, Jebel Hamra, Itzer, MA , Ifrane, Azrou, Bordj-Doumergue, Timhadite, Aguelmane; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 1993 ; Gagné 2010 (south of Morocco); Bruun et al. 2012 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Blastomyia Kieffer, 1913 Blastomyia origani (Tavares, 1901) Houard 1922 , MA , Col de Bouchtata, Zalagh (Mouret); Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 (south of Morocco); Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Braueriella Kieffer, 1896 Braueriella phillyreae (Löw, 1877) Houard 1922 , Rif , Jebel Kébir; Houard 1923 , AP , Cap Ghir (south of Morocco); Mimeur 1949 , Rif , Zoumi, Ouezzane, EM , Béni Snassen, AP , Larache, Zaërs, Mehdia, Sehoul, MA , Jebel Said, Taza, Tahala, Tadla; Mouna 1998 ; Skuhravá et al. 2017 – MNHN ( AP , Mehdia) Contarinia Rondani, 1860 Contarinia ilicis Kieffer, 1898 Houard 1919 , MA , Immouzer; Mimeur 1949 , EM , Béni Snassen, Ras Foughal, El-Harcha, MA , Ifrane, Aït Bou-Mzil, Monts Zaian, Agoumi-n´Aït Mguild; Skuhravá et al. 2017 Contarinia luteola Tavares, 1902 Mimeur 1949 , EM , El Harcha, MA , Ifrane, Djaba, Imouzzer-du-Kandar, Tafechna; Mouna 1998 ; Skuhravá et al. 2017 Contarinia nasturtii (Kieffer, 1888) Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 Contarinia pyrivora (Riley, 1886) = Diplosis pirivora Riley, in Mouna 1998 : 85 Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 Dasineura Rondani, 1840 Dasineura affinis (Kieffer, 1886) = Perrisia affinis (Kieffer), in Mouna 1998 : 85 Mimeur 1949 , Rif , Tanger, EM , Oujda, AP , Gharb, Port-Lyautey, Rabat, Fedala, Casablanca, Mazagan, Settat, Oued-Zem, Mogador, MA , Fès, Tahala, Marchand; Mouna 1998 ; Skuhravá et al. 2017 ; AP (Rabat) – MISR Dasineura asparagi (Tavares, 1902) Mimeur 1949 , AP , Rabat, Zaërs, HA , Chaouia; Skuhravá et al. 2017 Dasineura crataegi (Winnertz, 1853) Mimeur 1949 , EM , Chaouia des Béni Snassen, AP , Rabat, Zaërs, MA , Ifrane, Tahala Aguelmane de Sidi Ali, Fès, Agoumi-n´Aït Mguild, Tarhzirt, AA , Argana, Imi-n-Tanoute; Skuhravá et al. 2017 Dasineura ericaescopariae (Dufour, 1837) Houard 1912 , Rif , Cap Spartel; Rübsaamen 1899 ; Skuhravá et al. 2017 Dasineura helianthemi (Hardy, 1850) = Contarinia helianthemi (Hardy, 1850) Mimeur 1949 , AP , Gharb, Maâmora, Zaërs; Skuhravá et al. 2017 Dasineura napi (Loew, 1850) = Dasineura brassicae (Winnertz, 1853) in Mouna 1998 : 85 Mouna 1998 (no accurate locality); Skuhravá et al. 2017 Dasineura periclymeni (Rübsaamen, 1889) Mimeur 1949 , AP , Rabat, Chellah, Yquem, Grou, Korifla, EM , Berkane; Skuhravá et al. 2017 Dasineura plicatrix (Loew, 1850) Mimeur 1949 , EM , Massif des Béni Snassen, El-Harcha, AP , Larache, Korifla, Grou, Yquem, Malah, Rabat, MA , Azrou, Ifrane, Monts Zaian, Oulmés, Khénifra, HA , Tizi-Machou, AA , Bigoudine; Skuhravá et al. 2017 Dasineura rosae (Loew, 1850) = Wachtliella rosarum (Hardy, 1850) Mimeur 1949 , MA , Ifrane, Aguelmane de Sidi Ali, Aguelmane Azigza, Zaad, Bordj-Doumergue, Bekrite, HA , Tizi-Machou; Skuhravá et al. 2017 Dicrodiplosis Kieffer, 1895 Dicrodiplosis pseudococci (Felt, 1914) Harris 1968 , AP , Rabat, Salé, HA , Asni; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Dryomyia Kieffer, 1898 Dryomyia lichtensteinii (Löw, 1878) Mimeur 1949 , EM , Debdou, Ras Foughal, El-Harcha, MA , Taza, Oulmès, Azrou, Aguelmane Azigza, Ifrane, Imouzzer-du-Kandar, Tizi-n´Tretten, Ida-ou Tanane, Azilal, HA , Ayachi; Skuhravá et al. 1984 ; Skuhravá 1986 ; Oosterbroek 2007 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; MA (Oulmès) – MISR Etsuhoa Inouye, 1959 Etsuhoa thuriferae Skuhravá, 1996 Mimeur 1949 , HA , Sidi-Chamharouch, Aït Bou-Jafar; Skuhravá 1995 ; Skuhravá et al. 2017 Feltiella Rübsaamen, 1910 Feltiella acarisuga (Vallot, 1827) Gagné 1995 , AP , Rabat; Osborne et al. 2002 (productive areas of cereals); Gagné 2004 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Gephyraulus Rübsaamen, 1916 Gephyraulus diplotaxis (Solinas, 1982) Houard 1922 , EM , Oasis de Figuig, Jebel Ouazzani; Mimeur 1949 , AP , Vallée de l´Oued Korifla, Sidi Bouknadel; Skuhravá et al. 2017 Gephyraulus raphanistri (Kieffer, 1886) Houard 1923 , AP , Mogador; Mimeur 1949 , AP , Gharb, Skhirat, Bouznika; Skuhravá et al. 2017 Houardiella Kieffer, 1912 Houardiella salicorniae Kieffer, 1912 Trotter 1904 , Rif , Tingis; Houard 1912 , Rif , Tanger; Mimeur 1949 , AP , Loukous, Larache, Ksob, Ameur, Mogador, Rabat, Salé, Bou-Regreg, EM , Moulouya, AA , Agadir; Skuhravá et al. 2017 Iteomyia Kieffer, 1913 Iteomyia major (Kieffer, 1889) Mimeur 1949 , AP , Korifla, Grou, MA , Leghzel; Skuhravá et al. 2017 Jaapiella Rübsaamen, 1916 Jaapiella bryoniae (Bouché, 1847) Mimeur 1949 , Rif , Ouezzane, AP , Sidi Bouknadel, Mehdia, Temara, Skhirat, Pont-Blondin, Larache, Monod, Boulhaut, Safi, MA , Tahala, Fès, Khemisset, Marchand, AA , Agadir; Skuhravá et al. 2017 Lasioptera Meigen, 1818 Lasioptera berlesiana Paoli, 1907 Mimeur 1949 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Lasioptera rubi (Schrank, 1803) Mimeur 1949 , AP , Tinkert, MA , Ifrane, Taza, Tarhzirt, HA , N´Fis, Oumer-Rbia, Jebel Tardema; Skuhravá et al. 2017 Lasioptera thapsiae Kieffer, 1898 = Lasioptera carophila F. Löw, in Houard 1923 : 699 Houard 1921 ; Houard 1923 , Rif , Tanger; Mimeur 1949 , EM , Béni Snassen, Guercif, AP , Settat, MA , Tiddas; Mouna 1998 ; Skuhravá et al. 2017 Lestodiplosis Kieffer, 1894 Lestodiplosis aonidiellae Harris, 1968 Harris 1968 , EM , Oujda, AP , Rabat, MA , Fès; Skuhravá et al. 2017 Mayetiola Kieffer, 1896 Mayetiola avenae (Marchal, 1895) Mouna 1998 ; Lahloui et al. 2005, AP , Settat, Safi, El Jadida, MA , Béni Mellal, Khouribga, HA , Marrakech, El Kelaâ; Skuhravá et al. 2017 Mayetiola destructor (Say, 1817) Vayssière 1920 , EM , Oujda; Jourdan 1937 ; Hudault and Zelensky 1939 ; Mimeur 1949 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné et al. 1991 ; Amri et al. 1992 (productive areas of cereals); El Bouhssini et al. 1992a , 1992b ; Lhaloui et al. 1992 ; El Bouhssini et al. 1996a , 1996b ; Khalifi et al. 1996 ; Azzam et al. 1997 ; El Bouhssini et al. 1997 , 1999 ; El Bouhssini et al. 1998 ; Mouna 1998 ; Naber et al. 2000 , 2003 ; Lhaloui et al. 2001 ; Lhaloui et al. 2005 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; AP (Rabat), MA – MISR Mayetiola hordei Kieffer, 1909 Mouna 1998 ; Gagné et al. 1991 ; Lhaloui et al. 2005 , AP , Settat, Safi, El Jadida, MA , Béni Mellal, Khouribga, HA , Marrakech, El Kelaâ; Skuhravá et al. 2005 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Oligotrophus Latreille, 1805 Oligotrophus panteli Kieffer, 1898 Mimeur 1949 , EM , Béni Snassen, MA , Aguelmane Azigza, Azrou, HA , Ayachi, Aït Bou-Jafar; Skuhravá et al. 2017 Oligotrophus valerii (Tavares, 1904) = Arceuthomyia valerii (Tavares, 1904) Mimeur 1949 , HA , Ayachi, Aït Bou-Jafar; Skuhravá et al. 2017 Orseolia Kieffer & Massalongo, 1902 Orseolia cynodontis Kieffer & Massalongo, 1902 Houard 1922 , Rif , Tanger, Aïn Dalia ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Phyllodiplosis Kieffer, 1912 Phyllodiplosis cocciferae (Tavares, 1902) = Blastodiplosis cocciferae (Tavares, 1902) Trotter 1904 , Rif , Cap Spartel; Houard 1912 ; Mimeur 1949 , Rif , Cap Spartel, Ouezzane, EM , Béni Snassen, El-Harcha, AP , Larache, Maâmora, Boulhaut, Zaërs, MA , Taza, Djaba, Ifrane, Imouzzer-du-Kandar, Dayet Achlaf, Dayet Ifrah, Michlifen, Bordj-Doumergue, Zaad, Bekrite, Aguelmane Azigza, Agoumi-n´Aït Mguild, Bou-Mzil, HA , Bou-Jafar, Jebel Tardema; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 Psectrosema Kieffer, 1904 Psectrosema tamaricum (Kieffer, 1912) = Amblardiella tamaricum Kieffer, in Mouna 1998 : 85 Houard 1922 , EM , Zousfana, near Sidi Youssef; Mimeur 1949 , Morocco Mediterranean, continental and sub-saharan, EM , Berkane, Moulouya, AP , MA , Tadla, HA , Haouz, AA , Tafilalet, SA , Agad; Skuhravá et al. 1984 ; Skuhravá 1986 ; Mouna 1998 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Resseliella Seitner, 1906 Resseliella oleisuga (Targioni-Tozzetti, 1887) = Clinodiplosis oleisuga Targioni-Tozzetti, in Mouna 1998 : 85 Hafraoui 1966 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Mouna 1998 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Rhopalomyia Rübsaamen, 1892 Rhopalomyia navasi Tavares, 1904 Houard 1922 , EM , Jebel Mais, Djahifa, HA , Aït Ameli; Houard 1923 ; Mimeur 1949 , EM , Figuig, Jebel Nokra, Ifkern, Haute Moulouya, MA , Tadla; Skuhravá et al. 1993 ; Skuhravá et al. 2014b ; Skuhravá et al. 2017 Schizomyia Kieffer, 1889 Schizomyia buboniae (Frauenfeld, 1859) Houard 1917 , EM , Djorf de Taouriet; Houard 1922 , EM , Jebel Tagla, Aïn Yalon; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Stefaniella Kieffer, 1898 Stefaniella trinacriae De Stefani, 1900 Mimeur 1949 , AP , Oualidia, Zima, Casablanca; Skuhravá et al. 2017 Stefaniola Kieffer, 1913 Stefaniola africana Möhn, 1971 Mimeur 1949 , AP , Rabat, Salé, Bou-Regreg; Skuhravá et al. 2017 Stefaniola bilobata (Kieffer, 1913) Houard 1922 – 1923 ; Mimeur 1949 , MA , Tadla, HA , Ksar-es-Souk, AA , Haouz; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 1993 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Stefaniola opulenta Möhn, 1971 Möhn 1971 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; Pape and Thompson 2019 Stefaniola ventriosa Möhn, 1971 Möhn 1971 , MA , Oued Gheris; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Thecodiplosis Kieffer, 1895 Thecodiplosis brachyntera (Schwägrichen, 1835) Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 Lestremiinae Lestremia Macquart, 1826 Lestremia parvostylia Jaschhof, 1994 Jaschhof 1994 , SA , Abeino, 15 km N Goulimine; Papp 2007 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Micromyinae Campylomyza Meigen, 1818 Campylomyza flavipes Meigen, 1818 Jaschhof 1998 , HA , Telouet; Skuhravá et al. 2017 Campylomyza fusca Winnertz, 1853 Jaschhof 1998 , HA , Telouet; Skuhravá et al. 2017 Campylomyza mohrigi Jaschhof, 2009 Jaschhof 2009 (south Morocco); Gagné 2010 ; Skuhravá et al. 2017 Monardia Kieffer, 1895 Monardia ( Xylopriona ) toxicodendri (Felt, 1907) Jaschhof 1998 (South Morocco); Jaschhof 2009; Gagné 2010 ; Skuhravá et al. 2017 Cecidomyiinae Asphondylia Loew, 1850 Asphondylia capparis Rübsaamen, 1893 Houard 1921 , MA , Fès; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Asphondylia cytisi Frauenfeld, 1873 Mimeur 1949 , HA , Tanzat (1800 m), AA , Jebel Sargho, Amalou Bou Mansour (2000 m); Skuhravá et al. 2017 Asphondylia punica Marchal, 1897 = Asphondylia conglomerata De Stefani, 1900 Houard 1922 , EM , Zousfana (Jebel Tagla); Mimeur 1949 , AA , Agdz; Mouna 1998 ; Skuhravá et al. 2017 Asphondylia scrophulariae Schiner, 1856 Mimeur 1949 , AP , Rabat, Arcilla; Skuhravá et al. 2017 Asphondylia verbasci (Vallot, 1827) Mimeur 1949 , AP , Maâmora, Rabat, Zaërs; Skuhravá et al. 2017 Baldratia Kieffer, 1897 Baldratia salicorniae Kieffer, 1897 Mimeur 1949 , AP , Rabat, Salé, Bou-Regreg; Möhn 1966 , EM , Melilla, Bocona, AP , Rabat; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Bayeriola Gagné, 1991 Bayeriola thymicola (Kieffer, 1888) Houard 1923 , HA , Réghaya; Mimeur 1949 , EM , Sidi Ali Oujda, Jebel Hamra, Itzer, MA , Ifrane, Azrou, Bordj-Doumergue, Timhadite, Aguelmane; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 1993 ; Gagné 2010 (south of Morocco); Bruun et al. 2012 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Blastomyia Kieffer, 1913 Blastomyia origani (Tavares, 1901) Houard 1922 , MA , Col de Bouchtata, Zalagh (Mouret); Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 (south of Morocco); Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Braueriella Kieffer, 1896 Braueriella phillyreae (Löw, 1877) Houard 1922 , Rif , Jebel Kébir; Houard 1923 , AP , Cap Ghir (south of Morocco); Mimeur 1949 , Rif , Zoumi, Ouezzane, EM , Béni Snassen, AP , Larache, Zaërs, Mehdia, Sehoul, MA , Jebel Said, Taza, Tahala, Tadla; Mouna 1998 ; Skuhravá et al. 2017 – MNHN ( AP , Mehdia) Contarinia Rondani, 1860 Contarinia ilicis Kieffer, 1898 Houard 1919 , MA , Immouzer; Mimeur 1949 , EM , Béni Snassen, Ras Foughal, El-Harcha, MA , Ifrane, Aït Bou-Mzil, Monts Zaian, Agoumi-n´Aït Mguild; Skuhravá et al. 2017 Contarinia luteola Tavares, 1902 Mimeur 1949 , EM , El Harcha, MA , Ifrane, Djaba, Imouzzer-du-Kandar, Tafechna; Mouna 1998 ; Skuhravá et al. 2017 Contarinia nasturtii (Kieffer, 1888) Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 Contarinia pyrivora (Riley, 1886) = Diplosis pirivora Riley, in Mouna 1998 : 85 Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 Dasineura Rondani, 1840 Dasineura affinis (Kieffer, 1886) = Perrisia affinis (Kieffer), in Mouna 1998 : 85 Mimeur 1949 , Rif , Tanger, EM , Oujda, AP , Gharb, Port-Lyautey, Rabat, Fedala, Casablanca, Mazagan, Settat, Oued-Zem, Mogador, MA , Fès, Tahala, Marchand; Mouna 1998 ; Skuhravá et al. 2017 ; AP (Rabat) – MISR Dasineura asparagi (Tavares, 1902) Mimeur 1949 , AP , Rabat, Zaërs, HA , Chaouia; Skuhravá et al. 2017 Dasineura crataegi (Winnertz, 1853) Mimeur 1949 , EM , Chaouia des Béni Snassen, AP , Rabat, Zaërs, MA , Ifrane, Tahala Aguelmane de Sidi Ali, Fès, Agoumi-n´Aït Mguild, Tarhzirt, AA , Argana, Imi-n-Tanoute; Skuhravá et al. 2017 Dasineura ericaescopariae (Dufour, 1837) Houard 1912 , Rif , Cap Spartel; Rübsaamen 1899 ; Skuhravá et al. 2017 Dasineura helianthemi (Hardy, 1850) = Contarinia helianthemi (Hardy, 1850) Mimeur 1949 , AP , Gharb, Maâmora, Zaërs; Skuhravá et al. 2017 Dasineura napi (Loew, 1850) = Dasineura brassicae (Winnertz, 1853) in Mouna 1998 : 85 Mouna 1998 (no accurate locality); Skuhravá et al. 2017 Dasineura periclymeni (Rübsaamen, 1889) Mimeur 1949 , AP , Rabat, Chellah, Yquem, Grou, Korifla, EM , Berkane; Skuhravá et al. 2017 Dasineura plicatrix (Loew, 1850) Mimeur 1949 , EM , Massif des Béni Snassen, El-Harcha, AP , Larache, Korifla, Grou, Yquem, Malah, Rabat, MA , Azrou, Ifrane, Monts Zaian, Oulmés, Khénifra, HA , Tizi-Machou, AA , Bigoudine; Skuhravá et al. 2017 Dasineura rosae (Loew, 1850) = Wachtliella rosarum (Hardy, 1850) Mimeur 1949 , MA , Ifrane, Aguelmane de Sidi Ali, Aguelmane Azigza, Zaad, Bordj-Doumergue, Bekrite, HA , Tizi-Machou; Skuhravá et al. 2017 Dicrodiplosis Kieffer, 1895 Dicrodiplosis pseudococci (Felt, 1914) Harris 1968 , AP , Rabat, Salé, HA , Asni; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Dryomyia Kieffer, 1898 Dryomyia lichtensteinii (Löw, 1878) Mimeur 1949 , EM , Debdou, Ras Foughal, El-Harcha, MA , Taza, Oulmès, Azrou, Aguelmane Azigza, Ifrane, Imouzzer-du-Kandar, Tizi-n´Tretten, Ida-ou Tanane, Azilal, HA , Ayachi; Skuhravá et al. 1984 ; Skuhravá 1986 ; Oosterbroek 2007 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; MA (Oulmès) – MISR Etsuhoa Inouye, 1959 Etsuhoa thuriferae Skuhravá, 1996 Mimeur 1949 , HA , Sidi-Chamharouch, Aït Bou-Jafar; Skuhravá 1995 ; Skuhravá et al. 2017 Feltiella Rübsaamen, 1910 Feltiella acarisuga (Vallot, 1827) Gagné 1995 , AP , Rabat; Osborne et al. 2002 (productive areas of cereals); Gagné 2004 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Gephyraulus Rübsaamen, 1916 Gephyraulus diplotaxis (Solinas, 1982) Houard 1922 , EM , Oasis de Figuig, Jebel Ouazzani; Mimeur 1949 , AP , Vallée de l´Oued Korifla, Sidi Bouknadel; Skuhravá et al. 2017 Gephyraulus raphanistri (Kieffer, 1886) Houard 1923 , AP , Mogador; Mimeur 1949 , AP , Gharb, Skhirat, Bouznika; Skuhravá et al. 2017 Houardiella Kieffer, 1912 Houardiella salicorniae Kieffer, 1912 Trotter 1904 , Rif , Tingis; Houard 1912 , Rif , Tanger; Mimeur 1949 , AP , Loukous, Larache, Ksob, Ameur, Mogador, Rabat, Salé, Bou-Regreg, EM , Moulouya, AA , Agadir; Skuhravá et al. 2017 Iteomyia Kieffer, 1913 Iteomyia major (Kieffer, 1889) Mimeur 1949 , AP , Korifla, Grou, MA , Leghzel; Skuhravá et al. 2017 Jaapiella Rübsaamen, 1916 Jaapiella bryoniae (Bouché, 1847) Mimeur 1949 , Rif , Ouezzane, AP , Sidi Bouknadel, Mehdia, Temara, Skhirat, Pont-Blondin, Larache, Monod, Boulhaut, Safi, MA , Tahala, Fès, Khemisset, Marchand, AA , Agadir; Skuhravá et al. 2017 Lasioptera Meigen, 1818 Lasioptera berlesiana Paoli, 1907 Mimeur 1949 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 2017 Lasioptera rubi (Schrank, 1803) Mimeur 1949 , AP , Tinkert, MA , Ifrane, Taza, Tarhzirt, HA , N´Fis, Oumer-Rbia, Jebel Tardema; Skuhravá et al. 2017 Lasioptera thapsiae Kieffer, 1898 = Lasioptera carophila F. Löw, in Houard 1923 : 699 Houard 1921 ; Houard 1923 , Rif , Tanger; Mimeur 1949 , EM , Béni Snassen, Guercif, AP , Settat, MA , Tiddas; Mouna 1998 ; Skuhravá et al. 2017 Lestodiplosis Kieffer, 1894 Lestodiplosis aonidiellae Harris, 1968 Harris 1968 , EM , Oujda, AP , Rabat, MA , Fès; Skuhravá et al. 2017 Mayetiola Kieffer, 1896 Mayetiola avenae (Marchal, 1895) Mouna 1998 ; Lahloui et al. 2005, AP , Settat, Safi, El Jadida, MA , Béni Mellal, Khouribga, HA , Marrakech, El Kelaâ; Skuhravá et al. 2017 Mayetiola destructor (Say, 1817) Vayssière 1920 , EM , Oujda; Jourdan 1937 ; Hudault and Zelensky 1939 ; Mimeur 1949 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné et al. 1991 ; Amri et al. 1992 (productive areas of cereals); El Bouhssini et al. 1992a , 1992b ; Lhaloui et al. 1992 ; El Bouhssini et al. 1996a , 1996b ; Khalifi et al. 1996 ; Azzam et al. 1997 ; El Bouhssini et al. 1997 , 1999 ; El Bouhssini et al. 1998 ; Mouna 1998 ; Naber et al. 2000 , 2003 ; Lhaloui et al. 2001 ; Lhaloui et al. 2005 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; AP (Rabat), MA – MISR Mayetiola hordei Kieffer, 1909 Mouna 1998 ; Gagné et al. 1991 ; Lhaloui et al. 2005 , AP , Settat, Safi, El Jadida, MA , Béni Mellal, Khouribga, HA , Marrakech, El Kelaâ; Skuhravá et al. 2005 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Oligotrophus Latreille, 1805 Oligotrophus panteli Kieffer, 1898 Mimeur 1949 , EM , Béni Snassen, MA , Aguelmane Azigza, Azrou, HA , Ayachi, Aït Bou-Jafar; Skuhravá et al. 2017 Oligotrophus valerii (Tavares, 1904) = Arceuthomyia valerii (Tavares, 1904) Mimeur 1949 , HA , Ayachi, Aït Bou-Jafar; Skuhravá et al. 2017 Orseolia Kieffer & Massalongo, 1902 Orseolia cynodontis Kieffer & Massalongo, 1902 Houard 1922 , Rif , Tanger, Aïn Dalia ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Phyllodiplosis Kieffer, 1912 Phyllodiplosis cocciferae (Tavares, 1902) = Blastodiplosis cocciferae (Tavares, 1902) Trotter 1904 , Rif , Cap Spartel; Houard 1912 ; Mimeur 1949 , Rif , Cap Spartel, Ouezzane, EM , Béni Snassen, El-Harcha, AP , Larache, Maâmora, Boulhaut, Zaërs, MA , Taza, Djaba, Ifrane, Imouzzer-du-Kandar, Dayet Achlaf, Dayet Ifrah, Michlifen, Bordj-Doumergue, Zaad, Bekrite, Aguelmane Azigza, Agoumi-n´Aït Mguild, Bou-Mzil, HA , Bou-Jafar, Jebel Tardema; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 Psectrosema Kieffer, 1904 Psectrosema tamaricum (Kieffer, 1912) = Amblardiella tamaricum Kieffer, in Mouna 1998 : 85 Houard 1922 , EM , Zousfana, near Sidi Youssef; Mimeur 1949 , Morocco Mediterranean, continental and sub-saharan, EM , Berkane, Moulouya, AP , MA , Tadla, HA , Haouz, AA , Tafilalet, SA , Agad; Skuhravá et al. 1984 ; Skuhravá 1986 ; Mouna 1998 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Resseliella Seitner, 1906 Resseliella oleisuga (Targioni-Tozzetti, 1887) = Clinodiplosis oleisuga Targioni-Tozzetti, in Mouna 1998 : 85 Hafraoui 1966 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Mouna 1998 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Rhopalomyia Rübsaamen, 1892 Rhopalomyia navasi Tavares, 1904 Houard 1922 , EM , Jebel Mais, Djahifa, HA , Aït Ameli; Houard 1923 ; Mimeur 1949 , EM , Figuig, Jebel Nokra, Ifkern, Haute Moulouya, MA , Tadla; Skuhravá et al. 1993 ; Skuhravá et al. 2014b ; Skuhravá et al. 2017 Schizomyia Kieffer, 1889 Schizomyia buboniae (Frauenfeld, 1859) Houard 1917 , EM , Djorf de Taouriet; Houard 1922 , EM , Jebel Tagla, Aïn Yalon; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Stefaniella Kieffer, 1898 Stefaniella trinacriae De Stefani, 1900 Mimeur 1949 , AP , Oualidia, Zima, Casablanca; Skuhravá et al. 2017 Stefaniola Kieffer, 1913 Stefaniola africana Möhn, 1971 Mimeur 1949 , AP , Rabat, Salé, Bou-Regreg; Skuhravá et al. 2017 Stefaniola bilobata (Kieffer, 1913) Houard 1922 – 1923 ; Mimeur 1949 , MA , Tadla, HA , Ksar-es-Souk, AA , Haouz; Skuhravá et al. 1984 ; Skuhravá 1986 ; Skuhravá et al. 1993 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 Stefaniola opulenta Möhn, 1971 Möhn 1971 ; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2017 ; Pape and Thompson 2019 Stefaniola ventriosa Möhn, 1971 Möhn 1971 , MA , Oued Gheris; Skuhravá et al. 1984 ; Skuhravá 1986 ; Gagné 2010 ; Gagné and Jaschhof 2014 ; Skuhravá et al. 2014a ; Skuhravá et al. 2017 Thecodiplosis Kieffer, 1895 Thecodiplosis brachyntera (Schwägrichen, 1835) Mouna 1998 : 85 (no accurate locality); Skuhravá et al. 2017 KEROPLATIDAE K. Kettani, P.J. Chandler Number of species: 2 . Expected: 50 Faunistic knowledge of the family in Morocco: poor Keroplatinae Keroplatus Bosc, 1792 Keroplatus reaumurii (Dufour, 1839) Matile 1986 ; Chandler et al. 2005 ; Evenhuis 2006 Macrocera Meigen, 1803 Macrocera fasciata Meigen, 1804 Becker and Stein 1913 , Rif , Tanger; Chandler and Ribeiro 1995 ; Evenhuis 2006 Keroplatinae Keroplatus Bosc, 1792 Keroplatus reaumurii (Dufour, 1839) Matile 1986 ; Chandler et al. 2005 ; Evenhuis 2006 Macrocera Meigen, 1803 Macrocera fasciata Meigen, 1804 Becker and Stein 1913 , Rif , Tanger; Chandler and Ribeiro 1995 ; Evenhuis 2006 MYCETOPHILIDAE K. Kettani, P.J. Chandler Number of species: 64 . Expected: 250 Faunistic knowledge of the family in Morocco: poor Mycetophilinae Exechiini Allodiopsis Tuomikoski, 1966 Allodiopsis rustica Edwards, 1941 Banamar et al. 2020 , Rif , Dayat Tazia Anatella Winnertz, 1864 Anatella concava Plassmann, 1990 Banamar et al. 2020 , Rif , Oued Aârate Brevicornu Marshall, 1896 Brevicornu intermedium (Santos Abréu, 1920) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Tisgris, Oued Aârate, Dayat Tazia, Oued Maggou (Maggou Village), Douar Tizga, maison forestière de Talassemtane, Dayat Jebel Zemzem, Aïn Takhninjoute, Forêt Adrou, Dayat Amsemlil Brevicornu griseicolle Staeger, 1840 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Aârate, Dayat Tazia, Oued Maggou (Maggou Village), Aïn El Malaâb, maison forestière de Talassemtane, Douar Tizga, Aïn Takhninjoute, Dayat Amsemlil, Dayat Lemtahane, Dayat avant Taida Brevicornu sericoma (Meigen, 1830) Banamar et al. 2020 , Rif , Chefchaouen, Dayat Fifi, Forêt Jebel Lakraâ, Dayat Fifi, Oued Maggou (Maggou Village), maison forestière de Talassemtane, Douar Tizga, Oued Amsemlil, Grotte d'Hercule, Forêt Adrou, Cascade Chrafate, Oued Aârate, Bab el Karn, Dayat Amsemlil – MNHN (coll. J. Beaucournu) Brevicornu verralli (Edwards, 1925) Banamar et al. 2020 , Rif , Dayat Afersiw, Forêt Jebel Lakraâ Cordyla Meigen, 1803 Cordyla crassicornis Meigen, 1818 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Douar Abou Boubnar, maison forestière de Talassemtane, Forêt Adrou, Oued Sidi Yahia Aârab, EM , Oued Tafoughalt; Rif (Chefchaouen) – MNHN Cordyla insons Laštovka & Matile, 1974 Banamar et al. 2020 , Rif , Oued Maggou Cordyla murina Winnertz, 1864 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba Cordyla styliforceps (Bukowski, 1934) Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Oued Tkaraâ, Oued Sidi Yahia Aârab Exechia Winnertz, 1863 Exechia bicincta (Staeger, 1840) Banamar et al. 2020 , Rif , Oued Kelaâ, Oued Aârate, EM , Grotte des Pigeons, Oued Tafoughalt Exechia dorsalis (Staeger, 1840) Banamar et al. 2020 , Rif , Bab el Karn Exechia fulva Santos Abreu, 1920 = Rymosia exornata Séguy, in Séguy 1941a : 26 Séguy 1941a , HA , Toubkal; Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Oued Kelaâ, Forêt Aïn Boughaba, Forêt Jebel Lakraâ, Dayat Fifi, Aïn Sidi Brahim Ben Arrif, Oued Maggou (Maggou Village), Aïn Takhninjoute, maison forestière de Talassemtane, Oued Amsemlil, Dayat Jebel Zemzem, Aïn Takhninjoute, Bab el Karn, Dayat Fifi, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN ; Rif (20 km west of Targuist, coll. A.M. Hutson) – NHMUK Exechia fusca (Meigen, 1804) Banamar et al. 2020 , Rif , Oued Kelaâ, Forêt Jebel Lakraâ, Douar Kitane, Dayat Amsemlil Exechiopsis Tuomikoski, 1966 Exechiopsis coremura (Edwards, 1928) Banamar et al. 2020 , Rif , Cascade Chrafate Pseudexechia Tuomikoski, 1966 Pseudexechia tuomikoskii (Kjærandsen, 2009) Banamar et al. 2020 , Rif , Source Aheramen Rymosia Winnertz, 1864 Rymosia affinis Winnertz, 1864 Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Amsemlil, Aïn Tiouila, Dayat Fifi Rymosia beaucournui Matile, 1963 Chandler 1994 ; Chandler and Ribeiro 1995 ; Chandler et al. 2005 ; Banamar et al. 2020 , EM , Grotte des Pigeons; AP (Oued y Kern, coll. H. Choumara) – MNHN Rymosia pseudocretensis Burghele-Balacesco, 1966 Chandler 1994 ; Chandler et al. 2005 ; Banamar et al. 2020 ; AP (Oued y Kern, coll. H. Choumara) – MNHN Stigmatomeria Tuomikoski, 1966 Stigmatomeria crassicornis (Stannius, 1831) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Dayat Fifi, Aïn Takhninjoute, maison forestière de Talassemtane, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Tarnania Tuomikoski, 1966 Tarnania dziedzickii (Edwards, 1941) Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Amsemlil, Cascade Chrafate, Dayat Amsemlil, Grotte Aïn El-Aouda – MNHN Mycetophilini Mycetophila Meigen, 1804 Mycetophila alea Laffoon, 1965 Banamar et al. 2020 , Rif , Aïn el Ma Bared, maison forestière de Talassemtane, Oued Aârate, Bab el Karn; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila britannica Laštovka & Kidd, 1975 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, Forêt Jebel Lakraâ, Douar Kitane, Aïn El Malaâb, oued à 15 km de Fifi, Aïn El Ma Bared, maison forestière de Talassemtane, Oued Maggou (Maggou Village), Aïn Takhninjoute, Grotte d'Hercule, Oued Aârate, Bab el Karn, Dayat Amsemlil, Dayat Lemtahane, Dayat avant Taida, Forêt Taghzout – MNHN Mycetophila deflexa Chandler, 2001 Banamar et al. 2020 , Rif , Forêt Taghzout, route Ksar el Kebir–Chefchaouen Mycetophila edwardsi Lundström, 1913 Banamar et al. 2020 , Rif , Dayat Fifi, Dayat Tazia, Oued Tkarae, Forêt Jebel Lakraâ, Forêt Taghzout, Grotte d'Hercule, EM , Béni Snassen Mycetophila formosa Lundström, 1911 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Amsemlil, Dayat Amsemlil, Dayat avant Taida Mycetophila marginata Winnertz, 1864 Banamar et al. 2020 , Rif , Aïn Ras el Ma, Oued Maggou (Maggou Village), maison forestière de Talassemtane, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila perpallida Chandler, 1993 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, Aïn El Malaâb, maison forestière de Talassemtane, Bab el Karn, Dayat Amsemlil, Douar Kitane Mycetophila pictula Meigen, 1830 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Maggou (Maggou Village); Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila sordida van der Wulp, 1874 Chandler 1994 ; Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ; Oued Maggou (Maggou Village); MA (Khenolap-el-Ouaer, 1580 m, coll. J. Beaucournu) – MNHN Mycetophila spectabilis Winnertz, 1864 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Forêt Aïn Boughaba, oued à 15 km de Fifi, Aïn El Malaâb Mycetophila strigatoides (Landrock, 1927) Banamar et al. 2020 , Rif , Forêt Taghzout, Oued Aârate Mycetophila unicolor Stannius, 1831 Banamar et al. 2020 , Rif , Oued Kelaâ Mycetophila vittipes Zetterstedt, 1852 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Dayat Amsemlil Phronia Winnertz, 1863 Phronia biarcuata (Becker, 1908) Chandler 1994 ; Chandler and Ribeiro 1995 ; Chandler et al. 2005 ; Banamar et al. 2020 , Rif , Dayat Tazia, Aïn el Ma Bared, Aïn El Malaâb, Aïn Takhninjoute, maison forestière de Talassemtane, Grotte d'Hercule, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Phronia cinerascens Winnertz, 1864 Banamar et al. 2020 , Rif , Dayat Amsemlil Phronia nitidiventris (van der Wulp, 1858) Banamar et al. 2020 , Rif , Dayat Afersiw, Oued Aârate Phronia tenuis Winnertz, 1864 Banamar et al. 2020 , Rif , Oued Kelaâ, Oued Aârate, Oued Maggou (Maggou Village), Aïn Takhninjoute, Grotte d'Hercule Phronia tyrrhenica Edwards, 1928 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Dayat Amsemlil, Aïn Takhninjoute Phronia willistoni Dziedzicki, 1889 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Aârate, maison forestière de Talassemtane, Dayat Amsemlil, Cascade Chrafate, Bab el Karn Sceptonia Winnertz, 1864 Sceptonia intestata Plassmann & Schacht, 1990 Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Aïn El Malaâb Sceptonia membranacea Edwards, 1925 Banamar et al. 2020 , Rif , oued à 15 km de Fifi, Oued Aârate Trichonta Winnertz, 1864 Trichonta foeda Loew, 1869 Banamar et al. 2020 , Rif , Aïn Takhninjoute, Bab el Karn, Dayat Amsemlil, Oued Tkarae Trichonta icenica Edwards, 1925 Banamar et al. 2020 , EM , Grotte du Chameau Trichonta vitta (Meigen, 1830) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Bab el Karn Trichonta vulcani Dziedzicki, 1889 Banamar et al. 2020 , EM , Grotte du Chameau Zygomyia Winnertz, 1864 Zygomyia humeralis (Wiedemann, 1817) Banamar et al. 2020 , Rif , maison forestière de Talassemtane Zygomyia valida Winnertz, 1864 Banamar et al. 2020 , Rif , Aïn Ras el Ma, Oued Aârate Leiinae Docosia Winnertz, 1864 Docosia gilvipes (Walker, 1856) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Aïn Takhninjoute, Dayat Amsemlil Leia Meigen, 1818 Leia arsona Hutson, 1978 Banamar et al. 2020 , Rif , Oued Maggou (Maggou village), Oued Sidi Yahia Aârab, MA , Aïn Walili Leia beckeri Landrock, 1940 Banamar et al. 2020 , Rif , Aïn Ras el Ma Leia bimaculata (Meigen, 1804) Chandler 1994 ; Chandler et al. 2005 ; Banamar et al. 2020 , Rif , Dayat Tazia, maison forestière de Talassemtane, Aïn el Ma Bared, MA , Forêt 3.5 km S Azrou; MA (Forêt Ifrane, coll. P.N. Lawrence) – NHMUK Novakia Strobl, 1893 Novakia scatopsiformis Strobl, 1893 Banamar et al. 2020 , Rif , maison forestière de Talassemtane Novakia simillima Strobl, 1910 Banamar et al. 2020 , Rif , Oued Aârate, maison forestière de Talassemtane Gnoristinae Boletina Staeger, 1840 Boletina gripha Dziedzicki, 1885 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Aârate, Aïn Sidi Brahim Ben Arrif, Dayat Amsemlil, Grotte d'Hercule, Cascade Chrafate, Oued Sidi Yahia Aârab, Aïn el Ma Bared, Bab el Karn, MA , forêt 3.5 km S Azrou Coelosia Winnertz, 1864 Coelosia fusca Bezzi, 1892 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Kelaâ, Oued Amsemlil, Dayat Amsemlil, Aïn Takhninjoute, Cascade Chrafate, Bab el Karn, Dayat avant Taida Synapha Meigen, 1818 Synapha fasciata Meigen, 1818 Banamar et al. 2020 , Rif , Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Forêt Adrou, Dayat Amsemlil, Dayat avant Taida Synapha vitripennis (Meigen, 1818) Banamar et al. 2020 , Rif , Dayat Amsemlil Tetragoneura Winnertz, 1846 Tetragoneura ambigua Grzegorzek, 1885 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, EM , Oued Tafoughalt Mycomyinae Mycomya Rondani, 1856 Mycomya flavicollis (Zetterstedt, 1852) Banamar et al. 2020 , Rif , Aïn El Malaâb, maison forestière de Talassemtane Mycomya pygmalion Väisänen, 1984 Banamar et al. 2020 , Rif , Oued Amsemlil, Aïn Sidi Brahim Ben Arrif Mycomya tumida (Winnertz, 1864) Banamar et al. 2020 , Rif , Dayat Fifi Sciophilinae Azana Walker, 1856 Azana anomala Staeger, 1840 Banamar et al. 2020 , Rif , Oued Maggou (Maggou village), maison forestière de Talassemtane Sciophila Meigen, 1818 Sciophila iberolutea Chandler & Blasco-Zumeta, 2001 Chandler and Gatt 2000 , AP , Oued y Kern; Chandler and Blasco-Zumeta 2001 , AP , Oued y Kern; Bechev and Koç 2006 ; Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Jebel Zemzem, Oued Sidi Yahia Aârab, Bab el Karn, Marabout el Khaloua; AP (Oued y Kern, coll. H. Choumara) – MNHN Mycetophilinae Exechiini Allodiopsis Tuomikoski, 1966 Allodiopsis rustica Edwards, 1941 Banamar et al. 2020 , Rif , Dayat Tazia Anatella Winnertz, 1864 Anatella concava Plassmann, 1990 Banamar et al. 2020 , Rif , Oued Aârate Brevicornu Marshall, 1896 Brevicornu intermedium (Santos Abréu, 1920) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Tisgris, Oued Aârate, Dayat Tazia, Oued Maggou (Maggou Village), Douar Tizga, maison forestière de Talassemtane, Dayat Jebel Zemzem, Aïn Takhninjoute, Forêt Adrou, Dayat Amsemlil Brevicornu griseicolle Staeger, 1840 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Aârate, Dayat Tazia, Oued Maggou (Maggou Village), Aïn El Malaâb, maison forestière de Talassemtane, Douar Tizga, Aïn Takhninjoute, Dayat Amsemlil, Dayat Lemtahane, Dayat avant Taida Brevicornu sericoma (Meigen, 1830) Banamar et al. 2020 , Rif , Chefchaouen, Dayat Fifi, Forêt Jebel Lakraâ, Dayat Fifi, Oued Maggou (Maggou Village), maison forestière de Talassemtane, Douar Tizga, Oued Amsemlil, Grotte d'Hercule, Forêt Adrou, Cascade Chrafate, Oued Aârate, Bab el Karn, Dayat Amsemlil – MNHN (coll. J. Beaucournu) Brevicornu verralli (Edwards, 1925) Banamar et al. 2020 , Rif , Dayat Afersiw, Forêt Jebel Lakraâ Cordyla Meigen, 1803 Cordyla crassicornis Meigen, 1818 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Douar Abou Boubnar, maison forestière de Talassemtane, Forêt Adrou, Oued Sidi Yahia Aârab, EM , Oued Tafoughalt; Rif (Chefchaouen) – MNHN Cordyla insons Laštovka & Matile, 1974 Banamar et al. 2020 , Rif , Oued Maggou Cordyla murina Winnertz, 1864 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba Cordyla styliforceps (Bukowski, 1934) Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Oued Tkaraâ, Oued Sidi Yahia Aârab Exechia Winnertz, 1863 Exechia bicincta (Staeger, 1840) Banamar et al. 2020 , Rif , Oued Kelaâ, Oued Aârate, EM , Grotte des Pigeons, Oued Tafoughalt Exechia dorsalis (Staeger, 1840) Banamar et al. 2020 , Rif , Bab el Karn Exechia fulva Santos Abreu, 1920 = Rymosia exornata Séguy, in Séguy 1941a : 26 Séguy 1941a , HA , Toubkal; Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Oued Kelaâ, Forêt Aïn Boughaba, Forêt Jebel Lakraâ, Dayat Fifi, Aïn Sidi Brahim Ben Arrif, Oued Maggou (Maggou Village), Aïn Takhninjoute, maison forestière de Talassemtane, Oued Amsemlil, Dayat Jebel Zemzem, Aïn Takhninjoute, Bab el Karn, Dayat Fifi, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN ; Rif (20 km west of Targuist, coll. A.M. Hutson) – NHMUK Exechia fusca (Meigen, 1804) Banamar et al. 2020 , Rif , Oued Kelaâ, Forêt Jebel Lakraâ, Douar Kitane, Dayat Amsemlil Exechiopsis Tuomikoski, 1966 Exechiopsis coremura (Edwards, 1928) Banamar et al. 2020 , Rif , Cascade Chrafate Pseudexechia Tuomikoski, 1966 Pseudexechia tuomikoskii (Kjærandsen, 2009) Banamar et al. 2020 , Rif , Source Aheramen Rymosia Winnertz, 1864 Rymosia affinis Winnertz, 1864 Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Amsemlil, Aïn Tiouila, Dayat Fifi Rymosia beaucournui Matile, 1963 Chandler 1994 ; Chandler and Ribeiro 1995 ; Chandler et al. 2005 ; Banamar et al. 2020 , EM , Grotte des Pigeons; AP (Oued y Kern, coll. H. Choumara) – MNHN Rymosia pseudocretensis Burghele-Balacesco, 1966 Chandler 1994 ; Chandler et al. 2005 ; Banamar et al. 2020 ; AP (Oued y Kern, coll. H. Choumara) – MNHN Stigmatomeria Tuomikoski, 1966 Stigmatomeria crassicornis (Stannius, 1831) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Dayat Fifi, Aïn Takhninjoute, maison forestière de Talassemtane, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Tarnania Tuomikoski, 1966 Tarnania dziedzickii (Edwards, 1941) Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Amsemlil, Cascade Chrafate, Dayat Amsemlil, Grotte Aïn El-Aouda – MNHN Mycetophilini Mycetophila Meigen, 1804 Mycetophila alea Laffoon, 1965 Banamar et al. 2020 , Rif , Aïn el Ma Bared, maison forestière de Talassemtane, Oued Aârate, Bab el Karn; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila britannica Laštovka & Kidd, 1975 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, Forêt Jebel Lakraâ, Douar Kitane, Aïn El Malaâb, oued à 15 km de Fifi, Aïn El Ma Bared, maison forestière de Talassemtane, Oued Maggou (Maggou Village), Aïn Takhninjoute, Grotte d'Hercule, Oued Aârate, Bab el Karn, Dayat Amsemlil, Dayat Lemtahane, Dayat avant Taida, Forêt Taghzout – MNHN Mycetophila deflexa Chandler, 2001 Banamar et al. 2020 , Rif , Forêt Taghzout, route Ksar el Kebir–Chefchaouen Mycetophila edwardsi Lundström, 1913 Banamar et al. 2020 , Rif , Dayat Fifi, Dayat Tazia, Oued Tkarae, Forêt Jebel Lakraâ, Forêt Taghzout, Grotte d'Hercule, EM , Béni Snassen Mycetophila formosa Lundström, 1911 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Amsemlil, Dayat Amsemlil, Dayat avant Taida Mycetophila marginata Winnertz, 1864 Banamar et al. 2020 , Rif , Aïn Ras el Ma, Oued Maggou (Maggou Village), maison forestière de Talassemtane, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila perpallida Chandler, 1993 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, Aïn El Malaâb, maison forestière de Talassemtane, Bab el Karn, Dayat Amsemlil, Douar Kitane Mycetophila pictula Meigen, 1830 Chandler and Ribeiro 1995 ; Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Maggou (Maggou Village); Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Mycetophila sordida van der Wulp, 1874 Chandler 1994 ; Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ; Oued Maggou (Maggou Village); MA (Khenolap-el-Ouaer, 1580 m, coll. J. Beaucournu) – MNHN Mycetophila spectabilis Winnertz, 1864 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Forêt Aïn Boughaba, oued à 15 km de Fifi, Aïn El Malaâb Mycetophila strigatoides (Landrock, 1927) Banamar et al. 2020 , Rif , Forêt Taghzout, Oued Aârate Mycetophila unicolor Stannius, 1831 Banamar et al. 2020 , Rif , Oued Kelaâ Mycetophila vittipes Zetterstedt, 1852 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Dayat Amsemlil Phronia Winnertz, 1863 Phronia biarcuata (Becker, 1908) Chandler 1994 ; Chandler and Ribeiro 1995 ; Chandler et al. 2005 ; Banamar et al. 2020 , Rif , Dayat Tazia, Aïn el Ma Bared, Aïn El Malaâb, Aïn Takhninjoute, maison forestière de Talassemtane, Grotte d'Hercule, Dayat Amsemlil; Rif (Chefchaouen, coll. J. Beaucournu) – MNHN Phronia cinerascens Winnertz, 1864 Banamar et al. 2020 , Rif , Dayat Amsemlil Phronia nitidiventris (van der Wulp, 1858) Banamar et al. 2020 , Rif , Dayat Afersiw, Oued Aârate Phronia tenuis Winnertz, 1864 Banamar et al. 2020 , Rif , Oued Kelaâ, Oued Aârate, Oued Maggou (Maggou Village), Aïn Takhninjoute, Grotte d'Hercule Phronia tyrrhenica Edwards, 1928 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Dayat Amsemlil, Aïn Takhninjoute Phronia willistoni Dziedzicki, 1889 Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Oued Aârate, maison forestière de Talassemtane, Dayat Amsemlil, Cascade Chrafate, Bab el Karn Sceptonia Winnertz, 1864 Sceptonia intestata Plassmann & Schacht, 1990 Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Aïn El Malaâb Sceptonia membranacea Edwards, 1925 Banamar et al. 2020 , Rif , oued à 15 km de Fifi, Oued Aârate Trichonta Winnertz, 1864 Trichonta foeda Loew, 1869 Banamar et al. 2020 , Rif , Aïn Takhninjoute, Bab el Karn, Dayat Amsemlil, Oued Tkarae Trichonta icenica Edwards, 1925 Banamar et al. 2020 , EM , Grotte du Chameau Trichonta vitta (Meigen, 1830) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, Bab el Karn Trichonta vulcani Dziedzicki, 1889 Banamar et al. 2020 , EM , Grotte du Chameau Zygomyia Winnertz, 1864 Zygomyia humeralis (Wiedemann, 1817) Banamar et al. 2020 , Rif , maison forestière de Talassemtane Zygomyia valida Winnertz, 1864 Banamar et al. 2020 , Rif , Aïn Ras el Ma, Oued Aârate Leiinae Docosia Winnertz, 1864 Docosia gilvipes (Walker, 1856) Banamar et al. 2020 , Rif , Forêt Jebel Lakraâ, maison forestière de Talassemtane, Aïn Takhninjoute, Dayat Amsemlil Leia Meigen, 1818 Leia arsona Hutson, 1978 Banamar et al. 2020 , Rif , Oued Maggou (Maggou village), Oued Sidi Yahia Aârab, MA , Aïn Walili Leia beckeri Landrock, 1940 Banamar et al. 2020 , Rif , Aïn Ras el Ma Leia bimaculata (Meigen, 1804) Chandler 1994 ; Chandler et al. 2005 ; Banamar et al. 2020 , Rif , Dayat Tazia, maison forestière de Talassemtane, Aïn el Ma Bared, MA , Forêt 3.5 km S Azrou; MA (Forêt Ifrane, coll. P.N. Lawrence) – NHMUK Novakia Strobl, 1893 Novakia scatopsiformis Strobl, 1893 Banamar et al. 2020 , Rif , maison forestière de Talassemtane Novakia simillima Strobl, 1910 Banamar et al. 2020 , Rif , Oued Aârate, maison forestière de Talassemtane Gnoristinae Boletina Staeger, 1840 Boletina gripha Dziedzicki, 1885 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Aârate, Aïn Sidi Brahim Ben Arrif, Dayat Amsemlil, Grotte d'Hercule, Cascade Chrafate, Oued Sidi Yahia Aârab, Aïn el Ma Bared, Bab el Karn, MA , forêt 3.5 km S Azrou Coelosia Winnertz, 1864 Coelosia fusca Bezzi, 1892 Banamar et al. 2020 , Rif , Dayat Fifi, Oued Kelaâ, Oued Amsemlil, Dayat Amsemlil, Aïn Takhninjoute, Cascade Chrafate, Bab el Karn, Dayat avant Taida Synapha Meigen, 1818 Synapha fasciata Meigen, 1818 Banamar et al. 2020 , Rif , Aïn Sidi Brahim Ben Arrif, Dayat Tazia, Forêt Adrou, Dayat Amsemlil, Dayat avant Taida Synapha vitripennis (Meigen, 1818) Banamar et al. 2020 , Rif , Dayat Amsemlil Tetragoneura Winnertz, 1846 Tetragoneura ambigua Grzegorzek, 1885 Banamar et al. 2020 , Rif , Forêt Aïn Boughaba, EM , Oued Tafoughalt Mycomyinae Mycomya Rondani, 1856 Mycomya flavicollis (Zetterstedt, 1852) Banamar et al. 2020 , Rif , Aïn El Malaâb, maison forestière de Talassemtane Mycomya pygmalion Väisänen, 1984 Banamar et al. 2020 , Rif , Oued Amsemlil, Aïn Sidi Brahim Ben Arrif Mycomya tumida (Winnertz, 1864) Banamar et al. 2020 , Rif , Dayat Fifi Sciophilinae Azana Walker, 1856 Azana anomala Staeger, 1840 Banamar et al. 2020 , Rif , Oued Maggou (Maggou village), maison forestière de Talassemtane Sciophila Meigen, 1818 Sciophila iberolutea Chandler & Blasco-Zumeta, 2001 Chandler and Gatt 2000 , AP , Oued y Kern; Chandler and Blasco-Zumeta 2001 , AP , Oued y Kern; Bechev and Koç 2006 ; Banamar et al. 2020 , Rif , maison forestière de Talassemtane, Dayat Jebel Zemzem, Oued Sidi Yahia Aârab, Bab el Karn, Marabout el Khaloua; AP (Oued y Kern, coll. H. Choumara) – MNHN SCIARIDAE K. Kettani, K. Heller Number of species: 70 . Expected: 200–500 Faunistic knowledge of the family in Morocco: poor Austrosciara Schmitz & Mjöberg, 1924 Austrosciara hyalipennis (Meigen, 1804) El Ouazzani et al. 2019 , Rif , Douar El Hamma, MA , Lac Ouiouane Bradysia Winnertz, 1867 Bradysia alpicola (Winnertz, 1867) El Ouazzani et al. 2019 , MA , Lac Ouiouane Bradysia bulbigera Mohrig & Kauschke, 1994 El Ouazzani et al. 2019 , Rif , Oued Ouara, Oued Ametrasse, Merzouk Bni Salah, Dayat Bayn widane, HA , Ouirgane Bradysia cavernicola Mohrig & Eckert, 1999 Menzel and Heller 2004 , HA , Ouirgane Bradysia cinerascens (Grzegorzek, 1884) El Ouazzani et al. 2019 , Rif , Issaguen, Anissar ( PNPB ) Bradysia crinita Mohrig, 1992 El Ouazzani et al. 2019 , Rif , Issaguen, Douar El Hamma Bradysia fenestralis (Zetterstedt, 1838) El Ouazzani et al. 2019 , Rif , Forêt R'milat Bradysia fenestrata (Meigen, 1818) El Ouazzani et al. 2019 , Rif , Jebel Zemzem, Oued Tkarâa, Ben Karrich, Dayat Tazia, Perdicaris Park, Tourbière Amsemlil, Dayat Tazia Bradysia flavipila Tuomikoski, 1960 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia iberiana Rudzinski & Baumjohann, 2009 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia lembkei Mohrig & Menzel, 1990 El Ouazzani et al. 2019 , Rif , Oued Maggou, Dayat Tazia, AP , Forêt Maâmora, HA , Ouirgane, Gerifodene Bradysia lucichaeta Mohrig & Krivosheina, 1989 Mohrig et al. 1997 , AA , Sidi Rbat (40 km S Agadir) Bradysia mediterranea Mohrig & Menzel, 1992 Mohrig and Menzel 1992 , HA Bradysia nigrispina Menzel, 2006 El Ouazzani et al. 2019 , HA , Gerifodene Bradysia pectoralis (Staeger, 1840) El Ouazzani et al. 2019 , MA , Lac Ouiouane, HA , Ouirgane Bradysia placida (Winnertz, 1867) El Ouazzani et al. 2019 , HA , Ouirgane Bradysia promissa Mohrig & Röschmann, 1999 El Ouazzani et al. 2019 , Rif , Beni Barou, Anissar ( PNPB ), Oued Tkarâa ( PNPB ), Taida, Marabout Moulay Abdelsalam Bradysia reflexa Tuomikoski, 1960 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia regularis (Lengersdorf, 1934) El Ouazzani et al. 2019 , Rif , Talassemtane (maison forestière), HA , Ouirgane Bradysia ruginosa Mohrig, 1994 Mohrig et al. 1997 , SA , Ablino (15 km N Goulimine); El Ouazzani et al. 2019 , Rif , Jebel Lakraâ, Aïn El Fakir, HA , Amizmiz Bradysia santorina Mohrig & Menzel, 1992 Mohrig et al. 1997 , AA , Sidi Rbat (40 km S Agadir) Bradysia scabricornis Tuomikoski, 1960 El Ouazzani et al. 2019 , Rif , Oued Ouara, Maggou, Oued Azla, Douar El Hamma, HA , Ouirgane, Setti Fatma, Oued Imlil Bradysia subrufescens Mohrig & Krivosheina, 1989 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia subsantorina Mohrig & Kauschke, 1997 El Ouazzani et al. 2019 , HA , Ouirgane Bradysia tilicola (Loew, 1850) El Ouazzani et al. 2019 , Rif , Douar Tissouka, MA , Lac Ouiouane, HA , Ouirgane Bradysia transitata Rudzinski & Baumjohann, 2013 El Ouazzani et al. 2019 , Rif , Oued Ez-Zarka, Oued Tkarâa ( PNPB ), Oued Laou, AP , Larache (Strawberry farm), Forêt Maâmora, AA , Barrage Aoulouz, Assif Tifnout Bradysia trivittata (Staeger, 1840) Mohrig and Röschmann 1993, HA , AA , Sidi Rbat (40 km S Agadir); El Ouazzani et al. 2019 , Rif , Aïn Tayattine, Oued Ez-Zarka, Oued Bayine, Beni Barou, Douar El Hamma, Douar Mouarâa, Tétouan, MA , Lac Ouiouane, HA , Télouet, Ouirgane Bradysia vagans (Winnertz, 1868) misidentified as Bradysia rufescens (Zetterstedt, 1852) in Röschmann and Mohrig 1993 : 111 Röschmann and Mohrig 1993 , HA , Talouete; El Ouazzani et al. 2019 , Rif , Aïn Fouara, HA , Ouirgane Bradysia xenoreflexa Mohrig & Menzel, 1993 El Ouazzani et al. 2019 , AP , Forêt Maâmora Bradysiopsis Tuomikoski, 1960 Bradysiopsis vittata (Meigen, 1830) El Ouazzani et al. 2019 , HA , Setti Fatma Camptochaeta Hippa & Vilkamaa, 1994 Camptochaeta jeskei (Mohrig & Röschmann, 1993) = Corynoptera jeskei Mohrig and Röschmann, in Röschmann and Mohrig 1993 : 109 Röschmann and Mohrig 1993 , HA , Talouete (1800 m) Corynoptera Winnertz, 1867 Corynoptera andalusica Hippa, Vilkamaa & Heller, 2010 El Ouazzani et al. 2019 , MA , Lac Ouiouane, HA , Ouirgane Corynoptera bicuspidata (Lengersdorf, 1926) Hippa et al. 2010 , HA , Ouirgane, Lac Ouiouane Corynoptera bispinulosa Mohrig & Dimitrova, 1992 El Ouazzani et al. 2019 , EM , Tafoughalt Corynoptera caesula Hippa & Menzel, 2004 El Ouazzani et al. 2019 , Rif , Aïn Kchour, AP , Forêt Maâmora Corynoptera cincinnata Mohrig & Blasco-Zumeta, 1996 Hippa et al. 2010 , HA , Ouirgane Corynoptera dentiforceps (Bukowski & Lengersdorf, 1936) El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera deserta Heller & Menzel, 2006 El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera fatigans (Johannsen, 1912) = Corynoptera perpusilla Winnertz, 1867, in Mohrig et al. 1997 : 384 Mohrig et al. 1997 , HA , Anezal; Hippa et al. 2010 [for nomenclature see Mohrig et al. 2013 ] Corynoptera gemina (Hippa & Vilkamaa, 1994) El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera globiformis (Frey, 1945) El Ouazzani et al. 2019 , Rif , Talassemtane (maison forestière) Corynoptera hemiacantha Mohrig & Mamaev, 1992 El Ouazzani et al. 2019 , HA , Ouirgane Corynoptera iberica Hippa, Vilkamaa & Heller, 2010 El Ouazzani et al. 2019 , AP , Forêt Maâmora, Sidi Boughaba Corynoptera inclinata Hippa, Vilkamaa & Heller, 2010 Hippa et al. 2010 , HA , Ouirgane Corynoptera irmgardis (Lengersdorf, 1930) Hippa et al. 2010 , HA , Ouirgane Corynoptera postglobiformis Mohrig, 1993 El Ouazzani et al. 2019 , Rif , Talassemtane (maison forestière), HA , Ouirgane Corynoptera praeparvula Mohrig & Krivosheina, 1983 El Ouazzani et al. 2019 , HA , Ouirgane, Amizmiz, Gerifodene Corynoptera saccata Tuomikoski, 1960 Hippa et al. 2010 , SA , Goulimine; Mohrig et al. 2012 Corynoptera semipedestris Mohrig & Blasco-Zumeta, 1996 Mohrig et al. 1997 , SA , Ablino (15 km N Goulimine) Corynoptera spiciceps Hippa, Vilkamaa & Heller, 2010 Hippa et al. 2010 , HA , Ouirgane Corynoptera stipidaria Mohrig, 1994 Hippa et al. 2010 , HA , Ouirgane Corynoptera subcavipes Menzel & Smith, 2007 El Ouazzani et al. 2019 , Rif , Douar El Hamma, Talassemtane (maison forestière) Corynoptera subparvula Tuomikoski, 1960 El Ouazzani et al. 2019 , HA , Ouirgane Epidapus Haliday, 1851 Epidapus atomarius (De Geer, 1778) El Ouazzani et al. 2019 , Rif , Aïn el Ma Bared (Fifi) Leptosciarella Tuomikoski, 1960 Leptosciarella dives (Johannsen, 1912) Mohrig et al. 2012 , HA , Ouirgane Leptosciarella parcepilosa (Strobl, 1900) El Ouazzani et al. 2019 , AP , Sidi Boughaba Leptosciarella subviatica Mohrig & Menzel, 1997 El Ouazzani et al. 2019 , HA , Ouirgane Leptosciarella tomentosa (Mohrig & Kauschke, 1994) El Ouazzani et al. 2019 , AP , Forêt Maâmora Lycoriella Frey, 1942 Lycoriella agraria (Felt, 1898) El Ouazzani et al. 2019 , Rif , Douar Tissouka Lycoriella sativae (Johannsen, 1912) El Ouazzani et al. 2019 , Rif , M'Diq, Oued Zaouya, AP , Larache (strawberry farm), HA , Ouirgane Pseudolycoriella Menzel & Mohrig, 1998 Pseudolycoriella morenae (Strobl, 1900) El Ouazzani et al. 2019 , Rif , Perdicaris Park, Bab Tariouant, Oued Maggou, EM , Zegzel, HA , Ouirgane Scatopsciara Edwards, 1927 Scatopsciara ( Scatopsciara ) atomaria (Zetterstedt, 1851) = Scatopsciara vivida (Winnertz, 1867), in Röschmann and Mohrig 1993 : 111 Röschmann and Mohrig 1993 , HA , Talouete; El Ouazzani et al. 2019 , Rif , Oued Ez-Zarka, Oued Tkarâa ( PNPB ), Oued Guallet, Marécage Lemtahane ( PNPB ), Oued Ametrasse, Issaguen, Talassemtane (maison forestière), Douar El Hamma, Aïn Fouara, Oued Souk El Had, Merzouk Bni Salah, Oued Maggou, AP , Sidi Boughaba, HA , Assif Tifnout, Gerifodene, Armed, Amzmiz, Lac Tislit, AA , Barrage Mokhtar Soussi Scatopsciara ( Scatopsciara ) maroccoensis Mohrig & Jaschhof, 1997 Mohrig et al. 1997 , AA , Sidi Rbat (40 km S Agadir) Scatopsiara ( Scatopsciara ) nana (Winnertz, 1871) El Ouazzani et al. 2019 , Rif , Oued Ez-Zarka, Oued Maâmala, Oued Aârkob, Ben Karrich, Merja Sidi Lhaj Merzouk, Tétouan, HA , Ouirgane, Anafgou Scatopsciara ( Scatopsciara ) vitripennis (Meigen, 1818) El Ouazzani et al. 2019 , Rif , Oued Ouarra, Oued Tkarâa ( PNPB ), Oued Aoudour, Oued Ametrasse, Oued Aârkob, Oued Boumarouil, Ben Karrich, Dayat Tazia, Aïn El Fakir, Azib de Khmis Mdik, Merzouk Bni Salah, Oued Souk El Had, Oued Maggou, El Malâab (Talassemtane), AP , Sidi Boughaba, MA , Lac Ouiouane, HA , Ouirgane, AA , Barrage Aoulouz, Assif Tifnout, Barrage Mokhtar Soussi Scatopsciara ( Xenopygina ) curvilinea (Lengersdorf, 1934) El Ouazzani et al. 2019 , AP , Sidi Boughaba, HA , Aïn Taferaout, Amzmiz, AA , Assif Tifnout, Barrage Mokhtar Soussi Scatopsciara ( Xenopygina ) subarmata Mohrig & Mamaev, 1983 El Ouazzani et al. 2019 , Rif , Oued Amsa, AP , Larache, MA , Mont Habri, HA , Ouirgane, AA , Id Aissa, Tissint Schwenckfeldina Frey, 1942 Schwenckfeldina carbonaria (Meigen, 1830) = Sciara carbonaria Meigen, in Séguy 1941d : 2 Séguy 1941d , HA , Tizi-n'Test (2000 m) Sciara Meigen, 1803 Sciara flavimana Zetterstedt, 1851 El Ouazzani et al. 2019 , Rif , Douar El Hamma Sciara hemerobioides (Scopoli, 1763) = Lycoria ( Sciara ) thomae Linnaeus, in Becker and Stein 1913 : 85 Becker and Stein 1913 , Rif , Tanger Suborder BRACHYCERA Stratiomyoidea STRATIOMYIDAE K. Kettani, N. Woodley Number of species: 40 . Expected: 50–60 Faunistic knowledge of the family in Morocco: moderate Beridinae Beris Latreille, 1802 Beris rozkosnyi Kassebeer, 1996 Kassebeer 1996 ; Woodley 2001 , MA , Meknès, Ifrane; Kehlmaier 2004 ; Yimlahi et al. 2017 Chorisops Rondani, 1856 Chorisops tunisiae (Becker, 1915) Haenni 1990 , Rif , Tanger; Woodley 2001 ; Kehlmaier 2004 ; Mason et al. 2006 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 ; Lebard et al. 2020 Clitellariinae Pycnomalla Gerstaecker, 1857 Pycnomalla aterrima Sack, 1912 Séguy 1930a , MA , Tizi-s'Tkrine (1700 m); Séguy 1953a , MA , Dayat Aoua; Woodley 2001 ; Yimlahi et al. 2017 Pycnomalla auriflua (Erichson, 1841) Séguy 1930a , MA , Soufouloud (1900–2100 m), Boulhaut; Duisit 1960 , AP , Cap Cantin; Woodley 2001 Pycnomalla splendens (Fabricius, 1787) Séguy 1930a ; Séguy 1953a , MA , Dayat Aoua; Duisit 1960 , AP , forest of Maâmora, Casablanca, Cap Cantin; Rozkošný 1983, AP , Cap Cantin; Woodley 2001 ; Kehlmaier 2004 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 Nemotelinae Lasiopa Brullé, 1832 Lasiopa benoisti Séguy, 1930 Séguy 1930a , MA , Meknès; Duisit 1960 , EM , Aïn Guettara (Middle Moulouya); Woodley 2001 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 Lasiopa pantherina Séguy, 1930 Séguy 1930a , EM , Maharidja; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus Geoffroy, 1762 Nemotelus ( Camptopelta ) nigrinus Fallén, 1817 Duisit 1960 , AP , Khatouat (S Rabat); Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) atriceps Loew, 1856 Yimlahi et al. 2017 , AA , village Massa Nemotelus ( Nemotelus ) cingulatus Dufour, 1852 Rozkošný 1977 , AP , Skhirat; Rozkošný 1983, Rif , Tanger; Woodley 2001 ; Yimlahi et al. 2017 , Rif , Dayat Afrate, Oued Koub Nemotelus ( Nemotelus ) cylindricornis Rozkošný, 1977 Faucheux 2009 , AP , Oualidia Nemotelus ( Nemotelus ) danielssoni Mason, 1989 Yimlahi et al. 2017 , Rif , Oued Izelfane (Beni Boufrah) Nemotelus ( Nemotelus ) latiusculus Loew, 1871 Lindner 1949 ; Rozkošný 1977 ; Rozkošný 1983, Rif , Tanger; Woodley 2001 ; Kehlmaier 2004 ; Yimlahi et al. 2017 , Rif , Barrage Moulay Bouchta Nemotelus ( Nemotelus ) longirostris (Wiedemann, 1824) Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Duisit 1960 , AP , Mechra-bel-Ksiri (Gharb), EM , Saïdia, Rozkošný 1977 ; Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) maculiventris Bigot, 1861 Yimlahi et al. 2017 , Rif , Oued Zandoula Nemotelus ( Nemotelus ) nigrifrons Loew, 1846 Linder 1936; Becker and Stein 1912 , Rif , Tanger; Rozkošný 1977 , Rif , Tanger; Woodley 2001 ; Yimlahi et al. 2017 , Rif , affluent Tarmast ( NPH ) Nemotelus ( Nemotelus ) pantherinus (Linnaeus, 1758) Séguy 1930a , Rif , Tanger; Duisit 1960 , AP , Zëar; Rozkošný 1977 ; Dakki 1997 ; Woodley 2001 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) proboscideus Loew, 1846 Linder 1936; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) subuliginosus Rozkošný, 1974 Rozkošný 1977 ; Woodley 2001 , Rif , Tanger; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) ventralis Meigen, 1830 Woodley 2001 , AP , Essaouira; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) uliginosus (Linnaeus, 1767) Duisit 1960 , AP , Dradek Pachygastrinae Pachygaster Meigen, 1803 Pachygaster atra Panzer, 1798 Yimlahi et al. 2017 , Rif , Dayat Mezine Pachygaster maura Lindner, 1939 Lindner 1939 ; Woodley 2001 , MA , Tagzirt; Yimlahi et al. 2017 Sarginae Chloromyia Duncan, 1837 Chloromyia formosa (Scopoli, 1763) Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , HA , M'Rassine; Duisit 1960 , AP , Rabat, Korifla, Khatouat; Woodley 2001 ; Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 ; Yimlahi et al. 2017 , Rif , Taghbalout, Lac Ametrasse, Douar Kitane Stratiomyinae Oxycerini Oxycera Meigen, 1803 Oxycera germanica (Szilády, 1932) = Hermione dorieri var. barbarica , in Vaillant 1956b : 232, 237, 242 Vaillant 1956b , HA , Lac Tamhda (Anremer), Tahanaout, Sidi Chamarouch, Aguelmous Oxycera ochracea (Vaillant, 1950) = Hermione ochracea Vaillant, in Vaillant 1956b : 237, 244 Vaillant 1956b , HA , Lac Tamhda (Anremer), Imi-N'Ifri Oxycera pardalina (Meigen, 1822) Yimlahi et al. 2017 , Rif , Oued Abou Bnar ( NPT ), Oued Maggou, Oued Achekrade, Ruisseau maison forestière ( NPT ), MA , Cascade Aïn Vittel, Mchacha Aïn Vittel Oxycera rara (Scopoli, 1763) = Hermione pulchella var. similis Vaillant, in Vaillant 1956b : 242 Vaillant 1956b , HA , Sidi Chamarouch, Imi-N'Ifri Oxycera terminata Meigen, 1822 Yimlahi et al. 2017 , Rif , Cascade Chrafate Oxycera torrentium (Vaillant, 1950) = Hermione torrentium Vaillant, in Vaillant 1956b : 240, 241 Vaillant 1956b , HA , Izourar Oxycera trilineata (Linnaeus, 1767) = Hermione bucheti Séguy, in Séguy 1939: 62 = Hermione trilineata var. algira Vaillant, in Vaillant 1956b : 244 Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a ; Villeneuve 1933 ; Vaillant 1956b , HA , Imi-N'Ifri; Woodley 2001 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 , Rif , Dayat Aïn Jdioui Vanoyia Villeneuve, 1908 Vanoyia tenuicornis (Macquart, 1834) Lindner 1936 ; Woodley 2001 , Rif , Tanger; Yimlahi et al. 2017 Stratiomyini Odontomyia Meigen, 1803 Odontomyia alolena (Séguy, 1930) = Eulalia alolena Séguy, in Séguy 1930a : 65 Séguy 1930a , Rif , Tanger, EM , Maharidja, AP , Casablanca, MA , Aïn Leuh (1400–1500 m); Dakki 1997 ; Woodley 2001 , Rif , Tanger, EM , Maharidja, AP , Casablanca, MA , Aïn Leuh; Yimlahi et al. 2017 Odontomyia angulata (Panzer, 1798) Becker and Stein 1912 , 1913 , Rif , Tanger; Woodley 2001 ; Koçak and Kemal 2010 ; Mohammadi and Khaghaninia 2015 ; Yimlahi et al. 2017 Odontomyia discolor (Loew, 1846) Becker and Stein 1912 , 1913 , Rif , Tanger; Rozkošný 1982 , Rif , Tanger; Woodley 2001 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 Odontomyia flavissima (Rossi, 1790) = Hadracantha flavissina nigripes Pleske, in Pleske 1925: 27, 32; Séguy 1930a : 66 = Eulalia flavissima Rossi, in Duisit 1960 : 121 Séguy 1926 a; Séguy 1930a ; Duisit 1960 , MA , Boulhaut; Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Odontomyia limbata (Wiedemann, 1822) = Eulalia limbata Wiedemann, in Séguy 1930a : 65 Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, MA , Meskedell (1800–1900 m); Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 , Rif , Lac Ametrasse, Aïn Sidi Brahim Ben Arrif, Dayat Afrate, ruisseau mai­son forestière ( NPT ), Aïn El Malaâb ( NPT ), Dayat Rmali El Malaâb ( NPT ), Dayat Tazia; Rif (Tahaddart) – MISR Odontomyia microcera (Séguy, 1930) = Eulalia microcera Séguy, in Séguy 1930a : 65 Séguy 1930a ; Dakki 1997 ; Woodley 2001 , MA , Meknès (550 m); Yimlahi et al. 2017 Stratiomys Geoffroy, 1762 Stratiomys cenisia Meigen, 1822 Séguy 1930a , Rif , Tanger, AP , Rabat, Sidi Bettache, MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m), Meknès, Aïn Sferguila, HA , Timhadit; Duisit 1960 , AP , Maâmora, MA , Arhbala (1700 m); Dakki 1997 ; Woodley 2001 ; Koçak and Kemal 2010 ; Mohammadi and Khaghaninia 2015 ; Yimlahi et al. 2017 Stratiomys longicornis (Scopoli, 1763) = Stratiomys ( Hirtea ) anubis Wiedemann, in Séguy 1930a : 63 Séguy 1930a , EM , Itzer (Haute Moulouya), AP , Chellah (Rabat), Casablanca, MA , Ras el Ksar (1900 m), HA , Marrakech; Duisit 1960 , AP , Rabat, MA , Aguelmane Azigza (1800 m); Dakki 1997 ; Woodley 2001 ; Barták and Kubik 2005 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 Tabanoidea ATHERICIDAE K. Kettani, M. Mouna Number of species: 2 . Expected: 2 Faunistic knowledge of the family in Morocco: good Athericinae Atherix Meigen, 1803 Atherix amicorum (Thomas, 1985) = Ibisia amicorum Thomas, in Thomas 1985 : 89 Thomas 1985 , HA , Oued Réghaya near Marabout Sidi Chamarouch (Toubkal, 2310 m); Boumezzough and Thomas 1987 Atherix maroccana (Séguy, 1930) = Ibisia maroccana Séguy, in Thomas et al. 1995 : 64 Séguy 1930a , MA , Oued Tigrigra; Thomas et al. 1995 , MA , Oued Tigrigra (900 m), Timahdit (1830 m), HA , Asif Aït Bou Guemmaz (1900 m); Dakki 1997 RHAGIONIDAE K. Kettani, M.J. Ebejer Number of species: 4 . Expected: 7 Faunistic knowledge of the family in Morocco: poor Rhagioninae Chrysopilus Macquart, 1826 Chrysopilus asiliformis (Preyssler, 1791) = Chrysopilus aureus (Meigen, 1804), in Séguy 1941a : 29; Dakki 1997 : 62 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Dakki 1997 Chrysopilus pullus Loew, 1869 Ebejer et al. 2019 , Rif , Jebel Lakraâ ( NPT , 1377–1541 m), Adrou ( PNPB , 556 m) Chrysopilus splendidus (Meigen, 1820) Ebejer et al. 2019 , Rif , Oued Kbir (Béni Ratene, 157 m) Chrysopilus tsacasi Thomas, 1979 Thomas 1979 , HA , Jebel Toubkal (Tachdirt, 2500 m); Boumezzough and Thomas 1987 , HA , Oued Réghaya (Imlil, 1750 m), l'azib Oukaimeden (2730 m); Dakki 1997 ; Kerr 2004 TABANIDAE K. Kettani Number of species: 69 . Expected: 75 Faunistic knowledge of the family in Morocco: good Chrysopsinae Chrysopsini Chrysops Meigen, 1803 Chrysops caecutiens Linnaeus, 1758 El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Samsa, Oued Laou (Afertane), Oued Jnane Niche, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart Chrysops connexus Loew, 1858 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Timhadit, Oued Yquem, Volubilis, Kenitra, Meknès, Rif , Tanger; Leclercq 1967 ; Leclercq and Maldès 1987 ; Chvála et al. 1972 ; SA (Guelmim) – MISR Chrysops flavipes Meigen, 1804 = Heterochrysops perspicillaris Loew, in Séguy 1930a : 79 = Chrysops punctifer Loew, in Séguy 1930a , 79 Séguy 1930a , AP , Mogador, EM , Haute Moulouya, MA , Fès, Volubilis, AA , Taroudant; Leclercq 1967 , AA , Agadir-Tissint (Rocade du Draa); Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Kiliç 1999 ; Müller et al. 2012 Chrysops italicus Meigen, 1804 Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Müller et al. 2012 Chrysops mauritanicus Costa, 1893 Séguy 1930a , AP , Rabat, Fedhala, Larache, MA , Itzer, HA , Haute Réghaya; Leclercq 1967 , AP , Rabat (salt marshes on Salicornia ); Chvála et al. 1972 ; Leclercq and Maldès 1987 ; AP (Kénitra) – MISR Chrysops pallidiventris Kröber, 1922 Séguy 1930a , AP , Mogador, MA , Fès; Leclercq 1967 ; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Pape and Thompson 2019 Chrysops relictus Meigen, 1820 Chvála et al. 1972 ; El Haouari and Kettani 2014 , Rif , Oued Kbir (Tamuda), Oued Kelaâ (Talembote), Oued Bou Ahmed, Oued Jnane Niche, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès, Setti Fatma, Tafza Chrysops viduatus (Fabricius, 1794) El Haouari et al. 2014 , HA , Setti Fatma Silvius Meigen, 1920 Silvius algirus Meigen, 1830 Séguy 1930a ; Leclercq and Maldès 1987 ; Kiliç 1999 Silvius alpinus (Scopoli, 1763) = Silvius vituli Fabricius, 1805, in Séguy 1930a : 78 Séguy 1930a , MA , Meknès, Forêt Zaers; Leclercq and Maldès 1987 , AP , Rabat, EM , Béni Snassen, Haute Moulouya, MA , Aïn Leuh Silvius variegatus (Fabricius, 1805) = Diachlorus maroccanus Bigot, in Surcouf 1921 : 143; Séguy 1930a : 78 Surcouf 1921 , Rif , Tanger; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Rabat, EM , Haute Moulouya; Leclercq 1960 , Rif , Tanger, AP , Larache, Rabat, Salé EM , Haute Moulouya; Leclercq 1967 ; Leclercq and Maldès 1987 ; Koçak and Kemal 2013a ; AP (Rabat, Larache) – MISR Pangoniinae Pangoniini Pangonius Latreille, 1802 Pangonius alluaudi Séguy, 1930 Séguy 1930a , MA , Azrou, Aïn Leuh, Timhadit, Tasrah des Ighrezrane, Talzent, Aharmoumou; Leclercq 1967 , MA , Ifrane; Leclercq and Maldès 1987 ; MA – MISR Pangonius brevicornis (Kröber, 1921) Leclercq 1967 ; Leclercq and Maldès 1987 Pangonius hassani (Leclercq, 1968) Leclercq 1968 , MA , Ifrane, Dayat Aoua; Leclercq and Olsufjev 1975 ; Leclercq and Maldès 1987 , MA , Sidi Allal El Bahraoui Pangonius haustellatus (Fabricius, 1781) = Pangonius marginata (Fabricius, 1805), in Séguy 1930a : 74 = Pangonius aterrima Dufour 1853, in Séguy 1930a : 74 = Pangonius funebris Macquart, 1846, in Séguy 1930a : 74 Séguy 1930a , MA , Volubilis, Tizi-s'Tkrine, Aïn Leuh, Azrou, HA , Asni; Leclercq 1961b , MA , Ifrane; Leclercq 1967 , 1968 ; Chvála and Lyneborg 1970 ; Leclercq and Maldès 1987 , MA , Sidi Allal El Bahraoui (forest of Quercus suber of Maâmora); Müller et al. 2012 ; AP (Dradek near Rabat, Kénitra), MA (wide distribution between Azrou and Ras el Ma), HA – MISR Pangonius mauritanus (Linnaeus, 1767) = Pangonius funebris Fabricius, 1794, in Séguy 1930a : 76 = Pangonius maculatus (Fabricius), in Séguy 1953a : 78 Séguy 1930a ; Séguy 1953a , AP , Cap Ghir; Séguy 1949a , AA , Guelmim; Leclercq 1967 ; Leclercq and Maldès 1987 , MA , Maamar (800 m); AP (Dradek, El Maazi, Mazagan) – MISR Pangonius micans Meigen, 1820 Leclercq and Maldès 1987 Pangonius powelli Séguy, 1930 12 = Pangonius sobradieli Séguy, 1934e: 21 Séguy 1930a , MA , Bekrit, Tizi-s'Tkrine, Soufouloud; Séguy 1934e ; Séguy 1949a , AA , Guelmim; Leclercq and Maldès 1987 Pangonius raclinae Leclercq, 1960 Leclercq 1960 , HA , Tifni by Demnate; Leclercq 1967 ; Leclercq and Maldès 1987 ; Bisby et al. 2011 Philolichini Ectinocerella Séguy, 1929 Ectinocerella surcoufi Séguy, 1929 = Pangonius ectinocerella surcoufi Séguy, in Séguy 1929a : 100 Séguy 1929a , MA , Azrou, Ank El Djemel AA , Agadir; Séguy 1930a , MA , Azrou, AA , Agadir; Leclercq and Maldès 1987 , MA , From Meknès to Khemisset, near Beth river; Leclercq 1967 ; HA (Tifni) – MISR Tabaninae Diachlorini Dasybasis Macquart, 1847 Dasybasis barbata Coscaron & Philip, 1967 = Surcoufia barbata Bigot, 1892, in Séguy 1930a : 78 = Surcoufia paradoxa Kröber, 1925, in Séguy 1930a : 78 Séguy 1930a , Rif , Tanger Dasyrhamphis Enderlein, 1922 Dasyrhamphis algirus (Macquart, 1838) = Atylotus algirus Auct, in Séguy 1930a : 82 Séguy 1930a , AP , Dradek, EM , Oujda, HA , Talouet Glaoua; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; AP (Sibara) – MISR Dasyrhamphis anthracinus (Meigen, 1820) = Atylotus anthracinus Surcouf, 1924, in Séguy 1930a : 82 Séguy 1930a , AP , Rabat, Sidi Bettache, MA , M'Rirt, Aïn Sferguila, Volubilis Dasyrhamphis ater (Rossi, 1790) = Tabanus ater (Rossi, 1790), in Becker and Stein 1913 : 77 = Atylotus ater Barotte, 1926, in Séguy 1930a : 82 = Dasyrhamphis ater (Rossi, 1790), in Leclercq and Maldès 1987 : 80 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA ; Leclercq 1967 , MA , Ifrane; Leclercq and Maldès 1987 , HA , Jebel Tazzeka, Bab Ahzar (1200 m), Idni (1700 m); MA – MISR Dasyrhamphis tomentosus (Macquart, 1846) = Atylotus tomentosus Macquart, in Séguy 1930a : 84 Séguy 1930a , AP , Rabat, Oued Cherrat, MA , Azrou, El Hajeb, Meknès, Aïn Leuh, Tizi-S'Tkrine, Forêt Tiffert, Talzent, Tazarine, Meskedall; Séguy 1949a , AA , Guelmim; Leclercq and Maldès 1987 Dasyrhamphis villosus (Macquart, 1838) = Atylotus villosus Macquart, 1838, in Séguy 1930a : 84 Séguy 1930a , MA , Tameghilt; Leclercq 1967 ; Leclercq and Maldès 1987 ; MA – MISR Dasyrhamphis nigritus (Fabricius, 1794) = Therioplectes alexandrinus Wiedemann, 1830, in Séguy 1930a : 83 Séguy 1930a , MA , Aïn Leuh, El Hajeb, M'Rirt, Dar M'Tougui, Dar Kaid M'Tougui, EM , Oujda; Leclercq and Maldès 1987 Haematopotini Haematopota Meigen, 1803 Haematopota algira Kröber, 1922 Séguy 1930a ; Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 ; Leclercq 1968 , MA , Bab Ferrich, Dayat Aoua; Leclercq and Maldès 1987 Haematopota benoisti Séguy, 1930 Séguy 1930a , AP , Rabat, MA , M'Rirt; Leclercq 1967 ; Leclercq and Maldès 1987 ; Pape and Thompson 2019 Haematopota bigoti Gobert, 1880 Séguy 1930a , MA , Volubilis; Séguy 1926 a; Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 ; MA (Ifrane) – MISR Haematopota crassicornis Wahlberg, 1848 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a Haematopota fuscicornis Becker, 1914 = Chrysozona fuscicornis Povolny, in Becker and Stein 1913 : 78 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ( sic! fusicornis ), MA , Fès; Chvála et al. 1972 ; Leclercq and Maldès 1987 Haematopota grandis Meigen, 1820 Leclercq 1967 , AP , Kénitra; Chvála et al. 1972 ; Leclercq and Maldès 1987 Haematopota italica Meigen, 1804 = Haematopota tenuicornis Macquart, 1834, in Séguy 1930a : 81 = Haematopota longicornis Macquart, 1834, in Séguy 1930a : 81 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ; Chvála et al. 1972 Haematopota lambi Villeneuve, 1921 Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 , 1968 ; Leclercq and Maldès 1987 Haematopota ocelligera (Kröber, 1922) Leclercq 1961b , MA , Dayat Aoua (on a horse); Leclercq 1967 , AP , Sidi Yahia du Gharb (on Juncus acutus ); Leclercq 1968 , MA , Azrou; Leclercq and Maldès 1987 Haematopota pluvialis (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Leclercq and Maldès 1987 ; El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Smir, Oued Kbir (Tamuda), Oued Moukhlata (Boujdad), Oued Azla (Mokdassen Oulya), Oued Moulay Bouchta, Oued Jnane Niche, Oued Koudiat Shiba; El Haouari et al. 2014 , HA , Oulmès, Tafza Haematopota pseudolusitanica Szilády, 1923 = Chrysozona lusitanica Guérin, 1835, in Séguy 1930a : 81 Séguy 1930a , MA , M'Rirt, Sebou; Leclercq 1967 , MA , Sebou Haematopota subcylindrica Pandellé, 1888 Leclercq 1967 , AP , Sidi Yahia du Gharb; El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Boumarouil, Oued Jnane Niche; El Haouari et al. 2014 , HA , Tafza Heptatoma Meigen, 1803 Heptatoma pellucens (Fabricuis, 1779) El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Achiar (Bounezzal), Oued Azla (Mokdassen Oulya), Oued Azla (Mokdassen soufla), Oued Imsa (Centre Imsa), bog of Amsemlil, Oued Ouara (Khizana), Oued Boumarouil, Oued Laou (Siflaou), Oued Talembote, Oued Laou (Afertane), Oued Tizharine, Oued Bouhya (Kanar), Bab Tariouant, Oued Taysra (Ketama), Oued Srâ (Ketama); El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès Tabanini Atylotus Osten-Sacken, 1876 Atylotus agrestis (Wiedemann, 1828) Ovazza et al. 1968 ; Pape and Thompson 2019 Atylotus agricola Wiedemann, 1828 = Tabanus agricola var. Kröberi Surcouf, in Séguy 1953a : 78 Séguy 1953a , SA , entre Tagounit et Zegdou Atylotus fulvus (Meigen, 1804) Leclercq 1961b , MA , Aïn Leuh, Bordj Doumergue; Leclercq 1967 , Rif , Ketama; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Barták and Kubik 2005 Atylotus latistriatus (Brauer, 1880) = Dasystipia nigrifacies Gobert, 1881, in Séguy 1930a : 84 Séguy 1930a , MA , Aïn Leuh; Chvála et al. 1972 ; Kiliç 1999 Atylotus loewianus (Villeneuve, 1920) Leclercq 1967 , MA , Aguelmane Azigza (marshy meadow), Aguelmane de Sidi Ali Leclercq 1968 ; Chvála et al. 1972 Atylotus pulchellus Loew, 1858 Becker and Stein 1913 Rif , Tanger; Chvála et al. 1972 Atylotus quadrifarius (Loew, 1874) Chvála et al. 1972 ; Müller et al. 2011 Atylotus sublunaticornis (Zetterstedt, 1842) El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Kbir (Koudiat Krikra), Oued Martil, Oued Khizana, Oued Laou (Ifansa), Oued Bou Ahmed, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès Hybomitra Enderlein, 1922 Hybomitra arpadi Szilády 1923 El Haouari et al. 2014 , HA , Oulmès Hybomitra bimaculata Macquart, 1826 Ježek 1995; El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Lemtahane ( PNPB ), Oued Raouz, Oued Zarka, Oued Mokhlata (Boujdad), Oued Amsa (Er-Rifiyine), bog of Amsemlil, Oued Talembote (Talembote), Oued Jnane Niche, Oued Biyada, Oued Aârkob, Oued Sidi Yahia Aârab; El Haouari et al. 2014 , HA , Oulmès Hybomitra distinguenda (Verrall, 1909) Ježek et al. 2012 ; El Haouari et al. 2014 , HA , Imi-n'Tadart Hybomitra vittata (Fabricius, 1794) = Straba vittata Fabricius, 1794, in Séguy 1930a : 83 = Tabanus spectabilis Loew, 1858, in Séguy 1930a : 83 Séguy 1930a , Rif , Tanger AP , Maâmora, Rabat, Casablanca, MA , Oued Yquem, M'Rirt; Chvála and Lyneborg 1970 , Rif , Tanger, EM , Haute Moulouya; Leclercq and Maldès 1987 Tabanus Linnaeus, 1758 Tabanus autumnalis Linnaeus, 1761 = Straba autumnalis Linnaeus, 1761, in Séguy 1930a : 82 = Straba automnalis var. brunnescens Szilády, 1941, in Séguy 1930a : 82 = Straba automnalis var. molestans Becker, 1914, in Séguy 1930a : 83 Loew 1860 ; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat, EM , Béni Snassen, Itzer (Haute Moulouya), MA , Aïn Leuh; Séguy 1953a , HA , Ksar-es-Souk; Leclercq 1961b , MA , Dayat Aoua, Aïn Leuh, Immouzer Kander; Leclercq 1967 , AP , Kénitra (on Mimosa grove), Gharb (Sidi Yahia, Sidi Allal Tazi), MA , Adjir by Khenifra (on livestock), Leclercq 1968 , MA , Dayat Aoua; Leclercq and Maldès 1987 , HA , edges near river Tessaout, Kelaâ des Sraghna; Pârvu et al. 2006 , AP , Merja Zerga, MA , Kasba Tadla; El Haouari et al. 2014 , HA , Setti Fatma; MA (Allal Tazi) – MISR Tabanus barbarus Coquebert, 1804 Becker and Stein 1913 , Rif , Tanger; Leclercq 1961b Rif , Azib de Ketama; Chvála and Lyneborg 1970 , Rif , Tanger; Portillo 1982; Leclercq and Maldès 1987 , MA , marshes around Kasba Tadla; Popescu-Mirceni 2011 , AP , Merja Zerga Tabanus bifarius Loew, 1858 = Atylotus bifarius Loew, 1858, in Séguy 1930a : 83 Séguy 1930a , EM , Haute Moulouya Tabanus bovinus Linnaeus, 1758 Loew 1860 ; Séguy 1930a ; Leclercq 1967 ; Portillo 1982; Leclercq and Maldès 1987 ; Barták and Kubik 2005 , MA , M'Rirt; El Haouari and Kettani 2014 , Rif , Oued Khemis (Khemis Anjra), Oued Kelaâ (Akchour) Tabanus bromius Linnaeus, 1758 = Straba bromius Linnaeus, 1758, in Séguy 1930a : 83 Loew 1860 ; Séguy 1930a , EM , Haute Moulouya, MA , Aïn Leuh, Ras El Ksar; Leclercq 1961b , Rif , Azib de Ketama, MA , Ifrane, Immouzer, Azrou, Bordj Doumergue, Dayat Aoua, Aïn Leuh; Leclercq 1967 , MA , Immouzer des Marmoucha, Adjir by Khenifra; Leclercq 1968 , Rif , Ketama, MA , Bab-Bou-Idir, Bab Ferrich, Ifrane, Col du Zad; Pernot-Visentin and Beaucournu-Saguez 1974 , Rif ; Leclercq and Maldès 1987 ; El Haouari and Kettani 2014 , Rif , Oued Samsa, Oued Raouz, Oued Zarka, Oued Khemis (Khemis Anjra), Oued Azla (Mokdassen Oulya), Oued Kelaâ (Akchour), Oued Laou (Sifalaou), Oued Jnane Niche, Oued Berranda; El Haouari et al. 2014 , HA , Oulmès; AA (Errachidia) – MISR Tabanus choumarae Leclercq, 1967 Leclercq 1967 , AA , Aouinet Torkoz (down Draa); Leclercq and Olsufjev 1975 ; Leclercq and Maldès 1987 ; Pârvu et al. 2006 , AA , Ouarzazate Tabanus cordiger Meigen, 1820 Leclercq 1961b , MA , Bordj Doumergue, Dayat Aoua; Leclercq 1967 , Rif , Had el Rouadi, MA , Arbala par Ksiba, Boulemane, AA , Agadir-Tissint (Rocade du Draa), Akka; Leclercq 1968 , Rif , Ketama, MA , Bab Termas, Taza, Doniet; Leclercq 1986 ; Leclercq and Maldès 1987 , AA , Tinmal, edges of Draa river, Agdz, Ourika near Ouarzazate; Barták and Kubik 2005 ; Müller et al. 2011 ; Popescu-Mirceni 2011 , HA , Ouarzazate; El Haouari and Kettani 2014 , Rif , Oued Berranda; El Haouari et al. 2014 , Rif , Oued Oueghra Tabanus darimonti Leclercq, 1964 Leclercq 1967 , MA , Aïn Leuh; Leclercq 1968 , MA , Bab Boudir, forêt Bab-Azhar, Immouzer Kander; Portillo 1982; Leclercq and Maldès 1987 ; Mikuška et al. 2008 Tabanus eggeri Schiner, 1868 = Tabanus intermedius Egger, 1859, in Séguy 1930a : 83 Séguy 1930a , MA , M'Rirt; Leclercq 1968 , Rif , M'Diq, MA , Bab Boudir, El Hajeb, Azrou, Miscliffen; Portillo 1982; Leclercq and Maldès 1987 ; Mikuška et al. 2008 Tabanus leleani Austen, 1920 = Atylotus leleani Austen, 1920, in Séguy 1930a : 84 Séguy 1930a , HA , upstream of Réghaya; Leclercq 1967 ; Leclercq and Maldès 1987 ; MA – MISR Tabanus lunatus Fabricius, 1794 = Atylotus lunatus Fabricius, 1974, in Séguy 1930a : 84 Séguy 1930a , EM , Haute Moulouya, MA , Meknès; Leclercq 1961b , MA , Ifrane, Immouzer, Bordj Doumergue, Dayat Aoua, Aïn Leuh; Leclercq 1967 , EM , Haute Moulouya, MA , Ajdir by Khenifra (on livestock); Leclercq 1968 , Rif , Ketama, MA , Bab Bouder, Immouzer Kander, El Hajeb, Mishliffen, Jebel Hebri; Portillo 1982; Leclercq and Maldès 1987 , MA , Afourer (800 m) – MISR Tabanus maculicornis Zetterstedt, 1842 El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Lemtahane, Oued Boumarouil, Oued Laou (Dardara), Oued Kanar, Oued Jnane Niche; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès, Tafza Tabanus miki Brauer, 1880 El Haouari and Kettani 2014 , Rif , Oued Khemis, Oued Boumarouil, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Setti Fatma Tabanus nemoralis Meigen, 1820 Leclercq 1968 , Rif , Ketama; Portillo 1982; Leclercq and Maldès 1987 Tabanus quatuornotatus Meigen, 1820 Portillo 1982; Leclercq and Maldès 1987 ; Koçak and Kemal 2010 ; Müller et al. 2011 ; El Haouari and Kettani 2014 , Rif , Oued Maggou, Oued Ouara (Khizana); El Haouari et al. 2014 , HA , Setti Fatma; Rif (Talassemtane) – MISR Tabanus sudeticus Zeller, 1842 Leclercq 1967 ; Portillo 1982; Leclercq and Maldès 1987 ; Gioia Martins-Neto 2003 Tabanus tinctus Walker, 1850 Leclercq 1961b , MA , forêt Bab Azhar, Aïn Leuh; Pernot-Visentin and Beaucournu-Saguez 1974 , MA , HA (2300 m); Portillo 1982; Leclercq and Maldès 1987 ; MA (Azrou) – MISR VERMILEONIDAE K. Kettani, M.J. Ebejer Number of species: 6 . Expected: 6 Faunistic knowledge of the family in Morocco: good Vermileoninae Lampromyia Macquart, 1835 Lampromyia cylindrica (Fabricius, 1794) Stuckenberg 1998 , HA Lampromyia lecerfi Séguy, 1928 = Lampromyia Le Cerfi Séguy, in Séguy 1928a : 45 Séguy 1928a , HA , Tinmel (Goundafa), Asni; Stuckenberg 1998 , HA ; Kirk-Spriggs and McGregor 2009 ; Kehlmaier 2014 , HA ; AP (Tamri) – MHNN Lampromyia nigripennis Séguy, 1930 Séguy 1930a , EM , Berkane, grove in Tlet n'Rhohr; Stuckenberg 1998 , MA ; Kirk-Spriggs and McGregor 2009 ; Kehlmaier 2014 , MA , south of Azrou (1500 m), HA Lampromyia pallida Macquart, 1835 Stuckenberg 1998 , HA Vermileo Macquart, 1834 Vermileo vermileo (Linnaeus, 1758) 13 = Vermileo degeeri Macquart 1834, in Séguy 1953a : 78 Séguy 1953a , AP , Rabat Vermileo nigriventris Strobl, 1906 Ebejer et al. 2019 , Rif , Cap Spartel (Tanger, 15 m), Anissar ( PNPB , 987 m) Nemestrinoidea ACROCERIDAE K. Kettani, E.P. Nartshuk Number of species: 13 . Expected: 25 Faunistic knowledge of the family in Morocco: poor Acrocerinae Acrocera Meigen, 1803 Acrocera orbicula (Fabricius, 1787) Weinberg and Bächli 2002 , HA , Marrakech, Ouirgane (1000 m) Cyrtus Latreille, 1796 Cyrtus gibbus (Fabricius, 1794) Becker and Stein 1913 , Rif , Tanger; Séguy 1926 , Rif ; Pleske 1930 , Rif , Tanger; Schlinger 1972 ; Mouna 1998 Cyrtus maroccanus Séguy, 1930 Schlinger 1972 , Rif ; Mouna 1998 Cyrtus pallidus Gil Collado, 1929 Gil Collado 1929b , Rif , Tanger; Schlinger 1972 ; Mouna 1998 Cyrtus pusillus Macquart, 1834 Pleske 1930 , Rif , Tanger; Schlinger 1972 , Rif , Tanger; Mouna 1998 Ogcodes Latreille, 1797 Ogcodes zonatus (Erichson, 1840) Becker and Stein 1913 , Rif , Tanger; Séguy 1926 ; Pleske 1930 Opsebius Costa, 1856 Opsebius cyrtus Séguy, 1930 Mouna 1998 ; HA (Lac Ifni) – MISR Opsebius formosus Loew, 1871 Becker and Stein 1913 , Rif , Tanger Opsebius inclinatus Séguy, 1930 Mouna 1998 Opsebius inflatus (Loew, 1857) Weinberg and Bächli 2002 , MA , Azrou, Timahdit Ighboula (1850 m), HA , Marrakech, Ouirgane (1000 m) Opsebius pepo Loew, 1870 Pleske 1930 , Rif , Tanger Panopinae Astomella Latreille, 1809 Astomella hispaniae Lamarck, 1816 Gil Collado 1929b , AP , Mogador; Mouna 1998 Physegastrella Brunetti, 1926 Physegastrella maroccana Brunetti, 1926 Brunetti 1926 ; Pleske 1930 ; Mouna 1998 NEMESTRINIDAE K. Kettani, D. Barraclough Number of species: 13 Faunistic knowledge of the family in Morocco: poor Nemestrininae Nemestrinus Latreille, 1802 Nemestrinus aegyptiacus (Wiedemann, 1828) Timon-David 1951 ; Séguy 1953a , HA , Amsed; Bernardi 1973 ; Mouna 1998 ; SA (Oued el Ma) – MISR Nemestrinus ater (Olivier, 1810) Mouna 1998 ; EM (Zaouillet El Atenf) – MISR Nemestrinus escalerai Arias, 1913 Paramonow 1945; Bernardi 1973 , HA , Marrakech; Mouna 1998 Nemestrinus exalbidus (Lichtwardt, 1907) Mouna 1998 Nemestrinus fasciatus (Olivier, 1810) = Rhynchocephalus fasciatus Olivier, in Mouna 1998 : 86 Bernardi 1973 ; Mouna 1998 ; MA (Immouzer) – MISR Nemestrinus nigrovillosus Lichtwardt, 1909 Arias 1913 ; Séguy 1930a , MA , Ras el Ma, Azrou, Forêt Tiffert (2000–2200 m), HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Séguy 1941d , HA , Tizi-n'Test; Timon-David 1951 , MA , Tizi-n'Tretten; Bernardi 1973 ; Mouna 1998 Nemestrinus pieltaini (Gil Collado, 1934) = Nemestrellus pieltaini Gil Collado, in Gil Collado 1934 : 325 Gil Collado 1934 , Rif , Imasinen, Bab Chiquer, Bab Bagla; Bernardi 1973 ; Mouna 1998 Nemestrinus ruficornis (Macquart, 1840) Mouna 1998 Nemestrinus rufipes (Olivier, 1810) Mouna 1998 ; MA (Timahdit) – MISR Nemestrinus striatus (Lichtwardt, 1907) Mouna 1998 Trichopsideinae Fallenia Meigen, 1820 Fallenia fasciata (Fabricius, 1805) Arias 1913 ; Séguy 1930a , AP , Casablanca, Rabat, Bou Knadel, MA , M'Rirt (1200 m); Timon-David 1951 , AP , Forêt Maâmora; Mouna 1998 ; AP (Rabat, Bou Knadel), MA (Aïn Leuh) – MISR Neorhynchocephalus Lichtwardt, 1909 Neorhynchocephalus tauscheri (Fisher, 1812) = Rhynchocephalus tauscheri Fischer, in Mouna 1998 : 86 Mouna 1998 Trichopsidea Westwood, 1839 Trichopsidea costata (Loew, 1857) = Symmictus costatus Loew, in Arias 1913 : 26, Séguy 1930a : 89 Arias 1913 ; Séguy 1930a , MA , Tameghilt (1900 m); Mouna 1998 Asiloidea ASILIDAE K. Kettani, G. Tomasovic Number of species: 131 . Expected: 230 Faunistic knowledge of the family in Morocco: moderate Apocleinae Apoclea Macquart, 1838 Apoclea algira (Linnaeus, 1767) Séguy 1953a , AA , Tata; Tomasovic 1997 ; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Apoclea micracantha Loew, 1856 Tomasovic 1997 , HA , Sidi Mhejmed Ou Said; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Eremonotus Theodor, 1980 Eremonotus hauseri Geller-Grimm & Hradský, 1998 Geller-Grimm and Hradský 1998, HA ; Geller-Grimm 2007, AA , Agadir Asilinae Afroepitriptus Lehr, 1992 Afroepitriptus beckeri Lehr, 1992 Geller-Grimm 2007; Koçak and Kemal 2010 Antiphrisson Loew, 1849 Antiphrisson trifarius Loew, 1849 Tomasovic 1997 , HA , Errachidia, Ziz, Oasis Zouala; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 ; HA (Asni) – MISR Asilus Linnaeus, 1758 Asilus barbarus Linnaeus, 1758 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ; Séguy 1941a , HA , Tizi-Tamatert (Toubkal, 2250 m); Mouna 1998 ; Weinberg and Blasco-Zumeta 2004 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Asilus crabroniformis Linnaeus, 1758 Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Asilus tingitanus Boisduval, 1835 Geller-Grimm 2007, Rif , Tanger Dysmachus Loew, 1860 Dysmachus albisetosus (Macquart, 1850) Geller-Grimm 2007 Dysmachus cochleatus (Loew, 1854) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dysmachus cristatus (Wiedemann, 1820) = Dysmachus dasynotus Loew, in Becker and Stein 1913 : 72, Timon-David 1951 : 138 Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Rabat, Harcha, Salé, Oued Ksab, MA , Ifrane; Mouna 1998 ; Geller-Grimm 2007; AP (Rabat, Cap Cantia) – MISR Dysmachus digitulus Becker, 1923 Geller-Grimm 2007 Dysmachus elapsus Villeneuve, 1933 Villeneuve 1933 , AP , Mazagan, Mogador; Mouna 1998 ; Tomasovic 2001b ; Geller-Grimm 2007; AP (Cap Cantia) – MISR Dysmachus evanescens Villeneuve, 1912 Timon-David 1951 , AP , Sehoul; Mouna 1998 ; Geller-Grimm 2007 Dysmachus trigonus (Meigen, 1804) Timon-David 1951 , AP , Rabat, Chellah, Forêt Maâmora, Ras el Arba, Sehoul, Zaër Eccoptopus Loew, 1860 Eccoptopus longitarsis (Macquart, 1838) Timon-David 1951 , AA , Zagora; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Engelepogon Lehr, 1992 Engelepogon brunnipes (Fabricius, 1794) = Heligmoneura brunnipes Fabricius, in Séguy 1930a : 125 = Acanthopleura brunnipes Fabricius, in Timon-David 1951 : 137 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Meknès; Timon-David 1951 , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Epitriptus Loew, 1849 Epitriptus cingulatus (Fabricius, 1871) Séguy 1941a , AA , Agadir; Mouna 1998 Eremisca Hull, 1962 Eremisca heleni heleni (Efflatoun, 1934) Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Eremisca osiris (Wiedemann, 1828) Geller-Grimm 2007; El Hawagry 2011 Eutolmus Loew, 1848 Eutolmus wahisi Tomasovic, 2001 Tomasovic 2001a , Rif , Tétouan (Jebel Tazout, 1650 m); Geller-Grimm 2007; Koçak and Kemal 2010 Filiolus Lehr, 1967 Filiolus apicalis (Becker in Becker & Stein, 1913) = Eutolmus apicalis Becker, in Becker and Stein 1913 : 75 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Machimus Loew, 1849 Machimus cribratus (Loew, 1849) Geller-Grimm 2007; AP (Cap Cantia) – MISR Machimus fimbriatus (Meigen, 1804) Geller-Grimm 2007 Machimus fortis (Loew, 1849) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat; Mouna 1998 ; Geller-Grimm 2007 Machimus gonatistes (Zeller, 1840) Geller-Grimm 2007 Machimus mauritanicus Bequaert, 1964 Tomasovic 2003 ; Geller-Grimm 2007; AP (Forêt Boulhaut, Salé) – MISR Machimus nigrosetosus Séguy, 1941 Séguy 1941d AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Machimus perplexus Becker, 1915 Geller-Grimm 2007 Machimus pilipes (Meigen, 1820) = Eutolmus hispanus Loew, in Becker and Stein 1913 : 74 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Forêt Zaers, Tizi-n'Bouftene (2300 m), HA , bords de l'Imminen (Tachdirt: 2400–2600 m); Geller-Grimm 2007 Machimus pseudogonatistes Villeneuve, 1930 = Machimus ermineus Becker, in Mouna 1998 : 84 Villeneuve 1933 ; Mouna 1998 ; Geller-Grimm 2007 Neoepitriptus Lehr, 1992 Neoepitriptus inconstans (Wiedemann in Meigen, 1820) = Machimus micropyga Becker, in Becker and Stein 1913 : 74 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 , Rif , Tanger Neoepitriptus minusculus (Bezzi, 1898) = Machimus minusculus Bezzi, in Timon-David 1951 : 138, Mouna 1998 : 84 Timon-David 1951 , MA , Ifrane; Mouna 1998 ; Geller-Grimm 2007 Neomochtherus Osten-Sacken, 1878 Neomochterus brevipennis Séguy, 1932 Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Neomochtherus grandicollis (Becker, 1914) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Neomochterus ochriventris (Loew, 1854) Timon-David 1951 , AP , Sidi Moussa el Harati; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Pashtshenkoa Lehr, 1995 Pashtshenkoa clypeatus maroccanus (Tsacas, 1968) Geller-Grimm 2007 Phileris Tsacas & Weinberg, 1976 Phileris haplopygus Tsacas & Weinberg, 1976 Geller-Grimm 2007 Phileris pilosus Tsacas & Weinberg, 1976 Geller-Grimm 2007 Satanas Jacobson, 1908 Satanas gigas (Eversmann, 1855) Maldès 2000 , ME , Oujda, HA , Errachidia, Meski Turka Őzdikmen, 2008 Turka cervinus (Loew, 1856) = Stenopogon cervinus Loew, in Séguy 1930a : 122 Séguy 1930a , MA , pont de l'Oued Korifla (Zaers), HA , Sidi Bou Rziguine; Geller-Grimm 2007; Hayat et al. 2008 ; Özdikmen 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Dasypogoninae Dasypogon Meigen, 1803 Dasypogon atratus (Fabricius, 1794) = Selidopogon atratus Meigen, in Séguy 1930a : 118 = Selidopogon atratus Fabricius, in Timon-David 1951 : 136 Séguy 1930a , MA ; Timon-David 1951 , Rif , Ouezzane, AP , Rabat MA , Oued Beth; Mouna 1998 ; Geller-Grimm 2007 Dasypogon auripilus (Séguy, 1934) Mouna 1998 ; Geller-Grimm 2007; AP (Casablanca) – MISR Dasypogon crassus Macquart in Lucas, 1849 = Selidopogon crassus Macquart, in Séguy 1930a : 119, Timon-David 1951 : 136 Séguy 1930a , Rif , Tanger, MA , Meknès; Timon-David 1951 , AP , M'Soun, Guerrouaou; Mouna 1998 ; Geller-Grimm 2007 Dasypogon diadema (Fabricius, 1781) = Selidopogon cylindricus Fabricius, in Séguy 1930a : 119 = Selidopogon diadema Fabricius, in Séguy 1930a : 118 = Selidopogon sicanus Costa, 1853, in Hayat et al. 2008 : 183 Séguy 1930a , AP , Dar Salem, Tarfaya, Oued Korifla (Zaers), HA , Bou Tazzert; Timon-David 1951 , AP , Port Lyautey; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Dasypogon gougeleti (Bigot, 1878) = Selidopogon gougeleti Bigot, in Timon-David 1951 : 136 Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Geller-Grimm 2007 Dasypogon olcesci (Bigot, 1878) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dasypogon rubinipes (Becker in Becker & Stein, 1913) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dasypogon ruficauda (Fabricius, 1805) Geller-Grimm 2007 Saropogon Loew, 1847 Saropogon aretalogus Séguy, 1953 Séguy 1953a , MA , Ifrane; Geller-Grimm 2007 Saropogon aurifrons (Macquart in Lucas, 1850) Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Saropogon clausus Becker, 1906 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , EM , Itzer, Moulay Aïn Djemine (Haute Moulouya); Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Saropogon jugulum (Loew, 1847) Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Saropogon leucocephalus (Meigen, 1820) Séguy 1930a , MA , Forêt Tiffert (2000–2200 m); Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 ; Ghahari et al. 2014 Saropogon maroccanus Séguy, 1930 Séguy 1930a , MA , Ras El Ksar (1900 m); Séguy 1949a , SA , Goulimine; Mouna 1998 ; Carles-Tolrá 2002 ; Geller-Grimm 2007 Saropogon obscuripennis (Macquart in Lucas, 1849) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat, MA , Aïn Leuh, Tizi-s'Tkrine (1700 m), HA , Imi-M'Tanout, Dar M'Tougui; Séguy 1941d , AA , Agadir; Timon-David 1951 , EM , Guenfouda; Mouna 1998 ; Geller-Grimm 2007 Saropogon philocalus Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Saropogon rufipes (Gimmerthal, 1847) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Saropogon tassilaensis Séguy, 1953 Séguy 1953a , AA , Tassila (Souss); Geller-Grimm 2007 Dioctriinae Dioctria Meigen, 1803 Dioctria atrorubens Séguy, 1930 Séguy 1930a , MA , Tizi-s'Tkine (1700 m); Villeneuve 1933 ; Mouna 1998 ; Geller-Grimm 2007 Dioctria cothurnata Meigen, 1820 Ebejer et al. 2019 , Rif , Dardara (484 m) Dioctria fuscipes Macquart, 1834 Timon-David 1951 , MA , Aguelmane Sidi Ali (2070 m) Dioctria gagates Wiedemann in Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dioctria notha Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Dioctria rufa Strobl, 1906 Ebejer et al. 2019 , Rif , Dardara (484 m) Dioctria rungsi Timon-David, 1951 Timon-David 1951 , MA , Ifrane (1650 m); Mouna 1998 ; Geller-Grimm 2007 Laphriinae Glyphotriclis Hermann, 1920 Glyphotriclis ornatus (Schiner, 1868) = Triclis ornatus Schiner, in Becker and Stein 1913 : 67 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Marrakech; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Laphria Meigen, 1803 Laphria bomboides Macquart, 1849 = Laphria praelusia Séguy, in Séguy 1930a : 124 Séguy 1930a , MA , Soufouloud (1900–2100 m); Mouna 1998 ; MA (Meghraona, Tamtraekt) – MISR Pogonosoma Rondani, 1856 Pogonosoma maroccanum (Fabricius, 1794) Loew 1860 ; Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Carles-Tolrá 2002 ; Geller-Grimm 2004 ; Geller-Grimm 2007; Ghahari et al. 2007 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013a ; Ghahari et al. 2014 Stiphrolamyra Engel, 1928 Stiphrolamyra rubicunda Oldroyd, 1947 Timon-David 1951 , AP , Sidi Moussa el Harati; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 ; Ghahari et al. 2014 Stiphrolamyra vitai Hradský & Geller-Grimm, 1997 Hradský and Geller-Grimm 1997 , HA , Taroudant; Geller-Grimm 2007 Laphystiinae Perasis Hermann, 1905 Perasis sareptana Hermann, 1906 Séguy 1930a , HA , Asni; Mouna 1998 Scytomedes Röder, 1882 Scytomedes haemorrhoidalis (Fabricius, 1794) = Triclis haemorrhoidalis Fabricius, in Mouna 1998 : 84 Séguy 1930a , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Trichardis Hermann, 1906 Trichardis leucocomus (Van der Wulp, 1899) = Trichardis leucicoma Van der Wulp, in Timon-David 1951 : 132 Timon-David 1951 , AA , Tata, piste de Fask Tahrjicht; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Leptogastrinae Leptogaster Meigen, 1803 Leptogaster cylindrica (De Geer, 1776) = Leptogaster hispanica Meigen, in Séguy 1930a : 117 Séguy 1930a , MA , Meknès; Mouna 1998 ; Tomasovic 2006 , Rif ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Leptogaster pedunculata Loew, 1847 = Gonypes pedunculatus Loew, in Becker and Stein 1913 : 72 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Haute Réghaya; Mouna 1998 Leptogaster straminea Becker, 1907 Timon-David 1951 , MA , Aguelmane Sidi Ali (2070 m); Mouna 1998 ; Geller-Grimm 2007 Stenopogoninae Afroholopogon Londt, 1994 Afroholopogon waltlii (Meigen, 1838) = Heteropogon waltlii Meigen, in Séguy 1930a : 123 Séguy 1930a , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Amphisbetetus Hermann, 1906 Amphisbetetus sexspinus Tomasovic, 2008 Tomasovic and Weyer 2008 , AA , Imsouane (Agadir); Geller-Grimm 2007 Ancylorhynchus Berthold in Latreille, 1827 Ancylorrhyncus gummigutta (Becker, 1906) Séguy 1930a , Rif , Tanger; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Ancylorrhyncus limbatus (Fabricius, 1794) Séguy 1930a , MA , Meknès, Timhadit (2000 m); Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Ancylorrhyncus vultur Séguy, 1930 Séguy 1930a , MA , Timhadit (2000 m); Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Eriopogon Loew, 1847 Eriopogon jubatus Becker, 1906 Timon-David 1951 , Rif , Tanger, AP , Rabat; Hradský and Hüttinger 1995 , AP , Rabat; Mouna 1998 ; Geller-Grimm 2007; AP (Forêt Temara) – MISR Eriopogon laniger Meigen, 1804 = Holopogon flavescens Jaennicke, in Séguy 1930a : 123 Séguy 1930a , HA , Aguergour; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Eriopogon spatenkai Hradský & Hüttinger, 1995 Geller-Grimm 2007; Hradský and Hüttinger 1995 , MA , Mishliffen Galactopogon Engel, 1929 Galactopogon hispidus Engel, 1929 Ebejer et al. 2019 , AA , 23 km S of Rich (Errachidia, 2012 m) Habropogon Loew, 1847 Habropogon aerivagus (Séguy, 1953) Séguy 1953a , SA , Aouletis Habropogon appendiculatus Schiner, 1867 Timon-David 1951 , AA , Aïn Chaïb; Mouna 1998 ; Weinberg and Blasco-Zumeta 2004 ; Hradský and Geller-Grimm 2005 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 Habropogon bacescui Weinberg & Tsacas, 1973 Geller-Grimm 2007; Koçak and Kemal 2010 Habropogon distipilosus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon hauseri Hradský & Geller-Grimm, 2005 Hradský and Geller-Grimm 2005 , HA , Tizi-n'Test; Geller-Grimm 2007; Koçak and Kemal 2010 Habropogon odontophallus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon parappendiculatus Weinberg & Tsacas, 1973 Hradský and Geller-Grimm 2005 , HA , Aït Saoun; Geller-Grimm 2007; Kirk-Spriggs and McGregor 2009 , HA ; Koçak and Kemal 2010 Habropogon prionophallus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon pyrrhophaeus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon rubriventris Macquart, 1849 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Aïn el Hadjar (near Mogador), MA , Meknès, Tlet n'Rhohr, EM , Berkane (1350–1400 m); Mouna 1998 Habropogon senilis Wulp, 1899 Geller-Grimm 2007 Habropogon spissipes Hermann, 1909 Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Habropogon striatus (Fabricius, 1794) = Habropogon heteroneurus Timon-David, in Timon-David: 135 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 , AP , Rabat Heteropogon Loew, 1847 Heteropogon biplex Becker, in Becker & Stein 1913: 65 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Heteropogon manicatus (Meigen, 1820) Séguy 1930a , MA , Azrou, Meknès, Aïn Leuh, HA , Asni; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; MA (Ifrane) – MISR Heteropogon nubilus (Meigen, 1820) = Isopogon brevis Schiner, in Becker and Stein 1913 : 64 = Sisyrnodytes brevis Macquart, in Timon-David 1951 : 134, Séguy 1953a : 79 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AA , Imiter; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Shoeibi and Karimpour 2010 ; Ghahari et al. 2014 Holopogon Loew, 1847 Holopogon dimidiatus (Meigen, 1820) Séguy 1941d , AA , Agadir Holopogon dusmeti Strobl in Czerny & Strobl 1909 = Eriopogon dusmeti Strobl, in Timon-David 1951 : 132 Timon-David 1951 , EM , Guenfouda, HA , Tifni; Mouna 1998 ; Geller-Grimm 2007; HA (Tifni Demnat) – MISR Holopogon melaleucus (Meigen, 1820) Séguy 1930a , AP , Forêt Maâmora, Dar Salem (Rabat); Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Holopogon pusillus (Macquart, 1838) = Habropogon pusillus (Macquart), in Séguy 1949a : 154 Séguy 1949a , SA , Goulimine; Mouna 1998 Holopogon quadrinotatus Séguy, 1953 Séguy 1953a , SA , Amguilli Sguelma Acnephalum Macquart, 1838 = Pycnopogon Loew, 1847 in Londt 2010 Acnephalum apiformis (Macquart in Lucas, 1849) Séguy 1930a , MA , Timhadit (2000 m), Meskedall (1800–1900 m); Timon-David 1951 , MA , Ifrane (1650 m); Mouna 1998 ; Geller-Grimm 2007 Acnephalum denudatus (Séguy, 1949) = Stenopogon denudatus Loew, in Séguy 1930a : 123, Séguy 1934b : 162 Séguy 1930a , MA , Tizi-n'Tkrine; Séguy 1934b , HA , Haute Réghaya; Séguy 1949b , AP , Bou Tazzert near Mogador; Séguy 1953a , AA , Oasis du Ferkla; Mouna 1998 ; Geller-Grimm 2007 Acnephalum fasciculatus (Loew, 1847) Séguy 1930a , MA , Azrou, Timelilt, Sidi Bettache, HA , Asni, bords Imminen (Tachdirt), Likount (2500–2800 m), Lac Ifni (Skoutana), SA , Béni Mgild; Timon-David 1951 , AP , Oued Korifla, MA , Lac Aguelmane Sidi Ali (2070 m), Oued N'Zala; Mouna 1998 ; Geller-Grimm 2007; Lehr et al. 2007 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 ; MA (Ras el Ma) – MISR Stenopogon Loew, 1847 Stenopogon costatus Loew, 1871 = Stenopogon costarus Loew, in Mouna 1998 : 84 Séguy 1930a , MA , Tizi-n'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 Stenopogon gracilis (Macquart, 1838) = Stenopogon fumipenis Becker, in Becker and Stein 1913 : 68 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Stenopogon heteroneurus (Macquart, 1838) Timon-David 1951 , AP , Forêt Maâmora, Oued Akreuch, HA , Mouldikht; Mouna 1998 ; Hayat et al. 2008 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Stenopogon iphippus Séguy, 1932 Séguy 1932b , MA , Volubilis; Mouna 1998 ; Geller-Grimm 2007 Stenopogon iphis Séguy, 1932 Séguy 1932b , MA , Azrou; Timon-David 1951 , Rif , Plateau de Tisserouine (2000 m), MA , Ifrane (1650 m), Ito (Rabat); Geller-Grimm 2007 Stenopogon ischyrus Séguy, 1932 Séguy 1932b , MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 ; Geller-Grimm 2007 Stenopogon junceus (Wiedemann in Meigen, 1820) Timon-David 1951 , AP , Oued Akreuch, Zaër, MA , Sefrou; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Stenopogon kocheri Timon-David, 1951 Timon-David 1951 , HA , Tifni; Mouna 1998 ; Geller-Grimm 2007 Stenopogon porcus Loew, 1871 Séguy 1949a , AA , Akka; Mouna 1998 ; Geller-Grimm 2007 Stenopogon werneri Engel, 1933 Geller-Grimm 2007; Koçak and Kemal 2010 , MA , Fès, Zalagh Sisyrnodytes Loew, 1856 Sisyrnodytes leucophaetus Séguy, 1930 Séguy 1930a , MA , Béni Berberi; Mouna 1998 ; Geller-Grimm 2007; Londt 2009 Sisyrnodytes nilicola (Rondani, 1850) Oldroyd 1980 ; Londt 1987; Geller-Grimm 2007; Londt 2009 , AA , Ifni, Tiznit, Tata, Tazegzout; El Hawagry 2011 ; Samin et al. 2011 ; Ghahari et al. 2014 Stichopogoninae Stichopogon Loew, 1847 Stichopogon albellus Loew, 1856 Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 Stichopogon albofasciatus (Meigen, 1820) Séguy 1930a , HA , Kasba Taguendaft (Goundafa); Mouna 1998 ; Geller-Grimm 2007 Stichopogon elegantulus (Wiedemann, 1820) Séguy 1930a , HA , Kasba Taguendaft (Goundafa), Aguerd el Had, AA , Talekjount (Souss); Mouna 1998 ; Geller-Grimm 2007; Ghahari et al. 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 ; AP (Kénitra) – MISR Stichopogon inaequalis Loew, 1847 Séguy 1930a , HA , Aguerd el Had, AA , Talekjount (Souss); Mouna 1998 ; Geller-Grimm 2007 Stichopogon maroccanus (Becker, 1914) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007; El Hawagry 2011 , Rif , Tanger Stichopogon punctiferus Bigot, 1878 Geller-Grimm 2007 Stichopogon pusio (Macquart in Lucas, 1849) Séguy 1930a , HA , Kasba Taguendaft (Goundafa); Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Stichopogon schineri Koch, 1872 Timon-David 1951 , AA , Backkoum (Jebel Siroua); Mouna 1998 ; Hayat et al. 2008 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 BOMBYLIIDAE K. Kettani, M.J. Ebejer, J. Dils Number of species: 248 . Expected: 270 Faunistic knowledge of the family in Morocco: good Usiinae Apolysis Loew, 1860 Apolysis eremophila Loew, 1873 = Usia tomentosa Engel, in Paramonow 1947: 209 = Parageron ornata Engel, in Zaitzev 2007 : 160 Paramonow 1947; Mouna 1998 ; Zaitzev 2007 , AP (south), Tamri; Koçak and Kemal 2010 ; El Hawagri 2011; Evenhuis and Greathead 2015 Parageron Paramonov, 1929 Parageron gratus (Loew, 1856) = Usia grata Loew, in Timon-David 1951 : 143 Timon-David 1951 , AP , Oued Grou; Mouna 1998 ; Bader and Arabyat 2004 ; Zaitzev 2007 , SA ; Koçak and Kemal 2010 ; El Hawagri 2011; Evenhuis and Greathead 2015 – MISR Parageron griseus Paramonov, 1947 Zaitzev 2007 , SA Parageron hyalipennis (Séguy, 1941) = Oligodranes hyalipennis Séguy, in Séguy 1941d : 9 Séguy 1941d , AA , Agadir (Forêt Admine); Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , HA Parageron incisus (Wiedemann, 1830) = Usia incisa Wiedemann, in Timon-David 1951 : 143 Séguy 1930a , AP , Mogador, Casablanca, Sidi Bettache, Aïn Sferguila, MA , Forêt Zaers, Ras el Ma, Aïn Leuh, HA , Tenfecht; Timon-David 1951 , AP , Rabat, Sehoul, MA , Ifrane; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Parageron major Macquart, 1840 Mouna 1998 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 , AP , Rabat, Nkheila; Evenhuis and Greathead 2015 Usia Latreille, 1802 Usia ( Micrusia ) aurata (Fabricius, 1794) = Usia ( Micrusia ) taeniolata Costa, 1883, in, Koçak and Kemal 2010 Séguy 1930a , Rif , Tanger, AP , Rabat, Sidi Bettache; Paramonov 1950 , Rif , Tanger; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) crispa Gibbs, 2011 Gibbs 2011 , HA , Marrakech, AA , Agadir, Taroudant, Tafraoute, Ouarzazate Usia ( Micrusia ) cryptocrispa Gibbs, 2011 Gibbs 2011 , AP , Ben Slimane, Rabat Usia ( Micrusia ) dilsi Gibbs, 2011 Gibbs 2011 , Rif , Tétouan, Al Hoceima, HA , Taourirt Usia ( Micrusia ) echinus Gibbs, 2011 Gibbs 2011 , AP , Agadir, Guelmim, Sidi Ifni, Cap Ghir, MA , Tafraoute, HA , Marrakech, Tizi-n-Test, AA , Tafingoult Usia ( Micrusia ) falcata Gibbs, 2011 Gibbs 2011 , MA , Azrou, Ifrane Usia ( Micrusia ) forcipata Brullé, 1833 14 Mouna 1998 : 84 Usia ( Micrusia ) globicauda Gibbs, 2011 Gibbs 2011 , AP , Essaouira Usia ( Micrusia ) loewi Becker, 1906 Zaitzev 2007 , Rif , Tanger, AP , Skhirate, Nkheila, MA , Taferiate, HA , Taourirt Usia ( Micrusia ) novakii Strobl, 1902 Séguy 1941d , HA , Tizi-n'Test; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) parascripa Gibbs, 2011 Gibbs 2011 , MA , Ifrane, Mischliffen (2200 m) Usia ( Micrusia ) pusilla Meigen, 1820 Séguy 1930a , AP , Rabat, MA , Azrou, HA , Tafingoult (Goundafa, 1500–1600 m); Séguy 1934b , AP , Rabat (on Calendula ); Séguy 1953a , AP , Cap Ghir; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) versicolor (Fabricius, 1787) Séguy 1930a , AP , Berrchid, Casablanca, M'Rassine; Timon-David 1951 , AP , Rabat, MA , Oulmès; Mouna 1998 ; Koçak and Kemal 2010 ; Gibbs 2011 , HA ; Evenhuis and Greathead 2015 ; EM (Oujda) – MNHNR Usia ( Usia ) aenea (Rossi, 1794) Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 ; Bader and Arabyat 2004 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Usia ) angustifrons Becker, 1906 Mouna 1998 Usia ( Usia ) atrata (Fabricius, 1798) = Voluccella atrata Fabricius, in Fabricius 1798: 570 = Usia claripennis Macquart, in Macquart 1840: 105 Meigen 1820 ; Séguy 1930a , Rif , Tanger, MA , Aïn Leuh; Timon-David 1951 , AP , Rabat, Guerrouaou; Mouna 1998 ; Zaitzev 2007 , HA , Tizi-n'Tichka; Koçak and Kemal 2010 ; Gibbs 2014 , AP , Mogador, Arbaa-Sahel (320 m), Tamri (215 m), MA , Khemisset, Oulmès (700 m), Ifrane, El Merabtine, HA , Marrakech, Aït Ourirr (530 m), Oukaimeden (2200 m), Tizi-n'Test (1450 m), Timzit (1700 m), AA , Agadir, Tiznit, Igherm (1660 m), Taroudant, Tata, Iguiour (1260 m), SA , Bou Jarif, Goulimine; Evenhuis and Greathead 2015 Usia ( Usia ) bicolor Macquart, 1855 15 Mouna 1998 : 84 Usia ( Usia ) cornigera Gibbs, 2014 Gibbs 2014 , Rif , Tanger, AP , Sidi Bettache, Rabat, MA , Meknès (550 m), Aïn Leuh (1350 m), HA , Dar Kaid M'tougui Usia ( Usia ) florea (Fabricius, 1794) = Volucella florea Fabricius, in Becker 1906: 203 = Usia cuprea Macquart, 1834, in Becker 1906: 203 Becker 1906a ; Séguy 1930a , Rif , Tanger, AP , Mogador, Sidi Bettache, HA , Tinmel (Goundafa), around (Skoutana); Timon-David 1951 , EM , Oued Moulouya; Mouna 1998 ; Pârvu and Zaharia 2007 ; Koçak and Kemal 2010 ; Gibbs 2014 ; Evenhuis and Greathead 2015 Usia ( Usia ) ignorata Becker, 1906 Becker 1906a ; Mouna 1998 ; Bader and Arabyat 2004 ; Pârvu and Zaharia 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 Usia ( Usia ) maghrebensis Gibbs, 2014 Gibbs 2014 , Rif , Tanger, Tétouan, El Biutz (150 m), AP , Mogador, MA , Aïn Leuh (1350 m) Usia ( Usia ) vestita Macquart, 1846 Mouna 1998 : 84; Gibbs 2011 Phthiriinae Phthiria Meigen, 1802 Phthiria albogilva Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Evenhuis and Greathead 2015 Phthiria gaedii Wiedemann in Meigen, 1820 Séguy 1930a , MA , Foum Keneg; Timon-David 1951 , AP , Zaers, MA , Ifrane; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 – MISR ( MA , Ifrane) Phthiria maroccana Zaitzev, 2005 = Phthiria maroccana Zaitzev, in Zaitzev 2005 : 667 Zaitzev 2005 , MA , Taferiate, HA , Taourirt; Zaitzev 2007 , MA , Taferiate, HA , Taourirt Phthiria merlei Zaitzev, 2005 = Phthiria merlei Zaitzev, in Zaitzev 2005 : 665 Zaitzev 2005 , AP (south), Tamri, Inchaden (south of Aït Melloul); Zaitzev 2007 , AP (south), Tamri, Inchaden (south of Aït Melloul) Phthiria minuta (Fabricius, 1805) Séguy 1930a , HA , Tenfecht, AA , Souss; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 Phthiria pulicaria var. flavofasciata Strobl in Morge, 1976 Mouna 1998 ; Zaitzev 2007 , AA , Tizi-n'Tiniggigt (1600 m); Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Phthiria scutellaris Wiedemann in Meigen, 1820 Séguy 1930a , MA , Meknès; Séguy 1941a , MA , Meknès, HA , Imi-n'Ouaka (1500 m); Mouna 1998 ; Evenhuis and Greathead 2015 ; Rif (Sapinière Talassemtane) – MISR Phthiria simonyi Becker, 1908 Séguy 1949a , SA , Guelmim; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , MA , Meknès Phthiria umbripennis Loew, 1846 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , MA , Meknès Phthiria vagans Loew, 1846 Zaitzev 2007 , HA , Taourirt, AA , Tizi-n'Taratine Toxophorinae Geron Meigen, 1820 Geron intonsus Bezzi, 1925* MA , HA Geron macquarti Greathead in Evenhuis & Greathead 1999 Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 Geron subflavofemoratus Andréu Rubio, 1959 Andréu Rubio 1959 ; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Toxophora Meigen, 1803 Toxophora fasciculata (Villers, 1789) Séguy 1930a , AP , Rabat; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Toxophora fuscipennis (Macquart, 1840) Mouna 1998 : 84 Toxophora pauli Zaitzev, 2005 Zaitzev 2005 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate); Zaitzev 2007 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) Toxophora shelkovnikovi Paramonov, 1933 Zaitzev 2007 , AA , Ouarzazate Heterotropinae Heterotropus Loew, 1873 Heterotropus atlanticus Séguy, 1930 Séguy 1930a , AP , Mogador; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Heterotropus longitarsus Séguy, 1930 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Heterotropus maroccanus Zaitzev, 2003 Zaitzev 2003 , AA , Jebel Tighermine (SE of Ouarzazate); Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Bombyliinae Anastoechus Osten-Sacken, 1877 Anastoechus bahirae Becker, 1915 Mouna 1998 ; Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Anastoechus hyrcanus Pallas & Wiedemann in Wiedemann, 1818 Mouna 1998 : 84 Anastoechus latifrons (Macquart, 1839) Timon-David 1951 , AP , Dradek; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Anastoechus nitidulus ssp. nitidulus Fabricius, 1794 Mouna 1998 : 84 Anastoechus stramineus Wiedemann in Meigen, 1820 Mouna 1998 : 84 Anastoechus trisignatus (Portschinsky, 1881) Bader and Arabyat 2004 ; Ziatzev 2007, AP , Rabat, AA , Jebel Tighermine (SE of Ouarzazate), AA , 20 km SW Goulmima, SA , Tan-Tan, Between Guelmim and Tan-Tan (90 km from Guelmim), Taganint (south of Bou-Izakarn); Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombomyia Greathead, 1995 Bombomyia discoidea (Fabricius, 1794) Séguy 1930a , AP , Oued Korifla (Zaers), Sidi Bettache; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombomyia stictica Boisduval, 1835 Zaitzev 2007 , MA , col de Zeggota (N Meknès), Oulmès Bombomyia vertebralis (Dufour, 1833) = Bombylius punctatus Fabricius, in Timon-David 1951 : 144 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a AP , Dradek near Rabat; Timon-David 1951 , MA , Volubilis; Mouna 1998 ; Bader and Arabyat 2004 ; Koçak and Kemal 2010 Evenhuis and Greathead 2015 ; AP (Dradek, Casablanca), EM (Oujda), MA (Volubilis, Aïn Leuh) – MISR Bombylisoma Rondani, 1856 Bombylisoma algirum (Macquart, 1840) = Bombylius nigrifrons Becker, in Becker and Stein 1913 : 83 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylisoma breviusculum (Loew, 1855) Dils and Özbek 2006 ; Zaitzev 2007 ; Evenhuis and Greathead 2015 Bombylisoma flavibarbum Loew, 1855 Mouna 1998 : 84 Bombylisoma melanocephalum Fabricius, 1794 Zaitzev 2007 , HA , Taourirt, south of Tizi-n'Test Bombylius Linnaeus, 1758 Bombylius ( Bombylius ) albaminis Séguy, 1949 Séguy 1949a , HA , Alnif; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) ambustus Pallas & Wiedemann, 1818 Mouna 1998 : 84 Bombylius ( Bombylius ) analis (Olivier, 1789) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Oued Korifla, Rabat, Sidi Bettache, Aïn Sferguila; Timon-David 1951 , AP , Rabat; Mouna 1998 ; Zaitzev 2007 , MA , route Fès-Sidi Kacem (30 km from Fès); Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , AP (Rabat, Casablanca) – MISR Bombylius ( Bombylius ) audcenti Bowden, 1984 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) canescens Mikan, 1796 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , Rif , Cap Malabata (Tanger), MA , Tachguelt, route Fès-Sidi Kacem (30 km from Fès) Bombylius ( Bombylius ) cinerascens Mikan, 1796 Mouna 1998 ; Bader and Arabyat 2004 Bombylius ( Bombylius ) discolor Mikan, 1796 Mouna 1998 ; Zaitzev 2007 , MA , route El Hajeb-Ifrane (1 km from Ifrane) Bombylius ( Bombylius ) eploceus Séguy, 1949 Séguy 1949a , SA , Guelmim; Mouna 1998 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) fimbriatus Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, Jebel Ahmar (1700 m); Mouna 1998 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) flavipes Wiedemann, 1828 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) fulvescens Wiedemann in Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AP , Cap Ghir; Séguy 1941d , AA , Agadir; Mouna 1998 Bombylius ( Bombylius ) fuscus Fabricius, 1781 Mouna 1998 : 84 Bombylius ( Bombylius ) major (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , MA , Oulmès; Bader and Arabyat 2004 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Kénitra) – MISR Bombylius ( Bombylius ) mauritanus Olivier, 1789 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , HA Bombylius ( Bombylius ) medius (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Sehoul; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Oued Yquem, Dradek) – MISR Bombylius (Unplaced) megacephalus Portschinsky, 1887* EM , AA Bombylius ( Bombylius ) minor Linnaeus, 1758 Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; MA (Aguelmane Azigza), SA – MISR Bombylius ( Bombylius ) mus Bigot, 1862 Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) niveus Meigen, 1804 Mouna 1998 : 84; AP (Mogador) – MISR Bombylius ( Bombylius ) numidus Macquart, 1846 Séguy 1953a , MA , Ifrane; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) pauli Zaitzev, 2003 Zaitzev 2003 , MA , route Fès-Sidi Kacem (30 km from Fès); Zaitzev 2007 , MA , route Fès-Sidi Kacem (30 km from Fès) Bombylius ( Bombylius ) posticus (Fabricius, 1805) Dils and Özbek 2006 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) postversicolor Evenhuis & Greathead, 1999 = Bombylius versicolor Fabricius, 1805 Meigen 1820 ; Bezzi 1906 ; Séguy 1930a , AP , Mogador; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) pumilus Meigen, 1820 Mouna 1998 : 84 Bombylius ( Bombylius ) semifuscus (Meigen, 1820) Séguy 1953a , AP , Cap Ghir; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) torquatus Loew, 1855 Séguy 1930a , HA , Ouaounzert (Glaoua), Arround (Skoutana), Tachdirt (bord de l'Imminen, 2400–2600 m); Timon-David 1951 , AP , Rabat; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Mogador) – MISR Bombylius ( Bombylius ) undatus Mikan, 1796 Pârvu and Zaharia 2007 Bombylius ( Bombylius ) vagans Meigen, 1830 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) venosus Mikan, 1796 Mouna 1998 ; Zaitzev 2007 , AP , El Koudia (30 km SW from Rabat); AP (Dradek) – MISR Bombylius ( Zephyrectes ) cruciatus Fabricius, 1798 Séguy 1930a , MA , Aharmoumou (1100 m), Azrou, Ras el Ma, HA , Tizi-n'Test, Jebel Imdress (Goundafa, 2000–2450 m); Mouna 1998 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 ; Rif (Talassemtane), AP (Mogador), MA (Sefrou) – MISR Bombylius ( Zephyrectes ) leucopygus (Macquart, 1846) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , AP , Larache, MA , Moulay Idris (900 m); Evenhuis and Greathead 2015 , SA , Erfoud; MA (Ifrane) – MISR Conophorus Meigen, 1803 Conophorus bellus Becker, 1906* HA Conophorus fuliginosus (Wiedemann in Meigen, 1820) = Ploas fuliginisa (Meigen), in Séguy 1953a : 83 Séguy 1930a , MA , Aharmoumou (1100 m); Séguy 1953a , MA , Ahermoumou (1100 m); Timon-David 1951 , AP , Dradek, MA , Sefrou, HA , Marrakech; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; Evenhuis and Greathead 2015 ; AP (Salé, Mogador) – MISR Conophorus fuscipennis (Macquart, 1840) Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Mouna 1998 ; Evenhuis and Greathead 2015 Conophorus griseus (Fabricius, 1787) Mouna 1998 ; Zaitzev 2007 ; Evenhuis and Greathead 2015 Conophorus hamilkar Paramonov, 1929 Timon-David 1951 , AP , Mogador; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Mogador) – MISR Conophorus macroglossus (Dufour, 1852) Mouna 1998 ; Zaitzev 2007 ; Evenhuis and Greathead 201; AP (Mogador) – MISR Conophorus mauritanicus Bigot, 1892 = Conophorus heteropilosus Timon-David, in Timon-David 1951 : 141; Mouna 1998 : 84; Evenhuis and Greathead 2015 : 192 Timon-David 1951 , MA , Oulmès; Mouna 1998 ; Zaitzev 2007 , AP , El Koudia (30 km SW from Rabat), Forêt Zaer (35 km SW from Rabat), N Tretten; Koçak and Kemal 2010 ; Dils 2013 , MA , Mrirt; Evenhuis and Greathead 2015 Conophorus rossicus Paramonow, 1929 Dils and Özbek 2006 Dischistus Loew, 1855 Dischistus albatus (Séguy, 1934) = Acanthogeron albatus Séguy, 1934, in Séguy 1934d : 73; Zaitzev 2007 : 162 Séguy 1934d ; Zaitzev 2007 , SA , 30 km S Tata Dischistus auripilus (Séguy, 1930) = Acanthogeron auripilus Séguy, 1930, in Séguy 1930a : 104 Séguy 1930a , AP , Mogador; Séguy 1934b , AP , Zaers; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Evenhuis and Greathead 2015 Dischistus maroccanus (Séguy, 1930) = Acanthogeron maroccanus Séguy, 1930, in Séguy 1930a : 106 Séguy 1930a , AP , Mogador; Mouna 1998 ; Zaitzev 2007 , HA , Tazzarine; Evenhuis and Greathead 2015 Dischistus mittrei (Séguy, 1930) = Acanthogeron mittrei Séguy, in Séguy 1930a : 105 Séguy 1930a , AP , Mogador; Mouna 1998 ; Evenhuis and Greathead 2015 Dischistus perniveus (Bezzi, 1925) = Acanthogeron perniveus Bezzi, in Timon-David 1951 : 143 Timon-David 1951 , AP , Djamda de M'Tal; Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Dischistus senex (Wiedemann in Meigen, 1820) = Acanthogeron senex Meigen, 1820, in Séguy 1953a : 83, Mouna 1998 : 84, Zaitzev 2007 : 162 Séguy 1930a , HA , Tafingoult (Goundafa, 1500–1600 m); Villeneuve 1933 ; Séguy 1953a , HA , Aït Ourir; Mouna 1998 ; Zaitzev 2003 , HA , Taourirt; Zaitzev 2007 , HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Dradek), MA (Sefrou) – MISR Dischistus separatus (Becker, 1906) = Acanthogeron talboti Séguy, 1930, in Séguy 1930a : 106 Evenhuis and Greathead 2015 Efflatounia Bezzi, 1925 Efflatounia berbera Bowden, 1973 Ebejer et al. 2019 , AA , Agadir – NMWC Legnotomyia Bezzi, 1902 Legnotomyia fascipennis Bezzi, 1925* SA Merleus Zaitzev, 2003 Merleus punctipennis Zaitzev, 2003 = Merleus punctipennis Zaitzev 2003 : 599 Zaitzev 2003 , AP , Skhirate; Zaitzev 2007 , AP , Skhirate Prorachthes Loew, 1869 Prorachthes crassipalpis Villeneuve, 1930 Evenhuis and Greathead 2015 Systoechus Loew, 1855 Systoechus ctenopterus (Mikan, 1796) Timon-David 1951 , MA , Ifrane; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2007 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Systoechus gomezmenori Andréu Rubio, 1959 Carles-Tolrá 2002 ; Evenhuis and Greathead 2015 Systoechus gradatus (Wiedemann in Meigen, 1820) Timon-David 1951 , AP , Mouldikht; Mouna 1998 ; Zaitzev 2007 , MA , Taferiat, HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 Systoechus mixtus Wiedemann, 1821 = Bombylius stylicornis Macquart in Séguy 1941: 10 Séguy 1941d , AA , Agadir; Mouna 1998 Systoechus pumilio Becker, 1915 Mouna 1998 : 84 Triplasius Loew, 1855 Triplasius boghariensis (Lucas, 1852) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , EM , Oujda; Mouna 1998 ; Pârvu and Zaharia 2007 ; Evenhuis and Greathead 2015 Triplasius maculipennis (Macquart, 1846) = Bombylius maculipennis var. melanopus Timon-David, in Timon-David 1951 : 144 = Bombylius ( Triplasius ) maculipennis Macquart, 1849, in Zaitzev 2007 : 166 Timon-David 1951 , MA , Azrou; Zaitzev 2007 , MA , route El Hachef, Criosement route Raubei Idris-Merhassine; Evenhuis and Greathead 2015 Ecliminae Eclimus Loew, 1844 Eclimus gracilis Loew, 1844 Séguy 1930a , MA , Ras el Ma; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2007 , MA , Maaziz; Evenhuis and Greathead 2015 Thevenetimyia Bigot, 1875 Thevenetimyia quedenfeldti (Engel, 1885)* Rif , AP , MA Crocidiinae Crocidium Loew, 1860 Crocidium aegyptiacum Bezzi, 1925* SA Crocidium nudum Efflatoun, 1945* EM , AA Semiramis Becker in Becker and Stein 1914 Semiramis punctipennis Becker, 1914 Zaitzev 2007 , AA , Aoulouz Cythereinae Amictus Wiedemann, 1817 Amictus castaneus (Macquart, 1840) Séguy 1930a , AP , Rabat, HA , Ank el Djemal; Mouna 1998 ; Evenhuis and Greathead 2015 Amictus compressus (Fabricius, 1805) Evenhuis and Greathead 2015 Amictus heteropterus Macquart, 1838 Zaitzev 2007 , Rif , Tanger, AP , Rabat, HA , S Tizi-n'Test, AA , Tizi-n'Taratine Amictus oblongus (Fabricius, 1805) = Bombylius oblongus Fabricius, in Macquart 1834: 390 Macquart 1834 Amictus pulchellus Macquart, 1846 Séguy 1930a , AP , Rabat, Maâmora; Mouna 1998 ; Zaitzev 2007 , HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Amictus setosus Loew, 1869* AP Amictus tener Becker, 1906 Zaitzev 2007 , AP , Rabat Amictus validus Loew, 1869 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Amictus variegatus Meigen in Waltl, 1835 Mouna 1998 : 84 Chalcochiton Loew, 1844 Chalcochiton argentifrons (Macquart in Lucas, 1849) Séguy 1953a , AP , Cap Ghir, Salé, Sidi Battache, MA , Tizi-n'Bou Zabal (2300 m), AA , Aïn Chaïb (Souss); Evenhuis and Greathead 2015 Chalcochiton argyrocephalus (Macquart, 1840) = Chalcochiton ( Anthrax ) argyrocephala (Macquart), in Engel 1938 : 328 Engel 1938 ; Séguy 1953a , AA , Agadir; El Hawagry 2011 ; Evenhuis and Greathead 2015 Chalcochiton atlantica Dils, 2008 Dils 2008 , SA , Guelmim Chalcochiton holosericeus (Fabricius, 1794) = Chalcochiton semiargentaea Macquart, in Zaitsev 2007: 172 Séguy 1930a , AP , Maâmora, Sidi Bettache, HA , Tizi-n'Test, Jebal Imdress (2000–2450 m), Tafingoult (Goundafa, 1500–1600 m); Séguy 1941d ; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger, AP , Skhirate, EM , Taourirt, MA , Taferiat, Meknès-Moulay Idriss, Merhassine, AA , Agadir, Ouarzazate; Evenhuis and Greathead 2015 ; AP (Salé, Forêt Temara, Oued Yquem, Meshra) – MISR Chalcochiton maghrebi Dils, 2017 Dils 2017 , Rif , Souk El Kolla, Bab Taza, 10 km S of Mjara, AP , Sidi Bettache, Temsia, Imsouane, Mansouria, Rommani, Béni Slimane, Tioulit, EM , El Aioun, MA , Béni Mellal, el Ksiba, 10 km SE Bir Tamtam, Merchouch, Mrirt, Fès, HA , Azilal, Asni, Tizi-Mlil, AA , Taroudant, Tizi-n'Test, Tiznit, Agouim, Sidi Ifni, Mesti, Tafinegoult, Tizi-n'Tinififft, El Mrabtine, SA , Semara Chalcochiton maroccanus Zaitzev, 2006 Séguy 1953a , HA , Tafingoult (Goundafa, 1500–1600 m); Zaitzev 2006 , AP (south), Aït Melloul; Zaitzev 2007 , AP (south), Aït Melloul Chalcochiton merlei Zaitzev, 2006 Zaitzev 2006 , AP , Skhirate; Zaitzev 2007 , AP , Skhirate Chalcochiton pallasii Loew, 1856 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2007 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Callostoma Macquart, 1840 Callostoma fascipenne Macquart, 1840 Bader and Arabyat 2004 Cyllenia Latreille, 1802 Cyllenia rustica Rossi, 1790 Mouna 1998 ; Zaitzev 2007 ; AP (Mogador) – MISR Cytherea Fabricius, 1794 Cytherea albolineata Bezzi, 1925* SA Cytherea alexandrina Becker, 1902 Becker 1902 : 30; Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Cytherea aurea (Fabricius, 1794) Séguy 1930a , AP , Rabat, HA ; Mouna 1998 ; Bader and Arabyat 2004 ; Zaitzev 2007 , AA , Tizi-n'Taratine; El Hawagry 2011 ; Evenhuis and Greathead 2015 , HA , Tafingoult (Goundafa, 1500–1600 m); AP (Rabat, Oued Cherrat) – MISR Cytherea cinerea Fabricius, 1805 = Mulio delicatus Becker, 1906 Becker 1906b : 153; Timon-David 1951 , AP , Meshra; Mouna 1998 ; Bader and Arabyat 2004 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Cytherea delicata Becker, 1906 Zaitzev 2007 , HA , S Tizi-n'Test, AA , Zagora, Taroudant, Tizi-n'Taratine Cytherea dispar (Loew, 1873) Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Cytherea fenestrata (Loew, 1873) Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Cytherea infuscata (Meigen, 1820) Séguy 1930a , EM , Itzr (Haute Moulouya), MA , Forêt Timelilt (1900 m), HA , Aït el Hadj, Marrakech; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Meskara) – MISR Cytherea maroccana (Becker, 1903) = Mulio maroccanus Becker, in Becker 1903 : 89 Becker 1903 , Rif , Tanger; Bezzi 1906 : 249; Timon-David 1951 , AP , Azemmour; Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Cytherea obscura Fabricius, 1794 Séguy 1930a , EM , Haute Moulouya, AP , Sidi Bettache, HA , Ouaouenzert; Séguy 1941d ; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2007 , MA , Taferiat, AA , Agadir, Amredi, Jebel Tighermine (SE of Ouarzazate), Tizi-n'Tiniggigt, Tizi-n'Taratine, Tizi-n'Bachkoun; Karimpour 2012 ; Evenhuis and Greathead 2015 Cytherea rungsi Timon-David, 1951 Timon-David 1951 , EM , Guenfouda; Mouna 1998 ; Evenhuis and Greathead 2015 Cytherea thyridophora (Bezzi, 1925) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Jebel Bouhachem, 965 m) Cytherea trifaria (Becker, 1906) Evenhuis and Greathead 2015 Lomatiinae Lomatia Meigen, 1820 Lomatia abbreviata Villeneuve, 1911 Séguy 1930a , MA , Forêt Zaers; Timon-David 1951 , EM , Guercif; Mouna 1998 ; Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 ; AP (Maâmora, Oued Cherrat, Dradek), HA – MISR Lomatia belzebul paramonovi Fabricius, 1794 Séguy 1930a , AP , Dar Salem, MA , Timhadit, Meknès, Aïn Leuh; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Lomatia erynnis (Loew, 1869) Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 , AP , Rabat; Evenhuis and Greathead 2015 Lomatia hamifera Becker, 1915 Mouna 1998 : 84 Lomatia lachesis Egger, 1859 Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Lomatia lateralis (Meigen, 1820) Séguy 1930a , MA , Ras el Ma, HA , Forêt Timelilt; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Rabat), MA (Volubilis, Ras el Ma) – MISR Lomatia obscuripennis Loew, 1869 Zaitzev 2008 , AP , Nkheila; Evenhuis and Greathead 2015 Lomatia sabaea (Fabricius, 1781) Mouna 1998 : 84 Lomatia tysiphone Loew, 1860 Zaitzev 2008 , MA , Azrou, AA , Tizi-n'Taratine Antoniinae Antonia Loew, 1856 Antonia bouillonae Séguy, 1932 Evenhuis and Greathead 2015 Anthracinae Aphoebantini Aphoebantus Loew, 1872 Aphoebantus wadensis Becker, 1925* SA Anthracini Anthrax Scopoli, 1763 Anthrax aethiops (Fabricius, 1781) Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 ; AP (Forêt Maâmora) – MISR Anthrax anthrax (Schrank, 1781) = Argyramoeba anthrax Schrank, in Séguy 1930a : 93 Séguy 1930a , MA , Aïn Leuh, Soufouloud (1900–2100 m), HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Timon-David 1951 , MA , El Ksiba, Ifrane; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Anthrax binotatus (Wiedemann in Meigen, 1820) = Argyramoeba binotata Meigen, in Séguy 1926 : 209, Séguy 1930a : 94 Séguy 1926 ; Séguy 1930a , AP , Rabat, HA , Tizi-n'Test, Jebel Imdress (2000–2450 m); Séguy 1949a , HA , Alnif; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Anthrax dentatus (Becker, 1906) Bader and Arabyat 2004 ; Zaitzev 2008 , AA , Tizi-n'Tiniggigt; El Hawagry 2011 ; Evenhuis and Greathead 2015 Anthrax hemimelas Speiser, 1910 Zaitzev 2008 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) Anthrax kiritshenkoi Paramonov, 1935 Evenhuis and Greathead 2015 Anthrax lucidus (Becker, 1902) Ebejer et al. 2019 , AA , Ziz river (13 km N of Erfoud, 800 m) Anthrax morio Fabricius, 1775 Mouna 1998 ; MA (Ifrane, Azrou) – MISR Anthrax trifasciatus (Meigen, 1804) = Argyramoeba trifasciata Meigen, in Timon-David 1951 : 139 Séguy 1930a , MA , Meknès; Timon-David 1951 , AP , south of Rabat; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Anthrax varius Fabricius, 1794 Séguy 1930a , AP , Rabat; Mouna 1998 ; Evenhuis and Greathead 2015 – MISR Anthrax virgo Egger, 1859 = Argyramoeba virgo Egger, in Séguy 1930a : 94 Séguy 1930a , AP , Rabat; Zaitzev 2008 , MA , Taferiat, AA , Jebel Tighermine (SE of Ouarzazate) Cononedys Hermann, 1907 Cononedys efflatouni (Bezzi, 1925)* SA Cononedys escheri Bezzi, 1908 Zaitzev 2008 , AP , Skhirate, Rabat Cononedys lyneborgi (François, 1969) Evenhuis and Greathead 2015 Cononedys scutellatus Meigen, 1835 Zaitzev 2008 , Rif , Jebala, Haouta el Kazdir, AA , Aouzlida near Aoulouz Satyramoeba Sack, 1909 Satyramoeba hetrusca (Fabricius, 1794) Mouna 1998 : 84 Spogostylum Macquart, 1840 Spogostylum isis (Meigen, 1820) Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Spogostylum trinotatum Dufour, 1852 Mouna 1998 : 84 Spogostylum tripunctatum (Pallas in Wiedemann, 1818) Timon-David 1951 , HA , Aït Mhamed Sgatt; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate); El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 ; HA (Aïn Mhamed Sgatt) – MISR Turkmeniella Paramonov, 1940 Turkmeniella crosi (Villeneuve, 1910) Evenhuis and Greathead 2015 Exoprosopa Macquart, 1840 Exoprosopa aeacus Meigen, 1804 Mouna 1998 : 84 Exoprosopa baccha Loew, 1869 Mouna 1998 ; Zaitzev 1999 ; Dils and Özbek 2006 ; Zaitzev 2008 ; Evenhuis and Greathead 2015 Exoprosopa capucina (Fabricius, 1871) Mouna 1998 : 84 Exoprosopa circeoides Paramonov, 1928 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate) Exoprosopa cleomene Egger, 1859 Mouna 1998 : 84 Exoprosopa decrepita (Wiedemann, 1828) Zaitzev 2008 , AA , Zagora Exoprosopa efflatouni Bezzi, 1925 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate), Ouarzazate, SA , Taganint (south of Bou-Izakarn) Exoprosopa grandis Wiedemann in Meigen, 1820 Mouna 1998 ; Zaitzev 2008 , HA , Tishka (2200 m) Exoprosopa italica (Rossi, 1794) Zaitzev 2008 , HA , Taourirt, AA , Tizi-n'Taratine, Jebel Tighermine (SE of Ouarzazate), SA , Taganint (south of Bou-Izakarn) Exoprosopa jacchus (Fabricius, 1805) Séguy 1930a , AP , Mogador, Sidi Taibi, MA , Tizi-s'Tkrine (1700 m), Dar Salem, Aïn Leuh, HA , Bou Tazzert; Mouna 1998 ; Mirceni and Pârvu 2009 ; Evenhuis and Greathead 2015 ; Rif (Talassemtane, Forêt Izarine, road of Jebha, Zoumi) – MISR Exoprosopa minos (Meigen, 1804) Séguy 1949a , AA , Tata; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2008 , MA , Taferiat; El Hawagry 2011 ; El Hawagry and Dhafer 2015 ; Evenhuis and Greathead 2015 ; MA (Jebel Lachhab) – MISR Exoprosopa pandora (Fabricius, 1805) Greathead 2001 ; Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Exoprosopa rutila (Pallas & Wiedemann, 1818) Evenhuis and Greathead 2015 Micomitra Bowden, 1964 Micomitra stupida Rossi, 1790 = Exoprosopa stupida Rossi, in Mouna 1998 : 84 Mouna 1998 Plesiocera Macquart, 1840 Plesiocera algira (Macquart, 1840) Zaitzev 2008 , MA , Taferiat; Evenhuis and Greathead 2015 Heteralonia Rondani, 1863 Heteralonia ( Homolonia ) megerlei (Hoffmansegg in Wiedemann, 1818) Zaitzev 2008 , SA , Goulimine Heteralonia ( Mesoclis ) pygmalion (Fabricius, 1805) = Exoprosopa pygmalion Fabricius, in Timon-David 1951 : 139 = Mesoclis pygmalion Fabricius, 1805, in Zaitzev 2008 : 191 Séguy 1930a , Rif , Tanger, AP , Maâmora, Rabat, MA , Aïn Leuh; Timon-David 1951 , AP , Temara; Mouna 1998 ; Zaitzev 2008 , AP , Cherrat El Hawagry 2011 ; Evenhuis and Greathead 2015 Heteralonia ( Zygodipla ) algira (Fabricius, 1794) Séguy 1930a , Rif , Tanger, AP , Mogador, HA , Bou Tazzert; Mouna 1998 ; Zaitzev 2008 , HA , Tifnite (south of Aït Melloul); El Hawagry 2011 ; Evenhuis and Greathead 2015 Heteralonia ( Zygodipla ) bagdadensi s (Macquart, 1840) Zaitzev 2008 , AA , Zagora Heteralonia ( Zygodipla ) singularis (Macquart, 1840) Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Heteralonia arenacea Becker, 1906 Evenhuis and Greathead 2015 Heteralonia dispar (Loew, 1869) = Exoprosopa dispar Loew, in Timon-David 1951 : 139 Timon-David 1951 , HA , Marrakech; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Heteralonia rivularis (Meigen, 1820) = Exoprosopa rivularis Meigen, in Timon-David 1951 : 139 Séguy 1930a , AP , Rabat, Maâmora; Timon-David 1951 , AP , Oued Akreuch; Mouna 1998 ; Zaitzev 1999 ; Bader and Arabyat 2004 ; Zaitzev 2008 , AP , Rabat Oestranthrax Bezzi, 1921 Oestranthrax brunnescens (Loew, 1857) Bader and Arabyat 2004 Oestranthrax pallifrons Bezzi, 1926 Evenhuis and Greathead 2015 Pachyanthrax François, 1964 Pachyanthrax albosegmentatus (Engel, 1936) Zaitzev 2008 , AA , Jebel Tighermine (south of Ouarzazate) Pachyanthrax nomadorum (Greathead, 1970) Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Exhyalanthrax Becker, 1916 Exhyalanthrax afer (Fabricius, 1794) = Anthrax tangerinus Bigot, 1892 Bezzi 1906 ; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2008 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 , Rif , Tanger; MA (Ifrane) – MISR Hemipenthes Loew, 1869 Hemipenthes morio (Linnaeus, 1758) Séguy 1930a , MA , Azrou, HA , Arround (Skoutana, 2000–2400 m); Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 ; MA (Azrou, Ifrane) – MISR Hemipenthes velutinus (Meigen, 1820) Séguy 1930a , MA , Azrou; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Thyridanthrax Osten-Sacken, 1886 Thyridanthrax alphonsi Sánchez Terrón and Roldan Bravo, 2000 Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax elegans ssp. elegans (Wiedemann in Meigen, 1820) Séguy 1930a , AP , Rabat; Mouna 1998 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Oued Cherrat, Rabat), MA (Volubilis) – MISR Thyridanthrax fenestratus (Fallén, 1814) Séguy 1926 ; Séguy 1930a , EM , Berkane (1350–1400 m); Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; Rif (Tomorot) – MISR Thyridanthrax griseolus Klug, 1832 Zaitzev 2008 , SA , Taganint (south of Bou-Izakarn) Thyridanthrax hispanus (Loew, 1869) Becker and Stein 1913 , Rif , Tanger; Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax incanus (Klug, 1832) = Anthrax incana Klug, 1832, in Séguy 1953a : 83 Séguy 1930a , AP , Oued Korifla (Zaers); Timon-David 1951 , AP , Zaer; Séguy 1953a , MA , Tarda; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Thyridanthrax loustaui Andréu Rubio, 1961 Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax maroccanus Dils, 2012 Dils 2012 , AA , Ouarzazate, Skoura (1250 m), Amerzgane (1350 m) Thyridanthrax mutilus (Loew, 1869)* AA Thyridanthrax nebulosus (Dufour, 1852) Becker and Stein 1913 , Rif , Tanger; Andréu Rubio 1959 ; Mouna 1998 ; Sánchez Terrón and Roldan Bravo 2000 , Rif , Benibuifrur, Melilla, Restinga; Evenhuis and Greathead 2015 Thyridanthrax perspicillaris ssp. perspicillaris (Loew, 1869) Séguy 1930a , MA , Aïn Leuh, Forêt Azrou, HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Thyridanthrax polyphemus (Hoffmansegg, 1819) Séguy 1930a , MA , Volubilis (400 m); Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Veribubo Evenhuis, 1978 Veribubo angusteoculatus (Becker, 1902) Zaitzev 2008 , AA , Zagora Veribubo saudensis (François, 1970)* AA Veribubo tabaninus (François, 1970)* AA , SA Villa Lioy, 1864 Villa brunnea Becker, 1916 Mouna 1998 : 84 Villa ceballosi Andréu Rubio, 1959 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa cingulata Meigen, 1804 Mouna 1998 ; AP (Rabat, Casablanca), MA (Volubilis, Fès) – MISR Villa distincta (Meigen in Waltl, 1835) Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa fasciata (Meigen, 1804) = Villa circumdata (Meigen), in Séguy 1941a : 29 Séguy 1930a , AP , Rabat; Séguy 1941a , AP , Rabat, HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa hottentotta (Linnaeus, 1758) = Anthrax hottentotus Linnaeus, in Séguy 1926 : 198, Séguy 1930a : 92, Bléton and Fleuzet 1939: 64 Séguy 1930a , AP , Rabat, MA , Aïn Leuh; Bléton and Fleuzet 1939, MA , Fès; Séguy 1941d , HA , Tizi-n'Test; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 – MISR Villa ixion (Fabricius, 1794) Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Villa laevis Becker, 1915 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa leucostoma (Meigen, 1820) Mouna 1998 : 84; AP (Bou-Regreg) – MISR Villa luculenta Séguy, 1941 Séguy 1941d , AA , Taroudant; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa niphobleta (Loew, 1869) Bader and Arabyat 2004 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Villa venusta (Meigen, 1820) Mouna 1998 : 84 Desmatoneura Williston, 1895 Desmatoneura albifacies (Macquart, 1840) Ebejer et al. 2019 , AA , Merzouga (714 m) Desmatoneura flavifrons Becker, 1915 Zaitzev 2008 , AA , Ouarzazate, Taroudant, Jebel Tighermine (SE of Ouarzazate) Petrorossia Bezzi, 1908 Petrorossia albula Zaitzev, 1962 Zaitzev 1999 ; Bader and Arabyat 2004 ; Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate); El Hawagry 2011 ; Evenhuis and Greathead 2015 Petrorossia freidbergi Zaitzev, 1999 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate) Petrorossia hespera (Rossi, 1790) Séguy 1949a , AA , Tata; Mouna 1998 ; Zaitzev 1999 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Bou-Regreg), MA (Timahdit) – MISR Petrorossia margaritae Zaitzev, 1999 Zaitzev 2008 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) New records for Morocco Amictus setosus Loew, 1869 Atlantic Plain: Rommani, Marmouch, 33.568°N, 06.533°W , 400 m, 1♂1♀, Dils J.- Faes J., coll: PCJD . Aphoebantus wadensis Becker, 1925 Sahara: Tata, 9 km. W Tissint, 29.851°N, 07.265°W , 535 m, 1♂1♀, 03.iii.2007, Dils J.- Faes J., coll: PCJD . Bombylius (Unplaced) megacephalus Portschinsky, 1887 Eastern Morocco: Figuig, Abbou Lakhal, 32.1587°N, 01.507°W , 1050 m, 1♀, 07.iii.2009, Dils J.- Faes J., coll: PCJD . Anti Atlas: Tiznit, 84 km. SSE Guelmim, 28.631°N, 10.75522°W , 235 m, 1♂, 27.ii.2007, Dils J.- Faes J., coll: PCJD ; Tiznit, Abaynou, 29.057°N, 10.026°W , 360 m, 1♀, 13.iii.2009, Dils J.- Faes J., coll: PCJD . Cononedys efflatouni Bezzi, 1925 Sahara: Guelmim, Souk Tnine Nouaday, 29.166°N, 09.279°W , 680 m, 2♂3♀, 07.iv.2015, Dils J.- Faes J., coll: PCJD . Conophorus bellus Becker, 1906 High Atlas: Marrakech, Oukaimeden, 31.233°N, 07.817°W , 2200 m, 3♂, 06.iv.2006, Dils J.- Faes J., coll: PCJD . Crocidium aegyptiacum Bezzi, 1925 Anti Atlas: Tiznit, Mesti, 29.274°N, 10.139°W , 280 m, 1♂, 23.iii.2006, Dils J.- Faes J., coll: PCJD . Sahara: Tata, 28 km E of Tachjicht, 29.106°N, 09.149°W , 700 m, 1♀, 02.iii.2007, Dils J.- Faes J., coll: PCJD . Crocidium nudum Efflatoun, 1945 Eastern Morocco: Oujda, Plateau du Rekkam, 33.839°N, 02.55781°W , 1150 m, 1♀, 25.iv.2010, Dils J.- Faes J., coll: PCJD . Anti Atlas: Agadir, Imsouane, 30.885°N, 09.780°W , 270 m, 3♂13♀, 09.iv.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.024°N, 07.223°W , 1370 m, 17♂12♀, 31.iii.2006, Dils J.- Faes J., coll: PCJD ; Taliouine, 18 km. W of Taliouine, 30.6003°N, 08.082°W , 900 m, 2♀, 24.iii.2009, Dils J.- Faes J., coll: PCJD ; Taroudant, Tafinegoult, 30.734°N, 08.430°W , 680 m, 3♀, 24.iii.2009, Dils J.- Faes J., coll: PCJD ; Tiznit, Arbaa Sahel, 29.657°N, 09.869°W , 320 m, 11♂26♀, 21.iii.2006, Dils J.- Faes J., coll: PCJD . Cytherea albolineata Bezzi, 1925 Sahara: Guelmim, Tainzirt, 29.121°N, 09.333°W , 670 m, 1♀, 31.iii.2010, Dils J.- Faes J., coll: PCJD . Geron intonsus Bezzi, 1925 Middle Atlas: Khenifra, Boulôjoul, 32.873°N, 04.945°W , 1500 m, 7♂10♀, 26.iv.2008, Dils J.- Faes J., coll: PCJD . High Atlas: Midelt, 32.680°N, 04.677°W , 1400 m, 2♂2♀, 20.iv.2015, Dils J.- Faes J., coll: PCJD ; Midelt, Zeïda, 32.781°N, 04.964°W , 1500 m, 9♂11♀, 24.iv.2015, Dils J.- Faes J., coll: PCJD . Legnotomyia fascipennis Bezzi, 1925 Anti Atlas: Zagora, Tazarinne, 30.798°N, 05.584°W , 900 m, 1♂, 07.iii.2007, Dils J.- Faes J., coll: PCJD . Sahara: Tata, 9 km W of Tissint, 29.851°N, 07.265°W , 535 m, 2♂1♀, 03.iii.2007, Dils J.- Faes J., coll: PCJD . Thevenetimyia quedenfeldti (Engel, 1885) Rif: Tanger-Tétouan, Souk El Kolla (Quolla), 35.083°N, 05.538°W , 150 m, 5♂4♀, 30.iv.2017, Dils J.- Faes J., coll: PCJD . Atlantic Plain: Rommani, Merchouch, 33.568°N, 06.753°W , 400 m, 5♂22♀, 04.v.2010, Dils J.- Faes J., coll: PCJD . Middle Atlas: Tadla-Azilal, Afourer, 32.180°N, 06.520°W , 1150 m, 5♂10♀, 07.v.2008, Dils J.- Faes J., coll: PCJD ; Béni Mellal, El Ksiba, 32.576°N, 06.050°W , 870 m, 7♂23♀, 23.iv.2008, Dils J.- Faes J., coll: PCJD . Thyridanthrax mutilus Loew, 1869 Anti Atlas: Tiznit, Sidi Ifni, 29.384°N, 10.172°W , 0 m, 7♂1♀, 10.iv.2008, Dils J.- Faes J., coll: PCJD . Veribubo saudensis François, 1970 Anti Atlas: Erfoud, Tikkert-N-Ouchane, 31.223°N, 04.784°W , 830 m, 1♂3♀, 03.iv.2009, Dils J.- Faes J., coll: PCJD . Veribubo tabaninus François, 1970 Anti Atlas: Ouarzazate, Amerzgane, 31.024°N, 07.223°W , 1370 m, 2♂9♀, 31.iii.2006, Dils J.- Faes J., coll: PCJD ; Erfoud, Tikkert-N-Ouchane, 31.250°N, 04.617°W , 860 m, 1♀, 07.iii.2007, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.017°N, 07.229°W , 1350 m, 6♂27♀, 25.iii.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.017°N, 07.229°W , 1350 m, 12♂8♀, 25.iii.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, 30.847°N, 06.817°W , 1200 m, 1♀, 30.iii.2009, Dils J.- Faes J., coll: PCJD . Sahara: Guelmim, Tainzirt, 29.121°N, 09.333°W , 670 m, 22♀, 31.iii.2010, Dils J.- Faes J., coll: PCJD . MYDIDAE K. Kettani, T. Dikow Number of species: 9 . Expected: 10 Faunistic knowledge of the family in Morocco: moderate Leptomydinae Leptomydas Gerstaecker, 1868 Leptomydas lusitanicus (Wiedemann, 1820) Mouna 1998 Rhopaliinae Rhopalia Macquart, 1838 Rhopalia berlandi Séguy, 1949a: 153 Séguy 1949a , AA , Tagounit, Asni; Mouna 1998 ; Dikow 2017 Perissocerus Gerstaecker, 1868 Perissocerus rungsi Séguy, 1953 Séguy 1953a , SA Syllegomydinae Syllegomydini Syllegomydas Becker, 1906 Syllegomydas algiricus (Gerstaecker, 1868) = Rhopalia algirica Gerstaecker, in Séguy 1928c : 149 Gerstaecker 1868 , AP , Casablanca; Séguy 1928c , AP , Rabat; Séguy 1930a , AP , Rabat; Mouna 1998 ; El Hawagry 2011 ; Dikow 2017 Syllegomydas berlandi (Séguy, 1941) Séguy 1941, AA , Agadir; Dikow 2017 Syllegomydas bueni Arias, 1914 Arias 1914 , AA , Tafilalt; Séguy 1928c ; Séguy 1930a ; Carles-Tolrá 2015 ; Carles-Tolrá 2017 , EM , Mariouri, Trifa; Dikow 2017 Syllegomydas cinctus Macquart, 1835 Macquart 1835, MA , Immouzer road, AA , Agadir, Taroudant; Séguy 1930a ; Mouna 1998 ; Carles-Tolrá 2017 , EM , Quebdana, douar Shila, AA , Agadir coast; Dikow 2017 Syllegomydas maroccanus Séguy, 1928 Séguy 1928c , AP , Kénitra, Rabat, Oued Korifla, Forêt Zaers; Séguy 1930a , AP , Rabat, Temara, Oued Korifla, Forêt Zaers; Séguy 1932c ; Mouna 1998 ; Carles-Tolrá 2017 , AP , Larache, Ras Remel; Dikow 2017 ; AP (Kénitra) – MISR Syllegomydas merceti Arias, 1914 Arias 1914 , AP , Mogador; Séguy 1930a ; Mouna 1998 ; El Hawagry 2011 , AP , Mogador; Dikow 2017 MYTHICOMYIIDAE K. Kettani, N. Evenhuis Number of species: 8 . Expected: 15 Faunistic knowledge of the family in Morocco: poor Empidideicinae Empidideicus Becker, 1907 Empidideicus crocea Séguy, 1949 = Cyrtosia crocea Séguy, in Séguy 1949a : 85 Séguy 1949a , SA , Guelmim; Séguy 1949c ; Mouna 1998 ; Evenhuis 2002 Glabellulinae Glabellula Bezzi, 1902 Glabellula maroccana Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , Rif , Adrou ( PNPB ) – BPBM, MISR Leylaiya Efflatoun, 1945 Leylaiya pellea Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , AA , Tiznit – BPBM Mythicomyiinae Mythenteles Hall & Evenhuis, 1991 Mythenteles signifera Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , Rif , Talassemtane (maison forestière, 1699 m) – BPBM, MISR Platypyginae Cyrtisiopsis Séguy, 1930 Cyrtisiopsis melleus (Loew, 1856) Evenhuis 2002 ; Zaitzav 2008, AA , Jebel Tighermine (SE Ouarzazate); Koçak and Kemal 2010 ; El Hawagry 2011 Cyrtisiopsis singularis Séguy, 1930 Evenhuis 2002 Cyrtosia Perris, 1839 Cyrtosia aglota Séguy, 1930 Evenhuis 2002 Cyrtosia marginata Perris, 1839 Séguy 1930a , HA ; Mouna 1998 ; Evenhuis 2002 ; Evenhuis and David 2004 SCENOPINIDAE K. Kettani, M. Carles-Tolrá Number of species: 8 (+3 unidentified). Expected: 12 Faunistic knowledge of the family in Morocco: good Scenopininae Scenopinus Latreille, 1802 Scenopinus albicinctus (Rossi, 1794) = Omphrale albicincta Rossi, in Séguy 1930a : 110 Séguy 1930a ; Mouna 1998 Scenopinus fenestralis (Linnaeus, 1758) = Omphrale fenestralis Linnaeus, in Séguy 1930a : 110 Séguy 1930a ; Mouna 1998 Scenopinus glabrifrons Meigen, 1824 = Omphrale glabrifrons Meigen, in Séguy 1930a : 110 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 Scenopinus niger (De Geer, 1776) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 Scenopinus parallelus Kelsey, 1969 Kelsey 1969 , AP , Villa Cisneros (Dakhla), SA , Río de Oro (Oued Eddahab) Scenopinus physadius (Séguy, 1930) = Omphrale physadia Séguy, in Séguy 1930a : 111 Séguy, 1930, EM , Bou Denib; Kelsey 1969 , EM , Bou Denib; Mouna 1998 Scenopinus pilosus (Séguy, 1930) = Omphrale pilosa Séguy, in Séguy 1930a : 111 Séguy 1930a , AP , Bou Knadel; Kelsey 1969 , AP , Bou Knadel; Mouna 1998 ; Carles-Tolrá 2001 Scenopinus undescribed sp. 1 Ebejer et al. 2019 , Rif , Martil (9 m) Scenopinus undescribed sp. 2 Ebejer et al. 2019 , Rif , Adrou (556 m) Stenomphrale Kröber, 1937 Stenomphrale teutankhameni (Kröber, 1923) Ebejer et al. 2019 , AP , forest of Maâmora (56 m) Stenomphrale sp. AP (Essaouira (J.-P. Haenni leg.)) – MHNN Neuchâtel THEREVIDAE K. Kettani, M. Hauser Number of species: 27 . Faunistic knowledge of the family in Morocco: moderate Phycusinae Phycusini Actorthia Kröber, 1912 Actorthia micans (Kröber, 1924) Kröber 1924 , AA , Errachidia (45 km S Erfoud), Merzouga Phycus Walker, 1850 Phycus lacteipennis Lyneborg, 2002 Lyneborg 2002 , AA , 25 km S Goulmima (1000 m), SA , Mekn s-Tafilalet; Winterton et al. 2012 ; Badrawy and Mohammad 2013 Salentia Costa, 1857 Salentia anancitis (Séguy, 1941) = Apioeicoceras anancitis Séguy, in Séguy 1941d : 10 Séguy 1941d , AA , Agadir; Mouna 1998 Salentia costalis (Wiedemann, 1824) = Apioeicoceras costalis Wiedemann, in Séguy 1930a : 108 Wiedemann 1824 , AP , Mogador, HA , Marrakech-Tensift-Al Haouz; Séguy 1930a ; Mouna 1998 ; Koçak and Kemal 2010 Salentia fuscipennis Costa, 1857 Costa 1857 , Rif , Tanger, AA , Tagadirt (Agadir); Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Bou Knadel, Tinmel; Mouna 1998 Therevinae Therevini Acanthothereva Séguy, 1935 Acanthothereva rungsi Séguy, 1935 Séguy 1935b , AP , Mehdia (20 km S Rabat); Lyneborg 1968 ; Koçak and Kemal 2010 ; Rif (Cap Spartel) – MISR Acrosathe Irwin & Lyneborg, 1981 Acrosathe annulata (Fabricius, 1805) Ebejer et al. 2019 , Rif , Oued Kbir (Béni Ratene, 157 m) Chrysanthemyia Becker, 1912 Chrysanthemyia chrysanthemi (Fabricius, 1787) Fabricius 1787 , EM , Béni Snassen Mountains, Tafouralt (800 m); Séguy 1930a , MA , Meknès, Berrechid; Mouna 1998 ; MA (Oued Grou, Timahdit) – MISR Chrysanthemyia velutinifrons (Becker, 1912) = Chrysanthemyia lucidifrons Becker 1912 : 81 = Oedicera velutinifrons Becker, in Becker and Stein 1913 : 82 Becker 1912 , Rif , Region de Tanger-Tétouan, Cercle d'Ouezzane (300 m), AP , 3 km S Settat, EM , Figuig, MA , Fès-Boulmane, Sidi Harazem (223 m); Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Meknès; Mouna 1998 ; MA (Oued Grou) – MISR Hoplosathe Lyneborg & Zaitsev, 1980 Hoplosathe distincta Lyneborg & Zaitsev, 1980 Lyneborg and Zaitsev 1980, HA , Oued Tensift (Marrakech) Neotherevella Lyneborg, 1978 Neotherevella macularis (Wiedemann, 1828) Hauser et al. 2017 , AA , Tifnite (10 Km S Agadir), Merzouga (45 Km S Erfoud) Thereva Latreille, 1797 Thereva atra Kröber, 1913 El Hawagry 2011 Thereva aureoscutellata Kröber, 1914 Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m) Thereva binotata Loew, 1847 Koçak and Kemal 2010 Thereva bipunctata Meigen, 1820 16 Ebejer et al. 2019 , MA , Khénifra (17 km SW of Midelt, 1940 m; 17 km NW of Zaida, 1878 m; 28 km S of Timahdit, 2100 m), Lac Aguelmane Afennourir (30 km SW of Azrou, 2050 m) Thereva brevicornis Loew, 1847 17 Pârvu et al. 2006 , AP , Cap Sim; Popescu-Mirceni 2011 Thereva chrysargyrea Séguy, 1953 Séguy 1953a , SA , Amguilli Sguelma Thereva cincta Meigen, 1820 Ebejer et al. 2019 , Rif , Oued Aliane (Ksar Sghir, 1 m) Thereva funebris Meigen, 1820 18 = Thereva lugubris Meigen Mouna 1998 ; AP (Rabat), MA (Ifrane) – MISR Thereva graeca Kröber, 1912 3 Séguy 1930a , Rif , Tanger; Mouna 1998 Thereva plebeja (Linnaeus, 1758) 3 Séguy 1953a , AA , Tifnit (Souss); Mouna 1998 ; MA (Ras el Ma) – MISR Thereva powelli Séguy, 1930 Séguy 1930a , MA , Forêt Azrou; MA (Ras el Ma) – MISR Thereva spiloptera Wiedemann, 1824 Wiedemann 1824 , HA , Ouirgane (Marrakech, 1000 m); Séguy 1930a , Rif , Tanger, AP , Mogador, MA , Meknès; Séguy 1953a , AP , Temara; Mouna 1998 ; El Hawagry 2011 Thereva spinulosa Loew, 1847 Loew 1847 , AP , Maâmora, MA , Khemisset Thereva stigmatica Kröber, 1912 EL-Hawagy 2011, Rif , Tanger Thereva strigata (Fabricius, 1794) 19 Koçak and Kemal 2010 Thereva tuberculata Loew, 1847 = Thereva algirica Kröber, 1913, in Séguy 1953a : 83 Loew 1847 , AP , Salé; Séguy 1930a , MA , Meknès; Séguy 1953a , AP , Salé; Mouna 1998 ; Koçak and Kemal 2010 Acknowledgments We gratefully acknowledge Martin Ebejer (UK) for material, comments and cooperation, as well as Gail Kampmeier (USA) for sharing data of Moroccan Therevidae out of the mandala database ( http://wwx.inhs.illinois.edu/research/mandala/about/ ). Empidoidea ATELESTIDAE K. Kettani, P. Gatt Number of species: 1 . Expected: 1 Faunistic knowledge of the family in Morocco: poor Atelestus Walker, 1837 Atelestus sp. nov. Ebejer et al. 2019 , Rif , Dardara (484–730 m), Aïn Tissemlal (Azilane, 1255 m), Douar El Hamma (338 m), Chrabkha pond (Al Manzla, 58 m) EMPIDIDAE K. Kettani, C. Daugeron Number of species: 40 . Expected: 100 Faunistic knowledge of the family in Morocco: poor Clinocerinae Clinocera Meigen, 1803 Clinocera maroccana (Séguy, 1941): 29 (= Hydrodromia ) = Atalanta ( Hydrodromia ) algira (Vaillant, 1952): 65 Séguy 1941a , HA , Anrhemer (Toubkal, 2500 m); Vaillant 1956b , HA , Sidi Chamarouch; Vaillant 1964 ; Dakki 1997 Clinocera megalatlantica (Vaillant, 1957): 65 (= Atalanta ( Atalanta ) ) Vaillant 1956b , HA ; Dakki 1997 Clinocera nigra Meigen, 1804: 292 = Heleodromia unicolor (Curtis, 1834): plate 513, Paramesia roberti (Macquart, 1835): 657 Vaillant 1956b , HA , Izourar, Sidi Chamarouch, Aguelmous; Dakki 1997 Dolichocephala Meigen, 1803 Dolichocephala ocellata (Costa, 1858): 7 (= Ardoptera ) = oculata (Loew, 1858~7): 7 (= Ardoptera ) = novemguttata (Strobl, 1893): 98 (= Ardoptera ) = albohalterata (Strobl, 1898): 399 (= Ardoptera ) = barbarica Vaillant, 1952: 65 = algira Vaillant, 1957: 64 Vaillant 1956b , HA , Imi-N'Ifri; Dakki 1997 Dolichocephala pavonica Vaillant & Gagneur, 1998: 380 Vaillant and Gagneur 1998 , HA , Demnat Kowarzia Mik, 1881 Kowarzia barbatula (Mik, 1880): 347 (= Clinocera ) = Clinocera dorieri (Vaillant, 1968): 88 Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Aguelmous, Sidi Chamarouch, Izourar, Oukaimeden; Dakki 1997 ; Vaillant and Moubayed 1998 Kowarzia bipunctata (Haliday, 1833) (= Heleodromia ) Vaillant 1956b , HA , Oukaimeden; Vaillant 1964 ; Dakki 1997 Kowarzia dieuzedei Vaillant, 1953: 60 Vaillant 1956b , HA , Lac Tamhda (Anremer); Vaillant 1964 , HA ; Dakki 1997 Kowarzia madicola (Vaillant, 1965): 152 (= Atalanta ) Vaillant 1956b , HA , Tahanaout Kowarzia tenella (Wahlberg, 1844): 107 (= Parmesia ) = Heleodromia zetterstedti (Walker, 1851): 105 = Wiedemannia securigera (Engel, 1918): 70 Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Aguelmous, Sidi Chamarouch, Izourar, Oukaimeden; Dakki 1997 ; Vaillant and Moubayed 1998 Wiedemannia Zetterstedt, 1838 Wiedemannia ( Chamaedipsia ) beckeri (Mik, 1889): 71 (= Chamaedipsia ) = jugorum (Strobl, 1893): 105 (= Chamaedipsia ) = crinita Engel, 1918: 217 (as var. of W. beckeri ) = alticola Vaillant, 195l: 54 = alpina Vaillant, 1967: 274 (as ssp. of W. beckeri ) = glaciola Wagner, 1985: 86 (as ssp. of W. beckeri ; new name for W. beckeri alpina Vaillant) Dakki 1997 Wiedemannia ( Chamaedipsia ) mgounica Vaillant, 1957: 69 Vaillant 1956a , HA , M'Goum; Dakki 1997 Wiedemannia ( Philolutra ) azurea (Vaillant, 1951): 3 (= Philolutra ) El Mezdi and Giudicelli 1985 , HA , Khettaras Marrakech; Dakki 1997 ; Vaillant and Moubayed 1998 Wiedemannia ( Philolutra ) fallaciosa Loew, 1873: 44 Vaillant 1964 , HA ; Dakki 1997 Wiedemannia ( Philolutra ) fallaciosa ssp. litardierei Vaillant, 1957: 67 Vaillant 1956a , HA ; Dakki 1997 Wiedemannia ( Roederella ) ouedorum Vaillant, 1952: 371 = ovedorum (error) Vaillant, 1978: 469 Vaillant 1964 , HA ; Dakki 1997 Hemerodromiinae Hemerodromia Meigen, 1822 Hemerodromia bethiana Vaillant & Gagneur, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Hemerodromia subapicalis Yang, Zhang & Zhang, 2007 = Hemerodromia apicalis Vaillant and Gagneur 1998 : 372 (preoccupied by Smith, 1969) Vaillant and Gagneur 1998 , HA , Oum-er-Rbia; Yang et al. 2007 Hemerodromia tigrigrana Vaillant & Gagneur, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Hemerodromia todrhana (Vaillant, 1956) Vaillant 1956, HA , Todrha; El Mezdi and Guidicelli 1985; Dakki 1997 ; Vaillant and Gagneur 1998 Hemerodromia zarcana Vaillant & Moubayed, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Empidinae Empidini Empis Linnaeus, 1758 Empis ( Coptophlebia ) confluens Becker, 1907 MA (Meknès), 19.v.1997, K. Deneš leg. – OUMNH Empis ( Empis ) decora Meigen, 1822 Bahid 2018 , Rif , Oued Tkarae ( PNPB ); Ebejer et al. 2019 , Rif , Oued Nakhla, Moulay Abdelsalam Empis ( Empis ) nikita Shamshev, 2018 Shamshev 2018 , AP , Essaouira Empis ( Kritempis ) taffertensis Daugeron, 2009 Daugeron 2009 , MA , forest of Taffert; Bahid 2018 Empis ( Leptempis ) tenuis Bahid & Daugeron, 2017 Bahid et al. 2017 , MA , Tizi-s'Tkrine (Jebel Amar, 1760 m); Bahid 2018 Empis ( Pachymeria ) suberis Becker, 1907 Ebejer et al. 2019 , Rif , Moulay Abdelsalam, Issaguen, Bab Berred, Jebel Lakraâ Empis ( Polyblepharis ) eumera Loew, 1866 Ebejer et al. 2019 , MA , Ifrane Empis ( Xanthempis ) chopardi Daugeron, 1997 Daugeron 1997 , MA , Ifrane; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) edithae Daugeron, 1997 Daugeron 1997 , HA , High Imminen, Tachdirt; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) ifranensis Daugeron, 1997 Daugeron 1997 , MA , Ifrane; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) styriaca (Strobl, 1893) Chvála and Wagner 1989 , HA [doubtful record]; Bahid 2018 Empis ( Xanthempis ) widanensis Bahid & Daugeron, 2018 Bahid et al. 2018 , Rif , Dayat Bayan Widane, Aïn Sedraouia, Tazia, Anissar, Lalla Outka Rhamphomyia Meigen, 1822 Rhamphomyia ( Rhamphomyia ) maroccana Collin, 2009 Chvála and Wagner 1989 , MA , Ifrane; Collin 2009 , MA , Ifrane; Bahid 2018 , Rif , Oued Akrir (Fifi) Rhamphomyia ( Holoclera ) tenuipes Becker, 1907* AP , Haenni pers. comm. Hilarini Hilara Meigen, 1822 Hilara algecirasensis Strobl, 1899 Ebejer et al. 2019 , MA , Lac Aguelmane Afennourir, HA , Ziz river Hilara almeriensis Strobl, 1906 Chvála 2008 , AA , Tifoultoute (1146 m) Hilara fusitibia Strobl, 1899 Chvála 2008 , MA , Ifrane (Forêt de Cédres, 1500 m) Hilara longeciliata Strobl, 1906 Chvála 2008 , AP , Rabat (near Oued Bou-Regreg, 0–10 m) Hilara schachti Chvála, 2008 Chvála 2008 , MA , Ifrane (Ghabat al Behar, 1650–1700 m); Bahid 2018 New record for Morocco Rhamphomyia ( Holoclera ) tenuipes Becker, 1907 Atlantic Plain: Essaouira, 6 km W, 3.iv.2002, Forêt de genévriers pâturée, 1♂1♀, J.-P. Haenni leg., coll. MHNN . Acknowledgements We are very grateful to Bradley Sinclair (Canadian Food Inspection Agency, Canada) and Adrian Plant (Mahasarakham University, Thailand) for reviewing parts of this family. DOLICHOPODIDAE K. Kettani, I.Ya. Grichanov, O.P. Negrobov Number of species: 112 . Expected: 300 Faunistic knowledge of the family in Morocco: poor Diaphorinae Argyra Macquart, 1834 Argyra argentina Meigen, 1824 Parent 1924 , Rif , Cap Spartel, Tétouan; Parent 1927 , Rif , Tétouan Argyra argyria (Meigen, 1824) Parent 1924 (females only), Rif , Cap Spartel, Tétouan; Kechev and Ivanova 2015 Argyra biseta Parent, 1929 Parent 1929a , Rif , Tanger; Vaillant 1955a Argyra grata Loew, 1857 20 Negrobov 1991 (no material provided) Asyndetus Loew, 1869 Asyndetus separatus (Becker, 1902) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Chrysotus Meigen, 1824 Chrysotus albibarbus Loew, 1857 Ebejer et al. 2019 , MA , Lac Aguelmane Afennourir (30 km SW of Azrou, 1760 m); Grichanov 2019 , AA , Aït Melloul Chrysotus gramineus (Fallén, 1823) Parent 1924 , Rif , Tanger; Parent 1927 Chrysotus larachensis Grichanov, Nourti & Kettani, 2020 Grichanov et al. 2020b , Rif , El Hamma (338 m) Chrysotus pennatus Lichtwardt, 1902 Ebejer et al. 2019 , Rif , Smir Barrage (145 m), AA , 1 km N of Tarda (Errachidia, 1023 m) Chrysotus suavis Loew, 1857 Grichanov 2009 , HA , Asni area (1100–1400 m); Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), MA , Dayat Ifrane (1607 m), HA , Tahanout (956 m); Dawah et al. 2020 Diaphorus Meigen, 1824 Diaphorus africus Parent, 1924 Parent 1924 , Rif , Tétouan, Tanger; Parent 1927 , Rif , Tanger; Ebejer et al. 2019 , Rif , Oued Siflaou (281 m); Grichanov 2019 , AA , Ouarzazate (1100 m) Diaphorus vitripennis Loew, 1859 Grichanov 2019 , AA , Aït Melloul Dolichopodinae Dolichopus Latreille, 1796 Dolichopus andalusiacus (Strobl, 1899) Ebejer et al. 2019 , AP , Loukous marsh (2 m); Nourti et al. 2019a , Rif , plage Stihat (0 m) Dolichopus griseipennis Stannius, 1831 Parent 1924 , Rif , Tanger; Parent 1927 ; Séguy 1930a , Rif , Tanger; Nourti et al. 2019a , Rif , Adrou ( PNPB , 556 m) Dolichopus sabinus Haliday, 1838 Ebejer et al. 2019 , Rif , Martil (9 m), Oued Laou (2 m); Nourti et al. 2019a , Rif , plage Stihat (4 m) Dolichopus scutopilosus Parent, 1933 Parent 1933 , HA , Arround Dolichopus signifer Haliday, 1832 Parent 1929b , "Maroc"; Séguy 1930a , MA , Ras el Ma; Grichanov 2019 , HA , Oukaimeden (2600 m) Dolichopus strigipes Verrall, 1875 Grichanov 2009 , AP , 40 km S Larache (0–20 m) Gymnopternus Loew, 1857 Gymnopternus assimilis (Staeger, 1842) Nourti et al. 2019a , Rif , Amsemlil (1067 m) Hercostomus Loew, 1857 Hercostomus apollo (Loew, 1869) Nourti et al. 2019a , Rif , Talassemtane (1696 m), Adrou ( PNPB , 556 m), Amsemlil ( PNPB , 1067 m) Hercostomus canariensis Santos Abreu, 1929 Grichanov et al. 2020a , Rif , Pont de Dieu (Akchour, 536 m); Nourti et al. 2019a (as H. aff. exarticulatoides Stackelberg, 1949) Hercostomus chetifer (Haliday, 1849) Ebejer et al. 2019 , Rif , Sidi Yahia Aarab (377 m) Hercostomus discriminatus Parent, 1925 Parent 1925 , Rif , "Favier, Environs de Tanger"; Parent 1927 , Rif , Tanger; Vaillant 1950 , Rif , Tanger Hercostomus exarticulatus (Loew, 1857) Vaillant 1956b , HA , Lac Tamhda (Anremer), Aguelmous; Grichanov et al. 2020a Hercostomus excipiens Becker, 1907 Parent 1924 , Rif , Tétouan; Parent 1927 ; Séguy 1930a , Rif , Oued Judios (Tanger); Nourti et al. 2019a , Rif , Talembote (440 m) Hercostomus germanus (Wiedemann, 1817) Parent 1924 , Rif , Cap Spartel; Parent 1927 ; Kettani and Negrobov 2016 , Rif , Chefchaouen, Ketama – MISR ( Rif , Ketama) Hercostomus longiventris (Loew, 1857) Vaillant 1956b , HA , Lac Tamhda (Anremer), Izourar Muscidideicus Becker, 1917 Muscidideicus praetextatus (Haliday, 1855) Grichanov 2019 , AP , Oualidia lagune Ortochile Berthold, 1827 Ortochile morenae (Strobl, 1899) = Hercostomus morenae (Strobl, 1899), in Becker 1917 : 225; Nourti et al. 2019a : 124 Grichanov and Nourti 2021 ; Nourti et al. 2019a , Rif , Mnezla (74 m), Talassemtane (980 m), Oued Ametrasse (841 m), estuary Tahaddart (dune marshland, 0 m) Ortochile nigrocaerulea Latreille, 1779 Parent 1924 , Rif , Tanger, Cap Spartel, Tétouan, Béni Hozmar; Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Oued Judios (Tanger); Grichanov 2009 , Rif , Ouezzane (300 m); Nourti et al. 2019a , Rif , Douar El Hamma (338 m), Triwa Bni Hassane (654 m), Taida (501 m) Platyopsis Parent, 1929 Platyopsis maroccanus (Parent, 1929) Parent 1929b , Rif , Tanger; Vaillant 1950 , Rif , Tanger Poecilobothrus Mik, 1878 Poecilobothrus appendiculatus (Loew, 1859) = Hercostomus appendiculatus (Loew): Ebejer et al. 2019 : 146 Parent 1924 , Rif , Tanger, Cap Spartel; Ebejer et al. 2019 , Rif , Oued Nakhla (200 m), Moulay Abdelsalam (965 m), Dardara (730 m), Cap Spartel (155 m); Nourti et al. 2019a , Rif , Perdicaris Park (223 m) Poecilobothrus infuscatus (Stannius, 1831) Ebejer et al. 2019 , Rif , Tahaddart (2 m); Rif (Tahaddart) – MISR Sybistroma Meigen, 1824 Sybistroma dufouri Macquart, 1838 = Haltericerus spathulatus Loew, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger Sybistroma obscurellum Fallén, 182320 = Hypophyllus obscurellus Fallén, in Dakki 1997 : 61 Dakki 1997 (no material provided) Sybistroma quadrifilatum (Strobl, 1899) = Sybistroma parvulum (Parent, 1927), in Grichanov and Nourti 2021 : 190 Grichanov and Nourti 2021 , Rif , Fahs Anjra (372 m) Sybistroma theodori Grichanov & Nourti, 2021 Grichanov and Nourti 2021 , Rif , Moulay Abdelsalam (649 m) Tachytrechus Haliday, 1851 Tachytrechus consobrinus (Haliday, 1851)20 Parent 1938 (no material provided) Tachytrechus goudoti (Macquart, 1842) = Dolichopus goudoti Macquart, 1842 Macquart 1842 , Rif , Tanger; Parent 1926 (redescription), 1927 Tachytrechus insignis (Stannius, 1831) Parent 1927 , "Maroc"; Séguy 1930a , Rif , Tanger, HA , Aguerd el Had, Talekjount (1000–1100 m); Vaillant 1956b , HA , Lac Tamhda (Anremer); Popescu-Mirceni 2011 , AP , Merja Zerga; Grichanov 2019 , AP , Essaouira Tachytrechus notatus (Stannius, 1831) Vaillant 1950 (no material provided), 1956b, HA , Lac Tamhda (Anremer); Grichanov 2009 , AA , 15 km SW Tazenakcht; Dawah et al. 2020 Tachytrechus planitarsis Becker, 1907 Vaillant 1950 , HA , Touggourt; Grichanov 2009 , AA , 15 km SW Tazenakcht; Grichanov 2019 , AA , Ouarzazate (1100 m) Hydrophorinae Anahydrophorus Becker, 1917 Anahydrophorus cinereus (Fabricius, 1805) = Scatophaga cinerea Fabricius, 1805: 205 Fabricius 1805 , AP , Mogador (Essaouira); Séguy 1930a , Rif , Tanger; Vaillant 1955a , AP , Temara, Port-Lyautey; Boumezzough and Vaillant 1986a , AP , beach of Rabat; Kettani and Negrobov 2016 , AP , Skhirat; AP (Skhirat) – MISR Aphrosylus Haliday, 1851 Aphrosylus maroccanus Vaillant, 1955 Vaillant 1955a , AP , Port Lyautey Aphrosylus mitis Verrall, 1912 Grichanov 2019 , AP , Oualidia lagune Aphrosylus raptor luteipes Parent, 1929 Parent 1929b , AP , Mogador (as a variation of Aphrosylus raptor Haliday, 1851); Vaillant 1955a ; Negrobov, 1979 (as a subspecies of Aphrosylus raptor Haliday, 1851); Kettani and Negrobov 2016 (as Aphrosylus raptor Haliday, 1851); Grichanov 2019 , AP , Oualidia lagune Aphrosylus temaranus Vaillant, 1955 Vaillant 1955a , AP , Temara; Grichanov 2019 , AP , Oualidia lagune Aphrosylus venator Loew, 1857 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger Epithalassius Mik, 1891 Epithalassius corsicanus Becker, 1910 Pârvu 2008 , AP , Merja Zergha, Cap Sim, Essaouira Hydrophoprus Fallén, 1823 Hydrophorus balticus (Meigen, 1824) Vaillant 1956b , HA , Jebel Toubkal, Lac Tamhda (Anremer), Oukaimeden, Izourar; Boumezzough and Vaillant 1986a , HA , Jebel Toubkal (3100 m); Grichanov 2019 , HA , Aguelmouss (2050 m), Oukaimeden (2600 m); Nourti et al. 2019a , MA , Mont Habri (2071 m) Hydrophorus nilicola Parent, 1927 = Hydrophorus viridis nilicola Parent, in Boumezzough and Vaillant 1986a : 297 Boumezzough and Vaillant 1986a , MA , Tizi-n'Imdrhas (1800 m), HA , Oued N'fis (650 m), AA , near Agadir N'oussbai (400 m); Grichanov 2019 , AP , Essaouira Hydrophorus oceanus (Macquart, 1838) = Hydrophorus bisetus Loew, 1857, in Parent 1927 , Séguy 1930a , Grichanov 2019 Parent 1927 , AP , Rabat; Séguy 1930a , Rif , Tandja el Balia (Tanger) ( Hydrophorus bisetus Loew); Vaillant 1955a , AP , Port-Lyautey; Boumezzough and Vaillant 1986a , AP , beach of Rabat Hydrophorus praecox (Lehmann, 1822) Parent 1924 , Rif , Cap Spartel, de Tanger à Tétouan, Rincón de Medik, Dar Riffien (Ceuta); Parent 1927 ; Séguy 1941a , HA , Toubkal (2500 m); Boumezzough and Vaillant 1986a , HA , Lac Tamhda, Lac Tamdhanit (Massif Anremer, 2900 m), Lac Izourar (Massif Azourki, 2650 m) Hydrophorus viridis (Meigen, 1824) 21 Parent 1927 , AP , Rabat; Boumezzough and Vaillant 1986a : 297 Liancalus Loew, 1857 Liancalus virens (Scopoli, 1763) Vaillant 1956b , HA , Toubkal, Assif Tassouat (M'Goum), Aguelmous, Sidi Chamarouch, Imi-N'Ifri; Boumezzough and Vaillant 1986a , HA , Tahanaout (750 m), Adrar Anremer (2900 m), AA , Jebel Siroua (3000 m); Kettani and Negrobov 2016 , AP , S-Tifni; Nourti et al. 2019a , Rif , Amsemlil env. (1059 m) – MISR ( AP , S Tifni) Machaerium Haliday, 1832 Machaerium maritimae Haliday, 1832 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Kettani and Negrobov 2016 , AP , Oued Bou-Regreg; Grichanov 2019 , AP , Oualidia lagune; AP (Oued Bou-Regreg) – MISR Orthoceratium Schrank, 1803 Orthoceratium sabulosum (Becker, 1907) = Orthoceratium lacustre (Scopoli, 1763) 22 , in Ebejer et al. 2019 : 146 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Thinophilus Wahlberg, 1844 Thinophilus ( Thinophilus ) flavipalpis (Zetterstedt, 1843) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Grichanov 2009 , AP , 40 km S Larache (0–20 m) Thinophilus ( Thinophilus ) indigenus Becker, 1902 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m), Oued Laou (2 m); Grichanov 2019 , AA , Ouarzazate (572 m) Thinophilus ( Thinophilus ) mirandus Becker, 1907 Negrobov 1971 , Rif , Tanger; Grichanov 2019 , AA , Ouarzazate (572 m) Thinophilus ( Schoenophilus ) versutus Haliday, 1851 = Schoenophilus versutus (Haliday, 1851), in Parent 1924 , 1927 Parent 1924 , Rif , Cap Spartel, Tétouan; Parent 1927 ; Pârvu et al. 2006 , AP , Merja Zerga; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), Dayat Tazia (733 m) Medeterinae Medetera Fisher, 1819 Medetera diadema (Linnaeus, 1767) = Medeterus (cf. diadema ), in Séguy 1953a : 84 Parent 1938 (no material provided); Séguy 1953a , AP , Rabat; Nourti et al. 2019a , Rif , Kitane (49 m), Rif , plage Stihat (beach) Medetera media Parent, 1925 Nourti et al. 2019a , Rif , Faculty of Sciences of Tétouan (garden: on trunk of olive tree, 14 m) Medetera micacea Loew, 1857 Ebejer et al. 2019 , Rif , Dardara (730 m); Nourti et al. 2019a , Rif , Issaguen (1547 m), HA , Lac Tislit (Imilchil, 2254 m) Medetera pallipes (Zetterstedt, 1843) 23 Nourti et al. 2019a , Rif , Douar El Hamma (338 m) Medetera petrophila Kowarz, 187720 Parent 1938 (no material provided) Medetera petrophiloides Parent, 1925 Nourti et al. 2019a , Rif , Issaguen (1547 m) Medetera aff. roghii Rampini & Canzoneri, 1979 Nourti et al. 2019a , Rif , Douar El Hamma (338 m) Medetera truncorum Meigen, 1824 24 Ebejer et al. 2019 a, Rif , Dardara (484 m) Medetera varvara Grichanov & Vikhrev, 2009 Grichanov and Vikhrev 2009 , AP , Essaouira Thrypticus Gerstäcker, 1864 Thrypticus bellus Loew, 1869 Séguy 1930a , EM , Dayat Sidi Kacem; Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), estuary Oued Tahaddart (0 m), Barrage 9 Avril, plage Stihat (0 m) Microphorinae Schistostoma Becker, 1902 Schistostoma eremita Becker, 1902 Ebejer et al. 2019 , AA , Ziz river (12 km S of Rissani, 737 m), Lac Tiffert (4 km W of Merzouga, 702 m) Neurigoninae Neurigona Rondani, 1856 Neurigona solodovnikovi Grichanov, 2010 = Neurigona punctifera Becker, 1907, in Kettani and Negrobov 2016 (misidentification) Grichanov 2010, AP , 40 km S Larache; Kettani and Negrobov 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m); Nourti et al. 2019a , Rif , Perdicaris Park (223 m), Taria Bni Faghloum (894 m), Chrafate (832 m) Parathalassiinae Microphorella Becker, 1909 Microphorella ulrichi Gatt, 2003 Gatt 2003 , Rif , Tanger, Oued Armal (Ksar Sghir) Parathalassius Mik, 1891 Parathalassius blasigii Mik, 1891 Ebejer et al. 2019 , AP , Larache (5 m) Peloropeodinae Chrysotimus Loew, 1857 Chrysotimus molliculoides Parent, 1937 Parent 1937a , MA , Ifrane (1600 m); Parent 1937b ; Nourti et al. 2019a , Rif , Dayat Tazia (733 m) Micromorphus Mik, 1878 Micromorphus albipes (Zetterstedt, 1843) Parent 1924 , Rif , Béni Hozmar (Tétouan); Parent 1927 , Rif , Tétouan; Kechev and Ivanova 2015 ; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), estuary Oued Tahaddart (0 m), Barrage 9 Avril, Kharouba, plage Stihat (0 m), Aïn Jdioui, EM , Bouanane (Figuig, 855 m), AA , Boudnib (951 m); Dawah et al. 2020 Micromorphus minusculus Negrobov, 2000 Nourti et al. 2019a , EM , Bouanane (Figuig, 855 m), AA , Taliouine (Taroudant, 1014 m); Grichanov 2019 , AA , Ouarzazate Rhaphiinae Rhaphium Meigen, 1803 Rhaphium appendiculatum Zetterstedt, 184920 = Rhaphium macrocerum (Parent, 1925), in Parent 1938 , Grichanov 2019 Parent 1938 (no material provided) Rhaphium brevicorne Curtis, 1835 = Xiphandrium pectinatum Becker, in Vaillant 1956b : 112, Grichanov 2019 Vaillant 1956b , AP , Rabat, HA , Oukaimeden; Kettani and Negrobov 2016 , AP , Rabat; Grichanov 2019 , HA , Oukaimeden (1000 m); Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Dayat Tazia (733 m), Moulay Abdelsalam (1177 m); AP (Rabat) – MISR Rhaphium caliginosum Meigen, 1824 = Raphium lanceolatum Loew, 1850, in Kazerani et al. 2013 Parent 1924 , Rif , Cap Spartel; Kazerani et al. 2013 (no material provided); Kechev 2017 ; Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), Dbani (Bni Selmane, 1046 m) Rhaphium fascipes (Meigen, 1824) = Porphyrops fascipes Meigen, 1824, in Parent 1927 Parent 1927 , Rif , Tétouan Rhaphium fissum Loew, 1850 Grichanov 2009 , AA , Tizi-n'Test pass (2100 m) Sciapodinae Sciapus Zeller, 1842 Sciapus adumbratus Becker, 1902 Grichanov and Negrobov 2014 , AP , near Essaouira, AA , Oued Souss, near Ouarzazate (1100 m) Sciapus costae (Mik, 1890) Negrobov 1991 (no material provided); Ebejer et al. 2019 , Rif , Oued Laou (30 m) Sciapus euzonus (Loew, 1859) = Psilopus euzonus Loew, in Dakki 1997 : 62 Parent 1927 , Rif , El Mahadi; Séguy 1930a , Rif , Tanger; Séguy 1941d , AA , Taroudant Sciapus glaucescens (Loew, 1856) Grichanov and Negrobov 2014 , AP , Oualidia Sciapus heteropygus Parent, 1926 Nourti et al. 2019c , Rif , Talassemtane National Park (1696 m) Sciapus holoxanthos Parent, 1926 Nourti et al. 2019c , Rif , Jbel Bouhachem, Adrou (556 m), Talassemtane National Park (1696 m) Sciapus laetus (Meigen, 1838) Grichanov 2009 , AA , 40 km S Larache (0–20 m); Ebejer et al. 2019 , Rif , Martil (9 m) Sciapus longulus (Fallén, 1823) 25 Pârvu et al. 2006 , AP , Merja Zerga Sciapus aff. negrobovi Naglis & Barták, 2015 Nourti et al. 2019a , Rif , plage Stihat (0 m), Kitane (49 m) (as Sciapus aff. negrobovi ); Nourti et al. 2019c Sympycninae Campsicnemus Haliday in Walker, 1851 Campsicnemus crinitarsis Strobl, 1906 Dakki 1997 (no material provided); Grichanov 2012 , AP , Essaouira; Kettani and Negrobov 2016 , Rif , Oued Amsa Campsicnemus curvipes (Fallén, 1823) 26 Frey 1936 (no material provided) Campsicnemus filipes Loew, 1859 Grichanov 2009 , AA , 40 km S Larache (0–20 m) Campsicnemus loripes (Haliday, 1832) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m) Campsicnemus magius (Loew, 1845) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Grichanov 2019 , AP , Essaouira Campsicnemus simplicissimus Strobl, 1906 Nourti et al. 2019a , Rif , plage Stihat (0 m) Sympycnus Loew, 1857 Sympycnus pulicarius (Fallén, 1823) Nourti et al. 2019a , HA , Aïn Taferaout (Sidi Masali, 1237 m) Syntormon Loew, 1857 Syntormon aulicus Meigen, 1824 = Eutarsus aulicus Meigen, in Parent 1924 , 1927 ; Séguy 1930a : 125 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Vaillant 1952 , 1956b , HA , Imi-N'Ifri Syntormon codinai Parent, 1924 Parent 1924 , Rif , Cap Spartel, Tanger; Parent, 1927, Rif , Tanger Syntormon denticulatus (Zetterstedt, 1843) = Syntormon pumilus Parent, 1925 (nec Meigen, 1824; misidentification), in Pârvu et al. 2006 , Grichanov 2019 Parent 1924 , Rif , Tétouan; Parent 1927 , Rif , Tétouan; Séguy 1930a , Rif , Tanger; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Nourti et al. 2019a , Rif , plage Stihat (0 m), Amlay (294 m), Koudiat Taifour (100 m), Amsemlil env. (1067 m), Dayat Tazia (733 m), Oued Souk Lhad (613 m), HA , Aïn Taferaout (Sidi Masali, 1237 m), AA , Taliouine (1014 m) Syntormon mikii Strobl, 1899 Parent 1927 , "Maroc"; Kechev and Ivanova 2015 ; Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Moulay Abdelsalam (1177 m) Syntormon monilis (Haliday, 1851) Parent 1924 , Rif , Cap Spartel; Parent 1927 , Rif , Cap Spartel Syntormon pallipes (Fabricius, 1794) Parent 1924 , Rif , Cap Spartel, Tétouan, Chefchaouen; Parent 1927 , Rif , Cap Spartel; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), Talassemtane (339 m), Perdicaris Park (223 m), Amsemlil env. (1067 m); Dawah et al. 2020 Syntormon pilitibia Grichanov, 2013 Nourti et al. 2019a , Rif , Amsemlil ( PNPB , 1067 m) Syntormon pumilus (Meigen, 1824) = Syntormon rufipes auctt. (nec Meigen, 1824; misidentification), in Pârvu et al. 2006 ; Grichanov 2019 Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate Syntormon zelleri (Loew, 1850) Vaillant 1956b , HA , Oukaimeden, Izourar; Pârvu et al. 2006 , AP , Merja Zerga; Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Amsemlil env. (1059–1067 m) Teuchophorus Loew, 1857 Teuchophorus cristulatus Mueffels & Grootaert, 1990 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Teuchophorus rifensis Nourti, Grichanov & Kettani, 2019 Nourti et al. 2019b , Rif , Oued Souk Lhad (613 m) Teuchophorus spinigerellus (Zetterstedt, 1843) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate Xanthochlorinae Xanthochlorus Loew, 1857 Xanthochlorus tenellus (Wiedemann, 1817) Grichanov 2009 , AA , 40 km S Larache (0–20 m); Ebejer et al. 2019 , Rif , Moulay Abdelsalam (1180 m) HYBOTIDAE K. Kettani, P. Gatt Number of species: 44 . Expected: 120 Faunistic knowledge of the family in Morocco: poor Ocydromiinae Bicellariini Bicellaria Macquart, 1823 Bicellaria spuria Fallén, 1816 Becker and Stein 1913 , Rif , Tanger Ocydromiini Ocydromia Meigen, 1820 Ocydromia glabricula (Fallén, 1816) Becker and Stein 1913 , Rif , Tanger Oropezella Collin, 1926 Oropezella sphenoptera (Loew, 1873) Cassar et al. 2005 , Rif , lagoon Smir Tachydromiinae Drapetini Chersodromia Haliday in Walker, 1851 Chersodromia albopilosa Chvála, 1970 Cassar et al. 2008 , Rif , Basin Laou Chersodromia nigrosetosa Chvála, 1970 Chvála 1981 (Ceuta); Chvála and Kovalev 1989 Chersodromia pseudohirta Chvála, 1970 Ebejer et al. 2019 , Rif , Kabila beach Crossopalpus Bigot, 1857 Crossopalpus aeneus (Walker, 1871) 27 Shamshev et al. 2005 , HA , Marrakech, Ouirgane Crossopalpus dilutipes (Strobl, 1906) Ebejer et al. 2019 , AP , 9 km SE of Aïn Chouk (Lower Loukous marsh, 6 m) Crossopalpus nigritellus (Zetterstedt, 1842) Ebejer et al. 2019 , Rif , Talassemtane (maison forestière), Issaguen (1620 m) Crossopalpus setiger (Loew, 1859) Ebejer et al. 2019 Rif , Smir lagoon Drapetis Meigen, 1822 Drapetis disparilis Frey, 1936 Chvála 1981 (Ceuta); Chvála and Kovalev 1989 Drapetis laevis Becker, 1914 Becker and Stein 1913 , Rif , Tanger; Kovalev 1970 ; Chvála and Kovalev 1989 Elaphropeza Macquart, 1827 Elaphropeza boergei Chvála, 1971 Ebejer et al. 2019 , Rif , Smir lagoon, Oued Laou (saltmarsh) Elaphropeza hutsoni Smith, 1967 Ebejer et al. 2019 , Rif , Jnane Niche (46 m) Stilpon Loew, 1859 Stilpon demnatensis Vaillant, 1956 28 Vaillant 1956b : 244, HA , Imi-N'Ifri Stilpon moroccensis Grootaert & Zouhair, 2021 Grootaert et al. 2021 , Rif , beach of Stehat, Bab Tariouant, Amsemlil, EM , Bouanane Stilpon subnubilus Chvála, 1988 Ebejer et al. 2019 , Rif , Smir lagoon, M'Diq (Kabila beach and dunes), Martil (beach and dunes) Tachydromiini Platypalpus Macquart, 1827 Platypalpus alluaudi Grootaert & Chvála, 1992 Grootaert and Chvála 1992 , HA , Chichaoua; Vaňhara and Rozkošný 1997 Platypalpus anomalicerus (Becker, 1902) = Coryneta aerivaga Séguy, in Séguy 1941d : 11 Séguy 1941d , EM , Oued Guir, AA , Agadir; Grootaert and Chvála 1992 , EM , Oued Guir, AA , Agadir Platypalpus annulatus (Fallén, 1815) Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 Platypalpus anomalitarsis Chvála & Kovalev, 1974 Ebejer et al. 2019 , MA , 10 km S of Azrou (1775 m), 10 km S of Azrou (1720 m), AA , Ziz river (30 km N of Erfoud, 894 m) Platypalpus approximatus (Becker, 1902) Grootaert and Chvála 1992 , AP , Casablanca Platypalpus asniensis Grootaert & Chvála, 1992 Grootaert and Chvála 1992 , HA , Asni; Vaňhara and Rozkošný 1997 Platypalpus calceatus (Meigen, 1822) Pârvu et al. 2006 , MA , Meknès, AA , Foum Zghouig Platypalpus chillcotti Chvála, 1981 Grootaert and Chvála 1992 , MA , Ifrane Platypalpus chrysonotus (Strobl, 1899) Ebejer et al. 2019 , Rif , Oued Laou (saltmarsh) Platypalpus desertorum (Becker, 1907) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Platypalpus distichus Grootaert & Chvála, 1992 Ebejer et al. 2019 , AP , 9 km SE of Aïn Chouk (Lower Loukous marsh, 6 m), AA , Ziz river (10 km S of Errachidia, 1008 m) Platypalpus flavicornis (Meigen, 1822) Ebejer et al. 2019 , AA , 2 km N of Erfoud (818 m), Ziz river (30 km N of Erfoud, 894 m) Platypalpus longicauda Grootaert & Chvála, 1992 Ebejer et al. 2019 , Rif , Smir lagoon Platypalpus lyneborgi Chvála, 1981 Grootaert and Chvála 1992 , AP , Dradek, MA , Azrou Platypalpus nigritarsis (Fallén, 1816) Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 Platypalpus obscuripes (Strobl, 1899) Ebejer et al. 2019 , Rif , Martil (9 m), AP , Larache (Loukous marsh, 2 m) Platypalpus ostiorum (Becker, 1902) Grootaert and Chvála 1992 Platypalpus pachycerus (Collin, 1949) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Platypalpus pallidiventris (Meigen, 1822) Grootaert and Chvála 1992 , MA , Ifrane; Maarouf 2003 , HA , Chaouia Platypalpus pseudoexiguus (Strobl, 1909) Ebejer et al. 2019 , Rif , Oued Laou (saltmarsh) Platypalpus pseudounguiculatus (Strobl, 1909) = Tachydromia pseudounguiculata Strobl 1909 Grootaert and Chvála 1992 Platypalpus riojaensis Chvála, 1981 Grootaert and Chvála 1992 , EM , Oujda, MA , Meknès, HA , Chichaoua Platypalpus turgidus (Becker, 1907) Grootaert and Chvála 1992 , MA , Takkat-n- Sountat Platypalpus vockerothi Chvála, 1981 Grootaert and Chvála 1992 , HA , Asni Tachydromia Meigen, 1803 Tachydromia arrogans (Linnaeus, 1761) Ebejer et al. 2019 , Rif , Oued Laou, El-Fahsa (maquis) Tachydromia annulimana Meigen, 1822 29 = Tachista annulimana Meigen, in Becker and Stein 1913 : 84 Becker and Stein 1913 , Rif , Tanger Tachydromia undulata (Strobl, 1906) Chvála 1969 Platypezoidea PHORIDAE K. Kettani, H. Disney Number of species: 3 . Expected: >400 Faunistic knowledge of the family in Morocco: very poor Diplonevra Lioy, 1864 Diplonerva crassicornis (Meigen, 1830) = Phora crassicornis Meigen, in Meigen 1830 : 220 = Dohrniphora dudai Schmitz, in Schmitz 1920 : 100 Raclin 1957 ; Mouna 1998 ; AP (Rabat) – MISR Diplonevra tangeriana (Becker, 1913) 30 = Phora tangeriana Becker and Stein, in Becker and Stein 1913 : 90; Schmitz 1949 : 238 ( Species incerta ) Becker and Stein 1913 , Rif , Tanger; Schmitz 1949 Megaselia Rondani, 1856 Megaselia minor (Zetterstedt, 1848) = Trineura minor Zetterstedt, in Zetterstedt 1848 : 2864; Wood 1912 : 167 = Aphiochaeta angustifrons Wood, in Wood 1912 : 167; Disney 1984 : 239 = Phora minor Shob, in Mouna 1998 : 86 Zetterstedt 1848 ; Wood 1912 ; Disney 1984 ; Mouna 1998 ; AP (Rabat) – MISR PLATYPEZIDAE K. Kettani, M.J. Ebejer Number of species: 3 . Expected: 6 Faunistic knowledge of the family in Morocco: poor Platypezinae Lindneromyia Meigen, 1804 Lindneromyia dorsalis (Meigen, 1804) Chandler 2001 , Rif , Chefchaouen (600 m), AP , Rabat, Maâmora; Tkoč and Roháček 2014 ; AP (Maâmora) – MISR Microsania Zetterstedt, 1837 Microsania raclinae Collart 31 Mouna 1998 : 86 Protoclythia Kessel, 1949 Protoclythia rufa (Meigen, 1830) Ebejer et al. 2019 , Rif , Tahaddart (1 m) LONCHOPTERIDAE K. Kettani, M. Barták Number of species: 4 . Expected: 5 Faunistic knowledge of the family in Morocco: moderate Lonchopterinae Lonchoptera Meigen, 1803 Lonchoptera bifurcata (Fallén, 1810) = Dispa furcata Fallén, in Vaillant 1989 : 217 = Muscidora furcata Fallén, in Mouna 1998 : 86 Vaillant 1989 ; Mouna 1998 Lonchoptera fallax De Meijere, 1906 = Muscidora fallax Meigen, in Mouna 1998 : 86 Mouna 1998 Lonchoptera lutea Panzer, 1809 = Muscidora lutea Panzer, in Mouna 1998 : 86 Vaillant 1989 , HA (>3000 m); Mouna 1998 ; Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 ; MA – MISR Lonchoptera tristis Meigen, 1824 = Muscidora tristis Meigen, in Mouna 1998 : 86 Mouna 1998 Syrphoidea PIPUNCULIDAE K. Kettani, M.J. Ebejer Number of species: 16 . Expected: 50 Faunistic knowledge of the family in Morocco: poor Chalarinae Chalarus Walker, 1834 Chalarus brevicaudis Jervis, 1992 Ebejer and Kettani 2019b , Rif , Dardara (730 m), Azilane (1255 m), El Hamma (338 m) Chalarus sp. aff. brevicaudis Jervis, 1992 Ebejer and Kettani 2019b , Rif , Azilane (1255 m) Pipunculinae Eudorylini Claraeola Aczél, 1940 Claraeola sp. aff. halterata (Meigen, 1838) Ebejer and Kettani 2019b , Rif , Akchour (424 m) Clistoabdominalis Skevington, 2001 Clistoabdominalis dilatatus (De Meyer, 1997) Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m), El Hamma (338 m) Dasydorylas Skevington, 2001 Dasydorylas setosus (Becker, 1908) Kehlmaier 2005 ; Motamedinia et al. 2017 Eudorylas Aczél, 1940 Eudorylas ibericus Kehlmaier, 2005 Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m) Pipunculini Pipunculus Latreille, 1802 Pipunculus carlestolrai Kuznetzov, 1993 Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m) Tomosvaryellini Tomosvaryella Aczél, 1939 Tomosvaryella cilifemorata (Becker, 1907) Ebejer and Kettani 2019b , Rif , Adrou ( PNPB , 556 m) Tomosvaryella debruyni De Meyer, 1995 Ebejer and Kettani 2019b , Rif , Adrou ( PNPB , 556 m) Tomosvaryella frontata (Becker, 1897) Ebejer and Kettani 2019b , Rif , Oued Mhajrate (Ben Karrich, 67 m) Tomosvaryella geniculata (Meigen, 1824) Ebejer and Kettani 2019b , HA , Lac Tislite (Imilchil, 2254 m) Tomosvaryella kuthyi Aczél, 1944 Ebejer and Kettani 2019b , Rif , El Hamma (338 m), Akchour (424 m), Barrage Smir (27 m) Tomosvaryella minima (Becker, 1897) Ebejer and Kettani 2019b , Rif , Koudiat Taifour (100 m) Tomosvaryella mutata (Becker, 1898) Ebejer and Kettani 2019b , Rif , Jebel Lakraâ (Talassemtane, 1596 m) Tomosvaryella trichotibialis De Meyer, 1995 Ebejer and Kettani 2019b , Rif , Koudiat Taifour (100 m) Tomosvaryella sp. subvirescens group Ebejer and Kettani 2019b , AP , Loukous marsh (Larache) SYRPHIDAE K. Kettani, M.C.D. Speight Number of species: 166 . Expected: more than 200 Faunistic knowledge of the family in Morocco: moderate Eristalinae Brachyopini Brachyopa Meigen, 1822 Brachyopa atlantea Kassebeer, 2000 Kassebeer 2000 , HA , Ouirgane (1000 m); Speight 2018 ; Sahib et al. 2020 Chrysogaster Meigen, 1803 Chrysogaster basalis Loew, 1857 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Kassebeer 1999d ; Sahib et al. 2020 Ighboulomyia Kassebeer, 1999 Ighboulomyia atlasi Kassebeer, 1999 Kassebeer 1999c , MA , Azrou, Umgebung, Timahdit, Ighböula Ulaichuor, Quellteich; Sahib et al. 2020 Myolepta Newman, 1838 Myolepta difformis Strobl in Czerny & Strobl 1909 = Myolepta philonis Séguy, 1961, in Dirickx 1994 : 93 Dirickx 1994 , HA ; Reemer et al. 2004 , MA , HA ; Speight 2013 , 2018 ; Sahib et al. 2020 Neoascia Williston, 1886 Neoascia clausseni Hauser & Kassebeer, 1998 = Neoascia podagrica (Fabricius, 1775), in Gil Collado 1929: 40; Claussen 1989b: 373 Gil Collado 1929a ; Claußen 1989b ; Dirickx 1994 ; Hauser and Kassebeer 1998 , MA , HA , Taroudant (1800 m); Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; Sahib et al. 2020 , Rif , Oued Jnane Niche, Oued Maggou Orthonevra Macquart, 1829 Orthonevra bouazzai Kassebeer, 1999 Kassebeer 1999d , MA ; Sahib et al. 2020 Orthonevra brevicornis (Loew, 1843) 32 Sahib et al. 2020 , Rif , Aïn Afersiw Orthonevra elegans (Meigen, 1822) Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Orthonevra schachti Claußen, 1989b Claußen 1989b , HA , Oukaimeden (2600 m); Dirickx 1994 ; Schmid 1995 ; Kassebeer 1999d , MA ; Sahib et al. 2020 Riponnensia Maibach, Goeldlin & Speight, 1994 Riponnensia longicornis (Loew, 1843) = Orthonevra longicornis Loew, in Kanervo 1939 : 2; Séguy 1961 : 23; Kassebeer 1999c : 162 Kanervo 1939 ; Séguy 1961 , AP ; Claußen 1989b ; Kassebeer 1999c , MA , HA ; Dirickx 1994 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 Riponnensia splendens (Meigen, 1822) = Chrysogaster splendens (Meigen), in Gil Collado 1929: 405 = Orthonevra splendens (Meigen), in Claußen 1989b : 363, 373; Dirickx 1994 : 97 Gil Collado 1929a , Rif ; Mouna 1998 ; Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Kassebeer 1999c , MA ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh; AP (Dradek) – MISR Callicerini Callicera Panzer, 1806 Callicera fagesi Guérin-Meneville, 1844 Kassebeer 1998a , HA , Ouirgane (1000 m); Sahib et al. 2020 Callicera rufa Schummel, 1842 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Cerioidini Ceriana Rafinesque, 1815 Ceriana conopsoides (Linnaeus, 1758) = Cerioides conopsoides Linnaeus, in Séguy 1930a : 131 Séguy 1930a , MA , Ras El Ksar; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Maghrawa, Maâmora) – MISR Ceriana vespiformis (Latreille, 1804) = Cerioides vespiformis Latreille, in Becker and Stein 1913 : 88; Gil Collado 1929: 414; Séguy 1930a : 131; Kanervo 1939 : 5; Leclercq 1961: 242 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , HA ; Séguy 1930a , AP , Rabat, Casablanca, MA , Tizi-s'Tkrine, Aïn Leuh, Meknès; Kanervo 1939 , MA ; Leclercq 1961a , Rif , Melillia, MA , Dayat Aoua, Aïn Leuh; Claußen 1989b ; Dirickx 1994 ; Steenis et al. 2016 ; Speight 2018 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , 1 km after Dardara, Meadow Mizoghar, Oued Achekrade Sphiximorpha Rondani, 1850 Sphiximorpha subsessilis (Illiger in Rossi, 1807) Steenis et al. 2016 Eristalini Anasimyia Schiner, 1864 Anasimyia contracta Caußen & Torp, 1980 Kassebeer 1998a , MA , Timahdit (1850 m); Sahib et al. 2020 Eristalinus Rondani, 1845 Eristalinus aeneus (Scopoli, 1763) = Lumpetia aenea (Scopoli), in Becker and Stein 1913 : 86 = Eristalis aeneus (Scopoli), in Gil Collado 1929: 406, 407; Leclercq 1961: 242 = Lathyrophtalmus aeneus (Scopoli), in Séguy 1930: 129; Kanervo 1939 : 5 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , Tanger, AP , Mogador; Séguy 1930a , AP , Casablanca; Kanervo 1939 ; Timon-David 1951 , AP , Rabat; Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , Jumb Kitane, HA , vicinity of Asni Eristalinus megacephalus (Rossi, 1794) = Eristalis quinquelineatus Fabricius, in Becker and Stein 1913 : 85, Gil Collado 1929: 407, = Lathyrophthalmus quinquelineatus Fabricius, in Séguy 1930a : 129 Gil Collado 1929a , Rif , Tanger; Séguy 1930a , AP , Rabat, Oued Korifla, Sidi Bettache; Séguy 1961 ; Dakki 1997 ; Claußen 1989b ; Dirickx 1994 ; Dousti and Hayat 2006 ; Sahib et al. 2020 ; AP (Rabat) – MISR Eristalinus sepulchralis (Linnaeus, 1758) = Eristalis sepuleralis Linnaeus, in Becker and Stein 1913 : 85, Gil Collado 1929: 406 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , AP , Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eristalinus taeniops (Wiedemann, 1818) = Eristalis taeniops Wiedemann, in Becker and Stein 1913 : 85 = Eristalodes taeniops Wiedemann, in Séguy 1930a : 130 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Oued Korifla, Rabat, HA , Tenfecht (Takeljount); Leclercq 1961a , MA , Dayet Aoua; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; Dousti and Hayat 2006 ; Koçak and Kemal 2010 ; Speight 2013 , 2018 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Oued Martil, Halouma Kitane, Oued Sidi Yahia Aârab, MA , bridge Oued Oum-er-Rbia (Douar Ahl Souss), HA , Lac Oukaimeden; AP (Rabat), MA (Volubilis) – MISR Eristalis Latreille, 1804 Eristalis arbustorum (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1930a , MA , Aïn Leuh, Ras El Ksar, forest of Taffert; Kanervo 1939 , Rif ; Timon-David 1951 , AP , Rabat, MA , Ifrane; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Aïn Sidi Brahim Ben Arrif, MA , vicinity of Ifrane, HA , vicinity of Asni, Lac Oukaimeden, AA , Douar Issafen, Douar Issafen; MA , HA – MISR Eristalis jugorum Egger, 1858 33 Dakki 1997 Eristalis pertinax (Scopoli, 1763) Séguy 1930a , AP , Oued Korifla, Sidi Bettache, MA , Forêt de Timelilt; Claußen 1989b ; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 Eristalis similis (Fallén, 1817) = Eristalis pratorum Meigen, in Gil Collado 1929: 407 Gil Collado 1929a , Rif ; Séguy 1961 ; Claußen 1989b , HA , Oukaimeden (2600 m); Dirickx 1994 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , maison forestière, HA , Douar Zaouiat Cheikh, Lac Oukaimeden Eristalis tenax (Linnaeus, 1758) = Eristalomyia tenax Linnaeus, in Séguy 1930a : 130; Timon-David 1951 : 146 Gil Collado 1929a , Rif , AP ; Kanervo 1939 , MA ; Séguy 1949a , AA ; Timon-David 1951 ; Leclercq 1961a ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 , Rif , Village Sebt Zinnat, Belyounech, Aïn Takhninjoute, maison forestière, Jumb Kitane, meadow Fahs Lmhar, HA , Douar Zaouiat Cheikh, vicinity of Asni, Lac Oukaimeden Helophilus Meigen, 1822 Helophilus trivittatus (Fabricius, 1805) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Mallota Meigen, 1822 Mallota cimbiciformis (Fallén, 1817) = Mallota eristaloides Loew, in Becker and Stein 1913 : 85 Becker and Stein 1913 , Rif , Tange; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Mallota dusmeti Andreu, 1926 Kassebeer 1998a , HA , Ouirgane; Sahib et al. 2020 Melanogaster Rondani, 1857 Melanogaster lindbergi Kassebeer, 1999 = Chrysogaster macquardti Loew, in Becker and Stein 1913 : 87 = Chrysogaster viduata Meigen, in Kanervo 1939 : 2, Séguy 1961 : 27, 28 = Chrysogaster lucida (Scopoli), in Claußen 1989b : 372 Becker and Stein 1913 , Rif ; Kanervo 1939 , MA ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1999d , MA ; Popov et al. 2020 ; Sahib et al. 2020 Myathropa Rondani, 1845 Myathropa florea (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , MA , Aïn Leuh; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Oued à 15 km de Fifi Parhelophilus Girschner, 1897 Parhelophilus versicolor (Fabricius, 1794) = Helophilus versicolor (Fabricius), in Gil Collado 1929: 407 Gil Collado 1929a , AP , Oulad Mesbah; Claußen 1989b ; Dirickx 1994 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eumerini Eumerus Meigen, 1822 Eumerus amoenus Loew, 1848 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 , AA , Douar Aourir, beach of Tamelallt Eumerus barbarus (Coquebert, 1804) = Eumerus australis Meigen, in Gil Collado 1929: 412 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1961 ; Claußen 1989b , MA , Azrou (1900 m); Dirickx 1994 ; Mouna 1998 ; Speight 2013 , 2018 ; Steenis et al. 2017 , AA , 11 km NW Taliouine, S Aït-Baha, 10 km NE Tafraoute; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 ; AP (Cap Cantin) – MISR Eumerus basalis Loew, 1848 = Eumerus angusticornis Rondani, in Séguy 1930a : 130 = Eumerus basalis Loew, in Mouna 1998 : 86 Séguy 1930a , MA , forest of Timelilt; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus caballeroi Gil Collado, 1929 Gil Collado 1929a , AP , Laguna Gedira; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus hungaricus Szilády, 1940 Speight 2018 Eumerus lunatus (Fabricius, 1794) = Eumerus lunulatus Fabricius, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger; Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus melotus (Séguy, 1941) = Lampetia melota Séguy, in Séguy 1941d : 13 Séguy 1941d , AA , Agadir; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus nudus Loew, 1848 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus obliquus (Fabricius, 1805) Sahib et al. 2020 , Rif , Oued Jnane Niche, EM , Oued Khemis Eumerus ornatus Meigen, 1822 Séguy 1930a , MA , Aïn Leuh, forest of Timelilt; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Oued Cherrat) – MISR Eumerus pulchellus Loew, 1848 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Eumerus punctifrons Loew, 1857 Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus pusillus Loew, 1848 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Sahib et al. 2020 Eumerus sabulonum (Fallén, 1817) Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Eumerus schmideggeri Steenis, Hauser & Zuijen, 2017 Steenis et al. 2017 , AA , Sidi R'bat (37 km S Agadir); Sahib et al. 2020 Eumerus strigatus (Fallén, 1817) 34 Kanervo 1939 , Rif , HA ; Timon-David 1951 , AP , Rabat, Sehoul; Claußen 1989b , MA , Azrou (1700 m), HA , Oukaimeden (2200 m), Ansegmir-Tal W Midelt; Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Speight 2013 ; Sahib et al. 2020 Eumerus subornatus Claußen, 1989b Claußen 1989b , HA , Tizi-n'Test (1900 m); Schmid 1995 ; Speight 2013 , 2018 ; Dirickx 1994 ; Sahib et al. 2020 Eumerus truncatus Rondani, 1868 Steenis et al. 2017 , AA , S. Aït-Baha, 11 km NW Taliouine, 25 km NE Tizinit, 20 km E Tizinit, Assaka; Speight 2018 ; Sahib et al. 2020 Merodon Meigen, 1803 35 Merodon aberrans Egger, 1860 = Lampetia aberrans Egger, in Séguy 1961 : 174 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Vujic et al. 2011; Speight 2013 , 2018 ; Sahib et al. 2020 Merodon aeneus Meigen, 1822 36 = Lampetia aenea Meigen, in Becker and Stein 1913 : 86; Kanervo 1939 : 5; Timon-David 1951 : 146 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , AP , Vallée Oued Korifla, MA , Tizi-s'Tkrine; Kanervo 1939 , Rif ; Timon-David 1951 ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Merodon arrasus Becker, 1921 37 Becker 1921 , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon aurifer Loew, 1862 = Merodon distinctus Palma, 1864 = Lampetia distincta Palm, in Timon-David 1951 : 146 Timon-David 1951 , AP , Zaer, MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; Vujić et al. 2021c Merodon avidus Rossi, 1782 38 = Lampetia spinipes (Fabricius), in Becker and Stein 1913 : 86; Timon-David 1951 : 146 = Merodon spinipes (Fabricius), in Gil Collado 1929: 409 = Lampetia avida Rossi, in Séguy 1961 : 176 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , HA ; Séguy 1930a , AP , Chellah (Rabat), Oued Korifla, MA , Aïn Leuh; Kanervo 1939 ; Timon-David 1951 , AP ; Séguy 1961 ; Claußen 1989b , HA , Oukaimeden (2600 m); Hurkmans 1993 ; Dirickx 1994 ; Mouna 1998 ; Koçak and Kemal 2010 ; Likov et al. 2020 ; Sahib et al. 2020 , HA , Lac Oukaimeden Merodon bequaerti Hurkmans, 1993 Vujić et al. 2020a , EM , Mountain of Beni-Snassen, MA , Azrou Merodon cabanerensis Marcos-García, Vujić & Mengual, 2007 Vujić et al. 2018 , HA , Ait Mhamed (Azilal, 1700 m); Speight 2018 ; Sahib et al. 2020 ; Vujić et al. 2021 Merodon calcaratus (Fabricius, 1794) Vujić et al. 2021b , EM , Mountains of Béni Snassen, near Nador, AP , 38 km SW of El Jadida, Garbouz Merodon chalybeus Wiedemann in Meigen, 1822 = Lampetia spicata Becker, in Timon-David 1951 : 146 = Merodon spicatus Becker, in Claußen 1989b : 365, 373 Timon-David 1951 , AP , forest of Maâmora; Claußen 1989b , HA (2500 m); Dirickx 1994 ; Mouna 1998 ; Marcos-Garcia et al. 2007 ; Speight 2013 , 2018 ; Sahib et al. 2020 ; AP (Cap Cantin) – MISR Merodon clavipes (Fabricius, 1781) Hurkmans 1993 , MA ; Marcos-García et al. 2007; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon eques Fabricius, 1805 = Lampetia eques (Fabricius), in Séguy 1961 : 178 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Ebejer and Bensusan 2010 , AA ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Merodon equestris (Fabricius, 1794) = Eristalis ferrugineus (Fabricius), in Fabricius 1805 : 240 = Lampetia equestris (Fabricius), in Séguy 1961 : 178, 179 Fabricius 1805 , AP ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon escalerai Gil Collado, 1929 Gil Collado 1929a , AP , Essaouira; Claußen 1989b ; Dirickx 1994 ; Speight 2018 ; Sahib et al. 2020 Merodon femoratus Sack, 1913 = Merodon biarcuatus Curran, 1939, in Curran 1939: 6, 7; Claußen 1989: 373; Dirickx 1994 : 79; Koçak and Kemal 2010 : 1199; Speight 2018 : 137 = Merodon elegans Hurkmans, 1993, in Hurkmans 1993 : 195; Schmid 1995 ; Marcos-Garcia et al. 2007 : 553; Speight 2018 : 141 Curran 1939, AP , forest of Maâmora (Rabat); Claußen 1989b ; Hurkmans 1993 , AP ; Dirickx 1994 ; Schmid 1995 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Speight 2018 ; Likov et al. 2020 Merodon geniculatus Strobl, 1909 Gil Collado 1929a , Rif ; Claußen 1989b , HA (2500 m); Dirickx 1994 ; Mouna 1998 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Speight 2013 , 2018 ; Sahib et al. 2020 , HA , Lac Oukaimeden – MISR Merodon hurkmansi Marcos-García, Vujić & Mengual, 2007 Marcos-García et al. 2007 Merodon ibericus Vujić, 2015 = Merodon bicolor Gil Collado, 1930 Popović et al. 2015 , MA , Azrou, Ifrane; Acanski et al. 2016; Speight 2018 ; Sahib et al. 2020 Merodon italicus Rondani, 1845 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 Merodon longicornis Sack, 1913 Claußen and Hauser 1990 , MA ; Dirickx 1994 ; Sahib et al. 2020 Merodon maroccanus Gil Collado, 1929 Gil Collado 1929a , AP , Essaouira; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon minutus Strobl, 1893 = Lampetia minutus Strobl, in Séguy 1961 : 180 Séguy 1961 ; Leclercq 1961a ; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Merodon monticolus Villeneuve, 1924 Kassebeer 1998a , HA , Ouirgane, Taftraoute, Taliouine; Sahib et al. 2020 Merodon murorum (Fabricius, 1794) = Syrphus murorum (Fabricius), in Fabricius 1794 : 288 = Merodon auripilus (Meigen), in Meigen 1830 : 354 = Lampetia auripila Meigen, in Séguy 1941d : 13; Mouna 1998 : 86 Fabricius 1794 ; Meigen 1830 ; Séguy 1941d , AA , Agadir; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Vujić et al. 2018 , AP , Essaouira; Sahib et al. 2020 Merodon pruni (Rossi, 1790) = Lampetia pruni (Rossi), in Becker and Stein 1913 : 86 = Merodon pruni var. obscurus Gil Collado, in Gil Collado 1929: 407, 408 Becker and Stein 1913 ; Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Hurkmans, 1993; Dirickx 1994 ; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon pumilus Macquart, 1849 Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m); Sahib et al. 2020 Merodon rufus Meigen, 1838 = Lampetia rufa Meigen, 1838, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon segetum (Fabricius, 1794) Peck 1988 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon serrulatus Wiedemann in Meigen, 1822 Hurkmans 1993 ; Speight 2013 , 2018 ; Sahib et al. 2020 ; Vujić et al. 2020a Merodon sophron Hurkmans, 1993 Hurkmans 1993 , MA , Azrou; Schmid 1995 ; Koçak and Kemal 2010 ; Vujić et al. 2020a , MA , Azrou Merodon tangerensis Hurkmans, 1993 Hurkmans 1993 , Rif , Tanger; Schmid 1995 ; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon tricinctus Sack, 1913 = Lampetia tricincta Sack, in Timon-David 1951 : 146 Timon-David 1951 , MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Merodon unguicornis Strobl, 1909 Ebejer et al. 2019 , MA , 10 km S of Azrou (1775 m); Sahib et al. 2020 , Rif , maison forestière Platynochaetus Wiedemann, 1830 Platynochaetus rufus Macquart, 1835 Gil Collado 1929a , AP , Mogador; Dirickx 1994 ; Sahib et al. 2020 Platynochaetus setosus (Fabricius, 1794) Gil Collado 1929a , HA , Marrakech; Séguy 1953a , AA , Souss: Aïn Chaib; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Milesiini Milesia Latreille, 1804 Milesia crabroniformis (Fabricius, 1775) Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Sahib et al. 2020 Spilomyia Meigen, 1803 Spilomyia maroccana Kuznetzov, 1997 = Spilomyia digitata (Rondani), in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b , HA , Tizi-n'Test (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Kuznetzov 1997 ; Dirickx 1994 ; Kassebeer 1999d ; Steenis 2000 ; Sahib et al. 2020 Syritta Le Peletier & Serville, 1828 Syritta flaviventris Macquart, 1842 Claußen 1989b , AP , Kénitra; Dirickx 1994 ; Sahib et al. 2020 , EM , farm Saf-Saf Syritta pipiens (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gill Collado 1929a; Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès; Kanervo 1939 ; Timon-David 1951 , AP , Rabat, AA , Agdz; Leclercq 1961a , EM , Melilla; Claußen 1989b , HA , Tizi-n'Test (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; Sahib et al. 2020 , Rif , Bni Maaden, dam Moulay Bouchta, Oued Koub, Douar Kitane, Oued Maggou, EM , farm Saf-Saf, MA , vicinity of Ifrane, HA , vicinity of Asni, Tizi-n'Test, AA , Oued Assa; AP (Rabat), MA (Aïn Leuh), SA – MISR Temnostoma Le Peletier & Serville, 1828 Temnostoma bombylans (Fabricius, 1805) Séguy 1961 , MA ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xylota Meigen, 1822 Xylota segnis (Linnaeus, 1758) = Zelima ( Xylota ) segnis Linnaeus, in Becker and Stein 1913 : 86; Timon-David 1951 : 147 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Timon-David 1951 , HA , Zaouia Ahansal; Leclercq 1961a , Rif , Azib de Ketama; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Aïn El Maounzil, Oued Koub, Oued Sidi Ben Saâda, HA , vicinity of Asni; HA (Zaouiet Ahansal) – MISR Rhingiini Cheilosia Meigen, 1822 Cheilosia brunnipennis Becker, 1894 = Chilosia flavipes (Panzer), in Kanervo 1939 : 2 Kanervo 1939 , HA ; Kassebeer, 1998c, MA ; Séguy 1961 ; Claußen 1989b ; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia flavipes (Panzer, 1798) Mouna 1998 : 86 Cheilosia grossa (Fallén, 1817) Kassebeer 1998c , HA , Asif Mellah, Tizi-n'Tichka; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia latifrons (Zetterstedt, 1843) = Cheilosia intonsa Loew, in Timon-David 1951 : 144 Timon-David 1951 , AP , Sehoul; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Kassebeer 1998c , Rif , Ouezzane, AP , Oued Loukous, Larache, MA , Ifrane, HA , Oukaimeden; Sahib et al. 2020 Cheilosia mutabilis (Fallén, 1817) Speight 2013 , 2018 Cheilosia griseiventris Loew, 1857 = Chilosia marokkana Becker, in Becker 1894: 395; Becker and Stein 1913 : 87 = Cheilosia maroccana Becker, 1894, in Gil Collado 1929: 405 Becker 1894; Becker and Stein 1913 ; Gil Collado 1929a ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1998c , MA , HA ; Sahib et al. 2020 Cheilosia paralobi Malski, 1962 = Cheilosia longula (Zetterstedt, 1838), in Gil Collado 1929: 405 Gil Collado 1929a ; Claußen 1989b , MA , Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Kassebeer 1998c , HA ; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia parva Kassebeer, 1998 Kassebeer 1998c , MA , Azrou, Ifrane (1650 m); Claußen and Speight 2007 ; Sahib et al. 2020 Cheilosia rodgersi Wainwright, 1911 Becker and Stein 1913 , Rif , Tanger; Claußen 1989a ; Dirickx 1994 ; Kassebeer 1998c , Rif , Tanger; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia scutellata (Fallén, 1817) Gil Collado 1929a , Rif ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1998c , Rif , Chefchaouen, MA , Ouiouane, Ifrane; Sahib et al. 2020 Cheilosia soror (Zetterstedt, 1843) = Cheilosia rufipes (Preyssler, 1793), in Claussen and Hauser 1990: 436, Kassebeer 1998c : 65 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Kassebeer 1998c , MA , Ifrane; Dirickx 1994 ; Sahib et al. 2020 Cheilosia variabilis (Panzer, 1798) Kassebeer 1998c , MA , Ifrane; Speight 2013 , 2018 ; Khaganinia and Kazerani 2014 ; Sahib et al. 2020 Ferdinandea Rondani, 1844 Ferdinandea fumipennis Kassebeer, 1999 Kassebeer 1999b , MA , Ifrane, Azrou, HA , Marrakech, Ouirgane; Speight 2013 , 2018 ; Sahib et al. 2020 Volucellini Volucella Geoffroy, 1762 Volucella inanis (Linnaeus, 1758) Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 Volucella liquida Erichson, 1841 Gil Collado 1929a , Rif ; Séguy 1930a , AP , Mogador, MA , Azrou, Bekrit; Kanervo 1939 , MA ; Séguy 1953a , MA , Ifrane; Timon-David 1951 , MA , Ifrane, Azrou, HA , Aït Mohamed Sgatt; Leclercq 1961a , Rif , Azib de Ketama, MA , Dayat Aoua, Ifrane, Azrou; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , jumb Kitane – MISR Volucella zonaria Poda, 1761 Séguy 1930a , AP , Casablanca; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Rabat, Casablanca) – MISR Brachypalpus Macquart, 1834 Brachypalpus valgus (Panzer, 1798) Kassebeer 1998a , MA , Ifrane, HA , Ouirgane; Sahib et al. 2020 Psilotini Psilota Fallén, 1823 Psilota atra (Fallén, 1817) = Psilota toubkalana Kassebeer, 1995, in Kassebeer 1995 : 395–400 Kassebeer 1995 , HA ; Smit and Vujic 2008, HA , Ouirgane, Marrakech; Speight 2018 ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh Pipizinae Pipizini Heringia Rondani, 1856 Heringia heringi (Zetterstedt, 1843) Kassebeer 1998a , HA , Tahanaout, Ouirgane; Sahib et al. 2020 Pipizella Rondani, 1856 Pipizella thapsiana Kassebeer, 1995 Kassebeer 1995 , HA (1000 m); Speight 2013 , 2018 ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh Triglyphus Loew, 1840 Triglyphus escalerai Gil Collado, 1929 Gil Collado 1929a , Rif , Tanger; Dirickx 1994 ; Speight 2013 ; Sahib et al. 2020 Syrphinae Bacchini Melanostoma Schiner, 1860 Melanostoma mellinum (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Séguy 1934b , AP , Korifla; Timon-David 1951 , MA , Ifrane, Azrou, Aïn Leuh, HA , Aït Mizane; Leclercq 1961a , MA , Ifrane; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , Dayat Rahrah, Aïn el Ma Bared, Oued Dardara, Dayat El Ânassar, Dayat Lemtahane, Garden Ksar Al Rimal, tributary Oued Tazarine, Oued Farda, Dayat El Birdiyel, maison forestière, stream at 1 km from Sidi Yahia Aârab, Dayat Amsemlil, EM , farm Saf-Saf, MA , Oued d'Ifrane, HA , Lac Oukaimeden – MISR Melanostoma mundum Czerny & Strobl, 1909 Dakki 1997 ; Mouna 1998 Melanostoma scalare (Fabricius, 1794) Gil Collado 1929a , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 , Rif , Aïn el Ma Bared, Oued Mezine, Aïn Quanquben, Aïn Takhninjoute, Oued Koub Platycheirus Le Peletier & Serville, 1828 Platycheirus albimanus (Fabricius, 1781) 39 Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress; Mouna 1998 ; Kassebeer 1998b Platycheirus ambiguus (Fallén, 1817) Kassebeer 1998b , HA ; Sahib et al. 2020 Platycheirus atlasi Kassebeer, 1998 Kassebeer 1998b , MA , Azrou, Ifrane; Sahib et al. 2020 Platycheirus fulviventris (Macquart, 1829) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m); Sahib et al. 2020 Platycheirus manicatus (Meigen, 1822) Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Kassebeer 1998b , HA , Toubkal; Sahib et al. 2020 Platycheirus marokkanus Kassebeer, 1998 Kassebeer 1998b , MA , HA ; Speight 2018 ; Sahib et al. 2020 , Rif , Aïn Takhninjoute, HA , Douar Akhlij Tnine Ourika Xanthandrus Verrall, 1901 Xanthandrus comtus (Harris, 1776) Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Paragini Paragus Latreille, 1804 Paragus albifrons (Fallén, 1817) Kanervo 1939 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Paragus atlasi Claußen, 1989 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Schmid 1995 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus bicolor (Fabricius, 1794) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, forest of Timelilt, Azrou; Kanervo 1939 , AP , HA ; Séguy 1949a , SA , Guelmim; Timon-David 1951 , MA , Ifrane; Claußen 1989b ; Claußen and Hauser 1990 , HA , Tizi-n'Test; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Douar Dacheryène, HA , vicinity of Asni; MA (Fès, Ifrane, Azrou) – MISR Paragus cinctus Schiner & Egger, 1853 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 , HA , vicinity of Asni Paragus coadunatus Rondani, 1847 Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus flammeus Goeldlin, 1971 Claußen and Hauser 1990 ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus haemorrhous Meigen, 1844 Claußen 1989b , MA , Azrou (1700 m); Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Mharhar Paragus hermonensis Kaplan, 1981 Claußen 1989b , MA , Azrou (1700 m); Dirickx 1994 ; Sahib et al. 2020 Paragus quadrifasciatus Meigen, 1822 = Paragus pulcherrimus Strobl, in Timon-David 1951 : 144 Timon-David 1951 , MA , Ifrane; Claußen 1989b , AP , Kénitra; Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Douar Kitane, AA , Agadir airport; MA (Ifrane) – MISR Paragus majoranae Rondani, 1857 Claußen and Hauser 1990 , MA ; Dirickx 1994 ; Sahib et al. 2020 Paragus pecchiolii Rondani, 1857 Claußen and Hauser 1990 , MA , Ifrane (1750 m), Hajeb Paragus strigatus Meigen, 1822 = Paragus bimaculatus Meigen, in Wiedemann 1824 : 33 Wiedemann 1824 , AP ; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m), Hajeb; Dirickx 1994 ; Speight 2018 ; Sahib et al. 2020 Paragus tibialis (Fallén, 1817) = Paragus tibialis meridionalis Becker, in Becker and Stein 1913 : 88; Gil Collado 1929: 403; Leclercq 1961: 241 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Kanervo 1939 ; Séguy 1949a , SA , Guelmim; Leclercq 1961a , Rif , Bab Taza, MA , Taza; Claußen 1989b ; Claußen and Hauser 1990 , HA , Tizi-n'Test (2000 m); Dirickx 1994 ; Dakki 1997 ; Grabener 2017 ; Sahib et al. 2020 , HA , vicinity of Asni, Ijoukak vicinity; AP (Rabat) – MISR Paragus vandergooti Marcos-Garcia, 1986 Claußen 1989b , HA , Tizi-n'Test à (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Syrphini Chrysotoxum Meigen, 1803 Chrysotoxum bicinctum (Linnaeus, 1758) Timon-David 1951 , MA , Ifrane, HA , Haute Réghaya; Leclercq 1961a , MA , Mischliffen (2019 m); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; MA , HA – MISR Chrysotoxum intermedium Meigen, 1822 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , MA , Tizi-S'Tkrine, Aïn Leuh, forest of Taffert, HA , Tizi-n'Test, Goundafa; Kanervo 1939 ; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Pârvu et al. 2006 , AP , Cap Bedouza; Dousti and Hayat 2006 ; Pârvu and Zaharia 2007 ; Kazerani et al. 2013b ; Sahib et al. 2020 , Rif , 1 km after Dardara, Oued Azila, Dayat Jebel Zemzem, Aïn El Maounzil, Oued Tafoughalt, MA , Douar Zaouiat Cheikh, HA , vicinity of Asni, AA Douar Issafen; AP (Rabat), MA (Aïn Leuh), HA (Réghaya) – MISR Chrysotoxum volaticum Séguy, 1961 Séguy 1961 , MA ; Claußen 1989b , HA , Oukaimeden (2600 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 Dasysyrphus Enderlein, 1938 Dasysyrphus albostriatus (Fallén, 1817) Kassebeer 1998a , HA , Bin-el-Ouidane, Ouirgane, Imlil, Asni; Sahib et al. 2020 Epistrophe Walker, 1852 Epistrophe eligans (Harris, 1780) = Syrphus ochrostoma (Zetterstedt), in Becker and Stein 1913 : 88; Claußen 1989b : 372 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b ; Kassebeer 1998a , MA , Ifrane, HA , Imlil, Asni, Ouirgane; Sahib et al. 2020 , Rif , Dayat Tazia Epistrophe eligans (Harris, 1870) var. trifasciata Strobl Ebejer and Bensusan 2010 , AA ; Djellab et al. 2013 Episyrphus Matsumura & Adachi, 1917 Episyrphus balteatus (De Geer, 1776) = Syrphus balteatus De Geer, in Becker and Stein 1913 : 88; Séguy 1930a : 129 = Epistrophe balteata (De Geer), in Gil Collado 1929: 406; Kanervo 1939 : 3; Timon-David 1951 : 144 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , AP , Rabat, MA , Aïn Sferguila; Timon-David 1951 , AP , forest of Maâmora, Rabat, HA , Marrakech; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Sebt Zinate, Aïn Sidi Brahim Ben Arrif, dam Nakhla, Aïn Boughaba, Garden Ksar Al Rimal, Oued Aârkoub, Dayat Rahrah, Oued Sahel, dam Moulay Bouchta, maison forestière, Ksar El Kébir, Dayat Jebel Zemzem, Oued Taida, stream at 1 km from Sidi Yahia Aârab, Aïn Quanquben, Oued Maggou, Forest Bab El Karn, Douar Kitane, forest El Mahfoura, HA , Aïn Zarka of Meski, AA , Douar Zaouia; AP (Rabat) – MISR Eupeodes Osten-Sacken, 1877 Eupeodes corollae (Fabricius, 1794) = Syrphus berber Bigot, in Bigot 1884 : 88 = Syrphus corollae Meigen, in Becker and Stein 1913 : 88; Séguy 1930a : 129, Kanervo 1939 : 3; Gil Collado 1929: 406 = Syrphus corollae Fabricius, in Timon-David 1951 : 144 = Metasyrphus corollae (Fabricius), in Dirickx 1994 : 89 Bigot 1884 ; Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress, Goundafa; Kanervo 1939 ; Timon-David 1951 , AP , forest of Maâmora, Rabat, Sidi Taibi, HA , Réghaya, Tazzarine, AA , Agdz, Plaine de Souss (Taroudant); Leclercq 1961a , MA , Ifrane; Claußen and Hauser 1990 , MA , Ifrane (1750 m), HA , Oukaimeden (3200 m), AA , Tan-Tan; Dirickx 1994 ; Mouna 1998 ; Pârvu and Zaharia 2007 ; Grabener 2017 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Village Sebt Zinate, Aïn el Ma Bared, Garden Ksar Al Rimal, Oued Bin EL Ouidane, Oued Sahel, Aïn Takhninjoute, stream at 1 km from Sidi Yahia Aârab, Oued Jnane Niche, dam Smir, Oued Boumarouil, Dayat Jebel Zemzem, Oued Maggou, Meadow Fahs Lmhar, Douar Kitane, forest El Mahfoura, MA , Douar Zaouiat Cheikh, HA , vicinity of Asni, Ijoukak vicinity, Lac Oukaimeden; AA Agdz – MISR Eupeodes latifasciatus (Macquart, 1829) = Syrphus latifasciatus Macquart, in Séguy 1949: 156; Séguy 1953a : 84 = Metasyrphus latifasciatus (Macquart), in Dirickx 1994 : 89 Séguy 1949a , SA , Guelmim; Séguy 1953a , AA , Oued Khoref; Claußen 1989b ; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Douar Kitane, maison forestière, Oued Ametrasse Eupeodes luniger (Meigen, 1822) = Metasyrphus luniger (Meigen), in Dirickx 1994 : 90, 237 = Syrphus luniger Meigen, in Gil Collado 1929: 406 Gil Collado 1929a , AP ; Claussen 1989b; Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Maggou, Oued Martil, Belyounech, Douar Kitane, MA , Douar Ben Smim, HA , vicinity of Asni Eupeodes nuba (Wiedemann, 1830) = Syrphus rufinasutus Bigot, in Bigot 1884 : 88, Séguy 1961 : 107 = Metasyrphus nuba (Wiedemann), in Dirickx 1994 : 90, 238 Bigot 1884 ; Séguy 1961 ; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; Dousti and Hayat 2006 ; Ehteshamnia et al. 2010 ; Naderloo et al. 2011; Speight 2013 , 2018 ; Kazerani et al. 2013b ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eupeodes punctifer (Frey, 1934) 40 Mouna 1998 : 86 Ischiodon Sack, 1913 Ischiodon aegyptius (Wiedemann, 1830) = Simosyrphus aegyptius (Wiedemann, 1830) Gil Collado 1929a ; Timon-David 1951 , AP , Rabat, AA , Agdz; Mouna 1998 ; Grabener 2017 ; Mengual 2018 ; Sahib et al. 2020 ; AP (Rabat) – MISR Lapposyrphus Dušek & Láska, 1967 Lapposyrphus lapponicus (Zetterstedt, 1838) = Syrphus arcuatus Fallén, 1817, in Becker and Stein 1913 : 88 = Metasyrphus lapponicus (Zetterstedt, 1838), in Dirickx 1994 : 89 = Eupeodes lapponicus (Zetterstedt, 1838), in Claußen 1989b : 372 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Meliscaeva Frey, 1946 Meliscaeva auricollis (Meigen, 1822) = Epistrophe auricollis Meigen, in Becker and Stein 1913 : 89; Gil Collado 1929: 406; Timon-David 1951 : 145 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Timon-David 1951 , AP , Oued Korifla, Zaers, Rabat; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Aïn el Ma Bared, Dayat El Ânassar, Belyounech, Oued Mezine, dam Moulay Bouchta, Aïn Afersiw, dam Entrasol, Oued à 15 km de Fifi, jumb Kitane, Oued Maggou, Douar Kitane, Oued Sahel, MA , Aïn Ouilili; AP (Rabat, Zaers) – MISR Meliscaeva cinctella (Zetterstedt, 1843) = Syrphus cinctellus Zeterstedt, in Séguy 1934b : 162 Séguy 1934b , MA , Oued Leben (Taounate); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 – MISR Scaeva Fabricius, 1850 Scaeva albomaculata (Macquart, 1842) = Lasiopticus albomaculata (Macquart), in Gil Collado 1929: 405; Timon-David 1951 : 145 = Lasiophthicus albomaculatus Macquart, in Séguy 1953a : 84 Gil Collado 1929a ; Séguy 1953a , MA , Immouzer; Timon-David 1951 , AP , Rabat, MA , El Harcha; Leclercq 1961a , MA , Azrou; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane; Dirickx 1994 ; Mouna 1998 ; Dousti and Hayat 2006 ; Ehteshamnia et al. 2010 ; Naderloo et al. 2011; Speight 2013 , 2018 ; Kazerani et al. 2013b ; Grabener 2017 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Belyounech, HA , Aïn Zarka of Meski, Lac Oukaimeden; AP (Rabat, Cap Cantin), EM (Debdou) – MISR Scaeva dignota (Rondani, 1857) Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Maggou, Dayat Lemtahane Scaeva mecogramma (Bigot, 1860) Dirickx 1994 ; Kassebeer 1998a , Rif , Chefchaouen, AP , Kénitra, HA , Ouirgane; Sahib et al. 2020 Scaeva pyrastri (Linnaeus, 1758) = Catabomba pyrastri Linnaeus, in Becker and Stein 1913 : 88 = Lasiophthicus pyrastri Linnaeus, in Séguy 1930a : 128 = Lasiopticus pyrastri Linnaeus, in Gil Collado 1929: 405, Timon-David 1951 : 145 Becker and Stein 1913 , Rif ; Gil Collado 1929a , AP ; Séguy 1930a , AP , forest of Zaers, forest of Maâmora, MA , Tizi-s'Tkrine; Timon-David 1951 , AP , Rabat, MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Dayat Jebel Zemzem, stream at 1 km from Oued Sidi Yahia Aârab, AA , 1 km before Douar Aïn Lahmar; AP (Rabat, Cap Cantin) – MISR Scaeva selenitica (Meigen, 1822) = Lasiophthicus seleniticus Meigen, in Séguy 1930a : 128 Séguy 1930a , HA , Aguerd el Had, AA , Talekjount (Souss); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Sphaerophoria Le Peletier & Serville, 1828 Sphaerophoria interrupta (Fabricius, 1805) = Sphaerophoria menthastri (Linnaeus), in Becker and Stein 1913 : 87; Kanervo 1939 : 3; Timon-David 1951 : 145; Séguy 1961 : 109 Kanervo 1939 , AP , MA , HA ; Becker and Stein 1913 , Rif ; Timon-David 1951 , AP , Kénitra, Rabat, MA , Harcha, Ifrane, Sefrou, HA , Agdz; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 ; AP (Rabat), MA (Ifrane, Meknès) – MISR Sphaerophoria rueppelli (Wiedemann, 1830) Kanervo 1939 , Rif , HA ; Timon-David 1951 , AP , Rabat, AA , Agadir, Agdz, Zagora; Séguy 1961 ; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Tarmast tributary, Oued Sidi Ben Saâda, Dayat Amsemlil, EM , farm Saf-Saf, MA , Oued d'Ifrane; AP (Rabat), AA (Agadir, Agdz, Zagora) – MISR Sphaerophoria scripta (Linnaeus, 1758) = Sphaerophoria dispar (Meigen), in Timon-David 1951 : 145 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1930a , AP , Tlet n'Rhohr, MA , forest of Timelilt, Aïn Leuh, EM , Berkane; Kanervo 1939 , Rif , AP , MA , HA ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Timon-David 1951 , AP , Rabat, MA , Harcha, Sefrou, Ifrane; Leclercq 1961a , MA , Dayat Aoua, Ifrane, Azrou; Claußen 1989b , MA , Azrou (1700 m), HA , Oukaimeden, Tizi-n'Test (1900 m), Ansegmir-Tal W midelt (1400 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb (1750 m), HA , Oukaimeden (3200 m); Dirickx 1994 ; Mouna 1998 ; Pârvu and Zaharia 2007 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Oued Aârkoub, dam Nakhla, meadow Mizoghar, Oued Dardara, 1 km after Dardara, Dayat El Birdiyel, palm grove Igrane, Aïn Quanquben, maison forestière, Oued Sidi Ben Saâda, Dayat Lemtahane, Dayat Amsemlil, forest El Mahfoura, EM , farm Saf-Saf, MA , Oued d'Ifrane, HA , vicinity of Asni, Lac Oukaimeden, AA , Msidira – MISR Sphaerophoria taeniata (Meigen, 1822) = Sphaerophoria menthastri var. taeniata Meigen, in Timon-David 1951 : 10 Timon-David 1951 , AP , Rabat, saline mud; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 ; MA (Aïn Leuh, Azrou, Timahdit) – MISR Syrphus Fabricius, 1775 Syrphus ribesii (Linnaeus, 1758) Kassebeer 1998a , Rif , Chefchaouen, Tétouan; Sahib et al. 2020 , Rif , Douar Kitane Syrphus vitripennis Meigen, 1822 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xanthogramma Schiner, 1860 Xanthogramma dives (Rondani, 1857) Ebejer et al. 2019 , AA , 29 km N of Rich (Errachidia, 1570 m); Sahib et al. 2020 Xanthogramma evanescens Becker, 1913 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xanthogramma marginale (Loew, 1854) = Xanthogramma marginale var. morenae Loew, in Becker and Stein 1913 : 86, Gil Collado 1929: 406 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Kanervo 1939 , Rif , HA ; Séguy 1961 ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 ; Claußen 1989b ; Dirickx 1994 ; Ebejer and Bensusan 2010 , AA ; Speight 2013 , 2018 ; Sahib et al. 2020 , Rif , village Sebt Zinate, Oued Maggou, Oued Ametrasse, Douar Kitane Xanthogramma pedissequum (Harris, 1776) = Xanthogramma ornatum (Meigen, 1822), in Gil Collado 1929: 406 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Conopoidea CONOPIDAE 41 K. Kettani Number of species: 34 . Expected: 40 Faunistic knowledge of the family in Morocco: good Conopinae Conopini Conops Linnaeus, 1758 Conops aegyptiacus (Rondani, 1850) Kröber 1915 , 1928 Conops ceriaeformis Meigen, 1804 = Conops acuticornis Loew, 1847, in Becker and Stein 1913 : 89 Becker and Stein 1913 , Rif , Tanger; Kröber 1924 , 1928 , Rif , Tanger Conops djanetianus Séguy, 1938 Mouna 1998 : 85 Conops elegans Meigen, 1824 = Conops semifumosus Adams, in Séguy 1934b : 162 = Conops ruficornis Becker, 1913, in Becker and Stein 1913 : 89; Kröber 1924 : 69 Becker and Stein 1913 , Rif , Tanger; Kröber 1924 , 1927 , AP , Casablanca; Séguy 1934b , Rif , Tanger; Stuke 2016 ; El Hawagry et al. 2021 Conops nubeculipennis Bezzi, 1901 = Conops atrogonius Séguy, 1930, in Séguy 1930a : 134; Séguy 1953a : 85; Mouna 1998 : 85 Séguy 1930a , AP , Rabat, Mogador; Séguy 1953a , MA , Dayat Ifrah; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , 1.5 km S of Tissint, 14 km NW of Icht; Stuke 2016 ; El Hawagry et al. 2021 Conops theryi Séguy, 1928 Séguy 1928d ; Séguy 1930a , AP , Rabat, Casablanca; Mouna 1998 Conops tifedarius Séguy, 1928 Séguy 1928d ; Séguy 1930a , AP , Rabat; Mouna 1998 Leopoldius Rondani, 1843 Leopoldius coronatus (Rondani, 1857) = Brachyglossum coronatum Rondani, in Séguy 1930a : 132; Mouna 1998 : 85 Séguy 1930a , MA , Aïn Leuh (1400–1500 m); Mouna 1998 Physocephalini Physocephala Schiner, 1861 Physocephala chrysorrhoea (Meigen, 1824) Séguy 1930a , AP , Sidi Bettache; Mouna 1998 ; El Hawagry et al. 2021 Physocephala laticincta (Brullé, 1832) Séguy 1930a , MA , Aïn Leuh (1400–1500 m); Bei-Bienko and Steyskal 1989 ; Mouna 1998 Physocephala maculigera Kröber, 1915 Séguy 1941d , AA , Agadir; Mouna 1998 Physocephala nigra (De Geer, 1776) Séguy 1930a , AP , Sidi Bettache Physocephala pusilla (Meigen, 1804) Séguy 1928d ; Séguy 1930a , MA , Meknès, Ras el Ksar (1900 m), HA , Asni; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Aoulouz, 2 km NW of Tissint; El Hawagry et al. 2021 Physocephala rufipes (Fabricius, 1781) Mouna 1998 ; AP (Tagulet (Essaouira)) – MISR Physocephala vittata (Fabricius, 1794) Séguy 1928d ; Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès, Forêt Zaers; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Aoersi (15 km NE of Agadir), AA , Oued near beach (19 km W of Tiznit); El Hawagry et al. 2021 ; HA – MISR Pseudophysocephala Kröber, 1940 Pseudophysocephala bouvieri (Séguy, 1936) = Conops bouvieri Séguy, in Séguy 1936a : 299 Séguy 1936a , MA , Meknès (550 m); Sidney 2001 , MA , Meknès Dalmanninae Dalmannini Dalmannia Robineau-Desvoidy, 1830 Dalmannia aculeata (Linnaeus, 1761) Séguy 1928d ; Séguy 1930a , MA , Aïn Leuh, Meknès; Mouna 1998 – MISR Dalmannia dorsalis (Fabricius, 1794) = Dalmannia flavescens (Meigen), in Becker and Stein 1913 : 90 Becker and Stein 1913 , Rif , Tanger; Stuke and Kehlmaier 2008 , MA , Fès Myopinae Myopini Melanosoma Robineau-Desvoidy, 1853 Melanosoma bicolor (Meigen, 1824) Séguy 1941d , AA , Agadir (Admine forest); Mouna 1998 Melanosoma mundum Czerny & Strobl, 1909 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Tafingoult (Goundafa, 1500–1600 m), AA , Tenfeht (Souss, 1000–1500 m); Séguy 1953a , HA , Aït Ourir; Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , SE of Awir (10 km NNW of Agadir), Talmakant (80 km NE of Agadir), AA , 19 km W of Tiznit, Massa river (25 km NE of Tiznit), Imitek (30 km WSW of Tata), Issafen (55 km WNW of Tata); Grabener 2017 Myopa Camras, 1953 Myopa buccata (Linnaeus, 1758) Mouna 1998 ; MA (Oulmès) – MISR Myopa dorsalis Fabricius, 1794 Séguy 1930a , MA , Jebel Ahmar (1750 m); Mouna 1998 ; MA (Oulmès); El Hawagry et al. 2021 – MISR Myopa hirsuta Stuke & Clements, 2008 Stuke and Clements 2008 , MA , Azrou, Ifrane Myopa nigrita Wiedemann, 1824 Wiedemann 1824 , 1830 ; Kröber 1916 , 1928 Myopa pellucida Robineau-Desvoidy, 1830 Stuke and Clements 2008 , Rif , Chefchaouen, HA , Ourika Myopa picta Panzer, 1798 Séguy 1930a , AP , Casablanca, MA , Meknès; Mouna 1998 ; El Hawagry et al. 2021 Myopa stigma Meigen, 1824 Becker and Stein 1913 , Rif , Tanger; El Hawagry et al. 2021 Myopa testacea (Linnaeus, 1767) Séguy 1930a , AP , Casablanca, Fouarat, HA , Arround (Skoutana: 2000–2400 m), Tachdirt, Jebel Likount; Mouna 1998 ; AP (Fouarat) – MISR Myopa palliceps (Bigot, 1887) = Myopa minor Strobl, in Clements 2000 : 239 = Myopa vaulogeri Séguy, in Séguy 1930a : 136; Clements 2000 : 236 Séguy 1930a , AP , Casablanca; Villeneuve 1933 ; Mouna 1998 ; Clements 2000 , AP , Casablanca, HA , Marrakech, Ourigane (1000 m), AA , Ammelental (10 km NE of Tafraoute) Thecophora Rondani, 1845 Thecophora atra (Fabricius, 1775) = Occemyia atra Fabricius, in Séguy 1930a : 137; Becker and Stein 1913 : 90 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Sidi Bettache, MA , Meknès, HA , Asni, AA , Taroudant (Souss); Mouna 1998 ; HA (Mouldikht (Marrakech)); El Hawagry et al. 2021 – MISR Thecophora cinerascens (Meigen, 1804) = Thecophora pusilla (Meigen, 1824), in Pârvu et al. 2006 : 276; Popescu-Mirceni 2011 : 35 Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 , MA , Ifrane Sicini Sicus Scopoli, 1763 Sicus ferrugineus (Linnaeus, 1761) Séguy 1930a , MA , Aïn Leuh; Zimina 1975 ; Mouna 1998 ; MA (Tizi-n'Ifrah, Guisser (1400 m)) – MISR Zodioninae Zodionini Zodion Latreille, 1797 Zodion cinereum (Fabricius, 1794) Séguy 1930a , MA , Meknès, HA , Around (Skoutana); Mouna 1998 ; Grabener 2017 Zodion erythrurum Rondani, 1865 Séguy 1930a , AP , Casablanca, HA , Dar Caïd M'Tougui; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Tamzergoute (10 km N of Agadir); El Hawagry et al. 2021 Nerioidea MICROPEZIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Micropeza Meigen, 1803 Micropeza kettaniae Ebejer, 2019 Ebejer 2019b , Rif , Oued Kbir (Béni Ratene, 157 m), Dayat Tazia (Tazia, 733 m) – MISR , NHMUK Tanypezoidea PSILIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 5 Faunistic knowledge of the family in Morocco: poor Chamaepsila Hendel, 1917 Chamaepsila nigricornis (Meigen, 1826) Ebejer et al. 2019 , Rif , Tétouan, Onsar Lile (349 m), Aïn Tissemlal (Azilane, 1255 m), MA , 17 km SW of Midelt (1940 m), Lac Aguelmane Afennourir (30 km SW of Azrou, 2050 m) Tephritoidea LONCHAEIDAE K. Kettani, I. MacGowan Number of species: 5 . Expected: 30 Faunistic knowledge of the family in Morocco: poor Dasiopinae Dasiops Rondani, 1856 Dasiops latifrons (Meigen, 1826) Séguy 1934a ; Mouna 1998 ; MacGowan and Freidberg 2008 ; AP (Rabat) – MISR ; Rif (Tanger), AP (Rabat) – MHNP Lonchaeinae Lamprolonchaea Bezzi, 1920 Lamprolonchaea smaragdi (Walker, 1849) = Lonchea aurea Macquart, 1851, in Séguy 1953a : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1934a ; Séguy 1953a , AP , Rabat, MA , Fès; Rungs 1952 , HA , Arganier; Mouna 1998 Lonchaea Fallén, 1820 Lonchaea tarsata Fallén, 1820 MacGowan and Freidberg 2008 Lonchaea sp. = Recorded as Lonchaea laticornis Meigen, 1826 but almost certainly not this species Becker and Stein 1913 , Rif , Tanger Silba Macquart, 1851 Silba adipata McAlpine, 1956 = Lonchaea aristella Becker, 1903, in Séguy 1934b : 162 Séguy 1934b , AP , Rabat; MacGowan and Freidberg 2008 ; AP (Rabat) – MNHN PALLOPTERIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Palloptera Fallén, 1820 Palloptera ustulata (Fallén, 1820) Ebejer et al. 2019 , MA , Zaouia d'Ifrane (Ifrane, 1603 m) PIOPHILIDAE K. Kettani, M.J. Ebejer Number of species: 3 . Expected: 6 Faunistic knowledge of the family in Morocco: poor Piophilinae Mycetaulus Loew, 1845 Mycetaulus hispanicus Duda, 1927 Mouna 1998 ; Carles-Tolrá 2002 Piophila Fallén, 1810 Piophila casei (Linnaeus, 1758) Séguy 1930a , Rif , Tanger, AP , Rabat, MA , Meknès; Mouna 1998 ; Rif (Tanger), AP (Rabat), MA (Meknès) – MISR Prochyliza Walker, 1849 Prochyliza nigrimana (Meigen 1826) Ebejer et al. 2019 , Rif , Aïn Tissemlal (Azilane, 1255 m) PLATYSTOMATIDAE K. Kettani, G.V. Popov Number of species: 4 . Expected: 4 Faunistic knowledge of the family in Morocco: moderate Platystomatinae Platystoma Meigen, 1803 Platystoma idia Séguy, 1934 42 Séguy 1934a , AP , Aïn Sferguila (Forêt Zaers); Hennig 1945 , AP , Aïn Sferguila (Forêt Zaers); Soós 1984a Platystoma meridionale Hendel, 1913 = Platystoma seminationis (Fabricius), in Becker 1907 : 385 Becker 1907 ; Hendel 1913 , AP , Mogador; Hennig 1945 , AP , Mogador; Soós 1984a , AP , Mogador Rivellia Robineau-Desvoidy, 1830 Rivellia hispanica Lyneborg, 1969 Ebejer et al. 2019 , EM , Tafoughalt Rivellia syngenesiae (Fabricius, 1781) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; MA (Oulmès, Meknès) – MISR TEPHRITIDAE K. Kettani, A.L. Norrbom Number of species: 69 . Expected: 75 Faunistic knowledge of the family in Morocco: moderate Dacinae Ceratitidini Capparimyia Bezzi, 1920 Capparimyia savastanii (Martelli, 1911) = Capparimyia savastanii (Martelli), in Séguy 1953a : 85 Séguy 1953a , AA , Tiznit (on Capparis spinosa ); El Harym and Belqat 2017 Ceratitis MacLeay, 1829 Ceratitis capitata (Wiedemann, 1824) Becker and Stein 1913 , Rif , Tanger; Vayssière 1920 , AP , Rabat; Séguy 1930a (common in all of Morocco); Rungs 1952 , HA , Arganier; Harris et al. 1980 ; Mouna 1998 ; De Meyer 2000 , AP , Ouadj-Ouli-Mohamed, env. Settat, Insgane; Vidal et al. 2008 ; Aboussaid et al. 2009 ; Koçak and Kemal 2010 ; El Harym and Belqat 2017 , Rif , Kitane, El Haouta, MA , Sensla, AA , Environs Massa, Oued Massa, Douar Sidi Abou, Douar Tighrimt, Douar Zaouia; Elaini and Mazih 2018 ; Elaini et al. 2019 , HA , Arganeraie; AP (Safi) – MHNNR Dacini Bactrocera Macquart, 1835 Bactrocera oleae (Rossi, 1790) = Dacus oleae Rossi, in Becker and Stein 1913 : 94, Vayssière 1920 : 256 = Dacus oleae Gmelin, in Séguy 1930a : 168 Becker and Stein 1913 , Rif , Tanger; Vayssière 1920 , EM , Oujda; Séguy 1930a ; Mouna 1998 ; Savio 2011 , EM , Oujda; El Harym and Belqat 2017 , Rif , El Haouta, Oued Maâza, Cascade Chrafate, Koudiat El Aouinate, Lâazaba, Dhar Sbagh Mâasra, El Hajria, MA , Sensla, AA , route Bab El Khemis Dacus Fabricius, 1805 Dacus frontalis (Becker, 1922) El Harym and Belqat 2017 , AA , Oued Foum Ziguid (Douar Ouaiftoute), Oued Draa (Ikhf Mezrou), Isdaoun Dacus longistylus (Wiedemann, 1830) El Harym and Belqat 2017 , AA , Oued Tata, Douar Tighrimt, Oued Foum Ziguid (Douar Ouaiftoute) Tephritinae Dithrycini Oedaspis Loew, 1862 Oedaspis daphnea Séguy, 1930 Séguy 1930a , AP , El Mers (Rabat); Foote 1984 ; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 Oedaspis multifasciata (Loew, 1850) = Oedaspis multifasciatus (Loew), in Séguy 1953a : 85 Séguy 1953a , EM , Itzer (Haute Moulouya); El Harym and Belqat 2017 Oedaspis trotteriana Bezzi, 1913 Soós 1984b ; Ribera and Blasco-Zumeta 1998; Norrbom et al. 1999 ; El Harym and Belqat 2017 Myopitini Myopites Blot, 1827 Myopites cypriacus Hering 1938 El Harym et al. 2020 , Rif , Douar Halila, Dam Nakhla, Marabout Sidi Bou Hadjel Myopites inulaedyssentericae Blot, 1827 43 = Myopites apicatus Freidberg, 1980, in El Harym and Belqat 2017 : 140 El Harym and Belqat 2017 , Rif , affluent Tarmast, Aïn Afersiw Myopites longirostris (Loew, 1846) El Harym et al. 2020 , Rif , Oued Tahaddart, Douar Kouf, Mkhinak Myopites stylatus Fabricius, 1794 El Harym and Belqat 2017 , Rif , affluent Tarmast, Oued El Hamma, El Haouta Myopites variofasciatus Becker, 1903 = Myopites variofasciata Becker, in Séguy 1941a : 32 Séguy 1941a , HA , Imi-n'Ouaka (1500 m), Mouna 1998 Urophora Robineau-Desvoidy, 1830 Urophora congrua Loew, 1862 44 = Euribia congrua Loew, in Séguy 1941d : 14 Séguy 1941d , AA , Taroudant; Mouna 1998 ; El Harym and Belqat 2017 Urophora jaculata Rondani, 1870 = Urophora mauritanica Macquart, in El Harym and Belqat 2017 : 149 (misidentification) Urophora mauritanica Macquart, 1851 = Euribia algira Macquart, in Séguy 1930a : 169 = Urophora algira Macquart, in Séguy 1934a : 98, Mouna 1998 : 87 = Urophora macrura Loew, in Séguy 1934a : 100, Mouna 1998 : 87 Séguy 1930a , HA , Imi-N'Takandout, Dar Kaid M'Tougui; Séguy 1934a ; White and Korneyev 1989 , HA , Ito; Mouna 1998 ; Norrbom et al. 1999 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013b Urophora quadrifasciata algerica (Hering, 1941) = Euribia quadrifasciata Meigen, in Séguy 1930a : 169 Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 El Harym et al. 2020 , MA , Douar Oulad Abdoune, Mlakite, Tirra, Douar Oulad Amar, Tihli, Douar Oulad Amar Urophora solstitialis (Linnaeus, 1758) = Euribia solstitialis Linnaeus, in Séguy 1930a : 169 Séguy 1930a , HA , Haute Réghaya; Mouna 1998 ; El Harym and Belqat 2017 Noeetini Ensina Robineau-Desvoidy, 1830 Ensina sonchi (Linnaeus, 1767) El Harym and Belqat 2017 , Rif , Ksar Rimal, Douar Tizga, Oued Kbir, MA , Sensla, AA , Oued Massa (Pont Aghbalou), Centre Sidi Ouassay, Aïn Boharroch, Atbane, Oued Tamanarne, Oued Draa (Tahtah), Jnane Makadir, Kasbah Asma, Ait Aissa O Brahim, Oued Ziz (Pont Errachidia), Oued Ouarzazate; AA (Tafraout (Al Ourir, 12 km E)) – MHNNR Hypenidium Loew, 1862 Hypenidium graecum Loew, 1862 = Stephanaciura bipartita Séguy, in Séguy 1930a : 171 Séguy 1930a , MA , Tiffert (2000–2200 m); Villeneuve 1933 ; Soós 1984b ; Mouna 1998 : 87; Norrbom et al. 1999 ; El Harym and Belqat 2017 Tephrellini Aciura Robineau-Desvoidy Aciura coryli (Rossi, 1790) = Aciura Powelli Séguy, in Séguy 1930a : 170, Séguy 1953a : 85, Mouna 1998 : 87 Séguy 1930a , MA , Azrou (larvae from Phlomis crinita Cav.); Séguy 1953a , AP , Korifla; Mouna 1998 ; Norrbom et al. 1999 , MA , Azrou; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Arhil Oxyaciura Hendel, 1927 Oxyaciura tibialis (Robineau-Desvoidy, 1830) = Aciura tibialis Robineau-Desvoidy, in Becker and Stein 1913 : 94 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Sker; Séguy 1934a ; Mouna 1998 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013b ; El Harym and Belqat 2017 , Rif , Dayat El Birdiyel, Oued Azila, maison forestière; Oued Maâza (Tarik El Ouasâa); AP (Tamri, 10 km S) – MHNNR Sphaeniscus Becker, 1908 Sphaeniscus filiolus (Loew, 1869) = Spheniscomyia filiola Loew, in Séguy 1930a : 170; Mouna 1998 : 87 = Spheniscomyia aegyptiaca Efflatoun, in Séguy 1949a : 157; Mouna 1998 : 87 Séguy 1930a ; Séguy 1949a , SA , Guelmim; Mouna 1998 ; El Harym and Belqat 2017 , Rif , affluent Tarmast, Oued Maâza (Tarik El Ouasâa) Tephritini Acanthiophilus Becker, 1908 Acanthiophilus helianthi (Rossi, 1790) = Tephritis eluta Meigen, in Becker and Stein 1913 : 94 = Orellia eluta Meigen, in Mouna 1998 : 87 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger (Sarf, route Spartel), MA , Tizi s'Tkrine (Jebel Ahmar, 1700 m); Séguy 1949a , AA , Alnif, Foum-el-Hassan, SA , Guelmim; Mouna 1998 ; Koçak and Kemal 2013b ; El Harym and Belqat 2017 , Rif , Oued Zinat, Ksar Rimal, Oued Dardara, affluent Tarmast, Dayat El Birdiyel, Oued Azila, Dayat Jebel Zemzem, Oued Boumarouil, Douar Abou Boubnar (Mara­bout Sidi Gile), El Haouta, Oued Maâza (Tarik El Ouasâa), Douar Tizga, Dayat Afrate, Oued Mezine, Aïn El Malaâb, Oued Jnane Niche, MA , Oued Oum-er-Rbia, AA , Centre Sidi Ouassay, Avant Sidi Bin­zarne, route Bab El Khemis, airport Sidi Ifni, Oued Tisla, Oued Sayad, Oued Tamanarne, Oued Foum Ziguid (Douar Ouaiftoute), Jnane Makadir, Douar Rggaga, Oued Tinghir, Oued Ouarzazate; AP (Essaouira (Cap Hadid), Tamri, 10 km S) – MHNNR Campiglossa Rondani, 1870 Campiglossa martii (Becker, 1908) El Harym and Belqat 2017 , Rif , Oued Kbir, AA , Centre Sidi Ouassay Campiglossa producta (Loew, 1844) = Oxyna tessellata Loew, in Becker and Stein 1913 : 94 (misidentification) = Paroxyna tessellata (Loew), in Séguy 1930a : 174; Mouna 1998 : 87; Koçak and Kemal 2013b : 38 (misidentification) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Mogador, HA , Telouet Glaoua; Séguy 1934a ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Koçak and Kemal 2013b , HA ; El Harym and Belqat 2017 , Rif , Oued Al Mizzine, Aïn El Malaâb, Dayat El Hajjami Campiglossa sororcula (Wiedemann, 1830) = Dioxyna sororcula (Wiedemann), in El Harym and Belqat 2017 : 155 El Harym and Belqat 2017 , Rif , Ksar Rimal, Oued Jnane Niche, Oued Halila, Oued Zarka, Oued Martil, Oued Amsa, Oued Sahel, Dayat Jebel Zemzem, Oued Maggou, Dhar Sbagh Mâasra, Douar Kitane Capitites Foote & Freidberg, 1981 Capitites augur (Frauenfeld, 1857) = Trypanea augur (Frauenfeld), in Séguy 1930a : 176; Mouna 1998 : 87 Séguy 1930a , MA , Forêt Azrou, Tizi-s'Tkrine (Jebel Ahmar, 1700 m), HA , Tenfecht, AA , Souss; Mouna 1998 ; Pârvu et al. 2006 , AA , Ouarzazate, Lac Edehby; Popescu-Mirceni 2011 , MA , Tizi-s'Tkrine, Azrou, HA , Tenfecht, AA , Ouarzazate Capitites ramulosa (Loew, 1844) = Acanthiophilus ramulosus Loew, in Séguy 1930a : 177; Séguy 1941d : 15; Séguy 1949a : 157; Mouna 1998 : 87 Séguy 1930a , MA , Timelilt, HA , Tizi-n'Test (Jebel Imdress, 2000–2450 m); Séguy 1941d , AA , Taroudant; Séguy 1949a , AA , Foum-el-Hassan, Akka, Agdz, Alnif; Mouna 1998 ; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Amsa, Koudiat Taifour Desmella Munro, 1957 Desmella rostellata (Séguy, 1941) = Paroxyna rostellata Séguy, in Séguy 1941d : 14; Mouna 1998 : 87 Séguy 1941d , AP , Agadir; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 , AP , Agadir; El Harym and Belqat 2017 Euaresta Loew, 1873 Euaresta bullans (Wiedemann, 1830) Herman and Dirlbek 2006 , AA , Tiznit environs, Sidi Moussa d'Aglou; El Harym and Belqat 2017 , AA , Msidira Goniurellia Hendel, 1927 Goniurellia longicauda Freidberg, 1980 Freidberg 1980 , MA , Tizi-s'Tkrine (1700 m), Azrou, AA , Taroudant; Soós 1984b ; Freidberg and Kugler 1989 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 , AA , airport Sidi Ifni, Oued Tisla, Oued Tamanarne, Douar Zaouiet, Oued Tata, Douar Tighrimt, Ksibat Elhdeb, Oued Ziz (Pont Errachidia), Oued Ouarzazate Goniurellia persignata Freidberg, 1980 Freidberg 1980 , EM , Defilia, near Figuig; Soós 1984b ; Freidberg and Kugler 1989 ; Norrbom et al. 1999 ; Herman and Dirlbek 2006 , AA , Tiffoultoute (1146 m); El Harym and Belqat 2017 , Rif , Dhar Sbagh Mâasra, AA , Douar Zaouiet, Oued Ouarzazate Spathulina Rondani, 1856 Spathulina sicula Rondani, 1856 = Spathulina tristis Loew, in Séguy 1930a : 174; Mouna 1998 : 87 Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Barrage Smir Sphenella Robineau-Desvoidy, 1830 Sphenella marginata (Fallén, 1814) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Oued Judios (Tanger); Mouna 1998 ; Cassar et al. 2005 , Rif , lagoon Smir; Koçak and Kemal 2013b , HA ; El Harym and Belqat 2017 , Rif , affluent Tarmast, Dayat Jebel Zemzem, El Malaâb, Oued Maâza (Âachira), Dayat Aïn Jdioui, Oued Maggou, Dayat Afrate Tephritis Latreille, 1804 Tephritis carmen Hering, 1937 El Harym et al. 2020 , Rif , forest house of National Park of Talassemtane Tephritis dioscurea (Loew, 1856) Séguy 1930a , MA , El Hajeb; Mouna 1998 ; El Harym and Belqat 2017 Tephritis divisa (Rondani, 1871) El Harym and Belqat 2017 , Rif , Dayat Amsemlil Tephritis formosa (Loew, 1844) Séguy 1930a , HA , Asni; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Oued Abou Bnar, Oued Sidi Ben Saâda, Oued Achekrade, Oued El Kanar Tephritis leontodontis (De Geer, 1776) Séguy 1930a (All North Africa); Mouna 1998 ; El Harym and Belqat 2017 Tephritis matricariae (Loew, 1844) Séguy 1930a (all North Africa); Mouna 1998 ; El Harym and Belqat 2017 , Rif , affluent Oued Amsemlil, El Haouta, Dayat Jebel Zemzem, Dayat Amsemlil, Oued El Hamma Tephritis nigricauda (Loew, 1856) Séguy 1930a , AP , Berrechid; Séguy 1934a ; Séguy 1941d , AP , Agadir, Berrechid; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Dayat Jebel Zemzem, Oued Maâza (Tarik El Ouasâa), Aïn El Maounzil, Dayat Tazia, Dayat Amsemlil, Douar Tamakoute Tephritis postica (Loew, 1844) Herman and Dirlbek 2006 , MA , Volubilis (358 m); El Harym and Belqat 2017 , AA , Ksibat Elhdeb, Oued Tinghir Tephritis praecox (Loew, 1844) Séguy 1930a , Rif , Oued Judios (Tanger), MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 ; Herman and Dirlbek 2006 , MA , Ifrane, Azrou National Park (1743 m); El Harym and Belqat 2017 , Rif , Dayat El Ânassar, Dayat Amsemlil, affluent Oued Amsemlil, Douar Dacheryène, Douar Taghbaloute, Barrage Nakhla, Oued Sa­hel, Daya Jebel Zemzem, Douar Kitane, Oued El Hamma, Oued Kbir, Aïn el Ma Bared, Aïn El Malaâb, Douar Abou Boubnar (Marabout Sidi Gile), maison forestière, Douar Tizga, Oued Aïn Jdioui (Touaret), Dayat Afrate, Oued Jbara, Aïn El Maounzil, Dayat Tazia, Oued Jnane Niche, Oued Maggou, Aïn Tiouila, Dayat Lemtahane, Lâazaba, Dhar Sbagh Mâasra, El Hajria, Aïn Boharroch, Douar Tamakout, Douar Ouslaf, EM , Oued Béni Ouaklane (Béni Snassen); AP (Essaouira) – MHNNR Tephritis pulchra (Loew, 1844) Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 Tephritis simplex (Loew, 1844) Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Aïn Soualah, Aïn El Maounzil Tephritis stictica Loew, 1862 Séguy 1930a , AP , Rabat; Mouna 1998 ; El Harym and Belqat 2017 Tephritis theryi Séguy, 1930 Séguy 1930a , HA , Marrakech, Asni; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 Tephritis vespertina (Loew, 1844) El Harym and Belqat 2017 , Rif , Dayat Lemtahane, Dhar Sbagh Mâasra Tephritomyia Hendel, 1927 Tephritomyia lauta (Loew, 1869) = Acanthiophilus lauta Loew, in Séguy 1930a : 177; Mouna 1998 : 87 Séguy 1930a , HA , Tachdirt (Imminen, 2400–2600 m); Freidberg and Kugler 1989 ; Mouna 1998 ; Yaran and Kütük 2012 ; Morgulis et al. 2015, HA , Tizi-n'Tichka; El Harym and Belqat 2017 , Rif , Dayat El Birdiyel, Dayat Amsemlil, Lâazaba, AA , Msidira, Oued Ouarzazate Trupanea Schrank, 1795 Trupanea amoena (Frauenfeld, 1857) = Trypanea amoena Frauenfeld, in Séguy 1930a : 176; Mouna 1998 : 87 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Ksar Rimal, Oued Jnane niche, affluent Tarmast, Oued Martil (Tamouda), Oued Amsa, Oued El Hamma, Oued Boumarouil, Oued Sidi Yahia Aârab, Aïn Tiouila, AA , Oued Massa (Pont Aghbalou), Centre Sidi Ouassay, Avant Sidi Binzarne, Oued Tisla, Douar Tighrimt, Oued Draa (Tahtah), Jnane Makadir, Douar Rggaga, Aït Aissa O Brahim, Oued Draa (Ikhf Mezrou), Isdaoun, Ksibat Elhdeb, Oued Tinghir Trupanea guimari (Becker, 1908) El Harym and Belqat 2017 , AA , Centre Sidi Ouassay, Msidi­ra, Jnane Makadir, Aït Aissa O Brahim, Ksibat Elhdeb; Norrbom 2004 : 06600136 – INHS ( AA , 5 km W Ouarzazate) Trupanea stellata (Fuesslin, 1775) Séguy 1930a , MA , Timelilt (1900 m); Séguy 1949a , SA , Guelmim; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Mizoghar, Oued Maâza (Tarik El Ouasâa), Dayat Afrate, Aïn El Malâab, Oued Tkarae, AA , Centre Sidi Ouassay; AA (Taliouine) – MHNNR Terellini Chaetorellia Hendel, 1927 Chaetorellia conjuncta (Becker, 1912) El Harym and Belqat 2017 , AA , Airport Sidi Ifni, Oued Assa, Oued Sayad, Oued Foum Ziguid (Douar Ouaiftoute), Ksibat Elhdeb, Oued Ziz (Pont Errachidia), Oued Ouarzazate Chaetorellia hestia Hering, 1937 = Chaetorellia hexachaeta Loew, in Séguy 1930a : 174 [probably a misidentification] = Orellia hexachaeta Loew, in Séguy 1934a : 135 [misidentification, see White and Macquart 1989: 476]; Mouna 1998 : 87 Séguy 1930a , AP , Mogador; Séguy 1934a ; Mouna 1998 ; El Harym and Belqat 2017 , AA , Centre Sidi Ouassay Chaetorellia succinea (Costa, 1844) El Harym et al. 2020 , MA , Douar Oulad Abdoune Chaetostomella Hendel, 1927 Chaetostomella cylindrica (Robineau-Desvoidy, 1830) El Harym et al. 2020 , Rif , Marabout Douar Halila, Mkhinak, Douar Kitane Terellia Robineau-Desvoidy, 1830 Terellia colon (Meigen, 1826) = Orellia colon (Meigen), in Séguy 1930a : 173 Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 Terellia fuscicornis (Loew, 1844) Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 Terellia longicauda (Meigen, 1838) Séguy 1930a , MA , Aïn Leuh (1200–1400 m), HA , Tizi-n'Test, Goundafa (Jebel Imdress, 2000–2450 m); Séguy1934a ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; El Harym and Belqat 2017 Terellia luteola (Wiedemann, 1830) El Harym et al. 2020 , Rif , Bakrim, Aforidane, Aïn Siyed Terellia oasis (Hering, 1938) El Harym et al. 2020 , Rif , Douar Halila Terellia ptilostemi El Harym et al. 2021 El Harym et al. 2021 , Rif , Douar Chourdane (908 m), Aïn Akorian (1610 m), Aïn Elma Sefli (1345 m), Forest house of the Talassemtane National Park (1674 m) Terellia serratulae (Linnaeus, 1758) = Tephritis pallens Wiedemann, in Wiedemann 1824 : 54 = Trypeta serratula Linnaeus, in Becker and Stein 1913 : 94 = Terellia serratulae Linnaeus, in Séguy 1930a : 173 Wiedemann 1824 , Rif , Tanger; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a (all North Africa); Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 , Rif , Dayat Jebel Zemzem, Oued Maâza (Tarik El Ouasâa) Terellia virens (Loew, 1846) Séguy 1930a ; White 1989 , HA , Jebel Ayachi; Mouna 1998 ; Korneyev et al. 2013 , HA , Tizi-n'Talrhemt; El Harym and Belqat 2017 , AA , airport Sidi Ifni, Oued Ouarzazate Trypetinae Carpomyini Carpomya Costa, 1854 Carpomya incompleta (Becker, 1903) El Harym and Belqat 2017 , AA , Douar Zaouia Euleia Walker, 1835 Euleia heraclei (Linnaeus, 1758) = Acidia heraclei Linnaeus, in Séguy 1953a : 85 Séguy 1953a , MA , Sidi Slimane; Freidberg and Kugler 1989 ; Koçak and Kemal 2010 ; El Harym and Belqat 2017 , Rif , Oued Boumarouil, Aïn El Âakba Larbaâ Euleia marmorea (Fabricius, 1805) 45 = Philophylla flavescens Fabricius, in Séguy 1930a : 170; Mouna 1998 : 87 = Euleia flavescens Fabricius, in Soós 1984b : 88 Séguy 1930a , Rif , Tanger; Zimsen 1964 ; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 , Rif , Tanger; El Harym and Belqat 2017 Trypetini Chetostoma Rondani, 1856 Chetostoma curvinerve Rondani, 1856 El Harym and Belqat 2017 , Rif , Oued Kelaâ, Bab el Karn Acknowledgments We gratefully acknowledge the assistance and cooperation of Valery Korneyev and the late Amnon Freidberg who contributed to the revision of this family. ULIDIIDAE K. Kettani, M.J. Ebejer Number of species: 13 . Expected: 18 Faunistic knowledge of the family in Morocco: good Otitinae Ceroxys Macquart, 1835 Ceroxys urticae Linnaeus, 1758 Ebejer et al. 2019 , AP , Lower Loukous (6 m), Larache (5 m) Dorycera Meigen, 1830 Dorycera griseipennis (Becker, 1907) Soós 1984b Herina Robineau-Desvoidy, 1830 Herina ghilianii Rondani, 1869 Kameneva 2007 , HA , Ansegmir-Tal, W Midelt (1400 m); Ebejer 2015 Herina lacustris (Meigen, 1826) Kameneva 2007 , Rif , Baie de Tanger Herina oscillans (Meigen, 1826) = Herina schlueteri Becker, in Becker and Stein 1913 : 92; Soós 1984b : 56 Becker and Stein 1913 , Rif , Tanger; Soós 1984b ; Kameneva 2007 , Rif , Tanger Melieria Robineau-Desvoidy, 1830 Melieria nigritarsis Becker, 1903 Ebejer et al. 2019 , AA , Merzouga (714 m) Otites Latreille, 1804 Otites tangeriana Becker, 1913 = Otites tangeriana Becker, in Becker and Stein 1913 , 1918: 92 Becker and Stein 1913 , 1918, Rif , Tanger; Soós 1984b , Rif , Tanger Tetanops Fallén, 1820 Tetanops flavescens Macquart, 1835 Séguy 1930a , Rif , Tanger; Mouna 1998 Ulidiinae Physiphora Fallén, 1810 Physiphora alceae (Preyssler, 1791) = Chrysomyza demandata (Fabricius, 1798), in Séguy 1953a : 85; Mouna 1998 : 87 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Goundafa; Séguy 1953a , SA , El Aöun du Draa; Séguy 1949a , AA , Agdz; Mouna 1998 ; Koçak and Kemal 2010 ; Kameneva and Korneyev 2016 , AA , Tizi-n'Bachkoun (1600 m); Rif (M'Diq farm) – MISR ; Rif (Tanger) – MfN ; AP (Casablanca) – ZSSM Physiphora smaragdina (Loew, 1852) Kameneva and Korneyev 2016 , AA , 25 km S Goulmima (100 m) – NHMD Ulidia Meigen, 1826 Ulidia apicalis (Meigen, 1826) Séguy 1930a , MA , Meknès, HA , Skoutana; Séguy 1934a ; Lyneborg 1969 ; Soós 1984b ; Mouna 1998 Ulidia erythrophthalma Meigen, 1826 Becker and Stein 1913 , Rif , Tanger; Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 Ulidia megacephala Loew, 1845 Soós 1984b ; Zaitzev 1984 ; Koçak and Kemal 2010 Lauxanioidea CHAMAEMYIIDAE K. Kettani, M.J. Ebejer Number of species: 18 . Expected: 24 Faunistic knowledge of the family in Morocco: moderate Chamaemyiinae Chamaemyia Meigen, 1803 Chamaemyia aridella (Fallén, 1823) Ebejer 2016a , Rif , Moulay Abdelsalam (1180 m), Issaguen (1620 m); HA (Jebel Ayachi, Mikdane, maison forestière, MR Jaffar, Tizi-n'Zou) – NHMUK Chamaemyia flavicornis (Strobl, 1902) Ebejer 2016a , Rif , Martil beach and dunes (on human faeces), MA , Khénifra (17 km NW of Zaida, 1878 m), AA , Errachidia (29 km N of Rich, 1570 m); HA (Asni near Alrene, W Imlil) – NHMUK Chamaemyia flavipalpis (Haliday, 1838) = Chamaemyia maritima Zetterstedt, in Mouna 1998 : 85 Mouna 1998 ; Ebejer 2016a , Rif , Ksar Sghir, Oued Araml, Ksar Sghir, AP , Larache, Merja Zerga, Loukous marsh Chamaemyia herbarum (Robineau-Desvoidy, 1830) Ebejer 2016a , MA , Khénifra, 17 km SW of Midelt (1940 m), AA , Errachidia, 29 km N of Rich (1570 m); HA (Jebel Ayachi, Mikdane, MR Jaffar, Tizi-n'Zou) – NHMUK Chamaemyia juncorum (Fallén, 1958) Ebejer 2016a , MA , Khénifra (17 km SW of Midelt, 1940 m), AA , Errachidia (29 km N of Rich, 1570 m) Chamaemyia polystigma (Meigen, 1830) Becker and Stein 1913 , Rif , Tanger; Ebejer 2016a , Rif , Moulay Abdelsalam (1180 m), Sidi Yahia Aarab (377 m); HA (Asni) – NHMUK Melanochthiphila Frey, 1958 Melanochthiphila sp. Ebejer, 2016 Ebejer 2016a , AA , Errachidia (30 km W of Errachidia, 1065 m) Parochthiphila ( Euestelia ) Enderlein, 1927 Parochthiphila ( Euestelia ) coronata (Loew, 1858) Carles-Tolrá 1993 ; Mouna 1998 ; Ebejer 2016a , AP , Larache (Lower Loukous, 2 m); HA (Jebel Ayachi, Mikdane, MR Jaffar) – NHMUK Parochthiphila frontella (Rondani, 1874) Ebejer 2016a ; HA (Jebel Ayachi, Mikdane, Tizi-n'Zou, MR Jaffar) – NHMUK Parochthiphila inconstans Becker, 1903 Ebejer 2016a , AA , Ziz river (9.5 km SE of Rich, 1285 m), Errachidia (6 km N of Errachidia, 1010 m) Parochthiphila ( Euestelia ) nigripes Strobl, 1900 Ebejer 2016a , Rif , Ksar Sghir, Dardara Leucopinae Leucopis Meigen, 1803 Leucopis annulipes Zetterstedt, 1848 Mouna 1998 ; AP (Maâmora) – MISR Leucopis griseola (Fallén, 1823) Mouna 1998 ; AP (Rabat), MA (Meknès), HA (Marrakech) – MISR Leucopis formosana Hennig, 1938 Ebejer 2016a , AP , Sidi Smail, Oued Tensift (estuary) Leucopis glyphinivora Tanasijtshuk, 1958 Ebejer 2016a , AP , Ksar Sghir, Azemmour, Oued Tensift (estuary), AA , Errachidia (30 km W of Errachidia) Leucopis kerzhneri Tanasijtshuk, 1970 Ebejer 2016a ; HA (Jebel Ayachi) – NHMUK Leucopis palumbii Rondani, 1872 Ebejer 2016a , Rif , Ksar Sghir, Oued Aliane Lipoleucopis de Meijere, 1928 Lipoleucopis pulchra Raspi, 2008 Ebejer 2016a , AA , Errachidia, Ziz river (12 km S of Rissani) LAUXANIIDAE K. Kettani, M.J. Ebejer Number of species: 27 . Expected: 45 Faunistic knowledge of the family in Morocco: good Homoneurinae Homoneura Wulp, 1891 Homoneura ericpoli Carles-Tolrá, 1993 Ebejer and Kettani 2019a , Rif , Azilane (1255 m), Adrou (556 m), HA , Lac Tislit (Imlchil, 2254 m) Homoneura licina Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Schacht et al. 2004 ; Ebejer and Kettani 2019a , Rif , Oued Laou (2 m), AP , Merja Zerga, Sidi Smaine, Safi (estuary of Oued Tensift), Azemmour (El Jadida), Diabat (Essaouira), Larache (5 m) Homoneura transversa (Wiedemann, 1830) Wiedemann 1830 ; Ebejer and Kettani 2019a Prosopomyia Loew, 1856 Prosopomyia pallida Loew, 1856 Vaillant 1956b , HA , Oukaimeden, Imi-N'Ifri; Carles-Tolrá 1993 ; Mouna 1998 ; Ebejer and Kettani 2019a , Rif , Adrou (556 m), Issaguen (1547 m), HA , Jebel Ayachi – NHMUK Lauxaniinae Calliopum Strand, 1928 Calliopum oosterbroeki Shatalkin, 2000 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m), Moulay Abdelsalam (1180 m), Ketama, Rahbat Amlay (284 m), Ikadjiouen (294 m), Mechkralla ( NPT , 981 m), Kharouba (roadside meadow between Chefchaouen and Tétouan, 385 m), Dbani (Béni Selmane, 1046 m), Aïn Ben Ali (Béni Selmane, 1014 m), HA , Lalla Takrkoust (Oued N'fis, 1141 m) – NHMUK Calliopum tuberculosum (Becker, 1895) Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 135 m), Azilane (1255 m), Perdicaris Park (Tanger, 223 m), cascade Chrafate (859 m) Meiosimyza Hendel, 1925 Meiosimyza ( Lyciella ) rorida (Fallén, 1820) = Homoneura rorida Fallén, in Mouna 1998 : 85 Mouna 1998 ; MA (Aïn Leuh) – MISR Minettia Robineau-Desvoidy, 1830 Minettia aenigmatica Ebejer, 2019 Ebejer 2019a , Rif , Perdicaris Park (223 m), Oued Khmis (Khmis Anjra, 61 m), HA , Mikdane (Jebel Ayachi); Ebejer and Kettani 2019a Minettia biseriata (Loew, 1847) Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Jnane Niche (46 m), Oued Kbir (Béni Ratene ( PNPB ), 157 m) Minettia cantolraensis Carles-Tolrá, 1998 Ebejer and Kettani 2019a , Rif , Oued Laou, El-Fahsa, Oued Guallet (Bni Boufrah, 942 m), Oued Ouringa (2 m), Maggou ( NPT , 962 m), Ksar el-Kbir road to Chefchaouen at bridge near oued Azla (80 m), Oued Siflaou (281 m), Dardara (484 m), Azilane (1255 m), Adrou (556 m), Perdicaris Park (Tanger, 223 m), Bni Bahlou (986 m), El Hamma (936 m), Amaghouse (Oued Laou), Oued Koub (Laghdir, 148 m), EM , Aïn Sfa (Berkane, 638 m), Tafoughalt (Berkane, 788 m), HA , Lalla Takrkoust (Oued N'fis, 1141 m), AA , Taliouine (Taroudant, 1049 m) Minettia fasciata (Fallén, 1826) Merz 2004 , AP , Rabat; Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Ksar el-Kbir road to Chefchaouen at bridge near oued Azla (80 m), Azilane (1255 m), Adrou (556 m), Issaguen (1547 m), Oued Taida (Al Andalous, 503 m), Perdicaris Park (Tanger, 223 m), El Hamma (936 m), EM , Aïn Sfa (Berkane, 683 m) Minettia flavipalpis (Loew, 1847) 46 = Sapromyza flavipalpis Loew, 1847, in Becker and Stein 1913 : 93 ( Rif , Tanger), Ebejer and Kettani 2019a : 144 (???) Minettia flaviventris (Costa, 1844) Ebejer and Kettani 2019a , Rif , Azilane (1255 m) Minettia longiseta (Loew, 1847) Ebejer and Kettani 2019a , Rif , Maggou ( NPT , 962 m), Adrou ( PNPB , 556 m) Minettia plumicornis (Fallén, 1820)46 Schacht et al. 2004 ; Ebejer and Kettani 2019a Minettia subvittata (Loew, 1847) Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Azilane (1255 m) Minettia suillorum (Robineau-Desvoidy, 1830) = Minettia muricata Becker, 1895, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2019a , Rif , Adrou (556 m), Perdicaris Park (Tanger, 223 m), HA , Mikdane (Jebel Ayachi) – NHMUK Minettia tabidiventris (Rondani, 1877) Ebejer and Kettani 2019a , Rif , Adrou (556 m), Oued Kbir (Béni Ratene ( PNPB ), 157 m), Oued Koub (Laghdir, 148 m), Rahbat Amlay (284 m) Pachycerina Macquart, 1835 Pachycerina pulchra Loew, 1850 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m) Peplominettia Szilády, 1943 Peplominettia codinai (Hennig, 1951) Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m), Azilane (1255 m) Peplominettia striata Szilády, 1943 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m) Sapromyza Fallén, 1810 Sapromyza apicalis Loew, 1847 Schacht et al. 2004 ; Ebejer and Kettani 2019a , HA , Jebel Ayachi, Mikdane (stream), Lac Tislit (Imlchil, 2254 m) – NHMUK Sapromyza gozmanyi Papp, 1981 Ebejer and Kettani 2019a , Rif , Oued Kbir (Béni Ratene ( PNPB ), 157 m), Perdicaris Park (Tanger, 223 m), Barrage Smir (M'Diq, 27 m) Sapromyza obscuripennis Loew, 1847 Ebejer and Kettani 2019a , Rif , Jebel Talassemtane (1554 m), Jebel Lakraâ (1541 m) Sapromyza unizona Hendel, 1908 Ebejer and Kettani 2019a , Rif , Perdicaris Park (Tanger, 223 m), AP , Larache (5 m) Sapromyza ( Sapromyzosoma ) laevatrispina Carles-Tolrá, 1992 Ebejer and Kettani 2019a , Rif , Adrou (556 m), Issaguen (maison morestière, 1547 m) Sapromyza ( Sapromyzosoma ) parallela Carles-Tolrá, 1992 Ebejer and Kettani 2019a , Rif , Adrou (556 m), Azilane (1255 m), Talassemtane (maison forestière, 1699 m), HA , Jaffar river (Jebel Ayachi, at maison forestière), Mikdane (Jebel Ayachi) – NHMUK Sciomyzoidea COELOPIDAE K. Kettani Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: moderate Coelopinae Coelopa Meigen, 1830 Coelopa pilipes Haliday, 1838 Séguy 1930a , Rif , Tanger; Dakki 1997 ; Lair 2013 DRYOMYZIDAE K. Kettani Number of species: 1 . Expected: 1 Faunistic knowledge of the family in Morocco: poor Dryomyzinae Dryope Robineau-Desvoidy, 1830 Dryope flaveola (Fabricius, 1794) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1596 m) HELCOMYZIDAE K. Kettani Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Helcomyzinae Helcomyza Curtis, 1825 Helcomyza ustulata Curtis, 1825 Cassar et al. 2005 , Rif , Smir lagoon SCIOMYZIDAE K. Kettani, J-C. Vala Number of species: 25 . Expected: 40 Faunistic knowledge of the family in Morocco: good Sciomyzinae Sciomyzini Ditaeniella Sack, 1939 Ditaeniella grisescens (Meigen, 1830) Vala and Ghamizi 1991 , MA , Ras el Ma, AA , Agadir; Kassebeer 1999a , MA , HA ; Knutson and Vala 2011 Pherbellia Robineau-Desvoidy, 1830 Pherbellia cinerella (Fallén, 1820) = Ditaenia cinerella Fallén, in Séguy 1941a : 31 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m), Imi-n'Ouaka (1500 m); Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt, HA , Tizi-n'Test (1900 m); Rozkošný 1987 , HA ; Vala and Ghamizi 1991 , HA , MA ; Carles-Tolrá 1993 ; Kassebeer 1999a , MA , HA ; Rif (Talassemtane) – MISR Pherbellia dorsata (Zetterstedt, 1846) Kassebeer 1999a , MA ; Knutson and Vala 2011 Pherbellia griseola (Fallén, 1820) Kassebeer 1999a , MA ; Knutson and Vala 2011 Pherbellia hermonensis Knutson & Freidberg, 1983 Kassebeer 1999a , HA ; Knutson and Vala 2011 Pherbellia nana (Fallén, 1820) = Pherbellia villiersi Séguy, in Séguy 1941a : 31; Knutson 1981 : 336 (new comb.) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Knutson 1981 , HA , Tachdirt; Leclercq and Schacht 1987 , HA ; Vala and Ghamizi 1989, HA ; Kassebeer 1999a , MA , HA ; Knutson and Vala 2011 ; HA – MISR Tetanocerini Dichaetophora Rondani, 1868 Dichaetophora obliterata (Fabricius, 1805) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Leclercq and Schacht 1987 ; Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 ; Kassebeer 1999a Elgiva Meigen, 1838 Elgiva cucularia (Linnaeus, 1767) Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Vala 1989 ; Kassebeer 1999a Euthycera Latreille, 1829 Euthycera algira (Macquart, 1849) Vala and Reidenbach 1982 , Rif , Tanger; Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 ; Kassebeer 1999a , Rif , Tanger Euthycera soror (Robineau-Desvoidy, 1830) = Euthycera alaris Vala, 1983, syn. nov.Vala (pers. comm.) Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 , MA ; Carles-Tolrá 1993 ; Kassebeer 1999a , MA , HA Euthycera stichospila (Czerny, 1909) = Euthycera leclercqi Vala & Reidenbach, 1982, syn. by Rozkošný (1987) Czerny and Strobl 1909 ; Vala and Reidenbach 1982 ; Rozkošný 1987 Euthycera zelleri (Loew, 1847) Kassebeer 1999a , Rif , Tanger Hydromya Robineau-Desvoidy, 1830 Hydromya dorsalis (Fabricius, 1775) Séguy 1930a , Rif , Tanger; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m), HA , Oukaimeden (2600 m); Dakki 1997 , Rif , Ketama; Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 , MA , HA ; Kassebeer 1999a , MA , HA ; Pârvu et al. 2006 , AP , Merja Zerga Ilione Haliday in Curtis, 1837 Ilione albiseta (Scopoli, 1763) Leclercq and Schacht 1987 , HA ; Vala 1989 ; Kassebeer 1999a Ilione trifaria (Loew, 1820) Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Vala 1989 , MA , Dayat Aoua; Vala and Ghamizi 1991 , EM , MA , HA ; Carles-Tolrá 1993 Ilione unipunctata (Macquart, 1849) Leclercq and Schacht 1987 , HA ; Rozkošný 1987 ; Vala 1989 ; Kassebeer 1999a Oligolimnia Mayer, 1953 Oligolimnia zernyi Mayer, 1953 Rozkošný 1987 , HA , Tachdirt; Leclercq and Schacht 1987 , HA ; Vala 1989 , HA ; Kassebeer 1999a , HA Pherbina Robineau-Desvoidy, 1830 Pherbina coryleti (Scopoli, 1763) Séguy 1930a , Rif , Tanger; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; HA (Issougane n'Ouagouns) – MISR Pherbina mediterranea Mayer, 1953 Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 , AP , Sidi Boughaba, HA , south of Marrakech; Carles-Tolrá 1993 ; Kassebeer 1999a , MA Psacadina Enderlein, 1939 Psacadina disjecta Enderlein, 1939 Verbeke 1964 , AA , Tlata Reisana; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Kassebeer 1999a , MA Psacadina verbekei Rozkošný, 1975 Vala and Ghamizi 1991 , HA , south of Marrakech; Kassebeer 1999a Sepedon Latreille, 1804 Sepedon hispanica Loew, 1862 Verbeke 1964 ; Vala 1989 , AP , Mohammedia; Vala and Ghamizi 1991 , AP , Mohammedia, HA , Oued Tissaout (Kelaâ Sraghna); Kassebeer 1999a Sepedon sphegea (Fabricius, 1775) Séguy 1930a Rif , Tanger; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 , HA , Tamesloht (south of Marrakech), AA , Bou Acheiba (Agadir); Carles-Tolrá 1993 ; Dakki 1997 ; Kassebeer 1999a Sepedon spinipes (Scopoli, 1763) Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Vala and Ghamizi 1991 , MA , Imouzzer, Tifounassine; Carles-Tolrá 1993 ; Kassebeer 1999a , MA ; Knutson and Vala 2011 Trypetoptera Hendel, 1900 Trypetoptera punctulata (Scopoli, 1763) Leclercq and Schacht 1987 , HA ; Vala 1989 ; Kassebeer 1999a ; Knutson and Vala 2011 SEPSIDAE K. Kettani, J.-P. Haenni Number of species: 12 . Expected: 20 Faunistic knowledge of the family in Morocco: poor Sepsinae Saltellini Saltella Robineau-Desvoidy, 1830 Saltella sphondylii (Schrank, 1803) Ebejer et al. 2019 , Rif , Barrage Smir (145 m), Oued Mhajrate (Ben Karrich, 180 m) Sepsini Nemopoda Robineau-Desvoidy, 1830 Nemopoda nitidula (Fallén, 1820) Mouna 1998 (no locality given) Sepsis Fallén, 1810 Sepsis biflexuosa Strobl, 1893 Zuska and Pont 1984; Mouna 1998 ; Pont and Meier 2002 ; Ozerov 2005 ; HA (Asni) – NHMUK Sepsis cynipsea (Linnaeus, 1758) Séguy 1930a , AP , Rabat, MA , HA , Marrakech; Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Zuska and Pont 1984; Mouna 1998 Sepsis flavimana Meigen, 1826 Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Sepsis fulgens Meigen, 1826 Séguy 1941d , HA , Tizi-n'Test (2000 m), AA , Agadir; Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 , MA , Ifrane; MA (Ifrane), HA (Asni, near Alrene, Mikdane) – NHMUK Sepsis lateralis Wiedemann, 1830 Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Zuska and Pont 1984; Mouna 1998 ; AP (Sale Tropical Garden), HA (Marrakech) – NHMUK Sepsis punctum (Fabricius, 1794) Séguy 1941a , HA , Aït Souka (Toubkal); Zuska and Pont 1984; Mouna 1998 ; Pont and Meier 2002 , Rif , Tanger, AP , Rabat; Pârvu et al. 2006 , AP , Merja Zerga – MISR ; MA (Ifrane), HA (Asni, Mikdane), AA (Souss Massa, Agadir) – NHMUK Sepsis thoracica (Robineau-Desvoidy, 1830) Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; MA (near Azrou), HA (Mikdane, Marrakech, Amizmiz) – NHMUK Sepsis violacea Meigen, 1826 = Sepsis ciliforceps Duda, 1926, in Mouna 1998 : 86 Becker and Stein 1913 , Rif , Tanger; Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 – MISR Themira Robineau-Desvoidy, 1830 Themira minor (Haliday, 1833) Zuska and Pont 1984; Mouna 1998 (no locality given); HA (Mikdane) – NHMUK Themira paludosa Elberg, 1963 47 Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 An additional species, Australosepsis niveipennis (Becker, 1903) is reported from Morocco in major catalogues (Pont and Meier 1984, Ozerov 2005 ). However this report is based upon a misinterpretation by Zuska (1968) of a name of locality given in Duda (1926b: 2): "Marako" [a locality in Mali, or possibly another in Ethiopia] was wrongly understood for "Marokko", the German name of Morocco. Both Adrian Pont and Andrey Ozerov have confirmed (pers. comm. 27.xi.15) that they have not seen any Moroccan specimen of A. niveipennis . The Moroccan records from NHMUK were checked by Adrian Pont to whom we express our grateful thanks for his kind help. Opomyzoidea AGROMYZIDAE K. Kettani, M. Černý Number of species: 62 . Expected: 150 Faunistic knowledge of the family in Morocco: poor Agromyzinae Agromyza Fallén, 1810 Agromyza abiens Zetterstedt, 1848 Spencer 1967 , HA , Ourika Valley, Marrakech; Černý and Merz 2006 Agromyza albipennis Meigen, 1830 Séguy 1936b ; Mouna 1998 Agromyza ambigua Fallén, 1823 = Domomyza ambigua Fallén, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Agromyza bicaudata (Hendel, 1920) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza frontella (Rondani, 1875) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza frontosa (Becker, 1908) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza hiemalis Becker, 1908 Spencer 1967 , HA , Marrakech; Černý 2019 ; Černý et al. 2020 Agromyza intermittens (Becker, 1907) = Phytomyza secalina Hering, 1925, in Maarouf 2003 : 42 Maarouf 2003 , HA , Chaouia Agromyza luteitarsis (Rondani, 1875) Maarouf 2003 , HA , Chaouia Agromyza megalopsis Hering, 1933 Černý et al. 2020 , AA , Agadir Id Aissa (western end of gorge and village Amtoudi, 854 m) Agromyza nana Meigen, 1830 Spencer 1967 , 1973 , AP , Casablanca; Černý and Merz 2006 Agromyza nigrociliata (Hendel, 1931) Maarouf 2003 , HA , Chaouia Agromyza rondensis Strobl, 1900 Černý et al. 2020 , AA , Agadir Id Aissa (western end of gorge and village Amtoudi, 854 m), River Aoulouz (= Asif Tifnout, 697 m) Agromyza spenceri Griffiths, 1963 Černý and Merz 2006 , HA , Asni; Černý 2013 Melanagromyza Hendel, 1920 Melanagromyza lappae (Loew, 1850) Mouna 1998 ; AP (Rabat) – MISR Melanagromyza verbasci Spencer, 1957 Spencer 1967 , HA Ophiomyia Braschnikov, 1897 Ophiomyia beckeri (Hendel, 1923) Spencer 1967 , HA ; Černý and Merz 2006 ; Černý 2009 ; Černý and Tschirnhaus 2014 Ophiomyia curvipalpis (Zetterstedt, 1848) = Ophiomyia proboscidea (Strobl, 1900), in Mouna 1998 : 84 Spencer 1967 , HA ; Mouna 1998 ; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2019 Ophiomyia melandryi de Meijere, 1924 Spencer 1967 , HA ; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2009 Ophiomyia vimmeri Černý, 1994 Černý and Merz 2006 , AP , Maâmora (Rabat); Černý and Merz 2007 ; Černý 2018 Phytomyzinae Amauromyza Hendel, 1931 Amauromyza ( Amauromyza ) morionella (Zetterstedt, 1848) = Agromyza morionella Zetterstedt, in Becker and Stein 1913 : 95 Aulagromyza Enderlein, 1936 Aulagromyza atlantidis (Spencer, 1967) = Paraphytomyza atlantidis Spencer, 1967, in Mouna 1998 : 84 Spencer 1967 , HA , Asni; Mouna 1998 Aulagromyza cydoniae (Hendel, 1936) = Phytagromyza cydoniae Hendel, 1936, in Hendel 1931–1936: 518 Hendel 1931–1936, AP , Rabat Aulagromyza hamata (Hendel, 1932)* HA , Asni-Ouirgane Cerodontha Rondani, 1861 Cerodontha ( Cerodontha ) denticornis (Panzer, 1806) Spencer 1967 , 1973 , HA ; Černý and Merz 2006 ; Černý 2009 Cerodontha ( Cerodontha ) fulvipes (Meigen, 1830) Mouna 1998 Cerodontha ( Dizygomyza ) brisiaca Nowakowski, 1973 Černý and Merz 2006 , MA , Azrou, Ifrane Cerodontha ( Icteromyza ) capitata (Zetterstedt, 1848) Černý and Merz 2006 , Rif , Chefchaouen Cerodontha ( Icteromyza ) rozkosnyi Černý, 2007 Černý 2007 , AA , SW Tazenakht (1000 m); Černý 2011 Cerodontha ( Poemyza ) incisa (Meigen, 1830) = Dizygomyza incisa Meigen, in Séguy 1936b : 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Cerodontha ( Poemyza ) lateralis (Macquart, 1835) = Dizygomyza lateralis Macquart, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 ; Černý et al. 2020 Cerodontha ( Poemyza ) pygmaea (Meigen, 1830) = Dizygomyza pygmaea Meigen, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Chromatomyia Hardy, 1849 Chromatomyia aprilina (Goureau, 1851) Griffiths 1974 ; Spencer 1967 , Rif , Tanger (mountains) Chromatomyia horticola (Goureau, 1851) = Phytomyza horticola Goureau, in Griffiths 1967 : 14 Griffiths 1967 , AP , Casablanca; Spencer 1973 ; Černý 2009 Chromatomyia milii (Kaltenbach, 1864) = Phytomyza milii Kaltenbach, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Spencer 1967 , HA , Ourika Valley; Griffiths 1980 ; Mouna 1998 ; Černý and Merz 2006 Chromatomyia syngenesiae Hardy, 1849 = Phytomyza atricornis Meigen, in Kozlowsky and Rungs 1932 : 66; Mouna 1998 : 84 Kozlowsky and Rungs 1932 , AP , Rabat, Kénitra, Casablanca; Mouna 1998 ; AP (Rabat) – MISR Liriomyza Mik, 1894 Liriomyza bryoniae (Kaltenbach, 1858) Spencer 1967 , 1973 , AP , Casablanca; Ayoub 2002 , AA , Souss Massa, Agadir; Černý and Merz 2006 Liriomyza cicerina (Rondani, 1874) Spencer 1973 , HA ; Lahmar and Zeouienne 1992 ; Mouna 1998 Liriomyza congesta (Becker, 1903) Černý et al. 2020 , AA , River bed of Ougni, 0.6 km N Akka N'Ait Sidi and 1.8 km NW Tissint (582 m), River Aoulouz (= Asif Tifnout), bridge of road (697 m), River Oued Draa near hotel, Gardin Oued Tamnougalt (911 m) Liriomyza huidobrensis (Blanchard, 1926) Hanafi and Schnitzler 2004 , AA , Souss Valley Liriomyza orbona (Meigen, 1830) = Agromyza fuscolimbata Strobl, 1900, in Maarouf 2003 : 43 = Liriomyza orbonella Hendel, 1931, in Maarouf 2003 : 43 Maarouf 2003 , HA , Chaouia Liriomyza pedestris Hendel, 1931 Spencer 1967 , Rif , Tanger; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2019 ; Černý et al. 2020 Liriomyza pusilla (Meigen, 1830) = Agromyza pusilla Meigen, in Mouna 1998 : 84 Mouna 1998 Liriomyza sonchi Hendel, 1931 Spencer 1967 , AP , Casablanca Liriomyza trifolii (Burgess in Comstock, 1880) Hanafi and Schnitzler 2004 , AA , Souss Valley Napomyza Westwood, 1840 Napomyza cichorii Spencer, 1966 Černý and Merz 2006 , MA , Ifrane; Černý 2009 Napomyza lateralis (Fallén, 1823) Černý and Merz 2006 , MA , Ifrane Napomyza scrophulariae Spencer, 1966 Černý and Merz 2006 , MA , Ifrane; Černý and Merz 2007 ; Černý 2012 , 2013 Phytoliriomyza Hendel, 1931 Phytoliriomyza immoderata Spencer, 1963 Černý and Merz 2006 , MA , Azrou, Ifrane; Černý 2009 ; Černý 2019 ; Černý et al. 2020 Phytoliriomyza oasis (Becker, 1907) Spencer 1967 , HA ; Černý and Merz 2006 Phytomyza Fallén, 1810 Phytomyza conyzae Hendel, 1920 Spencer 1967 , Rif , Tanger; Černý 2009 Phytomyza ferulae Hering, 1927 Spencer 1967 , Rif , Tanger; Černý 2013 ; Černý et al. 2020 Phytomyza gymnostoma Loew, 1858 Mouna 1998 ; AP (Mechra el kettane) – MISR Phytomyza orobanchia Kaltenbach, 1864 Geipert 1993 ; Geipert et al. 1994 , Rif ; Klein 1995 ; Klein et al. 1995 ; Boumezzough 1996 , MA , Saiss, Rommani; Boughdad et al. 1997 ; Klein et al. 1999 ; Kroschel and Klein 1999 ; Yazough and Klein 1999 ; Kroschel and Klein 2003 Phytomyza phillyreae Hering in Buhr, 1930 = Phytomyza unedo Séguy, 1953, in Séguy 1953b : 72 Séguy 1953b , AP , Korifla; Spencer 1967 , HA , Ourika Valley; Černý and Merz 2006 ; Černý 2009 Phytomyza ranunculi (Schrank, 1803) Spencer 1967 , HA ; Černý and Merz 2006 ; Černý 2009 , 2013 Phytomyza wahlgreni Rydén, 1944 Černý and Merz 2006 , MA , Azrou, Ifrane; Černý et al. 2020 Pseudonapomyza Hendel, 1920 Pseudonapomyza atra (Meigen, 1830) = Phytomyza acuticornis Loew, 1858, in Maarouf 2003 : 43 Maarouf 2003 , HA , Chaouia Pseudonapomyza atratula Zlobin, 2003 Černý et al. 2020 , AA , 6 km ESE Quijjane, 85 km S Agadir (353 m) Pseudonapomyza bifida Zlobin, 2003 Černý et al. 2020 , AA , Road no. 109/165 from Akna to Taroudant (841 m) Pseudonapomyza spicata (Malloch, 1914) Černý et al. 2020 , AA , River bed of Ougni, 0.6 km N Akka N'Ait Sidi and 1.8 km NW Tissint (582 m) Pseudonapomyza spinosa Spencer, 1973 Maarouf 2003 , HA , Chaouia New record for Morocco Aulagromyza hamata (Hendel, 1932) High Atlas: Asni-Ouirgane, 31°13'52"N, 8°00'8"W , 1282 m a.s.l., 1♂, 24.iv.2014, river valley, V. Vrabec leg., M. Barták coll. and M. Černý det. ANTHOMYZIDAE K. Kettani, M.J. Ebejer Number of species: 2 . Expected: 4 Faunistic knowledge of the family in Morocco: poor Amygdalops Lamb, 1914 Amygdalops thomasseti Lamb, 1914 Ebejer et al. 2019 , Rif , Stehat (0 m) Anagnota Becker, 1902 Anagnota major Roháček & Freidberg, 1993 Roháček 2006 , HA , Marrakech (1000 m) ASTEIIDAE K. Kettani, M.J. Ebejer Number of species: 5 . Expected: 10 Faunistic knowledge of the family in Morocco: poor Asteiinae Asteia Meigen, 1830 Asteia amoena Meigen, 1830 Mouna 1998 ; AP (Rabat) – MISR Asteia ibizana (Enderlein, 1935) Ebejer et al. 2019 , AP , Larache (2 m) Asteia mahunkai Papp, 1979 Ebejer et al. 2019 , AP , Larache (2 m) Phlebosotera Duda, 1927 Phlebosotera clypeata Freidberg & Carles-Tolrá, 2010 Freidberg and Carles-Tolrá 2010 , HA , Jaffar river Phlebosotera mirabilis Papp, 1972 Ebejer et al. 2019 , AA , 12 km S of Rissani (737 m) AULACIGASTRIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Aulacigaster Macquart, 1835 Aulacigaster leucopeza (Meigen, 1830) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) CLUSIIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Clusiodes Coquillett, 1904 Clusioides verticalis (Collin, 1912) Ebejer et al. 2019 , Rif , Amsemlil bog ( PNPB , 1067 m) ODINIIDAE K. Kettani, M.J. Ebejer Number of species: 2 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Odiniinae Odinia Robineau-Desvoidy, 1830 Odinia Boletina (Zetterstedt, 1848) Séguy 1934a ; Mouna 1998 ; Gaimari and Mathis 2011 Odinia meijerei Collin, 1952 Ebejer et al. 2019 , Rif , Adrou ( PNPB , 556 m) OPOMYZIDAE K. Kettani, M.J. Ebejer Number of species: 5 . Expected: 6 Faunistic knowledge of the family in Morocco: moderate Geomyza Fallén, 1810 Geomyza apicalis (Meigen, 1830) Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 , AP , Merja Zerga Geomyza combinata (Linnaeus, 1767) Mouna 1998 ; AP (Maâmora) – MISR Geomyza tripunctata (Fallén, 1823) Maarouf 2003 , HA , Chaouia Opomyza Fallén, 1820 Opomyza florum (Fabricius, 1794) Maarouf 2003 , HA , Chaouia Opomyza petrei Mesnil, 1934 Ebejer et al. 2019 , Rif , Aïn Tissemlal (Azilane, 1255 m) Carnoidea CANACIDAE K. Kettani, L. Munari Number of species: 15 . Expected: 25 Faunistic knowledge of the family in Morocco: poor Canacinae Canace Haliday in Curtis, 1837 Canace actites Mathis, 1982 Ebejer et al. 2019 , AP , Loukous (2 m) Canace nasica Haliday, 1839 Dahl 1964 , AA , Tamri (north of Agadir); Mouna 1998 Xanthocanace Hendel, 1914 Xanthocanace ranula (Loew, 1874) Munari 2010 , AP , 40 km S Larache; Munari and Mathis 2010 ; Munari 2011 ; Munari and Bramuzzo 2018 , Rif , Briyech, AP , Azemmour, El Khaoucha, Fedala, Oued Nefifikh, Oued Loukous, 40 km S Larache Tethininae Tethina Haliday in Curtis, 1837 Tethina alboguttata (Strobl, 1900) Freidberg and Beschovski 1996 , AP , Aïn Diab, Essaouira, Safi, AA , Agadir, Ouarzazate; Mathis and Munari 1996 ; Munari 2002 , 2004 , 2005 , 2011 ; Munari and Mathis 2010 ; Koçak and Kemal 2010 ; Munari and Bramuzzo 2018 , Rif , Briyech, AP , Agadir, Tamri, Essaouira, Cap Hadid, Larache, Loukous, Safi, Dar Caïd-Hadji Tethina albosetulosa (Strobl, 1900) Cassar et al. 2008 , Rif , Oued Laou Basin Tethina flavigenis (Hendel, 1934) Munari and Bramuzzo 2018 , AP , Oued Oum-er-Rbia Tethina grossipes (Becker, 1908) Munari 2004 , AA , Agadir; Munari and Mathis 2010 ; Munari and Bramuzzo 2018 , AP , Tamri Tethina incisuralis (Macquart, 1851) = Tethina pictipes ( Becker 1903 ); Séguy 1941d : 18, Mouna 1998 : 87 Munari 1997 , AA , Erfoud, Rissani (900 m); Munari 2002 , 2010 , 2011 ; Munari and Mathis 2010 ; Munari and Bramuzzo 2018 , AA , Erfoud, Rissani Tethina longirostris (Loew, 1865) Cassar et al. 2008 , Rif , Oued Laou Basin Tethina mariae Munari, 1997 Munari 1997 , AP , 40 km S Larache; Munari and Báez 2000 ; Munari 2002 , 2004 , 2010 , 2011 ; Munari and Mathis 2010 – NHMD ( HT ♂) Tethina pallipes (Loew, 1865) = Tethina ochracea (Hendel, 1913) Cassar et al. 2008 , Rif , Oued Laou Tethina pictipennis Freidberg & Beschovski, 1996 Freidberg and Beschovski 1996 , AP , 40 km south of Larache; Mathis and Munari 1996 , Munari 2002 ; Munari 2004 ; Munari and Mathis 2010 ; Munari 2011 , AA , Agadir (Tamri); Munari and Bramuzzo 2018 , Rif , Briyech, AP , Tamri, Larache, Loukous – NHMD ( HT ♂) Tethina strobliana (Mercier, 1923) Munari and Bramuzzo 2018 , AP , Oued Abou, Rehouna (Rabat) Tethina yaromi Freidberg & Beschovski, 1996 Cassar et al. 2005 , Rif , Smir lagoon Tethina sp. near salinicola Beschovski, 1998 Munari 2004 , AA , Agadir, Ouarzazate; Munari 2005 ; Munari and Bramuzzo 2018 , AP , Tamri CARNIDAE K. Kettani, M.J. Ebejer Number of species: 3 . Expected: >10 Faunistic knowledge of the family in Morocco: poor Meoneura Rondani , 1856 Meoneura hungarica Papp, 1977 Ebejer et al. 2019 , Rif , Adrou ( PNPB , 556 m) Meoneura prima Becker, 1905 Brake 2011 Meoneura triangularis Collin, 1930 Brake 2011 CHLOROPIDAE 48 K. Kettani, M. von Tschirnhaus, M.J. Ebejer Number of species: 74 . Expected: 140 Faunistic knowledge of the family in Morocco: moderate Chloropinae Assuania Becker, 1903 Assuania melanoleuca (Séguy, 1949) comb. nov. 49 = Chlorops melanoleuca Séguy, in Séguy 1949a : 158 Séguy 1949a , AA , Agdz; Nartshuk 1984 ; Mouna 1998 Assuania thalhammeri (Strobl, 1893) Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Camarota Latreille, 1805 Camarota curvipennis (Latreille, 1805) = Camarota curvipennis (Latreille), in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Oued Laou Capnoptera Loew, 1866 Capnoptera pilosa Loew, 1866 Duda 1933; Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1288 m), Aïn Jdioui (Tahaddart, 8 m) Capnoptera scutata (Rossi, 1790) = Eristalis rufipes Fabricius, in Fabricius 1805 : 245 Ebejer and Kettani 2016 , AP , Larache (2 m) Cetema Hendel, 1907 Cetema maroccanum Nartshuk, 1995 = Cetema maroccana Nartshuk, in Nartshuk 1995 : 277–280 Nartshuk 1995 , HA , Oukaimeden; Ebejer and Kettani 2016 Cetema monticulum Becker, 1910 = Cetema monticula Becker, in Séguy 1941a : 33 = Cetema monticula Rossi, in Mouna 1998 : 85 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Ebejer and Kettani 2016 Chlorops Meigen, 1803 Chlorops interruptus Meigen, 1830 = Chlorops interrupta Meigen, in Séguy 1953a : 86 Séguy 1953a , AP , Oued Yquem (near Rabat); Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1288 m), Oued Aliane (Ksar Sghir, 1 m), Cap Spartel (155 m) Chlorops limbatus Meigen, 1830 Ebejer and Kettani 2016 , Rif , Jebel Moussa (800 m) Chlorops pumilionis (Bjerkander, 1778) = Chlorops nasuta Schrank, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2016 Chlorops serenus Loew, 1866 Ebejer and Kettani 2016 , MA , 17 km NW of Zaida (Khénifra, 1878 m) Cryptonevra Lioy, 1864 Cryptonevra flavitarsis (Meigen, 1830) = Haplegis flavitarsis (Meigen), in Séguy 1949a : 158; Séguy 1953a : 86; Mouna 1998 : 85 Séguy 1949a , AA , Agdz; Séguy 1953a , AP , Oued Yquem (near Rabat); Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 Eurina Meigen, 1830 Eurina lurida Meigen, 1830 Ebejer and Kettani 2016 , Rif , M'Diq, Smir, Kabila beach and dunes Eutropha Loew, 1866 Eutropha albipilosa (Becker, 1908) Duda 1930; Deeming and Al-Dhafer 2012 , AP , 17 km N of Larache, AA , Azemmour, estuary of Oued Tensift; Ebejer and Kettani 2016 , AP , Larache (5 m) Eutropha fulvifrons (Haliday, 1833) Séguy 1941d , AA , Agadir; Mouna 1998 ; Cassar et al. 2005 , Rif , Smir lagoon; Ebejer and Kettani 2016 , AP , Larache (5 m) Lagaroceras Becker, 1903 Lagaroceras andalusiaca (Strobl, 1899) Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m), Oued Aliane (Ksar Sghir, 1 m), AP , Larache (2 m), AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Lasiosina Becker, 1910 Lasiosina herpini (Guérin-Méneville, 1843) = Lasiosina cinctipes Meigen, in Mouna 1998 : 85 Ebejer and Kettani 2016 , Rif , Smir lagoon Lasiosina lindbergi (Duda, 1933) = Steleocerus lindbergi (Duda), in Duda 1933: 142 Duda 1933, MA ; Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Meromyza Meigen, 1830 Meromyza athletica Fedoseeva, 1974 = Meromyza variegata Meigen, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2016 Meromyza curvinervis (Zetterstedt, 1848) = Oxinis curvinervis Latreille, in Mouna 1998 : 85 Mouna 1998 Meromyza nigriventris Macquart, 1835 Ebejer and Kettani 2016 , MA , 17 km SW of Midelt (Khénifra, 1940 m), AA , 29 km N of Rich (Errachidia, 1570 m) Meromyza pratorum Meigen, 1830 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Ebejer and Kettani 2016 Metopostigma Becker, 1903 Metopostigma sabulona Becker, 1910 Ebejer and Kettani 2016 , AA , Oued Ziz (10 km S of Errachidia, 1008 m), Merzouga (714 m) Platycephala Fallén, 1820 Platycephala scapularum (Becker, 1907) Nartshuk 1984 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 ; Ebejer and Kettani 2016 Pseudopachychaeta Strobl, 1902 Pseudopachychaeta pachycera Strobl, 1902 Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m), Oued Ziz (1052 m) Thaumatomyia Zenker, 1833 Thaumatomyia elongatula (Becker, 1910) Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil, 3 m) Thaumatomyia glabra (Meigen, 1830) Ebejer and Kettani 2016 , Rif , Martil (9 m) Thaumatomyia notata (Meigen, 1830) = Chloropisca notata (Meigen), in Séguy 1930a : 179, 1941a : 33; Mouna 1998 : 85 Séguy 1930a , Rif , Tanger; Séguy 1941a , HA , Tachdirt (Jebel Toubkal, 2500 m), Canyon Tessaout (M'Goum, 3000–3200 m); Harris et al. 1980 : 229, HA , Chichaoua; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Laou estuary, Oued Amsa (Amsa, 14 m), Jnane Niche (27 m), Oued Maggou (Maggou, 786 m), Marj Khayl (Beni Leit ( PNPB ), 1088 m), Oued Boumarioul (Aïn Hamra, 560 m), Aïn Tissemlal (Azilane, 1255 m), Oued Bouhya (Bou Ahmed, 19 m), Souk Khemis Anjra (Oued Kbir, 55 m), Zinat (231 m), Oued Zarka (Yarghite, 135 m), Oued Talembote (Usine électrique, 120 m), Oued Kelâa (Akoumi, 400 m), Issaguen (maison forestière Issaguen, 1543 m), Bni Boufrah (94 m), Oued Guallet (Bni Boufrah, 946 m), Oued Tabandoute (Bni Boufrah, 540 m), Oued Taâouniya (Koudiat Ajira, 1536 m), Talankramte (Site sacré Sidi Gneiss ( PNPB ): 461 m), AP , Larache (5 m) Thaumatomyia sulcifrons (Becker, 1907) = Chlorops sulcifrons Becker, in Séguy 1949a : 158 = Chloropisca sulcifrons Zetterstedt, in Mouna 1998 : 85 Séguy 1934a ; Séguy 1949a , AA , Foum-el-Hassan, Akka, Agdz; Nartshuk 1984 ; Mouna 1998 ; Dawah and Abdullah 2006 ; Ebejer and Kettani 2016 Oscinellinae Aphanotrigonum Duda, 1932 Aphanotrigonum cinctellum (Zetterstedt, 1848) Ebejer and Kettani 2016 , AP , Larache (5 m) Aphanotrigonum femorellum Collin, 1946 Ebejer and Kettani 2016 , AP , Larache Aphanotrigonum inerme Collin, 1946 Ebejer and Kettani 2016 , Rif , M'Diq (5 m), AP , Larache (Loukous marsh, 2 m) Aphanotrigonum parahastatum Dely-Draskovits, 1981* AA Calamoncosis Enderlein, 1911 Calamoncosis duinensis (Strobl, 1909) Ebejer and Kettani 2016 , AP , Rabat (on Phragmites ) – MISR Conioscinella Duda, 1929 Conioscinella frontella (Fallén, 1810) Ebejer and Kettani 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m) Conioscinella sordidella (Zetterstedt, 1848) Ebejer and Kettani 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m) Elachiptera Macquart, 1835 Elachiptera bimaculata (Loew, 1845) De Lépiney and Mimeur 1932: 110, Rabat; Séguy 1934a : 479; Bléton and Fieuzet 1943 ; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil: 3 m), Jnane Niche (46 m), Oued Mhannech (18 m), Souk Khemis Anjra (55 m), Boujdad (7 m); AP (Rabat) – MISR Elachiptera cornuta (Fallén, 1820) Mouna 1998 : 85 Elachiptera diastema Collin, 1946 Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m), Oued Tiffert (Tiffert, 1230 m), Issaguen (1543 m), AP , Larache (5 m) Elachiptera graeca Becker, 1910 Séguy 1941, HA , Imi-n'Ouaka (1500 m); Nartshuk 1984 ; Mouna 1998 Elachiptera megaspis (Loew, 1858) Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Dardara (730 m), Aïn Jdioui (Tahaddart, 8 m), Jnane Niche (46 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m); AP (Rabat) – MISR Elachiptera orizae Séguy, 1949* AA Elachiptera rufescens (Walker, 1871) Deeming and Al-Dhafer 2012 , AA , 2 km N Erfoud (818 m); Ebejer and Kettani 2016 , AA , 2 km N Erfoud (818 m) Elachiptera rufifrons Duda, 1932 Ebejer and Kettani 2016 , AP , 9 km SE Aïn Chouk (6 m), Larache (5 m) Elachiptera sarda Nartshuk, 2009 Ebejer and Kettani 2016 , AA , 14 km E of Rich (Errachidia, 1278 m) Elachiptera scrobiculata (Strobl, 1901) = Elachiptera trapezina (Corti, 1909), in Bléton and Fieuzet 1943 : 116 Bléton and Fieuzet 1943 ; Ebejer and Kettani 2016 Elachiptera strobli (Corti, 1909) Ebejer and Kettani 2016 , Rif , Oued Boumarioul (Aïn Hamra, 560 m), Oued Aliane (Ksar Sghir, 1 m); Cap Spartel (155 m), Oued Sidi Ben Saâda (Laghdir, 242 m) Epimadiza Becker, 1910 Epimadiza nigrescens Duda, 1933 50 = Oscinosoma anthracias Séguy, in Séguy 1949a : 158, Mouna 1998 : 85 = Oxinosoma anthracias Séguy, in Mouna 1998 : 85 = " Siphonella oscinina (Fallén)", in Nartshuk 1984 : 236 Séguy 1949a , AA , Alnif; Sabrosky 1965 : 406; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 Hapleginella Duda, 1933 Hapleginella laevifrons (Loew, 1858) El Hassani et al. 1986: 8–11, Rif , Nord Occidental, MA Lasiochaeta Corti, 1909 Lasiochaeta pubescens (Thalhammer, 1898) = Elachiptera pubescens (Thalhammer), var. rufithorax Duda, 1932 in Duda 1932: 31 = Melanochaeta pubescens Thalh., in De Lépiney and Mimeur 1932: 110 = Melanochaeta pubescens (Thalhammer), in Mouna 1998 : 85 De Lépiney and Mimeur 1932, AP , Rabat; Duda 1932: 31, HA ; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Sidi Yahia Aarab (Sidi Yahia Aarab, 178 m), Beni Maâdene (Oued Martil, 3 m), Oued Mhannech (Tamuda, 18 m), Oued Amsa (Amsa, 14 m), Zinat (231 m), Oued Maggou (Maggou, 786 m), Oued Guallet (Bni Boufrah, 946 m); AP (Rabat) – MISR Oscinella Becker, 1909 Oscinella cariciphila Collin, 1946 Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil, 3 m), AA , Merzouga (714 m) Oscinella frit (Linnaeus, 1758) = Oxinosoma frit Linnaeus, in Mouna 1998 : 85 De Lépiney and Mimeur 1932: 109, AP , Rabat; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Issaguen (1543 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m), Oued Guallet (Bni Boufrah, 946 m), Oued Ametrasse (Ametrasse, 841 m), AP , Lower Loukous saltmarsh (2 m), MA , Khénifra (17 km SW of Midelt, 1940 m), Lac Aguelmane Afennourir (30 km SW of Azrou, 1490 m); AP (Rabat) – MISR Oscinella nartshukiana Beschovski, 1978 Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m), AP , Larache Oscinella nitidigenis (Becker, 1908) Ebejer and Kettani 2016 , AA , 6 km N of Errachidia (1010 m), Oued Ziz (1052 m) Oscinella nitidissima (Meigen, 1838) Ebejer and Kettani 2016 , Rif , Dardara (730 m), Moulay Abdelsalam (965 m), Cap Spartel (155 m), Oued Laou (dunes, 2 m), AP , Larache (5 m), MA , Lac Aguelmane Afennourir (30 km SW of Azrou, 1490 m) Oscinella pusilla (Meigen, 1830) Ebejer and Kettani 2016 , AA , Lac Tiffert (4 km W of Merzouga, 702 m) Oscinella ventricosi Nartshuk, 1955 Ebejer and Kettani 2016 , Rif , Oued Boumarioul (Aïn Hamra, 560 m), AP , Larache (5 m) Oscinella vindicata (Meigen, 1830) Ebejer and Kettani 2016 , AP , Larache (5 m), AA , Merzouga (agriculture under date palms, 714 m) Oscinimorpha Lioy, 1864 Oscinimorpha arcuata (Duda, 1932) Ebejer and Kettani 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), Oued Jnane Azaghar (Bni Boufrah, 997 m) Oscinimorpha longirostris (Loew, 1858) Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m) Oscinimorpha minutissima (Strobl, 1900) = Siphonella minutissima Strobl, in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Dardara (484 m) Oscinimorpha novakii (Strobl, 1893) = Conioscinella novakii (Strobl), in Duda 1933: 58 Duda 1933; Ebejer and Kettani 2016 Oscinisoma Lioy, 1864 Oscinisoma cognatum (Meigen, 1830) = Oscinis rufipes Meigen, 1830, with homonym Oscinis rufipes Wiedemann 1830 : 580, in Wiedemann 1830 : 580, Tanger; synonymy and probable specific identity of homonyms established by Becker 1910 : 166, but without considering the similar Oscinisoma gilvipes (Loew, 1858). Polyodaspis Duda, 1933 51 Polyodaspis sulcicollis (Meigen, 1838) = Siphonella sulcicollis (Meigen), in Kroschel and Klein 1999 : 138 Kroschel and Klein 1999 ; Ebejer and Kettani 2016 , MA , Khénifra (17 km SW of Midelt, 1940 m) Pselaphia Becker, 1911 Pselaphia dimidiocera Ebejer & Kettani, 2016 Ebejer and Kettani 2016 , Rif , Adrou (Taghzout, 556 m) – NMWC Rhodesiella Adams, 1905 Rhodesiella fedtshenkoi Nartshuk, 1978 Deeming and Al-Dhafer 2012 , SA , Goulimine (Bou Jarif); Ebejer and Kettani 2016 , Rif , Smir lagoon, Oued Laou (saltmarsh), Jnane Niche (46 m) Sabroskyina Beschovski, 1987 Sabroskyina aharonii (Duda, 1933) Ebejer and Kettani 2016 , AA , 2 km N Erfoud (818 m) Siphunculina Rondani, 1856 Siphunculina ornatifrons (Loew, 1858) = Microneurum ornatifrons (Loew), in Duda 1933: 98 Becker and Stein 1913 , Rif , Tanger; Duda 1933; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Martil (9 m), Oued Mhannech (Tamuda, 18 m), Jnane Niche (27 m), AP , Aïn Chouk 9 km SE (6 m), Larache (5 m) Trachysiphonella Enderlein, 1936 Trachysiphonella ruficeps (Macquart, 1835) Ebejer and Kettani 2016 , AA , 29 km N of Rich (Errachidia, 1570 m) Tricimba Lioy, 1864 Tricimba cincta (Meigen, 1830) Ebejer and Kettani 2016 , Rif , Dardara (484 m) Tricimba heratica Dely-Draskovits, 1983* AA Tricimba humeralis (Loew, 1858) = Tricimba punctifrons Becker, in Becker and Stein 1913 : 93 = Tricimba humeralis (Loew), in Séguy 1941d : 18, 1957 : 273; Mouna 1998 : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1941d , AA , Agadir; Séguy 1957 , AA , Agadir; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Mhannech (Tamuda, 18 m), Aïn Tissemlal (Azilane, 1255 m), Oued Bouhya (Bou Ahmed, 19 m), AA , Oued Ziz (9.5 km SE of Rich, 1285 m), Oued Ziz (13 km N of Erfoud, 800 m) Siphonellopsinae Apotropina Hendel, 1907 Apotropina longepilosa (Strobl, 1893) Ebejer and Kettani 2016 , Rif , Oued Sidi Ben Saâda (Laghdir, 242 m), Oued Kbir (Dardara, 345 m), Oued Siflaou (281 m), Jnane Niche (46 m) Siphonellopsis Strobl, 1906 Siphonellopsis lacteibasis Strobl, 1906 Séguy 1934a : 482; Nartshuk 1984 ; Mouna 1998 New records for Morocco Aphanotrigonum parahastatum Dely-Draskovits, 1981 Anti Atlas: Ougui river, 1.8 km NW Tissint, 29°55'03"N, 7°19'56"W , 582 m, 30.xii.2016, 3♂♂3♀♀, M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). Elachiptera orizae Séguy, 1949 Anti Atlas: river Aoulouz up stream of Idrgane, 30°44'11"N, 7°59'13"W , 866 m, 31.xii.2016, 1♂, M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). Tricimba heratica Dely-Draskovits, 1983 Anti Atlas: Oued Souss, streamup of Idrgane, 30°44'11"N, 7°59'13"W , 866 m, 31.xii.2016, 1♀ (males are needed to confirm the identification), M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). MILICHIIDAE K. Kettani Number of species: 8 . Expected: 14 Faunistic knowledge of the family in Morocco: moderate Madizinae Madizini Desmometopa Loew, 1866 Desmometopa m-nigrum (Zetterstedt, 1848) Séguy 1949a , SA , Guelmim; Mouna 1998 ; Rif (M'Diq), AP (Larache), HA (Tazzarin) – MISR Desmometopa varipalpis Malloch, 1927 Ebejer et al. 2019 , AA , 6 km N of Errachidia (1010 m) Leptometopa Becker, 1903 Leptometopa rufifrons Becker, 1903 Ebejer et al. 2019 , AA , Merzouga (714 m); AA (Merzouga) – MISR Madiza Fallén, 1810 Madiza glabra Fallén, 1820 Mouna 1998 ; Ebejer et al. 2019 , HA , Anafgou ( NPHAO , 2271 m) Milichiinae Milichiini Milichia Meigen, 1830 Milichia albomaculata (Strobl, 1900) Pont and Singh 1965 ; Mouna 1998 ; Brake 2000 ; Koçak and Kemal 2010 ; HA – NHMUK Milichia speciosa Meigen, 1830 Séguy 1930a , HA , Arround (Skoutana) Milichiella Giglio-Tos, 1895 Milichiella lacteipennis (Loew, 1866) Brake 2000 ; Raspi et al. 2009 ; Rif (M'Diq farm) – MISR Phyllomyzinae Phyllomyzini Phyllomyza Fallén, 1810 Phyllomyza sp. aff. equitans (Hendel, 1919) Ebejer et al. 2019 , AA , Oued Ziz (12 km S of Rissani, 737 m) Acknowledgement We gratefully acknowledge the cooperation of Martin J. Ebejer who contributed to the revision of this family. Sphaeroceroidea CHYROMYIDAE K. Kettani, M.J. Ebejer Number of species: 22 . Expected: 30 Faunistic knowledge of the family in Morocco: good Aphaniosominae Aphaniosoma Becker, 1903 Aphaniosoma approximatum Becker, 1903 Ebejer 2018 , SA , Oued Ougni (0.6 km N of Akka, N'Aït Sidi and 1.8 km NW Tissint, 582 m) Aphaniosoma claridgei Ebejer, 1995 Ebejer 2016b , Rif , Smir lagoon Aphaniosoma collini Lyneborg, 1973 Ebejer 2016b , AP , Larache (5 m) Aphaniosoma forcipatum Ebejer, 1998 Ebejer 2016b , AA , Lac de Tiffert (4 km W of Merzouga, 702 m), Erfoud (30 km N, 894 m), Ziz river (14 km E of Rich, 1278 m) Aphaniosoma gatti Ebejer, 2016 Ebejer 2016b , AP , El Jadida, Azemmour; AP (El Jadida Azemmour) – MISR Aphaniosoma melitense Ebejer, 1993 Andrade and Almeida 2010 , Rif , Mediterranean coast; Ebejer 2016b , AP , Larache (2 m) Aphaniosoma nigricauda Ebejer, 1998 Ebejer 2016b , AA , Lac de Tiffert (4 km W of Merzouga, 702 m), Merzouga (714 m), Ziz river (10 km S of Errachidia, 1008 m; 13 km N of Erfoud, 800 m) Aphaniosoma nigripes Ebejer, 2016 Ebejer 2016b , AA , Ziz river (9.5 km SE of Rich, 1285 m), Erfoud (30 km N, 894 m; AA (Ziz river) – MISR Aphaniosoma nigrum Ebejer, 1998 Ebejer 2016b , AA , Ziz river (Errachidia, 1052 m; 9.5 km SE of Rich, 1285 m), Erfoud (30 km N, 894 m); AA (Ziz river) – MISR Aphaniosoma nitidum Ebejer, 2016 Ebejer 2016b , AA , Ziz river (30 km N of Erfoud, 894 m) Aphaniosoma propinquans Collin, 1949 Ebejer 2016b , Rif , Smir lagoon, Oued Aliane (Ksar Sghir), AP , Larache (2 m); Rif (Oued Aliane) – MISR Aphaniosoma proximum Ebejer, 1998 Ebejer 2016b , AP , Larache (5 m), Sidi Smail (El Jadida), Azemmour, HA , Oued Tensift Aphaniosoma quadrinotatum (Becker, 1904) Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Aphaniosoma rufum Frey, 1935 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Aphaniosoma soror Ebejer, 2016 Ebejer 2016b , AA , Errachidia (14 km E of Rich, 1278 m) Aphaniosoma trisetum Ebejer, 2016 Ebejer 2016b , AA , Ziz river (9.5 km SE of Rich, 1285 m, 13 km N of Erfoud, 800 m, Errachidia, 1052 m), Merzouga (714 m); AA (Ziz river) – MISR Aphaniosoma zizense Ebejer, 2016 Ebejer 2016b , AA , Ziz river (Errachidia, 1052 m; 9.5 km SE of Rich, 1285 m; 10 km S of Errachidia, 1008 m); AA (Ziz river) – MISR Chyromyinae Chyromya Robineau-Desvoidy, 1830 Chyromya robusta Hendel, 1931 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Gymnochiromyia Hendel, 1933 Gymnochiromyia flavella (Zetterstedt, 1848) Ebejer 2016b , Rif , Ksar El Kebir (13 m) Gymnochiromyia homobifida Carles-Tolrá, 2001 Ebejer 2016b , Rif , maison forestière de Talassemtane ( NPT ) Gymnochiromyia inermis (Collin, 1933) Ebejer 1998b ; Ebejer 2016b Gymnochiromyia mihalyii Soós, 1979 Ebejer 2016b , Rif , Oued Siflaou (281 m), Aïn Tissemlal (Azilane, 1255 m), AP , Larache (5 m) Gymnochiromyia tschirnhausi Ebejer, 2018 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Gymnochiromyia zernyi (Czerny, 1929) Ebejer 1998b ; Ebejer 2016b HELEOMYZIDAE K. Kettani, A.J. Woźnica Number of species: 19 . Expected: 30 Faunistic knowledge of the family in Morocco: moderate Heleomyzinae Gymnomus Loew, 1863 Gymnomus atlasicus Woźnica, 2011 Woźnica 2011 , HA , Tazzeka Gymnomus caesius (Meigen, 1830) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Neoleria Malloch, 1919 Neoleria sp.* Rif Heteromyzinae Tephrochlamys Loew, 1862 Tephrochlamys rufiventris (Meigen, 1830)* AA Suilliinae Suillia Robineau-Desvoidy, 1830 Suillia bicolor (Zetterstedt, 1838)* Rif Suillia bistrigata (Meigen, 1830) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Bouhachem, 1098 m), Jebel Lakraâ (Talassemtane, 1541 m), MA , 6 km S of Azrou (1610 m), 20 km S of Azrou (1720 m) Suillia humilis (Meigen, 1830) = Helomyza similis Meigen, in Mouna 1998 : 85 Mouna 1998 ; AP (Maâmora) – MISR Suillia notata (Meigen, 1830) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Bouhachem, 1180 m), Jebel Lakraâ (Talassemtane, 1541 m), Oued Kbir (Béni Ratene, 157 m) Suillia oxyphora (Mik, 1900)* Rif Suillia pallida (Fallén, 1820) Séguy 1941d , HA , Tizi-n'Test (2200 m); Mouna 1998 Suillia tuberiperda (Rondani, 1876) Ebejer et al. 2019 , MA , 6 km S of Azrou (1610 m) Suillia variegata (Loew, 1862) = Helomyza variegata Loew, in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AP , Sidi Yahia du Gharb; Mouna 1998 Trixoscelidinae Trixoscelis Rondani, 1856 Trixoscelis approximata (Loew, 1865) Cassar et al. 2008 , Rif , Smir lagoon; Rif , Tétouan Trixoscelis baliogastra (Černý, 1909) Hackman 1970 Trixoscelis canescens (Loew, 1865) Ebejer et al. 2019 , Rif , Aïn Tissemlal (Azilane, 1255 m) Trixoscelis curvata Carles-Tolrá, 1993 Ebejer et al. 2019 , AP , Larache (5 m) Trixoscelis laeta (Becker, 1907) Becker 1907 ; Séguy 1941d , AA , Agadir; Hackman 1970 ; Mouna 1998 ; Cassar et al. 2008 , Rif , Smir lagoon Trixoscelis mendizabali Hackman, 1970 Hackman 1970 , AA , Aït Melloul (Oued Souss) Trixoscelis pedestris (Loew, 1865) Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 76 m) New records for Morocco Neoleria sp. Rif: Tétouan, Jebel Bouhachem ( PNPB ), Amsemlil, 35.26234°N, 5.43341°W , 1067 m, 11.xii.15, forest ( Pinus pinaster ), K. Kettani leg. (1♀), A.J. Woźnica det. Tephrochlamys rufiventris (Meigen, 1830) Rif: Tétouan, Jebel Bouhachem ( PNPB ), Remla, 35.236865, -5.408025, 961 m, 22.iv.18, forest ( Pinus pinaster ), K. Kettani leg. (1♀), A.J. Woźnica det. Anti Atlas: Errachidia, 13 km E of Goulmima, 31°44.568N, 4°51.945W , dry stony steppe 1100 m, 3.v.2012, M.J. Ebejer leg. (1♂), A.J. Woźnica det. Suillia bicolor (Zetterstedt, 1838) Rif: Talassemtane, Jebel Lakraâ, 35°06.913N, 5°08.034W , 1541 m, 12.vi.2013, meadow in mixed forest, M.J. Ebejer leg. (1♀); Jebel Bouhachem, Taghzout, Adrou, 556 m, 35°22.39N, 5°32.28W , 25.iv.2015, M.J. Ebejer leg. (2♀♀), A.J. Woźnica det. Suillia oxyphora (Mik, 1900) Rif: Jebel Talassemtane, 35°07'N, 5°07'W , 1554 m, 13.iv.2009, Fir forest ( Abies maroccana ), A. Taheri leg. (1♂3♀♀); Azilane, Aïn Tissemlal, 35°11.67N, 5°15.20W , 1255 m, 4.vii.13–13.viii.2013, K. Kettani leg. (1♀), Fir forest ( Abies maroccana ), A.J. Woźnica det. SPHAEROCERIDAE K. Kettani, P. Gatt Number of species: 67 . Expected: 130 Faunistic knowledge of the family in Morocco: poor Copromyzinae Borborillus Duda, 1923 Borborillus vitripennis (Meigen, 1830) Becker and Stein 1913 , Rif , Tanger; Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Copromyza Fallén, 1810 Copromyza equina Fallén, 1820 Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Crumomyia Macquart, 1835 Crumomyia glabrifrons (Meigen, 1830) Gatt et al. 2016 , Rif , Tariouma (El Anasser, 1383 m), MA , Lac Aguelmane Sidi Ali (2050 m) Lotophila Lioy, 1864 Lotophila atra (Meigen, 1830) Gatt 2006 , Rif , Tanger; Marshall et al. 2011 Norrbomia Papp, 1988 Norrbomia hispanica (Duda, 1923) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Norrbomia marginatis (Adams, 1905) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Norrbomia nilotica (Becker, 1903) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia niveipennis (Duda, 1923) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia somogyii (Papp, 1973) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia sordida (Zetterstedt, 1847) Gatt et al. 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), AP , Larache, Loukous Marsh Limosininae Bifronsina Roháček, 1983 Bifronsina bifrons (Stenhammar, 1855) Roháček et al. 2001 ; Marshall et al. 2011 Ceroptera Macquart, 1835 Ceroptera rufitarsis (Meigen, 1830) = Limosina picta Becker, in Becker and Stein 1913 : 94 Becker and Stein 1913 , Rif , Tanger; Papp 1984a ; Roháček et al. 2001 , Rif , Tanger Chaetopodella Duda, 1920 Chaetopodella scutellaris (Haliday, 1836) = Leptocera scutellaris (Haliday), in Séguy 1941a : 32 Séguy 1941a , HA , Imi-n'Ouaka (1500 m) Coproica Rondani, 1861 Coproica digitata (Duda, 1918) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Coproica ferruginata (Stenhammar, 1855) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Coproica hirticula Collin, 1956 Gatt et al. 2016 , Rif , Dardara (730 m), Oued Laou (30 m), Zarka waterfall (Yarghit, 135 m), Oued Ouara (Beni Zid, 440 m), Oued Jnane Azaghar (Bni Boufrah, 997 m), Oued Mhannech (125 m), Adrou (Taghzout, 556 m), Oued Jnane Niche (Jnane Niche, 27 m) Coproica hirtula (Rondani, 1880) Gatt et al. 2016 , Rif , Zarka waterfall (Yarghit, 135 m), AA , 14 km N Errachidia (Errachidia, 1214 m) Coproica lugubris (Haliday, 1836) Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Coproica pusio (Zetterstedt. 1847) Gatt et al. 2016 , Rif , Tamrabete (Oued Laou, 203 m), Smir lagoon (5 m) Coproica rohaceki Carles-Tolrá, 1990 Gatt et al. 2016 , Rif , Oued Amsa (Amsa, 14 m), Oued Moulay Bouchta (Dar Akobaâ, 285 m) Coproica rufifrons Hayashi, 1991 Gatt et al. 2016 , Rif , Issaguen (1543 m) Coproica vagans (Haliday, 1833) Gatt et al. 2016 , AA , 14 km N Errachidia (1214 m), AA , Oued Ziz (13 km N Erfoud, 800 m), SA , Merzouga (698 m) Eulimosina ochripes (Meigen, 1830) Gatt et al. 2016 , Rif , Issaguen (1543 m), Ksar el Kebir (20 m), MA , Lac Aguelamane Afenourrir (30 km SW Azrou, 1760 m) Leptocera Olivier, 1813 Leptocera caenosa (Rondani, 1880) Gatt et al. 2016 , Rif , Dardara (484 m) Leptocera fontinalis (Fallén, 1826) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Leptocera nigra Olivier, 1813 Munari 1993 , MA , Azrou; Roháček et al. 2001 ; Marshall et al. 2011 Limosina Macquart, 1835 Limosina silvatica (Meigen, 1830) Gatt et al. 2016 , Rif , Issaguen (1543 m), Talassemtane ( NPT , 1696 m), MA , 3,5 km S Azrou (1450 m), 17 km NW Zaida (Khénifra, 1878 m) Minilimosina Roháček, 1983 Minilimosina ( Svarciella ) vitripennis (Zetterstedt, 1847) Gatt et al. 2016 , MA , 3,5 km S Azrou (1450 m) Opacifrons Duda, 1918 Opacifrons coxata (Stenhammar, 1855) Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Opacifrons maculifrons (Becker, 1907) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Opalimosina Roháček, 1983 Opalimosina ( Opalimosina ) mirabilis (Collin, 1902) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Paralimosina Papp, 1973 Paralimosina fucata (Rondani, 1880) Gatt et al. 2016 , Rif , 5 km W Dardara (730 m), Dardara (484 m), Oued Azla (Mokdassen Oulya, 186 m), Oued Talembote (Talembote, 320 m), Oued El Khizana (El Khizana, 980 m), Jebel Kelaâ (Talassemtane, 1554 m), Adrou (Taghzout, 556 m) Phthitia Enderlein, 1938 Phthitia ( Kimosina ) sicana (Munari, 1988) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Phthitia ( Kimosina ) ciliata (Duda, 1918) Gatt et al. 2016 , SA , Bou Jarif (Goulimine) Phthitia ( Kimosina ) plumosula (Rondani, 1880) Gatt et al. 2016 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), Jebel Kelaâ (Talassemtane, 1554 m), Zaouiet and Habtiyène (Maggou, 1213 m), Oued Arozane (Bni Moussa, 317 m), Issaguen (1543 m), Zarka waterfall (Yarghit, 135 m), Taghramt (Bine El Ouidane, 276 m) Phthitia ( Kimosina ) pteremoides (Papp, 1973) Gatt et al. 2016 , SA , Bou Jarif (Goulimine) Poecilosomella Duda, 1925 Poecilosomella angulata (Thomson, 1869) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Pseudocollinella Duda, 1924 Pseudocollinella jorlii (Carles-Tolrá, 1990) Munari 1993 , MA , Aguelmane; Roháček et al. 2001 ; Marshall et al. 2011 Pullimosina Roháček, 1983 Pullimosina ( Pullimosina ) heteroneura (Haliday, 1836) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Pullimosina ( Pullimosina ) zayensis Marshall, 1986 Roháček et al. 2001 ; Marshall et al. 2011 Puncticorpus Duda, 1918 Puncticorpus lusitanicum (Richards, 1963) Papp 1984a ; Roháček et al. 2001 Rachispoda Lioy, 1864 Rachispoda acrosticalis (Becker, 1903) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda brevior (Roháček, 1991) Roháček 1991 , AP , Oued Bou-Regreg; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda duodecimseta (Papp, 1973) Roháček 1991 , EM , Guercif-Oued Moulouya; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda fuscipennis (Haliday, 1833) Roháček 1991 , EM , Oued Bou-Regreg; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda gel (Papp, 1978) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda kabuli (Papp, 1978) Roháček 1991 , MA , Taza, Oued Fès; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda lagura (Roháček, 1991) Gatt et al. 2016 , Rif , Ksar el Kebir (20 m), AA , Oued Ziz (12 km S Rissani, 737 m), Oued Ziz (13 km N Erfoud, 800 m), Oued Ziz (30 km N Erfoud, 894 m) Rachispoda lutosoidea (Duda, 1938) Roháček 1991 , MA , Azrou, Aguelmane, Oued Sebou; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda modesta (Duda, 1924) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda uniseta (Roháček, 1991) Roháček 1991 , MA , Taza, Oued Fès; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 ; Vaňhara and Rozkošný 1997 Rachispoda varicornis (Strobl, 1900) Roháček 1991 , AP , Oued Bou-Regreg, MA , Azrou; Munari 1993 , MA , Oued Sebou; Roháček et al. 2001 ; Marshall et al. 2011 Spelobia Spuler, 1924 Spelobia baezi (Papp, 1977) Roháček et al. 2001 ; Marshall et al. 2011 Spelobia clunipes (Meigen, 1830) Gatt et al. 2016 , Rif , 5 km W Dardara (730 m), Martil (beach and dunes), Oued Mhannech (18 m), Oued Afertane (Afertane, 56 m), Maggou waterfall (Maggou, 786 m), Onsar Akboul (NPB, 1315 m), Oued Ametrasse (Ametrasse, 841 m), Oued Tahaddart (Tahaddart, 87 m), Issaguen (1543 m), Lemtahane ( PNPB , 1088 m) Spelobia hungarica (Villeneuve, 1917) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Spelobia quaesita Roháček, 1983 Roháček et al. 2001 ; Marshall et al. 2011 Spinilimosina Roháček, 1983 Spinilimosina brevicostata (Duda, 1918) Roháček et al. 2001 ; Marshall et al. 2011 Telomerina Roháček, 1983 Telomerina pseudoleucoptera (Duda, 1924) Gatt et al. 2016 , Rif , Maggou waterfall (Maggou, 786 m) Thoracochaeta Duda, 1918 Thoracochaeta brachystoma (Stenhammar, 1855) Gatt 2006 ; Cassar et al. 2008 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Thoracochaeta erectiseta Carles-Tolrá, 1994 Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Trachyopella Duda, 1918 Trachyopella ( Trachyopella ) coprina (Duda, 1918) Gatt et al. 2016 , Rif , Oued Jnane Niche (Jnane Niche, 27 m) Trachyopella ( Trachyopella ) melania (Haliday, 1836) Gatt et al. 2016 , Rif , Oued Nwawel (Azla, 57 m), M'Diq (5 m) Sphaerocerinae Ischiolepta Lioy, 1864 Ischiolepta pusilla (Fallén, 1820) Gatt et al. 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), Aïn Tissemlal (Azilane, 1255 m) Ischiolepta vaporariorum (Haliday, 1836) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Lotobia Lioy, 1864 Lotobia africana (Becker, 1907) Roháček et al. 2001 ; Marshall et al. 2011 Lotobia pallidiventris (Meigen, 1830) Gatt 2006 , Rif , Tétouan, M'Diq, Oued Laou; Marshall et al. 2011 Sphaerocera Latreille, 1804 Sphaerocera curvipes Latreille, 1805 Roháček et al. 2001 ; Marshall et al. 2011 Ephydroidea BRAULIDAE K. Kettani Number of species: 1 . Expected: 1 Faunistic knowledge of the family in Morocco: good Braulinae Braula Nitzsch, 1818 Braula coeca Nitzsch, 1818 Séguy 1930a ; Mouna 1998 ; Howard et al. 2000 CAMILLIDAE K. Kettani, M.J. Ebejer Number of species: 4 . Expected: 5 Faunistic knowledge of the family in Morocco: poor Camilla Haliday in Curtis, 1837 Camilla acutipennis (Loew, 1865) Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 76 m), Chrabkha pond (Al Manzla, 58 m) Camilla flavicauda Duda, 1922 Mouna 1998 ; Pârvu et al. 2006 , AP , Cap Sim; Popescu-Mirceni 2011 Camilla glabra (Fallén, 1823) Séguy 1941d , HA , Tizi-n'Test; Mouna 1998 Camilla pruinosa Duda, 1934 Ebejer et al. 2019 , AP , Larache (5 m) CRYPTOCHETIDAE K. Kettani, E.P. Nartshuk Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Cryptochetum Rondani, 1875 Cryptochetum (as Cryptochaetum ) mimeuri Séguy, 1953 Séguy 1953c , MA , Ifrane; Nartshuk 1979 , MA , Ifrane DIASTATIDAE K. Kettani, M.J. Ebejer Number of species: 2 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Campichoetinae Campichoeta Macquart, 1835 Campichoeta obscuripennis (Meigen, 1830) Ebejer et al. 2019 , HA , Lalla Takrkoust (628 m) Diastatinae Diastata Meigen, 1830 Diastata adusta Meigen, 1830 = Diastata unipunctata Zetterstedt, 1847 Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 DROSOPHILIDAE K. Kettani, G. Bächli Number of species: 26 . Expected: 30 Faunistic knowledge of the family in Morocco: good Drosophilinae Drosophilini Drosophila Fallén, 1823 Drosophila ( Drosophila ) busckii Coquillett, 1901 = Drosophila rubrostriata Becker, 1908, in Séguy 1953a : 85 Séguy 1953a , AP , Rabat, Sidi Yahia du Gharb; Prevosti 1974 , HA , Asni; Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech – MISR Drosophila ( Drosophila ) buzzatii Patterson & Wheeler, 1942 Prevosti 1974 , HA , Asni (Admin forest); Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Drosophila ( Drosophila ) funebris (Fabricius, 1787) Prevosti 1974 , HA , Asni; Mouna 1998 ; AP (Maâmora) – MISR Drosophila ( Drosophila ) hydei Sturtevant, 1921 Staiger and Gloor 1952 , AP , Rabat; Gloor and Satiger 1954, AP , Rabat; Prevosti 1974 , HA , Asni (Admin forest); Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech Drosophila ( Drosophila ) immigrans Sturtevant, 1921 Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Drosophila ( Drosophila ) kuntzei Duda, 1924 Mouna 1998 ; Prevosti 1974 , AP , Essaouira, HA , Asni Drosophila ( Drosophila ) mercatorum Patterson & Wheeler, 1942 Prevosti 1974 , AA , Agadir (Admin forest near Agadir); Mouna 1998 Drosophila ( Drosophila ) phalerata Meigen, 1830 Prevosti 1974 , AP , Essaouira, HA , Asni; Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger), MA (Ifrane) – ZSM Drosophila ( Drosophila ) repleta Wollaston, 1858 Séguy 1953a , AP , Rabat, MA , Fès Drosophila ( Drosophila ) tsigana Burla & Gloor, 1952 Suwito et al. 2014 , MA , Ifrane Drosophila ( Sophophora ) ambigua Pomini, 1940 Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger), MA (Ifrane) – ZSM Drosophila ( Sophophora ) melanogaster Meigen, 1830 Medioni 1958 ; David and Bocquet 1973 , HA , Marrakech, AA , Agadir, Ouarzazate, Taroudant, AA , Zagora; Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Jousset and Plus 1975 ; Plus et al. 1975 , HA , Marrakech, AA , Agadir, Ouarzazate, Taroudant, AA , Zagora; Prevosti et al. 1975 , Rif , Tanger; Allemand and David 1976 , AA , Ouarzazate; Ashburner and Lemeunier 1976 , HA , Marrakech, AA , Agadir, Taroudant, AA , Zagora; Fleuriet 1976 , AA , Zagora; Plus et al. 1976 , AA , Ouarzazate, Zagora; Jousset 1976 , AA , Ouarzazate; Plus and Scotti 1984 , AA , Ouarzazate; Thomas-Orillard 1984 , AA , Ouarzazate; Afonso et al. 1985 , HA , Asni; Prevosti et al. 1985 , AP , Essaouira, AA , Agadir; David et al. 1986 , AP , Rabat, Casablanca; Ayala et al. 1989 , Rif , Chefchaouen; Boulétreau et al. 1992, AA , Agadir; Costa et al. 1992 , AP , Casablanca; Capy et al. 1993 , AP , Casablanca, Rabat; Ritchie et al. 1994 , AP , Casablanca; Mouna 1998 ; Bonnivard and Higuet 1999 , AA , Agadir; Chakir et al. 2002 , 2007 , 2008 , 2011 , HA , Marrakech; Ayrinhac et al. 2004 , HA , Marrakech; Catania et al. 2004 , HA , Marrakech AA , Agadir; Rohmer et al. 2004 , HA , Marrakech; Dieringer et al. 2005 , HA , Marrakech, AA , Agadir; Yassin and Orgogozo 2013 , HA , Marrakech; Bächli 2015 (TaxoDros) – HNHM , AP (Rabat), HA (Marrakech) – MISR , MA (Ifrane) – ZSM Drosophila ( Sophophora ) simulans Sturtevant, 1919 Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Baba-Aissa et al. 1988 , AP , Rabat; Nigro 1988 , AP , Larache; Capy et al. 1990, 1992 , 1993 , AP , Rabat, MA , Béni Mellal AA , Agadir; Chakrani et al. 1993 , AA , Agadir; Mouna 1998 ; Charlat et al. 2003 , AA , Agadir; Biémont et al. 2003 , HA , Marrakech; Chakir et al. 2002 , 2007 , 2008 , 2011 , HA , Marrakech; Nardon et al. 2005 , HA , Marrakech; Yassin and Orgogozo 2013 , HA , Marrakech; Bächli 2015 (TaxoDros); AA (Agadir) – ZSM Drosophila ( Sophophora ) subobscura Collin, 1936 Götz 1965 , Rif , Tanger; Prevosti 1971a , Rif , Tanger; Prevosti 1971b , AP , Essaouira, HA , Asni, AA , Ait-Melloul; Prevosti 1971c ; Gonzalez-Duarte et al. 1973, AP , Essaouira, HA , Asni AA , Agadir; Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Prevosti et al. 1975 , AA , Agadir; Duarte 1976, AP , Essaouira, HA , Asni AA , Agadir; Gonzalez 1976, AP , Essaouira, HA , Asni, AA , Agadir; Prevosti 1978 , Rif , Tanger, AP , Essaouira, HA , Asni AA , Agadir; Krimbas and Loukas 1980 , AA , Agadir; Cabrera et al. 1983 , Rif , Chefchaouen; Larruga et al. 1983 , Rif , Chefchaouen; Prevosti et al. 1985 , AA , Agadir; Pascual et al. 1986 , Rif , Chefchaouen; Latorre et al. 1986 , Rif , Chefchaouen; Constanti et al. 1986 , Rif , Chefchaouen; Ayala et al. 1989 , Rif , Chefchaouen; Afonso et al. 1990 , Rif , Chefchaouen; Pascual et al. 1990 , Rif , Chefchaouen; Paricio et al. 1991 , Rif , Chefchaouen; Menozzi and Krimbas 1992 , AA , Agadir; Latorre et al. 1992 , Rif , Chefchaouen; Fain et al. 1993 , AA , Agadir; Alberola and Frutos 1993; Alberola and Frutos 1996 ; Pinto et al. 1997 , HA , Asni, Marrakech AA , Agadir; Mouna 1998 ; David et al. 2003 , HA , Marrakech; Brehm et al. 2004 , AA , Agadir; Nardon et al. 2005 , HA , Marrakech; Bächli 2015 (TaxoDros); MA (Azrou) – NHMD Drosophila ( Sophophora ) suzukii (Matsumura, 1931) Landolt et al. 2012 , Rif , north-eastern Morocco Hirtodrosophila Duda, 1924 Hirtodrosophila cameraria (Haliday, 1833) Ebejer et al. 2019 , Rif , Aïn Ras el Ma, ruisseau maison forestière (Talassemtane) Lordiphosa Basden, 1961 Lordiphosa andalusiaca (Strobl, 1906) = Lordiphosa forcipata (Collin, 1952), in Hackman 1960 : 102 Hackman 1960 ; Bächli 2015 (TaxoDros); AP (Rabat) – NHMD Scaptomyza Hardy, 1849 Scaptomyza adusta (Loew, 1862) Ebejer et al. 2019 , Rif , Dardara (730 m), AP , Loukous marsh (2 m) Scaptomyza flava (Fallén, 1823) = Scaptomyza flaveola (Meigen, 1830), in Kozlowsky and Rungs 1932 : 66 Kozlowsky and Rungs 1932 , AP , Rabat Scaptomyza graminum (Fallén, 1823) Maarouf 2003 , HA , Chaouia; Bächli 2015 (TaxoDros); HA (Asni, Tinerhir) – NHMD Scaptomyza ( Parascaptomyza ) pallida (Zetterstedt, 1847) Ibn Jilali 1988 (agricultural areas); Maarouf 2003 , HA , Chaouia; Bächli 2015 (TaxoDros); AP (Essaouira) MHNNR, MA (Azrou) – NHMD Scaptodrosophila Duda, 1923 Scaptodrosophila rufifrons (Loew, 1873) Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Zaprionus Coquillett, 1901 Zaprionus indianus Gupta, 1970 Yassin and David 2010 Steganinae Gitonini Gitona Meigen, 1830 Gitona microchaeta Séguy, 1941 Séguy 1941d , AA , Agadir; Bächli 1982 , AA , Agadir; Bächli and Rocha Pité 1984 , AA , Agadir; Mouna 1998 Phortica Fallén, 1823 Phortica variegata (Fallén, 1823) Ebejer et al. 2019 , Rif , Bab Berred (1433 m), Jebel Lakraâ (Talassemtane, 1541 m) Steganini Leucophenga Mik, 1886 Leucophenga maculata (Dufour, 1839) Séguy 1934b , AP , Port-Liautey (Maâmora) EPHYDRIDAE K. Kettani, T. Zatwarnicki Number of species: 117 . Expected: 140 Faunistic knowledge of the family in Morocco: good Discomyzinae Discomyzini Actocetor Becker, 1903 Actocetor indicus (Wiedemann, 1824) = Actocetor margaritatus (Wiedemann, 1830), in Séguy 1934b : 162, 1953a : 86 Séguy 1934b , Rif , Béni Aross; Séguy 1953a , SA , Tindouf Discomyza Meigen, 1830 Discomyza incurva (Fallén, 1823) = Discomyza italica Séguy, 1929, in Vitte 1991 : 3; Dakki 1997 : 63 Cresson 1939 ; Vitte 1991 , AP , Atlantic coast and Plains; Dakki 1997 Psilopini Clanoneurum Becker, 1903 Clanoneurum cimiciforme (Haliday, 1855) Séguy 1941d , AA , Agadir; Dahl 1964 ; Vitte 1991 , AP , Rabat; Dakki 1997 Diasemocera Bezzi, 1895 Diasemocera aequalipes (Becker, 1907) = Psilopa aequalipes (Becker, 1907), in Ebejer et al. 2019 : 147 Zatwarnicki 2018 ; Ebejer et al. 2019 , AA , Lac Tiffert (4 km W of Merzouga, 702 m), Ziz river (13 km N of Erfoud, 800 m) Diasemocera biskrae (Becker, 1907) = Psilopa biskrae (Becker, 1907), in Vitte 1991 : 32 Vitte 1991 , AP , M'Diq; Dakki 1997 Diasemocera composita (Becker, 1903) = Psilopa composita (Becker, 1903), in Vitte 1991 : 32, Dakki 1997 : 63 Vitte 1991 , AP , Rabat; Dakki 1997 Diasemocera fratella (Becker, 1903) = Psilopa fratella (Becker, 1903), in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Errachidia (1 km N of Tarda, 1023 m), AA , Ziz river (13 km N of Erfoud, 800 m) Diasemocera glabricula (Fallén, 1813) = Psilopa nigritella Stenhammar, 1844, in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Diasemocera leucostoma (Meigen, 1830) = Psilopa leucostoma (Meigen, 1830), in Séguy 1941d : 18 Séguy 1941d , AA , Agadir Diasemocera maritima (Perris, 1847) = Psilopa maritima (Perris, 1847), in Cassar et al. 2008 : 25 Cassar et al. 2008 , Rif , Laou Basin Diasemocera nana (Loew, 1860) = Psilopa nana Loew, 1860, in Vitte 1991 : 32, Dakki 1997 : 63 Vitte 1991 , Rif , AP , Atlantic coast; Dakki 1997 Diasemocera rufithorax (Becker, 1903) = Psilopa rufithorax (Becker, 1903), in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Merzouga (714 m) Psilopa Fallén, 1823 Psilopa clara (Wollaston, 1858) = Psilopa rutilans Canzoneri & Meneghini, 1972, in Cassar et al. 2005 : 69 Cassar et al. 2005 , Rif , Smir lagoon; Zatwarnicki 2018 , AP , Larache (Lower Loukous), Safi, AA , Sidi Moussa, D'Agion (0–50 m) Psilopa meneghinii Canzoneri, 1986 Vitte 1991 , AP , Atlantic coast; Dakki 1997 Psilopa compta (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Psilopa nilotica (Becker, 1903) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m), 1 km N of Tarda (Errachidia, 1023 m), Merzouga (714 m), 2 km N Erfoud (818 m) Psilopa nitidula (Fallén, 1813) Becker and Stein 1913 , Rif , Tanger; Séguy 1941a , HA , Toubkal; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Zatwarnicki 1991 , Rif , Tanger, Tétouan, MA , Ifrane; Dakki 1997 Psilopa obscuripes Loew, 1860 Ebejer et al. 2019 , Rif , Oued Azla (near bridge, 80 m), AP , Larache (5 m), Lower Loukous saltmarsh (2 m), MA , Khénifra (28 km S of Timahdit, 2100 m) Psilopa polita (Macquart, 1835) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Risini Achaetorisa Papp, 1980 Achaetorisa brevicornis Papp, 1980 Papp 1980, HA , Ouirgane Ephydrinae Dagini Brachydeutera Leow, 1862 Brachydeutera meridionalis (Rondani, 1856) = Brachydeutera ibari Ninomyia, 1929, in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , Rif , Oued Martil (Taboula, 14 m) Parydrini Parydra Stenhammar, 1844 Parydra ( Chaetoapnaea ) fossarum (Haliday, 1833) Séguy 1930a , AP , Rabat, MA , Meknès, HA , Marrakech; Vitte 1991 ; Dakki 1997 ; Vitte 1988 , MA , lakes of Middle Atlas; MA (Jebel Khazzane) – MISR Parydra ( Chaetoapnaea ) hecate (Haliday, 1833) = Napaea hecate (Haliday), in Vaillant 1956b : 244 Vaillant 1956b , HA , Imi-N'Ifri Parydra ( Chaetoapnaea ) quadripunctata (Meigen, 1830) Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 , AP , Merja Zerga Parydra ( Paranapaea ) pubera Loew, 1860 Dahl 1964 , AA , Aït Melloul, Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Parydra ( Parydra ) aquila (Fallén, 1813) Vitte 1991 , Rif , Ouezzane, Ketama; Dakki 1997 Parydra ( Parydra ) coarctata (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Ksar el Kbir; Dakki 1997 Parydra ( Parydra ) cognata Loew, 1860 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Parydra ( Parydra ) flavitarsis Dahl, 1964 Vitte 1988 , Rif , MA , lakes of Middle Atlas; Vitte 1991 , Rif , MA , Fès; Dakki 1997 ; Gatt and Ebejer 2003 ; Dakki et al. 2003; Chillasse and Dakki 2004 , Rif , MA ; Dakki and Himmi 2008 , MA , Oued Sebou Parydra ( Parydra ) littoralis (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Tétouan, Ketama; Dakki 1997 Parydra ( Parydra ) nigritarsis Strobl, 1893 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Ketama; Dakki 1997 Parydra ( Parydra ) nubecula Becker, 1896 Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 , AP , Merja Zerga Parydra ( Parydra ) quinquemaculata Becker, 1896 Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Ephydrini Ephydra Fallén, 1823 Ephydra bivittata Loew, 1860 Vitte 1991 ; Dakki 1997 Ephydra flavipes (Macquart, 1843) Vitte 1991 , AP , Atlantic coast; Dakki 1997 Ephydra glauca Meigen, 1830 Vitte 1991 ; Dakki 1997 Ephydra macellaria Egger, 1862 Dahl 1964 , AA , Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 ; Vitte 1991 , AP , Atlantic coast Halmopota Haliday, 1856 Halmopota mediterranea Loew, 1860 Dahl 1964 , AA , Aït Melloul; Dakki 1997 ; Vitte 1991 , Rif , Asilah, Chefchaouen Paracoenia Cresson, 1935 Paracoenia fumosa (Stenhammar, 1844) Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Setacera Cresson, 1930 Setacera breviventris (Loew, 1860) Vitte 1991 , Rif , Ksar el Kbir; Dakki 1997 Scatellini Haloscatella Mathis, 1979 Haloscatella dichaeta (Loew, 1860) = Scatella dichaeta Loew, 1860, in Vitte 1988 : 392, 1991 : 26; Dakki 1997 : 62 Vitte 1988 , MA , Khemisset, Oued Beth, Dayat Aoua; Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Lamproscatella Hendel, 1917 Lamproscatella sibilans (Haliday, 1833) Ebejer et al. 2019 , AA , 1 km N of Tarda (Errachidia, 1023 m) Limnellia Malloch, 1925 Limnellia quadrata (Fallén, 1813) Vitte 1991 , Rif , Central Rif; Dakki 1997 Philotelma Becker, 1896 Philotelma nigripenne (Meigen, 1830) = Scatella nigripennis (Meigen, 1830), in Vitte 1988 : 391; Dakki 1997 : 62 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Scatella Robineau-Desvoidy, 1830 Scatella ( Neoscatella ) subguttata (Meigen, 1830) Vitte 1991 , AP , Atlantic and Mediterranean coast, Smir lagoon; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 Scatella ( Scatella ) ciliata Collin, 1930 Vitte 1991 , AP , Moulay Bousselham, Asilah; Dakki 1997 Scatella ( Scatella ) lacustris (Meigen, 1830) = Scatella ( Scatella ) tenuicosta Collin, 1930, in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Ziz river (13 km N of Erfoud, 800 m) Scatella ( Scatella ) lutosa (Haliday, 1833) Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Scatella ( Scatella ) obsoleta Loew, 1861 = Scatella callosicosta Bezzi, 1895, in Vitte 1988 : 392, 1991 : 26; Dakki 1997 : 62 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , M'Diq; Dakki 1997 Scatella ( Scatella ) paludum (Meigen, 1830) Dahl 1964 , AP , Oued Korifla; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatella ( Scatella ) rufipes Strobl, 1906 = Scatella rubida Becker, 1907, in Olafsson 1991 : 21; Vitte 1991 : 26; Dakki 1997 : 62 Olafsson 1991 , EM , Figuig, Defilia; Vitte 1991 , Rif , AP (Atlantic coast); Dakki 1997 ; Gatt and Ebejer 2003 Scatella ( Scatella ) stagnalis (Fallén, 1813) Séguy 1941a , HA , Toubkal; Dahl 1964 , AA , Aït Melloul, Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatophila Becker, 1896 Scatophila caviceps (Stenhammar, 1844) Vitte 1988 , AP , Rabat, Temara, MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatophila despecta (Haliday, 1839) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Khemisset, Oued Beth; Dakki 1997 Scatophila farinae Becker, 1903 Zatwarnicki 1987 , HA , Vallée de l'Ait Mizane; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Taounate; Dakki 1997 ; Gatt and Ebejer 2003 Scatophila modesta Becker, 1908 Vitte 1991 , Rif , Tétouan; Dakki 1997 Scatophila unicornis Czerny, 1900 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Gymnomyzinae Discocerinini Diclasiopa Hendel, 1917 Diclasiopa galactoptera (Becker, 1903) = Discocerina galactoptera Becker, in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 ; Kirk-Spriggs and McGregor 2009 Diclasiopa lacteipennis (Loew, 1862) = Discocerina lacteipennis Loew, 1862, in Vitte 1988 : 394, 1991 : 32; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Rabat; Vitte 1991 , MA , Khemisset, Taounate; Dakki 1997 Diclasiopa niveipennis (Becker, 1896) = Discocerina niveipennis (Becker, 1896), in Vitte 1988 : 394, 1991 : 32; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Discocerina Macquart, 1835 Discocerina obscurella (Fallén, 1813) Vitte 1988 , AP , MA , lakes of Middle Atlas; Vitte 1991 , MA , Fès, Taounate; Mathis 1997 ; Dakki 1997 ; Wolff et al. 2016 Ditrichophora Cresson, 1924 Ditrichophora calceata (Meigen, 1830) = Discocerina calceata (Meigen, 1830), in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Ditrichophora mauritanica (Vitte, 1991) = Discocerina mauritanica Vitte, 1991, in Vitte 1991 : 33 Vitte 1991 , Rif , MA , Azrou; Dakki 1997 ; Chillasse and Dakki 2004 , Rif , MA ; Dakki and Himmi 2008 , MA , Oued Sebou Gymnoclasiopa Hendel, 1930 Gymnoclasiopa plumosa (Fallén, 1823) = Discocerina plumosa (Fallén, 1823), in Vitte 1988 : 394, 1991 : 33; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Gymnoclasiopa pulchella (Meigen, 1830) = Discocerina pulchella (Meigen, 1830), in Vitte 1991 : 33; Dakki 1997 : 63 Vitte 1991 , Rif , Ketama, Ouezzane; Dakki 1997 Hecamedoides Hendel, 1917 Hecamedoides glaucellus (Stenhammar, 1844) = Discocerina glaucella (Stenhammar, 1844), in Vitte 1991 : 33 Vitte 1991 , Rif , AP Polytrichophora Cresson, 1924 Polytrichophora duplosetosa (Becker, 1896) AP (Rabat) – MISR Gymnomyzini Athyroglossa Loew, 1860 Athyroglossa ( Athyroglossa ) glabra (Meigen, 1830) Vitte 1988 , Rif , MA , lakes of Middle Atlas; Dakki 1997 Athyroglossa ( Athyroglossa ) nudiuscula Loew, 1860 Vitte 1991 , Rif ; Dakki 1997 Athyroglossa ( Parathyroglossa ) ordinata Becker, 1896 Vitte 1991 , Rif ; Vitte 1988 , MA , lakes of Middle Atlas; Mathis and Zatwarnicki 1990 , MA , Ifrane; Dakki 1997 Chlorichaeta Becker, 1922 Chlorichaeta albipennis (Loew, 1848) Vitte 1991 ; Dakki 1997 Mosillus Latreille, 1804 Mosillus subsultans (Fabricius, 1794) = Gymnopa subsultans Fabricius, in Séguy 1930a : 181 Séguy 1930a , MA , M'Rirt, HA , Imminen (Tachidirt); Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Mathis et al. 1993 , MA , Ifrane (1650 m), maison forestière (cedar forest: 2700 m), Oued Jaffar (N of source, 0–1500 m), HA , Mikdane (Jebel Ayachi); Dakki 1997 ; Koçak and Kemal 2010 Hecamedini Allotrichoma Becker, 1896 Allotrichoma laterale (Loew, 1860) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Allotrichoma leotoni Vitte, 1992 Vitte 1992 , Rif , Ouezzane, Boured Allotrichoma quadripectinatum (Becker, 1896) = Allotrichoma bellicosum Giordani Soika, 1956, in Vitte 1991 : 30; Dakki 1997 : 63 Vitte 1991 , Rif , AP ; Dakki 1997 Allotrichoma simplex (Loew, 1861) = Allotrichoma filiforme Becker, 1896, in Vitte 1988 : 393, 1991 : 30; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Khemisset, Oued Sebou; Dakki 1997 Elephantinosoma Becker, 1903 Elephantinosoma chnumi Becker, 1903 Gatt and Ebejer 2003 ; Kirk-Spriggs and McGregor 2009 Hecamede Haliday, 1837 Hecamede albicans (Meigen, 1830) Vitte 1991 , AP ; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Cassar et al. 2008 , Rif , Smir Lagoon; Popescu-Mirceni 2011 Lipochaetini Glenanthe Haliday, 1839 Glenanthe ripicola (Haliday, 1839) Vitte 1991 , AP ; Dakki 1997 ; Cassar et al. 2008 , Rif , Laou Basin; Zatwarnicki and Mathis 2011 , AA , Tarfaya – HNHM Homalometopus Becker, 1903 Homalometopus sp. Vitte 1991 Ochtherini Ochthera Latreille, 1802 Ochthera manicata (Fabricius, 1794) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Ochthera pilimana Becker, 1903 Dakki 1997 Ochthera schembrii Rondani, 1847 = Ochthera mantispa Loew, 1847, in Vitte 1988 : 394, 1991 : 28; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , AP ; Dakki 1997 Hydrelliinae Atissini Asmeringa Becker, 1903 Asmeringa inermis Becker, 1903 Vitte 1991 , AP , Rabat; Dakki 1997 ; Gatt and Ebejer 2003 Atissa Haliday, 1839 Atissa durrenbergensis Loew, 1864 Vitte 1991 , AP ; Dakki 1997 Atissa hepaticoloris Becker, 1903 Vitte 1991 , AP ; Dakki 1997 ; Gatt and Ebejer 2003 Atissa limosina Becker, 1896 Vitte 1991 , AP , Rabat, MA , Fès; Dakki 1997 Atissa pygmaea (Haliday, 1839) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 Ptilomyia Coquillett, 1900 Ptilomyia angustigenis (Becker, 1926) = Atissa angustigenis Becker, in Vitte 1988 : 393, 1991 : 30; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Gatt and Ebejer 2003 Dryxini Dryxo Robineau-Desvoidy, 1830 Dryxo ornata (Macquart, 1843) Mathis and Zatwarnicki 2002 , AA , Aoulouz Hydrelliini Hydrellia Robineau-Desvoidy, 1830 Hydrellia albifrons (Fallén, 1813) Vitte 1991 , AP , Rif ; Dakki 1997 Hydrellia argyrogenis Becker, 1896 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , Atlantic coast and Plains; Dakki 1997 Hydrellia armata Canzoneri & Meneghini, 1976 Vitte 1991 , Rif , Ksar el Kbir, MA , Fès; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou Hydrellia atlas Vitte, 1989 Vitte 1989 , MA , Dayat Aoua; Dakki 1997 ; Dakki et al. 2003; Chilasse and Dakki 2004, MA Hydrellia griseola (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Wolff et al. 2016 ; Rif (Oued Laou) – MISR Hydrellia maculiventris Becker, 1896 Vitte 1991 , Rif , AP , Atlantic coast; Dakki 1997 ; Gatt and Ebejer 2003 Hydrellia maura Meigen, 1838 = Hydrellia modesta Loew, 1860, in Vitte 1988 : 392, 1991 : 29; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Zatwarnicki 1988 , MA , Ifrane, HA , Vallée de l'Aït Mizane; Vitte 1991 ; Dakki 1997 Hydrellia nigricans (Stenhammar, 1844) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Hydrellia obscura (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 ; Rif (Aïn Jdioui) – MISR Hydrellia pubescen s Becker, 1926 = Hydrellia nasturtii Collin, 1928, in Vitte 1991 : 29; Dakki 1997 : 63 Vitte 1991 , MA , Fès; Dakki 1997 ; Gatt and Ebejer 2003 Hydrellia ranunculi (Haliday, 1838) Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Hydrellia rharbia Vitte, 1991 Vitte 1989 , AP , Merja Halloufa (near Moulay Bousselham); Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou Hydrellia subalbiceps Collin, 1966 Vitte 1991 , Rif , Ketama; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Notiphilini Notiphila Fallén, 1810 Notiphila ( Notiphila ) annulipes Stenhammar, 1844 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Notiphila ( Notiphila ) cinerea Fallén, 1830 Séguy 1930a , MA , Meknès; Séguy 1941a , HA , Imi-n'Ouaka; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Kirk-Spriggs and McGregor 2009 ; Rif (Talassemtane, Aïn Jdioui) – MISR Notiphila ( Notiphila ) cogani Canzoneri & Meneghini, 1979 Vitte 1988 , MA , lakes of Middle Atlas; Krivosheina 1998 ; Vitte 1991 ; Dakki 1997 ; Rif (Aïn Jdioui) – MISR Notiphila ( Notiphila ) dorsata Stenhammar, 1844 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , coastal lake areas; Dakki 1997 Notiphila ( Notiphila ) maculata Stenhammar, 1844 Vitte 1991 , Rif , AP , coastal plains; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Notiphila ( Notiphila ) riparia Meigen, 1830 Krivosheina 1998 , AA , Aït Melloul (Souss); Vitte 1988 , MA , lakes of Middle Atlas and reedbeds; Vitte 1991 ; Dakki 1997 Notiphila ( Notiphila ) stagnicola (Robineau-Desvoidy, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , AP , coastal plains; Dakki 1997 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 , AA , Ouarzazate Ilytheinae Hyadinini Hyadina Haliday, 1837 Hyadina guttata (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Hyadina pollinosa Oldenberg, 1923 Vitte 1991 , MA , Fès; Dakki 1997 Hyadina rufipes (Meigen, 1830) = Hyadina nitida (Macquart, 1835), in Vitte 1991 : 27; Dakki 1997 : 63 Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Nostima Coquillett, 1900 Nostima picta (Fallén, 1813) Vitte 1991 , AP ; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Pelina Haliday, 1837 Pelina aenea (Fallén, 1813) Vitte 1991 , Rif , Ouezzane; Dakki 1997 Pelina subpunctata Becker, 1896 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Philygria Stenhammar, 1844 Philygria posticata (Meigen, 1830) Ebejer et al. 2019 , MA , Khénifra (17 km SW of Midelt, 1940 m) Acknowledgements We gratefully acknowledge the assistance and cooperation of Martin J. Ebejer who contributed to the revision of this family. Hippoboscoidea HIPPOBOSCIDAE K. Kettani, B. Droz Number of species: 17 . Expected: 25 Faunistic knowledge of the family in Morocco: moderate Hippoboscinae Hippoboscini Crataerina von Olfers, 1816 Crataerina acutipennis Austen, 1926 Mouna 1998 : 85 Crataerina pallida (Latreille, 1811) Maa 1969 ; Mouna 1998 ; AA (Tiznit) – MISR Hippobosca Linnaeus, 1758 Hippobosca camelina Leach, 1817 = Hippobosca dromedarina Speiser, in Séguy 1930a : 184 Séguy 1930a ; Bequaert 1939 , AP , Rabat, EM , Taourit, HA ; Séguy 1953a , AA , Zegdou, SA , Oued Agouidir; Mouna 1998 ; EM (Oued el Maa) – MISR Hippobosca equina (Linnaeus, 1758) Séguy 1930a , AP , Rabat, Settat, Mogador; Séguy 1953a , AA , Inzegane; Maa 1969 ; Bequaert 1939 , MA , Aguelmane, HA , Ijoukak – MISR Hippobosca fulva Austen, 1912 Bequaert 1939 , EM , Taourirt (Ebner), Tendrara (Ebner); Maa 1963 Hippobosca longipennis Fabricius, 1805 = Hippobosca capensis Olfers, in Bequaert 1939 : 78 Séguy 1930a , MA , Meknès; Bequaert 1939 , HA , Marrakech; Maa 1963 , 1969 ; Mouna 1998 ; AA (Agdz) – MISR Hippobosca variegata Megerle, 1803 = Hippobosca maculata Leach, in Séguy 1930a : 184 Séguy 1930a ; Moussiaux and Desmecht 2008 , HA (south) Icosta Speiser, 1905 Icosta minor (Bigot, 1858) = Lynchia minor Bigot, in Mouna 1998 : 85 Báez 1978 ; Mouna 1998 Ornithoica Rondani, 1878 Ornithoica turdi (Olivier in Latreille, 1811) Maa 1966 , EM , Figuig; Maa 1969 ; Mouna 1998 ; Droz and Haenni 2011 Ornithomyia Latreille, 1802 Ornithomyia avicularia (Linnaeus, 1758) Séguy 1930a ; Mouna 1998 Ornithomyia fringillina (Curtis, 1836) Séguy 1930a , MA , Meknès; Mouna 1998 Ornithophila Rondani, 1879 Ornithophila gestroi (Rondani, 1878) = Ornitheza gestroi Rondani, in Mouna 1998 : 85 Mouna 1998 ; Pape and Thompson 2019 Ornithophila metallica (Schiner, 1864) = Ornitheza metallica Schiner Mouna 1998 : 85 Pseudolynchia Bequaert, 1926 Pseudolynchia canariensis (Macquart, 1839) = Pseudolynchia maura Bigot, in Séguy 1930a : 184 Séguy 1930a ; Mouna 1998 Stenepteryx Leach, 1817 Stenepteryx hirundinis (Linnaeus, 1758) = Crataerina hirundinis Linnaeus, in Mouna 1998 : 85 Summer 1978 , MA , Midelt; Mouna 1998 Lipopteninae Lipoptena Nitzsch, 1818 Lipoptena capreoli Rondani, 1878 Mouna 1998 : 85 Melophagus Latreille, 1802 Melophagus ovinus (Linnaeus, 1758) = Melanophagus ovinus Linnaeus, in Mouna 1998 : 85 Séguy 1930a ; Maa 1969 ; Mouna 1998 NYCTERIBIIDAE K. Kettani, G. Graciolli Number of species: 8 . Expected: 18 Faunistic knowledge of the family in Morocco: poor Nycteribiinae Basilia Miranda-Ribeiro, 1903 Basilia italica Theodor, 1954 Aellen 1955 Nycteribia Latreille, 1796 Nycteribia ( Acrocholidia ) vexata Westwood, 1835 Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (hosts: Myotis oxygnathus Monticelli, 1885, Miniopterus shreibersii (Kuhl, 1817) and Rhinolophus mehelyi (Matschie, 1901)), MA , Grotte de Ras el Oued (host: Myotis oxygnathus and Miniopterus shreibersii ; Aellen 1963 ; Theodor 1967 , MA , Oued Mellah; Mouna 1998 Nycteribia ( Nycteribia ) latreillei (Leach, 1817) Séguy 1930a , Rif , Samsa (Tétouan); Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (host: Miniopterus shreibersii (Kuhl, 1817)), MA , Grotte de Ras el Oued (hosts: Myotis oxygnathus Monticelli, 1885 and Miniopterus shreibersii ); Aellen 1963 ; Theodor 1967 , AP , Mazagan (host: Myotis myotis (Bourhausen, 1797)); Mouna 1998 Nycteribia ( Nycteribia ) pedicularia Latreille, 1805 = Listropodia pedicularia Latreille, in Séguy 1930a : 185 Falcoz 1924 , Rif , Caverne d'Hercule; Séguy 1930a , Rif , Caverne d'Hercule; Mouna 1998 Nycteribia ( Nycteribia ) schmidtlii Schiner, 1853 = Listropodia schmidli Schiner, in Séguy 1930a : 186 Falcoz 1924 , Rif , Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne de Samsa, SA ; Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (undetermined bat), MA , Grotte de Ras el Ma (host: Rhinolophus ferrumequinum (Schreber, 1774)), Grotte de Ras el Oued (hosts: Miniopterus shreibersii (Kuhl, 1817) and Myotis oxygnathus Monticelli, 1885); Aellen 1963 ; Theodor 1967 (host: Rhinolophus ferrumequinum ); Mouna 1998 Penicillidia Kolenati, 1963 Penicillidia ( Penicillidia ) conspicua Speiser, 1901 Falcoz 1924 , Rif , Caverne d'Hercule, Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne d'Hercule, Caverne de Samsa; Aellen 1952 ; Aellen 1955 , MA , Grotte de Ras el Oued, AP , Grotte de Sidi Bou Knadel; Aellen 1963 ; Mouna 1998 ; Koçak and Kemal 2010 Penicillidia ( Penicillidia ) dufouri (Westwood, 1835) Falcoz 1924 , Rif , Caverne d'Hercule; Séguy 1930a , Rif , Caverne d'Hercule; Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel, MA , Grotte de Ras el Oued, AA , Oulad Teima; Aellen 1963 ; Theodor 1967 , AP , Mazagan ( Myotis myotis (Bourhausen, 1797)); Mouna 1998 ; Koçak and Kemal 2010 Phthiridium Hermann, 1804 Phthiridium biarticulatum Hermann, 1804 = Stylidia biarticulata Herman, in Falcoz 1924 : 310; Séguy 1930a : 185 Falcoz 1924 , Rif , Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne de Samsa; Aellen 1952 ; Aellen 1955 , MA , Grotte de Ras el Ma (host: Rhinolophus ferrumequinum (Schreber, 1774)), AA , Oulad Teima (host: Rhinolophus ferrumequinum ); Aellen 1963 ; Theodor 1967 (host: Rhinolophus ferrumequinum ); Mouna 1998 STREBLIDAE K. Kettani, G. Graciolli Number of species: 2 . Expected: 7 Faunistic knowledge of the family in Morocco: poor Brachytarsininae Brachytarsina Macquart, 1851 Brachytarsina flavipennis Macquart, 1851 = Nycteribosca kollari Frauenfeld, in Falcóz 1924: 226; Aellen 1955 : 100 Falcóz 1924, Rif , caverne d'Hercule, caverne de Samsa, près Tétouan (host: Rhinolophus ferrumequinum (Schreber, 1774); Séguy 1930a , Rif , caverne d'Hercule, caverne de Samsa; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (hosts: Rhinolophus mehelyi (Matschie, 1901) and Myotis oxygnathus Monticelli, 1885); Mouna 1998 ; Koçak and Kemal 2010 Raymondia Frauenfeld, 1855 Raymondia huberi Frauenfeld, 1855 = Raymondia setosa Jobling, 1930 Beaucournu et al. 1985 , AA , Assa (Bas Draa) (host: Asellia tridens (E. Geoffroy, 1813)) Muscoidea ANTHOMYIIDAE K. Kettani, D.M. Ackland Number of species: 36 . Expected: Many more, especially in the mountains Faunistic knowledge of the family in Morocco: poor Anthomyiinae Adia Robineau-Desvoidy, 1830 Adia cinerella (Fallén, 1825) = Chortophila cinerella Fallén, in Séguy 1930a : 162 = Hylemyia cinerella Fallén, in Séguy 1941d : 18 Séguy 1941d , AA , Agadir; Séguy 1930a ; Mouna 1998 ; AP (Rabat), HA (Marrakech), AA (Tifnit (south of Agadir)) – MISR Anthomyia Meigen, 1803 Anthomyia imbrida Rondani, 1866 Séguy 1930a , MA , Meknès; Mouna 1998 Anthomyia liturata (Robineau-Desvoidy, 1830) = Hylemyia pullula Zetterstedt, in Séguy 1930a : 162 Séguy 1930a , MA , Ras el Ksar (1900 m), Tameghilt (1700–1800 m), Forêt Tiffert (2000–2200 m); Mouna 1998 Anthomyia quinquemaculata Macquart, 1839 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), MA , 3.5 km S of Azrou (1450 m) Anthomyia pluvialis (Linnaeus, 1758) Séguy 1929b ; Séguy 1930a , MA , Meknès; Michelsen 1980 , AP , Aïn Diab; Mouna 1998 ; Ackland 1987 , 2001 ; Pârvu et al. 2006 , AA , Foum Zguid (Tata); Popescu-Mirceni 2011 – MISR Anthomyia procellaris Rondani, 1866 Séguy 1929b ; Séguy 1930a , MA , Meknès, Berkane (1350–1400 m), Tlet n'Rhohr; Mouna 1998 Anthomyia tempestatum Wiedemann, 1830 Michelsen and Báez 1985 , HA ; Ackland 2001 ; Grabener 2017 Botanophila Lioy, 1864 Botanophila dissecta (Meigen, 1826) Mouna 1998 ; MA (Meknès) – MISR Botanophila varicolor (Meigen, 1826) Ebejer et al. 2019 , MA , Lac Aguelmane Sidi Ali (2052 m) Delia Robineau-Desvoidy, 1830 Delia antiqua (Meigen, 1826) Mouna 1998 Delia coarctata (Fallén, 1825) Mouna 1998 Delia flavibasis (Stein, 1903) = Hylemyia hordeacea Séguy, in Séguy 1941d : 18 Séguy 1934a , AP , Casablanca; Séguy 1936b , AP , Rabat; Séguy 1941d , AA , Taroudant; Mouna 1998 ; Ackland 2008 Delia flavogrisea (Ringdahl, 1926) 52 Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 Delia planipalpis (Stein, 1898) = Chortophila pilipyga Villeneuve, in Mouna 1998 : 85 Mouna 1998 ; AP (Mazagan) – MISR Delia platura (Meigen, 1826) = Chortophila cilicrura Rondani, in Séguy 1930a : 162 Séguy 1949a , SA , Goulimine; Mouna 1998 ; Singh et al. 2014 ; Grabener 2017 Delia radicum Linnaeus, 1758 = Chortophila brassicae Bouché, in Séguy 1934b : 162, Mouna 1998 : 85 Séguy 1934b , AP , Rabat; Mouna 1998 ; Biron et al. 2000 , AP , Rabat; Andreassen 2007 – MISR Fucellia Robineau-Desvoidy, 1842 Fucellia maritima (Haliday, 1838) Séguy 1930a , Rif , Agla near Cap Spartel (on Fucus ); Cassar et al. 2008 , Rif , Smir lagoon; Mouna 1998 Hylemya Robineau-Desvoidy, 1830 Hylemya vagans (Panzer, 1798) Mouna 1998 – MISR Leucophora Robineau-Desvoidy, 1830 Leucophora cinerea Robineau-Desvoidy, 1830 Ebejer et al. 2019 , MA , 17 km SW of Midelt (Khénifra, 1940 m) Leucophora dissimilis (Villeneuve, 1920) Ebejer et al. 2019 , MA , 17 km NW of Zaida (Khénifra, 1878 m) Paregle Schnabl, 1911 Paregle audacula (Harris, 1780) Pârvu and Zaharia 2007 Paregle pilipes (Stein, 1916) Mouna 1998 Phorbia Robineau-Desvoidy, 1830 Phorbia fumigata (Meigen, 1826) = Phorbia securis Tiensuu, in Maarouf et al. 1996 : 17 Balachowsky and Mesnil 1935 ; Bleuton 1938; Jourdan 1938 ; Maarouf and Chemseddine 1995 , AP , Chaouia, Doukkala, Abda; Maarouf and Chemseddine 1995 , HA , Chaouia, Doukkala, Abda; Maarouf et al. 1996 , AP , Safi, Settat, Sidi El Aydi, Jemaa Riah, Berrechid, Médiouna, Mohammédia, Bouznika, Skhirat, Kénitra, Sidi Allal Tazi, Souk Larbaa du Gharb, MA , Khernisset, Meknès, Douiyat, Fès, Sefrou, Annaceur, Oulad Saïd, El Aounate, Sidi Bennbur, Zemarnra, Chemmaïa, Jemaa, des Shaïm, Tlet Sidi Bouguedra, Khemisset Chaouïa, Béni Mellal, HA , Skhour Rehamna, Ben Guérir, Marrakech, Tamellalet, Kelaâ des Sraghna, Oulad Ayad, Afourér, Azilal, El Borouj, Guisser; Lhaloui et al. 1998 Phorbia sepia (Meigen, 1826) Bleuton 1938, MA , Fès, Meknès, Taza; Jourdan 1938 ; Mouna 1998 Subhylemyia Ringdahl, 1933 Subhylemyia longula (Fallén, 1824) Mouna 1998 ; AP (Cap Cantin) – MISR Pegomyinae Calythea Schnabl in Schnabl and Dziedzicki 1911 Calythea nigricans (Robineau-Desvoidy, 1830) = Calythea albicincta Fallén, in Séguy 1930a : 161 Séguy 1930a , MA , Meknès, Aïn Leuh; Mouna 1998 Mycophaga Rondani, 1856 Mycophaga testacea (Gimmerthal, 1834) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Pegomya Robineau-Desvoidy, 1830 Pegomya betae (Curtis, 1847) Rungs 1962 , AP ; Mouna 1998 Pegomya bicolor (Wiedemann, 1817) Séguy 1934b , AP , Rabat; Koçak and Kemal 2010 ; Pitkin et al. 2011 Pegomya hyoscyami (Panzer, 1809) Rungs 1962 , AP ; Mouna 1998 ; AP (Rabat) – MISR Pegomya rufina (Fallén, 1825) Mouna 1998 Pegomya testacea (De Geer, 1776) = Pegomya silacea Meigen, in Mouna 1998 : 85 Séguy 1930a , MA , Forêt Tiffert (2000–2200 m); Mouna 1998 Pegomya solennis (Meigen, 1826) = Pegomyia nigritarsis Fallén, in Séguy 1935a : 119 Séguy 1935a , AA , Oued Draa (Taffagount); Rungs 1962 ; Mouna 1998 Pegomya terminalis Rondani, 1866 Ebejer et al. 2019 , Rif , Adrou (556 m), Jebel Lakraâ (Talassemtane, 1541 m) Pegomya winthemi (Meigen, 1826) Mouna 1998 Pegoplata Schnabl & Dziedzicki, 1911 Pegoplata annulata (Pandellé, 1899) = Pegoplata virginea auctt, not Meigen AP (Rabat) – MISR FANNIIDAE K. Kettani, A.C. Pont Number of species: 10 . Expected: 17 Faunistic knowledge of the family in Morocco: poor Fannia Robineau-Desvoidy, 1830 Fannia canicularis (Linnaeus, 1761) Becker and Stein 1913 , Rif , Tanger; Charrier 1927 ; Séguy 1930a , HA , Tenfecht; Séguy 1932a ; Séguy 1941a ; Séguy 1941d , HA ; Séguy 1953a , AP , Rabat; Pont 1986a ; Mouna 1998 ; Pont pers. comm., MA , Azrou; AP (Dradek) – MISR Fannia cothurnata (Loew, 1873) 53 Mouna 1998 Fannia krimensis Ringdahl, 1934 Pont 1983 , HA , Jebel Ayachi; Pont 1986a ; Mouna 1998 Fannia lepida (Wiedemann, 1817) Pont pers. comm. Fannia leucosticta (Meigen, 1838) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA ; Pont 1986a ; Mouna 1998 Fannia monilis (Haliday, 1838) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), Dardara (484 m) Fannia norvegica Ringdahl, 1934 Pont 1986a ; Pont pers. comm., HA , Jebel Ayachi Fannia pallidibasis Pont, 1983 Pont 1983 , HA , Jebel Ayachi; Mouna 1998 Fannia scalaris (Fabricius, 1794) Charrier 1927 , Rif , Tanger; Séguy 1930a ; Séguy 1941a , HA ; Séguy 1953a , AP , Rabat; Pont 1986a ; Mouna 1998 ; Rif (Tanger): Caverne d'Hercule (Pont pers. comm.) – MHNP Fannia sociella (Zetterstedt, 1845) 54 Mouna 1998 MUSCIDAE K. Kettani, A.C. Pont Number of species: 115 . Expected: 140 Faunistic knowledge of the family in Morocco: moderate Atherigoninae Atherigona Rondani, 1856 Atherigona humeralis (Wiedemann, 1830) Pont pers. comm., AP , Casablanca Atherigona pulla (Wiedemann, 1830) Pont 1986b ; Pont pers. comm., AP , Larache, Rabat, HA , Asni, AA , Agadir, Taroudant Atherigona soccata Rondani, 1871 = Atherigona varia (Meigen, 1826) (misidentifications of authors) in Séguy 1930a : 159 Bléton and Fieuzet 1943 , MA , Fès; Séguy 1953a , AP , Rabat, Sidi Yahia du Gharb; Mouna 1998 ; Pont 1986b Atherigona varia (Meigen, 1826) = Atherigona quadripunctata Rossi, in Séguy 1949a : 159 Séguy 1930a , Rif , Tanger; Séguy 1941d ; Séguy 1949a , AA , Akka, Alnif; Bléton and Fieuzet 1943 , MA , Meknès, Gharb, Fouarat; Séguy 1953a , AP , Sidi Yahia du Gharb, Rabat; Pont 1986b ; Mouna 1998 ; Pont pers. comm., Rif , Meloussa, Tanger, AP , Larache, Rabat, HA , Jebel Ayachi, AA , Akka Atherigona ( Acritochaeta ) yorki Deeming, 1971 Pont 1986b , 1991a ; Dike 1990 ; Pont pers. comm., AP , Rabat Azeliinae Azeliini Azelia Robineau-Desvoidy, 1830 Azelia parva Rondani, 1866 Michelsen pers. comm., Rif , Ouezzane Hydrotaea Robineau-Desvoidy, 1830 Hydrotaea aenescens (Wiedemann, 1830) Morocco, first record 1989; Pont et al. 2007 Hydrotaea armipes (Fallén, 1825) Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi Hydrotaea capensis (Wiedemann, 1818) = Ophyra anthrax (Meigen, 1826), in Séguy 1941d : 20 Séguy 1934b , AP , Chellah; Séguy 1941d , AA , Agadir; Pont 1986b ; Hernández-Moreno and Peris 1989 , Rif , Tanger; Mouna 1998 ; Turchetto et al. 2003 ; Pont pers. comm., HA , Jebel Ayachi; Rif (Environ de Tanger (Pont pers. comm.)) – MHNP Hydrotaea cinerea Robineau-Desvoidy, 1830 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea dentipes (Fabricius, 1805) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea floccosa Macquart, 1835 = Hydrotaea armipes (Fallén, 1825) (misidentifications of authors) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger; Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi Hydrotaea glabricula (Fallén, 1825) Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora Hydrotaea ignava (Harris, 1780) = Ophyra leucostoma (Wiedemann, 1817), in Séguy 1953a : 87 Séguy 1953a , HA , Tadla; Pont 1986b ; Hernández-Moreno and Peris 1989 , Rif , Tanger; Pont pers. comm., AP , Casablanca Hydrotaea pellucens Portschinsky, 1879 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea tuberculata Rondani, 1866 Michelsen pers. comm., AP , Rabat Hydrotaea velutina Robineau-Desvoidy, 1830 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Thricops Rondani, 1856 Thricops simplex (Wiedemann, 1817) Pont 1986b , 1991b ; Pont pers. comm., HA , Jebel Ayachi Reinwardtiini Muscina Robineau-Desvoidy, 1830 Muscina levida (Harris, 1780) = Muscina assimilis (Fallén, 1823) Mouna 1998 – MHNP (no locality, on Boletus nigrescens (Pont pers. comm.)); AP (Rabat) – MISR Muscina prolapsa (Harris, 1780) = Muscina pabulorum (Fallén, 1817) Mouna 1998 ; Michelsen pers. comm., AP , Rabat, Larache; AP (Sidi Yahia) – MISR Muscina stabulans (Fallén, 1817) Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat, Mogador, MA , Meknès, HA , Aguerd El Had, Souss; Séguy 1932, HA , Taroudant; Séguy 1934b , AP , Rabat; Séguy 1953a , AP , Rabat, Salé, Forêt Maâmora, Salé; Pont 1986b ; Mouna 1998 ; Pont pers. comm., AP , Casablanca, MA , El Kebab, HA , Jebel Ayachi; AP (Casablanca, Rabat (Pont pers. comm.)) – MHNP; MISR Coenosiinae Coenosiini Coenosia Meigen, 1826 Coenosia antennata (Zetterstedt, 1849) Michelsen pers. comm., AP , Larache Coenosia atra Meigen, 1830 Pont 1986b ; Barták et al. 2004 ; Barták and Kubik 2005 ; Pont pers. comm., HA , Jebel Ayachi Coenosia attenuata Stein, 1903 Pont 1986b ; Pont pers. comm., EM , Figuig Coenosia humilis Meigen, 1826 Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Imlil, Asni, AA , Agadir Coenosia mixta Schnabl, 1911 Pont 1986b Coenosia nevadensis Lyneborg, 1970 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Coenosia pedella (Fallén, 1825) = Coenosia decipiens Meigen 1826 (certainly a misidentification) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger Coenosia praetexta Pandellé, 1899 Michelsen pers. comm., AP , Larache, Rabat Coenosia pumila (Fallén, 1825) 55 Pont 1986b ; Mouna 1998 ; Gregor et al. 2002 Coenosia testacea (Robineau-Desvoidy, 1830) Pont 1986b ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi Coenosia tigrina (Fabricius, 1775) Séguy 1930a , AP , Rabat, MA , Meknès; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi; MA (Ifrane) – MISR Lispocephala Pokorny, 1893 Lispocephala brachialis (Rondani, 1877) Pont 1986b ; Gregor et al. 2002 ; Barták and Kubik 2005 ; Pont pers. comm., HA , Jebel Ayachi Lispocephala mikii (Strobl, 1893) Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Jebel Ayachi, AA , Agadir Lispocephala ungulata (Rondani, 1866) Ackland and Pont 1966 , HA , Jebel Ayachi; Pont 1986b Orchisia Rondani, 1877 Orchisia costata (Meigen, 1826) Charrier 1927 , Rif , Tanger; Pont 1986b Schoenomyza Haliday, 1833 Schoenomyza litorella (Fallén, 1823) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Pont 1986b ; Mouna 1998 ; Pont pers. comm., EM , Figuig, HA , Asni, Imlil, Jebel Ayachi Limnophorini Limnophora Robineau-Desvoidy, 1830 Limnophora bipunctata Stein, 1908 Pont 1986b ; Pont pers. comm., EM , Figuig Limnophora flavitarsis Stein, 1908 Pont pers. comm., EM , Figuig, HA , Jebel Ayachi Limnophora mediterranea Pont, 2012 Pont pers. comm., HA , Jebel Ayachi Limnophora obsignata (Rondani, 1866) Séguy 1930a , MA , Aïn Leuh, HA , Aguerd El Had, Souss (Talekjount); Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi, AA , Agadir Limnophora olympiae Lyneborg, 1965 Pont 1986b ; Pont et al. 2012 a; Pont pers. comm., HA , Jebel Ayachi Limnophora pandellei Séguy, 1923 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Limnophora pollinifrons Stein, 1916 Pont 1986b ; Pont et al. 2012 a; Pont pers. comm., MA , Aïn El Orma Limnophora riparia (Fallén 1824) = Melanochelia riparia Fallén, in Séguy 1930a : 160 Séguy 1930a , AA , Souss (Tenfecht); Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Sidi Chamarouch; Pont 1986b ; Mouna 1998 Limnophora rufimana (Strobl, 1893) Séguy 1930a , MA , Aïn Leuh; Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Jebel Ayachi Limnophora tigrina (Am Stein, 1860) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Lispe Latreille, 1797 Lispe assimilis Wiedemann, 1824 = Lispe inexpectata Canzoneri & Meneghini, 1966 Canzoneri and Meneghini 1966 , MA , Oued Fès (Taza); Pont 1986b ; Vikhrev 2012b , AP , Essaouira, HA , Marrakech, SA , Tan-Tan Lispe apicalis Mik, 1869 Canzoneri and Meneghini 1966 , MA , Oued Sebou, EM , Guercif (Oued Moulouya); Canzoneri and Meneghini 1972 , MA , Taza, Oued Fès; Pont 1986b ; Koçak and Kemal 2010 Lispe bengalensis (Robineau-Desvoidy, 1830) = Lispe berlandi Séguy, in Séguy 1940 : 341 Séguy 1940 , AA , Rio de Oro (Oued Eddahab) (type locality of berlandi ) Lispe caesia Meigen, 1826 = Lispe microchaeta Séguy, in Séguy 1940 : 342 Séguy 1940 , AA , Rio de Oro (Oued Eddahab) (type locality of microchaeta ); Canzoneri and Meneghini 1966 , AP , Fedhala (Oued Nefifikh), Saline di Sète; Pont 1986b ; Mouna 1998 ; Koçak and Kemal 2010 ; Vikhrev et al. 2016 , AP , Oualidia lagoon, Essaouira, SA , Tan-Tan (salt lagoon); AP (Chellah) – MISR ; ZMUM Lispe candicans Kowarz, 1892 Séguy 1940 , AA , Rio de Oro (Villa Cisneros) Lispe cilitarsis Loew, 1856 Vikhrev 2012b , SA , Tan-Tan province Lispe draperi Séguy, 1930 Canzoneri and Meneghini 1966 , MA , Azrou, Aguelmane, Fès (Oued Sebou); Vikhrev 2011a , AP , Essaouira, HA , Oued N'fis (east of Marrakech) Lispe halophora Becker, 1903 Vikhrev pers. comm., SA , Tan-Tan province Lispe kowarzi Becker, 1903 Vikhrev 2012c , AP , Essaouira Lispe loewi Ringdahl, 1922 = Lispe litorea Fallén, 1825 (misidentification of authors) in Séguy 1930a : 160 Séguy 1930a , AP , saline mud in Mediterranean region; Canzoneri and Meneghini 1966 , AP , Fedhala; Pont 1986b ; Dakki 1997 ; Mouna 1998 Lispe marina Becker, 1913 Michelsen pers. comm., AP , Larache Lispe melaleuca Loew, 1847 Canzoneri and Meneghini 1966 , MA , Azrou (Aguelmane); Pont 1986b , 1991b Lispe modesta Stein, 1913 Vikhrev 2012b , AP , Essaouira, HA , Marrakech, SA , Tan-Tan Lispe nana Macquart, 1835 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès), Fès (Oued Sebou), Azrou (Aguelmane), EM , Guercif (Oued Moulouya); Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Aïn el Orma, EM , Figuig, HA , Jebel Ayachi; Rif (Oued Laou dunes), AP (Rabat) – MISR Lispe nivalis Wiedemann, 1830 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès); Pont 1986b , 1991; Vikhrev 2012c , AP , Essaouira, HA , Ouarzazate province, SA , Tan-Tan province; Pont pers. comm., MA , Aïn El Orma Lispe pectinipes Becker, 1903 = Lispa mixticia Séguy, in Séguy 1941d : 19 Séguy 1941d , HA , Taroudant (type locality of mixticia ); Pont 1986b ; Mouna 1998 ; Kirk-Spriggs and McGregor 2009 ; Vikhrev 2011b , AP , Essaouira; Pont pers. comm., HA , Jebel Ayachi Lispe pygmaea Fallén, 1825 Canzoneri and Meneghini 1966 , MA , Azrou (Aguelmane); Pont 1986b ; Vikhrev 2012a , AP , Essaouira Lispe rigida Becker, 1903 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès); Pont 1986b , 1991; Vikhrev 2012c , HA , Ouarzazate Lispe scalaris Loew, 1847 = Lispe maroccana Canzoneri & Meneghini, 1966 (as scalaris ssp.) = Lispe persica Becker, 1904 in Kirk-Spriggs and McGregor 2009 Canzoneri and Meneghini 1966 , AP , Fedhala, Dielfa (Oued Tadmid), EM , Guercif (Oued Moulouya), MA , Fès (Oued Sebou); Pont 1986b ; Kirk-Spriggs and McGregor 2009 ; Vikhrev 2012a , HA , Ouarzazate province Lispe tentaculata (De Geer, 1776) Séguy 1930a , HA , Kasba Taguendaft (Goundafa), Skoutana (Arround); Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Kirk-Spriggs and McGregor 2009 ; Pont pers. comm., MA , Aïn el Orma, EM , Figuig, HA , Jebel Ayachi; AP (Rabat), HA (Tizi-n'Tichka) – MISR Muscinae Muscini Dasyphora Robineau-Desvoidy, 1830 Dasyphora albofasciata (Macquart, 1839) = Dasiphora saltuum Rondani, 1862 Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Dasyphora penicillata (Egger, 1856) Pont 1986b ; Koçak and Kemal 2010 ; Pont pers. comm., HA , Jebel Ayachi Dasyphora cyanella (Meigen, 1826) Peris and Llorente 1963 , Rif , Tanger; Mouna 1998 Morellia Robineau-Desvoidy, 1830 Morellia asetosa Baranov, 1925 = Morellia simplex (Loew, 1857) (misidentification of authors) in Peris and Llorente 1963 , Rif , Tanger; Pont 1986b 56 Musca Linnaeus, 1758 Musca autumnalis De Geer, 1776 = Musca corvina Fabricius, 1781 in Séguy 1930a : 156 Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat, MA , Oued Korifla, Meknès; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; MA (Volubilis), AA (Tifnit) – MISR Musca biseta Hough, 1898 Pont 1986b ; Pont pers. comm., AP , Temara, MA , Timhadit, Meknès, EM , near Figuig Musca domestica Linnaeus, 1758 Charrier 1927 , Rif , Tanger; Séguy 1930a , 1932, 1934b , AP , Casablanca; Séguy 1934c , AP , Casablanca; Séguy 1941a , HA ; Peris and Llorente 1963 , Rif , Tanger, Melilla, Bab Taza, El Ajmas, Yebala; Pont 1986b ; Mouna 1998 ; Pârvu et al. 2006 , AA , Tiggane Tata; Popescu-Mirceni 2011 ; Pont pers. comm., AP , Temara, MA , Timhadit, Orionane, Lixus , Meknès, HA , Jebel Ayachi; Grabener 2017 ; HA (Jebel Tachdirt, 3100 m, Tachdirt (Bords Imminen), 2400–2600 m, Kasba Taguendaft (Goundafa), Andjera (Pont pers. comm.)) – MHNP Musca larvipara Portschinsky, 1910 Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora Musca osiris Wiedemann, 1830 = Musca vitripennis Meigen, 1826 (misidentifications of authors) in Séguy 1930a : 157 Séguy 1941d , AA , Agadir; Pont 1986b Musca sorbens Wiedemann, 1830 = Musca angustifrons Thomson, 1869 (misidentification of authors) in Séguy 1930a : 156, 1940 : 245, 1953a : 88 Séguy 1930a , MA , Oued Korifla, HA , Talingoult (Goundafa), Souss; Séguy 1940 , AA , Rio de Oro (Villa Cisneros); Séguy 1941d , AA , Agadir; Séguy 1949a , AA , from Foum Zguid to Zagora; Saccà 1952 ; Séguy 1953a , AP , Rabat; Peris and Llorente 1963 ; Pont 1986b , Rif , Melilla, Tanger, AP , Mogador; Mouna 1998 ; Koçak and Kemal 2010 ; Pont pers. comm., MA , Timhadit, HA ; Grabener 2017 – MISR Musca tempestiva Fallén, 1817 Séguy 1941d ; Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora, HA , Asni Musca vitripennis Meigen, 1926 = Plaxemyia vitripennis Meigen, 1826 in Becker and Stein 1913 : 91 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Ras el Ksar, Aïn Leuh, HA , Tinmel (Goundafa), Arround (Skoutana); Séguy 1941d , AA , Agadir; Peris and Llorent 1963, Rif , Tanger, Melilla, AP , Mogador; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; Rif (environs de Tanger, Sart. route de Spartel (Pont pers. comm.)) – MHNP; AP (Cap Cantin, Dradek), MA (Azrou) – MISR Neomyia Walker, 1859 Neomyia cornicina (Fabricius, 1781) = Cryptolucilia caesarion (Meigen, 1826) in Séguy 1930a : 156 Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger (Oued Judios), AP , Rabat, MA , M'Rirt, Aïn Leuh, Tizi-s'Tkrine, Forêt Zaers, Forêt Tiffert, HA , Arround (Skoutana), Tachdirt; Séguy 1941d (very common); Peris and Llorente 1963 , Rif , Tanger, AP , Mogador, Tzalatza, Reisana, Desembocadura del Lixus ; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi – MISR Neomyia viridescens (Robineau-Desvoidy, 1830) = Orthellia cornicina (Fabricius, 1781) (misidentifications of authors) Charrier 1927 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Polietes Rondani, 1866 Polietes meridionalis Peris & Llorente, 1963 Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Pyrellia Robineau-Desvoidy, 1830 Pyrellia vivida Robineau-Desvoidy, 1830 = Pyrellia cadaverina (Linnaeus, 1758) (misidentifications of authors) in Charrier 1927 : 620; Séguy 1930a : 156; Peris and Llorente 1963 : 252 = Pyrellia serena (Meigen, 1826) (misidentification of authors) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger; Séguy 1930a , MA , Aïn Leuh; Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Stomoxyini Haematobia Le Peletier & Serville, 1828 Haematobia irritans (Linnaeus, 1758) = Lyperosia irritans (Linnaeus, 1758) in Séguy 1930a : 157 Séguy 1930a , MA , Meknès; Pont 1986b ; Mouna 1998 Stomoxys Geoffroy, 1762 Stomoxys calcitrans (Linnaeus, 1758) Charrier 1927 , Rif , Tanger; Séguy 1930a ; Séguy 1941d ; Peris 1951 , Rif , Tanger; Pont 1986b ; Mouna 1998 ; Pârvu et al. 2006 , AA , Tiggane Tata; Dsouli 2009 ; Popescu-Mirceni 2011 ; Pont pers. comm., MA , Meknès, Timhadit, Azrou, Sidi Mjber, Neguerett, Tazekka, HA , Jebel Ayachi, El Kebab, AA , Figuig; HA (Haute Réghaya (Pont pers. comm.)) – MHNP; AP (Rabat), MA (Volubilis), HA – MISR Mydaeinae Graphomya Robineau-Desvoidy, 1830 Graphomya maculata (Scopoli, 1763) Séguy 1930a , MA , Forêt Zaers, Aïn Leuh; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; MA (Volubilis) – MISR Gymnodia Robineau-Desvoidy, 1863 Gymnodia eremophila (Brauer & Bergenstamm, 1894) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Gymnodia polystigma (Meigen, 1826) = Limnophora polystigma (Meigen, 1826) = Brontaea polystigma (Meigen, 1826) Mouna 1998 ; AP (Rabat) – MISR Gymnodia genurufa (Pandellé, 1899) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Gymnodia tonitrui (Wiedemann, 1824) = Limnophora tonitrui (Wiedemann, 1824) = Brontaea tonitrui (Wiedemann, 1824) Séguy 1949a , AA , Tata; Saccà 1952 , AP , Rabat; Pont 1986b ; Mouna 1998 Hebecnema Schnabl, 1889 Hebecnema fumosa (Meigen, 1826) Séguy 1930a , AP , Casablanca; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; Rif (Tanger), AP (Mogador (Pont pers. comm.)) – MHNP Hebecnema nigra (Robineau-Desvoidy, 1830) = Hebecnema vespertina (Fallén, 1823) (misidentifications of authors) in Séguy 1930a : 159 Séguy 1930a , AP , Casablanca; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Hebecnema umbratica (Meigen, 1826) Pont 1986b Myospila Rondani, 1856 Myospila meditabunda (Fabricius, 1781) Séguy 1941d , AA , Agadir; Pont 1970 ; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; AP (Cap Cantin) – MISR Phaoniinae Helina Robineau-Desvoidy, 1830 Helina clara (Meigen, 1826) = Mydaea clara (Meigen, 1826) in Séguy 1930a : 159 Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat; Pont 1986b ; Mouna 1998 ; AP (Rabat) – MISR Helina czernyi Lyneborg, 1970 Michelsen pers. comm., Rif , Chefchaouen, Ouezzane, MA , Azrou, HA , Asni Helina evecta (Harris, 1780) = Mydaea lucorum (Fallén, 1823) in Séguy 1930a : 159 Séguy 1930a ; Werner 1938 , EM , Oudjda-Berguent; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi Helina nevadensis Lyneborg, 1970 Pont 1986b ; Pont pers. comm., Rif , Talassemtane, HA , Jebel Ayachi Helina parcepilosa (Stein, 1907) Michelsen pers. comm., AP , Rabat, HA , Tinerhir Helina quadrum (Fabricius, 1805) = Mydaea quadrum "Fallén" in Séguy 1930a : 159 Séguy 1930a , AP , Rabat; Pont, 1986b; Mouna 1998 Helina reversio (Harris, 1780) Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; MA (Forêt Timelilt, 1650–1900 m (Pont pers. comm.)) – MNHN Helina richardi Pont, 2012 Pont 2012b , Rif , Ras el Ma, MA , Azrou, HA , Jebel Ayachi Helina sexmaculata (Preyssler, 1791) = Mydaea uliginosa (Fallén, 1825) Mouna 1998 ; Grabener 2017 ; MA (Aguelmane Azigza) – MISR Helina vockerothi Lyneborg, 1970 Michelsen pers. comm., HA , Tizi-n'Test (2100 m), Asni Phaonia Robineau-Desvoidy, 1830 Phaonia cincta (Zetterstedt, 1846) 57 Charrier 1927 , Rif , Tanger; Pont 1986b (record queried); Koçak and Kemal 2010 Phaonia errans (Meigen, 1826) Mouna 1998 ; Michelsen pers. comm., MA , Azrou; AP (Chellah), MA (Ifrane) – MISR Phaonia exoleta (Meigen, 1826) Michelsen pers. comm., AP , Larache Phaonia mediterranea Hennig, 1963 Pont 1973 , HA , Jebel Ayachi; Pont 1986b ; Mouna 1998 ; Gregor et al. 2002 Phaonia rufipalpis (Macquart, 1835) Michelsen pers. comm., Rif , Ouezzane Phaonia scutellata (Zetterstedt, 1845) Michelsen pers. comm., Rif , Ouezzane, HA , Asni, Tizi-n'Test, AA , Aoulouz Phaonia subventa (Harris, 1780) Vikhrev and Erofeeva 2018 , HA , Oukaimeden (2000 m); Rif (environs de Tanger (Pont pers. comm.)) – MHNP Phaonia trimaculata (Bouché, 1834) Séguy 1930a , AP , Casablanca; Séguy 1934b , AP , Maâmora; Séguy 1953a , AP , Port Lyautey, Maâmora, Rabat; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., AP , Forêt Maâmora, HA , Jebel Ayachi; AP (Rabat) – MISR Phaonia tuguriorum (Scopoli, 1763) = Phaonia signata (Meigen, 1826) in Séguy 1930a : 158 Séguy 1930a , MA , Forêt Timlilt; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Phaonia valida (Harris, 1780) = Phaonia erratica (Fallén, 1825) (misidentifications of authors) in Séguy 1953a : 87 Séguy 1953a , MA , Ifrane; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi; MA (Ifrane (Pont pers. comm.)) – MHNP Phaonia sp. near szelenyii Mihályi, 1974 HA (Haute Réghaya (Pont pers. comm.)) – MHNP SCATHOPHAGIDAE K. Kettani Number of species: 3 . Expected: 4 Faunistic knowledge of the family in Morocco: poor Scathophaginae Norellia Robineau-Desvoidy, 1830 Norellia tipularia (Fabricius, 1794) Ebejer et al. 2019 , Rif , Dardara (730 m) Scathophaga Meigen, 1803 Scathophaga stercoraria (Linnaeus, 1758) = Scathophaga merdaria Fabricius, 1794, in Séguy 1930a : 163 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, MA , Meknès; Mouna 1998 ; Koçak and Kemal 2010 ; AP (Azemour, Oued Yakem, Cap Cantin) – MISR Scathophaga lutaria (Fabricius, 1794) Ebejer et al. 2019 , Rif , Talassemtane (1554 m), Jebel Lakraâ (1541 m) Oestroidea CALLIPHORIDAE K. Kettani, K. Rognes Number of species: 8 . Expected: 10 Faunistic knowledge of the family in Morocco: moderate Calliphorinae Bellardia Robineau-Desvoidy, 1863 Bellardia maroccana (Villeneuve, 1941) = Onesia maroccana Villeneuve, in Villeneuve 1932 : 123 Villeuneuve 1932b; Schumann 1974 , AP , Aïn Diab (Casablanca); Verves 2004 ; Koçak and Kemal 2010 Bellardia mascariensis (Villeneuve, 1926) Schumann 1974 , HA , Marrakech; Verves 2004 ; Koçak and Kemal 2010 Calliphora Robineau-Desvoidy, 1863 Calliphora vicina Robineau-Desvoidy, 1830 = Calliphora erythrocephala Meigen, in Séguy 1930a : 154 Séguy 1930a ; Maurice 1947 , Rif , Tanger; AP (Casablanca, Rabat), MA (Azrou, Ifrane) – MNHN , MISR Calliphora vomitoria (Linnaeus, 1758) Séguy 1930a ; Maurice 1947 , Rif , Tanger; Kurahashi 1971 ; Kurahashi and Magpayo 2000 ; Mouna 1998 ; AP (Rabat), HA – MISR , NHMD Chrysomyinae Chrysomya Robineau-Desvoidy, 1863 Chrysomya albiceps (Wiedemann, 1819) Maurice 1947 ; Séguy 1949a , HA , Alnif; Gonzales-Mora and Peris 1988; Mouna 1998 ; Verves 2003 ; Grabener 2017 ; Dawah et al. 2019 ; EM (Oujda) – MISR , AP (Rabat) – MNHN Luciliinae Lucilia Robineau-Desvoidy, 1863 Lucilia sericata (Meigen, 1824) Séguy 1930a , Rif , Tanger, EM , Berkane (1350–1400 m), MA , Meknès, Berrechid, HA , Tizi-n'Test, Goundafa (Jebel Imdress, 2000–2450 m); Séguy 1953a , AP , Rabat; Mouna 1998 ; Grabener 2017 ; AP (Dradek, Salé), MA (Azrou, Volubilis) – MISR , AP (Temara), MA (Ifrane) – MNHN Melanomyinae Melinda Robineau-Desvoidy, 1863 Melinda gentilis (Robineau-Desvoidy, 1830) MA (Ifrane) – MNHN Melinda viridicyanea (Robineau-Desvoidy, 1830) MA (Ifrane) – MNHN OESTRIDAE K. Kettani, T. Pape Number of species: 10 . Expected: 12 Faunistic knowledge of the family in Morocco: moderate Gastrophilinae Gasterophilus Leach, 1817 Gasterophilus flavipes (Olivier, 1811) Séguy 1928d , EM , Haute Moulouya; Séguy 1930a , EM , Itzer (Haute Moulouya); Mouna 1998 ; Li et al. 2019 Gasterophilus haemorrhoidalis (Linnaeus, 1758) Maurice 1947 ; Mouna 1998 ; AP (Rabat) – MISR Gasterophilus intestinalis (De Geer, 1776) Séguy 1930a ; Mouna 1998 ; Pandey et al. 1992 Gasterophilus nasalis (Linnaeus, 1758) = Gasterophilus veterinus Clark, in Mouna 1998 : 85 Maurice 1947 ; Pandey et al. 1992 ; Mouna 1998 Gasterophilus pecorum (Fabricius, 1794) Séguy 1930a ; Mouna 1998 Hypodermatinae Hypoderma Rondani, 1856 Hypoderma bovis (Linnaeus, 1758) Maurice 1947 ; Mouna 1998 Hypoderma lineatum (De Villers, 1789) Dakkok et al. 1978 , MA , Benslimane; Mouna 1998 Oestrinae Cephalopina Strand, 1928 Cephalopina titillator (Clark, 1797) = Cephalopsis titillator Clark, in Séguy 1930a : 139 Séguy 1930a , Sub- SA (camel breeding region); Mouna 1998 Oestrus Linnaeus, 1758 Oestrus ovis Linnaeus, 1761 Séguy 1930a , AP , Rabat (sheep breeding area), HA , Marrakech, Bou Tazzert; Maurice 1947 ; Mouna 1998 – MISR Rhinoestrus Brauer, 1886 Rhinoestrus purpureus (Brauer, 1858) Maurice 1947 ; Mouna 1998 POLLENIIDAE K. Kettani, K. Rognes Number of species: 12 . Expected: 15 Faunistic knowledge of the family in Morocco: moderate Pollenia Fabricius, 1794 Pollenia amentaria (Scopoli, 1763) Séguy 1949a , AA , Foum-el-Hassan; Séguy 1953a , MA , Sidi Allal Tazi; Mouna 1998 ; Pârvu et al. 2006 , SA , Foum Zghouig; Popescu-Mirceni 2011 ; Gisondi et al. 2020 Pollenia bicolor Robineau-Desvoidy, 1830 Rognes 1991a , HA , Mikdane (Jebel Ayachi); Rognes 1991b , HA , Mikdane (Jebel Ayachi); Gisondi et al. 2020 ; HA (Mikdane) – NHMUK Pollenia contempta Robineau-Desvoidy, 1863 Séguy 1930a , MA , Meknès, from M'Rirt to El Hajeb; Mouna 1998 ; Rognes 1992 Pollenia haeretica Séguy, 1928 Séguy 1934b , AP , Rabat; Rognes 2010 ; AP (40 km S Larache) – NHMD Pollenia ibalia Séguy, 1930 = Pollenia funebris Villeneuve, in Villeneuve 1932 : 284 = Pollenia rungsi Séguy, in Séguy 1953a : 88 Séguy 1930a , EM , Berkane, MA , Tlet n'Rhohr (in garden), Douar Ras el Ksar (900 m); Villeneuve 1932 , HA , Marrakech; Séguy 1953a , AP , Rabat; Séguy 1941a , HA , cañon Tessaout (M'Goum, 3000–3200 m); Mouna 1998 ; Rognes 2010 ; Grabener 2017 ; Gisondi et al. 2020 ; HA (Asni) – NHMUK ; HA (Ijoukak) – MNHN ; HA (15 km SW Tazenakht) – NHMD Pollenia leclercqiana (Lehrer, 1978) Rognes 2010 , 2011 ; Gisondi et al. 2020 Pollenia luteovillosa Rognes, 1987 Gisondi et al. 2020 , HA , Mikdane (Jbel Ayachi) Pollenia ponti Rognes, 1991 Rognes 1991b , HA , Jaffar river (Jebel Ayachi); Gisondi et al. 2020 ; HA (Jebel Ayachi) – NHMUK Pollenia rudis (Fabricius, 1794) Séguy 1930a , EM , Itzer (Haute Moulouya), Berkane (1350 m), AP , Casablanca, MA , Meknès, Aharmoumou (1100 m), Tlet n'Rhohr (in garden), Douar Ras el Ksar (1900 m), HA , Tizi-n'Test, Goundafa (Jebel Imdress, 2000–2450 m); Maurice 1947 , Rif , Tanger; Rognes 1987 , AP , Aïn Diab, Larache, HA , Asni, Jebel Ayachi, Mikdane; Grabener 2017 ; Gisondi et al. 2020 ; AP (Casablanca, Rabat), MA (Ifrane), HA (Haute Réghaya) – MNHN Pollenia ruficrura Rondani, 1862 Rognes 2011 ; Gisondi et al. 2020 Pollenia stigi (Rognes, 1992) Rognes 1992 , MA , Ifrane, Azrou; Gisondi et al. 2020 Pollenia vagabunda (Meigen, 1826) = Pollenia hasei Séguy, in Séguy 1928e : 370, Séguy 1930a : 147 Séguy 1928e ; Séguy 1930a , AP , Casablanca; Mouna 1998 ; Rognes 1992 ; Gisondi et al. 2020 RHINIIDAE K. Kettani, K. Rognes Number of species: 17 . Expected: ~20 Faunistic knowledge of the family in Morocco: moderate Cosmina Robineau-Desvoidy, 1830 Cosmina claripennis Robineau-Desvoidy, 1830 = Cosmina bezziana Villeneuve, in Villeneuve 1932 a: 285 Villeneuve 1932 a, AP , Mogador; Schumann 1986 ; Mouna 1998 ; Rognes 2002 Cosmina maroccana Séguy, 1949 Séguy 1949a , AA , Tenfecht, Vallée du Guir, SA , Guelmim; Séguy 1953a , AA , Tenfecht, Vallée du Guir, SA , Guelmim; Mouna 1998 ; Rognes 2002 ; Grabener 2017 Cosmina punctulata Robineau-Desvoidy, 1830 Séguy 1930a , AA , Tenfecht (Souss, 1000–1500 m) Cosmina viridis (Townsend, 1917) Ebejer et al. 2019 , AA , 13 km E of Goulmima (1100 m) Rhinia Robineau-Desvoidy, 1830 Rhinia apicalis (Wiedemann, 1830) Séguy 1930a , AP , Rabat, MA , Forêt Zaers; Zumpt 1956 ; Gonzales-Mora and Peris 1988; Mouna 1998 ; Verves 2003 ; Lehrer 2007 Rhyncomya Robineau-Desvoidy, 1830 Rhyncomya callopis (Loew, 1856) 58 Séguy 1941d , AA , Agadir; Séguy 1953a , AP , Sidi Ifni, SA , Tindouf, Amguilli Sguelma, Guelta des Zemmours; Schumann 1986 ; Mouna 1998 ; Rognes 1992 ; Dawah et al. 2019 ; El Hawagry and El-Azab 2019; MA (Tafilalt) – MISR Rhyncomya columbina (Meigen, 1824) Peris 1951 , Rif , Tanger, MA , Ifrane; Schumann 1986 ; Gonzalez-Mora and Peris 1988 Rhyncomya cyanescens (Loew, 1844) = Rhyncomya hemisia Séguy, in Séguy 1930a : 150 Séguy 1930a , EM , Berkane (1350–1400 m); Gonzales-Mora and Peris 1988; Rognes 2002 , MA Rhyncomya desertica (Peris, 1951) Grabener 2017 ; El Hawagry and El-Azab 2019 Rhyncomya impavida (Rossi, 1790) Séguy 1930a , Rif , Tanger, EM , Berkane (1350–1400 m), MA , Forêt Tiffert (2000–2200 m); Schumann 1986 ; Mouna 1998 Rhyncomya nigripes (Séguy, 1933) Grabener 2017 ; El Hawagry and El-Azab 2019 Rhyncomya ursina Séguy 1928 = Rhynchomyia ursina Séguy, in Séguy 1928b : 152 Séguy 1928b , AP , Atlantic coast of SA Rhyncomya yahavensis Rognes, 2002 Grabener 2017 ; Ebejer et al. 2019 , AA , 30 km W of Errachidia (1065 m) Rhyncomya zernyana Villeneuve, 1926 Zumpt 1956 ; Gonzales-Mora and Peris 1988 Stomorhina Rondani, 1861 Stomorhina lunata (Fabricius, 1805) Séguy 1930a ; Peris 1951 ; Gonzales-Mora and Peris 1988; Mouna 1998 ; AP (Rabat) – MISR Villeneuviella Austen, 1914 Villeneuviella icadion (Séguy, 1953) = Rhynchoestrus icadion Séguy, in Séguy 1953a : 89 Séguy 1953a , SA , Tindouf Villeneuviella weissi (Séguy, 1926) = Rhynchoestrus weissi Séguy, in Séguy 1934b : 162 Séguy 1934b , EM , Berkane Zobzit (1100 m); Séguy 1953a , SA , Mader Bergat RHINOPHORIDAE K. Kettani, T. Pape Number of species: 8 . Expected: 15 Faunistic knowledge of the family in Morocco: poor Rhinophorinae Melanophora Meigen, 1803 Melanophora roralis (Linnaeus, 1758) Peris 1963 , Rif , Tanger, AP , Larache; Mouna 1998 Oplisa Rondani, 1862 Oplisa aterrima (Strobl, 1899) = Hoplisa aterrima Strobl, in Peris 1963 : 602 Peris 1963 , Rif , Tanger, Zoco de Taleta, Ketama; Mouna 1998 ; Cerretti et al. 2020 Paykullia Robineau-Desvoidy, 1830 Paykullia carmela (Peris, 1963) = Chaetostevenia carmela Peris, in Peris 1963 : 606 Peris 1963 , Rif , Tanger; Cerretti et al. 2020 ; Pape and Thompson 2019 – MNCN Phyto Robineau-Desvoidy, 1830 Phyto atrior (Villeneuve, 1941) = Styloneuria atrior Villeneuve, in Villeneuve 1941 : 122 Villeneuve 1941 , AP , Rabat; Cerretti et al. 2020 ; Pape and Thompson 2019 – IRSNB Phyto discrepans Pandellé, 1896 Cerretti et al. 2020 , Rif , Chefchaouen (600 m), Ouezzane (300 m), MA , 40 km N Fès (1150 m) – NHMD Phyto melanocephala (Meigen, 1824) Ebejer et al. 2019 , Rif , Barrage Smir (145 m); Cerretti et al. 2020 Stevenia Robineau-Desvoidy, 1830 Stevenia deceptoria (Loew, 1847) Mulieri et al. 2010 ; Cerretti et al. 2020 Tricogena Rondani, 1856 Tricogena rubricosa (Meigen, 1824) = Frauenfeldia rubricosa Meigen, in Peris 1963 : 603 Peris 1963 , Rif , Tanger; Mouna 1998 ; Cerretti et al. 2020 – NHMD SARCOPHAGIDAE K. Kettani, D. Whitmore, T. Pape Number of species: 66 . Expected: ~150 Faunistic knowledge of the family in Morocco: poor Miltogramminae Amobia Robineau-Desvoidy, 1830 Amobia signata (Meigen, 1824) Pape 1996 ; Verves 2019 Apodacra Macquart, 1854 Apodacra africana Rohdendorf, 1930 Pape 1996 , Rif , Tanger; Verves 2019 Craticulina Pandellé, 1895 Craticulina antachates (Séguy, 1949) = Apodacra antachates Séguy, in Séguy 1949a : 160 Séguy 1949a , AA , Zagora; Pape 1996 , AA , Zagora; Mouna 1998 Craticulina tabaniformis (Fabricius, 1805) Fabricius 1805 , AP , Mogador; Séguy 1930a , AP , Mogador; Séguy 1935a , AP , beach of Rabat; Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Dolichotachina Villeneuve, 1913 Dolichotachina marginella (Wiedemann, 1930) Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Macronychia Rondani, 1859 Macronychia lemariei Jacentkovský, 1941* AP Macronychia polyodon (Meigen, 1824) Pape 1996 ; Verves 2019 Metopia Meigen, 1803 Metopia argyrocephala (Meigen, 1824) = Metopia leucocephala (Rossi), in Mouna 1998 : 86 Mouna 1998 Miltogramma Meigen, 1803 Miltogramma aurifrons Dufour, 1850 Séguy 1930a , AP , Rabat, MA , Meknès; Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019; Verves 2019 Miltogramma germari Meigen, 1824 Séguy 1930a , MA , Meknès, from M'Rirt to El Hajeb, Sidi Taibi; Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019; Verves 2019 Miltogramma maroccana (Séguy, 1941) = Sphecapatodes maroccana Séguy, in Séguy 1941d : 22 Séguy 1941d , AA , Taroudant; Pape and Szpila 2012 Miltogramma murina Meigen, 1824 Pape 1996 ; Verves 2019 Miltogramma oestracea (Fallén, 1820) Ebejer et al. 2019 , Rif , Belwazen (M'Diq, 200 m), AP , Lower Loukous saltmarsh (2 m) Miltogramma rutilans Meigen, 1824 Ebejer et al. 2019 , Rif , Oued Mhajrate (Ben Karrich, 180 m) Miltogramma testaceifrons (Roser, 1840) = Miltogramma pilitarsis Rondani, in Séguy 1930a : 145; Mouna 1998 : 86 Séguy 1930a , MA , Aïn Leuh; Pape 1996 ; Mouna 1998 Protomiltogramma Townsend, 1916 Protomiltogramma fasciata (Meigen, 1824) = Setulia fasciata (Meigen), in Mouna 1998 : 86 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Senotainia Macquart, 1846 Senotainia albifrons (Rondani, 1859) = Sphecapata albifrons Rondani, in Séguy 1930a : 145 Séguy 1930a Taxigramma Macquart, 1850 Taxigramma heteroneura (Meigen, 1830) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Taxigramma pluriseta (Pandellé, 1895) Ebejer et al. 2019 , Rif , Oued Mhajrate (Ben Karrich, 180 m), AP , Lower Loukous saltmarsh (2 m) Paramacronychiinae Nyctia Robineau-Desvoidy, 1830 Nyctia halterata (Panzer, 1798) = Musca maura Fabricius, in Fabricius 1805 : 302 Fabricius 1805 , Rif , Tanger; Pape 1996 , Rif , Tanger; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Nyctia lugubris (Macquart, 1834) Ebejer et al. 2019 , AP , Lower Loukous saltmarsh (2 m) Sarcophila Rondani, 1856 Sarcophila latifrons (Fallén, 1817) 59 Séguy 1930a , AP , Maâmora, HA , Skoutana (Arround, 2000–2400 m); Mouna 1998 Wohlfahrtia Brauer & Bergenstamm, 1889 Wohlfahrtia bella (Macquart, 1839) = Disjunctio bella (Macquart), in Séguy 1930a : 144 Séguy 1930a , MA , Aïn Leuh; Pape 1996 ; Mouna 1998 ; Hall et al. 2009 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia indigens Villeneuve, 1928 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia magnifica (Schiner, 1862) Delanoë 1922 , AP , Doukkala; Séguy 1930a , AP , Maâmora; Séguy 1941a , HA , Tizi-n'Icheden (3000 m); Maurice 1947 ; Pape 1996 ; Mouna 1998 ; Lmimouni et al. 2004 ; Tliqui et al. 2007 ; Farkas et al. 2009 , Rif , Al Hoceima, Taguidit, Tafensa, EM , Aghbal; Hall et al. 2009 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia nuba (Wiedemann, 1830) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia trina (Wiedemann, 1830) Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019 Sarcophaginae Blaesoxipha Loew, 1861 Blaesoxipha ( Blaesoxipha ) lapidosa Pape, 1994 Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019 Blaesoxipha ( Blaesoxipha ) litoralis (Villeneuve, 1911) Pape 1996 ; Verves 2019 Blaesoxipha ( Blaesoxipha ) pygmaea (Zetterstedt, 1844) Pape 1996 ; Verves 2019 Blaesoxipha ( Servaisia ) rossica Villeneuve, 1912 Pape 1996 ; Verves 2019 Ravinia Robineau-Desvoidy, 1863 Ravinia pernix (Harris, 1780) = Gesneriodes disjuncta Séguy, in Séguy 1938 : 43 = Sarcophaga striata (Fabricius), in Séguy 1941d : 22, Séguy 1949a : 159 Séguy 1938 , HA , Skoutana; Séguy 1941d , AA , Taroudant; Séguy 1949a , AA , Akka; Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga Meigen, 1826 Sarcophaga ( Bercaea ) africa (Wiedemann, 1824) Pape 1996 ; Abkari et al. 1998 ; Verves 2003 , 2019 ; Grabener 2017 ; El Hawagry and El-Azab 2019 Sarcophaga ( Helicophagella ) maculata Meigen, 1835 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Helicophagella ) melanura Meigen, 1826 El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Helicophagella ) novercoides Böttcher, 1913* Rif , HA , AA Sarcophaga ( Heteronychia ) balanina Pandellé, 1896 Whitmore et al. 2013 , AP , Larache; Fendane et al. 2018 , AP , Diabat (Essaouira), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Heteronychia ) cucullans Pandellé, 1896 Séguy 1930a , HA , Maharidja; Mouna 1998 Sarcophaga ( Heteronychia ) ferox Villeneuve, 1908 Whitmore 2011 , Rif , Ouezzane, AP , Larache, MA , Béni Mellal, Afourer, AA , Aoulouz; Whitmore et al. 2013 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Heteronychia ) filia Rondani, 1860 Whitmore 2011 , MA , Azrou, Timahdit; Whitmore et al. 2013 ; Verves 2019 Sarcophaga ( Heteronychia ) javita (Peris, González-Mora & Mingo, 1998)* AA Sarcophaga ( Heteronychia ) longestylata Strobl, 1906 Pape 1996 ; Whitmore et al. 2013 , MA , Ifrane, Azrou; Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Heteronychia ) minima Rondani, 1862 Whitmore 2011 , MA , Azrou, Ifrane, Afourer (Béni Mellal), HA , Ijoukak, Ouirgane (Marrakech), AA , Oulma (Agadir); Whitmore et al. 2013 ; Fendane et al. 2018 , AP , Smimou (Essaouira), El Akarta (Oualidia); Verves 2019 Sarcophaga ( Heteronychia ) obvia (Povolný, 2004) Whitmore et al. 2013 , MA , Afourer (Béni Mellal), HA , Ait Lekak (Marrakech), S Asni (Imlil, Marrakech), Tagadirt, Quirgane, AA , Oulma Ort (Agadir) Sarcophaga ( Heteronychia ) pandellei (Rohdendorf, 1937) Séguy 1930a , MA , Tizi-s'Tkrine; Mouna 1998 ; Whitmore et al. 2013 , MA , Azrou, Ifrane, Afourer (Béni Mellal) Sarcophaga ( Heteronychia ) tangerensis Whitmore, 2011 = Heteronychia ( Heteronychia ) amica Peris, González-Mora & Mingo, in Peris et al. 1998 : 173 Peris et al. 1998 , Rif , Tanger; Whitmore 2011 , Rif , Tanger Sarcophaga ( Heteronychia ) villeneuveana (Enderlein, 1928) = Pierretia ( Bercaea ) maroccana Rohdendorf, in Rohdendorf 1937 : 325 = Sarcophaga ( Heteronychia ) penicillata Villeneuve, in Coupland and Barker 2004 : 113 (misidentification) Rohdendorf 1937 , MA , Aïn Defali; Coupland and Barker 2004 ; Whitmore 2009 ; Fendane et al. 2018 , AP , Diabat (Essaouira), Ghabat Tansift (Souiria), Lalla Fatna (Safi), Laatoutate (Safi), El Akarta (Oualidia), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Heteronychia ) uncicurva Pandellé, 1896 Fendane et al. 2018 , AP , Smimou (Essaouira), Diabat (Essaouira), Lalla Fatna (Safi), Laatoutate (Safi), Bir Retma (Casablanca) Sarcophaga ( Liopygia ) argyrostoma (Robineau-Desvoidy, 1830)* HA Sarcophaga ( Liopygia ) crassipalpis Macquart, 1839 Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) aegyptica Salem, 1935 Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Liosarcophaga ) dux Thomson, 1869* SA Sarcophaga ( Liosarcophaga ) jacobsoni (Rohdendorf, 1937) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) marshalli Parker, 1923 Fendane et al. 2018 , AP , Smimou (Essaouira), Diabat (Essaouira), Ghabat Tansift (Souiria), Laatoutate (Safi); El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) pharaonis Rohdendorf, 1934 Carles-Tolrá 2002 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) tibialis Macquart, 1851 = Sarcophaga beckeri Villeneuve, in Maurice 1947 : 57; Mouna 1998 : 86 Maurice 1947 ; Mouna 1998 ; Fendane et al. 2018 , AP , Laatoutate (Safi) Sarcophaga ( Liosarcophaga ) teretirostris Pandellé, 1896 = Parasarcophaga decellei Lehrer, in Lehrer 1976 : 3 Lehrer 1976 , MA , Kandar, Imouzzer; Pape 1996 , MA , Kandar, Imouzzer; Verves 2019 Sarcophaga ( Liosarcophaga ) tuberosa Pandellé, 1896 60 Mouna 1998 Sarcophaga ( Myorhina ) nigriventris Meigen, 1826 Pape 1996 ; Mouna 1998 ; Fendane et al. 2018 , AP , Ghabat Tansift (Souiria), Laatoutate (Safi), El Akarta (Oualidia), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Myorhina ) soror Rondani, 1860 Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Pandelleana ) protuberans Pandellé, 1896 Séguy 1949a , AA , Agadir Tissint, SA , Guelmim, Foum-el-Hassan, Tata; Mouna 1998 Sarcophaga ( Parasarcophaga ) hirtipes Wiedemann, 1830 Pape 1996 ; Verves 2003 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Sarcophaga ) lehmanni Müller, 1922 Pape 1996 ; Cassar et al. 2005 , Rif , Smir lagoon; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Sarcophaga ) marcelleclercqi Lehrer, 1975 Lehrer 1975 ; Pape 1996 , MA , Azrou; Verves 2019 Sarcophaga ( Thyrsocnema ) belgiana (Lehrer, 1976) Lehrer 1976 ; Pape 1996 Sarcophaga ( Thyrsocnema ) sp. 61 Ebejer et al. 2019 [as incisilobata , misidentification], Rif , Tahaddart (8 m) New records for Morocco Macronychia lemariei Jacentkovský, 1941 Atlantic plain: Rabat, Forêt de Maâmora, 100 m, 25–26.iv.1989, 1♂1♀, Zoological Museum of Copenhagen Expedition (NMHD). Sarcophaga ( Helicophagella ) novercoides Böttcher, 1913 Rif: Ouezzane, 300 m, 21–22.iv.1989, 1♂, Zoological Museum of Copenhagen Expedition (NMHD). High Atlas: Marrakech, Ouirgane, 1000 m, 1–9.iv.1997 Mai, 1♂, C. Kassebeer leg. (NMHD). Anti Atlas: 30 km NW Aoulouz, 1400 m, 10.iv.1989, 1♂, Zoological Museum of Copenhagen Expedition (NMHD). Sarcophaga ( Heteronychia ) javita (Peris, González-Mora & Mingo, 1998) Anti Atlas: Agadir, S Oulma, 30°31'N, 9°09'W , 200 m, 21.iv.1997, 1♂, C. Kassebeer leg. (NMHD). Sarcophaga ( Liopygia ) argyrostoma (Robineau-Desvoidy, 1830) High Atlas: Marrakech, Tagadirt, Ouirgane, 1000 m, 1.x.1994, 1♀, C. Kassebeer leg. (NMHD). Sarcophaga ( Liosarcophaga ) dux Thomson, 1869 Sahara: Erfoud, Rissani area, 900 m, 13–14.iv.1989, Zoological Museum of Copenhagen Expedition ( NHMD ). TACHINIDAE K. Kettani, P. Cerretti, H.-P. Tschorsnig Number of species: 147 . Expected: 200 Faunistic knowledge of the family in Morocco: poor Dexiinae Dexiini Billaea Robineau-Desvoidy, 1830 Billaea lata (Macquart, 1849) = Rhynchodinera lata Macquart, in Séguy 1930a : 143 Séguy 1930a , MA , Aharmoumou, Camp Boulhout, Sidi Bettache, Aïn Sferguila, Meknès; Mouna 1998 ; AP (Mehdia) – MISR ; AP (Essaouira, 4 km E Ounara), HA (Marrakech, Lakhdar, N Demnate) – PCPT Estheria Robineau-Desvoidy, 1830 Estheria atripes Villeneuve, 1920 Cerretti and Tschorsnig 2012 Estheria iberica Tschorsnig, 2003* MA Estheria nigripes (Villeneuve, 1920) Cerretti and Tschorsnig 2012 ; MA (Béni Mellal, El Ksiba), AA (Agadir, Oulma) – PCPT Estheria picta (Meigen, 1826) 62 Moutia 1940 Zeuxia Meigen, 1826 Zeuxia aberrans (Loew, 1847) = Zeuxia nigripes Meigen, in Séguy 1941d : 23 Brémond 1938 ; Séguy 1941d , AP , Rabat, MA , Volubilis, AA , Agadir; IOBC-List 11; Mesnil 1980; Mouna 1998 ; Tschorsnig 2017; AA (10 km NW Aït-Baha) – PCPT Dufouriini Dufouria Robineau-Desvoidy, 1830 Dufouria nigrita (Fallén, 1810) Ebejer et al. 2019 , AP , Larache (Lower Loukous saltmarsh, 2 m); MA (Ouzoud) – PCPT Voriini Athrycia Robineau-Desvoidy, 1830 Athrycia trepida (Meigen, 1824)* MA Cyrtophloeba Rondani, 1856 Cyrtophloeba ruricola (Meigen, 1824) = Plagia ruricola Meigen, in Séguy 1935a : 120, in Rungs 1940 : 14 Séguy 1935a , MA , Ifrane; Rungs 1940 , MA (Cédraie); Mouna 1998 ; HA (Tizi-n'Test), AA (Taroudant) – PCPT Eriothrix Meigen, 1830 Eriothrix apennina (Rondani, 1862) Herting and Dely-Draskovits 1993 ; Koçak and Kemal 2010 Eriothrix rufomaculata (De Geer, 1776)* MA , HA Hypovoria Villeneuve, 1913 Hypovoria hilaris Villeneuve, 1912 Séguy 1935a , AP , Oued Beth; Séguy 1953a , AP , Sehoul; Mouna 1998 ; AA (10 km SE Aït-Ourir) – PCPT Hypovoria pilibasis (Villeneuve, 1922) Zeegers 2010; HA (Tizi-n'Test), AA (Taroudant) – PCPT Kirbya Robineau-Desvoidy, 1830 Kirbya moerens (Meigen, 1830)* MA Nanoplagia Villeneuve, 1929 Nanoplagia sinaica (Villeneuve in Hermann & Villeneuve 1909) Cerretti 2009; Grabener 2017 ; HA (Marrakech, 8 km N Ouirgane), AA (40 km SW Ouarzazate, 10 km SW Tazenakht, NE Agadir, 12 km W Oulma) – PCPT Periscepsia Gistel, 1848 Periscepsia meyeri (Villeneuve, 1930) Ebejer et al. 2019 , Rif , Adrou ( PNPB , 556 m) Stomina Robineau-Desvoidy, 1830 Stomina caliendrata (Rondani, 1862) = Morphomyia caliendrata Rondani, in Séguy 1930a : 143 Séguy 1930a , MA , from M'Rirt to Hajeb, HA , Kasba Taguendaft (Gounfada); Mouna 1998 ; HA (Massif Toubkal) – PCPT Thelaira Robineau-Desvoidy, 1830 Thelaira haematodes (Meigen, 1824) 63 = Phoenicella haematodes Meigen: Séguy 1930a : 142 Séguy 1930a , HA , Arround; Mouna 1998 Uclesia Girschner, 1901 Uclesia fumipennis Girschner, 1901 Séguy 1934b ; Herting and Dely-Draskovits 1993 ; Mouna 1998 ; HA (Marrakech) – MISR Voria Robineau-Desvoidy, 1830 Voria ruralis (Fallén, 1810) Jourdan 1935c Wagneria Robineau-Desvoidy, 1830 Wagneria dilatata Kugler, 1977 Kugler 1977 Exoristinae Acemyini Ceracia Rondani, 1865 Ceracia mucronifera Rondani, 1865 = Myothyria benoisti (Mesnil), in Mesnil 1959 : 20 Mesnil 1959 , MA , Forêt Maâmora near Tiflet; Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 Blondeliini Compsilura Bouché, 1834 Compsilura concinnata (Meigen, 1824) IOBC-list 1 (1956); Hérard and Fraval 1980 Istocheta Rondani, 1859 Istocheta cinerea (Macquart, 1850) Herting 1960 Istocheta longicornis (Fallén, 1810) = Latigena longicornis Fallén, in Séguy 1953a : 91 Séguy 1953a , AP , Forêt Zaers Lomachantha Rondani, 1859 Lomachantha parra Rondani, 1859 Efetov and Tarmann 1999 Robinaldia Herting, 1983 Robinaldia angustata (Villeneuve, 1933) Herting and Dely-Draskovits 1993 ; Tschorsnig and Herting 1994 Zaira Robineau-Desvoidy, 1830 Zaira cinerea (Fallén, 1820) Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 8 m) Eryciini Alsomyia Brauer & Bergenstamm, 1891 Alsomyia olfaciens (Pandellé, 1896) IOBC-List 12 (1993) Amphicestonia Villeneuve, 1939 Amphicestonia dispar (Villeneuve, 1922) Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 ; MA (Ifrane) – PCPT Aplomyia Robineau-Desvoidy, 1830 Aplomyia confinis (Fallén, 1820) Ebejer et al. 2019 , Rif , Dardara (484 m) Carcelia Robineau-Desvoidy, 1830 Carcelia dilaticornis Mesnil, 1950 Mesnil 1950 Carcelia iliaca (Ratzeburg, 1840)63 Mouna 1998 Carcelia lucorum (Meigen, 1824)* HA Drino Robineau-Desvoidy, 1863 Drino atropivora (Robineau-Desvoidy, 1830) = Sturmia atropivora Robineau-Desvoidy, in Bléton and Fieuzet 1939 : 64 De Lépiney and Mimeur 1932; Bléton and Fieuzet 1939 , MA , Fès; Mouna 1998 ; Tschorsnig 2017; AP (Rabat), MA (Bel Lakssiri) – MISR Drino galii (Brauer & Bergenstamm, 1891)* HA , AA Drino gilva (Hartig, 1838)63 = Sturmia gilva Hartig Mouna 1998 – MISR (no locality given) Drino imberbis (Wiedemann, 1830)63 Rungs 1954 ; Grabener 2017 Drino inconspicua (Meigen, 1830) Séguy 1935a , AP , Sehoul (Rabat); Bléton and Fieuzet 1939 , MA , Dayat Achleff; Mouna 1998 ; MA (Meknès, Béni Mellal) – MISR Drino maroccana Mesnil, 1951 De Lépiney 1930 ; De Lépiney and Mimeur 1932 (probably misidentified as Sturmia inconspicua ); Mesnil 1951 ; Herting and Dely-Draskovits 1993 ; Ziegler 2011 Drino triplaca Herting, 1979 Herting 1979 , AP , Rabat Drino vicina (Zetterstedt, 1849) = Sturmia vicina Zetterstedt, 1849 De Lépiney and Mimeur 1932; Bouclier-Maurin 1923 ; AP (Rabat) – MISR Gymnophryxe Villeneuve, 1922 Gymnophryxe carthaginiensis (Bischof, 1900) Mesnil 1956 Nilea Robineau-Desvoidy, 1863 Nilea innoxia Robineau-Desvoidy, 1863 Bléton and Fieuzet 1939 Phryxe Robineau-Desvoidy, 1830 Phryxe caudata (Rondani, 1859) Biliotti 1956 ; El Yousfi 1994 ; IOBC-list 11 (1989) Phryxe setifacies (Villeneuve, 1910) IOBC-list 11 (1989); IOBC-list 12 (1993) Phryxe vulgaris (Fallén, 1810) Séguy 1953a , MA , Tamrabta (1700 m) – PCPT ( HA , Marrakech, Imlil, S Asni) Ptesiomyia Brauer & Bergenstamm, 1893 Ptesiomyia microstoma Brauer & Bergenstamm, 1893 Séguy 1953a , AP , Rabat; EM (Mte des Béni Snassen, Taforalt), MA (Béni Mellal, Bin-el-Ouidane; Meknès, Ifrane ( NPI )), HA (Marrakech, Oukaimeden) – PCPT Senometopia Macquart, 1834 Senometopia separata (Rondani, 1859) Hérard and Fraval 1980 Tryphera Meigen, 1838 Tryphera lugubris (Meigen, 1824) IOBC-List 1 (1956) Ethillini Atylomyia Brauer, 1898 Atylomyia albifrons Villeneuve, 1911 = Atylomyia rungsi Mesnil, in Mesnil 1962: 778 Mesnil 1962, AA (near Agadir), Aït Melloul; Herting and Dely-Draskovits 1993 Exoristini Bessa Robineau-Desvoidy, 1863 Bessa parallela (Meigen, 1824) Séguy 1935a (probably misidentified as Bessa selecta ); Tschorsnig 2017 Chetogena Rondani, 1856 Chetogena filipalpis Rondani, 1859 Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 8 m); MA (Fès, Sidi Harazem, Ifrane, Forêt de Cèdres) – PCPT Chetogena mageritensis (Villeneuve & Mesnil, 1936) Herting and Dely-Draskovits 1993 Chetogena media Rondani, 1859* MA Chetogena nigrofasciata (Strobl, 1902) = Chetogena repanda (Mesnil, 1939), in Herting and Dely-Draskovits 1993 (type locality: Skel): 17 Gheibi et al. 2010 Chetogena obliquata (Fallén, 1810) De Lépiney and Mimeur 1932; Herting 1960; IOBC-list 12 (1993); HA (Marrakech, Oukaimeden) – PCPT Exorista Meigen, 1803 Exorista deligata Pandellé, 1896 Mesnil 1946 , AP , Sidi Taibi near Kénitra; Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 ; Gheibi et al. 2010 Exorista grandis (Zetterstedt, 1844) Ebejer et al. 2019 , Rif , Dardara (484 m) Exorista larvarum (Linnaeus, 1758) Mouna 1998 Exorista nova (Rondani, 1859) Tschorsnig 2017 Exorista rendina (Herting, 1975)* AA Exorista segregata (Rondani, 1859) Mouna 1998 ; AA (Agadir, Oulma) – PCPT Goniini Anurophylla Villeneuve, 1938 Anurophylla aprica (Villeneuve, 1912)* MA Baumhaueria Meigen, 1838 Baumhaueria goniaeformis (Meigen, 1824) De Lépiney and Mimeur 1932; AP (Maâmora) – MISR ; HA (Marrakech, Oukaimeden) – PCPT Blepharipa Rondani, 1856 Blepharipa pratensis (Meigen, 1824)* HA Ceratochaetops Mesnil, 1970 Ceratochaetops triseta (Villeneuve, 1922) = Ceratochoeta triseta Villeneuve, in Rungs 1940 : 15 Rungs 1940 , MA (Cédraie); Mouna 1998 ; MA (Khénifra, El-Herri, Ifrane ( NPI ), Meknès), HA (Marrakech, 8 km N Ouirgane) – PCPT Ceromasia Rondani, 1856 Ceromasia rubrifrons (Macquart, 1834) IOBC-list 11 (1989); IOBC-list 12 (1993); HA (Marrakech, Ouirgane, Tagadirt, S Asni) – PCPT Clemelis Robineau-Desvoidy, 1863 Clemelis pullata (Meigen, 1824) IOBC-list 13 (1997) Gaedia Meigen, 1838 Gaedia connexa Meigen, 1824 Séguy 1953a , EM , Berkane Gonia Meigen, 1803 Gonia aterrima Tschorsnig, 1991 Tschorsnig 1991 Gonia atra Meigen, 1826 Séguy 1930a , MA , Tizi-n'Bouftene, between Azrou and Ras el Ma, Forêt Azrou, HA , Arround (Skoutana), Jebel Likount, Asni; Mouna 1998 ; Grabener 2017 ; MA (Tighassaline, El-Herri, Aïn Leuh-Tagounit, Meknès, Ifrane ( NPI )), HA (40 km SW Ouarzazate, Marrakech, Ouirgane, Lakhdar, N Demnate, Oukaimeden), AA (Agadir, Oulma) – PCPT Gonia bimaculata Wiedemann, 1819 = Gonia cilipeda Rondani, in Séguy 1953a : 91 Séguy 1930a , AP , Rabat, MA , Tizi-s'Tkrine, Tizi-n'Bouftene, between Azrou and Ras el Ma, Forêt Azrou, Berkane, HA , Arround (Skoutana), Jebel Likount, Asni, Ouaouzert (Glaoua); Séguy 1953a , AP , Temara; AP (Rabat) – MISR ; HA (S Asni Ouirgane, Marrakech), AA (80 km N Taroudant, Aoulouz), AA (15 km NW Zagora) – PCPT Gonia capitata (De Geer, 1776) Mouna 1998 ; MA (Ifrane) – MISR Gonia maculipennis Egger, 1862* MA Gonia ornata Meigen, 1826 Séguy 1953a , AP , Rabat, MA , Ifrane, SA , Kelaâ M'Goum; MA (Ifrane, Forêt de Cèdres), HA (Oukaimeden (2600 m), Marrakech) – PCPT Gonia vacua Meigen, 1826* MA Pales Robineau-Desvoidy, 1830 Pales pavida (Meigen, 1824) IOBC-list 1 (1956); Cerretti 2005 ; MA (Fès, Sidi Harazem) – PCPT Platymya Robineau-Desvoidy, 1830 Platymya antennata (Brauer & Bergenstamm, 1891) Efetov and Tarmann 1999 Pseudogonia Brauer and von Bergenstamm, 1889 Pseudogonia rufifrons (Wiedemann, 1830) De Lépiney and Mimeur 1932; IOBC-list 1 (1956); Mouna 1998 ; Tschorsnig 2017; Grabener 2017 ; AA (S Tafraoute, Aït Mansur, Agadir, Oulma) – PCPT Sturmia Robineau-Desvoidy, 1830 Sturmia bella (Meigen, 1824) Stefanescu et al. 2012 ; Tschorsnig 2017 Winthemiini Nemorilla Rondani, 1856 Nemorilla maculosa (Meigen, 1824) Kozlovsky and Rungs 1933 ; Brémond and Rungs 1938 [as N. floralis ; probable misidentification]; IOBC list 1 (1956); Mouna 1998 ; EM (Oujda, Col de Jerada) – PCPT Phasiinae Cylindromyiini Besseria Robineau-Desvoidy, 1830 Besseria lateritia (Meigen, 1824)* HA Cylindromyia Meigen, 1803 Cylindromyia bicolor (Olivier, 1812) Séguy 1930a , AP , Rabat; Mouna 1998 Cylindromyia brassicaria (Fabricius, 1775) Séguy 1930a , EM , Soufouloud, MA , Aharmoumou, Berrechid, Meknès, Sidi Bettache, Berkane, HA , Tenfecht, Ouaounzert, Marrakech, Asni; Séguy 1935a , AP , Gharb; Séguy 1941a , HA , Jebel Ayachi; Séguy 1949, SA , Guelmim; Dupuis 1963 ; Mouna 1998 ; MA (Sefrou) – MISR ; AP (Essaouira, 4 km E Ounara), HA (10 km W Chichaoua, Oukaimeden), AA (140 km E Agadir, Aoulouz, Tizi-n'Tichka) – PCPT Cylindromyia intermedia Meigen, 1824 Becker and Stein 1913 , Rif , Tanger; HA (Ouirgane, Imlil, Tizi-n'Test), AA (10 km SE Ouarzazate (oasis), Taroudant) – PCPT Cylindromyia maroccana Tschorsnig, 1997 Tschorsnig 1997 , HA , Ouirgane, Tagadirt (1000 m) Cylindromyia pilipes Loew, 1844 Becker and Stein 1913 , Rif , Tanger; Herting and Dely-Draskovits 1993 ; Gilasian et al. 2013 Phania Meigen, 1824 Phania albisquama (Villeneuve, 1924)* MA Gymnosomatini Clytiomya Rondani, 1861 Clytiomya continua (Panzer, 1798) = Clytiomyia dalmatica Robineau-Desvoidy, in Séguy 1935a : 119 Séguy 1935a , AP , Gharb; Mouna 1998 ; AP (Rabat) – MISR Clytiomya sola (Rondani, 1861) Séguy 1935a ; Dupuis 1963 ; MA (Khénifra, Tighassaline) – PCPT Ectophasia Townsend, 1912 Ectophasia crassipennis (Fabricius, 1794) = Phasia crassipennis Fabricius, in Séguy 1930a : 141 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 Eliozeta Rondani, 1856 Eliozeta helluo (Fabricius, 1805) = Clytiomyia helluo Fabricius, in Séguy 1935: 119 Séguy 1930a , MA , Meknès (Aïn Sferguila); Jourdan 1935; Séguy 1935a , AP , Gharb; Thompson 1950 ; Dupuis 1963 ; Mouna 1998 ; Tschorsnig 2017 Gymnosoma Meigen, 1803 Gymnosoma carpocoridis Dupuis, 1961 Dupuis 1963 ; Herting and Dely-Draskovits 1993 Gymnosoma clavatum (Rohdendorf, 1947) Dupuis 1963 ; Grabener 2017 ; MA (Meknès, Ifrane ( NPI )), AA (140 km E Agadir, Aoulouz), AA (80 km S Zagora, Oued Draa, Mhamid) – PCPT Gymnosoma dolycoridis Dupuis, 1960 Dupuis 1963 ; MA (Fès, Sidi Harazem) – PCPT Gymnosoma rotundatum Linnaeus, 1758 64 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Mogador, MA , Tizi-s'Tkrine, Sidi Bettache, Moulay Aïn Djemine, HA , Asni; Séguy 1934b ; Séguy 1935a ; Thompson 1950 ; Mouna 1998 ; AP (Mogador), MA (Maghrawa) – MISR ; MA (Meknès, Ifrane ( NPI )) – PCPT Gymnosoma rungsi (Mesnil, 1952) = Rhodogyne rungsi (Mesnil), in Mesnil 1952 : 151 Mesnil 1952 , AP , Rabat; Dupuis 1963 ; Herting and Dely-Draskovits 1993 Leucostomatini Clairvillia Robineau-Desvoidy, 1830 Clairvillia biguttata (Meigen, 1824)* HA Dionomelia Kugler, 1978 Dionomelia hennigi Kugler, 1978* SA Leucostoma Meigen, 1803 Leucostoma abbreviatum Herting, 1971 Ziegler 2012 Leucostoma crassum Kugler, 1966* HA Leucostoma obsidianum (Wiedemann, 1830) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Leucostoma tetraptera (Meigen, 1824) Dupuis 1953 ; Ebejer et al. 2019 , Rif , Barrage Smir (27 m) Weberia Robineau-Desvoidy, 1830 Weberia digramma (Meigen, 1824)* AA Phasiini Elomya Robineau-Desvoidy, 1830 Elomya lateralis (Meigen, 1824) Séguy 1930a , AP , From Zarjoulea to Larache, MA , Berkane; Dupuis 1952 ; Dupuis 1963 ; Herting and Dely-Draskovits 1993 ; Mouna 1998 ; Cerretti and Ziegler 2004 ; MA (Khénifra, Tighassaline, Meknès, Ifrane ( NPI )), HA (Marrakech, Ouirgane) – PCPT Phasia Latreille, 1804 Phasia mesnili (Draber-Monko, 1965) Sun and Marshall 2003 , HA ; AP (10 km E Essaouira), AA (S Tafraoute, Aït Mansur, S Aït-Baha) – PCPT Phasia obesa (Fabricius, 1798) Sun and Marshall 2003 , HA , Asni Phasia pusilla Meigen, 1824 Dupuis 1963 ; Sun and Marshall 2003 , MA Phasia subcoleoptrata (Linnaeus, 1767) Dupuis 1963 ; Herting and Dely-Draskovits 1993 ; Sun and Marshall 2003 , MA ; Cerretti and Ziegler 2004 Phasia venturii (Draber-Monko, 1965) Sun and Marshall 2003 , HA , Asni; AP (Essaouira, 4 km E Ounara), AA (11 km NW Taliouine, 10 km SE Aït-Ourir) – PCPT Trichopodini Trichopoda Berthold, 1827 Trichopoda pennipes (Fabricius, 1794) Ebejer et al. 2019 , Rif , Tahaddart (8 m) Xystini Xysta Meigen, 1824 Xysta holosericea (Fabricius, 1805)* HA Tachininae Graphogastrini Graphogaster Rondani, 1868 Graphogaster vestita Rondani, 1868* MA Phytomyptera Rondani, 1845 Phytomyptera nigrina (Meigen, 1824) = Phytomyptera nitidiventris Rondani, in Bléton and Fieuzet 1939 : 64 Bléton and Fieuzet 1939 ; Mouna 1998 Leskiini Aphria Robineau-Desvoidy, 1830 Aphria longirostris (Meigen, 1824) Ebejer et al. 2019 , Rif , Jnane Niche (46 m) Bithia Robineau-Desvoidy, 1863 Bithia demotica (Egger, 1861) Tschorsnig and Bläsius 2001 ; IOBC-list 14 (2005); Tschorsnig 2017 Bithia modesta (Meigen, 1824) Tschorsnig and Bläsius 2001 ; Tschorsnig 2017 Linnaemyini + Ernestiini Gymnochaeta Robineau-Desvoidy, 1830 Gymnochaeta viridis Fallén, 1810 Séguy 1930a , HA , Arround (Skoutana); Mouna 1998 Linnaemya Robineau-Desvoidy, 1830 Linnaemya soror Zimin, 1954* MA , HA , AA Loewia Egger, 1856 Loewia setibarba Egger, 1856 Becker and Stein 1913 , Rif , Tanger Panzeria Robineau-Desvoidy, 1830 Panzeria castellana (Strobl, 1906)* HA Panzeria nemorum (Meigen, 1824)* MA Zophomyia Macquart, 1835 Zophomyia temula (Scopoli, 1763) Séguy 1930a , AP , Casablanca, MA , Meknès; Mouna 1998 ; MA (Khénifra, Tighassaline, Meknès, Ifrane ( NPI )) – PCPT Macquartiini Macquartia Robineau-Desvoidy, 1830 Macquartia chalconota (Meigen, 1824) Ebejer et al. 2019 , Rif , Smir lagoon; HA (Marrakech, Ouirgane, Tizi-n'Test), AA (Taroudant) – PCPT Macquartia macularis Villeneuve, 1926 Herting and Dely-Draskovits 1993 Macquartia tessellum (Meigen, 1824) = Macquartia brevicornis Macquart, in Séguy 1941d : 23 Séguy 1941d , MA , Meknès, HA , Tizi-n'Test; Mouna 1998 ; HA (Imlil, S Asni, Tizi-n'Test), AA (Taroudant) – PCPT Megaprosopini Microphthalma Macquart, 1844 Microphthalma europaea Egger, 1860 Ebejer et al. 2019 , AA , Ziz river (30 km N of Erfoud, 894 m) Minthoini Hyperaea Robineau-Desvoidy, 1863 Hyperaea femoralis (Meigen, 1824) Herting and Dely-Draskovits 1993 Mintho Robineau-Desvoidy, 1830 Mintho compressa (Fabricius, 1787) Walker 1849 ; Herting and Dely-Draskovits 1993 Mintho rufiventris (Fallén, 1817) = Mintho praeceps (Scopoli, 1763), in Séguy 1930a : 143; Séguy 1953a : 91 Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès; Séguy 1953a , SA , El Aïoun du Draa; Séguy 1949a , AA , Tata; Mouna 1998 ; Dawah 2011 ; AP (Rabat, Salé), MA (Meknès) – MISR ; MA (Meknès, Ifrane ( NPI )), AA (11 km NW Taliouine) – PCPT Minthodes Brauer & Bergenstamm, 1889 Minthodes numidica Villeneuve, 1932* AA Minthodes setifacies Mesnil, 1939 = Minthodes ( Myxominthodes ) setifacies Mesnil, in Mesnil 1939 : 211 Mesnil 1939 , MA , Forêt Azrou; Herting and Dely-Draskovits 1993 Plesina Meigen, 1838 Plesina phalerata (Meigen, 1824) Herting and Dely-Draskovits 1993 ; Cerretti and Tschorsnig 2008 Pseudomintho Brauer & Bergenstamm, 1889 Pseudomintho diversipes (Strobl, 1889) Ebejer et al. 2019 , Rif , Moulay Abdelsalam ( PNPB , 965 m); AP (Essaouira, 4 km E Ounara) – PCPT Siphonini Actia Robineau-Desvoidy, 1830 Actia infantula (Zetterstedt, 1844) Ebejer et al. 2019 , Rif , Tanger (Douar Dakchire forest, 320 m) Peribaea Robineau-Desvoidy, 1863 Peribaea apicalis Robineau-Desvoidy, 1863 Ebejer et al. 2019 , Rif , Dardara (484 m) Peribaea tibialis (Robineau-Desvoidy, 1851) Draber-Mońko 2011 Siphona Meigen, 1803 Siphona geniculata (De Geer, 1776) 65 Séguy 1930a , HA ; Mouna 1998 ; HA (Vallée Oued N'fis) – MISR Siphona maroccana Cerretti & Tschorsnig, 2007 Cerretti and Tshorsnig 2007, HA , Asif Mellah, W Tizi-n'Tichka Siphona variata Andersen, 1982 Ebejer et al. 2019 , Rif , Sidi Yahia Aârab (377 m), Oued Kbir ( PNPB , 157 m) Tachinini Germaria Robineau-Desvoidy, 1830 Germaria barbara Mesnil, 1963* HA Peleteria Robineau-Desvoidy, 1830 Peleteria ruficornis (Macquart, 1835)* HA , AA Tachina Meigen, 1803 Tachina corsicana (Villeneuve, 1931)* HA , AA Tachina fera (Linnaeus, 1761) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, Forêt Tiffert, Sidi Bettache, HA , Arround (Skoutana), Jebel Likount; Séguy 1953a , MA , Ifrane (1650 m); Mouna 1998 ; Rif (fir forest of Talassemtane), AP (Dradek) – MISR ( MA , Meknès); MA (Ifrane ( NPI )), Béni Mellal, Bin-el-Ouidane), HA (Marrakech, Ouirgane) – PCPT Tachina magnicornis (Zetterstedt, 1844) Séguy 1930a , MA , Ras el Ksar, Aïn Leuh, Sidi Bettache; Mouna 1998 ; MA (Béni Mellal, El Ksiba, 5 km N) – PCPT Tachina praeceps Meigen, 1824 HA , AA Triarthriini Lissoglossa Villeneuve, 1912 Lissoglossa bequaerti Villeneuve, 1912 Herting and Dely-Draskovits 1993 New records for Morocco The data added under the abbreviation "PCPT" (for "personal communication Hans-Peter Tschorsnig") are based on (unpublished) material which was identified by HPT for several collectors (M. Hauser, M. Hradský, C.F. Kassebeer, U. Koschwitz, J.A.W. Lucas, G. Miksch, H. and T. v. Oorschot, C. Schmid-Egger, M. Schwarz, K. Špatenka, V. Vrabec) during the last ~ 30 years. Usually only a few duplicate specimens were retained in the collection of SMNS . The main part was sent back to the collectors, but the data were noted by HPT on handwritten lists. Estheria iberica Tschorsnig, 2003 Middle Atlas: Ifrane, National Park of Ifrane, 19.ix.1989, K. Špatenka leg, 1 specimen, PCPT. Athrycia trepida (Meigen, 1824) Middle Atlas: Meknès; Ifrane, National Park of Ifrane, 22.v.1995, C. Kassebeer leg., 1 specimen, PCPT. Eriothrix rufomaculata (De Geer, 1776) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. High Atlas: Marrakech, Oukaimeden, 19.v.1995, C. Kassebeer leg., 7 specimens, PCPT. Kirbya moerens (Meigen, 1830) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. Carcelia lucorum (Meigen, 1824) High Atlas: Marrakech, Imlil, S Asni, 24.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Drino galii (Brauer & Bergenstamm, 1891) High Atlas: Marrakech, Ouirgane, 24.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Anti Atlas: 11 km NW Taliouine; Agadir, Ameskroud, 17.v.1995, C. Kassebeer leg., 2 specimens, PCPT. Chetogena media Rondani, 1859 Middle Atlas: Béni Mellal, El Ksiba, 30.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Exorista rendina (Herting, 1975) Anti Atlas: 11 km NW Taliouine, 15.iii.1997, M. Hauser leg., 1 male in SMNS ; 10 km NE Tafraoute, 14.iii.1997, G. Miksch leg., 1 male in SMNS , PCPT. Anurophylla aprica (Villeneuve, 1912) Middle Atlas: Béni Mellal, Afourer, 28.iii.1995, C. Kassebeer leg., 1 female in SMNS , PCPT. Blepharipa pratensis (Meigen, 1824) High Atlas: Tizi-n'Test, 2000 m, 21.v.1995, M. Hauser leg., 2 specimens, PCPT. Anti Atlas: Taroudant, PCPT. Gonia maculipennis Egger, 1862 Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 female in SMNS , PCPT. Gonia vacua Meigen, 1826 Middle Atlas: Meknès; Ifrane, National Park of Ifrane, 29.iii.1995 and 22.v.1995, C. Kassebeerleg., 2 specimens, PCPT. Besseria lateritia (Meigen, 1824) High Atlas: SE Asni, Imlil, 23.v.1995, M. Hauser leg., 2 specimens; Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Phania albisquama (Villeneuve, 1924) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec and L. Vrabcová leg., 1 specimen, PCPT. Clairvillia biguttata (Meigen, 1824) High Atlas: Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg, 1 specimen, PCPT. Dionomelia hennigi Kugler, 1978 SA : Boujdour, 8.v.1999, V. Vrabec leg, 1 male in SMNS , PCPT. Leucostoma crassum Kugler, 1966 High Atlas: Tizi-n'Test, pass 23.vi.1996, U. Koschwitz leg., 1 male in SMN, PCPT. Weberia digramma (Meigen, 1824) Anti Atlas: 10 km NW Aït-Baha, PCPT. Xysta holosericea (Fabricius, 1805) High Atlas: Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg, 1 specimen, PCPT. Linnaemya soror Zimin, 1954 Middle Atlas: Béni Mellal, El Ksiba, 5 km N; Béni Mellal, Afourer; Khénifra, Tighassaline; Meknès; National Park of Ifrane. High Atlas: Marrakech, Ouirgane; Marrakech, Tagaddirt, S Asni; Marrakech, Lakhdar, N Demnate. Anti Atlas: 11 km NW Taliouine, all C. Kassebeer leg., 57 specimens (collected between 25.iii. and 23.v.1995), PCPT. Panzeria castellana (Strobl, 1906) High Atlas: Marrakech, Ouirgane, 26.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Panzeria nemorum (Meigen, 1824) Middle Atlas: Meknès; National Park of Ifrane, 22.v.1995, C. Kassebeer leg., 1 specimen, PCPT. Graphogaster vestita Rondani, 1868 Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. Minthodes numidica Villeneuve, 1932 Anti Atlas: S Aït-Baha, PCPT. Germaria barbara Mesnil, 1963 High Atlas: S Tizi-n'Test, 1900 m, PCPT. Peleteria ruficornis (Macquart, 1835) High Atlas: Marrakech, Ouirgane; Marrakech, Tagaddirt, S Asni; Tizi-n'Test. Anti Atlas: Taroudant, all C. Kassebeer leg., 6 specimens (collected between 28.ix.1994 and 1.iv.1995), PCPT. Tachina corsicana (Villeneuve, 1931) High Atlas: Marrakech, Oukaimeden, 19.v.1995, C. Kassebeer leg., 1 specimen; Tizi-n'Test. Anti Atlas: Taroudant, 21.v.1995, M. Hauser leg., 2 specimens, PCPT. Tachina praeceps Meigen, 1824 High Atlas: Marrakech, Oukaimeden, 2500 m, 27.vi.1987, M. Schwarz leg., 1 specimen; Tizi-n'Test. Anti Atlas: Taroudant, 29.vi.1987, M. Schwarz leg., 1 specimen, PCPT. Stratiomyoidea STRATIOMYIDAE K. Kettani, N. Woodley Number of species: 40 . Expected: 50–60 Faunistic knowledge of the family in Morocco: moderate Beridinae Beris Latreille, 1802 Beris rozkosnyi Kassebeer, 1996 Kassebeer 1996 ; Woodley 2001 , MA , Meknès, Ifrane; Kehlmaier 2004 ; Yimlahi et al. 2017 Chorisops Rondani, 1856 Chorisops tunisiae (Becker, 1915) Haenni 1990 , Rif , Tanger; Woodley 2001 ; Kehlmaier 2004 ; Mason et al. 2006 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 ; Lebard et al. 2020 Clitellariinae Pycnomalla Gerstaecker, 1857 Pycnomalla aterrima Sack, 1912 Séguy 1930a , MA , Tizi-s'Tkrine (1700 m); Séguy 1953a , MA , Dayat Aoua; Woodley 2001 ; Yimlahi et al. 2017 Pycnomalla auriflua (Erichson, 1841) Séguy 1930a , MA , Soufouloud (1900–2100 m), Boulhaut; Duisit 1960 , AP , Cap Cantin; Woodley 2001 Pycnomalla splendens (Fabricius, 1787) Séguy 1930a ; Séguy 1953a , MA , Dayat Aoua; Duisit 1960 , AP , forest of Maâmora, Casablanca, Cap Cantin; Rozkošný 1983, AP , Cap Cantin; Woodley 2001 ; Kehlmaier 2004 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 Nemotelinae Lasiopa Brullé, 1832 Lasiopa benoisti Séguy, 1930 Séguy 1930a , MA , Meknès; Duisit 1960 , EM , Aïn Guettara (Middle Moulouya); Woodley 2001 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 Lasiopa pantherina Séguy, 1930 Séguy 1930a , EM , Maharidja; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus Geoffroy, 1762 Nemotelus ( Camptopelta ) nigrinus Fallén, 1817 Duisit 1960 , AP , Khatouat (S Rabat); Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) atriceps Loew, 1856 Yimlahi et al. 2017 , AA , village Massa Nemotelus ( Nemotelus ) cingulatus Dufour, 1852 Rozkošný 1977 , AP , Skhirat; Rozkošný 1983, Rif , Tanger; Woodley 2001 ; Yimlahi et al. 2017 , Rif , Dayat Afrate, Oued Koub Nemotelus ( Nemotelus ) cylindricornis Rozkošný, 1977 Faucheux 2009 , AP , Oualidia Nemotelus ( Nemotelus ) danielssoni Mason, 1989 Yimlahi et al. 2017 , Rif , Oued Izelfane (Beni Boufrah) Nemotelus ( Nemotelus ) latiusculus Loew, 1871 Lindner 1949 ; Rozkošný 1977 ; Rozkošný 1983, Rif , Tanger; Woodley 2001 ; Kehlmaier 2004 ; Yimlahi et al. 2017 , Rif , Barrage Moulay Bouchta Nemotelus ( Nemotelus ) longirostris (Wiedemann, 1824) Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Duisit 1960 , AP , Mechra-bel-Ksiri (Gharb), EM , Saïdia, Rozkošný 1977 ; Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) maculiventris Bigot, 1861 Yimlahi et al. 2017 , Rif , Oued Zandoula Nemotelus ( Nemotelus ) nigrifrons Loew, 1846 Linder 1936; Becker and Stein 1912 , Rif , Tanger; Rozkošný 1977 , Rif , Tanger; Woodley 2001 ; Yimlahi et al. 2017 , Rif , affluent Tarmast ( NPH ) Nemotelus ( Nemotelus ) pantherinus (Linnaeus, 1758) Séguy 1930a , Rif , Tanger; Duisit 1960 , AP , Zëar; Rozkošný 1977 ; Dakki 1997 ; Woodley 2001 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) proboscideus Loew, 1846 Linder 1936; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) subuliginosus Rozkošný, 1974 Rozkošný 1977 ; Woodley 2001 , Rif , Tanger; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) ventralis Meigen, 1830 Woodley 2001 , AP , Essaouira; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) uliginosus (Linnaeus, 1767) Duisit 1960 , AP , Dradek Pachygastrinae Pachygaster Meigen, 1803 Pachygaster atra Panzer, 1798 Yimlahi et al. 2017 , Rif , Dayat Mezine Pachygaster maura Lindner, 1939 Lindner 1939 ; Woodley 2001 , MA , Tagzirt; Yimlahi et al. 2017 Sarginae Chloromyia Duncan, 1837 Chloromyia formosa (Scopoli, 1763) Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , HA , M'Rassine; Duisit 1960 , AP , Rabat, Korifla, Khatouat; Woodley 2001 ; Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 ; Yimlahi et al. 2017 , Rif , Taghbalout, Lac Ametrasse, Douar Kitane Stratiomyinae Oxycerini Oxycera Meigen, 1803 Oxycera germanica (Szilády, 1932) = Hermione dorieri var. barbarica , in Vaillant 1956b : 232, 237, 242 Vaillant 1956b , HA , Lac Tamhda (Anremer), Tahanaout, Sidi Chamarouch, Aguelmous Oxycera ochracea (Vaillant, 1950) = Hermione ochracea Vaillant, in Vaillant 1956b : 237, 244 Vaillant 1956b , HA , Lac Tamhda (Anremer), Imi-N'Ifri Oxycera pardalina (Meigen, 1822) Yimlahi et al. 2017 , Rif , Oued Abou Bnar ( NPT ), Oued Maggou, Oued Achekrade, Ruisseau maison forestière ( NPT ), MA , Cascade Aïn Vittel, Mchacha Aïn Vittel Oxycera rara (Scopoli, 1763) = Hermione pulchella var. similis Vaillant, in Vaillant 1956b : 242 Vaillant 1956b , HA , Sidi Chamarouch, Imi-N'Ifri Oxycera terminata Meigen, 1822 Yimlahi et al. 2017 , Rif , Cascade Chrafate Oxycera torrentium (Vaillant, 1950) = Hermione torrentium Vaillant, in Vaillant 1956b : 240, 241 Vaillant 1956b , HA , Izourar Oxycera trilineata (Linnaeus, 1767) = Hermione bucheti Séguy, in Séguy 1939: 62 = Hermione trilineata var. algira Vaillant, in Vaillant 1956b : 244 Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a ; Villeneuve 1933 ; Vaillant 1956b , HA , Imi-N'Ifri; Woodley 2001 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 , Rif , Dayat Aïn Jdioui Vanoyia Villeneuve, 1908 Vanoyia tenuicornis (Macquart, 1834) Lindner 1936 ; Woodley 2001 , Rif , Tanger; Yimlahi et al. 2017 Stratiomyini Odontomyia Meigen, 1803 Odontomyia alolena (Séguy, 1930) = Eulalia alolena Séguy, in Séguy 1930a : 65 Séguy 1930a , Rif , Tanger, EM , Maharidja, AP , Casablanca, MA , Aïn Leuh (1400–1500 m); Dakki 1997 ; Woodley 2001 , Rif , Tanger, EM , Maharidja, AP , Casablanca, MA , Aïn Leuh; Yimlahi et al. 2017 Odontomyia angulata (Panzer, 1798) Becker and Stein 1912 , 1913 , Rif , Tanger; Woodley 2001 ; Koçak and Kemal 2010 ; Mohammadi and Khaghaninia 2015 ; Yimlahi et al. 2017 Odontomyia discolor (Loew, 1846) Becker and Stein 1912 , 1913 , Rif , Tanger; Rozkošný 1982 , Rif , Tanger; Woodley 2001 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 Odontomyia flavissima (Rossi, 1790) = Hadracantha flavissina nigripes Pleske, in Pleske 1925: 27, 32; Séguy 1930a : 66 = Eulalia flavissima Rossi, in Duisit 1960 : 121 Séguy 1926 a; Séguy 1930a ; Duisit 1960 , MA , Boulhaut; Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Odontomyia limbata (Wiedemann, 1822) = Eulalia limbata Wiedemann, in Séguy 1930a : 65 Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, MA , Meskedell (1800–1900 m); Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 , Rif , Lac Ametrasse, Aïn Sidi Brahim Ben Arrif, Dayat Afrate, ruisseau mai­son forestière ( NPT ), Aïn El Malaâb ( NPT ), Dayat Rmali El Malaâb ( NPT ), Dayat Tazia; Rif (Tahaddart) – MISR Odontomyia microcera (Séguy, 1930) = Eulalia microcera Séguy, in Séguy 1930a : 65 Séguy 1930a ; Dakki 1997 ; Woodley 2001 , MA , Meknès (550 m); Yimlahi et al. 2017 Stratiomys Geoffroy, 1762 Stratiomys cenisia Meigen, 1822 Séguy 1930a , Rif , Tanger, AP , Rabat, Sidi Bettache, MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m), Meknès, Aïn Sferguila, HA , Timhadit; Duisit 1960 , AP , Maâmora, MA , Arhbala (1700 m); Dakki 1997 ; Woodley 2001 ; Koçak and Kemal 2010 ; Mohammadi and Khaghaninia 2015 ; Yimlahi et al. 2017 Stratiomys longicornis (Scopoli, 1763) = Stratiomys ( Hirtea ) anubis Wiedemann, in Séguy 1930a : 63 Séguy 1930a , EM , Itzer (Haute Moulouya), AP , Chellah (Rabat), Casablanca, MA , Ras el Ksar (1900 m), HA , Marrakech; Duisit 1960 , AP , Rabat, MA , Aguelmane Azigza (1800 m); Dakki 1997 ; Woodley 2001 ; Barták and Kubik 2005 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 STRATIOMYIDAE K. Kettani, N. Woodley Number of species: 40 . Expected: 50–60 Faunistic knowledge of the family in Morocco: moderate Beridinae Beris Latreille, 1802 Beris rozkosnyi Kassebeer, 1996 Kassebeer 1996 ; Woodley 2001 , MA , Meknès, Ifrane; Kehlmaier 2004 ; Yimlahi et al. 2017 Chorisops Rondani, 1856 Chorisops tunisiae (Becker, 1915) Haenni 1990 , Rif , Tanger; Woodley 2001 ; Kehlmaier 2004 ; Mason et al. 2006 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 ; Lebard et al. 2020 Clitellariinae Pycnomalla Gerstaecker, 1857 Pycnomalla aterrima Sack, 1912 Séguy 1930a , MA , Tizi-s'Tkrine (1700 m); Séguy 1953a , MA , Dayat Aoua; Woodley 2001 ; Yimlahi et al. 2017 Pycnomalla auriflua (Erichson, 1841) Séguy 1930a , MA , Soufouloud (1900–2100 m), Boulhaut; Duisit 1960 , AP , Cap Cantin; Woodley 2001 Pycnomalla splendens (Fabricius, 1787) Séguy 1930a ; Séguy 1953a , MA , Dayat Aoua; Duisit 1960 , AP , forest of Maâmora, Casablanca, Cap Cantin; Rozkošný 1983, AP , Cap Cantin; Woodley 2001 ; Kehlmaier 2004 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 Nemotelinae Lasiopa Brullé, 1832 Lasiopa benoisti Séguy, 1930 Séguy 1930a , MA , Meknès; Duisit 1960 , EM , Aïn Guettara (Middle Moulouya); Woodley 2001 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 Lasiopa pantherina Séguy, 1930 Séguy 1930a , EM , Maharidja; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus Geoffroy, 1762 Nemotelus ( Camptopelta ) nigrinus Fallén, 1817 Duisit 1960 , AP , Khatouat (S Rabat); Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) atriceps Loew, 1856 Yimlahi et al. 2017 , AA , village Massa Nemotelus ( Nemotelus ) cingulatus Dufour, 1852 Rozkošný 1977 , AP , Skhirat; Rozkošný 1983, Rif , Tanger; Woodley 2001 ; Yimlahi et al. 2017 , Rif , Dayat Afrate, Oued Koub Nemotelus ( Nemotelus ) cylindricornis Rozkošný, 1977 Faucheux 2009 , AP , Oualidia Nemotelus ( Nemotelus ) danielssoni Mason, 1989 Yimlahi et al. 2017 , Rif , Oued Izelfane (Beni Boufrah) Nemotelus ( Nemotelus ) latiusculus Loew, 1871 Lindner 1949 ; Rozkošný 1977 ; Rozkošný 1983, Rif , Tanger; Woodley 2001 ; Kehlmaier 2004 ; Yimlahi et al. 2017 , Rif , Barrage Moulay Bouchta Nemotelus ( Nemotelus ) longirostris (Wiedemann, 1824) Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Duisit 1960 , AP , Mechra-bel-Ksiri (Gharb), EM , Saïdia, Rozkošný 1977 ; Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) maculiventris Bigot, 1861 Yimlahi et al. 2017 , Rif , Oued Zandoula Nemotelus ( Nemotelus ) nigrifrons Loew, 1846 Linder 1936; Becker and Stein 1912 , Rif , Tanger; Rozkošný 1977 , Rif , Tanger; Woodley 2001 ; Yimlahi et al. 2017 , Rif , affluent Tarmast ( NPH ) Nemotelus ( Nemotelus ) pantherinus (Linnaeus, 1758) Séguy 1930a , Rif , Tanger; Duisit 1960 , AP , Zëar; Rozkošný 1977 ; Dakki 1997 ; Woodley 2001 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) proboscideus Loew, 1846 Linder 1936; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) subuliginosus Rozkošný, 1974 Rozkošný 1977 ; Woodley 2001 , Rif , Tanger; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) ventralis Meigen, 1830 Woodley 2001 , AP , Essaouira; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) uliginosus (Linnaeus, 1767) Duisit 1960 , AP , Dradek Pachygastrinae Pachygaster Meigen, 1803 Pachygaster atra Panzer, 1798 Yimlahi et al. 2017 , Rif , Dayat Mezine Pachygaster maura Lindner, 1939 Lindner 1939 ; Woodley 2001 , MA , Tagzirt; Yimlahi et al. 2017 Sarginae Chloromyia Duncan, 1837 Chloromyia formosa (Scopoli, 1763) Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , HA , M'Rassine; Duisit 1960 , AP , Rabat, Korifla, Khatouat; Woodley 2001 ; Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 ; Yimlahi et al. 2017 , Rif , Taghbalout, Lac Ametrasse, Douar Kitane Stratiomyinae Oxycerini Oxycera Meigen, 1803 Oxycera germanica (Szilády, 1932) = Hermione dorieri var. barbarica , in Vaillant 1956b : 232, 237, 242 Vaillant 1956b , HA , Lac Tamhda (Anremer), Tahanaout, Sidi Chamarouch, Aguelmous Oxycera ochracea (Vaillant, 1950) = Hermione ochracea Vaillant, in Vaillant 1956b : 237, 244 Vaillant 1956b , HA , Lac Tamhda (Anremer), Imi-N'Ifri Oxycera pardalina (Meigen, 1822) Yimlahi et al. 2017 , Rif , Oued Abou Bnar ( NPT ), Oued Maggou, Oued Achekrade, Ruisseau maison forestière ( NPT ), MA , Cascade Aïn Vittel, Mchacha Aïn Vittel Oxycera rara (Scopoli, 1763) = Hermione pulchella var. similis Vaillant, in Vaillant 1956b : 242 Vaillant 1956b , HA , Sidi Chamarouch, Imi-N'Ifri Oxycera terminata Meigen, 1822 Yimlahi et al. 2017 , Rif , Cascade Chrafate Oxycera torrentium (Vaillant, 1950) = Hermione torrentium Vaillant, in Vaillant 1956b : 240, 241 Vaillant 1956b , HA , Izourar Oxycera trilineata (Linnaeus, 1767) = Hermione bucheti Séguy, in Séguy 1939: 62 = Hermione trilineata var. algira Vaillant, in Vaillant 1956b : 244 Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a ; Villeneuve 1933 ; Vaillant 1956b , HA , Imi-N'Ifri; Woodley 2001 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 , Rif , Dayat Aïn Jdioui Vanoyia Villeneuve, 1908 Vanoyia tenuicornis (Macquart, 1834) Lindner 1936 ; Woodley 2001 , Rif , Tanger; Yimlahi et al. 2017 Stratiomyini Odontomyia Meigen, 1803 Odontomyia alolena (Séguy, 1930) = Eulalia alolena Séguy, in Séguy 1930a : 65 Séguy 1930a , Rif , Tanger, EM , Maharidja, AP , Casablanca, MA , Aïn Leuh (1400–1500 m); Dakki 1997 ; Woodley 2001 , Rif , Tanger, EM , Maharidja, AP , Casablanca, MA , Aïn Leuh; Yimlahi et al. 2017 Odontomyia angulata (Panzer, 1798) Becker and Stein 1912 , 1913 , Rif , Tanger; Woodley 2001 ; Koçak and Kemal 2010 ; Mohammadi and Khaghaninia 2015 ; Yimlahi et al. 2017 Odontomyia discolor (Loew, 1846) Becker and Stein 1912 , 1913 , Rif , Tanger; Rozkošný 1982 , Rif , Tanger; Woodley 2001 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 Odontomyia flavissima (Rossi, 1790) = Hadracantha flavissina nigripes Pleske, in Pleske 1925: 27, 32; Séguy 1930a : 66 = Eulalia flavissima Rossi, in Duisit 1960 : 121 Séguy 1926 a; Séguy 1930a ; Duisit 1960 , MA , Boulhaut; Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Odontomyia limbata (Wiedemann, 1822) = Eulalia limbata Wiedemann, in Séguy 1930a : 65 Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, MA , Meskedell (1800–1900 m); Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 , Rif , Lac Ametrasse, Aïn Sidi Brahim Ben Arrif, Dayat Afrate, ruisseau mai­son forestière ( NPT ), Aïn El Malaâb ( NPT ), Dayat Rmali El Malaâb ( NPT ), Dayat Tazia; Rif (Tahaddart) – MISR Odontomyia microcera (Séguy, 1930) = Eulalia microcera Séguy, in Séguy 1930a : 65 Séguy 1930a ; Dakki 1997 ; Woodley 2001 , MA , Meknès (550 m); Yimlahi et al. 2017 Stratiomys Geoffroy, 1762 Stratiomys cenisia Meigen, 1822 Séguy 1930a , Rif , Tanger, AP , Rabat, Sidi Bettache, MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m), Meknès, Aïn Sferguila, HA , Timhadit; Duisit 1960 , AP , Maâmora, MA , Arhbala (1700 m); Dakki 1997 ; Woodley 2001 ; Koçak and Kemal 2010 ; Mohammadi and Khaghaninia 2015 ; Yimlahi et al. 2017 Stratiomys longicornis (Scopoli, 1763) = Stratiomys ( Hirtea ) anubis Wiedemann, in Séguy 1930a : 63 Séguy 1930a , EM , Itzer (Haute Moulouya), AP , Chellah (Rabat), Casablanca, MA , Ras el Ksar (1900 m), HA , Marrakech; Duisit 1960 , AP , Rabat, MA , Aguelmane Azigza (1800 m); Dakki 1997 ; Woodley 2001 ; Barták and Kubik 2005 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 Beridinae Beris Latreille, 1802 Beris rozkosnyi Kassebeer, 1996 Kassebeer 1996 ; Woodley 2001 , MA , Meknès, Ifrane; Kehlmaier 2004 ; Yimlahi et al. 2017 Chorisops Rondani, 1856 Chorisops tunisiae (Becker, 1915) Haenni 1990 , Rif , Tanger; Woodley 2001 ; Kehlmaier 2004 ; Mason et al. 2006 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 ; Lebard et al. 2020 Clitellariinae Pycnomalla Gerstaecker, 1857 Pycnomalla aterrima Sack, 1912 Séguy 1930a , MA , Tizi-s'Tkrine (1700 m); Séguy 1953a , MA , Dayat Aoua; Woodley 2001 ; Yimlahi et al. 2017 Pycnomalla auriflua (Erichson, 1841) Séguy 1930a , MA , Soufouloud (1900–2100 m), Boulhaut; Duisit 1960 , AP , Cap Cantin; Woodley 2001 Pycnomalla splendens (Fabricius, 1787) Séguy 1930a ; Séguy 1953a , MA , Dayat Aoua; Duisit 1960 , AP , forest of Maâmora, Casablanca, Cap Cantin; Rozkošný 1983, AP , Cap Cantin; Woodley 2001 ; Kehlmaier 2004 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 Nemotelinae Lasiopa Brullé, 1832 Lasiopa benoisti Séguy, 1930 Séguy 1930a , MA , Meknès; Duisit 1960 , EM , Aïn Guettara (Middle Moulouya); Woodley 2001 ; Koçak and Kemal 2010 ; Yimlahi et al. 2017 Lasiopa pantherina Séguy, 1930 Séguy 1930a , EM , Maharidja; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus Geoffroy, 1762 Nemotelus ( Camptopelta ) nigrinus Fallén, 1817 Duisit 1960 , AP , Khatouat (S Rabat); Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) atriceps Loew, 1856 Yimlahi et al. 2017 , AA , village Massa Nemotelus ( Nemotelus ) cingulatus Dufour, 1852 Rozkošný 1977 , AP , Skhirat; Rozkošný 1983, Rif , Tanger; Woodley 2001 ; Yimlahi et al. 2017 , Rif , Dayat Afrate, Oued Koub Nemotelus ( Nemotelus ) cylindricornis Rozkošný, 1977 Faucheux 2009 , AP , Oualidia Nemotelus ( Nemotelus ) danielssoni Mason, 1989 Yimlahi et al. 2017 , Rif , Oued Izelfane (Beni Boufrah) Nemotelus ( Nemotelus ) latiusculus Loew, 1871 Lindner 1949 ; Rozkošný 1977 ; Rozkošný 1983, Rif , Tanger; Woodley 2001 ; Kehlmaier 2004 ; Yimlahi et al. 2017 , Rif , Barrage Moulay Bouchta Nemotelus ( Nemotelus ) longirostris (Wiedemann, 1824) Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Duisit 1960 , AP , Mechra-bel-Ksiri (Gharb), EM , Saïdia, Rozkošný 1977 ; Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) maculiventris Bigot, 1861 Yimlahi et al. 2017 , Rif , Oued Zandoula Nemotelus ( Nemotelus ) nigrifrons Loew, 1846 Linder 1936; Becker and Stein 1912 , Rif , Tanger; Rozkošný 1977 , Rif , Tanger; Woodley 2001 ; Yimlahi et al. 2017 , Rif , affluent Tarmast ( NPH ) Nemotelus ( Nemotelus ) pantherinus (Linnaeus, 1758) Séguy 1930a , Rif , Tanger; Duisit 1960 , AP , Zëar; Rozkošný 1977 ; Dakki 1997 ; Woodley 2001 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) proboscideus Loew, 1846 Linder 1936; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) subuliginosus Rozkošný, 1974 Rozkošný 1977 ; Woodley 2001 , Rif , Tanger; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) ventralis Meigen, 1830 Woodley 2001 , AP , Essaouira; Yimlahi et al. 2017 Nemotelus ( Nemotelus ) uliginosus (Linnaeus, 1767) Duisit 1960 , AP , Dradek Pachygastrinae Pachygaster Meigen, 1803 Pachygaster atra Panzer, 1798 Yimlahi et al. 2017 , Rif , Dayat Mezine Pachygaster maura Lindner, 1939 Lindner 1939 ; Woodley 2001 , MA , Tagzirt; Yimlahi et al. 2017 Sarginae Chloromyia Duncan, 1837 Chloromyia formosa (Scopoli, 1763) Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , HA , M'Rassine; Duisit 1960 , AP , Rabat, Korifla, Khatouat; Woodley 2001 ; Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 ; Yimlahi et al. 2017 , Rif , Taghbalout, Lac Ametrasse, Douar Kitane Stratiomyinae Oxycerini Oxycera Meigen, 1803 Oxycera germanica (Szilády, 1932) = Hermione dorieri var. barbarica , in Vaillant 1956b : 232, 237, 242 Vaillant 1956b , HA , Lac Tamhda (Anremer), Tahanaout, Sidi Chamarouch, Aguelmous Oxycera ochracea (Vaillant, 1950) = Hermione ochracea Vaillant, in Vaillant 1956b : 237, 244 Vaillant 1956b , HA , Lac Tamhda (Anremer), Imi-N'Ifri Oxycera pardalina (Meigen, 1822) Yimlahi et al. 2017 , Rif , Oued Abou Bnar ( NPT ), Oued Maggou, Oued Achekrade, Ruisseau maison forestière ( NPT ), MA , Cascade Aïn Vittel, Mchacha Aïn Vittel Oxycera rara (Scopoli, 1763) = Hermione pulchella var. similis Vaillant, in Vaillant 1956b : 242 Vaillant 1956b , HA , Sidi Chamarouch, Imi-N'Ifri Oxycera terminata Meigen, 1822 Yimlahi et al. 2017 , Rif , Cascade Chrafate Oxycera torrentium (Vaillant, 1950) = Hermione torrentium Vaillant, in Vaillant 1956b : 240, 241 Vaillant 1956b , HA , Izourar Oxycera trilineata (Linnaeus, 1767) = Hermione bucheti Séguy, in Séguy 1939: 62 = Hermione trilineata var. algira Vaillant, in Vaillant 1956b : 244 Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a ; Villeneuve 1933 ; Vaillant 1956b , HA , Imi-N'Ifri; Woodley 2001 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 , Rif , Dayat Aïn Jdioui Vanoyia Villeneuve, 1908 Vanoyia tenuicornis (Macquart, 1834) Lindner 1936 ; Woodley 2001 , Rif , Tanger; Yimlahi et al. 2017 Stratiomyini Odontomyia Meigen, 1803 Odontomyia alolena (Séguy, 1930) = Eulalia alolena Séguy, in Séguy 1930a : 65 Séguy 1930a , Rif , Tanger, EM , Maharidja, AP , Casablanca, MA , Aïn Leuh (1400–1500 m); Dakki 1997 ; Woodley 2001 , Rif , Tanger, EM , Maharidja, AP , Casablanca, MA , Aïn Leuh; Yimlahi et al. 2017 Odontomyia angulata (Panzer, 1798) Becker and Stein 1912 , 1913 , Rif , Tanger; Woodley 2001 ; Koçak and Kemal 2010 ; Mohammadi and Khaghaninia 2015 ; Yimlahi et al. 2017 Odontomyia discolor (Loew, 1846) Becker and Stein 1912 , 1913 , Rif , Tanger; Rozkošný 1982 , Rif , Tanger; Woodley 2001 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 Odontomyia flavissima (Rossi, 1790) = Hadracantha flavissina nigripes Pleske, in Pleske 1925: 27, 32; Séguy 1930a : 66 = Eulalia flavissima Rossi, in Duisit 1960 : 121 Séguy 1926 a; Séguy 1930a ; Duisit 1960 , MA , Boulhaut; Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 Odontomyia limbata (Wiedemann, 1822) = Eulalia limbata Wiedemann, in Séguy 1930a : 65 Becker and Stein 1912 , 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, MA , Meskedell (1800–1900 m); Dakki 1997 ; Woodley 2001 ; Yimlahi et al. 2017 , Rif , Lac Ametrasse, Aïn Sidi Brahim Ben Arrif, Dayat Afrate, ruisseau mai­son forestière ( NPT ), Aïn El Malaâb ( NPT ), Dayat Rmali El Malaâb ( NPT ), Dayat Tazia; Rif (Tahaddart) – MISR Odontomyia microcera (Séguy, 1930) = Eulalia microcera Séguy, in Séguy 1930a : 65 Séguy 1930a ; Dakki 1997 ; Woodley 2001 , MA , Meknès (550 m); Yimlahi et al. 2017 Stratiomys Geoffroy, 1762 Stratiomys cenisia Meigen, 1822 Séguy 1930a , Rif , Tanger, AP , Rabat, Sidi Bettache, MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m), Meknès, Aïn Sferguila, HA , Timhadit; Duisit 1960 , AP , Maâmora, MA , Arhbala (1700 m); Dakki 1997 ; Woodley 2001 ; Koçak and Kemal 2010 ; Mohammadi and Khaghaninia 2015 ; Yimlahi et al. 2017 Stratiomys longicornis (Scopoli, 1763) = Stratiomys ( Hirtea ) anubis Wiedemann, in Séguy 1930a : 63 Séguy 1930a , EM , Itzer (Haute Moulouya), AP , Chellah (Rabat), Casablanca, MA , Ras el Ksar (1900 m), HA , Marrakech; Duisit 1960 , AP , Rabat, MA , Aguelmane Azigza (1800 m); Dakki 1997 ; Woodley 2001 ; Barták and Kubik 2005 ; Koçak and Kemal 2010 ; Üstüner and Hasbenli 2013 ; Yimlahi et al. 2017 Tabanoidea ATHERICIDAE K. Kettani, M. Mouna Number of species: 2 . Expected: 2 Faunistic knowledge of the family in Morocco: good Athericinae Atherix Meigen, 1803 Atherix amicorum (Thomas, 1985) = Ibisia amicorum Thomas, in Thomas 1985 : 89 Thomas 1985 , HA , Oued Réghaya near Marabout Sidi Chamarouch (Toubkal, 2310 m); Boumezzough and Thomas 1987 Atherix maroccana (Séguy, 1930) = Ibisia maroccana Séguy, in Thomas et al. 1995 : 64 Séguy 1930a , MA , Oued Tigrigra; Thomas et al. 1995 , MA , Oued Tigrigra (900 m), Timahdit (1830 m), HA , Asif Aït Bou Guemmaz (1900 m); Dakki 1997 RHAGIONIDAE K. Kettani, M.J. Ebejer Number of species: 4 . Expected: 7 Faunistic knowledge of the family in Morocco: poor Rhagioninae Chrysopilus Macquart, 1826 Chrysopilus asiliformis (Preyssler, 1791) = Chrysopilus aureus (Meigen, 1804), in Séguy 1941a : 29; Dakki 1997 : 62 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Dakki 1997 Chrysopilus pullus Loew, 1869 Ebejer et al. 2019 , Rif , Jebel Lakraâ ( NPT , 1377–1541 m), Adrou ( PNPB , 556 m) Chrysopilus splendidus (Meigen, 1820) Ebejer et al. 2019 , Rif , Oued Kbir (Béni Ratene, 157 m) Chrysopilus tsacasi Thomas, 1979 Thomas 1979 , HA , Jebel Toubkal (Tachdirt, 2500 m); Boumezzough and Thomas 1987 , HA , Oued Réghaya (Imlil, 1750 m), l'azib Oukaimeden (2730 m); Dakki 1997 ; Kerr 2004 TABANIDAE K. Kettani Number of species: 69 . Expected: 75 Faunistic knowledge of the family in Morocco: good Chrysopsinae Chrysopsini Chrysops Meigen, 1803 Chrysops caecutiens Linnaeus, 1758 El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Samsa, Oued Laou (Afertane), Oued Jnane Niche, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart Chrysops connexus Loew, 1858 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Timhadit, Oued Yquem, Volubilis, Kenitra, Meknès, Rif , Tanger; Leclercq 1967 ; Leclercq and Maldès 1987 ; Chvála et al. 1972 ; SA (Guelmim) – MISR Chrysops flavipes Meigen, 1804 = Heterochrysops perspicillaris Loew, in Séguy 1930a : 79 = Chrysops punctifer Loew, in Séguy 1930a , 79 Séguy 1930a , AP , Mogador, EM , Haute Moulouya, MA , Fès, Volubilis, AA , Taroudant; Leclercq 1967 , AA , Agadir-Tissint (Rocade du Draa); Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Kiliç 1999 ; Müller et al. 2012 Chrysops italicus Meigen, 1804 Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Müller et al. 2012 Chrysops mauritanicus Costa, 1893 Séguy 1930a , AP , Rabat, Fedhala, Larache, MA , Itzer, HA , Haute Réghaya; Leclercq 1967 , AP , Rabat (salt marshes on Salicornia ); Chvála et al. 1972 ; Leclercq and Maldès 1987 ; AP (Kénitra) – MISR Chrysops pallidiventris Kröber, 1922 Séguy 1930a , AP , Mogador, MA , Fès; Leclercq 1967 ; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Pape and Thompson 2019 Chrysops relictus Meigen, 1820 Chvála et al. 1972 ; El Haouari and Kettani 2014 , Rif , Oued Kbir (Tamuda), Oued Kelaâ (Talembote), Oued Bou Ahmed, Oued Jnane Niche, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès, Setti Fatma, Tafza Chrysops viduatus (Fabricius, 1794) El Haouari et al. 2014 , HA , Setti Fatma Silvius Meigen, 1920 Silvius algirus Meigen, 1830 Séguy 1930a ; Leclercq and Maldès 1987 ; Kiliç 1999 Silvius alpinus (Scopoli, 1763) = Silvius vituli Fabricius, 1805, in Séguy 1930a : 78 Séguy 1930a , MA , Meknès, Forêt Zaers; Leclercq and Maldès 1987 , AP , Rabat, EM , Béni Snassen, Haute Moulouya, MA , Aïn Leuh Silvius variegatus (Fabricius, 1805) = Diachlorus maroccanus Bigot, in Surcouf 1921 : 143; Séguy 1930a : 78 Surcouf 1921 , Rif , Tanger; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Rabat, EM , Haute Moulouya; Leclercq 1960 , Rif , Tanger, AP , Larache, Rabat, Salé EM , Haute Moulouya; Leclercq 1967 ; Leclercq and Maldès 1987 ; Koçak and Kemal 2013a ; AP (Rabat, Larache) – MISR Pangoniinae Pangoniini Pangonius Latreille, 1802 Pangonius alluaudi Séguy, 1930 Séguy 1930a , MA , Azrou, Aïn Leuh, Timhadit, Tasrah des Ighrezrane, Talzent, Aharmoumou; Leclercq 1967 , MA , Ifrane; Leclercq and Maldès 1987 ; MA – MISR Pangonius brevicornis (Kröber, 1921) Leclercq 1967 ; Leclercq and Maldès 1987 Pangonius hassani (Leclercq, 1968) Leclercq 1968 , MA , Ifrane, Dayat Aoua; Leclercq and Olsufjev 1975 ; Leclercq and Maldès 1987 , MA , Sidi Allal El Bahraoui Pangonius haustellatus (Fabricius, 1781) = Pangonius marginata (Fabricius, 1805), in Séguy 1930a : 74 = Pangonius aterrima Dufour 1853, in Séguy 1930a : 74 = Pangonius funebris Macquart, 1846, in Séguy 1930a : 74 Séguy 1930a , MA , Volubilis, Tizi-s'Tkrine, Aïn Leuh, Azrou, HA , Asni; Leclercq 1961b , MA , Ifrane; Leclercq 1967 , 1968 ; Chvála and Lyneborg 1970 ; Leclercq and Maldès 1987 , MA , Sidi Allal El Bahraoui (forest of Quercus suber of Maâmora); Müller et al. 2012 ; AP (Dradek near Rabat, Kénitra), MA (wide distribution between Azrou and Ras el Ma), HA – MISR Pangonius mauritanus (Linnaeus, 1767) = Pangonius funebris Fabricius, 1794, in Séguy 1930a : 76 = Pangonius maculatus (Fabricius), in Séguy 1953a : 78 Séguy 1930a ; Séguy 1953a , AP , Cap Ghir; Séguy 1949a , AA , Guelmim; Leclercq 1967 ; Leclercq and Maldès 1987 , MA , Maamar (800 m); AP (Dradek, El Maazi, Mazagan) – MISR Pangonius micans Meigen, 1820 Leclercq and Maldès 1987 Pangonius powelli Séguy, 1930 12 = Pangonius sobradieli Séguy, 1934e: 21 Séguy 1930a , MA , Bekrit, Tizi-s'Tkrine, Soufouloud; Séguy 1934e ; Séguy 1949a , AA , Guelmim; Leclercq and Maldès 1987 Pangonius raclinae Leclercq, 1960 Leclercq 1960 , HA , Tifni by Demnate; Leclercq 1967 ; Leclercq and Maldès 1987 ; Bisby et al. 2011 Philolichini Ectinocerella Séguy, 1929 Ectinocerella surcoufi Séguy, 1929 = Pangonius ectinocerella surcoufi Séguy, in Séguy 1929a : 100 Séguy 1929a , MA , Azrou, Ank El Djemel AA , Agadir; Séguy 1930a , MA , Azrou, AA , Agadir; Leclercq and Maldès 1987 , MA , From Meknès to Khemisset, near Beth river; Leclercq 1967 ; HA (Tifni) – MISR Tabaninae Diachlorini Dasybasis Macquart, 1847 Dasybasis barbata Coscaron & Philip, 1967 = Surcoufia barbata Bigot, 1892, in Séguy 1930a : 78 = Surcoufia paradoxa Kröber, 1925, in Séguy 1930a : 78 Séguy 1930a , Rif , Tanger Dasyrhamphis Enderlein, 1922 Dasyrhamphis algirus (Macquart, 1838) = Atylotus algirus Auct, in Séguy 1930a : 82 Séguy 1930a , AP , Dradek, EM , Oujda, HA , Talouet Glaoua; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; AP (Sibara) – MISR Dasyrhamphis anthracinus (Meigen, 1820) = Atylotus anthracinus Surcouf, 1924, in Séguy 1930a : 82 Séguy 1930a , AP , Rabat, Sidi Bettache, MA , M'Rirt, Aïn Sferguila, Volubilis Dasyrhamphis ater (Rossi, 1790) = Tabanus ater (Rossi, 1790), in Becker and Stein 1913 : 77 = Atylotus ater Barotte, 1926, in Séguy 1930a : 82 = Dasyrhamphis ater (Rossi, 1790), in Leclercq and Maldès 1987 : 80 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA ; Leclercq 1967 , MA , Ifrane; Leclercq and Maldès 1987 , HA , Jebel Tazzeka, Bab Ahzar (1200 m), Idni (1700 m); MA – MISR Dasyrhamphis tomentosus (Macquart, 1846) = Atylotus tomentosus Macquart, in Séguy 1930a : 84 Séguy 1930a , AP , Rabat, Oued Cherrat, MA , Azrou, El Hajeb, Meknès, Aïn Leuh, Tizi-S'Tkrine, Forêt Tiffert, Talzent, Tazarine, Meskedall; Séguy 1949a , AA , Guelmim; Leclercq and Maldès 1987 Dasyrhamphis villosus (Macquart, 1838) = Atylotus villosus Macquart, 1838, in Séguy 1930a : 84 Séguy 1930a , MA , Tameghilt; Leclercq 1967 ; Leclercq and Maldès 1987 ; MA – MISR Dasyrhamphis nigritus (Fabricius, 1794) = Therioplectes alexandrinus Wiedemann, 1830, in Séguy 1930a : 83 Séguy 1930a , MA , Aïn Leuh, El Hajeb, M'Rirt, Dar M'Tougui, Dar Kaid M'Tougui, EM , Oujda; Leclercq and Maldès 1987 Haematopotini Haematopota Meigen, 1803 Haematopota algira Kröber, 1922 Séguy 1930a ; Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 ; Leclercq 1968 , MA , Bab Ferrich, Dayat Aoua; Leclercq and Maldès 1987 Haematopota benoisti Séguy, 1930 Séguy 1930a , AP , Rabat, MA , M'Rirt; Leclercq 1967 ; Leclercq and Maldès 1987 ; Pape and Thompson 2019 Haematopota bigoti Gobert, 1880 Séguy 1930a , MA , Volubilis; Séguy 1926 a; Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 ; MA (Ifrane) – MISR Haematopota crassicornis Wahlberg, 1848 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a Haematopota fuscicornis Becker, 1914 = Chrysozona fuscicornis Povolny, in Becker and Stein 1913 : 78 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ( sic! fusicornis ), MA , Fès; Chvála et al. 1972 ; Leclercq and Maldès 1987 Haematopota grandis Meigen, 1820 Leclercq 1967 , AP , Kénitra; Chvála et al. 1972 ; Leclercq and Maldès 1987 Haematopota italica Meigen, 1804 = Haematopota tenuicornis Macquart, 1834, in Séguy 1930a : 81 = Haematopota longicornis Macquart, 1834, in Séguy 1930a : 81 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ; Chvála et al. 1972 Haematopota lambi Villeneuve, 1921 Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 , 1968 ; Leclercq and Maldès 1987 Haematopota ocelligera (Kröber, 1922) Leclercq 1961b , MA , Dayat Aoua (on a horse); Leclercq 1967 , AP , Sidi Yahia du Gharb (on Juncus acutus ); Leclercq 1968 , MA , Azrou; Leclercq and Maldès 1987 Haematopota pluvialis (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Leclercq and Maldès 1987 ; El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Smir, Oued Kbir (Tamuda), Oued Moukhlata (Boujdad), Oued Azla (Mokdassen Oulya), Oued Moulay Bouchta, Oued Jnane Niche, Oued Koudiat Shiba; El Haouari et al. 2014 , HA , Oulmès, Tafza Haematopota pseudolusitanica Szilády, 1923 = Chrysozona lusitanica Guérin, 1835, in Séguy 1930a : 81 Séguy 1930a , MA , M'Rirt, Sebou; Leclercq 1967 , MA , Sebou Haematopota subcylindrica Pandellé, 1888 Leclercq 1967 , AP , Sidi Yahia du Gharb; El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Boumarouil, Oued Jnane Niche; El Haouari et al. 2014 , HA , Tafza Heptatoma Meigen, 1803 Heptatoma pellucens (Fabricuis, 1779) El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Achiar (Bounezzal), Oued Azla (Mokdassen Oulya), Oued Azla (Mokdassen soufla), Oued Imsa (Centre Imsa), bog of Amsemlil, Oued Ouara (Khizana), Oued Boumarouil, Oued Laou (Siflaou), Oued Talembote, Oued Laou (Afertane), Oued Tizharine, Oued Bouhya (Kanar), Bab Tariouant, Oued Taysra (Ketama), Oued Srâ (Ketama); El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès Tabanini Atylotus Osten-Sacken, 1876 Atylotus agrestis (Wiedemann, 1828) Ovazza et al. 1968 ; Pape and Thompson 2019 Atylotus agricola Wiedemann, 1828 = Tabanus agricola var. Kröberi Surcouf, in Séguy 1953a : 78 Séguy 1953a , SA , entre Tagounit et Zegdou Atylotus fulvus (Meigen, 1804) Leclercq 1961b , MA , Aïn Leuh, Bordj Doumergue; Leclercq 1967 , Rif , Ketama; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Barták and Kubik 2005 Atylotus latistriatus (Brauer, 1880) = Dasystipia nigrifacies Gobert, 1881, in Séguy 1930a : 84 Séguy 1930a , MA , Aïn Leuh; Chvála et al. 1972 ; Kiliç 1999 Atylotus loewianus (Villeneuve, 1920) Leclercq 1967 , MA , Aguelmane Azigza (marshy meadow), Aguelmane de Sidi Ali Leclercq 1968 ; Chvála et al. 1972 Atylotus pulchellus Loew, 1858 Becker and Stein 1913 Rif , Tanger; Chvála et al. 1972 Atylotus quadrifarius (Loew, 1874) Chvála et al. 1972 ; Müller et al. 2011 Atylotus sublunaticornis (Zetterstedt, 1842) El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Kbir (Koudiat Krikra), Oued Martil, Oued Khizana, Oued Laou (Ifansa), Oued Bou Ahmed, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès Hybomitra Enderlein, 1922 Hybomitra arpadi Szilády 1923 El Haouari et al. 2014 , HA , Oulmès Hybomitra bimaculata Macquart, 1826 Ježek 1995; El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Lemtahane ( PNPB ), Oued Raouz, Oued Zarka, Oued Mokhlata (Boujdad), Oued Amsa (Er-Rifiyine), bog of Amsemlil, Oued Talembote (Talembote), Oued Jnane Niche, Oued Biyada, Oued Aârkob, Oued Sidi Yahia Aârab; El Haouari et al. 2014 , HA , Oulmès Hybomitra distinguenda (Verrall, 1909) Ježek et al. 2012 ; El Haouari et al. 2014 , HA , Imi-n'Tadart Hybomitra vittata (Fabricius, 1794) = Straba vittata Fabricius, 1794, in Séguy 1930a : 83 = Tabanus spectabilis Loew, 1858, in Séguy 1930a : 83 Séguy 1930a , Rif , Tanger AP , Maâmora, Rabat, Casablanca, MA , Oued Yquem, M'Rirt; Chvála and Lyneborg 1970 , Rif , Tanger, EM , Haute Moulouya; Leclercq and Maldès 1987 Tabanus Linnaeus, 1758 Tabanus autumnalis Linnaeus, 1761 = Straba autumnalis Linnaeus, 1761, in Séguy 1930a : 82 = Straba automnalis var. brunnescens Szilády, 1941, in Séguy 1930a : 82 = Straba automnalis var. molestans Becker, 1914, in Séguy 1930a : 83 Loew 1860 ; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat, EM , Béni Snassen, Itzer (Haute Moulouya), MA , Aïn Leuh; Séguy 1953a , HA , Ksar-es-Souk; Leclercq 1961b , MA , Dayat Aoua, Aïn Leuh, Immouzer Kander; Leclercq 1967 , AP , Kénitra (on Mimosa grove), Gharb (Sidi Yahia, Sidi Allal Tazi), MA , Adjir by Khenifra (on livestock), Leclercq 1968 , MA , Dayat Aoua; Leclercq and Maldès 1987 , HA , edges near river Tessaout, Kelaâ des Sraghna; Pârvu et al. 2006 , AP , Merja Zerga, MA , Kasba Tadla; El Haouari et al. 2014 , HA , Setti Fatma; MA (Allal Tazi) – MISR Tabanus barbarus Coquebert, 1804 Becker and Stein 1913 , Rif , Tanger; Leclercq 1961b Rif , Azib de Ketama; Chvála and Lyneborg 1970 , Rif , Tanger; Portillo 1982; Leclercq and Maldès 1987 , MA , marshes around Kasba Tadla; Popescu-Mirceni 2011 , AP , Merja Zerga Tabanus bifarius Loew, 1858 = Atylotus bifarius Loew, 1858, in Séguy 1930a : 83 Séguy 1930a , EM , Haute Moulouya Tabanus bovinus Linnaeus, 1758 Loew 1860 ; Séguy 1930a ; Leclercq 1967 ; Portillo 1982; Leclercq and Maldès 1987 ; Barták and Kubik 2005 , MA , M'Rirt; El Haouari and Kettani 2014 , Rif , Oued Khemis (Khemis Anjra), Oued Kelaâ (Akchour) Tabanus bromius Linnaeus, 1758 = Straba bromius Linnaeus, 1758, in Séguy 1930a : 83 Loew 1860 ; Séguy 1930a , EM , Haute Moulouya, MA , Aïn Leuh, Ras El Ksar; Leclercq 1961b , Rif , Azib de Ketama, MA , Ifrane, Immouzer, Azrou, Bordj Doumergue, Dayat Aoua, Aïn Leuh; Leclercq 1967 , MA , Immouzer des Marmoucha, Adjir by Khenifra; Leclercq 1968 , Rif , Ketama, MA , Bab-Bou-Idir, Bab Ferrich, Ifrane, Col du Zad; Pernot-Visentin and Beaucournu-Saguez 1974 , Rif ; Leclercq and Maldès 1987 ; El Haouari and Kettani 2014 , Rif , Oued Samsa, Oued Raouz, Oued Zarka, Oued Khemis (Khemis Anjra), Oued Azla (Mokdassen Oulya), Oued Kelaâ (Akchour), Oued Laou (Sifalaou), Oued Jnane Niche, Oued Berranda; El Haouari et al. 2014 , HA , Oulmès; AA (Errachidia) – MISR Tabanus choumarae Leclercq, 1967 Leclercq 1967 , AA , Aouinet Torkoz (down Draa); Leclercq and Olsufjev 1975 ; Leclercq and Maldès 1987 ; Pârvu et al. 2006 , AA , Ouarzazate Tabanus cordiger Meigen, 1820 Leclercq 1961b , MA , Bordj Doumergue, Dayat Aoua; Leclercq 1967 , Rif , Had el Rouadi, MA , Arbala par Ksiba, Boulemane, AA , Agadir-Tissint (Rocade du Draa), Akka; Leclercq 1968 , Rif , Ketama, MA , Bab Termas, Taza, Doniet; Leclercq 1986 ; Leclercq and Maldès 1987 , AA , Tinmal, edges of Draa river, Agdz, Ourika near Ouarzazate; Barták and Kubik 2005 ; Müller et al. 2011 ; Popescu-Mirceni 2011 , HA , Ouarzazate; El Haouari and Kettani 2014 , Rif , Oued Berranda; El Haouari et al. 2014 , Rif , Oued Oueghra Tabanus darimonti Leclercq, 1964 Leclercq 1967 , MA , Aïn Leuh; Leclercq 1968 , MA , Bab Boudir, forêt Bab-Azhar, Immouzer Kander; Portillo 1982; Leclercq and Maldès 1987 ; Mikuška et al. 2008 Tabanus eggeri Schiner, 1868 = Tabanus intermedius Egger, 1859, in Séguy 1930a : 83 Séguy 1930a , MA , M'Rirt; Leclercq 1968 , Rif , M'Diq, MA , Bab Boudir, El Hajeb, Azrou, Miscliffen; Portillo 1982; Leclercq and Maldès 1987 ; Mikuška et al. 2008 Tabanus leleani Austen, 1920 = Atylotus leleani Austen, 1920, in Séguy 1930a : 84 Séguy 1930a , HA , upstream of Réghaya; Leclercq 1967 ; Leclercq and Maldès 1987 ; MA – MISR Tabanus lunatus Fabricius, 1794 = Atylotus lunatus Fabricius, 1974, in Séguy 1930a : 84 Séguy 1930a , EM , Haute Moulouya, MA , Meknès; Leclercq 1961b , MA , Ifrane, Immouzer, Bordj Doumergue, Dayat Aoua, Aïn Leuh; Leclercq 1967 , EM , Haute Moulouya, MA , Ajdir by Khenifra (on livestock); Leclercq 1968 , Rif , Ketama, MA , Bab Bouder, Immouzer Kander, El Hajeb, Mishliffen, Jebel Hebri; Portillo 1982; Leclercq and Maldès 1987 , MA , Afourer (800 m) – MISR Tabanus maculicornis Zetterstedt, 1842 El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Lemtahane, Oued Boumarouil, Oued Laou (Dardara), Oued Kanar, Oued Jnane Niche; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès, Tafza Tabanus miki Brauer, 1880 El Haouari and Kettani 2014 , Rif , Oued Khemis, Oued Boumarouil, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Setti Fatma Tabanus nemoralis Meigen, 1820 Leclercq 1968 , Rif , Ketama; Portillo 1982; Leclercq and Maldès 1987 Tabanus quatuornotatus Meigen, 1820 Portillo 1982; Leclercq and Maldès 1987 ; Koçak and Kemal 2010 ; Müller et al. 2011 ; El Haouari and Kettani 2014 , Rif , Oued Maggou, Oued Ouara (Khizana); El Haouari et al. 2014 , HA , Setti Fatma; Rif (Talassemtane) – MISR Tabanus sudeticus Zeller, 1842 Leclercq 1967 ; Portillo 1982; Leclercq and Maldès 1987 ; Gioia Martins-Neto 2003 Tabanus tinctus Walker, 1850 Leclercq 1961b , MA , forêt Bab Azhar, Aïn Leuh; Pernot-Visentin and Beaucournu-Saguez 1974 , MA , HA (2300 m); Portillo 1982; Leclercq and Maldès 1987 ; MA (Azrou) – MISR VERMILEONIDAE K. Kettani, M.J. Ebejer Number of species: 6 . Expected: 6 Faunistic knowledge of the family in Morocco: good Vermileoninae Lampromyia Macquart, 1835 Lampromyia cylindrica (Fabricius, 1794) Stuckenberg 1998 , HA Lampromyia lecerfi Séguy, 1928 = Lampromyia Le Cerfi Séguy, in Séguy 1928a : 45 Séguy 1928a , HA , Tinmel (Goundafa), Asni; Stuckenberg 1998 , HA ; Kirk-Spriggs and McGregor 2009 ; Kehlmaier 2014 , HA ; AP (Tamri) – MHNN Lampromyia nigripennis Séguy, 1930 Séguy 1930a , EM , Berkane, grove in Tlet n'Rhohr; Stuckenberg 1998 , MA ; Kirk-Spriggs and McGregor 2009 ; Kehlmaier 2014 , MA , south of Azrou (1500 m), HA Lampromyia pallida Macquart, 1835 Stuckenberg 1998 , HA Vermileo Macquart, 1834 Vermileo vermileo (Linnaeus, 1758) 13 = Vermileo degeeri Macquart 1834, in Séguy 1953a : 78 Séguy 1953a , AP , Rabat Vermileo nigriventris Strobl, 1906 Ebejer et al. 2019 , Rif , Cap Spartel (Tanger, 15 m), Anissar ( PNPB , 987 m) ATHERICIDAE K. Kettani, M. Mouna Number of species: 2 . Expected: 2 Faunistic knowledge of the family in Morocco: good Athericinae Atherix Meigen, 1803 Atherix amicorum (Thomas, 1985) = Ibisia amicorum Thomas, in Thomas 1985 : 89 Thomas 1985 , HA , Oued Réghaya near Marabout Sidi Chamarouch (Toubkal, 2310 m); Boumezzough and Thomas 1987 Atherix maroccana (Séguy, 1930) = Ibisia maroccana Séguy, in Thomas et al. 1995 : 64 Séguy 1930a , MA , Oued Tigrigra; Thomas et al. 1995 , MA , Oued Tigrigra (900 m), Timahdit (1830 m), HA , Asif Aït Bou Guemmaz (1900 m); Dakki 1997 Athericinae Atherix Meigen, 1803 Atherix amicorum (Thomas, 1985) = Ibisia amicorum Thomas, in Thomas 1985 : 89 Thomas 1985 , HA , Oued Réghaya near Marabout Sidi Chamarouch (Toubkal, 2310 m); Boumezzough and Thomas 1987 Atherix maroccana (Séguy, 1930) = Ibisia maroccana Séguy, in Thomas et al. 1995 : 64 Séguy 1930a , MA , Oued Tigrigra; Thomas et al. 1995 , MA , Oued Tigrigra (900 m), Timahdit (1830 m), HA , Asif Aït Bou Guemmaz (1900 m); Dakki 1997 RHAGIONIDAE K. Kettani, M.J. Ebejer Number of species: 4 . Expected: 7 Faunistic knowledge of the family in Morocco: poor Rhagioninae Chrysopilus Macquart, 1826 Chrysopilus asiliformis (Preyssler, 1791) = Chrysopilus aureus (Meigen, 1804), in Séguy 1941a : 29; Dakki 1997 : 62 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Dakki 1997 Chrysopilus pullus Loew, 1869 Ebejer et al. 2019 , Rif , Jebel Lakraâ ( NPT , 1377–1541 m), Adrou ( PNPB , 556 m) Chrysopilus splendidus (Meigen, 1820) Ebejer et al. 2019 , Rif , Oued Kbir (Béni Ratene, 157 m) Chrysopilus tsacasi Thomas, 1979 Thomas 1979 , HA , Jebel Toubkal (Tachdirt, 2500 m); Boumezzough and Thomas 1987 , HA , Oued Réghaya (Imlil, 1750 m), l'azib Oukaimeden (2730 m); Dakki 1997 ; Kerr 2004 Rhagioninae Chrysopilus Macquart, 1826 Chrysopilus asiliformis (Preyssler, 1791) = Chrysopilus aureus (Meigen, 1804), in Séguy 1941a : 29; Dakki 1997 : 62 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Dakki 1997 Chrysopilus pullus Loew, 1869 Ebejer et al. 2019 , Rif , Jebel Lakraâ ( NPT , 1377–1541 m), Adrou ( PNPB , 556 m) Chrysopilus splendidus (Meigen, 1820) Ebejer et al. 2019 , Rif , Oued Kbir (Béni Ratene, 157 m) Chrysopilus tsacasi Thomas, 1979 Thomas 1979 , HA , Jebel Toubkal (Tachdirt, 2500 m); Boumezzough and Thomas 1987 , HA , Oued Réghaya (Imlil, 1750 m), l'azib Oukaimeden (2730 m); Dakki 1997 ; Kerr 2004 TABANIDAE K. Kettani Number of species: 69 . Expected: 75 Faunistic knowledge of the family in Morocco: good Chrysopsinae Chrysopsini Chrysops Meigen, 1803 Chrysops caecutiens Linnaeus, 1758 El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Samsa, Oued Laou (Afertane), Oued Jnane Niche, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart Chrysops connexus Loew, 1858 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Timhadit, Oued Yquem, Volubilis, Kenitra, Meknès, Rif , Tanger; Leclercq 1967 ; Leclercq and Maldès 1987 ; Chvála et al. 1972 ; SA (Guelmim) – MISR Chrysops flavipes Meigen, 1804 = Heterochrysops perspicillaris Loew, in Séguy 1930a : 79 = Chrysops punctifer Loew, in Séguy 1930a , 79 Séguy 1930a , AP , Mogador, EM , Haute Moulouya, MA , Fès, Volubilis, AA , Taroudant; Leclercq 1967 , AA , Agadir-Tissint (Rocade du Draa); Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Kiliç 1999 ; Müller et al. 2012 Chrysops italicus Meigen, 1804 Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Müller et al. 2012 Chrysops mauritanicus Costa, 1893 Séguy 1930a , AP , Rabat, Fedhala, Larache, MA , Itzer, HA , Haute Réghaya; Leclercq 1967 , AP , Rabat (salt marshes on Salicornia ); Chvála et al. 1972 ; Leclercq and Maldès 1987 ; AP (Kénitra) – MISR Chrysops pallidiventris Kröber, 1922 Séguy 1930a , AP , Mogador, MA , Fès; Leclercq 1967 ; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Pape and Thompson 2019 Chrysops relictus Meigen, 1820 Chvála et al. 1972 ; El Haouari and Kettani 2014 , Rif , Oued Kbir (Tamuda), Oued Kelaâ (Talembote), Oued Bou Ahmed, Oued Jnane Niche, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès, Setti Fatma, Tafza Chrysops viduatus (Fabricius, 1794) El Haouari et al. 2014 , HA , Setti Fatma Silvius Meigen, 1920 Silvius algirus Meigen, 1830 Séguy 1930a ; Leclercq and Maldès 1987 ; Kiliç 1999 Silvius alpinus (Scopoli, 1763) = Silvius vituli Fabricius, 1805, in Séguy 1930a : 78 Séguy 1930a , MA , Meknès, Forêt Zaers; Leclercq and Maldès 1987 , AP , Rabat, EM , Béni Snassen, Haute Moulouya, MA , Aïn Leuh Silvius variegatus (Fabricius, 1805) = Diachlorus maroccanus Bigot, in Surcouf 1921 : 143; Séguy 1930a : 78 Surcouf 1921 , Rif , Tanger; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Rabat, EM , Haute Moulouya; Leclercq 1960 , Rif , Tanger, AP , Larache, Rabat, Salé EM , Haute Moulouya; Leclercq 1967 ; Leclercq and Maldès 1987 ; Koçak and Kemal 2013a ; AP (Rabat, Larache) – MISR Pangoniinae Pangoniini Pangonius Latreille, 1802 Pangonius alluaudi Séguy, 1930 Séguy 1930a , MA , Azrou, Aïn Leuh, Timhadit, Tasrah des Ighrezrane, Talzent, Aharmoumou; Leclercq 1967 , MA , Ifrane; Leclercq and Maldès 1987 ; MA – MISR Pangonius brevicornis (Kröber, 1921) Leclercq 1967 ; Leclercq and Maldès 1987 Pangonius hassani (Leclercq, 1968) Leclercq 1968 , MA , Ifrane, Dayat Aoua; Leclercq and Olsufjev 1975 ; Leclercq and Maldès 1987 , MA , Sidi Allal El Bahraoui Pangonius haustellatus (Fabricius, 1781) = Pangonius marginata (Fabricius, 1805), in Séguy 1930a : 74 = Pangonius aterrima Dufour 1853, in Séguy 1930a : 74 = Pangonius funebris Macquart, 1846, in Séguy 1930a : 74 Séguy 1930a , MA , Volubilis, Tizi-s'Tkrine, Aïn Leuh, Azrou, HA , Asni; Leclercq 1961b , MA , Ifrane; Leclercq 1967 , 1968 ; Chvála and Lyneborg 1970 ; Leclercq and Maldès 1987 , MA , Sidi Allal El Bahraoui (forest of Quercus suber of Maâmora); Müller et al. 2012 ; AP (Dradek near Rabat, Kénitra), MA (wide distribution between Azrou and Ras el Ma), HA – MISR Pangonius mauritanus (Linnaeus, 1767) = Pangonius funebris Fabricius, 1794, in Séguy 1930a : 76 = Pangonius maculatus (Fabricius), in Séguy 1953a : 78 Séguy 1930a ; Séguy 1953a , AP , Cap Ghir; Séguy 1949a , AA , Guelmim; Leclercq 1967 ; Leclercq and Maldès 1987 , MA , Maamar (800 m); AP (Dradek, El Maazi, Mazagan) – MISR Pangonius micans Meigen, 1820 Leclercq and Maldès 1987 Pangonius powelli Séguy, 1930 12 = Pangonius sobradieli Séguy, 1934e: 21 Séguy 1930a , MA , Bekrit, Tizi-s'Tkrine, Soufouloud; Séguy 1934e ; Séguy 1949a , AA , Guelmim; Leclercq and Maldès 1987 Pangonius raclinae Leclercq, 1960 Leclercq 1960 , HA , Tifni by Demnate; Leclercq 1967 ; Leclercq and Maldès 1987 ; Bisby et al. 2011 Philolichini Ectinocerella Séguy, 1929 Ectinocerella surcoufi Séguy, 1929 = Pangonius ectinocerella surcoufi Séguy, in Séguy 1929a : 100 Séguy 1929a , MA , Azrou, Ank El Djemel AA , Agadir; Séguy 1930a , MA , Azrou, AA , Agadir; Leclercq and Maldès 1987 , MA , From Meknès to Khemisset, near Beth river; Leclercq 1967 ; HA (Tifni) – MISR Tabaninae Diachlorini Dasybasis Macquart, 1847 Dasybasis barbata Coscaron & Philip, 1967 = Surcoufia barbata Bigot, 1892, in Séguy 1930a : 78 = Surcoufia paradoxa Kröber, 1925, in Séguy 1930a : 78 Séguy 1930a , Rif , Tanger Dasyrhamphis Enderlein, 1922 Dasyrhamphis algirus (Macquart, 1838) = Atylotus algirus Auct, in Séguy 1930a : 82 Séguy 1930a , AP , Dradek, EM , Oujda, HA , Talouet Glaoua; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; AP (Sibara) – MISR Dasyrhamphis anthracinus (Meigen, 1820) = Atylotus anthracinus Surcouf, 1924, in Séguy 1930a : 82 Séguy 1930a , AP , Rabat, Sidi Bettache, MA , M'Rirt, Aïn Sferguila, Volubilis Dasyrhamphis ater (Rossi, 1790) = Tabanus ater (Rossi, 1790), in Becker and Stein 1913 : 77 = Atylotus ater Barotte, 1926, in Séguy 1930a : 82 = Dasyrhamphis ater (Rossi, 1790), in Leclercq and Maldès 1987 : 80 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA ; Leclercq 1967 , MA , Ifrane; Leclercq and Maldès 1987 , HA , Jebel Tazzeka, Bab Ahzar (1200 m), Idni (1700 m); MA – MISR Dasyrhamphis tomentosus (Macquart, 1846) = Atylotus tomentosus Macquart, in Séguy 1930a : 84 Séguy 1930a , AP , Rabat, Oued Cherrat, MA , Azrou, El Hajeb, Meknès, Aïn Leuh, Tizi-S'Tkrine, Forêt Tiffert, Talzent, Tazarine, Meskedall; Séguy 1949a , AA , Guelmim; Leclercq and Maldès 1987 Dasyrhamphis villosus (Macquart, 1838) = Atylotus villosus Macquart, 1838, in Séguy 1930a : 84 Séguy 1930a , MA , Tameghilt; Leclercq 1967 ; Leclercq and Maldès 1987 ; MA – MISR Dasyrhamphis nigritus (Fabricius, 1794) = Therioplectes alexandrinus Wiedemann, 1830, in Séguy 1930a : 83 Séguy 1930a , MA , Aïn Leuh, El Hajeb, M'Rirt, Dar M'Tougui, Dar Kaid M'Tougui, EM , Oujda; Leclercq and Maldès 1987 Haematopotini Haematopota Meigen, 1803 Haematopota algira Kröber, 1922 Séguy 1930a ; Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 ; Leclercq 1968 , MA , Bab Ferrich, Dayat Aoua; Leclercq and Maldès 1987 Haematopota benoisti Séguy, 1930 Séguy 1930a , AP , Rabat, MA , M'Rirt; Leclercq 1967 ; Leclercq and Maldès 1987 ; Pape and Thompson 2019 Haematopota bigoti Gobert, 1880 Séguy 1930a , MA , Volubilis; Séguy 1926 a; Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 ; MA (Ifrane) – MISR Haematopota crassicornis Wahlberg, 1848 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a Haematopota fuscicornis Becker, 1914 = Chrysozona fuscicornis Povolny, in Becker and Stein 1913 : 78 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ( sic! fusicornis ), MA , Fès; Chvála et al. 1972 ; Leclercq and Maldès 1987 Haematopota grandis Meigen, 1820 Leclercq 1967 , AP , Kénitra; Chvála et al. 1972 ; Leclercq and Maldès 1987 Haematopota italica Meigen, 1804 = Haematopota tenuicornis Macquart, 1834, in Séguy 1930a : 81 = Haematopota longicornis Macquart, 1834, in Séguy 1930a : 81 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ; Chvála et al. 1972 Haematopota lambi Villeneuve, 1921 Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 , 1968 ; Leclercq and Maldès 1987 Haematopota ocelligera (Kröber, 1922) Leclercq 1961b , MA , Dayat Aoua (on a horse); Leclercq 1967 , AP , Sidi Yahia du Gharb (on Juncus acutus ); Leclercq 1968 , MA , Azrou; Leclercq and Maldès 1987 Haematopota pluvialis (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Leclercq and Maldès 1987 ; El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Smir, Oued Kbir (Tamuda), Oued Moukhlata (Boujdad), Oued Azla (Mokdassen Oulya), Oued Moulay Bouchta, Oued Jnane Niche, Oued Koudiat Shiba; El Haouari et al. 2014 , HA , Oulmès, Tafza Haematopota pseudolusitanica Szilády, 1923 = Chrysozona lusitanica Guérin, 1835, in Séguy 1930a : 81 Séguy 1930a , MA , M'Rirt, Sebou; Leclercq 1967 , MA , Sebou Haematopota subcylindrica Pandellé, 1888 Leclercq 1967 , AP , Sidi Yahia du Gharb; El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Boumarouil, Oued Jnane Niche; El Haouari et al. 2014 , HA , Tafza Heptatoma Meigen, 1803 Heptatoma pellucens (Fabricuis, 1779) El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Achiar (Bounezzal), Oued Azla (Mokdassen Oulya), Oued Azla (Mokdassen soufla), Oued Imsa (Centre Imsa), bog of Amsemlil, Oued Ouara (Khizana), Oued Boumarouil, Oued Laou (Siflaou), Oued Talembote, Oued Laou (Afertane), Oued Tizharine, Oued Bouhya (Kanar), Bab Tariouant, Oued Taysra (Ketama), Oued Srâ (Ketama); El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès Tabanini Atylotus Osten-Sacken, 1876 Atylotus agrestis (Wiedemann, 1828) Ovazza et al. 1968 ; Pape and Thompson 2019 Atylotus agricola Wiedemann, 1828 = Tabanus agricola var. Kröberi Surcouf, in Séguy 1953a : 78 Séguy 1953a , SA , entre Tagounit et Zegdou Atylotus fulvus (Meigen, 1804) Leclercq 1961b , MA , Aïn Leuh, Bordj Doumergue; Leclercq 1967 , Rif , Ketama; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Barták and Kubik 2005 Atylotus latistriatus (Brauer, 1880) = Dasystipia nigrifacies Gobert, 1881, in Séguy 1930a : 84 Séguy 1930a , MA , Aïn Leuh; Chvála et al. 1972 ; Kiliç 1999 Atylotus loewianus (Villeneuve, 1920) Leclercq 1967 , MA , Aguelmane Azigza (marshy meadow), Aguelmane de Sidi Ali Leclercq 1968 ; Chvála et al. 1972 Atylotus pulchellus Loew, 1858 Becker and Stein 1913 Rif , Tanger; Chvála et al. 1972 Atylotus quadrifarius (Loew, 1874) Chvála et al. 1972 ; Müller et al. 2011 Atylotus sublunaticornis (Zetterstedt, 1842) El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Kbir (Koudiat Krikra), Oued Martil, Oued Khizana, Oued Laou (Ifansa), Oued Bou Ahmed, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès Hybomitra Enderlein, 1922 Hybomitra arpadi Szilády 1923 El Haouari et al. 2014 , HA , Oulmès Hybomitra bimaculata Macquart, 1826 Ježek 1995; El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Lemtahane ( PNPB ), Oued Raouz, Oued Zarka, Oued Mokhlata (Boujdad), Oued Amsa (Er-Rifiyine), bog of Amsemlil, Oued Talembote (Talembote), Oued Jnane Niche, Oued Biyada, Oued Aârkob, Oued Sidi Yahia Aârab; El Haouari et al. 2014 , HA , Oulmès Hybomitra distinguenda (Verrall, 1909) Ježek et al. 2012 ; El Haouari et al. 2014 , HA , Imi-n'Tadart Hybomitra vittata (Fabricius, 1794) = Straba vittata Fabricius, 1794, in Séguy 1930a : 83 = Tabanus spectabilis Loew, 1858, in Séguy 1930a : 83 Séguy 1930a , Rif , Tanger AP , Maâmora, Rabat, Casablanca, MA , Oued Yquem, M'Rirt; Chvála and Lyneborg 1970 , Rif , Tanger, EM , Haute Moulouya; Leclercq and Maldès 1987 Tabanus Linnaeus, 1758 Tabanus autumnalis Linnaeus, 1761 = Straba autumnalis Linnaeus, 1761, in Séguy 1930a : 82 = Straba automnalis var. brunnescens Szilády, 1941, in Séguy 1930a : 82 = Straba automnalis var. molestans Becker, 1914, in Séguy 1930a : 83 Loew 1860 ; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat, EM , Béni Snassen, Itzer (Haute Moulouya), MA , Aïn Leuh; Séguy 1953a , HA , Ksar-es-Souk; Leclercq 1961b , MA , Dayat Aoua, Aïn Leuh, Immouzer Kander; Leclercq 1967 , AP , Kénitra (on Mimosa grove), Gharb (Sidi Yahia, Sidi Allal Tazi), MA , Adjir by Khenifra (on livestock), Leclercq 1968 , MA , Dayat Aoua; Leclercq and Maldès 1987 , HA , edges near river Tessaout, Kelaâ des Sraghna; Pârvu et al. 2006 , AP , Merja Zerga, MA , Kasba Tadla; El Haouari et al. 2014 , HA , Setti Fatma; MA (Allal Tazi) – MISR Tabanus barbarus Coquebert, 1804 Becker and Stein 1913 , Rif , Tanger; Leclercq 1961b Rif , Azib de Ketama; Chvála and Lyneborg 1970 , Rif , Tanger; Portillo 1982; Leclercq and Maldès 1987 , MA , marshes around Kasba Tadla; Popescu-Mirceni 2011 , AP , Merja Zerga Tabanus bifarius Loew, 1858 = Atylotus bifarius Loew, 1858, in Séguy 1930a : 83 Séguy 1930a , EM , Haute Moulouya Tabanus bovinus Linnaeus, 1758 Loew 1860 ; Séguy 1930a ; Leclercq 1967 ; Portillo 1982; Leclercq and Maldès 1987 ; Barták and Kubik 2005 , MA , M'Rirt; El Haouari and Kettani 2014 , Rif , Oued Khemis (Khemis Anjra), Oued Kelaâ (Akchour) Tabanus bromius Linnaeus, 1758 = Straba bromius Linnaeus, 1758, in Séguy 1930a : 83 Loew 1860 ; Séguy 1930a , EM , Haute Moulouya, MA , Aïn Leuh, Ras El Ksar; Leclercq 1961b , Rif , Azib de Ketama, MA , Ifrane, Immouzer, Azrou, Bordj Doumergue, Dayat Aoua, Aïn Leuh; Leclercq 1967 , MA , Immouzer des Marmoucha, Adjir by Khenifra; Leclercq 1968 , Rif , Ketama, MA , Bab-Bou-Idir, Bab Ferrich, Ifrane, Col du Zad; Pernot-Visentin and Beaucournu-Saguez 1974 , Rif ; Leclercq and Maldès 1987 ; El Haouari and Kettani 2014 , Rif , Oued Samsa, Oued Raouz, Oued Zarka, Oued Khemis (Khemis Anjra), Oued Azla (Mokdassen Oulya), Oued Kelaâ (Akchour), Oued Laou (Sifalaou), Oued Jnane Niche, Oued Berranda; El Haouari et al. 2014 , HA , Oulmès; AA (Errachidia) – MISR Tabanus choumarae Leclercq, 1967 Leclercq 1967 , AA , Aouinet Torkoz (down Draa); Leclercq and Olsufjev 1975 ; Leclercq and Maldès 1987 ; Pârvu et al. 2006 , AA , Ouarzazate Tabanus cordiger Meigen, 1820 Leclercq 1961b , MA , Bordj Doumergue, Dayat Aoua; Leclercq 1967 , Rif , Had el Rouadi, MA , Arbala par Ksiba, Boulemane, AA , Agadir-Tissint (Rocade du Draa), Akka; Leclercq 1968 , Rif , Ketama, MA , Bab Termas, Taza, Doniet; Leclercq 1986 ; Leclercq and Maldès 1987 , AA , Tinmal, edges of Draa river, Agdz, Ourika near Ouarzazate; Barták and Kubik 2005 ; Müller et al. 2011 ; Popescu-Mirceni 2011 , HA , Ouarzazate; El Haouari and Kettani 2014 , Rif , Oued Berranda; El Haouari et al. 2014 , Rif , Oued Oueghra Tabanus darimonti Leclercq, 1964 Leclercq 1967 , MA , Aïn Leuh; Leclercq 1968 , MA , Bab Boudir, forêt Bab-Azhar, Immouzer Kander; Portillo 1982; Leclercq and Maldès 1987 ; Mikuška et al. 2008 Tabanus eggeri Schiner, 1868 = Tabanus intermedius Egger, 1859, in Séguy 1930a : 83 Séguy 1930a , MA , M'Rirt; Leclercq 1968 , Rif , M'Diq, MA , Bab Boudir, El Hajeb, Azrou, Miscliffen; Portillo 1982; Leclercq and Maldès 1987 ; Mikuška et al. 2008 Tabanus leleani Austen, 1920 = Atylotus leleani Austen, 1920, in Séguy 1930a : 84 Séguy 1930a , HA , upstream of Réghaya; Leclercq 1967 ; Leclercq and Maldès 1987 ; MA – MISR Tabanus lunatus Fabricius, 1794 = Atylotus lunatus Fabricius, 1974, in Séguy 1930a : 84 Séguy 1930a , EM , Haute Moulouya, MA , Meknès; Leclercq 1961b , MA , Ifrane, Immouzer, Bordj Doumergue, Dayat Aoua, Aïn Leuh; Leclercq 1967 , EM , Haute Moulouya, MA , Ajdir by Khenifra (on livestock); Leclercq 1968 , Rif , Ketama, MA , Bab Bouder, Immouzer Kander, El Hajeb, Mishliffen, Jebel Hebri; Portillo 1982; Leclercq and Maldès 1987 , MA , Afourer (800 m) – MISR Tabanus maculicornis Zetterstedt, 1842 El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Lemtahane, Oued Boumarouil, Oued Laou (Dardara), Oued Kanar, Oued Jnane Niche; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès, Tafza Tabanus miki Brauer, 1880 El Haouari and Kettani 2014 , Rif , Oued Khemis, Oued Boumarouil, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Setti Fatma Tabanus nemoralis Meigen, 1820 Leclercq 1968 , Rif , Ketama; Portillo 1982; Leclercq and Maldès 1987 Tabanus quatuornotatus Meigen, 1820 Portillo 1982; Leclercq and Maldès 1987 ; Koçak and Kemal 2010 ; Müller et al. 2011 ; El Haouari and Kettani 2014 , Rif , Oued Maggou, Oued Ouara (Khizana); El Haouari et al. 2014 , HA , Setti Fatma; Rif (Talassemtane) – MISR Tabanus sudeticus Zeller, 1842 Leclercq 1967 ; Portillo 1982; Leclercq and Maldès 1987 ; Gioia Martins-Neto 2003 Tabanus tinctus Walker, 1850 Leclercq 1961b , MA , forêt Bab Azhar, Aïn Leuh; Pernot-Visentin and Beaucournu-Saguez 1974 , MA , HA (2300 m); Portillo 1982; Leclercq and Maldès 1987 ; MA (Azrou) – MISR Chrysopsinae Chrysopsini Chrysops Meigen, 1803 Chrysops caecutiens Linnaeus, 1758 El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Samsa, Oued Laou (Afertane), Oued Jnane Niche, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart Chrysops connexus Loew, 1858 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Timhadit, Oued Yquem, Volubilis, Kenitra, Meknès, Rif , Tanger; Leclercq 1967 ; Leclercq and Maldès 1987 ; Chvála et al. 1972 ; SA (Guelmim) – MISR Chrysops flavipes Meigen, 1804 = Heterochrysops perspicillaris Loew, in Séguy 1930a : 79 = Chrysops punctifer Loew, in Séguy 1930a , 79 Séguy 1930a , AP , Mogador, EM , Haute Moulouya, MA , Fès, Volubilis, AA , Taroudant; Leclercq 1967 , AA , Agadir-Tissint (Rocade du Draa); Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Kiliç 1999 ; Müller et al. 2012 Chrysops italicus Meigen, 1804 Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Müller et al. 2012 Chrysops mauritanicus Costa, 1893 Séguy 1930a , AP , Rabat, Fedhala, Larache, MA , Itzer, HA , Haute Réghaya; Leclercq 1967 , AP , Rabat (salt marshes on Salicornia ); Chvála et al. 1972 ; Leclercq and Maldès 1987 ; AP (Kénitra) – MISR Chrysops pallidiventris Kröber, 1922 Séguy 1930a , AP , Mogador, MA , Fès; Leclercq 1967 ; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Pape and Thompson 2019 Chrysops relictus Meigen, 1820 Chvála et al. 1972 ; El Haouari and Kettani 2014 , Rif , Oued Kbir (Tamuda), Oued Kelaâ (Talembote), Oued Bou Ahmed, Oued Jnane Niche, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès, Setti Fatma, Tafza Chrysops viduatus (Fabricius, 1794) El Haouari et al. 2014 , HA , Setti Fatma Silvius Meigen, 1920 Silvius algirus Meigen, 1830 Séguy 1930a ; Leclercq and Maldès 1987 ; Kiliç 1999 Silvius alpinus (Scopoli, 1763) = Silvius vituli Fabricius, 1805, in Séguy 1930a : 78 Séguy 1930a , MA , Meknès, Forêt Zaers; Leclercq and Maldès 1987 , AP , Rabat, EM , Béni Snassen, Haute Moulouya, MA , Aïn Leuh Silvius variegatus (Fabricius, 1805) = Diachlorus maroccanus Bigot, in Surcouf 1921 : 143; Séguy 1930a : 78 Surcouf 1921 , Rif , Tanger; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Rabat, EM , Haute Moulouya; Leclercq 1960 , Rif , Tanger, AP , Larache, Rabat, Salé EM , Haute Moulouya; Leclercq 1967 ; Leclercq and Maldès 1987 ; Koçak and Kemal 2013a ; AP (Rabat, Larache) – MISR Pangoniinae Pangoniini Pangonius Latreille, 1802 Pangonius alluaudi Séguy, 1930 Séguy 1930a , MA , Azrou, Aïn Leuh, Timhadit, Tasrah des Ighrezrane, Talzent, Aharmoumou; Leclercq 1967 , MA , Ifrane; Leclercq and Maldès 1987 ; MA – MISR Pangonius brevicornis (Kröber, 1921) Leclercq 1967 ; Leclercq and Maldès 1987 Pangonius hassani (Leclercq, 1968) Leclercq 1968 , MA , Ifrane, Dayat Aoua; Leclercq and Olsufjev 1975 ; Leclercq and Maldès 1987 , MA , Sidi Allal El Bahraoui Pangonius haustellatus (Fabricius, 1781) = Pangonius marginata (Fabricius, 1805), in Séguy 1930a : 74 = Pangonius aterrima Dufour 1853, in Séguy 1930a : 74 = Pangonius funebris Macquart, 1846, in Séguy 1930a : 74 Séguy 1930a , MA , Volubilis, Tizi-s'Tkrine, Aïn Leuh, Azrou, HA , Asni; Leclercq 1961b , MA , Ifrane; Leclercq 1967 , 1968 ; Chvála and Lyneborg 1970 ; Leclercq and Maldès 1987 , MA , Sidi Allal El Bahraoui (forest of Quercus suber of Maâmora); Müller et al. 2012 ; AP (Dradek near Rabat, Kénitra), MA (wide distribution between Azrou and Ras el Ma), HA – MISR Pangonius mauritanus (Linnaeus, 1767) = Pangonius funebris Fabricius, 1794, in Séguy 1930a : 76 = Pangonius maculatus (Fabricius), in Séguy 1953a : 78 Séguy 1930a ; Séguy 1953a , AP , Cap Ghir; Séguy 1949a , AA , Guelmim; Leclercq 1967 ; Leclercq and Maldès 1987 , MA , Maamar (800 m); AP (Dradek, El Maazi, Mazagan) – MISR Pangonius micans Meigen, 1820 Leclercq and Maldès 1987 Pangonius powelli Séguy, 1930 12 = Pangonius sobradieli Séguy, 1934e: 21 Séguy 1930a , MA , Bekrit, Tizi-s'Tkrine, Soufouloud; Séguy 1934e ; Séguy 1949a , AA , Guelmim; Leclercq and Maldès 1987 Pangonius raclinae Leclercq, 1960 Leclercq 1960 , HA , Tifni by Demnate; Leclercq 1967 ; Leclercq and Maldès 1987 ; Bisby et al. 2011 Philolichini Ectinocerella Séguy, 1929 Ectinocerella surcoufi Séguy, 1929 = Pangonius ectinocerella surcoufi Séguy, in Séguy 1929a : 100 Séguy 1929a , MA , Azrou, Ank El Djemel AA , Agadir; Séguy 1930a , MA , Azrou, AA , Agadir; Leclercq and Maldès 1987 , MA , From Meknès to Khemisset, near Beth river; Leclercq 1967 ; HA (Tifni) – MISR Tabaninae Diachlorini Dasybasis Macquart, 1847 Dasybasis barbata Coscaron & Philip, 1967 = Surcoufia barbata Bigot, 1892, in Séguy 1930a : 78 = Surcoufia paradoxa Kröber, 1925, in Séguy 1930a : 78 Séguy 1930a , Rif , Tanger Dasyrhamphis Enderlein, 1922 Dasyrhamphis algirus (Macquart, 1838) = Atylotus algirus Auct, in Séguy 1930a : 82 Séguy 1930a , AP , Dradek, EM , Oujda, HA , Talouet Glaoua; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; AP (Sibara) – MISR Dasyrhamphis anthracinus (Meigen, 1820) = Atylotus anthracinus Surcouf, 1924, in Séguy 1930a : 82 Séguy 1930a , AP , Rabat, Sidi Bettache, MA , M'Rirt, Aïn Sferguila, Volubilis Dasyrhamphis ater (Rossi, 1790) = Tabanus ater (Rossi, 1790), in Becker and Stein 1913 : 77 = Atylotus ater Barotte, 1926, in Séguy 1930a : 82 = Dasyrhamphis ater (Rossi, 1790), in Leclercq and Maldès 1987 : 80 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA ; Leclercq 1967 , MA , Ifrane; Leclercq and Maldès 1987 , HA , Jebel Tazzeka, Bab Ahzar (1200 m), Idni (1700 m); MA – MISR Dasyrhamphis tomentosus (Macquart, 1846) = Atylotus tomentosus Macquart, in Séguy 1930a : 84 Séguy 1930a , AP , Rabat, Oued Cherrat, MA , Azrou, El Hajeb, Meknès, Aïn Leuh, Tizi-S'Tkrine, Forêt Tiffert, Talzent, Tazarine, Meskedall; Séguy 1949a , AA , Guelmim; Leclercq and Maldès 1987 Dasyrhamphis villosus (Macquart, 1838) = Atylotus villosus Macquart, 1838, in Séguy 1930a : 84 Séguy 1930a , MA , Tameghilt; Leclercq 1967 ; Leclercq and Maldès 1987 ; MA – MISR Dasyrhamphis nigritus (Fabricius, 1794) = Therioplectes alexandrinus Wiedemann, 1830, in Séguy 1930a : 83 Séguy 1930a , MA , Aïn Leuh, El Hajeb, M'Rirt, Dar M'Tougui, Dar Kaid M'Tougui, EM , Oujda; Leclercq and Maldès 1987 Haematopotini Haematopota Meigen, 1803 Haematopota algira Kröber, 1922 Séguy 1930a ; Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 ; Leclercq 1968 , MA , Bab Ferrich, Dayat Aoua; Leclercq and Maldès 1987 Haematopota benoisti Séguy, 1930 Séguy 1930a , AP , Rabat, MA , M'Rirt; Leclercq 1967 ; Leclercq and Maldès 1987 ; Pape and Thompson 2019 Haematopota bigoti Gobert, 1880 Séguy 1930a , MA , Volubilis; Séguy 1926 a; Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 ; MA (Ifrane) – MISR Haematopota crassicornis Wahlberg, 1848 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a Haematopota fuscicornis Becker, 1914 = Chrysozona fuscicornis Povolny, in Becker and Stein 1913 : 78 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ( sic! fusicornis ), MA , Fès; Chvála et al. 1972 ; Leclercq and Maldès 1987 Haematopota grandis Meigen, 1820 Leclercq 1967 , AP , Kénitra; Chvála et al. 1972 ; Leclercq and Maldès 1987 Haematopota italica Meigen, 1804 = Haematopota tenuicornis Macquart, 1834, in Séguy 1930a : 81 = Haematopota longicornis Macquart, 1834, in Séguy 1930a : 81 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ; Chvála et al. 1972 Haematopota lambi Villeneuve, 1921 Leclercq 1961b , MA , Dayat Aoua; Leclercq 1967 , 1968 ; Leclercq and Maldès 1987 Haematopota ocelligera (Kröber, 1922) Leclercq 1961b , MA , Dayat Aoua (on a horse); Leclercq 1967 , AP , Sidi Yahia du Gharb (on Juncus acutus ); Leclercq 1968 , MA , Azrou; Leclercq and Maldès 1987 Haematopota pluvialis (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Leclercq and Maldès 1987 ; El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Smir, Oued Kbir (Tamuda), Oued Moukhlata (Boujdad), Oued Azla (Mokdassen Oulya), Oued Moulay Bouchta, Oued Jnane Niche, Oued Koudiat Shiba; El Haouari et al. 2014 , HA , Oulmès, Tafza Haematopota pseudolusitanica Szilády, 1923 = Chrysozona lusitanica Guérin, 1835, in Séguy 1930a : 81 Séguy 1930a , MA , M'Rirt, Sebou; Leclercq 1967 , MA , Sebou Haematopota subcylindrica Pandellé, 1888 Leclercq 1967 , AP , Sidi Yahia du Gharb; El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Boumarouil, Oued Jnane Niche; El Haouari et al. 2014 , HA , Tafza Heptatoma Meigen, 1803 Heptatoma pellucens (Fabricuis, 1779) El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Achiar (Bounezzal), Oued Azla (Mokdassen Oulya), Oued Azla (Mokdassen soufla), Oued Imsa (Centre Imsa), bog of Amsemlil, Oued Ouara (Khizana), Oued Boumarouil, Oued Laou (Siflaou), Oued Talembote, Oued Laou (Afertane), Oued Tizharine, Oued Bouhya (Kanar), Bab Tariouant, Oued Taysra (Ketama), Oued Srâ (Ketama); El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès Tabanini Atylotus Osten-Sacken, 1876 Atylotus agrestis (Wiedemann, 1828) Ovazza et al. 1968 ; Pape and Thompson 2019 Atylotus agricola Wiedemann, 1828 = Tabanus agricola var. Kröberi Surcouf, in Séguy 1953a : 78 Séguy 1953a , SA , entre Tagounit et Zegdou Atylotus fulvus (Meigen, 1804) Leclercq 1961b , MA , Aïn Leuh, Bordj Doumergue; Leclercq 1967 , Rif , Ketama; Chvála et al. 1972 ; Leclercq and Maldès 1987 ; Barták and Kubik 2005 Atylotus latistriatus (Brauer, 1880) = Dasystipia nigrifacies Gobert, 1881, in Séguy 1930a : 84 Séguy 1930a , MA , Aïn Leuh; Chvála et al. 1972 ; Kiliç 1999 Atylotus loewianus (Villeneuve, 1920) Leclercq 1967 , MA , Aguelmane Azigza (marshy meadow), Aguelmane de Sidi Ali Leclercq 1968 ; Chvála et al. 1972 Atylotus pulchellus Loew, 1858 Becker and Stein 1913 Rif , Tanger; Chvála et al. 1972 Atylotus quadrifarius (Loew, 1874) Chvála et al. 1972 ; Müller et al. 2011 Atylotus sublunaticornis (Zetterstedt, 1842) El Haouari and Kettani 2014 , Rif , Oued Rha, Oued Kbir (Koudiat Krikra), Oued Martil, Oued Khizana, Oued Laou (Ifansa), Oued Bou Ahmed, Oued Biyada; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès Hybomitra Enderlein, 1922 Hybomitra arpadi Szilády 1923 El Haouari et al. 2014 , HA , Oulmès Hybomitra bimaculata Macquart, 1826 Ježek 1995; El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Lemtahane ( PNPB ), Oued Raouz, Oued Zarka, Oued Mokhlata (Boujdad), Oued Amsa (Er-Rifiyine), bog of Amsemlil, Oued Talembote (Talembote), Oued Jnane Niche, Oued Biyada, Oued Aârkob, Oued Sidi Yahia Aârab; El Haouari et al. 2014 , HA , Oulmès Hybomitra distinguenda (Verrall, 1909) Ježek et al. 2012 ; El Haouari et al. 2014 , HA , Imi-n'Tadart Hybomitra vittata (Fabricius, 1794) = Straba vittata Fabricius, 1794, in Séguy 1930a : 83 = Tabanus spectabilis Loew, 1858, in Séguy 1930a : 83 Séguy 1930a , Rif , Tanger AP , Maâmora, Rabat, Casablanca, MA , Oued Yquem, M'Rirt; Chvála and Lyneborg 1970 , Rif , Tanger, EM , Haute Moulouya; Leclercq and Maldès 1987 Tabanus Linnaeus, 1758 Tabanus autumnalis Linnaeus, 1761 = Straba autumnalis Linnaeus, 1761, in Séguy 1930a : 82 = Straba automnalis var. brunnescens Szilády, 1941, in Séguy 1930a : 82 = Straba automnalis var. molestans Becker, 1914, in Séguy 1930a : 83 Loew 1860 ; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat, EM , Béni Snassen, Itzer (Haute Moulouya), MA , Aïn Leuh; Séguy 1953a , HA , Ksar-es-Souk; Leclercq 1961b , MA , Dayat Aoua, Aïn Leuh, Immouzer Kander; Leclercq 1967 , AP , Kénitra (on Mimosa grove), Gharb (Sidi Yahia, Sidi Allal Tazi), MA , Adjir by Khenifra (on livestock), Leclercq 1968 , MA , Dayat Aoua; Leclercq and Maldès 1987 , HA , edges near river Tessaout, Kelaâ des Sraghna; Pârvu et al. 2006 , AP , Merja Zerga, MA , Kasba Tadla; El Haouari et al. 2014 , HA , Setti Fatma; MA (Allal Tazi) – MISR Tabanus barbarus Coquebert, 1804 Becker and Stein 1913 , Rif , Tanger; Leclercq 1961b Rif , Azib de Ketama; Chvála and Lyneborg 1970 , Rif , Tanger; Portillo 1982; Leclercq and Maldès 1987 , MA , marshes around Kasba Tadla; Popescu-Mirceni 2011 , AP , Merja Zerga Tabanus bifarius Loew, 1858 = Atylotus bifarius Loew, 1858, in Séguy 1930a : 83 Séguy 1930a , EM , Haute Moulouya Tabanus bovinus Linnaeus, 1758 Loew 1860 ; Séguy 1930a ; Leclercq 1967 ; Portillo 1982; Leclercq and Maldès 1987 ; Barták and Kubik 2005 , MA , M'Rirt; El Haouari and Kettani 2014 , Rif , Oued Khemis (Khemis Anjra), Oued Kelaâ (Akchour) Tabanus bromius Linnaeus, 1758 = Straba bromius Linnaeus, 1758, in Séguy 1930a : 83 Loew 1860 ; Séguy 1930a , EM , Haute Moulouya, MA , Aïn Leuh, Ras El Ksar; Leclercq 1961b , Rif , Azib de Ketama, MA , Ifrane, Immouzer, Azrou, Bordj Doumergue, Dayat Aoua, Aïn Leuh; Leclercq 1967 , MA , Immouzer des Marmoucha, Adjir by Khenifra; Leclercq 1968 , Rif , Ketama, MA , Bab-Bou-Idir, Bab Ferrich, Ifrane, Col du Zad; Pernot-Visentin and Beaucournu-Saguez 1974 , Rif ; Leclercq and Maldès 1987 ; El Haouari and Kettani 2014 , Rif , Oued Samsa, Oued Raouz, Oued Zarka, Oued Khemis (Khemis Anjra), Oued Azla (Mokdassen Oulya), Oued Kelaâ (Akchour), Oued Laou (Sifalaou), Oued Jnane Niche, Oued Berranda; El Haouari et al. 2014 , HA , Oulmès; AA (Errachidia) – MISR Tabanus choumarae Leclercq, 1967 Leclercq 1967 , AA , Aouinet Torkoz (down Draa); Leclercq and Olsufjev 1975 ; Leclercq and Maldès 1987 ; Pârvu et al. 2006 , AA , Ouarzazate Tabanus cordiger Meigen, 1820 Leclercq 1961b , MA , Bordj Doumergue, Dayat Aoua; Leclercq 1967 , Rif , Had el Rouadi, MA , Arbala par Ksiba, Boulemane, AA , Agadir-Tissint (Rocade du Draa), Akka; Leclercq 1968 , Rif , Ketama, MA , Bab Termas, Taza, Doniet; Leclercq 1986 ; Leclercq and Maldès 1987 , AA , Tinmal, edges of Draa river, Agdz, Ourika near Ouarzazate; Barták and Kubik 2005 ; Müller et al. 2011 ; Popescu-Mirceni 2011 , HA , Ouarzazate; El Haouari and Kettani 2014 , Rif , Oued Berranda; El Haouari et al. 2014 , Rif , Oued Oueghra Tabanus darimonti Leclercq, 1964 Leclercq 1967 , MA , Aïn Leuh; Leclercq 1968 , MA , Bab Boudir, forêt Bab-Azhar, Immouzer Kander; Portillo 1982; Leclercq and Maldès 1987 ; Mikuška et al. 2008 Tabanus eggeri Schiner, 1868 = Tabanus intermedius Egger, 1859, in Séguy 1930a : 83 Séguy 1930a , MA , M'Rirt; Leclercq 1968 , Rif , M'Diq, MA , Bab Boudir, El Hajeb, Azrou, Miscliffen; Portillo 1982; Leclercq and Maldès 1987 ; Mikuška et al. 2008 Tabanus leleani Austen, 1920 = Atylotus leleani Austen, 1920, in Séguy 1930a : 84 Séguy 1930a , HA , upstream of Réghaya; Leclercq 1967 ; Leclercq and Maldès 1987 ; MA – MISR Tabanus lunatus Fabricius, 1794 = Atylotus lunatus Fabricius, 1974, in Séguy 1930a : 84 Séguy 1930a , EM , Haute Moulouya, MA , Meknès; Leclercq 1961b , MA , Ifrane, Immouzer, Bordj Doumergue, Dayat Aoua, Aïn Leuh; Leclercq 1967 , EM , Haute Moulouya, MA , Ajdir by Khenifra (on livestock); Leclercq 1968 , Rif , Ketama, MA , Bab Bouder, Immouzer Kander, El Hajeb, Mishliffen, Jebel Hebri; Portillo 1982; Leclercq and Maldès 1987 , MA , Afourer (800 m) – MISR Tabanus maculicornis Zetterstedt, 1842 El Haouari and Kettani 2014 , Rif , Oued Rha, marshes of Lemtahane, Oued Boumarouil, Oued Laou (Dardara), Oued Kanar, Oued Jnane Niche; El Haouari et al. 2014 , HA , Imi-n'Tadart, Oulmès, Tafza Tabanus miki Brauer, 1880 El Haouari and Kettani 2014 , Rif , Oued Khemis, Oued Boumarouil, Oued Berranda, Oued Biyada; El Haouari et al. 2014 , HA , Setti Fatma Tabanus nemoralis Meigen, 1820 Leclercq 1968 , Rif , Ketama; Portillo 1982; Leclercq and Maldès 1987 Tabanus quatuornotatus Meigen, 1820 Portillo 1982; Leclercq and Maldès 1987 ; Koçak and Kemal 2010 ; Müller et al. 2011 ; El Haouari and Kettani 2014 , Rif , Oued Maggou, Oued Ouara (Khizana); El Haouari et al. 2014 , HA , Setti Fatma; Rif (Talassemtane) – MISR Tabanus sudeticus Zeller, 1842 Leclercq 1967 ; Portillo 1982; Leclercq and Maldès 1987 ; Gioia Martins-Neto 2003 Tabanus tinctus Walker, 1850 Leclercq 1961b , MA , forêt Bab Azhar, Aïn Leuh; Pernot-Visentin and Beaucournu-Saguez 1974 , MA , HA (2300 m); Portillo 1982; Leclercq and Maldès 1987 ; MA (Azrou) – MISR VERMILEONIDAE K. Kettani, M.J. Ebejer Number of species: 6 . Expected: 6 Faunistic knowledge of the family in Morocco: good Vermileoninae Lampromyia Macquart, 1835 Lampromyia cylindrica (Fabricius, 1794) Stuckenberg 1998 , HA Lampromyia lecerfi Séguy, 1928 = Lampromyia Le Cerfi Séguy, in Séguy 1928a : 45 Séguy 1928a , HA , Tinmel (Goundafa), Asni; Stuckenberg 1998 , HA ; Kirk-Spriggs and McGregor 2009 ; Kehlmaier 2014 , HA ; AP (Tamri) – MHNN Lampromyia nigripennis Séguy, 1930 Séguy 1930a , EM , Berkane, grove in Tlet n'Rhohr; Stuckenberg 1998 , MA ; Kirk-Spriggs and McGregor 2009 ; Kehlmaier 2014 , MA , south of Azrou (1500 m), HA Lampromyia pallida Macquart, 1835 Stuckenberg 1998 , HA Vermileo Macquart, 1834 Vermileo vermileo (Linnaeus, 1758) 13 = Vermileo degeeri Macquart 1834, in Séguy 1953a : 78 Séguy 1953a , AP , Rabat Vermileo nigriventris Strobl, 1906 Ebejer et al. 2019 , Rif , Cap Spartel (Tanger, 15 m), Anissar ( PNPB , 987 m) Vermileoninae Lampromyia Macquart, 1835 Lampromyia cylindrica (Fabricius, 1794) Stuckenberg 1998 , HA Lampromyia lecerfi Séguy, 1928 = Lampromyia Le Cerfi Séguy, in Séguy 1928a : 45 Séguy 1928a , HA , Tinmel (Goundafa), Asni; Stuckenberg 1998 , HA ; Kirk-Spriggs and McGregor 2009 ; Kehlmaier 2014 , HA ; AP (Tamri) – MHNN Lampromyia nigripennis Séguy, 1930 Séguy 1930a , EM , Berkane, grove in Tlet n'Rhohr; Stuckenberg 1998 , MA ; Kirk-Spriggs and McGregor 2009 ; Kehlmaier 2014 , MA , south of Azrou (1500 m), HA Lampromyia pallida Macquart, 1835 Stuckenberg 1998 , HA Vermileo Macquart, 1834 Vermileo vermileo (Linnaeus, 1758) 13 = Vermileo degeeri Macquart 1834, in Séguy 1953a : 78 Séguy 1953a , AP , Rabat Vermileo nigriventris Strobl, 1906 Ebejer et al. 2019 , Rif , Cap Spartel (Tanger, 15 m), Anissar ( PNPB , 987 m) Nemestrinoidea ACROCERIDAE K. Kettani, E.P. Nartshuk Number of species: 13 . Expected: 25 Faunistic knowledge of the family in Morocco: poor Acrocerinae Acrocera Meigen, 1803 Acrocera orbicula (Fabricius, 1787) Weinberg and Bächli 2002 , HA , Marrakech, Ouirgane (1000 m) Cyrtus Latreille, 1796 Cyrtus gibbus (Fabricius, 1794) Becker and Stein 1913 , Rif , Tanger; Séguy 1926 , Rif ; Pleske 1930 , Rif , Tanger; Schlinger 1972 ; Mouna 1998 Cyrtus maroccanus Séguy, 1930 Schlinger 1972 , Rif ; Mouna 1998 Cyrtus pallidus Gil Collado, 1929 Gil Collado 1929b , Rif , Tanger; Schlinger 1972 ; Mouna 1998 Cyrtus pusillus Macquart, 1834 Pleske 1930 , Rif , Tanger; Schlinger 1972 , Rif , Tanger; Mouna 1998 Ogcodes Latreille, 1797 Ogcodes zonatus (Erichson, 1840) Becker and Stein 1913 , Rif , Tanger; Séguy 1926 ; Pleske 1930 Opsebius Costa, 1856 Opsebius cyrtus Séguy, 1930 Mouna 1998 ; HA (Lac Ifni) – MISR Opsebius formosus Loew, 1871 Becker and Stein 1913 , Rif , Tanger Opsebius inclinatus Séguy, 1930 Mouna 1998 Opsebius inflatus (Loew, 1857) Weinberg and Bächli 2002 , MA , Azrou, Timahdit Ighboula (1850 m), HA , Marrakech, Ouirgane (1000 m) Opsebius pepo Loew, 1870 Pleske 1930 , Rif , Tanger Panopinae Astomella Latreille, 1809 Astomella hispaniae Lamarck, 1816 Gil Collado 1929b , AP , Mogador; Mouna 1998 Physegastrella Brunetti, 1926 Physegastrella maroccana Brunetti, 1926 Brunetti 1926 ; Pleske 1930 ; Mouna 1998 NEMESTRINIDAE K. Kettani, D. Barraclough Number of species: 13 Faunistic knowledge of the family in Morocco: poor Nemestrininae Nemestrinus Latreille, 1802 Nemestrinus aegyptiacus (Wiedemann, 1828) Timon-David 1951 ; Séguy 1953a , HA , Amsed; Bernardi 1973 ; Mouna 1998 ; SA (Oued el Ma) – MISR Nemestrinus ater (Olivier, 1810) Mouna 1998 ; EM (Zaouillet El Atenf) – MISR Nemestrinus escalerai Arias, 1913 Paramonow 1945; Bernardi 1973 , HA , Marrakech; Mouna 1998 Nemestrinus exalbidus (Lichtwardt, 1907) Mouna 1998 Nemestrinus fasciatus (Olivier, 1810) = Rhynchocephalus fasciatus Olivier, in Mouna 1998 : 86 Bernardi 1973 ; Mouna 1998 ; MA (Immouzer) – MISR Nemestrinus nigrovillosus Lichtwardt, 1909 Arias 1913 ; Séguy 1930a , MA , Ras el Ma, Azrou, Forêt Tiffert (2000–2200 m), HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Séguy 1941d , HA , Tizi-n'Test; Timon-David 1951 , MA , Tizi-n'Tretten; Bernardi 1973 ; Mouna 1998 Nemestrinus pieltaini (Gil Collado, 1934) = Nemestrellus pieltaini Gil Collado, in Gil Collado 1934 : 325 Gil Collado 1934 , Rif , Imasinen, Bab Chiquer, Bab Bagla; Bernardi 1973 ; Mouna 1998 Nemestrinus ruficornis (Macquart, 1840) Mouna 1998 Nemestrinus rufipes (Olivier, 1810) Mouna 1998 ; MA (Timahdit) – MISR Nemestrinus striatus (Lichtwardt, 1907) Mouna 1998 Trichopsideinae Fallenia Meigen, 1820 Fallenia fasciata (Fabricius, 1805) Arias 1913 ; Séguy 1930a , AP , Casablanca, Rabat, Bou Knadel, MA , M'Rirt (1200 m); Timon-David 1951 , AP , Forêt Maâmora; Mouna 1998 ; AP (Rabat, Bou Knadel), MA (Aïn Leuh) – MISR Neorhynchocephalus Lichtwardt, 1909 Neorhynchocephalus tauscheri (Fisher, 1812) = Rhynchocephalus tauscheri Fischer, in Mouna 1998 : 86 Mouna 1998 Trichopsidea Westwood, 1839 Trichopsidea costata (Loew, 1857) = Symmictus costatus Loew, in Arias 1913 : 26, Séguy 1930a : 89 Arias 1913 ; Séguy 1930a , MA , Tameghilt (1900 m); Mouna 1998 ACROCERIDAE K. Kettani, E.P. Nartshuk Number of species: 13 . Expected: 25 Faunistic knowledge of the family in Morocco: poor Acrocerinae Acrocera Meigen, 1803 Acrocera orbicula (Fabricius, 1787) Weinberg and Bächli 2002 , HA , Marrakech, Ouirgane (1000 m) Cyrtus Latreille, 1796 Cyrtus gibbus (Fabricius, 1794) Becker and Stein 1913 , Rif , Tanger; Séguy 1926 , Rif ; Pleske 1930 , Rif , Tanger; Schlinger 1972 ; Mouna 1998 Cyrtus maroccanus Séguy, 1930 Schlinger 1972 , Rif ; Mouna 1998 Cyrtus pallidus Gil Collado, 1929 Gil Collado 1929b , Rif , Tanger; Schlinger 1972 ; Mouna 1998 Cyrtus pusillus Macquart, 1834 Pleske 1930 , Rif , Tanger; Schlinger 1972 , Rif , Tanger; Mouna 1998 Ogcodes Latreille, 1797 Ogcodes zonatus (Erichson, 1840) Becker and Stein 1913 , Rif , Tanger; Séguy 1926 ; Pleske 1930 Opsebius Costa, 1856 Opsebius cyrtus Séguy, 1930 Mouna 1998 ; HA (Lac Ifni) – MISR Opsebius formosus Loew, 1871 Becker and Stein 1913 , Rif , Tanger Opsebius inclinatus Séguy, 1930 Mouna 1998 Opsebius inflatus (Loew, 1857) Weinberg and Bächli 2002 , MA , Azrou, Timahdit Ighboula (1850 m), HA , Marrakech, Ouirgane (1000 m) Opsebius pepo Loew, 1870 Pleske 1930 , Rif , Tanger Panopinae Astomella Latreille, 1809 Astomella hispaniae Lamarck, 1816 Gil Collado 1929b , AP , Mogador; Mouna 1998 Physegastrella Brunetti, 1926 Physegastrella maroccana Brunetti, 1926 Brunetti 1926 ; Pleske 1930 ; Mouna 1998 Acrocerinae Acrocera Meigen, 1803 Acrocera orbicula (Fabricius, 1787) Weinberg and Bächli 2002 , HA , Marrakech, Ouirgane (1000 m) Cyrtus Latreille, 1796 Cyrtus gibbus (Fabricius, 1794) Becker and Stein 1913 , Rif , Tanger; Séguy 1926 , Rif ; Pleske 1930 , Rif , Tanger; Schlinger 1972 ; Mouna 1998 Cyrtus maroccanus Séguy, 1930 Schlinger 1972 , Rif ; Mouna 1998 Cyrtus pallidus Gil Collado, 1929 Gil Collado 1929b , Rif , Tanger; Schlinger 1972 ; Mouna 1998 Cyrtus pusillus Macquart, 1834 Pleske 1930 , Rif , Tanger; Schlinger 1972 , Rif , Tanger; Mouna 1998 Ogcodes Latreille, 1797 Ogcodes zonatus (Erichson, 1840) Becker and Stein 1913 , Rif , Tanger; Séguy 1926 ; Pleske 1930 Opsebius Costa, 1856 Opsebius cyrtus Séguy, 1930 Mouna 1998 ; HA (Lac Ifni) – MISR Opsebius formosus Loew, 1871 Becker and Stein 1913 , Rif , Tanger Opsebius inclinatus Séguy, 1930 Mouna 1998 Opsebius inflatus (Loew, 1857) Weinberg and Bächli 2002 , MA , Azrou, Timahdit Ighboula (1850 m), HA , Marrakech, Ouirgane (1000 m) Opsebius pepo Loew, 1870 Pleske 1930 , Rif , Tanger Panopinae Astomella Latreille, 1809 Astomella hispaniae Lamarck, 1816 Gil Collado 1929b , AP , Mogador; Mouna 1998 Physegastrella Brunetti, 1926 Physegastrella maroccana Brunetti, 1926 Brunetti 1926 ; Pleske 1930 ; Mouna 1998 NEMESTRINIDAE K. Kettani, D. Barraclough Number of species: 13 Faunistic knowledge of the family in Morocco: poor Nemestrininae Nemestrinus Latreille, 1802 Nemestrinus aegyptiacus (Wiedemann, 1828) Timon-David 1951 ; Séguy 1953a , HA , Amsed; Bernardi 1973 ; Mouna 1998 ; SA (Oued el Ma) – MISR Nemestrinus ater (Olivier, 1810) Mouna 1998 ; EM (Zaouillet El Atenf) – MISR Nemestrinus escalerai Arias, 1913 Paramonow 1945; Bernardi 1973 , HA , Marrakech; Mouna 1998 Nemestrinus exalbidus (Lichtwardt, 1907) Mouna 1998 Nemestrinus fasciatus (Olivier, 1810) = Rhynchocephalus fasciatus Olivier, in Mouna 1998 : 86 Bernardi 1973 ; Mouna 1998 ; MA (Immouzer) – MISR Nemestrinus nigrovillosus Lichtwardt, 1909 Arias 1913 ; Séguy 1930a , MA , Ras el Ma, Azrou, Forêt Tiffert (2000–2200 m), HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Séguy 1941d , HA , Tizi-n'Test; Timon-David 1951 , MA , Tizi-n'Tretten; Bernardi 1973 ; Mouna 1998 Nemestrinus pieltaini (Gil Collado, 1934) = Nemestrellus pieltaini Gil Collado, in Gil Collado 1934 : 325 Gil Collado 1934 , Rif , Imasinen, Bab Chiquer, Bab Bagla; Bernardi 1973 ; Mouna 1998 Nemestrinus ruficornis (Macquart, 1840) Mouna 1998 Nemestrinus rufipes (Olivier, 1810) Mouna 1998 ; MA (Timahdit) – MISR Nemestrinus striatus (Lichtwardt, 1907) Mouna 1998 Trichopsideinae Fallenia Meigen, 1820 Fallenia fasciata (Fabricius, 1805) Arias 1913 ; Séguy 1930a , AP , Casablanca, Rabat, Bou Knadel, MA , M'Rirt (1200 m); Timon-David 1951 , AP , Forêt Maâmora; Mouna 1998 ; AP (Rabat, Bou Knadel), MA (Aïn Leuh) – MISR Neorhynchocephalus Lichtwardt, 1909 Neorhynchocephalus tauscheri (Fisher, 1812) = Rhynchocephalus tauscheri Fischer, in Mouna 1998 : 86 Mouna 1998 Trichopsidea Westwood, 1839 Trichopsidea costata (Loew, 1857) = Symmictus costatus Loew, in Arias 1913 : 26, Séguy 1930a : 89 Arias 1913 ; Séguy 1930a , MA , Tameghilt (1900 m); Mouna 1998 Nemestrininae Nemestrinus Latreille, 1802 Nemestrinus aegyptiacus (Wiedemann, 1828) Timon-David 1951 ; Séguy 1953a , HA , Amsed; Bernardi 1973 ; Mouna 1998 ; SA (Oued el Ma) – MISR Nemestrinus ater (Olivier, 1810) Mouna 1998 ; EM (Zaouillet El Atenf) – MISR Nemestrinus escalerai Arias, 1913 Paramonow 1945; Bernardi 1973 , HA , Marrakech; Mouna 1998 Nemestrinus exalbidus (Lichtwardt, 1907) Mouna 1998 Nemestrinus fasciatus (Olivier, 1810) = Rhynchocephalus fasciatus Olivier, in Mouna 1998 : 86 Bernardi 1973 ; Mouna 1998 ; MA (Immouzer) – MISR Nemestrinus nigrovillosus Lichtwardt, 1909 Arias 1913 ; Séguy 1930a , MA , Ras el Ma, Azrou, Forêt Tiffert (2000–2200 m), HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Séguy 1941d , HA , Tizi-n'Test; Timon-David 1951 , MA , Tizi-n'Tretten; Bernardi 1973 ; Mouna 1998 Nemestrinus pieltaini (Gil Collado, 1934) = Nemestrellus pieltaini Gil Collado, in Gil Collado 1934 : 325 Gil Collado 1934 , Rif , Imasinen, Bab Chiquer, Bab Bagla; Bernardi 1973 ; Mouna 1998 Nemestrinus ruficornis (Macquart, 1840) Mouna 1998 Nemestrinus rufipes (Olivier, 1810) Mouna 1998 ; MA (Timahdit) – MISR Nemestrinus striatus (Lichtwardt, 1907) Mouna 1998 Trichopsideinae Fallenia Meigen, 1820 Fallenia fasciata (Fabricius, 1805) Arias 1913 ; Séguy 1930a , AP , Casablanca, Rabat, Bou Knadel, MA , M'Rirt (1200 m); Timon-David 1951 , AP , Forêt Maâmora; Mouna 1998 ; AP (Rabat, Bou Knadel), MA (Aïn Leuh) – MISR Neorhynchocephalus Lichtwardt, 1909 Neorhynchocephalus tauscheri (Fisher, 1812) = Rhynchocephalus tauscheri Fischer, in Mouna 1998 : 86 Mouna 1998 Trichopsidea Westwood, 1839 Trichopsidea costata (Loew, 1857) = Symmictus costatus Loew, in Arias 1913 : 26, Séguy 1930a : 89 Arias 1913 ; Séguy 1930a , MA , Tameghilt (1900 m); Mouna 1998 Asiloidea ASILIDAE K. Kettani, G. Tomasovic Number of species: 131 . Expected: 230 Faunistic knowledge of the family in Morocco: moderate Apocleinae Apoclea Macquart, 1838 Apoclea algira (Linnaeus, 1767) Séguy 1953a , AA , Tata; Tomasovic 1997 ; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Apoclea micracantha Loew, 1856 Tomasovic 1997 , HA , Sidi Mhejmed Ou Said; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Eremonotus Theodor, 1980 Eremonotus hauseri Geller-Grimm & Hradský, 1998 Geller-Grimm and Hradský 1998, HA ; Geller-Grimm 2007, AA , Agadir Asilinae Afroepitriptus Lehr, 1992 Afroepitriptus beckeri Lehr, 1992 Geller-Grimm 2007; Koçak and Kemal 2010 Antiphrisson Loew, 1849 Antiphrisson trifarius Loew, 1849 Tomasovic 1997 , HA , Errachidia, Ziz, Oasis Zouala; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 ; HA (Asni) – MISR Asilus Linnaeus, 1758 Asilus barbarus Linnaeus, 1758 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ; Séguy 1941a , HA , Tizi-Tamatert (Toubkal, 2250 m); Mouna 1998 ; Weinberg and Blasco-Zumeta 2004 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Asilus crabroniformis Linnaeus, 1758 Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Asilus tingitanus Boisduval, 1835 Geller-Grimm 2007, Rif , Tanger Dysmachus Loew, 1860 Dysmachus albisetosus (Macquart, 1850) Geller-Grimm 2007 Dysmachus cochleatus (Loew, 1854) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dysmachus cristatus (Wiedemann, 1820) = Dysmachus dasynotus Loew, in Becker and Stein 1913 : 72, Timon-David 1951 : 138 Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Rabat, Harcha, Salé, Oued Ksab, MA , Ifrane; Mouna 1998 ; Geller-Grimm 2007; AP (Rabat, Cap Cantia) – MISR Dysmachus digitulus Becker, 1923 Geller-Grimm 2007 Dysmachus elapsus Villeneuve, 1933 Villeneuve 1933 , AP , Mazagan, Mogador; Mouna 1998 ; Tomasovic 2001b ; Geller-Grimm 2007; AP (Cap Cantia) – MISR Dysmachus evanescens Villeneuve, 1912 Timon-David 1951 , AP , Sehoul; Mouna 1998 ; Geller-Grimm 2007 Dysmachus trigonus (Meigen, 1804) Timon-David 1951 , AP , Rabat, Chellah, Forêt Maâmora, Ras el Arba, Sehoul, Zaër Eccoptopus Loew, 1860 Eccoptopus longitarsis (Macquart, 1838) Timon-David 1951 , AA , Zagora; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Engelepogon Lehr, 1992 Engelepogon brunnipes (Fabricius, 1794) = Heligmoneura brunnipes Fabricius, in Séguy 1930a : 125 = Acanthopleura brunnipes Fabricius, in Timon-David 1951 : 137 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Meknès; Timon-David 1951 , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Epitriptus Loew, 1849 Epitriptus cingulatus (Fabricius, 1871) Séguy 1941a , AA , Agadir; Mouna 1998 Eremisca Hull, 1962 Eremisca heleni heleni (Efflatoun, 1934) Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Eremisca osiris (Wiedemann, 1828) Geller-Grimm 2007; El Hawagry 2011 Eutolmus Loew, 1848 Eutolmus wahisi Tomasovic, 2001 Tomasovic 2001a , Rif , Tétouan (Jebel Tazout, 1650 m); Geller-Grimm 2007; Koçak and Kemal 2010 Filiolus Lehr, 1967 Filiolus apicalis (Becker in Becker & Stein, 1913) = Eutolmus apicalis Becker, in Becker and Stein 1913 : 75 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Machimus Loew, 1849 Machimus cribratus (Loew, 1849) Geller-Grimm 2007; AP (Cap Cantia) – MISR Machimus fimbriatus (Meigen, 1804) Geller-Grimm 2007 Machimus fortis (Loew, 1849) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat; Mouna 1998 ; Geller-Grimm 2007 Machimus gonatistes (Zeller, 1840) Geller-Grimm 2007 Machimus mauritanicus Bequaert, 1964 Tomasovic 2003 ; Geller-Grimm 2007; AP (Forêt Boulhaut, Salé) – MISR Machimus nigrosetosus Séguy, 1941 Séguy 1941d AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Machimus perplexus Becker, 1915 Geller-Grimm 2007 Machimus pilipes (Meigen, 1820) = Eutolmus hispanus Loew, in Becker and Stein 1913 : 74 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Forêt Zaers, Tizi-n'Bouftene (2300 m), HA , bords de l'Imminen (Tachdirt: 2400–2600 m); Geller-Grimm 2007 Machimus pseudogonatistes Villeneuve, 1930 = Machimus ermineus Becker, in Mouna 1998 : 84 Villeneuve 1933 ; Mouna 1998 ; Geller-Grimm 2007 Neoepitriptus Lehr, 1992 Neoepitriptus inconstans (Wiedemann in Meigen, 1820) = Machimus micropyga Becker, in Becker and Stein 1913 : 74 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 , Rif , Tanger Neoepitriptus minusculus (Bezzi, 1898) = Machimus minusculus Bezzi, in Timon-David 1951 : 138, Mouna 1998 : 84 Timon-David 1951 , MA , Ifrane; Mouna 1998 ; Geller-Grimm 2007 Neomochtherus Osten-Sacken, 1878 Neomochterus brevipennis Séguy, 1932 Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Neomochtherus grandicollis (Becker, 1914) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Neomochterus ochriventris (Loew, 1854) Timon-David 1951 , AP , Sidi Moussa el Harati; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Pashtshenkoa Lehr, 1995 Pashtshenkoa clypeatus maroccanus (Tsacas, 1968) Geller-Grimm 2007 Phileris Tsacas & Weinberg, 1976 Phileris haplopygus Tsacas & Weinberg, 1976 Geller-Grimm 2007 Phileris pilosus Tsacas & Weinberg, 1976 Geller-Grimm 2007 Satanas Jacobson, 1908 Satanas gigas (Eversmann, 1855) Maldès 2000 , ME , Oujda, HA , Errachidia, Meski Turka Őzdikmen, 2008 Turka cervinus (Loew, 1856) = Stenopogon cervinus Loew, in Séguy 1930a : 122 Séguy 1930a , MA , pont de l'Oued Korifla (Zaers), HA , Sidi Bou Rziguine; Geller-Grimm 2007; Hayat et al. 2008 ; Özdikmen 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Dasypogoninae Dasypogon Meigen, 1803 Dasypogon atratus (Fabricius, 1794) = Selidopogon atratus Meigen, in Séguy 1930a : 118 = Selidopogon atratus Fabricius, in Timon-David 1951 : 136 Séguy 1930a , MA ; Timon-David 1951 , Rif , Ouezzane, AP , Rabat MA , Oued Beth; Mouna 1998 ; Geller-Grimm 2007 Dasypogon auripilus (Séguy, 1934) Mouna 1998 ; Geller-Grimm 2007; AP (Casablanca) – MISR Dasypogon crassus Macquart in Lucas, 1849 = Selidopogon crassus Macquart, in Séguy 1930a : 119, Timon-David 1951 : 136 Séguy 1930a , Rif , Tanger, MA , Meknès; Timon-David 1951 , AP , M'Soun, Guerrouaou; Mouna 1998 ; Geller-Grimm 2007 Dasypogon diadema (Fabricius, 1781) = Selidopogon cylindricus Fabricius, in Séguy 1930a : 119 = Selidopogon diadema Fabricius, in Séguy 1930a : 118 = Selidopogon sicanus Costa, 1853, in Hayat et al. 2008 : 183 Séguy 1930a , AP , Dar Salem, Tarfaya, Oued Korifla (Zaers), HA , Bou Tazzert; Timon-David 1951 , AP , Port Lyautey; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Dasypogon gougeleti (Bigot, 1878) = Selidopogon gougeleti Bigot, in Timon-David 1951 : 136 Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Geller-Grimm 2007 Dasypogon olcesci (Bigot, 1878) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dasypogon rubinipes (Becker in Becker & Stein, 1913) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dasypogon ruficauda (Fabricius, 1805) Geller-Grimm 2007 Saropogon Loew, 1847 Saropogon aretalogus Séguy, 1953 Séguy 1953a , MA , Ifrane; Geller-Grimm 2007 Saropogon aurifrons (Macquart in Lucas, 1850) Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Saropogon clausus Becker, 1906 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , EM , Itzer, Moulay Aïn Djemine (Haute Moulouya); Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Saropogon jugulum (Loew, 1847) Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Saropogon leucocephalus (Meigen, 1820) Séguy 1930a , MA , Forêt Tiffert (2000–2200 m); Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 ; Ghahari et al. 2014 Saropogon maroccanus Séguy, 1930 Séguy 1930a , MA , Ras El Ksar (1900 m); Séguy 1949a , SA , Goulimine; Mouna 1998 ; Carles-Tolrá 2002 ; Geller-Grimm 2007 Saropogon obscuripennis (Macquart in Lucas, 1849) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat, MA , Aïn Leuh, Tizi-s'Tkrine (1700 m), HA , Imi-M'Tanout, Dar M'Tougui; Séguy 1941d , AA , Agadir; Timon-David 1951 , EM , Guenfouda; Mouna 1998 ; Geller-Grimm 2007 Saropogon philocalus Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Saropogon rufipes (Gimmerthal, 1847) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Saropogon tassilaensis Séguy, 1953 Séguy 1953a , AA , Tassila (Souss); Geller-Grimm 2007 Dioctriinae Dioctria Meigen, 1803 Dioctria atrorubens Séguy, 1930 Séguy 1930a , MA , Tizi-s'Tkine (1700 m); Villeneuve 1933 ; Mouna 1998 ; Geller-Grimm 2007 Dioctria cothurnata Meigen, 1820 Ebejer et al. 2019 , Rif , Dardara (484 m) Dioctria fuscipes Macquart, 1834 Timon-David 1951 , MA , Aguelmane Sidi Ali (2070 m) Dioctria gagates Wiedemann in Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dioctria notha Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Dioctria rufa Strobl, 1906 Ebejer et al. 2019 , Rif , Dardara (484 m) Dioctria rungsi Timon-David, 1951 Timon-David 1951 , MA , Ifrane (1650 m); Mouna 1998 ; Geller-Grimm 2007 Laphriinae Glyphotriclis Hermann, 1920 Glyphotriclis ornatus (Schiner, 1868) = Triclis ornatus Schiner, in Becker and Stein 1913 : 67 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Marrakech; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Laphria Meigen, 1803 Laphria bomboides Macquart, 1849 = Laphria praelusia Séguy, in Séguy 1930a : 124 Séguy 1930a , MA , Soufouloud (1900–2100 m); Mouna 1998 ; MA (Meghraona, Tamtraekt) – MISR Pogonosoma Rondani, 1856 Pogonosoma maroccanum (Fabricius, 1794) Loew 1860 ; Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Carles-Tolrá 2002 ; Geller-Grimm 2004 ; Geller-Grimm 2007; Ghahari et al. 2007 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013a ; Ghahari et al. 2014 Stiphrolamyra Engel, 1928 Stiphrolamyra rubicunda Oldroyd, 1947 Timon-David 1951 , AP , Sidi Moussa el Harati; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 ; Ghahari et al. 2014 Stiphrolamyra vitai Hradský & Geller-Grimm, 1997 Hradský and Geller-Grimm 1997 , HA , Taroudant; Geller-Grimm 2007 Laphystiinae Perasis Hermann, 1905 Perasis sareptana Hermann, 1906 Séguy 1930a , HA , Asni; Mouna 1998 Scytomedes Röder, 1882 Scytomedes haemorrhoidalis (Fabricius, 1794) = Triclis haemorrhoidalis Fabricius, in Mouna 1998 : 84 Séguy 1930a , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Trichardis Hermann, 1906 Trichardis leucocomus (Van der Wulp, 1899) = Trichardis leucicoma Van der Wulp, in Timon-David 1951 : 132 Timon-David 1951 , AA , Tata, piste de Fask Tahrjicht; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Leptogastrinae Leptogaster Meigen, 1803 Leptogaster cylindrica (De Geer, 1776) = Leptogaster hispanica Meigen, in Séguy 1930a : 117 Séguy 1930a , MA , Meknès; Mouna 1998 ; Tomasovic 2006 , Rif ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Leptogaster pedunculata Loew, 1847 = Gonypes pedunculatus Loew, in Becker and Stein 1913 : 72 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Haute Réghaya; Mouna 1998 Leptogaster straminea Becker, 1907 Timon-David 1951 , MA , Aguelmane Sidi Ali (2070 m); Mouna 1998 ; Geller-Grimm 2007 Stenopogoninae Afroholopogon Londt, 1994 Afroholopogon waltlii (Meigen, 1838) = Heteropogon waltlii Meigen, in Séguy 1930a : 123 Séguy 1930a , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Amphisbetetus Hermann, 1906 Amphisbetetus sexspinus Tomasovic, 2008 Tomasovic and Weyer 2008 , AA , Imsouane (Agadir); Geller-Grimm 2007 Ancylorhynchus Berthold in Latreille, 1827 Ancylorrhyncus gummigutta (Becker, 1906) Séguy 1930a , Rif , Tanger; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Ancylorrhyncus limbatus (Fabricius, 1794) Séguy 1930a , MA , Meknès, Timhadit (2000 m); Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Ancylorrhyncus vultur Séguy, 1930 Séguy 1930a , MA , Timhadit (2000 m); Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Eriopogon Loew, 1847 Eriopogon jubatus Becker, 1906 Timon-David 1951 , Rif , Tanger, AP , Rabat; Hradský and Hüttinger 1995 , AP , Rabat; Mouna 1998 ; Geller-Grimm 2007; AP (Forêt Temara) – MISR Eriopogon laniger Meigen, 1804 = Holopogon flavescens Jaennicke, in Séguy 1930a : 123 Séguy 1930a , HA , Aguergour; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Eriopogon spatenkai Hradský & Hüttinger, 1995 Geller-Grimm 2007; Hradský and Hüttinger 1995 , MA , Mishliffen Galactopogon Engel, 1929 Galactopogon hispidus Engel, 1929 Ebejer et al. 2019 , AA , 23 km S of Rich (Errachidia, 2012 m) Habropogon Loew, 1847 Habropogon aerivagus (Séguy, 1953) Séguy 1953a , SA , Aouletis Habropogon appendiculatus Schiner, 1867 Timon-David 1951 , AA , Aïn Chaïb; Mouna 1998 ; Weinberg and Blasco-Zumeta 2004 ; Hradský and Geller-Grimm 2005 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 Habropogon bacescui Weinberg & Tsacas, 1973 Geller-Grimm 2007; Koçak and Kemal 2010 Habropogon distipilosus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon hauseri Hradský & Geller-Grimm, 2005 Hradský and Geller-Grimm 2005 , HA , Tizi-n'Test; Geller-Grimm 2007; Koçak and Kemal 2010 Habropogon odontophallus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon parappendiculatus Weinberg & Tsacas, 1973 Hradský and Geller-Grimm 2005 , HA , Aït Saoun; Geller-Grimm 2007; Kirk-Spriggs and McGregor 2009 , HA ; Koçak and Kemal 2010 Habropogon prionophallus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon pyrrhophaeus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon rubriventris Macquart, 1849 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Aïn el Hadjar (near Mogador), MA , Meknès, Tlet n'Rhohr, EM , Berkane (1350–1400 m); Mouna 1998 Habropogon senilis Wulp, 1899 Geller-Grimm 2007 Habropogon spissipes Hermann, 1909 Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Habropogon striatus (Fabricius, 1794) = Habropogon heteroneurus Timon-David, in Timon-David: 135 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 , AP , Rabat Heteropogon Loew, 1847 Heteropogon biplex Becker, in Becker & Stein 1913: 65 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Heteropogon manicatus (Meigen, 1820) Séguy 1930a , MA , Azrou, Meknès, Aïn Leuh, HA , Asni; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; MA (Ifrane) – MISR Heteropogon nubilus (Meigen, 1820) = Isopogon brevis Schiner, in Becker and Stein 1913 : 64 = Sisyrnodytes brevis Macquart, in Timon-David 1951 : 134, Séguy 1953a : 79 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AA , Imiter; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Shoeibi and Karimpour 2010 ; Ghahari et al. 2014 Holopogon Loew, 1847 Holopogon dimidiatus (Meigen, 1820) Séguy 1941d , AA , Agadir Holopogon dusmeti Strobl in Czerny & Strobl 1909 = Eriopogon dusmeti Strobl, in Timon-David 1951 : 132 Timon-David 1951 , EM , Guenfouda, HA , Tifni; Mouna 1998 ; Geller-Grimm 2007; HA (Tifni Demnat) – MISR Holopogon melaleucus (Meigen, 1820) Séguy 1930a , AP , Forêt Maâmora, Dar Salem (Rabat); Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Holopogon pusillus (Macquart, 1838) = Habropogon pusillus (Macquart), in Séguy 1949a : 154 Séguy 1949a , SA , Goulimine; Mouna 1998 Holopogon quadrinotatus Séguy, 1953 Séguy 1953a , SA , Amguilli Sguelma Acnephalum Macquart, 1838 = Pycnopogon Loew, 1847 in Londt 2010 Acnephalum apiformis (Macquart in Lucas, 1849) Séguy 1930a , MA , Timhadit (2000 m), Meskedall (1800–1900 m); Timon-David 1951 , MA , Ifrane (1650 m); Mouna 1998 ; Geller-Grimm 2007 Acnephalum denudatus (Séguy, 1949) = Stenopogon denudatus Loew, in Séguy 1930a : 123, Séguy 1934b : 162 Séguy 1930a , MA , Tizi-n'Tkrine; Séguy 1934b , HA , Haute Réghaya; Séguy 1949b , AP , Bou Tazzert near Mogador; Séguy 1953a , AA , Oasis du Ferkla; Mouna 1998 ; Geller-Grimm 2007 Acnephalum fasciculatus (Loew, 1847) Séguy 1930a , MA , Azrou, Timelilt, Sidi Bettache, HA , Asni, bords Imminen (Tachdirt), Likount (2500–2800 m), Lac Ifni (Skoutana), SA , Béni Mgild; Timon-David 1951 , AP , Oued Korifla, MA , Lac Aguelmane Sidi Ali (2070 m), Oued N'Zala; Mouna 1998 ; Geller-Grimm 2007; Lehr et al. 2007 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 ; MA (Ras el Ma) – MISR Stenopogon Loew, 1847 Stenopogon costatus Loew, 1871 = Stenopogon costarus Loew, in Mouna 1998 : 84 Séguy 1930a , MA , Tizi-n'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 Stenopogon gracilis (Macquart, 1838) = Stenopogon fumipenis Becker, in Becker and Stein 1913 : 68 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Stenopogon heteroneurus (Macquart, 1838) Timon-David 1951 , AP , Forêt Maâmora, Oued Akreuch, HA , Mouldikht; Mouna 1998 ; Hayat et al. 2008 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Stenopogon iphippus Séguy, 1932 Séguy 1932b , MA , Volubilis; Mouna 1998 ; Geller-Grimm 2007 Stenopogon iphis Séguy, 1932 Séguy 1932b , MA , Azrou; Timon-David 1951 , Rif , Plateau de Tisserouine (2000 m), MA , Ifrane (1650 m), Ito (Rabat); Geller-Grimm 2007 Stenopogon ischyrus Séguy, 1932 Séguy 1932b , MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 ; Geller-Grimm 2007 Stenopogon junceus (Wiedemann in Meigen, 1820) Timon-David 1951 , AP , Oued Akreuch, Zaër, MA , Sefrou; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Stenopogon kocheri Timon-David, 1951 Timon-David 1951 , HA , Tifni; Mouna 1998 ; Geller-Grimm 2007 Stenopogon porcus Loew, 1871 Séguy 1949a , AA , Akka; Mouna 1998 ; Geller-Grimm 2007 Stenopogon werneri Engel, 1933 Geller-Grimm 2007; Koçak and Kemal 2010 , MA , Fès, Zalagh Sisyrnodytes Loew, 1856 Sisyrnodytes leucophaetus Séguy, 1930 Séguy 1930a , MA , Béni Berberi; Mouna 1998 ; Geller-Grimm 2007; Londt 2009 Sisyrnodytes nilicola (Rondani, 1850) Oldroyd 1980 ; Londt 1987; Geller-Grimm 2007; Londt 2009 , AA , Ifni, Tiznit, Tata, Tazegzout; El Hawagry 2011 ; Samin et al. 2011 ; Ghahari et al. 2014 Stichopogoninae Stichopogon Loew, 1847 Stichopogon albellus Loew, 1856 Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 Stichopogon albofasciatus (Meigen, 1820) Séguy 1930a , HA , Kasba Taguendaft (Goundafa); Mouna 1998 ; Geller-Grimm 2007 Stichopogon elegantulus (Wiedemann, 1820) Séguy 1930a , HA , Kasba Taguendaft (Goundafa), Aguerd el Had, AA , Talekjount (Souss); Mouna 1998 ; Geller-Grimm 2007; Ghahari et al. 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 ; AP (Kénitra) – MISR Stichopogon inaequalis Loew, 1847 Séguy 1930a , HA , Aguerd el Had, AA , Talekjount (Souss); Mouna 1998 ; Geller-Grimm 2007 Stichopogon maroccanus (Becker, 1914) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007; El Hawagry 2011 , Rif , Tanger Stichopogon punctiferus Bigot, 1878 Geller-Grimm 2007 Stichopogon pusio (Macquart in Lucas, 1849) Séguy 1930a , HA , Kasba Taguendaft (Goundafa); Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Stichopogon schineri Koch, 1872 Timon-David 1951 , AA , Backkoum (Jebel Siroua); Mouna 1998 ; Hayat et al. 2008 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 BOMBYLIIDAE K. Kettani, M.J. Ebejer, J. Dils Number of species: 248 . Expected: 270 Faunistic knowledge of the family in Morocco: good Usiinae Apolysis Loew, 1860 Apolysis eremophila Loew, 1873 = Usia tomentosa Engel, in Paramonow 1947: 209 = Parageron ornata Engel, in Zaitzev 2007 : 160 Paramonow 1947; Mouna 1998 ; Zaitzev 2007 , AP (south), Tamri; Koçak and Kemal 2010 ; El Hawagri 2011; Evenhuis and Greathead 2015 Parageron Paramonov, 1929 Parageron gratus (Loew, 1856) = Usia grata Loew, in Timon-David 1951 : 143 Timon-David 1951 , AP , Oued Grou; Mouna 1998 ; Bader and Arabyat 2004 ; Zaitzev 2007 , SA ; Koçak and Kemal 2010 ; El Hawagri 2011; Evenhuis and Greathead 2015 – MISR Parageron griseus Paramonov, 1947 Zaitzev 2007 , SA Parageron hyalipennis (Séguy, 1941) = Oligodranes hyalipennis Séguy, in Séguy 1941d : 9 Séguy 1941d , AA , Agadir (Forêt Admine); Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , HA Parageron incisus (Wiedemann, 1830) = Usia incisa Wiedemann, in Timon-David 1951 : 143 Séguy 1930a , AP , Mogador, Casablanca, Sidi Bettache, Aïn Sferguila, MA , Forêt Zaers, Ras el Ma, Aïn Leuh, HA , Tenfecht; Timon-David 1951 , AP , Rabat, Sehoul, MA , Ifrane; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Parageron major Macquart, 1840 Mouna 1998 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 , AP , Rabat, Nkheila; Evenhuis and Greathead 2015 Usia Latreille, 1802 Usia ( Micrusia ) aurata (Fabricius, 1794) = Usia ( Micrusia ) taeniolata Costa, 1883, in, Koçak and Kemal 2010 Séguy 1930a , Rif , Tanger, AP , Rabat, Sidi Bettache; Paramonov 1950 , Rif , Tanger; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) crispa Gibbs, 2011 Gibbs 2011 , HA , Marrakech, AA , Agadir, Taroudant, Tafraoute, Ouarzazate Usia ( Micrusia ) cryptocrispa Gibbs, 2011 Gibbs 2011 , AP , Ben Slimane, Rabat Usia ( Micrusia ) dilsi Gibbs, 2011 Gibbs 2011 , Rif , Tétouan, Al Hoceima, HA , Taourirt Usia ( Micrusia ) echinus Gibbs, 2011 Gibbs 2011 , AP , Agadir, Guelmim, Sidi Ifni, Cap Ghir, MA , Tafraoute, HA , Marrakech, Tizi-n-Test, AA , Tafingoult Usia ( Micrusia ) falcata Gibbs, 2011 Gibbs 2011 , MA , Azrou, Ifrane Usia ( Micrusia ) forcipata Brullé, 1833 14 Mouna 1998 : 84 Usia ( Micrusia ) globicauda Gibbs, 2011 Gibbs 2011 , AP , Essaouira Usia ( Micrusia ) loewi Becker, 1906 Zaitzev 2007 , Rif , Tanger, AP , Skhirate, Nkheila, MA , Taferiate, HA , Taourirt Usia ( Micrusia ) novakii Strobl, 1902 Séguy 1941d , HA , Tizi-n'Test; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) parascripa Gibbs, 2011 Gibbs 2011 , MA , Ifrane, Mischliffen (2200 m) Usia ( Micrusia ) pusilla Meigen, 1820 Séguy 1930a , AP , Rabat, MA , Azrou, HA , Tafingoult (Goundafa, 1500–1600 m); Séguy 1934b , AP , Rabat (on Calendula ); Séguy 1953a , AP , Cap Ghir; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) versicolor (Fabricius, 1787) Séguy 1930a , AP , Berrchid, Casablanca, M'Rassine; Timon-David 1951 , AP , Rabat, MA , Oulmès; Mouna 1998 ; Koçak and Kemal 2010 ; Gibbs 2011 , HA ; Evenhuis and Greathead 2015 ; EM (Oujda) – MNHNR Usia ( Usia ) aenea (Rossi, 1794) Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 ; Bader and Arabyat 2004 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Usia ) angustifrons Becker, 1906 Mouna 1998 Usia ( Usia ) atrata (Fabricius, 1798) = Voluccella atrata Fabricius, in Fabricius 1798: 570 = Usia claripennis Macquart, in Macquart 1840: 105 Meigen 1820 ; Séguy 1930a , Rif , Tanger, MA , Aïn Leuh; Timon-David 1951 , AP , Rabat, Guerrouaou; Mouna 1998 ; Zaitzev 2007 , HA , Tizi-n'Tichka; Koçak and Kemal 2010 ; Gibbs 2014 , AP , Mogador, Arbaa-Sahel (320 m), Tamri (215 m), MA , Khemisset, Oulmès (700 m), Ifrane, El Merabtine, HA , Marrakech, Aït Ourirr (530 m), Oukaimeden (2200 m), Tizi-n'Test (1450 m), Timzit (1700 m), AA , Agadir, Tiznit, Igherm (1660 m), Taroudant, Tata, Iguiour (1260 m), SA , Bou Jarif, Goulimine; Evenhuis and Greathead 2015 Usia ( Usia ) bicolor Macquart, 1855 15 Mouna 1998 : 84 Usia ( Usia ) cornigera Gibbs, 2014 Gibbs 2014 , Rif , Tanger, AP , Sidi Bettache, Rabat, MA , Meknès (550 m), Aïn Leuh (1350 m), HA , Dar Kaid M'tougui Usia ( Usia ) florea (Fabricius, 1794) = Volucella florea Fabricius, in Becker 1906: 203 = Usia cuprea Macquart, 1834, in Becker 1906: 203 Becker 1906a ; Séguy 1930a , Rif , Tanger, AP , Mogador, Sidi Bettache, HA , Tinmel (Goundafa), around (Skoutana); Timon-David 1951 , EM , Oued Moulouya; Mouna 1998 ; Pârvu and Zaharia 2007 ; Koçak and Kemal 2010 ; Gibbs 2014 ; Evenhuis and Greathead 2015 Usia ( Usia ) ignorata Becker, 1906 Becker 1906a ; Mouna 1998 ; Bader and Arabyat 2004 ; Pârvu and Zaharia 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 Usia ( Usia ) maghrebensis Gibbs, 2014 Gibbs 2014 , Rif , Tanger, Tétouan, El Biutz (150 m), AP , Mogador, MA , Aïn Leuh (1350 m) Usia ( Usia ) vestita Macquart, 1846 Mouna 1998 : 84; Gibbs 2011 Phthiriinae Phthiria Meigen, 1802 Phthiria albogilva Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Evenhuis and Greathead 2015 Phthiria gaedii Wiedemann in Meigen, 1820 Séguy 1930a , MA , Foum Keneg; Timon-David 1951 , AP , Zaers, MA , Ifrane; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 – MISR ( MA , Ifrane) Phthiria maroccana Zaitzev, 2005 = Phthiria maroccana Zaitzev, in Zaitzev 2005 : 667 Zaitzev 2005 , MA , Taferiate, HA , Taourirt; Zaitzev 2007 , MA , Taferiate, HA , Taourirt Phthiria merlei Zaitzev, 2005 = Phthiria merlei Zaitzev, in Zaitzev 2005 : 665 Zaitzev 2005 , AP (south), Tamri, Inchaden (south of Aït Melloul); Zaitzev 2007 , AP (south), Tamri, Inchaden (south of Aït Melloul) Phthiria minuta (Fabricius, 1805) Séguy 1930a , HA , Tenfecht, AA , Souss; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 Phthiria pulicaria var. flavofasciata Strobl in Morge, 1976 Mouna 1998 ; Zaitzev 2007 , AA , Tizi-n'Tiniggigt (1600 m); Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Phthiria scutellaris Wiedemann in Meigen, 1820 Séguy 1930a , MA , Meknès; Séguy 1941a , MA , Meknès, HA , Imi-n'Ouaka (1500 m); Mouna 1998 ; Evenhuis and Greathead 2015 ; Rif (Sapinière Talassemtane) – MISR Phthiria simonyi Becker, 1908 Séguy 1949a , SA , Guelmim; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , MA , Meknès Phthiria umbripennis Loew, 1846 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , MA , Meknès Phthiria vagans Loew, 1846 Zaitzev 2007 , HA , Taourirt, AA , Tizi-n'Taratine Toxophorinae Geron Meigen, 1820 Geron intonsus Bezzi, 1925* MA , HA Geron macquarti Greathead in Evenhuis & Greathead 1999 Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 Geron subflavofemoratus Andréu Rubio, 1959 Andréu Rubio 1959 ; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Toxophora Meigen, 1803 Toxophora fasciculata (Villers, 1789) Séguy 1930a , AP , Rabat; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Toxophora fuscipennis (Macquart, 1840) Mouna 1998 : 84 Toxophora pauli Zaitzev, 2005 Zaitzev 2005 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate); Zaitzev 2007 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) Toxophora shelkovnikovi Paramonov, 1933 Zaitzev 2007 , AA , Ouarzazate Heterotropinae Heterotropus Loew, 1873 Heterotropus atlanticus Séguy, 1930 Séguy 1930a , AP , Mogador; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Heterotropus longitarsus Séguy, 1930 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Heterotropus maroccanus Zaitzev, 2003 Zaitzev 2003 , AA , Jebel Tighermine (SE of Ouarzazate); Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Bombyliinae Anastoechus Osten-Sacken, 1877 Anastoechus bahirae Becker, 1915 Mouna 1998 ; Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Anastoechus hyrcanus Pallas & Wiedemann in Wiedemann, 1818 Mouna 1998 : 84 Anastoechus latifrons (Macquart, 1839) Timon-David 1951 , AP , Dradek; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Anastoechus nitidulus ssp. nitidulus Fabricius, 1794 Mouna 1998 : 84 Anastoechus stramineus Wiedemann in Meigen, 1820 Mouna 1998 : 84 Anastoechus trisignatus (Portschinsky, 1881) Bader and Arabyat 2004 ; Ziatzev 2007, AP , Rabat, AA , Jebel Tighermine (SE of Ouarzazate), AA , 20 km SW Goulmima, SA , Tan-Tan, Between Guelmim and Tan-Tan (90 km from Guelmim), Taganint (south of Bou-Izakarn); Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombomyia Greathead, 1995 Bombomyia discoidea (Fabricius, 1794) Séguy 1930a , AP , Oued Korifla (Zaers), Sidi Bettache; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombomyia stictica Boisduval, 1835 Zaitzev 2007 , MA , col de Zeggota (N Meknès), Oulmès Bombomyia vertebralis (Dufour, 1833) = Bombylius punctatus Fabricius, in Timon-David 1951 : 144 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a AP , Dradek near Rabat; Timon-David 1951 , MA , Volubilis; Mouna 1998 ; Bader and Arabyat 2004 ; Koçak and Kemal 2010 Evenhuis and Greathead 2015 ; AP (Dradek, Casablanca), EM (Oujda), MA (Volubilis, Aïn Leuh) – MISR Bombylisoma Rondani, 1856 Bombylisoma algirum (Macquart, 1840) = Bombylius nigrifrons Becker, in Becker and Stein 1913 : 83 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylisoma breviusculum (Loew, 1855) Dils and Özbek 2006 ; Zaitzev 2007 ; Evenhuis and Greathead 2015 Bombylisoma flavibarbum Loew, 1855 Mouna 1998 : 84 Bombylisoma melanocephalum Fabricius, 1794 Zaitzev 2007 , HA , Taourirt, south of Tizi-n'Test Bombylius Linnaeus, 1758 Bombylius ( Bombylius ) albaminis Séguy, 1949 Séguy 1949a , HA , Alnif; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) ambustus Pallas & Wiedemann, 1818 Mouna 1998 : 84 Bombylius ( Bombylius ) analis (Olivier, 1789) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Oued Korifla, Rabat, Sidi Bettache, Aïn Sferguila; Timon-David 1951 , AP , Rabat; Mouna 1998 ; Zaitzev 2007 , MA , route Fès-Sidi Kacem (30 km from Fès); Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , AP (Rabat, Casablanca) – MISR Bombylius ( Bombylius ) audcenti Bowden, 1984 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) canescens Mikan, 1796 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , Rif , Cap Malabata (Tanger), MA , Tachguelt, route Fès-Sidi Kacem (30 km from Fès) Bombylius ( Bombylius ) cinerascens Mikan, 1796 Mouna 1998 ; Bader and Arabyat 2004 Bombylius ( Bombylius ) discolor Mikan, 1796 Mouna 1998 ; Zaitzev 2007 , MA , route El Hajeb-Ifrane (1 km from Ifrane) Bombylius ( Bombylius ) eploceus Séguy, 1949 Séguy 1949a , SA , Guelmim; Mouna 1998 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) fimbriatus Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, Jebel Ahmar (1700 m); Mouna 1998 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) flavipes Wiedemann, 1828 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) fulvescens Wiedemann in Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AP , Cap Ghir; Séguy 1941d , AA , Agadir; Mouna 1998 Bombylius ( Bombylius ) fuscus Fabricius, 1781 Mouna 1998 : 84 Bombylius ( Bombylius ) major (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , MA , Oulmès; Bader and Arabyat 2004 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Kénitra) – MISR Bombylius ( Bombylius ) mauritanus Olivier, 1789 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , HA Bombylius ( Bombylius ) medius (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Sehoul; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Oued Yquem, Dradek) – MISR Bombylius (Unplaced) megacephalus Portschinsky, 1887* EM , AA Bombylius ( Bombylius ) minor Linnaeus, 1758 Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; MA (Aguelmane Azigza), SA – MISR Bombylius ( Bombylius ) mus Bigot, 1862 Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) niveus Meigen, 1804 Mouna 1998 : 84; AP (Mogador) – MISR Bombylius ( Bombylius ) numidus Macquart, 1846 Séguy 1953a , MA , Ifrane; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) pauli Zaitzev, 2003 Zaitzev 2003 , MA , route Fès-Sidi Kacem (30 km from Fès); Zaitzev 2007 , MA , route Fès-Sidi Kacem (30 km from Fès) Bombylius ( Bombylius ) posticus (Fabricius, 1805) Dils and Özbek 2006 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) postversicolor Evenhuis & Greathead, 1999 = Bombylius versicolor Fabricius, 1805 Meigen 1820 ; Bezzi 1906 ; Séguy 1930a , AP , Mogador; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) pumilus Meigen, 1820 Mouna 1998 : 84 Bombylius ( Bombylius ) semifuscus (Meigen, 1820) Séguy 1953a , AP , Cap Ghir; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) torquatus Loew, 1855 Séguy 1930a , HA , Ouaounzert (Glaoua), Arround (Skoutana), Tachdirt (bord de l'Imminen, 2400–2600 m); Timon-David 1951 , AP , Rabat; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Mogador) – MISR Bombylius ( Bombylius ) undatus Mikan, 1796 Pârvu and Zaharia 2007 Bombylius ( Bombylius ) vagans Meigen, 1830 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) venosus Mikan, 1796 Mouna 1998 ; Zaitzev 2007 , AP , El Koudia (30 km SW from Rabat); AP (Dradek) – MISR Bombylius ( Zephyrectes ) cruciatus Fabricius, 1798 Séguy 1930a , MA , Aharmoumou (1100 m), Azrou, Ras el Ma, HA , Tizi-n'Test, Jebel Imdress (Goundafa, 2000–2450 m); Mouna 1998 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 ; Rif (Talassemtane), AP (Mogador), MA (Sefrou) – MISR Bombylius ( Zephyrectes ) leucopygus (Macquart, 1846) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , AP , Larache, MA , Moulay Idris (900 m); Evenhuis and Greathead 2015 , SA , Erfoud; MA (Ifrane) – MISR Conophorus Meigen, 1803 Conophorus bellus Becker, 1906* HA Conophorus fuliginosus (Wiedemann in Meigen, 1820) = Ploas fuliginisa (Meigen), in Séguy 1953a : 83 Séguy 1930a , MA , Aharmoumou (1100 m); Séguy 1953a , MA , Ahermoumou (1100 m); Timon-David 1951 , AP , Dradek, MA , Sefrou, HA , Marrakech; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; Evenhuis and Greathead 2015 ; AP (Salé, Mogador) – MISR Conophorus fuscipennis (Macquart, 1840) Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Mouna 1998 ; Evenhuis and Greathead 2015 Conophorus griseus (Fabricius, 1787) Mouna 1998 ; Zaitzev 2007 ; Evenhuis and Greathead 2015 Conophorus hamilkar Paramonov, 1929 Timon-David 1951 , AP , Mogador; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Mogador) – MISR Conophorus macroglossus (Dufour, 1852) Mouna 1998 ; Zaitzev 2007 ; Evenhuis and Greathead 201; AP (Mogador) – MISR Conophorus mauritanicus Bigot, 1892 = Conophorus heteropilosus Timon-David, in Timon-David 1951 : 141; Mouna 1998 : 84; Evenhuis and Greathead 2015 : 192 Timon-David 1951 , MA , Oulmès; Mouna 1998 ; Zaitzev 2007 , AP , El Koudia (30 km SW from Rabat), Forêt Zaer (35 km SW from Rabat), N Tretten; Koçak and Kemal 2010 ; Dils 2013 , MA , Mrirt; Evenhuis and Greathead 2015 Conophorus rossicus Paramonow, 1929 Dils and Özbek 2006 Dischistus Loew, 1855 Dischistus albatus (Séguy, 1934) = Acanthogeron albatus Séguy, 1934, in Séguy 1934d : 73; Zaitzev 2007 : 162 Séguy 1934d ; Zaitzev 2007 , SA , 30 km S Tata Dischistus auripilus (Séguy, 1930) = Acanthogeron auripilus Séguy, 1930, in Séguy 1930a : 104 Séguy 1930a , AP , Mogador; Séguy 1934b , AP , Zaers; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Evenhuis and Greathead 2015 Dischistus maroccanus (Séguy, 1930) = Acanthogeron maroccanus Séguy, 1930, in Séguy 1930a : 106 Séguy 1930a , AP , Mogador; Mouna 1998 ; Zaitzev 2007 , HA , Tazzarine; Evenhuis and Greathead 2015 Dischistus mittrei (Séguy, 1930) = Acanthogeron mittrei Séguy, in Séguy 1930a : 105 Séguy 1930a , AP , Mogador; Mouna 1998 ; Evenhuis and Greathead 2015 Dischistus perniveus (Bezzi, 1925) = Acanthogeron perniveus Bezzi, in Timon-David 1951 : 143 Timon-David 1951 , AP , Djamda de M'Tal; Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Dischistus senex (Wiedemann in Meigen, 1820) = Acanthogeron senex Meigen, 1820, in Séguy 1953a : 83, Mouna 1998 : 84, Zaitzev 2007 : 162 Séguy 1930a , HA , Tafingoult (Goundafa, 1500–1600 m); Villeneuve 1933 ; Séguy 1953a , HA , Aït Ourir; Mouna 1998 ; Zaitzev 2003 , HA , Taourirt; Zaitzev 2007 , HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Dradek), MA (Sefrou) – MISR Dischistus separatus (Becker, 1906) = Acanthogeron talboti Séguy, 1930, in Séguy 1930a : 106 Evenhuis and Greathead 2015 Efflatounia Bezzi, 1925 Efflatounia berbera Bowden, 1973 Ebejer et al. 2019 , AA , Agadir – NMWC Legnotomyia Bezzi, 1902 Legnotomyia fascipennis Bezzi, 1925* SA Merleus Zaitzev, 2003 Merleus punctipennis Zaitzev, 2003 = Merleus punctipennis Zaitzev 2003 : 599 Zaitzev 2003 , AP , Skhirate; Zaitzev 2007 , AP , Skhirate Prorachthes Loew, 1869 Prorachthes crassipalpis Villeneuve, 1930 Evenhuis and Greathead 2015 Systoechus Loew, 1855 Systoechus ctenopterus (Mikan, 1796) Timon-David 1951 , MA , Ifrane; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2007 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Systoechus gomezmenori Andréu Rubio, 1959 Carles-Tolrá 2002 ; Evenhuis and Greathead 2015 Systoechus gradatus (Wiedemann in Meigen, 1820) Timon-David 1951 , AP , Mouldikht; Mouna 1998 ; Zaitzev 2007 , MA , Taferiat, HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 Systoechus mixtus Wiedemann, 1821 = Bombylius stylicornis Macquart in Séguy 1941: 10 Séguy 1941d , AA , Agadir; Mouna 1998 Systoechus pumilio Becker, 1915 Mouna 1998 : 84 Triplasius Loew, 1855 Triplasius boghariensis (Lucas, 1852) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , EM , Oujda; Mouna 1998 ; Pârvu and Zaharia 2007 ; Evenhuis and Greathead 2015 Triplasius maculipennis (Macquart, 1846) = Bombylius maculipennis var. melanopus Timon-David, in Timon-David 1951 : 144 = Bombylius ( Triplasius ) maculipennis Macquart, 1849, in Zaitzev 2007 : 166 Timon-David 1951 , MA , Azrou; Zaitzev 2007 , MA , route El Hachef, Criosement route Raubei Idris-Merhassine; Evenhuis and Greathead 2015 Ecliminae Eclimus Loew, 1844 Eclimus gracilis Loew, 1844 Séguy 1930a , MA , Ras el Ma; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2007 , MA , Maaziz; Evenhuis and Greathead 2015 Thevenetimyia Bigot, 1875 Thevenetimyia quedenfeldti (Engel, 1885)* Rif , AP , MA Crocidiinae Crocidium Loew, 1860 Crocidium aegyptiacum Bezzi, 1925* SA Crocidium nudum Efflatoun, 1945* EM , AA Semiramis Becker in Becker and Stein 1914 Semiramis punctipennis Becker, 1914 Zaitzev 2007 , AA , Aoulouz Cythereinae Amictus Wiedemann, 1817 Amictus castaneus (Macquart, 1840) Séguy 1930a , AP , Rabat, HA , Ank el Djemal; Mouna 1998 ; Evenhuis and Greathead 2015 Amictus compressus (Fabricius, 1805) Evenhuis and Greathead 2015 Amictus heteropterus Macquart, 1838 Zaitzev 2007 , Rif , Tanger, AP , Rabat, HA , S Tizi-n'Test, AA , Tizi-n'Taratine Amictus oblongus (Fabricius, 1805) = Bombylius oblongus Fabricius, in Macquart 1834: 390 Macquart 1834 Amictus pulchellus Macquart, 1846 Séguy 1930a , AP , Rabat, Maâmora; Mouna 1998 ; Zaitzev 2007 , HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Amictus setosus Loew, 1869* AP Amictus tener Becker, 1906 Zaitzev 2007 , AP , Rabat Amictus validus Loew, 1869 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Amictus variegatus Meigen in Waltl, 1835 Mouna 1998 : 84 Chalcochiton Loew, 1844 Chalcochiton argentifrons (Macquart in Lucas, 1849) Séguy 1953a , AP , Cap Ghir, Salé, Sidi Battache, MA , Tizi-n'Bou Zabal (2300 m), AA , Aïn Chaïb (Souss); Evenhuis and Greathead 2015 Chalcochiton argyrocephalus (Macquart, 1840) = Chalcochiton ( Anthrax ) argyrocephala (Macquart), in Engel 1938 : 328 Engel 1938 ; Séguy 1953a , AA , Agadir; El Hawagry 2011 ; Evenhuis and Greathead 2015 Chalcochiton atlantica Dils, 2008 Dils 2008 , SA , Guelmim Chalcochiton holosericeus (Fabricius, 1794) = Chalcochiton semiargentaea Macquart, in Zaitsev 2007: 172 Séguy 1930a , AP , Maâmora, Sidi Bettache, HA , Tizi-n'Test, Jebal Imdress (2000–2450 m), Tafingoult (Goundafa, 1500–1600 m); Séguy 1941d ; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger, AP , Skhirate, EM , Taourirt, MA , Taferiat, Meknès-Moulay Idriss, Merhassine, AA , Agadir, Ouarzazate; Evenhuis and Greathead 2015 ; AP (Salé, Forêt Temara, Oued Yquem, Meshra) – MISR Chalcochiton maghrebi Dils, 2017 Dils 2017 , Rif , Souk El Kolla, Bab Taza, 10 km S of Mjara, AP , Sidi Bettache, Temsia, Imsouane, Mansouria, Rommani, Béni Slimane, Tioulit, EM , El Aioun, MA , Béni Mellal, el Ksiba, 10 km SE Bir Tamtam, Merchouch, Mrirt, Fès, HA , Azilal, Asni, Tizi-Mlil, AA , Taroudant, Tizi-n'Test, Tiznit, Agouim, Sidi Ifni, Mesti, Tafinegoult, Tizi-n'Tinififft, El Mrabtine, SA , Semara Chalcochiton maroccanus Zaitzev, 2006 Séguy 1953a , HA , Tafingoult (Goundafa, 1500–1600 m); Zaitzev 2006 , AP (south), Aït Melloul; Zaitzev 2007 , AP (south), Aït Melloul Chalcochiton merlei Zaitzev, 2006 Zaitzev 2006 , AP , Skhirate; Zaitzev 2007 , AP , Skhirate Chalcochiton pallasii Loew, 1856 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2007 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Callostoma Macquart, 1840 Callostoma fascipenne Macquart, 1840 Bader and Arabyat 2004 Cyllenia Latreille, 1802 Cyllenia rustica Rossi, 1790 Mouna 1998 ; Zaitzev 2007 ; AP (Mogador) – MISR Cytherea Fabricius, 1794 Cytherea albolineata Bezzi, 1925* SA Cytherea alexandrina Becker, 1902 Becker 1902 : 30; Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Cytherea aurea (Fabricius, 1794) Séguy 1930a , AP , Rabat, HA ; Mouna 1998 ; Bader and Arabyat 2004 ; Zaitzev 2007 , AA , Tizi-n'Taratine; El Hawagry 2011 ; Evenhuis and Greathead 2015 , HA , Tafingoult (Goundafa, 1500–1600 m); AP (Rabat, Oued Cherrat) – MISR Cytherea cinerea Fabricius, 1805 = Mulio delicatus Becker, 1906 Becker 1906b : 153; Timon-David 1951 , AP , Meshra; Mouna 1998 ; Bader and Arabyat 2004 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Cytherea delicata Becker, 1906 Zaitzev 2007 , HA , S Tizi-n'Test, AA , Zagora, Taroudant, Tizi-n'Taratine Cytherea dispar (Loew, 1873) Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Cytherea fenestrata (Loew, 1873) Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Cytherea infuscata (Meigen, 1820) Séguy 1930a , EM , Itzr (Haute Moulouya), MA , Forêt Timelilt (1900 m), HA , Aït el Hadj, Marrakech; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Meskara) – MISR Cytherea maroccana (Becker, 1903) = Mulio maroccanus Becker, in Becker 1903 : 89 Becker 1903 , Rif , Tanger; Bezzi 1906 : 249; Timon-David 1951 , AP , Azemmour; Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Cytherea obscura Fabricius, 1794 Séguy 1930a , EM , Haute Moulouya, AP , Sidi Bettache, HA , Ouaouenzert; Séguy 1941d ; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2007 , MA , Taferiat, AA , Agadir, Amredi, Jebel Tighermine (SE of Ouarzazate), Tizi-n'Tiniggigt, Tizi-n'Taratine, Tizi-n'Bachkoun; Karimpour 2012 ; Evenhuis and Greathead 2015 Cytherea rungsi Timon-David, 1951 Timon-David 1951 , EM , Guenfouda; Mouna 1998 ; Evenhuis and Greathead 2015 Cytherea thyridophora (Bezzi, 1925) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Jebel Bouhachem, 965 m) Cytherea trifaria (Becker, 1906) Evenhuis and Greathead 2015 Lomatiinae Lomatia Meigen, 1820 Lomatia abbreviata Villeneuve, 1911 Séguy 1930a , MA , Forêt Zaers; Timon-David 1951 , EM , Guercif; Mouna 1998 ; Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 ; AP (Maâmora, Oued Cherrat, Dradek), HA – MISR Lomatia belzebul paramonovi Fabricius, 1794 Séguy 1930a , AP , Dar Salem, MA , Timhadit, Meknès, Aïn Leuh; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Lomatia erynnis (Loew, 1869) Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 , AP , Rabat; Evenhuis and Greathead 2015 Lomatia hamifera Becker, 1915 Mouna 1998 : 84 Lomatia lachesis Egger, 1859 Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Lomatia lateralis (Meigen, 1820) Séguy 1930a , MA , Ras el Ma, HA , Forêt Timelilt; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Rabat), MA (Volubilis, Ras el Ma) – MISR Lomatia obscuripennis Loew, 1869 Zaitzev 2008 , AP , Nkheila; Evenhuis and Greathead 2015 Lomatia sabaea (Fabricius, 1781) Mouna 1998 : 84 Lomatia tysiphone Loew, 1860 Zaitzev 2008 , MA , Azrou, AA , Tizi-n'Taratine Antoniinae Antonia Loew, 1856 Antonia bouillonae Séguy, 1932 Evenhuis and Greathead 2015 Anthracinae Aphoebantini Aphoebantus Loew, 1872 Aphoebantus wadensis Becker, 1925* SA Anthracini Anthrax Scopoli, 1763 Anthrax aethiops (Fabricius, 1781) Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 ; AP (Forêt Maâmora) – MISR Anthrax anthrax (Schrank, 1781) = Argyramoeba anthrax Schrank, in Séguy 1930a : 93 Séguy 1930a , MA , Aïn Leuh, Soufouloud (1900–2100 m), HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Timon-David 1951 , MA , El Ksiba, Ifrane; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Anthrax binotatus (Wiedemann in Meigen, 1820) = Argyramoeba binotata Meigen, in Séguy 1926 : 209, Séguy 1930a : 94 Séguy 1926 ; Séguy 1930a , AP , Rabat, HA , Tizi-n'Test, Jebel Imdress (2000–2450 m); Séguy 1949a , HA , Alnif; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Anthrax dentatus (Becker, 1906) Bader and Arabyat 2004 ; Zaitzev 2008 , AA , Tizi-n'Tiniggigt; El Hawagry 2011 ; Evenhuis and Greathead 2015 Anthrax hemimelas Speiser, 1910 Zaitzev 2008 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) Anthrax kiritshenkoi Paramonov, 1935 Evenhuis and Greathead 2015 Anthrax lucidus (Becker, 1902) Ebejer et al. 2019 , AA , Ziz river (13 km N of Erfoud, 800 m) Anthrax morio Fabricius, 1775 Mouna 1998 ; MA (Ifrane, Azrou) – MISR Anthrax trifasciatus (Meigen, 1804) = Argyramoeba trifasciata Meigen, in Timon-David 1951 : 139 Séguy 1930a , MA , Meknès; Timon-David 1951 , AP , south of Rabat; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Anthrax varius Fabricius, 1794 Séguy 1930a , AP , Rabat; Mouna 1998 ; Evenhuis and Greathead 2015 – MISR Anthrax virgo Egger, 1859 = Argyramoeba virgo Egger, in Séguy 1930a : 94 Séguy 1930a , AP , Rabat; Zaitzev 2008 , MA , Taferiat, AA , Jebel Tighermine (SE of Ouarzazate) Cononedys Hermann, 1907 Cononedys efflatouni (Bezzi, 1925)* SA Cononedys escheri Bezzi, 1908 Zaitzev 2008 , AP , Skhirate, Rabat Cononedys lyneborgi (François, 1969) Evenhuis and Greathead 2015 Cononedys scutellatus Meigen, 1835 Zaitzev 2008 , Rif , Jebala, Haouta el Kazdir, AA , Aouzlida near Aoulouz Satyramoeba Sack, 1909 Satyramoeba hetrusca (Fabricius, 1794) Mouna 1998 : 84 Spogostylum Macquart, 1840 Spogostylum isis (Meigen, 1820) Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Spogostylum trinotatum Dufour, 1852 Mouna 1998 : 84 Spogostylum tripunctatum (Pallas in Wiedemann, 1818) Timon-David 1951 , HA , Aït Mhamed Sgatt; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate); El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 ; HA (Aïn Mhamed Sgatt) – MISR Turkmeniella Paramonov, 1940 Turkmeniella crosi (Villeneuve, 1910) Evenhuis and Greathead 2015 Exoprosopa Macquart, 1840 Exoprosopa aeacus Meigen, 1804 Mouna 1998 : 84 Exoprosopa baccha Loew, 1869 Mouna 1998 ; Zaitzev 1999 ; Dils and Özbek 2006 ; Zaitzev 2008 ; Evenhuis and Greathead 2015 Exoprosopa capucina (Fabricius, 1871) Mouna 1998 : 84 Exoprosopa circeoides Paramonov, 1928 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate) Exoprosopa cleomene Egger, 1859 Mouna 1998 : 84 Exoprosopa decrepita (Wiedemann, 1828) Zaitzev 2008 , AA , Zagora Exoprosopa efflatouni Bezzi, 1925 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate), Ouarzazate, SA , Taganint (south of Bou-Izakarn) Exoprosopa grandis Wiedemann in Meigen, 1820 Mouna 1998 ; Zaitzev 2008 , HA , Tishka (2200 m) Exoprosopa italica (Rossi, 1794) Zaitzev 2008 , HA , Taourirt, AA , Tizi-n'Taratine, Jebel Tighermine (SE of Ouarzazate), SA , Taganint (south of Bou-Izakarn) Exoprosopa jacchus (Fabricius, 1805) Séguy 1930a , AP , Mogador, Sidi Taibi, MA , Tizi-s'Tkrine (1700 m), Dar Salem, Aïn Leuh, HA , Bou Tazzert; Mouna 1998 ; Mirceni and Pârvu 2009 ; Evenhuis and Greathead 2015 ; Rif (Talassemtane, Forêt Izarine, road of Jebha, Zoumi) – MISR Exoprosopa minos (Meigen, 1804) Séguy 1949a , AA , Tata; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2008 , MA , Taferiat; El Hawagry 2011 ; El Hawagry and Dhafer 2015 ; Evenhuis and Greathead 2015 ; MA (Jebel Lachhab) – MISR Exoprosopa pandora (Fabricius, 1805) Greathead 2001 ; Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Exoprosopa rutila (Pallas & Wiedemann, 1818) Evenhuis and Greathead 2015 Micomitra Bowden, 1964 Micomitra stupida Rossi, 1790 = Exoprosopa stupida Rossi, in Mouna 1998 : 84 Mouna 1998 Plesiocera Macquart, 1840 Plesiocera algira (Macquart, 1840) Zaitzev 2008 , MA , Taferiat; Evenhuis and Greathead 2015 Heteralonia Rondani, 1863 Heteralonia ( Homolonia ) megerlei (Hoffmansegg in Wiedemann, 1818) Zaitzev 2008 , SA , Goulimine Heteralonia ( Mesoclis ) pygmalion (Fabricius, 1805) = Exoprosopa pygmalion Fabricius, in Timon-David 1951 : 139 = Mesoclis pygmalion Fabricius, 1805, in Zaitzev 2008 : 191 Séguy 1930a , Rif , Tanger, AP , Maâmora, Rabat, MA , Aïn Leuh; Timon-David 1951 , AP , Temara; Mouna 1998 ; Zaitzev 2008 , AP , Cherrat El Hawagry 2011 ; Evenhuis and Greathead 2015 Heteralonia ( Zygodipla ) algira (Fabricius, 1794) Séguy 1930a , Rif , Tanger, AP , Mogador, HA , Bou Tazzert; Mouna 1998 ; Zaitzev 2008 , HA , Tifnite (south of Aït Melloul); El Hawagry 2011 ; Evenhuis and Greathead 2015 Heteralonia ( Zygodipla ) bagdadensi s (Macquart, 1840) Zaitzev 2008 , AA , Zagora Heteralonia ( Zygodipla ) singularis (Macquart, 1840) Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Heteralonia arenacea Becker, 1906 Evenhuis and Greathead 2015 Heteralonia dispar (Loew, 1869) = Exoprosopa dispar Loew, in Timon-David 1951 : 139 Timon-David 1951 , HA , Marrakech; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Heteralonia rivularis (Meigen, 1820) = Exoprosopa rivularis Meigen, in Timon-David 1951 : 139 Séguy 1930a , AP , Rabat, Maâmora; Timon-David 1951 , AP , Oued Akreuch; Mouna 1998 ; Zaitzev 1999 ; Bader and Arabyat 2004 ; Zaitzev 2008 , AP , Rabat Oestranthrax Bezzi, 1921 Oestranthrax brunnescens (Loew, 1857) Bader and Arabyat 2004 Oestranthrax pallifrons Bezzi, 1926 Evenhuis and Greathead 2015 Pachyanthrax François, 1964 Pachyanthrax albosegmentatus (Engel, 1936) Zaitzev 2008 , AA , Jebel Tighermine (south of Ouarzazate) Pachyanthrax nomadorum (Greathead, 1970) Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Exhyalanthrax Becker, 1916 Exhyalanthrax afer (Fabricius, 1794) = Anthrax tangerinus Bigot, 1892 Bezzi 1906 ; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2008 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 , Rif , Tanger; MA (Ifrane) – MISR Hemipenthes Loew, 1869 Hemipenthes morio (Linnaeus, 1758) Séguy 1930a , MA , Azrou, HA , Arround (Skoutana, 2000–2400 m); Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 ; MA (Azrou, Ifrane) – MISR Hemipenthes velutinus (Meigen, 1820) Séguy 1930a , MA , Azrou; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Thyridanthrax Osten-Sacken, 1886 Thyridanthrax alphonsi Sánchez Terrón and Roldan Bravo, 2000 Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax elegans ssp. elegans (Wiedemann in Meigen, 1820) Séguy 1930a , AP , Rabat; Mouna 1998 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Oued Cherrat, Rabat), MA (Volubilis) – MISR Thyridanthrax fenestratus (Fallén, 1814) Séguy 1926 ; Séguy 1930a , EM , Berkane (1350–1400 m); Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; Rif (Tomorot) – MISR Thyridanthrax griseolus Klug, 1832 Zaitzev 2008 , SA , Taganint (south of Bou-Izakarn) Thyridanthrax hispanus (Loew, 1869) Becker and Stein 1913 , Rif , Tanger; Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax incanus (Klug, 1832) = Anthrax incana Klug, 1832, in Séguy 1953a : 83 Séguy 1930a , AP , Oued Korifla (Zaers); Timon-David 1951 , AP , Zaer; Séguy 1953a , MA , Tarda; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Thyridanthrax loustaui Andréu Rubio, 1961 Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax maroccanus Dils, 2012 Dils 2012 , AA , Ouarzazate, Skoura (1250 m), Amerzgane (1350 m) Thyridanthrax mutilus (Loew, 1869)* AA Thyridanthrax nebulosus (Dufour, 1852) Becker and Stein 1913 , Rif , Tanger; Andréu Rubio 1959 ; Mouna 1998 ; Sánchez Terrón and Roldan Bravo 2000 , Rif , Benibuifrur, Melilla, Restinga; Evenhuis and Greathead 2015 Thyridanthrax perspicillaris ssp. perspicillaris (Loew, 1869) Séguy 1930a , MA , Aïn Leuh, Forêt Azrou, HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Thyridanthrax polyphemus (Hoffmansegg, 1819) Séguy 1930a , MA , Volubilis (400 m); Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Veribubo Evenhuis, 1978 Veribubo angusteoculatus (Becker, 1902) Zaitzev 2008 , AA , Zagora Veribubo saudensis (François, 1970)* AA Veribubo tabaninus (François, 1970)* AA , SA Villa Lioy, 1864 Villa brunnea Becker, 1916 Mouna 1998 : 84 Villa ceballosi Andréu Rubio, 1959 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa cingulata Meigen, 1804 Mouna 1998 ; AP (Rabat, Casablanca), MA (Volubilis, Fès) – MISR Villa distincta (Meigen in Waltl, 1835) Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa fasciata (Meigen, 1804) = Villa circumdata (Meigen), in Séguy 1941a : 29 Séguy 1930a , AP , Rabat; Séguy 1941a , AP , Rabat, HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa hottentotta (Linnaeus, 1758) = Anthrax hottentotus Linnaeus, in Séguy 1926 : 198, Séguy 1930a : 92, Bléton and Fleuzet 1939: 64 Séguy 1930a , AP , Rabat, MA , Aïn Leuh; Bléton and Fleuzet 1939, MA , Fès; Séguy 1941d , HA , Tizi-n'Test; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 – MISR Villa ixion (Fabricius, 1794) Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Villa laevis Becker, 1915 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa leucostoma (Meigen, 1820) Mouna 1998 : 84; AP (Bou-Regreg) – MISR Villa luculenta Séguy, 1941 Séguy 1941d , AA , Taroudant; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa niphobleta (Loew, 1869) Bader and Arabyat 2004 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Villa venusta (Meigen, 1820) Mouna 1998 : 84 Desmatoneura Williston, 1895 Desmatoneura albifacies (Macquart, 1840) Ebejer et al. 2019 , AA , Merzouga (714 m) Desmatoneura flavifrons Becker, 1915 Zaitzev 2008 , AA , Ouarzazate, Taroudant, Jebel Tighermine (SE of Ouarzazate) Petrorossia Bezzi, 1908 Petrorossia albula Zaitzev, 1962 Zaitzev 1999 ; Bader and Arabyat 2004 ; Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate); El Hawagry 2011 ; Evenhuis and Greathead 2015 Petrorossia freidbergi Zaitzev, 1999 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate) Petrorossia hespera (Rossi, 1790) Séguy 1949a , AA , Tata; Mouna 1998 ; Zaitzev 1999 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Bou-Regreg), MA (Timahdit) – MISR Petrorossia margaritae Zaitzev, 1999 Zaitzev 2008 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) New records for Morocco Amictus setosus Loew, 1869 Atlantic Plain: Rommani, Marmouch, 33.568°N, 06.533°W , 400 m, 1♂1♀, Dils J.- Faes J., coll: PCJD . Aphoebantus wadensis Becker, 1925 Sahara: Tata, 9 km. W Tissint, 29.851°N, 07.265°W , 535 m, 1♂1♀, 03.iii.2007, Dils J.- Faes J., coll: PCJD . Bombylius (Unplaced) megacephalus Portschinsky, 1887 Eastern Morocco: Figuig, Abbou Lakhal, 32.1587°N, 01.507°W , 1050 m, 1♀, 07.iii.2009, Dils J.- Faes J., coll: PCJD . Anti Atlas: Tiznit, 84 km. SSE Guelmim, 28.631°N, 10.75522°W , 235 m, 1♂, 27.ii.2007, Dils J.- Faes J., coll: PCJD ; Tiznit, Abaynou, 29.057°N, 10.026°W , 360 m, 1♀, 13.iii.2009, Dils J.- Faes J., coll: PCJD . Cononedys efflatouni Bezzi, 1925 Sahara: Guelmim, Souk Tnine Nouaday, 29.166°N, 09.279°W , 680 m, 2♂3♀, 07.iv.2015, Dils J.- Faes J., coll: PCJD . Conophorus bellus Becker, 1906 High Atlas: Marrakech, Oukaimeden, 31.233°N, 07.817°W , 2200 m, 3♂, 06.iv.2006, Dils J.- Faes J., coll: PCJD . Crocidium aegyptiacum Bezzi, 1925 Anti Atlas: Tiznit, Mesti, 29.274°N, 10.139°W , 280 m, 1♂, 23.iii.2006, Dils J.- Faes J., coll: PCJD . Sahara: Tata, 28 km E of Tachjicht, 29.106°N, 09.149°W , 700 m, 1♀, 02.iii.2007, Dils J.- Faes J., coll: PCJD . Crocidium nudum Efflatoun, 1945 Eastern Morocco: Oujda, Plateau du Rekkam, 33.839°N, 02.55781°W , 1150 m, 1♀, 25.iv.2010, Dils J.- Faes J., coll: PCJD . Anti Atlas: Agadir, Imsouane, 30.885°N, 09.780°W , 270 m, 3♂13♀, 09.iv.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.024°N, 07.223°W , 1370 m, 17♂12♀, 31.iii.2006, Dils J.- Faes J., coll: PCJD ; Taliouine, 18 km. W of Taliouine, 30.6003°N, 08.082°W , 900 m, 2♀, 24.iii.2009, Dils J.- Faes J., coll: PCJD ; Taroudant, Tafinegoult, 30.734°N, 08.430°W , 680 m, 3♀, 24.iii.2009, Dils J.- Faes J., coll: PCJD ; Tiznit, Arbaa Sahel, 29.657°N, 09.869°W , 320 m, 11♂26♀, 21.iii.2006, Dils J.- Faes J., coll: PCJD . Cytherea albolineata Bezzi, 1925 Sahara: Guelmim, Tainzirt, 29.121°N, 09.333°W , 670 m, 1♀, 31.iii.2010, Dils J.- Faes J., coll: PCJD . Geron intonsus Bezzi, 1925 Middle Atlas: Khenifra, Boulôjoul, 32.873°N, 04.945°W , 1500 m, 7♂10♀, 26.iv.2008, Dils J.- Faes J., coll: PCJD . High Atlas: Midelt, 32.680°N, 04.677°W , 1400 m, 2♂2♀, 20.iv.2015, Dils J.- Faes J., coll: PCJD ; Midelt, Zeïda, 32.781°N, 04.964°W , 1500 m, 9♂11♀, 24.iv.2015, Dils J.- Faes J., coll: PCJD . Legnotomyia fascipennis Bezzi, 1925 Anti Atlas: Zagora, Tazarinne, 30.798°N, 05.584°W , 900 m, 1♂, 07.iii.2007, Dils J.- Faes J., coll: PCJD . Sahara: Tata, 9 km W of Tissint, 29.851°N, 07.265°W , 535 m, 2♂1♀, 03.iii.2007, Dils J.- Faes J., coll: PCJD . Thevenetimyia quedenfeldti (Engel, 1885) Rif: Tanger-Tétouan, Souk El Kolla (Quolla), 35.083°N, 05.538°W , 150 m, 5♂4♀, 30.iv.2017, Dils J.- Faes J., coll: PCJD . Atlantic Plain: Rommani, Merchouch, 33.568°N, 06.753°W , 400 m, 5♂22♀, 04.v.2010, Dils J.- Faes J., coll: PCJD . Middle Atlas: Tadla-Azilal, Afourer, 32.180°N, 06.520°W , 1150 m, 5♂10♀, 07.v.2008, Dils J.- Faes J., coll: PCJD ; Béni Mellal, El Ksiba, 32.576°N, 06.050°W , 870 m, 7♂23♀, 23.iv.2008, Dils J.- Faes J., coll: PCJD . Thyridanthrax mutilus Loew, 1869 Anti Atlas: Tiznit, Sidi Ifni, 29.384°N, 10.172°W , 0 m, 7♂1♀, 10.iv.2008, Dils J.- Faes J., coll: PCJD . Veribubo saudensis François, 1970 Anti Atlas: Erfoud, Tikkert-N-Ouchane, 31.223°N, 04.784°W , 830 m, 1♂3♀, 03.iv.2009, Dils J.- Faes J., coll: PCJD . Veribubo tabaninus François, 1970 Anti Atlas: Ouarzazate, Amerzgane, 31.024°N, 07.223°W , 1370 m, 2♂9♀, 31.iii.2006, Dils J.- Faes J., coll: PCJD ; Erfoud, Tikkert-N-Ouchane, 31.250°N, 04.617°W , 860 m, 1♀, 07.iii.2007, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.017°N, 07.229°W , 1350 m, 6♂27♀, 25.iii.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.017°N, 07.229°W , 1350 m, 12♂8♀, 25.iii.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, 30.847°N, 06.817°W , 1200 m, 1♀, 30.iii.2009, Dils J.- Faes J., coll: PCJD . Sahara: Guelmim, Tainzirt, 29.121°N, 09.333°W , 670 m, 22♀, 31.iii.2010, Dils J.- Faes J., coll: PCJD . MYDIDAE K. Kettani, T. Dikow Number of species: 9 . Expected: 10 Faunistic knowledge of the family in Morocco: moderate Leptomydinae Leptomydas Gerstaecker, 1868 Leptomydas lusitanicus (Wiedemann, 1820) Mouna 1998 Rhopaliinae Rhopalia Macquart, 1838 Rhopalia berlandi Séguy, 1949a: 153 Séguy 1949a , AA , Tagounit, Asni; Mouna 1998 ; Dikow 2017 Perissocerus Gerstaecker, 1868 Perissocerus rungsi Séguy, 1953 Séguy 1953a , SA Syllegomydinae Syllegomydini Syllegomydas Becker, 1906 Syllegomydas algiricus (Gerstaecker, 1868) = Rhopalia algirica Gerstaecker, in Séguy 1928c : 149 Gerstaecker 1868 , AP , Casablanca; Séguy 1928c , AP , Rabat; Séguy 1930a , AP , Rabat; Mouna 1998 ; El Hawagry 2011 ; Dikow 2017 Syllegomydas berlandi (Séguy, 1941) Séguy 1941, AA , Agadir; Dikow 2017 Syllegomydas bueni Arias, 1914 Arias 1914 , AA , Tafilalt; Séguy 1928c ; Séguy 1930a ; Carles-Tolrá 2015 ; Carles-Tolrá 2017 , EM , Mariouri, Trifa; Dikow 2017 Syllegomydas cinctus Macquart, 1835 Macquart 1835, MA , Immouzer road, AA , Agadir, Taroudant; Séguy 1930a ; Mouna 1998 ; Carles-Tolrá 2017 , EM , Quebdana, douar Shila, AA , Agadir coast; Dikow 2017 Syllegomydas maroccanus Séguy, 1928 Séguy 1928c , AP , Kénitra, Rabat, Oued Korifla, Forêt Zaers; Séguy 1930a , AP , Rabat, Temara, Oued Korifla, Forêt Zaers; Séguy 1932c ; Mouna 1998 ; Carles-Tolrá 2017 , AP , Larache, Ras Remel; Dikow 2017 ; AP (Kénitra) – MISR Syllegomydas merceti Arias, 1914 Arias 1914 , AP , Mogador; Séguy 1930a ; Mouna 1998 ; El Hawagry 2011 , AP , Mogador; Dikow 2017 MYTHICOMYIIDAE K. Kettani, N. Evenhuis Number of species: 8 . Expected: 15 Faunistic knowledge of the family in Morocco: poor Empidideicinae Empidideicus Becker, 1907 Empidideicus crocea Séguy, 1949 = Cyrtosia crocea Séguy, in Séguy 1949a : 85 Séguy 1949a , SA , Guelmim; Séguy 1949c ; Mouna 1998 ; Evenhuis 2002 Glabellulinae Glabellula Bezzi, 1902 Glabellula maroccana Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , Rif , Adrou ( PNPB ) – BPBM, MISR Leylaiya Efflatoun, 1945 Leylaiya pellea Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , AA , Tiznit – BPBM Mythicomyiinae Mythenteles Hall & Evenhuis, 1991 Mythenteles signifera Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , Rif , Talassemtane (maison forestière, 1699 m) – BPBM, MISR Platypyginae Cyrtisiopsis Séguy, 1930 Cyrtisiopsis melleus (Loew, 1856) Evenhuis 2002 ; Zaitzav 2008, AA , Jebel Tighermine (SE Ouarzazate); Koçak and Kemal 2010 ; El Hawagry 2011 Cyrtisiopsis singularis Séguy, 1930 Evenhuis 2002 Cyrtosia Perris, 1839 Cyrtosia aglota Séguy, 1930 Evenhuis 2002 Cyrtosia marginata Perris, 1839 Séguy 1930a , HA ; Mouna 1998 ; Evenhuis 2002 ; Evenhuis and David 2004 SCENOPINIDAE K. Kettani, M. Carles-Tolrá Number of species: 8 (+3 unidentified). Expected: 12 Faunistic knowledge of the family in Morocco: good Scenopininae Scenopinus Latreille, 1802 Scenopinus albicinctus (Rossi, 1794) = Omphrale albicincta Rossi, in Séguy 1930a : 110 Séguy 1930a ; Mouna 1998 Scenopinus fenestralis (Linnaeus, 1758) = Omphrale fenestralis Linnaeus, in Séguy 1930a : 110 Séguy 1930a ; Mouna 1998 Scenopinus glabrifrons Meigen, 1824 = Omphrale glabrifrons Meigen, in Séguy 1930a : 110 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 Scenopinus niger (De Geer, 1776) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 Scenopinus parallelus Kelsey, 1969 Kelsey 1969 , AP , Villa Cisneros (Dakhla), SA , Río de Oro (Oued Eddahab) Scenopinus physadius (Séguy, 1930) = Omphrale physadia Séguy, in Séguy 1930a : 111 Séguy, 1930, EM , Bou Denib; Kelsey 1969 , EM , Bou Denib; Mouna 1998 Scenopinus pilosus (Séguy, 1930) = Omphrale pilosa Séguy, in Séguy 1930a : 111 Séguy 1930a , AP , Bou Knadel; Kelsey 1969 , AP , Bou Knadel; Mouna 1998 ; Carles-Tolrá 2001 Scenopinus undescribed sp. 1 Ebejer et al. 2019 , Rif , Martil (9 m) Scenopinus undescribed sp. 2 Ebejer et al. 2019 , Rif , Adrou (556 m) Stenomphrale Kröber, 1937 Stenomphrale teutankhameni (Kröber, 1923) Ebejer et al. 2019 , AP , forest of Maâmora (56 m) Stenomphrale sp. AP (Essaouira (J.-P. Haenni leg.)) – MHNN Neuchâtel THEREVIDAE K. Kettani, M. Hauser Number of species: 27 . Faunistic knowledge of the family in Morocco: moderate Phycusinae Phycusini Actorthia Kröber, 1912 Actorthia micans (Kröber, 1924) Kröber 1924 , AA , Errachidia (45 km S Erfoud), Merzouga Phycus Walker, 1850 Phycus lacteipennis Lyneborg, 2002 Lyneborg 2002 , AA , 25 km S Goulmima (1000 m), SA , Mekn s-Tafilalet; Winterton et al. 2012 ; Badrawy and Mohammad 2013 Salentia Costa, 1857 Salentia anancitis (Séguy, 1941) = Apioeicoceras anancitis Séguy, in Séguy 1941d : 10 Séguy 1941d , AA , Agadir; Mouna 1998 Salentia costalis (Wiedemann, 1824) = Apioeicoceras costalis Wiedemann, in Séguy 1930a : 108 Wiedemann 1824 , AP , Mogador, HA , Marrakech-Tensift-Al Haouz; Séguy 1930a ; Mouna 1998 ; Koçak and Kemal 2010 Salentia fuscipennis Costa, 1857 Costa 1857 , Rif , Tanger, AA , Tagadirt (Agadir); Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Bou Knadel, Tinmel; Mouna 1998 Therevinae Therevini Acanthothereva Séguy, 1935 Acanthothereva rungsi Séguy, 1935 Séguy 1935b , AP , Mehdia (20 km S Rabat); Lyneborg 1968 ; Koçak and Kemal 2010 ; Rif (Cap Spartel) – MISR Acrosathe Irwin & Lyneborg, 1981 Acrosathe annulata (Fabricius, 1805) Ebejer et al. 2019 , Rif , Oued Kbir (Béni Ratene, 157 m) Chrysanthemyia Becker, 1912 Chrysanthemyia chrysanthemi (Fabricius, 1787) Fabricius 1787 , EM , Béni Snassen Mountains, Tafouralt (800 m); Séguy 1930a , MA , Meknès, Berrechid; Mouna 1998 ; MA (Oued Grou, Timahdit) – MISR Chrysanthemyia velutinifrons (Becker, 1912) = Chrysanthemyia lucidifrons Becker 1912 : 81 = Oedicera velutinifrons Becker, in Becker and Stein 1913 : 82 Becker 1912 , Rif , Region de Tanger-Tétouan, Cercle d'Ouezzane (300 m), AP , 3 km S Settat, EM , Figuig, MA , Fès-Boulmane, Sidi Harazem (223 m); Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Meknès; Mouna 1998 ; MA (Oued Grou) – MISR Hoplosathe Lyneborg & Zaitsev, 1980 Hoplosathe distincta Lyneborg & Zaitsev, 1980 Lyneborg and Zaitsev 1980, HA , Oued Tensift (Marrakech) Neotherevella Lyneborg, 1978 Neotherevella macularis (Wiedemann, 1828) Hauser et al. 2017 , AA , Tifnite (10 Km S Agadir), Merzouga (45 Km S Erfoud) Thereva Latreille, 1797 Thereva atra Kröber, 1913 El Hawagry 2011 Thereva aureoscutellata Kröber, 1914 Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m) Thereva binotata Loew, 1847 Koçak and Kemal 2010 Thereva bipunctata Meigen, 1820 16 Ebejer et al. 2019 , MA , Khénifra (17 km SW of Midelt, 1940 m; 17 km NW of Zaida, 1878 m; 28 km S of Timahdit, 2100 m), Lac Aguelmane Afennourir (30 km SW of Azrou, 2050 m) Thereva brevicornis Loew, 1847 17 Pârvu et al. 2006 , AP , Cap Sim; Popescu-Mirceni 2011 Thereva chrysargyrea Séguy, 1953 Séguy 1953a , SA , Amguilli Sguelma Thereva cincta Meigen, 1820 Ebejer et al. 2019 , Rif , Oued Aliane (Ksar Sghir, 1 m) Thereva funebris Meigen, 1820 18 = Thereva lugubris Meigen Mouna 1998 ; AP (Rabat), MA (Ifrane) – MISR Thereva graeca Kröber, 1912 3 Séguy 1930a , Rif , Tanger; Mouna 1998 Thereva plebeja (Linnaeus, 1758) 3 Séguy 1953a , AA , Tifnit (Souss); Mouna 1998 ; MA (Ras el Ma) – MISR Thereva powelli Séguy, 1930 Séguy 1930a , MA , Forêt Azrou; MA (Ras el Ma) – MISR Thereva spiloptera Wiedemann, 1824 Wiedemann 1824 , HA , Ouirgane (Marrakech, 1000 m); Séguy 1930a , Rif , Tanger, AP , Mogador, MA , Meknès; Séguy 1953a , AP , Temara; Mouna 1998 ; El Hawagry 2011 Thereva spinulosa Loew, 1847 Loew 1847 , AP , Maâmora, MA , Khemisset Thereva stigmatica Kröber, 1912 EL-Hawagy 2011, Rif , Tanger Thereva strigata (Fabricius, 1794) 19 Koçak and Kemal 2010 Thereva tuberculata Loew, 1847 = Thereva algirica Kröber, 1913, in Séguy 1953a : 83 Loew 1847 , AP , Salé; Séguy 1930a , MA , Meknès; Séguy 1953a , AP , Salé; Mouna 1998 ; Koçak and Kemal 2010 Acknowledgments We gratefully acknowledge Martin Ebejer (UK) for material, comments and cooperation, as well as Gail Kampmeier (USA) for sharing data of Moroccan Therevidae out of the mandala database ( http://wwx.inhs.illinois.edu/research/mandala/about/ ). ASILIDAE K. Kettani, G. Tomasovic Number of species: 131 . Expected: 230 Faunistic knowledge of the family in Morocco: moderate Apocleinae Apoclea Macquart, 1838 Apoclea algira (Linnaeus, 1767) Séguy 1953a , AA , Tata; Tomasovic 1997 ; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Apoclea micracantha Loew, 1856 Tomasovic 1997 , HA , Sidi Mhejmed Ou Said; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Eremonotus Theodor, 1980 Eremonotus hauseri Geller-Grimm & Hradský, 1998 Geller-Grimm and Hradský 1998, HA ; Geller-Grimm 2007, AA , Agadir Asilinae Afroepitriptus Lehr, 1992 Afroepitriptus beckeri Lehr, 1992 Geller-Grimm 2007; Koçak and Kemal 2010 Antiphrisson Loew, 1849 Antiphrisson trifarius Loew, 1849 Tomasovic 1997 , HA , Errachidia, Ziz, Oasis Zouala; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 ; HA (Asni) – MISR Asilus Linnaeus, 1758 Asilus barbarus Linnaeus, 1758 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ; Séguy 1941a , HA , Tizi-Tamatert (Toubkal, 2250 m); Mouna 1998 ; Weinberg and Blasco-Zumeta 2004 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Asilus crabroniformis Linnaeus, 1758 Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Asilus tingitanus Boisduval, 1835 Geller-Grimm 2007, Rif , Tanger Dysmachus Loew, 1860 Dysmachus albisetosus (Macquart, 1850) Geller-Grimm 2007 Dysmachus cochleatus (Loew, 1854) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dysmachus cristatus (Wiedemann, 1820) = Dysmachus dasynotus Loew, in Becker and Stein 1913 : 72, Timon-David 1951 : 138 Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Rabat, Harcha, Salé, Oued Ksab, MA , Ifrane; Mouna 1998 ; Geller-Grimm 2007; AP (Rabat, Cap Cantia) – MISR Dysmachus digitulus Becker, 1923 Geller-Grimm 2007 Dysmachus elapsus Villeneuve, 1933 Villeneuve 1933 , AP , Mazagan, Mogador; Mouna 1998 ; Tomasovic 2001b ; Geller-Grimm 2007; AP (Cap Cantia) – MISR Dysmachus evanescens Villeneuve, 1912 Timon-David 1951 , AP , Sehoul; Mouna 1998 ; Geller-Grimm 2007 Dysmachus trigonus (Meigen, 1804) Timon-David 1951 , AP , Rabat, Chellah, Forêt Maâmora, Ras el Arba, Sehoul, Zaër Eccoptopus Loew, 1860 Eccoptopus longitarsis (Macquart, 1838) Timon-David 1951 , AA , Zagora; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Engelepogon Lehr, 1992 Engelepogon brunnipes (Fabricius, 1794) = Heligmoneura brunnipes Fabricius, in Séguy 1930a : 125 = Acanthopleura brunnipes Fabricius, in Timon-David 1951 : 137 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Meknès; Timon-David 1951 , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Epitriptus Loew, 1849 Epitriptus cingulatus (Fabricius, 1871) Séguy 1941a , AA , Agadir; Mouna 1998 Eremisca Hull, 1962 Eremisca heleni heleni (Efflatoun, 1934) Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Eremisca osiris (Wiedemann, 1828) Geller-Grimm 2007; El Hawagry 2011 Eutolmus Loew, 1848 Eutolmus wahisi Tomasovic, 2001 Tomasovic 2001a , Rif , Tétouan (Jebel Tazout, 1650 m); Geller-Grimm 2007; Koçak and Kemal 2010 Filiolus Lehr, 1967 Filiolus apicalis (Becker in Becker & Stein, 1913) = Eutolmus apicalis Becker, in Becker and Stein 1913 : 75 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Machimus Loew, 1849 Machimus cribratus (Loew, 1849) Geller-Grimm 2007; AP (Cap Cantia) – MISR Machimus fimbriatus (Meigen, 1804) Geller-Grimm 2007 Machimus fortis (Loew, 1849) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat; Mouna 1998 ; Geller-Grimm 2007 Machimus gonatistes (Zeller, 1840) Geller-Grimm 2007 Machimus mauritanicus Bequaert, 1964 Tomasovic 2003 ; Geller-Grimm 2007; AP (Forêt Boulhaut, Salé) – MISR Machimus nigrosetosus Séguy, 1941 Séguy 1941d AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Machimus perplexus Becker, 1915 Geller-Grimm 2007 Machimus pilipes (Meigen, 1820) = Eutolmus hispanus Loew, in Becker and Stein 1913 : 74 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Forêt Zaers, Tizi-n'Bouftene (2300 m), HA , bords de l'Imminen (Tachdirt: 2400–2600 m); Geller-Grimm 2007 Machimus pseudogonatistes Villeneuve, 1930 = Machimus ermineus Becker, in Mouna 1998 : 84 Villeneuve 1933 ; Mouna 1998 ; Geller-Grimm 2007 Neoepitriptus Lehr, 1992 Neoepitriptus inconstans (Wiedemann in Meigen, 1820) = Machimus micropyga Becker, in Becker and Stein 1913 : 74 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 , Rif , Tanger Neoepitriptus minusculus (Bezzi, 1898) = Machimus minusculus Bezzi, in Timon-David 1951 : 138, Mouna 1998 : 84 Timon-David 1951 , MA , Ifrane; Mouna 1998 ; Geller-Grimm 2007 Neomochtherus Osten-Sacken, 1878 Neomochterus brevipennis Séguy, 1932 Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Neomochtherus grandicollis (Becker, 1914) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Neomochterus ochriventris (Loew, 1854) Timon-David 1951 , AP , Sidi Moussa el Harati; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Pashtshenkoa Lehr, 1995 Pashtshenkoa clypeatus maroccanus (Tsacas, 1968) Geller-Grimm 2007 Phileris Tsacas & Weinberg, 1976 Phileris haplopygus Tsacas & Weinberg, 1976 Geller-Grimm 2007 Phileris pilosus Tsacas & Weinberg, 1976 Geller-Grimm 2007 Satanas Jacobson, 1908 Satanas gigas (Eversmann, 1855) Maldès 2000 , ME , Oujda, HA , Errachidia, Meski Turka Őzdikmen, 2008 Turka cervinus (Loew, 1856) = Stenopogon cervinus Loew, in Séguy 1930a : 122 Séguy 1930a , MA , pont de l'Oued Korifla (Zaers), HA , Sidi Bou Rziguine; Geller-Grimm 2007; Hayat et al. 2008 ; Özdikmen 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Dasypogoninae Dasypogon Meigen, 1803 Dasypogon atratus (Fabricius, 1794) = Selidopogon atratus Meigen, in Séguy 1930a : 118 = Selidopogon atratus Fabricius, in Timon-David 1951 : 136 Séguy 1930a , MA ; Timon-David 1951 , Rif , Ouezzane, AP , Rabat MA , Oued Beth; Mouna 1998 ; Geller-Grimm 2007 Dasypogon auripilus (Séguy, 1934) Mouna 1998 ; Geller-Grimm 2007; AP (Casablanca) – MISR Dasypogon crassus Macquart in Lucas, 1849 = Selidopogon crassus Macquart, in Séguy 1930a : 119, Timon-David 1951 : 136 Séguy 1930a , Rif , Tanger, MA , Meknès; Timon-David 1951 , AP , M'Soun, Guerrouaou; Mouna 1998 ; Geller-Grimm 2007 Dasypogon diadema (Fabricius, 1781) = Selidopogon cylindricus Fabricius, in Séguy 1930a : 119 = Selidopogon diadema Fabricius, in Séguy 1930a : 118 = Selidopogon sicanus Costa, 1853, in Hayat et al. 2008 : 183 Séguy 1930a , AP , Dar Salem, Tarfaya, Oued Korifla (Zaers), HA , Bou Tazzert; Timon-David 1951 , AP , Port Lyautey; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Dasypogon gougeleti (Bigot, 1878) = Selidopogon gougeleti Bigot, in Timon-David 1951 : 136 Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Geller-Grimm 2007 Dasypogon olcesci (Bigot, 1878) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dasypogon rubinipes (Becker in Becker & Stein, 1913) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dasypogon ruficauda (Fabricius, 1805) Geller-Grimm 2007 Saropogon Loew, 1847 Saropogon aretalogus Séguy, 1953 Séguy 1953a , MA , Ifrane; Geller-Grimm 2007 Saropogon aurifrons (Macquart in Lucas, 1850) Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Saropogon clausus Becker, 1906 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , EM , Itzer, Moulay Aïn Djemine (Haute Moulouya); Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Saropogon jugulum (Loew, 1847) Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Saropogon leucocephalus (Meigen, 1820) Séguy 1930a , MA , Forêt Tiffert (2000–2200 m); Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 ; Ghahari et al. 2014 Saropogon maroccanus Séguy, 1930 Séguy 1930a , MA , Ras El Ksar (1900 m); Séguy 1949a , SA , Goulimine; Mouna 1998 ; Carles-Tolrá 2002 ; Geller-Grimm 2007 Saropogon obscuripennis (Macquart in Lucas, 1849) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat, MA , Aïn Leuh, Tizi-s'Tkrine (1700 m), HA , Imi-M'Tanout, Dar M'Tougui; Séguy 1941d , AA , Agadir; Timon-David 1951 , EM , Guenfouda; Mouna 1998 ; Geller-Grimm 2007 Saropogon philocalus Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Saropogon rufipes (Gimmerthal, 1847) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Saropogon tassilaensis Séguy, 1953 Séguy 1953a , AA , Tassila (Souss); Geller-Grimm 2007 Dioctriinae Dioctria Meigen, 1803 Dioctria atrorubens Séguy, 1930 Séguy 1930a , MA , Tizi-s'Tkine (1700 m); Villeneuve 1933 ; Mouna 1998 ; Geller-Grimm 2007 Dioctria cothurnata Meigen, 1820 Ebejer et al. 2019 , Rif , Dardara (484 m) Dioctria fuscipes Macquart, 1834 Timon-David 1951 , MA , Aguelmane Sidi Ali (2070 m) Dioctria gagates Wiedemann in Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dioctria notha Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Dioctria rufa Strobl, 1906 Ebejer et al. 2019 , Rif , Dardara (484 m) Dioctria rungsi Timon-David, 1951 Timon-David 1951 , MA , Ifrane (1650 m); Mouna 1998 ; Geller-Grimm 2007 Laphriinae Glyphotriclis Hermann, 1920 Glyphotriclis ornatus (Schiner, 1868) = Triclis ornatus Schiner, in Becker and Stein 1913 : 67 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Marrakech; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Laphria Meigen, 1803 Laphria bomboides Macquart, 1849 = Laphria praelusia Séguy, in Séguy 1930a : 124 Séguy 1930a , MA , Soufouloud (1900–2100 m); Mouna 1998 ; MA (Meghraona, Tamtraekt) – MISR Pogonosoma Rondani, 1856 Pogonosoma maroccanum (Fabricius, 1794) Loew 1860 ; Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Carles-Tolrá 2002 ; Geller-Grimm 2004 ; Geller-Grimm 2007; Ghahari et al. 2007 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013a ; Ghahari et al. 2014 Stiphrolamyra Engel, 1928 Stiphrolamyra rubicunda Oldroyd, 1947 Timon-David 1951 , AP , Sidi Moussa el Harati; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 ; Ghahari et al. 2014 Stiphrolamyra vitai Hradský & Geller-Grimm, 1997 Hradský and Geller-Grimm 1997 , HA , Taroudant; Geller-Grimm 2007 Laphystiinae Perasis Hermann, 1905 Perasis sareptana Hermann, 1906 Séguy 1930a , HA , Asni; Mouna 1998 Scytomedes Röder, 1882 Scytomedes haemorrhoidalis (Fabricius, 1794) = Triclis haemorrhoidalis Fabricius, in Mouna 1998 : 84 Séguy 1930a , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Trichardis Hermann, 1906 Trichardis leucocomus (Van der Wulp, 1899) = Trichardis leucicoma Van der Wulp, in Timon-David 1951 : 132 Timon-David 1951 , AA , Tata, piste de Fask Tahrjicht; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Leptogastrinae Leptogaster Meigen, 1803 Leptogaster cylindrica (De Geer, 1776) = Leptogaster hispanica Meigen, in Séguy 1930a : 117 Séguy 1930a , MA , Meknès; Mouna 1998 ; Tomasovic 2006 , Rif ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Leptogaster pedunculata Loew, 1847 = Gonypes pedunculatus Loew, in Becker and Stein 1913 : 72 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Haute Réghaya; Mouna 1998 Leptogaster straminea Becker, 1907 Timon-David 1951 , MA , Aguelmane Sidi Ali (2070 m); Mouna 1998 ; Geller-Grimm 2007 Stenopogoninae Afroholopogon Londt, 1994 Afroholopogon waltlii (Meigen, 1838) = Heteropogon waltlii Meigen, in Séguy 1930a : 123 Séguy 1930a , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Amphisbetetus Hermann, 1906 Amphisbetetus sexspinus Tomasovic, 2008 Tomasovic and Weyer 2008 , AA , Imsouane (Agadir); Geller-Grimm 2007 Ancylorhynchus Berthold in Latreille, 1827 Ancylorrhyncus gummigutta (Becker, 1906) Séguy 1930a , Rif , Tanger; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Ancylorrhyncus limbatus (Fabricius, 1794) Séguy 1930a , MA , Meknès, Timhadit (2000 m); Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Ancylorrhyncus vultur Séguy, 1930 Séguy 1930a , MA , Timhadit (2000 m); Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Eriopogon Loew, 1847 Eriopogon jubatus Becker, 1906 Timon-David 1951 , Rif , Tanger, AP , Rabat; Hradský and Hüttinger 1995 , AP , Rabat; Mouna 1998 ; Geller-Grimm 2007; AP (Forêt Temara) – MISR Eriopogon laniger Meigen, 1804 = Holopogon flavescens Jaennicke, in Séguy 1930a : 123 Séguy 1930a , HA , Aguergour; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Eriopogon spatenkai Hradský & Hüttinger, 1995 Geller-Grimm 2007; Hradský and Hüttinger 1995 , MA , Mishliffen Galactopogon Engel, 1929 Galactopogon hispidus Engel, 1929 Ebejer et al. 2019 , AA , 23 km S of Rich (Errachidia, 2012 m) Habropogon Loew, 1847 Habropogon aerivagus (Séguy, 1953) Séguy 1953a , SA , Aouletis Habropogon appendiculatus Schiner, 1867 Timon-David 1951 , AA , Aïn Chaïb; Mouna 1998 ; Weinberg and Blasco-Zumeta 2004 ; Hradský and Geller-Grimm 2005 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 Habropogon bacescui Weinberg & Tsacas, 1973 Geller-Grimm 2007; Koçak and Kemal 2010 Habropogon distipilosus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon hauseri Hradský & Geller-Grimm, 2005 Hradský and Geller-Grimm 2005 , HA , Tizi-n'Test; Geller-Grimm 2007; Koçak and Kemal 2010 Habropogon odontophallus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon parappendiculatus Weinberg & Tsacas, 1973 Hradský and Geller-Grimm 2005 , HA , Aït Saoun; Geller-Grimm 2007; Kirk-Spriggs and McGregor 2009 , HA ; Koçak and Kemal 2010 Habropogon prionophallus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon pyrrhophaeus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon rubriventris Macquart, 1849 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Aïn el Hadjar (near Mogador), MA , Meknès, Tlet n'Rhohr, EM , Berkane (1350–1400 m); Mouna 1998 Habropogon senilis Wulp, 1899 Geller-Grimm 2007 Habropogon spissipes Hermann, 1909 Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Habropogon striatus (Fabricius, 1794) = Habropogon heteroneurus Timon-David, in Timon-David: 135 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 , AP , Rabat Heteropogon Loew, 1847 Heteropogon biplex Becker, in Becker & Stein 1913: 65 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Heteropogon manicatus (Meigen, 1820) Séguy 1930a , MA , Azrou, Meknès, Aïn Leuh, HA , Asni; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; MA (Ifrane) – MISR Heteropogon nubilus (Meigen, 1820) = Isopogon brevis Schiner, in Becker and Stein 1913 : 64 = Sisyrnodytes brevis Macquart, in Timon-David 1951 : 134, Séguy 1953a : 79 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AA , Imiter; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Shoeibi and Karimpour 2010 ; Ghahari et al. 2014 Holopogon Loew, 1847 Holopogon dimidiatus (Meigen, 1820) Séguy 1941d , AA , Agadir Holopogon dusmeti Strobl in Czerny & Strobl 1909 = Eriopogon dusmeti Strobl, in Timon-David 1951 : 132 Timon-David 1951 , EM , Guenfouda, HA , Tifni; Mouna 1998 ; Geller-Grimm 2007; HA (Tifni Demnat) – MISR Holopogon melaleucus (Meigen, 1820) Séguy 1930a , AP , Forêt Maâmora, Dar Salem (Rabat); Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Holopogon pusillus (Macquart, 1838) = Habropogon pusillus (Macquart), in Séguy 1949a : 154 Séguy 1949a , SA , Goulimine; Mouna 1998 Holopogon quadrinotatus Séguy, 1953 Séguy 1953a , SA , Amguilli Sguelma Acnephalum Macquart, 1838 = Pycnopogon Loew, 1847 in Londt 2010 Acnephalum apiformis (Macquart in Lucas, 1849) Séguy 1930a , MA , Timhadit (2000 m), Meskedall (1800–1900 m); Timon-David 1951 , MA , Ifrane (1650 m); Mouna 1998 ; Geller-Grimm 2007 Acnephalum denudatus (Séguy, 1949) = Stenopogon denudatus Loew, in Séguy 1930a : 123, Séguy 1934b : 162 Séguy 1930a , MA , Tizi-n'Tkrine; Séguy 1934b , HA , Haute Réghaya; Séguy 1949b , AP , Bou Tazzert near Mogador; Séguy 1953a , AA , Oasis du Ferkla; Mouna 1998 ; Geller-Grimm 2007 Acnephalum fasciculatus (Loew, 1847) Séguy 1930a , MA , Azrou, Timelilt, Sidi Bettache, HA , Asni, bords Imminen (Tachdirt), Likount (2500–2800 m), Lac Ifni (Skoutana), SA , Béni Mgild; Timon-David 1951 , AP , Oued Korifla, MA , Lac Aguelmane Sidi Ali (2070 m), Oued N'Zala; Mouna 1998 ; Geller-Grimm 2007; Lehr et al. 2007 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 ; MA (Ras el Ma) – MISR Stenopogon Loew, 1847 Stenopogon costatus Loew, 1871 = Stenopogon costarus Loew, in Mouna 1998 : 84 Séguy 1930a , MA , Tizi-n'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 Stenopogon gracilis (Macquart, 1838) = Stenopogon fumipenis Becker, in Becker and Stein 1913 : 68 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Stenopogon heteroneurus (Macquart, 1838) Timon-David 1951 , AP , Forêt Maâmora, Oued Akreuch, HA , Mouldikht; Mouna 1998 ; Hayat et al. 2008 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Stenopogon iphippus Séguy, 1932 Séguy 1932b , MA , Volubilis; Mouna 1998 ; Geller-Grimm 2007 Stenopogon iphis Séguy, 1932 Séguy 1932b , MA , Azrou; Timon-David 1951 , Rif , Plateau de Tisserouine (2000 m), MA , Ifrane (1650 m), Ito (Rabat); Geller-Grimm 2007 Stenopogon ischyrus Séguy, 1932 Séguy 1932b , MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 ; Geller-Grimm 2007 Stenopogon junceus (Wiedemann in Meigen, 1820) Timon-David 1951 , AP , Oued Akreuch, Zaër, MA , Sefrou; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Stenopogon kocheri Timon-David, 1951 Timon-David 1951 , HA , Tifni; Mouna 1998 ; Geller-Grimm 2007 Stenopogon porcus Loew, 1871 Séguy 1949a , AA , Akka; Mouna 1998 ; Geller-Grimm 2007 Stenopogon werneri Engel, 1933 Geller-Grimm 2007; Koçak and Kemal 2010 , MA , Fès, Zalagh Sisyrnodytes Loew, 1856 Sisyrnodytes leucophaetus Séguy, 1930 Séguy 1930a , MA , Béni Berberi; Mouna 1998 ; Geller-Grimm 2007; Londt 2009 Sisyrnodytes nilicola (Rondani, 1850) Oldroyd 1980 ; Londt 1987; Geller-Grimm 2007; Londt 2009 , AA , Ifni, Tiznit, Tata, Tazegzout; El Hawagry 2011 ; Samin et al. 2011 ; Ghahari et al. 2014 Stichopogoninae Stichopogon Loew, 1847 Stichopogon albellus Loew, 1856 Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 Stichopogon albofasciatus (Meigen, 1820) Séguy 1930a , HA , Kasba Taguendaft (Goundafa); Mouna 1998 ; Geller-Grimm 2007 Stichopogon elegantulus (Wiedemann, 1820) Séguy 1930a , HA , Kasba Taguendaft (Goundafa), Aguerd el Had, AA , Talekjount (Souss); Mouna 1998 ; Geller-Grimm 2007; Ghahari et al. 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 ; AP (Kénitra) – MISR Stichopogon inaequalis Loew, 1847 Séguy 1930a , HA , Aguerd el Had, AA , Talekjount (Souss); Mouna 1998 ; Geller-Grimm 2007 Stichopogon maroccanus (Becker, 1914) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007; El Hawagry 2011 , Rif , Tanger Stichopogon punctiferus Bigot, 1878 Geller-Grimm 2007 Stichopogon pusio (Macquart in Lucas, 1849) Séguy 1930a , HA , Kasba Taguendaft (Goundafa); Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Stichopogon schineri Koch, 1872 Timon-David 1951 , AA , Backkoum (Jebel Siroua); Mouna 1998 ; Hayat et al. 2008 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Apocleinae Apoclea Macquart, 1838 Apoclea algira (Linnaeus, 1767) Séguy 1953a , AA , Tata; Tomasovic 1997 ; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Apoclea micracantha Loew, 1856 Tomasovic 1997 , HA , Sidi Mhejmed Ou Said; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Eremonotus Theodor, 1980 Eremonotus hauseri Geller-Grimm & Hradský, 1998 Geller-Grimm and Hradský 1998, HA ; Geller-Grimm 2007, AA , Agadir Asilinae Afroepitriptus Lehr, 1992 Afroepitriptus beckeri Lehr, 1992 Geller-Grimm 2007; Koçak and Kemal 2010 Antiphrisson Loew, 1849 Antiphrisson trifarius Loew, 1849 Tomasovic 1997 , HA , Errachidia, Ziz, Oasis Zouala; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 ; HA (Asni) – MISR Asilus Linnaeus, 1758 Asilus barbarus Linnaeus, 1758 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a ; Séguy 1941a , HA , Tizi-Tamatert (Toubkal, 2250 m); Mouna 1998 ; Weinberg and Blasco-Zumeta 2004 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Asilus crabroniformis Linnaeus, 1758 Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Asilus tingitanus Boisduval, 1835 Geller-Grimm 2007, Rif , Tanger Dysmachus Loew, 1860 Dysmachus albisetosus (Macquart, 1850) Geller-Grimm 2007 Dysmachus cochleatus (Loew, 1854) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dysmachus cristatus (Wiedemann, 1820) = Dysmachus dasynotus Loew, in Becker and Stein 1913 : 72, Timon-David 1951 : 138 Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Rabat, Harcha, Salé, Oued Ksab, MA , Ifrane; Mouna 1998 ; Geller-Grimm 2007; AP (Rabat, Cap Cantia) – MISR Dysmachus digitulus Becker, 1923 Geller-Grimm 2007 Dysmachus elapsus Villeneuve, 1933 Villeneuve 1933 , AP , Mazagan, Mogador; Mouna 1998 ; Tomasovic 2001b ; Geller-Grimm 2007; AP (Cap Cantia) – MISR Dysmachus evanescens Villeneuve, 1912 Timon-David 1951 , AP , Sehoul; Mouna 1998 ; Geller-Grimm 2007 Dysmachus trigonus (Meigen, 1804) Timon-David 1951 , AP , Rabat, Chellah, Forêt Maâmora, Ras el Arba, Sehoul, Zaër Eccoptopus Loew, 1860 Eccoptopus longitarsis (Macquart, 1838) Timon-David 1951 , AA , Zagora; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Engelepogon Lehr, 1992 Engelepogon brunnipes (Fabricius, 1794) = Heligmoneura brunnipes Fabricius, in Séguy 1930a : 125 = Acanthopleura brunnipes Fabricius, in Timon-David 1951 : 137 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Meknès; Timon-David 1951 , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Epitriptus Loew, 1849 Epitriptus cingulatus (Fabricius, 1871) Séguy 1941a , AA , Agadir; Mouna 1998 Eremisca Hull, 1962 Eremisca heleni heleni (Efflatoun, 1934) Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Eremisca osiris (Wiedemann, 1828) Geller-Grimm 2007; El Hawagry 2011 Eutolmus Loew, 1848 Eutolmus wahisi Tomasovic, 2001 Tomasovic 2001a , Rif , Tétouan (Jebel Tazout, 1650 m); Geller-Grimm 2007; Koçak and Kemal 2010 Filiolus Lehr, 1967 Filiolus apicalis (Becker in Becker & Stein, 1913) = Eutolmus apicalis Becker, in Becker and Stein 1913 : 75 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Machimus Loew, 1849 Machimus cribratus (Loew, 1849) Geller-Grimm 2007; AP (Cap Cantia) – MISR Machimus fimbriatus (Meigen, 1804) Geller-Grimm 2007 Machimus fortis (Loew, 1849) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat; Mouna 1998 ; Geller-Grimm 2007 Machimus gonatistes (Zeller, 1840) Geller-Grimm 2007 Machimus mauritanicus Bequaert, 1964 Tomasovic 2003 ; Geller-Grimm 2007; AP (Forêt Boulhaut, Salé) – MISR Machimus nigrosetosus Séguy, 1941 Séguy 1941d AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Machimus perplexus Becker, 1915 Geller-Grimm 2007 Machimus pilipes (Meigen, 1820) = Eutolmus hispanus Loew, in Becker and Stein 1913 : 74 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Forêt Zaers, Tizi-n'Bouftene (2300 m), HA , bords de l'Imminen (Tachdirt: 2400–2600 m); Geller-Grimm 2007 Machimus pseudogonatistes Villeneuve, 1930 = Machimus ermineus Becker, in Mouna 1998 : 84 Villeneuve 1933 ; Mouna 1998 ; Geller-Grimm 2007 Neoepitriptus Lehr, 1992 Neoepitriptus inconstans (Wiedemann in Meigen, 1820) = Machimus micropyga Becker, in Becker and Stein 1913 : 74 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 , Rif , Tanger Neoepitriptus minusculus (Bezzi, 1898) = Machimus minusculus Bezzi, in Timon-David 1951 : 138, Mouna 1998 : 84 Timon-David 1951 , MA , Ifrane; Mouna 1998 ; Geller-Grimm 2007 Neomochtherus Osten-Sacken, 1878 Neomochterus brevipennis Séguy, 1932 Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Neomochtherus grandicollis (Becker, 1914) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Neomochterus ochriventris (Loew, 1854) Timon-David 1951 , AP , Sidi Moussa el Harati; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Pashtshenkoa Lehr, 1995 Pashtshenkoa clypeatus maroccanus (Tsacas, 1968) Geller-Grimm 2007 Phileris Tsacas & Weinberg, 1976 Phileris haplopygus Tsacas & Weinberg, 1976 Geller-Grimm 2007 Phileris pilosus Tsacas & Weinberg, 1976 Geller-Grimm 2007 Satanas Jacobson, 1908 Satanas gigas (Eversmann, 1855) Maldès 2000 , ME , Oujda, HA , Errachidia, Meski Turka Őzdikmen, 2008 Turka cervinus (Loew, 1856) = Stenopogon cervinus Loew, in Séguy 1930a : 122 Séguy 1930a , MA , pont de l'Oued Korifla (Zaers), HA , Sidi Bou Rziguine; Geller-Grimm 2007; Hayat et al. 2008 ; Özdikmen 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Dasypogoninae Dasypogon Meigen, 1803 Dasypogon atratus (Fabricius, 1794) = Selidopogon atratus Meigen, in Séguy 1930a : 118 = Selidopogon atratus Fabricius, in Timon-David 1951 : 136 Séguy 1930a , MA ; Timon-David 1951 , Rif , Ouezzane, AP , Rabat MA , Oued Beth; Mouna 1998 ; Geller-Grimm 2007 Dasypogon auripilus (Séguy, 1934) Mouna 1998 ; Geller-Grimm 2007; AP (Casablanca) – MISR Dasypogon crassus Macquart in Lucas, 1849 = Selidopogon crassus Macquart, in Séguy 1930a : 119, Timon-David 1951 : 136 Séguy 1930a , Rif , Tanger, MA , Meknès; Timon-David 1951 , AP , M'Soun, Guerrouaou; Mouna 1998 ; Geller-Grimm 2007 Dasypogon diadema (Fabricius, 1781) = Selidopogon cylindricus Fabricius, in Séguy 1930a : 119 = Selidopogon diadema Fabricius, in Séguy 1930a : 118 = Selidopogon sicanus Costa, 1853, in Hayat et al. 2008 : 183 Séguy 1930a , AP , Dar Salem, Tarfaya, Oued Korifla (Zaers), HA , Bou Tazzert; Timon-David 1951 , AP , Port Lyautey; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Dasypogon gougeleti (Bigot, 1878) = Selidopogon gougeleti Bigot, in Timon-David 1951 : 136 Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Geller-Grimm 2007 Dasypogon olcesci (Bigot, 1878) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dasypogon rubinipes (Becker in Becker & Stein, 1913) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dasypogon ruficauda (Fabricius, 1805) Geller-Grimm 2007 Saropogon Loew, 1847 Saropogon aretalogus Séguy, 1953 Séguy 1953a , MA , Ifrane; Geller-Grimm 2007 Saropogon aurifrons (Macquart in Lucas, 1850) Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Saropogon clausus Becker, 1906 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , EM , Itzer, Moulay Aïn Djemine (Haute Moulouya); Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 Saropogon jugulum (Loew, 1847) Timon-David 1951 , AP , Zaers; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Saropogon leucocephalus (Meigen, 1820) Séguy 1930a , MA , Forêt Tiffert (2000–2200 m); Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 ; Ghahari et al. 2014 Saropogon maroccanus Séguy, 1930 Séguy 1930a , MA , Ras El Ksar (1900 m); Séguy 1949a , SA , Goulimine; Mouna 1998 ; Carles-Tolrá 2002 ; Geller-Grimm 2007 Saropogon obscuripennis (Macquart in Lucas, 1849) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Rabat, MA , Aïn Leuh, Tizi-s'Tkrine (1700 m), HA , Imi-M'Tanout, Dar M'Tougui; Séguy 1941d , AA , Agadir; Timon-David 1951 , EM , Guenfouda; Mouna 1998 ; Geller-Grimm 2007 Saropogon philocalus Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Saropogon rufipes (Gimmerthal, 1847) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Saropogon tassilaensis Séguy, 1953 Séguy 1953a , AA , Tassila (Souss); Geller-Grimm 2007 Dioctriinae Dioctria Meigen, 1803 Dioctria atrorubens Séguy, 1930 Séguy 1930a , MA , Tizi-s'Tkine (1700 m); Villeneuve 1933 ; Mouna 1998 ; Geller-Grimm 2007 Dioctria cothurnata Meigen, 1820 Ebejer et al. 2019 , Rif , Dardara (484 m) Dioctria fuscipes Macquart, 1834 Timon-David 1951 , MA , Aguelmane Sidi Ali (2070 m) Dioctria gagates Wiedemann in Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Dioctria notha Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Dioctria rufa Strobl, 1906 Ebejer et al. 2019 , Rif , Dardara (484 m) Dioctria rungsi Timon-David, 1951 Timon-David 1951 , MA , Ifrane (1650 m); Mouna 1998 ; Geller-Grimm 2007 Laphriinae Glyphotriclis Hermann, 1920 Glyphotriclis ornatus (Schiner, 1868) = Triclis ornatus Schiner, in Becker and Stein 1913 : 67 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Marrakech; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Laphria Meigen, 1803 Laphria bomboides Macquart, 1849 = Laphria praelusia Séguy, in Séguy 1930a : 124 Séguy 1930a , MA , Soufouloud (1900–2100 m); Mouna 1998 ; MA (Meghraona, Tamtraekt) – MISR Pogonosoma Rondani, 1856 Pogonosoma maroccanum (Fabricius, 1794) Loew 1860 ; Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Carles-Tolrá 2002 ; Geller-Grimm 2004 ; Geller-Grimm 2007; Ghahari et al. 2007 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013a ; Ghahari et al. 2014 Stiphrolamyra Engel, 1928 Stiphrolamyra rubicunda Oldroyd, 1947 Timon-David 1951 , AP , Sidi Moussa el Harati; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 ; Ghahari et al. 2014 Stiphrolamyra vitai Hradský & Geller-Grimm, 1997 Hradský and Geller-Grimm 1997 , HA , Taroudant; Geller-Grimm 2007 Laphystiinae Perasis Hermann, 1905 Perasis sareptana Hermann, 1906 Séguy 1930a , HA , Asni; Mouna 1998 Scytomedes Röder, 1882 Scytomedes haemorrhoidalis (Fabricius, 1794) = Triclis haemorrhoidalis Fabricius, in Mouna 1998 : 84 Séguy 1930a , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Trichardis Hermann, 1906 Trichardis leucocomus (Van der Wulp, 1899) = Trichardis leucicoma Van der Wulp, in Timon-David 1951 : 132 Timon-David 1951 , AA , Tata, piste de Fask Tahrjicht; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Leptogastrinae Leptogaster Meigen, 1803 Leptogaster cylindrica (De Geer, 1776) = Leptogaster hispanica Meigen, in Séguy 1930a : 117 Séguy 1930a , MA , Meknès; Mouna 1998 ; Tomasovic 2006 , Rif ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Leptogaster pedunculata Loew, 1847 = Gonypes pedunculatus Loew, in Becker and Stein 1913 : 72 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Haute Réghaya; Mouna 1998 Leptogaster straminea Becker, 1907 Timon-David 1951 , MA , Aguelmane Sidi Ali (2070 m); Mouna 1998 ; Geller-Grimm 2007 Stenopogoninae Afroholopogon Londt, 1994 Afroholopogon waltlii (Meigen, 1838) = Heteropogon waltlii Meigen, in Séguy 1930a : 123 Séguy 1930a , MA , Meknès; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Amphisbetetus Hermann, 1906 Amphisbetetus sexspinus Tomasovic, 2008 Tomasovic and Weyer 2008 , AA , Imsouane (Agadir); Geller-Grimm 2007 Ancylorhynchus Berthold in Latreille, 1827 Ancylorrhyncus gummigutta (Becker, 1906) Séguy 1930a , Rif , Tanger; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Ancylorrhyncus limbatus (Fabricius, 1794) Séguy 1930a , MA , Meknès, Timhadit (2000 m); Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Ancylorrhyncus vultur Séguy, 1930 Séguy 1930a , MA , Timhadit (2000 m); Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 Eriopogon Loew, 1847 Eriopogon jubatus Becker, 1906 Timon-David 1951 , Rif , Tanger, AP , Rabat; Hradský and Hüttinger 1995 , AP , Rabat; Mouna 1998 ; Geller-Grimm 2007; AP (Forêt Temara) – MISR Eriopogon laniger Meigen, 1804 = Holopogon flavescens Jaennicke, in Séguy 1930a : 123 Séguy 1930a , HA , Aguergour; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Eriopogon spatenkai Hradský & Hüttinger, 1995 Geller-Grimm 2007; Hradský and Hüttinger 1995 , MA , Mishliffen Galactopogon Engel, 1929 Galactopogon hispidus Engel, 1929 Ebejer et al. 2019 , AA , 23 km S of Rich (Errachidia, 2012 m) Habropogon Loew, 1847 Habropogon aerivagus (Séguy, 1953) Séguy 1953a , SA , Aouletis Habropogon appendiculatus Schiner, 1867 Timon-David 1951 , AA , Aïn Chaïb; Mouna 1998 ; Weinberg and Blasco-Zumeta 2004 ; Hradský and Geller-Grimm 2005 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 Habropogon bacescui Weinberg & Tsacas, 1973 Geller-Grimm 2007; Koçak and Kemal 2010 Habropogon distipilosus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon hauseri Hradský & Geller-Grimm, 2005 Hradský and Geller-Grimm 2005 , HA , Tizi-n'Test; Geller-Grimm 2007; Koçak and Kemal 2010 Habropogon odontophallus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon parappendiculatus Weinberg & Tsacas, 1973 Hradský and Geller-Grimm 2005 , HA , Aït Saoun; Geller-Grimm 2007; Kirk-Spriggs and McGregor 2009 , HA ; Koçak and Kemal 2010 Habropogon prionophallus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon pyrrhophaeus Weinberg & Tsacas, 1973 Geller-Grimm 2007 Habropogon rubriventris Macquart, 1849 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Aïn el Hadjar (near Mogador), MA , Meknès, Tlet n'Rhohr, EM , Berkane (1350–1400 m); Mouna 1998 Habropogon senilis Wulp, 1899 Geller-Grimm 2007 Habropogon spissipes Hermann, 1909 Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Habropogon striatus (Fabricius, 1794) = Habropogon heteroneurus Timon-David, in Timon-David: 135 Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 , AP , Rabat Heteropogon Loew, 1847 Heteropogon biplex Becker, in Becker & Stein 1913: 65 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Heteropogon manicatus (Meigen, 1820) Séguy 1930a , MA , Azrou, Meknès, Aïn Leuh, HA , Asni; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; MA (Ifrane) – MISR Heteropogon nubilus (Meigen, 1820) = Isopogon brevis Schiner, in Becker and Stein 1913 : 64 = Sisyrnodytes brevis Macquart, in Timon-David 1951 : 134, Séguy 1953a : 79 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AA , Imiter; Mouna 1998 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Shoeibi and Karimpour 2010 ; Ghahari et al. 2014 Holopogon Loew, 1847 Holopogon dimidiatus (Meigen, 1820) Séguy 1941d , AA , Agadir Holopogon dusmeti Strobl in Czerny & Strobl 1909 = Eriopogon dusmeti Strobl, in Timon-David 1951 : 132 Timon-David 1951 , EM , Guenfouda, HA , Tifni; Mouna 1998 ; Geller-Grimm 2007; HA (Tifni Demnat) – MISR Holopogon melaleucus (Meigen, 1820) Séguy 1930a , AP , Forêt Maâmora, Dar Salem (Rabat); Séguy 1941d , AA , Agadir; Mouna 1998 ; Geller-Grimm 2007 Holopogon pusillus (Macquart, 1838) = Habropogon pusillus (Macquart), in Séguy 1949a : 154 Séguy 1949a , SA , Goulimine; Mouna 1998 Holopogon quadrinotatus Séguy, 1953 Séguy 1953a , SA , Amguilli Sguelma Acnephalum Macquart, 1838 = Pycnopogon Loew, 1847 in Londt 2010 Acnephalum apiformis (Macquart in Lucas, 1849) Séguy 1930a , MA , Timhadit (2000 m), Meskedall (1800–1900 m); Timon-David 1951 , MA , Ifrane (1650 m); Mouna 1998 ; Geller-Grimm 2007 Acnephalum denudatus (Séguy, 1949) = Stenopogon denudatus Loew, in Séguy 1930a : 123, Séguy 1934b : 162 Séguy 1930a , MA , Tizi-n'Tkrine; Séguy 1934b , HA , Haute Réghaya; Séguy 1949b , AP , Bou Tazzert near Mogador; Séguy 1953a , AA , Oasis du Ferkla; Mouna 1998 ; Geller-Grimm 2007 Acnephalum fasciculatus (Loew, 1847) Séguy 1930a , MA , Azrou, Timelilt, Sidi Bettache, HA , Asni, bords Imminen (Tachdirt), Likount (2500–2800 m), Lac Ifni (Skoutana), SA , Béni Mgild; Timon-David 1951 , AP , Oued Korifla, MA , Lac Aguelmane Sidi Ali (2070 m), Oued N'Zala; Mouna 1998 ; Geller-Grimm 2007; Lehr et al. 2007 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 ; MA (Ras el Ma) – MISR Stenopogon Loew, 1847 Stenopogon costatus Loew, 1871 = Stenopogon costarus Loew, in Mouna 1998 : 84 Séguy 1930a , MA , Tizi-n'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 Stenopogon gracilis (Macquart, 1838) = Stenopogon fumipenis Becker, in Becker and Stein 1913 : 68 Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007 Stenopogon heteroneurus (Macquart, 1838) Timon-David 1951 , AP , Forêt Maâmora, Oued Akreuch, HA , Mouldikht; Mouna 1998 ; Hayat et al. 2008 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 Stenopogon iphippus Séguy, 1932 Séguy 1932b , MA , Volubilis; Mouna 1998 ; Geller-Grimm 2007 Stenopogon iphis Séguy, 1932 Séguy 1932b , MA , Azrou; Timon-David 1951 , Rif , Plateau de Tisserouine (2000 m), MA , Ifrane (1650 m), Ito (Rabat); Geller-Grimm 2007 Stenopogon ischyrus Séguy, 1932 Séguy 1932b , MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 ; Geller-Grimm 2007 Stenopogon junceus (Wiedemann in Meigen, 1820) Timon-David 1951 , AP , Oued Akreuch, Zaër, MA , Sefrou; Mouna 1998 ; Geller-Grimm 2007; Hayat et al. 2008 ; Koçak and Kemal 2010 ; Ghahari et al. 2014 Stenopogon kocheri Timon-David, 1951 Timon-David 1951 , HA , Tifni; Mouna 1998 ; Geller-Grimm 2007 Stenopogon porcus Loew, 1871 Séguy 1949a , AA , Akka; Mouna 1998 ; Geller-Grimm 2007 Stenopogon werneri Engel, 1933 Geller-Grimm 2007; Koçak and Kemal 2010 , MA , Fès, Zalagh Sisyrnodytes Loew, 1856 Sisyrnodytes leucophaetus Séguy, 1930 Séguy 1930a , MA , Béni Berberi; Mouna 1998 ; Geller-Grimm 2007; Londt 2009 Sisyrnodytes nilicola (Rondani, 1850) Oldroyd 1980 ; Londt 1987; Geller-Grimm 2007; Londt 2009 , AA , Ifni, Tiznit, Tata, Tazegzout; El Hawagry 2011 ; Samin et al. 2011 ; Ghahari et al. 2014 Stichopogoninae Stichopogon Loew, 1847 Stichopogon albellus Loew, 1856 Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 Stichopogon albofasciatus (Meigen, 1820) Séguy 1930a , HA , Kasba Taguendaft (Goundafa); Mouna 1998 ; Geller-Grimm 2007 Stichopogon elegantulus (Wiedemann, 1820) Séguy 1930a , HA , Kasba Taguendaft (Goundafa), Aguerd el Had, AA , Talekjount (Souss); Mouna 1998 ; Geller-Grimm 2007; Ghahari et al. 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Ghahari et al. 2014 ; AP (Kénitra) – MISR Stichopogon inaequalis Loew, 1847 Séguy 1930a , HA , Aguerd el Had, AA , Talekjount (Souss); Mouna 1998 ; Geller-Grimm 2007 Stichopogon maroccanus (Becker, 1914) Becker and Stein 1913 , Rif , Tanger; Geller-Grimm 2007; El Hawagry 2011 , Rif , Tanger Stichopogon punctiferus Bigot, 1878 Geller-Grimm 2007 Stichopogon pusio (Macquart in Lucas, 1849) Séguy 1930a , HA , Kasba Taguendaft (Goundafa); Mouna 1998 ; Geller-Grimm 2007; El Hawagry 2011 Stichopogon schineri Koch, 1872 Timon-David 1951 , AA , Backkoum (Jebel Siroua); Mouna 1998 ; Hayat et al. 2008 ; Geller-Grimm 2007; Koçak and Kemal 2010 ; Ghahari et al. 2014 BOMBYLIIDAE K. Kettani, M.J. Ebejer, J. Dils Number of species: 248 . Expected: 270 Faunistic knowledge of the family in Morocco: good Usiinae Apolysis Loew, 1860 Apolysis eremophila Loew, 1873 = Usia tomentosa Engel, in Paramonow 1947: 209 = Parageron ornata Engel, in Zaitzev 2007 : 160 Paramonow 1947; Mouna 1998 ; Zaitzev 2007 , AP (south), Tamri; Koçak and Kemal 2010 ; El Hawagri 2011; Evenhuis and Greathead 2015 Parageron Paramonov, 1929 Parageron gratus (Loew, 1856) = Usia grata Loew, in Timon-David 1951 : 143 Timon-David 1951 , AP , Oued Grou; Mouna 1998 ; Bader and Arabyat 2004 ; Zaitzev 2007 , SA ; Koçak and Kemal 2010 ; El Hawagri 2011; Evenhuis and Greathead 2015 – MISR Parageron griseus Paramonov, 1947 Zaitzev 2007 , SA Parageron hyalipennis (Séguy, 1941) = Oligodranes hyalipennis Séguy, in Séguy 1941d : 9 Séguy 1941d , AA , Agadir (Forêt Admine); Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , HA Parageron incisus (Wiedemann, 1830) = Usia incisa Wiedemann, in Timon-David 1951 : 143 Séguy 1930a , AP , Mogador, Casablanca, Sidi Bettache, Aïn Sferguila, MA , Forêt Zaers, Ras el Ma, Aïn Leuh, HA , Tenfecht; Timon-David 1951 , AP , Rabat, Sehoul, MA , Ifrane; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Parageron major Macquart, 1840 Mouna 1998 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 , AP , Rabat, Nkheila; Evenhuis and Greathead 2015 Usia Latreille, 1802 Usia ( Micrusia ) aurata (Fabricius, 1794) = Usia ( Micrusia ) taeniolata Costa, 1883, in, Koçak and Kemal 2010 Séguy 1930a , Rif , Tanger, AP , Rabat, Sidi Bettache; Paramonov 1950 , Rif , Tanger; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) crispa Gibbs, 2011 Gibbs 2011 , HA , Marrakech, AA , Agadir, Taroudant, Tafraoute, Ouarzazate Usia ( Micrusia ) cryptocrispa Gibbs, 2011 Gibbs 2011 , AP , Ben Slimane, Rabat Usia ( Micrusia ) dilsi Gibbs, 2011 Gibbs 2011 , Rif , Tétouan, Al Hoceima, HA , Taourirt Usia ( Micrusia ) echinus Gibbs, 2011 Gibbs 2011 , AP , Agadir, Guelmim, Sidi Ifni, Cap Ghir, MA , Tafraoute, HA , Marrakech, Tizi-n-Test, AA , Tafingoult Usia ( Micrusia ) falcata Gibbs, 2011 Gibbs 2011 , MA , Azrou, Ifrane Usia ( Micrusia ) forcipata Brullé, 1833 14 Mouna 1998 : 84 Usia ( Micrusia ) globicauda Gibbs, 2011 Gibbs 2011 , AP , Essaouira Usia ( Micrusia ) loewi Becker, 1906 Zaitzev 2007 , Rif , Tanger, AP , Skhirate, Nkheila, MA , Taferiate, HA , Taourirt Usia ( Micrusia ) novakii Strobl, 1902 Séguy 1941d , HA , Tizi-n'Test; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) parascripa Gibbs, 2011 Gibbs 2011 , MA , Ifrane, Mischliffen (2200 m) Usia ( Micrusia ) pusilla Meigen, 1820 Séguy 1930a , AP , Rabat, MA , Azrou, HA , Tafingoult (Goundafa, 1500–1600 m); Séguy 1934b , AP , Rabat (on Calendula ); Séguy 1953a , AP , Cap Ghir; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) versicolor (Fabricius, 1787) Séguy 1930a , AP , Berrchid, Casablanca, M'Rassine; Timon-David 1951 , AP , Rabat, MA , Oulmès; Mouna 1998 ; Koçak and Kemal 2010 ; Gibbs 2011 , HA ; Evenhuis and Greathead 2015 ; EM (Oujda) – MNHNR Usia ( Usia ) aenea (Rossi, 1794) Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 ; Bader and Arabyat 2004 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Usia ) angustifrons Becker, 1906 Mouna 1998 Usia ( Usia ) atrata (Fabricius, 1798) = Voluccella atrata Fabricius, in Fabricius 1798: 570 = Usia claripennis Macquart, in Macquart 1840: 105 Meigen 1820 ; Séguy 1930a , Rif , Tanger, MA , Aïn Leuh; Timon-David 1951 , AP , Rabat, Guerrouaou; Mouna 1998 ; Zaitzev 2007 , HA , Tizi-n'Tichka; Koçak and Kemal 2010 ; Gibbs 2014 , AP , Mogador, Arbaa-Sahel (320 m), Tamri (215 m), MA , Khemisset, Oulmès (700 m), Ifrane, El Merabtine, HA , Marrakech, Aït Ourirr (530 m), Oukaimeden (2200 m), Tizi-n'Test (1450 m), Timzit (1700 m), AA , Agadir, Tiznit, Igherm (1660 m), Taroudant, Tata, Iguiour (1260 m), SA , Bou Jarif, Goulimine; Evenhuis and Greathead 2015 Usia ( Usia ) bicolor Macquart, 1855 15 Mouna 1998 : 84 Usia ( Usia ) cornigera Gibbs, 2014 Gibbs 2014 , Rif , Tanger, AP , Sidi Bettache, Rabat, MA , Meknès (550 m), Aïn Leuh (1350 m), HA , Dar Kaid M'tougui Usia ( Usia ) florea (Fabricius, 1794) = Volucella florea Fabricius, in Becker 1906: 203 = Usia cuprea Macquart, 1834, in Becker 1906: 203 Becker 1906a ; Séguy 1930a , Rif , Tanger, AP , Mogador, Sidi Bettache, HA , Tinmel (Goundafa), around (Skoutana); Timon-David 1951 , EM , Oued Moulouya; Mouna 1998 ; Pârvu and Zaharia 2007 ; Koçak and Kemal 2010 ; Gibbs 2014 ; Evenhuis and Greathead 2015 Usia ( Usia ) ignorata Becker, 1906 Becker 1906a ; Mouna 1998 ; Bader and Arabyat 2004 ; Pârvu and Zaharia 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 Usia ( Usia ) maghrebensis Gibbs, 2014 Gibbs 2014 , Rif , Tanger, Tétouan, El Biutz (150 m), AP , Mogador, MA , Aïn Leuh (1350 m) Usia ( Usia ) vestita Macquart, 1846 Mouna 1998 : 84; Gibbs 2011 Phthiriinae Phthiria Meigen, 1802 Phthiria albogilva Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Evenhuis and Greathead 2015 Phthiria gaedii Wiedemann in Meigen, 1820 Séguy 1930a , MA , Foum Keneg; Timon-David 1951 , AP , Zaers, MA , Ifrane; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 – MISR ( MA , Ifrane) Phthiria maroccana Zaitzev, 2005 = Phthiria maroccana Zaitzev, in Zaitzev 2005 : 667 Zaitzev 2005 , MA , Taferiate, HA , Taourirt; Zaitzev 2007 , MA , Taferiate, HA , Taourirt Phthiria merlei Zaitzev, 2005 = Phthiria merlei Zaitzev, in Zaitzev 2005 : 665 Zaitzev 2005 , AP (south), Tamri, Inchaden (south of Aït Melloul); Zaitzev 2007 , AP (south), Tamri, Inchaden (south of Aït Melloul) Phthiria minuta (Fabricius, 1805) Séguy 1930a , HA , Tenfecht, AA , Souss; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 Phthiria pulicaria var. flavofasciata Strobl in Morge, 1976 Mouna 1998 ; Zaitzev 2007 , AA , Tizi-n'Tiniggigt (1600 m); Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Phthiria scutellaris Wiedemann in Meigen, 1820 Séguy 1930a , MA , Meknès; Séguy 1941a , MA , Meknès, HA , Imi-n'Ouaka (1500 m); Mouna 1998 ; Evenhuis and Greathead 2015 ; Rif (Sapinière Talassemtane) – MISR Phthiria simonyi Becker, 1908 Séguy 1949a , SA , Guelmim; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , MA , Meknès Phthiria umbripennis Loew, 1846 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , MA , Meknès Phthiria vagans Loew, 1846 Zaitzev 2007 , HA , Taourirt, AA , Tizi-n'Taratine Toxophorinae Geron Meigen, 1820 Geron intonsus Bezzi, 1925* MA , HA Geron macquarti Greathead in Evenhuis & Greathead 1999 Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 Geron subflavofemoratus Andréu Rubio, 1959 Andréu Rubio 1959 ; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Toxophora Meigen, 1803 Toxophora fasciculata (Villers, 1789) Séguy 1930a , AP , Rabat; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Toxophora fuscipennis (Macquart, 1840) Mouna 1998 : 84 Toxophora pauli Zaitzev, 2005 Zaitzev 2005 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate); Zaitzev 2007 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) Toxophora shelkovnikovi Paramonov, 1933 Zaitzev 2007 , AA , Ouarzazate Heterotropinae Heterotropus Loew, 1873 Heterotropus atlanticus Séguy, 1930 Séguy 1930a , AP , Mogador; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Heterotropus longitarsus Séguy, 1930 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Heterotropus maroccanus Zaitzev, 2003 Zaitzev 2003 , AA , Jebel Tighermine (SE of Ouarzazate); Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Bombyliinae Anastoechus Osten-Sacken, 1877 Anastoechus bahirae Becker, 1915 Mouna 1998 ; Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Anastoechus hyrcanus Pallas & Wiedemann in Wiedemann, 1818 Mouna 1998 : 84 Anastoechus latifrons (Macquart, 1839) Timon-David 1951 , AP , Dradek; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Anastoechus nitidulus ssp. nitidulus Fabricius, 1794 Mouna 1998 : 84 Anastoechus stramineus Wiedemann in Meigen, 1820 Mouna 1998 : 84 Anastoechus trisignatus (Portschinsky, 1881) Bader and Arabyat 2004 ; Ziatzev 2007, AP , Rabat, AA , Jebel Tighermine (SE of Ouarzazate), AA , 20 km SW Goulmima, SA , Tan-Tan, Between Guelmim and Tan-Tan (90 km from Guelmim), Taganint (south of Bou-Izakarn); Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombomyia Greathead, 1995 Bombomyia discoidea (Fabricius, 1794) Séguy 1930a , AP , Oued Korifla (Zaers), Sidi Bettache; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombomyia stictica Boisduval, 1835 Zaitzev 2007 , MA , col de Zeggota (N Meknès), Oulmès Bombomyia vertebralis (Dufour, 1833) = Bombylius punctatus Fabricius, in Timon-David 1951 : 144 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a AP , Dradek near Rabat; Timon-David 1951 , MA , Volubilis; Mouna 1998 ; Bader and Arabyat 2004 ; Koçak and Kemal 2010 Evenhuis and Greathead 2015 ; AP (Dradek, Casablanca), EM (Oujda), MA (Volubilis, Aïn Leuh) – MISR Bombylisoma Rondani, 1856 Bombylisoma algirum (Macquart, 1840) = Bombylius nigrifrons Becker, in Becker and Stein 1913 : 83 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylisoma breviusculum (Loew, 1855) Dils and Özbek 2006 ; Zaitzev 2007 ; Evenhuis and Greathead 2015 Bombylisoma flavibarbum Loew, 1855 Mouna 1998 : 84 Bombylisoma melanocephalum Fabricius, 1794 Zaitzev 2007 , HA , Taourirt, south of Tizi-n'Test Bombylius Linnaeus, 1758 Bombylius ( Bombylius ) albaminis Séguy, 1949 Séguy 1949a , HA , Alnif; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) ambustus Pallas & Wiedemann, 1818 Mouna 1998 : 84 Bombylius ( Bombylius ) analis (Olivier, 1789) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Oued Korifla, Rabat, Sidi Bettache, Aïn Sferguila; Timon-David 1951 , AP , Rabat; Mouna 1998 ; Zaitzev 2007 , MA , route Fès-Sidi Kacem (30 km from Fès); Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , AP (Rabat, Casablanca) – MISR Bombylius ( Bombylius ) audcenti Bowden, 1984 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) canescens Mikan, 1796 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , Rif , Cap Malabata (Tanger), MA , Tachguelt, route Fès-Sidi Kacem (30 km from Fès) Bombylius ( Bombylius ) cinerascens Mikan, 1796 Mouna 1998 ; Bader and Arabyat 2004 Bombylius ( Bombylius ) discolor Mikan, 1796 Mouna 1998 ; Zaitzev 2007 , MA , route El Hajeb-Ifrane (1 km from Ifrane) Bombylius ( Bombylius ) eploceus Séguy, 1949 Séguy 1949a , SA , Guelmim; Mouna 1998 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) fimbriatus Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, Jebel Ahmar (1700 m); Mouna 1998 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) flavipes Wiedemann, 1828 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) fulvescens Wiedemann in Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AP , Cap Ghir; Séguy 1941d , AA , Agadir; Mouna 1998 Bombylius ( Bombylius ) fuscus Fabricius, 1781 Mouna 1998 : 84 Bombylius ( Bombylius ) major (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , MA , Oulmès; Bader and Arabyat 2004 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Kénitra) – MISR Bombylius ( Bombylius ) mauritanus Olivier, 1789 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , HA Bombylius ( Bombylius ) medius (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Sehoul; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Oued Yquem, Dradek) – MISR Bombylius (Unplaced) megacephalus Portschinsky, 1887* EM , AA Bombylius ( Bombylius ) minor Linnaeus, 1758 Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; MA (Aguelmane Azigza), SA – MISR Bombylius ( Bombylius ) mus Bigot, 1862 Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) niveus Meigen, 1804 Mouna 1998 : 84; AP (Mogador) – MISR Bombylius ( Bombylius ) numidus Macquart, 1846 Séguy 1953a , MA , Ifrane; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) pauli Zaitzev, 2003 Zaitzev 2003 , MA , route Fès-Sidi Kacem (30 km from Fès); Zaitzev 2007 , MA , route Fès-Sidi Kacem (30 km from Fès) Bombylius ( Bombylius ) posticus (Fabricius, 1805) Dils and Özbek 2006 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) postversicolor Evenhuis & Greathead, 1999 = Bombylius versicolor Fabricius, 1805 Meigen 1820 ; Bezzi 1906 ; Séguy 1930a , AP , Mogador; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) pumilus Meigen, 1820 Mouna 1998 : 84 Bombylius ( Bombylius ) semifuscus (Meigen, 1820) Séguy 1953a , AP , Cap Ghir; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) torquatus Loew, 1855 Séguy 1930a , HA , Ouaounzert (Glaoua), Arround (Skoutana), Tachdirt (bord de l'Imminen, 2400–2600 m); Timon-David 1951 , AP , Rabat; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Mogador) – MISR Bombylius ( Bombylius ) undatus Mikan, 1796 Pârvu and Zaharia 2007 Bombylius ( Bombylius ) vagans Meigen, 1830 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) venosus Mikan, 1796 Mouna 1998 ; Zaitzev 2007 , AP , El Koudia (30 km SW from Rabat); AP (Dradek) – MISR Bombylius ( Zephyrectes ) cruciatus Fabricius, 1798 Séguy 1930a , MA , Aharmoumou (1100 m), Azrou, Ras el Ma, HA , Tizi-n'Test, Jebel Imdress (Goundafa, 2000–2450 m); Mouna 1998 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 ; Rif (Talassemtane), AP (Mogador), MA (Sefrou) – MISR Bombylius ( Zephyrectes ) leucopygus (Macquart, 1846) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , AP , Larache, MA , Moulay Idris (900 m); Evenhuis and Greathead 2015 , SA , Erfoud; MA (Ifrane) – MISR Conophorus Meigen, 1803 Conophorus bellus Becker, 1906* HA Conophorus fuliginosus (Wiedemann in Meigen, 1820) = Ploas fuliginisa (Meigen), in Séguy 1953a : 83 Séguy 1930a , MA , Aharmoumou (1100 m); Séguy 1953a , MA , Ahermoumou (1100 m); Timon-David 1951 , AP , Dradek, MA , Sefrou, HA , Marrakech; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; Evenhuis and Greathead 2015 ; AP (Salé, Mogador) – MISR Conophorus fuscipennis (Macquart, 1840) Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Mouna 1998 ; Evenhuis and Greathead 2015 Conophorus griseus (Fabricius, 1787) Mouna 1998 ; Zaitzev 2007 ; Evenhuis and Greathead 2015 Conophorus hamilkar Paramonov, 1929 Timon-David 1951 , AP , Mogador; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Mogador) – MISR Conophorus macroglossus (Dufour, 1852) Mouna 1998 ; Zaitzev 2007 ; Evenhuis and Greathead 201; AP (Mogador) – MISR Conophorus mauritanicus Bigot, 1892 = Conophorus heteropilosus Timon-David, in Timon-David 1951 : 141; Mouna 1998 : 84; Evenhuis and Greathead 2015 : 192 Timon-David 1951 , MA , Oulmès; Mouna 1998 ; Zaitzev 2007 , AP , El Koudia (30 km SW from Rabat), Forêt Zaer (35 km SW from Rabat), N Tretten; Koçak and Kemal 2010 ; Dils 2013 , MA , Mrirt; Evenhuis and Greathead 2015 Conophorus rossicus Paramonow, 1929 Dils and Özbek 2006 Dischistus Loew, 1855 Dischistus albatus (Séguy, 1934) = Acanthogeron albatus Séguy, 1934, in Séguy 1934d : 73; Zaitzev 2007 : 162 Séguy 1934d ; Zaitzev 2007 , SA , 30 km S Tata Dischistus auripilus (Séguy, 1930) = Acanthogeron auripilus Séguy, 1930, in Séguy 1930a : 104 Séguy 1930a , AP , Mogador; Séguy 1934b , AP , Zaers; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Evenhuis and Greathead 2015 Dischistus maroccanus (Séguy, 1930) = Acanthogeron maroccanus Séguy, 1930, in Séguy 1930a : 106 Séguy 1930a , AP , Mogador; Mouna 1998 ; Zaitzev 2007 , HA , Tazzarine; Evenhuis and Greathead 2015 Dischistus mittrei (Séguy, 1930) = Acanthogeron mittrei Séguy, in Séguy 1930a : 105 Séguy 1930a , AP , Mogador; Mouna 1998 ; Evenhuis and Greathead 2015 Dischistus perniveus (Bezzi, 1925) = Acanthogeron perniveus Bezzi, in Timon-David 1951 : 143 Timon-David 1951 , AP , Djamda de M'Tal; Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Dischistus senex (Wiedemann in Meigen, 1820) = Acanthogeron senex Meigen, 1820, in Séguy 1953a : 83, Mouna 1998 : 84, Zaitzev 2007 : 162 Séguy 1930a , HA , Tafingoult (Goundafa, 1500–1600 m); Villeneuve 1933 ; Séguy 1953a , HA , Aït Ourir; Mouna 1998 ; Zaitzev 2003 , HA , Taourirt; Zaitzev 2007 , HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Dradek), MA (Sefrou) – MISR Dischistus separatus (Becker, 1906) = Acanthogeron talboti Séguy, 1930, in Séguy 1930a : 106 Evenhuis and Greathead 2015 Efflatounia Bezzi, 1925 Efflatounia berbera Bowden, 1973 Ebejer et al. 2019 , AA , Agadir – NMWC Legnotomyia Bezzi, 1902 Legnotomyia fascipennis Bezzi, 1925* SA Merleus Zaitzev, 2003 Merleus punctipennis Zaitzev, 2003 = Merleus punctipennis Zaitzev 2003 : 599 Zaitzev 2003 , AP , Skhirate; Zaitzev 2007 , AP , Skhirate Prorachthes Loew, 1869 Prorachthes crassipalpis Villeneuve, 1930 Evenhuis and Greathead 2015 Systoechus Loew, 1855 Systoechus ctenopterus (Mikan, 1796) Timon-David 1951 , MA , Ifrane; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2007 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Systoechus gomezmenori Andréu Rubio, 1959 Carles-Tolrá 2002 ; Evenhuis and Greathead 2015 Systoechus gradatus (Wiedemann in Meigen, 1820) Timon-David 1951 , AP , Mouldikht; Mouna 1998 ; Zaitzev 2007 , MA , Taferiat, HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 Systoechus mixtus Wiedemann, 1821 = Bombylius stylicornis Macquart in Séguy 1941: 10 Séguy 1941d , AA , Agadir; Mouna 1998 Systoechus pumilio Becker, 1915 Mouna 1998 : 84 Triplasius Loew, 1855 Triplasius boghariensis (Lucas, 1852) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , EM , Oujda; Mouna 1998 ; Pârvu and Zaharia 2007 ; Evenhuis and Greathead 2015 Triplasius maculipennis (Macquart, 1846) = Bombylius maculipennis var. melanopus Timon-David, in Timon-David 1951 : 144 = Bombylius ( Triplasius ) maculipennis Macquart, 1849, in Zaitzev 2007 : 166 Timon-David 1951 , MA , Azrou; Zaitzev 2007 , MA , route El Hachef, Criosement route Raubei Idris-Merhassine; Evenhuis and Greathead 2015 Ecliminae Eclimus Loew, 1844 Eclimus gracilis Loew, 1844 Séguy 1930a , MA , Ras el Ma; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2007 , MA , Maaziz; Evenhuis and Greathead 2015 Thevenetimyia Bigot, 1875 Thevenetimyia quedenfeldti (Engel, 1885)* Rif , AP , MA Crocidiinae Crocidium Loew, 1860 Crocidium aegyptiacum Bezzi, 1925* SA Crocidium nudum Efflatoun, 1945* EM , AA Semiramis Becker in Becker and Stein 1914 Semiramis punctipennis Becker, 1914 Zaitzev 2007 , AA , Aoulouz Cythereinae Amictus Wiedemann, 1817 Amictus castaneus (Macquart, 1840) Séguy 1930a , AP , Rabat, HA , Ank el Djemal; Mouna 1998 ; Evenhuis and Greathead 2015 Amictus compressus (Fabricius, 1805) Evenhuis and Greathead 2015 Amictus heteropterus Macquart, 1838 Zaitzev 2007 , Rif , Tanger, AP , Rabat, HA , S Tizi-n'Test, AA , Tizi-n'Taratine Amictus oblongus (Fabricius, 1805) = Bombylius oblongus Fabricius, in Macquart 1834: 390 Macquart 1834 Amictus pulchellus Macquart, 1846 Séguy 1930a , AP , Rabat, Maâmora; Mouna 1998 ; Zaitzev 2007 , HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Amictus setosus Loew, 1869* AP Amictus tener Becker, 1906 Zaitzev 2007 , AP , Rabat Amictus validus Loew, 1869 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Amictus variegatus Meigen in Waltl, 1835 Mouna 1998 : 84 Chalcochiton Loew, 1844 Chalcochiton argentifrons (Macquart in Lucas, 1849) Séguy 1953a , AP , Cap Ghir, Salé, Sidi Battache, MA , Tizi-n'Bou Zabal (2300 m), AA , Aïn Chaïb (Souss); Evenhuis and Greathead 2015 Chalcochiton argyrocephalus (Macquart, 1840) = Chalcochiton ( Anthrax ) argyrocephala (Macquart), in Engel 1938 : 328 Engel 1938 ; Séguy 1953a , AA , Agadir; El Hawagry 2011 ; Evenhuis and Greathead 2015 Chalcochiton atlantica Dils, 2008 Dils 2008 , SA , Guelmim Chalcochiton holosericeus (Fabricius, 1794) = Chalcochiton semiargentaea Macquart, in Zaitsev 2007: 172 Séguy 1930a , AP , Maâmora, Sidi Bettache, HA , Tizi-n'Test, Jebal Imdress (2000–2450 m), Tafingoult (Goundafa, 1500–1600 m); Séguy 1941d ; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger, AP , Skhirate, EM , Taourirt, MA , Taferiat, Meknès-Moulay Idriss, Merhassine, AA , Agadir, Ouarzazate; Evenhuis and Greathead 2015 ; AP (Salé, Forêt Temara, Oued Yquem, Meshra) – MISR Chalcochiton maghrebi Dils, 2017 Dils 2017 , Rif , Souk El Kolla, Bab Taza, 10 km S of Mjara, AP , Sidi Bettache, Temsia, Imsouane, Mansouria, Rommani, Béni Slimane, Tioulit, EM , El Aioun, MA , Béni Mellal, el Ksiba, 10 km SE Bir Tamtam, Merchouch, Mrirt, Fès, HA , Azilal, Asni, Tizi-Mlil, AA , Taroudant, Tizi-n'Test, Tiznit, Agouim, Sidi Ifni, Mesti, Tafinegoult, Tizi-n'Tinififft, El Mrabtine, SA , Semara Chalcochiton maroccanus Zaitzev, 2006 Séguy 1953a , HA , Tafingoult (Goundafa, 1500–1600 m); Zaitzev 2006 , AP (south), Aït Melloul; Zaitzev 2007 , AP (south), Aït Melloul Chalcochiton merlei Zaitzev, 2006 Zaitzev 2006 , AP , Skhirate; Zaitzev 2007 , AP , Skhirate Chalcochiton pallasii Loew, 1856 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2007 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Callostoma Macquart, 1840 Callostoma fascipenne Macquart, 1840 Bader and Arabyat 2004 Cyllenia Latreille, 1802 Cyllenia rustica Rossi, 1790 Mouna 1998 ; Zaitzev 2007 ; AP (Mogador) – MISR Cytherea Fabricius, 1794 Cytherea albolineata Bezzi, 1925* SA Cytherea alexandrina Becker, 1902 Becker 1902 : 30; Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Cytherea aurea (Fabricius, 1794) Séguy 1930a , AP , Rabat, HA ; Mouna 1998 ; Bader and Arabyat 2004 ; Zaitzev 2007 , AA , Tizi-n'Taratine; El Hawagry 2011 ; Evenhuis and Greathead 2015 , HA , Tafingoult (Goundafa, 1500–1600 m); AP (Rabat, Oued Cherrat) – MISR Cytherea cinerea Fabricius, 1805 = Mulio delicatus Becker, 1906 Becker 1906b : 153; Timon-David 1951 , AP , Meshra; Mouna 1998 ; Bader and Arabyat 2004 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Cytherea delicata Becker, 1906 Zaitzev 2007 , HA , S Tizi-n'Test, AA , Zagora, Taroudant, Tizi-n'Taratine Cytherea dispar (Loew, 1873) Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Cytherea fenestrata (Loew, 1873) Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Cytherea infuscata (Meigen, 1820) Séguy 1930a , EM , Itzr (Haute Moulouya), MA , Forêt Timelilt (1900 m), HA , Aït el Hadj, Marrakech; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Meskara) – MISR Cytherea maroccana (Becker, 1903) = Mulio maroccanus Becker, in Becker 1903 : 89 Becker 1903 , Rif , Tanger; Bezzi 1906 : 249; Timon-David 1951 , AP , Azemmour; Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Cytherea obscura Fabricius, 1794 Séguy 1930a , EM , Haute Moulouya, AP , Sidi Bettache, HA , Ouaouenzert; Séguy 1941d ; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2007 , MA , Taferiat, AA , Agadir, Amredi, Jebel Tighermine (SE of Ouarzazate), Tizi-n'Tiniggigt, Tizi-n'Taratine, Tizi-n'Bachkoun; Karimpour 2012 ; Evenhuis and Greathead 2015 Cytherea rungsi Timon-David, 1951 Timon-David 1951 , EM , Guenfouda; Mouna 1998 ; Evenhuis and Greathead 2015 Cytherea thyridophora (Bezzi, 1925) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Jebel Bouhachem, 965 m) Cytherea trifaria (Becker, 1906) Evenhuis and Greathead 2015 Lomatiinae Lomatia Meigen, 1820 Lomatia abbreviata Villeneuve, 1911 Séguy 1930a , MA , Forêt Zaers; Timon-David 1951 , EM , Guercif; Mouna 1998 ; Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 ; AP (Maâmora, Oued Cherrat, Dradek), HA – MISR Lomatia belzebul paramonovi Fabricius, 1794 Séguy 1930a , AP , Dar Salem, MA , Timhadit, Meknès, Aïn Leuh; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Lomatia erynnis (Loew, 1869) Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 , AP , Rabat; Evenhuis and Greathead 2015 Lomatia hamifera Becker, 1915 Mouna 1998 : 84 Lomatia lachesis Egger, 1859 Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Lomatia lateralis (Meigen, 1820) Séguy 1930a , MA , Ras el Ma, HA , Forêt Timelilt; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Rabat), MA (Volubilis, Ras el Ma) – MISR Lomatia obscuripennis Loew, 1869 Zaitzev 2008 , AP , Nkheila; Evenhuis and Greathead 2015 Lomatia sabaea (Fabricius, 1781) Mouna 1998 : 84 Lomatia tysiphone Loew, 1860 Zaitzev 2008 , MA , Azrou, AA , Tizi-n'Taratine Antoniinae Antonia Loew, 1856 Antonia bouillonae Séguy, 1932 Evenhuis and Greathead 2015 Anthracinae Aphoebantini Aphoebantus Loew, 1872 Aphoebantus wadensis Becker, 1925* SA Anthracini Anthrax Scopoli, 1763 Anthrax aethiops (Fabricius, 1781) Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 ; AP (Forêt Maâmora) – MISR Anthrax anthrax (Schrank, 1781) = Argyramoeba anthrax Schrank, in Séguy 1930a : 93 Séguy 1930a , MA , Aïn Leuh, Soufouloud (1900–2100 m), HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Timon-David 1951 , MA , El Ksiba, Ifrane; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Anthrax binotatus (Wiedemann in Meigen, 1820) = Argyramoeba binotata Meigen, in Séguy 1926 : 209, Séguy 1930a : 94 Séguy 1926 ; Séguy 1930a , AP , Rabat, HA , Tizi-n'Test, Jebel Imdress (2000–2450 m); Séguy 1949a , HA , Alnif; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Anthrax dentatus (Becker, 1906) Bader and Arabyat 2004 ; Zaitzev 2008 , AA , Tizi-n'Tiniggigt; El Hawagry 2011 ; Evenhuis and Greathead 2015 Anthrax hemimelas Speiser, 1910 Zaitzev 2008 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) Anthrax kiritshenkoi Paramonov, 1935 Evenhuis and Greathead 2015 Anthrax lucidus (Becker, 1902) Ebejer et al. 2019 , AA , Ziz river (13 km N of Erfoud, 800 m) Anthrax morio Fabricius, 1775 Mouna 1998 ; MA (Ifrane, Azrou) – MISR Anthrax trifasciatus (Meigen, 1804) = Argyramoeba trifasciata Meigen, in Timon-David 1951 : 139 Séguy 1930a , MA , Meknès; Timon-David 1951 , AP , south of Rabat; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Anthrax varius Fabricius, 1794 Séguy 1930a , AP , Rabat; Mouna 1998 ; Evenhuis and Greathead 2015 – MISR Anthrax virgo Egger, 1859 = Argyramoeba virgo Egger, in Séguy 1930a : 94 Séguy 1930a , AP , Rabat; Zaitzev 2008 , MA , Taferiat, AA , Jebel Tighermine (SE of Ouarzazate) Cononedys Hermann, 1907 Cononedys efflatouni (Bezzi, 1925)* SA Cononedys escheri Bezzi, 1908 Zaitzev 2008 , AP , Skhirate, Rabat Cononedys lyneborgi (François, 1969) Evenhuis and Greathead 2015 Cononedys scutellatus Meigen, 1835 Zaitzev 2008 , Rif , Jebala, Haouta el Kazdir, AA , Aouzlida near Aoulouz Satyramoeba Sack, 1909 Satyramoeba hetrusca (Fabricius, 1794) Mouna 1998 : 84 Spogostylum Macquart, 1840 Spogostylum isis (Meigen, 1820) Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Spogostylum trinotatum Dufour, 1852 Mouna 1998 : 84 Spogostylum tripunctatum (Pallas in Wiedemann, 1818) Timon-David 1951 , HA , Aït Mhamed Sgatt; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate); El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 ; HA (Aïn Mhamed Sgatt) – MISR Turkmeniella Paramonov, 1940 Turkmeniella crosi (Villeneuve, 1910) Evenhuis and Greathead 2015 Exoprosopa Macquart, 1840 Exoprosopa aeacus Meigen, 1804 Mouna 1998 : 84 Exoprosopa baccha Loew, 1869 Mouna 1998 ; Zaitzev 1999 ; Dils and Özbek 2006 ; Zaitzev 2008 ; Evenhuis and Greathead 2015 Exoprosopa capucina (Fabricius, 1871) Mouna 1998 : 84 Exoprosopa circeoides Paramonov, 1928 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate) Exoprosopa cleomene Egger, 1859 Mouna 1998 : 84 Exoprosopa decrepita (Wiedemann, 1828) Zaitzev 2008 , AA , Zagora Exoprosopa efflatouni Bezzi, 1925 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate), Ouarzazate, SA , Taganint (south of Bou-Izakarn) Exoprosopa grandis Wiedemann in Meigen, 1820 Mouna 1998 ; Zaitzev 2008 , HA , Tishka (2200 m) Exoprosopa italica (Rossi, 1794) Zaitzev 2008 , HA , Taourirt, AA , Tizi-n'Taratine, Jebel Tighermine (SE of Ouarzazate), SA , Taganint (south of Bou-Izakarn) Exoprosopa jacchus (Fabricius, 1805) Séguy 1930a , AP , Mogador, Sidi Taibi, MA , Tizi-s'Tkrine (1700 m), Dar Salem, Aïn Leuh, HA , Bou Tazzert; Mouna 1998 ; Mirceni and Pârvu 2009 ; Evenhuis and Greathead 2015 ; Rif (Talassemtane, Forêt Izarine, road of Jebha, Zoumi) – MISR Exoprosopa minos (Meigen, 1804) Séguy 1949a , AA , Tata; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2008 , MA , Taferiat; El Hawagry 2011 ; El Hawagry and Dhafer 2015 ; Evenhuis and Greathead 2015 ; MA (Jebel Lachhab) – MISR Exoprosopa pandora (Fabricius, 1805) Greathead 2001 ; Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Exoprosopa rutila (Pallas & Wiedemann, 1818) Evenhuis and Greathead 2015 Micomitra Bowden, 1964 Micomitra stupida Rossi, 1790 = Exoprosopa stupida Rossi, in Mouna 1998 : 84 Mouna 1998 Plesiocera Macquart, 1840 Plesiocera algira (Macquart, 1840) Zaitzev 2008 , MA , Taferiat; Evenhuis and Greathead 2015 Heteralonia Rondani, 1863 Heteralonia ( Homolonia ) megerlei (Hoffmansegg in Wiedemann, 1818) Zaitzev 2008 , SA , Goulimine Heteralonia ( Mesoclis ) pygmalion (Fabricius, 1805) = Exoprosopa pygmalion Fabricius, in Timon-David 1951 : 139 = Mesoclis pygmalion Fabricius, 1805, in Zaitzev 2008 : 191 Séguy 1930a , Rif , Tanger, AP , Maâmora, Rabat, MA , Aïn Leuh; Timon-David 1951 , AP , Temara; Mouna 1998 ; Zaitzev 2008 , AP , Cherrat El Hawagry 2011 ; Evenhuis and Greathead 2015 Heteralonia ( Zygodipla ) algira (Fabricius, 1794) Séguy 1930a , Rif , Tanger, AP , Mogador, HA , Bou Tazzert; Mouna 1998 ; Zaitzev 2008 , HA , Tifnite (south of Aït Melloul); El Hawagry 2011 ; Evenhuis and Greathead 2015 Heteralonia ( Zygodipla ) bagdadensi s (Macquart, 1840) Zaitzev 2008 , AA , Zagora Heteralonia ( Zygodipla ) singularis (Macquart, 1840) Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Heteralonia arenacea Becker, 1906 Evenhuis and Greathead 2015 Heteralonia dispar (Loew, 1869) = Exoprosopa dispar Loew, in Timon-David 1951 : 139 Timon-David 1951 , HA , Marrakech; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Heteralonia rivularis (Meigen, 1820) = Exoprosopa rivularis Meigen, in Timon-David 1951 : 139 Séguy 1930a , AP , Rabat, Maâmora; Timon-David 1951 , AP , Oued Akreuch; Mouna 1998 ; Zaitzev 1999 ; Bader and Arabyat 2004 ; Zaitzev 2008 , AP , Rabat Oestranthrax Bezzi, 1921 Oestranthrax brunnescens (Loew, 1857) Bader and Arabyat 2004 Oestranthrax pallifrons Bezzi, 1926 Evenhuis and Greathead 2015 Pachyanthrax François, 1964 Pachyanthrax albosegmentatus (Engel, 1936) Zaitzev 2008 , AA , Jebel Tighermine (south of Ouarzazate) Pachyanthrax nomadorum (Greathead, 1970) Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Exhyalanthrax Becker, 1916 Exhyalanthrax afer (Fabricius, 1794) = Anthrax tangerinus Bigot, 1892 Bezzi 1906 ; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2008 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 , Rif , Tanger; MA (Ifrane) – MISR Hemipenthes Loew, 1869 Hemipenthes morio (Linnaeus, 1758) Séguy 1930a , MA , Azrou, HA , Arround (Skoutana, 2000–2400 m); Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 ; MA (Azrou, Ifrane) – MISR Hemipenthes velutinus (Meigen, 1820) Séguy 1930a , MA , Azrou; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Thyridanthrax Osten-Sacken, 1886 Thyridanthrax alphonsi Sánchez Terrón and Roldan Bravo, 2000 Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax elegans ssp. elegans (Wiedemann in Meigen, 1820) Séguy 1930a , AP , Rabat; Mouna 1998 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Oued Cherrat, Rabat), MA (Volubilis) – MISR Thyridanthrax fenestratus (Fallén, 1814) Séguy 1926 ; Séguy 1930a , EM , Berkane (1350–1400 m); Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; Rif (Tomorot) – MISR Thyridanthrax griseolus Klug, 1832 Zaitzev 2008 , SA , Taganint (south of Bou-Izakarn) Thyridanthrax hispanus (Loew, 1869) Becker and Stein 1913 , Rif , Tanger; Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax incanus (Klug, 1832) = Anthrax incana Klug, 1832, in Séguy 1953a : 83 Séguy 1930a , AP , Oued Korifla (Zaers); Timon-David 1951 , AP , Zaer; Séguy 1953a , MA , Tarda; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Thyridanthrax loustaui Andréu Rubio, 1961 Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax maroccanus Dils, 2012 Dils 2012 , AA , Ouarzazate, Skoura (1250 m), Amerzgane (1350 m) Thyridanthrax mutilus (Loew, 1869)* AA Thyridanthrax nebulosus (Dufour, 1852) Becker and Stein 1913 , Rif , Tanger; Andréu Rubio 1959 ; Mouna 1998 ; Sánchez Terrón and Roldan Bravo 2000 , Rif , Benibuifrur, Melilla, Restinga; Evenhuis and Greathead 2015 Thyridanthrax perspicillaris ssp. perspicillaris (Loew, 1869) Séguy 1930a , MA , Aïn Leuh, Forêt Azrou, HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Thyridanthrax polyphemus (Hoffmansegg, 1819) Séguy 1930a , MA , Volubilis (400 m); Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Veribubo Evenhuis, 1978 Veribubo angusteoculatus (Becker, 1902) Zaitzev 2008 , AA , Zagora Veribubo saudensis (François, 1970)* AA Veribubo tabaninus (François, 1970)* AA , SA Villa Lioy, 1864 Villa brunnea Becker, 1916 Mouna 1998 : 84 Villa ceballosi Andréu Rubio, 1959 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa cingulata Meigen, 1804 Mouna 1998 ; AP (Rabat, Casablanca), MA (Volubilis, Fès) – MISR Villa distincta (Meigen in Waltl, 1835) Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa fasciata (Meigen, 1804) = Villa circumdata (Meigen), in Séguy 1941a : 29 Séguy 1930a , AP , Rabat; Séguy 1941a , AP , Rabat, HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa hottentotta (Linnaeus, 1758) = Anthrax hottentotus Linnaeus, in Séguy 1926 : 198, Séguy 1930a : 92, Bléton and Fleuzet 1939: 64 Séguy 1930a , AP , Rabat, MA , Aïn Leuh; Bléton and Fleuzet 1939, MA , Fès; Séguy 1941d , HA , Tizi-n'Test; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 – MISR Villa ixion (Fabricius, 1794) Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Villa laevis Becker, 1915 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa leucostoma (Meigen, 1820) Mouna 1998 : 84; AP (Bou-Regreg) – MISR Villa luculenta Séguy, 1941 Séguy 1941d , AA , Taroudant; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa niphobleta (Loew, 1869) Bader and Arabyat 2004 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Villa venusta (Meigen, 1820) Mouna 1998 : 84 Desmatoneura Williston, 1895 Desmatoneura albifacies (Macquart, 1840) Ebejer et al. 2019 , AA , Merzouga (714 m) Desmatoneura flavifrons Becker, 1915 Zaitzev 2008 , AA , Ouarzazate, Taroudant, Jebel Tighermine (SE of Ouarzazate) Petrorossia Bezzi, 1908 Petrorossia albula Zaitzev, 1962 Zaitzev 1999 ; Bader and Arabyat 2004 ; Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate); El Hawagry 2011 ; Evenhuis and Greathead 2015 Petrorossia freidbergi Zaitzev, 1999 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate) Petrorossia hespera (Rossi, 1790) Séguy 1949a , AA , Tata; Mouna 1998 ; Zaitzev 1999 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Bou-Regreg), MA (Timahdit) – MISR Petrorossia margaritae Zaitzev, 1999 Zaitzev 2008 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) New records for Morocco Amictus setosus Loew, 1869 Atlantic Plain: Rommani, Marmouch, 33.568°N, 06.533°W , 400 m, 1♂1♀, Dils J.- Faes J., coll: PCJD . Aphoebantus wadensis Becker, 1925 Sahara: Tata, 9 km. W Tissint, 29.851°N, 07.265°W , 535 m, 1♂1♀, 03.iii.2007, Dils J.- Faes J., coll: PCJD . Bombylius (Unplaced) megacephalus Portschinsky, 1887 Eastern Morocco: Figuig, Abbou Lakhal, 32.1587°N, 01.507°W , 1050 m, 1♀, 07.iii.2009, Dils J.- Faes J., coll: PCJD . Anti Atlas: Tiznit, 84 km. SSE Guelmim, 28.631°N, 10.75522°W , 235 m, 1♂, 27.ii.2007, Dils J.- Faes J., coll: PCJD ; Tiznit, Abaynou, 29.057°N, 10.026°W , 360 m, 1♀, 13.iii.2009, Dils J.- Faes J., coll: PCJD . Cononedys efflatouni Bezzi, 1925 Sahara: Guelmim, Souk Tnine Nouaday, 29.166°N, 09.279°W , 680 m, 2♂3♀, 07.iv.2015, Dils J.- Faes J., coll: PCJD . Conophorus bellus Becker, 1906 High Atlas: Marrakech, Oukaimeden, 31.233°N, 07.817°W , 2200 m, 3♂, 06.iv.2006, Dils J.- Faes J., coll: PCJD . Crocidium aegyptiacum Bezzi, 1925 Anti Atlas: Tiznit, Mesti, 29.274°N, 10.139°W , 280 m, 1♂, 23.iii.2006, Dils J.- Faes J., coll: PCJD . Sahara: Tata, 28 km E of Tachjicht, 29.106°N, 09.149°W , 700 m, 1♀, 02.iii.2007, Dils J.- Faes J., coll: PCJD . Crocidium nudum Efflatoun, 1945 Eastern Morocco: Oujda, Plateau du Rekkam, 33.839°N, 02.55781°W , 1150 m, 1♀, 25.iv.2010, Dils J.- Faes J., coll: PCJD . Anti Atlas: Agadir, Imsouane, 30.885°N, 09.780°W , 270 m, 3♂13♀, 09.iv.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.024°N, 07.223°W , 1370 m, 17♂12♀, 31.iii.2006, Dils J.- Faes J., coll: PCJD ; Taliouine, 18 km. W of Taliouine, 30.6003°N, 08.082°W , 900 m, 2♀, 24.iii.2009, Dils J.- Faes J., coll: PCJD ; Taroudant, Tafinegoult, 30.734°N, 08.430°W , 680 m, 3♀, 24.iii.2009, Dils J.- Faes J., coll: PCJD ; Tiznit, Arbaa Sahel, 29.657°N, 09.869°W , 320 m, 11♂26♀, 21.iii.2006, Dils J.- Faes J., coll: PCJD . Cytherea albolineata Bezzi, 1925 Sahara: Guelmim, Tainzirt, 29.121°N, 09.333°W , 670 m, 1♀, 31.iii.2010, Dils J.- Faes J., coll: PCJD . Geron intonsus Bezzi, 1925 Middle Atlas: Khenifra, Boulôjoul, 32.873°N, 04.945°W , 1500 m, 7♂10♀, 26.iv.2008, Dils J.- Faes J., coll: PCJD . High Atlas: Midelt, 32.680°N, 04.677°W , 1400 m, 2♂2♀, 20.iv.2015, Dils J.- Faes J., coll: PCJD ; Midelt, Zeïda, 32.781°N, 04.964°W , 1500 m, 9♂11♀, 24.iv.2015, Dils J.- Faes J., coll: PCJD . Legnotomyia fascipennis Bezzi, 1925 Anti Atlas: Zagora, Tazarinne, 30.798°N, 05.584°W , 900 m, 1♂, 07.iii.2007, Dils J.- Faes J., coll: PCJD . Sahara: Tata, 9 km W of Tissint, 29.851°N, 07.265°W , 535 m, 2♂1♀, 03.iii.2007, Dils J.- Faes J., coll: PCJD . Thevenetimyia quedenfeldti (Engel, 1885) Rif: Tanger-Tétouan, Souk El Kolla (Quolla), 35.083°N, 05.538°W , 150 m, 5♂4♀, 30.iv.2017, Dils J.- Faes J., coll: PCJD . Atlantic Plain: Rommani, Merchouch, 33.568°N, 06.753°W , 400 m, 5♂22♀, 04.v.2010, Dils J.- Faes J., coll: PCJD . Middle Atlas: Tadla-Azilal, Afourer, 32.180°N, 06.520°W , 1150 m, 5♂10♀, 07.v.2008, Dils J.- Faes J., coll: PCJD ; Béni Mellal, El Ksiba, 32.576°N, 06.050°W , 870 m, 7♂23♀, 23.iv.2008, Dils J.- Faes J., coll: PCJD . Thyridanthrax mutilus Loew, 1869 Anti Atlas: Tiznit, Sidi Ifni, 29.384°N, 10.172°W , 0 m, 7♂1♀, 10.iv.2008, Dils J.- Faes J., coll: PCJD . Veribubo saudensis François, 1970 Anti Atlas: Erfoud, Tikkert-N-Ouchane, 31.223°N, 04.784°W , 830 m, 1♂3♀, 03.iv.2009, Dils J.- Faes J., coll: PCJD . Veribubo tabaninus François, 1970 Anti Atlas: Ouarzazate, Amerzgane, 31.024°N, 07.223°W , 1370 m, 2♂9♀, 31.iii.2006, Dils J.- Faes J., coll: PCJD ; Erfoud, Tikkert-N-Ouchane, 31.250°N, 04.617°W , 860 m, 1♀, 07.iii.2007, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.017°N, 07.229°W , 1350 m, 6♂27♀, 25.iii.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.017°N, 07.229°W , 1350 m, 12♂8♀, 25.iii.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, 30.847°N, 06.817°W , 1200 m, 1♀, 30.iii.2009, Dils J.- Faes J., coll: PCJD . Sahara: Guelmim, Tainzirt, 29.121°N, 09.333°W , 670 m, 22♀, 31.iii.2010, Dils J.- Faes J., coll: PCJD . Usiinae Apolysis Loew, 1860 Apolysis eremophila Loew, 1873 = Usia tomentosa Engel, in Paramonow 1947: 209 = Parageron ornata Engel, in Zaitzev 2007 : 160 Paramonow 1947; Mouna 1998 ; Zaitzev 2007 , AP (south), Tamri; Koçak and Kemal 2010 ; El Hawagri 2011; Evenhuis and Greathead 2015 Parageron Paramonov, 1929 Parageron gratus (Loew, 1856) = Usia grata Loew, in Timon-David 1951 : 143 Timon-David 1951 , AP , Oued Grou; Mouna 1998 ; Bader and Arabyat 2004 ; Zaitzev 2007 , SA ; Koçak and Kemal 2010 ; El Hawagri 2011; Evenhuis and Greathead 2015 – MISR Parageron griseus Paramonov, 1947 Zaitzev 2007 , SA Parageron hyalipennis (Séguy, 1941) = Oligodranes hyalipennis Séguy, in Séguy 1941d : 9 Séguy 1941d , AA , Agadir (Forêt Admine); Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , HA Parageron incisus (Wiedemann, 1830) = Usia incisa Wiedemann, in Timon-David 1951 : 143 Séguy 1930a , AP , Mogador, Casablanca, Sidi Bettache, Aïn Sferguila, MA , Forêt Zaers, Ras el Ma, Aïn Leuh, HA , Tenfecht; Timon-David 1951 , AP , Rabat, Sehoul, MA , Ifrane; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Parageron major Macquart, 1840 Mouna 1998 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 , AP , Rabat, Nkheila; Evenhuis and Greathead 2015 Usia Latreille, 1802 Usia ( Micrusia ) aurata (Fabricius, 1794) = Usia ( Micrusia ) taeniolata Costa, 1883, in, Koçak and Kemal 2010 Séguy 1930a , Rif , Tanger, AP , Rabat, Sidi Bettache; Paramonov 1950 , Rif , Tanger; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) crispa Gibbs, 2011 Gibbs 2011 , HA , Marrakech, AA , Agadir, Taroudant, Tafraoute, Ouarzazate Usia ( Micrusia ) cryptocrispa Gibbs, 2011 Gibbs 2011 , AP , Ben Slimane, Rabat Usia ( Micrusia ) dilsi Gibbs, 2011 Gibbs 2011 , Rif , Tétouan, Al Hoceima, HA , Taourirt Usia ( Micrusia ) echinus Gibbs, 2011 Gibbs 2011 , AP , Agadir, Guelmim, Sidi Ifni, Cap Ghir, MA , Tafraoute, HA , Marrakech, Tizi-n-Test, AA , Tafingoult Usia ( Micrusia ) falcata Gibbs, 2011 Gibbs 2011 , MA , Azrou, Ifrane Usia ( Micrusia ) forcipata Brullé, 1833 14 Mouna 1998 : 84 Usia ( Micrusia ) globicauda Gibbs, 2011 Gibbs 2011 , AP , Essaouira Usia ( Micrusia ) loewi Becker, 1906 Zaitzev 2007 , Rif , Tanger, AP , Skhirate, Nkheila, MA , Taferiate, HA , Taourirt Usia ( Micrusia ) novakii Strobl, 1902 Séguy 1941d , HA , Tizi-n'Test; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) parascripa Gibbs, 2011 Gibbs 2011 , MA , Ifrane, Mischliffen (2200 m) Usia ( Micrusia ) pusilla Meigen, 1820 Séguy 1930a , AP , Rabat, MA , Azrou, HA , Tafingoult (Goundafa, 1500–1600 m); Séguy 1934b , AP , Rabat (on Calendula ); Séguy 1953a , AP , Cap Ghir; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Micrusia ) versicolor (Fabricius, 1787) Séguy 1930a , AP , Berrchid, Casablanca, M'Rassine; Timon-David 1951 , AP , Rabat, MA , Oulmès; Mouna 1998 ; Koçak and Kemal 2010 ; Gibbs 2011 , HA ; Evenhuis and Greathead 2015 ; EM (Oujda) – MNHNR Usia ( Usia ) aenea (Rossi, 1794) Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 ; Bader and Arabyat 2004 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Usia ( Usia ) angustifrons Becker, 1906 Mouna 1998 Usia ( Usia ) atrata (Fabricius, 1798) = Voluccella atrata Fabricius, in Fabricius 1798: 570 = Usia claripennis Macquart, in Macquart 1840: 105 Meigen 1820 ; Séguy 1930a , Rif , Tanger, MA , Aïn Leuh; Timon-David 1951 , AP , Rabat, Guerrouaou; Mouna 1998 ; Zaitzev 2007 , HA , Tizi-n'Tichka; Koçak and Kemal 2010 ; Gibbs 2014 , AP , Mogador, Arbaa-Sahel (320 m), Tamri (215 m), MA , Khemisset, Oulmès (700 m), Ifrane, El Merabtine, HA , Marrakech, Aït Ourirr (530 m), Oukaimeden (2200 m), Tizi-n'Test (1450 m), Timzit (1700 m), AA , Agadir, Tiznit, Igherm (1660 m), Taroudant, Tata, Iguiour (1260 m), SA , Bou Jarif, Goulimine; Evenhuis and Greathead 2015 Usia ( Usia ) bicolor Macquart, 1855 15 Mouna 1998 : 84 Usia ( Usia ) cornigera Gibbs, 2014 Gibbs 2014 , Rif , Tanger, AP , Sidi Bettache, Rabat, MA , Meknès (550 m), Aïn Leuh (1350 m), HA , Dar Kaid M'tougui Usia ( Usia ) florea (Fabricius, 1794) = Volucella florea Fabricius, in Becker 1906: 203 = Usia cuprea Macquart, 1834, in Becker 1906: 203 Becker 1906a ; Séguy 1930a , Rif , Tanger, AP , Mogador, Sidi Bettache, HA , Tinmel (Goundafa), around (Skoutana); Timon-David 1951 , EM , Oued Moulouya; Mouna 1998 ; Pârvu and Zaharia 2007 ; Koçak and Kemal 2010 ; Gibbs 2014 ; Evenhuis and Greathead 2015 Usia ( Usia ) ignorata Becker, 1906 Becker 1906a ; Mouna 1998 ; Bader and Arabyat 2004 ; Pârvu and Zaharia 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 Usia ( Usia ) maghrebensis Gibbs, 2014 Gibbs 2014 , Rif , Tanger, Tétouan, El Biutz (150 m), AP , Mogador, MA , Aïn Leuh (1350 m) Usia ( Usia ) vestita Macquart, 1846 Mouna 1998 : 84; Gibbs 2011 Phthiriinae Phthiria Meigen, 1802 Phthiria albogilva Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Evenhuis and Greathead 2015 Phthiria gaedii Wiedemann in Meigen, 1820 Séguy 1930a , MA , Foum Keneg; Timon-David 1951 , AP , Zaers, MA , Ifrane; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 – MISR ( MA , Ifrane) Phthiria maroccana Zaitzev, 2005 = Phthiria maroccana Zaitzev, in Zaitzev 2005 : 667 Zaitzev 2005 , MA , Taferiate, HA , Taourirt; Zaitzev 2007 , MA , Taferiate, HA , Taourirt Phthiria merlei Zaitzev, 2005 = Phthiria merlei Zaitzev, in Zaitzev 2005 : 665 Zaitzev 2005 , AP (south), Tamri, Inchaden (south of Aït Melloul); Zaitzev 2007 , AP (south), Tamri, Inchaden (south of Aït Melloul) Phthiria minuta (Fabricius, 1805) Séguy 1930a , HA , Tenfecht, AA , Souss; Mouna 1998 ; Koçak and Kemal 2010 ; El Hawagry 2011 Phthiria pulicaria var. flavofasciata Strobl in Morge, 1976 Mouna 1998 ; Zaitzev 2007 , AA , Tizi-n'Tiniggigt (1600 m); Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Phthiria scutellaris Wiedemann in Meigen, 1820 Séguy 1930a , MA , Meknès; Séguy 1941a , MA , Meknès, HA , Imi-n'Ouaka (1500 m); Mouna 1998 ; Evenhuis and Greathead 2015 ; Rif (Sapinière Talassemtane) – MISR Phthiria simonyi Becker, 1908 Séguy 1949a , SA , Guelmim; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , MA , Meknès Phthiria umbripennis Loew, 1846 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , MA , Meknès Phthiria vagans Loew, 1846 Zaitzev 2007 , HA , Taourirt, AA , Tizi-n'Taratine Toxophorinae Geron Meigen, 1820 Geron intonsus Bezzi, 1925* MA , HA Geron macquarti Greathead in Evenhuis & Greathead 1999 Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 Geron subflavofemoratus Andréu Rubio, 1959 Andréu Rubio 1959 ; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Toxophora Meigen, 1803 Toxophora fasciculata (Villers, 1789) Séguy 1930a , AP , Rabat; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Toxophora fuscipennis (Macquart, 1840) Mouna 1998 : 84 Toxophora pauli Zaitzev, 2005 Zaitzev 2005 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate); Zaitzev 2007 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) Toxophora shelkovnikovi Paramonov, 1933 Zaitzev 2007 , AA , Ouarzazate Heterotropinae Heterotropus Loew, 1873 Heterotropus atlanticus Séguy, 1930 Séguy 1930a , AP , Mogador; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Heterotropus longitarsus Séguy, 1930 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Heterotropus maroccanus Zaitzev, 2003 Zaitzev 2003 , AA , Jebel Tighermine (SE of Ouarzazate); Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Bombyliinae Anastoechus Osten-Sacken, 1877 Anastoechus bahirae Becker, 1915 Mouna 1998 ; Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Anastoechus hyrcanus Pallas & Wiedemann in Wiedemann, 1818 Mouna 1998 : 84 Anastoechus latifrons (Macquart, 1839) Timon-David 1951 , AP , Dradek; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Anastoechus nitidulus ssp. nitidulus Fabricius, 1794 Mouna 1998 : 84 Anastoechus stramineus Wiedemann in Meigen, 1820 Mouna 1998 : 84 Anastoechus trisignatus (Portschinsky, 1881) Bader and Arabyat 2004 ; Ziatzev 2007, AP , Rabat, AA , Jebel Tighermine (SE of Ouarzazate), AA , 20 km SW Goulmima, SA , Tan-Tan, Between Guelmim and Tan-Tan (90 km from Guelmim), Taganint (south of Bou-Izakarn); Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombomyia Greathead, 1995 Bombomyia discoidea (Fabricius, 1794) Séguy 1930a , AP , Oued Korifla (Zaers), Sidi Bettache; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombomyia stictica Boisduval, 1835 Zaitzev 2007 , MA , col de Zeggota (N Meknès), Oulmès Bombomyia vertebralis (Dufour, 1833) = Bombylius punctatus Fabricius, in Timon-David 1951 : 144 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a AP , Dradek near Rabat; Timon-David 1951 , MA , Volubilis; Mouna 1998 ; Bader and Arabyat 2004 ; Koçak and Kemal 2010 Evenhuis and Greathead 2015 ; AP (Dradek, Casablanca), EM (Oujda), MA (Volubilis, Aïn Leuh) – MISR Bombylisoma Rondani, 1856 Bombylisoma algirum (Macquart, 1840) = Bombylius nigrifrons Becker, in Becker and Stein 1913 : 83 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylisoma breviusculum (Loew, 1855) Dils and Özbek 2006 ; Zaitzev 2007 ; Evenhuis and Greathead 2015 Bombylisoma flavibarbum Loew, 1855 Mouna 1998 : 84 Bombylisoma melanocephalum Fabricius, 1794 Zaitzev 2007 , HA , Taourirt, south of Tizi-n'Test Bombylius Linnaeus, 1758 Bombylius ( Bombylius ) albaminis Séguy, 1949 Séguy 1949a , HA , Alnif; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) ambustus Pallas & Wiedemann, 1818 Mouna 1998 : 84 Bombylius ( Bombylius ) analis (Olivier, 1789) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Oued Korifla, Rabat, Sidi Bettache, Aïn Sferguila; Timon-David 1951 , AP , Rabat; Mouna 1998 ; Zaitzev 2007 , MA , route Fès-Sidi Kacem (30 km from Fès); Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , AP (Rabat, Casablanca) – MISR Bombylius ( Bombylius ) audcenti Bowden, 1984 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) canescens Mikan, 1796 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , Rif , Cap Malabata (Tanger), MA , Tachguelt, route Fès-Sidi Kacem (30 km from Fès) Bombylius ( Bombylius ) cinerascens Mikan, 1796 Mouna 1998 ; Bader and Arabyat 2004 Bombylius ( Bombylius ) discolor Mikan, 1796 Mouna 1998 ; Zaitzev 2007 , MA , route El Hajeb-Ifrane (1 km from Ifrane) Bombylius ( Bombylius ) eploceus Séguy, 1949 Séguy 1949a , SA , Guelmim; Mouna 1998 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) fimbriatus Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, Jebel Ahmar (1700 m); Mouna 1998 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) flavipes Wiedemann, 1828 Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) fulvescens Wiedemann in Meigen, 1820 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AP , Cap Ghir; Séguy 1941d , AA , Agadir; Mouna 1998 Bombylius ( Bombylius ) fuscus Fabricius, 1781 Mouna 1998 : 84 Bombylius ( Bombylius ) major (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , MA , Oulmès; Bader and Arabyat 2004 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Kénitra) – MISR Bombylius ( Bombylius ) mauritanus Olivier, 1789 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 , HA Bombylius ( Bombylius ) medius (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Timon-David 1951 , AP , Sehoul; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Pârvu and Zaharia 2007 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Oued Yquem, Dradek) – MISR Bombylius (Unplaced) megacephalus Portschinsky, 1887* EM , AA Bombylius ( Bombylius ) minor Linnaeus, 1758 Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; MA (Aguelmane Azigza), SA – MISR Bombylius ( Bombylius ) mus Bigot, 1862 Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) niveus Meigen, 1804 Mouna 1998 : 84; AP (Mogador) – MISR Bombylius ( Bombylius ) numidus Macquart, 1846 Séguy 1953a , MA , Ifrane; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) pauli Zaitzev, 2003 Zaitzev 2003 , MA , route Fès-Sidi Kacem (30 km from Fès); Zaitzev 2007 , MA , route Fès-Sidi Kacem (30 km from Fès) Bombylius ( Bombylius ) posticus (Fabricius, 1805) Dils and Özbek 2006 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) postversicolor Evenhuis & Greathead, 1999 = Bombylius versicolor Fabricius, 1805 Meigen 1820 ; Bezzi 1906 ; Séguy 1930a , AP , Mogador; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) pumilus Meigen, 1820 Mouna 1998 : 84 Bombylius ( Bombylius ) semifuscus (Meigen, 1820) Séguy 1953a , AP , Cap Ghir; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) torquatus Loew, 1855 Séguy 1930a , HA , Ouaounzert (Glaoua), Arround (Skoutana), Tachdirt (bord de l'Imminen, 2400–2600 m); Timon-David 1951 , AP , Rabat; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Mogador) – MISR Bombylius ( Bombylius ) undatus Mikan, 1796 Pârvu and Zaharia 2007 Bombylius ( Bombylius ) vagans Meigen, 1830 Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Bombylius ( Bombylius ) venosus Mikan, 1796 Mouna 1998 ; Zaitzev 2007 , AP , El Koudia (30 km SW from Rabat); AP (Dradek) – MISR Bombylius ( Zephyrectes ) cruciatus Fabricius, 1798 Séguy 1930a , MA , Aharmoumou (1100 m), Azrou, Ras el Ma, HA , Tizi-n'Test, Jebel Imdress (Goundafa, 2000–2450 m); Mouna 1998 ; Zaitzev 2007 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 ; Rif (Talassemtane), AP (Mogador), MA (Sefrou) – MISR Bombylius ( Zephyrectes ) leucopygus (Macquart, 1846) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; Zaitzev 2007 , AP , Larache, MA , Moulay Idris (900 m); Evenhuis and Greathead 2015 , SA , Erfoud; MA (Ifrane) – MISR Conophorus Meigen, 1803 Conophorus bellus Becker, 1906* HA Conophorus fuliginosus (Wiedemann in Meigen, 1820) = Ploas fuliginisa (Meigen), in Séguy 1953a : 83 Séguy 1930a , MA , Aharmoumou (1100 m); Séguy 1953a , MA , Ahermoumou (1100 m); Timon-David 1951 , AP , Dradek, MA , Sefrou, HA , Marrakech; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger; Evenhuis and Greathead 2015 ; AP (Salé, Mogador) – MISR Conophorus fuscipennis (Macquart, 1840) Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Mouna 1998 ; Evenhuis and Greathead 2015 Conophorus griseus (Fabricius, 1787) Mouna 1998 ; Zaitzev 2007 ; Evenhuis and Greathead 2015 Conophorus hamilkar Paramonov, 1929 Timon-David 1951 , AP , Mogador; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Mogador) – MISR Conophorus macroglossus (Dufour, 1852) Mouna 1998 ; Zaitzev 2007 ; Evenhuis and Greathead 201; AP (Mogador) – MISR Conophorus mauritanicus Bigot, 1892 = Conophorus heteropilosus Timon-David, in Timon-David 1951 : 141; Mouna 1998 : 84; Evenhuis and Greathead 2015 : 192 Timon-David 1951 , MA , Oulmès; Mouna 1998 ; Zaitzev 2007 , AP , El Koudia (30 km SW from Rabat), Forêt Zaer (35 km SW from Rabat), N Tretten; Koçak and Kemal 2010 ; Dils 2013 , MA , Mrirt; Evenhuis and Greathead 2015 Conophorus rossicus Paramonow, 1929 Dils and Özbek 2006 Dischistus Loew, 1855 Dischistus albatus (Séguy, 1934) = Acanthogeron albatus Séguy, 1934, in Séguy 1934d : 73; Zaitzev 2007 : 162 Séguy 1934d ; Zaitzev 2007 , SA , 30 km S Tata Dischistus auripilus (Séguy, 1930) = Acanthogeron auripilus Séguy, 1930, in Séguy 1930a : 104 Séguy 1930a , AP , Mogador; Séguy 1934b , AP , Zaers; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Evenhuis and Greathead 2015 Dischistus maroccanus (Séguy, 1930) = Acanthogeron maroccanus Séguy, 1930, in Séguy 1930a : 106 Séguy 1930a , AP , Mogador; Mouna 1998 ; Zaitzev 2007 , HA , Tazzarine; Evenhuis and Greathead 2015 Dischistus mittrei (Séguy, 1930) = Acanthogeron mittrei Séguy, in Séguy 1930a : 105 Séguy 1930a , AP , Mogador; Mouna 1998 ; Evenhuis and Greathead 2015 Dischistus perniveus (Bezzi, 1925) = Acanthogeron perniveus Bezzi, in Timon-David 1951 : 143 Timon-David 1951 , AP , Djamda de M'Tal; Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Dischistus senex (Wiedemann in Meigen, 1820) = Acanthogeron senex Meigen, 1820, in Séguy 1953a : 83, Mouna 1998 : 84, Zaitzev 2007 : 162 Séguy 1930a , HA , Tafingoult (Goundafa, 1500–1600 m); Villeneuve 1933 ; Séguy 1953a , HA , Aït Ourir; Mouna 1998 ; Zaitzev 2003 , HA , Taourirt; Zaitzev 2007 , HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Dradek), MA (Sefrou) – MISR Dischistus separatus (Becker, 1906) = Acanthogeron talboti Séguy, 1930, in Séguy 1930a : 106 Evenhuis and Greathead 2015 Efflatounia Bezzi, 1925 Efflatounia berbera Bowden, 1973 Ebejer et al. 2019 , AA , Agadir – NMWC Legnotomyia Bezzi, 1902 Legnotomyia fascipennis Bezzi, 1925* SA Merleus Zaitzev, 2003 Merleus punctipennis Zaitzev, 2003 = Merleus punctipennis Zaitzev 2003 : 599 Zaitzev 2003 , AP , Skhirate; Zaitzev 2007 , AP , Skhirate Prorachthes Loew, 1869 Prorachthes crassipalpis Villeneuve, 1930 Evenhuis and Greathead 2015 Systoechus Loew, 1855 Systoechus ctenopterus (Mikan, 1796) Timon-David 1951 , MA , Ifrane; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2007 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Systoechus gomezmenori Andréu Rubio, 1959 Carles-Tolrá 2002 ; Evenhuis and Greathead 2015 Systoechus gradatus (Wiedemann in Meigen, 1820) Timon-David 1951 , AP , Mouldikht; Mouna 1998 ; Zaitzev 2007 , MA , Taferiat, HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 Systoechus mixtus Wiedemann, 1821 = Bombylius stylicornis Macquart in Séguy 1941: 10 Séguy 1941d , AA , Agadir; Mouna 1998 Systoechus pumilio Becker, 1915 Mouna 1998 : 84 Triplasius Loew, 1855 Triplasius boghariensis (Lucas, 1852) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , EM , Oujda; Mouna 1998 ; Pârvu and Zaharia 2007 ; Evenhuis and Greathead 2015 Triplasius maculipennis (Macquart, 1846) = Bombylius maculipennis var. melanopus Timon-David, in Timon-David 1951 : 144 = Bombylius ( Triplasius ) maculipennis Macquart, 1849, in Zaitzev 2007 : 166 Timon-David 1951 , MA , Azrou; Zaitzev 2007 , MA , route El Hachef, Criosement route Raubei Idris-Merhassine; Evenhuis and Greathead 2015 Ecliminae Eclimus Loew, 1844 Eclimus gracilis Loew, 1844 Séguy 1930a , MA , Ras el Ma; Timon-David 1951 , AP , Oued Korifla; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2007 , MA , Maaziz; Evenhuis and Greathead 2015 Thevenetimyia Bigot, 1875 Thevenetimyia quedenfeldti (Engel, 1885)* Rif , AP , MA Crocidiinae Crocidium Loew, 1860 Crocidium aegyptiacum Bezzi, 1925* SA Crocidium nudum Efflatoun, 1945* EM , AA Semiramis Becker in Becker and Stein 1914 Semiramis punctipennis Becker, 1914 Zaitzev 2007 , AA , Aoulouz Cythereinae Amictus Wiedemann, 1817 Amictus castaneus (Macquart, 1840) Séguy 1930a , AP , Rabat, HA , Ank el Djemal; Mouna 1998 ; Evenhuis and Greathead 2015 Amictus compressus (Fabricius, 1805) Evenhuis and Greathead 2015 Amictus heteropterus Macquart, 1838 Zaitzev 2007 , Rif , Tanger, AP , Rabat, HA , S Tizi-n'Test, AA , Tizi-n'Taratine Amictus oblongus (Fabricius, 1805) = Bombylius oblongus Fabricius, in Macquart 1834: 390 Macquart 1834 Amictus pulchellus Macquart, 1846 Séguy 1930a , AP , Rabat, Maâmora; Mouna 1998 ; Zaitzev 2007 , HA , Taourirt; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Amictus setosus Loew, 1869* AP Amictus tener Becker, 1906 Zaitzev 2007 , AP , Rabat Amictus validus Loew, 1869 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Amictus variegatus Meigen in Waltl, 1835 Mouna 1998 : 84 Chalcochiton Loew, 1844 Chalcochiton argentifrons (Macquart in Lucas, 1849) Séguy 1953a , AP , Cap Ghir, Salé, Sidi Battache, MA , Tizi-n'Bou Zabal (2300 m), AA , Aïn Chaïb (Souss); Evenhuis and Greathead 2015 Chalcochiton argyrocephalus (Macquart, 1840) = Chalcochiton ( Anthrax ) argyrocephala (Macquart), in Engel 1938 : 328 Engel 1938 ; Séguy 1953a , AA , Agadir; El Hawagry 2011 ; Evenhuis and Greathead 2015 Chalcochiton atlantica Dils, 2008 Dils 2008 , SA , Guelmim Chalcochiton holosericeus (Fabricius, 1794) = Chalcochiton semiargentaea Macquart, in Zaitsev 2007: 172 Séguy 1930a , AP , Maâmora, Sidi Bettache, HA , Tizi-n'Test, Jebal Imdress (2000–2450 m), Tafingoult (Goundafa, 1500–1600 m); Séguy 1941d ; Mouna 1998 ; Zaitzev 2007 , Rif , Tanger, AP , Skhirate, EM , Taourirt, MA , Taferiat, Meknès-Moulay Idriss, Merhassine, AA , Agadir, Ouarzazate; Evenhuis and Greathead 2015 ; AP (Salé, Forêt Temara, Oued Yquem, Meshra) – MISR Chalcochiton maghrebi Dils, 2017 Dils 2017 , Rif , Souk El Kolla, Bab Taza, 10 km S of Mjara, AP , Sidi Bettache, Temsia, Imsouane, Mansouria, Rommani, Béni Slimane, Tioulit, EM , El Aioun, MA , Béni Mellal, el Ksiba, 10 km SE Bir Tamtam, Merchouch, Mrirt, Fès, HA , Azilal, Asni, Tizi-Mlil, AA , Taroudant, Tizi-n'Test, Tiznit, Agouim, Sidi Ifni, Mesti, Tafinegoult, Tizi-n'Tinififft, El Mrabtine, SA , Semara Chalcochiton maroccanus Zaitzev, 2006 Séguy 1953a , HA , Tafingoult (Goundafa, 1500–1600 m); Zaitzev 2006 , AP (south), Aït Melloul; Zaitzev 2007 , AP (south), Aït Melloul Chalcochiton merlei Zaitzev, 2006 Zaitzev 2006 , AP , Skhirate; Zaitzev 2007 , AP , Skhirate Chalcochiton pallasii Loew, 1856 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2007 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Callostoma Macquart, 1840 Callostoma fascipenne Macquart, 1840 Bader and Arabyat 2004 Cyllenia Latreille, 1802 Cyllenia rustica Rossi, 1790 Mouna 1998 ; Zaitzev 2007 ; AP (Mogador) – MISR Cytherea Fabricius, 1794 Cytherea albolineata Bezzi, 1925* SA Cytherea alexandrina Becker, 1902 Becker 1902 : 30; Zaitzev 2007 , AA , Jebel Tighermine (SE of Ouarzazate) Cytherea aurea (Fabricius, 1794) Séguy 1930a , AP , Rabat, HA ; Mouna 1998 ; Bader and Arabyat 2004 ; Zaitzev 2007 , AA , Tizi-n'Taratine; El Hawagry 2011 ; Evenhuis and Greathead 2015 , HA , Tafingoult (Goundafa, 1500–1600 m); AP (Rabat, Oued Cherrat) – MISR Cytherea cinerea Fabricius, 1805 = Mulio delicatus Becker, 1906 Becker 1906b : 153; Timon-David 1951 , AP , Meshra; Mouna 1998 ; Bader and Arabyat 2004 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Cytherea delicata Becker, 1906 Zaitzev 2007 , HA , S Tizi-n'Test, AA , Zagora, Taroudant, Tizi-n'Taratine Cytherea dispar (Loew, 1873) Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Cytherea fenestrata (Loew, 1873) Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Cytherea infuscata (Meigen, 1820) Séguy 1930a , EM , Itzr (Haute Moulouya), MA , Forêt Timelilt (1900 m), HA , Aït el Hadj, Marrakech; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Meskara) – MISR Cytherea maroccana (Becker, 1903) = Mulio maroccanus Becker, in Becker 1903 : 89 Becker 1903 , Rif , Tanger; Bezzi 1906 : 249; Timon-David 1951 , AP , Azemmour; Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Cytherea obscura Fabricius, 1794 Séguy 1930a , EM , Haute Moulouya, AP , Sidi Bettache, HA , Ouaouenzert; Séguy 1941d ; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2007 , MA , Taferiat, AA , Agadir, Amredi, Jebel Tighermine (SE of Ouarzazate), Tizi-n'Tiniggigt, Tizi-n'Taratine, Tizi-n'Bachkoun; Karimpour 2012 ; Evenhuis and Greathead 2015 Cytherea rungsi Timon-David, 1951 Timon-David 1951 , EM , Guenfouda; Mouna 1998 ; Evenhuis and Greathead 2015 Cytherea thyridophora (Bezzi, 1925) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Jebel Bouhachem, 965 m) Cytherea trifaria (Becker, 1906) Evenhuis and Greathead 2015 Lomatiinae Lomatia Meigen, 1820 Lomatia abbreviata Villeneuve, 1911 Séguy 1930a , MA , Forêt Zaers; Timon-David 1951 , EM , Guercif; Mouna 1998 ; Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 ; AP (Maâmora, Oued Cherrat, Dradek), HA – MISR Lomatia belzebul paramonovi Fabricius, 1794 Séguy 1930a , AP , Dar Salem, MA , Timhadit, Meknès, Aïn Leuh; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Lomatia erynnis (Loew, 1869) Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 , AP , Rabat; Evenhuis and Greathead 2015 Lomatia hamifera Becker, 1915 Mouna 1998 : 84 Lomatia lachesis Egger, 1859 Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Lomatia lateralis (Meigen, 1820) Séguy 1930a , MA , Ras el Ma, HA , Forêt Timelilt; Mouna 1998 ; Evenhuis and Greathead 2015 ; AP (Rabat), MA (Volubilis, Ras el Ma) – MISR Lomatia obscuripennis Loew, 1869 Zaitzev 2008 , AP , Nkheila; Evenhuis and Greathead 2015 Lomatia sabaea (Fabricius, 1781) Mouna 1998 : 84 Lomatia tysiphone Loew, 1860 Zaitzev 2008 , MA , Azrou, AA , Tizi-n'Taratine Antoniinae Antonia Loew, 1856 Antonia bouillonae Séguy, 1932 Evenhuis and Greathead 2015 Anthracinae Aphoebantini Aphoebantus Loew, 1872 Aphoebantus wadensis Becker, 1925* SA Anthracini Anthrax Scopoli, 1763 Anthrax aethiops (Fabricius, 1781) Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 ; AP (Forêt Maâmora) – MISR Anthrax anthrax (Schrank, 1781) = Argyramoeba anthrax Schrank, in Séguy 1930a : 93 Séguy 1930a , MA , Aïn Leuh, Soufouloud (1900–2100 m), HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Timon-David 1951 , MA , El Ksiba, Ifrane; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Anthrax binotatus (Wiedemann in Meigen, 1820) = Argyramoeba binotata Meigen, in Séguy 1926 : 209, Séguy 1930a : 94 Séguy 1926 ; Séguy 1930a , AP , Rabat, HA , Tizi-n'Test, Jebel Imdress (2000–2450 m); Séguy 1949a , HA , Alnif; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Rabat) – MISR Anthrax dentatus (Becker, 1906) Bader and Arabyat 2004 ; Zaitzev 2008 , AA , Tizi-n'Tiniggigt; El Hawagry 2011 ; Evenhuis and Greathead 2015 Anthrax hemimelas Speiser, 1910 Zaitzev 2008 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) Anthrax kiritshenkoi Paramonov, 1935 Evenhuis and Greathead 2015 Anthrax lucidus (Becker, 1902) Ebejer et al. 2019 , AA , Ziz river (13 km N of Erfoud, 800 m) Anthrax morio Fabricius, 1775 Mouna 1998 ; MA (Ifrane, Azrou) – MISR Anthrax trifasciatus (Meigen, 1804) = Argyramoeba trifasciata Meigen, in Timon-David 1951 : 139 Séguy 1930a , MA , Meknès; Timon-David 1951 , AP , south of Rabat; Mouna 1998 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Anthrax varius Fabricius, 1794 Séguy 1930a , AP , Rabat; Mouna 1998 ; Evenhuis and Greathead 2015 – MISR Anthrax virgo Egger, 1859 = Argyramoeba virgo Egger, in Séguy 1930a : 94 Séguy 1930a , AP , Rabat; Zaitzev 2008 , MA , Taferiat, AA , Jebel Tighermine (SE of Ouarzazate) Cononedys Hermann, 1907 Cononedys efflatouni (Bezzi, 1925)* SA Cononedys escheri Bezzi, 1908 Zaitzev 2008 , AP , Skhirate, Rabat Cononedys lyneborgi (François, 1969) Evenhuis and Greathead 2015 Cononedys scutellatus Meigen, 1835 Zaitzev 2008 , Rif , Jebala, Haouta el Kazdir, AA , Aouzlida near Aoulouz Satyramoeba Sack, 1909 Satyramoeba hetrusca (Fabricius, 1794) Mouna 1998 : 84 Spogostylum Macquart, 1840 Spogostylum isis (Meigen, 1820) Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Spogostylum trinotatum Dufour, 1852 Mouna 1998 : 84 Spogostylum tripunctatum (Pallas in Wiedemann, 1818) Timon-David 1951 , HA , Aït Mhamed Sgatt; Mouna 1998 ; Dils and Özbek 2006 ; Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate); El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 ; HA (Aïn Mhamed Sgatt) – MISR Turkmeniella Paramonov, 1940 Turkmeniella crosi (Villeneuve, 1910) Evenhuis and Greathead 2015 Exoprosopa Macquart, 1840 Exoprosopa aeacus Meigen, 1804 Mouna 1998 : 84 Exoprosopa baccha Loew, 1869 Mouna 1998 ; Zaitzev 1999 ; Dils and Özbek 2006 ; Zaitzev 2008 ; Evenhuis and Greathead 2015 Exoprosopa capucina (Fabricius, 1871) Mouna 1998 : 84 Exoprosopa circeoides Paramonov, 1928 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate) Exoprosopa cleomene Egger, 1859 Mouna 1998 : 84 Exoprosopa decrepita (Wiedemann, 1828) Zaitzev 2008 , AA , Zagora Exoprosopa efflatouni Bezzi, 1925 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate), Ouarzazate, SA , Taganint (south of Bou-Izakarn) Exoprosopa grandis Wiedemann in Meigen, 1820 Mouna 1998 ; Zaitzev 2008 , HA , Tishka (2200 m) Exoprosopa italica (Rossi, 1794) Zaitzev 2008 , HA , Taourirt, AA , Tizi-n'Taratine, Jebel Tighermine (SE of Ouarzazate), SA , Taganint (south of Bou-Izakarn) Exoprosopa jacchus (Fabricius, 1805) Séguy 1930a , AP , Mogador, Sidi Taibi, MA , Tizi-s'Tkrine (1700 m), Dar Salem, Aïn Leuh, HA , Bou Tazzert; Mouna 1998 ; Mirceni and Pârvu 2009 ; Evenhuis and Greathead 2015 ; Rif (Talassemtane, Forêt Izarine, road of Jebha, Zoumi) – MISR Exoprosopa minos (Meigen, 1804) Séguy 1949a , AA , Tata; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2008 , MA , Taferiat; El Hawagry 2011 ; El Hawagry and Dhafer 2015 ; Evenhuis and Greathead 2015 ; MA (Jebel Lachhab) – MISR Exoprosopa pandora (Fabricius, 1805) Greathead 2001 ; Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Exoprosopa rutila (Pallas & Wiedemann, 1818) Evenhuis and Greathead 2015 Micomitra Bowden, 1964 Micomitra stupida Rossi, 1790 = Exoprosopa stupida Rossi, in Mouna 1998 : 84 Mouna 1998 Plesiocera Macquart, 1840 Plesiocera algira (Macquart, 1840) Zaitzev 2008 , MA , Taferiat; Evenhuis and Greathead 2015 Heteralonia Rondani, 1863 Heteralonia ( Homolonia ) megerlei (Hoffmansegg in Wiedemann, 1818) Zaitzev 2008 , SA , Goulimine Heteralonia ( Mesoclis ) pygmalion (Fabricius, 1805) = Exoprosopa pygmalion Fabricius, in Timon-David 1951 : 139 = Mesoclis pygmalion Fabricius, 1805, in Zaitzev 2008 : 191 Séguy 1930a , Rif , Tanger, AP , Maâmora, Rabat, MA , Aïn Leuh; Timon-David 1951 , AP , Temara; Mouna 1998 ; Zaitzev 2008 , AP , Cherrat El Hawagry 2011 ; Evenhuis and Greathead 2015 Heteralonia ( Zygodipla ) algira (Fabricius, 1794) Séguy 1930a , Rif , Tanger, AP , Mogador, HA , Bou Tazzert; Mouna 1998 ; Zaitzev 2008 , HA , Tifnite (south of Aït Melloul); El Hawagry 2011 ; Evenhuis and Greathead 2015 Heteralonia ( Zygodipla ) bagdadensi s (Macquart, 1840) Zaitzev 2008 , AA , Zagora Heteralonia ( Zygodipla ) singularis (Macquart, 1840) Bader and Arabyat 2004 ; Evenhuis and Greathead 2015 Heteralonia arenacea Becker, 1906 Evenhuis and Greathead 2015 Heteralonia dispar (Loew, 1869) = Exoprosopa dispar Loew, in Timon-David 1951 : 139 Timon-David 1951 , HA , Marrakech; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 Heteralonia rivularis (Meigen, 1820) = Exoprosopa rivularis Meigen, in Timon-David 1951 : 139 Séguy 1930a , AP , Rabat, Maâmora; Timon-David 1951 , AP , Oued Akreuch; Mouna 1998 ; Zaitzev 1999 ; Bader and Arabyat 2004 ; Zaitzev 2008 , AP , Rabat Oestranthrax Bezzi, 1921 Oestranthrax brunnescens (Loew, 1857) Bader and Arabyat 2004 Oestranthrax pallifrons Bezzi, 1926 Evenhuis and Greathead 2015 Pachyanthrax François, 1964 Pachyanthrax albosegmentatus (Engel, 1936) Zaitzev 2008 , AA , Jebel Tighermine (south of Ouarzazate) Pachyanthrax nomadorum (Greathead, 1970) Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Exhyalanthrax Becker, 1916 Exhyalanthrax afer (Fabricius, 1794) = Anthrax tangerinus Bigot, 1892 Bezzi 1906 ; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Zaitzev 2008 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 , Rif , Tanger; MA (Ifrane) – MISR Hemipenthes Loew, 1869 Hemipenthes morio (Linnaeus, 1758) Séguy 1930a , MA , Azrou, HA , Arround (Skoutana, 2000–2400 m); Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 ; MA (Azrou, Ifrane) – MISR Hemipenthes velutinus (Meigen, 1820) Séguy 1930a , MA , Azrou; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Thyridanthrax Osten-Sacken, 1886 Thyridanthrax alphonsi Sánchez Terrón and Roldan Bravo, 2000 Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax elegans ssp. elegans (Wiedemann in Meigen, 1820) Séguy 1930a , AP , Rabat; Mouna 1998 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Oued Cherrat, Rabat), MA (Volubilis) – MISR Thyridanthrax fenestratus (Fallén, 1814) Séguy 1926 ; Séguy 1930a , EM , Berkane (1350–1400 m); Mouna 1998 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; Rif (Tomorot) – MISR Thyridanthrax griseolus Klug, 1832 Zaitzev 2008 , SA , Taganint (south of Bou-Izakarn) Thyridanthrax hispanus (Loew, 1869) Becker and Stein 1913 , Rif , Tanger; Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax incanus (Klug, 1832) = Anthrax incana Klug, 1832, in Séguy 1953a : 83 Séguy 1930a , AP , Oued Korifla (Zaers); Timon-David 1951 , AP , Zaer; Séguy 1953a , MA , Tarda; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Thyridanthrax loustaui Andréu Rubio, 1961 Sánchez Terrón and Roldan Bravo 2000 Thyridanthrax maroccanus Dils, 2012 Dils 2012 , AA , Ouarzazate, Skoura (1250 m), Amerzgane (1350 m) Thyridanthrax mutilus (Loew, 1869)* AA Thyridanthrax nebulosus (Dufour, 1852) Becker and Stein 1913 , Rif , Tanger; Andréu Rubio 1959 ; Mouna 1998 ; Sánchez Terrón and Roldan Bravo 2000 , Rif , Benibuifrur, Melilla, Restinga; Evenhuis and Greathead 2015 Thyridanthrax perspicillaris ssp. perspicillaris (Loew, 1869) Séguy 1930a , MA , Aïn Leuh, Forêt Azrou, HA , Tizi-n'Test, Jebel Imdress (2000–2450 m), Goundafa; Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 Thyridanthrax polyphemus (Hoffmansegg, 1819) Séguy 1930a , MA , Volubilis (400 m); Mouna 1998 ; Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Veribubo Evenhuis, 1978 Veribubo angusteoculatus (Becker, 1902) Zaitzev 2008 , AA , Zagora Veribubo saudensis (François, 1970)* AA Veribubo tabaninus (François, 1970)* AA , SA Villa Lioy, 1864 Villa brunnea Becker, 1916 Mouna 1998 : 84 Villa ceballosi Andréu Rubio, 1959 Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa cingulata Meigen, 1804 Mouna 1998 ; AP (Rabat, Casablanca), MA (Volubilis, Fès) – MISR Villa distincta (Meigen in Waltl, 1835) Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa fasciata (Meigen, 1804) = Villa circumdata (Meigen), in Séguy 1941a : 29 Séguy 1930a , AP , Rabat; Séguy 1941a , AP , Rabat, HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa hottentotta (Linnaeus, 1758) = Anthrax hottentotus Linnaeus, in Séguy 1926 : 198, Séguy 1930a : 92, Bléton and Fleuzet 1939: 64 Séguy 1930a , AP , Rabat, MA , Aïn Leuh; Bléton and Fleuzet 1939, MA , Fès; Séguy 1941d , HA , Tizi-n'Test; Mouna 1998 ; Dils and Özbek 2006 ; Evenhuis and Greathead 2015 – MISR Villa ixion (Fabricius, 1794) Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Villa laevis Becker, 1915 Bader and Arabyat 2004 ; Dils and Özbek 2006 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa leucostoma (Meigen, 1820) Mouna 1998 : 84; AP (Bou-Regreg) – MISR Villa luculenta Séguy, 1941 Séguy 1941d , AA , Taroudant; Mouna 1998 ; Koçak and Kemal 2010 ; Evenhuis and Greathead 2015 Villa niphobleta (Loew, 1869) Bader and Arabyat 2004 ; Koçak and Kemal 2010 ; Karimpour 2012 ; Evenhuis and Greathead 2015 Villa venusta (Meigen, 1820) Mouna 1998 : 84 Desmatoneura Williston, 1895 Desmatoneura albifacies (Macquart, 1840) Ebejer et al. 2019 , AA , Merzouga (714 m) Desmatoneura flavifrons Becker, 1915 Zaitzev 2008 , AA , Ouarzazate, Taroudant, Jebel Tighermine (SE of Ouarzazate) Petrorossia Bezzi, 1908 Petrorossia albula Zaitzev, 1962 Zaitzev 1999 ; Bader and Arabyat 2004 ; Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate); El Hawagry 2011 ; Evenhuis and Greathead 2015 Petrorossia freidbergi Zaitzev, 1999 Zaitzev 2008 , AA , Jebel Tighermine (SE of Ouarzazate) Petrorossia hespera (Rossi, 1790) Séguy 1949a , AA , Tata; Mouna 1998 ; Zaitzev 1999 ; Dils and Özbek 2006 ; El Hawagry 2011 ; Evenhuis and Greathead 2015 ; AP (Bou-Regreg), MA (Timahdit) – MISR Petrorossia margaritae Zaitzev, 1999 Zaitzev 2008 , AA , Ouarzazate, Jebel Tighermine (SE of Ouarzazate) New records for Morocco Amictus setosus Loew, 1869 Atlantic Plain: Rommani, Marmouch, 33.568°N, 06.533°W , 400 m, 1♂1♀, Dils J.- Faes J., coll: PCJD . Aphoebantus wadensis Becker, 1925 Sahara: Tata, 9 km. W Tissint, 29.851°N, 07.265°W , 535 m, 1♂1♀, 03.iii.2007, Dils J.- Faes J., coll: PCJD . Bombylius (Unplaced) megacephalus Portschinsky, 1887 Eastern Morocco: Figuig, Abbou Lakhal, 32.1587°N, 01.507°W , 1050 m, 1♀, 07.iii.2009, Dils J.- Faes J., coll: PCJD . Anti Atlas: Tiznit, 84 km. SSE Guelmim, 28.631°N, 10.75522°W , 235 m, 1♂, 27.ii.2007, Dils J.- Faes J., coll: PCJD ; Tiznit, Abaynou, 29.057°N, 10.026°W , 360 m, 1♀, 13.iii.2009, Dils J.- Faes J., coll: PCJD . Cononedys efflatouni Bezzi, 1925 Sahara: Guelmim, Souk Tnine Nouaday, 29.166°N, 09.279°W , 680 m, 2♂3♀, 07.iv.2015, Dils J.- Faes J., coll: PCJD . Conophorus bellus Becker, 1906 High Atlas: Marrakech, Oukaimeden, 31.233°N, 07.817°W , 2200 m, 3♂, 06.iv.2006, Dils J.- Faes J., coll: PCJD . Crocidium aegyptiacum Bezzi, 1925 Anti Atlas: Tiznit, Mesti, 29.274°N, 10.139°W , 280 m, 1♂, 23.iii.2006, Dils J.- Faes J., coll: PCJD . Sahara: Tata, 28 km E of Tachjicht, 29.106°N, 09.149°W , 700 m, 1♀, 02.iii.2007, Dils J.- Faes J., coll: PCJD . Crocidium nudum Efflatoun, 1945 Eastern Morocco: Oujda, Plateau du Rekkam, 33.839°N, 02.55781°W , 1150 m, 1♀, 25.iv.2010, Dils J.- Faes J., coll: PCJD . Anti Atlas: Agadir, Imsouane, 30.885°N, 09.780°W , 270 m, 3♂13♀, 09.iv.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.024°N, 07.223°W , 1370 m, 17♂12♀, 31.iii.2006, Dils J.- Faes J., coll: PCJD ; Taliouine, 18 km. W of Taliouine, 30.6003°N, 08.082°W , 900 m, 2♀, 24.iii.2009, Dils J.- Faes J., coll: PCJD ; Taroudant, Tafinegoult, 30.734°N, 08.430°W , 680 m, 3♀, 24.iii.2009, Dils J.- Faes J., coll: PCJD ; Tiznit, Arbaa Sahel, 29.657°N, 09.869°W , 320 m, 11♂26♀, 21.iii.2006, Dils J.- Faes J., coll: PCJD . Cytherea albolineata Bezzi, 1925 Sahara: Guelmim, Tainzirt, 29.121°N, 09.333°W , 670 m, 1♀, 31.iii.2010, Dils J.- Faes J., coll: PCJD . Geron intonsus Bezzi, 1925 Middle Atlas: Khenifra, Boulôjoul, 32.873°N, 04.945°W , 1500 m, 7♂10♀, 26.iv.2008, Dils J.- Faes J., coll: PCJD . High Atlas: Midelt, 32.680°N, 04.677°W , 1400 m, 2♂2♀, 20.iv.2015, Dils J.- Faes J., coll: PCJD ; Midelt, Zeïda, 32.781°N, 04.964°W , 1500 m, 9♂11♀, 24.iv.2015, Dils J.- Faes J., coll: PCJD . Legnotomyia fascipennis Bezzi, 1925 Anti Atlas: Zagora, Tazarinne, 30.798°N, 05.584°W , 900 m, 1♂, 07.iii.2007, Dils J.- Faes J., coll: PCJD . Sahara: Tata, 9 km W of Tissint, 29.851°N, 07.265°W , 535 m, 2♂1♀, 03.iii.2007, Dils J.- Faes J., coll: PCJD . Thevenetimyia quedenfeldti (Engel, 1885) Rif: Tanger-Tétouan, Souk El Kolla (Quolla), 35.083°N, 05.538°W , 150 m, 5♂4♀, 30.iv.2017, Dils J.- Faes J., coll: PCJD . Atlantic Plain: Rommani, Merchouch, 33.568°N, 06.753°W , 400 m, 5♂22♀, 04.v.2010, Dils J.- Faes J., coll: PCJD . Middle Atlas: Tadla-Azilal, Afourer, 32.180°N, 06.520°W , 1150 m, 5♂10♀, 07.v.2008, Dils J.- Faes J., coll: PCJD ; Béni Mellal, El Ksiba, 32.576°N, 06.050°W , 870 m, 7♂23♀, 23.iv.2008, Dils J.- Faes J., coll: PCJD . Thyridanthrax mutilus Loew, 1869 Anti Atlas: Tiznit, Sidi Ifni, 29.384°N, 10.172°W , 0 m, 7♂1♀, 10.iv.2008, Dils J.- Faes J., coll: PCJD . Veribubo saudensis François, 1970 Anti Atlas: Erfoud, Tikkert-N-Ouchane, 31.223°N, 04.784°W , 830 m, 1♂3♀, 03.iv.2009, Dils J.- Faes J., coll: PCJD . Veribubo tabaninus François, 1970 Anti Atlas: Ouarzazate, Amerzgane, 31.024°N, 07.223°W , 1370 m, 2♂9♀, 31.iii.2006, Dils J.- Faes J., coll: PCJD ; Erfoud, Tikkert-N-Ouchane, 31.250°N, 04.617°W , 860 m, 1♀, 07.iii.2007, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.017°N, 07.229°W , 1350 m, 6♂27♀, 25.iii.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, Amerzgane, 31.017°N, 07.229°W , 1350 m, 12♂8♀, 25.iii.2009, Dils J.- Faes J., coll: PCJD ; Ouarzazate, 30.847°N, 06.817°W , 1200 m, 1♀, 30.iii.2009, Dils J.- Faes J., coll: PCJD . Sahara: Guelmim, Tainzirt, 29.121°N, 09.333°W , 670 m, 22♀, 31.iii.2010, Dils J.- Faes J., coll: PCJD . MYDIDAE K. Kettani, T. Dikow Number of species: 9 . Expected: 10 Faunistic knowledge of the family in Morocco: moderate Leptomydinae Leptomydas Gerstaecker, 1868 Leptomydas lusitanicus (Wiedemann, 1820) Mouna 1998 Rhopaliinae Rhopalia Macquart, 1838 Rhopalia berlandi Séguy, 1949a: 153 Séguy 1949a , AA , Tagounit, Asni; Mouna 1998 ; Dikow 2017 Perissocerus Gerstaecker, 1868 Perissocerus rungsi Séguy, 1953 Séguy 1953a , SA Syllegomydinae Syllegomydini Syllegomydas Becker, 1906 Syllegomydas algiricus (Gerstaecker, 1868) = Rhopalia algirica Gerstaecker, in Séguy 1928c : 149 Gerstaecker 1868 , AP , Casablanca; Séguy 1928c , AP , Rabat; Séguy 1930a , AP , Rabat; Mouna 1998 ; El Hawagry 2011 ; Dikow 2017 Syllegomydas berlandi (Séguy, 1941) Séguy 1941, AA , Agadir; Dikow 2017 Syllegomydas bueni Arias, 1914 Arias 1914 , AA , Tafilalt; Séguy 1928c ; Séguy 1930a ; Carles-Tolrá 2015 ; Carles-Tolrá 2017 , EM , Mariouri, Trifa; Dikow 2017 Syllegomydas cinctus Macquart, 1835 Macquart 1835, MA , Immouzer road, AA , Agadir, Taroudant; Séguy 1930a ; Mouna 1998 ; Carles-Tolrá 2017 , EM , Quebdana, douar Shila, AA , Agadir coast; Dikow 2017 Syllegomydas maroccanus Séguy, 1928 Séguy 1928c , AP , Kénitra, Rabat, Oued Korifla, Forêt Zaers; Séguy 1930a , AP , Rabat, Temara, Oued Korifla, Forêt Zaers; Séguy 1932c ; Mouna 1998 ; Carles-Tolrá 2017 , AP , Larache, Ras Remel; Dikow 2017 ; AP (Kénitra) – MISR Syllegomydas merceti Arias, 1914 Arias 1914 , AP , Mogador; Séguy 1930a ; Mouna 1998 ; El Hawagry 2011 , AP , Mogador; Dikow 2017 Leptomydinae Leptomydas Gerstaecker, 1868 Leptomydas lusitanicus (Wiedemann, 1820) Mouna 1998 Rhopaliinae Rhopalia Macquart, 1838 Rhopalia berlandi Séguy, 1949a: 153 Séguy 1949a , AA , Tagounit, Asni; Mouna 1998 ; Dikow 2017 Perissocerus Gerstaecker, 1868 Perissocerus rungsi Séguy, 1953 Séguy 1953a , SA Syllegomydinae Syllegomydini Syllegomydas Becker, 1906 Syllegomydas algiricus (Gerstaecker, 1868) = Rhopalia algirica Gerstaecker, in Séguy 1928c : 149 Gerstaecker 1868 , AP , Casablanca; Séguy 1928c , AP , Rabat; Séguy 1930a , AP , Rabat; Mouna 1998 ; El Hawagry 2011 ; Dikow 2017 Syllegomydas berlandi (Séguy, 1941) Séguy 1941, AA , Agadir; Dikow 2017 Syllegomydas bueni Arias, 1914 Arias 1914 , AA , Tafilalt; Séguy 1928c ; Séguy 1930a ; Carles-Tolrá 2015 ; Carles-Tolrá 2017 , EM , Mariouri, Trifa; Dikow 2017 Syllegomydas cinctus Macquart, 1835 Macquart 1835, MA , Immouzer road, AA , Agadir, Taroudant; Séguy 1930a ; Mouna 1998 ; Carles-Tolrá 2017 , EM , Quebdana, douar Shila, AA , Agadir coast; Dikow 2017 Syllegomydas maroccanus Séguy, 1928 Séguy 1928c , AP , Kénitra, Rabat, Oued Korifla, Forêt Zaers; Séguy 1930a , AP , Rabat, Temara, Oued Korifla, Forêt Zaers; Séguy 1932c ; Mouna 1998 ; Carles-Tolrá 2017 , AP , Larache, Ras Remel; Dikow 2017 ; AP (Kénitra) – MISR Syllegomydas merceti Arias, 1914 Arias 1914 , AP , Mogador; Séguy 1930a ; Mouna 1998 ; El Hawagry 2011 , AP , Mogador; Dikow 2017 MYTHICOMYIIDAE K. Kettani, N. Evenhuis Number of species: 8 . Expected: 15 Faunistic knowledge of the family in Morocco: poor Empidideicinae Empidideicus Becker, 1907 Empidideicus crocea Séguy, 1949 = Cyrtosia crocea Séguy, in Séguy 1949a : 85 Séguy 1949a , SA , Guelmim; Séguy 1949c ; Mouna 1998 ; Evenhuis 2002 Glabellulinae Glabellula Bezzi, 1902 Glabellula maroccana Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , Rif , Adrou ( PNPB ) – BPBM, MISR Leylaiya Efflatoun, 1945 Leylaiya pellea Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , AA , Tiznit – BPBM Mythicomyiinae Mythenteles Hall & Evenhuis, 1991 Mythenteles signifera Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , Rif , Talassemtane (maison forestière, 1699 m) – BPBM, MISR Platypyginae Cyrtisiopsis Séguy, 1930 Cyrtisiopsis melleus (Loew, 1856) Evenhuis 2002 ; Zaitzav 2008, AA , Jebel Tighermine (SE Ouarzazate); Koçak and Kemal 2010 ; El Hawagry 2011 Cyrtisiopsis singularis Séguy, 1930 Evenhuis 2002 Cyrtosia Perris, 1839 Cyrtosia aglota Séguy, 1930 Evenhuis 2002 Cyrtosia marginata Perris, 1839 Séguy 1930a , HA ; Mouna 1998 ; Evenhuis 2002 ; Evenhuis and David 2004 Empidideicinae Empidideicus Becker, 1907 Empidideicus crocea Séguy, 1949 = Cyrtosia crocea Séguy, in Séguy 1949a : 85 Séguy 1949a , SA , Guelmim; Séguy 1949c ; Mouna 1998 ; Evenhuis 2002 Glabellulinae Glabellula Bezzi, 1902 Glabellula maroccana Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , Rif , Adrou ( PNPB ) – BPBM, MISR Leylaiya Efflatoun, 1945 Leylaiya pellea Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , AA , Tiznit – BPBM Mythicomyiinae Mythenteles Hall & Evenhuis, 1991 Mythenteles signifera Evenhuis & Kettani, 2018 Evenhuis and Kettani 2018 , Rif , Talassemtane (maison forestière, 1699 m) – BPBM, MISR Platypyginae Cyrtisiopsis Séguy, 1930 Cyrtisiopsis melleus (Loew, 1856) Evenhuis 2002 ; Zaitzav 2008, AA , Jebel Tighermine (SE Ouarzazate); Koçak and Kemal 2010 ; El Hawagry 2011 Cyrtisiopsis singularis Séguy, 1930 Evenhuis 2002 Cyrtosia Perris, 1839 Cyrtosia aglota Séguy, 1930 Evenhuis 2002 Cyrtosia marginata Perris, 1839 Séguy 1930a , HA ; Mouna 1998 ; Evenhuis 2002 ; Evenhuis and David 2004 SCENOPINIDAE K. Kettani, M. Carles-Tolrá Number of species: 8 (+3 unidentified). Expected: 12 Faunistic knowledge of the family in Morocco: good Scenopininae Scenopinus Latreille, 1802 Scenopinus albicinctus (Rossi, 1794) = Omphrale albicincta Rossi, in Séguy 1930a : 110 Séguy 1930a ; Mouna 1998 Scenopinus fenestralis (Linnaeus, 1758) = Omphrale fenestralis Linnaeus, in Séguy 1930a : 110 Séguy 1930a ; Mouna 1998 Scenopinus glabrifrons Meigen, 1824 = Omphrale glabrifrons Meigen, in Séguy 1930a : 110 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 Scenopinus niger (De Geer, 1776) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 Scenopinus parallelus Kelsey, 1969 Kelsey 1969 , AP , Villa Cisneros (Dakhla), SA , Río de Oro (Oued Eddahab) Scenopinus physadius (Séguy, 1930) = Omphrale physadia Séguy, in Séguy 1930a : 111 Séguy, 1930, EM , Bou Denib; Kelsey 1969 , EM , Bou Denib; Mouna 1998 Scenopinus pilosus (Séguy, 1930) = Omphrale pilosa Séguy, in Séguy 1930a : 111 Séguy 1930a , AP , Bou Knadel; Kelsey 1969 , AP , Bou Knadel; Mouna 1998 ; Carles-Tolrá 2001 Scenopinus undescribed sp. 1 Ebejer et al. 2019 , Rif , Martil (9 m) Scenopinus undescribed sp. 2 Ebejer et al. 2019 , Rif , Adrou (556 m) Stenomphrale Kröber, 1937 Stenomphrale teutankhameni (Kröber, 1923) Ebejer et al. 2019 , AP , forest of Maâmora (56 m) Stenomphrale sp. AP (Essaouira (J.-P. Haenni leg.)) – MHNN Neuchâtel Scenopininae Scenopinus Latreille, 1802 Scenopinus albicinctus (Rossi, 1794) = Omphrale albicincta Rossi, in Séguy 1930a : 110 Séguy 1930a ; Mouna 1998 Scenopinus fenestralis (Linnaeus, 1758) = Omphrale fenestralis Linnaeus, in Séguy 1930a : 110 Séguy 1930a ; Mouna 1998 Scenopinus glabrifrons Meigen, 1824 = Omphrale glabrifrons Meigen, in Séguy 1930a : 110 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 Scenopinus niger (De Geer, 1776) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 Scenopinus parallelus Kelsey, 1969 Kelsey 1969 , AP , Villa Cisneros (Dakhla), SA , Río de Oro (Oued Eddahab) Scenopinus physadius (Séguy, 1930) = Omphrale physadia Séguy, in Séguy 1930a : 111 Séguy, 1930, EM , Bou Denib; Kelsey 1969 , EM , Bou Denib; Mouna 1998 Scenopinus pilosus (Séguy, 1930) = Omphrale pilosa Séguy, in Séguy 1930a : 111 Séguy 1930a , AP , Bou Knadel; Kelsey 1969 , AP , Bou Knadel; Mouna 1998 ; Carles-Tolrá 2001 Scenopinus undescribed sp. 1 Ebejer et al. 2019 , Rif , Martil (9 m) Scenopinus undescribed sp. 2 Ebejer et al. 2019 , Rif , Adrou (556 m) Stenomphrale Kröber, 1937 Stenomphrale teutankhameni (Kröber, 1923) Ebejer et al. 2019 , AP , forest of Maâmora (56 m) Stenomphrale sp. AP (Essaouira (J.-P. Haenni leg.)) – MHNN Neuchâtel THEREVIDAE K. Kettani, M. Hauser Number of species: 27 . Faunistic knowledge of the family in Morocco: moderate Phycusinae Phycusini Actorthia Kröber, 1912 Actorthia micans (Kröber, 1924) Kröber 1924 , AA , Errachidia (45 km S Erfoud), Merzouga Phycus Walker, 1850 Phycus lacteipennis Lyneborg, 2002 Lyneborg 2002 , AA , 25 km S Goulmima (1000 m), SA , Mekn s-Tafilalet; Winterton et al. 2012 ; Badrawy and Mohammad 2013 Salentia Costa, 1857 Salentia anancitis (Séguy, 1941) = Apioeicoceras anancitis Séguy, in Séguy 1941d : 10 Séguy 1941d , AA , Agadir; Mouna 1998 Salentia costalis (Wiedemann, 1824) = Apioeicoceras costalis Wiedemann, in Séguy 1930a : 108 Wiedemann 1824 , AP , Mogador, HA , Marrakech-Tensift-Al Haouz; Séguy 1930a ; Mouna 1998 ; Koçak and Kemal 2010 Salentia fuscipennis Costa, 1857 Costa 1857 , Rif , Tanger, AA , Tagadirt (Agadir); Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Bou Knadel, Tinmel; Mouna 1998 Therevinae Therevini Acanthothereva Séguy, 1935 Acanthothereva rungsi Séguy, 1935 Séguy 1935b , AP , Mehdia (20 km S Rabat); Lyneborg 1968 ; Koçak and Kemal 2010 ; Rif (Cap Spartel) – MISR Acrosathe Irwin & Lyneborg, 1981 Acrosathe annulata (Fabricius, 1805) Ebejer et al. 2019 , Rif , Oued Kbir (Béni Ratene, 157 m) Chrysanthemyia Becker, 1912 Chrysanthemyia chrysanthemi (Fabricius, 1787) Fabricius 1787 , EM , Béni Snassen Mountains, Tafouralt (800 m); Séguy 1930a , MA , Meknès, Berrechid; Mouna 1998 ; MA (Oued Grou, Timahdit) – MISR Chrysanthemyia velutinifrons (Becker, 1912) = Chrysanthemyia lucidifrons Becker 1912 : 81 = Oedicera velutinifrons Becker, in Becker and Stein 1913 : 82 Becker 1912 , Rif , Region de Tanger-Tétouan, Cercle d'Ouezzane (300 m), AP , 3 km S Settat, EM , Figuig, MA , Fès-Boulmane, Sidi Harazem (223 m); Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Meknès; Mouna 1998 ; MA (Oued Grou) – MISR Hoplosathe Lyneborg & Zaitsev, 1980 Hoplosathe distincta Lyneborg & Zaitsev, 1980 Lyneborg and Zaitsev 1980, HA , Oued Tensift (Marrakech) Neotherevella Lyneborg, 1978 Neotherevella macularis (Wiedemann, 1828) Hauser et al. 2017 , AA , Tifnite (10 Km S Agadir), Merzouga (45 Km S Erfoud) Thereva Latreille, 1797 Thereva atra Kröber, 1913 El Hawagry 2011 Thereva aureoscutellata Kröber, 1914 Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m) Thereva binotata Loew, 1847 Koçak and Kemal 2010 Thereva bipunctata Meigen, 1820 16 Ebejer et al. 2019 , MA , Khénifra (17 km SW of Midelt, 1940 m; 17 km NW of Zaida, 1878 m; 28 km S of Timahdit, 2100 m), Lac Aguelmane Afennourir (30 km SW of Azrou, 2050 m) Thereva brevicornis Loew, 1847 17 Pârvu et al. 2006 , AP , Cap Sim; Popescu-Mirceni 2011 Thereva chrysargyrea Séguy, 1953 Séguy 1953a , SA , Amguilli Sguelma Thereva cincta Meigen, 1820 Ebejer et al. 2019 , Rif , Oued Aliane (Ksar Sghir, 1 m) Thereva funebris Meigen, 1820 18 = Thereva lugubris Meigen Mouna 1998 ; AP (Rabat), MA (Ifrane) – MISR Thereva graeca Kröber, 1912 3 Séguy 1930a , Rif , Tanger; Mouna 1998 Thereva plebeja (Linnaeus, 1758) 3 Séguy 1953a , AA , Tifnit (Souss); Mouna 1998 ; MA (Ras el Ma) – MISR Thereva powelli Séguy, 1930 Séguy 1930a , MA , Forêt Azrou; MA (Ras el Ma) – MISR Thereva spiloptera Wiedemann, 1824 Wiedemann 1824 , HA , Ouirgane (Marrakech, 1000 m); Séguy 1930a , Rif , Tanger, AP , Mogador, MA , Meknès; Séguy 1953a , AP , Temara; Mouna 1998 ; El Hawagry 2011 Thereva spinulosa Loew, 1847 Loew 1847 , AP , Maâmora, MA , Khemisset Thereva stigmatica Kröber, 1912 EL-Hawagy 2011, Rif , Tanger Thereva strigata (Fabricius, 1794) 19 Koçak and Kemal 2010 Thereva tuberculata Loew, 1847 = Thereva algirica Kröber, 1913, in Séguy 1953a : 83 Loew 1847 , AP , Salé; Séguy 1930a , MA , Meknès; Séguy 1953a , AP , Salé; Mouna 1998 ; Koçak and Kemal 2010 Acknowledgments We gratefully acknowledge Martin Ebejer (UK) for material, comments and cooperation, as well as Gail Kampmeier (USA) for sharing data of Moroccan Therevidae out of the mandala database ( http://wwx.inhs.illinois.edu/research/mandala/about/ ). Phycusinae Phycusini Actorthia Kröber, 1912 Actorthia micans (Kröber, 1924) Kröber 1924 , AA , Errachidia (45 km S Erfoud), Merzouga Phycus Walker, 1850 Phycus lacteipennis Lyneborg, 2002 Lyneborg 2002 , AA , 25 km S Goulmima (1000 m), SA , Mekn s-Tafilalet; Winterton et al. 2012 ; Badrawy and Mohammad 2013 Salentia Costa, 1857 Salentia anancitis (Séguy, 1941) = Apioeicoceras anancitis Séguy, in Séguy 1941d : 10 Séguy 1941d , AA , Agadir; Mouna 1998 Salentia costalis (Wiedemann, 1824) = Apioeicoceras costalis Wiedemann, in Séguy 1930a : 108 Wiedemann 1824 , AP , Mogador, HA , Marrakech-Tensift-Al Haouz; Séguy 1930a ; Mouna 1998 ; Koçak and Kemal 2010 Salentia fuscipennis Costa, 1857 Costa 1857 , Rif , Tanger, AA , Tagadirt (Agadir); Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Bou Knadel, Tinmel; Mouna 1998 Therevinae Therevini Acanthothereva Séguy, 1935 Acanthothereva rungsi Séguy, 1935 Séguy 1935b , AP , Mehdia (20 km S Rabat); Lyneborg 1968 ; Koçak and Kemal 2010 ; Rif (Cap Spartel) – MISR Acrosathe Irwin & Lyneborg, 1981 Acrosathe annulata (Fabricius, 1805) Ebejer et al. 2019 , Rif , Oued Kbir (Béni Ratene, 157 m) Chrysanthemyia Becker, 1912 Chrysanthemyia chrysanthemi (Fabricius, 1787) Fabricius 1787 , EM , Béni Snassen Mountains, Tafouralt (800 m); Séguy 1930a , MA , Meknès, Berrechid; Mouna 1998 ; MA (Oued Grou, Timahdit) – MISR Chrysanthemyia velutinifrons (Becker, 1912) = Chrysanthemyia lucidifrons Becker 1912 : 81 = Oedicera velutinifrons Becker, in Becker and Stein 1913 : 82 Becker 1912 , Rif , Region de Tanger-Tétouan, Cercle d'Ouezzane (300 m), AP , 3 km S Settat, EM , Figuig, MA , Fès-Boulmane, Sidi Harazem (223 m); Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Meknès; Mouna 1998 ; MA (Oued Grou) – MISR Hoplosathe Lyneborg & Zaitsev, 1980 Hoplosathe distincta Lyneborg & Zaitsev, 1980 Lyneborg and Zaitsev 1980, HA , Oued Tensift (Marrakech) Neotherevella Lyneborg, 1978 Neotherevella macularis (Wiedemann, 1828) Hauser et al. 2017 , AA , Tifnite (10 Km S Agadir), Merzouga (45 Km S Erfoud) Thereva Latreille, 1797 Thereva atra Kröber, 1913 El Hawagry 2011 Thereva aureoscutellata Kröber, 1914 Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m) Thereva binotata Loew, 1847 Koçak and Kemal 2010 Thereva bipunctata Meigen, 1820 16 Ebejer et al. 2019 , MA , Khénifra (17 km SW of Midelt, 1940 m; 17 km NW of Zaida, 1878 m; 28 km S of Timahdit, 2100 m), Lac Aguelmane Afennourir (30 km SW of Azrou, 2050 m) Thereva brevicornis Loew, 1847 17 Pârvu et al. 2006 , AP , Cap Sim; Popescu-Mirceni 2011 Thereva chrysargyrea Séguy, 1953 Séguy 1953a , SA , Amguilli Sguelma Thereva cincta Meigen, 1820 Ebejer et al. 2019 , Rif , Oued Aliane (Ksar Sghir, 1 m) Thereva funebris Meigen, 1820 18 = Thereva lugubris Meigen Mouna 1998 ; AP (Rabat), MA (Ifrane) – MISR Thereva graeca Kröber, 1912 3 Séguy 1930a , Rif , Tanger; Mouna 1998 Thereva plebeja (Linnaeus, 1758) 3 Séguy 1953a , AA , Tifnit (Souss); Mouna 1998 ; MA (Ras el Ma) – MISR Thereva powelli Séguy, 1930 Séguy 1930a , MA , Forêt Azrou; MA (Ras el Ma) – MISR Thereva spiloptera Wiedemann, 1824 Wiedemann 1824 , HA , Ouirgane (Marrakech, 1000 m); Séguy 1930a , Rif , Tanger, AP , Mogador, MA , Meknès; Séguy 1953a , AP , Temara; Mouna 1998 ; El Hawagry 2011 Thereva spinulosa Loew, 1847 Loew 1847 , AP , Maâmora, MA , Khemisset Thereva stigmatica Kröber, 1912 EL-Hawagy 2011, Rif , Tanger Thereva strigata (Fabricius, 1794) 19 Koçak and Kemal 2010 Thereva tuberculata Loew, 1847 = Thereva algirica Kröber, 1913, in Séguy 1953a : 83 Loew 1847 , AP , Salé; Séguy 1930a , MA , Meknès; Séguy 1953a , AP , Salé; Mouna 1998 ; Koçak and Kemal 2010 Acknowledgments We gratefully acknowledge Martin Ebejer (UK) for material, comments and cooperation, as well as Gail Kampmeier (USA) for sharing data of Moroccan Therevidae out of the mandala database ( http://wwx.inhs.illinois.edu/research/mandala/about/ ). Empidoidea ATELESTIDAE K. Kettani, P. Gatt Number of species: 1 . Expected: 1 Faunistic knowledge of the family in Morocco: poor Atelestus Walker, 1837 Atelestus sp. nov. Ebejer et al. 2019 , Rif , Dardara (484–730 m), Aïn Tissemlal (Azilane, 1255 m), Douar El Hamma (338 m), Chrabkha pond (Al Manzla, 58 m) EMPIDIDAE K. Kettani, C. Daugeron Number of species: 40 . Expected: 100 Faunistic knowledge of the family in Morocco: poor Clinocerinae Clinocera Meigen, 1803 Clinocera maroccana (Séguy, 1941): 29 (= Hydrodromia ) = Atalanta ( Hydrodromia ) algira (Vaillant, 1952): 65 Séguy 1941a , HA , Anrhemer (Toubkal, 2500 m); Vaillant 1956b , HA , Sidi Chamarouch; Vaillant 1964 ; Dakki 1997 Clinocera megalatlantica (Vaillant, 1957): 65 (= Atalanta ( Atalanta ) ) Vaillant 1956b , HA ; Dakki 1997 Clinocera nigra Meigen, 1804: 292 = Heleodromia unicolor (Curtis, 1834): plate 513, Paramesia roberti (Macquart, 1835): 657 Vaillant 1956b , HA , Izourar, Sidi Chamarouch, Aguelmous; Dakki 1997 Dolichocephala Meigen, 1803 Dolichocephala ocellata (Costa, 1858): 7 (= Ardoptera ) = oculata (Loew, 1858~7): 7 (= Ardoptera ) = novemguttata (Strobl, 1893): 98 (= Ardoptera ) = albohalterata (Strobl, 1898): 399 (= Ardoptera ) = barbarica Vaillant, 1952: 65 = algira Vaillant, 1957: 64 Vaillant 1956b , HA , Imi-N'Ifri; Dakki 1997 Dolichocephala pavonica Vaillant & Gagneur, 1998: 380 Vaillant and Gagneur 1998 , HA , Demnat Kowarzia Mik, 1881 Kowarzia barbatula (Mik, 1880): 347 (= Clinocera ) = Clinocera dorieri (Vaillant, 1968): 88 Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Aguelmous, Sidi Chamarouch, Izourar, Oukaimeden; Dakki 1997 ; Vaillant and Moubayed 1998 Kowarzia bipunctata (Haliday, 1833) (= Heleodromia ) Vaillant 1956b , HA , Oukaimeden; Vaillant 1964 ; Dakki 1997 Kowarzia dieuzedei Vaillant, 1953: 60 Vaillant 1956b , HA , Lac Tamhda (Anremer); Vaillant 1964 , HA ; Dakki 1997 Kowarzia madicola (Vaillant, 1965): 152 (= Atalanta ) Vaillant 1956b , HA , Tahanaout Kowarzia tenella (Wahlberg, 1844): 107 (= Parmesia ) = Heleodromia zetterstedti (Walker, 1851): 105 = Wiedemannia securigera (Engel, 1918): 70 Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Aguelmous, Sidi Chamarouch, Izourar, Oukaimeden; Dakki 1997 ; Vaillant and Moubayed 1998 Wiedemannia Zetterstedt, 1838 Wiedemannia ( Chamaedipsia ) beckeri (Mik, 1889): 71 (= Chamaedipsia ) = jugorum (Strobl, 1893): 105 (= Chamaedipsia ) = crinita Engel, 1918: 217 (as var. of W. beckeri ) = alticola Vaillant, 195l: 54 = alpina Vaillant, 1967: 274 (as ssp. of W. beckeri ) = glaciola Wagner, 1985: 86 (as ssp. of W. beckeri ; new name for W. beckeri alpina Vaillant) Dakki 1997 Wiedemannia ( Chamaedipsia ) mgounica Vaillant, 1957: 69 Vaillant 1956a , HA , M'Goum; Dakki 1997 Wiedemannia ( Philolutra ) azurea (Vaillant, 1951): 3 (= Philolutra ) El Mezdi and Giudicelli 1985 , HA , Khettaras Marrakech; Dakki 1997 ; Vaillant and Moubayed 1998 Wiedemannia ( Philolutra ) fallaciosa Loew, 1873: 44 Vaillant 1964 , HA ; Dakki 1997 Wiedemannia ( Philolutra ) fallaciosa ssp. litardierei Vaillant, 1957: 67 Vaillant 1956a , HA ; Dakki 1997 Wiedemannia ( Roederella ) ouedorum Vaillant, 1952: 371 = ovedorum (error) Vaillant, 1978: 469 Vaillant 1964 , HA ; Dakki 1997 Hemerodromiinae Hemerodromia Meigen, 1822 Hemerodromia bethiana Vaillant & Gagneur, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Hemerodromia subapicalis Yang, Zhang & Zhang, 2007 = Hemerodromia apicalis Vaillant and Gagneur 1998 : 372 (preoccupied by Smith, 1969) Vaillant and Gagneur 1998 , HA , Oum-er-Rbia; Yang et al. 2007 Hemerodromia tigrigrana Vaillant & Gagneur, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Hemerodromia todrhana (Vaillant, 1956) Vaillant 1956, HA , Todrha; El Mezdi and Guidicelli 1985; Dakki 1997 ; Vaillant and Gagneur 1998 Hemerodromia zarcana Vaillant & Moubayed, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Empidinae Empidini Empis Linnaeus, 1758 Empis ( Coptophlebia ) confluens Becker, 1907 MA (Meknès), 19.v.1997, K. Deneš leg. – OUMNH Empis ( Empis ) decora Meigen, 1822 Bahid 2018 , Rif , Oued Tkarae ( PNPB ); Ebejer et al. 2019 , Rif , Oued Nakhla, Moulay Abdelsalam Empis ( Empis ) nikita Shamshev, 2018 Shamshev 2018 , AP , Essaouira Empis ( Kritempis ) taffertensis Daugeron, 2009 Daugeron 2009 , MA , forest of Taffert; Bahid 2018 Empis ( Leptempis ) tenuis Bahid & Daugeron, 2017 Bahid et al. 2017 , MA , Tizi-s'Tkrine (Jebel Amar, 1760 m); Bahid 2018 Empis ( Pachymeria ) suberis Becker, 1907 Ebejer et al. 2019 , Rif , Moulay Abdelsalam, Issaguen, Bab Berred, Jebel Lakraâ Empis ( Polyblepharis ) eumera Loew, 1866 Ebejer et al. 2019 , MA , Ifrane Empis ( Xanthempis ) chopardi Daugeron, 1997 Daugeron 1997 , MA , Ifrane; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) edithae Daugeron, 1997 Daugeron 1997 , HA , High Imminen, Tachdirt; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) ifranensis Daugeron, 1997 Daugeron 1997 , MA , Ifrane; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) styriaca (Strobl, 1893) Chvála and Wagner 1989 , HA [doubtful record]; Bahid 2018 Empis ( Xanthempis ) widanensis Bahid & Daugeron, 2018 Bahid et al. 2018 , Rif , Dayat Bayan Widane, Aïn Sedraouia, Tazia, Anissar, Lalla Outka Rhamphomyia Meigen, 1822 Rhamphomyia ( Rhamphomyia ) maroccana Collin, 2009 Chvála and Wagner 1989 , MA , Ifrane; Collin 2009 , MA , Ifrane; Bahid 2018 , Rif , Oued Akrir (Fifi) Rhamphomyia ( Holoclera ) tenuipes Becker, 1907* AP , Haenni pers. comm. Hilarini Hilara Meigen, 1822 Hilara algecirasensis Strobl, 1899 Ebejer et al. 2019 , MA , Lac Aguelmane Afennourir, HA , Ziz river Hilara almeriensis Strobl, 1906 Chvála 2008 , AA , Tifoultoute (1146 m) Hilara fusitibia Strobl, 1899 Chvála 2008 , MA , Ifrane (Forêt de Cédres, 1500 m) Hilara longeciliata Strobl, 1906 Chvála 2008 , AP , Rabat (near Oued Bou-Regreg, 0–10 m) Hilara schachti Chvála, 2008 Chvála 2008 , MA , Ifrane (Ghabat al Behar, 1650–1700 m); Bahid 2018 New record for Morocco Rhamphomyia ( Holoclera ) tenuipes Becker, 1907 Atlantic Plain: Essaouira, 6 km W, 3.iv.2002, Forêt de genévriers pâturée, 1♂1♀, J.-P. Haenni leg., coll. MHNN . Acknowledgements We are very grateful to Bradley Sinclair (Canadian Food Inspection Agency, Canada) and Adrian Plant (Mahasarakham University, Thailand) for reviewing parts of this family. DOLICHOPODIDAE K. Kettani, I.Ya. Grichanov, O.P. Negrobov Number of species: 112 . Expected: 300 Faunistic knowledge of the family in Morocco: poor Diaphorinae Argyra Macquart, 1834 Argyra argentina Meigen, 1824 Parent 1924 , Rif , Cap Spartel, Tétouan; Parent 1927 , Rif , Tétouan Argyra argyria (Meigen, 1824) Parent 1924 (females only), Rif , Cap Spartel, Tétouan; Kechev and Ivanova 2015 Argyra biseta Parent, 1929 Parent 1929a , Rif , Tanger; Vaillant 1955a Argyra grata Loew, 1857 20 Negrobov 1991 (no material provided) Asyndetus Loew, 1869 Asyndetus separatus (Becker, 1902) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Chrysotus Meigen, 1824 Chrysotus albibarbus Loew, 1857 Ebejer et al. 2019 , MA , Lac Aguelmane Afennourir (30 km SW of Azrou, 1760 m); Grichanov 2019 , AA , Aït Melloul Chrysotus gramineus (Fallén, 1823) Parent 1924 , Rif , Tanger; Parent 1927 Chrysotus larachensis Grichanov, Nourti & Kettani, 2020 Grichanov et al. 2020b , Rif , El Hamma (338 m) Chrysotus pennatus Lichtwardt, 1902 Ebejer et al. 2019 , Rif , Smir Barrage (145 m), AA , 1 km N of Tarda (Errachidia, 1023 m) Chrysotus suavis Loew, 1857 Grichanov 2009 , HA , Asni area (1100–1400 m); Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), MA , Dayat Ifrane (1607 m), HA , Tahanout (956 m); Dawah et al. 2020 Diaphorus Meigen, 1824 Diaphorus africus Parent, 1924 Parent 1924 , Rif , Tétouan, Tanger; Parent 1927 , Rif , Tanger; Ebejer et al. 2019 , Rif , Oued Siflaou (281 m); Grichanov 2019 , AA , Ouarzazate (1100 m) Diaphorus vitripennis Loew, 1859 Grichanov 2019 , AA , Aït Melloul Dolichopodinae Dolichopus Latreille, 1796 Dolichopus andalusiacus (Strobl, 1899) Ebejer et al. 2019 , AP , Loukous marsh (2 m); Nourti et al. 2019a , Rif , plage Stihat (0 m) Dolichopus griseipennis Stannius, 1831 Parent 1924 , Rif , Tanger; Parent 1927 ; Séguy 1930a , Rif , Tanger; Nourti et al. 2019a , Rif , Adrou ( PNPB , 556 m) Dolichopus sabinus Haliday, 1838 Ebejer et al. 2019 , Rif , Martil (9 m), Oued Laou (2 m); Nourti et al. 2019a , Rif , plage Stihat (4 m) Dolichopus scutopilosus Parent, 1933 Parent 1933 , HA , Arround Dolichopus signifer Haliday, 1832 Parent 1929b , "Maroc"; Séguy 1930a , MA , Ras el Ma; Grichanov 2019 , HA , Oukaimeden (2600 m) Dolichopus strigipes Verrall, 1875 Grichanov 2009 , AP , 40 km S Larache (0–20 m) Gymnopternus Loew, 1857 Gymnopternus assimilis (Staeger, 1842) Nourti et al. 2019a , Rif , Amsemlil (1067 m) Hercostomus Loew, 1857 Hercostomus apollo (Loew, 1869) Nourti et al. 2019a , Rif , Talassemtane (1696 m), Adrou ( PNPB , 556 m), Amsemlil ( PNPB , 1067 m) Hercostomus canariensis Santos Abreu, 1929 Grichanov et al. 2020a , Rif , Pont de Dieu (Akchour, 536 m); Nourti et al. 2019a (as H. aff. exarticulatoides Stackelberg, 1949) Hercostomus chetifer (Haliday, 1849) Ebejer et al. 2019 , Rif , Sidi Yahia Aarab (377 m) Hercostomus discriminatus Parent, 1925 Parent 1925 , Rif , "Favier, Environs de Tanger"; Parent 1927 , Rif , Tanger; Vaillant 1950 , Rif , Tanger Hercostomus exarticulatus (Loew, 1857) Vaillant 1956b , HA , Lac Tamhda (Anremer), Aguelmous; Grichanov et al. 2020a Hercostomus excipiens Becker, 1907 Parent 1924 , Rif , Tétouan; Parent 1927 ; Séguy 1930a , Rif , Oued Judios (Tanger); Nourti et al. 2019a , Rif , Talembote (440 m) Hercostomus germanus (Wiedemann, 1817) Parent 1924 , Rif , Cap Spartel; Parent 1927 ; Kettani and Negrobov 2016 , Rif , Chefchaouen, Ketama – MISR ( Rif , Ketama) Hercostomus longiventris (Loew, 1857) Vaillant 1956b , HA , Lac Tamhda (Anremer), Izourar Muscidideicus Becker, 1917 Muscidideicus praetextatus (Haliday, 1855) Grichanov 2019 , AP , Oualidia lagune Ortochile Berthold, 1827 Ortochile morenae (Strobl, 1899) = Hercostomus morenae (Strobl, 1899), in Becker 1917 : 225; Nourti et al. 2019a : 124 Grichanov and Nourti 2021 ; Nourti et al. 2019a , Rif , Mnezla (74 m), Talassemtane (980 m), Oued Ametrasse (841 m), estuary Tahaddart (dune marshland, 0 m) Ortochile nigrocaerulea Latreille, 1779 Parent 1924 , Rif , Tanger, Cap Spartel, Tétouan, Béni Hozmar; Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Oued Judios (Tanger); Grichanov 2009 , Rif , Ouezzane (300 m); Nourti et al. 2019a , Rif , Douar El Hamma (338 m), Triwa Bni Hassane (654 m), Taida (501 m) Platyopsis Parent, 1929 Platyopsis maroccanus (Parent, 1929) Parent 1929b , Rif , Tanger; Vaillant 1950 , Rif , Tanger Poecilobothrus Mik, 1878 Poecilobothrus appendiculatus (Loew, 1859) = Hercostomus appendiculatus (Loew): Ebejer et al. 2019 : 146 Parent 1924 , Rif , Tanger, Cap Spartel; Ebejer et al. 2019 , Rif , Oued Nakhla (200 m), Moulay Abdelsalam (965 m), Dardara (730 m), Cap Spartel (155 m); Nourti et al. 2019a , Rif , Perdicaris Park (223 m) Poecilobothrus infuscatus (Stannius, 1831) Ebejer et al. 2019 , Rif , Tahaddart (2 m); Rif (Tahaddart) – MISR Sybistroma Meigen, 1824 Sybistroma dufouri Macquart, 1838 = Haltericerus spathulatus Loew, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger Sybistroma obscurellum Fallén, 182320 = Hypophyllus obscurellus Fallén, in Dakki 1997 : 61 Dakki 1997 (no material provided) Sybistroma quadrifilatum (Strobl, 1899) = Sybistroma parvulum (Parent, 1927), in Grichanov and Nourti 2021 : 190 Grichanov and Nourti 2021 , Rif , Fahs Anjra (372 m) Sybistroma theodori Grichanov & Nourti, 2021 Grichanov and Nourti 2021 , Rif , Moulay Abdelsalam (649 m) Tachytrechus Haliday, 1851 Tachytrechus consobrinus (Haliday, 1851)20 Parent 1938 (no material provided) Tachytrechus goudoti (Macquart, 1842) = Dolichopus goudoti Macquart, 1842 Macquart 1842 , Rif , Tanger; Parent 1926 (redescription), 1927 Tachytrechus insignis (Stannius, 1831) Parent 1927 , "Maroc"; Séguy 1930a , Rif , Tanger, HA , Aguerd el Had, Talekjount (1000–1100 m); Vaillant 1956b , HA , Lac Tamhda (Anremer); Popescu-Mirceni 2011 , AP , Merja Zerga; Grichanov 2019 , AP , Essaouira Tachytrechus notatus (Stannius, 1831) Vaillant 1950 (no material provided), 1956b, HA , Lac Tamhda (Anremer); Grichanov 2009 , AA , 15 km SW Tazenakcht; Dawah et al. 2020 Tachytrechus planitarsis Becker, 1907 Vaillant 1950 , HA , Touggourt; Grichanov 2009 , AA , 15 km SW Tazenakcht; Grichanov 2019 , AA , Ouarzazate (1100 m) Hydrophorinae Anahydrophorus Becker, 1917 Anahydrophorus cinereus (Fabricius, 1805) = Scatophaga cinerea Fabricius, 1805: 205 Fabricius 1805 , AP , Mogador (Essaouira); Séguy 1930a , Rif , Tanger; Vaillant 1955a , AP , Temara, Port-Lyautey; Boumezzough and Vaillant 1986a , AP , beach of Rabat; Kettani and Negrobov 2016 , AP , Skhirat; AP (Skhirat) – MISR Aphrosylus Haliday, 1851 Aphrosylus maroccanus Vaillant, 1955 Vaillant 1955a , AP , Port Lyautey Aphrosylus mitis Verrall, 1912 Grichanov 2019 , AP , Oualidia lagune Aphrosylus raptor luteipes Parent, 1929 Parent 1929b , AP , Mogador (as a variation of Aphrosylus raptor Haliday, 1851); Vaillant 1955a ; Negrobov, 1979 (as a subspecies of Aphrosylus raptor Haliday, 1851); Kettani and Negrobov 2016 (as Aphrosylus raptor Haliday, 1851); Grichanov 2019 , AP , Oualidia lagune Aphrosylus temaranus Vaillant, 1955 Vaillant 1955a , AP , Temara; Grichanov 2019 , AP , Oualidia lagune Aphrosylus venator Loew, 1857 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger Epithalassius Mik, 1891 Epithalassius corsicanus Becker, 1910 Pârvu 2008 , AP , Merja Zergha, Cap Sim, Essaouira Hydrophoprus Fallén, 1823 Hydrophorus balticus (Meigen, 1824) Vaillant 1956b , HA , Jebel Toubkal, Lac Tamhda (Anremer), Oukaimeden, Izourar; Boumezzough and Vaillant 1986a , HA , Jebel Toubkal (3100 m); Grichanov 2019 , HA , Aguelmouss (2050 m), Oukaimeden (2600 m); Nourti et al. 2019a , MA , Mont Habri (2071 m) Hydrophorus nilicola Parent, 1927 = Hydrophorus viridis nilicola Parent, in Boumezzough and Vaillant 1986a : 297 Boumezzough and Vaillant 1986a , MA , Tizi-n'Imdrhas (1800 m), HA , Oued N'fis (650 m), AA , near Agadir N'oussbai (400 m); Grichanov 2019 , AP , Essaouira Hydrophorus oceanus (Macquart, 1838) = Hydrophorus bisetus Loew, 1857, in Parent 1927 , Séguy 1930a , Grichanov 2019 Parent 1927 , AP , Rabat; Séguy 1930a , Rif , Tandja el Balia (Tanger) ( Hydrophorus bisetus Loew); Vaillant 1955a , AP , Port-Lyautey; Boumezzough and Vaillant 1986a , AP , beach of Rabat Hydrophorus praecox (Lehmann, 1822) Parent 1924 , Rif , Cap Spartel, de Tanger à Tétouan, Rincón de Medik, Dar Riffien (Ceuta); Parent 1927 ; Séguy 1941a , HA , Toubkal (2500 m); Boumezzough and Vaillant 1986a , HA , Lac Tamhda, Lac Tamdhanit (Massif Anremer, 2900 m), Lac Izourar (Massif Azourki, 2650 m) Hydrophorus viridis (Meigen, 1824) 21 Parent 1927 , AP , Rabat; Boumezzough and Vaillant 1986a : 297 Liancalus Loew, 1857 Liancalus virens (Scopoli, 1763) Vaillant 1956b , HA , Toubkal, Assif Tassouat (M'Goum), Aguelmous, Sidi Chamarouch, Imi-N'Ifri; Boumezzough and Vaillant 1986a , HA , Tahanaout (750 m), Adrar Anremer (2900 m), AA , Jebel Siroua (3000 m); Kettani and Negrobov 2016 , AP , S-Tifni; Nourti et al. 2019a , Rif , Amsemlil env. (1059 m) – MISR ( AP , S Tifni) Machaerium Haliday, 1832 Machaerium maritimae Haliday, 1832 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Kettani and Negrobov 2016 , AP , Oued Bou-Regreg; Grichanov 2019 , AP , Oualidia lagune; AP (Oued Bou-Regreg) – MISR Orthoceratium Schrank, 1803 Orthoceratium sabulosum (Becker, 1907) = Orthoceratium lacustre (Scopoli, 1763) 22 , in Ebejer et al. 2019 : 146 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Thinophilus Wahlberg, 1844 Thinophilus ( Thinophilus ) flavipalpis (Zetterstedt, 1843) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Grichanov 2009 , AP , 40 km S Larache (0–20 m) Thinophilus ( Thinophilus ) indigenus Becker, 1902 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m), Oued Laou (2 m); Grichanov 2019 , AA , Ouarzazate (572 m) Thinophilus ( Thinophilus ) mirandus Becker, 1907 Negrobov 1971 , Rif , Tanger; Grichanov 2019 , AA , Ouarzazate (572 m) Thinophilus ( Schoenophilus ) versutus Haliday, 1851 = Schoenophilus versutus (Haliday, 1851), in Parent 1924 , 1927 Parent 1924 , Rif , Cap Spartel, Tétouan; Parent 1927 ; Pârvu et al. 2006 , AP , Merja Zerga; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), Dayat Tazia (733 m) Medeterinae Medetera Fisher, 1819 Medetera diadema (Linnaeus, 1767) = Medeterus (cf. diadema ), in Séguy 1953a : 84 Parent 1938 (no material provided); Séguy 1953a , AP , Rabat; Nourti et al. 2019a , Rif , Kitane (49 m), Rif , plage Stihat (beach) Medetera media Parent, 1925 Nourti et al. 2019a , Rif , Faculty of Sciences of Tétouan (garden: on trunk of olive tree, 14 m) Medetera micacea Loew, 1857 Ebejer et al. 2019 , Rif , Dardara (730 m); Nourti et al. 2019a , Rif , Issaguen (1547 m), HA , Lac Tislit (Imilchil, 2254 m) Medetera pallipes (Zetterstedt, 1843) 23 Nourti et al. 2019a , Rif , Douar El Hamma (338 m) Medetera petrophila Kowarz, 187720 Parent 1938 (no material provided) Medetera petrophiloides Parent, 1925 Nourti et al. 2019a , Rif , Issaguen (1547 m) Medetera aff. roghii Rampini & Canzoneri, 1979 Nourti et al. 2019a , Rif , Douar El Hamma (338 m) Medetera truncorum Meigen, 1824 24 Ebejer et al. 2019 a, Rif , Dardara (484 m) Medetera varvara Grichanov & Vikhrev, 2009 Grichanov and Vikhrev 2009 , AP , Essaouira Thrypticus Gerstäcker, 1864 Thrypticus bellus Loew, 1869 Séguy 1930a , EM , Dayat Sidi Kacem; Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), estuary Oued Tahaddart (0 m), Barrage 9 Avril, plage Stihat (0 m) Microphorinae Schistostoma Becker, 1902 Schistostoma eremita Becker, 1902 Ebejer et al. 2019 , AA , Ziz river (12 km S of Rissani, 737 m), Lac Tiffert (4 km W of Merzouga, 702 m) Neurigoninae Neurigona Rondani, 1856 Neurigona solodovnikovi Grichanov, 2010 = Neurigona punctifera Becker, 1907, in Kettani and Negrobov 2016 (misidentification) Grichanov 2010, AP , 40 km S Larache; Kettani and Negrobov 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m); Nourti et al. 2019a , Rif , Perdicaris Park (223 m), Taria Bni Faghloum (894 m), Chrafate (832 m) Parathalassiinae Microphorella Becker, 1909 Microphorella ulrichi Gatt, 2003 Gatt 2003 , Rif , Tanger, Oued Armal (Ksar Sghir) Parathalassius Mik, 1891 Parathalassius blasigii Mik, 1891 Ebejer et al. 2019 , AP , Larache (5 m) Peloropeodinae Chrysotimus Loew, 1857 Chrysotimus molliculoides Parent, 1937 Parent 1937a , MA , Ifrane (1600 m); Parent 1937b ; Nourti et al. 2019a , Rif , Dayat Tazia (733 m) Micromorphus Mik, 1878 Micromorphus albipes (Zetterstedt, 1843) Parent 1924 , Rif , Béni Hozmar (Tétouan); Parent 1927 , Rif , Tétouan; Kechev and Ivanova 2015 ; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), estuary Oued Tahaddart (0 m), Barrage 9 Avril, Kharouba, plage Stihat (0 m), Aïn Jdioui, EM , Bouanane (Figuig, 855 m), AA , Boudnib (951 m); Dawah et al. 2020 Micromorphus minusculus Negrobov, 2000 Nourti et al. 2019a , EM , Bouanane (Figuig, 855 m), AA , Taliouine (Taroudant, 1014 m); Grichanov 2019 , AA , Ouarzazate Rhaphiinae Rhaphium Meigen, 1803 Rhaphium appendiculatum Zetterstedt, 184920 = Rhaphium macrocerum (Parent, 1925), in Parent 1938 , Grichanov 2019 Parent 1938 (no material provided) Rhaphium brevicorne Curtis, 1835 = Xiphandrium pectinatum Becker, in Vaillant 1956b : 112, Grichanov 2019 Vaillant 1956b , AP , Rabat, HA , Oukaimeden; Kettani and Negrobov 2016 , AP , Rabat; Grichanov 2019 , HA , Oukaimeden (1000 m); Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Dayat Tazia (733 m), Moulay Abdelsalam (1177 m); AP (Rabat) – MISR Rhaphium caliginosum Meigen, 1824 = Raphium lanceolatum Loew, 1850, in Kazerani et al. 2013 Parent 1924 , Rif , Cap Spartel; Kazerani et al. 2013 (no material provided); Kechev 2017 ; Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), Dbani (Bni Selmane, 1046 m) Rhaphium fascipes (Meigen, 1824) = Porphyrops fascipes Meigen, 1824, in Parent 1927 Parent 1927 , Rif , Tétouan Rhaphium fissum Loew, 1850 Grichanov 2009 , AA , Tizi-n'Test pass (2100 m) Sciapodinae Sciapus Zeller, 1842 Sciapus adumbratus Becker, 1902 Grichanov and Negrobov 2014 , AP , near Essaouira, AA , Oued Souss, near Ouarzazate (1100 m) Sciapus costae (Mik, 1890) Negrobov 1991 (no material provided); Ebejer et al. 2019 , Rif , Oued Laou (30 m) Sciapus euzonus (Loew, 1859) = Psilopus euzonus Loew, in Dakki 1997 : 62 Parent 1927 , Rif , El Mahadi; Séguy 1930a , Rif , Tanger; Séguy 1941d , AA , Taroudant Sciapus glaucescens (Loew, 1856) Grichanov and Negrobov 2014 , AP , Oualidia Sciapus heteropygus Parent, 1926 Nourti et al. 2019c , Rif , Talassemtane National Park (1696 m) Sciapus holoxanthos Parent, 1926 Nourti et al. 2019c , Rif , Jbel Bouhachem, Adrou (556 m), Talassemtane National Park (1696 m) Sciapus laetus (Meigen, 1838) Grichanov 2009 , AA , 40 km S Larache (0–20 m); Ebejer et al. 2019 , Rif , Martil (9 m) Sciapus longulus (Fallén, 1823) 25 Pârvu et al. 2006 , AP , Merja Zerga Sciapus aff. negrobovi Naglis & Barták, 2015 Nourti et al. 2019a , Rif , plage Stihat (0 m), Kitane (49 m) (as Sciapus aff. negrobovi ); Nourti et al. 2019c Sympycninae Campsicnemus Haliday in Walker, 1851 Campsicnemus crinitarsis Strobl, 1906 Dakki 1997 (no material provided); Grichanov 2012 , AP , Essaouira; Kettani and Negrobov 2016 , Rif , Oued Amsa Campsicnemus curvipes (Fallén, 1823) 26 Frey 1936 (no material provided) Campsicnemus filipes Loew, 1859 Grichanov 2009 , AA , 40 km S Larache (0–20 m) Campsicnemus loripes (Haliday, 1832) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m) Campsicnemus magius (Loew, 1845) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Grichanov 2019 , AP , Essaouira Campsicnemus simplicissimus Strobl, 1906 Nourti et al. 2019a , Rif , plage Stihat (0 m) Sympycnus Loew, 1857 Sympycnus pulicarius (Fallén, 1823) Nourti et al. 2019a , HA , Aïn Taferaout (Sidi Masali, 1237 m) Syntormon Loew, 1857 Syntormon aulicus Meigen, 1824 = Eutarsus aulicus Meigen, in Parent 1924 , 1927 ; Séguy 1930a : 125 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Vaillant 1952 , 1956b , HA , Imi-N'Ifri Syntormon codinai Parent, 1924 Parent 1924 , Rif , Cap Spartel, Tanger; Parent, 1927, Rif , Tanger Syntormon denticulatus (Zetterstedt, 1843) = Syntormon pumilus Parent, 1925 (nec Meigen, 1824; misidentification), in Pârvu et al. 2006 , Grichanov 2019 Parent 1924 , Rif , Tétouan; Parent 1927 , Rif , Tétouan; Séguy 1930a , Rif , Tanger; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Nourti et al. 2019a , Rif , plage Stihat (0 m), Amlay (294 m), Koudiat Taifour (100 m), Amsemlil env. (1067 m), Dayat Tazia (733 m), Oued Souk Lhad (613 m), HA , Aïn Taferaout (Sidi Masali, 1237 m), AA , Taliouine (1014 m) Syntormon mikii Strobl, 1899 Parent 1927 , "Maroc"; Kechev and Ivanova 2015 ; Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Moulay Abdelsalam (1177 m) Syntormon monilis (Haliday, 1851) Parent 1924 , Rif , Cap Spartel; Parent 1927 , Rif , Cap Spartel Syntormon pallipes (Fabricius, 1794) Parent 1924 , Rif , Cap Spartel, Tétouan, Chefchaouen; Parent 1927 , Rif , Cap Spartel; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), Talassemtane (339 m), Perdicaris Park (223 m), Amsemlil env. (1067 m); Dawah et al. 2020 Syntormon pilitibia Grichanov, 2013 Nourti et al. 2019a , Rif , Amsemlil ( PNPB , 1067 m) Syntormon pumilus (Meigen, 1824) = Syntormon rufipes auctt. (nec Meigen, 1824; misidentification), in Pârvu et al. 2006 ; Grichanov 2019 Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate Syntormon zelleri (Loew, 1850) Vaillant 1956b , HA , Oukaimeden, Izourar; Pârvu et al. 2006 , AP , Merja Zerga; Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Amsemlil env. (1059–1067 m) Teuchophorus Loew, 1857 Teuchophorus cristulatus Mueffels & Grootaert, 1990 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Teuchophorus rifensis Nourti, Grichanov & Kettani, 2019 Nourti et al. 2019b , Rif , Oued Souk Lhad (613 m) Teuchophorus spinigerellus (Zetterstedt, 1843) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate Xanthochlorinae Xanthochlorus Loew, 1857 Xanthochlorus tenellus (Wiedemann, 1817) Grichanov 2009 , AA , 40 km S Larache (0–20 m); Ebejer et al. 2019 , Rif , Moulay Abdelsalam (1180 m) HYBOTIDAE K. Kettani, P. Gatt Number of species: 44 . Expected: 120 Faunistic knowledge of the family in Morocco: poor Ocydromiinae Bicellariini Bicellaria Macquart, 1823 Bicellaria spuria Fallén, 1816 Becker and Stein 1913 , Rif , Tanger Ocydromiini Ocydromia Meigen, 1820 Ocydromia glabricula (Fallén, 1816) Becker and Stein 1913 , Rif , Tanger Oropezella Collin, 1926 Oropezella sphenoptera (Loew, 1873) Cassar et al. 2005 , Rif , lagoon Smir Tachydromiinae Drapetini Chersodromia Haliday in Walker, 1851 Chersodromia albopilosa Chvála, 1970 Cassar et al. 2008 , Rif , Basin Laou Chersodromia nigrosetosa Chvála, 1970 Chvála 1981 (Ceuta); Chvála and Kovalev 1989 Chersodromia pseudohirta Chvála, 1970 Ebejer et al. 2019 , Rif , Kabila beach Crossopalpus Bigot, 1857 Crossopalpus aeneus (Walker, 1871) 27 Shamshev et al. 2005 , HA , Marrakech, Ouirgane Crossopalpus dilutipes (Strobl, 1906) Ebejer et al. 2019 , AP , 9 km SE of Aïn Chouk (Lower Loukous marsh, 6 m) Crossopalpus nigritellus (Zetterstedt, 1842) Ebejer et al. 2019 , Rif , Talassemtane (maison forestière), Issaguen (1620 m) Crossopalpus setiger (Loew, 1859) Ebejer et al. 2019 Rif , Smir lagoon Drapetis Meigen, 1822 Drapetis disparilis Frey, 1936 Chvála 1981 (Ceuta); Chvála and Kovalev 1989 Drapetis laevis Becker, 1914 Becker and Stein 1913 , Rif , Tanger; Kovalev 1970 ; Chvála and Kovalev 1989 Elaphropeza Macquart, 1827 Elaphropeza boergei Chvála, 1971 Ebejer et al. 2019 , Rif , Smir lagoon, Oued Laou (saltmarsh) Elaphropeza hutsoni Smith, 1967 Ebejer et al. 2019 , Rif , Jnane Niche (46 m) Stilpon Loew, 1859 Stilpon demnatensis Vaillant, 1956 28 Vaillant 1956b : 244, HA , Imi-N'Ifri Stilpon moroccensis Grootaert & Zouhair, 2021 Grootaert et al. 2021 , Rif , beach of Stehat, Bab Tariouant, Amsemlil, EM , Bouanane Stilpon subnubilus Chvála, 1988 Ebejer et al. 2019 , Rif , Smir lagoon, M'Diq (Kabila beach and dunes), Martil (beach and dunes) Tachydromiini Platypalpus Macquart, 1827 Platypalpus alluaudi Grootaert & Chvála, 1992 Grootaert and Chvála 1992 , HA , Chichaoua; Vaňhara and Rozkošný 1997 Platypalpus anomalicerus (Becker, 1902) = Coryneta aerivaga Séguy, in Séguy 1941d : 11 Séguy 1941d , EM , Oued Guir, AA , Agadir; Grootaert and Chvála 1992 , EM , Oued Guir, AA , Agadir Platypalpus annulatus (Fallén, 1815) Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 Platypalpus anomalitarsis Chvála & Kovalev, 1974 Ebejer et al. 2019 , MA , 10 km S of Azrou (1775 m), 10 km S of Azrou (1720 m), AA , Ziz river (30 km N of Erfoud, 894 m) Platypalpus approximatus (Becker, 1902) Grootaert and Chvála 1992 , AP , Casablanca Platypalpus asniensis Grootaert & Chvála, 1992 Grootaert and Chvála 1992 , HA , Asni; Vaňhara and Rozkošný 1997 Platypalpus calceatus (Meigen, 1822) Pârvu et al. 2006 , MA , Meknès, AA , Foum Zghouig Platypalpus chillcotti Chvála, 1981 Grootaert and Chvála 1992 , MA , Ifrane Platypalpus chrysonotus (Strobl, 1899) Ebejer et al. 2019 , Rif , Oued Laou (saltmarsh) Platypalpus desertorum (Becker, 1907) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Platypalpus distichus Grootaert & Chvála, 1992 Ebejer et al. 2019 , AP , 9 km SE of Aïn Chouk (Lower Loukous marsh, 6 m), AA , Ziz river (10 km S of Errachidia, 1008 m) Platypalpus flavicornis (Meigen, 1822) Ebejer et al. 2019 , AA , 2 km N of Erfoud (818 m), Ziz river (30 km N of Erfoud, 894 m) Platypalpus longicauda Grootaert & Chvála, 1992 Ebejer et al. 2019 , Rif , Smir lagoon Platypalpus lyneborgi Chvála, 1981 Grootaert and Chvála 1992 , AP , Dradek, MA , Azrou Platypalpus nigritarsis (Fallén, 1816) Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 Platypalpus obscuripes (Strobl, 1899) Ebejer et al. 2019 , Rif , Martil (9 m), AP , Larache (Loukous marsh, 2 m) Platypalpus ostiorum (Becker, 1902) Grootaert and Chvála 1992 Platypalpus pachycerus (Collin, 1949) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Platypalpus pallidiventris (Meigen, 1822) Grootaert and Chvála 1992 , MA , Ifrane; Maarouf 2003 , HA , Chaouia Platypalpus pseudoexiguus (Strobl, 1909) Ebejer et al. 2019 , Rif , Oued Laou (saltmarsh) Platypalpus pseudounguiculatus (Strobl, 1909) = Tachydromia pseudounguiculata Strobl 1909 Grootaert and Chvála 1992 Platypalpus riojaensis Chvála, 1981 Grootaert and Chvála 1992 , EM , Oujda, MA , Meknès, HA , Chichaoua Platypalpus turgidus (Becker, 1907) Grootaert and Chvála 1992 , MA , Takkat-n- Sountat Platypalpus vockerothi Chvála, 1981 Grootaert and Chvála 1992 , HA , Asni Tachydromia Meigen, 1803 Tachydromia arrogans (Linnaeus, 1761) Ebejer et al. 2019 , Rif , Oued Laou, El-Fahsa (maquis) Tachydromia annulimana Meigen, 1822 29 = Tachista annulimana Meigen, in Becker and Stein 1913 : 84 Becker and Stein 1913 , Rif , Tanger Tachydromia undulata (Strobl, 1906) Chvála 1969 ATELESTIDAE K. Kettani, P. Gatt Number of species: 1 . Expected: 1 Faunistic knowledge of the family in Morocco: poor Atelestus Walker, 1837 Atelestus sp. nov. Ebejer et al. 2019 , Rif , Dardara (484–730 m), Aïn Tissemlal (Azilane, 1255 m), Douar El Hamma (338 m), Chrabkha pond (Al Manzla, 58 m) EMPIDIDAE K. Kettani, C. Daugeron Number of species: 40 . Expected: 100 Faunistic knowledge of the family in Morocco: poor Clinocerinae Clinocera Meigen, 1803 Clinocera maroccana (Séguy, 1941): 29 (= Hydrodromia ) = Atalanta ( Hydrodromia ) algira (Vaillant, 1952): 65 Séguy 1941a , HA , Anrhemer (Toubkal, 2500 m); Vaillant 1956b , HA , Sidi Chamarouch; Vaillant 1964 ; Dakki 1997 Clinocera megalatlantica (Vaillant, 1957): 65 (= Atalanta ( Atalanta ) ) Vaillant 1956b , HA ; Dakki 1997 Clinocera nigra Meigen, 1804: 292 = Heleodromia unicolor (Curtis, 1834): plate 513, Paramesia roberti (Macquart, 1835): 657 Vaillant 1956b , HA , Izourar, Sidi Chamarouch, Aguelmous; Dakki 1997 Dolichocephala Meigen, 1803 Dolichocephala ocellata (Costa, 1858): 7 (= Ardoptera ) = oculata (Loew, 1858~7): 7 (= Ardoptera ) = novemguttata (Strobl, 1893): 98 (= Ardoptera ) = albohalterata (Strobl, 1898): 399 (= Ardoptera ) = barbarica Vaillant, 1952: 65 = algira Vaillant, 1957: 64 Vaillant 1956b , HA , Imi-N'Ifri; Dakki 1997 Dolichocephala pavonica Vaillant & Gagneur, 1998: 380 Vaillant and Gagneur 1998 , HA , Demnat Kowarzia Mik, 1881 Kowarzia barbatula (Mik, 1880): 347 (= Clinocera ) = Clinocera dorieri (Vaillant, 1968): 88 Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Aguelmous, Sidi Chamarouch, Izourar, Oukaimeden; Dakki 1997 ; Vaillant and Moubayed 1998 Kowarzia bipunctata (Haliday, 1833) (= Heleodromia ) Vaillant 1956b , HA , Oukaimeden; Vaillant 1964 ; Dakki 1997 Kowarzia dieuzedei Vaillant, 1953: 60 Vaillant 1956b , HA , Lac Tamhda (Anremer); Vaillant 1964 , HA ; Dakki 1997 Kowarzia madicola (Vaillant, 1965): 152 (= Atalanta ) Vaillant 1956b , HA , Tahanaout Kowarzia tenella (Wahlberg, 1844): 107 (= Parmesia ) = Heleodromia zetterstedti (Walker, 1851): 105 = Wiedemannia securigera (Engel, 1918): 70 Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Aguelmous, Sidi Chamarouch, Izourar, Oukaimeden; Dakki 1997 ; Vaillant and Moubayed 1998 Wiedemannia Zetterstedt, 1838 Wiedemannia ( Chamaedipsia ) beckeri (Mik, 1889): 71 (= Chamaedipsia ) = jugorum (Strobl, 1893): 105 (= Chamaedipsia ) = crinita Engel, 1918: 217 (as var. of W. beckeri ) = alticola Vaillant, 195l: 54 = alpina Vaillant, 1967: 274 (as ssp. of W. beckeri ) = glaciola Wagner, 1985: 86 (as ssp. of W. beckeri ; new name for W. beckeri alpina Vaillant) Dakki 1997 Wiedemannia ( Chamaedipsia ) mgounica Vaillant, 1957: 69 Vaillant 1956a , HA , M'Goum; Dakki 1997 Wiedemannia ( Philolutra ) azurea (Vaillant, 1951): 3 (= Philolutra ) El Mezdi and Giudicelli 1985 , HA , Khettaras Marrakech; Dakki 1997 ; Vaillant and Moubayed 1998 Wiedemannia ( Philolutra ) fallaciosa Loew, 1873: 44 Vaillant 1964 , HA ; Dakki 1997 Wiedemannia ( Philolutra ) fallaciosa ssp. litardierei Vaillant, 1957: 67 Vaillant 1956a , HA ; Dakki 1997 Wiedemannia ( Roederella ) ouedorum Vaillant, 1952: 371 = ovedorum (error) Vaillant, 1978: 469 Vaillant 1964 , HA ; Dakki 1997 Hemerodromiinae Hemerodromia Meigen, 1822 Hemerodromia bethiana Vaillant & Gagneur, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Hemerodromia subapicalis Yang, Zhang & Zhang, 2007 = Hemerodromia apicalis Vaillant and Gagneur 1998 : 372 (preoccupied by Smith, 1969) Vaillant and Gagneur 1998 , HA , Oum-er-Rbia; Yang et al. 2007 Hemerodromia tigrigrana Vaillant & Gagneur, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Hemerodromia todrhana (Vaillant, 1956) Vaillant 1956, HA , Todrha; El Mezdi and Guidicelli 1985; Dakki 1997 ; Vaillant and Gagneur 1998 Hemerodromia zarcana Vaillant & Moubayed, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Empidinae Empidini Empis Linnaeus, 1758 Empis ( Coptophlebia ) confluens Becker, 1907 MA (Meknès), 19.v.1997, K. Deneš leg. – OUMNH Empis ( Empis ) decora Meigen, 1822 Bahid 2018 , Rif , Oued Tkarae ( PNPB ); Ebejer et al. 2019 , Rif , Oued Nakhla, Moulay Abdelsalam Empis ( Empis ) nikita Shamshev, 2018 Shamshev 2018 , AP , Essaouira Empis ( Kritempis ) taffertensis Daugeron, 2009 Daugeron 2009 , MA , forest of Taffert; Bahid 2018 Empis ( Leptempis ) tenuis Bahid & Daugeron, 2017 Bahid et al. 2017 , MA , Tizi-s'Tkrine (Jebel Amar, 1760 m); Bahid 2018 Empis ( Pachymeria ) suberis Becker, 1907 Ebejer et al. 2019 , Rif , Moulay Abdelsalam, Issaguen, Bab Berred, Jebel Lakraâ Empis ( Polyblepharis ) eumera Loew, 1866 Ebejer et al. 2019 , MA , Ifrane Empis ( Xanthempis ) chopardi Daugeron, 1997 Daugeron 1997 , MA , Ifrane; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) edithae Daugeron, 1997 Daugeron 1997 , HA , High Imminen, Tachdirt; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) ifranensis Daugeron, 1997 Daugeron 1997 , MA , Ifrane; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) styriaca (Strobl, 1893) Chvála and Wagner 1989 , HA [doubtful record]; Bahid 2018 Empis ( Xanthempis ) widanensis Bahid & Daugeron, 2018 Bahid et al. 2018 , Rif , Dayat Bayan Widane, Aïn Sedraouia, Tazia, Anissar, Lalla Outka Rhamphomyia Meigen, 1822 Rhamphomyia ( Rhamphomyia ) maroccana Collin, 2009 Chvála and Wagner 1989 , MA , Ifrane; Collin 2009 , MA , Ifrane; Bahid 2018 , Rif , Oued Akrir (Fifi) Rhamphomyia ( Holoclera ) tenuipes Becker, 1907* AP , Haenni pers. comm. Hilarini Hilara Meigen, 1822 Hilara algecirasensis Strobl, 1899 Ebejer et al. 2019 , MA , Lac Aguelmane Afennourir, HA , Ziz river Hilara almeriensis Strobl, 1906 Chvála 2008 , AA , Tifoultoute (1146 m) Hilara fusitibia Strobl, 1899 Chvála 2008 , MA , Ifrane (Forêt de Cédres, 1500 m) Hilara longeciliata Strobl, 1906 Chvála 2008 , AP , Rabat (near Oued Bou-Regreg, 0–10 m) Hilara schachti Chvála, 2008 Chvála 2008 , MA , Ifrane (Ghabat al Behar, 1650–1700 m); Bahid 2018 New record for Morocco Rhamphomyia ( Holoclera ) tenuipes Becker, 1907 Atlantic Plain: Essaouira, 6 km W, 3.iv.2002, Forêt de genévriers pâturée, 1♂1♀, J.-P. Haenni leg., coll. MHNN . Acknowledgements We are very grateful to Bradley Sinclair (Canadian Food Inspection Agency, Canada) and Adrian Plant (Mahasarakham University, Thailand) for reviewing parts of this family. Clinocerinae Clinocera Meigen, 1803 Clinocera maroccana (Séguy, 1941): 29 (= Hydrodromia ) = Atalanta ( Hydrodromia ) algira (Vaillant, 1952): 65 Séguy 1941a , HA , Anrhemer (Toubkal, 2500 m); Vaillant 1956b , HA , Sidi Chamarouch; Vaillant 1964 ; Dakki 1997 Clinocera megalatlantica (Vaillant, 1957): 65 (= Atalanta ( Atalanta ) ) Vaillant 1956b , HA ; Dakki 1997 Clinocera nigra Meigen, 1804: 292 = Heleodromia unicolor (Curtis, 1834): plate 513, Paramesia roberti (Macquart, 1835): 657 Vaillant 1956b , HA , Izourar, Sidi Chamarouch, Aguelmous; Dakki 1997 Dolichocephala Meigen, 1803 Dolichocephala ocellata (Costa, 1858): 7 (= Ardoptera ) = oculata (Loew, 1858~7): 7 (= Ardoptera ) = novemguttata (Strobl, 1893): 98 (= Ardoptera ) = albohalterata (Strobl, 1898): 399 (= Ardoptera ) = barbarica Vaillant, 1952: 65 = algira Vaillant, 1957: 64 Vaillant 1956b , HA , Imi-N'Ifri; Dakki 1997 Dolichocephala pavonica Vaillant & Gagneur, 1998: 380 Vaillant and Gagneur 1998 , HA , Demnat Kowarzia Mik, 1881 Kowarzia barbatula (Mik, 1880): 347 (= Clinocera ) = Clinocera dorieri (Vaillant, 1968): 88 Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Aguelmous, Sidi Chamarouch, Izourar, Oukaimeden; Dakki 1997 ; Vaillant and Moubayed 1998 Kowarzia bipunctata (Haliday, 1833) (= Heleodromia ) Vaillant 1956b , HA , Oukaimeden; Vaillant 1964 ; Dakki 1997 Kowarzia dieuzedei Vaillant, 1953: 60 Vaillant 1956b , HA , Lac Tamhda (Anremer); Vaillant 1964 , HA ; Dakki 1997 Kowarzia madicola (Vaillant, 1965): 152 (= Atalanta ) Vaillant 1956b , HA , Tahanaout Kowarzia tenella (Wahlberg, 1844): 107 (= Parmesia ) = Heleodromia zetterstedti (Walker, 1851): 105 = Wiedemannia securigera (Engel, 1918): 70 Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Aguelmous, Sidi Chamarouch, Izourar, Oukaimeden; Dakki 1997 ; Vaillant and Moubayed 1998 Wiedemannia Zetterstedt, 1838 Wiedemannia ( Chamaedipsia ) beckeri (Mik, 1889): 71 (= Chamaedipsia ) = jugorum (Strobl, 1893): 105 (= Chamaedipsia ) = crinita Engel, 1918: 217 (as var. of W. beckeri ) = alticola Vaillant, 195l: 54 = alpina Vaillant, 1967: 274 (as ssp. of W. beckeri ) = glaciola Wagner, 1985: 86 (as ssp. of W. beckeri ; new name for W. beckeri alpina Vaillant) Dakki 1997 Wiedemannia ( Chamaedipsia ) mgounica Vaillant, 1957: 69 Vaillant 1956a , HA , M'Goum; Dakki 1997 Wiedemannia ( Philolutra ) azurea (Vaillant, 1951): 3 (= Philolutra ) El Mezdi and Giudicelli 1985 , HA , Khettaras Marrakech; Dakki 1997 ; Vaillant and Moubayed 1998 Wiedemannia ( Philolutra ) fallaciosa Loew, 1873: 44 Vaillant 1964 , HA ; Dakki 1997 Wiedemannia ( Philolutra ) fallaciosa ssp. litardierei Vaillant, 1957: 67 Vaillant 1956a , HA ; Dakki 1997 Wiedemannia ( Roederella ) ouedorum Vaillant, 1952: 371 = ovedorum (error) Vaillant, 1978: 469 Vaillant 1964 , HA ; Dakki 1997 Hemerodromiinae Hemerodromia Meigen, 1822 Hemerodromia bethiana Vaillant & Gagneur, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Hemerodromia subapicalis Yang, Zhang & Zhang, 2007 = Hemerodromia apicalis Vaillant and Gagneur 1998 : 372 (preoccupied by Smith, 1969) Vaillant and Gagneur 1998 , HA , Oum-er-Rbia; Yang et al. 2007 Hemerodromia tigrigrana Vaillant & Gagneur, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Hemerodromia todrhana (Vaillant, 1956) Vaillant 1956, HA , Todrha; El Mezdi and Guidicelli 1985; Dakki 1997 ; Vaillant and Gagneur 1998 Hemerodromia zarcana Vaillant & Moubayed, 1998 Vaillant and Gagneur 1998 , MA , Tigrigra (Azrou) Empidinae Empidini Empis Linnaeus, 1758 Empis ( Coptophlebia ) confluens Becker, 1907 MA (Meknès), 19.v.1997, K. Deneš leg. – OUMNH Empis ( Empis ) decora Meigen, 1822 Bahid 2018 , Rif , Oued Tkarae ( PNPB ); Ebejer et al. 2019 , Rif , Oued Nakhla, Moulay Abdelsalam Empis ( Empis ) nikita Shamshev, 2018 Shamshev 2018 , AP , Essaouira Empis ( Kritempis ) taffertensis Daugeron, 2009 Daugeron 2009 , MA , forest of Taffert; Bahid 2018 Empis ( Leptempis ) tenuis Bahid & Daugeron, 2017 Bahid et al. 2017 , MA , Tizi-s'Tkrine (Jebel Amar, 1760 m); Bahid 2018 Empis ( Pachymeria ) suberis Becker, 1907 Ebejer et al. 2019 , Rif , Moulay Abdelsalam, Issaguen, Bab Berred, Jebel Lakraâ Empis ( Polyblepharis ) eumera Loew, 1866 Ebejer et al. 2019 , MA , Ifrane Empis ( Xanthempis ) chopardi Daugeron, 1997 Daugeron 1997 , MA , Ifrane; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) edithae Daugeron, 1997 Daugeron 1997 , HA , High Imminen, Tachdirt; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) ifranensis Daugeron, 1997 Daugeron 1997 , MA , Ifrane; Daugeron 2000 ; Bahid 2018 Empis ( Xanthempis ) styriaca (Strobl, 1893) Chvála and Wagner 1989 , HA [doubtful record]; Bahid 2018 Empis ( Xanthempis ) widanensis Bahid & Daugeron, 2018 Bahid et al. 2018 , Rif , Dayat Bayan Widane, Aïn Sedraouia, Tazia, Anissar, Lalla Outka Rhamphomyia Meigen, 1822 Rhamphomyia ( Rhamphomyia ) maroccana Collin, 2009 Chvála and Wagner 1989 , MA , Ifrane; Collin 2009 , MA , Ifrane; Bahid 2018 , Rif , Oued Akrir (Fifi) Rhamphomyia ( Holoclera ) tenuipes Becker, 1907* AP , Haenni pers. comm. Hilarini Hilara Meigen, 1822 Hilara algecirasensis Strobl, 1899 Ebejer et al. 2019 , MA , Lac Aguelmane Afennourir, HA , Ziz river Hilara almeriensis Strobl, 1906 Chvála 2008 , AA , Tifoultoute (1146 m) Hilara fusitibia Strobl, 1899 Chvála 2008 , MA , Ifrane (Forêt de Cédres, 1500 m) Hilara longeciliata Strobl, 1906 Chvála 2008 , AP , Rabat (near Oued Bou-Regreg, 0–10 m) Hilara schachti Chvála, 2008 Chvála 2008 , MA , Ifrane (Ghabat al Behar, 1650–1700 m); Bahid 2018 New record for Morocco Rhamphomyia ( Holoclera ) tenuipes Becker, 1907 Atlantic Plain: Essaouira, 6 km W, 3.iv.2002, Forêt de genévriers pâturée, 1♂1♀, J.-P. Haenni leg., coll. MHNN . Acknowledgements We are very grateful to Bradley Sinclair (Canadian Food Inspection Agency, Canada) and Adrian Plant (Mahasarakham University, Thailand) for reviewing parts of this family. DOLICHOPODIDAE K. Kettani, I.Ya. Grichanov, O.P. Negrobov Number of species: 112 . Expected: 300 Faunistic knowledge of the family in Morocco: poor Diaphorinae Argyra Macquart, 1834 Argyra argentina Meigen, 1824 Parent 1924 , Rif , Cap Spartel, Tétouan; Parent 1927 , Rif , Tétouan Argyra argyria (Meigen, 1824) Parent 1924 (females only), Rif , Cap Spartel, Tétouan; Kechev and Ivanova 2015 Argyra biseta Parent, 1929 Parent 1929a , Rif , Tanger; Vaillant 1955a Argyra grata Loew, 1857 20 Negrobov 1991 (no material provided) Asyndetus Loew, 1869 Asyndetus separatus (Becker, 1902) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Chrysotus Meigen, 1824 Chrysotus albibarbus Loew, 1857 Ebejer et al. 2019 , MA , Lac Aguelmane Afennourir (30 km SW of Azrou, 1760 m); Grichanov 2019 , AA , Aït Melloul Chrysotus gramineus (Fallén, 1823) Parent 1924 , Rif , Tanger; Parent 1927 Chrysotus larachensis Grichanov, Nourti & Kettani, 2020 Grichanov et al. 2020b , Rif , El Hamma (338 m) Chrysotus pennatus Lichtwardt, 1902 Ebejer et al. 2019 , Rif , Smir Barrage (145 m), AA , 1 km N of Tarda (Errachidia, 1023 m) Chrysotus suavis Loew, 1857 Grichanov 2009 , HA , Asni area (1100–1400 m); Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), MA , Dayat Ifrane (1607 m), HA , Tahanout (956 m); Dawah et al. 2020 Diaphorus Meigen, 1824 Diaphorus africus Parent, 1924 Parent 1924 , Rif , Tétouan, Tanger; Parent 1927 , Rif , Tanger; Ebejer et al. 2019 , Rif , Oued Siflaou (281 m); Grichanov 2019 , AA , Ouarzazate (1100 m) Diaphorus vitripennis Loew, 1859 Grichanov 2019 , AA , Aït Melloul Dolichopodinae Dolichopus Latreille, 1796 Dolichopus andalusiacus (Strobl, 1899) Ebejer et al. 2019 , AP , Loukous marsh (2 m); Nourti et al. 2019a , Rif , plage Stihat (0 m) Dolichopus griseipennis Stannius, 1831 Parent 1924 , Rif , Tanger; Parent 1927 ; Séguy 1930a , Rif , Tanger; Nourti et al. 2019a , Rif , Adrou ( PNPB , 556 m) Dolichopus sabinus Haliday, 1838 Ebejer et al. 2019 , Rif , Martil (9 m), Oued Laou (2 m); Nourti et al. 2019a , Rif , plage Stihat (4 m) Dolichopus scutopilosus Parent, 1933 Parent 1933 , HA , Arround Dolichopus signifer Haliday, 1832 Parent 1929b , "Maroc"; Séguy 1930a , MA , Ras el Ma; Grichanov 2019 , HA , Oukaimeden (2600 m) Dolichopus strigipes Verrall, 1875 Grichanov 2009 , AP , 40 km S Larache (0–20 m) Gymnopternus Loew, 1857 Gymnopternus assimilis (Staeger, 1842) Nourti et al. 2019a , Rif , Amsemlil (1067 m) Hercostomus Loew, 1857 Hercostomus apollo (Loew, 1869) Nourti et al. 2019a , Rif , Talassemtane (1696 m), Adrou ( PNPB , 556 m), Amsemlil ( PNPB , 1067 m) Hercostomus canariensis Santos Abreu, 1929 Grichanov et al. 2020a , Rif , Pont de Dieu (Akchour, 536 m); Nourti et al. 2019a (as H. aff. exarticulatoides Stackelberg, 1949) Hercostomus chetifer (Haliday, 1849) Ebejer et al. 2019 , Rif , Sidi Yahia Aarab (377 m) Hercostomus discriminatus Parent, 1925 Parent 1925 , Rif , "Favier, Environs de Tanger"; Parent 1927 , Rif , Tanger; Vaillant 1950 , Rif , Tanger Hercostomus exarticulatus (Loew, 1857) Vaillant 1956b , HA , Lac Tamhda (Anremer), Aguelmous; Grichanov et al. 2020a Hercostomus excipiens Becker, 1907 Parent 1924 , Rif , Tétouan; Parent 1927 ; Séguy 1930a , Rif , Oued Judios (Tanger); Nourti et al. 2019a , Rif , Talembote (440 m) Hercostomus germanus (Wiedemann, 1817) Parent 1924 , Rif , Cap Spartel; Parent 1927 ; Kettani and Negrobov 2016 , Rif , Chefchaouen, Ketama – MISR ( Rif , Ketama) Hercostomus longiventris (Loew, 1857) Vaillant 1956b , HA , Lac Tamhda (Anremer), Izourar Muscidideicus Becker, 1917 Muscidideicus praetextatus (Haliday, 1855) Grichanov 2019 , AP , Oualidia lagune Ortochile Berthold, 1827 Ortochile morenae (Strobl, 1899) = Hercostomus morenae (Strobl, 1899), in Becker 1917 : 225; Nourti et al. 2019a : 124 Grichanov and Nourti 2021 ; Nourti et al. 2019a , Rif , Mnezla (74 m), Talassemtane (980 m), Oued Ametrasse (841 m), estuary Tahaddart (dune marshland, 0 m) Ortochile nigrocaerulea Latreille, 1779 Parent 1924 , Rif , Tanger, Cap Spartel, Tétouan, Béni Hozmar; Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Oued Judios (Tanger); Grichanov 2009 , Rif , Ouezzane (300 m); Nourti et al. 2019a , Rif , Douar El Hamma (338 m), Triwa Bni Hassane (654 m), Taida (501 m) Platyopsis Parent, 1929 Platyopsis maroccanus (Parent, 1929) Parent 1929b , Rif , Tanger; Vaillant 1950 , Rif , Tanger Poecilobothrus Mik, 1878 Poecilobothrus appendiculatus (Loew, 1859) = Hercostomus appendiculatus (Loew): Ebejer et al. 2019 : 146 Parent 1924 , Rif , Tanger, Cap Spartel; Ebejer et al. 2019 , Rif , Oued Nakhla (200 m), Moulay Abdelsalam (965 m), Dardara (730 m), Cap Spartel (155 m); Nourti et al. 2019a , Rif , Perdicaris Park (223 m) Poecilobothrus infuscatus (Stannius, 1831) Ebejer et al. 2019 , Rif , Tahaddart (2 m); Rif (Tahaddart) – MISR Sybistroma Meigen, 1824 Sybistroma dufouri Macquart, 1838 = Haltericerus spathulatus Loew, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger Sybistroma obscurellum Fallén, 182320 = Hypophyllus obscurellus Fallén, in Dakki 1997 : 61 Dakki 1997 (no material provided) Sybistroma quadrifilatum (Strobl, 1899) = Sybistroma parvulum (Parent, 1927), in Grichanov and Nourti 2021 : 190 Grichanov and Nourti 2021 , Rif , Fahs Anjra (372 m) Sybistroma theodori Grichanov & Nourti, 2021 Grichanov and Nourti 2021 , Rif , Moulay Abdelsalam (649 m) Tachytrechus Haliday, 1851 Tachytrechus consobrinus (Haliday, 1851)20 Parent 1938 (no material provided) Tachytrechus goudoti (Macquart, 1842) = Dolichopus goudoti Macquart, 1842 Macquart 1842 , Rif , Tanger; Parent 1926 (redescription), 1927 Tachytrechus insignis (Stannius, 1831) Parent 1927 , "Maroc"; Séguy 1930a , Rif , Tanger, HA , Aguerd el Had, Talekjount (1000–1100 m); Vaillant 1956b , HA , Lac Tamhda (Anremer); Popescu-Mirceni 2011 , AP , Merja Zerga; Grichanov 2019 , AP , Essaouira Tachytrechus notatus (Stannius, 1831) Vaillant 1950 (no material provided), 1956b, HA , Lac Tamhda (Anremer); Grichanov 2009 , AA , 15 km SW Tazenakcht; Dawah et al. 2020 Tachytrechus planitarsis Becker, 1907 Vaillant 1950 , HA , Touggourt; Grichanov 2009 , AA , 15 km SW Tazenakcht; Grichanov 2019 , AA , Ouarzazate (1100 m) Hydrophorinae Anahydrophorus Becker, 1917 Anahydrophorus cinereus (Fabricius, 1805) = Scatophaga cinerea Fabricius, 1805: 205 Fabricius 1805 , AP , Mogador (Essaouira); Séguy 1930a , Rif , Tanger; Vaillant 1955a , AP , Temara, Port-Lyautey; Boumezzough and Vaillant 1986a , AP , beach of Rabat; Kettani and Negrobov 2016 , AP , Skhirat; AP (Skhirat) – MISR Aphrosylus Haliday, 1851 Aphrosylus maroccanus Vaillant, 1955 Vaillant 1955a , AP , Port Lyautey Aphrosylus mitis Verrall, 1912 Grichanov 2019 , AP , Oualidia lagune Aphrosylus raptor luteipes Parent, 1929 Parent 1929b , AP , Mogador (as a variation of Aphrosylus raptor Haliday, 1851); Vaillant 1955a ; Negrobov, 1979 (as a subspecies of Aphrosylus raptor Haliday, 1851); Kettani and Negrobov 2016 (as Aphrosylus raptor Haliday, 1851); Grichanov 2019 , AP , Oualidia lagune Aphrosylus temaranus Vaillant, 1955 Vaillant 1955a , AP , Temara; Grichanov 2019 , AP , Oualidia lagune Aphrosylus venator Loew, 1857 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger Epithalassius Mik, 1891 Epithalassius corsicanus Becker, 1910 Pârvu 2008 , AP , Merja Zergha, Cap Sim, Essaouira Hydrophoprus Fallén, 1823 Hydrophorus balticus (Meigen, 1824) Vaillant 1956b , HA , Jebel Toubkal, Lac Tamhda (Anremer), Oukaimeden, Izourar; Boumezzough and Vaillant 1986a , HA , Jebel Toubkal (3100 m); Grichanov 2019 , HA , Aguelmouss (2050 m), Oukaimeden (2600 m); Nourti et al. 2019a , MA , Mont Habri (2071 m) Hydrophorus nilicola Parent, 1927 = Hydrophorus viridis nilicola Parent, in Boumezzough and Vaillant 1986a : 297 Boumezzough and Vaillant 1986a , MA , Tizi-n'Imdrhas (1800 m), HA , Oued N'fis (650 m), AA , near Agadir N'oussbai (400 m); Grichanov 2019 , AP , Essaouira Hydrophorus oceanus (Macquart, 1838) = Hydrophorus bisetus Loew, 1857, in Parent 1927 , Séguy 1930a , Grichanov 2019 Parent 1927 , AP , Rabat; Séguy 1930a , Rif , Tandja el Balia (Tanger) ( Hydrophorus bisetus Loew); Vaillant 1955a , AP , Port-Lyautey; Boumezzough and Vaillant 1986a , AP , beach of Rabat Hydrophorus praecox (Lehmann, 1822) Parent 1924 , Rif , Cap Spartel, de Tanger à Tétouan, Rincón de Medik, Dar Riffien (Ceuta); Parent 1927 ; Séguy 1941a , HA , Toubkal (2500 m); Boumezzough and Vaillant 1986a , HA , Lac Tamhda, Lac Tamdhanit (Massif Anremer, 2900 m), Lac Izourar (Massif Azourki, 2650 m) Hydrophorus viridis (Meigen, 1824) 21 Parent 1927 , AP , Rabat; Boumezzough and Vaillant 1986a : 297 Liancalus Loew, 1857 Liancalus virens (Scopoli, 1763) Vaillant 1956b , HA , Toubkal, Assif Tassouat (M'Goum), Aguelmous, Sidi Chamarouch, Imi-N'Ifri; Boumezzough and Vaillant 1986a , HA , Tahanaout (750 m), Adrar Anremer (2900 m), AA , Jebel Siroua (3000 m); Kettani and Negrobov 2016 , AP , S-Tifni; Nourti et al. 2019a , Rif , Amsemlil env. (1059 m) – MISR ( AP , S Tifni) Machaerium Haliday, 1832 Machaerium maritimae Haliday, 1832 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Kettani and Negrobov 2016 , AP , Oued Bou-Regreg; Grichanov 2019 , AP , Oualidia lagune; AP (Oued Bou-Regreg) – MISR Orthoceratium Schrank, 1803 Orthoceratium sabulosum (Becker, 1907) = Orthoceratium lacustre (Scopoli, 1763) 22 , in Ebejer et al. 2019 : 146 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Thinophilus Wahlberg, 1844 Thinophilus ( Thinophilus ) flavipalpis (Zetterstedt, 1843) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Grichanov 2009 , AP , 40 km S Larache (0–20 m) Thinophilus ( Thinophilus ) indigenus Becker, 1902 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m), Oued Laou (2 m); Grichanov 2019 , AA , Ouarzazate (572 m) Thinophilus ( Thinophilus ) mirandus Becker, 1907 Negrobov 1971 , Rif , Tanger; Grichanov 2019 , AA , Ouarzazate (572 m) Thinophilus ( Schoenophilus ) versutus Haliday, 1851 = Schoenophilus versutus (Haliday, 1851), in Parent 1924 , 1927 Parent 1924 , Rif , Cap Spartel, Tétouan; Parent 1927 ; Pârvu et al. 2006 , AP , Merja Zerga; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), Dayat Tazia (733 m) Medeterinae Medetera Fisher, 1819 Medetera diadema (Linnaeus, 1767) = Medeterus (cf. diadema ), in Séguy 1953a : 84 Parent 1938 (no material provided); Séguy 1953a , AP , Rabat; Nourti et al. 2019a , Rif , Kitane (49 m), Rif , plage Stihat (beach) Medetera media Parent, 1925 Nourti et al. 2019a , Rif , Faculty of Sciences of Tétouan (garden: on trunk of olive tree, 14 m) Medetera micacea Loew, 1857 Ebejer et al. 2019 , Rif , Dardara (730 m); Nourti et al. 2019a , Rif , Issaguen (1547 m), HA , Lac Tislit (Imilchil, 2254 m) Medetera pallipes (Zetterstedt, 1843) 23 Nourti et al. 2019a , Rif , Douar El Hamma (338 m) Medetera petrophila Kowarz, 187720 Parent 1938 (no material provided) Medetera petrophiloides Parent, 1925 Nourti et al. 2019a , Rif , Issaguen (1547 m) Medetera aff. roghii Rampini & Canzoneri, 1979 Nourti et al. 2019a , Rif , Douar El Hamma (338 m) Medetera truncorum Meigen, 1824 24 Ebejer et al. 2019 a, Rif , Dardara (484 m) Medetera varvara Grichanov & Vikhrev, 2009 Grichanov and Vikhrev 2009 , AP , Essaouira Thrypticus Gerstäcker, 1864 Thrypticus bellus Loew, 1869 Séguy 1930a , EM , Dayat Sidi Kacem; Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), estuary Oued Tahaddart (0 m), Barrage 9 Avril, plage Stihat (0 m) Microphorinae Schistostoma Becker, 1902 Schistostoma eremita Becker, 1902 Ebejer et al. 2019 , AA , Ziz river (12 km S of Rissani, 737 m), Lac Tiffert (4 km W of Merzouga, 702 m) Neurigoninae Neurigona Rondani, 1856 Neurigona solodovnikovi Grichanov, 2010 = Neurigona punctifera Becker, 1907, in Kettani and Negrobov 2016 (misidentification) Grichanov 2010, AP , 40 km S Larache; Kettani and Negrobov 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m); Nourti et al. 2019a , Rif , Perdicaris Park (223 m), Taria Bni Faghloum (894 m), Chrafate (832 m) Parathalassiinae Microphorella Becker, 1909 Microphorella ulrichi Gatt, 2003 Gatt 2003 , Rif , Tanger, Oued Armal (Ksar Sghir) Parathalassius Mik, 1891 Parathalassius blasigii Mik, 1891 Ebejer et al. 2019 , AP , Larache (5 m) Peloropeodinae Chrysotimus Loew, 1857 Chrysotimus molliculoides Parent, 1937 Parent 1937a , MA , Ifrane (1600 m); Parent 1937b ; Nourti et al. 2019a , Rif , Dayat Tazia (733 m) Micromorphus Mik, 1878 Micromorphus albipes (Zetterstedt, 1843) Parent 1924 , Rif , Béni Hozmar (Tétouan); Parent 1927 , Rif , Tétouan; Kechev and Ivanova 2015 ; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), estuary Oued Tahaddart (0 m), Barrage 9 Avril, Kharouba, plage Stihat (0 m), Aïn Jdioui, EM , Bouanane (Figuig, 855 m), AA , Boudnib (951 m); Dawah et al. 2020 Micromorphus minusculus Negrobov, 2000 Nourti et al. 2019a , EM , Bouanane (Figuig, 855 m), AA , Taliouine (Taroudant, 1014 m); Grichanov 2019 , AA , Ouarzazate Rhaphiinae Rhaphium Meigen, 1803 Rhaphium appendiculatum Zetterstedt, 184920 = Rhaphium macrocerum (Parent, 1925), in Parent 1938 , Grichanov 2019 Parent 1938 (no material provided) Rhaphium brevicorne Curtis, 1835 = Xiphandrium pectinatum Becker, in Vaillant 1956b : 112, Grichanov 2019 Vaillant 1956b , AP , Rabat, HA , Oukaimeden; Kettani and Negrobov 2016 , AP , Rabat; Grichanov 2019 , HA , Oukaimeden (1000 m); Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Dayat Tazia (733 m), Moulay Abdelsalam (1177 m); AP (Rabat) – MISR Rhaphium caliginosum Meigen, 1824 = Raphium lanceolatum Loew, 1850, in Kazerani et al. 2013 Parent 1924 , Rif , Cap Spartel; Kazerani et al. 2013 (no material provided); Kechev 2017 ; Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), Dbani (Bni Selmane, 1046 m) Rhaphium fascipes (Meigen, 1824) = Porphyrops fascipes Meigen, 1824, in Parent 1927 Parent 1927 , Rif , Tétouan Rhaphium fissum Loew, 1850 Grichanov 2009 , AA , Tizi-n'Test pass (2100 m) Sciapodinae Sciapus Zeller, 1842 Sciapus adumbratus Becker, 1902 Grichanov and Negrobov 2014 , AP , near Essaouira, AA , Oued Souss, near Ouarzazate (1100 m) Sciapus costae (Mik, 1890) Negrobov 1991 (no material provided); Ebejer et al. 2019 , Rif , Oued Laou (30 m) Sciapus euzonus (Loew, 1859) = Psilopus euzonus Loew, in Dakki 1997 : 62 Parent 1927 , Rif , El Mahadi; Séguy 1930a , Rif , Tanger; Séguy 1941d , AA , Taroudant Sciapus glaucescens (Loew, 1856) Grichanov and Negrobov 2014 , AP , Oualidia Sciapus heteropygus Parent, 1926 Nourti et al. 2019c , Rif , Talassemtane National Park (1696 m) Sciapus holoxanthos Parent, 1926 Nourti et al. 2019c , Rif , Jbel Bouhachem, Adrou (556 m), Talassemtane National Park (1696 m) Sciapus laetus (Meigen, 1838) Grichanov 2009 , AA , 40 km S Larache (0–20 m); Ebejer et al. 2019 , Rif , Martil (9 m) Sciapus longulus (Fallén, 1823) 25 Pârvu et al. 2006 , AP , Merja Zerga Sciapus aff. negrobovi Naglis & Barták, 2015 Nourti et al. 2019a , Rif , plage Stihat (0 m), Kitane (49 m) (as Sciapus aff. negrobovi ); Nourti et al. 2019c Sympycninae Campsicnemus Haliday in Walker, 1851 Campsicnemus crinitarsis Strobl, 1906 Dakki 1997 (no material provided); Grichanov 2012 , AP , Essaouira; Kettani and Negrobov 2016 , Rif , Oued Amsa Campsicnemus curvipes (Fallén, 1823) 26 Frey 1936 (no material provided) Campsicnemus filipes Loew, 1859 Grichanov 2009 , AA , 40 km S Larache (0–20 m) Campsicnemus loripes (Haliday, 1832) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m) Campsicnemus magius (Loew, 1845) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Grichanov 2019 , AP , Essaouira Campsicnemus simplicissimus Strobl, 1906 Nourti et al. 2019a , Rif , plage Stihat (0 m) Sympycnus Loew, 1857 Sympycnus pulicarius (Fallén, 1823) Nourti et al. 2019a , HA , Aïn Taferaout (Sidi Masali, 1237 m) Syntormon Loew, 1857 Syntormon aulicus Meigen, 1824 = Eutarsus aulicus Meigen, in Parent 1924 , 1927 ; Séguy 1930a : 125 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Vaillant 1952 , 1956b , HA , Imi-N'Ifri Syntormon codinai Parent, 1924 Parent 1924 , Rif , Cap Spartel, Tanger; Parent, 1927, Rif , Tanger Syntormon denticulatus (Zetterstedt, 1843) = Syntormon pumilus Parent, 1925 (nec Meigen, 1824; misidentification), in Pârvu et al. 2006 , Grichanov 2019 Parent 1924 , Rif , Tétouan; Parent 1927 , Rif , Tétouan; Séguy 1930a , Rif , Tanger; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Nourti et al. 2019a , Rif , plage Stihat (0 m), Amlay (294 m), Koudiat Taifour (100 m), Amsemlil env. (1067 m), Dayat Tazia (733 m), Oued Souk Lhad (613 m), HA , Aïn Taferaout (Sidi Masali, 1237 m), AA , Taliouine (1014 m) Syntormon mikii Strobl, 1899 Parent 1927 , "Maroc"; Kechev and Ivanova 2015 ; Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Moulay Abdelsalam (1177 m) Syntormon monilis (Haliday, 1851) Parent 1924 , Rif , Cap Spartel; Parent 1927 , Rif , Cap Spartel Syntormon pallipes (Fabricius, 1794) Parent 1924 , Rif , Cap Spartel, Tétouan, Chefchaouen; Parent 1927 , Rif , Cap Spartel; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), Talassemtane (339 m), Perdicaris Park (223 m), Amsemlil env. (1067 m); Dawah et al. 2020 Syntormon pilitibia Grichanov, 2013 Nourti et al. 2019a , Rif , Amsemlil ( PNPB , 1067 m) Syntormon pumilus (Meigen, 1824) = Syntormon rufipes auctt. (nec Meigen, 1824; misidentification), in Pârvu et al. 2006 ; Grichanov 2019 Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate Syntormon zelleri (Loew, 1850) Vaillant 1956b , HA , Oukaimeden, Izourar; Pârvu et al. 2006 , AP , Merja Zerga; Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Amsemlil env. (1059–1067 m) Teuchophorus Loew, 1857 Teuchophorus cristulatus Mueffels & Grootaert, 1990 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Teuchophorus rifensis Nourti, Grichanov & Kettani, 2019 Nourti et al. 2019b , Rif , Oued Souk Lhad (613 m) Teuchophorus spinigerellus (Zetterstedt, 1843) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate Xanthochlorinae Xanthochlorus Loew, 1857 Xanthochlorus tenellus (Wiedemann, 1817) Grichanov 2009 , AA , 40 km S Larache (0–20 m); Ebejer et al. 2019 , Rif , Moulay Abdelsalam (1180 m) Diaphorinae Argyra Macquart, 1834 Argyra argentina Meigen, 1824 Parent 1924 , Rif , Cap Spartel, Tétouan; Parent 1927 , Rif , Tétouan Argyra argyria (Meigen, 1824) Parent 1924 (females only), Rif , Cap Spartel, Tétouan; Kechev and Ivanova 2015 Argyra biseta Parent, 1929 Parent 1929a , Rif , Tanger; Vaillant 1955a Argyra grata Loew, 1857 20 Negrobov 1991 (no material provided) Asyndetus Loew, 1869 Asyndetus separatus (Becker, 1902) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Chrysotus Meigen, 1824 Chrysotus albibarbus Loew, 1857 Ebejer et al. 2019 , MA , Lac Aguelmane Afennourir (30 km SW of Azrou, 1760 m); Grichanov 2019 , AA , Aït Melloul Chrysotus gramineus (Fallén, 1823) Parent 1924 , Rif , Tanger; Parent 1927 Chrysotus larachensis Grichanov, Nourti & Kettani, 2020 Grichanov et al. 2020b , Rif , El Hamma (338 m) Chrysotus pennatus Lichtwardt, 1902 Ebejer et al. 2019 , Rif , Smir Barrage (145 m), AA , 1 km N of Tarda (Errachidia, 1023 m) Chrysotus suavis Loew, 1857 Grichanov 2009 , HA , Asni area (1100–1400 m); Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), MA , Dayat Ifrane (1607 m), HA , Tahanout (956 m); Dawah et al. 2020 Diaphorus Meigen, 1824 Diaphorus africus Parent, 1924 Parent 1924 , Rif , Tétouan, Tanger; Parent 1927 , Rif , Tanger; Ebejer et al. 2019 , Rif , Oued Siflaou (281 m); Grichanov 2019 , AA , Ouarzazate (1100 m) Diaphorus vitripennis Loew, 1859 Grichanov 2019 , AA , Aït Melloul Dolichopodinae Dolichopus Latreille, 1796 Dolichopus andalusiacus (Strobl, 1899) Ebejer et al. 2019 , AP , Loukous marsh (2 m); Nourti et al. 2019a , Rif , plage Stihat (0 m) Dolichopus griseipennis Stannius, 1831 Parent 1924 , Rif , Tanger; Parent 1927 ; Séguy 1930a , Rif , Tanger; Nourti et al. 2019a , Rif , Adrou ( PNPB , 556 m) Dolichopus sabinus Haliday, 1838 Ebejer et al. 2019 , Rif , Martil (9 m), Oued Laou (2 m); Nourti et al. 2019a , Rif , plage Stihat (4 m) Dolichopus scutopilosus Parent, 1933 Parent 1933 , HA , Arround Dolichopus signifer Haliday, 1832 Parent 1929b , "Maroc"; Séguy 1930a , MA , Ras el Ma; Grichanov 2019 , HA , Oukaimeden (2600 m) Dolichopus strigipes Verrall, 1875 Grichanov 2009 , AP , 40 km S Larache (0–20 m) Gymnopternus Loew, 1857 Gymnopternus assimilis (Staeger, 1842) Nourti et al. 2019a , Rif , Amsemlil (1067 m) Hercostomus Loew, 1857 Hercostomus apollo (Loew, 1869) Nourti et al. 2019a , Rif , Talassemtane (1696 m), Adrou ( PNPB , 556 m), Amsemlil ( PNPB , 1067 m) Hercostomus canariensis Santos Abreu, 1929 Grichanov et al. 2020a , Rif , Pont de Dieu (Akchour, 536 m); Nourti et al. 2019a (as H. aff. exarticulatoides Stackelberg, 1949) Hercostomus chetifer (Haliday, 1849) Ebejer et al. 2019 , Rif , Sidi Yahia Aarab (377 m) Hercostomus discriminatus Parent, 1925 Parent 1925 , Rif , "Favier, Environs de Tanger"; Parent 1927 , Rif , Tanger; Vaillant 1950 , Rif , Tanger Hercostomus exarticulatus (Loew, 1857) Vaillant 1956b , HA , Lac Tamhda (Anremer), Aguelmous; Grichanov et al. 2020a Hercostomus excipiens Becker, 1907 Parent 1924 , Rif , Tétouan; Parent 1927 ; Séguy 1930a , Rif , Oued Judios (Tanger); Nourti et al. 2019a , Rif , Talembote (440 m) Hercostomus germanus (Wiedemann, 1817) Parent 1924 , Rif , Cap Spartel; Parent 1927 ; Kettani and Negrobov 2016 , Rif , Chefchaouen, Ketama – MISR ( Rif , Ketama) Hercostomus longiventris (Loew, 1857) Vaillant 1956b , HA , Lac Tamhda (Anremer), Izourar Muscidideicus Becker, 1917 Muscidideicus praetextatus (Haliday, 1855) Grichanov 2019 , AP , Oualidia lagune Ortochile Berthold, 1827 Ortochile morenae (Strobl, 1899) = Hercostomus morenae (Strobl, 1899), in Becker 1917 : 225; Nourti et al. 2019a : 124 Grichanov and Nourti 2021 ; Nourti et al. 2019a , Rif , Mnezla (74 m), Talassemtane (980 m), Oued Ametrasse (841 m), estuary Tahaddart (dune marshland, 0 m) Ortochile nigrocaerulea Latreille, 1779 Parent 1924 , Rif , Tanger, Cap Spartel, Tétouan, Béni Hozmar; Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Oued Judios (Tanger); Grichanov 2009 , Rif , Ouezzane (300 m); Nourti et al. 2019a , Rif , Douar El Hamma (338 m), Triwa Bni Hassane (654 m), Taida (501 m) Platyopsis Parent, 1929 Platyopsis maroccanus (Parent, 1929) Parent 1929b , Rif , Tanger; Vaillant 1950 , Rif , Tanger Poecilobothrus Mik, 1878 Poecilobothrus appendiculatus (Loew, 1859) = Hercostomus appendiculatus (Loew): Ebejer et al. 2019 : 146 Parent 1924 , Rif , Tanger, Cap Spartel; Ebejer et al. 2019 , Rif , Oued Nakhla (200 m), Moulay Abdelsalam (965 m), Dardara (730 m), Cap Spartel (155 m); Nourti et al. 2019a , Rif , Perdicaris Park (223 m) Poecilobothrus infuscatus (Stannius, 1831) Ebejer et al. 2019 , Rif , Tahaddart (2 m); Rif (Tahaddart) – MISR Sybistroma Meigen, 1824 Sybistroma dufouri Macquart, 1838 = Haltericerus spathulatus Loew, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger Sybistroma obscurellum Fallén, 182320 = Hypophyllus obscurellus Fallén, in Dakki 1997 : 61 Dakki 1997 (no material provided) Sybistroma quadrifilatum (Strobl, 1899) = Sybistroma parvulum (Parent, 1927), in Grichanov and Nourti 2021 : 190 Grichanov and Nourti 2021 , Rif , Fahs Anjra (372 m) Sybistroma theodori Grichanov & Nourti, 2021 Grichanov and Nourti 2021 , Rif , Moulay Abdelsalam (649 m) Tachytrechus Haliday, 1851 Tachytrechus consobrinus (Haliday, 1851)20 Parent 1938 (no material provided) Tachytrechus goudoti (Macquart, 1842) = Dolichopus goudoti Macquart, 1842 Macquart 1842 , Rif , Tanger; Parent 1926 (redescription), 1927 Tachytrechus insignis (Stannius, 1831) Parent 1927 , "Maroc"; Séguy 1930a , Rif , Tanger, HA , Aguerd el Had, Talekjount (1000–1100 m); Vaillant 1956b , HA , Lac Tamhda (Anremer); Popescu-Mirceni 2011 , AP , Merja Zerga; Grichanov 2019 , AP , Essaouira Tachytrechus notatus (Stannius, 1831) Vaillant 1950 (no material provided), 1956b, HA , Lac Tamhda (Anremer); Grichanov 2009 , AA , 15 km SW Tazenakcht; Dawah et al. 2020 Tachytrechus planitarsis Becker, 1907 Vaillant 1950 , HA , Touggourt; Grichanov 2009 , AA , 15 km SW Tazenakcht; Grichanov 2019 , AA , Ouarzazate (1100 m) Hydrophorinae Anahydrophorus Becker, 1917 Anahydrophorus cinereus (Fabricius, 1805) = Scatophaga cinerea Fabricius, 1805: 205 Fabricius 1805 , AP , Mogador (Essaouira); Séguy 1930a , Rif , Tanger; Vaillant 1955a , AP , Temara, Port-Lyautey; Boumezzough and Vaillant 1986a , AP , beach of Rabat; Kettani and Negrobov 2016 , AP , Skhirat; AP (Skhirat) – MISR Aphrosylus Haliday, 1851 Aphrosylus maroccanus Vaillant, 1955 Vaillant 1955a , AP , Port Lyautey Aphrosylus mitis Verrall, 1912 Grichanov 2019 , AP , Oualidia lagune Aphrosylus raptor luteipes Parent, 1929 Parent 1929b , AP , Mogador (as a variation of Aphrosylus raptor Haliday, 1851); Vaillant 1955a ; Negrobov, 1979 (as a subspecies of Aphrosylus raptor Haliday, 1851); Kettani and Negrobov 2016 (as Aphrosylus raptor Haliday, 1851); Grichanov 2019 , AP , Oualidia lagune Aphrosylus temaranus Vaillant, 1955 Vaillant 1955a , AP , Temara; Grichanov 2019 , AP , Oualidia lagune Aphrosylus venator Loew, 1857 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger Epithalassius Mik, 1891 Epithalassius corsicanus Becker, 1910 Pârvu 2008 , AP , Merja Zergha, Cap Sim, Essaouira Hydrophoprus Fallén, 1823 Hydrophorus balticus (Meigen, 1824) Vaillant 1956b , HA , Jebel Toubkal, Lac Tamhda (Anremer), Oukaimeden, Izourar; Boumezzough and Vaillant 1986a , HA , Jebel Toubkal (3100 m); Grichanov 2019 , HA , Aguelmouss (2050 m), Oukaimeden (2600 m); Nourti et al. 2019a , MA , Mont Habri (2071 m) Hydrophorus nilicola Parent, 1927 = Hydrophorus viridis nilicola Parent, in Boumezzough and Vaillant 1986a : 297 Boumezzough and Vaillant 1986a , MA , Tizi-n'Imdrhas (1800 m), HA , Oued N'fis (650 m), AA , near Agadir N'oussbai (400 m); Grichanov 2019 , AP , Essaouira Hydrophorus oceanus (Macquart, 1838) = Hydrophorus bisetus Loew, 1857, in Parent 1927 , Séguy 1930a , Grichanov 2019 Parent 1927 , AP , Rabat; Séguy 1930a , Rif , Tandja el Balia (Tanger) ( Hydrophorus bisetus Loew); Vaillant 1955a , AP , Port-Lyautey; Boumezzough and Vaillant 1986a , AP , beach of Rabat Hydrophorus praecox (Lehmann, 1822) Parent 1924 , Rif , Cap Spartel, de Tanger à Tétouan, Rincón de Medik, Dar Riffien (Ceuta); Parent 1927 ; Séguy 1941a , HA , Toubkal (2500 m); Boumezzough and Vaillant 1986a , HA , Lac Tamhda, Lac Tamdhanit (Massif Anremer, 2900 m), Lac Izourar (Massif Azourki, 2650 m) Hydrophorus viridis (Meigen, 1824) 21 Parent 1927 , AP , Rabat; Boumezzough and Vaillant 1986a : 297 Liancalus Loew, 1857 Liancalus virens (Scopoli, 1763) Vaillant 1956b , HA , Toubkal, Assif Tassouat (M'Goum), Aguelmous, Sidi Chamarouch, Imi-N'Ifri; Boumezzough and Vaillant 1986a , HA , Tahanaout (750 m), Adrar Anremer (2900 m), AA , Jebel Siroua (3000 m); Kettani and Negrobov 2016 , AP , S-Tifni; Nourti et al. 2019a , Rif , Amsemlil env. (1059 m) – MISR ( AP , S Tifni) Machaerium Haliday, 1832 Machaerium maritimae Haliday, 1832 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Kettani and Negrobov 2016 , AP , Oued Bou-Regreg; Grichanov 2019 , AP , Oualidia lagune; AP (Oued Bou-Regreg) – MISR Orthoceratium Schrank, 1803 Orthoceratium sabulosum (Becker, 1907) = Orthoceratium lacustre (Scopoli, 1763) 22 , in Ebejer et al. 2019 : 146 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Thinophilus Wahlberg, 1844 Thinophilus ( Thinophilus ) flavipalpis (Zetterstedt, 1843) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Grichanov 2009 , AP , 40 km S Larache (0–20 m) Thinophilus ( Thinophilus ) indigenus Becker, 1902 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m), Oued Laou (2 m); Grichanov 2019 , AA , Ouarzazate (572 m) Thinophilus ( Thinophilus ) mirandus Becker, 1907 Negrobov 1971 , Rif , Tanger; Grichanov 2019 , AA , Ouarzazate (572 m) Thinophilus ( Schoenophilus ) versutus Haliday, 1851 = Schoenophilus versutus (Haliday, 1851), in Parent 1924 , 1927 Parent 1924 , Rif , Cap Spartel, Tétouan; Parent 1927 ; Pârvu et al. 2006 , AP , Merja Zerga; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), Dayat Tazia (733 m) Medeterinae Medetera Fisher, 1819 Medetera diadema (Linnaeus, 1767) = Medeterus (cf. diadema ), in Séguy 1953a : 84 Parent 1938 (no material provided); Séguy 1953a , AP , Rabat; Nourti et al. 2019a , Rif , Kitane (49 m), Rif , plage Stihat (beach) Medetera media Parent, 1925 Nourti et al. 2019a , Rif , Faculty of Sciences of Tétouan (garden: on trunk of olive tree, 14 m) Medetera micacea Loew, 1857 Ebejer et al. 2019 , Rif , Dardara (730 m); Nourti et al. 2019a , Rif , Issaguen (1547 m), HA , Lac Tislit (Imilchil, 2254 m) Medetera pallipes (Zetterstedt, 1843) 23 Nourti et al. 2019a , Rif , Douar El Hamma (338 m) Medetera petrophila Kowarz, 187720 Parent 1938 (no material provided) Medetera petrophiloides Parent, 1925 Nourti et al. 2019a , Rif , Issaguen (1547 m) Medetera aff. roghii Rampini & Canzoneri, 1979 Nourti et al. 2019a , Rif , Douar El Hamma (338 m) Medetera truncorum Meigen, 1824 24 Ebejer et al. 2019 a, Rif , Dardara (484 m) Medetera varvara Grichanov & Vikhrev, 2009 Grichanov and Vikhrev 2009 , AP , Essaouira Thrypticus Gerstäcker, 1864 Thrypticus bellus Loew, 1869 Séguy 1930a , EM , Dayat Sidi Kacem; Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), estuary Oued Tahaddart (0 m), Barrage 9 Avril, plage Stihat (0 m) Microphorinae Schistostoma Becker, 1902 Schistostoma eremita Becker, 1902 Ebejer et al. 2019 , AA , Ziz river (12 km S of Rissani, 737 m), Lac Tiffert (4 km W of Merzouga, 702 m) Neurigoninae Neurigona Rondani, 1856 Neurigona solodovnikovi Grichanov, 2010 = Neurigona punctifera Becker, 1907, in Kettani and Negrobov 2016 (misidentification) Grichanov 2010, AP , 40 km S Larache; Kettani and Negrobov 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m); Nourti et al. 2019a , Rif , Perdicaris Park (223 m), Taria Bni Faghloum (894 m), Chrafate (832 m) Parathalassiinae Microphorella Becker, 1909 Microphorella ulrichi Gatt, 2003 Gatt 2003 , Rif , Tanger, Oued Armal (Ksar Sghir) Parathalassius Mik, 1891 Parathalassius blasigii Mik, 1891 Ebejer et al. 2019 , AP , Larache (5 m) Peloropeodinae Chrysotimus Loew, 1857 Chrysotimus molliculoides Parent, 1937 Parent 1937a , MA , Ifrane (1600 m); Parent 1937b ; Nourti et al. 2019a , Rif , Dayat Tazia (733 m) Micromorphus Mik, 1878 Micromorphus albipes (Zetterstedt, 1843) Parent 1924 , Rif , Béni Hozmar (Tétouan); Parent 1927 , Rif , Tétouan; Kechev and Ivanova 2015 ; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), estuary Oued Tahaddart (0 m), Barrage 9 Avril, Kharouba, plage Stihat (0 m), Aïn Jdioui, EM , Bouanane (Figuig, 855 m), AA , Boudnib (951 m); Dawah et al. 2020 Micromorphus minusculus Negrobov, 2000 Nourti et al. 2019a , EM , Bouanane (Figuig, 855 m), AA , Taliouine (Taroudant, 1014 m); Grichanov 2019 , AA , Ouarzazate Rhaphiinae Rhaphium Meigen, 1803 Rhaphium appendiculatum Zetterstedt, 184920 = Rhaphium macrocerum (Parent, 1925), in Parent 1938 , Grichanov 2019 Parent 1938 (no material provided) Rhaphium brevicorne Curtis, 1835 = Xiphandrium pectinatum Becker, in Vaillant 1956b : 112, Grichanov 2019 Vaillant 1956b , AP , Rabat, HA , Oukaimeden; Kettani and Negrobov 2016 , AP , Rabat; Grichanov 2019 , HA , Oukaimeden (1000 m); Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Dayat Tazia (733 m), Moulay Abdelsalam (1177 m); AP (Rabat) – MISR Rhaphium caliginosum Meigen, 1824 = Raphium lanceolatum Loew, 1850, in Kazerani et al. 2013 Parent 1924 , Rif , Cap Spartel; Kazerani et al. 2013 (no material provided); Kechev 2017 ; Nourti et al. 2019a , Rif , Amsemlil env. (1067 m), Dbani (Bni Selmane, 1046 m) Rhaphium fascipes (Meigen, 1824) = Porphyrops fascipes Meigen, 1824, in Parent 1927 Parent 1927 , Rif , Tétouan Rhaphium fissum Loew, 1850 Grichanov 2009 , AA , Tizi-n'Test pass (2100 m) Sciapodinae Sciapus Zeller, 1842 Sciapus adumbratus Becker, 1902 Grichanov and Negrobov 2014 , AP , near Essaouira, AA , Oued Souss, near Ouarzazate (1100 m) Sciapus costae (Mik, 1890) Negrobov 1991 (no material provided); Ebejer et al. 2019 , Rif , Oued Laou (30 m) Sciapus euzonus (Loew, 1859) = Psilopus euzonus Loew, in Dakki 1997 : 62 Parent 1927 , Rif , El Mahadi; Séguy 1930a , Rif , Tanger; Séguy 1941d , AA , Taroudant Sciapus glaucescens (Loew, 1856) Grichanov and Negrobov 2014 , AP , Oualidia Sciapus heteropygus Parent, 1926 Nourti et al. 2019c , Rif , Talassemtane National Park (1696 m) Sciapus holoxanthos Parent, 1926 Nourti et al. 2019c , Rif , Jbel Bouhachem, Adrou (556 m), Talassemtane National Park (1696 m) Sciapus laetus (Meigen, 1838) Grichanov 2009 , AA , 40 km S Larache (0–20 m); Ebejer et al. 2019 , Rif , Martil (9 m) Sciapus longulus (Fallén, 1823) 25 Pârvu et al. 2006 , AP , Merja Zerga Sciapus aff. negrobovi Naglis & Barták, 2015 Nourti et al. 2019a , Rif , plage Stihat (0 m), Kitane (49 m) (as Sciapus aff. negrobovi ); Nourti et al. 2019c Sympycninae Campsicnemus Haliday in Walker, 1851 Campsicnemus crinitarsis Strobl, 1906 Dakki 1997 (no material provided); Grichanov 2012 , AP , Essaouira; Kettani and Negrobov 2016 , Rif , Oued Amsa Campsicnemus curvipes (Fallén, 1823) 26 Frey 1936 (no material provided) Campsicnemus filipes Loew, 1859 Grichanov 2009 , AA , 40 km S Larache (0–20 m) Campsicnemus loripes (Haliday, 1832) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m) Campsicnemus magius (Loew, 1845) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Grichanov 2019 , AP , Essaouira Campsicnemus simplicissimus Strobl, 1906 Nourti et al. 2019a , Rif , plage Stihat (0 m) Sympycnus Loew, 1857 Sympycnus pulicarius (Fallén, 1823) Nourti et al. 2019a , HA , Aïn Taferaout (Sidi Masali, 1237 m) Syntormon Loew, 1857 Syntormon aulicus Meigen, 1824 = Eutarsus aulicus Meigen, in Parent 1924 , 1927 ; Séguy 1930a : 125 Parent 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger; Vaillant 1952 , 1956b , HA , Imi-N'Ifri Syntormon codinai Parent, 1924 Parent 1924 , Rif , Cap Spartel, Tanger; Parent, 1927, Rif , Tanger Syntormon denticulatus (Zetterstedt, 1843) = Syntormon pumilus Parent, 1925 (nec Meigen, 1824; misidentification), in Pârvu et al. 2006 , Grichanov 2019 Parent 1924 , Rif , Tétouan; Parent 1927 , Rif , Tétouan; Séguy 1930a , Rif , Tanger; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Nourti et al. 2019a , Rif , plage Stihat (0 m), Amlay (294 m), Koudiat Taifour (100 m), Amsemlil env. (1067 m), Dayat Tazia (733 m), Oued Souk Lhad (613 m), HA , Aïn Taferaout (Sidi Masali, 1237 m), AA , Taliouine (1014 m) Syntormon mikii Strobl, 1899 Parent 1927 , "Maroc"; Kechev and Ivanova 2015 ; Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Moulay Abdelsalam (1177 m) Syntormon monilis (Haliday, 1851) Parent 1924 , Rif , Cap Spartel; Parent 1927 , Rif , Cap Spartel Syntormon pallipes (Fabricius, 1794) Parent 1924 , Rif , Cap Spartel, Tétouan, Chefchaouen; Parent 1927 , Rif , Cap Spartel; Nourti et al. 2019a , Rif , Oued Souk Lhad (613 m), Talassemtane (339 m), Perdicaris Park (223 m), Amsemlil env. (1067 m); Dawah et al. 2020 Syntormon pilitibia Grichanov, 2013 Nourti et al. 2019a , Rif , Amsemlil ( PNPB , 1067 m) Syntormon pumilus (Meigen, 1824) = Syntormon rufipes auctt. (nec Meigen, 1824; misidentification), in Pârvu et al. 2006 ; Grichanov 2019 Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate Syntormon zelleri (Loew, 1850) Vaillant 1956b , HA , Oukaimeden, Izourar; Pârvu et al. 2006 , AP , Merja Zerga; Nourti et al. 2019a , Rif , Pont de Dieu (Akchour, 536 m), Amsemlil env. (1059–1067 m) Teuchophorus Loew, 1857 Teuchophorus cristulatus Mueffels & Grootaert, 1990 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Teuchophorus rifensis Nourti, Grichanov & Kettani, 2019 Nourti et al. 2019b , Rif , Oued Souk Lhad (613 m) Teuchophorus spinigerellus (Zetterstedt, 1843) Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate Xanthochlorinae Xanthochlorus Loew, 1857 Xanthochlorus tenellus (Wiedemann, 1817) Grichanov 2009 , AA , 40 km S Larache (0–20 m); Ebejer et al. 2019 , Rif , Moulay Abdelsalam (1180 m) HYBOTIDAE K. Kettani, P. Gatt Number of species: 44 . Expected: 120 Faunistic knowledge of the family in Morocco: poor Ocydromiinae Bicellariini Bicellaria Macquart, 1823 Bicellaria spuria Fallén, 1816 Becker and Stein 1913 , Rif , Tanger Ocydromiini Ocydromia Meigen, 1820 Ocydromia glabricula (Fallén, 1816) Becker and Stein 1913 , Rif , Tanger Oropezella Collin, 1926 Oropezella sphenoptera (Loew, 1873) Cassar et al. 2005 , Rif , lagoon Smir Tachydromiinae Drapetini Chersodromia Haliday in Walker, 1851 Chersodromia albopilosa Chvála, 1970 Cassar et al. 2008 , Rif , Basin Laou Chersodromia nigrosetosa Chvála, 1970 Chvála 1981 (Ceuta); Chvála and Kovalev 1989 Chersodromia pseudohirta Chvála, 1970 Ebejer et al. 2019 , Rif , Kabila beach Crossopalpus Bigot, 1857 Crossopalpus aeneus (Walker, 1871) 27 Shamshev et al. 2005 , HA , Marrakech, Ouirgane Crossopalpus dilutipes (Strobl, 1906) Ebejer et al. 2019 , AP , 9 km SE of Aïn Chouk (Lower Loukous marsh, 6 m) Crossopalpus nigritellus (Zetterstedt, 1842) Ebejer et al. 2019 , Rif , Talassemtane (maison forestière), Issaguen (1620 m) Crossopalpus setiger (Loew, 1859) Ebejer et al. 2019 Rif , Smir lagoon Drapetis Meigen, 1822 Drapetis disparilis Frey, 1936 Chvála 1981 (Ceuta); Chvála and Kovalev 1989 Drapetis laevis Becker, 1914 Becker and Stein 1913 , Rif , Tanger; Kovalev 1970 ; Chvála and Kovalev 1989 Elaphropeza Macquart, 1827 Elaphropeza boergei Chvála, 1971 Ebejer et al. 2019 , Rif , Smir lagoon, Oued Laou (saltmarsh) Elaphropeza hutsoni Smith, 1967 Ebejer et al. 2019 , Rif , Jnane Niche (46 m) Stilpon Loew, 1859 Stilpon demnatensis Vaillant, 1956 28 Vaillant 1956b : 244, HA , Imi-N'Ifri Stilpon moroccensis Grootaert & Zouhair, 2021 Grootaert et al. 2021 , Rif , beach of Stehat, Bab Tariouant, Amsemlil, EM , Bouanane Stilpon subnubilus Chvála, 1988 Ebejer et al. 2019 , Rif , Smir lagoon, M'Diq (Kabila beach and dunes), Martil (beach and dunes) Tachydromiini Platypalpus Macquart, 1827 Platypalpus alluaudi Grootaert & Chvála, 1992 Grootaert and Chvála 1992 , HA , Chichaoua; Vaňhara and Rozkošný 1997 Platypalpus anomalicerus (Becker, 1902) = Coryneta aerivaga Séguy, in Séguy 1941d : 11 Séguy 1941d , EM , Oued Guir, AA , Agadir; Grootaert and Chvála 1992 , EM , Oued Guir, AA , Agadir Platypalpus annulatus (Fallén, 1815) Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 Platypalpus anomalitarsis Chvála & Kovalev, 1974 Ebejer et al. 2019 , MA , 10 km S of Azrou (1775 m), 10 km S of Azrou (1720 m), AA , Ziz river (30 km N of Erfoud, 894 m) Platypalpus approximatus (Becker, 1902) Grootaert and Chvála 1992 , AP , Casablanca Platypalpus asniensis Grootaert & Chvála, 1992 Grootaert and Chvála 1992 , HA , Asni; Vaňhara and Rozkošný 1997 Platypalpus calceatus (Meigen, 1822) Pârvu et al. 2006 , MA , Meknès, AA , Foum Zghouig Platypalpus chillcotti Chvála, 1981 Grootaert and Chvála 1992 , MA , Ifrane Platypalpus chrysonotus (Strobl, 1899) Ebejer et al. 2019 , Rif , Oued Laou (saltmarsh) Platypalpus desertorum (Becker, 1907) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Platypalpus distichus Grootaert & Chvála, 1992 Ebejer et al. 2019 , AP , 9 km SE of Aïn Chouk (Lower Loukous marsh, 6 m), AA , Ziz river (10 km S of Errachidia, 1008 m) Platypalpus flavicornis (Meigen, 1822) Ebejer et al. 2019 , AA , 2 km N of Erfoud (818 m), Ziz river (30 km N of Erfoud, 894 m) Platypalpus longicauda Grootaert & Chvála, 1992 Ebejer et al. 2019 , Rif , Smir lagoon Platypalpus lyneborgi Chvála, 1981 Grootaert and Chvála 1992 , AP , Dradek, MA , Azrou Platypalpus nigritarsis (Fallén, 1816) Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 Platypalpus obscuripes (Strobl, 1899) Ebejer et al. 2019 , Rif , Martil (9 m), AP , Larache (Loukous marsh, 2 m) Platypalpus ostiorum (Becker, 1902) Grootaert and Chvála 1992 Platypalpus pachycerus (Collin, 1949) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Platypalpus pallidiventris (Meigen, 1822) Grootaert and Chvála 1992 , MA , Ifrane; Maarouf 2003 , HA , Chaouia Platypalpus pseudoexiguus (Strobl, 1909) Ebejer et al. 2019 , Rif , Oued Laou (saltmarsh) Platypalpus pseudounguiculatus (Strobl, 1909) = Tachydromia pseudounguiculata Strobl 1909 Grootaert and Chvála 1992 Platypalpus riojaensis Chvála, 1981 Grootaert and Chvála 1992 , EM , Oujda, MA , Meknès, HA , Chichaoua Platypalpus turgidus (Becker, 1907) Grootaert and Chvála 1992 , MA , Takkat-n- Sountat Platypalpus vockerothi Chvála, 1981 Grootaert and Chvála 1992 , HA , Asni Tachydromia Meigen, 1803 Tachydromia arrogans (Linnaeus, 1761) Ebejer et al. 2019 , Rif , Oued Laou, El-Fahsa (maquis) Tachydromia annulimana Meigen, 1822 29 = Tachista annulimana Meigen, in Becker and Stein 1913 : 84 Becker and Stein 1913 , Rif , Tanger Tachydromia undulata (Strobl, 1906) Chvála 1969 Ocydromiinae Bicellariini Bicellaria Macquart, 1823 Bicellaria spuria Fallén, 1816 Becker and Stein 1913 , Rif , Tanger Ocydromiini Ocydromia Meigen, 1820 Ocydromia glabricula (Fallén, 1816) Becker and Stein 1913 , Rif , Tanger Oropezella Collin, 1926 Oropezella sphenoptera (Loew, 1873) Cassar et al. 2005 , Rif , lagoon Smir Tachydromiinae Drapetini Chersodromia Haliday in Walker, 1851 Chersodromia albopilosa Chvála, 1970 Cassar et al. 2008 , Rif , Basin Laou Chersodromia nigrosetosa Chvála, 1970 Chvála 1981 (Ceuta); Chvála and Kovalev 1989 Chersodromia pseudohirta Chvála, 1970 Ebejer et al. 2019 , Rif , Kabila beach Crossopalpus Bigot, 1857 Crossopalpus aeneus (Walker, 1871) 27 Shamshev et al. 2005 , HA , Marrakech, Ouirgane Crossopalpus dilutipes (Strobl, 1906) Ebejer et al. 2019 , AP , 9 km SE of Aïn Chouk (Lower Loukous marsh, 6 m) Crossopalpus nigritellus (Zetterstedt, 1842) Ebejer et al. 2019 , Rif , Talassemtane (maison forestière), Issaguen (1620 m) Crossopalpus setiger (Loew, 1859) Ebejer et al. 2019 Rif , Smir lagoon Drapetis Meigen, 1822 Drapetis disparilis Frey, 1936 Chvála 1981 (Ceuta); Chvála and Kovalev 1989 Drapetis laevis Becker, 1914 Becker and Stein 1913 , Rif , Tanger; Kovalev 1970 ; Chvála and Kovalev 1989 Elaphropeza Macquart, 1827 Elaphropeza boergei Chvála, 1971 Ebejer et al. 2019 , Rif , Smir lagoon, Oued Laou (saltmarsh) Elaphropeza hutsoni Smith, 1967 Ebejer et al. 2019 , Rif , Jnane Niche (46 m) Stilpon Loew, 1859 Stilpon demnatensis Vaillant, 1956 28 Vaillant 1956b : 244, HA , Imi-N'Ifri Stilpon moroccensis Grootaert & Zouhair, 2021 Grootaert et al. 2021 , Rif , beach of Stehat, Bab Tariouant, Amsemlil, EM , Bouanane Stilpon subnubilus Chvála, 1988 Ebejer et al. 2019 , Rif , Smir lagoon, M'Diq (Kabila beach and dunes), Martil (beach and dunes) Tachydromiini Platypalpus Macquart, 1827 Platypalpus alluaudi Grootaert & Chvála, 1992 Grootaert and Chvála 1992 , HA , Chichaoua; Vaňhara and Rozkošný 1997 Platypalpus anomalicerus (Becker, 1902) = Coryneta aerivaga Séguy, in Séguy 1941d : 11 Séguy 1941d , EM , Oued Guir, AA , Agadir; Grootaert and Chvála 1992 , EM , Oued Guir, AA , Agadir Platypalpus annulatus (Fallén, 1815) Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 Platypalpus anomalitarsis Chvála & Kovalev, 1974 Ebejer et al. 2019 , MA , 10 km S of Azrou (1775 m), 10 km S of Azrou (1720 m), AA , Ziz river (30 km N of Erfoud, 894 m) Platypalpus approximatus (Becker, 1902) Grootaert and Chvála 1992 , AP , Casablanca Platypalpus asniensis Grootaert & Chvála, 1992 Grootaert and Chvála 1992 , HA , Asni; Vaňhara and Rozkošný 1997 Platypalpus calceatus (Meigen, 1822) Pârvu et al. 2006 , MA , Meknès, AA , Foum Zghouig Platypalpus chillcotti Chvála, 1981 Grootaert and Chvála 1992 , MA , Ifrane Platypalpus chrysonotus (Strobl, 1899) Ebejer et al. 2019 , Rif , Oued Laou (saltmarsh) Platypalpus desertorum (Becker, 1907) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Platypalpus distichus Grootaert & Chvála, 1992 Ebejer et al. 2019 , AP , 9 km SE of Aïn Chouk (Lower Loukous marsh, 6 m), AA , Ziz river (10 km S of Errachidia, 1008 m) Platypalpus flavicornis (Meigen, 1822) Ebejer et al. 2019 , AA , 2 km N of Erfoud (818 m), Ziz river (30 km N of Erfoud, 894 m) Platypalpus longicauda Grootaert & Chvála, 1992 Ebejer et al. 2019 , Rif , Smir lagoon Platypalpus lyneborgi Chvála, 1981 Grootaert and Chvála 1992 , AP , Dradek, MA , Azrou Platypalpus nigritarsis (Fallén, 1816) Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 Platypalpus obscuripes (Strobl, 1899) Ebejer et al. 2019 , Rif , Martil (9 m), AP , Larache (Loukous marsh, 2 m) Platypalpus ostiorum (Becker, 1902) Grootaert and Chvála 1992 Platypalpus pachycerus (Collin, 1949) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Platypalpus pallidiventris (Meigen, 1822) Grootaert and Chvála 1992 , MA , Ifrane; Maarouf 2003 , HA , Chaouia Platypalpus pseudoexiguus (Strobl, 1909) Ebejer et al. 2019 , Rif , Oued Laou (saltmarsh) Platypalpus pseudounguiculatus (Strobl, 1909) = Tachydromia pseudounguiculata Strobl 1909 Grootaert and Chvála 1992 Platypalpus riojaensis Chvála, 1981 Grootaert and Chvála 1992 , EM , Oujda, MA , Meknès, HA , Chichaoua Platypalpus turgidus (Becker, 1907) Grootaert and Chvála 1992 , MA , Takkat-n- Sountat Platypalpus vockerothi Chvála, 1981 Grootaert and Chvála 1992 , HA , Asni Tachydromia Meigen, 1803 Tachydromia arrogans (Linnaeus, 1761) Ebejer et al. 2019 , Rif , Oued Laou, El-Fahsa (maquis) Tachydromia annulimana Meigen, 1822 29 = Tachista annulimana Meigen, in Becker and Stein 1913 : 84 Becker and Stein 1913 , Rif , Tanger Tachydromia undulata (Strobl, 1906) Chvála 1969 Platypezoidea PHORIDAE K. Kettani, H. Disney Number of species: 3 . Expected: >400 Faunistic knowledge of the family in Morocco: very poor Diplonevra Lioy, 1864 Diplonerva crassicornis (Meigen, 1830) = Phora crassicornis Meigen, in Meigen 1830 : 220 = Dohrniphora dudai Schmitz, in Schmitz 1920 : 100 Raclin 1957 ; Mouna 1998 ; AP (Rabat) – MISR Diplonevra tangeriana (Becker, 1913) 30 = Phora tangeriana Becker and Stein, in Becker and Stein 1913 : 90; Schmitz 1949 : 238 ( Species incerta ) Becker and Stein 1913 , Rif , Tanger; Schmitz 1949 Megaselia Rondani, 1856 Megaselia minor (Zetterstedt, 1848) = Trineura minor Zetterstedt, in Zetterstedt 1848 : 2864; Wood 1912 : 167 = Aphiochaeta angustifrons Wood, in Wood 1912 : 167; Disney 1984 : 239 = Phora minor Shob, in Mouna 1998 : 86 Zetterstedt 1848 ; Wood 1912 ; Disney 1984 ; Mouna 1998 ; AP (Rabat) – MISR PLATYPEZIDAE K. Kettani, M.J. Ebejer Number of species: 3 . Expected: 6 Faunistic knowledge of the family in Morocco: poor Platypezinae Lindneromyia Meigen, 1804 Lindneromyia dorsalis (Meigen, 1804) Chandler 2001 , Rif , Chefchaouen (600 m), AP , Rabat, Maâmora; Tkoč and Roháček 2014 ; AP (Maâmora) – MISR Microsania Zetterstedt, 1837 Microsania raclinae Collart 31 Mouna 1998 : 86 Protoclythia Kessel, 1949 Protoclythia rufa (Meigen, 1830) Ebejer et al. 2019 , Rif , Tahaddart (1 m) LONCHOPTERIDAE K. Kettani, M. Barták Number of species: 4 . Expected: 5 Faunistic knowledge of the family in Morocco: moderate Lonchopterinae Lonchoptera Meigen, 1803 Lonchoptera bifurcata (Fallén, 1810) = Dispa furcata Fallén, in Vaillant 1989 : 217 = Muscidora furcata Fallén, in Mouna 1998 : 86 Vaillant 1989 ; Mouna 1998 Lonchoptera fallax De Meijere, 1906 = Muscidora fallax Meigen, in Mouna 1998 : 86 Mouna 1998 Lonchoptera lutea Panzer, 1809 = Muscidora lutea Panzer, in Mouna 1998 : 86 Vaillant 1989 , HA (>3000 m); Mouna 1998 ; Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 ; MA – MISR Lonchoptera tristis Meigen, 1824 = Muscidora tristis Meigen, in Mouna 1998 : 86 Mouna 1998 PHORIDAE K. Kettani, H. Disney Number of species: 3 . Expected: >400 Faunistic knowledge of the family in Morocco: very poor Diplonevra Lioy, 1864 Diplonerva crassicornis (Meigen, 1830) = Phora crassicornis Meigen, in Meigen 1830 : 220 = Dohrniphora dudai Schmitz, in Schmitz 1920 : 100 Raclin 1957 ; Mouna 1998 ; AP (Rabat) – MISR Diplonevra tangeriana (Becker, 1913) 30 = Phora tangeriana Becker and Stein, in Becker and Stein 1913 : 90; Schmitz 1949 : 238 ( Species incerta ) Becker and Stein 1913 , Rif , Tanger; Schmitz 1949 Megaselia Rondani, 1856 Megaselia minor (Zetterstedt, 1848) = Trineura minor Zetterstedt, in Zetterstedt 1848 : 2864; Wood 1912 : 167 = Aphiochaeta angustifrons Wood, in Wood 1912 : 167; Disney 1984 : 239 = Phora minor Shob, in Mouna 1998 : 86 Zetterstedt 1848 ; Wood 1912 ; Disney 1984 ; Mouna 1998 ; AP (Rabat) – MISR PLATYPEZIDAE K. Kettani, M.J. Ebejer Number of species: 3 . Expected: 6 Faunistic knowledge of the family in Morocco: poor Platypezinae Lindneromyia Meigen, 1804 Lindneromyia dorsalis (Meigen, 1804) Chandler 2001 , Rif , Chefchaouen (600 m), AP , Rabat, Maâmora; Tkoč and Roháček 2014 ; AP (Maâmora) – MISR Microsania Zetterstedt, 1837 Microsania raclinae Collart 31 Mouna 1998 : 86 Protoclythia Kessel, 1949 Protoclythia rufa (Meigen, 1830) Ebejer et al. 2019 , Rif , Tahaddart (1 m) Platypezinae Lindneromyia Meigen, 1804 Lindneromyia dorsalis (Meigen, 1804) Chandler 2001 , Rif , Chefchaouen (600 m), AP , Rabat, Maâmora; Tkoč and Roháček 2014 ; AP (Maâmora) – MISR Microsania Zetterstedt, 1837 Microsania raclinae Collart 31 Mouna 1998 : 86 Protoclythia Kessel, 1949 Protoclythia rufa (Meigen, 1830) Ebejer et al. 2019 , Rif , Tahaddart (1 m) LONCHOPTERIDAE K. Kettani, M. Barták Number of species: 4 . Expected: 5 Faunistic knowledge of the family in Morocco: moderate Lonchopterinae Lonchoptera Meigen, 1803 Lonchoptera bifurcata (Fallén, 1810) = Dispa furcata Fallén, in Vaillant 1989 : 217 = Muscidora furcata Fallén, in Mouna 1998 : 86 Vaillant 1989 ; Mouna 1998 Lonchoptera fallax De Meijere, 1906 = Muscidora fallax Meigen, in Mouna 1998 : 86 Mouna 1998 Lonchoptera lutea Panzer, 1809 = Muscidora lutea Panzer, in Mouna 1998 : 86 Vaillant 1989 , HA (>3000 m); Mouna 1998 ; Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 ; MA – MISR Lonchoptera tristis Meigen, 1824 = Muscidora tristis Meigen, in Mouna 1998 : 86 Mouna 1998 Lonchopterinae Lonchoptera Meigen, 1803 Lonchoptera bifurcata (Fallén, 1810) = Dispa furcata Fallén, in Vaillant 1989 : 217 = Muscidora furcata Fallén, in Mouna 1998 : 86 Vaillant 1989 ; Mouna 1998 Lonchoptera fallax De Meijere, 1906 = Muscidora fallax Meigen, in Mouna 1998 : 86 Mouna 1998 Lonchoptera lutea Panzer, 1809 = Muscidora lutea Panzer, in Mouna 1998 : 86 Vaillant 1989 , HA (>3000 m); Mouna 1998 ; Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 ; MA – MISR Lonchoptera tristis Meigen, 1824 = Muscidora tristis Meigen, in Mouna 1998 : 86 Mouna 1998 Syrphoidea PIPUNCULIDAE K. Kettani, M.J. Ebejer Number of species: 16 . Expected: 50 Faunistic knowledge of the family in Morocco: poor Chalarinae Chalarus Walker, 1834 Chalarus brevicaudis Jervis, 1992 Ebejer and Kettani 2019b , Rif , Dardara (730 m), Azilane (1255 m), El Hamma (338 m) Chalarus sp. aff. brevicaudis Jervis, 1992 Ebejer and Kettani 2019b , Rif , Azilane (1255 m) Pipunculinae Eudorylini Claraeola Aczél, 1940 Claraeola sp. aff. halterata (Meigen, 1838) Ebejer and Kettani 2019b , Rif , Akchour (424 m) Clistoabdominalis Skevington, 2001 Clistoabdominalis dilatatus (De Meyer, 1997) Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m), El Hamma (338 m) Dasydorylas Skevington, 2001 Dasydorylas setosus (Becker, 1908) Kehlmaier 2005 ; Motamedinia et al. 2017 Eudorylas Aczél, 1940 Eudorylas ibericus Kehlmaier, 2005 Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m) Pipunculini Pipunculus Latreille, 1802 Pipunculus carlestolrai Kuznetzov, 1993 Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m) Tomosvaryellini Tomosvaryella Aczél, 1939 Tomosvaryella cilifemorata (Becker, 1907) Ebejer and Kettani 2019b , Rif , Adrou ( PNPB , 556 m) Tomosvaryella debruyni De Meyer, 1995 Ebejer and Kettani 2019b , Rif , Adrou ( PNPB , 556 m) Tomosvaryella frontata (Becker, 1897) Ebejer and Kettani 2019b , Rif , Oued Mhajrate (Ben Karrich, 67 m) Tomosvaryella geniculata (Meigen, 1824) Ebejer and Kettani 2019b , HA , Lac Tislite (Imilchil, 2254 m) Tomosvaryella kuthyi Aczél, 1944 Ebejer and Kettani 2019b , Rif , El Hamma (338 m), Akchour (424 m), Barrage Smir (27 m) Tomosvaryella minima (Becker, 1897) Ebejer and Kettani 2019b , Rif , Koudiat Taifour (100 m) Tomosvaryella mutata (Becker, 1898) Ebejer and Kettani 2019b , Rif , Jebel Lakraâ (Talassemtane, 1596 m) Tomosvaryella trichotibialis De Meyer, 1995 Ebejer and Kettani 2019b , Rif , Koudiat Taifour (100 m) Tomosvaryella sp. subvirescens group Ebejer and Kettani 2019b , AP , Loukous marsh (Larache) SYRPHIDAE K. Kettani, M.C.D. Speight Number of species: 166 . Expected: more than 200 Faunistic knowledge of the family in Morocco: moderate Eristalinae Brachyopini Brachyopa Meigen, 1822 Brachyopa atlantea Kassebeer, 2000 Kassebeer 2000 , HA , Ouirgane (1000 m); Speight 2018 ; Sahib et al. 2020 Chrysogaster Meigen, 1803 Chrysogaster basalis Loew, 1857 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Kassebeer 1999d ; Sahib et al. 2020 Ighboulomyia Kassebeer, 1999 Ighboulomyia atlasi Kassebeer, 1999 Kassebeer 1999c , MA , Azrou, Umgebung, Timahdit, Ighböula Ulaichuor, Quellteich; Sahib et al. 2020 Myolepta Newman, 1838 Myolepta difformis Strobl in Czerny & Strobl 1909 = Myolepta philonis Séguy, 1961, in Dirickx 1994 : 93 Dirickx 1994 , HA ; Reemer et al. 2004 , MA , HA ; Speight 2013 , 2018 ; Sahib et al. 2020 Neoascia Williston, 1886 Neoascia clausseni Hauser & Kassebeer, 1998 = Neoascia podagrica (Fabricius, 1775), in Gil Collado 1929: 40; Claussen 1989b: 373 Gil Collado 1929a ; Claußen 1989b ; Dirickx 1994 ; Hauser and Kassebeer 1998 , MA , HA , Taroudant (1800 m); Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; Sahib et al. 2020 , Rif , Oued Jnane Niche, Oued Maggou Orthonevra Macquart, 1829 Orthonevra bouazzai Kassebeer, 1999 Kassebeer 1999d , MA ; Sahib et al. 2020 Orthonevra brevicornis (Loew, 1843) 32 Sahib et al. 2020 , Rif , Aïn Afersiw Orthonevra elegans (Meigen, 1822) Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Orthonevra schachti Claußen, 1989b Claußen 1989b , HA , Oukaimeden (2600 m); Dirickx 1994 ; Schmid 1995 ; Kassebeer 1999d , MA ; Sahib et al. 2020 Riponnensia Maibach, Goeldlin & Speight, 1994 Riponnensia longicornis (Loew, 1843) = Orthonevra longicornis Loew, in Kanervo 1939 : 2; Séguy 1961 : 23; Kassebeer 1999c : 162 Kanervo 1939 ; Séguy 1961 , AP ; Claußen 1989b ; Kassebeer 1999c , MA , HA ; Dirickx 1994 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 Riponnensia splendens (Meigen, 1822) = Chrysogaster splendens (Meigen), in Gil Collado 1929: 405 = Orthonevra splendens (Meigen), in Claußen 1989b : 363, 373; Dirickx 1994 : 97 Gil Collado 1929a , Rif ; Mouna 1998 ; Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Kassebeer 1999c , MA ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh; AP (Dradek) – MISR Callicerini Callicera Panzer, 1806 Callicera fagesi Guérin-Meneville, 1844 Kassebeer 1998a , HA , Ouirgane (1000 m); Sahib et al. 2020 Callicera rufa Schummel, 1842 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Cerioidini Ceriana Rafinesque, 1815 Ceriana conopsoides (Linnaeus, 1758) = Cerioides conopsoides Linnaeus, in Séguy 1930a : 131 Séguy 1930a , MA , Ras El Ksar; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Maghrawa, Maâmora) – MISR Ceriana vespiformis (Latreille, 1804) = Cerioides vespiformis Latreille, in Becker and Stein 1913 : 88; Gil Collado 1929: 414; Séguy 1930a : 131; Kanervo 1939 : 5; Leclercq 1961: 242 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , HA ; Séguy 1930a , AP , Rabat, Casablanca, MA , Tizi-s'Tkrine, Aïn Leuh, Meknès; Kanervo 1939 , MA ; Leclercq 1961a , Rif , Melillia, MA , Dayat Aoua, Aïn Leuh; Claußen 1989b ; Dirickx 1994 ; Steenis et al. 2016 ; Speight 2018 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , 1 km after Dardara, Meadow Mizoghar, Oued Achekrade Sphiximorpha Rondani, 1850 Sphiximorpha subsessilis (Illiger in Rossi, 1807) Steenis et al. 2016 Eristalini Anasimyia Schiner, 1864 Anasimyia contracta Caußen & Torp, 1980 Kassebeer 1998a , MA , Timahdit (1850 m); Sahib et al. 2020 Eristalinus Rondani, 1845 Eristalinus aeneus (Scopoli, 1763) = Lumpetia aenea (Scopoli), in Becker and Stein 1913 : 86 = Eristalis aeneus (Scopoli), in Gil Collado 1929: 406, 407; Leclercq 1961: 242 = Lathyrophtalmus aeneus (Scopoli), in Séguy 1930: 129; Kanervo 1939 : 5 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , Tanger, AP , Mogador; Séguy 1930a , AP , Casablanca; Kanervo 1939 ; Timon-David 1951 , AP , Rabat; Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , Jumb Kitane, HA , vicinity of Asni Eristalinus megacephalus (Rossi, 1794) = Eristalis quinquelineatus Fabricius, in Becker and Stein 1913 : 85, Gil Collado 1929: 407, = Lathyrophthalmus quinquelineatus Fabricius, in Séguy 1930a : 129 Gil Collado 1929a , Rif , Tanger; Séguy 1930a , AP , Rabat, Oued Korifla, Sidi Bettache; Séguy 1961 ; Dakki 1997 ; Claußen 1989b ; Dirickx 1994 ; Dousti and Hayat 2006 ; Sahib et al. 2020 ; AP (Rabat) – MISR Eristalinus sepulchralis (Linnaeus, 1758) = Eristalis sepuleralis Linnaeus, in Becker and Stein 1913 : 85, Gil Collado 1929: 406 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , AP , Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eristalinus taeniops (Wiedemann, 1818) = Eristalis taeniops Wiedemann, in Becker and Stein 1913 : 85 = Eristalodes taeniops Wiedemann, in Séguy 1930a : 130 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Oued Korifla, Rabat, HA , Tenfecht (Takeljount); Leclercq 1961a , MA , Dayet Aoua; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; Dousti and Hayat 2006 ; Koçak and Kemal 2010 ; Speight 2013 , 2018 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Oued Martil, Halouma Kitane, Oued Sidi Yahia Aârab, MA , bridge Oued Oum-er-Rbia (Douar Ahl Souss), HA , Lac Oukaimeden; AP (Rabat), MA (Volubilis) – MISR Eristalis Latreille, 1804 Eristalis arbustorum (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1930a , MA , Aïn Leuh, Ras El Ksar, forest of Taffert; Kanervo 1939 , Rif ; Timon-David 1951 , AP , Rabat, MA , Ifrane; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Aïn Sidi Brahim Ben Arrif, MA , vicinity of Ifrane, HA , vicinity of Asni, Lac Oukaimeden, AA , Douar Issafen, Douar Issafen; MA , HA – MISR Eristalis jugorum Egger, 1858 33 Dakki 1997 Eristalis pertinax (Scopoli, 1763) Séguy 1930a , AP , Oued Korifla, Sidi Bettache, MA , Forêt de Timelilt; Claußen 1989b ; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 Eristalis similis (Fallén, 1817) = Eristalis pratorum Meigen, in Gil Collado 1929: 407 Gil Collado 1929a , Rif ; Séguy 1961 ; Claußen 1989b , HA , Oukaimeden (2600 m); Dirickx 1994 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , maison forestière, HA , Douar Zaouiat Cheikh, Lac Oukaimeden Eristalis tenax (Linnaeus, 1758) = Eristalomyia tenax Linnaeus, in Séguy 1930a : 130; Timon-David 1951 : 146 Gil Collado 1929a , Rif , AP ; Kanervo 1939 , MA ; Séguy 1949a , AA ; Timon-David 1951 ; Leclercq 1961a ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 , Rif , Village Sebt Zinnat, Belyounech, Aïn Takhninjoute, maison forestière, Jumb Kitane, meadow Fahs Lmhar, HA , Douar Zaouiat Cheikh, vicinity of Asni, Lac Oukaimeden Helophilus Meigen, 1822 Helophilus trivittatus (Fabricius, 1805) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Mallota Meigen, 1822 Mallota cimbiciformis (Fallén, 1817) = Mallota eristaloides Loew, in Becker and Stein 1913 : 85 Becker and Stein 1913 , Rif , Tange; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Mallota dusmeti Andreu, 1926 Kassebeer 1998a , HA , Ouirgane; Sahib et al. 2020 Melanogaster Rondani, 1857 Melanogaster lindbergi Kassebeer, 1999 = Chrysogaster macquardti Loew, in Becker and Stein 1913 : 87 = Chrysogaster viduata Meigen, in Kanervo 1939 : 2, Séguy 1961 : 27, 28 = Chrysogaster lucida (Scopoli), in Claußen 1989b : 372 Becker and Stein 1913 , Rif ; Kanervo 1939 , MA ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1999d , MA ; Popov et al. 2020 ; Sahib et al. 2020 Myathropa Rondani, 1845 Myathropa florea (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , MA , Aïn Leuh; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Oued à 15 km de Fifi Parhelophilus Girschner, 1897 Parhelophilus versicolor (Fabricius, 1794) = Helophilus versicolor (Fabricius), in Gil Collado 1929: 407 Gil Collado 1929a , AP , Oulad Mesbah; Claußen 1989b ; Dirickx 1994 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eumerini Eumerus Meigen, 1822 Eumerus amoenus Loew, 1848 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 , AA , Douar Aourir, beach of Tamelallt Eumerus barbarus (Coquebert, 1804) = Eumerus australis Meigen, in Gil Collado 1929: 412 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1961 ; Claußen 1989b , MA , Azrou (1900 m); Dirickx 1994 ; Mouna 1998 ; Speight 2013 , 2018 ; Steenis et al. 2017 , AA , 11 km NW Taliouine, S Aït-Baha, 10 km NE Tafraoute; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 ; AP (Cap Cantin) – MISR Eumerus basalis Loew, 1848 = Eumerus angusticornis Rondani, in Séguy 1930a : 130 = Eumerus basalis Loew, in Mouna 1998 : 86 Séguy 1930a , MA , forest of Timelilt; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus caballeroi Gil Collado, 1929 Gil Collado 1929a , AP , Laguna Gedira; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus hungaricus Szilády, 1940 Speight 2018 Eumerus lunatus (Fabricius, 1794) = Eumerus lunulatus Fabricius, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger; Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus melotus (Séguy, 1941) = Lampetia melota Séguy, in Séguy 1941d : 13 Séguy 1941d , AA , Agadir; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus nudus Loew, 1848 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus obliquus (Fabricius, 1805) Sahib et al. 2020 , Rif , Oued Jnane Niche, EM , Oued Khemis Eumerus ornatus Meigen, 1822 Séguy 1930a , MA , Aïn Leuh, forest of Timelilt; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Oued Cherrat) – MISR Eumerus pulchellus Loew, 1848 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Eumerus punctifrons Loew, 1857 Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus pusillus Loew, 1848 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Sahib et al. 2020 Eumerus sabulonum (Fallén, 1817) Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Eumerus schmideggeri Steenis, Hauser & Zuijen, 2017 Steenis et al. 2017 , AA , Sidi R'bat (37 km S Agadir); Sahib et al. 2020 Eumerus strigatus (Fallén, 1817) 34 Kanervo 1939 , Rif , HA ; Timon-David 1951 , AP , Rabat, Sehoul; Claußen 1989b , MA , Azrou (1700 m), HA , Oukaimeden (2200 m), Ansegmir-Tal W Midelt; Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Speight 2013 ; Sahib et al. 2020 Eumerus subornatus Claußen, 1989b Claußen 1989b , HA , Tizi-n'Test (1900 m); Schmid 1995 ; Speight 2013 , 2018 ; Dirickx 1994 ; Sahib et al. 2020 Eumerus truncatus Rondani, 1868 Steenis et al. 2017 , AA , S. Aït-Baha, 11 km NW Taliouine, 25 km NE Tizinit, 20 km E Tizinit, Assaka; Speight 2018 ; Sahib et al. 2020 Merodon Meigen, 1803 35 Merodon aberrans Egger, 1860 = Lampetia aberrans Egger, in Séguy 1961 : 174 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Vujic et al. 2011; Speight 2013 , 2018 ; Sahib et al. 2020 Merodon aeneus Meigen, 1822 36 = Lampetia aenea Meigen, in Becker and Stein 1913 : 86; Kanervo 1939 : 5; Timon-David 1951 : 146 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , AP , Vallée Oued Korifla, MA , Tizi-s'Tkrine; Kanervo 1939 , Rif ; Timon-David 1951 ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Merodon arrasus Becker, 1921 37 Becker 1921 , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon aurifer Loew, 1862 = Merodon distinctus Palma, 1864 = Lampetia distincta Palm, in Timon-David 1951 : 146 Timon-David 1951 , AP , Zaer, MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; Vujić et al. 2021c Merodon avidus Rossi, 1782 38 = Lampetia spinipes (Fabricius), in Becker and Stein 1913 : 86; Timon-David 1951 : 146 = Merodon spinipes (Fabricius), in Gil Collado 1929: 409 = Lampetia avida Rossi, in Séguy 1961 : 176 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , HA ; Séguy 1930a , AP , Chellah (Rabat), Oued Korifla, MA , Aïn Leuh; Kanervo 1939 ; Timon-David 1951 , AP ; Séguy 1961 ; Claußen 1989b , HA , Oukaimeden (2600 m); Hurkmans 1993 ; Dirickx 1994 ; Mouna 1998 ; Koçak and Kemal 2010 ; Likov et al. 2020 ; Sahib et al. 2020 , HA , Lac Oukaimeden Merodon bequaerti Hurkmans, 1993 Vujić et al. 2020a , EM , Mountain of Beni-Snassen, MA , Azrou Merodon cabanerensis Marcos-García, Vujić & Mengual, 2007 Vujić et al. 2018 , HA , Ait Mhamed (Azilal, 1700 m); Speight 2018 ; Sahib et al. 2020 ; Vujić et al. 2021 Merodon calcaratus (Fabricius, 1794) Vujić et al. 2021b , EM , Mountains of Béni Snassen, near Nador, AP , 38 km SW of El Jadida, Garbouz Merodon chalybeus Wiedemann in Meigen, 1822 = Lampetia spicata Becker, in Timon-David 1951 : 146 = Merodon spicatus Becker, in Claußen 1989b : 365, 373 Timon-David 1951 , AP , forest of Maâmora; Claußen 1989b , HA (2500 m); Dirickx 1994 ; Mouna 1998 ; Marcos-Garcia et al. 2007 ; Speight 2013 , 2018 ; Sahib et al. 2020 ; AP (Cap Cantin) – MISR Merodon clavipes (Fabricius, 1781) Hurkmans 1993 , MA ; Marcos-García et al. 2007; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon eques Fabricius, 1805 = Lampetia eques (Fabricius), in Séguy 1961 : 178 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Ebejer and Bensusan 2010 , AA ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Merodon equestris (Fabricius, 1794) = Eristalis ferrugineus (Fabricius), in Fabricius 1805 : 240 = Lampetia equestris (Fabricius), in Séguy 1961 : 178, 179 Fabricius 1805 , AP ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon escalerai Gil Collado, 1929 Gil Collado 1929a , AP , Essaouira; Claußen 1989b ; Dirickx 1994 ; Speight 2018 ; Sahib et al. 2020 Merodon femoratus Sack, 1913 = Merodon biarcuatus Curran, 1939, in Curran 1939: 6, 7; Claußen 1989: 373; Dirickx 1994 : 79; Koçak and Kemal 2010 : 1199; Speight 2018 : 137 = Merodon elegans Hurkmans, 1993, in Hurkmans 1993 : 195; Schmid 1995 ; Marcos-Garcia et al. 2007 : 553; Speight 2018 : 141 Curran 1939, AP , forest of Maâmora (Rabat); Claußen 1989b ; Hurkmans 1993 , AP ; Dirickx 1994 ; Schmid 1995 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Speight 2018 ; Likov et al. 2020 Merodon geniculatus Strobl, 1909 Gil Collado 1929a , Rif ; Claußen 1989b , HA (2500 m); Dirickx 1994 ; Mouna 1998 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Speight 2013 , 2018 ; Sahib et al. 2020 , HA , Lac Oukaimeden – MISR Merodon hurkmansi Marcos-García, Vujić & Mengual, 2007 Marcos-García et al. 2007 Merodon ibericus Vujić, 2015 = Merodon bicolor Gil Collado, 1930 Popović et al. 2015 , MA , Azrou, Ifrane; Acanski et al. 2016; Speight 2018 ; Sahib et al. 2020 Merodon italicus Rondani, 1845 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 Merodon longicornis Sack, 1913 Claußen and Hauser 1990 , MA ; Dirickx 1994 ; Sahib et al. 2020 Merodon maroccanus Gil Collado, 1929 Gil Collado 1929a , AP , Essaouira; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon minutus Strobl, 1893 = Lampetia minutus Strobl, in Séguy 1961 : 180 Séguy 1961 ; Leclercq 1961a ; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Merodon monticolus Villeneuve, 1924 Kassebeer 1998a , HA , Ouirgane, Taftraoute, Taliouine; Sahib et al. 2020 Merodon murorum (Fabricius, 1794) = Syrphus murorum (Fabricius), in Fabricius 1794 : 288 = Merodon auripilus (Meigen), in Meigen 1830 : 354 = Lampetia auripila Meigen, in Séguy 1941d : 13; Mouna 1998 : 86 Fabricius 1794 ; Meigen 1830 ; Séguy 1941d , AA , Agadir; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Vujić et al. 2018 , AP , Essaouira; Sahib et al. 2020 Merodon pruni (Rossi, 1790) = Lampetia pruni (Rossi), in Becker and Stein 1913 : 86 = Merodon pruni var. obscurus Gil Collado, in Gil Collado 1929: 407, 408 Becker and Stein 1913 ; Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Hurkmans, 1993; Dirickx 1994 ; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon pumilus Macquart, 1849 Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m); Sahib et al. 2020 Merodon rufus Meigen, 1838 = Lampetia rufa Meigen, 1838, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon segetum (Fabricius, 1794) Peck 1988 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon serrulatus Wiedemann in Meigen, 1822 Hurkmans 1993 ; Speight 2013 , 2018 ; Sahib et al. 2020 ; Vujić et al. 2020a Merodon sophron Hurkmans, 1993 Hurkmans 1993 , MA , Azrou; Schmid 1995 ; Koçak and Kemal 2010 ; Vujić et al. 2020a , MA , Azrou Merodon tangerensis Hurkmans, 1993 Hurkmans 1993 , Rif , Tanger; Schmid 1995 ; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon tricinctus Sack, 1913 = Lampetia tricincta Sack, in Timon-David 1951 : 146 Timon-David 1951 , MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Merodon unguicornis Strobl, 1909 Ebejer et al. 2019 , MA , 10 km S of Azrou (1775 m); Sahib et al. 2020 , Rif , maison forestière Platynochaetus Wiedemann, 1830 Platynochaetus rufus Macquart, 1835 Gil Collado 1929a , AP , Mogador; Dirickx 1994 ; Sahib et al. 2020 Platynochaetus setosus (Fabricius, 1794) Gil Collado 1929a , HA , Marrakech; Séguy 1953a , AA , Souss: Aïn Chaib; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Milesiini Milesia Latreille, 1804 Milesia crabroniformis (Fabricius, 1775) Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Sahib et al. 2020 Spilomyia Meigen, 1803 Spilomyia maroccana Kuznetzov, 1997 = Spilomyia digitata (Rondani), in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b , HA , Tizi-n'Test (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Kuznetzov 1997 ; Dirickx 1994 ; Kassebeer 1999d ; Steenis 2000 ; Sahib et al. 2020 Syritta Le Peletier & Serville, 1828 Syritta flaviventris Macquart, 1842 Claußen 1989b , AP , Kénitra; Dirickx 1994 ; Sahib et al. 2020 , EM , farm Saf-Saf Syritta pipiens (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gill Collado 1929a; Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès; Kanervo 1939 ; Timon-David 1951 , AP , Rabat, AA , Agdz; Leclercq 1961a , EM , Melilla; Claußen 1989b , HA , Tizi-n'Test (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; Sahib et al. 2020 , Rif , Bni Maaden, dam Moulay Bouchta, Oued Koub, Douar Kitane, Oued Maggou, EM , farm Saf-Saf, MA , vicinity of Ifrane, HA , vicinity of Asni, Tizi-n'Test, AA , Oued Assa; AP (Rabat), MA (Aïn Leuh), SA – MISR Temnostoma Le Peletier & Serville, 1828 Temnostoma bombylans (Fabricius, 1805) Séguy 1961 , MA ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xylota Meigen, 1822 Xylota segnis (Linnaeus, 1758) = Zelima ( Xylota ) segnis Linnaeus, in Becker and Stein 1913 : 86; Timon-David 1951 : 147 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Timon-David 1951 , HA , Zaouia Ahansal; Leclercq 1961a , Rif , Azib de Ketama; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Aïn El Maounzil, Oued Koub, Oued Sidi Ben Saâda, HA , vicinity of Asni; HA (Zaouiet Ahansal) – MISR Rhingiini Cheilosia Meigen, 1822 Cheilosia brunnipennis Becker, 1894 = Chilosia flavipes (Panzer), in Kanervo 1939 : 2 Kanervo 1939 , HA ; Kassebeer, 1998c, MA ; Séguy 1961 ; Claußen 1989b ; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia flavipes (Panzer, 1798) Mouna 1998 : 86 Cheilosia grossa (Fallén, 1817) Kassebeer 1998c , HA , Asif Mellah, Tizi-n'Tichka; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia latifrons (Zetterstedt, 1843) = Cheilosia intonsa Loew, in Timon-David 1951 : 144 Timon-David 1951 , AP , Sehoul; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Kassebeer 1998c , Rif , Ouezzane, AP , Oued Loukous, Larache, MA , Ifrane, HA , Oukaimeden; Sahib et al. 2020 Cheilosia mutabilis (Fallén, 1817) Speight 2013 , 2018 Cheilosia griseiventris Loew, 1857 = Chilosia marokkana Becker, in Becker 1894: 395; Becker and Stein 1913 : 87 = Cheilosia maroccana Becker, 1894, in Gil Collado 1929: 405 Becker 1894; Becker and Stein 1913 ; Gil Collado 1929a ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1998c , MA , HA ; Sahib et al. 2020 Cheilosia paralobi Malski, 1962 = Cheilosia longula (Zetterstedt, 1838), in Gil Collado 1929: 405 Gil Collado 1929a ; Claußen 1989b , MA , Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Kassebeer 1998c , HA ; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia parva Kassebeer, 1998 Kassebeer 1998c , MA , Azrou, Ifrane (1650 m); Claußen and Speight 2007 ; Sahib et al. 2020 Cheilosia rodgersi Wainwright, 1911 Becker and Stein 1913 , Rif , Tanger; Claußen 1989a ; Dirickx 1994 ; Kassebeer 1998c , Rif , Tanger; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia scutellata (Fallén, 1817) Gil Collado 1929a , Rif ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1998c , Rif , Chefchaouen, MA , Ouiouane, Ifrane; Sahib et al. 2020 Cheilosia soror (Zetterstedt, 1843) = Cheilosia rufipes (Preyssler, 1793), in Claussen and Hauser 1990: 436, Kassebeer 1998c : 65 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Kassebeer 1998c , MA , Ifrane; Dirickx 1994 ; Sahib et al. 2020 Cheilosia variabilis (Panzer, 1798) Kassebeer 1998c , MA , Ifrane; Speight 2013 , 2018 ; Khaganinia and Kazerani 2014 ; Sahib et al. 2020 Ferdinandea Rondani, 1844 Ferdinandea fumipennis Kassebeer, 1999 Kassebeer 1999b , MA , Ifrane, Azrou, HA , Marrakech, Ouirgane; Speight 2013 , 2018 ; Sahib et al. 2020 Volucellini Volucella Geoffroy, 1762 Volucella inanis (Linnaeus, 1758) Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 Volucella liquida Erichson, 1841 Gil Collado 1929a , Rif ; Séguy 1930a , AP , Mogador, MA , Azrou, Bekrit; Kanervo 1939 , MA ; Séguy 1953a , MA , Ifrane; Timon-David 1951 , MA , Ifrane, Azrou, HA , Aït Mohamed Sgatt; Leclercq 1961a , Rif , Azib de Ketama, MA , Dayat Aoua, Ifrane, Azrou; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , jumb Kitane – MISR Volucella zonaria Poda, 1761 Séguy 1930a , AP , Casablanca; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Rabat, Casablanca) – MISR Brachypalpus Macquart, 1834 Brachypalpus valgus (Panzer, 1798) Kassebeer 1998a , MA , Ifrane, HA , Ouirgane; Sahib et al. 2020 Psilotini Psilota Fallén, 1823 Psilota atra (Fallén, 1817) = Psilota toubkalana Kassebeer, 1995, in Kassebeer 1995 : 395–400 Kassebeer 1995 , HA ; Smit and Vujic 2008, HA , Ouirgane, Marrakech; Speight 2018 ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh Pipizinae Pipizini Heringia Rondani, 1856 Heringia heringi (Zetterstedt, 1843) Kassebeer 1998a , HA , Tahanaout, Ouirgane; Sahib et al. 2020 Pipizella Rondani, 1856 Pipizella thapsiana Kassebeer, 1995 Kassebeer 1995 , HA (1000 m); Speight 2013 , 2018 ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh Triglyphus Loew, 1840 Triglyphus escalerai Gil Collado, 1929 Gil Collado 1929a , Rif , Tanger; Dirickx 1994 ; Speight 2013 ; Sahib et al. 2020 Syrphinae Bacchini Melanostoma Schiner, 1860 Melanostoma mellinum (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Séguy 1934b , AP , Korifla; Timon-David 1951 , MA , Ifrane, Azrou, Aïn Leuh, HA , Aït Mizane; Leclercq 1961a , MA , Ifrane; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , Dayat Rahrah, Aïn el Ma Bared, Oued Dardara, Dayat El Ânassar, Dayat Lemtahane, Garden Ksar Al Rimal, tributary Oued Tazarine, Oued Farda, Dayat El Birdiyel, maison forestière, stream at 1 km from Sidi Yahia Aârab, Dayat Amsemlil, EM , farm Saf-Saf, MA , Oued d'Ifrane, HA , Lac Oukaimeden – MISR Melanostoma mundum Czerny & Strobl, 1909 Dakki 1997 ; Mouna 1998 Melanostoma scalare (Fabricius, 1794) Gil Collado 1929a , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 , Rif , Aïn el Ma Bared, Oued Mezine, Aïn Quanquben, Aïn Takhninjoute, Oued Koub Platycheirus Le Peletier & Serville, 1828 Platycheirus albimanus (Fabricius, 1781) 39 Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress; Mouna 1998 ; Kassebeer 1998b Platycheirus ambiguus (Fallén, 1817) Kassebeer 1998b , HA ; Sahib et al. 2020 Platycheirus atlasi Kassebeer, 1998 Kassebeer 1998b , MA , Azrou, Ifrane; Sahib et al. 2020 Platycheirus fulviventris (Macquart, 1829) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m); Sahib et al. 2020 Platycheirus manicatus (Meigen, 1822) Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Kassebeer 1998b , HA , Toubkal; Sahib et al. 2020 Platycheirus marokkanus Kassebeer, 1998 Kassebeer 1998b , MA , HA ; Speight 2018 ; Sahib et al. 2020 , Rif , Aïn Takhninjoute, HA , Douar Akhlij Tnine Ourika Xanthandrus Verrall, 1901 Xanthandrus comtus (Harris, 1776) Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Paragini Paragus Latreille, 1804 Paragus albifrons (Fallén, 1817) Kanervo 1939 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Paragus atlasi Claußen, 1989 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Schmid 1995 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus bicolor (Fabricius, 1794) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, forest of Timelilt, Azrou; Kanervo 1939 , AP , HA ; Séguy 1949a , SA , Guelmim; Timon-David 1951 , MA , Ifrane; Claußen 1989b ; Claußen and Hauser 1990 , HA , Tizi-n'Test; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Douar Dacheryène, HA , vicinity of Asni; MA (Fès, Ifrane, Azrou) – MISR Paragus cinctus Schiner & Egger, 1853 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 , HA , vicinity of Asni Paragus coadunatus Rondani, 1847 Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus flammeus Goeldlin, 1971 Claußen and Hauser 1990 ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus haemorrhous Meigen, 1844 Claußen 1989b , MA , Azrou (1700 m); Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Mharhar Paragus hermonensis Kaplan, 1981 Claußen 1989b , MA , Azrou (1700 m); Dirickx 1994 ; Sahib et al. 2020 Paragus quadrifasciatus Meigen, 1822 = Paragus pulcherrimus Strobl, in Timon-David 1951 : 144 Timon-David 1951 , MA , Ifrane; Claußen 1989b , AP , Kénitra; Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Douar Kitane, AA , Agadir airport; MA (Ifrane) – MISR Paragus majoranae Rondani, 1857 Claußen and Hauser 1990 , MA ; Dirickx 1994 ; Sahib et al. 2020 Paragus pecchiolii Rondani, 1857 Claußen and Hauser 1990 , MA , Ifrane (1750 m), Hajeb Paragus strigatus Meigen, 1822 = Paragus bimaculatus Meigen, in Wiedemann 1824 : 33 Wiedemann 1824 , AP ; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m), Hajeb; Dirickx 1994 ; Speight 2018 ; Sahib et al. 2020 Paragus tibialis (Fallén, 1817) = Paragus tibialis meridionalis Becker, in Becker and Stein 1913 : 88; Gil Collado 1929: 403; Leclercq 1961: 241 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Kanervo 1939 ; Séguy 1949a , SA , Guelmim; Leclercq 1961a , Rif , Bab Taza, MA , Taza; Claußen 1989b ; Claußen and Hauser 1990 , HA , Tizi-n'Test (2000 m); Dirickx 1994 ; Dakki 1997 ; Grabener 2017 ; Sahib et al. 2020 , HA , vicinity of Asni, Ijoukak vicinity; AP (Rabat) – MISR Paragus vandergooti Marcos-Garcia, 1986 Claußen 1989b , HA , Tizi-n'Test à (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Syrphini Chrysotoxum Meigen, 1803 Chrysotoxum bicinctum (Linnaeus, 1758) Timon-David 1951 , MA , Ifrane, HA , Haute Réghaya; Leclercq 1961a , MA , Mischliffen (2019 m); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; MA , HA – MISR Chrysotoxum intermedium Meigen, 1822 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , MA , Tizi-S'Tkrine, Aïn Leuh, forest of Taffert, HA , Tizi-n'Test, Goundafa; Kanervo 1939 ; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Pârvu et al. 2006 , AP , Cap Bedouza; Dousti and Hayat 2006 ; Pârvu and Zaharia 2007 ; Kazerani et al. 2013b ; Sahib et al. 2020 , Rif , 1 km after Dardara, Oued Azila, Dayat Jebel Zemzem, Aïn El Maounzil, Oued Tafoughalt, MA , Douar Zaouiat Cheikh, HA , vicinity of Asni, AA Douar Issafen; AP (Rabat), MA (Aïn Leuh), HA (Réghaya) – MISR Chrysotoxum volaticum Séguy, 1961 Séguy 1961 , MA ; Claußen 1989b , HA , Oukaimeden (2600 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 Dasysyrphus Enderlein, 1938 Dasysyrphus albostriatus (Fallén, 1817) Kassebeer 1998a , HA , Bin-el-Ouidane, Ouirgane, Imlil, Asni; Sahib et al. 2020 Epistrophe Walker, 1852 Epistrophe eligans (Harris, 1780) = Syrphus ochrostoma (Zetterstedt), in Becker and Stein 1913 : 88; Claußen 1989b : 372 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b ; Kassebeer 1998a , MA , Ifrane, HA , Imlil, Asni, Ouirgane; Sahib et al. 2020 , Rif , Dayat Tazia Epistrophe eligans (Harris, 1870) var. trifasciata Strobl Ebejer and Bensusan 2010 , AA ; Djellab et al. 2013 Episyrphus Matsumura & Adachi, 1917 Episyrphus balteatus (De Geer, 1776) = Syrphus balteatus De Geer, in Becker and Stein 1913 : 88; Séguy 1930a : 129 = Epistrophe balteata (De Geer), in Gil Collado 1929: 406; Kanervo 1939 : 3; Timon-David 1951 : 144 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , AP , Rabat, MA , Aïn Sferguila; Timon-David 1951 , AP , forest of Maâmora, Rabat, HA , Marrakech; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Sebt Zinate, Aïn Sidi Brahim Ben Arrif, dam Nakhla, Aïn Boughaba, Garden Ksar Al Rimal, Oued Aârkoub, Dayat Rahrah, Oued Sahel, dam Moulay Bouchta, maison forestière, Ksar El Kébir, Dayat Jebel Zemzem, Oued Taida, stream at 1 km from Sidi Yahia Aârab, Aïn Quanquben, Oued Maggou, Forest Bab El Karn, Douar Kitane, forest El Mahfoura, HA , Aïn Zarka of Meski, AA , Douar Zaouia; AP (Rabat) – MISR Eupeodes Osten-Sacken, 1877 Eupeodes corollae (Fabricius, 1794) = Syrphus berber Bigot, in Bigot 1884 : 88 = Syrphus corollae Meigen, in Becker and Stein 1913 : 88; Séguy 1930a : 129, Kanervo 1939 : 3; Gil Collado 1929: 406 = Syrphus corollae Fabricius, in Timon-David 1951 : 144 = Metasyrphus corollae (Fabricius), in Dirickx 1994 : 89 Bigot 1884 ; Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress, Goundafa; Kanervo 1939 ; Timon-David 1951 , AP , forest of Maâmora, Rabat, Sidi Taibi, HA , Réghaya, Tazzarine, AA , Agdz, Plaine de Souss (Taroudant); Leclercq 1961a , MA , Ifrane; Claußen and Hauser 1990 , MA , Ifrane (1750 m), HA , Oukaimeden (3200 m), AA , Tan-Tan; Dirickx 1994 ; Mouna 1998 ; Pârvu and Zaharia 2007 ; Grabener 2017 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Village Sebt Zinate, Aïn el Ma Bared, Garden Ksar Al Rimal, Oued Bin EL Ouidane, Oued Sahel, Aïn Takhninjoute, stream at 1 km from Sidi Yahia Aârab, Oued Jnane Niche, dam Smir, Oued Boumarouil, Dayat Jebel Zemzem, Oued Maggou, Meadow Fahs Lmhar, Douar Kitane, forest El Mahfoura, MA , Douar Zaouiat Cheikh, HA , vicinity of Asni, Ijoukak vicinity, Lac Oukaimeden; AA Agdz – MISR Eupeodes latifasciatus (Macquart, 1829) = Syrphus latifasciatus Macquart, in Séguy 1949: 156; Séguy 1953a : 84 = Metasyrphus latifasciatus (Macquart), in Dirickx 1994 : 89 Séguy 1949a , SA , Guelmim; Séguy 1953a , AA , Oued Khoref; Claußen 1989b ; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Douar Kitane, maison forestière, Oued Ametrasse Eupeodes luniger (Meigen, 1822) = Metasyrphus luniger (Meigen), in Dirickx 1994 : 90, 237 = Syrphus luniger Meigen, in Gil Collado 1929: 406 Gil Collado 1929a , AP ; Claussen 1989b; Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Maggou, Oued Martil, Belyounech, Douar Kitane, MA , Douar Ben Smim, HA , vicinity of Asni Eupeodes nuba (Wiedemann, 1830) = Syrphus rufinasutus Bigot, in Bigot 1884 : 88, Séguy 1961 : 107 = Metasyrphus nuba (Wiedemann), in Dirickx 1994 : 90, 238 Bigot 1884 ; Séguy 1961 ; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; Dousti and Hayat 2006 ; Ehteshamnia et al. 2010 ; Naderloo et al. 2011; Speight 2013 , 2018 ; Kazerani et al. 2013b ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eupeodes punctifer (Frey, 1934) 40 Mouna 1998 : 86 Ischiodon Sack, 1913 Ischiodon aegyptius (Wiedemann, 1830) = Simosyrphus aegyptius (Wiedemann, 1830) Gil Collado 1929a ; Timon-David 1951 , AP , Rabat, AA , Agdz; Mouna 1998 ; Grabener 2017 ; Mengual 2018 ; Sahib et al. 2020 ; AP (Rabat) – MISR Lapposyrphus Dušek & Láska, 1967 Lapposyrphus lapponicus (Zetterstedt, 1838) = Syrphus arcuatus Fallén, 1817, in Becker and Stein 1913 : 88 = Metasyrphus lapponicus (Zetterstedt, 1838), in Dirickx 1994 : 89 = Eupeodes lapponicus (Zetterstedt, 1838), in Claußen 1989b : 372 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Meliscaeva Frey, 1946 Meliscaeva auricollis (Meigen, 1822) = Epistrophe auricollis Meigen, in Becker and Stein 1913 : 89; Gil Collado 1929: 406; Timon-David 1951 : 145 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Timon-David 1951 , AP , Oued Korifla, Zaers, Rabat; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Aïn el Ma Bared, Dayat El Ânassar, Belyounech, Oued Mezine, dam Moulay Bouchta, Aïn Afersiw, dam Entrasol, Oued à 15 km de Fifi, jumb Kitane, Oued Maggou, Douar Kitane, Oued Sahel, MA , Aïn Ouilili; AP (Rabat, Zaers) – MISR Meliscaeva cinctella (Zetterstedt, 1843) = Syrphus cinctellus Zeterstedt, in Séguy 1934b : 162 Séguy 1934b , MA , Oued Leben (Taounate); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 – MISR Scaeva Fabricius, 1850 Scaeva albomaculata (Macquart, 1842) = Lasiopticus albomaculata (Macquart), in Gil Collado 1929: 405; Timon-David 1951 : 145 = Lasiophthicus albomaculatus Macquart, in Séguy 1953a : 84 Gil Collado 1929a ; Séguy 1953a , MA , Immouzer; Timon-David 1951 , AP , Rabat, MA , El Harcha; Leclercq 1961a , MA , Azrou; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane; Dirickx 1994 ; Mouna 1998 ; Dousti and Hayat 2006 ; Ehteshamnia et al. 2010 ; Naderloo et al. 2011; Speight 2013 , 2018 ; Kazerani et al. 2013b ; Grabener 2017 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Belyounech, HA , Aïn Zarka of Meski, Lac Oukaimeden; AP (Rabat, Cap Cantin), EM (Debdou) – MISR Scaeva dignota (Rondani, 1857) Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Maggou, Dayat Lemtahane Scaeva mecogramma (Bigot, 1860) Dirickx 1994 ; Kassebeer 1998a , Rif , Chefchaouen, AP , Kénitra, HA , Ouirgane; Sahib et al. 2020 Scaeva pyrastri (Linnaeus, 1758) = Catabomba pyrastri Linnaeus, in Becker and Stein 1913 : 88 = Lasiophthicus pyrastri Linnaeus, in Séguy 1930a : 128 = Lasiopticus pyrastri Linnaeus, in Gil Collado 1929: 405, Timon-David 1951 : 145 Becker and Stein 1913 , Rif ; Gil Collado 1929a , AP ; Séguy 1930a , AP , forest of Zaers, forest of Maâmora, MA , Tizi-s'Tkrine; Timon-David 1951 , AP , Rabat, MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Dayat Jebel Zemzem, stream at 1 km from Oued Sidi Yahia Aârab, AA , 1 km before Douar Aïn Lahmar; AP (Rabat, Cap Cantin) – MISR Scaeva selenitica (Meigen, 1822) = Lasiophthicus seleniticus Meigen, in Séguy 1930a : 128 Séguy 1930a , HA , Aguerd el Had, AA , Talekjount (Souss); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Sphaerophoria Le Peletier & Serville, 1828 Sphaerophoria interrupta (Fabricius, 1805) = Sphaerophoria menthastri (Linnaeus), in Becker and Stein 1913 : 87; Kanervo 1939 : 3; Timon-David 1951 : 145; Séguy 1961 : 109 Kanervo 1939 , AP , MA , HA ; Becker and Stein 1913 , Rif ; Timon-David 1951 , AP , Kénitra, Rabat, MA , Harcha, Ifrane, Sefrou, HA , Agdz; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 ; AP (Rabat), MA (Ifrane, Meknès) – MISR Sphaerophoria rueppelli (Wiedemann, 1830) Kanervo 1939 , Rif , HA ; Timon-David 1951 , AP , Rabat, AA , Agadir, Agdz, Zagora; Séguy 1961 ; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Tarmast tributary, Oued Sidi Ben Saâda, Dayat Amsemlil, EM , farm Saf-Saf, MA , Oued d'Ifrane; AP (Rabat), AA (Agadir, Agdz, Zagora) – MISR Sphaerophoria scripta (Linnaeus, 1758) = Sphaerophoria dispar (Meigen), in Timon-David 1951 : 145 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1930a , AP , Tlet n'Rhohr, MA , forest of Timelilt, Aïn Leuh, EM , Berkane; Kanervo 1939 , Rif , AP , MA , HA ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Timon-David 1951 , AP , Rabat, MA , Harcha, Sefrou, Ifrane; Leclercq 1961a , MA , Dayat Aoua, Ifrane, Azrou; Claußen 1989b , MA , Azrou (1700 m), HA , Oukaimeden, Tizi-n'Test (1900 m), Ansegmir-Tal W midelt (1400 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb (1750 m), HA , Oukaimeden (3200 m); Dirickx 1994 ; Mouna 1998 ; Pârvu and Zaharia 2007 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Oued Aârkoub, dam Nakhla, meadow Mizoghar, Oued Dardara, 1 km after Dardara, Dayat El Birdiyel, palm grove Igrane, Aïn Quanquben, maison forestière, Oued Sidi Ben Saâda, Dayat Lemtahane, Dayat Amsemlil, forest El Mahfoura, EM , farm Saf-Saf, MA , Oued d'Ifrane, HA , vicinity of Asni, Lac Oukaimeden, AA , Msidira – MISR Sphaerophoria taeniata (Meigen, 1822) = Sphaerophoria menthastri var. taeniata Meigen, in Timon-David 1951 : 10 Timon-David 1951 , AP , Rabat, saline mud; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 ; MA (Aïn Leuh, Azrou, Timahdit) – MISR Syrphus Fabricius, 1775 Syrphus ribesii (Linnaeus, 1758) Kassebeer 1998a , Rif , Chefchaouen, Tétouan; Sahib et al. 2020 , Rif , Douar Kitane Syrphus vitripennis Meigen, 1822 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xanthogramma Schiner, 1860 Xanthogramma dives (Rondani, 1857) Ebejer et al. 2019 , AA , 29 km N of Rich (Errachidia, 1570 m); Sahib et al. 2020 Xanthogramma evanescens Becker, 1913 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xanthogramma marginale (Loew, 1854) = Xanthogramma marginale var. morenae Loew, in Becker and Stein 1913 : 86, Gil Collado 1929: 406 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Kanervo 1939 , Rif , HA ; Séguy 1961 ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 ; Claußen 1989b ; Dirickx 1994 ; Ebejer and Bensusan 2010 , AA ; Speight 2013 , 2018 ; Sahib et al. 2020 , Rif , village Sebt Zinate, Oued Maggou, Oued Ametrasse, Douar Kitane Xanthogramma pedissequum (Harris, 1776) = Xanthogramma ornatum (Meigen, 1822), in Gil Collado 1929: 406 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 PIPUNCULIDAE K. Kettani, M.J. Ebejer Number of species: 16 . Expected: 50 Faunistic knowledge of the family in Morocco: poor Chalarinae Chalarus Walker, 1834 Chalarus brevicaudis Jervis, 1992 Ebejer and Kettani 2019b , Rif , Dardara (730 m), Azilane (1255 m), El Hamma (338 m) Chalarus sp. aff. brevicaudis Jervis, 1992 Ebejer and Kettani 2019b , Rif , Azilane (1255 m) Pipunculinae Eudorylini Claraeola Aczél, 1940 Claraeola sp. aff. halterata (Meigen, 1838) Ebejer and Kettani 2019b , Rif , Akchour (424 m) Clistoabdominalis Skevington, 2001 Clistoabdominalis dilatatus (De Meyer, 1997) Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m), El Hamma (338 m) Dasydorylas Skevington, 2001 Dasydorylas setosus (Becker, 1908) Kehlmaier 2005 ; Motamedinia et al. 2017 Eudorylas Aczél, 1940 Eudorylas ibericus Kehlmaier, 2005 Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m) Pipunculini Pipunculus Latreille, 1802 Pipunculus carlestolrai Kuznetzov, 1993 Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m) Tomosvaryellini Tomosvaryella Aczél, 1939 Tomosvaryella cilifemorata (Becker, 1907) Ebejer and Kettani 2019b , Rif , Adrou ( PNPB , 556 m) Tomosvaryella debruyni De Meyer, 1995 Ebejer and Kettani 2019b , Rif , Adrou ( PNPB , 556 m) Tomosvaryella frontata (Becker, 1897) Ebejer and Kettani 2019b , Rif , Oued Mhajrate (Ben Karrich, 67 m) Tomosvaryella geniculata (Meigen, 1824) Ebejer and Kettani 2019b , HA , Lac Tislite (Imilchil, 2254 m) Tomosvaryella kuthyi Aczél, 1944 Ebejer and Kettani 2019b , Rif , El Hamma (338 m), Akchour (424 m), Barrage Smir (27 m) Tomosvaryella minima (Becker, 1897) Ebejer and Kettani 2019b , Rif , Koudiat Taifour (100 m) Tomosvaryella mutata (Becker, 1898) Ebejer and Kettani 2019b , Rif , Jebel Lakraâ (Talassemtane, 1596 m) Tomosvaryella trichotibialis De Meyer, 1995 Ebejer and Kettani 2019b , Rif , Koudiat Taifour (100 m) Tomosvaryella sp. subvirescens group Ebejer and Kettani 2019b , AP , Loukous marsh (Larache) Chalarinae Chalarus Walker, 1834 Chalarus brevicaudis Jervis, 1992 Ebejer and Kettani 2019b , Rif , Dardara (730 m), Azilane (1255 m), El Hamma (338 m) Chalarus sp. aff. brevicaudis Jervis, 1992 Ebejer and Kettani 2019b , Rif , Azilane (1255 m) Pipunculinae Eudorylini Claraeola Aczél, 1940 Claraeola sp. aff. halterata (Meigen, 1838) Ebejer and Kettani 2019b , Rif , Akchour (424 m) Clistoabdominalis Skevington, 2001 Clistoabdominalis dilatatus (De Meyer, 1997) Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m), El Hamma (338 m) Dasydorylas Skevington, 2001 Dasydorylas setosus (Becker, 1908) Kehlmaier 2005 ; Motamedinia et al. 2017 Eudorylas Aczél, 1940 Eudorylas ibericus Kehlmaier, 2005 Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m) Pipunculini Pipunculus Latreille, 1802 Pipunculus carlestolrai Kuznetzov, 1993 Ebejer and Kettani 2019b , Rif , Jebel Talassemtane (1546 m) Tomosvaryellini Tomosvaryella Aczél, 1939 Tomosvaryella cilifemorata (Becker, 1907) Ebejer and Kettani 2019b , Rif , Adrou ( PNPB , 556 m) Tomosvaryella debruyni De Meyer, 1995 Ebejer and Kettani 2019b , Rif , Adrou ( PNPB , 556 m) Tomosvaryella frontata (Becker, 1897) Ebejer and Kettani 2019b , Rif , Oued Mhajrate (Ben Karrich, 67 m) Tomosvaryella geniculata (Meigen, 1824) Ebejer and Kettani 2019b , HA , Lac Tislite (Imilchil, 2254 m) Tomosvaryella kuthyi Aczél, 1944 Ebejer and Kettani 2019b , Rif , El Hamma (338 m), Akchour (424 m), Barrage Smir (27 m) Tomosvaryella minima (Becker, 1897) Ebejer and Kettani 2019b , Rif , Koudiat Taifour (100 m) Tomosvaryella mutata (Becker, 1898) Ebejer and Kettani 2019b , Rif , Jebel Lakraâ (Talassemtane, 1596 m) Tomosvaryella trichotibialis De Meyer, 1995 Ebejer and Kettani 2019b , Rif , Koudiat Taifour (100 m) Tomosvaryella sp. subvirescens group Ebejer and Kettani 2019b , AP , Loukous marsh (Larache) SYRPHIDAE K. Kettani, M.C.D. Speight Number of species: 166 . Expected: more than 200 Faunistic knowledge of the family in Morocco: moderate Eristalinae Brachyopini Brachyopa Meigen, 1822 Brachyopa atlantea Kassebeer, 2000 Kassebeer 2000 , HA , Ouirgane (1000 m); Speight 2018 ; Sahib et al. 2020 Chrysogaster Meigen, 1803 Chrysogaster basalis Loew, 1857 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Kassebeer 1999d ; Sahib et al. 2020 Ighboulomyia Kassebeer, 1999 Ighboulomyia atlasi Kassebeer, 1999 Kassebeer 1999c , MA , Azrou, Umgebung, Timahdit, Ighböula Ulaichuor, Quellteich; Sahib et al. 2020 Myolepta Newman, 1838 Myolepta difformis Strobl in Czerny & Strobl 1909 = Myolepta philonis Séguy, 1961, in Dirickx 1994 : 93 Dirickx 1994 , HA ; Reemer et al. 2004 , MA , HA ; Speight 2013 , 2018 ; Sahib et al. 2020 Neoascia Williston, 1886 Neoascia clausseni Hauser & Kassebeer, 1998 = Neoascia podagrica (Fabricius, 1775), in Gil Collado 1929: 40; Claussen 1989b: 373 Gil Collado 1929a ; Claußen 1989b ; Dirickx 1994 ; Hauser and Kassebeer 1998 , MA , HA , Taroudant (1800 m); Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; Sahib et al. 2020 , Rif , Oued Jnane Niche, Oued Maggou Orthonevra Macquart, 1829 Orthonevra bouazzai Kassebeer, 1999 Kassebeer 1999d , MA ; Sahib et al. 2020 Orthonevra brevicornis (Loew, 1843) 32 Sahib et al. 2020 , Rif , Aïn Afersiw Orthonevra elegans (Meigen, 1822) Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Orthonevra schachti Claußen, 1989b Claußen 1989b , HA , Oukaimeden (2600 m); Dirickx 1994 ; Schmid 1995 ; Kassebeer 1999d , MA ; Sahib et al. 2020 Riponnensia Maibach, Goeldlin & Speight, 1994 Riponnensia longicornis (Loew, 1843) = Orthonevra longicornis Loew, in Kanervo 1939 : 2; Séguy 1961 : 23; Kassebeer 1999c : 162 Kanervo 1939 ; Séguy 1961 , AP ; Claußen 1989b ; Kassebeer 1999c , MA , HA ; Dirickx 1994 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 Riponnensia splendens (Meigen, 1822) = Chrysogaster splendens (Meigen), in Gil Collado 1929: 405 = Orthonevra splendens (Meigen), in Claußen 1989b : 363, 373; Dirickx 1994 : 97 Gil Collado 1929a , Rif ; Mouna 1998 ; Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Kassebeer 1999c , MA ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh; AP (Dradek) – MISR Callicerini Callicera Panzer, 1806 Callicera fagesi Guérin-Meneville, 1844 Kassebeer 1998a , HA , Ouirgane (1000 m); Sahib et al. 2020 Callicera rufa Schummel, 1842 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Cerioidini Ceriana Rafinesque, 1815 Ceriana conopsoides (Linnaeus, 1758) = Cerioides conopsoides Linnaeus, in Séguy 1930a : 131 Séguy 1930a , MA , Ras El Ksar; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Maghrawa, Maâmora) – MISR Ceriana vespiformis (Latreille, 1804) = Cerioides vespiformis Latreille, in Becker and Stein 1913 : 88; Gil Collado 1929: 414; Séguy 1930a : 131; Kanervo 1939 : 5; Leclercq 1961: 242 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , HA ; Séguy 1930a , AP , Rabat, Casablanca, MA , Tizi-s'Tkrine, Aïn Leuh, Meknès; Kanervo 1939 , MA ; Leclercq 1961a , Rif , Melillia, MA , Dayat Aoua, Aïn Leuh; Claußen 1989b ; Dirickx 1994 ; Steenis et al. 2016 ; Speight 2018 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , 1 km after Dardara, Meadow Mizoghar, Oued Achekrade Sphiximorpha Rondani, 1850 Sphiximorpha subsessilis (Illiger in Rossi, 1807) Steenis et al. 2016 Eristalini Anasimyia Schiner, 1864 Anasimyia contracta Caußen & Torp, 1980 Kassebeer 1998a , MA , Timahdit (1850 m); Sahib et al. 2020 Eristalinus Rondani, 1845 Eristalinus aeneus (Scopoli, 1763) = Lumpetia aenea (Scopoli), in Becker and Stein 1913 : 86 = Eristalis aeneus (Scopoli), in Gil Collado 1929: 406, 407; Leclercq 1961: 242 = Lathyrophtalmus aeneus (Scopoli), in Séguy 1930: 129; Kanervo 1939 : 5 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , Tanger, AP , Mogador; Séguy 1930a , AP , Casablanca; Kanervo 1939 ; Timon-David 1951 , AP , Rabat; Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , Jumb Kitane, HA , vicinity of Asni Eristalinus megacephalus (Rossi, 1794) = Eristalis quinquelineatus Fabricius, in Becker and Stein 1913 : 85, Gil Collado 1929: 407, = Lathyrophthalmus quinquelineatus Fabricius, in Séguy 1930a : 129 Gil Collado 1929a , Rif , Tanger; Séguy 1930a , AP , Rabat, Oued Korifla, Sidi Bettache; Séguy 1961 ; Dakki 1997 ; Claußen 1989b ; Dirickx 1994 ; Dousti and Hayat 2006 ; Sahib et al. 2020 ; AP (Rabat) – MISR Eristalinus sepulchralis (Linnaeus, 1758) = Eristalis sepuleralis Linnaeus, in Becker and Stein 1913 : 85, Gil Collado 1929: 406 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , AP , Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eristalinus taeniops (Wiedemann, 1818) = Eristalis taeniops Wiedemann, in Becker and Stein 1913 : 85 = Eristalodes taeniops Wiedemann, in Séguy 1930a : 130 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Oued Korifla, Rabat, HA , Tenfecht (Takeljount); Leclercq 1961a , MA , Dayet Aoua; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; Dousti and Hayat 2006 ; Koçak and Kemal 2010 ; Speight 2013 , 2018 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Oued Martil, Halouma Kitane, Oued Sidi Yahia Aârab, MA , bridge Oued Oum-er-Rbia (Douar Ahl Souss), HA , Lac Oukaimeden; AP (Rabat), MA (Volubilis) – MISR Eristalis Latreille, 1804 Eristalis arbustorum (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1930a , MA , Aïn Leuh, Ras El Ksar, forest of Taffert; Kanervo 1939 , Rif ; Timon-David 1951 , AP , Rabat, MA , Ifrane; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Aïn Sidi Brahim Ben Arrif, MA , vicinity of Ifrane, HA , vicinity of Asni, Lac Oukaimeden, AA , Douar Issafen, Douar Issafen; MA , HA – MISR Eristalis jugorum Egger, 1858 33 Dakki 1997 Eristalis pertinax (Scopoli, 1763) Séguy 1930a , AP , Oued Korifla, Sidi Bettache, MA , Forêt de Timelilt; Claußen 1989b ; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 Eristalis similis (Fallén, 1817) = Eristalis pratorum Meigen, in Gil Collado 1929: 407 Gil Collado 1929a , Rif ; Séguy 1961 ; Claußen 1989b , HA , Oukaimeden (2600 m); Dirickx 1994 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , maison forestière, HA , Douar Zaouiat Cheikh, Lac Oukaimeden Eristalis tenax (Linnaeus, 1758) = Eristalomyia tenax Linnaeus, in Séguy 1930a : 130; Timon-David 1951 : 146 Gil Collado 1929a , Rif , AP ; Kanervo 1939 , MA ; Séguy 1949a , AA ; Timon-David 1951 ; Leclercq 1961a ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 , Rif , Village Sebt Zinnat, Belyounech, Aïn Takhninjoute, maison forestière, Jumb Kitane, meadow Fahs Lmhar, HA , Douar Zaouiat Cheikh, vicinity of Asni, Lac Oukaimeden Helophilus Meigen, 1822 Helophilus trivittatus (Fabricius, 1805) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Mallota Meigen, 1822 Mallota cimbiciformis (Fallén, 1817) = Mallota eristaloides Loew, in Becker and Stein 1913 : 85 Becker and Stein 1913 , Rif , Tange; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Mallota dusmeti Andreu, 1926 Kassebeer 1998a , HA , Ouirgane; Sahib et al. 2020 Melanogaster Rondani, 1857 Melanogaster lindbergi Kassebeer, 1999 = Chrysogaster macquardti Loew, in Becker and Stein 1913 : 87 = Chrysogaster viduata Meigen, in Kanervo 1939 : 2, Séguy 1961 : 27, 28 = Chrysogaster lucida (Scopoli), in Claußen 1989b : 372 Becker and Stein 1913 , Rif ; Kanervo 1939 , MA ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1999d , MA ; Popov et al. 2020 ; Sahib et al. 2020 Myathropa Rondani, 1845 Myathropa florea (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , MA , Aïn Leuh; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Oued à 15 km de Fifi Parhelophilus Girschner, 1897 Parhelophilus versicolor (Fabricius, 1794) = Helophilus versicolor (Fabricius), in Gil Collado 1929: 407 Gil Collado 1929a , AP , Oulad Mesbah; Claußen 1989b ; Dirickx 1994 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eumerini Eumerus Meigen, 1822 Eumerus amoenus Loew, 1848 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 , AA , Douar Aourir, beach of Tamelallt Eumerus barbarus (Coquebert, 1804) = Eumerus australis Meigen, in Gil Collado 1929: 412 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1961 ; Claußen 1989b , MA , Azrou (1900 m); Dirickx 1994 ; Mouna 1998 ; Speight 2013 , 2018 ; Steenis et al. 2017 , AA , 11 km NW Taliouine, S Aït-Baha, 10 km NE Tafraoute; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 ; AP (Cap Cantin) – MISR Eumerus basalis Loew, 1848 = Eumerus angusticornis Rondani, in Séguy 1930a : 130 = Eumerus basalis Loew, in Mouna 1998 : 86 Séguy 1930a , MA , forest of Timelilt; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus caballeroi Gil Collado, 1929 Gil Collado 1929a , AP , Laguna Gedira; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus hungaricus Szilády, 1940 Speight 2018 Eumerus lunatus (Fabricius, 1794) = Eumerus lunulatus Fabricius, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger; Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus melotus (Séguy, 1941) = Lampetia melota Séguy, in Séguy 1941d : 13 Séguy 1941d , AA , Agadir; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus nudus Loew, 1848 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus obliquus (Fabricius, 1805) Sahib et al. 2020 , Rif , Oued Jnane Niche, EM , Oued Khemis Eumerus ornatus Meigen, 1822 Séguy 1930a , MA , Aïn Leuh, forest of Timelilt; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Oued Cherrat) – MISR Eumerus pulchellus Loew, 1848 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Eumerus punctifrons Loew, 1857 Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus pusillus Loew, 1848 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Sahib et al. 2020 Eumerus sabulonum (Fallén, 1817) Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Eumerus schmideggeri Steenis, Hauser & Zuijen, 2017 Steenis et al. 2017 , AA , Sidi R'bat (37 km S Agadir); Sahib et al. 2020 Eumerus strigatus (Fallén, 1817) 34 Kanervo 1939 , Rif , HA ; Timon-David 1951 , AP , Rabat, Sehoul; Claußen 1989b , MA , Azrou (1700 m), HA , Oukaimeden (2200 m), Ansegmir-Tal W Midelt; Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Speight 2013 ; Sahib et al. 2020 Eumerus subornatus Claußen, 1989b Claußen 1989b , HA , Tizi-n'Test (1900 m); Schmid 1995 ; Speight 2013 , 2018 ; Dirickx 1994 ; Sahib et al. 2020 Eumerus truncatus Rondani, 1868 Steenis et al. 2017 , AA , S. Aït-Baha, 11 km NW Taliouine, 25 km NE Tizinit, 20 km E Tizinit, Assaka; Speight 2018 ; Sahib et al. 2020 Merodon Meigen, 1803 35 Merodon aberrans Egger, 1860 = Lampetia aberrans Egger, in Séguy 1961 : 174 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Vujic et al. 2011; Speight 2013 , 2018 ; Sahib et al. 2020 Merodon aeneus Meigen, 1822 36 = Lampetia aenea Meigen, in Becker and Stein 1913 : 86; Kanervo 1939 : 5; Timon-David 1951 : 146 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , AP , Vallée Oued Korifla, MA , Tizi-s'Tkrine; Kanervo 1939 , Rif ; Timon-David 1951 ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Merodon arrasus Becker, 1921 37 Becker 1921 , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon aurifer Loew, 1862 = Merodon distinctus Palma, 1864 = Lampetia distincta Palm, in Timon-David 1951 : 146 Timon-David 1951 , AP , Zaer, MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; Vujić et al. 2021c Merodon avidus Rossi, 1782 38 = Lampetia spinipes (Fabricius), in Becker and Stein 1913 : 86; Timon-David 1951 : 146 = Merodon spinipes (Fabricius), in Gil Collado 1929: 409 = Lampetia avida Rossi, in Séguy 1961 : 176 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , HA ; Séguy 1930a , AP , Chellah (Rabat), Oued Korifla, MA , Aïn Leuh; Kanervo 1939 ; Timon-David 1951 , AP ; Séguy 1961 ; Claußen 1989b , HA , Oukaimeden (2600 m); Hurkmans 1993 ; Dirickx 1994 ; Mouna 1998 ; Koçak and Kemal 2010 ; Likov et al. 2020 ; Sahib et al. 2020 , HA , Lac Oukaimeden Merodon bequaerti Hurkmans, 1993 Vujić et al. 2020a , EM , Mountain of Beni-Snassen, MA , Azrou Merodon cabanerensis Marcos-García, Vujić & Mengual, 2007 Vujić et al. 2018 , HA , Ait Mhamed (Azilal, 1700 m); Speight 2018 ; Sahib et al. 2020 ; Vujić et al. 2021 Merodon calcaratus (Fabricius, 1794) Vujić et al. 2021b , EM , Mountains of Béni Snassen, near Nador, AP , 38 km SW of El Jadida, Garbouz Merodon chalybeus Wiedemann in Meigen, 1822 = Lampetia spicata Becker, in Timon-David 1951 : 146 = Merodon spicatus Becker, in Claußen 1989b : 365, 373 Timon-David 1951 , AP , forest of Maâmora; Claußen 1989b , HA (2500 m); Dirickx 1994 ; Mouna 1998 ; Marcos-Garcia et al. 2007 ; Speight 2013 , 2018 ; Sahib et al. 2020 ; AP (Cap Cantin) – MISR Merodon clavipes (Fabricius, 1781) Hurkmans 1993 , MA ; Marcos-García et al. 2007; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon eques Fabricius, 1805 = Lampetia eques (Fabricius), in Séguy 1961 : 178 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Ebejer and Bensusan 2010 , AA ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Merodon equestris (Fabricius, 1794) = Eristalis ferrugineus (Fabricius), in Fabricius 1805 : 240 = Lampetia equestris (Fabricius), in Séguy 1961 : 178, 179 Fabricius 1805 , AP ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon escalerai Gil Collado, 1929 Gil Collado 1929a , AP , Essaouira; Claußen 1989b ; Dirickx 1994 ; Speight 2018 ; Sahib et al. 2020 Merodon femoratus Sack, 1913 = Merodon biarcuatus Curran, 1939, in Curran 1939: 6, 7; Claußen 1989: 373; Dirickx 1994 : 79; Koçak and Kemal 2010 : 1199; Speight 2018 : 137 = Merodon elegans Hurkmans, 1993, in Hurkmans 1993 : 195; Schmid 1995 ; Marcos-Garcia et al. 2007 : 553; Speight 2018 : 141 Curran 1939, AP , forest of Maâmora (Rabat); Claußen 1989b ; Hurkmans 1993 , AP ; Dirickx 1994 ; Schmid 1995 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Speight 2018 ; Likov et al. 2020 Merodon geniculatus Strobl, 1909 Gil Collado 1929a , Rif ; Claußen 1989b , HA (2500 m); Dirickx 1994 ; Mouna 1998 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Speight 2013 , 2018 ; Sahib et al. 2020 , HA , Lac Oukaimeden – MISR Merodon hurkmansi Marcos-García, Vujić & Mengual, 2007 Marcos-García et al. 2007 Merodon ibericus Vujić, 2015 = Merodon bicolor Gil Collado, 1930 Popović et al. 2015 , MA , Azrou, Ifrane; Acanski et al. 2016; Speight 2018 ; Sahib et al. 2020 Merodon italicus Rondani, 1845 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 Merodon longicornis Sack, 1913 Claußen and Hauser 1990 , MA ; Dirickx 1994 ; Sahib et al. 2020 Merodon maroccanus Gil Collado, 1929 Gil Collado 1929a , AP , Essaouira; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon minutus Strobl, 1893 = Lampetia minutus Strobl, in Séguy 1961 : 180 Séguy 1961 ; Leclercq 1961a ; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Merodon monticolus Villeneuve, 1924 Kassebeer 1998a , HA , Ouirgane, Taftraoute, Taliouine; Sahib et al. 2020 Merodon murorum (Fabricius, 1794) = Syrphus murorum (Fabricius), in Fabricius 1794 : 288 = Merodon auripilus (Meigen), in Meigen 1830 : 354 = Lampetia auripila Meigen, in Séguy 1941d : 13; Mouna 1998 : 86 Fabricius 1794 ; Meigen 1830 ; Séguy 1941d , AA , Agadir; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Vujić et al. 2018 , AP , Essaouira; Sahib et al. 2020 Merodon pruni (Rossi, 1790) = Lampetia pruni (Rossi), in Becker and Stein 1913 : 86 = Merodon pruni var. obscurus Gil Collado, in Gil Collado 1929: 407, 408 Becker and Stein 1913 ; Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Hurkmans, 1993; Dirickx 1994 ; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon pumilus Macquart, 1849 Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m); Sahib et al. 2020 Merodon rufus Meigen, 1838 = Lampetia rufa Meigen, 1838, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon segetum (Fabricius, 1794) Peck 1988 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon serrulatus Wiedemann in Meigen, 1822 Hurkmans 1993 ; Speight 2013 , 2018 ; Sahib et al. 2020 ; Vujić et al. 2020a Merodon sophron Hurkmans, 1993 Hurkmans 1993 , MA , Azrou; Schmid 1995 ; Koçak and Kemal 2010 ; Vujić et al. 2020a , MA , Azrou Merodon tangerensis Hurkmans, 1993 Hurkmans 1993 , Rif , Tanger; Schmid 1995 ; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon tricinctus Sack, 1913 = Lampetia tricincta Sack, in Timon-David 1951 : 146 Timon-David 1951 , MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Merodon unguicornis Strobl, 1909 Ebejer et al. 2019 , MA , 10 km S of Azrou (1775 m); Sahib et al. 2020 , Rif , maison forestière Platynochaetus Wiedemann, 1830 Platynochaetus rufus Macquart, 1835 Gil Collado 1929a , AP , Mogador; Dirickx 1994 ; Sahib et al. 2020 Platynochaetus setosus (Fabricius, 1794) Gil Collado 1929a , HA , Marrakech; Séguy 1953a , AA , Souss: Aïn Chaib; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Milesiini Milesia Latreille, 1804 Milesia crabroniformis (Fabricius, 1775) Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Sahib et al. 2020 Spilomyia Meigen, 1803 Spilomyia maroccana Kuznetzov, 1997 = Spilomyia digitata (Rondani), in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b , HA , Tizi-n'Test (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Kuznetzov 1997 ; Dirickx 1994 ; Kassebeer 1999d ; Steenis 2000 ; Sahib et al. 2020 Syritta Le Peletier & Serville, 1828 Syritta flaviventris Macquart, 1842 Claußen 1989b , AP , Kénitra; Dirickx 1994 ; Sahib et al. 2020 , EM , farm Saf-Saf Syritta pipiens (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gill Collado 1929a; Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès; Kanervo 1939 ; Timon-David 1951 , AP , Rabat, AA , Agdz; Leclercq 1961a , EM , Melilla; Claußen 1989b , HA , Tizi-n'Test (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; Sahib et al. 2020 , Rif , Bni Maaden, dam Moulay Bouchta, Oued Koub, Douar Kitane, Oued Maggou, EM , farm Saf-Saf, MA , vicinity of Ifrane, HA , vicinity of Asni, Tizi-n'Test, AA , Oued Assa; AP (Rabat), MA (Aïn Leuh), SA – MISR Temnostoma Le Peletier & Serville, 1828 Temnostoma bombylans (Fabricius, 1805) Séguy 1961 , MA ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xylota Meigen, 1822 Xylota segnis (Linnaeus, 1758) = Zelima ( Xylota ) segnis Linnaeus, in Becker and Stein 1913 : 86; Timon-David 1951 : 147 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Timon-David 1951 , HA , Zaouia Ahansal; Leclercq 1961a , Rif , Azib de Ketama; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Aïn El Maounzil, Oued Koub, Oued Sidi Ben Saâda, HA , vicinity of Asni; HA (Zaouiet Ahansal) – MISR Rhingiini Cheilosia Meigen, 1822 Cheilosia brunnipennis Becker, 1894 = Chilosia flavipes (Panzer), in Kanervo 1939 : 2 Kanervo 1939 , HA ; Kassebeer, 1998c, MA ; Séguy 1961 ; Claußen 1989b ; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia flavipes (Panzer, 1798) Mouna 1998 : 86 Cheilosia grossa (Fallén, 1817) Kassebeer 1998c , HA , Asif Mellah, Tizi-n'Tichka; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia latifrons (Zetterstedt, 1843) = Cheilosia intonsa Loew, in Timon-David 1951 : 144 Timon-David 1951 , AP , Sehoul; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Kassebeer 1998c , Rif , Ouezzane, AP , Oued Loukous, Larache, MA , Ifrane, HA , Oukaimeden; Sahib et al. 2020 Cheilosia mutabilis (Fallén, 1817) Speight 2013 , 2018 Cheilosia griseiventris Loew, 1857 = Chilosia marokkana Becker, in Becker 1894: 395; Becker and Stein 1913 : 87 = Cheilosia maroccana Becker, 1894, in Gil Collado 1929: 405 Becker 1894; Becker and Stein 1913 ; Gil Collado 1929a ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1998c , MA , HA ; Sahib et al. 2020 Cheilosia paralobi Malski, 1962 = Cheilosia longula (Zetterstedt, 1838), in Gil Collado 1929: 405 Gil Collado 1929a ; Claußen 1989b , MA , Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Kassebeer 1998c , HA ; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia parva Kassebeer, 1998 Kassebeer 1998c , MA , Azrou, Ifrane (1650 m); Claußen and Speight 2007 ; Sahib et al. 2020 Cheilosia rodgersi Wainwright, 1911 Becker and Stein 1913 , Rif , Tanger; Claußen 1989a ; Dirickx 1994 ; Kassebeer 1998c , Rif , Tanger; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia scutellata (Fallén, 1817) Gil Collado 1929a , Rif ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1998c , Rif , Chefchaouen, MA , Ouiouane, Ifrane; Sahib et al. 2020 Cheilosia soror (Zetterstedt, 1843) = Cheilosia rufipes (Preyssler, 1793), in Claussen and Hauser 1990: 436, Kassebeer 1998c : 65 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Kassebeer 1998c , MA , Ifrane; Dirickx 1994 ; Sahib et al. 2020 Cheilosia variabilis (Panzer, 1798) Kassebeer 1998c , MA , Ifrane; Speight 2013 , 2018 ; Khaganinia and Kazerani 2014 ; Sahib et al. 2020 Ferdinandea Rondani, 1844 Ferdinandea fumipennis Kassebeer, 1999 Kassebeer 1999b , MA , Ifrane, Azrou, HA , Marrakech, Ouirgane; Speight 2013 , 2018 ; Sahib et al. 2020 Volucellini Volucella Geoffroy, 1762 Volucella inanis (Linnaeus, 1758) Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 Volucella liquida Erichson, 1841 Gil Collado 1929a , Rif ; Séguy 1930a , AP , Mogador, MA , Azrou, Bekrit; Kanervo 1939 , MA ; Séguy 1953a , MA , Ifrane; Timon-David 1951 , MA , Ifrane, Azrou, HA , Aït Mohamed Sgatt; Leclercq 1961a , Rif , Azib de Ketama, MA , Dayat Aoua, Ifrane, Azrou; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , jumb Kitane – MISR Volucella zonaria Poda, 1761 Séguy 1930a , AP , Casablanca; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Rabat, Casablanca) – MISR Brachypalpus Macquart, 1834 Brachypalpus valgus (Panzer, 1798) Kassebeer 1998a , MA , Ifrane, HA , Ouirgane; Sahib et al. 2020 Psilotini Psilota Fallén, 1823 Psilota atra (Fallén, 1817) = Psilota toubkalana Kassebeer, 1995, in Kassebeer 1995 : 395–400 Kassebeer 1995 , HA ; Smit and Vujic 2008, HA , Ouirgane, Marrakech; Speight 2018 ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh Pipizinae Pipizini Heringia Rondani, 1856 Heringia heringi (Zetterstedt, 1843) Kassebeer 1998a , HA , Tahanaout, Ouirgane; Sahib et al. 2020 Pipizella Rondani, 1856 Pipizella thapsiana Kassebeer, 1995 Kassebeer 1995 , HA (1000 m); Speight 2013 , 2018 ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh Triglyphus Loew, 1840 Triglyphus escalerai Gil Collado, 1929 Gil Collado 1929a , Rif , Tanger; Dirickx 1994 ; Speight 2013 ; Sahib et al. 2020 Syrphinae Bacchini Melanostoma Schiner, 1860 Melanostoma mellinum (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Séguy 1934b , AP , Korifla; Timon-David 1951 , MA , Ifrane, Azrou, Aïn Leuh, HA , Aït Mizane; Leclercq 1961a , MA , Ifrane; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , Dayat Rahrah, Aïn el Ma Bared, Oued Dardara, Dayat El Ânassar, Dayat Lemtahane, Garden Ksar Al Rimal, tributary Oued Tazarine, Oued Farda, Dayat El Birdiyel, maison forestière, stream at 1 km from Sidi Yahia Aârab, Dayat Amsemlil, EM , farm Saf-Saf, MA , Oued d'Ifrane, HA , Lac Oukaimeden – MISR Melanostoma mundum Czerny & Strobl, 1909 Dakki 1997 ; Mouna 1998 Melanostoma scalare (Fabricius, 1794) Gil Collado 1929a , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 , Rif , Aïn el Ma Bared, Oued Mezine, Aïn Quanquben, Aïn Takhninjoute, Oued Koub Platycheirus Le Peletier & Serville, 1828 Platycheirus albimanus (Fabricius, 1781) 39 Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress; Mouna 1998 ; Kassebeer 1998b Platycheirus ambiguus (Fallén, 1817) Kassebeer 1998b , HA ; Sahib et al. 2020 Platycheirus atlasi Kassebeer, 1998 Kassebeer 1998b , MA , Azrou, Ifrane; Sahib et al. 2020 Platycheirus fulviventris (Macquart, 1829) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m); Sahib et al. 2020 Platycheirus manicatus (Meigen, 1822) Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Kassebeer 1998b , HA , Toubkal; Sahib et al. 2020 Platycheirus marokkanus Kassebeer, 1998 Kassebeer 1998b , MA , HA ; Speight 2018 ; Sahib et al. 2020 , Rif , Aïn Takhninjoute, HA , Douar Akhlij Tnine Ourika Xanthandrus Verrall, 1901 Xanthandrus comtus (Harris, 1776) Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Paragini Paragus Latreille, 1804 Paragus albifrons (Fallén, 1817) Kanervo 1939 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Paragus atlasi Claußen, 1989 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Schmid 1995 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus bicolor (Fabricius, 1794) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, forest of Timelilt, Azrou; Kanervo 1939 , AP , HA ; Séguy 1949a , SA , Guelmim; Timon-David 1951 , MA , Ifrane; Claußen 1989b ; Claußen and Hauser 1990 , HA , Tizi-n'Test; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Douar Dacheryène, HA , vicinity of Asni; MA (Fès, Ifrane, Azrou) – MISR Paragus cinctus Schiner & Egger, 1853 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 , HA , vicinity of Asni Paragus coadunatus Rondani, 1847 Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus flammeus Goeldlin, 1971 Claußen and Hauser 1990 ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus haemorrhous Meigen, 1844 Claußen 1989b , MA , Azrou (1700 m); Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Mharhar Paragus hermonensis Kaplan, 1981 Claußen 1989b , MA , Azrou (1700 m); Dirickx 1994 ; Sahib et al. 2020 Paragus quadrifasciatus Meigen, 1822 = Paragus pulcherrimus Strobl, in Timon-David 1951 : 144 Timon-David 1951 , MA , Ifrane; Claußen 1989b , AP , Kénitra; Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Douar Kitane, AA , Agadir airport; MA (Ifrane) – MISR Paragus majoranae Rondani, 1857 Claußen and Hauser 1990 , MA ; Dirickx 1994 ; Sahib et al. 2020 Paragus pecchiolii Rondani, 1857 Claußen and Hauser 1990 , MA , Ifrane (1750 m), Hajeb Paragus strigatus Meigen, 1822 = Paragus bimaculatus Meigen, in Wiedemann 1824 : 33 Wiedemann 1824 , AP ; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m), Hajeb; Dirickx 1994 ; Speight 2018 ; Sahib et al. 2020 Paragus tibialis (Fallén, 1817) = Paragus tibialis meridionalis Becker, in Becker and Stein 1913 : 88; Gil Collado 1929: 403; Leclercq 1961: 241 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Kanervo 1939 ; Séguy 1949a , SA , Guelmim; Leclercq 1961a , Rif , Bab Taza, MA , Taza; Claußen 1989b ; Claußen and Hauser 1990 , HA , Tizi-n'Test (2000 m); Dirickx 1994 ; Dakki 1997 ; Grabener 2017 ; Sahib et al. 2020 , HA , vicinity of Asni, Ijoukak vicinity; AP (Rabat) – MISR Paragus vandergooti Marcos-Garcia, 1986 Claußen 1989b , HA , Tizi-n'Test à (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Syrphini Chrysotoxum Meigen, 1803 Chrysotoxum bicinctum (Linnaeus, 1758) Timon-David 1951 , MA , Ifrane, HA , Haute Réghaya; Leclercq 1961a , MA , Mischliffen (2019 m); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; MA , HA – MISR Chrysotoxum intermedium Meigen, 1822 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , MA , Tizi-S'Tkrine, Aïn Leuh, forest of Taffert, HA , Tizi-n'Test, Goundafa; Kanervo 1939 ; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Pârvu et al. 2006 , AP , Cap Bedouza; Dousti and Hayat 2006 ; Pârvu and Zaharia 2007 ; Kazerani et al. 2013b ; Sahib et al. 2020 , Rif , 1 km after Dardara, Oued Azila, Dayat Jebel Zemzem, Aïn El Maounzil, Oued Tafoughalt, MA , Douar Zaouiat Cheikh, HA , vicinity of Asni, AA Douar Issafen; AP (Rabat), MA (Aïn Leuh), HA (Réghaya) – MISR Chrysotoxum volaticum Séguy, 1961 Séguy 1961 , MA ; Claußen 1989b , HA , Oukaimeden (2600 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 Dasysyrphus Enderlein, 1938 Dasysyrphus albostriatus (Fallén, 1817) Kassebeer 1998a , HA , Bin-el-Ouidane, Ouirgane, Imlil, Asni; Sahib et al. 2020 Epistrophe Walker, 1852 Epistrophe eligans (Harris, 1780) = Syrphus ochrostoma (Zetterstedt), in Becker and Stein 1913 : 88; Claußen 1989b : 372 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b ; Kassebeer 1998a , MA , Ifrane, HA , Imlil, Asni, Ouirgane; Sahib et al. 2020 , Rif , Dayat Tazia Epistrophe eligans (Harris, 1870) var. trifasciata Strobl Ebejer and Bensusan 2010 , AA ; Djellab et al. 2013 Episyrphus Matsumura & Adachi, 1917 Episyrphus balteatus (De Geer, 1776) = Syrphus balteatus De Geer, in Becker and Stein 1913 : 88; Séguy 1930a : 129 = Epistrophe balteata (De Geer), in Gil Collado 1929: 406; Kanervo 1939 : 3; Timon-David 1951 : 144 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , AP , Rabat, MA , Aïn Sferguila; Timon-David 1951 , AP , forest of Maâmora, Rabat, HA , Marrakech; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Sebt Zinate, Aïn Sidi Brahim Ben Arrif, dam Nakhla, Aïn Boughaba, Garden Ksar Al Rimal, Oued Aârkoub, Dayat Rahrah, Oued Sahel, dam Moulay Bouchta, maison forestière, Ksar El Kébir, Dayat Jebel Zemzem, Oued Taida, stream at 1 km from Sidi Yahia Aârab, Aïn Quanquben, Oued Maggou, Forest Bab El Karn, Douar Kitane, forest El Mahfoura, HA , Aïn Zarka of Meski, AA , Douar Zaouia; AP (Rabat) – MISR Eupeodes Osten-Sacken, 1877 Eupeodes corollae (Fabricius, 1794) = Syrphus berber Bigot, in Bigot 1884 : 88 = Syrphus corollae Meigen, in Becker and Stein 1913 : 88; Séguy 1930a : 129, Kanervo 1939 : 3; Gil Collado 1929: 406 = Syrphus corollae Fabricius, in Timon-David 1951 : 144 = Metasyrphus corollae (Fabricius), in Dirickx 1994 : 89 Bigot 1884 ; Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress, Goundafa; Kanervo 1939 ; Timon-David 1951 , AP , forest of Maâmora, Rabat, Sidi Taibi, HA , Réghaya, Tazzarine, AA , Agdz, Plaine de Souss (Taroudant); Leclercq 1961a , MA , Ifrane; Claußen and Hauser 1990 , MA , Ifrane (1750 m), HA , Oukaimeden (3200 m), AA , Tan-Tan; Dirickx 1994 ; Mouna 1998 ; Pârvu and Zaharia 2007 ; Grabener 2017 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Village Sebt Zinate, Aïn el Ma Bared, Garden Ksar Al Rimal, Oued Bin EL Ouidane, Oued Sahel, Aïn Takhninjoute, stream at 1 km from Sidi Yahia Aârab, Oued Jnane Niche, dam Smir, Oued Boumarouil, Dayat Jebel Zemzem, Oued Maggou, Meadow Fahs Lmhar, Douar Kitane, forest El Mahfoura, MA , Douar Zaouiat Cheikh, HA , vicinity of Asni, Ijoukak vicinity, Lac Oukaimeden; AA Agdz – MISR Eupeodes latifasciatus (Macquart, 1829) = Syrphus latifasciatus Macquart, in Séguy 1949: 156; Séguy 1953a : 84 = Metasyrphus latifasciatus (Macquart), in Dirickx 1994 : 89 Séguy 1949a , SA , Guelmim; Séguy 1953a , AA , Oued Khoref; Claußen 1989b ; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Douar Kitane, maison forestière, Oued Ametrasse Eupeodes luniger (Meigen, 1822) = Metasyrphus luniger (Meigen), in Dirickx 1994 : 90, 237 = Syrphus luniger Meigen, in Gil Collado 1929: 406 Gil Collado 1929a , AP ; Claussen 1989b; Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Maggou, Oued Martil, Belyounech, Douar Kitane, MA , Douar Ben Smim, HA , vicinity of Asni Eupeodes nuba (Wiedemann, 1830) = Syrphus rufinasutus Bigot, in Bigot 1884 : 88, Séguy 1961 : 107 = Metasyrphus nuba (Wiedemann), in Dirickx 1994 : 90, 238 Bigot 1884 ; Séguy 1961 ; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; Dousti and Hayat 2006 ; Ehteshamnia et al. 2010 ; Naderloo et al. 2011; Speight 2013 , 2018 ; Kazerani et al. 2013b ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eupeodes punctifer (Frey, 1934) 40 Mouna 1998 : 86 Ischiodon Sack, 1913 Ischiodon aegyptius (Wiedemann, 1830) = Simosyrphus aegyptius (Wiedemann, 1830) Gil Collado 1929a ; Timon-David 1951 , AP , Rabat, AA , Agdz; Mouna 1998 ; Grabener 2017 ; Mengual 2018 ; Sahib et al. 2020 ; AP (Rabat) – MISR Lapposyrphus Dušek & Láska, 1967 Lapposyrphus lapponicus (Zetterstedt, 1838) = Syrphus arcuatus Fallén, 1817, in Becker and Stein 1913 : 88 = Metasyrphus lapponicus (Zetterstedt, 1838), in Dirickx 1994 : 89 = Eupeodes lapponicus (Zetterstedt, 1838), in Claußen 1989b : 372 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Meliscaeva Frey, 1946 Meliscaeva auricollis (Meigen, 1822) = Epistrophe auricollis Meigen, in Becker and Stein 1913 : 89; Gil Collado 1929: 406; Timon-David 1951 : 145 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Timon-David 1951 , AP , Oued Korifla, Zaers, Rabat; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Aïn el Ma Bared, Dayat El Ânassar, Belyounech, Oued Mezine, dam Moulay Bouchta, Aïn Afersiw, dam Entrasol, Oued à 15 km de Fifi, jumb Kitane, Oued Maggou, Douar Kitane, Oued Sahel, MA , Aïn Ouilili; AP (Rabat, Zaers) – MISR Meliscaeva cinctella (Zetterstedt, 1843) = Syrphus cinctellus Zeterstedt, in Séguy 1934b : 162 Séguy 1934b , MA , Oued Leben (Taounate); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 – MISR Scaeva Fabricius, 1850 Scaeva albomaculata (Macquart, 1842) = Lasiopticus albomaculata (Macquart), in Gil Collado 1929: 405; Timon-David 1951 : 145 = Lasiophthicus albomaculatus Macquart, in Séguy 1953a : 84 Gil Collado 1929a ; Séguy 1953a , MA , Immouzer; Timon-David 1951 , AP , Rabat, MA , El Harcha; Leclercq 1961a , MA , Azrou; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane; Dirickx 1994 ; Mouna 1998 ; Dousti and Hayat 2006 ; Ehteshamnia et al. 2010 ; Naderloo et al. 2011; Speight 2013 , 2018 ; Kazerani et al. 2013b ; Grabener 2017 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Belyounech, HA , Aïn Zarka of Meski, Lac Oukaimeden; AP (Rabat, Cap Cantin), EM (Debdou) – MISR Scaeva dignota (Rondani, 1857) Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Maggou, Dayat Lemtahane Scaeva mecogramma (Bigot, 1860) Dirickx 1994 ; Kassebeer 1998a , Rif , Chefchaouen, AP , Kénitra, HA , Ouirgane; Sahib et al. 2020 Scaeva pyrastri (Linnaeus, 1758) = Catabomba pyrastri Linnaeus, in Becker and Stein 1913 : 88 = Lasiophthicus pyrastri Linnaeus, in Séguy 1930a : 128 = Lasiopticus pyrastri Linnaeus, in Gil Collado 1929: 405, Timon-David 1951 : 145 Becker and Stein 1913 , Rif ; Gil Collado 1929a , AP ; Séguy 1930a , AP , forest of Zaers, forest of Maâmora, MA , Tizi-s'Tkrine; Timon-David 1951 , AP , Rabat, MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Dayat Jebel Zemzem, stream at 1 km from Oued Sidi Yahia Aârab, AA , 1 km before Douar Aïn Lahmar; AP (Rabat, Cap Cantin) – MISR Scaeva selenitica (Meigen, 1822) = Lasiophthicus seleniticus Meigen, in Séguy 1930a : 128 Séguy 1930a , HA , Aguerd el Had, AA , Talekjount (Souss); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Sphaerophoria Le Peletier & Serville, 1828 Sphaerophoria interrupta (Fabricius, 1805) = Sphaerophoria menthastri (Linnaeus), in Becker and Stein 1913 : 87; Kanervo 1939 : 3; Timon-David 1951 : 145; Séguy 1961 : 109 Kanervo 1939 , AP , MA , HA ; Becker and Stein 1913 , Rif ; Timon-David 1951 , AP , Kénitra, Rabat, MA , Harcha, Ifrane, Sefrou, HA , Agdz; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 ; AP (Rabat), MA (Ifrane, Meknès) – MISR Sphaerophoria rueppelli (Wiedemann, 1830) Kanervo 1939 , Rif , HA ; Timon-David 1951 , AP , Rabat, AA , Agadir, Agdz, Zagora; Séguy 1961 ; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Tarmast tributary, Oued Sidi Ben Saâda, Dayat Amsemlil, EM , farm Saf-Saf, MA , Oued d'Ifrane; AP (Rabat), AA (Agadir, Agdz, Zagora) – MISR Sphaerophoria scripta (Linnaeus, 1758) = Sphaerophoria dispar (Meigen), in Timon-David 1951 : 145 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1930a , AP , Tlet n'Rhohr, MA , forest of Timelilt, Aïn Leuh, EM , Berkane; Kanervo 1939 , Rif , AP , MA , HA ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Timon-David 1951 , AP , Rabat, MA , Harcha, Sefrou, Ifrane; Leclercq 1961a , MA , Dayat Aoua, Ifrane, Azrou; Claußen 1989b , MA , Azrou (1700 m), HA , Oukaimeden, Tizi-n'Test (1900 m), Ansegmir-Tal W midelt (1400 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb (1750 m), HA , Oukaimeden (3200 m); Dirickx 1994 ; Mouna 1998 ; Pârvu and Zaharia 2007 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Oued Aârkoub, dam Nakhla, meadow Mizoghar, Oued Dardara, 1 km after Dardara, Dayat El Birdiyel, palm grove Igrane, Aïn Quanquben, maison forestière, Oued Sidi Ben Saâda, Dayat Lemtahane, Dayat Amsemlil, forest El Mahfoura, EM , farm Saf-Saf, MA , Oued d'Ifrane, HA , vicinity of Asni, Lac Oukaimeden, AA , Msidira – MISR Sphaerophoria taeniata (Meigen, 1822) = Sphaerophoria menthastri var. taeniata Meigen, in Timon-David 1951 : 10 Timon-David 1951 , AP , Rabat, saline mud; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 ; MA (Aïn Leuh, Azrou, Timahdit) – MISR Syrphus Fabricius, 1775 Syrphus ribesii (Linnaeus, 1758) Kassebeer 1998a , Rif , Chefchaouen, Tétouan; Sahib et al. 2020 , Rif , Douar Kitane Syrphus vitripennis Meigen, 1822 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xanthogramma Schiner, 1860 Xanthogramma dives (Rondani, 1857) Ebejer et al. 2019 , AA , 29 km N of Rich (Errachidia, 1570 m); Sahib et al. 2020 Xanthogramma evanescens Becker, 1913 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xanthogramma marginale (Loew, 1854) = Xanthogramma marginale var. morenae Loew, in Becker and Stein 1913 : 86, Gil Collado 1929: 406 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Kanervo 1939 , Rif , HA ; Séguy 1961 ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 ; Claußen 1989b ; Dirickx 1994 ; Ebejer and Bensusan 2010 , AA ; Speight 2013 , 2018 ; Sahib et al. 2020 , Rif , village Sebt Zinate, Oued Maggou, Oued Ametrasse, Douar Kitane Xanthogramma pedissequum (Harris, 1776) = Xanthogramma ornatum (Meigen, 1822), in Gil Collado 1929: 406 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Eristalinae Brachyopini Brachyopa Meigen, 1822 Brachyopa atlantea Kassebeer, 2000 Kassebeer 2000 , HA , Ouirgane (1000 m); Speight 2018 ; Sahib et al. 2020 Chrysogaster Meigen, 1803 Chrysogaster basalis Loew, 1857 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Kassebeer 1999d ; Sahib et al. 2020 Ighboulomyia Kassebeer, 1999 Ighboulomyia atlasi Kassebeer, 1999 Kassebeer 1999c , MA , Azrou, Umgebung, Timahdit, Ighböula Ulaichuor, Quellteich; Sahib et al. 2020 Myolepta Newman, 1838 Myolepta difformis Strobl in Czerny & Strobl 1909 = Myolepta philonis Séguy, 1961, in Dirickx 1994 : 93 Dirickx 1994 , HA ; Reemer et al. 2004 , MA , HA ; Speight 2013 , 2018 ; Sahib et al. 2020 Neoascia Williston, 1886 Neoascia clausseni Hauser & Kassebeer, 1998 = Neoascia podagrica (Fabricius, 1775), in Gil Collado 1929: 40; Claussen 1989b: 373 Gil Collado 1929a ; Claußen 1989b ; Dirickx 1994 ; Hauser and Kassebeer 1998 , MA , HA , Taroudant (1800 m); Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; Sahib et al. 2020 , Rif , Oued Jnane Niche, Oued Maggou Orthonevra Macquart, 1829 Orthonevra bouazzai Kassebeer, 1999 Kassebeer 1999d , MA ; Sahib et al. 2020 Orthonevra brevicornis (Loew, 1843) 32 Sahib et al. 2020 , Rif , Aïn Afersiw Orthonevra elegans (Meigen, 1822) Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Orthonevra schachti Claußen, 1989b Claußen 1989b , HA , Oukaimeden (2600 m); Dirickx 1994 ; Schmid 1995 ; Kassebeer 1999d , MA ; Sahib et al. 2020 Riponnensia Maibach, Goeldlin & Speight, 1994 Riponnensia longicornis (Loew, 1843) = Orthonevra longicornis Loew, in Kanervo 1939 : 2; Séguy 1961 : 23; Kassebeer 1999c : 162 Kanervo 1939 ; Séguy 1961 , AP ; Claußen 1989b ; Kassebeer 1999c , MA , HA ; Dirickx 1994 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 Riponnensia splendens (Meigen, 1822) = Chrysogaster splendens (Meigen), in Gil Collado 1929: 405 = Orthonevra splendens (Meigen), in Claußen 1989b : 363, 373; Dirickx 1994 : 97 Gil Collado 1929a , Rif ; Mouna 1998 ; Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Kassebeer 1999c , MA ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh; AP (Dradek) – MISR Callicerini Callicera Panzer, 1806 Callicera fagesi Guérin-Meneville, 1844 Kassebeer 1998a , HA , Ouirgane (1000 m); Sahib et al. 2020 Callicera rufa Schummel, 1842 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Cerioidini Ceriana Rafinesque, 1815 Ceriana conopsoides (Linnaeus, 1758) = Cerioides conopsoides Linnaeus, in Séguy 1930a : 131 Séguy 1930a , MA , Ras El Ksar; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Maghrawa, Maâmora) – MISR Ceriana vespiformis (Latreille, 1804) = Cerioides vespiformis Latreille, in Becker and Stein 1913 : 88; Gil Collado 1929: 414; Séguy 1930a : 131; Kanervo 1939 : 5; Leclercq 1961: 242 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , HA ; Séguy 1930a , AP , Rabat, Casablanca, MA , Tizi-s'Tkrine, Aïn Leuh, Meknès; Kanervo 1939 , MA ; Leclercq 1961a , Rif , Melillia, MA , Dayat Aoua, Aïn Leuh; Claußen 1989b ; Dirickx 1994 ; Steenis et al. 2016 ; Speight 2018 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , 1 km after Dardara, Meadow Mizoghar, Oued Achekrade Sphiximorpha Rondani, 1850 Sphiximorpha subsessilis (Illiger in Rossi, 1807) Steenis et al. 2016 Eristalini Anasimyia Schiner, 1864 Anasimyia contracta Caußen & Torp, 1980 Kassebeer 1998a , MA , Timahdit (1850 m); Sahib et al. 2020 Eristalinus Rondani, 1845 Eristalinus aeneus (Scopoli, 1763) = Lumpetia aenea (Scopoli), in Becker and Stein 1913 : 86 = Eristalis aeneus (Scopoli), in Gil Collado 1929: 406, 407; Leclercq 1961: 242 = Lathyrophtalmus aeneus (Scopoli), in Séguy 1930: 129; Kanervo 1939 : 5 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , Tanger, AP , Mogador; Séguy 1930a , AP , Casablanca; Kanervo 1939 ; Timon-David 1951 , AP , Rabat; Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , Jumb Kitane, HA , vicinity of Asni Eristalinus megacephalus (Rossi, 1794) = Eristalis quinquelineatus Fabricius, in Becker and Stein 1913 : 85, Gil Collado 1929: 407, = Lathyrophthalmus quinquelineatus Fabricius, in Séguy 1930a : 129 Gil Collado 1929a , Rif , Tanger; Séguy 1930a , AP , Rabat, Oued Korifla, Sidi Bettache; Séguy 1961 ; Dakki 1997 ; Claußen 1989b ; Dirickx 1994 ; Dousti and Hayat 2006 ; Sahib et al. 2020 ; AP (Rabat) – MISR Eristalinus sepulchralis (Linnaeus, 1758) = Eristalis sepuleralis Linnaeus, in Becker and Stein 1913 : 85, Gil Collado 1929: 406 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , AP , Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eristalinus taeniops (Wiedemann, 1818) = Eristalis taeniops Wiedemann, in Becker and Stein 1913 : 85 = Eristalodes taeniops Wiedemann, in Séguy 1930a : 130 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Oued Korifla, Rabat, HA , Tenfecht (Takeljount); Leclercq 1961a , MA , Dayet Aoua; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; Dousti and Hayat 2006 ; Koçak and Kemal 2010 ; Speight 2013 , 2018 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Oued Martil, Halouma Kitane, Oued Sidi Yahia Aârab, MA , bridge Oued Oum-er-Rbia (Douar Ahl Souss), HA , Lac Oukaimeden; AP (Rabat), MA (Volubilis) – MISR Eristalis Latreille, 1804 Eristalis arbustorum (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1930a , MA , Aïn Leuh, Ras El Ksar, forest of Taffert; Kanervo 1939 , Rif ; Timon-David 1951 , AP , Rabat, MA , Ifrane; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Aïn Sidi Brahim Ben Arrif, MA , vicinity of Ifrane, HA , vicinity of Asni, Lac Oukaimeden, AA , Douar Issafen, Douar Issafen; MA , HA – MISR Eristalis jugorum Egger, 1858 33 Dakki 1997 Eristalis pertinax (Scopoli, 1763) Séguy 1930a , AP , Oued Korifla, Sidi Bettache, MA , Forêt de Timelilt; Claußen 1989b ; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 Eristalis similis (Fallén, 1817) = Eristalis pratorum Meigen, in Gil Collado 1929: 407 Gil Collado 1929a , Rif ; Séguy 1961 ; Claußen 1989b , HA , Oukaimeden (2600 m); Dirickx 1994 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , maison forestière, HA , Douar Zaouiat Cheikh, Lac Oukaimeden Eristalis tenax (Linnaeus, 1758) = Eristalomyia tenax Linnaeus, in Séguy 1930a : 130; Timon-David 1951 : 146 Gil Collado 1929a , Rif , AP ; Kanervo 1939 , MA ; Séguy 1949a , AA ; Timon-David 1951 ; Leclercq 1961a ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 , Rif , Village Sebt Zinnat, Belyounech, Aïn Takhninjoute, maison forestière, Jumb Kitane, meadow Fahs Lmhar, HA , Douar Zaouiat Cheikh, vicinity of Asni, Lac Oukaimeden Helophilus Meigen, 1822 Helophilus trivittatus (Fabricius, 1805) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Mallota Meigen, 1822 Mallota cimbiciformis (Fallén, 1817) = Mallota eristaloides Loew, in Becker and Stein 1913 : 85 Becker and Stein 1913 , Rif , Tange; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Mallota dusmeti Andreu, 1926 Kassebeer 1998a , HA , Ouirgane; Sahib et al. 2020 Melanogaster Rondani, 1857 Melanogaster lindbergi Kassebeer, 1999 = Chrysogaster macquardti Loew, in Becker and Stein 1913 : 87 = Chrysogaster viduata Meigen, in Kanervo 1939 : 2, Séguy 1961 : 27, 28 = Chrysogaster lucida (Scopoli), in Claußen 1989b : 372 Becker and Stein 1913 , Rif ; Kanervo 1939 , MA ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1999d , MA ; Popov et al. 2020 ; Sahib et al. 2020 Myathropa Rondani, 1845 Myathropa florea (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , MA , Aïn Leuh; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Oued à 15 km de Fifi Parhelophilus Girschner, 1897 Parhelophilus versicolor (Fabricius, 1794) = Helophilus versicolor (Fabricius), in Gil Collado 1929: 407 Gil Collado 1929a , AP , Oulad Mesbah; Claußen 1989b ; Dirickx 1994 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eumerini Eumerus Meigen, 1822 Eumerus amoenus Loew, 1848 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 , AA , Douar Aourir, beach of Tamelallt Eumerus barbarus (Coquebert, 1804) = Eumerus australis Meigen, in Gil Collado 1929: 412 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1961 ; Claußen 1989b , MA , Azrou (1900 m); Dirickx 1994 ; Mouna 1998 ; Speight 2013 , 2018 ; Steenis et al. 2017 , AA , 11 km NW Taliouine, S Aït-Baha, 10 km NE Tafraoute; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 ; AP (Cap Cantin) – MISR Eumerus basalis Loew, 1848 = Eumerus angusticornis Rondani, in Séguy 1930a : 130 = Eumerus basalis Loew, in Mouna 1998 : 86 Séguy 1930a , MA , forest of Timelilt; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus caballeroi Gil Collado, 1929 Gil Collado 1929a , AP , Laguna Gedira; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus hungaricus Szilády, 1940 Speight 2018 Eumerus lunatus (Fabricius, 1794) = Eumerus lunulatus Fabricius, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger; Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Dousti and Hayat 2006 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus melotus (Séguy, 1941) = Lampetia melota Séguy, in Séguy 1941d : 13 Séguy 1941d , AA , Agadir; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus nudus Loew, 1848 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Eumerus obliquus (Fabricius, 1805) Sahib et al. 2020 , Rif , Oued Jnane Niche, EM , Oued Khemis Eumerus ornatus Meigen, 1822 Séguy 1930a , MA , Aïn Leuh, forest of Timelilt; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Oued Cherrat) – MISR Eumerus pulchellus Loew, 1848 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Eumerus punctifrons Loew, 1857 Leclercq 1961a , EM , Melilla; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Eumerus pusillus Loew, 1848 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Sahib et al. 2020 Eumerus sabulonum (Fallén, 1817) Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Eumerus schmideggeri Steenis, Hauser & Zuijen, 2017 Steenis et al. 2017 , AA , Sidi R'bat (37 km S Agadir); Sahib et al. 2020 Eumerus strigatus (Fallén, 1817) 34 Kanervo 1939 , Rif , HA ; Timon-David 1951 , AP , Rabat, Sehoul; Claußen 1989b , MA , Azrou (1700 m), HA , Oukaimeden (2200 m), Ansegmir-Tal W Midelt; Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Speight 2013 ; Sahib et al. 2020 Eumerus subornatus Claußen, 1989b Claußen 1989b , HA , Tizi-n'Test (1900 m); Schmid 1995 ; Speight 2013 , 2018 ; Dirickx 1994 ; Sahib et al. 2020 Eumerus truncatus Rondani, 1868 Steenis et al. 2017 , AA , S. Aït-Baha, 11 km NW Taliouine, 25 km NE Tizinit, 20 km E Tizinit, Assaka; Speight 2018 ; Sahib et al. 2020 Merodon Meigen, 1803 35 Merodon aberrans Egger, 1860 = Lampetia aberrans Egger, in Séguy 1961 : 174 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Vujic et al. 2011; Speight 2013 , 2018 ; Sahib et al. 2020 Merodon aeneus Meigen, 1822 36 = Lampetia aenea Meigen, in Becker and Stein 1913 : 86; Kanervo 1939 : 5; Timon-David 1951 : 146 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , AP , Vallée Oued Korifla, MA , Tizi-s'Tkrine; Kanervo 1939 , Rif ; Timon-David 1951 ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Merodon arrasus Becker, 1921 37 Becker 1921 , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon aurifer Loew, 1862 = Merodon distinctus Palma, 1864 = Lampetia distincta Palm, in Timon-David 1951 : 146 Timon-David 1951 , AP , Zaer, MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; Vujić et al. 2021c Merodon avidus Rossi, 1782 38 = Lampetia spinipes (Fabricius), in Becker and Stein 1913 : 86; Timon-David 1951 : 146 = Merodon spinipes (Fabricius), in Gil Collado 1929: 409 = Lampetia avida Rossi, in Séguy 1961 : 176 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif , HA ; Séguy 1930a , AP , Chellah (Rabat), Oued Korifla, MA , Aïn Leuh; Kanervo 1939 ; Timon-David 1951 , AP ; Séguy 1961 ; Claußen 1989b , HA , Oukaimeden (2600 m); Hurkmans 1993 ; Dirickx 1994 ; Mouna 1998 ; Koçak and Kemal 2010 ; Likov et al. 2020 ; Sahib et al. 2020 , HA , Lac Oukaimeden Merodon bequaerti Hurkmans, 1993 Vujić et al. 2020a , EM , Mountain of Beni-Snassen, MA , Azrou Merodon cabanerensis Marcos-García, Vujić & Mengual, 2007 Vujić et al. 2018 , HA , Ait Mhamed (Azilal, 1700 m); Speight 2018 ; Sahib et al. 2020 ; Vujić et al. 2021 Merodon calcaratus (Fabricius, 1794) Vujić et al. 2021b , EM , Mountains of Béni Snassen, near Nador, AP , 38 km SW of El Jadida, Garbouz Merodon chalybeus Wiedemann in Meigen, 1822 = Lampetia spicata Becker, in Timon-David 1951 : 146 = Merodon spicatus Becker, in Claußen 1989b : 365, 373 Timon-David 1951 , AP , forest of Maâmora; Claußen 1989b , HA (2500 m); Dirickx 1994 ; Mouna 1998 ; Marcos-Garcia et al. 2007 ; Speight 2013 , 2018 ; Sahib et al. 2020 ; AP (Cap Cantin) – MISR Merodon clavipes (Fabricius, 1781) Hurkmans 1993 , MA ; Marcos-García et al. 2007; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon eques Fabricius, 1805 = Lampetia eques (Fabricius), in Séguy 1961 : 178 Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Ebejer and Bensusan 2010 , AA ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Merodon equestris (Fabricius, 1794) = Eristalis ferrugineus (Fabricius), in Fabricius 1805 : 240 = Lampetia equestris (Fabricius), in Séguy 1961 : 178, 179 Fabricius 1805 , AP ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon escalerai Gil Collado, 1929 Gil Collado 1929a , AP , Essaouira; Claußen 1989b ; Dirickx 1994 ; Speight 2018 ; Sahib et al. 2020 Merodon femoratus Sack, 1913 = Merodon biarcuatus Curran, 1939, in Curran 1939: 6, 7; Claußen 1989: 373; Dirickx 1994 : 79; Koçak and Kemal 2010 : 1199; Speight 2018 : 137 = Merodon elegans Hurkmans, 1993, in Hurkmans 1993 : 195; Schmid 1995 ; Marcos-Garcia et al. 2007 : 553; Speight 2018 : 141 Curran 1939, AP , forest of Maâmora (Rabat); Claußen 1989b ; Hurkmans 1993 , AP ; Dirickx 1994 ; Schmid 1995 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Speight 2018 ; Likov et al. 2020 Merodon geniculatus Strobl, 1909 Gil Collado 1929a , Rif ; Claußen 1989b , HA (2500 m); Dirickx 1994 ; Mouna 1998 ; Marcos-Garcia et al. 2007 ; Koçak and Kemal 2010 ; Speight 2013 , 2018 ; Sahib et al. 2020 , HA , Lac Oukaimeden – MISR Merodon hurkmansi Marcos-García, Vujić & Mengual, 2007 Marcos-García et al. 2007 Merodon ibericus Vujić, 2015 = Merodon bicolor Gil Collado, 1930 Popović et al. 2015 , MA , Azrou, Ifrane; Acanski et al. 2016; Speight 2018 ; Sahib et al. 2020 Merodon italicus Rondani, 1845 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 Merodon longicornis Sack, 1913 Claußen and Hauser 1990 , MA ; Dirickx 1994 ; Sahib et al. 2020 Merodon maroccanus Gil Collado, 1929 Gil Collado 1929a , AP , Essaouira; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon minutus Strobl, 1893 = Lampetia minutus Strobl, in Séguy 1961 : 180 Séguy 1961 ; Leclercq 1961a ; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Merodon monticolus Villeneuve, 1924 Kassebeer 1998a , HA , Ouirgane, Taftraoute, Taliouine; Sahib et al. 2020 Merodon murorum (Fabricius, 1794) = Syrphus murorum (Fabricius), in Fabricius 1794 : 288 = Merodon auripilus (Meigen), in Meigen 1830 : 354 = Lampetia auripila Meigen, in Séguy 1941d : 13; Mouna 1998 : 86 Fabricius 1794 ; Meigen 1830 ; Séguy 1941d , AA , Agadir; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Vujić et al. 2018 , AP , Essaouira; Sahib et al. 2020 Merodon pruni (Rossi, 1790) = Lampetia pruni (Rossi), in Becker and Stein 1913 : 86 = Merodon pruni var. obscurus Gil Collado, in Gil Collado 1929: 407, 408 Becker and Stein 1913 ; Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Hurkmans, 1993; Dirickx 1994 ; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon pumilus Macquart, 1849 Ebejer et al. 2019 , Rif , Moulay Abdelsalam (965 m); Sahib et al. 2020 Merodon rufus Meigen, 1838 = Lampetia rufa Meigen, 1838, in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon segetum (Fabricius, 1794) Peck 1988 ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Merodon serrulatus Wiedemann in Meigen, 1822 Hurkmans 1993 ; Speight 2013 , 2018 ; Sahib et al. 2020 ; Vujić et al. 2020a Merodon sophron Hurkmans, 1993 Hurkmans 1993 , MA , Azrou; Schmid 1995 ; Koçak and Kemal 2010 ; Vujić et al. 2020a , MA , Azrou Merodon tangerensis Hurkmans, 1993 Hurkmans 1993 , Rif , Tanger; Schmid 1995 ; Koçak and Kemal 2010 ; Sahib et al. 2020 Merodon tricinctus Sack, 1913 = Lampetia tricincta Sack, in Timon-David 1951 : 146 Timon-David 1951 , MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Merodon unguicornis Strobl, 1909 Ebejer et al. 2019 , MA , 10 km S of Azrou (1775 m); Sahib et al. 2020 , Rif , maison forestière Platynochaetus Wiedemann, 1830 Platynochaetus rufus Macquart, 1835 Gil Collado 1929a , AP , Mogador; Dirickx 1994 ; Sahib et al. 2020 Platynochaetus setosus (Fabricius, 1794) Gil Collado 1929a , HA , Marrakech; Séguy 1953a , AA , Souss: Aïn Chaib; Claußen 1989b ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Milesiini Milesia Latreille, 1804 Milesia crabroniformis (Fabricius, 1775) Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Sahib et al. 2020 Spilomyia Meigen, 1803 Spilomyia maroccana Kuznetzov, 1997 = Spilomyia digitata (Rondani), in Becker and Stein 1913 : 86 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b , HA , Tizi-n'Test (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Kuznetzov 1997 ; Dirickx 1994 ; Kassebeer 1999d ; Steenis 2000 ; Sahib et al. 2020 Syritta Le Peletier & Serville, 1828 Syritta flaviventris Macquart, 1842 Claußen 1989b , AP , Kénitra; Dirickx 1994 ; Sahib et al. 2020 , EM , farm Saf-Saf Syritta pipiens (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Gill Collado 1929a; Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès; Kanervo 1939 ; Timon-David 1951 , AP , Rabat, AA , Agdz; Leclercq 1961a , EM , Melilla; Claußen 1989b , HA , Tizi-n'Test (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; Sahib et al. 2020 , Rif , Bni Maaden, dam Moulay Bouchta, Oued Koub, Douar Kitane, Oued Maggou, EM , farm Saf-Saf, MA , vicinity of Ifrane, HA , vicinity of Asni, Tizi-n'Test, AA , Oued Assa; AP (Rabat), MA (Aïn Leuh), SA – MISR Temnostoma Le Peletier & Serville, 1828 Temnostoma bombylans (Fabricius, 1805) Séguy 1961 , MA ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xylota Meigen, 1822 Xylota segnis (Linnaeus, 1758) = Zelima ( Xylota ) segnis Linnaeus, in Becker and Stein 1913 : 86; Timon-David 1951 : 147 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Timon-David 1951 , HA , Zaouia Ahansal; Leclercq 1961a , Rif , Azib de Ketama; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Aïn El Maounzil, Oued Koub, Oued Sidi Ben Saâda, HA , vicinity of Asni; HA (Zaouiet Ahansal) – MISR Rhingiini Cheilosia Meigen, 1822 Cheilosia brunnipennis Becker, 1894 = Chilosia flavipes (Panzer), in Kanervo 1939 : 2 Kanervo 1939 , HA ; Kassebeer, 1998c, MA ; Séguy 1961 ; Claußen 1989b ; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia flavipes (Panzer, 1798) Mouna 1998 : 86 Cheilosia grossa (Fallén, 1817) Kassebeer 1998c , HA , Asif Mellah, Tizi-n'Tichka; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia latifrons (Zetterstedt, 1843) = Cheilosia intonsa Loew, in Timon-David 1951 : 144 Timon-David 1951 , AP , Sehoul; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Kassebeer 1998c , Rif , Ouezzane, AP , Oued Loukous, Larache, MA , Ifrane, HA , Oukaimeden; Sahib et al. 2020 Cheilosia mutabilis (Fallén, 1817) Speight 2013 , 2018 Cheilosia griseiventris Loew, 1857 = Chilosia marokkana Becker, in Becker 1894: 395; Becker and Stein 1913 : 87 = Cheilosia maroccana Becker, 1894, in Gil Collado 1929: 405 Becker 1894; Becker and Stein 1913 ; Gil Collado 1929a ; Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1998c , MA , HA ; Sahib et al. 2020 Cheilosia paralobi Malski, 1962 = Cheilosia longula (Zetterstedt, 1838), in Gil Collado 1929: 405 Gil Collado 1929a ; Claußen 1989b , MA , Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Kassebeer 1998c , HA ; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia parva Kassebeer, 1998 Kassebeer 1998c , MA , Azrou, Ifrane (1650 m); Claußen and Speight 2007 ; Sahib et al. 2020 Cheilosia rodgersi Wainwright, 1911 Becker and Stein 1913 , Rif , Tanger; Claußen 1989a ; Dirickx 1994 ; Kassebeer 1998c , Rif , Tanger; Speight 2013 , 2018 ; Sahib et al. 2020 Cheilosia scutellata (Fallén, 1817) Gil Collado 1929a , Rif ; Claußen 1989b ; Dirickx 1994 ; Kassebeer 1998c , Rif , Chefchaouen, MA , Ouiouane, Ifrane; Sahib et al. 2020 Cheilosia soror (Zetterstedt, 1843) = Cheilosia rufipes (Preyssler, 1793), in Claussen and Hauser 1990: 436, Kassebeer 1998c : 65 Claußen and Hauser 1990 , MA , Ifrane (1750 m); Kassebeer 1998c , MA , Ifrane; Dirickx 1994 ; Sahib et al. 2020 Cheilosia variabilis (Panzer, 1798) Kassebeer 1998c , MA , Ifrane; Speight 2013 , 2018 ; Khaganinia and Kazerani 2014 ; Sahib et al. 2020 Ferdinandea Rondani, 1844 Ferdinandea fumipennis Kassebeer, 1999 Kassebeer 1999b , MA , Ifrane, Azrou, HA , Marrakech, Ouirgane; Speight 2013 , 2018 ; Sahib et al. 2020 Volucellini Volucella Geoffroy, 1762 Volucella inanis (Linnaeus, 1758) Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 Volucella liquida Erichson, 1841 Gil Collado 1929a , Rif ; Séguy 1930a , AP , Mogador, MA , Azrou, Bekrit; Kanervo 1939 , MA ; Séguy 1953a , MA , Ifrane; Timon-David 1951 , MA , Ifrane, Azrou, HA , Aït Mohamed Sgatt; Leclercq 1961a , Rif , Azib de Ketama, MA , Dayat Aoua, Ifrane, Azrou; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , jumb Kitane – MISR Volucella zonaria Poda, 1761 Séguy 1930a , AP , Casablanca; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; AP (Rabat, Casablanca) – MISR Brachypalpus Macquart, 1834 Brachypalpus valgus (Panzer, 1798) Kassebeer 1998a , MA , Ifrane, HA , Ouirgane; Sahib et al. 2020 Psilotini Psilota Fallén, 1823 Psilota atra (Fallén, 1817) = Psilota toubkalana Kassebeer, 1995, in Kassebeer 1995 : 395–400 Kassebeer 1995 , HA ; Smit and Vujic 2008, HA , Ouirgane, Marrakech; Speight 2018 ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh Pipizinae Pipizini Heringia Rondani, 1856 Heringia heringi (Zetterstedt, 1843) Kassebeer 1998a , HA , Tahanaout, Ouirgane; Sahib et al. 2020 Pipizella Rondani, 1856 Pipizella thapsiana Kassebeer, 1995 Kassebeer 1995 , HA (1000 m); Speight 2013 , 2018 ; Sahib et al. 2020 , MA , Douar Zaouiat Cheikh Triglyphus Loew, 1840 Triglyphus escalerai Gil Collado, 1929 Gil Collado 1929a , Rif , Tanger; Dirickx 1994 ; Speight 2013 ; Sahib et al. 2020 Syrphinae Bacchini Melanostoma Schiner, 1860 Melanostoma mellinum (Linnaeus, 1758) Becker and Stein 1913 , Rif , Tanger; Séguy 1934b , AP , Korifla; Timon-David 1951 , MA , Ifrane, Azrou, Aïn Leuh, HA , Aït Mizane; Leclercq 1961a , MA , Ifrane; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 ; Pârvu and Zaharia 2007 ; Sahib et al. 2020 , Rif , Dayat Rahrah, Aïn el Ma Bared, Oued Dardara, Dayat El Ânassar, Dayat Lemtahane, Garden Ksar Al Rimal, tributary Oued Tazarine, Oued Farda, Dayat El Birdiyel, maison forestière, stream at 1 km from Sidi Yahia Aârab, Dayat Amsemlil, EM , farm Saf-Saf, MA , Oued d'Ifrane, HA , Lac Oukaimeden – MISR Melanostoma mundum Czerny & Strobl, 1909 Dakki 1997 ; Mouna 1998 Melanostoma scalare (Fabricius, 1794) Gil Collado 1929a , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 , Rif , Aïn el Ma Bared, Oued Mezine, Aïn Quanquben, Aïn Takhninjoute, Oued Koub Platycheirus Le Peletier & Serville, 1828 Platycheirus albimanus (Fabricius, 1781) 39 Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress; Mouna 1998 ; Kassebeer 1998b Platycheirus ambiguus (Fallén, 1817) Kassebeer 1998b , HA ; Sahib et al. 2020 Platycheirus atlasi Kassebeer, 1998 Kassebeer 1998b , MA , Azrou, Ifrane; Sahib et al. 2020 Platycheirus fulviventris (Macquart, 1829) Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m); Sahib et al. 2020 Platycheirus manicatus (Meigen, 1822) Séguy 1961 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Kassebeer 1998b , HA , Toubkal; Sahib et al. 2020 Platycheirus marokkanus Kassebeer, 1998 Kassebeer 1998b , MA , HA ; Speight 2018 ; Sahib et al. 2020 , Rif , Aïn Takhninjoute, HA , Douar Akhlij Tnine Ourika Xanthandrus Verrall, 1901 Xanthandrus comtus (Harris, 1776) Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Paragini Paragus Latreille, 1804 Paragus albifrons (Fallén, 1817) Kanervo 1939 ; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Paragus atlasi Claußen, 1989 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Schmid 1995 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus bicolor (Fabricius, 1794) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, forest of Timelilt, Azrou; Kanervo 1939 , AP , HA ; Séguy 1949a , SA , Guelmim; Timon-David 1951 , MA , Ifrane; Claußen 1989b ; Claußen and Hauser 1990 , HA , Tizi-n'Test; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Douar Dacheryène, HA , vicinity of Asni; MA (Fès, Ifrane, Azrou) – MISR Paragus cinctus Schiner & Egger, 1853 Claußen 1989b , HA , Tizi-n'Test (1900 m); Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 , HA , vicinity of Asni Paragus coadunatus Rondani, 1847 Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus flammeus Goeldlin, 1971 Claußen and Hauser 1990 ; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Paragus haemorrhous Meigen, 1844 Claußen 1989b , MA , Azrou (1700 m); Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Mharhar Paragus hermonensis Kaplan, 1981 Claußen 1989b , MA , Azrou (1700 m); Dirickx 1994 ; Sahib et al. 2020 Paragus quadrifasciatus Meigen, 1822 = Paragus pulcherrimus Strobl, in Timon-David 1951 : 144 Timon-David 1951 , MA , Ifrane; Claußen 1989b , AP , Kénitra; Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Douar Kitane, AA , Agadir airport; MA (Ifrane) – MISR Paragus majoranae Rondani, 1857 Claußen and Hauser 1990 , MA ; Dirickx 1994 ; Sahib et al. 2020 Paragus pecchiolii Rondani, 1857 Claußen and Hauser 1990 , MA , Ifrane (1750 m), Hajeb Paragus strigatus Meigen, 1822 = Paragus bimaculatus Meigen, in Wiedemann 1824 : 33 Wiedemann 1824 , AP ; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m), Hajeb; Dirickx 1994 ; Speight 2018 ; Sahib et al. 2020 Paragus tibialis (Fallén, 1817) = Paragus tibialis meridionalis Becker, in Becker and Stein 1913 : 88; Gil Collado 1929: 403; Leclercq 1961: 241 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Kanervo 1939 ; Séguy 1949a , SA , Guelmim; Leclercq 1961a , Rif , Bab Taza, MA , Taza; Claußen 1989b ; Claußen and Hauser 1990 , HA , Tizi-n'Test (2000 m); Dirickx 1994 ; Dakki 1997 ; Grabener 2017 ; Sahib et al. 2020 , HA , vicinity of Asni, Ijoukak vicinity; AP (Rabat) – MISR Paragus vandergooti Marcos-Garcia, 1986 Claußen 1989b , HA , Tizi-n'Test à (1900 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb; Dirickx 1994 ; Speight 2013 , 2018 ; Sahib et al. 2020 Syrphini Chrysotoxum Meigen, 1803 Chrysotoxum bicinctum (Linnaeus, 1758) Timon-David 1951 , MA , Ifrane, HA , Haute Réghaya; Leclercq 1961a , MA , Mischliffen (2019 m); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 ; MA , HA – MISR Chrysotoxum intermedium Meigen, 1822 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , MA , Tizi-S'Tkrine, Aïn Leuh, forest of Taffert, HA , Tizi-n'Test, Goundafa; Kanervo 1939 ; Claußen 1989b ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Mouna 1998 ; Pârvu et al. 2006 , AP , Cap Bedouza; Dousti and Hayat 2006 ; Pârvu and Zaharia 2007 ; Kazerani et al. 2013b ; Sahib et al. 2020 , Rif , 1 km after Dardara, Oued Azila, Dayat Jebel Zemzem, Aïn El Maounzil, Oued Tafoughalt, MA , Douar Zaouiat Cheikh, HA , vicinity of Asni, AA Douar Issafen; AP (Rabat), MA (Aïn Leuh), HA (Réghaya) – MISR Chrysotoxum volaticum Séguy, 1961 Séguy 1961 , MA ; Claußen 1989b , HA , Oukaimeden (2600 m); Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 Dasysyrphus Enderlein, 1938 Dasysyrphus albostriatus (Fallén, 1817) Kassebeer 1998a , HA , Bin-el-Ouidane, Ouirgane, Imlil, Asni; Sahib et al. 2020 Epistrophe Walker, 1852 Epistrophe eligans (Harris, 1780) = Syrphus ochrostoma (Zetterstedt), in Becker and Stein 1913 : 88; Claußen 1989b : 372 Becker and Stein 1913 , Rif , Tanger; Claußen 1989b ; Kassebeer 1998a , MA , Ifrane, HA , Imlil, Asni, Ouirgane; Sahib et al. 2020 , Rif , Dayat Tazia Epistrophe eligans (Harris, 1870) var. trifasciata Strobl Ebejer and Bensusan 2010 , AA ; Djellab et al. 2013 Episyrphus Matsumura & Adachi, 1917 Episyrphus balteatus (De Geer, 1776) = Syrphus balteatus De Geer, in Becker and Stein 1913 : 88; Séguy 1930a : 129 = Epistrophe balteata (De Geer), in Gil Collado 1929: 406; Kanervo 1939 : 3; Timon-David 1951 : 144 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , AP , Rabat, MA , Aïn Sferguila; Timon-David 1951 , AP , forest of Maâmora, Rabat, HA , Marrakech; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Sebt Zinate, Aïn Sidi Brahim Ben Arrif, dam Nakhla, Aïn Boughaba, Garden Ksar Al Rimal, Oued Aârkoub, Dayat Rahrah, Oued Sahel, dam Moulay Bouchta, maison forestière, Ksar El Kébir, Dayat Jebel Zemzem, Oued Taida, stream at 1 km from Sidi Yahia Aârab, Aïn Quanquben, Oued Maggou, Forest Bab El Karn, Douar Kitane, forest El Mahfoura, HA , Aïn Zarka of Meski, AA , Douar Zaouia; AP (Rabat) – MISR Eupeodes Osten-Sacken, 1877 Eupeodes corollae (Fabricius, 1794) = Syrphus berber Bigot, in Bigot 1884 : 88 = Syrphus corollae Meigen, in Becker and Stein 1913 : 88; Séguy 1930a : 129, Kanervo 1939 : 3; Gil Collado 1929: 406 = Syrphus corollae Fabricius, in Timon-David 1951 : 144 = Metasyrphus corollae (Fabricius), in Dirickx 1994 : 89 Bigot 1884 ; Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Séguy 1930a , HA , Tizi-n'Test, Jebel Imdress, Goundafa; Kanervo 1939 ; Timon-David 1951 , AP , forest of Maâmora, Rabat, Sidi Taibi, HA , Réghaya, Tazzarine, AA , Agdz, Plaine de Souss (Taroudant); Leclercq 1961a , MA , Ifrane; Claußen and Hauser 1990 , MA , Ifrane (1750 m), HA , Oukaimeden (3200 m), AA , Tan-Tan; Dirickx 1994 ; Mouna 1998 ; Pârvu and Zaharia 2007 ; Grabener 2017 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Village Sebt Zinate, Aïn el Ma Bared, Garden Ksar Al Rimal, Oued Bin EL Ouidane, Oued Sahel, Aïn Takhninjoute, stream at 1 km from Sidi Yahia Aârab, Oued Jnane Niche, dam Smir, Oued Boumarouil, Dayat Jebel Zemzem, Oued Maggou, Meadow Fahs Lmhar, Douar Kitane, forest El Mahfoura, MA , Douar Zaouiat Cheikh, HA , vicinity of Asni, Ijoukak vicinity, Lac Oukaimeden; AA Agdz – MISR Eupeodes latifasciatus (Macquart, 1829) = Syrphus latifasciatus Macquart, in Séguy 1949: 156; Séguy 1953a : 84 = Metasyrphus latifasciatus (Macquart), in Dirickx 1994 : 89 Séguy 1949a , SA , Guelmim; Séguy 1953a , AA , Oued Khoref; Claußen 1989b ; Dirickx 1994 ; Dakki 1997 ; Sahib et al. 2020 , Rif , Douar Kitane, maison forestière, Oued Ametrasse Eupeodes luniger (Meigen, 1822) = Metasyrphus luniger (Meigen), in Dirickx 1994 : 90, 237 = Syrphus luniger Meigen, in Gil Collado 1929: 406 Gil Collado 1929a , AP ; Claussen 1989b; Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Maggou, Oued Martil, Belyounech, Douar Kitane, MA , Douar Ben Smim, HA , vicinity of Asni Eupeodes nuba (Wiedemann, 1830) = Syrphus rufinasutus Bigot, in Bigot 1884 : 88, Séguy 1961 : 107 = Metasyrphus nuba (Wiedemann), in Dirickx 1994 : 90, 238 Bigot 1884 ; Séguy 1961 ; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; Dousti and Hayat 2006 ; Ehteshamnia et al. 2010 ; Naderloo et al. 2011; Speight 2013 , 2018 ; Kazerani et al. 2013b ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 Eupeodes punctifer (Frey, 1934) 40 Mouna 1998 : 86 Ischiodon Sack, 1913 Ischiodon aegyptius (Wiedemann, 1830) = Simosyrphus aegyptius (Wiedemann, 1830) Gil Collado 1929a ; Timon-David 1951 , AP , Rabat, AA , Agdz; Mouna 1998 ; Grabener 2017 ; Mengual 2018 ; Sahib et al. 2020 ; AP (Rabat) – MISR Lapposyrphus Dušek & Láska, 1967 Lapposyrphus lapponicus (Zetterstedt, 1838) = Syrphus arcuatus Fallén, 1817, in Becker and Stein 1913 : 88 = Metasyrphus lapponicus (Zetterstedt, 1838), in Dirickx 1994 : 89 = Eupeodes lapponicus (Zetterstedt, 1838), in Claußen 1989b : 372 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Meliscaeva Frey, 1946 Meliscaeva auricollis (Meigen, 1822) = Epistrophe auricollis Meigen, in Becker and Stein 1913 : 89; Gil Collado 1929: 406; Timon-David 1951 : 145 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a ; Timon-David 1951 , AP , Oued Korifla, Zaers, Rabat; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Aïn el Ma Bared, Dayat El Ânassar, Belyounech, Oued Mezine, dam Moulay Bouchta, Aïn Afersiw, dam Entrasol, Oued à 15 km de Fifi, jumb Kitane, Oued Maggou, Douar Kitane, Oued Sahel, MA , Aïn Ouilili; AP (Rabat, Zaers) – MISR Meliscaeva cinctella (Zetterstedt, 1843) = Syrphus cinctellus Zeterstedt, in Séguy 1934b : 162 Séguy 1934b , MA , Oued Leben (Taounate); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 – MISR Scaeva Fabricius, 1850 Scaeva albomaculata (Macquart, 1842) = Lasiopticus albomaculata (Macquart), in Gil Collado 1929: 405; Timon-David 1951 : 145 = Lasiophthicus albomaculatus Macquart, in Séguy 1953a : 84 Gil Collado 1929a ; Séguy 1953a , MA , Immouzer; Timon-David 1951 , AP , Rabat, MA , El Harcha; Leclercq 1961a , MA , Azrou; Claußen 1989b , MA , Azrou (1700 m); Claußen and Hauser 1990 , MA , Ifrane; Dirickx 1994 ; Mouna 1998 ; Dousti and Hayat 2006 ; Ehteshamnia et al. 2010 ; Naderloo et al. 2011; Speight 2013 , 2018 ; Kazerani et al. 2013b ; Grabener 2017 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Belyounech, HA , Aïn Zarka of Meski, Lac Oukaimeden; AP (Rabat, Cap Cantin), EM (Debdou) – MISR Scaeva dignota (Rondani, 1857) Claußen and Hauser 1990 , MA , Ifrane (1750 m); Dirickx 1994 ; Sahib et al. 2020 , Rif , Oued Maggou, Dayat Lemtahane Scaeva mecogramma (Bigot, 1860) Dirickx 1994 ; Kassebeer 1998a , Rif , Chefchaouen, AP , Kénitra, HA , Ouirgane; Sahib et al. 2020 Scaeva pyrastri (Linnaeus, 1758) = Catabomba pyrastri Linnaeus, in Becker and Stein 1913 : 88 = Lasiophthicus pyrastri Linnaeus, in Séguy 1930a : 128 = Lasiopticus pyrastri Linnaeus, in Gil Collado 1929: 405, Timon-David 1951 : 145 Becker and Stein 1913 , Rif ; Gil Collado 1929a , AP ; Séguy 1930a , AP , forest of Zaers, forest of Maâmora, MA , Tizi-s'Tkrine; Timon-David 1951 , AP , Rabat, MA , Ifrane; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 , Rif , Dayat Jebel Zemzem, stream at 1 km from Oued Sidi Yahia Aârab, AA , 1 km before Douar Aïn Lahmar; AP (Rabat, Cap Cantin) – MISR Scaeva selenitica (Meigen, 1822) = Lasiophthicus seleniticus Meigen, in Séguy 1930a : 128 Séguy 1930a , HA , Aguerd el Had, AA , Talekjount (Souss); Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; Sahib et al. 2020 Sphaerophoria Le Peletier & Serville, 1828 Sphaerophoria interrupta (Fabricius, 1805) = Sphaerophoria menthastri (Linnaeus), in Becker and Stein 1913 : 87; Kanervo 1939 : 3; Timon-David 1951 : 145; Séguy 1961 : 109 Kanervo 1939 , AP , MA , HA ; Becker and Stein 1913 , Rif ; Timon-David 1951 , AP , Kénitra, Rabat, MA , Harcha, Ifrane, Sefrou, HA , Agdz; Claußen 1989b ; Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 ; AP (Rabat), MA (Ifrane, Meknès) – MISR Sphaerophoria rueppelli (Wiedemann, 1830) Kanervo 1939 , Rif , HA ; Timon-David 1951 , AP , Rabat, AA , Agadir, Agdz, Zagora; Séguy 1961 ; Claußen 1989b , HA , Ansegmir-Tal W Midelt (1400 m); Dirickx 1994 ; Mouna 1998 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Tarmast tributary, Oued Sidi Ben Saâda, Dayat Amsemlil, EM , farm Saf-Saf, MA , Oued d'Ifrane; AP (Rabat), AA (Agadir, Agdz, Zagora) – MISR Sphaerophoria scripta (Linnaeus, 1758) = Sphaerophoria dispar (Meigen), in Timon-David 1951 : 145 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Séguy 1930a , AP , Tlet n'Rhohr, MA , forest of Timelilt, Aïn Leuh, EM , Berkane; Kanervo 1939 , Rif , AP , MA , HA ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Timon-David 1951 , AP , Rabat, MA , Harcha, Sefrou, Ifrane; Leclercq 1961a , MA , Dayat Aoua, Ifrane, Azrou; Claußen 1989b , MA , Azrou (1700 m), HA , Oukaimeden, Tizi-n'Test (1900 m), Ansegmir-Tal W midelt (1400 m); Claußen and Hauser 1990 , MA , Ifrane, Hajeb (1750 m), HA , Oukaimeden (3200 m); Dirickx 1994 ; Mouna 1998 ; Pârvu and Zaharia 2007 ; El Hawagry and Gilbert 2019 ; Sahib et al. 2020 , Rif , Oued Aârkoub, dam Nakhla, meadow Mizoghar, Oued Dardara, 1 km after Dardara, Dayat El Birdiyel, palm grove Igrane, Aïn Quanquben, maison forestière, Oued Sidi Ben Saâda, Dayat Lemtahane, Dayat Amsemlil, forest El Mahfoura, EM , farm Saf-Saf, MA , Oued d'Ifrane, HA , vicinity of Asni, Lac Oukaimeden, AA , Msidira – MISR Sphaerophoria taeniata (Meigen, 1822) = Sphaerophoria menthastri var. taeniata Meigen, in Timon-David 1951 : 10 Timon-David 1951 , AP , Rabat, saline mud; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 ; MA (Aïn Leuh, Azrou, Timahdit) – MISR Syrphus Fabricius, 1775 Syrphus ribesii (Linnaeus, 1758) Kassebeer 1998a , Rif , Chefchaouen, Tétouan; Sahib et al. 2020 , Rif , Douar Kitane Syrphus vitripennis Meigen, 1822 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xanthogramma Schiner, 1860 Xanthogramma dives (Rondani, 1857) Ebejer et al. 2019 , AA , 29 km N of Rich (Errachidia, 1570 m); Sahib et al. 2020 Xanthogramma evanescens Becker, 1913 Becker and Stein 1913 , Rif ; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Xanthogramma marginale (Loew, 1854) = Xanthogramma marginale var. morenae Loew, in Becker and Stein 1913 : 86, Gil Collado 1929: 406 Becker and Stein 1913 , Rif , Tanger; Gil Collado 1929a , Rif ; Kanervo 1939 , Rif , HA ; Séguy 1961 ; Claußen and Hauser 1990 , MA , Ifrane (1750 m); Mouna 1998 ; Claußen 1989b ; Dirickx 1994 ; Ebejer and Bensusan 2010 , AA ; Speight 2013 , 2018 ; Sahib et al. 2020 , Rif , village Sebt Zinate, Oued Maggou, Oued Ametrasse, Douar Kitane Xanthogramma pedissequum (Harris, 1776) = Xanthogramma ornatum (Meigen, 1822), in Gil Collado 1929: 406 Gil Collado 1929a , Rif , Tanger; Claußen 1989b ; Dirickx 1994 ; Sahib et al. 2020 Conopoidea CONOPIDAE 41 K. Kettani Number of species: 34 . Expected: 40 Faunistic knowledge of the family in Morocco: good Conopinae Conopini Conops Linnaeus, 1758 Conops aegyptiacus (Rondani, 1850) Kröber 1915 , 1928 Conops ceriaeformis Meigen, 1804 = Conops acuticornis Loew, 1847, in Becker and Stein 1913 : 89 Becker and Stein 1913 , Rif , Tanger; Kröber 1924 , 1928 , Rif , Tanger Conops djanetianus Séguy, 1938 Mouna 1998 : 85 Conops elegans Meigen, 1824 = Conops semifumosus Adams, in Séguy 1934b : 162 = Conops ruficornis Becker, 1913, in Becker and Stein 1913 : 89; Kröber 1924 : 69 Becker and Stein 1913 , Rif , Tanger; Kröber 1924 , 1927 , AP , Casablanca; Séguy 1934b , Rif , Tanger; Stuke 2016 ; El Hawagry et al. 2021 Conops nubeculipennis Bezzi, 1901 = Conops atrogonius Séguy, 1930, in Séguy 1930a : 134; Séguy 1953a : 85; Mouna 1998 : 85 Séguy 1930a , AP , Rabat, Mogador; Séguy 1953a , MA , Dayat Ifrah; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , 1.5 km S of Tissint, 14 km NW of Icht; Stuke 2016 ; El Hawagry et al. 2021 Conops theryi Séguy, 1928 Séguy 1928d ; Séguy 1930a , AP , Rabat, Casablanca; Mouna 1998 Conops tifedarius Séguy, 1928 Séguy 1928d ; Séguy 1930a , AP , Rabat; Mouna 1998 Leopoldius Rondani, 1843 Leopoldius coronatus (Rondani, 1857) = Brachyglossum coronatum Rondani, in Séguy 1930a : 132; Mouna 1998 : 85 Séguy 1930a , MA , Aïn Leuh (1400–1500 m); Mouna 1998 Physocephalini Physocephala Schiner, 1861 Physocephala chrysorrhoea (Meigen, 1824) Séguy 1930a , AP , Sidi Bettache; Mouna 1998 ; El Hawagry et al. 2021 Physocephala laticincta (Brullé, 1832) Séguy 1930a , MA , Aïn Leuh (1400–1500 m); Bei-Bienko and Steyskal 1989 ; Mouna 1998 Physocephala maculigera Kröber, 1915 Séguy 1941d , AA , Agadir; Mouna 1998 Physocephala nigra (De Geer, 1776) Séguy 1930a , AP , Sidi Bettache Physocephala pusilla (Meigen, 1804) Séguy 1928d ; Séguy 1930a , MA , Meknès, Ras el Ksar (1900 m), HA , Asni; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Aoulouz, 2 km NW of Tissint; El Hawagry et al. 2021 Physocephala rufipes (Fabricius, 1781) Mouna 1998 ; AP (Tagulet (Essaouira)) – MISR Physocephala vittata (Fabricius, 1794) Séguy 1928d ; Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès, Forêt Zaers; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Aoersi (15 km NE of Agadir), AA , Oued near beach (19 km W of Tiznit); El Hawagry et al. 2021 ; HA – MISR Pseudophysocephala Kröber, 1940 Pseudophysocephala bouvieri (Séguy, 1936) = Conops bouvieri Séguy, in Séguy 1936a : 299 Séguy 1936a , MA , Meknès (550 m); Sidney 2001 , MA , Meknès Dalmanninae Dalmannini Dalmannia Robineau-Desvoidy, 1830 Dalmannia aculeata (Linnaeus, 1761) Séguy 1928d ; Séguy 1930a , MA , Aïn Leuh, Meknès; Mouna 1998 – MISR Dalmannia dorsalis (Fabricius, 1794) = Dalmannia flavescens (Meigen), in Becker and Stein 1913 : 90 Becker and Stein 1913 , Rif , Tanger; Stuke and Kehlmaier 2008 , MA , Fès Myopinae Myopini Melanosoma Robineau-Desvoidy, 1853 Melanosoma bicolor (Meigen, 1824) Séguy 1941d , AA , Agadir (Admine forest); Mouna 1998 Melanosoma mundum Czerny & Strobl, 1909 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Tafingoult (Goundafa, 1500–1600 m), AA , Tenfeht (Souss, 1000–1500 m); Séguy 1953a , HA , Aït Ourir; Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , SE of Awir (10 km NNW of Agadir), Talmakant (80 km NE of Agadir), AA , 19 km W of Tiznit, Massa river (25 km NE of Tiznit), Imitek (30 km WSW of Tata), Issafen (55 km WNW of Tata); Grabener 2017 Myopa Camras, 1953 Myopa buccata (Linnaeus, 1758) Mouna 1998 ; MA (Oulmès) – MISR Myopa dorsalis Fabricius, 1794 Séguy 1930a , MA , Jebel Ahmar (1750 m); Mouna 1998 ; MA (Oulmès); El Hawagry et al. 2021 – MISR Myopa hirsuta Stuke & Clements, 2008 Stuke and Clements 2008 , MA , Azrou, Ifrane Myopa nigrita Wiedemann, 1824 Wiedemann 1824 , 1830 ; Kröber 1916 , 1928 Myopa pellucida Robineau-Desvoidy, 1830 Stuke and Clements 2008 , Rif , Chefchaouen, HA , Ourika Myopa picta Panzer, 1798 Séguy 1930a , AP , Casablanca, MA , Meknès; Mouna 1998 ; El Hawagry et al. 2021 Myopa stigma Meigen, 1824 Becker and Stein 1913 , Rif , Tanger; El Hawagry et al. 2021 Myopa testacea (Linnaeus, 1767) Séguy 1930a , AP , Casablanca, Fouarat, HA , Arround (Skoutana: 2000–2400 m), Tachdirt, Jebel Likount; Mouna 1998 ; AP (Fouarat) – MISR Myopa palliceps (Bigot, 1887) = Myopa minor Strobl, in Clements 2000 : 239 = Myopa vaulogeri Séguy, in Séguy 1930a : 136; Clements 2000 : 236 Séguy 1930a , AP , Casablanca; Villeneuve 1933 ; Mouna 1998 ; Clements 2000 , AP , Casablanca, HA , Marrakech, Ourigane (1000 m), AA , Ammelental (10 km NE of Tafraoute) Thecophora Rondani, 1845 Thecophora atra (Fabricius, 1775) = Occemyia atra Fabricius, in Séguy 1930a : 137; Becker and Stein 1913 : 90 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Sidi Bettache, MA , Meknès, HA , Asni, AA , Taroudant (Souss); Mouna 1998 ; HA (Mouldikht (Marrakech)); El Hawagry et al. 2021 – MISR Thecophora cinerascens (Meigen, 1804) = Thecophora pusilla (Meigen, 1824), in Pârvu et al. 2006 : 276; Popescu-Mirceni 2011 : 35 Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 , MA , Ifrane Sicini Sicus Scopoli, 1763 Sicus ferrugineus (Linnaeus, 1761) Séguy 1930a , MA , Aïn Leuh; Zimina 1975 ; Mouna 1998 ; MA (Tizi-n'Ifrah, Guisser (1400 m)) – MISR Zodioninae Zodionini Zodion Latreille, 1797 Zodion cinereum (Fabricius, 1794) Séguy 1930a , MA , Meknès, HA , Around (Skoutana); Mouna 1998 ; Grabener 2017 Zodion erythrurum Rondani, 1865 Séguy 1930a , AP , Casablanca, HA , Dar Caïd M'Tougui; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Tamzergoute (10 km N of Agadir); El Hawagry et al. 2021 CONOPIDAE 41 K. Kettani Number of species: 34 . Expected: 40 Faunistic knowledge of the family in Morocco: good Conopinae Conopini Conops Linnaeus, 1758 Conops aegyptiacus (Rondani, 1850) Kröber 1915 , 1928 Conops ceriaeformis Meigen, 1804 = Conops acuticornis Loew, 1847, in Becker and Stein 1913 : 89 Becker and Stein 1913 , Rif , Tanger; Kröber 1924 , 1928 , Rif , Tanger Conops djanetianus Séguy, 1938 Mouna 1998 : 85 Conops elegans Meigen, 1824 = Conops semifumosus Adams, in Séguy 1934b : 162 = Conops ruficornis Becker, 1913, in Becker and Stein 1913 : 89; Kröber 1924 : 69 Becker and Stein 1913 , Rif , Tanger; Kröber 1924 , 1927 , AP , Casablanca; Séguy 1934b , Rif , Tanger; Stuke 2016 ; El Hawagry et al. 2021 Conops nubeculipennis Bezzi, 1901 = Conops atrogonius Séguy, 1930, in Séguy 1930a : 134; Séguy 1953a : 85; Mouna 1998 : 85 Séguy 1930a , AP , Rabat, Mogador; Séguy 1953a , MA , Dayat Ifrah; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , 1.5 km S of Tissint, 14 km NW of Icht; Stuke 2016 ; El Hawagry et al. 2021 Conops theryi Séguy, 1928 Séguy 1928d ; Séguy 1930a , AP , Rabat, Casablanca; Mouna 1998 Conops tifedarius Séguy, 1928 Séguy 1928d ; Séguy 1930a , AP , Rabat; Mouna 1998 Leopoldius Rondani, 1843 Leopoldius coronatus (Rondani, 1857) = Brachyglossum coronatum Rondani, in Séguy 1930a : 132; Mouna 1998 : 85 Séguy 1930a , MA , Aïn Leuh (1400–1500 m); Mouna 1998 Physocephalini Physocephala Schiner, 1861 Physocephala chrysorrhoea (Meigen, 1824) Séguy 1930a , AP , Sidi Bettache; Mouna 1998 ; El Hawagry et al. 2021 Physocephala laticincta (Brullé, 1832) Séguy 1930a , MA , Aïn Leuh (1400–1500 m); Bei-Bienko and Steyskal 1989 ; Mouna 1998 Physocephala maculigera Kröber, 1915 Séguy 1941d , AA , Agadir; Mouna 1998 Physocephala nigra (De Geer, 1776) Séguy 1930a , AP , Sidi Bettache Physocephala pusilla (Meigen, 1804) Séguy 1928d ; Séguy 1930a , MA , Meknès, Ras el Ksar (1900 m), HA , Asni; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Aoulouz, 2 km NW of Tissint; El Hawagry et al. 2021 Physocephala rufipes (Fabricius, 1781) Mouna 1998 ; AP (Tagulet (Essaouira)) – MISR Physocephala vittata (Fabricius, 1794) Séguy 1928d ; Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès, Forêt Zaers; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Aoersi (15 km NE of Agadir), AA , Oued near beach (19 km W of Tiznit); El Hawagry et al. 2021 ; HA – MISR Pseudophysocephala Kröber, 1940 Pseudophysocephala bouvieri (Séguy, 1936) = Conops bouvieri Séguy, in Séguy 1936a : 299 Séguy 1936a , MA , Meknès (550 m); Sidney 2001 , MA , Meknès Dalmanninae Dalmannini Dalmannia Robineau-Desvoidy, 1830 Dalmannia aculeata (Linnaeus, 1761) Séguy 1928d ; Séguy 1930a , MA , Aïn Leuh, Meknès; Mouna 1998 – MISR Dalmannia dorsalis (Fabricius, 1794) = Dalmannia flavescens (Meigen), in Becker and Stein 1913 : 90 Becker and Stein 1913 , Rif , Tanger; Stuke and Kehlmaier 2008 , MA , Fès Myopinae Myopini Melanosoma Robineau-Desvoidy, 1853 Melanosoma bicolor (Meigen, 1824) Séguy 1941d , AA , Agadir (Admine forest); Mouna 1998 Melanosoma mundum Czerny & Strobl, 1909 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Tafingoult (Goundafa, 1500–1600 m), AA , Tenfeht (Souss, 1000–1500 m); Séguy 1953a , HA , Aït Ourir; Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , SE of Awir (10 km NNW of Agadir), Talmakant (80 km NE of Agadir), AA , 19 km W of Tiznit, Massa river (25 km NE of Tiznit), Imitek (30 km WSW of Tata), Issafen (55 km WNW of Tata); Grabener 2017 Myopa Camras, 1953 Myopa buccata (Linnaeus, 1758) Mouna 1998 ; MA (Oulmès) – MISR Myopa dorsalis Fabricius, 1794 Séguy 1930a , MA , Jebel Ahmar (1750 m); Mouna 1998 ; MA (Oulmès); El Hawagry et al. 2021 – MISR Myopa hirsuta Stuke & Clements, 2008 Stuke and Clements 2008 , MA , Azrou, Ifrane Myopa nigrita Wiedemann, 1824 Wiedemann 1824 , 1830 ; Kröber 1916 , 1928 Myopa pellucida Robineau-Desvoidy, 1830 Stuke and Clements 2008 , Rif , Chefchaouen, HA , Ourika Myopa picta Panzer, 1798 Séguy 1930a , AP , Casablanca, MA , Meknès; Mouna 1998 ; El Hawagry et al. 2021 Myopa stigma Meigen, 1824 Becker and Stein 1913 , Rif , Tanger; El Hawagry et al. 2021 Myopa testacea (Linnaeus, 1767) Séguy 1930a , AP , Casablanca, Fouarat, HA , Arround (Skoutana: 2000–2400 m), Tachdirt, Jebel Likount; Mouna 1998 ; AP (Fouarat) – MISR Myopa palliceps (Bigot, 1887) = Myopa minor Strobl, in Clements 2000 : 239 = Myopa vaulogeri Séguy, in Séguy 1930a : 136; Clements 2000 : 236 Séguy 1930a , AP , Casablanca; Villeneuve 1933 ; Mouna 1998 ; Clements 2000 , AP , Casablanca, HA , Marrakech, Ourigane (1000 m), AA , Ammelental (10 km NE of Tafraoute) Thecophora Rondani, 1845 Thecophora atra (Fabricius, 1775) = Occemyia atra Fabricius, in Séguy 1930a : 137; Becker and Stein 1913 : 90 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Sidi Bettache, MA , Meknès, HA , Asni, AA , Taroudant (Souss); Mouna 1998 ; HA (Mouldikht (Marrakech)); El Hawagry et al. 2021 – MISR Thecophora cinerascens (Meigen, 1804) = Thecophora pusilla (Meigen, 1824), in Pârvu et al. 2006 : 276; Popescu-Mirceni 2011 : 35 Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 , MA , Ifrane Sicini Sicus Scopoli, 1763 Sicus ferrugineus (Linnaeus, 1761) Séguy 1930a , MA , Aïn Leuh; Zimina 1975 ; Mouna 1998 ; MA (Tizi-n'Ifrah, Guisser (1400 m)) – MISR Zodioninae Zodionini Zodion Latreille, 1797 Zodion cinereum (Fabricius, 1794) Séguy 1930a , MA , Meknès, HA , Around (Skoutana); Mouna 1998 ; Grabener 2017 Zodion erythrurum Rondani, 1865 Séguy 1930a , AP , Casablanca, HA , Dar Caïd M'Tougui; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Tamzergoute (10 km N of Agadir); El Hawagry et al. 2021 Conopinae Conopini Conops Linnaeus, 1758 Conops aegyptiacus (Rondani, 1850) Kröber 1915 , 1928 Conops ceriaeformis Meigen, 1804 = Conops acuticornis Loew, 1847, in Becker and Stein 1913 : 89 Becker and Stein 1913 , Rif , Tanger; Kröber 1924 , 1928 , Rif , Tanger Conops djanetianus Séguy, 1938 Mouna 1998 : 85 Conops elegans Meigen, 1824 = Conops semifumosus Adams, in Séguy 1934b : 162 = Conops ruficornis Becker, 1913, in Becker and Stein 1913 : 89; Kröber 1924 : 69 Becker and Stein 1913 , Rif , Tanger; Kröber 1924 , 1927 , AP , Casablanca; Séguy 1934b , Rif , Tanger; Stuke 2016 ; El Hawagry et al. 2021 Conops nubeculipennis Bezzi, 1901 = Conops atrogonius Séguy, 1930, in Séguy 1930a : 134; Séguy 1953a : 85; Mouna 1998 : 85 Séguy 1930a , AP , Rabat, Mogador; Séguy 1953a , MA , Dayat Ifrah; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , 1.5 km S of Tissint, 14 km NW of Icht; Stuke 2016 ; El Hawagry et al. 2021 Conops theryi Séguy, 1928 Séguy 1928d ; Séguy 1930a , AP , Rabat, Casablanca; Mouna 1998 Conops tifedarius Séguy, 1928 Séguy 1928d ; Séguy 1930a , AP , Rabat; Mouna 1998 Leopoldius Rondani, 1843 Leopoldius coronatus (Rondani, 1857) = Brachyglossum coronatum Rondani, in Séguy 1930a : 132; Mouna 1998 : 85 Séguy 1930a , MA , Aïn Leuh (1400–1500 m); Mouna 1998 Physocephalini Physocephala Schiner, 1861 Physocephala chrysorrhoea (Meigen, 1824) Séguy 1930a , AP , Sidi Bettache; Mouna 1998 ; El Hawagry et al. 2021 Physocephala laticincta (Brullé, 1832) Séguy 1930a , MA , Aïn Leuh (1400–1500 m); Bei-Bienko and Steyskal 1989 ; Mouna 1998 Physocephala maculigera Kröber, 1915 Séguy 1941d , AA , Agadir; Mouna 1998 Physocephala nigra (De Geer, 1776) Séguy 1930a , AP , Sidi Bettache Physocephala pusilla (Meigen, 1804) Séguy 1928d ; Séguy 1930a , MA , Meknès, Ras el Ksar (1900 m), HA , Asni; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Aoulouz, 2 km NW of Tissint; El Hawagry et al. 2021 Physocephala rufipes (Fabricius, 1781) Mouna 1998 ; AP (Tagulet (Essaouira)) – MISR Physocephala vittata (Fabricius, 1794) Séguy 1928d ; Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès, Forêt Zaers; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Aoersi (15 km NE of Agadir), AA , Oued near beach (19 km W of Tiznit); El Hawagry et al. 2021 ; HA – MISR Pseudophysocephala Kröber, 1940 Pseudophysocephala bouvieri (Séguy, 1936) = Conops bouvieri Séguy, in Séguy 1936a : 299 Séguy 1936a , MA , Meknès (550 m); Sidney 2001 , MA , Meknès Dalmanninae Dalmannini Dalmannia Robineau-Desvoidy, 1830 Dalmannia aculeata (Linnaeus, 1761) Séguy 1928d ; Séguy 1930a , MA , Aïn Leuh, Meknès; Mouna 1998 – MISR Dalmannia dorsalis (Fabricius, 1794) = Dalmannia flavescens (Meigen), in Becker and Stein 1913 : 90 Becker and Stein 1913 , Rif , Tanger; Stuke and Kehlmaier 2008 , MA , Fès Myopinae Myopini Melanosoma Robineau-Desvoidy, 1853 Melanosoma bicolor (Meigen, 1824) Séguy 1941d , AA , Agadir (Admine forest); Mouna 1998 Melanosoma mundum Czerny & Strobl, 1909 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Tafingoult (Goundafa, 1500–1600 m), AA , Tenfeht (Souss, 1000–1500 m); Séguy 1953a , HA , Aït Ourir; Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , SE of Awir (10 km NNW of Agadir), Talmakant (80 km NE of Agadir), AA , 19 km W of Tiznit, Massa river (25 km NE of Tiznit), Imitek (30 km WSW of Tata), Issafen (55 km WNW of Tata); Grabener 2017 Myopa Camras, 1953 Myopa buccata (Linnaeus, 1758) Mouna 1998 ; MA (Oulmès) – MISR Myopa dorsalis Fabricius, 1794 Séguy 1930a , MA , Jebel Ahmar (1750 m); Mouna 1998 ; MA (Oulmès); El Hawagry et al. 2021 – MISR Myopa hirsuta Stuke & Clements, 2008 Stuke and Clements 2008 , MA , Azrou, Ifrane Myopa nigrita Wiedemann, 1824 Wiedemann 1824 , 1830 ; Kröber 1916 , 1928 Myopa pellucida Robineau-Desvoidy, 1830 Stuke and Clements 2008 , Rif , Chefchaouen, HA , Ourika Myopa picta Panzer, 1798 Séguy 1930a , AP , Casablanca, MA , Meknès; Mouna 1998 ; El Hawagry et al. 2021 Myopa stigma Meigen, 1824 Becker and Stein 1913 , Rif , Tanger; El Hawagry et al. 2021 Myopa testacea (Linnaeus, 1767) Séguy 1930a , AP , Casablanca, Fouarat, HA , Arround (Skoutana: 2000–2400 m), Tachdirt, Jebel Likount; Mouna 1998 ; AP (Fouarat) – MISR Myopa palliceps (Bigot, 1887) = Myopa minor Strobl, in Clements 2000 : 239 = Myopa vaulogeri Séguy, in Séguy 1930a : 136; Clements 2000 : 236 Séguy 1930a , AP , Casablanca; Villeneuve 1933 ; Mouna 1998 ; Clements 2000 , AP , Casablanca, HA , Marrakech, Ourigane (1000 m), AA , Ammelental (10 km NE of Tafraoute) Thecophora Rondani, 1845 Thecophora atra (Fabricius, 1775) = Occemyia atra Fabricius, in Séguy 1930a : 137; Becker and Stein 1913 : 90 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , AP , Sidi Bettache, MA , Meknès, HA , Asni, AA , Taroudant (Souss); Mouna 1998 ; HA (Mouldikht (Marrakech)); El Hawagry et al. 2021 – MISR Thecophora cinerascens (Meigen, 1804) = Thecophora pusilla (Meigen, 1824), in Pârvu et al. 2006 : 276; Popescu-Mirceni 2011 : 35 Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 , MA , Ifrane Sicini Sicus Scopoli, 1763 Sicus ferrugineus (Linnaeus, 1761) Séguy 1930a , MA , Aïn Leuh; Zimina 1975 ; Mouna 1998 ; MA (Tizi-n'Ifrah, Guisser (1400 m)) – MISR Zodioninae Zodionini Zodion Latreille, 1797 Zodion cinereum (Fabricius, 1794) Séguy 1930a , MA , Meknès, HA , Around (Skoutana); Mouna 1998 ; Grabener 2017 Zodion erythrurum Rondani, 1865 Séguy 1930a , AP , Casablanca, HA , Dar Caïd M'Tougui; Mouna 1998 ; Stuke and Schmid-Egger 2015 , AA , Tamzergoute (10 km N of Agadir); El Hawagry et al. 2021 Nerioidea MICROPEZIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Micropeza Meigen, 1803 Micropeza kettaniae Ebejer, 2019 Ebejer 2019b , Rif , Oued Kbir (Béni Ratene, 157 m), Dayat Tazia (Tazia, 733 m) – MISR , NHMUK MICROPEZIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Micropeza Meigen, 1803 Micropeza kettaniae Ebejer, 2019 Ebejer 2019b , Rif , Oued Kbir (Béni Ratene, 157 m), Dayat Tazia (Tazia, 733 m) – MISR , NHMUK Tanypezoidea PSILIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 5 Faunistic knowledge of the family in Morocco: poor Chamaepsila Hendel, 1917 Chamaepsila nigricornis (Meigen, 1826) Ebejer et al. 2019 , Rif , Tétouan, Onsar Lile (349 m), Aïn Tissemlal (Azilane, 1255 m), MA , 17 km SW of Midelt (1940 m), Lac Aguelmane Afennourir (30 km SW of Azrou, 2050 m) PSILIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 5 Faunistic knowledge of the family in Morocco: poor Chamaepsila Hendel, 1917 Chamaepsila nigricornis (Meigen, 1826) Ebejer et al. 2019 , Rif , Tétouan, Onsar Lile (349 m), Aïn Tissemlal (Azilane, 1255 m), MA , 17 km SW of Midelt (1940 m), Lac Aguelmane Afennourir (30 km SW of Azrou, 2050 m) Tephritoidea LONCHAEIDAE K. Kettani, I. MacGowan Number of species: 5 . Expected: 30 Faunistic knowledge of the family in Morocco: poor Dasiopinae Dasiops Rondani, 1856 Dasiops latifrons (Meigen, 1826) Séguy 1934a ; Mouna 1998 ; MacGowan and Freidberg 2008 ; AP (Rabat) – MISR ; Rif (Tanger), AP (Rabat) – MHNP Lonchaeinae Lamprolonchaea Bezzi, 1920 Lamprolonchaea smaragdi (Walker, 1849) = Lonchea aurea Macquart, 1851, in Séguy 1953a : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1934a ; Séguy 1953a , AP , Rabat, MA , Fès; Rungs 1952 , HA , Arganier; Mouna 1998 Lonchaea Fallén, 1820 Lonchaea tarsata Fallén, 1820 MacGowan and Freidberg 2008 Lonchaea sp. = Recorded as Lonchaea laticornis Meigen, 1826 but almost certainly not this species Becker and Stein 1913 , Rif , Tanger Silba Macquart, 1851 Silba adipata McAlpine, 1956 = Lonchaea aristella Becker, 1903, in Séguy 1934b : 162 Séguy 1934b , AP , Rabat; MacGowan and Freidberg 2008 ; AP (Rabat) – MNHN PALLOPTERIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Palloptera Fallén, 1820 Palloptera ustulata (Fallén, 1820) Ebejer et al. 2019 , MA , Zaouia d'Ifrane (Ifrane, 1603 m) PIOPHILIDAE K. Kettani, M.J. Ebejer Number of species: 3 . Expected: 6 Faunistic knowledge of the family in Morocco: poor Piophilinae Mycetaulus Loew, 1845 Mycetaulus hispanicus Duda, 1927 Mouna 1998 ; Carles-Tolrá 2002 Piophila Fallén, 1810 Piophila casei (Linnaeus, 1758) Séguy 1930a , Rif , Tanger, AP , Rabat, MA , Meknès; Mouna 1998 ; Rif (Tanger), AP (Rabat), MA (Meknès) – MISR Prochyliza Walker, 1849 Prochyliza nigrimana (Meigen 1826) Ebejer et al. 2019 , Rif , Aïn Tissemlal (Azilane, 1255 m) PLATYSTOMATIDAE K. Kettani, G.V. Popov Number of species: 4 . Expected: 4 Faunistic knowledge of the family in Morocco: moderate Platystomatinae Platystoma Meigen, 1803 Platystoma idia Séguy, 1934 42 Séguy 1934a , AP , Aïn Sferguila (Forêt Zaers); Hennig 1945 , AP , Aïn Sferguila (Forêt Zaers); Soós 1984a Platystoma meridionale Hendel, 1913 = Platystoma seminationis (Fabricius), in Becker 1907 : 385 Becker 1907 ; Hendel 1913 , AP , Mogador; Hennig 1945 , AP , Mogador; Soós 1984a , AP , Mogador Rivellia Robineau-Desvoidy, 1830 Rivellia hispanica Lyneborg, 1969 Ebejer et al. 2019 , EM , Tafoughalt Rivellia syngenesiae (Fabricius, 1781) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; MA (Oulmès, Meknès) – MISR TEPHRITIDAE K. Kettani, A.L. Norrbom Number of species: 69 . Expected: 75 Faunistic knowledge of the family in Morocco: moderate Dacinae Ceratitidini Capparimyia Bezzi, 1920 Capparimyia savastanii (Martelli, 1911) = Capparimyia savastanii (Martelli), in Séguy 1953a : 85 Séguy 1953a , AA , Tiznit (on Capparis spinosa ); El Harym and Belqat 2017 Ceratitis MacLeay, 1829 Ceratitis capitata (Wiedemann, 1824) Becker and Stein 1913 , Rif , Tanger; Vayssière 1920 , AP , Rabat; Séguy 1930a (common in all of Morocco); Rungs 1952 , HA , Arganier; Harris et al. 1980 ; Mouna 1998 ; De Meyer 2000 , AP , Ouadj-Ouli-Mohamed, env. Settat, Insgane; Vidal et al. 2008 ; Aboussaid et al. 2009 ; Koçak and Kemal 2010 ; El Harym and Belqat 2017 , Rif , Kitane, El Haouta, MA , Sensla, AA , Environs Massa, Oued Massa, Douar Sidi Abou, Douar Tighrimt, Douar Zaouia; Elaini and Mazih 2018 ; Elaini et al. 2019 , HA , Arganeraie; AP (Safi) – MHNNR Dacini Bactrocera Macquart, 1835 Bactrocera oleae (Rossi, 1790) = Dacus oleae Rossi, in Becker and Stein 1913 : 94, Vayssière 1920 : 256 = Dacus oleae Gmelin, in Séguy 1930a : 168 Becker and Stein 1913 , Rif , Tanger; Vayssière 1920 , EM , Oujda; Séguy 1930a ; Mouna 1998 ; Savio 2011 , EM , Oujda; El Harym and Belqat 2017 , Rif , El Haouta, Oued Maâza, Cascade Chrafate, Koudiat El Aouinate, Lâazaba, Dhar Sbagh Mâasra, El Hajria, MA , Sensla, AA , route Bab El Khemis Dacus Fabricius, 1805 Dacus frontalis (Becker, 1922) El Harym and Belqat 2017 , AA , Oued Foum Ziguid (Douar Ouaiftoute), Oued Draa (Ikhf Mezrou), Isdaoun Dacus longistylus (Wiedemann, 1830) El Harym and Belqat 2017 , AA , Oued Tata, Douar Tighrimt, Oued Foum Ziguid (Douar Ouaiftoute) Tephritinae Dithrycini Oedaspis Loew, 1862 Oedaspis daphnea Séguy, 1930 Séguy 1930a , AP , El Mers (Rabat); Foote 1984 ; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 Oedaspis multifasciata (Loew, 1850) = Oedaspis multifasciatus (Loew), in Séguy 1953a : 85 Séguy 1953a , EM , Itzer (Haute Moulouya); El Harym and Belqat 2017 Oedaspis trotteriana Bezzi, 1913 Soós 1984b ; Ribera and Blasco-Zumeta 1998; Norrbom et al. 1999 ; El Harym and Belqat 2017 Myopitini Myopites Blot, 1827 Myopites cypriacus Hering 1938 El Harym et al. 2020 , Rif , Douar Halila, Dam Nakhla, Marabout Sidi Bou Hadjel Myopites inulaedyssentericae Blot, 1827 43 = Myopites apicatus Freidberg, 1980, in El Harym and Belqat 2017 : 140 El Harym and Belqat 2017 , Rif , affluent Tarmast, Aïn Afersiw Myopites longirostris (Loew, 1846) El Harym et al. 2020 , Rif , Oued Tahaddart, Douar Kouf, Mkhinak Myopites stylatus Fabricius, 1794 El Harym and Belqat 2017 , Rif , affluent Tarmast, Oued El Hamma, El Haouta Myopites variofasciatus Becker, 1903 = Myopites variofasciata Becker, in Séguy 1941a : 32 Séguy 1941a , HA , Imi-n'Ouaka (1500 m), Mouna 1998 Urophora Robineau-Desvoidy, 1830 Urophora congrua Loew, 1862 44 = Euribia congrua Loew, in Séguy 1941d : 14 Séguy 1941d , AA , Taroudant; Mouna 1998 ; El Harym and Belqat 2017 Urophora jaculata Rondani, 1870 = Urophora mauritanica Macquart, in El Harym and Belqat 2017 : 149 (misidentification) Urophora mauritanica Macquart, 1851 = Euribia algira Macquart, in Séguy 1930a : 169 = Urophora algira Macquart, in Séguy 1934a : 98, Mouna 1998 : 87 = Urophora macrura Loew, in Séguy 1934a : 100, Mouna 1998 : 87 Séguy 1930a , HA , Imi-N'Takandout, Dar Kaid M'Tougui; Séguy 1934a ; White and Korneyev 1989 , HA , Ito; Mouna 1998 ; Norrbom et al. 1999 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013b Urophora quadrifasciata algerica (Hering, 1941) = Euribia quadrifasciata Meigen, in Séguy 1930a : 169 Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 El Harym et al. 2020 , MA , Douar Oulad Abdoune, Mlakite, Tirra, Douar Oulad Amar, Tihli, Douar Oulad Amar Urophora solstitialis (Linnaeus, 1758) = Euribia solstitialis Linnaeus, in Séguy 1930a : 169 Séguy 1930a , HA , Haute Réghaya; Mouna 1998 ; El Harym and Belqat 2017 Noeetini Ensina Robineau-Desvoidy, 1830 Ensina sonchi (Linnaeus, 1767) El Harym and Belqat 2017 , Rif , Ksar Rimal, Douar Tizga, Oued Kbir, MA , Sensla, AA , Oued Massa (Pont Aghbalou), Centre Sidi Ouassay, Aïn Boharroch, Atbane, Oued Tamanarne, Oued Draa (Tahtah), Jnane Makadir, Kasbah Asma, Ait Aissa O Brahim, Oued Ziz (Pont Errachidia), Oued Ouarzazate; AA (Tafraout (Al Ourir, 12 km E)) – MHNNR Hypenidium Loew, 1862 Hypenidium graecum Loew, 1862 = Stephanaciura bipartita Séguy, in Séguy 1930a : 171 Séguy 1930a , MA , Tiffert (2000–2200 m); Villeneuve 1933 ; Soós 1984b ; Mouna 1998 : 87; Norrbom et al. 1999 ; El Harym and Belqat 2017 Tephrellini Aciura Robineau-Desvoidy Aciura coryli (Rossi, 1790) = Aciura Powelli Séguy, in Séguy 1930a : 170, Séguy 1953a : 85, Mouna 1998 : 87 Séguy 1930a , MA , Azrou (larvae from Phlomis crinita Cav.); Séguy 1953a , AP , Korifla; Mouna 1998 ; Norrbom et al. 1999 , MA , Azrou; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Arhil Oxyaciura Hendel, 1927 Oxyaciura tibialis (Robineau-Desvoidy, 1830) = Aciura tibialis Robineau-Desvoidy, in Becker and Stein 1913 : 94 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Sker; Séguy 1934a ; Mouna 1998 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013b ; El Harym and Belqat 2017 , Rif , Dayat El Birdiyel, Oued Azila, maison forestière; Oued Maâza (Tarik El Ouasâa); AP (Tamri, 10 km S) – MHNNR Sphaeniscus Becker, 1908 Sphaeniscus filiolus (Loew, 1869) = Spheniscomyia filiola Loew, in Séguy 1930a : 170; Mouna 1998 : 87 = Spheniscomyia aegyptiaca Efflatoun, in Séguy 1949a : 157; Mouna 1998 : 87 Séguy 1930a ; Séguy 1949a , SA , Guelmim; Mouna 1998 ; El Harym and Belqat 2017 , Rif , affluent Tarmast, Oued Maâza (Tarik El Ouasâa) Tephritini Acanthiophilus Becker, 1908 Acanthiophilus helianthi (Rossi, 1790) = Tephritis eluta Meigen, in Becker and Stein 1913 : 94 = Orellia eluta Meigen, in Mouna 1998 : 87 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger (Sarf, route Spartel), MA , Tizi s'Tkrine (Jebel Ahmar, 1700 m); Séguy 1949a , AA , Alnif, Foum-el-Hassan, SA , Guelmim; Mouna 1998 ; Koçak and Kemal 2013b ; El Harym and Belqat 2017 , Rif , Oued Zinat, Ksar Rimal, Oued Dardara, affluent Tarmast, Dayat El Birdiyel, Oued Azila, Dayat Jebel Zemzem, Oued Boumarouil, Douar Abou Boubnar (Mara­bout Sidi Gile), El Haouta, Oued Maâza (Tarik El Ouasâa), Douar Tizga, Dayat Afrate, Oued Mezine, Aïn El Malaâb, Oued Jnane Niche, MA , Oued Oum-er-Rbia, AA , Centre Sidi Ouassay, Avant Sidi Bin­zarne, route Bab El Khemis, airport Sidi Ifni, Oued Tisla, Oued Sayad, Oued Tamanarne, Oued Foum Ziguid (Douar Ouaiftoute), Jnane Makadir, Douar Rggaga, Oued Tinghir, Oued Ouarzazate; AP (Essaouira (Cap Hadid), Tamri, 10 km S) – MHNNR Campiglossa Rondani, 1870 Campiglossa martii (Becker, 1908) El Harym and Belqat 2017 , Rif , Oued Kbir, AA , Centre Sidi Ouassay Campiglossa producta (Loew, 1844) = Oxyna tessellata Loew, in Becker and Stein 1913 : 94 (misidentification) = Paroxyna tessellata (Loew), in Séguy 1930a : 174; Mouna 1998 : 87; Koçak and Kemal 2013b : 38 (misidentification) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Mogador, HA , Telouet Glaoua; Séguy 1934a ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Koçak and Kemal 2013b , HA ; El Harym and Belqat 2017 , Rif , Oued Al Mizzine, Aïn El Malaâb, Dayat El Hajjami Campiglossa sororcula (Wiedemann, 1830) = Dioxyna sororcula (Wiedemann), in El Harym and Belqat 2017 : 155 El Harym and Belqat 2017 , Rif , Ksar Rimal, Oued Jnane Niche, Oued Halila, Oued Zarka, Oued Martil, Oued Amsa, Oued Sahel, Dayat Jebel Zemzem, Oued Maggou, Dhar Sbagh Mâasra, Douar Kitane Capitites Foote & Freidberg, 1981 Capitites augur (Frauenfeld, 1857) = Trypanea augur (Frauenfeld), in Séguy 1930a : 176; Mouna 1998 : 87 Séguy 1930a , MA , Forêt Azrou, Tizi-s'Tkrine (Jebel Ahmar, 1700 m), HA , Tenfecht, AA , Souss; Mouna 1998 ; Pârvu et al. 2006 , AA , Ouarzazate, Lac Edehby; Popescu-Mirceni 2011 , MA , Tizi-s'Tkrine, Azrou, HA , Tenfecht, AA , Ouarzazate Capitites ramulosa (Loew, 1844) = Acanthiophilus ramulosus Loew, in Séguy 1930a : 177; Séguy 1941d : 15; Séguy 1949a : 157; Mouna 1998 : 87 Séguy 1930a , MA , Timelilt, HA , Tizi-n'Test (Jebel Imdress, 2000–2450 m); Séguy 1941d , AA , Taroudant; Séguy 1949a , AA , Foum-el-Hassan, Akka, Agdz, Alnif; Mouna 1998 ; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Amsa, Koudiat Taifour Desmella Munro, 1957 Desmella rostellata (Séguy, 1941) = Paroxyna rostellata Séguy, in Séguy 1941d : 14; Mouna 1998 : 87 Séguy 1941d , AP , Agadir; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 , AP , Agadir; El Harym and Belqat 2017 Euaresta Loew, 1873 Euaresta bullans (Wiedemann, 1830) Herman and Dirlbek 2006 , AA , Tiznit environs, Sidi Moussa d'Aglou; El Harym and Belqat 2017 , AA , Msidira Goniurellia Hendel, 1927 Goniurellia longicauda Freidberg, 1980 Freidberg 1980 , MA , Tizi-s'Tkrine (1700 m), Azrou, AA , Taroudant; Soós 1984b ; Freidberg and Kugler 1989 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 , AA , airport Sidi Ifni, Oued Tisla, Oued Tamanarne, Douar Zaouiet, Oued Tata, Douar Tighrimt, Ksibat Elhdeb, Oued Ziz (Pont Errachidia), Oued Ouarzazate Goniurellia persignata Freidberg, 1980 Freidberg 1980 , EM , Defilia, near Figuig; Soós 1984b ; Freidberg and Kugler 1989 ; Norrbom et al. 1999 ; Herman and Dirlbek 2006 , AA , Tiffoultoute (1146 m); El Harym and Belqat 2017 , Rif , Dhar Sbagh Mâasra, AA , Douar Zaouiet, Oued Ouarzazate Spathulina Rondani, 1856 Spathulina sicula Rondani, 1856 = Spathulina tristis Loew, in Séguy 1930a : 174; Mouna 1998 : 87 Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Barrage Smir Sphenella Robineau-Desvoidy, 1830 Sphenella marginata (Fallén, 1814) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Oued Judios (Tanger); Mouna 1998 ; Cassar et al. 2005 , Rif , lagoon Smir; Koçak and Kemal 2013b , HA ; El Harym and Belqat 2017 , Rif , affluent Tarmast, Dayat Jebel Zemzem, El Malaâb, Oued Maâza (Âachira), Dayat Aïn Jdioui, Oued Maggou, Dayat Afrate Tephritis Latreille, 1804 Tephritis carmen Hering, 1937 El Harym et al. 2020 , Rif , forest house of National Park of Talassemtane Tephritis dioscurea (Loew, 1856) Séguy 1930a , MA , El Hajeb; Mouna 1998 ; El Harym and Belqat 2017 Tephritis divisa (Rondani, 1871) El Harym and Belqat 2017 , Rif , Dayat Amsemlil Tephritis formosa (Loew, 1844) Séguy 1930a , HA , Asni; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Oued Abou Bnar, Oued Sidi Ben Saâda, Oued Achekrade, Oued El Kanar Tephritis leontodontis (De Geer, 1776) Séguy 1930a (All North Africa); Mouna 1998 ; El Harym and Belqat 2017 Tephritis matricariae (Loew, 1844) Séguy 1930a (all North Africa); Mouna 1998 ; El Harym and Belqat 2017 , Rif , affluent Oued Amsemlil, El Haouta, Dayat Jebel Zemzem, Dayat Amsemlil, Oued El Hamma Tephritis nigricauda (Loew, 1856) Séguy 1930a , AP , Berrechid; Séguy 1934a ; Séguy 1941d , AP , Agadir, Berrechid; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Dayat Jebel Zemzem, Oued Maâza (Tarik El Ouasâa), Aïn El Maounzil, Dayat Tazia, Dayat Amsemlil, Douar Tamakoute Tephritis postica (Loew, 1844) Herman and Dirlbek 2006 , MA , Volubilis (358 m); El Harym and Belqat 2017 , AA , Ksibat Elhdeb, Oued Tinghir Tephritis praecox (Loew, 1844) Séguy 1930a , Rif , Oued Judios (Tanger), MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 ; Herman and Dirlbek 2006 , MA , Ifrane, Azrou National Park (1743 m); El Harym and Belqat 2017 , Rif , Dayat El Ânassar, Dayat Amsemlil, affluent Oued Amsemlil, Douar Dacheryène, Douar Taghbaloute, Barrage Nakhla, Oued Sa­hel, Daya Jebel Zemzem, Douar Kitane, Oued El Hamma, Oued Kbir, Aïn el Ma Bared, Aïn El Malaâb, Douar Abou Boubnar (Marabout Sidi Gile), maison forestière, Douar Tizga, Oued Aïn Jdioui (Touaret), Dayat Afrate, Oued Jbara, Aïn El Maounzil, Dayat Tazia, Oued Jnane Niche, Oued Maggou, Aïn Tiouila, Dayat Lemtahane, Lâazaba, Dhar Sbagh Mâasra, El Hajria, Aïn Boharroch, Douar Tamakout, Douar Ouslaf, EM , Oued Béni Ouaklane (Béni Snassen); AP (Essaouira) – MHNNR Tephritis pulchra (Loew, 1844) Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 Tephritis simplex (Loew, 1844) Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Aïn Soualah, Aïn El Maounzil Tephritis stictica Loew, 1862 Séguy 1930a , AP , Rabat; Mouna 1998 ; El Harym and Belqat 2017 Tephritis theryi Séguy, 1930 Séguy 1930a , HA , Marrakech, Asni; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 Tephritis vespertina (Loew, 1844) El Harym and Belqat 2017 , Rif , Dayat Lemtahane, Dhar Sbagh Mâasra Tephritomyia Hendel, 1927 Tephritomyia lauta (Loew, 1869) = Acanthiophilus lauta Loew, in Séguy 1930a : 177; Mouna 1998 : 87 Séguy 1930a , HA , Tachdirt (Imminen, 2400–2600 m); Freidberg and Kugler 1989 ; Mouna 1998 ; Yaran and Kütük 2012 ; Morgulis et al. 2015, HA , Tizi-n'Tichka; El Harym and Belqat 2017 , Rif , Dayat El Birdiyel, Dayat Amsemlil, Lâazaba, AA , Msidira, Oued Ouarzazate Trupanea Schrank, 1795 Trupanea amoena (Frauenfeld, 1857) = Trypanea amoena Frauenfeld, in Séguy 1930a : 176; Mouna 1998 : 87 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Ksar Rimal, Oued Jnane niche, affluent Tarmast, Oued Martil (Tamouda), Oued Amsa, Oued El Hamma, Oued Boumarouil, Oued Sidi Yahia Aârab, Aïn Tiouila, AA , Oued Massa (Pont Aghbalou), Centre Sidi Ouassay, Avant Sidi Binzarne, Oued Tisla, Douar Tighrimt, Oued Draa (Tahtah), Jnane Makadir, Douar Rggaga, Aït Aissa O Brahim, Oued Draa (Ikhf Mezrou), Isdaoun, Ksibat Elhdeb, Oued Tinghir Trupanea guimari (Becker, 1908) El Harym and Belqat 2017 , AA , Centre Sidi Ouassay, Msidi­ra, Jnane Makadir, Aït Aissa O Brahim, Ksibat Elhdeb; Norrbom 2004 : 06600136 – INHS ( AA , 5 km W Ouarzazate) Trupanea stellata (Fuesslin, 1775) Séguy 1930a , MA , Timelilt (1900 m); Séguy 1949a , SA , Guelmim; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Mizoghar, Oued Maâza (Tarik El Ouasâa), Dayat Afrate, Aïn El Malâab, Oued Tkarae, AA , Centre Sidi Ouassay; AA (Taliouine) – MHNNR Terellini Chaetorellia Hendel, 1927 Chaetorellia conjuncta (Becker, 1912) El Harym and Belqat 2017 , AA , Airport Sidi Ifni, Oued Assa, Oued Sayad, Oued Foum Ziguid (Douar Ouaiftoute), Ksibat Elhdeb, Oued Ziz (Pont Errachidia), Oued Ouarzazate Chaetorellia hestia Hering, 1937 = Chaetorellia hexachaeta Loew, in Séguy 1930a : 174 [probably a misidentification] = Orellia hexachaeta Loew, in Séguy 1934a : 135 [misidentification, see White and Macquart 1989: 476]; Mouna 1998 : 87 Séguy 1930a , AP , Mogador; Séguy 1934a ; Mouna 1998 ; El Harym and Belqat 2017 , AA , Centre Sidi Ouassay Chaetorellia succinea (Costa, 1844) El Harym et al. 2020 , MA , Douar Oulad Abdoune Chaetostomella Hendel, 1927 Chaetostomella cylindrica (Robineau-Desvoidy, 1830) El Harym et al. 2020 , Rif , Marabout Douar Halila, Mkhinak, Douar Kitane Terellia Robineau-Desvoidy, 1830 Terellia colon (Meigen, 1826) = Orellia colon (Meigen), in Séguy 1930a : 173 Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 Terellia fuscicornis (Loew, 1844) Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 Terellia longicauda (Meigen, 1838) Séguy 1930a , MA , Aïn Leuh (1200–1400 m), HA , Tizi-n'Test, Goundafa (Jebel Imdress, 2000–2450 m); Séguy1934a ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; El Harym and Belqat 2017 Terellia luteola (Wiedemann, 1830) El Harym et al. 2020 , Rif , Bakrim, Aforidane, Aïn Siyed Terellia oasis (Hering, 1938) El Harym et al. 2020 , Rif , Douar Halila Terellia ptilostemi El Harym et al. 2021 El Harym et al. 2021 , Rif , Douar Chourdane (908 m), Aïn Akorian (1610 m), Aïn Elma Sefli (1345 m), Forest house of the Talassemtane National Park (1674 m) Terellia serratulae (Linnaeus, 1758) = Tephritis pallens Wiedemann, in Wiedemann 1824 : 54 = Trypeta serratula Linnaeus, in Becker and Stein 1913 : 94 = Terellia serratulae Linnaeus, in Séguy 1930a : 173 Wiedemann 1824 , Rif , Tanger; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a (all North Africa); Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 , Rif , Dayat Jebel Zemzem, Oued Maâza (Tarik El Ouasâa) Terellia virens (Loew, 1846) Séguy 1930a ; White 1989 , HA , Jebel Ayachi; Mouna 1998 ; Korneyev et al. 2013 , HA , Tizi-n'Talrhemt; El Harym and Belqat 2017 , AA , airport Sidi Ifni, Oued Ouarzazate Trypetinae Carpomyini Carpomya Costa, 1854 Carpomya incompleta (Becker, 1903) El Harym and Belqat 2017 , AA , Douar Zaouia Euleia Walker, 1835 Euleia heraclei (Linnaeus, 1758) = Acidia heraclei Linnaeus, in Séguy 1953a : 85 Séguy 1953a , MA , Sidi Slimane; Freidberg and Kugler 1989 ; Koçak and Kemal 2010 ; El Harym and Belqat 2017 , Rif , Oued Boumarouil, Aïn El Âakba Larbaâ Euleia marmorea (Fabricius, 1805) 45 = Philophylla flavescens Fabricius, in Séguy 1930a : 170; Mouna 1998 : 87 = Euleia flavescens Fabricius, in Soós 1984b : 88 Séguy 1930a , Rif , Tanger; Zimsen 1964 ; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 , Rif , Tanger; El Harym and Belqat 2017 Trypetini Chetostoma Rondani, 1856 Chetostoma curvinerve Rondani, 1856 El Harym and Belqat 2017 , Rif , Oued Kelaâ, Bab el Karn Acknowledgments We gratefully acknowledge the assistance and cooperation of Valery Korneyev and the late Amnon Freidberg who contributed to the revision of this family. ULIDIIDAE K. Kettani, M.J. Ebejer Number of species: 13 . Expected: 18 Faunistic knowledge of the family in Morocco: good Otitinae Ceroxys Macquart, 1835 Ceroxys urticae Linnaeus, 1758 Ebejer et al. 2019 , AP , Lower Loukous (6 m), Larache (5 m) Dorycera Meigen, 1830 Dorycera griseipennis (Becker, 1907) Soós 1984b Herina Robineau-Desvoidy, 1830 Herina ghilianii Rondani, 1869 Kameneva 2007 , HA , Ansegmir-Tal, W Midelt (1400 m); Ebejer 2015 Herina lacustris (Meigen, 1826) Kameneva 2007 , Rif , Baie de Tanger Herina oscillans (Meigen, 1826) = Herina schlueteri Becker, in Becker and Stein 1913 : 92; Soós 1984b : 56 Becker and Stein 1913 , Rif , Tanger; Soós 1984b ; Kameneva 2007 , Rif , Tanger Melieria Robineau-Desvoidy, 1830 Melieria nigritarsis Becker, 1903 Ebejer et al. 2019 , AA , Merzouga (714 m) Otites Latreille, 1804 Otites tangeriana Becker, 1913 = Otites tangeriana Becker, in Becker and Stein 1913 , 1918: 92 Becker and Stein 1913 , 1918, Rif , Tanger; Soós 1984b , Rif , Tanger Tetanops Fallén, 1820 Tetanops flavescens Macquart, 1835 Séguy 1930a , Rif , Tanger; Mouna 1998 Ulidiinae Physiphora Fallén, 1810 Physiphora alceae (Preyssler, 1791) = Chrysomyza demandata (Fabricius, 1798), in Séguy 1953a : 85; Mouna 1998 : 87 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Goundafa; Séguy 1953a , SA , El Aöun du Draa; Séguy 1949a , AA , Agdz; Mouna 1998 ; Koçak and Kemal 2010 ; Kameneva and Korneyev 2016 , AA , Tizi-n'Bachkoun (1600 m); Rif (M'Diq farm) – MISR ; Rif (Tanger) – MfN ; AP (Casablanca) – ZSSM Physiphora smaragdina (Loew, 1852) Kameneva and Korneyev 2016 , AA , 25 km S Goulmima (100 m) – NHMD Ulidia Meigen, 1826 Ulidia apicalis (Meigen, 1826) Séguy 1930a , MA , Meknès, HA , Skoutana; Séguy 1934a ; Lyneborg 1969 ; Soós 1984b ; Mouna 1998 Ulidia erythrophthalma Meigen, 1826 Becker and Stein 1913 , Rif , Tanger; Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 Ulidia megacephala Loew, 1845 Soós 1984b ; Zaitzev 1984 ; Koçak and Kemal 2010 LONCHAEIDAE K. Kettani, I. MacGowan Number of species: 5 . Expected: 30 Faunistic knowledge of the family in Morocco: poor Dasiopinae Dasiops Rondani, 1856 Dasiops latifrons (Meigen, 1826) Séguy 1934a ; Mouna 1998 ; MacGowan and Freidberg 2008 ; AP (Rabat) – MISR ; Rif (Tanger), AP (Rabat) – MHNP Lonchaeinae Lamprolonchaea Bezzi, 1920 Lamprolonchaea smaragdi (Walker, 1849) = Lonchea aurea Macquart, 1851, in Séguy 1953a : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1934a ; Séguy 1953a , AP , Rabat, MA , Fès; Rungs 1952 , HA , Arganier; Mouna 1998 Lonchaea Fallén, 1820 Lonchaea tarsata Fallén, 1820 MacGowan and Freidberg 2008 Lonchaea sp. = Recorded as Lonchaea laticornis Meigen, 1826 but almost certainly not this species Becker and Stein 1913 , Rif , Tanger Silba Macquart, 1851 Silba adipata McAlpine, 1956 = Lonchaea aristella Becker, 1903, in Séguy 1934b : 162 Séguy 1934b , AP , Rabat; MacGowan and Freidberg 2008 ; AP (Rabat) – MNHN Dasiopinae Dasiops Rondani, 1856 Dasiops latifrons (Meigen, 1826) Séguy 1934a ; Mouna 1998 ; MacGowan and Freidberg 2008 ; AP (Rabat) – MISR ; Rif (Tanger), AP (Rabat) – MHNP Lonchaeinae Lamprolonchaea Bezzi, 1920 Lamprolonchaea smaragdi (Walker, 1849) = Lonchea aurea Macquart, 1851, in Séguy 1953a : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1934a ; Séguy 1953a , AP , Rabat, MA , Fès; Rungs 1952 , HA , Arganier; Mouna 1998 Lonchaea Fallén, 1820 Lonchaea tarsata Fallén, 1820 MacGowan and Freidberg 2008 Lonchaea sp. = Recorded as Lonchaea laticornis Meigen, 1826 but almost certainly not this species Becker and Stein 1913 , Rif , Tanger Silba Macquart, 1851 Silba adipata McAlpine, 1956 = Lonchaea aristella Becker, 1903, in Séguy 1934b : 162 Séguy 1934b , AP , Rabat; MacGowan and Freidberg 2008 ; AP (Rabat) – MNHN PALLOPTERIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Palloptera Fallén, 1820 Palloptera ustulata (Fallén, 1820) Ebejer et al. 2019 , MA , Zaouia d'Ifrane (Ifrane, 1603 m) PIOPHILIDAE K. Kettani, M.J. Ebejer Number of species: 3 . Expected: 6 Faunistic knowledge of the family in Morocco: poor Piophilinae Mycetaulus Loew, 1845 Mycetaulus hispanicus Duda, 1927 Mouna 1998 ; Carles-Tolrá 2002 Piophila Fallén, 1810 Piophila casei (Linnaeus, 1758) Séguy 1930a , Rif , Tanger, AP , Rabat, MA , Meknès; Mouna 1998 ; Rif (Tanger), AP (Rabat), MA (Meknès) – MISR Prochyliza Walker, 1849 Prochyliza nigrimana (Meigen 1826) Ebejer et al. 2019 , Rif , Aïn Tissemlal (Azilane, 1255 m) Piophilinae Mycetaulus Loew, 1845 Mycetaulus hispanicus Duda, 1927 Mouna 1998 ; Carles-Tolrá 2002 Piophila Fallén, 1810 Piophila casei (Linnaeus, 1758) Séguy 1930a , Rif , Tanger, AP , Rabat, MA , Meknès; Mouna 1998 ; Rif (Tanger), AP (Rabat), MA (Meknès) – MISR Prochyliza Walker, 1849 Prochyliza nigrimana (Meigen 1826) Ebejer et al. 2019 , Rif , Aïn Tissemlal (Azilane, 1255 m) PLATYSTOMATIDAE K. Kettani, G.V. Popov Number of species: 4 . Expected: 4 Faunistic knowledge of the family in Morocco: moderate Platystomatinae Platystoma Meigen, 1803 Platystoma idia Séguy, 1934 42 Séguy 1934a , AP , Aïn Sferguila (Forêt Zaers); Hennig 1945 , AP , Aïn Sferguila (Forêt Zaers); Soós 1984a Platystoma meridionale Hendel, 1913 = Platystoma seminationis (Fabricius), in Becker 1907 : 385 Becker 1907 ; Hendel 1913 , AP , Mogador; Hennig 1945 , AP , Mogador; Soós 1984a , AP , Mogador Rivellia Robineau-Desvoidy, 1830 Rivellia hispanica Lyneborg, 1969 Ebejer et al. 2019 , EM , Tafoughalt Rivellia syngenesiae (Fabricius, 1781) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; MA (Oulmès, Meknès) – MISR Platystomatinae Platystoma Meigen, 1803 Platystoma idia Séguy, 1934 42 Séguy 1934a , AP , Aïn Sferguila (Forêt Zaers); Hennig 1945 , AP , Aïn Sferguila (Forêt Zaers); Soós 1984a Platystoma meridionale Hendel, 1913 = Platystoma seminationis (Fabricius), in Becker 1907 : 385 Becker 1907 ; Hendel 1913 , AP , Mogador; Hennig 1945 , AP , Mogador; Soós 1984a , AP , Mogador Rivellia Robineau-Desvoidy, 1830 Rivellia hispanica Lyneborg, 1969 Ebejer et al. 2019 , EM , Tafoughalt Rivellia syngenesiae (Fabricius, 1781) Becker and Stein 1913 , Rif , Tanger; Mouna 1998 ; MA (Oulmès, Meknès) – MISR TEPHRITIDAE K. Kettani, A.L. Norrbom Number of species: 69 . Expected: 75 Faunistic knowledge of the family in Morocco: moderate Dacinae Ceratitidini Capparimyia Bezzi, 1920 Capparimyia savastanii (Martelli, 1911) = Capparimyia savastanii (Martelli), in Séguy 1953a : 85 Séguy 1953a , AA , Tiznit (on Capparis spinosa ); El Harym and Belqat 2017 Ceratitis MacLeay, 1829 Ceratitis capitata (Wiedemann, 1824) Becker and Stein 1913 , Rif , Tanger; Vayssière 1920 , AP , Rabat; Séguy 1930a (common in all of Morocco); Rungs 1952 , HA , Arganier; Harris et al. 1980 ; Mouna 1998 ; De Meyer 2000 , AP , Ouadj-Ouli-Mohamed, env. Settat, Insgane; Vidal et al. 2008 ; Aboussaid et al. 2009 ; Koçak and Kemal 2010 ; El Harym and Belqat 2017 , Rif , Kitane, El Haouta, MA , Sensla, AA , Environs Massa, Oued Massa, Douar Sidi Abou, Douar Tighrimt, Douar Zaouia; Elaini and Mazih 2018 ; Elaini et al. 2019 , HA , Arganeraie; AP (Safi) – MHNNR Dacini Bactrocera Macquart, 1835 Bactrocera oleae (Rossi, 1790) = Dacus oleae Rossi, in Becker and Stein 1913 : 94, Vayssière 1920 : 256 = Dacus oleae Gmelin, in Séguy 1930a : 168 Becker and Stein 1913 , Rif , Tanger; Vayssière 1920 , EM , Oujda; Séguy 1930a ; Mouna 1998 ; Savio 2011 , EM , Oujda; El Harym and Belqat 2017 , Rif , El Haouta, Oued Maâza, Cascade Chrafate, Koudiat El Aouinate, Lâazaba, Dhar Sbagh Mâasra, El Hajria, MA , Sensla, AA , route Bab El Khemis Dacus Fabricius, 1805 Dacus frontalis (Becker, 1922) El Harym and Belqat 2017 , AA , Oued Foum Ziguid (Douar Ouaiftoute), Oued Draa (Ikhf Mezrou), Isdaoun Dacus longistylus (Wiedemann, 1830) El Harym and Belqat 2017 , AA , Oued Tata, Douar Tighrimt, Oued Foum Ziguid (Douar Ouaiftoute) Tephritinae Dithrycini Oedaspis Loew, 1862 Oedaspis daphnea Séguy, 1930 Séguy 1930a , AP , El Mers (Rabat); Foote 1984 ; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 Oedaspis multifasciata (Loew, 1850) = Oedaspis multifasciatus (Loew), in Séguy 1953a : 85 Séguy 1953a , EM , Itzer (Haute Moulouya); El Harym and Belqat 2017 Oedaspis trotteriana Bezzi, 1913 Soós 1984b ; Ribera and Blasco-Zumeta 1998; Norrbom et al. 1999 ; El Harym and Belqat 2017 Myopitini Myopites Blot, 1827 Myopites cypriacus Hering 1938 El Harym et al. 2020 , Rif , Douar Halila, Dam Nakhla, Marabout Sidi Bou Hadjel Myopites inulaedyssentericae Blot, 1827 43 = Myopites apicatus Freidberg, 1980, in El Harym and Belqat 2017 : 140 El Harym and Belqat 2017 , Rif , affluent Tarmast, Aïn Afersiw Myopites longirostris (Loew, 1846) El Harym et al. 2020 , Rif , Oued Tahaddart, Douar Kouf, Mkhinak Myopites stylatus Fabricius, 1794 El Harym and Belqat 2017 , Rif , affluent Tarmast, Oued El Hamma, El Haouta Myopites variofasciatus Becker, 1903 = Myopites variofasciata Becker, in Séguy 1941a : 32 Séguy 1941a , HA , Imi-n'Ouaka (1500 m), Mouna 1998 Urophora Robineau-Desvoidy, 1830 Urophora congrua Loew, 1862 44 = Euribia congrua Loew, in Séguy 1941d : 14 Séguy 1941d , AA , Taroudant; Mouna 1998 ; El Harym and Belqat 2017 Urophora jaculata Rondani, 1870 = Urophora mauritanica Macquart, in El Harym and Belqat 2017 : 149 (misidentification) Urophora mauritanica Macquart, 1851 = Euribia algira Macquart, in Séguy 1930a : 169 = Urophora algira Macquart, in Séguy 1934a : 98, Mouna 1998 : 87 = Urophora macrura Loew, in Séguy 1934a : 100, Mouna 1998 : 87 Séguy 1930a , HA , Imi-N'Takandout, Dar Kaid M'Tougui; Séguy 1934a ; White and Korneyev 1989 , HA , Ito; Mouna 1998 ; Norrbom et al. 1999 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013b Urophora quadrifasciata algerica (Hering, 1941) = Euribia quadrifasciata Meigen, in Séguy 1930a : 169 Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 El Harym et al. 2020 , MA , Douar Oulad Abdoune, Mlakite, Tirra, Douar Oulad Amar, Tihli, Douar Oulad Amar Urophora solstitialis (Linnaeus, 1758) = Euribia solstitialis Linnaeus, in Séguy 1930a : 169 Séguy 1930a , HA , Haute Réghaya; Mouna 1998 ; El Harym and Belqat 2017 Noeetini Ensina Robineau-Desvoidy, 1830 Ensina sonchi (Linnaeus, 1767) El Harym and Belqat 2017 , Rif , Ksar Rimal, Douar Tizga, Oued Kbir, MA , Sensla, AA , Oued Massa (Pont Aghbalou), Centre Sidi Ouassay, Aïn Boharroch, Atbane, Oued Tamanarne, Oued Draa (Tahtah), Jnane Makadir, Kasbah Asma, Ait Aissa O Brahim, Oued Ziz (Pont Errachidia), Oued Ouarzazate; AA (Tafraout (Al Ourir, 12 km E)) – MHNNR Hypenidium Loew, 1862 Hypenidium graecum Loew, 1862 = Stephanaciura bipartita Séguy, in Séguy 1930a : 171 Séguy 1930a , MA , Tiffert (2000–2200 m); Villeneuve 1933 ; Soós 1984b ; Mouna 1998 : 87; Norrbom et al. 1999 ; El Harym and Belqat 2017 Tephrellini Aciura Robineau-Desvoidy Aciura coryli (Rossi, 1790) = Aciura Powelli Séguy, in Séguy 1930a : 170, Séguy 1953a : 85, Mouna 1998 : 87 Séguy 1930a , MA , Azrou (larvae from Phlomis crinita Cav.); Séguy 1953a , AP , Korifla; Mouna 1998 ; Norrbom et al. 1999 , MA , Azrou; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Arhil Oxyaciura Hendel, 1927 Oxyaciura tibialis (Robineau-Desvoidy, 1830) = Aciura tibialis Robineau-Desvoidy, in Becker and Stein 1913 : 94 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Sker; Séguy 1934a ; Mouna 1998 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013b ; El Harym and Belqat 2017 , Rif , Dayat El Birdiyel, Oued Azila, maison forestière; Oued Maâza (Tarik El Ouasâa); AP (Tamri, 10 km S) – MHNNR Sphaeniscus Becker, 1908 Sphaeniscus filiolus (Loew, 1869) = Spheniscomyia filiola Loew, in Séguy 1930a : 170; Mouna 1998 : 87 = Spheniscomyia aegyptiaca Efflatoun, in Séguy 1949a : 157; Mouna 1998 : 87 Séguy 1930a ; Séguy 1949a , SA , Guelmim; Mouna 1998 ; El Harym and Belqat 2017 , Rif , affluent Tarmast, Oued Maâza (Tarik El Ouasâa) Tephritini Acanthiophilus Becker, 1908 Acanthiophilus helianthi (Rossi, 1790) = Tephritis eluta Meigen, in Becker and Stein 1913 : 94 = Orellia eluta Meigen, in Mouna 1998 : 87 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger (Sarf, route Spartel), MA , Tizi s'Tkrine (Jebel Ahmar, 1700 m); Séguy 1949a , AA , Alnif, Foum-el-Hassan, SA , Guelmim; Mouna 1998 ; Koçak and Kemal 2013b ; El Harym and Belqat 2017 , Rif , Oued Zinat, Ksar Rimal, Oued Dardara, affluent Tarmast, Dayat El Birdiyel, Oued Azila, Dayat Jebel Zemzem, Oued Boumarouil, Douar Abou Boubnar (Mara­bout Sidi Gile), El Haouta, Oued Maâza (Tarik El Ouasâa), Douar Tizga, Dayat Afrate, Oued Mezine, Aïn El Malaâb, Oued Jnane Niche, MA , Oued Oum-er-Rbia, AA , Centre Sidi Ouassay, Avant Sidi Bin­zarne, route Bab El Khemis, airport Sidi Ifni, Oued Tisla, Oued Sayad, Oued Tamanarne, Oued Foum Ziguid (Douar Ouaiftoute), Jnane Makadir, Douar Rggaga, Oued Tinghir, Oued Ouarzazate; AP (Essaouira (Cap Hadid), Tamri, 10 km S) – MHNNR Campiglossa Rondani, 1870 Campiglossa martii (Becker, 1908) El Harym and Belqat 2017 , Rif , Oued Kbir, AA , Centre Sidi Ouassay Campiglossa producta (Loew, 1844) = Oxyna tessellata Loew, in Becker and Stein 1913 : 94 (misidentification) = Paroxyna tessellata (Loew), in Séguy 1930a : 174; Mouna 1998 : 87; Koçak and Kemal 2013b : 38 (misidentification) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Mogador, HA , Telouet Glaoua; Séguy 1934a ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Koçak and Kemal 2013b , HA ; El Harym and Belqat 2017 , Rif , Oued Al Mizzine, Aïn El Malaâb, Dayat El Hajjami Campiglossa sororcula (Wiedemann, 1830) = Dioxyna sororcula (Wiedemann), in El Harym and Belqat 2017 : 155 El Harym and Belqat 2017 , Rif , Ksar Rimal, Oued Jnane Niche, Oued Halila, Oued Zarka, Oued Martil, Oued Amsa, Oued Sahel, Dayat Jebel Zemzem, Oued Maggou, Dhar Sbagh Mâasra, Douar Kitane Capitites Foote & Freidberg, 1981 Capitites augur (Frauenfeld, 1857) = Trypanea augur (Frauenfeld), in Séguy 1930a : 176; Mouna 1998 : 87 Séguy 1930a , MA , Forêt Azrou, Tizi-s'Tkrine (Jebel Ahmar, 1700 m), HA , Tenfecht, AA , Souss; Mouna 1998 ; Pârvu et al. 2006 , AA , Ouarzazate, Lac Edehby; Popescu-Mirceni 2011 , MA , Tizi-s'Tkrine, Azrou, HA , Tenfecht, AA , Ouarzazate Capitites ramulosa (Loew, 1844) = Acanthiophilus ramulosus Loew, in Séguy 1930a : 177; Séguy 1941d : 15; Séguy 1949a : 157; Mouna 1998 : 87 Séguy 1930a , MA , Timelilt, HA , Tizi-n'Test (Jebel Imdress, 2000–2450 m); Séguy 1941d , AA , Taroudant; Séguy 1949a , AA , Foum-el-Hassan, Akka, Agdz, Alnif; Mouna 1998 ; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Amsa, Koudiat Taifour Desmella Munro, 1957 Desmella rostellata (Séguy, 1941) = Paroxyna rostellata Séguy, in Séguy 1941d : 14; Mouna 1998 : 87 Séguy 1941d , AP , Agadir; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 , AP , Agadir; El Harym and Belqat 2017 Euaresta Loew, 1873 Euaresta bullans (Wiedemann, 1830) Herman and Dirlbek 2006 , AA , Tiznit environs, Sidi Moussa d'Aglou; El Harym and Belqat 2017 , AA , Msidira Goniurellia Hendel, 1927 Goniurellia longicauda Freidberg, 1980 Freidberg 1980 , MA , Tizi-s'Tkrine (1700 m), Azrou, AA , Taroudant; Soós 1984b ; Freidberg and Kugler 1989 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 , AA , airport Sidi Ifni, Oued Tisla, Oued Tamanarne, Douar Zaouiet, Oued Tata, Douar Tighrimt, Ksibat Elhdeb, Oued Ziz (Pont Errachidia), Oued Ouarzazate Goniurellia persignata Freidberg, 1980 Freidberg 1980 , EM , Defilia, near Figuig; Soós 1984b ; Freidberg and Kugler 1989 ; Norrbom et al. 1999 ; Herman and Dirlbek 2006 , AA , Tiffoultoute (1146 m); El Harym and Belqat 2017 , Rif , Dhar Sbagh Mâasra, AA , Douar Zaouiet, Oued Ouarzazate Spathulina Rondani, 1856 Spathulina sicula Rondani, 1856 = Spathulina tristis Loew, in Séguy 1930a : 174; Mouna 1998 : 87 Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Barrage Smir Sphenella Robineau-Desvoidy, 1830 Sphenella marginata (Fallén, 1814) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Oued Judios (Tanger); Mouna 1998 ; Cassar et al. 2005 , Rif , lagoon Smir; Koçak and Kemal 2013b , HA ; El Harym and Belqat 2017 , Rif , affluent Tarmast, Dayat Jebel Zemzem, El Malaâb, Oued Maâza (Âachira), Dayat Aïn Jdioui, Oued Maggou, Dayat Afrate Tephritis Latreille, 1804 Tephritis carmen Hering, 1937 El Harym et al. 2020 , Rif , forest house of National Park of Talassemtane Tephritis dioscurea (Loew, 1856) Séguy 1930a , MA , El Hajeb; Mouna 1998 ; El Harym and Belqat 2017 Tephritis divisa (Rondani, 1871) El Harym and Belqat 2017 , Rif , Dayat Amsemlil Tephritis formosa (Loew, 1844) Séguy 1930a , HA , Asni; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Oued Abou Bnar, Oued Sidi Ben Saâda, Oued Achekrade, Oued El Kanar Tephritis leontodontis (De Geer, 1776) Séguy 1930a (All North Africa); Mouna 1998 ; El Harym and Belqat 2017 Tephritis matricariae (Loew, 1844) Séguy 1930a (all North Africa); Mouna 1998 ; El Harym and Belqat 2017 , Rif , affluent Oued Amsemlil, El Haouta, Dayat Jebel Zemzem, Dayat Amsemlil, Oued El Hamma Tephritis nigricauda (Loew, 1856) Séguy 1930a , AP , Berrechid; Séguy 1934a ; Séguy 1941d , AP , Agadir, Berrechid; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Dayat Jebel Zemzem, Oued Maâza (Tarik El Ouasâa), Aïn El Maounzil, Dayat Tazia, Dayat Amsemlil, Douar Tamakoute Tephritis postica (Loew, 1844) Herman and Dirlbek 2006 , MA , Volubilis (358 m); El Harym and Belqat 2017 , AA , Ksibat Elhdeb, Oued Tinghir Tephritis praecox (Loew, 1844) Séguy 1930a , Rif , Oued Judios (Tanger), MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 ; Herman and Dirlbek 2006 , MA , Ifrane, Azrou National Park (1743 m); El Harym and Belqat 2017 , Rif , Dayat El Ânassar, Dayat Amsemlil, affluent Oued Amsemlil, Douar Dacheryène, Douar Taghbaloute, Barrage Nakhla, Oued Sa­hel, Daya Jebel Zemzem, Douar Kitane, Oued El Hamma, Oued Kbir, Aïn el Ma Bared, Aïn El Malaâb, Douar Abou Boubnar (Marabout Sidi Gile), maison forestière, Douar Tizga, Oued Aïn Jdioui (Touaret), Dayat Afrate, Oued Jbara, Aïn El Maounzil, Dayat Tazia, Oued Jnane Niche, Oued Maggou, Aïn Tiouila, Dayat Lemtahane, Lâazaba, Dhar Sbagh Mâasra, El Hajria, Aïn Boharroch, Douar Tamakout, Douar Ouslaf, EM , Oued Béni Ouaklane (Béni Snassen); AP (Essaouira) – MHNNR Tephritis pulchra (Loew, 1844) Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 Tephritis simplex (Loew, 1844) Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Aïn Soualah, Aïn El Maounzil Tephritis stictica Loew, 1862 Séguy 1930a , AP , Rabat; Mouna 1998 ; El Harym and Belqat 2017 Tephritis theryi Séguy, 1930 Séguy 1930a , HA , Marrakech, Asni; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 Tephritis vespertina (Loew, 1844) El Harym and Belqat 2017 , Rif , Dayat Lemtahane, Dhar Sbagh Mâasra Tephritomyia Hendel, 1927 Tephritomyia lauta (Loew, 1869) = Acanthiophilus lauta Loew, in Séguy 1930a : 177; Mouna 1998 : 87 Séguy 1930a , HA , Tachdirt (Imminen, 2400–2600 m); Freidberg and Kugler 1989 ; Mouna 1998 ; Yaran and Kütük 2012 ; Morgulis et al. 2015, HA , Tizi-n'Tichka; El Harym and Belqat 2017 , Rif , Dayat El Birdiyel, Dayat Amsemlil, Lâazaba, AA , Msidira, Oued Ouarzazate Trupanea Schrank, 1795 Trupanea amoena (Frauenfeld, 1857) = Trypanea amoena Frauenfeld, in Séguy 1930a : 176; Mouna 1998 : 87 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Ksar Rimal, Oued Jnane niche, affluent Tarmast, Oued Martil (Tamouda), Oued Amsa, Oued El Hamma, Oued Boumarouil, Oued Sidi Yahia Aârab, Aïn Tiouila, AA , Oued Massa (Pont Aghbalou), Centre Sidi Ouassay, Avant Sidi Binzarne, Oued Tisla, Douar Tighrimt, Oued Draa (Tahtah), Jnane Makadir, Douar Rggaga, Aït Aissa O Brahim, Oued Draa (Ikhf Mezrou), Isdaoun, Ksibat Elhdeb, Oued Tinghir Trupanea guimari (Becker, 1908) El Harym and Belqat 2017 , AA , Centre Sidi Ouassay, Msidi­ra, Jnane Makadir, Aït Aissa O Brahim, Ksibat Elhdeb; Norrbom 2004 : 06600136 – INHS ( AA , 5 km W Ouarzazate) Trupanea stellata (Fuesslin, 1775) Séguy 1930a , MA , Timelilt (1900 m); Séguy 1949a , SA , Guelmim; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Mizoghar, Oued Maâza (Tarik El Ouasâa), Dayat Afrate, Aïn El Malâab, Oued Tkarae, AA , Centre Sidi Ouassay; AA (Taliouine) – MHNNR Terellini Chaetorellia Hendel, 1927 Chaetorellia conjuncta (Becker, 1912) El Harym and Belqat 2017 , AA , Airport Sidi Ifni, Oued Assa, Oued Sayad, Oued Foum Ziguid (Douar Ouaiftoute), Ksibat Elhdeb, Oued Ziz (Pont Errachidia), Oued Ouarzazate Chaetorellia hestia Hering, 1937 = Chaetorellia hexachaeta Loew, in Séguy 1930a : 174 [probably a misidentification] = Orellia hexachaeta Loew, in Séguy 1934a : 135 [misidentification, see White and Macquart 1989: 476]; Mouna 1998 : 87 Séguy 1930a , AP , Mogador; Séguy 1934a ; Mouna 1998 ; El Harym and Belqat 2017 , AA , Centre Sidi Ouassay Chaetorellia succinea (Costa, 1844) El Harym et al. 2020 , MA , Douar Oulad Abdoune Chaetostomella Hendel, 1927 Chaetostomella cylindrica (Robineau-Desvoidy, 1830) El Harym et al. 2020 , Rif , Marabout Douar Halila, Mkhinak, Douar Kitane Terellia Robineau-Desvoidy, 1830 Terellia colon (Meigen, 1826) = Orellia colon (Meigen), in Séguy 1930a : 173 Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 Terellia fuscicornis (Loew, 1844) Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 Terellia longicauda (Meigen, 1838) Séguy 1930a , MA , Aïn Leuh (1200–1400 m), HA , Tizi-n'Test, Goundafa (Jebel Imdress, 2000–2450 m); Séguy1934a ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; El Harym and Belqat 2017 Terellia luteola (Wiedemann, 1830) El Harym et al. 2020 , Rif , Bakrim, Aforidane, Aïn Siyed Terellia oasis (Hering, 1938) El Harym et al. 2020 , Rif , Douar Halila Terellia ptilostemi El Harym et al. 2021 El Harym et al. 2021 , Rif , Douar Chourdane (908 m), Aïn Akorian (1610 m), Aïn Elma Sefli (1345 m), Forest house of the Talassemtane National Park (1674 m) Terellia serratulae (Linnaeus, 1758) = Tephritis pallens Wiedemann, in Wiedemann 1824 : 54 = Trypeta serratula Linnaeus, in Becker and Stein 1913 : 94 = Terellia serratulae Linnaeus, in Séguy 1930a : 173 Wiedemann 1824 , Rif , Tanger; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a (all North Africa); Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 , Rif , Dayat Jebel Zemzem, Oued Maâza (Tarik El Ouasâa) Terellia virens (Loew, 1846) Séguy 1930a ; White 1989 , HA , Jebel Ayachi; Mouna 1998 ; Korneyev et al. 2013 , HA , Tizi-n'Talrhemt; El Harym and Belqat 2017 , AA , airport Sidi Ifni, Oued Ouarzazate Trypetinae Carpomyini Carpomya Costa, 1854 Carpomya incompleta (Becker, 1903) El Harym and Belqat 2017 , AA , Douar Zaouia Euleia Walker, 1835 Euleia heraclei (Linnaeus, 1758) = Acidia heraclei Linnaeus, in Séguy 1953a : 85 Séguy 1953a , MA , Sidi Slimane; Freidberg and Kugler 1989 ; Koçak and Kemal 2010 ; El Harym and Belqat 2017 , Rif , Oued Boumarouil, Aïn El Âakba Larbaâ Euleia marmorea (Fabricius, 1805) 45 = Philophylla flavescens Fabricius, in Séguy 1930a : 170; Mouna 1998 : 87 = Euleia flavescens Fabricius, in Soós 1984b : 88 Séguy 1930a , Rif , Tanger; Zimsen 1964 ; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 , Rif , Tanger; El Harym and Belqat 2017 Trypetini Chetostoma Rondani, 1856 Chetostoma curvinerve Rondani, 1856 El Harym and Belqat 2017 , Rif , Oued Kelaâ, Bab el Karn Acknowledgments We gratefully acknowledge the assistance and cooperation of Valery Korneyev and the late Amnon Freidberg who contributed to the revision of this family. Dacinae Ceratitidini Capparimyia Bezzi, 1920 Capparimyia savastanii (Martelli, 1911) = Capparimyia savastanii (Martelli), in Séguy 1953a : 85 Séguy 1953a , AA , Tiznit (on Capparis spinosa ); El Harym and Belqat 2017 Ceratitis MacLeay, 1829 Ceratitis capitata (Wiedemann, 1824) Becker and Stein 1913 , Rif , Tanger; Vayssière 1920 , AP , Rabat; Séguy 1930a (common in all of Morocco); Rungs 1952 , HA , Arganier; Harris et al. 1980 ; Mouna 1998 ; De Meyer 2000 , AP , Ouadj-Ouli-Mohamed, env. Settat, Insgane; Vidal et al. 2008 ; Aboussaid et al. 2009 ; Koçak and Kemal 2010 ; El Harym and Belqat 2017 , Rif , Kitane, El Haouta, MA , Sensla, AA , Environs Massa, Oued Massa, Douar Sidi Abou, Douar Tighrimt, Douar Zaouia; Elaini and Mazih 2018 ; Elaini et al. 2019 , HA , Arganeraie; AP (Safi) – MHNNR Dacini Bactrocera Macquart, 1835 Bactrocera oleae (Rossi, 1790) = Dacus oleae Rossi, in Becker and Stein 1913 : 94, Vayssière 1920 : 256 = Dacus oleae Gmelin, in Séguy 1930a : 168 Becker and Stein 1913 , Rif , Tanger; Vayssière 1920 , EM , Oujda; Séguy 1930a ; Mouna 1998 ; Savio 2011 , EM , Oujda; El Harym and Belqat 2017 , Rif , El Haouta, Oued Maâza, Cascade Chrafate, Koudiat El Aouinate, Lâazaba, Dhar Sbagh Mâasra, El Hajria, MA , Sensla, AA , route Bab El Khemis Dacus Fabricius, 1805 Dacus frontalis (Becker, 1922) El Harym and Belqat 2017 , AA , Oued Foum Ziguid (Douar Ouaiftoute), Oued Draa (Ikhf Mezrou), Isdaoun Dacus longistylus (Wiedemann, 1830) El Harym and Belqat 2017 , AA , Oued Tata, Douar Tighrimt, Oued Foum Ziguid (Douar Ouaiftoute) Tephritinae Dithrycini Oedaspis Loew, 1862 Oedaspis daphnea Séguy, 1930 Séguy 1930a , AP , El Mers (Rabat); Foote 1984 ; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 Oedaspis multifasciata (Loew, 1850) = Oedaspis multifasciatus (Loew), in Séguy 1953a : 85 Séguy 1953a , EM , Itzer (Haute Moulouya); El Harym and Belqat 2017 Oedaspis trotteriana Bezzi, 1913 Soós 1984b ; Ribera and Blasco-Zumeta 1998; Norrbom et al. 1999 ; El Harym and Belqat 2017 Myopitini Myopites Blot, 1827 Myopites cypriacus Hering 1938 El Harym et al. 2020 , Rif , Douar Halila, Dam Nakhla, Marabout Sidi Bou Hadjel Myopites inulaedyssentericae Blot, 1827 43 = Myopites apicatus Freidberg, 1980, in El Harym and Belqat 2017 : 140 El Harym and Belqat 2017 , Rif , affluent Tarmast, Aïn Afersiw Myopites longirostris (Loew, 1846) El Harym et al. 2020 , Rif , Oued Tahaddart, Douar Kouf, Mkhinak Myopites stylatus Fabricius, 1794 El Harym and Belqat 2017 , Rif , affluent Tarmast, Oued El Hamma, El Haouta Myopites variofasciatus Becker, 1903 = Myopites variofasciata Becker, in Séguy 1941a : 32 Séguy 1941a , HA , Imi-n'Ouaka (1500 m), Mouna 1998 Urophora Robineau-Desvoidy, 1830 Urophora congrua Loew, 1862 44 = Euribia congrua Loew, in Séguy 1941d : 14 Séguy 1941d , AA , Taroudant; Mouna 1998 ; El Harym and Belqat 2017 Urophora jaculata Rondani, 1870 = Urophora mauritanica Macquart, in El Harym and Belqat 2017 : 149 (misidentification) Urophora mauritanica Macquart, 1851 = Euribia algira Macquart, in Séguy 1930a : 169 = Urophora algira Macquart, in Séguy 1934a : 98, Mouna 1998 : 87 = Urophora macrura Loew, in Séguy 1934a : 100, Mouna 1998 : 87 Séguy 1930a , HA , Imi-N'Takandout, Dar Kaid M'Tougui; Séguy 1934a ; White and Korneyev 1989 , HA , Ito; Mouna 1998 ; Norrbom et al. 1999 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013b Urophora quadrifasciata algerica (Hering, 1941) = Euribia quadrifasciata Meigen, in Séguy 1930a : 169 Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 El Harym et al. 2020 , MA , Douar Oulad Abdoune, Mlakite, Tirra, Douar Oulad Amar, Tihli, Douar Oulad Amar Urophora solstitialis (Linnaeus, 1758) = Euribia solstitialis Linnaeus, in Séguy 1930a : 169 Séguy 1930a , HA , Haute Réghaya; Mouna 1998 ; El Harym and Belqat 2017 Noeetini Ensina Robineau-Desvoidy, 1830 Ensina sonchi (Linnaeus, 1767) El Harym and Belqat 2017 , Rif , Ksar Rimal, Douar Tizga, Oued Kbir, MA , Sensla, AA , Oued Massa (Pont Aghbalou), Centre Sidi Ouassay, Aïn Boharroch, Atbane, Oued Tamanarne, Oued Draa (Tahtah), Jnane Makadir, Kasbah Asma, Ait Aissa O Brahim, Oued Ziz (Pont Errachidia), Oued Ouarzazate; AA (Tafraout (Al Ourir, 12 km E)) – MHNNR Hypenidium Loew, 1862 Hypenidium graecum Loew, 1862 = Stephanaciura bipartita Séguy, in Séguy 1930a : 171 Séguy 1930a , MA , Tiffert (2000–2200 m); Villeneuve 1933 ; Soós 1984b ; Mouna 1998 : 87; Norrbom et al. 1999 ; El Harym and Belqat 2017 Tephrellini Aciura Robineau-Desvoidy Aciura coryli (Rossi, 1790) = Aciura Powelli Séguy, in Séguy 1930a : 170, Séguy 1953a : 85, Mouna 1998 : 87 Séguy 1930a , MA , Azrou (larvae from Phlomis crinita Cav.); Séguy 1953a , AP , Korifla; Mouna 1998 ; Norrbom et al. 1999 , MA , Azrou; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Arhil Oxyaciura Hendel, 1927 Oxyaciura tibialis (Robineau-Desvoidy, 1830) = Aciura tibialis Robineau-Desvoidy, in Becker and Stein 1913 : 94 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Sker; Séguy 1934a ; Mouna 1998 ; Koçak and Kemal 2010 ; Koçak and Kemal 2013b ; El Harym and Belqat 2017 , Rif , Dayat El Birdiyel, Oued Azila, maison forestière; Oued Maâza (Tarik El Ouasâa); AP (Tamri, 10 km S) – MHNNR Sphaeniscus Becker, 1908 Sphaeniscus filiolus (Loew, 1869) = Spheniscomyia filiola Loew, in Séguy 1930a : 170; Mouna 1998 : 87 = Spheniscomyia aegyptiaca Efflatoun, in Séguy 1949a : 157; Mouna 1998 : 87 Séguy 1930a ; Séguy 1949a , SA , Guelmim; Mouna 1998 ; El Harym and Belqat 2017 , Rif , affluent Tarmast, Oued Maâza (Tarik El Ouasâa) Tephritini Acanthiophilus Becker, 1908 Acanthiophilus helianthi (Rossi, 1790) = Tephritis eluta Meigen, in Becker and Stein 1913 : 94 = Orellia eluta Meigen, in Mouna 1998 : 87 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger (Sarf, route Spartel), MA , Tizi s'Tkrine (Jebel Ahmar, 1700 m); Séguy 1949a , AA , Alnif, Foum-el-Hassan, SA , Guelmim; Mouna 1998 ; Koçak and Kemal 2013b ; El Harym and Belqat 2017 , Rif , Oued Zinat, Ksar Rimal, Oued Dardara, affluent Tarmast, Dayat El Birdiyel, Oued Azila, Dayat Jebel Zemzem, Oued Boumarouil, Douar Abou Boubnar (Mara­bout Sidi Gile), El Haouta, Oued Maâza (Tarik El Ouasâa), Douar Tizga, Dayat Afrate, Oued Mezine, Aïn El Malaâb, Oued Jnane Niche, MA , Oued Oum-er-Rbia, AA , Centre Sidi Ouassay, Avant Sidi Bin­zarne, route Bab El Khemis, airport Sidi Ifni, Oued Tisla, Oued Sayad, Oued Tamanarne, Oued Foum Ziguid (Douar Ouaiftoute), Jnane Makadir, Douar Rggaga, Oued Tinghir, Oued Ouarzazate; AP (Essaouira (Cap Hadid), Tamri, 10 km S) – MHNNR Campiglossa Rondani, 1870 Campiglossa martii (Becker, 1908) El Harym and Belqat 2017 , Rif , Oued Kbir, AA , Centre Sidi Ouassay Campiglossa producta (Loew, 1844) = Oxyna tessellata Loew, in Becker and Stein 1913 : 94 (misidentification) = Paroxyna tessellata (Loew), in Séguy 1930a : 174; Mouna 1998 : 87; Koçak and Kemal 2013b : 38 (misidentification) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Mogador, HA , Telouet Glaoua; Séguy 1934a ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Koçak and Kemal 2013b , HA ; El Harym and Belqat 2017 , Rif , Oued Al Mizzine, Aïn El Malaâb, Dayat El Hajjami Campiglossa sororcula (Wiedemann, 1830) = Dioxyna sororcula (Wiedemann), in El Harym and Belqat 2017 : 155 El Harym and Belqat 2017 , Rif , Ksar Rimal, Oued Jnane Niche, Oued Halila, Oued Zarka, Oued Martil, Oued Amsa, Oued Sahel, Dayat Jebel Zemzem, Oued Maggou, Dhar Sbagh Mâasra, Douar Kitane Capitites Foote & Freidberg, 1981 Capitites augur (Frauenfeld, 1857) = Trypanea augur (Frauenfeld), in Séguy 1930a : 176; Mouna 1998 : 87 Séguy 1930a , MA , Forêt Azrou, Tizi-s'Tkrine (Jebel Ahmar, 1700 m), HA , Tenfecht, AA , Souss; Mouna 1998 ; Pârvu et al. 2006 , AA , Ouarzazate, Lac Edehby; Popescu-Mirceni 2011 , MA , Tizi-s'Tkrine, Azrou, HA , Tenfecht, AA , Ouarzazate Capitites ramulosa (Loew, 1844) = Acanthiophilus ramulosus Loew, in Séguy 1930a : 177; Séguy 1941d : 15; Séguy 1949a : 157; Mouna 1998 : 87 Séguy 1930a , MA , Timelilt, HA , Tizi-n'Test (Jebel Imdress, 2000–2450 m); Séguy 1941d , AA , Taroudant; Séguy 1949a , AA , Foum-el-Hassan, Akka, Agdz, Alnif; Mouna 1998 ; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Amsa, Koudiat Taifour Desmella Munro, 1957 Desmella rostellata (Séguy, 1941) = Paroxyna rostellata Séguy, in Séguy 1941d : 14; Mouna 1998 : 87 Séguy 1941d , AP , Agadir; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 , AP , Agadir; El Harym and Belqat 2017 Euaresta Loew, 1873 Euaresta bullans (Wiedemann, 1830) Herman and Dirlbek 2006 , AA , Tiznit environs, Sidi Moussa d'Aglou; El Harym and Belqat 2017 , AA , Msidira Goniurellia Hendel, 1927 Goniurellia longicauda Freidberg, 1980 Freidberg 1980 , MA , Tizi-s'Tkrine (1700 m), Azrou, AA , Taroudant; Soós 1984b ; Freidberg and Kugler 1989 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 , AA , airport Sidi Ifni, Oued Tisla, Oued Tamanarne, Douar Zaouiet, Oued Tata, Douar Tighrimt, Ksibat Elhdeb, Oued Ziz (Pont Errachidia), Oued Ouarzazate Goniurellia persignata Freidberg, 1980 Freidberg 1980 , EM , Defilia, near Figuig; Soós 1984b ; Freidberg and Kugler 1989 ; Norrbom et al. 1999 ; Herman and Dirlbek 2006 , AA , Tiffoultoute (1146 m); El Harym and Belqat 2017 , Rif , Dhar Sbagh Mâasra, AA , Douar Zaouiet, Oued Ouarzazate Spathulina Rondani, 1856 Spathulina sicula Rondani, 1856 = Spathulina tristis Loew, in Séguy 1930a : 174; Mouna 1998 : 87 Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Barrage Smir Sphenella Robineau-Desvoidy, 1830 Sphenella marginata (Fallén, 1814) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Oued Judios (Tanger); Mouna 1998 ; Cassar et al. 2005 , Rif , lagoon Smir; Koçak and Kemal 2013b , HA ; El Harym and Belqat 2017 , Rif , affluent Tarmast, Dayat Jebel Zemzem, El Malaâb, Oued Maâza (Âachira), Dayat Aïn Jdioui, Oued Maggou, Dayat Afrate Tephritis Latreille, 1804 Tephritis carmen Hering, 1937 El Harym et al. 2020 , Rif , forest house of National Park of Talassemtane Tephritis dioscurea (Loew, 1856) Séguy 1930a , MA , El Hajeb; Mouna 1998 ; El Harym and Belqat 2017 Tephritis divisa (Rondani, 1871) El Harym and Belqat 2017 , Rif , Dayat Amsemlil Tephritis formosa (Loew, 1844) Séguy 1930a , HA , Asni; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Oued Abou Bnar, Oued Sidi Ben Saâda, Oued Achekrade, Oued El Kanar Tephritis leontodontis (De Geer, 1776) Séguy 1930a (All North Africa); Mouna 1998 ; El Harym and Belqat 2017 Tephritis matricariae (Loew, 1844) Séguy 1930a (all North Africa); Mouna 1998 ; El Harym and Belqat 2017 , Rif , affluent Oued Amsemlil, El Haouta, Dayat Jebel Zemzem, Dayat Amsemlil, Oued El Hamma Tephritis nigricauda (Loew, 1856) Séguy 1930a , AP , Berrechid; Séguy 1934a ; Séguy 1941d , AP , Agadir, Berrechid; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Dayat Jebel Zemzem, Oued Maâza (Tarik El Ouasâa), Aïn El Maounzil, Dayat Tazia, Dayat Amsemlil, Douar Tamakoute Tephritis postica (Loew, 1844) Herman and Dirlbek 2006 , MA , Volubilis (358 m); El Harym and Belqat 2017 , AA , Ksibat Elhdeb, Oued Tinghir Tephritis praecox (Loew, 1844) Séguy 1930a , Rif , Oued Judios (Tanger), MA , Tizi-s'Tkrine (Jebel Ahmar, 1700 m); Mouna 1998 ; Herman and Dirlbek 2006 , MA , Ifrane, Azrou National Park (1743 m); El Harym and Belqat 2017 , Rif , Dayat El Ânassar, Dayat Amsemlil, affluent Oued Amsemlil, Douar Dacheryène, Douar Taghbaloute, Barrage Nakhla, Oued Sa­hel, Daya Jebel Zemzem, Douar Kitane, Oued El Hamma, Oued Kbir, Aïn el Ma Bared, Aïn El Malaâb, Douar Abou Boubnar (Marabout Sidi Gile), maison forestière, Douar Tizga, Oued Aïn Jdioui (Touaret), Dayat Afrate, Oued Jbara, Aïn El Maounzil, Dayat Tazia, Oued Jnane Niche, Oued Maggou, Aïn Tiouila, Dayat Lemtahane, Lâazaba, Dhar Sbagh Mâasra, El Hajria, Aïn Boharroch, Douar Tamakout, Douar Ouslaf, EM , Oued Béni Ouaklane (Béni Snassen); AP (Essaouira) – MHNNR Tephritis pulchra (Loew, 1844) Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 Tephritis simplex (Loew, 1844) Séguy 1930a ; Mouna 1998 ; El Harym and Belqat 2017 ; El Harym et al. 2020 , Rif , Aïn Soualah, Aïn El Maounzil Tephritis stictica Loew, 1862 Séguy 1930a , AP , Rabat; Mouna 1998 ; El Harym and Belqat 2017 Tephritis theryi Séguy, 1930 Séguy 1930a , HA , Marrakech, Asni; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 Tephritis vespertina (Loew, 1844) El Harym and Belqat 2017 , Rif , Dayat Lemtahane, Dhar Sbagh Mâasra Tephritomyia Hendel, 1927 Tephritomyia lauta (Loew, 1869) = Acanthiophilus lauta Loew, in Séguy 1930a : 177; Mouna 1998 : 87 Séguy 1930a , HA , Tachdirt (Imminen, 2400–2600 m); Freidberg and Kugler 1989 ; Mouna 1998 ; Yaran and Kütük 2012 ; Morgulis et al. 2015, HA , Tizi-n'Tichka; El Harym and Belqat 2017 , Rif , Dayat El Birdiyel, Dayat Amsemlil, Lâazaba, AA , Msidira, Oued Ouarzazate Trupanea Schrank, 1795 Trupanea amoena (Frauenfeld, 1857) = Trypanea amoena Frauenfeld, in Séguy 1930a : 176; Mouna 1998 : 87 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Ksar Rimal, Oued Jnane niche, affluent Tarmast, Oued Martil (Tamouda), Oued Amsa, Oued El Hamma, Oued Boumarouil, Oued Sidi Yahia Aârab, Aïn Tiouila, AA , Oued Massa (Pont Aghbalou), Centre Sidi Ouassay, Avant Sidi Binzarne, Oued Tisla, Douar Tighrimt, Oued Draa (Tahtah), Jnane Makadir, Douar Rggaga, Aït Aissa O Brahim, Oued Draa (Ikhf Mezrou), Isdaoun, Ksibat Elhdeb, Oued Tinghir Trupanea guimari (Becker, 1908) El Harym and Belqat 2017 , AA , Centre Sidi Ouassay, Msidi­ra, Jnane Makadir, Aït Aissa O Brahim, Ksibat Elhdeb; Norrbom 2004 : 06600136 – INHS ( AA , 5 km W Ouarzazate) Trupanea stellata (Fuesslin, 1775) Séguy 1930a , MA , Timelilt (1900 m); Séguy 1949a , SA , Guelmim; Mouna 1998 ; El Harym and Belqat 2017 , Rif , Mizoghar, Oued Maâza (Tarik El Ouasâa), Dayat Afrate, Aïn El Malâab, Oued Tkarae, AA , Centre Sidi Ouassay; AA (Taliouine) – MHNNR Terellini Chaetorellia Hendel, 1927 Chaetorellia conjuncta (Becker, 1912) El Harym and Belqat 2017 , AA , Airport Sidi Ifni, Oued Assa, Oued Sayad, Oued Foum Ziguid (Douar Ouaiftoute), Ksibat Elhdeb, Oued Ziz (Pont Errachidia), Oued Ouarzazate Chaetorellia hestia Hering, 1937 = Chaetorellia hexachaeta Loew, in Séguy 1930a : 174 [probably a misidentification] = Orellia hexachaeta Loew, in Séguy 1934a : 135 [misidentification, see White and Macquart 1989: 476]; Mouna 1998 : 87 Séguy 1930a , AP , Mogador; Séguy 1934a ; Mouna 1998 ; El Harym and Belqat 2017 , AA , Centre Sidi Ouassay Chaetorellia succinea (Costa, 1844) El Harym et al. 2020 , MA , Douar Oulad Abdoune Chaetostomella Hendel, 1927 Chaetostomella cylindrica (Robineau-Desvoidy, 1830) El Harym et al. 2020 , Rif , Marabout Douar Halila, Mkhinak, Douar Kitane Terellia Robineau-Desvoidy, 1830 Terellia colon (Meigen, 1826) = Orellia colon (Meigen), in Séguy 1930a : 173 Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 Terellia fuscicornis (Loew, 1844) Séguy 1930a (common in all North Africa); Mouna 1998 ; El Harym and Belqat 2017 Terellia longicauda (Meigen, 1838) Séguy 1930a , MA , Aïn Leuh (1200–1400 m), HA , Tizi-n'Test, Goundafa (Jebel Imdress, 2000–2450 m); Séguy1934a ; Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; El Harym and Belqat 2017 Terellia luteola (Wiedemann, 1830) El Harym et al. 2020 , Rif , Bakrim, Aforidane, Aïn Siyed Terellia oasis (Hering, 1938) El Harym et al. 2020 , Rif , Douar Halila Terellia ptilostemi El Harym et al. 2021 El Harym et al. 2021 , Rif , Douar Chourdane (908 m), Aïn Akorian (1610 m), Aïn Elma Sefli (1345 m), Forest house of the Talassemtane National Park (1674 m) Terellia serratulae (Linnaeus, 1758) = Tephritis pallens Wiedemann, in Wiedemann 1824 : 54 = Trypeta serratula Linnaeus, in Becker and Stein 1913 : 94 = Terellia serratulae Linnaeus, in Séguy 1930a : 173 Wiedemann 1824 , Rif , Tanger; Becker and Stein 1913 , Rif , Tanger; Séguy 1930a (all North Africa); Mouna 1998 ; Norrbom et al. 1999 ; El Harym and Belqat 2017 , Rif , Dayat Jebel Zemzem, Oued Maâza (Tarik El Ouasâa) Terellia virens (Loew, 1846) Séguy 1930a ; White 1989 , HA , Jebel Ayachi; Mouna 1998 ; Korneyev et al. 2013 , HA , Tizi-n'Talrhemt; El Harym and Belqat 2017 , AA , airport Sidi Ifni, Oued Ouarzazate Trypetinae Carpomyini Carpomya Costa, 1854 Carpomya incompleta (Becker, 1903) El Harym and Belqat 2017 , AA , Douar Zaouia Euleia Walker, 1835 Euleia heraclei (Linnaeus, 1758) = Acidia heraclei Linnaeus, in Séguy 1953a : 85 Séguy 1953a , MA , Sidi Slimane; Freidberg and Kugler 1989 ; Koçak and Kemal 2010 ; El Harym and Belqat 2017 , Rif , Oued Boumarouil, Aïn El Âakba Larbaâ Euleia marmorea (Fabricius, 1805) 45 = Philophylla flavescens Fabricius, in Séguy 1930a : 170; Mouna 1998 : 87 = Euleia flavescens Fabricius, in Soós 1984b : 88 Séguy 1930a , Rif , Tanger; Zimsen 1964 ; Soós 1984b ; Mouna 1998 ; Norrbom et al. 1999 , Rif , Tanger; El Harym and Belqat 2017 Trypetini Chetostoma Rondani, 1856 Chetostoma curvinerve Rondani, 1856 El Harym and Belqat 2017 , Rif , Oued Kelaâ, Bab el Karn Acknowledgments We gratefully acknowledge the assistance and cooperation of Valery Korneyev and the late Amnon Freidberg who contributed to the revision of this family. ULIDIIDAE K. Kettani, M.J. Ebejer Number of species: 13 . Expected: 18 Faunistic knowledge of the family in Morocco: good Otitinae Ceroxys Macquart, 1835 Ceroxys urticae Linnaeus, 1758 Ebejer et al. 2019 , AP , Lower Loukous (6 m), Larache (5 m) Dorycera Meigen, 1830 Dorycera griseipennis (Becker, 1907) Soós 1984b Herina Robineau-Desvoidy, 1830 Herina ghilianii Rondani, 1869 Kameneva 2007 , HA , Ansegmir-Tal, W Midelt (1400 m); Ebejer 2015 Herina lacustris (Meigen, 1826) Kameneva 2007 , Rif , Baie de Tanger Herina oscillans (Meigen, 1826) = Herina schlueteri Becker, in Becker and Stein 1913 : 92; Soós 1984b : 56 Becker and Stein 1913 , Rif , Tanger; Soós 1984b ; Kameneva 2007 , Rif , Tanger Melieria Robineau-Desvoidy, 1830 Melieria nigritarsis Becker, 1903 Ebejer et al. 2019 , AA , Merzouga (714 m) Otites Latreille, 1804 Otites tangeriana Becker, 1913 = Otites tangeriana Becker, in Becker and Stein 1913 , 1918: 92 Becker and Stein 1913 , 1918, Rif , Tanger; Soós 1984b , Rif , Tanger Tetanops Fallén, 1820 Tetanops flavescens Macquart, 1835 Séguy 1930a , Rif , Tanger; Mouna 1998 Ulidiinae Physiphora Fallén, 1810 Physiphora alceae (Preyssler, 1791) = Chrysomyza demandata (Fabricius, 1798), in Séguy 1953a : 85; Mouna 1998 : 87 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Goundafa; Séguy 1953a , SA , El Aöun du Draa; Séguy 1949a , AA , Agdz; Mouna 1998 ; Koçak and Kemal 2010 ; Kameneva and Korneyev 2016 , AA , Tizi-n'Bachkoun (1600 m); Rif (M'Diq farm) – MISR ; Rif (Tanger) – MfN ; AP (Casablanca) – ZSSM Physiphora smaragdina (Loew, 1852) Kameneva and Korneyev 2016 , AA , 25 km S Goulmima (100 m) – NHMD Ulidia Meigen, 1826 Ulidia apicalis (Meigen, 1826) Séguy 1930a , MA , Meknès, HA , Skoutana; Séguy 1934a ; Lyneborg 1969 ; Soós 1984b ; Mouna 1998 Ulidia erythrophthalma Meigen, 1826 Becker and Stein 1913 , Rif , Tanger; Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 Ulidia megacephala Loew, 1845 Soós 1984b ; Zaitzev 1984 ; Koçak and Kemal 2010 Otitinae Ceroxys Macquart, 1835 Ceroxys urticae Linnaeus, 1758 Ebejer et al. 2019 , AP , Lower Loukous (6 m), Larache (5 m) Dorycera Meigen, 1830 Dorycera griseipennis (Becker, 1907) Soós 1984b Herina Robineau-Desvoidy, 1830 Herina ghilianii Rondani, 1869 Kameneva 2007 , HA , Ansegmir-Tal, W Midelt (1400 m); Ebejer 2015 Herina lacustris (Meigen, 1826) Kameneva 2007 , Rif , Baie de Tanger Herina oscillans (Meigen, 1826) = Herina schlueteri Becker, in Becker and Stein 1913 : 92; Soós 1984b : 56 Becker and Stein 1913 , Rif , Tanger; Soós 1984b ; Kameneva 2007 , Rif , Tanger Melieria Robineau-Desvoidy, 1830 Melieria nigritarsis Becker, 1903 Ebejer et al. 2019 , AA , Merzouga (714 m) Otites Latreille, 1804 Otites tangeriana Becker, 1913 = Otites tangeriana Becker, in Becker and Stein 1913 , 1918: 92 Becker and Stein 1913 , 1918, Rif , Tanger; Soós 1984b , Rif , Tanger Tetanops Fallén, 1820 Tetanops flavescens Macquart, 1835 Séguy 1930a , Rif , Tanger; Mouna 1998 Ulidiinae Physiphora Fallén, 1810 Physiphora alceae (Preyssler, 1791) = Chrysomyza demandata (Fabricius, 1798), in Séguy 1953a : 85; Mouna 1998 : 87 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , HA , Goundafa; Séguy 1953a , SA , El Aöun du Draa; Séguy 1949a , AA , Agdz; Mouna 1998 ; Koçak and Kemal 2010 ; Kameneva and Korneyev 2016 , AA , Tizi-n'Bachkoun (1600 m); Rif (M'Diq farm) – MISR ; Rif (Tanger) – MfN ; AP (Casablanca) – ZSSM Physiphora smaragdina (Loew, 1852) Kameneva and Korneyev 2016 , AA , 25 km S Goulmima (100 m) – NHMD Ulidia Meigen, 1826 Ulidia apicalis (Meigen, 1826) Séguy 1930a , MA , Meknès, HA , Skoutana; Séguy 1934a ; Lyneborg 1969 ; Soós 1984b ; Mouna 1998 Ulidia erythrophthalma Meigen, 1826 Becker and Stein 1913 , Rif , Tanger; Séguy 1949a , AA , Foum-el-Hassan; Mouna 1998 Ulidia megacephala Loew, 1845 Soós 1984b ; Zaitzev 1984 ; Koçak and Kemal 2010 Lauxanioidea CHAMAEMYIIDAE K. Kettani, M.J. Ebejer Number of species: 18 . Expected: 24 Faunistic knowledge of the family in Morocco: moderate Chamaemyiinae Chamaemyia Meigen, 1803 Chamaemyia aridella (Fallén, 1823) Ebejer 2016a , Rif , Moulay Abdelsalam (1180 m), Issaguen (1620 m); HA (Jebel Ayachi, Mikdane, maison forestière, MR Jaffar, Tizi-n'Zou) – NHMUK Chamaemyia flavicornis (Strobl, 1902) Ebejer 2016a , Rif , Martil beach and dunes (on human faeces), MA , Khénifra (17 km NW of Zaida, 1878 m), AA , Errachidia (29 km N of Rich, 1570 m); HA (Asni near Alrene, W Imlil) – NHMUK Chamaemyia flavipalpis (Haliday, 1838) = Chamaemyia maritima Zetterstedt, in Mouna 1998 : 85 Mouna 1998 ; Ebejer 2016a , Rif , Ksar Sghir, Oued Araml, Ksar Sghir, AP , Larache, Merja Zerga, Loukous marsh Chamaemyia herbarum (Robineau-Desvoidy, 1830) Ebejer 2016a , MA , Khénifra, 17 km SW of Midelt (1940 m), AA , Errachidia, 29 km N of Rich (1570 m); HA (Jebel Ayachi, Mikdane, MR Jaffar, Tizi-n'Zou) – NHMUK Chamaemyia juncorum (Fallén, 1958) Ebejer 2016a , MA , Khénifra (17 km SW of Midelt, 1940 m), AA , Errachidia (29 km N of Rich, 1570 m) Chamaemyia polystigma (Meigen, 1830) Becker and Stein 1913 , Rif , Tanger; Ebejer 2016a , Rif , Moulay Abdelsalam (1180 m), Sidi Yahia Aarab (377 m); HA (Asni) – NHMUK Melanochthiphila Frey, 1958 Melanochthiphila sp. Ebejer, 2016 Ebejer 2016a , AA , Errachidia (30 km W of Errachidia, 1065 m) Parochthiphila ( Euestelia ) Enderlein, 1927 Parochthiphila ( Euestelia ) coronata (Loew, 1858) Carles-Tolrá 1993 ; Mouna 1998 ; Ebejer 2016a , AP , Larache (Lower Loukous, 2 m); HA (Jebel Ayachi, Mikdane, MR Jaffar) – NHMUK Parochthiphila frontella (Rondani, 1874) Ebejer 2016a ; HA (Jebel Ayachi, Mikdane, Tizi-n'Zou, MR Jaffar) – NHMUK Parochthiphila inconstans Becker, 1903 Ebejer 2016a , AA , Ziz river (9.5 km SE of Rich, 1285 m), Errachidia (6 km N of Errachidia, 1010 m) Parochthiphila ( Euestelia ) nigripes Strobl, 1900 Ebejer 2016a , Rif , Ksar Sghir, Dardara Leucopinae Leucopis Meigen, 1803 Leucopis annulipes Zetterstedt, 1848 Mouna 1998 ; AP (Maâmora) – MISR Leucopis griseola (Fallén, 1823) Mouna 1998 ; AP (Rabat), MA (Meknès), HA (Marrakech) – MISR Leucopis formosana Hennig, 1938 Ebejer 2016a , AP , Sidi Smail, Oued Tensift (estuary) Leucopis glyphinivora Tanasijtshuk, 1958 Ebejer 2016a , AP , Ksar Sghir, Azemmour, Oued Tensift (estuary), AA , Errachidia (30 km W of Errachidia) Leucopis kerzhneri Tanasijtshuk, 1970 Ebejer 2016a ; HA (Jebel Ayachi) – NHMUK Leucopis palumbii Rondani, 1872 Ebejer 2016a , Rif , Ksar Sghir, Oued Aliane Lipoleucopis de Meijere, 1928 Lipoleucopis pulchra Raspi, 2008 Ebejer 2016a , AA , Errachidia, Ziz river (12 km S of Rissani) LAUXANIIDAE K. Kettani, M.J. Ebejer Number of species: 27 . Expected: 45 Faunistic knowledge of the family in Morocco: good Homoneurinae Homoneura Wulp, 1891 Homoneura ericpoli Carles-Tolrá, 1993 Ebejer and Kettani 2019a , Rif , Azilane (1255 m), Adrou (556 m), HA , Lac Tislit (Imlchil, 2254 m) Homoneura licina Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Schacht et al. 2004 ; Ebejer and Kettani 2019a , Rif , Oued Laou (2 m), AP , Merja Zerga, Sidi Smaine, Safi (estuary of Oued Tensift), Azemmour (El Jadida), Diabat (Essaouira), Larache (5 m) Homoneura transversa (Wiedemann, 1830) Wiedemann 1830 ; Ebejer and Kettani 2019a Prosopomyia Loew, 1856 Prosopomyia pallida Loew, 1856 Vaillant 1956b , HA , Oukaimeden, Imi-N'Ifri; Carles-Tolrá 1993 ; Mouna 1998 ; Ebejer and Kettani 2019a , Rif , Adrou (556 m), Issaguen (1547 m), HA , Jebel Ayachi – NHMUK Lauxaniinae Calliopum Strand, 1928 Calliopum oosterbroeki Shatalkin, 2000 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m), Moulay Abdelsalam (1180 m), Ketama, Rahbat Amlay (284 m), Ikadjiouen (294 m), Mechkralla ( NPT , 981 m), Kharouba (roadside meadow between Chefchaouen and Tétouan, 385 m), Dbani (Béni Selmane, 1046 m), Aïn Ben Ali (Béni Selmane, 1014 m), HA , Lalla Takrkoust (Oued N'fis, 1141 m) – NHMUK Calliopum tuberculosum (Becker, 1895) Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 135 m), Azilane (1255 m), Perdicaris Park (Tanger, 223 m), cascade Chrafate (859 m) Meiosimyza Hendel, 1925 Meiosimyza ( Lyciella ) rorida (Fallén, 1820) = Homoneura rorida Fallén, in Mouna 1998 : 85 Mouna 1998 ; MA (Aïn Leuh) – MISR Minettia Robineau-Desvoidy, 1830 Minettia aenigmatica Ebejer, 2019 Ebejer 2019a , Rif , Perdicaris Park (223 m), Oued Khmis (Khmis Anjra, 61 m), HA , Mikdane (Jebel Ayachi); Ebejer and Kettani 2019a Minettia biseriata (Loew, 1847) Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Jnane Niche (46 m), Oued Kbir (Béni Ratene ( PNPB ), 157 m) Minettia cantolraensis Carles-Tolrá, 1998 Ebejer and Kettani 2019a , Rif , Oued Laou, El-Fahsa, Oued Guallet (Bni Boufrah, 942 m), Oued Ouringa (2 m), Maggou ( NPT , 962 m), Ksar el-Kbir road to Chefchaouen at bridge near oued Azla (80 m), Oued Siflaou (281 m), Dardara (484 m), Azilane (1255 m), Adrou (556 m), Perdicaris Park (Tanger, 223 m), Bni Bahlou (986 m), El Hamma (936 m), Amaghouse (Oued Laou), Oued Koub (Laghdir, 148 m), EM , Aïn Sfa (Berkane, 638 m), Tafoughalt (Berkane, 788 m), HA , Lalla Takrkoust (Oued N'fis, 1141 m), AA , Taliouine (Taroudant, 1049 m) Minettia fasciata (Fallén, 1826) Merz 2004 , AP , Rabat; Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Ksar el-Kbir road to Chefchaouen at bridge near oued Azla (80 m), Azilane (1255 m), Adrou (556 m), Issaguen (1547 m), Oued Taida (Al Andalous, 503 m), Perdicaris Park (Tanger, 223 m), El Hamma (936 m), EM , Aïn Sfa (Berkane, 683 m) Minettia flavipalpis (Loew, 1847) 46 = Sapromyza flavipalpis Loew, 1847, in Becker and Stein 1913 : 93 ( Rif , Tanger), Ebejer and Kettani 2019a : 144 (???) Minettia flaviventris (Costa, 1844) Ebejer and Kettani 2019a , Rif , Azilane (1255 m) Minettia longiseta (Loew, 1847) Ebejer and Kettani 2019a , Rif , Maggou ( NPT , 962 m), Adrou ( PNPB , 556 m) Minettia plumicornis (Fallén, 1820)46 Schacht et al. 2004 ; Ebejer and Kettani 2019a Minettia subvittata (Loew, 1847) Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Azilane (1255 m) Minettia suillorum (Robineau-Desvoidy, 1830) = Minettia muricata Becker, 1895, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2019a , Rif , Adrou (556 m), Perdicaris Park (Tanger, 223 m), HA , Mikdane (Jebel Ayachi) – NHMUK Minettia tabidiventris (Rondani, 1877) Ebejer and Kettani 2019a , Rif , Adrou (556 m), Oued Kbir (Béni Ratene ( PNPB ), 157 m), Oued Koub (Laghdir, 148 m), Rahbat Amlay (284 m) Pachycerina Macquart, 1835 Pachycerina pulchra Loew, 1850 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m) Peplominettia Szilády, 1943 Peplominettia codinai (Hennig, 1951) Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m), Azilane (1255 m) Peplominettia striata Szilády, 1943 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m) Sapromyza Fallén, 1810 Sapromyza apicalis Loew, 1847 Schacht et al. 2004 ; Ebejer and Kettani 2019a , HA , Jebel Ayachi, Mikdane (stream), Lac Tislit (Imlchil, 2254 m) – NHMUK Sapromyza gozmanyi Papp, 1981 Ebejer and Kettani 2019a , Rif , Oued Kbir (Béni Ratene ( PNPB ), 157 m), Perdicaris Park (Tanger, 223 m), Barrage Smir (M'Diq, 27 m) Sapromyza obscuripennis Loew, 1847 Ebejer and Kettani 2019a , Rif , Jebel Talassemtane (1554 m), Jebel Lakraâ (1541 m) Sapromyza unizona Hendel, 1908 Ebejer and Kettani 2019a , Rif , Perdicaris Park (Tanger, 223 m), AP , Larache (5 m) Sapromyza ( Sapromyzosoma ) laevatrispina Carles-Tolrá, 1992 Ebejer and Kettani 2019a , Rif , Adrou (556 m), Issaguen (maison morestière, 1547 m) Sapromyza ( Sapromyzosoma ) parallela Carles-Tolrá, 1992 Ebejer and Kettani 2019a , Rif , Adrou (556 m), Azilane (1255 m), Talassemtane (maison forestière, 1699 m), HA , Jaffar river (Jebel Ayachi, at maison forestière), Mikdane (Jebel Ayachi) – NHMUK CHAMAEMYIIDAE K. Kettani, M.J. Ebejer Number of species: 18 . Expected: 24 Faunistic knowledge of the family in Morocco: moderate Chamaemyiinae Chamaemyia Meigen, 1803 Chamaemyia aridella (Fallén, 1823) Ebejer 2016a , Rif , Moulay Abdelsalam (1180 m), Issaguen (1620 m); HA (Jebel Ayachi, Mikdane, maison forestière, MR Jaffar, Tizi-n'Zou) – NHMUK Chamaemyia flavicornis (Strobl, 1902) Ebejer 2016a , Rif , Martil beach and dunes (on human faeces), MA , Khénifra (17 km NW of Zaida, 1878 m), AA , Errachidia (29 km N of Rich, 1570 m); HA (Asni near Alrene, W Imlil) – NHMUK Chamaemyia flavipalpis (Haliday, 1838) = Chamaemyia maritima Zetterstedt, in Mouna 1998 : 85 Mouna 1998 ; Ebejer 2016a , Rif , Ksar Sghir, Oued Araml, Ksar Sghir, AP , Larache, Merja Zerga, Loukous marsh Chamaemyia herbarum (Robineau-Desvoidy, 1830) Ebejer 2016a , MA , Khénifra, 17 km SW of Midelt (1940 m), AA , Errachidia, 29 km N of Rich (1570 m); HA (Jebel Ayachi, Mikdane, MR Jaffar, Tizi-n'Zou) – NHMUK Chamaemyia juncorum (Fallén, 1958) Ebejer 2016a , MA , Khénifra (17 km SW of Midelt, 1940 m), AA , Errachidia (29 km N of Rich, 1570 m) Chamaemyia polystigma (Meigen, 1830) Becker and Stein 1913 , Rif , Tanger; Ebejer 2016a , Rif , Moulay Abdelsalam (1180 m), Sidi Yahia Aarab (377 m); HA (Asni) – NHMUK Melanochthiphila Frey, 1958 Melanochthiphila sp. Ebejer, 2016 Ebejer 2016a , AA , Errachidia (30 km W of Errachidia, 1065 m) Parochthiphila ( Euestelia ) Enderlein, 1927 Parochthiphila ( Euestelia ) coronata (Loew, 1858) Carles-Tolrá 1993 ; Mouna 1998 ; Ebejer 2016a , AP , Larache (Lower Loukous, 2 m); HA (Jebel Ayachi, Mikdane, MR Jaffar) – NHMUK Parochthiphila frontella (Rondani, 1874) Ebejer 2016a ; HA (Jebel Ayachi, Mikdane, Tizi-n'Zou, MR Jaffar) – NHMUK Parochthiphila inconstans Becker, 1903 Ebejer 2016a , AA , Ziz river (9.5 km SE of Rich, 1285 m), Errachidia (6 km N of Errachidia, 1010 m) Parochthiphila ( Euestelia ) nigripes Strobl, 1900 Ebejer 2016a , Rif , Ksar Sghir, Dardara Leucopinae Leucopis Meigen, 1803 Leucopis annulipes Zetterstedt, 1848 Mouna 1998 ; AP (Maâmora) – MISR Leucopis griseola (Fallén, 1823) Mouna 1998 ; AP (Rabat), MA (Meknès), HA (Marrakech) – MISR Leucopis formosana Hennig, 1938 Ebejer 2016a , AP , Sidi Smail, Oued Tensift (estuary) Leucopis glyphinivora Tanasijtshuk, 1958 Ebejer 2016a , AP , Ksar Sghir, Azemmour, Oued Tensift (estuary), AA , Errachidia (30 km W of Errachidia) Leucopis kerzhneri Tanasijtshuk, 1970 Ebejer 2016a ; HA (Jebel Ayachi) – NHMUK Leucopis palumbii Rondani, 1872 Ebejer 2016a , Rif , Ksar Sghir, Oued Aliane Lipoleucopis de Meijere, 1928 Lipoleucopis pulchra Raspi, 2008 Ebejer 2016a , AA , Errachidia, Ziz river (12 km S of Rissani) Chamaemyiinae Chamaemyia Meigen, 1803 Chamaemyia aridella (Fallén, 1823) Ebejer 2016a , Rif , Moulay Abdelsalam (1180 m), Issaguen (1620 m); HA (Jebel Ayachi, Mikdane, maison forestière, MR Jaffar, Tizi-n'Zou) – NHMUK Chamaemyia flavicornis (Strobl, 1902) Ebejer 2016a , Rif , Martil beach and dunes (on human faeces), MA , Khénifra (17 km NW of Zaida, 1878 m), AA , Errachidia (29 km N of Rich, 1570 m); HA (Asni near Alrene, W Imlil) – NHMUK Chamaemyia flavipalpis (Haliday, 1838) = Chamaemyia maritima Zetterstedt, in Mouna 1998 : 85 Mouna 1998 ; Ebejer 2016a , Rif , Ksar Sghir, Oued Araml, Ksar Sghir, AP , Larache, Merja Zerga, Loukous marsh Chamaemyia herbarum (Robineau-Desvoidy, 1830) Ebejer 2016a , MA , Khénifra, 17 km SW of Midelt (1940 m), AA , Errachidia, 29 km N of Rich (1570 m); HA (Jebel Ayachi, Mikdane, MR Jaffar, Tizi-n'Zou) – NHMUK Chamaemyia juncorum (Fallén, 1958) Ebejer 2016a , MA , Khénifra (17 km SW of Midelt, 1940 m), AA , Errachidia (29 km N of Rich, 1570 m) Chamaemyia polystigma (Meigen, 1830) Becker and Stein 1913 , Rif , Tanger; Ebejer 2016a , Rif , Moulay Abdelsalam (1180 m), Sidi Yahia Aarab (377 m); HA (Asni) – NHMUK Melanochthiphila Frey, 1958 Melanochthiphila sp. Ebejer, 2016 Ebejer 2016a , AA , Errachidia (30 km W of Errachidia, 1065 m) Parochthiphila ( Euestelia ) Enderlein, 1927 Parochthiphila ( Euestelia ) coronata (Loew, 1858) Carles-Tolrá 1993 ; Mouna 1998 ; Ebejer 2016a , AP , Larache (Lower Loukous, 2 m); HA (Jebel Ayachi, Mikdane, MR Jaffar) – NHMUK Parochthiphila frontella (Rondani, 1874) Ebejer 2016a ; HA (Jebel Ayachi, Mikdane, Tizi-n'Zou, MR Jaffar) – NHMUK Parochthiphila inconstans Becker, 1903 Ebejer 2016a , AA , Ziz river (9.5 km SE of Rich, 1285 m), Errachidia (6 km N of Errachidia, 1010 m) Parochthiphila ( Euestelia ) nigripes Strobl, 1900 Ebejer 2016a , Rif , Ksar Sghir, Dardara Leucopinae Leucopis Meigen, 1803 Leucopis annulipes Zetterstedt, 1848 Mouna 1998 ; AP (Maâmora) – MISR Leucopis griseola (Fallén, 1823) Mouna 1998 ; AP (Rabat), MA (Meknès), HA (Marrakech) – MISR Leucopis formosana Hennig, 1938 Ebejer 2016a , AP , Sidi Smail, Oued Tensift (estuary) Leucopis glyphinivora Tanasijtshuk, 1958 Ebejer 2016a , AP , Ksar Sghir, Azemmour, Oued Tensift (estuary), AA , Errachidia (30 km W of Errachidia) Leucopis kerzhneri Tanasijtshuk, 1970 Ebejer 2016a ; HA (Jebel Ayachi) – NHMUK Leucopis palumbii Rondani, 1872 Ebejer 2016a , Rif , Ksar Sghir, Oued Aliane Lipoleucopis de Meijere, 1928 Lipoleucopis pulchra Raspi, 2008 Ebejer 2016a , AA , Errachidia, Ziz river (12 km S of Rissani) LAUXANIIDAE K. Kettani, M.J. Ebejer Number of species: 27 . Expected: 45 Faunistic knowledge of the family in Morocco: good Homoneurinae Homoneura Wulp, 1891 Homoneura ericpoli Carles-Tolrá, 1993 Ebejer and Kettani 2019a , Rif , Azilane (1255 m), Adrou (556 m), HA , Lac Tislit (Imlchil, 2254 m) Homoneura licina Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Schacht et al. 2004 ; Ebejer and Kettani 2019a , Rif , Oued Laou (2 m), AP , Merja Zerga, Sidi Smaine, Safi (estuary of Oued Tensift), Azemmour (El Jadida), Diabat (Essaouira), Larache (5 m) Homoneura transversa (Wiedemann, 1830) Wiedemann 1830 ; Ebejer and Kettani 2019a Prosopomyia Loew, 1856 Prosopomyia pallida Loew, 1856 Vaillant 1956b , HA , Oukaimeden, Imi-N'Ifri; Carles-Tolrá 1993 ; Mouna 1998 ; Ebejer and Kettani 2019a , Rif , Adrou (556 m), Issaguen (1547 m), HA , Jebel Ayachi – NHMUK Lauxaniinae Calliopum Strand, 1928 Calliopum oosterbroeki Shatalkin, 2000 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m), Moulay Abdelsalam (1180 m), Ketama, Rahbat Amlay (284 m), Ikadjiouen (294 m), Mechkralla ( NPT , 981 m), Kharouba (roadside meadow between Chefchaouen and Tétouan, 385 m), Dbani (Béni Selmane, 1046 m), Aïn Ben Ali (Béni Selmane, 1014 m), HA , Lalla Takrkoust (Oued N'fis, 1141 m) – NHMUK Calliopum tuberculosum (Becker, 1895) Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 135 m), Azilane (1255 m), Perdicaris Park (Tanger, 223 m), cascade Chrafate (859 m) Meiosimyza Hendel, 1925 Meiosimyza ( Lyciella ) rorida (Fallén, 1820) = Homoneura rorida Fallén, in Mouna 1998 : 85 Mouna 1998 ; MA (Aïn Leuh) – MISR Minettia Robineau-Desvoidy, 1830 Minettia aenigmatica Ebejer, 2019 Ebejer 2019a , Rif , Perdicaris Park (223 m), Oued Khmis (Khmis Anjra, 61 m), HA , Mikdane (Jebel Ayachi); Ebejer and Kettani 2019a Minettia biseriata (Loew, 1847) Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Jnane Niche (46 m), Oued Kbir (Béni Ratene ( PNPB ), 157 m) Minettia cantolraensis Carles-Tolrá, 1998 Ebejer and Kettani 2019a , Rif , Oued Laou, El-Fahsa, Oued Guallet (Bni Boufrah, 942 m), Oued Ouringa (2 m), Maggou ( NPT , 962 m), Ksar el-Kbir road to Chefchaouen at bridge near oued Azla (80 m), Oued Siflaou (281 m), Dardara (484 m), Azilane (1255 m), Adrou (556 m), Perdicaris Park (Tanger, 223 m), Bni Bahlou (986 m), El Hamma (936 m), Amaghouse (Oued Laou), Oued Koub (Laghdir, 148 m), EM , Aïn Sfa (Berkane, 638 m), Tafoughalt (Berkane, 788 m), HA , Lalla Takrkoust (Oued N'fis, 1141 m), AA , Taliouine (Taroudant, 1049 m) Minettia fasciata (Fallén, 1826) Merz 2004 , AP , Rabat; Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Ksar el-Kbir road to Chefchaouen at bridge near oued Azla (80 m), Azilane (1255 m), Adrou (556 m), Issaguen (1547 m), Oued Taida (Al Andalous, 503 m), Perdicaris Park (Tanger, 223 m), El Hamma (936 m), EM , Aïn Sfa (Berkane, 683 m) Minettia flavipalpis (Loew, 1847) 46 = Sapromyza flavipalpis Loew, 1847, in Becker and Stein 1913 : 93 ( Rif , Tanger), Ebejer and Kettani 2019a : 144 (???) Minettia flaviventris (Costa, 1844) Ebejer and Kettani 2019a , Rif , Azilane (1255 m) Minettia longiseta (Loew, 1847) Ebejer and Kettani 2019a , Rif , Maggou ( NPT , 962 m), Adrou ( PNPB , 556 m) Minettia plumicornis (Fallén, 1820)46 Schacht et al. 2004 ; Ebejer and Kettani 2019a Minettia subvittata (Loew, 1847) Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Azilane (1255 m) Minettia suillorum (Robineau-Desvoidy, 1830) = Minettia muricata Becker, 1895, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2019a , Rif , Adrou (556 m), Perdicaris Park (Tanger, 223 m), HA , Mikdane (Jebel Ayachi) – NHMUK Minettia tabidiventris (Rondani, 1877) Ebejer and Kettani 2019a , Rif , Adrou (556 m), Oued Kbir (Béni Ratene ( PNPB ), 157 m), Oued Koub (Laghdir, 148 m), Rahbat Amlay (284 m) Pachycerina Macquart, 1835 Pachycerina pulchra Loew, 1850 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m) Peplominettia Szilády, 1943 Peplominettia codinai (Hennig, 1951) Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m), Azilane (1255 m) Peplominettia striata Szilády, 1943 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m) Sapromyza Fallén, 1810 Sapromyza apicalis Loew, 1847 Schacht et al. 2004 ; Ebejer and Kettani 2019a , HA , Jebel Ayachi, Mikdane (stream), Lac Tislit (Imlchil, 2254 m) – NHMUK Sapromyza gozmanyi Papp, 1981 Ebejer and Kettani 2019a , Rif , Oued Kbir (Béni Ratene ( PNPB ), 157 m), Perdicaris Park (Tanger, 223 m), Barrage Smir (M'Diq, 27 m) Sapromyza obscuripennis Loew, 1847 Ebejer and Kettani 2019a , Rif , Jebel Talassemtane (1554 m), Jebel Lakraâ (1541 m) Sapromyza unizona Hendel, 1908 Ebejer and Kettani 2019a , Rif , Perdicaris Park (Tanger, 223 m), AP , Larache (5 m) Sapromyza ( Sapromyzosoma ) laevatrispina Carles-Tolrá, 1992 Ebejer and Kettani 2019a , Rif , Adrou (556 m), Issaguen (maison morestière, 1547 m) Sapromyza ( Sapromyzosoma ) parallela Carles-Tolrá, 1992 Ebejer and Kettani 2019a , Rif , Adrou (556 m), Azilane (1255 m), Talassemtane (maison forestière, 1699 m), HA , Jaffar river (Jebel Ayachi, at maison forestière), Mikdane (Jebel Ayachi) – NHMUK Homoneurinae Homoneura Wulp, 1891 Homoneura ericpoli Carles-Tolrá, 1993 Ebejer and Kettani 2019a , Rif , Azilane (1255 m), Adrou (556 m), HA , Lac Tislit (Imlchil, 2254 m) Homoneura licina Séguy, 1941 Séguy 1941d , AA , Agadir; Mouna 1998 ; Schacht et al. 2004 ; Ebejer and Kettani 2019a , Rif , Oued Laou (2 m), AP , Merja Zerga, Sidi Smaine, Safi (estuary of Oued Tensift), Azemmour (El Jadida), Diabat (Essaouira), Larache (5 m) Homoneura transversa (Wiedemann, 1830) Wiedemann 1830 ; Ebejer and Kettani 2019a Prosopomyia Loew, 1856 Prosopomyia pallida Loew, 1856 Vaillant 1956b , HA , Oukaimeden, Imi-N'Ifri; Carles-Tolrá 1993 ; Mouna 1998 ; Ebejer and Kettani 2019a , Rif , Adrou (556 m), Issaguen (1547 m), HA , Jebel Ayachi – NHMUK Lauxaniinae Calliopum Strand, 1928 Calliopum oosterbroeki Shatalkin, 2000 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m), Moulay Abdelsalam (1180 m), Ketama, Rahbat Amlay (284 m), Ikadjiouen (294 m), Mechkralla ( NPT , 981 m), Kharouba (roadside meadow between Chefchaouen and Tétouan, 385 m), Dbani (Béni Selmane, 1046 m), Aïn Ben Ali (Béni Selmane, 1014 m), HA , Lalla Takrkoust (Oued N'fis, 1141 m) – NHMUK Calliopum tuberculosum (Becker, 1895) Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 135 m), Azilane (1255 m), Perdicaris Park (Tanger, 223 m), cascade Chrafate (859 m) Meiosimyza Hendel, 1925 Meiosimyza ( Lyciella ) rorida (Fallén, 1820) = Homoneura rorida Fallén, in Mouna 1998 : 85 Mouna 1998 ; MA (Aïn Leuh) – MISR Minettia Robineau-Desvoidy, 1830 Minettia aenigmatica Ebejer, 2019 Ebejer 2019a , Rif , Perdicaris Park (223 m), Oued Khmis (Khmis Anjra, 61 m), HA , Mikdane (Jebel Ayachi); Ebejer and Kettani 2019a Minettia biseriata (Loew, 1847) Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Jnane Niche (46 m), Oued Kbir (Béni Ratene ( PNPB ), 157 m) Minettia cantolraensis Carles-Tolrá, 1998 Ebejer and Kettani 2019a , Rif , Oued Laou, El-Fahsa, Oued Guallet (Bni Boufrah, 942 m), Oued Ouringa (2 m), Maggou ( NPT , 962 m), Ksar el-Kbir road to Chefchaouen at bridge near oued Azla (80 m), Oued Siflaou (281 m), Dardara (484 m), Azilane (1255 m), Adrou (556 m), Perdicaris Park (Tanger, 223 m), Bni Bahlou (986 m), El Hamma (936 m), Amaghouse (Oued Laou), Oued Koub (Laghdir, 148 m), EM , Aïn Sfa (Berkane, 638 m), Tafoughalt (Berkane, 788 m), HA , Lalla Takrkoust (Oued N'fis, 1141 m), AA , Taliouine (Taroudant, 1049 m) Minettia fasciata (Fallén, 1826) Merz 2004 , AP , Rabat; Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Ksar el-Kbir road to Chefchaouen at bridge near oued Azla (80 m), Azilane (1255 m), Adrou (556 m), Issaguen (1547 m), Oued Taida (Al Andalous, 503 m), Perdicaris Park (Tanger, 223 m), El Hamma (936 m), EM , Aïn Sfa (Berkane, 683 m) Minettia flavipalpis (Loew, 1847) 46 = Sapromyza flavipalpis Loew, 1847, in Becker and Stein 1913 : 93 ( Rif , Tanger), Ebejer and Kettani 2019a : 144 (???) Minettia flaviventris (Costa, 1844) Ebejer and Kettani 2019a , Rif , Azilane (1255 m) Minettia longiseta (Loew, 1847) Ebejer and Kettani 2019a , Rif , Maggou ( NPT , 962 m), Adrou ( PNPB , 556 m) Minettia plumicornis (Fallén, 1820)46 Schacht et al. 2004 ; Ebejer and Kettani 2019a Minettia subvittata (Loew, 1847) Ebejer and Kettani 2019a , Rif , Oued Khmis (Khmis Anjra, 61 m), Azilane (1255 m) Minettia suillorum (Robineau-Desvoidy, 1830) = Minettia muricata Becker, 1895, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2019a , Rif , Adrou (556 m), Perdicaris Park (Tanger, 223 m), HA , Mikdane (Jebel Ayachi) – NHMUK Minettia tabidiventris (Rondani, 1877) Ebejer and Kettani 2019a , Rif , Adrou (556 m), Oued Kbir (Béni Ratene ( PNPB ), 157 m), Oued Koub (Laghdir, 148 m), Rahbat Amlay (284 m) Pachycerina Macquart, 1835 Pachycerina pulchra Loew, 1850 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m) Peplominettia Szilády, 1943 Peplominettia codinai (Hennig, 1951) Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m), Azilane (1255 m) Peplominettia striata Szilády, 1943 Ebejer and Kettani 2019a , Rif , Oued Zarka (Yarghite, 137 m) Sapromyza Fallén, 1810 Sapromyza apicalis Loew, 1847 Schacht et al. 2004 ; Ebejer and Kettani 2019a , HA , Jebel Ayachi, Mikdane (stream), Lac Tislit (Imlchil, 2254 m) – NHMUK Sapromyza gozmanyi Papp, 1981 Ebejer and Kettani 2019a , Rif , Oued Kbir (Béni Ratene ( PNPB ), 157 m), Perdicaris Park (Tanger, 223 m), Barrage Smir (M'Diq, 27 m) Sapromyza obscuripennis Loew, 1847 Ebejer and Kettani 2019a , Rif , Jebel Talassemtane (1554 m), Jebel Lakraâ (1541 m) Sapromyza unizona Hendel, 1908 Ebejer and Kettani 2019a , Rif , Perdicaris Park (Tanger, 223 m), AP , Larache (5 m) Sapromyza ( Sapromyzosoma ) laevatrispina Carles-Tolrá, 1992 Ebejer and Kettani 2019a , Rif , Adrou (556 m), Issaguen (maison morestière, 1547 m) Sapromyza ( Sapromyzosoma ) parallela Carles-Tolrá, 1992 Ebejer and Kettani 2019a , Rif , Adrou (556 m), Azilane (1255 m), Talassemtane (maison forestière, 1699 m), HA , Jaffar river (Jebel Ayachi, at maison forestière), Mikdane (Jebel Ayachi) – NHMUK Sciomyzoidea COELOPIDAE K. Kettani Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: moderate Coelopinae Coelopa Meigen, 1830 Coelopa pilipes Haliday, 1838 Séguy 1930a , Rif , Tanger; Dakki 1997 ; Lair 2013 DRYOMYZIDAE K. Kettani Number of species: 1 . Expected: 1 Faunistic knowledge of the family in Morocco: poor Dryomyzinae Dryope Robineau-Desvoidy, 1830 Dryope flaveola (Fabricius, 1794) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1596 m) HELCOMYZIDAE K. Kettani Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Helcomyzinae Helcomyza Curtis, 1825 Helcomyza ustulata Curtis, 1825 Cassar et al. 2005 , Rif , Smir lagoon SCIOMYZIDAE K. Kettani, J-C. Vala Number of species: 25 . Expected: 40 Faunistic knowledge of the family in Morocco: good Sciomyzinae Sciomyzini Ditaeniella Sack, 1939 Ditaeniella grisescens (Meigen, 1830) Vala and Ghamizi 1991 , MA , Ras el Ma, AA , Agadir; Kassebeer 1999a , MA , HA ; Knutson and Vala 2011 Pherbellia Robineau-Desvoidy, 1830 Pherbellia cinerella (Fallén, 1820) = Ditaenia cinerella Fallén, in Séguy 1941a : 31 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m), Imi-n'Ouaka (1500 m); Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt, HA , Tizi-n'Test (1900 m); Rozkošný 1987 , HA ; Vala and Ghamizi 1991 , HA , MA ; Carles-Tolrá 1993 ; Kassebeer 1999a , MA , HA ; Rif (Talassemtane) – MISR Pherbellia dorsata (Zetterstedt, 1846) Kassebeer 1999a , MA ; Knutson and Vala 2011 Pherbellia griseola (Fallén, 1820) Kassebeer 1999a , MA ; Knutson and Vala 2011 Pherbellia hermonensis Knutson & Freidberg, 1983 Kassebeer 1999a , HA ; Knutson and Vala 2011 Pherbellia nana (Fallén, 1820) = Pherbellia villiersi Séguy, in Séguy 1941a : 31; Knutson 1981 : 336 (new comb.) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Knutson 1981 , HA , Tachdirt; Leclercq and Schacht 1987 , HA ; Vala and Ghamizi 1989, HA ; Kassebeer 1999a , MA , HA ; Knutson and Vala 2011 ; HA – MISR Tetanocerini Dichaetophora Rondani, 1868 Dichaetophora obliterata (Fabricius, 1805) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Leclercq and Schacht 1987 ; Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 ; Kassebeer 1999a Elgiva Meigen, 1838 Elgiva cucularia (Linnaeus, 1767) Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Vala 1989 ; Kassebeer 1999a Euthycera Latreille, 1829 Euthycera algira (Macquart, 1849) Vala and Reidenbach 1982 , Rif , Tanger; Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 ; Kassebeer 1999a , Rif , Tanger Euthycera soror (Robineau-Desvoidy, 1830) = Euthycera alaris Vala, 1983, syn. nov.Vala (pers. comm.) Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 , MA ; Carles-Tolrá 1993 ; Kassebeer 1999a , MA , HA Euthycera stichospila (Czerny, 1909) = Euthycera leclercqi Vala & Reidenbach, 1982, syn. by Rozkošný (1987) Czerny and Strobl 1909 ; Vala and Reidenbach 1982 ; Rozkošný 1987 Euthycera zelleri (Loew, 1847) Kassebeer 1999a , Rif , Tanger Hydromya Robineau-Desvoidy, 1830 Hydromya dorsalis (Fabricius, 1775) Séguy 1930a , Rif , Tanger; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m), HA , Oukaimeden (2600 m); Dakki 1997 , Rif , Ketama; Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 , MA , HA ; Kassebeer 1999a , MA , HA ; Pârvu et al. 2006 , AP , Merja Zerga Ilione Haliday in Curtis, 1837 Ilione albiseta (Scopoli, 1763) Leclercq and Schacht 1987 , HA ; Vala 1989 ; Kassebeer 1999a Ilione trifaria (Loew, 1820) Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Vala 1989 , MA , Dayat Aoua; Vala and Ghamizi 1991 , EM , MA , HA ; Carles-Tolrá 1993 Ilione unipunctata (Macquart, 1849) Leclercq and Schacht 1987 , HA ; Rozkošný 1987 ; Vala 1989 ; Kassebeer 1999a Oligolimnia Mayer, 1953 Oligolimnia zernyi Mayer, 1953 Rozkošný 1987 , HA , Tachdirt; Leclercq and Schacht 1987 , HA ; Vala 1989 , HA ; Kassebeer 1999a , HA Pherbina Robineau-Desvoidy, 1830 Pherbina coryleti (Scopoli, 1763) Séguy 1930a , Rif , Tanger; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; HA (Issougane n'Ouagouns) – MISR Pherbina mediterranea Mayer, 1953 Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 , AP , Sidi Boughaba, HA , south of Marrakech; Carles-Tolrá 1993 ; Kassebeer 1999a , MA Psacadina Enderlein, 1939 Psacadina disjecta Enderlein, 1939 Verbeke 1964 , AA , Tlata Reisana; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Kassebeer 1999a , MA Psacadina verbekei Rozkošný, 1975 Vala and Ghamizi 1991 , HA , south of Marrakech; Kassebeer 1999a Sepedon Latreille, 1804 Sepedon hispanica Loew, 1862 Verbeke 1964 ; Vala 1989 , AP , Mohammedia; Vala and Ghamizi 1991 , AP , Mohammedia, HA , Oued Tissaout (Kelaâ Sraghna); Kassebeer 1999a Sepedon sphegea (Fabricius, 1775) Séguy 1930a Rif , Tanger; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 , HA , Tamesloht (south of Marrakech), AA , Bou Acheiba (Agadir); Carles-Tolrá 1993 ; Dakki 1997 ; Kassebeer 1999a Sepedon spinipes (Scopoli, 1763) Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Vala and Ghamizi 1991 , MA , Imouzzer, Tifounassine; Carles-Tolrá 1993 ; Kassebeer 1999a , MA ; Knutson and Vala 2011 Trypetoptera Hendel, 1900 Trypetoptera punctulata (Scopoli, 1763) Leclercq and Schacht 1987 , HA ; Vala 1989 ; Kassebeer 1999a ; Knutson and Vala 2011 SEPSIDAE K. Kettani, J.-P. Haenni Number of species: 12 . Expected: 20 Faunistic knowledge of the family in Morocco: poor Sepsinae Saltellini Saltella Robineau-Desvoidy, 1830 Saltella sphondylii (Schrank, 1803) Ebejer et al. 2019 , Rif , Barrage Smir (145 m), Oued Mhajrate (Ben Karrich, 180 m) Sepsini Nemopoda Robineau-Desvoidy, 1830 Nemopoda nitidula (Fallén, 1820) Mouna 1998 (no locality given) Sepsis Fallén, 1810 Sepsis biflexuosa Strobl, 1893 Zuska and Pont 1984; Mouna 1998 ; Pont and Meier 2002 ; Ozerov 2005 ; HA (Asni) – NHMUK Sepsis cynipsea (Linnaeus, 1758) Séguy 1930a , AP , Rabat, MA , HA , Marrakech; Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Zuska and Pont 1984; Mouna 1998 Sepsis flavimana Meigen, 1826 Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Sepsis fulgens Meigen, 1826 Séguy 1941d , HA , Tizi-n'Test (2000 m), AA , Agadir; Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 , MA , Ifrane; MA (Ifrane), HA (Asni, near Alrene, Mikdane) – NHMUK Sepsis lateralis Wiedemann, 1830 Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Zuska and Pont 1984; Mouna 1998 ; AP (Sale Tropical Garden), HA (Marrakech) – NHMUK Sepsis punctum (Fabricius, 1794) Séguy 1941a , HA , Aït Souka (Toubkal); Zuska and Pont 1984; Mouna 1998 ; Pont and Meier 2002 , Rif , Tanger, AP , Rabat; Pârvu et al. 2006 , AP , Merja Zerga – MISR ; MA (Ifrane), HA (Asni, Mikdane), AA (Souss Massa, Agadir) – NHMUK Sepsis thoracica (Robineau-Desvoidy, 1830) Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; MA (near Azrou), HA (Mikdane, Marrakech, Amizmiz) – NHMUK Sepsis violacea Meigen, 1826 = Sepsis ciliforceps Duda, 1926, in Mouna 1998 : 86 Becker and Stein 1913 , Rif , Tanger; Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 – MISR Themira Robineau-Desvoidy, 1830 Themira minor (Haliday, 1833) Zuska and Pont 1984; Mouna 1998 (no locality given); HA (Mikdane) – NHMUK Themira paludosa Elberg, 1963 47 Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 An additional species, Australosepsis niveipennis (Becker, 1903) is reported from Morocco in major catalogues (Pont and Meier 1984, Ozerov 2005 ). However this report is based upon a misinterpretation by Zuska (1968) of a name of locality given in Duda (1926b: 2): "Marako" [a locality in Mali, or possibly another in Ethiopia] was wrongly understood for "Marokko", the German name of Morocco. Both Adrian Pont and Andrey Ozerov have confirmed (pers. comm. 27.xi.15) that they have not seen any Moroccan specimen of A. niveipennis . The Moroccan records from NHMUK were checked by Adrian Pont to whom we express our grateful thanks for his kind help. COELOPIDAE K. Kettani Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: moderate Coelopinae Coelopa Meigen, 1830 Coelopa pilipes Haliday, 1838 Séguy 1930a , Rif , Tanger; Dakki 1997 ; Lair 2013 Coelopinae Coelopa Meigen, 1830 Coelopa pilipes Haliday, 1838 Séguy 1930a , Rif , Tanger; Dakki 1997 ; Lair 2013 DRYOMYZIDAE K. Kettani Number of species: 1 . Expected: 1 Faunistic knowledge of the family in Morocco: poor Dryomyzinae Dryope Robineau-Desvoidy, 1830 Dryope flaveola (Fabricius, 1794) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1596 m) Dryomyzinae Dryope Robineau-Desvoidy, 1830 Dryope flaveola (Fabricius, 1794) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1596 m) HELCOMYZIDAE K. Kettani Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Helcomyzinae Helcomyza Curtis, 1825 Helcomyza ustulata Curtis, 1825 Cassar et al. 2005 , Rif , Smir lagoon Helcomyzinae Helcomyza Curtis, 1825 Helcomyza ustulata Curtis, 1825 Cassar et al. 2005 , Rif , Smir lagoon SCIOMYZIDAE K. Kettani, J-C. Vala Number of species: 25 . Expected: 40 Faunistic knowledge of the family in Morocco: good Sciomyzinae Sciomyzini Ditaeniella Sack, 1939 Ditaeniella grisescens (Meigen, 1830) Vala and Ghamizi 1991 , MA , Ras el Ma, AA , Agadir; Kassebeer 1999a , MA , HA ; Knutson and Vala 2011 Pherbellia Robineau-Desvoidy, 1830 Pherbellia cinerella (Fallén, 1820) = Ditaenia cinerella Fallén, in Séguy 1941a : 31 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m), Imi-n'Ouaka (1500 m); Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt, HA , Tizi-n'Test (1900 m); Rozkošný 1987 , HA ; Vala and Ghamizi 1991 , HA , MA ; Carles-Tolrá 1993 ; Kassebeer 1999a , MA , HA ; Rif (Talassemtane) – MISR Pherbellia dorsata (Zetterstedt, 1846) Kassebeer 1999a , MA ; Knutson and Vala 2011 Pherbellia griseola (Fallén, 1820) Kassebeer 1999a , MA ; Knutson and Vala 2011 Pherbellia hermonensis Knutson & Freidberg, 1983 Kassebeer 1999a , HA ; Knutson and Vala 2011 Pherbellia nana (Fallén, 1820) = Pherbellia villiersi Séguy, in Séguy 1941a : 31; Knutson 1981 : 336 (new comb.) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Knutson 1981 , HA , Tachdirt; Leclercq and Schacht 1987 , HA ; Vala and Ghamizi 1989, HA ; Kassebeer 1999a , MA , HA ; Knutson and Vala 2011 ; HA – MISR Tetanocerini Dichaetophora Rondani, 1868 Dichaetophora obliterata (Fabricius, 1805) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Leclercq and Schacht 1987 ; Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 ; Kassebeer 1999a Elgiva Meigen, 1838 Elgiva cucularia (Linnaeus, 1767) Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Vala 1989 ; Kassebeer 1999a Euthycera Latreille, 1829 Euthycera algira (Macquart, 1849) Vala and Reidenbach 1982 , Rif , Tanger; Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 ; Kassebeer 1999a , Rif , Tanger Euthycera soror (Robineau-Desvoidy, 1830) = Euthycera alaris Vala, 1983, syn. nov.Vala (pers. comm.) Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 , MA ; Carles-Tolrá 1993 ; Kassebeer 1999a , MA , HA Euthycera stichospila (Czerny, 1909) = Euthycera leclercqi Vala & Reidenbach, 1982, syn. by Rozkošný (1987) Czerny and Strobl 1909 ; Vala and Reidenbach 1982 ; Rozkošný 1987 Euthycera zelleri (Loew, 1847) Kassebeer 1999a , Rif , Tanger Hydromya Robineau-Desvoidy, 1830 Hydromya dorsalis (Fabricius, 1775) Séguy 1930a , Rif , Tanger; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m), HA , Oukaimeden (2600 m); Dakki 1997 , Rif , Ketama; Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 , MA , HA ; Kassebeer 1999a , MA , HA ; Pârvu et al. 2006 , AP , Merja Zerga Ilione Haliday in Curtis, 1837 Ilione albiseta (Scopoli, 1763) Leclercq and Schacht 1987 , HA ; Vala 1989 ; Kassebeer 1999a Ilione trifaria (Loew, 1820) Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Vala 1989 , MA , Dayat Aoua; Vala and Ghamizi 1991 , EM , MA , HA ; Carles-Tolrá 1993 Ilione unipunctata (Macquart, 1849) Leclercq and Schacht 1987 , HA ; Rozkošný 1987 ; Vala 1989 ; Kassebeer 1999a Oligolimnia Mayer, 1953 Oligolimnia zernyi Mayer, 1953 Rozkošný 1987 , HA , Tachdirt; Leclercq and Schacht 1987 , HA ; Vala 1989 , HA ; Kassebeer 1999a , HA Pherbina Robineau-Desvoidy, 1830 Pherbina coryleti (Scopoli, 1763) Séguy 1930a , Rif , Tanger; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; HA (Issougane n'Ouagouns) – MISR Pherbina mediterranea Mayer, 1953 Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 , AP , Sidi Boughaba, HA , south of Marrakech; Carles-Tolrá 1993 ; Kassebeer 1999a , MA Psacadina Enderlein, 1939 Psacadina disjecta Enderlein, 1939 Verbeke 1964 , AA , Tlata Reisana; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Kassebeer 1999a , MA Psacadina verbekei Rozkošný, 1975 Vala and Ghamizi 1991 , HA , south of Marrakech; Kassebeer 1999a Sepedon Latreille, 1804 Sepedon hispanica Loew, 1862 Verbeke 1964 ; Vala 1989 , AP , Mohammedia; Vala and Ghamizi 1991 , AP , Mohammedia, HA , Oued Tissaout (Kelaâ Sraghna); Kassebeer 1999a Sepedon sphegea (Fabricius, 1775) Séguy 1930a Rif , Tanger; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 , HA , Tamesloht (south of Marrakech), AA , Bou Acheiba (Agadir); Carles-Tolrá 1993 ; Dakki 1997 ; Kassebeer 1999a Sepedon spinipes (Scopoli, 1763) Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Vala and Ghamizi 1991 , MA , Imouzzer, Tifounassine; Carles-Tolrá 1993 ; Kassebeer 1999a , MA ; Knutson and Vala 2011 Trypetoptera Hendel, 1900 Trypetoptera punctulata (Scopoli, 1763) Leclercq and Schacht 1987 , HA ; Vala 1989 ; Kassebeer 1999a ; Knutson and Vala 2011 Sciomyzinae Sciomyzini Ditaeniella Sack, 1939 Ditaeniella grisescens (Meigen, 1830) Vala and Ghamizi 1991 , MA , Ras el Ma, AA , Agadir; Kassebeer 1999a , MA , HA ; Knutson and Vala 2011 Pherbellia Robineau-Desvoidy, 1830 Pherbellia cinerella (Fallén, 1820) = Ditaenia cinerella Fallén, in Séguy 1941a : 31 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m), Imi-n'Ouaka (1500 m); Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt, HA , Tizi-n'Test (1900 m); Rozkošný 1987 , HA ; Vala and Ghamizi 1991 , HA , MA ; Carles-Tolrá 1993 ; Kassebeer 1999a , MA , HA ; Rif (Talassemtane) – MISR Pherbellia dorsata (Zetterstedt, 1846) Kassebeer 1999a , MA ; Knutson and Vala 2011 Pherbellia griseola (Fallén, 1820) Kassebeer 1999a , MA ; Knutson and Vala 2011 Pherbellia hermonensis Knutson & Freidberg, 1983 Kassebeer 1999a , HA ; Knutson and Vala 2011 Pherbellia nana (Fallén, 1820) = Pherbellia villiersi Séguy, in Séguy 1941a : 31; Knutson 1981 : 336 (new comb.) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Knutson 1981 , HA , Tachdirt; Leclercq and Schacht 1987 , HA ; Vala and Ghamizi 1989, HA ; Kassebeer 1999a , MA , HA ; Knutson and Vala 2011 ; HA – MISR Tetanocerini Dichaetophora Rondani, 1868 Dichaetophora obliterata (Fabricius, 1805) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Leclercq and Schacht 1987 ; Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 ; Kassebeer 1999a Elgiva Meigen, 1838 Elgiva cucularia (Linnaeus, 1767) Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Vala 1989 ; Kassebeer 1999a Euthycera Latreille, 1829 Euthycera algira (Macquart, 1849) Vala and Reidenbach 1982 , Rif , Tanger; Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 ; Kassebeer 1999a , Rif , Tanger Euthycera soror (Robineau-Desvoidy, 1830) = Euthycera alaris Vala, 1983, syn. nov.Vala (pers. comm.) Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 , MA ; Carles-Tolrá 1993 ; Kassebeer 1999a , MA , HA Euthycera stichospila (Czerny, 1909) = Euthycera leclercqi Vala & Reidenbach, 1982, syn. by Rozkošný (1987) Czerny and Strobl 1909 ; Vala and Reidenbach 1982 ; Rozkošný 1987 Euthycera zelleri (Loew, 1847) Kassebeer 1999a , Rif , Tanger Hydromya Robineau-Desvoidy, 1830 Hydromya dorsalis (Fabricius, 1775) Séguy 1930a , Rif , Tanger; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m), HA , Oukaimeden (2600 m); Dakki 1997 , Rif , Ketama; Vala 1989 , Rif , Tanger; Vala and Ghamizi 1991 , MA , HA ; Kassebeer 1999a , MA , HA ; Pârvu et al. 2006 , AP , Merja Zerga Ilione Haliday in Curtis, 1837 Ilione albiseta (Scopoli, 1763) Leclercq and Schacht 1987 , HA ; Vala 1989 ; Kassebeer 1999a Ilione trifaria (Loew, 1820) Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Vala 1989 , MA , Dayat Aoua; Vala and Ghamizi 1991 , EM , MA , HA ; Carles-Tolrá 1993 Ilione unipunctata (Macquart, 1849) Leclercq and Schacht 1987 , HA ; Rozkošný 1987 ; Vala 1989 ; Kassebeer 1999a Oligolimnia Mayer, 1953 Oligolimnia zernyi Mayer, 1953 Rozkošný 1987 , HA , Tachdirt; Leclercq and Schacht 1987 , HA ; Vala 1989 , HA ; Kassebeer 1999a , HA Pherbina Robineau-Desvoidy, 1830 Pherbina coryleti (Scopoli, 1763) Séguy 1930a , Rif , Tanger; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; HA (Issougane n'Ouagouns) – MISR Pherbina mediterranea Mayer, 1953 Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 , AP , Sidi Boughaba, HA , south of Marrakech; Carles-Tolrá 1993 ; Kassebeer 1999a , MA Psacadina Enderlein, 1939 Psacadina disjecta Enderlein, 1939 Verbeke 1964 , AA , Tlata Reisana; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Kassebeer 1999a , MA Psacadina verbekei Rozkošný, 1975 Vala and Ghamizi 1991 , HA , south of Marrakech; Kassebeer 1999a Sepedon Latreille, 1804 Sepedon hispanica Loew, 1862 Verbeke 1964 ; Vala 1989 , AP , Mohammedia; Vala and Ghamizi 1991 , AP , Mohammedia, HA , Oued Tissaout (Kelaâ Sraghna); Kassebeer 1999a Sepedon sphegea (Fabricius, 1775) Séguy 1930a Rif , Tanger; Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Rozkošný 1987 ; Vala 1989 ; Vala and Ghamizi 1991 , HA , Tamesloht (south of Marrakech), AA , Bou Acheiba (Agadir); Carles-Tolrá 1993 ; Dakki 1997 ; Kassebeer 1999a Sepedon spinipes (Scopoli, 1763) Leclercq and Schacht 1987 , HA , Ansegmir-Tal, W Midelt (1400 m); Vala and Ghamizi 1991 , MA , Imouzzer, Tifounassine; Carles-Tolrá 1993 ; Kassebeer 1999a , MA ; Knutson and Vala 2011 Trypetoptera Hendel, 1900 Trypetoptera punctulata (Scopoli, 1763) Leclercq and Schacht 1987 , HA ; Vala 1989 ; Kassebeer 1999a ; Knutson and Vala 2011 SEPSIDAE K. Kettani, J.-P. Haenni Number of species: 12 . Expected: 20 Faunistic knowledge of the family in Morocco: poor Sepsinae Saltellini Saltella Robineau-Desvoidy, 1830 Saltella sphondylii (Schrank, 1803) Ebejer et al. 2019 , Rif , Barrage Smir (145 m), Oued Mhajrate (Ben Karrich, 180 m) Sepsini Nemopoda Robineau-Desvoidy, 1830 Nemopoda nitidula (Fallén, 1820) Mouna 1998 (no locality given) Sepsis Fallén, 1810 Sepsis biflexuosa Strobl, 1893 Zuska and Pont 1984; Mouna 1998 ; Pont and Meier 2002 ; Ozerov 2005 ; HA (Asni) – NHMUK Sepsis cynipsea (Linnaeus, 1758) Séguy 1930a , AP , Rabat, MA , HA , Marrakech; Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Zuska and Pont 1984; Mouna 1998 Sepsis flavimana Meigen, 1826 Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Sepsis fulgens Meigen, 1826 Séguy 1941d , HA , Tizi-n'Test (2000 m), AA , Agadir; Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 , MA , Ifrane; MA (Ifrane), HA (Asni, near Alrene, Mikdane) – NHMUK Sepsis lateralis Wiedemann, 1830 Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Zuska and Pont 1984; Mouna 1998 ; AP (Sale Tropical Garden), HA (Marrakech) – NHMUK Sepsis punctum (Fabricius, 1794) Séguy 1941a , HA , Aït Souka (Toubkal); Zuska and Pont 1984; Mouna 1998 ; Pont and Meier 2002 , Rif , Tanger, AP , Rabat; Pârvu et al. 2006 , AP , Merja Zerga – MISR ; MA (Ifrane), HA (Asni, Mikdane), AA (Souss Massa, Agadir) – NHMUK Sepsis thoracica (Robineau-Desvoidy, 1830) Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; MA (near Azrou), HA (Mikdane, Marrakech, Amizmiz) – NHMUK Sepsis violacea Meigen, 1826 = Sepsis ciliforceps Duda, 1926, in Mouna 1998 : 86 Becker and Stein 1913 , Rif , Tanger; Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 – MISR Themira Robineau-Desvoidy, 1830 Themira minor (Haliday, 1833) Zuska and Pont 1984; Mouna 1998 (no locality given); HA (Mikdane) – NHMUK Themira paludosa Elberg, 1963 47 Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 An additional species, Australosepsis niveipennis (Becker, 1903) is reported from Morocco in major catalogues (Pont and Meier 1984, Ozerov 2005 ). However this report is based upon a misinterpretation by Zuska (1968) of a name of locality given in Duda (1926b: 2): "Marako" [a locality in Mali, or possibly another in Ethiopia] was wrongly understood for "Marokko", the German name of Morocco. Both Adrian Pont and Andrey Ozerov have confirmed (pers. comm. 27.xi.15) that they have not seen any Moroccan specimen of A. niveipennis . The Moroccan records from NHMUK were checked by Adrian Pont to whom we express our grateful thanks for his kind help. Sepsinae Saltellini Saltella Robineau-Desvoidy, 1830 Saltella sphondylii (Schrank, 1803) Ebejer et al. 2019 , Rif , Barrage Smir (145 m), Oued Mhajrate (Ben Karrich, 180 m) Sepsini Nemopoda Robineau-Desvoidy, 1830 Nemopoda nitidula (Fallén, 1820) Mouna 1998 (no locality given) Sepsis Fallén, 1810 Sepsis biflexuosa Strobl, 1893 Zuska and Pont 1984; Mouna 1998 ; Pont and Meier 2002 ; Ozerov 2005 ; HA (Asni) – NHMUK Sepsis cynipsea (Linnaeus, 1758) Séguy 1930a , AP , Rabat, MA , HA , Marrakech; Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Zuska and Pont 1984; Mouna 1998 Sepsis flavimana Meigen, 1826 Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Sepsis fulgens Meigen, 1826 Séguy 1941d , HA , Tizi-n'Test (2000 m), AA , Agadir; Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 , MA , Ifrane; MA (Ifrane), HA (Asni, near Alrene, Mikdane) – NHMUK Sepsis lateralis Wiedemann, 1830 Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Zuska and Pont 1984; Mouna 1998 ; AP (Sale Tropical Garden), HA (Marrakech) – NHMUK Sepsis punctum (Fabricius, 1794) Séguy 1941a , HA , Aït Souka (Toubkal); Zuska and Pont 1984; Mouna 1998 ; Pont and Meier 2002 , Rif , Tanger, AP , Rabat; Pârvu et al. 2006 , AP , Merja Zerga – MISR ; MA (Ifrane), HA (Asni, Mikdane), AA (Souss Massa, Agadir) – NHMUK Sepsis thoracica (Robineau-Desvoidy, 1830) Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 ; MA (near Azrou), HA (Mikdane, Marrakech, Amizmiz) – NHMUK Sepsis violacea Meigen, 1826 = Sepsis ciliforceps Duda, 1926, in Mouna 1998 : 86 Becker and Stein 1913 , Rif , Tanger; Zuska and Pont 1984; Mouna 1998 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 – MISR Themira Robineau-Desvoidy, 1830 Themira minor (Haliday, 1833) Zuska and Pont 1984; Mouna 1998 (no locality given); HA (Mikdane) – NHMUK Themira paludosa Elberg, 1963 47 Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 An additional species, Australosepsis niveipennis (Becker, 1903) is reported from Morocco in major catalogues (Pont and Meier 1984, Ozerov 2005 ). However this report is based upon a misinterpretation by Zuska (1968) of a name of locality given in Duda (1926b: 2): "Marako" [a locality in Mali, or possibly another in Ethiopia] was wrongly understood for "Marokko", the German name of Morocco. Both Adrian Pont and Andrey Ozerov have confirmed (pers. comm. 27.xi.15) that they have not seen any Moroccan specimen of A. niveipennis . The Moroccan records from NHMUK were checked by Adrian Pont to whom we express our grateful thanks for his kind help. Opomyzoidea AGROMYZIDAE K. Kettani, M. Černý Number of species: 62 . Expected: 150 Faunistic knowledge of the family in Morocco: poor Agromyzinae Agromyza Fallén, 1810 Agromyza abiens Zetterstedt, 1848 Spencer 1967 , HA , Ourika Valley, Marrakech; Černý and Merz 2006 Agromyza albipennis Meigen, 1830 Séguy 1936b ; Mouna 1998 Agromyza ambigua Fallén, 1823 = Domomyza ambigua Fallén, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Agromyza bicaudata (Hendel, 1920) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza frontella (Rondani, 1875) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza frontosa (Becker, 1908) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza hiemalis Becker, 1908 Spencer 1967 , HA , Marrakech; Černý 2019 ; Černý et al. 2020 Agromyza intermittens (Becker, 1907) = Phytomyza secalina Hering, 1925, in Maarouf 2003 : 42 Maarouf 2003 , HA , Chaouia Agromyza luteitarsis (Rondani, 1875) Maarouf 2003 , HA , Chaouia Agromyza megalopsis Hering, 1933 Černý et al. 2020 , AA , Agadir Id Aissa (western end of gorge and village Amtoudi, 854 m) Agromyza nana Meigen, 1830 Spencer 1967 , 1973 , AP , Casablanca; Černý and Merz 2006 Agromyza nigrociliata (Hendel, 1931) Maarouf 2003 , HA , Chaouia Agromyza rondensis Strobl, 1900 Černý et al. 2020 , AA , Agadir Id Aissa (western end of gorge and village Amtoudi, 854 m), River Aoulouz (= Asif Tifnout, 697 m) Agromyza spenceri Griffiths, 1963 Černý and Merz 2006 , HA , Asni; Černý 2013 Melanagromyza Hendel, 1920 Melanagromyza lappae (Loew, 1850) Mouna 1998 ; AP (Rabat) – MISR Melanagromyza verbasci Spencer, 1957 Spencer 1967 , HA Ophiomyia Braschnikov, 1897 Ophiomyia beckeri (Hendel, 1923) Spencer 1967 , HA ; Černý and Merz 2006 ; Černý 2009 ; Černý and Tschirnhaus 2014 Ophiomyia curvipalpis (Zetterstedt, 1848) = Ophiomyia proboscidea (Strobl, 1900), in Mouna 1998 : 84 Spencer 1967 , HA ; Mouna 1998 ; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2019 Ophiomyia melandryi de Meijere, 1924 Spencer 1967 , HA ; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2009 Ophiomyia vimmeri Černý, 1994 Černý and Merz 2006 , AP , Maâmora (Rabat); Černý and Merz 2007 ; Černý 2018 Phytomyzinae Amauromyza Hendel, 1931 Amauromyza ( Amauromyza ) morionella (Zetterstedt, 1848) = Agromyza morionella Zetterstedt, in Becker and Stein 1913 : 95 Aulagromyza Enderlein, 1936 Aulagromyza atlantidis (Spencer, 1967) = Paraphytomyza atlantidis Spencer, 1967, in Mouna 1998 : 84 Spencer 1967 , HA , Asni; Mouna 1998 Aulagromyza cydoniae (Hendel, 1936) = Phytagromyza cydoniae Hendel, 1936, in Hendel 1931–1936: 518 Hendel 1931–1936, AP , Rabat Aulagromyza hamata (Hendel, 1932)* HA , Asni-Ouirgane Cerodontha Rondani, 1861 Cerodontha ( Cerodontha ) denticornis (Panzer, 1806) Spencer 1967 , 1973 , HA ; Černý and Merz 2006 ; Černý 2009 Cerodontha ( Cerodontha ) fulvipes (Meigen, 1830) Mouna 1998 Cerodontha ( Dizygomyza ) brisiaca Nowakowski, 1973 Černý and Merz 2006 , MA , Azrou, Ifrane Cerodontha ( Icteromyza ) capitata (Zetterstedt, 1848) Černý and Merz 2006 , Rif , Chefchaouen Cerodontha ( Icteromyza ) rozkosnyi Černý, 2007 Černý 2007 , AA , SW Tazenakht (1000 m); Černý 2011 Cerodontha ( Poemyza ) incisa (Meigen, 1830) = Dizygomyza incisa Meigen, in Séguy 1936b : 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Cerodontha ( Poemyza ) lateralis (Macquart, 1835) = Dizygomyza lateralis Macquart, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 ; Černý et al. 2020 Cerodontha ( Poemyza ) pygmaea (Meigen, 1830) = Dizygomyza pygmaea Meigen, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Chromatomyia Hardy, 1849 Chromatomyia aprilina (Goureau, 1851) Griffiths 1974 ; Spencer 1967 , Rif , Tanger (mountains) Chromatomyia horticola (Goureau, 1851) = Phytomyza horticola Goureau, in Griffiths 1967 : 14 Griffiths 1967 , AP , Casablanca; Spencer 1973 ; Černý 2009 Chromatomyia milii (Kaltenbach, 1864) = Phytomyza milii Kaltenbach, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Spencer 1967 , HA , Ourika Valley; Griffiths 1980 ; Mouna 1998 ; Černý and Merz 2006 Chromatomyia syngenesiae Hardy, 1849 = Phytomyza atricornis Meigen, in Kozlowsky and Rungs 1932 : 66; Mouna 1998 : 84 Kozlowsky and Rungs 1932 , AP , Rabat, Kénitra, Casablanca; Mouna 1998 ; AP (Rabat) – MISR Liriomyza Mik, 1894 Liriomyza bryoniae (Kaltenbach, 1858) Spencer 1967 , 1973 , AP , Casablanca; Ayoub 2002 , AA , Souss Massa, Agadir; Černý and Merz 2006 Liriomyza cicerina (Rondani, 1874) Spencer 1973 , HA ; Lahmar and Zeouienne 1992 ; Mouna 1998 Liriomyza congesta (Becker, 1903) Černý et al. 2020 , AA , River bed of Ougni, 0.6 km N Akka N'Ait Sidi and 1.8 km NW Tissint (582 m), River Aoulouz (= Asif Tifnout), bridge of road (697 m), River Oued Draa near hotel, Gardin Oued Tamnougalt (911 m) Liriomyza huidobrensis (Blanchard, 1926) Hanafi and Schnitzler 2004 , AA , Souss Valley Liriomyza orbona (Meigen, 1830) = Agromyza fuscolimbata Strobl, 1900, in Maarouf 2003 : 43 = Liriomyza orbonella Hendel, 1931, in Maarouf 2003 : 43 Maarouf 2003 , HA , Chaouia Liriomyza pedestris Hendel, 1931 Spencer 1967 , Rif , Tanger; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2019 ; Černý et al. 2020 Liriomyza pusilla (Meigen, 1830) = Agromyza pusilla Meigen, in Mouna 1998 : 84 Mouna 1998 Liriomyza sonchi Hendel, 1931 Spencer 1967 , AP , Casablanca Liriomyza trifolii (Burgess in Comstock, 1880) Hanafi and Schnitzler 2004 , AA , Souss Valley Napomyza Westwood, 1840 Napomyza cichorii Spencer, 1966 Černý and Merz 2006 , MA , Ifrane; Černý 2009 Napomyza lateralis (Fallén, 1823) Černý and Merz 2006 , MA , Ifrane Napomyza scrophulariae Spencer, 1966 Černý and Merz 2006 , MA , Ifrane; Černý and Merz 2007 ; Černý 2012 , 2013 Phytoliriomyza Hendel, 1931 Phytoliriomyza immoderata Spencer, 1963 Černý and Merz 2006 , MA , Azrou, Ifrane; Černý 2009 ; Černý 2019 ; Černý et al. 2020 Phytoliriomyza oasis (Becker, 1907) Spencer 1967 , HA ; Černý and Merz 2006 Phytomyza Fallén, 1810 Phytomyza conyzae Hendel, 1920 Spencer 1967 , Rif , Tanger; Černý 2009 Phytomyza ferulae Hering, 1927 Spencer 1967 , Rif , Tanger; Černý 2013 ; Černý et al. 2020 Phytomyza gymnostoma Loew, 1858 Mouna 1998 ; AP (Mechra el kettane) – MISR Phytomyza orobanchia Kaltenbach, 1864 Geipert 1993 ; Geipert et al. 1994 , Rif ; Klein 1995 ; Klein et al. 1995 ; Boumezzough 1996 , MA , Saiss, Rommani; Boughdad et al. 1997 ; Klein et al. 1999 ; Kroschel and Klein 1999 ; Yazough and Klein 1999 ; Kroschel and Klein 2003 Phytomyza phillyreae Hering in Buhr, 1930 = Phytomyza unedo Séguy, 1953, in Séguy 1953b : 72 Séguy 1953b , AP , Korifla; Spencer 1967 , HA , Ourika Valley; Černý and Merz 2006 ; Černý 2009 Phytomyza ranunculi (Schrank, 1803) Spencer 1967 , HA ; Černý and Merz 2006 ; Černý 2009 , 2013 Phytomyza wahlgreni Rydén, 1944 Černý and Merz 2006 , MA , Azrou, Ifrane; Černý et al. 2020 Pseudonapomyza Hendel, 1920 Pseudonapomyza atra (Meigen, 1830) = Phytomyza acuticornis Loew, 1858, in Maarouf 2003 : 43 Maarouf 2003 , HA , Chaouia Pseudonapomyza atratula Zlobin, 2003 Černý et al. 2020 , AA , 6 km ESE Quijjane, 85 km S Agadir (353 m) Pseudonapomyza bifida Zlobin, 2003 Černý et al. 2020 , AA , Road no. 109/165 from Akna to Taroudant (841 m) Pseudonapomyza spicata (Malloch, 1914) Černý et al. 2020 , AA , River bed of Ougni, 0.6 km N Akka N'Ait Sidi and 1.8 km NW Tissint (582 m) Pseudonapomyza spinosa Spencer, 1973 Maarouf 2003 , HA , Chaouia New record for Morocco Aulagromyza hamata (Hendel, 1932) High Atlas: Asni-Ouirgane, 31°13'52"N, 8°00'8"W , 1282 m a.s.l., 1♂, 24.iv.2014, river valley, V. Vrabec leg., M. Barták coll. and M. Černý det. ANTHOMYZIDAE K. Kettani, M.J. Ebejer Number of species: 2 . Expected: 4 Faunistic knowledge of the family in Morocco: poor Amygdalops Lamb, 1914 Amygdalops thomasseti Lamb, 1914 Ebejer et al. 2019 , Rif , Stehat (0 m) Anagnota Becker, 1902 Anagnota major Roháček & Freidberg, 1993 Roháček 2006 , HA , Marrakech (1000 m) ASTEIIDAE K. Kettani, M.J. Ebejer Number of species: 5 . Expected: 10 Faunistic knowledge of the family in Morocco: poor Asteiinae Asteia Meigen, 1830 Asteia amoena Meigen, 1830 Mouna 1998 ; AP (Rabat) – MISR Asteia ibizana (Enderlein, 1935) Ebejer et al. 2019 , AP , Larache (2 m) Asteia mahunkai Papp, 1979 Ebejer et al. 2019 , AP , Larache (2 m) Phlebosotera Duda, 1927 Phlebosotera clypeata Freidberg & Carles-Tolrá, 2010 Freidberg and Carles-Tolrá 2010 , HA , Jaffar river Phlebosotera mirabilis Papp, 1972 Ebejer et al. 2019 , AA , 12 km S of Rissani (737 m) AULACIGASTRIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Aulacigaster Macquart, 1835 Aulacigaster leucopeza (Meigen, 1830) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) CLUSIIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Clusiodes Coquillett, 1904 Clusioides verticalis (Collin, 1912) Ebejer et al. 2019 , Rif , Amsemlil bog ( PNPB , 1067 m) ODINIIDAE K. Kettani, M.J. Ebejer Number of species: 2 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Odiniinae Odinia Robineau-Desvoidy, 1830 Odinia Boletina (Zetterstedt, 1848) Séguy 1934a ; Mouna 1998 ; Gaimari and Mathis 2011 Odinia meijerei Collin, 1952 Ebejer et al. 2019 , Rif , Adrou ( PNPB , 556 m) OPOMYZIDAE K. Kettani, M.J. Ebejer Number of species: 5 . Expected: 6 Faunistic knowledge of the family in Morocco: moderate Geomyza Fallén, 1810 Geomyza apicalis (Meigen, 1830) Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 , AP , Merja Zerga Geomyza combinata (Linnaeus, 1767) Mouna 1998 ; AP (Maâmora) – MISR Geomyza tripunctata (Fallén, 1823) Maarouf 2003 , HA , Chaouia Opomyza Fallén, 1820 Opomyza florum (Fabricius, 1794) Maarouf 2003 , HA , Chaouia Opomyza petrei Mesnil, 1934 Ebejer et al. 2019 , Rif , Aïn Tissemlal (Azilane, 1255 m) AGROMYZIDAE K. Kettani, M. Černý Number of species: 62 . Expected: 150 Faunistic knowledge of the family in Morocco: poor Agromyzinae Agromyza Fallén, 1810 Agromyza abiens Zetterstedt, 1848 Spencer 1967 , HA , Ourika Valley, Marrakech; Černý and Merz 2006 Agromyza albipennis Meigen, 1830 Séguy 1936b ; Mouna 1998 Agromyza ambigua Fallén, 1823 = Domomyza ambigua Fallén, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Agromyza bicaudata (Hendel, 1920) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza frontella (Rondani, 1875) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza frontosa (Becker, 1908) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza hiemalis Becker, 1908 Spencer 1967 , HA , Marrakech; Černý 2019 ; Černý et al. 2020 Agromyza intermittens (Becker, 1907) = Phytomyza secalina Hering, 1925, in Maarouf 2003 : 42 Maarouf 2003 , HA , Chaouia Agromyza luteitarsis (Rondani, 1875) Maarouf 2003 , HA , Chaouia Agromyza megalopsis Hering, 1933 Černý et al. 2020 , AA , Agadir Id Aissa (western end of gorge and village Amtoudi, 854 m) Agromyza nana Meigen, 1830 Spencer 1967 , 1973 , AP , Casablanca; Černý and Merz 2006 Agromyza nigrociliata (Hendel, 1931) Maarouf 2003 , HA , Chaouia Agromyza rondensis Strobl, 1900 Černý et al. 2020 , AA , Agadir Id Aissa (western end of gorge and village Amtoudi, 854 m), River Aoulouz (= Asif Tifnout, 697 m) Agromyza spenceri Griffiths, 1963 Černý and Merz 2006 , HA , Asni; Černý 2013 Melanagromyza Hendel, 1920 Melanagromyza lappae (Loew, 1850) Mouna 1998 ; AP (Rabat) – MISR Melanagromyza verbasci Spencer, 1957 Spencer 1967 , HA Ophiomyia Braschnikov, 1897 Ophiomyia beckeri (Hendel, 1923) Spencer 1967 , HA ; Černý and Merz 2006 ; Černý 2009 ; Černý and Tschirnhaus 2014 Ophiomyia curvipalpis (Zetterstedt, 1848) = Ophiomyia proboscidea (Strobl, 1900), in Mouna 1998 : 84 Spencer 1967 , HA ; Mouna 1998 ; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2019 Ophiomyia melandryi de Meijere, 1924 Spencer 1967 , HA ; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2009 Ophiomyia vimmeri Černý, 1994 Černý and Merz 2006 , AP , Maâmora (Rabat); Černý and Merz 2007 ; Černý 2018 Phytomyzinae Amauromyza Hendel, 1931 Amauromyza ( Amauromyza ) morionella (Zetterstedt, 1848) = Agromyza morionella Zetterstedt, in Becker and Stein 1913 : 95 Aulagromyza Enderlein, 1936 Aulagromyza atlantidis (Spencer, 1967) = Paraphytomyza atlantidis Spencer, 1967, in Mouna 1998 : 84 Spencer 1967 , HA , Asni; Mouna 1998 Aulagromyza cydoniae (Hendel, 1936) = Phytagromyza cydoniae Hendel, 1936, in Hendel 1931–1936: 518 Hendel 1931–1936, AP , Rabat Aulagromyza hamata (Hendel, 1932)* HA , Asni-Ouirgane Cerodontha Rondani, 1861 Cerodontha ( Cerodontha ) denticornis (Panzer, 1806) Spencer 1967 , 1973 , HA ; Černý and Merz 2006 ; Černý 2009 Cerodontha ( Cerodontha ) fulvipes (Meigen, 1830) Mouna 1998 Cerodontha ( Dizygomyza ) brisiaca Nowakowski, 1973 Černý and Merz 2006 , MA , Azrou, Ifrane Cerodontha ( Icteromyza ) capitata (Zetterstedt, 1848) Černý and Merz 2006 , Rif , Chefchaouen Cerodontha ( Icteromyza ) rozkosnyi Černý, 2007 Černý 2007 , AA , SW Tazenakht (1000 m); Černý 2011 Cerodontha ( Poemyza ) incisa (Meigen, 1830) = Dizygomyza incisa Meigen, in Séguy 1936b : 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Cerodontha ( Poemyza ) lateralis (Macquart, 1835) = Dizygomyza lateralis Macquart, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 ; Černý et al. 2020 Cerodontha ( Poemyza ) pygmaea (Meigen, 1830) = Dizygomyza pygmaea Meigen, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Chromatomyia Hardy, 1849 Chromatomyia aprilina (Goureau, 1851) Griffiths 1974 ; Spencer 1967 , Rif , Tanger (mountains) Chromatomyia horticola (Goureau, 1851) = Phytomyza horticola Goureau, in Griffiths 1967 : 14 Griffiths 1967 , AP , Casablanca; Spencer 1973 ; Černý 2009 Chromatomyia milii (Kaltenbach, 1864) = Phytomyza milii Kaltenbach, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Spencer 1967 , HA , Ourika Valley; Griffiths 1980 ; Mouna 1998 ; Černý and Merz 2006 Chromatomyia syngenesiae Hardy, 1849 = Phytomyza atricornis Meigen, in Kozlowsky and Rungs 1932 : 66; Mouna 1998 : 84 Kozlowsky and Rungs 1932 , AP , Rabat, Kénitra, Casablanca; Mouna 1998 ; AP (Rabat) – MISR Liriomyza Mik, 1894 Liriomyza bryoniae (Kaltenbach, 1858) Spencer 1967 , 1973 , AP , Casablanca; Ayoub 2002 , AA , Souss Massa, Agadir; Černý and Merz 2006 Liriomyza cicerina (Rondani, 1874) Spencer 1973 , HA ; Lahmar and Zeouienne 1992 ; Mouna 1998 Liriomyza congesta (Becker, 1903) Černý et al. 2020 , AA , River bed of Ougni, 0.6 km N Akka N'Ait Sidi and 1.8 km NW Tissint (582 m), River Aoulouz (= Asif Tifnout), bridge of road (697 m), River Oued Draa near hotel, Gardin Oued Tamnougalt (911 m) Liriomyza huidobrensis (Blanchard, 1926) Hanafi and Schnitzler 2004 , AA , Souss Valley Liriomyza orbona (Meigen, 1830) = Agromyza fuscolimbata Strobl, 1900, in Maarouf 2003 : 43 = Liriomyza orbonella Hendel, 1931, in Maarouf 2003 : 43 Maarouf 2003 , HA , Chaouia Liriomyza pedestris Hendel, 1931 Spencer 1967 , Rif , Tanger; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2019 ; Černý et al. 2020 Liriomyza pusilla (Meigen, 1830) = Agromyza pusilla Meigen, in Mouna 1998 : 84 Mouna 1998 Liriomyza sonchi Hendel, 1931 Spencer 1967 , AP , Casablanca Liriomyza trifolii (Burgess in Comstock, 1880) Hanafi and Schnitzler 2004 , AA , Souss Valley Napomyza Westwood, 1840 Napomyza cichorii Spencer, 1966 Černý and Merz 2006 , MA , Ifrane; Černý 2009 Napomyza lateralis (Fallén, 1823) Černý and Merz 2006 , MA , Ifrane Napomyza scrophulariae Spencer, 1966 Černý and Merz 2006 , MA , Ifrane; Černý and Merz 2007 ; Černý 2012 , 2013 Phytoliriomyza Hendel, 1931 Phytoliriomyza immoderata Spencer, 1963 Černý and Merz 2006 , MA , Azrou, Ifrane; Černý 2009 ; Černý 2019 ; Černý et al. 2020 Phytoliriomyza oasis (Becker, 1907) Spencer 1967 , HA ; Černý and Merz 2006 Phytomyza Fallén, 1810 Phytomyza conyzae Hendel, 1920 Spencer 1967 , Rif , Tanger; Černý 2009 Phytomyza ferulae Hering, 1927 Spencer 1967 , Rif , Tanger; Černý 2013 ; Černý et al. 2020 Phytomyza gymnostoma Loew, 1858 Mouna 1998 ; AP (Mechra el kettane) – MISR Phytomyza orobanchia Kaltenbach, 1864 Geipert 1993 ; Geipert et al. 1994 , Rif ; Klein 1995 ; Klein et al. 1995 ; Boumezzough 1996 , MA , Saiss, Rommani; Boughdad et al. 1997 ; Klein et al. 1999 ; Kroschel and Klein 1999 ; Yazough and Klein 1999 ; Kroschel and Klein 2003 Phytomyza phillyreae Hering in Buhr, 1930 = Phytomyza unedo Séguy, 1953, in Séguy 1953b : 72 Séguy 1953b , AP , Korifla; Spencer 1967 , HA , Ourika Valley; Černý and Merz 2006 ; Černý 2009 Phytomyza ranunculi (Schrank, 1803) Spencer 1967 , HA ; Černý and Merz 2006 ; Černý 2009 , 2013 Phytomyza wahlgreni Rydén, 1944 Černý and Merz 2006 , MA , Azrou, Ifrane; Černý et al. 2020 Pseudonapomyza Hendel, 1920 Pseudonapomyza atra (Meigen, 1830) = Phytomyza acuticornis Loew, 1858, in Maarouf 2003 : 43 Maarouf 2003 , HA , Chaouia Pseudonapomyza atratula Zlobin, 2003 Černý et al. 2020 , AA , 6 km ESE Quijjane, 85 km S Agadir (353 m) Pseudonapomyza bifida Zlobin, 2003 Černý et al. 2020 , AA , Road no. 109/165 from Akna to Taroudant (841 m) Pseudonapomyza spicata (Malloch, 1914) Černý et al. 2020 , AA , River bed of Ougni, 0.6 km N Akka N'Ait Sidi and 1.8 km NW Tissint (582 m) Pseudonapomyza spinosa Spencer, 1973 Maarouf 2003 , HA , Chaouia New record for Morocco Aulagromyza hamata (Hendel, 1932) High Atlas: Asni-Ouirgane, 31°13'52"N, 8°00'8"W , 1282 m a.s.l., 1♂, 24.iv.2014, river valley, V. Vrabec leg., M. Barták coll. and M. Černý det. Agromyzinae Agromyza Fallén, 1810 Agromyza abiens Zetterstedt, 1848 Spencer 1967 , HA , Ourika Valley, Marrakech; Černý and Merz 2006 Agromyza albipennis Meigen, 1830 Séguy 1936b ; Mouna 1998 Agromyza ambigua Fallén, 1823 = Domomyza ambigua Fallén, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Agromyza bicaudata (Hendel, 1920) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza frontella (Rondani, 1875) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza frontosa (Becker, 1908) Černý and Merz 2006 , MA , Azrou, Ifrane Agromyza hiemalis Becker, 1908 Spencer 1967 , HA , Marrakech; Černý 2019 ; Černý et al. 2020 Agromyza intermittens (Becker, 1907) = Phytomyza secalina Hering, 1925, in Maarouf 2003 : 42 Maarouf 2003 , HA , Chaouia Agromyza luteitarsis (Rondani, 1875) Maarouf 2003 , HA , Chaouia Agromyza megalopsis Hering, 1933 Černý et al. 2020 , AA , Agadir Id Aissa (western end of gorge and village Amtoudi, 854 m) Agromyza nana Meigen, 1830 Spencer 1967 , 1973 , AP , Casablanca; Černý and Merz 2006 Agromyza nigrociliata (Hendel, 1931) Maarouf 2003 , HA , Chaouia Agromyza rondensis Strobl, 1900 Černý et al. 2020 , AA , Agadir Id Aissa (western end of gorge and village Amtoudi, 854 m), River Aoulouz (= Asif Tifnout, 697 m) Agromyza spenceri Griffiths, 1963 Černý and Merz 2006 , HA , Asni; Černý 2013 Melanagromyza Hendel, 1920 Melanagromyza lappae (Loew, 1850) Mouna 1998 ; AP (Rabat) – MISR Melanagromyza verbasci Spencer, 1957 Spencer 1967 , HA Ophiomyia Braschnikov, 1897 Ophiomyia beckeri (Hendel, 1923) Spencer 1967 , HA ; Černý and Merz 2006 ; Černý 2009 ; Černý and Tschirnhaus 2014 Ophiomyia curvipalpis (Zetterstedt, 1848) = Ophiomyia proboscidea (Strobl, 1900), in Mouna 1998 : 84 Spencer 1967 , HA ; Mouna 1998 ; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2019 Ophiomyia melandryi de Meijere, 1924 Spencer 1967 , HA ; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2009 Ophiomyia vimmeri Černý, 1994 Černý and Merz 2006 , AP , Maâmora (Rabat); Černý and Merz 2007 ; Černý 2018 Phytomyzinae Amauromyza Hendel, 1931 Amauromyza ( Amauromyza ) morionella (Zetterstedt, 1848) = Agromyza morionella Zetterstedt, in Becker and Stein 1913 : 95 Aulagromyza Enderlein, 1936 Aulagromyza atlantidis (Spencer, 1967) = Paraphytomyza atlantidis Spencer, 1967, in Mouna 1998 : 84 Spencer 1967 , HA , Asni; Mouna 1998 Aulagromyza cydoniae (Hendel, 1936) = Phytagromyza cydoniae Hendel, 1936, in Hendel 1931–1936: 518 Hendel 1931–1936, AP , Rabat Aulagromyza hamata (Hendel, 1932)* HA , Asni-Ouirgane Cerodontha Rondani, 1861 Cerodontha ( Cerodontha ) denticornis (Panzer, 1806) Spencer 1967 , 1973 , HA ; Černý and Merz 2006 ; Černý 2009 Cerodontha ( Cerodontha ) fulvipes (Meigen, 1830) Mouna 1998 Cerodontha ( Dizygomyza ) brisiaca Nowakowski, 1973 Černý and Merz 2006 , MA , Azrou, Ifrane Cerodontha ( Icteromyza ) capitata (Zetterstedt, 1848) Černý and Merz 2006 , Rif , Chefchaouen Cerodontha ( Icteromyza ) rozkosnyi Černý, 2007 Černý 2007 , AA , SW Tazenakht (1000 m); Černý 2011 Cerodontha ( Poemyza ) incisa (Meigen, 1830) = Dizygomyza incisa Meigen, in Séguy 1936b : 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Cerodontha ( Poemyza ) lateralis (Macquart, 1835) = Dizygomyza lateralis Macquart, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 ; Černý et al. 2020 Cerodontha ( Poemyza ) pygmaea (Meigen, 1830) = Dizygomyza pygmaea Meigen, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Mouna 1998 Chromatomyia Hardy, 1849 Chromatomyia aprilina (Goureau, 1851) Griffiths 1974 ; Spencer 1967 , Rif , Tanger (mountains) Chromatomyia horticola (Goureau, 1851) = Phytomyza horticola Goureau, in Griffiths 1967 : 14 Griffiths 1967 , AP , Casablanca; Spencer 1973 ; Černý 2009 Chromatomyia milii (Kaltenbach, 1864) = Phytomyza milii Kaltenbach, in Séguy 1936: 5; Mouna 1998 : 84 Séguy 1936b , AP , Rabat; Spencer 1967 , HA , Ourika Valley; Griffiths 1980 ; Mouna 1998 ; Černý and Merz 2006 Chromatomyia syngenesiae Hardy, 1849 = Phytomyza atricornis Meigen, in Kozlowsky and Rungs 1932 : 66; Mouna 1998 : 84 Kozlowsky and Rungs 1932 , AP , Rabat, Kénitra, Casablanca; Mouna 1998 ; AP (Rabat) – MISR Liriomyza Mik, 1894 Liriomyza bryoniae (Kaltenbach, 1858) Spencer 1967 , 1973 , AP , Casablanca; Ayoub 2002 , AA , Souss Massa, Agadir; Černý and Merz 2006 Liriomyza cicerina (Rondani, 1874) Spencer 1973 , HA ; Lahmar and Zeouienne 1992 ; Mouna 1998 Liriomyza congesta (Becker, 1903) Černý et al. 2020 , AA , River bed of Ougni, 0.6 km N Akka N'Ait Sidi and 1.8 km NW Tissint (582 m), River Aoulouz (= Asif Tifnout), bridge of road (697 m), River Oued Draa near hotel, Gardin Oued Tamnougalt (911 m) Liriomyza huidobrensis (Blanchard, 1926) Hanafi and Schnitzler 2004 , AA , Souss Valley Liriomyza orbona (Meigen, 1830) = Agromyza fuscolimbata Strobl, 1900, in Maarouf 2003 : 43 = Liriomyza orbonella Hendel, 1931, in Maarouf 2003 : 43 Maarouf 2003 , HA , Chaouia Liriomyza pedestris Hendel, 1931 Spencer 1967 , Rif , Tanger; Černý and Merz 2006 ; Černý and Merz 2007 ; Černý 2019 ; Černý et al. 2020 Liriomyza pusilla (Meigen, 1830) = Agromyza pusilla Meigen, in Mouna 1998 : 84 Mouna 1998 Liriomyza sonchi Hendel, 1931 Spencer 1967 , AP , Casablanca Liriomyza trifolii (Burgess in Comstock, 1880) Hanafi and Schnitzler 2004 , AA , Souss Valley Napomyza Westwood, 1840 Napomyza cichorii Spencer, 1966 Černý and Merz 2006 , MA , Ifrane; Černý 2009 Napomyza lateralis (Fallén, 1823) Černý and Merz 2006 , MA , Ifrane Napomyza scrophulariae Spencer, 1966 Černý and Merz 2006 , MA , Ifrane; Černý and Merz 2007 ; Černý 2012 , 2013 Phytoliriomyza Hendel, 1931 Phytoliriomyza immoderata Spencer, 1963 Černý and Merz 2006 , MA , Azrou, Ifrane; Černý 2009 ; Černý 2019 ; Černý et al. 2020 Phytoliriomyza oasis (Becker, 1907) Spencer 1967 , HA ; Černý and Merz 2006 Phytomyza Fallén, 1810 Phytomyza conyzae Hendel, 1920 Spencer 1967 , Rif , Tanger; Černý 2009 Phytomyza ferulae Hering, 1927 Spencer 1967 , Rif , Tanger; Černý 2013 ; Černý et al. 2020 Phytomyza gymnostoma Loew, 1858 Mouna 1998 ; AP (Mechra el kettane) – MISR Phytomyza orobanchia Kaltenbach, 1864 Geipert 1993 ; Geipert et al. 1994 , Rif ; Klein 1995 ; Klein et al. 1995 ; Boumezzough 1996 , MA , Saiss, Rommani; Boughdad et al. 1997 ; Klein et al. 1999 ; Kroschel and Klein 1999 ; Yazough and Klein 1999 ; Kroschel and Klein 2003 Phytomyza phillyreae Hering in Buhr, 1930 = Phytomyza unedo Séguy, 1953, in Séguy 1953b : 72 Séguy 1953b , AP , Korifla; Spencer 1967 , HA , Ourika Valley; Černý and Merz 2006 ; Černý 2009 Phytomyza ranunculi (Schrank, 1803) Spencer 1967 , HA ; Černý and Merz 2006 ; Černý 2009 , 2013 Phytomyza wahlgreni Rydén, 1944 Černý and Merz 2006 , MA , Azrou, Ifrane; Černý et al. 2020 Pseudonapomyza Hendel, 1920 Pseudonapomyza atra (Meigen, 1830) = Phytomyza acuticornis Loew, 1858, in Maarouf 2003 : 43 Maarouf 2003 , HA , Chaouia Pseudonapomyza atratula Zlobin, 2003 Černý et al. 2020 , AA , 6 km ESE Quijjane, 85 km S Agadir (353 m) Pseudonapomyza bifida Zlobin, 2003 Černý et al. 2020 , AA , Road no. 109/165 from Akna to Taroudant (841 m) Pseudonapomyza spicata (Malloch, 1914) Černý et al. 2020 , AA , River bed of Ougni, 0.6 km N Akka N'Ait Sidi and 1.8 km NW Tissint (582 m) Pseudonapomyza spinosa Spencer, 1973 Maarouf 2003 , HA , Chaouia New record for Morocco Aulagromyza hamata (Hendel, 1932) High Atlas: Asni-Ouirgane, 31°13'52"N, 8°00'8"W , 1282 m a.s.l., 1♂, 24.iv.2014, river valley, V. Vrabec leg., M. Barták coll. and M. Černý det. ANTHOMYZIDAE K. Kettani, M.J. Ebejer Number of species: 2 . Expected: 4 Faunistic knowledge of the family in Morocco: poor Amygdalops Lamb, 1914 Amygdalops thomasseti Lamb, 1914 Ebejer et al. 2019 , Rif , Stehat (0 m) Anagnota Becker, 1902 Anagnota major Roháček & Freidberg, 1993 Roháček 2006 , HA , Marrakech (1000 m) ASTEIIDAE K. Kettani, M.J. Ebejer Number of species: 5 . Expected: 10 Faunistic knowledge of the family in Morocco: poor Asteiinae Asteia Meigen, 1830 Asteia amoena Meigen, 1830 Mouna 1998 ; AP (Rabat) – MISR Asteia ibizana (Enderlein, 1935) Ebejer et al. 2019 , AP , Larache (2 m) Asteia mahunkai Papp, 1979 Ebejer et al. 2019 , AP , Larache (2 m) Phlebosotera Duda, 1927 Phlebosotera clypeata Freidberg & Carles-Tolrá, 2010 Freidberg and Carles-Tolrá 2010 , HA , Jaffar river Phlebosotera mirabilis Papp, 1972 Ebejer et al. 2019 , AA , 12 km S of Rissani (737 m) Asteiinae Asteia Meigen, 1830 Asteia amoena Meigen, 1830 Mouna 1998 ; AP (Rabat) – MISR Asteia ibizana (Enderlein, 1935) Ebejer et al. 2019 , AP , Larache (2 m) Asteia mahunkai Papp, 1979 Ebejer et al. 2019 , AP , Larache (2 m) Phlebosotera Duda, 1927 Phlebosotera clypeata Freidberg & Carles-Tolrá, 2010 Freidberg and Carles-Tolrá 2010 , HA , Jaffar river Phlebosotera mirabilis Papp, 1972 Ebejer et al. 2019 , AA , 12 km S of Rissani (737 m) AULACIGASTRIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Aulacigaster Macquart, 1835 Aulacigaster leucopeza (Meigen, 1830) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) CLUSIIDAE K. Kettani, M.J. Ebejer Number of species: 1 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Clusiodes Coquillett, 1904 Clusioides verticalis (Collin, 1912) Ebejer et al. 2019 , Rif , Amsemlil bog ( PNPB , 1067 m) ODINIIDAE K. Kettani, M.J. Ebejer Number of species: 2 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Odiniinae Odinia Robineau-Desvoidy, 1830 Odinia Boletina (Zetterstedt, 1848) Séguy 1934a ; Mouna 1998 ; Gaimari and Mathis 2011 Odinia meijerei Collin, 1952 Ebejer et al. 2019 , Rif , Adrou ( PNPB , 556 m) Odiniinae Odinia Robineau-Desvoidy, 1830 Odinia Boletina (Zetterstedt, 1848) Séguy 1934a ; Mouna 1998 ; Gaimari and Mathis 2011 Odinia meijerei Collin, 1952 Ebejer et al. 2019 , Rif , Adrou ( PNPB , 556 m) OPOMYZIDAE K. Kettani, M.J. Ebejer Number of species: 5 . Expected: 6 Faunistic knowledge of the family in Morocco: moderate Geomyza Fallén, 1810 Geomyza apicalis (Meigen, 1830) Pârvu et al. 2006 , MA , Ifrane; Popescu-Mirceni 2011 , AP , Merja Zerga Geomyza combinata (Linnaeus, 1767) Mouna 1998 ; AP (Maâmora) – MISR Geomyza tripunctata (Fallén, 1823) Maarouf 2003 , HA , Chaouia Opomyza Fallén, 1820 Opomyza florum (Fabricius, 1794) Maarouf 2003 , HA , Chaouia Opomyza petrei Mesnil, 1934 Ebejer et al. 2019 , Rif , Aïn Tissemlal (Azilane, 1255 m) Carnoidea CANACIDAE K. Kettani, L. Munari Number of species: 15 . Expected: 25 Faunistic knowledge of the family in Morocco: poor Canacinae Canace Haliday in Curtis, 1837 Canace actites Mathis, 1982 Ebejer et al. 2019 , AP , Loukous (2 m) Canace nasica Haliday, 1839 Dahl 1964 , AA , Tamri (north of Agadir); Mouna 1998 Xanthocanace Hendel, 1914 Xanthocanace ranula (Loew, 1874) Munari 2010 , AP , 40 km S Larache; Munari and Mathis 2010 ; Munari 2011 ; Munari and Bramuzzo 2018 , Rif , Briyech, AP , Azemmour, El Khaoucha, Fedala, Oued Nefifikh, Oued Loukous, 40 km S Larache Tethininae Tethina Haliday in Curtis, 1837 Tethina alboguttata (Strobl, 1900) Freidberg and Beschovski 1996 , AP , Aïn Diab, Essaouira, Safi, AA , Agadir, Ouarzazate; Mathis and Munari 1996 ; Munari 2002 , 2004 , 2005 , 2011 ; Munari and Mathis 2010 ; Koçak and Kemal 2010 ; Munari and Bramuzzo 2018 , Rif , Briyech, AP , Agadir, Tamri, Essaouira, Cap Hadid, Larache, Loukous, Safi, Dar Caïd-Hadji Tethina albosetulosa (Strobl, 1900) Cassar et al. 2008 , Rif , Oued Laou Basin Tethina flavigenis (Hendel, 1934) Munari and Bramuzzo 2018 , AP , Oued Oum-er-Rbia Tethina grossipes (Becker, 1908) Munari 2004 , AA , Agadir; Munari and Mathis 2010 ; Munari and Bramuzzo 2018 , AP , Tamri Tethina incisuralis (Macquart, 1851) = Tethina pictipes ( Becker 1903 ); Séguy 1941d : 18, Mouna 1998 : 87 Munari 1997 , AA , Erfoud, Rissani (900 m); Munari 2002 , 2010 , 2011 ; Munari and Mathis 2010 ; Munari and Bramuzzo 2018 , AA , Erfoud, Rissani Tethina longirostris (Loew, 1865) Cassar et al. 2008 , Rif , Oued Laou Basin Tethina mariae Munari, 1997 Munari 1997 , AP , 40 km S Larache; Munari and Báez 2000 ; Munari 2002 , 2004 , 2010 , 2011 ; Munari and Mathis 2010 – NHMD ( HT ♂) Tethina pallipes (Loew, 1865) = Tethina ochracea (Hendel, 1913) Cassar et al. 2008 , Rif , Oued Laou Tethina pictipennis Freidberg & Beschovski, 1996 Freidberg and Beschovski 1996 , AP , 40 km south of Larache; Mathis and Munari 1996 , Munari 2002 ; Munari 2004 ; Munari and Mathis 2010 ; Munari 2011 , AA , Agadir (Tamri); Munari and Bramuzzo 2018 , Rif , Briyech, AP , Tamri, Larache, Loukous – NHMD ( HT ♂) Tethina strobliana (Mercier, 1923) Munari and Bramuzzo 2018 , AP , Oued Abou, Rehouna (Rabat) Tethina yaromi Freidberg & Beschovski, 1996 Cassar et al. 2005 , Rif , Smir lagoon Tethina sp. near salinicola Beschovski, 1998 Munari 2004 , AA , Agadir, Ouarzazate; Munari 2005 ; Munari and Bramuzzo 2018 , AP , Tamri CARNIDAE K. Kettani, M.J. Ebejer Number of species: 3 . Expected: >10 Faunistic knowledge of the family in Morocco: poor Meoneura Rondani , 1856 Meoneura hungarica Papp, 1977 Ebejer et al. 2019 , Rif , Adrou ( PNPB , 556 m) Meoneura prima Becker, 1905 Brake 2011 Meoneura triangularis Collin, 1930 Brake 2011 CHLOROPIDAE 48 K. Kettani, M. von Tschirnhaus, M.J. Ebejer Number of species: 74 . Expected: 140 Faunistic knowledge of the family in Morocco: moderate Chloropinae Assuania Becker, 1903 Assuania melanoleuca (Séguy, 1949) comb. nov. 49 = Chlorops melanoleuca Séguy, in Séguy 1949a : 158 Séguy 1949a , AA , Agdz; Nartshuk 1984 ; Mouna 1998 Assuania thalhammeri (Strobl, 1893) Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Camarota Latreille, 1805 Camarota curvipennis (Latreille, 1805) = Camarota curvipennis (Latreille), in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Oued Laou Capnoptera Loew, 1866 Capnoptera pilosa Loew, 1866 Duda 1933; Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1288 m), Aïn Jdioui (Tahaddart, 8 m) Capnoptera scutata (Rossi, 1790) = Eristalis rufipes Fabricius, in Fabricius 1805 : 245 Ebejer and Kettani 2016 , AP , Larache (2 m) Cetema Hendel, 1907 Cetema maroccanum Nartshuk, 1995 = Cetema maroccana Nartshuk, in Nartshuk 1995 : 277–280 Nartshuk 1995 , HA , Oukaimeden; Ebejer and Kettani 2016 Cetema monticulum Becker, 1910 = Cetema monticula Becker, in Séguy 1941a : 33 = Cetema monticula Rossi, in Mouna 1998 : 85 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Ebejer and Kettani 2016 Chlorops Meigen, 1803 Chlorops interruptus Meigen, 1830 = Chlorops interrupta Meigen, in Séguy 1953a : 86 Séguy 1953a , AP , Oued Yquem (near Rabat); Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1288 m), Oued Aliane (Ksar Sghir, 1 m), Cap Spartel (155 m) Chlorops limbatus Meigen, 1830 Ebejer and Kettani 2016 , Rif , Jebel Moussa (800 m) Chlorops pumilionis (Bjerkander, 1778) = Chlorops nasuta Schrank, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2016 Chlorops serenus Loew, 1866 Ebejer and Kettani 2016 , MA , 17 km NW of Zaida (Khénifra, 1878 m) Cryptonevra Lioy, 1864 Cryptonevra flavitarsis (Meigen, 1830) = Haplegis flavitarsis (Meigen), in Séguy 1949a : 158; Séguy 1953a : 86; Mouna 1998 : 85 Séguy 1949a , AA , Agdz; Séguy 1953a , AP , Oued Yquem (near Rabat); Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 Eurina Meigen, 1830 Eurina lurida Meigen, 1830 Ebejer and Kettani 2016 , Rif , M'Diq, Smir, Kabila beach and dunes Eutropha Loew, 1866 Eutropha albipilosa (Becker, 1908) Duda 1930; Deeming and Al-Dhafer 2012 , AP , 17 km N of Larache, AA , Azemmour, estuary of Oued Tensift; Ebejer and Kettani 2016 , AP , Larache (5 m) Eutropha fulvifrons (Haliday, 1833) Séguy 1941d , AA , Agadir; Mouna 1998 ; Cassar et al. 2005 , Rif , Smir lagoon; Ebejer and Kettani 2016 , AP , Larache (5 m) Lagaroceras Becker, 1903 Lagaroceras andalusiaca (Strobl, 1899) Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m), Oued Aliane (Ksar Sghir, 1 m), AP , Larache (2 m), AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Lasiosina Becker, 1910 Lasiosina herpini (Guérin-Méneville, 1843) = Lasiosina cinctipes Meigen, in Mouna 1998 : 85 Ebejer and Kettani 2016 , Rif , Smir lagoon Lasiosina lindbergi (Duda, 1933) = Steleocerus lindbergi (Duda), in Duda 1933: 142 Duda 1933, MA ; Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Meromyza Meigen, 1830 Meromyza athletica Fedoseeva, 1974 = Meromyza variegata Meigen, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2016 Meromyza curvinervis (Zetterstedt, 1848) = Oxinis curvinervis Latreille, in Mouna 1998 : 85 Mouna 1998 Meromyza nigriventris Macquart, 1835 Ebejer and Kettani 2016 , MA , 17 km SW of Midelt (Khénifra, 1940 m), AA , 29 km N of Rich (Errachidia, 1570 m) Meromyza pratorum Meigen, 1830 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Ebejer and Kettani 2016 Metopostigma Becker, 1903 Metopostigma sabulona Becker, 1910 Ebejer and Kettani 2016 , AA , Oued Ziz (10 km S of Errachidia, 1008 m), Merzouga (714 m) Platycephala Fallén, 1820 Platycephala scapularum (Becker, 1907) Nartshuk 1984 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 ; Ebejer and Kettani 2016 Pseudopachychaeta Strobl, 1902 Pseudopachychaeta pachycera Strobl, 1902 Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m), Oued Ziz (1052 m) Thaumatomyia Zenker, 1833 Thaumatomyia elongatula (Becker, 1910) Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil, 3 m) Thaumatomyia glabra (Meigen, 1830) Ebejer and Kettani 2016 , Rif , Martil (9 m) Thaumatomyia notata (Meigen, 1830) = Chloropisca notata (Meigen), in Séguy 1930a : 179, 1941a : 33; Mouna 1998 : 85 Séguy 1930a , Rif , Tanger; Séguy 1941a , HA , Tachdirt (Jebel Toubkal, 2500 m), Canyon Tessaout (M'Goum, 3000–3200 m); Harris et al. 1980 : 229, HA , Chichaoua; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Laou estuary, Oued Amsa (Amsa, 14 m), Jnane Niche (27 m), Oued Maggou (Maggou, 786 m), Marj Khayl (Beni Leit ( PNPB ), 1088 m), Oued Boumarioul (Aïn Hamra, 560 m), Aïn Tissemlal (Azilane, 1255 m), Oued Bouhya (Bou Ahmed, 19 m), Souk Khemis Anjra (Oued Kbir, 55 m), Zinat (231 m), Oued Zarka (Yarghite, 135 m), Oued Talembote (Usine électrique, 120 m), Oued Kelâa (Akoumi, 400 m), Issaguen (maison forestière Issaguen, 1543 m), Bni Boufrah (94 m), Oued Guallet (Bni Boufrah, 946 m), Oued Tabandoute (Bni Boufrah, 540 m), Oued Taâouniya (Koudiat Ajira, 1536 m), Talankramte (Site sacré Sidi Gneiss ( PNPB ): 461 m), AP , Larache (5 m) Thaumatomyia sulcifrons (Becker, 1907) = Chlorops sulcifrons Becker, in Séguy 1949a : 158 = Chloropisca sulcifrons Zetterstedt, in Mouna 1998 : 85 Séguy 1934a ; Séguy 1949a , AA , Foum-el-Hassan, Akka, Agdz; Nartshuk 1984 ; Mouna 1998 ; Dawah and Abdullah 2006 ; Ebejer and Kettani 2016 Oscinellinae Aphanotrigonum Duda, 1932 Aphanotrigonum cinctellum (Zetterstedt, 1848) Ebejer and Kettani 2016 , AP , Larache (5 m) Aphanotrigonum femorellum Collin, 1946 Ebejer and Kettani 2016 , AP , Larache Aphanotrigonum inerme Collin, 1946 Ebejer and Kettani 2016 , Rif , M'Diq (5 m), AP , Larache (Loukous marsh, 2 m) Aphanotrigonum parahastatum Dely-Draskovits, 1981* AA Calamoncosis Enderlein, 1911 Calamoncosis duinensis (Strobl, 1909) Ebejer and Kettani 2016 , AP , Rabat (on Phragmites ) – MISR Conioscinella Duda, 1929 Conioscinella frontella (Fallén, 1810) Ebejer and Kettani 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m) Conioscinella sordidella (Zetterstedt, 1848) Ebejer and Kettani 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m) Elachiptera Macquart, 1835 Elachiptera bimaculata (Loew, 1845) De Lépiney and Mimeur 1932: 110, Rabat; Séguy 1934a : 479; Bléton and Fieuzet 1943 ; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil: 3 m), Jnane Niche (46 m), Oued Mhannech (18 m), Souk Khemis Anjra (55 m), Boujdad (7 m); AP (Rabat) – MISR Elachiptera cornuta (Fallén, 1820) Mouna 1998 : 85 Elachiptera diastema Collin, 1946 Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m), Oued Tiffert (Tiffert, 1230 m), Issaguen (1543 m), AP , Larache (5 m) Elachiptera graeca Becker, 1910 Séguy 1941, HA , Imi-n'Ouaka (1500 m); Nartshuk 1984 ; Mouna 1998 Elachiptera megaspis (Loew, 1858) Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Dardara (730 m), Aïn Jdioui (Tahaddart, 8 m), Jnane Niche (46 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m); AP (Rabat) – MISR Elachiptera orizae Séguy, 1949* AA Elachiptera rufescens (Walker, 1871) Deeming and Al-Dhafer 2012 , AA , 2 km N Erfoud (818 m); Ebejer and Kettani 2016 , AA , 2 km N Erfoud (818 m) Elachiptera rufifrons Duda, 1932 Ebejer and Kettani 2016 , AP , 9 km SE Aïn Chouk (6 m), Larache (5 m) Elachiptera sarda Nartshuk, 2009 Ebejer and Kettani 2016 , AA , 14 km E of Rich (Errachidia, 1278 m) Elachiptera scrobiculata (Strobl, 1901) = Elachiptera trapezina (Corti, 1909), in Bléton and Fieuzet 1943 : 116 Bléton and Fieuzet 1943 ; Ebejer and Kettani 2016 Elachiptera strobli (Corti, 1909) Ebejer and Kettani 2016 , Rif , Oued Boumarioul (Aïn Hamra, 560 m), Oued Aliane (Ksar Sghir, 1 m); Cap Spartel (155 m), Oued Sidi Ben Saâda (Laghdir, 242 m) Epimadiza Becker, 1910 Epimadiza nigrescens Duda, 1933 50 = Oscinosoma anthracias Séguy, in Séguy 1949a : 158, Mouna 1998 : 85 = Oxinosoma anthracias Séguy, in Mouna 1998 : 85 = " Siphonella oscinina (Fallén)", in Nartshuk 1984 : 236 Séguy 1949a , AA , Alnif; Sabrosky 1965 : 406; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 Hapleginella Duda, 1933 Hapleginella laevifrons (Loew, 1858) El Hassani et al. 1986: 8–11, Rif , Nord Occidental, MA Lasiochaeta Corti, 1909 Lasiochaeta pubescens (Thalhammer, 1898) = Elachiptera pubescens (Thalhammer), var. rufithorax Duda, 1932 in Duda 1932: 31 = Melanochaeta pubescens Thalh., in De Lépiney and Mimeur 1932: 110 = Melanochaeta pubescens (Thalhammer), in Mouna 1998 : 85 De Lépiney and Mimeur 1932, AP , Rabat; Duda 1932: 31, HA ; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Sidi Yahia Aarab (Sidi Yahia Aarab, 178 m), Beni Maâdene (Oued Martil, 3 m), Oued Mhannech (Tamuda, 18 m), Oued Amsa (Amsa, 14 m), Zinat (231 m), Oued Maggou (Maggou, 786 m), Oued Guallet (Bni Boufrah, 946 m); AP (Rabat) – MISR Oscinella Becker, 1909 Oscinella cariciphila Collin, 1946 Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil, 3 m), AA , Merzouga (714 m) Oscinella frit (Linnaeus, 1758) = Oxinosoma frit Linnaeus, in Mouna 1998 : 85 De Lépiney and Mimeur 1932: 109, AP , Rabat; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Issaguen (1543 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m), Oued Guallet (Bni Boufrah, 946 m), Oued Ametrasse (Ametrasse, 841 m), AP , Lower Loukous saltmarsh (2 m), MA , Khénifra (17 km SW of Midelt, 1940 m), Lac Aguelmane Afennourir (30 km SW of Azrou, 1490 m); AP (Rabat) – MISR Oscinella nartshukiana Beschovski, 1978 Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m), AP , Larache Oscinella nitidigenis (Becker, 1908) Ebejer and Kettani 2016 , AA , 6 km N of Errachidia (1010 m), Oued Ziz (1052 m) Oscinella nitidissima (Meigen, 1838) Ebejer and Kettani 2016 , Rif , Dardara (730 m), Moulay Abdelsalam (965 m), Cap Spartel (155 m), Oued Laou (dunes, 2 m), AP , Larache (5 m), MA , Lac Aguelmane Afennourir (30 km SW of Azrou, 1490 m) Oscinella pusilla (Meigen, 1830) Ebejer and Kettani 2016 , AA , Lac Tiffert (4 km W of Merzouga, 702 m) Oscinella ventricosi Nartshuk, 1955 Ebejer and Kettani 2016 , Rif , Oued Boumarioul (Aïn Hamra, 560 m), AP , Larache (5 m) Oscinella vindicata (Meigen, 1830) Ebejer and Kettani 2016 , AP , Larache (5 m), AA , Merzouga (agriculture under date palms, 714 m) Oscinimorpha Lioy, 1864 Oscinimorpha arcuata (Duda, 1932) Ebejer and Kettani 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), Oued Jnane Azaghar (Bni Boufrah, 997 m) Oscinimorpha longirostris (Loew, 1858) Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m) Oscinimorpha minutissima (Strobl, 1900) = Siphonella minutissima Strobl, in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Dardara (484 m) Oscinimorpha novakii (Strobl, 1893) = Conioscinella novakii (Strobl), in Duda 1933: 58 Duda 1933; Ebejer and Kettani 2016 Oscinisoma Lioy, 1864 Oscinisoma cognatum (Meigen, 1830) = Oscinis rufipes Meigen, 1830, with homonym Oscinis rufipes Wiedemann 1830 : 580, in Wiedemann 1830 : 580, Tanger; synonymy and probable specific identity of homonyms established by Becker 1910 : 166, but without considering the similar Oscinisoma gilvipes (Loew, 1858). Polyodaspis Duda, 1933 51 Polyodaspis sulcicollis (Meigen, 1838) = Siphonella sulcicollis (Meigen), in Kroschel and Klein 1999 : 138 Kroschel and Klein 1999 ; Ebejer and Kettani 2016 , MA , Khénifra (17 km SW of Midelt, 1940 m) Pselaphia Becker, 1911 Pselaphia dimidiocera Ebejer & Kettani, 2016 Ebejer and Kettani 2016 , Rif , Adrou (Taghzout, 556 m) – NMWC Rhodesiella Adams, 1905 Rhodesiella fedtshenkoi Nartshuk, 1978 Deeming and Al-Dhafer 2012 , SA , Goulimine (Bou Jarif); Ebejer and Kettani 2016 , Rif , Smir lagoon, Oued Laou (saltmarsh), Jnane Niche (46 m) Sabroskyina Beschovski, 1987 Sabroskyina aharonii (Duda, 1933) Ebejer and Kettani 2016 , AA , 2 km N Erfoud (818 m) Siphunculina Rondani, 1856 Siphunculina ornatifrons (Loew, 1858) = Microneurum ornatifrons (Loew), in Duda 1933: 98 Becker and Stein 1913 , Rif , Tanger; Duda 1933; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Martil (9 m), Oued Mhannech (Tamuda, 18 m), Jnane Niche (27 m), AP , Aïn Chouk 9 km SE (6 m), Larache (5 m) Trachysiphonella Enderlein, 1936 Trachysiphonella ruficeps (Macquart, 1835) Ebejer and Kettani 2016 , AA , 29 km N of Rich (Errachidia, 1570 m) Tricimba Lioy, 1864 Tricimba cincta (Meigen, 1830) Ebejer and Kettani 2016 , Rif , Dardara (484 m) Tricimba heratica Dely-Draskovits, 1983* AA Tricimba humeralis (Loew, 1858) = Tricimba punctifrons Becker, in Becker and Stein 1913 : 93 = Tricimba humeralis (Loew), in Séguy 1941d : 18, 1957 : 273; Mouna 1998 : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1941d , AA , Agadir; Séguy 1957 , AA , Agadir; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Mhannech (Tamuda, 18 m), Aïn Tissemlal (Azilane, 1255 m), Oued Bouhya (Bou Ahmed, 19 m), AA , Oued Ziz (9.5 km SE of Rich, 1285 m), Oued Ziz (13 km N of Erfoud, 800 m) Siphonellopsinae Apotropina Hendel, 1907 Apotropina longepilosa (Strobl, 1893) Ebejer and Kettani 2016 , Rif , Oued Sidi Ben Saâda (Laghdir, 242 m), Oued Kbir (Dardara, 345 m), Oued Siflaou (281 m), Jnane Niche (46 m) Siphonellopsis Strobl, 1906 Siphonellopsis lacteibasis Strobl, 1906 Séguy 1934a : 482; Nartshuk 1984 ; Mouna 1998 New records for Morocco Aphanotrigonum parahastatum Dely-Draskovits, 1981 Anti Atlas: Ougui river, 1.8 km NW Tissint, 29°55'03"N, 7°19'56"W , 582 m, 30.xii.2016, 3♂♂3♀♀, M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). Elachiptera orizae Séguy, 1949 Anti Atlas: river Aoulouz up stream of Idrgane, 30°44'11"N, 7°59'13"W , 866 m, 31.xii.2016, 1♂, M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). Tricimba heratica Dely-Draskovits, 1983 Anti Atlas: Oued Souss, streamup of Idrgane, 30°44'11"N, 7°59'13"W , 866 m, 31.xii.2016, 1♀ (males are needed to confirm the identification), M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). MILICHIIDAE K. Kettani Number of species: 8 . Expected: 14 Faunistic knowledge of the family in Morocco: moderate Madizinae Madizini Desmometopa Loew, 1866 Desmometopa m-nigrum (Zetterstedt, 1848) Séguy 1949a , SA , Guelmim; Mouna 1998 ; Rif (M'Diq), AP (Larache), HA (Tazzarin) – MISR Desmometopa varipalpis Malloch, 1927 Ebejer et al. 2019 , AA , 6 km N of Errachidia (1010 m) Leptometopa Becker, 1903 Leptometopa rufifrons Becker, 1903 Ebejer et al. 2019 , AA , Merzouga (714 m); AA (Merzouga) – MISR Madiza Fallén, 1810 Madiza glabra Fallén, 1820 Mouna 1998 ; Ebejer et al. 2019 , HA , Anafgou ( NPHAO , 2271 m) Milichiinae Milichiini Milichia Meigen, 1830 Milichia albomaculata (Strobl, 1900) Pont and Singh 1965 ; Mouna 1998 ; Brake 2000 ; Koçak and Kemal 2010 ; HA – NHMUK Milichia speciosa Meigen, 1830 Séguy 1930a , HA , Arround (Skoutana) Milichiella Giglio-Tos, 1895 Milichiella lacteipennis (Loew, 1866) Brake 2000 ; Raspi et al. 2009 ; Rif (M'Diq farm) – MISR Phyllomyzinae Phyllomyzini Phyllomyza Fallén, 1810 Phyllomyza sp. aff. equitans (Hendel, 1919) Ebejer et al. 2019 , AA , Oued Ziz (12 km S of Rissani, 737 m) Acknowledgement We gratefully acknowledge the cooperation of Martin J. Ebejer who contributed to the revision of this family. CANACIDAE K. Kettani, L. Munari Number of species: 15 . Expected: 25 Faunistic knowledge of the family in Morocco: poor Canacinae Canace Haliday in Curtis, 1837 Canace actites Mathis, 1982 Ebejer et al. 2019 , AP , Loukous (2 m) Canace nasica Haliday, 1839 Dahl 1964 , AA , Tamri (north of Agadir); Mouna 1998 Xanthocanace Hendel, 1914 Xanthocanace ranula (Loew, 1874) Munari 2010 , AP , 40 km S Larache; Munari and Mathis 2010 ; Munari 2011 ; Munari and Bramuzzo 2018 , Rif , Briyech, AP , Azemmour, El Khaoucha, Fedala, Oued Nefifikh, Oued Loukous, 40 km S Larache Tethininae Tethina Haliday in Curtis, 1837 Tethina alboguttata (Strobl, 1900) Freidberg and Beschovski 1996 , AP , Aïn Diab, Essaouira, Safi, AA , Agadir, Ouarzazate; Mathis and Munari 1996 ; Munari 2002 , 2004 , 2005 , 2011 ; Munari and Mathis 2010 ; Koçak and Kemal 2010 ; Munari and Bramuzzo 2018 , Rif , Briyech, AP , Agadir, Tamri, Essaouira, Cap Hadid, Larache, Loukous, Safi, Dar Caïd-Hadji Tethina albosetulosa (Strobl, 1900) Cassar et al. 2008 , Rif , Oued Laou Basin Tethina flavigenis (Hendel, 1934) Munari and Bramuzzo 2018 , AP , Oued Oum-er-Rbia Tethina grossipes (Becker, 1908) Munari 2004 , AA , Agadir; Munari and Mathis 2010 ; Munari and Bramuzzo 2018 , AP , Tamri Tethina incisuralis (Macquart, 1851) = Tethina pictipes ( Becker 1903 ); Séguy 1941d : 18, Mouna 1998 : 87 Munari 1997 , AA , Erfoud, Rissani (900 m); Munari 2002 , 2010 , 2011 ; Munari and Mathis 2010 ; Munari and Bramuzzo 2018 , AA , Erfoud, Rissani Tethina longirostris (Loew, 1865) Cassar et al. 2008 , Rif , Oued Laou Basin Tethina mariae Munari, 1997 Munari 1997 , AP , 40 km S Larache; Munari and Báez 2000 ; Munari 2002 , 2004 , 2010 , 2011 ; Munari and Mathis 2010 – NHMD ( HT ♂) Tethina pallipes (Loew, 1865) = Tethina ochracea (Hendel, 1913) Cassar et al. 2008 , Rif , Oued Laou Tethina pictipennis Freidberg & Beschovski, 1996 Freidberg and Beschovski 1996 , AP , 40 km south of Larache; Mathis and Munari 1996 , Munari 2002 ; Munari 2004 ; Munari and Mathis 2010 ; Munari 2011 , AA , Agadir (Tamri); Munari and Bramuzzo 2018 , Rif , Briyech, AP , Tamri, Larache, Loukous – NHMD ( HT ♂) Tethina strobliana (Mercier, 1923) Munari and Bramuzzo 2018 , AP , Oued Abou, Rehouna (Rabat) Tethina yaromi Freidberg & Beschovski, 1996 Cassar et al. 2005 , Rif , Smir lagoon Tethina sp. near salinicola Beschovski, 1998 Munari 2004 , AA , Agadir, Ouarzazate; Munari 2005 ; Munari and Bramuzzo 2018 , AP , Tamri Canacinae Canace Haliday in Curtis, 1837 Canace actites Mathis, 1982 Ebejer et al. 2019 , AP , Loukous (2 m) Canace nasica Haliday, 1839 Dahl 1964 , AA , Tamri (north of Agadir); Mouna 1998 Xanthocanace Hendel, 1914 Xanthocanace ranula (Loew, 1874) Munari 2010 , AP , 40 km S Larache; Munari and Mathis 2010 ; Munari 2011 ; Munari and Bramuzzo 2018 , Rif , Briyech, AP , Azemmour, El Khaoucha, Fedala, Oued Nefifikh, Oued Loukous, 40 km S Larache Tethininae Tethina Haliday in Curtis, 1837 Tethina alboguttata (Strobl, 1900) Freidberg and Beschovski 1996 , AP , Aïn Diab, Essaouira, Safi, AA , Agadir, Ouarzazate; Mathis and Munari 1996 ; Munari 2002 , 2004 , 2005 , 2011 ; Munari and Mathis 2010 ; Koçak and Kemal 2010 ; Munari and Bramuzzo 2018 , Rif , Briyech, AP , Agadir, Tamri, Essaouira, Cap Hadid, Larache, Loukous, Safi, Dar Caïd-Hadji Tethina albosetulosa (Strobl, 1900) Cassar et al. 2008 , Rif , Oued Laou Basin Tethina flavigenis (Hendel, 1934) Munari and Bramuzzo 2018 , AP , Oued Oum-er-Rbia Tethina grossipes (Becker, 1908) Munari 2004 , AA , Agadir; Munari and Mathis 2010 ; Munari and Bramuzzo 2018 , AP , Tamri Tethina incisuralis (Macquart, 1851) = Tethina pictipes ( Becker 1903 ); Séguy 1941d : 18, Mouna 1998 : 87 Munari 1997 , AA , Erfoud, Rissani (900 m); Munari 2002 , 2010 , 2011 ; Munari and Mathis 2010 ; Munari and Bramuzzo 2018 , AA , Erfoud, Rissani Tethina longirostris (Loew, 1865) Cassar et al. 2008 , Rif , Oued Laou Basin Tethina mariae Munari, 1997 Munari 1997 , AP , 40 km S Larache; Munari and Báez 2000 ; Munari 2002 , 2004 , 2010 , 2011 ; Munari and Mathis 2010 – NHMD ( HT ♂) Tethina pallipes (Loew, 1865) = Tethina ochracea (Hendel, 1913) Cassar et al. 2008 , Rif , Oued Laou Tethina pictipennis Freidberg & Beschovski, 1996 Freidberg and Beschovski 1996 , AP , 40 km south of Larache; Mathis and Munari 1996 , Munari 2002 ; Munari 2004 ; Munari and Mathis 2010 ; Munari 2011 , AA , Agadir (Tamri); Munari and Bramuzzo 2018 , Rif , Briyech, AP , Tamri, Larache, Loukous – NHMD ( HT ♂) Tethina strobliana (Mercier, 1923) Munari and Bramuzzo 2018 , AP , Oued Abou, Rehouna (Rabat) Tethina yaromi Freidberg & Beschovski, 1996 Cassar et al. 2005 , Rif , Smir lagoon Tethina sp. near salinicola Beschovski, 1998 Munari 2004 , AA , Agadir, Ouarzazate; Munari 2005 ; Munari and Bramuzzo 2018 , AP , Tamri CARNIDAE K. Kettani, M.J. Ebejer Number of species: 3 . Expected: >10 Faunistic knowledge of the family in Morocco: poor Meoneura Rondani , 1856 Meoneura hungarica Papp, 1977 Ebejer et al. 2019 , Rif , Adrou ( PNPB , 556 m) Meoneura prima Becker, 1905 Brake 2011 Meoneura triangularis Collin, 1930 Brake 2011 CHLOROPIDAE 48 K. Kettani, M. von Tschirnhaus, M.J. Ebejer Number of species: 74 . Expected: 140 Faunistic knowledge of the family in Morocco: moderate Chloropinae Assuania Becker, 1903 Assuania melanoleuca (Séguy, 1949) comb. nov. 49 = Chlorops melanoleuca Séguy, in Séguy 1949a : 158 Séguy 1949a , AA , Agdz; Nartshuk 1984 ; Mouna 1998 Assuania thalhammeri (Strobl, 1893) Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Camarota Latreille, 1805 Camarota curvipennis (Latreille, 1805) = Camarota curvipennis (Latreille), in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Oued Laou Capnoptera Loew, 1866 Capnoptera pilosa Loew, 1866 Duda 1933; Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1288 m), Aïn Jdioui (Tahaddart, 8 m) Capnoptera scutata (Rossi, 1790) = Eristalis rufipes Fabricius, in Fabricius 1805 : 245 Ebejer and Kettani 2016 , AP , Larache (2 m) Cetema Hendel, 1907 Cetema maroccanum Nartshuk, 1995 = Cetema maroccana Nartshuk, in Nartshuk 1995 : 277–280 Nartshuk 1995 , HA , Oukaimeden; Ebejer and Kettani 2016 Cetema monticulum Becker, 1910 = Cetema monticula Becker, in Séguy 1941a : 33 = Cetema monticula Rossi, in Mouna 1998 : 85 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Ebejer and Kettani 2016 Chlorops Meigen, 1803 Chlorops interruptus Meigen, 1830 = Chlorops interrupta Meigen, in Séguy 1953a : 86 Séguy 1953a , AP , Oued Yquem (near Rabat); Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1288 m), Oued Aliane (Ksar Sghir, 1 m), Cap Spartel (155 m) Chlorops limbatus Meigen, 1830 Ebejer and Kettani 2016 , Rif , Jebel Moussa (800 m) Chlorops pumilionis (Bjerkander, 1778) = Chlorops nasuta Schrank, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2016 Chlorops serenus Loew, 1866 Ebejer and Kettani 2016 , MA , 17 km NW of Zaida (Khénifra, 1878 m) Cryptonevra Lioy, 1864 Cryptonevra flavitarsis (Meigen, 1830) = Haplegis flavitarsis (Meigen), in Séguy 1949a : 158; Séguy 1953a : 86; Mouna 1998 : 85 Séguy 1949a , AA , Agdz; Séguy 1953a , AP , Oued Yquem (near Rabat); Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 Eurina Meigen, 1830 Eurina lurida Meigen, 1830 Ebejer and Kettani 2016 , Rif , M'Diq, Smir, Kabila beach and dunes Eutropha Loew, 1866 Eutropha albipilosa (Becker, 1908) Duda 1930; Deeming and Al-Dhafer 2012 , AP , 17 km N of Larache, AA , Azemmour, estuary of Oued Tensift; Ebejer and Kettani 2016 , AP , Larache (5 m) Eutropha fulvifrons (Haliday, 1833) Séguy 1941d , AA , Agadir; Mouna 1998 ; Cassar et al. 2005 , Rif , Smir lagoon; Ebejer and Kettani 2016 , AP , Larache (5 m) Lagaroceras Becker, 1903 Lagaroceras andalusiaca (Strobl, 1899) Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m), Oued Aliane (Ksar Sghir, 1 m), AP , Larache (2 m), AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Lasiosina Becker, 1910 Lasiosina herpini (Guérin-Méneville, 1843) = Lasiosina cinctipes Meigen, in Mouna 1998 : 85 Ebejer and Kettani 2016 , Rif , Smir lagoon Lasiosina lindbergi (Duda, 1933) = Steleocerus lindbergi (Duda), in Duda 1933: 142 Duda 1933, MA ; Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Meromyza Meigen, 1830 Meromyza athletica Fedoseeva, 1974 = Meromyza variegata Meigen, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2016 Meromyza curvinervis (Zetterstedt, 1848) = Oxinis curvinervis Latreille, in Mouna 1998 : 85 Mouna 1998 Meromyza nigriventris Macquart, 1835 Ebejer and Kettani 2016 , MA , 17 km SW of Midelt (Khénifra, 1940 m), AA , 29 km N of Rich (Errachidia, 1570 m) Meromyza pratorum Meigen, 1830 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Ebejer and Kettani 2016 Metopostigma Becker, 1903 Metopostigma sabulona Becker, 1910 Ebejer and Kettani 2016 , AA , Oued Ziz (10 km S of Errachidia, 1008 m), Merzouga (714 m) Platycephala Fallén, 1820 Platycephala scapularum (Becker, 1907) Nartshuk 1984 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 ; Ebejer and Kettani 2016 Pseudopachychaeta Strobl, 1902 Pseudopachychaeta pachycera Strobl, 1902 Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m), Oued Ziz (1052 m) Thaumatomyia Zenker, 1833 Thaumatomyia elongatula (Becker, 1910) Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil, 3 m) Thaumatomyia glabra (Meigen, 1830) Ebejer and Kettani 2016 , Rif , Martil (9 m) Thaumatomyia notata (Meigen, 1830) = Chloropisca notata (Meigen), in Séguy 1930a : 179, 1941a : 33; Mouna 1998 : 85 Séguy 1930a , Rif , Tanger; Séguy 1941a , HA , Tachdirt (Jebel Toubkal, 2500 m), Canyon Tessaout (M'Goum, 3000–3200 m); Harris et al. 1980 : 229, HA , Chichaoua; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Laou estuary, Oued Amsa (Amsa, 14 m), Jnane Niche (27 m), Oued Maggou (Maggou, 786 m), Marj Khayl (Beni Leit ( PNPB ), 1088 m), Oued Boumarioul (Aïn Hamra, 560 m), Aïn Tissemlal (Azilane, 1255 m), Oued Bouhya (Bou Ahmed, 19 m), Souk Khemis Anjra (Oued Kbir, 55 m), Zinat (231 m), Oued Zarka (Yarghite, 135 m), Oued Talembote (Usine électrique, 120 m), Oued Kelâa (Akoumi, 400 m), Issaguen (maison forestière Issaguen, 1543 m), Bni Boufrah (94 m), Oued Guallet (Bni Boufrah, 946 m), Oued Tabandoute (Bni Boufrah, 540 m), Oued Taâouniya (Koudiat Ajira, 1536 m), Talankramte (Site sacré Sidi Gneiss ( PNPB ): 461 m), AP , Larache (5 m) Thaumatomyia sulcifrons (Becker, 1907) = Chlorops sulcifrons Becker, in Séguy 1949a : 158 = Chloropisca sulcifrons Zetterstedt, in Mouna 1998 : 85 Séguy 1934a ; Séguy 1949a , AA , Foum-el-Hassan, Akka, Agdz; Nartshuk 1984 ; Mouna 1998 ; Dawah and Abdullah 2006 ; Ebejer and Kettani 2016 Oscinellinae Aphanotrigonum Duda, 1932 Aphanotrigonum cinctellum (Zetterstedt, 1848) Ebejer and Kettani 2016 , AP , Larache (5 m) Aphanotrigonum femorellum Collin, 1946 Ebejer and Kettani 2016 , AP , Larache Aphanotrigonum inerme Collin, 1946 Ebejer and Kettani 2016 , Rif , M'Diq (5 m), AP , Larache (Loukous marsh, 2 m) Aphanotrigonum parahastatum Dely-Draskovits, 1981* AA Calamoncosis Enderlein, 1911 Calamoncosis duinensis (Strobl, 1909) Ebejer and Kettani 2016 , AP , Rabat (on Phragmites ) – MISR Conioscinella Duda, 1929 Conioscinella frontella (Fallén, 1810) Ebejer and Kettani 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m) Conioscinella sordidella (Zetterstedt, 1848) Ebejer and Kettani 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m) Elachiptera Macquart, 1835 Elachiptera bimaculata (Loew, 1845) De Lépiney and Mimeur 1932: 110, Rabat; Séguy 1934a : 479; Bléton and Fieuzet 1943 ; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil: 3 m), Jnane Niche (46 m), Oued Mhannech (18 m), Souk Khemis Anjra (55 m), Boujdad (7 m); AP (Rabat) – MISR Elachiptera cornuta (Fallén, 1820) Mouna 1998 : 85 Elachiptera diastema Collin, 1946 Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m), Oued Tiffert (Tiffert, 1230 m), Issaguen (1543 m), AP , Larache (5 m) Elachiptera graeca Becker, 1910 Séguy 1941, HA , Imi-n'Ouaka (1500 m); Nartshuk 1984 ; Mouna 1998 Elachiptera megaspis (Loew, 1858) Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Dardara (730 m), Aïn Jdioui (Tahaddart, 8 m), Jnane Niche (46 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m); AP (Rabat) – MISR Elachiptera orizae Séguy, 1949* AA Elachiptera rufescens (Walker, 1871) Deeming and Al-Dhafer 2012 , AA , 2 km N Erfoud (818 m); Ebejer and Kettani 2016 , AA , 2 km N Erfoud (818 m) Elachiptera rufifrons Duda, 1932 Ebejer and Kettani 2016 , AP , 9 km SE Aïn Chouk (6 m), Larache (5 m) Elachiptera sarda Nartshuk, 2009 Ebejer and Kettani 2016 , AA , 14 km E of Rich (Errachidia, 1278 m) Elachiptera scrobiculata (Strobl, 1901) = Elachiptera trapezina (Corti, 1909), in Bléton and Fieuzet 1943 : 116 Bléton and Fieuzet 1943 ; Ebejer and Kettani 2016 Elachiptera strobli (Corti, 1909) Ebejer and Kettani 2016 , Rif , Oued Boumarioul (Aïn Hamra, 560 m), Oued Aliane (Ksar Sghir, 1 m); Cap Spartel (155 m), Oued Sidi Ben Saâda (Laghdir, 242 m) Epimadiza Becker, 1910 Epimadiza nigrescens Duda, 1933 50 = Oscinosoma anthracias Séguy, in Séguy 1949a : 158, Mouna 1998 : 85 = Oxinosoma anthracias Séguy, in Mouna 1998 : 85 = " Siphonella oscinina (Fallén)", in Nartshuk 1984 : 236 Séguy 1949a , AA , Alnif; Sabrosky 1965 : 406; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 Hapleginella Duda, 1933 Hapleginella laevifrons (Loew, 1858) El Hassani et al. 1986: 8–11, Rif , Nord Occidental, MA Lasiochaeta Corti, 1909 Lasiochaeta pubescens (Thalhammer, 1898) = Elachiptera pubescens (Thalhammer), var. rufithorax Duda, 1932 in Duda 1932: 31 = Melanochaeta pubescens Thalh., in De Lépiney and Mimeur 1932: 110 = Melanochaeta pubescens (Thalhammer), in Mouna 1998 : 85 De Lépiney and Mimeur 1932, AP , Rabat; Duda 1932: 31, HA ; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Sidi Yahia Aarab (Sidi Yahia Aarab, 178 m), Beni Maâdene (Oued Martil, 3 m), Oued Mhannech (Tamuda, 18 m), Oued Amsa (Amsa, 14 m), Zinat (231 m), Oued Maggou (Maggou, 786 m), Oued Guallet (Bni Boufrah, 946 m); AP (Rabat) – MISR Oscinella Becker, 1909 Oscinella cariciphila Collin, 1946 Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil, 3 m), AA , Merzouga (714 m) Oscinella frit (Linnaeus, 1758) = Oxinosoma frit Linnaeus, in Mouna 1998 : 85 De Lépiney and Mimeur 1932: 109, AP , Rabat; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Issaguen (1543 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m), Oued Guallet (Bni Boufrah, 946 m), Oued Ametrasse (Ametrasse, 841 m), AP , Lower Loukous saltmarsh (2 m), MA , Khénifra (17 km SW of Midelt, 1940 m), Lac Aguelmane Afennourir (30 km SW of Azrou, 1490 m); AP (Rabat) – MISR Oscinella nartshukiana Beschovski, 1978 Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m), AP , Larache Oscinella nitidigenis (Becker, 1908) Ebejer and Kettani 2016 , AA , 6 km N of Errachidia (1010 m), Oued Ziz (1052 m) Oscinella nitidissima (Meigen, 1838) Ebejer and Kettani 2016 , Rif , Dardara (730 m), Moulay Abdelsalam (965 m), Cap Spartel (155 m), Oued Laou (dunes, 2 m), AP , Larache (5 m), MA , Lac Aguelmane Afennourir (30 km SW of Azrou, 1490 m) Oscinella pusilla (Meigen, 1830) Ebejer and Kettani 2016 , AA , Lac Tiffert (4 km W of Merzouga, 702 m) Oscinella ventricosi Nartshuk, 1955 Ebejer and Kettani 2016 , Rif , Oued Boumarioul (Aïn Hamra, 560 m), AP , Larache (5 m) Oscinella vindicata (Meigen, 1830) Ebejer and Kettani 2016 , AP , Larache (5 m), AA , Merzouga (agriculture under date palms, 714 m) Oscinimorpha Lioy, 1864 Oscinimorpha arcuata (Duda, 1932) Ebejer and Kettani 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), Oued Jnane Azaghar (Bni Boufrah, 997 m) Oscinimorpha longirostris (Loew, 1858) Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m) Oscinimorpha minutissima (Strobl, 1900) = Siphonella minutissima Strobl, in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Dardara (484 m) Oscinimorpha novakii (Strobl, 1893) = Conioscinella novakii (Strobl), in Duda 1933: 58 Duda 1933; Ebejer and Kettani 2016 Oscinisoma Lioy, 1864 Oscinisoma cognatum (Meigen, 1830) = Oscinis rufipes Meigen, 1830, with homonym Oscinis rufipes Wiedemann 1830 : 580, in Wiedemann 1830 : 580, Tanger; synonymy and probable specific identity of homonyms established by Becker 1910 : 166, but without considering the similar Oscinisoma gilvipes (Loew, 1858). Polyodaspis Duda, 1933 51 Polyodaspis sulcicollis (Meigen, 1838) = Siphonella sulcicollis (Meigen), in Kroschel and Klein 1999 : 138 Kroschel and Klein 1999 ; Ebejer and Kettani 2016 , MA , Khénifra (17 km SW of Midelt, 1940 m) Pselaphia Becker, 1911 Pselaphia dimidiocera Ebejer & Kettani, 2016 Ebejer and Kettani 2016 , Rif , Adrou (Taghzout, 556 m) – NMWC Rhodesiella Adams, 1905 Rhodesiella fedtshenkoi Nartshuk, 1978 Deeming and Al-Dhafer 2012 , SA , Goulimine (Bou Jarif); Ebejer and Kettani 2016 , Rif , Smir lagoon, Oued Laou (saltmarsh), Jnane Niche (46 m) Sabroskyina Beschovski, 1987 Sabroskyina aharonii (Duda, 1933) Ebejer and Kettani 2016 , AA , 2 km N Erfoud (818 m) Siphunculina Rondani, 1856 Siphunculina ornatifrons (Loew, 1858) = Microneurum ornatifrons (Loew), in Duda 1933: 98 Becker and Stein 1913 , Rif , Tanger; Duda 1933; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Martil (9 m), Oued Mhannech (Tamuda, 18 m), Jnane Niche (27 m), AP , Aïn Chouk 9 km SE (6 m), Larache (5 m) Trachysiphonella Enderlein, 1936 Trachysiphonella ruficeps (Macquart, 1835) Ebejer and Kettani 2016 , AA , 29 km N of Rich (Errachidia, 1570 m) Tricimba Lioy, 1864 Tricimba cincta (Meigen, 1830) Ebejer and Kettani 2016 , Rif , Dardara (484 m) Tricimba heratica Dely-Draskovits, 1983* AA Tricimba humeralis (Loew, 1858) = Tricimba punctifrons Becker, in Becker and Stein 1913 : 93 = Tricimba humeralis (Loew), in Séguy 1941d : 18, 1957 : 273; Mouna 1998 : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1941d , AA , Agadir; Séguy 1957 , AA , Agadir; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Mhannech (Tamuda, 18 m), Aïn Tissemlal (Azilane, 1255 m), Oued Bouhya (Bou Ahmed, 19 m), AA , Oued Ziz (9.5 km SE of Rich, 1285 m), Oued Ziz (13 km N of Erfoud, 800 m) Siphonellopsinae Apotropina Hendel, 1907 Apotropina longepilosa (Strobl, 1893) Ebejer and Kettani 2016 , Rif , Oued Sidi Ben Saâda (Laghdir, 242 m), Oued Kbir (Dardara, 345 m), Oued Siflaou (281 m), Jnane Niche (46 m) Siphonellopsis Strobl, 1906 Siphonellopsis lacteibasis Strobl, 1906 Séguy 1934a : 482; Nartshuk 1984 ; Mouna 1998 New records for Morocco Aphanotrigonum parahastatum Dely-Draskovits, 1981 Anti Atlas: Ougui river, 1.8 km NW Tissint, 29°55'03"N, 7°19'56"W , 582 m, 30.xii.2016, 3♂♂3♀♀, M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). Elachiptera orizae Séguy, 1949 Anti Atlas: river Aoulouz up stream of Idrgane, 30°44'11"N, 7°59'13"W , 866 m, 31.xii.2016, 1♂, M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). Tricimba heratica Dely-Draskovits, 1983 Anti Atlas: Oued Souss, streamup of Idrgane, 30°44'11"N, 7°59'13"W , 866 m, 31.xii.2016, 1♀ (males are needed to confirm the identification), M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). Chloropinae Assuania Becker, 1903 Assuania melanoleuca (Séguy, 1949) comb. nov. 49 = Chlorops melanoleuca Séguy, in Séguy 1949a : 158 Séguy 1949a , AA , Agdz; Nartshuk 1984 ; Mouna 1998 Assuania thalhammeri (Strobl, 1893) Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Camarota Latreille, 1805 Camarota curvipennis (Latreille, 1805) = Camarota curvipennis (Latreille), in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Oued Laou Capnoptera Loew, 1866 Capnoptera pilosa Loew, 1866 Duda 1933; Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1288 m), Aïn Jdioui (Tahaddart, 8 m) Capnoptera scutata (Rossi, 1790) = Eristalis rufipes Fabricius, in Fabricius 1805 : 245 Ebejer and Kettani 2016 , AP , Larache (2 m) Cetema Hendel, 1907 Cetema maroccanum Nartshuk, 1995 = Cetema maroccana Nartshuk, in Nartshuk 1995 : 277–280 Nartshuk 1995 , HA , Oukaimeden; Ebejer and Kettani 2016 Cetema monticulum Becker, 1910 = Cetema monticula Becker, in Séguy 1941a : 33 = Cetema monticula Rossi, in Mouna 1998 : 85 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Ebejer and Kettani 2016 Chlorops Meigen, 1803 Chlorops interruptus Meigen, 1830 = Chlorops interrupta Meigen, in Séguy 1953a : 86 Séguy 1953a , AP , Oued Yquem (near Rabat); Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1288 m), Oued Aliane (Ksar Sghir, 1 m), Cap Spartel (155 m) Chlorops limbatus Meigen, 1830 Ebejer and Kettani 2016 , Rif , Jebel Moussa (800 m) Chlorops pumilionis (Bjerkander, 1778) = Chlorops nasuta Schrank, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2016 Chlorops serenus Loew, 1866 Ebejer and Kettani 2016 , MA , 17 km NW of Zaida (Khénifra, 1878 m) Cryptonevra Lioy, 1864 Cryptonevra flavitarsis (Meigen, 1830) = Haplegis flavitarsis (Meigen), in Séguy 1949a : 158; Séguy 1953a : 86; Mouna 1998 : 85 Séguy 1949a , AA , Agdz; Séguy 1953a , AP , Oued Yquem (near Rabat); Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 Eurina Meigen, 1830 Eurina lurida Meigen, 1830 Ebejer and Kettani 2016 , Rif , M'Diq, Smir, Kabila beach and dunes Eutropha Loew, 1866 Eutropha albipilosa (Becker, 1908) Duda 1930; Deeming and Al-Dhafer 2012 , AP , 17 km N of Larache, AA , Azemmour, estuary of Oued Tensift; Ebejer and Kettani 2016 , AP , Larache (5 m) Eutropha fulvifrons (Haliday, 1833) Séguy 1941d , AA , Agadir; Mouna 1998 ; Cassar et al. 2005 , Rif , Smir lagoon; Ebejer and Kettani 2016 , AP , Larache (5 m) Lagaroceras Becker, 1903 Lagaroceras andalusiaca (Strobl, 1899) Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m), Oued Aliane (Ksar Sghir, 1 m), AP , Larache (2 m), AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Lasiosina Becker, 1910 Lasiosina herpini (Guérin-Méneville, 1843) = Lasiosina cinctipes Meigen, in Mouna 1998 : 85 Ebejer and Kettani 2016 , Rif , Smir lagoon Lasiosina lindbergi (Duda, 1933) = Steleocerus lindbergi (Duda), in Duda 1933: 142 Duda 1933, MA ; Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m) Meromyza Meigen, 1830 Meromyza athletica Fedoseeva, 1974 = Meromyza variegata Meigen, in Mouna 1998 : 85 Mouna 1998 ; Ebejer and Kettani 2016 Meromyza curvinervis (Zetterstedt, 1848) = Oxinis curvinervis Latreille, in Mouna 1998 : 85 Mouna 1998 Meromyza nigriventris Macquart, 1835 Ebejer and Kettani 2016 , MA , 17 km SW of Midelt (Khénifra, 1940 m), AA , 29 km N of Rich (Errachidia, 1570 m) Meromyza pratorum Meigen, 1830 Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Mouna 1998 ; Ebejer and Kettani 2016 Metopostigma Becker, 1903 Metopostigma sabulona Becker, 1910 Ebejer and Kettani 2016 , AA , Oued Ziz (10 km S of Errachidia, 1008 m), Merzouga (714 m) Platycephala Fallén, 1820 Platycephala scapularum (Becker, 1907) Nartshuk 1984 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 ; Ebejer and Kettani 2016 Pseudopachychaeta Strobl, 1902 Pseudopachychaeta pachycera Strobl, 1902 Ebejer and Kettani 2016 , AA , Oued Ziz (9.5 km SE of Rich, 1285 m), Oued Ziz (1052 m) Thaumatomyia Zenker, 1833 Thaumatomyia elongatula (Becker, 1910) Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil, 3 m) Thaumatomyia glabra (Meigen, 1830) Ebejer and Kettani 2016 , Rif , Martil (9 m) Thaumatomyia notata (Meigen, 1830) = Chloropisca notata (Meigen), in Séguy 1930a : 179, 1941a : 33; Mouna 1998 : 85 Séguy 1930a , Rif , Tanger; Séguy 1941a , HA , Tachdirt (Jebel Toubkal, 2500 m), Canyon Tessaout (M'Goum, 3000–3200 m); Harris et al. 1980 : 229, HA , Chichaoua; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Laou estuary, Oued Amsa (Amsa, 14 m), Jnane Niche (27 m), Oued Maggou (Maggou, 786 m), Marj Khayl (Beni Leit ( PNPB ), 1088 m), Oued Boumarioul (Aïn Hamra, 560 m), Aïn Tissemlal (Azilane, 1255 m), Oued Bouhya (Bou Ahmed, 19 m), Souk Khemis Anjra (Oued Kbir, 55 m), Zinat (231 m), Oued Zarka (Yarghite, 135 m), Oued Talembote (Usine électrique, 120 m), Oued Kelâa (Akoumi, 400 m), Issaguen (maison forestière Issaguen, 1543 m), Bni Boufrah (94 m), Oued Guallet (Bni Boufrah, 946 m), Oued Tabandoute (Bni Boufrah, 540 m), Oued Taâouniya (Koudiat Ajira, 1536 m), Talankramte (Site sacré Sidi Gneiss ( PNPB ): 461 m), AP , Larache (5 m) Thaumatomyia sulcifrons (Becker, 1907) = Chlorops sulcifrons Becker, in Séguy 1949a : 158 = Chloropisca sulcifrons Zetterstedt, in Mouna 1998 : 85 Séguy 1934a ; Séguy 1949a , AA , Foum-el-Hassan, Akka, Agdz; Nartshuk 1984 ; Mouna 1998 ; Dawah and Abdullah 2006 ; Ebejer and Kettani 2016 Oscinellinae Aphanotrigonum Duda, 1932 Aphanotrigonum cinctellum (Zetterstedt, 1848) Ebejer and Kettani 2016 , AP , Larache (5 m) Aphanotrigonum femorellum Collin, 1946 Ebejer and Kettani 2016 , AP , Larache Aphanotrigonum inerme Collin, 1946 Ebejer and Kettani 2016 , Rif , M'Diq (5 m), AP , Larache (Loukous marsh, 2 m) Aphanotrigonum parahastatum Dely-Draskovits, 1981* AA Calamoncosis Enderlein, 1911 Calamoncosis duinensis (Strobl, 1909) Ebejer and Kettani 2016 , AP , Rabat (on Phragmites ) – MISR Conioscinella Duda, 1929 Conioscinella frontella (Fallén, 1810) Ebejer and Kettani 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m) Conioscinella sordidella (Zetterstedt, 1848) Ebejer and Kettani 2016 , Rif , Aïn Tissemlal (Azilane, 1255 m) Elachiptera Macquart, 1835 Elachiptera bimaculata (Loew, 1845) De Lépiney and Mimeur 1932: 110, Rabat; Séguy 1934a : 479; Bléton and Fieuzet 1943 ; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil: 3 m), Jnane Niche (46 m), Oued Mhannech (18 m), Souk Khemis Anjra (55 m), Boujdad (7 m); AP (Rabat) – MISR Elachiptera cornuta (Fallén, 1820) Mouna 1998 : 85 Elachiptera diastema Collin, 1946 Ebejer and Kettani 2016 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m), Oued Tiffert (Tiffert, 1230 m), Issaguen (1543 m), AP , Larache (5 m) Elachiptera graeca Becker, 1910 Séguy 1941, HA , Imi-n'Ouaka (1500 m); Nartshuk 1984 ; Mouna 1998 Elachiptera megaspis (Loew, 1858) Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Dardara (730 m), Aïn Jdioui (Tahaddart, 8 m), Jnane Niche (46 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m); AP (Rabat) – MISR Elachiptera orizae Séguy, 1949* AA Elachiptera rufescens (Walker, 1871) Deeming and Al-Dhafer 2012 , AA , 2 km N Erfoud (818 m); Ebejer and Kettani 2016 , AA , 2 km N Erfoud (818 m) Elachiptera rufifrons Duda, 1932 Ebejer and Kettani 2016 , AP , 9 km SE Aïn Chouk (6 m), Larache (5 m) Elachiptera sarda Nartshuk, 2009 Ebejer and Kettani 2016 , AA , 14 km E of Rich (Errachidia, 1278 m) Elachiptera scrobiculata (Strobl, 1901) = Elachiptera trapezina (Corti, 1909), in Bléton and Fieuzet 1943 : 116 Bléton and Fieuzet 1943 ; Ebejer and Kettani 2016 Elachiptera strobli (Corti, 1909) Ebejer and Kettani 2016 , Rif , Oued Boumarioul (Aïn Hamra, 560 m), Oued Aliane (Ksar Sghir, 1 m); Cap Spartel (155 m), Oued Sidi Ben Saâda (Laghdir, 242 m) Epimadiza Becker, 1910 Epimadiza nigrescens Duda, 1933 50 = Oscinosoma anthracias Séguy, in Séguy 1949a : 158, Mouna 1998 : 85 = Oxinosoma anthracias Séguy, in Mouna 1998 : 85 = " Siphonella oscinina (Fallén)", in Nartshuk 1984 : 236 Séguy 1949a , AA , Alnif; Sabrosky 1965 : 406; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 Hapleginella Duda, 1933 Hapleginella laevifrons (Loew, 1858) El Hassani et al. 1986: 8–11, Rif , Nord Occidental, MA Lasiochaeta Corti, 1909 Lasiochaeta pubescens (Thalhammer, 1898) = Elachiptera pubescens (Thalhammer), var. rufithorax Duda, 1932 in Duda 1932: 31 = Melanochaeta pubescens Thalh., in De Lépiney and Mimeur 1932: 110 = Melanochaeta pubescens (Thalhammer), in Mouna 1998 : 85 De Lépiney and Mimeur 1932, AP , Rabat; Duda 1932: 31, HA ; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Sidi Yahia Aarab (Sidi Yahia Aarab, 178 m), Beni Maâdene (Oued Martil, 3 m), Oued Mhannech (Tamuda, 18 m), Oued Amsa (Amsa, 14 m), Zinat (231 m), Oued Maggou (Maggou, 786 m), Oued Guallet (Bni Boufrah, 946 m); AP (Rabat) – MISR Oscinella Becker, 1909 Oscinella cariciphila Collin, 1946 Ebejer and Kettani 2016 , Rif , Beni Maâdene (Oued Martil, 3 m), AA , Merzouga (714 m) Oscinella frit (Linnaeus, 1758) = Oxinosoma frit Linnaeus, in Mouna 1998 : 85 De Lépiney and Mimeur 1932: 109, AP , Rabat; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Issaguen (1543 m), Aïn Tissemlal (Azilane, 1255 m), Oued Maggou (Maggou, 786 m), Oued Guallet (Bni Boufrah, 946 m), Oued Ametrasse (Ametrasse, 841 m), AP , Lower Loukous saltmarsh (2 m), MA , Khénifra (17 km SW of Midelt, 1940 m), Lac Aguelmane Afennourir (30 km SW of Azrou, 1490 m); AP (Rabat) – MISR Oscinella nartshukiana Beschovski, 1978 Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m), AP , Larache Oscinella nitidigenis (Becker, 1908) Ebejer and Kettani 2016 , AA , 6 km N of Errachidia (1010 m), Oued Ziz (1052 m) Oscinella nitidissima (Meigen, 1838) Ebejer and Kettani 2016 , Rif , Dardara (730 m), Moulay Abdelsalam (965 m), Cap Spartel (155 m), Oued Laou (dunes, 2 m), AP , Larache (5 m), MA , Lac Aguelmane Afennourir (30 km SW of Azrou, 1490 m) Oscinella pusilla (Meigen, 1830) Ebejer and Kettani 2016 , AA , Lac Tiffert (4 km W of Merzouga, 702 m) Oscinella ventricosi Nartshuk, 1955 Ebejer and Kettani 2016 , Rif , Oued Boumarioul (Aïn Hamra, 560 m), AP , Larache (5 m) Oscinella vindicata (Meigen, 1830) Ebejer and Kettani 2016 , AP , Larache (5 m), AA , Merzouga (agriculture under date palms, 714 m) Oscinimorpha Lioy, 1864 Oscinimorpha arcuata (Duda, 1932) Ebejer and Kettani 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), Oued Jnane Azaghar (Bni Boufrah, 997 m) Oscinimorpha longirostris (Loew, 1858) Ebejer and Kettani 2016 , Rif , Ksar El Kebir (13 m) Oscinimorpha minutissima (Strobl, 1900) = Siphonella minutissima Strobl, in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Dardara (484 m) Oscinimorpha novakii (Strobl, 1893) = Conioscinella novakii (Strobl), in Duda 1933: 58 Duda 1933; Ebejer and Kettani 2016 Oscinisoma Lioy, 1864 Oscinisoma cognatum (Meigen, 1830) = Oscinis rufipes Meigen, 1830, with homonym Oscinis rufipes Wiedemann 1830 : 580, in Wiedemann 1830 : 580, Tanger; synonymy and probable specific identity of homonyms established by Becker 1910 : 166, but without considering the similar Oscinisoma gilvipes (Loew, 1858). Polyodaspis Duda, 1933 51 Polyodaspis sulcicollis (Meigen, 1838) = Siphonella sulcicollis (Meigen), in Kroschel and Klein 1999 : 138 Kroschel and Klein 1999 ; Ebejer and Kettani 2016 , MA , Khénifra (17 km SW of Midelt, 1940 m) Pselaphia Becker, 1911 Pselaphia dimidiocera Ebejer & Kettani, 2016 Ebejer and Kettani 2016 , Rif , Adrou (Taghzout, 556 m) – NMWC Rhodesiella Adams, 1905 Rhodesiella fedtshenkoi Nartshuk, 1978 Deeming and Al-Dhafer 2012 , SA , Goulimine (Bou Jarif); Ebejer and Kettani 2016 , Rif , Smir lagoon, Oued Laou (saltmarsh), Jnane Niche (46 m) Sabroskyina Beschovski, 1987 Sabroskyina aharonii (Duda, 1933) Ebejer and Kettani 2016 , AA , 2 km N Erfoud (818 m) Siphunculina Rondani, 1856 Siphunculina ornatifrons (Loew, 1858) = Microneurum ornatifrons (Loew), in Duda 1933: 98 Becker and Stein 1913 , Rif , Tanger; Duda 1933; Nartshuk 1984 ; Ebejer and Kettani 2016 , Rif , Martil (9 m), Oued Mhannech (Tamuda, 18 m), Jnane Niche (27 m), AP , Aïn Chouk 9 km SE (6 m), Larache (5 m) Trachysiphonella Enderlein, 1936 Trachysiphonella ruficeps (Macquart, 1835) Ebejer and Kettani 2016 , AA , 29 km N of Rich (Errachidia, 1570 m) Tricimba Lioy, 1864 Tricimba cincta (Meigen, 1830) Ebejer and Kettani 2016 , Rif , Dardara (484 m) Tricimba heratica Dely-Draskovits, 1983* AA Tricimba humeralis (Loew, 1858) = Tricimba punctifrons Becker, in Becker and Stein 1913 : 93 = Tricimba humeralis (Loew), in Séguy 1941d : 18, 1957 : 273; Mouna 1998 : 85 Becker and Stein 1913 , Rif , Tanger; Séguy 1941d , AA , Agadir; Séguy 1957 , AA , Agadir; Nartshuk 1984 ; Mouna 1998 ; Ebejer and Kettani 2016 , Rif , Oued Mhannech (Tamuda, 18 m), Aïn Tissemlal (Azilane, 1255 m), Oued Bouhya (Bou Ahmed, 19 m), AA , Oued Ziz (9.5 km SE of Rich, 1285 m), Oued Ziz (13 km N of Erfoud, 800 m) Siphonellopsinae Apotropina Hendel, 1907 Apotropina longepilosa (Strobl, 1893) Ebejer and Kettani 2016 , Rif , Oued Sidi Ben Saâda (Laghdir, 242 m), Oued Kbir (Dardara, 345 m), Oued Siflaou (281 m), Jnane Niche (46 m) Siphonellopsis Strobl, 1906 Siphonellopsis lacteibasis Strobl, 1906 Séguy 1934a : 482; Nartshuk 1984 ; Mouna 1998 New records for Morocco Aphanotrigonum parahastatum Dely-Draskovits, 1981 Anti Atlas: Ougui river, 1.8 km NW Tissint, 29°55'03"N, 7°19'56"W , 582 m, 30.xii.2016, 3♂♂3♀♀, M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). Elachiptera orizae Séguy, 1949 Anti Atlas: river Aoulouz up stream of Idrgane, 30°44'11"N, 7°59'13"W , 866 m, 31.xii.2016, 1♂, M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). Tricimba heratica Dely-Draskovits, 1983 Anti Atlas: Oued Souss, streamup of Idrgane, 30°44'11"N, 7°59'13"W , 866 m, 31.xii.2016, 1♀ (males are needed to confirm the identification), M. von Tschirnhaus det. – ZSM (M. von Tschirnhaus leg.). MILICHIIDAE K. Kettani Number of species: 8 . Expected: 14 Faunistic knowledge of the family in Morocco: moderate Madizinae Madizini Desmometopa Loew, 1866 Desmometopa m-nigrum (Zetterstedt, 1848) Séguy 1949a , SA , Guelmim; Mouna 1998 ; Rif (M'Diq), AP (Larache), HA (Tazzarin) – MISR Desmometopa varipalpis Malloch, 1927 Ebejer et al. 2019 , AA , 6 km N of Errachidia (1010 m) Leptometopa Becker, 1903 Leptometopa rufifrons Becker, 1903 Ebejer et al. 2019 , AA , Merzouga (714 m); AA (Merzouga) – MISR Madiza Fallén, 1810 Madiza glabra Fallén, 1820 Mouna 1998 ; Ebejer et al. 2019 , HA , Anafgou ( NPHAO , 2271 m) Milichiinae Milichiini Milichia Meigen, 1830 Milichia albomaculata (Strobl, 1900) Pont and Singh 1965 ; Mouna 1998 ; Brake 2000 ; Koçak and Kemal 2010 ; HA – NHMUK Milichia speciosa Meigen, 1830 Séguy 1930a , HA , Arround (Skoutana) Milichiella Giglio-Tos, 1895 Milichiella lacteipennis (Loew, 1866) Brake 2000 ; Raspi et al. 2009 ; Rif (M'Diq farm) – MISR Phyllomyzinae Phyllomyzini Phyllomyza Fallén, 1810 Phyllomyza sp. aff. equitans (Hendel, 1919) Ebejer et al. 2019 , AA , Oued Ziz (12 km S of Rissani, 737 m) Acknowledgement We gratefully acknowledge the cooperation of Martin J. Ebejer who contributed to the revision of this family. Madizinae Madizini Desmometopa Loew, 1866 Desmometopa m-nigrum (Zetterstedt, 1848) Séguy 1949a , SA , Guelmim; Mouna 1998 ; Rif (M'Diq), AP (Larache), HA (Tazzarin) – MISR Desmometopa varipalpis Malloch, 1927 Ebejer et al. 2019 , AA , 6 km N of Errachidia (1010 m) Leptometopa Becker, 1903 Leptometopa rufifrons Becker, 1903 Ebejer et al. 2019 , AA , Merzouga (714 m); AA (Merzouga) – MISR Madiza Fallén, 1810 Madiza glabra Fallén, 1820 Mouna 1998 ; Ebejer et al. 2019 , HA , Anafgou ( NPHAO , 2271 m) Milichiinae Milichiini Milichia Meigen, 1830 Milichia albomaculata (Strobl, 1900) Pont and Singh 1965 ; Mouna 1998 ; Brake 2000 ; Koçak and Kemal 2010 ; HA – NHMUK Milichia speciosa Meigen, 1830 Séguy 1930a , HA , Arround (Skoutana) Milichiella Giglio-Tos, 1895 Milichiella lacteipennis (Loew, 1866) Brake 2000 ; Raspi et al. 2009 ; Rif (M'Diq farm) – MISR Phyllomyzinae Phyllomyzini Phyllomyza Fallén, 1810 Phyllomyza sp. aff. equitans (Hendel, 1919) Ebejer et al. 2019 , AA , Oued Ziz (12 km S of Rissani, 737 m) Acknowledgement We gratefully acknowledge the cooperation of Martin J. Ebejer who contributed to the revision of this family. Sphaeroceroidea CHYROMYIDAE K. Kettani, M.J. Ebejer Number of species: 22 . Expected: 30 Faunistic knowledge of the family in Morocco: good Aphaniosominae Aphaniosoma Becker, 1903 Aphaniosoma approximatum Becker, 1903 Ebejer 2018 , SA , Oued Ougni (0.6 km N of Akka, N'Aït Sidi and 1.8 km NW Tissint, 582 m) Aphaniosoma claridgei Ebejer, 1995 Ebejer 2016b , Rif , Smir lagoon Aphaniosoma collini Lyneborg, 1973 Ebejer 2016b , AP , Larache (5 m) Aphaniosoma forcipatum Ebejer, 1998 Ebejer 2016b , AA , Lac de Tiffert (4 km W of Merzouga, 702 m), Erfoud (30 km N, 894 m), Ziz river (14 km E of Rich, 1278 m) Aphaniosoma gatti Ebejer, 2016 Ebejer 2016b , AP , El Jadida, Azemmour; AP (El Jadida Azemmour) – MISR Aphaniosoma melitense Ebejer, 1993 Andrade and Almeida 2010 , Rif , Mediterranean coast; Ebejer 2016b , AP , Larache (2 m) Aphaniosoma nigricauda Ebejer, 1998 Ebejer 2016b , AA , Lac de Tiffert (4 km W of Merzouga, 702 m), Merzouga (714 m), Ziz river (10 km S of Errachidia, 1008 m; 13 km N of Erfoud, 800 m) Aphaniosoma nigripes Ebejer, 2016 Ebejer 2016b , AA , Ziz river (9.5 km SE of Rich, 1285 m), Erfoud (30 km N, 894 m; AA (Ziz river) – MISR Aphaniosoma nigrum Ebejer, 1998 Ebejer 2016b , AA , Ziz river (Errachidia, 1052 m; 9.5 km SE of Rich, 1285 m), Erfoud (30 km N, 894 m); AA (Ziz river) – MISR Aphaniosoma nitidum Ebejer, 2016 Ebejer 2016b , AA , Ziz river (30 km N of Erfoud, 894 m) Aphaniosoma propinquans Collin, 1949 Ebejer 2016b , Rif , Smir lagoon, Oued Aliane (Ksar Sghir), AP , Larache (2 m); Rif (Oued Aliane) – MISR Aphaniosoma proximum Ebejer, 1998 Ebejer 2016b , AP , Larache (5 m), Sidi Smail (El Jadida), Azemmour, HA , Oued Tensift Aphaniosoma quadrinotatum (Becker, 1904) Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Aphaniosoma rufum Frey, 1935 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Aphaniosoma soror Ebejer, 2016 Ebejer 2016b , AA , Errachidia (14 km E of Rich, 1278 m) Aphaniosoma trisetum Ebejer, 2016 Ebejer 2016b , AA , Ziz river (9.5 km SE of Rich, 1285 m, 13 km N of Erfoud, 800 m, Errachidia, 1052 m), Merzouga (714 m); AA (Ziz river) – MISR Aphaniosoma zizense Ebejer, 2016 Ebejer 2016b , AA , Ziz river (Errachidia, 1052 m; 9.5 km SE of Rich, 1285 m; 10 km S of Errachidia, 1008 m); AA (Ziz river) – MISR Chyromyinae Chyromya Robineau-Desvoidy, 1830 Chyromya robusta Hendel, 1931 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Gymnochiromyia Hendel, 1933 Gymnochiromyia flavella (Zetterstedt, 1848) Ebejer 2016b , Rif , Ksar El Kebir (13 m) Gymnochiromyia homobifida Carles-Tolrá, 2001 Ebejer 2016b , Rif , maison forestière de Talassemtane ( NPT ) Gymnochiromyia inermis (Collin, 1933) Ebejer 1998b ; Ebejer 2016b Gymnochiromyia mihalyii Soós, 1979 Ebejer 2016b , Rif , Oued Siflaou (281 m), Aïn Tissemlal (Azilane, 1255 m), AP , Larache (5 m) Gymnochiromyia tschirnhausi Ebejer, 2018 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Gymnochiromyia zernyi (Czerny, 1929) Ebejer 1998b ; Ebejer 2016b HELEOMYZIDAE K. Kettani, A.J. Woźnica Number of species: 19 . Expected: 30 Faunistic knowledge of the family in Morocco: moderate Heleomyzinae Gymnomus Loew, 1863 Gymnomus atlasicus Woźnica, 2011 Woźnica 2011 , HA , Tazzeka Gymnomus caesius (Meigen, 1830) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Neoleria Malloch, 1919 Neoleria sp.* Rif Heteromyzinae Tephrochlamys Loew, 1862 Tephrochlamys rufiventris (Meigen, 1830)* AA Suilliinae Suillia Robineau-Desvoidy, 1830 Suillia bicolor (Zetterstedt, 1838)* Rif Suillia bistrigata (Meigen, 1830) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Bouhachem, 1098 m), Jebel Lakraâ (Talassemtane, 1541 m), MA , 6 km S of Azrou (1610 m), 20 km S of Azrou (1720 m) Suillia humilis (Meigen, 1830) = Helomyza similis Meigen, in Mouna 1998 : 85 Mouna 1998 ; AP (Maâmora) – MISR Suillia notata (Meigen, 1830) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Bouhachem, 1180 m), Jebel Lakraâ (Talassemtane, 1541 m), Oued Kbir (Béni Ratene, 157 m) Suillia oxyphora (Mik, 1900)* Rif Suillia pallida (Fallén, 1820) Séguy 1941d , HA , Tizi-n'Test (2200 m); Mouna 1998 Suillia tuberiperda (Rondani, 1876) Ebejer et al. 2019 , MA , 6 km S of Azrou (1610 m) Suillia variegata (Loew, 1862) = Helomyza variegata Loew, in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AP , Sidi Yahia du Gharb; Mouna 1998 Trixoscelidinae Trixoscelis Rondani, 1856 Trixoscelis approximata (Loew, 1865) Cassar et al. 2008 , Rif , Smir lagoon; Rif , Tétouan Trixoscelis baliogastra (Černý, 1909) Hackman 1970 Trixoscelis canescens (Loew, 1865) Ebejer et al. 2019 , Rif , Aïn Tissemlal (Azilane, 1255 m) Trixoscelis curvata Carles-Tolrá, 1993 Ebejer et al. 2019 , AP , Larache (5 m) Trixoscelis laeta (Becker, 1907) Becker 1907 ; Séguy 1941d , AA , Agadir; Hackman 1970 ; Mouna 1998 ; Cassar et al. 2008 , Rif , Smir lagoon Trixoscelis mendizabali Hackman, 1970 Hackman 1970 , AA , Aït Melloul (Oued Souss) Trixoscelis pedestris (Loew, 1865) Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 76 m) New records for Morocco Neoleria sp. Rif: Tétouan, Jebel Bouhachem ( PNPB ), Amsemlil, 35.26234°N, 5.43341°W , 1067 m, 11.xii.15, forest ( Pinus pinaster ), K. Kettani leg. (1♀), A.J. Woźnica det. Tephrochlamys rufiventris (Meigen, 1830) Rif: Tétouan, Jebel Bouhachem ( PNPB ), Remla, 35.236865, -5.408025, 961 m, 22.iv.18, forest ( Pinus pinaster ), K. Kettani leg. (1♀), A.J. Woźnica det. Anti Atlas: Errachidia, 13 km E of Goulmima, 31°44.568N, 4°51.945W , dry stony steppe 1100 m, 3.v.2012, M.J. Ebejer leg. (1♂), A.J. Woźnica det. Suillia bicolor (Zetterstedt, 1838) Rif: Talassemtane, Jebel Lakraâ, 35°06.913N, 5°08.034W , 1541 m, 12.vi.2013, meadow in mixed forest, M.J. Ebejer leg. (1♀); Jebel Bouhachem, Taghzout, Adrou, 556 m, 35°22.39N, 5°32.28W , 25.iv.2015, M.J. Ebejer leg. (2♀♀), A.J. Woźnica det. Suillia oxyphora (Mik, 1900) Rif: Jebel Talassemtane, 35°07'N, 5°07'W , 1554 m, 13.iv.2009, Fir forest ( Abies maroccana ), A. Taheri leg. (1♂3♀♀); Azilane, Aïn Tissemlal, 35°11.67N, 5°15.20W , 1255 m, 4.vii.13–13.viii.2013, K. Kettani leg. (1♀), Fir forest ( Abies maroccana ), A.J. Woźnica det. SPHAEROCERIDAE K. Kettani, P. Gatt Number of species: 67 . Expected: 130 Faunistic knowledge of the family in Morocco: poor Copromyzinae Borborillus Duda, 1923 Borborillus vitripennis (Meigen, 1830) Becker and Stein 1913 , Rif , Tanger; Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Copromyza Fallén, 1810 Copromyza equina Fallén, 1820 Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Crumomyia Macquart, 1835 Crumomyia glabrifrons (Meigen, 1830) Gatt et al. 2016 , Rif , Tariouma (El Anasser, 1383 m), MA , Lac Aguelmane Sidi Ali (2050 m) Lotophila Lioy, 1864 Lotophila atra (Meigen, 1830) Gatt 2006 , Rif , Tanger; Marshall et al. 2011 Norrbomia Papp, 1988 Norrbomia hispanica (Duda, 1923) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Norrbomia marginatis (Adams, 1905) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Norrbomia nilotica (Becker, 1903) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia niveipennis (Duda, 1923) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia somogyii (Papp, 1973) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia sordida (Zetterstedt, 1847) Gatt et al. 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), AP , Larache, Loukous Marsh Limosininae Bifronsina Roháček, 1983 Bifronsina bifrons (Stenhammar, 1855) Roháček et al. 2001 ; Marshall et al. 2011 Ceroptera Macquart, 1835 Ceroptera rufitarsis (Meigen, 1830) = Limosina picta Becker, in Becker and Stein 1913 : 94 Becker and Stein 1913 , Rif , Tanger; Papp 1984a ; Roháček et al. 2001 , Rif , Tanger Chaetopodella Duda, 1920 Chaetopodella scutellaris (Haliday, 1836) = Leptocera scutellaris (Haliday), in Séguy 1941a : 32 Séguy 1941a , HA , Imi-n'Ouaka (1500 m) Coproica Rondani, 1861 Coproica digitata (Duda, 1918) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Coproica ferruginata (Stenhammar, 1855) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Coproica hirticula Collin, 1956 Gatt et al. 2016 , Rif , Dardara (730 m), Oued Laou (30 m), Zarka waterfall (Yarghit, 135 m), Oued Ouara (Beni Zid, 440 m), Oued Jnane Azaghar (Bni Boufrah, 997 m), Oued Mhannech (125 m), Adrou (Taghzout, 556 m), Oued Jnane Niche (Jnane Niche, 27 m) Coproica hirtula (Rondani, 1880) Gatt et al. 2016 , Rif , Zarka waterfall (Yarghit, 135 m), AA , 14 km N Errachidia (Errachidia, 1214 m) Coproica lugubris (Haliday, 1836) Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Coproica pusio (Zetterstedt. 1847) Gatt et al. 2016 , Rif , Tamrabete (Oued Laou, 203 m), Smir lagoon (5 m) Coproica rohaceki Carles-Tolrá, 1990 Gatt et al. 2016 , Rif , Oued Amsa (Amsa, 14 m), Oued Moulay Bouchta (Dar Akobaâ, 285 m) Coproica rufifrons Hayashi, 1991 Gatt et al. 2016 , Rif , Issaguen (1543 m) Coproica vagans (Haliday, 1833) Gatt et al. 2016 , AA , 14 km N Errachidia (1214 m), AA , Oued Ziz (13 km N Erfoud, 800 m), SA , Merzouga (698 m) Eulimosina ochripes (Meigen, 1830) Gatt et al. 2016 , Rif , Issaguen (1543 m), Ksar el Kebir (20 m), MA , Lac Aguelamane Afenourrir (30 km SW Azrou, 1760 m) Leptocera Olivier, 1813 Leptocera caenosa (Rondani, 1880) Gatt et al. 2016 , Rif , Dardara (484 m) Leptocera fontinalis (Fallén, 1826) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Leptocera nigra Olivier, 1813 Munari 1993 , MA , Azrou; Roháček et al. 2001 ; Marshall et al. 2011 Limosina Macquart, 1835 Limosina silvatica (Meigen, 1830) Gatt et al. 2016 , Rif , Issaguen (1543 m), Talassemtane ( NPT , 1696 m), MA , 3,5 km S Azrou (1450 m), 17 km NW Zaida (Khénifra, 1878 m) Minilimosina Roháček, 1983 Minilimosina ( Svarciella ) vitripennis (Zetterstedt, 1847) Gatt et al. 2016 , MA , 3,5 km S Azrou (1450 m) Opacifrons Duda, 1918 Opacifrons coxata (Stenhammar, 1855) Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Opacifrons maculifrons (Becker, 1907) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Opalimosina Roháček, 1983 Opalimosina ( Opalimosina ) mirabilis (Collin, 1902) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Paralimosina Papp, 1973 Paralimosina fucata (Rondani, 1880) Gatt et al. 2016 , Rif , 5 km W Dardara (730 m), Dardara (484 m), Oued Azla (Mokdassen Oulya, 186 m), Oued Talembote (Talembote, 320 m), Oued El Khizana (El Khizana, 980 m), Jebel Kelaâ (Talassemtane, 1554 m), Adrou (Taghzout, 556 m) Phthitia Enderlein, 1938 Phthitia ( Kimosina ) sicana (Munari, 1988) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Phthitia ( Kimosina ) ciliata (Duda, 1918) Gatt et al. 2016 , SA , Bou Jarif (Goulimine) Phthitia ( Kimosina ) plumosula (Rondani, 1880) Gatt et al. 2016 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), Jebel Kelaâ (Talassemtane, 1554 m), Zaouiet and Habtiyène (Maggou, 1213 m), Oued Arozane (Bni Moussa, 317 m), Issaguen (1543 m), Zarka waterfall (Yarghit, 135 m), Taghramt (Bine El Ouidane, 276 m) Phthitia ( Kimosina ) pteremoides (Papp, 1973) Gatt et al. 2016 , SA , Bou Jarif (Goulimine) Poecilosomella Duda, 1925 Poecilosomella angulata (Thomson, 1869) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Pseudocollinella Duda, 1924 Pseudocollinella jorlii (Carles-Tolrá, 1990) Munari 1993 , MA , Aguelmane; Roháček et al. 2001 ; Marshall et al. 2011 Pullimosina Roháček, 1983 Pullimosina ( Pullimosina ) heteroneura (Haliday, 1836) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Pullimosina ( Pullimosina ) zayensis Marshall, 1986 Roháček et al. 2001 ; Marshall et al. 2011 Puncticorpus Duda, 1918 Puncticorpus lusitanicum (Richards, 1963) Papp 1984a ; Roháček et al. 2001 Rachispoda Lioy, 1864 Rachispoda acrosticalis (Becker, 1903) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda brevior (Roháček, 1991) Roháček 1991 , AP , Oued Bou-Regreg; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda duodecimseta (Papp, 1973) Roháček 1991 , EM , Guercif-Oued Moulouya; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda fuscipennis (Haliday, 1833) Roháček 1991 , EM , Oued Bou-Regreg; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda gel (Papp, 1978) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda kabuli (Papp, 1978) Roháček 1991 , MA , Taza, Oued Fès; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda lagura (Roháček, 1991) Gatt et al. 2016 , Rif , Ksar el Kebir (20 m), AA , Oued Ziz (12 km S Rissani, 737 m), Oued Ziz (13 km N Erfoud, 800 m), Oued Ziz (30 km N Erfoud, 894 m) Rachispoda lutosoidea (Duda, 1938) Roháček 1991 , MA , Azrou, Aguelmane, Oued Sebou; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda modesta (Duda, 1924) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda uniseta (Roháček, 1991) Roháček 1991 , MA , Taza, Oued Fès; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 ; Vaňhara and Rozkošný 1997 Rachispoda varicornis (Strobl, 1900) Roháček 1991 , AP , Oued Bou-Regreg, MA , Azrou; Munari 1993 , MA , Oued Sebou; Roháček et al. 2001 ; Marshall et al. 2011 Spelobia Spuler, 1924 Spelobia baezi (Papp, 1977) Roháček et al. 2001 ; Marshall et al. 2011 Spelobia clunipes (Meigen, 1830) Gatt et al. 2016 , Rif , 5 km W Dardara (730 m), Martil (beach and dunes), Oued Mhannech (18 m), Oued Afertane (Afertane, 56 m), Maggou waterfall (Maggou, 786 m), Onsar Akboul (NPB, 1315 m), Oued Ametrasse (Ametrasse, 841 m), Oued Tahaddart (Tahaddart, 87 m), Issaguen (1543 m), Lemtahane ( PNPB , 1088 m) Spelobia hungarica (Villeneuve, 1917) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Spelobia quaesita Roháček, 1983 Roháček et al. 2001 ; Marshall et al. 2011 Spinilimosina Roháček, 1983 Spinilimosina brevicostata (Duda, 1918) Roháček et al. 2001 ; Marshall et al. 2011 Telomerina Roháček, 1983 Telomerina pseudoleucoptera (Duda, 1924) Gatt et al. 2016 , Rif , Maggou waterfall (Maggou, 786 m) Thoracochaeta Duda, 1918 Thoracochaeta brachystoma (Stenhammar, 1855) Gatt 2006 ; Cassar et al. 2008 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Thoracochaeta erectiseta Carles-Tolrá, 1994 Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Trachyopella Duda, 1918 Trachyopella ( Trachyopella ) coprina (Duda, 1918) Gatt et al. 2016 , Rif , Oued Jnane Niche (Jnane Niche, 27 m) Trachyopella ( Trachyopella ) melania (Haliday, 1836) Gatt et al. 2016 , Rif , Oued Nwawel (Azla, 57 m), M'Diq (5 m) Sphaerocerinae Ischiolepta Lioy, 1864 Ischiolepta pusilla (Fallén, 1820) Gatt et al. 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), Aïn Tissemlal (Azilane, 1255 m) Ischiolepta vaporariorum (Haliday, 1836) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Lotobia Lioy, 1864 Lotobia africana (Becker, 1907) Roháček et al. 2001 ; Marshall et al. 2011 Lotobia pallidiventris (Meigen, 1830) Gatt 2006 , Rif , Tétouan, M'Diq, Oued Laou; Marshall et al. 2011 Sphaerocera Latreille, 1804 Sphaerocera curvipes Latreille, 1805 Roháček et al. 2001 ; Marshall et al. 2011 CHYROMYIDAE K. Kettani, M.J. Ebejer Number of species: 22 . Expected: 30 Faunistic knowledge of the family in Morocco: good Aphaniosominae Aphaniosoma Becker, 1903 Aphaniosoma approximatum Becker, 1903 Ebejer 2018 , SA , Oued Ougni (0.6 km N of Akka, N'Aït Sidi and 1.8 km NW Tissint, 582 m) Aphaniosoma claridgei Ebejer, 1995 Ebejer 2016b , Rif , Smir lagoon Aphaniosoma collini Lyneborg, 1973 Ebejer 2016b , AP , Larache (5 m) Aphaniosoma forcipatum Ebejer, 1998 Ebejer 2016b , AA , Lac de Tiffert (4 km W of Merzouga, 702 m), Erfoud (30 km N, 894 m), Ziz river (14 km E of Rich, 1278 m) Aphaniosoma gatti Ebejer, 2016 Ebejer 2016b , AP , El Jadida, Azemmour; AP (El Jadida Azemmour) – MISR Aphaniosoma melitense Ebejer, 1993 Andrade and Almeida 2010 , Rif , Mediterranean coast; Ebejer 2016b , AP , Larache (2 m) Aphaniosoma nigricauda Ebejer, 1998 Ebejer 2016b , AA , Lac de Tiffert (4 km W of Merzouga, 702 m), Merzouga (714 m), Ziz river (10 km S of Errachidia, 1008 m; 13 km N of Erfoud, 800 m) Aphaniosoma nigripes Ebejer, 2016 Ebejer 2016b , AA , Ziz river (9.5 km SE of Rich, 1285 m), Erfoud (30 km N, 894 m; AA (Ziz river) – MISR Aphaniosoma nigrum Ebejer, 1998 Ebejer 2016b , AA , Ziz river (Errachidia, 1052 m; 9.5 km SE of Rich, 1285 m), Erfoud (30 km N, 894 m); AA (Ziz river) – MISR Aphaniosoma nitidum Ebejer, 2016 Ebejer 2016b , AA , Ziz river (30 km N of Erfoud, 894 m) Aphaniosoma propinquans Collin, 1949 Ebejer 2016b , Rif , Smir lagoon, Oued Aliane (Ksar Sghir), AP , Larache (2 m); Rif (Oued Aliane) – MISR Aphaniosoma proximum Ebejer, 1998 Ebejer 2016b , AP , Larache (5 m), Sidi Smail (El Jadida), Azemmour, HA , Oued Tensift Aphaniosoma quadrinotatum (Becker, 1904) Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Aphaniosoma rufum Frey, 1935 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Aphaniosoma soror Ebejer, 2016 Ebejer 2016b , AA , Errachidia (14 km E of Rich, 1278 m) Aphaniosoma trisetum Ebejer, 2016 Ebejer 2016b , AA , Ziz river (9.5 km SE of Rich, 1285 m, 13 km N of Erfoud, 800 m, Errachidia, 1052 m), Merzouga (714 m); AA (Ziz river) – MISR Aphaniosoma zizense Ebejer, 2016 Ebejer 2016b , AA , Ziz river (Errachidia, 1052 m; 9.5 km SE of Rich, 1285 m; 10 km S of Errachidia, 1008 m); AA (Ziz river) – MISR Chyromyinae Chyromya Robineau-Desvoidy, 1830 Chyromya robusta Hendel, 1931 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Gymnochiromyia Hendel, 1933 Gymnochiromyia flavella (Zetterstedt, 1848) Ebejer 2016b , Rif , Ksar El Kebir (13 m) Gymnochiromyia homobifida Carles-Tolrá, 2001 Ebejer 2016b , Rif , maison forestière de Talassemtane ( NPT ) Gymnochiromyia inermis (Collin, 1933) Ebejer 1998b ; Ebejer 2016b Gymnochiromyia mihalyii Soós, 1979 Ebejer 2016b , Rif , Oued Siflaou (281 m), Aïn Tissemlal (Azilane, 1255 m), AP , Larache (5 m) Gymnochiromyia tschirnhausi Ebejer, 2018 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Gymnochiromyia zernyi (Czerny, 1929) Ebejer 1998b ; Ebejer 2016b Aphaniosominae Aphaniosoma Becker, 1903 Aphaniosoma approximatum Becker, 1903 Ebejer 2018 , SA , Oued Ougni (0.6 km N of Akka, N'Aït Sidi and 1.8 km NW Tissint, 582 m) Aphaniosoma claridgei Ebejer, 1995 Ebejer 2016b , Rif , Smir lagoon Aphaniosoma collini Lyneborg, 1973 Ebejer 2016b , AP , Larache (5 m) Aphaniosoma forcipatum Ebejer, 1998 Ebejer 2016b , AA , Lac de Tiffert (4 km W of Merzouga, 702 m), Erfoud (30 km N, 894 m), Ziz river (14 km E of Rich, 1278 m) Aphaniosoma gatti Ebejer, 2016 Ebejer 2016b , AP , El Jadida, Azemmour; AP (El Jadida Azemmour) – MISR Aphaniosoma melitense Ebejer, 1993 Andrade and Almeida 2010 , Rif , Mediterranean coast; Ebejer 2016b , AP , Larache (2 m) Aphaniosoma nigricauda Ebejer, 1998 Ebejer 2016b , AA , Lac de Tiffert (4 km W of Merzouga, 702 m), Merzouga (714 m), Ziz river (10 km S of Errachidia, 1008 m; 13 km N of Erfoud, 800 m) Aphaniosoma nigripes Ebejer, 2016 Ebejer 2016b , AA , Ziz river (9.5 km SE of Rich, 1285 m), Erfoud (30 km N, 894 m; AA (Ziz river) – MISR Aphaniosoma nigrum Ebejer, 1998 Ebejer 2016b , AA , Ziz river (Errachidia, 1052 m; 9.5 km SE of Rich, 1285 m), Erfoud (30 km N, 894 m); AA (Ziz river) – MISR Aphaniosoma nitidum Ebejer, 2016 Ebejer 2016b , AA , Ziz river (30 km N of Erfoud, 894 m) Aphaniosoma propinquans Collin, 1949 Ebejer 2016b , Rif , Smir lagoon, Oued Aliane (Ksar Sghir), AP , Larache (2 m); Rif (Oued Aliane) – MISR Aphaniosoma proximum Ebejer, 1998 Ebejer 2016b , AP , Larache (5 m), Sidi Smail (El Jadida), Azemmour, HA , Oued Tensift Aphaniosoma quadrinotatum (Becker, 1904) Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Aphaniosoma rufum Frey, 1935 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Aphaniosoma soror Ebejer, 2016 Ebejer 2016b , AA , Errachidia (14 km E of Rich, 1278 m) Aphaniosoma trisetum Ebejer, 2016 Ebejer 2016b , AA , Ziz river (9.5 km SE of Rich, 1285 m, 13 km N of Erfoud, 800 m, Errachidia, 1052 m), Merzouga (714 m); AA (Ziz river) – MISR Aphaniosoma zizense Ebejer, 2016 Ebejer 2016b , AA , Ziz river (Errachidia, 1052 m; 9.5 km SE of Rich, 1285 m; 10 km S of Errachidia, 1008 m); AA (Ziz river) – MISR Chyromyinae Chyromya Robineau-Desvoidy, 1830 Chyromya robusta Hendel, 1931 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Gymnochiromyia Hendel, 1933 Gymnochiromyia flavella (Zetterstedt, 1848) Ebejer 2016b , Rif , Ksar El Kebir (13 m) Gymnochiromyia homobifida Carles-Tolrá, 2001 Ebejer 2016b , Rif , maison forestière de Talassemtane ( NPT ) Gymnochiromyia inermis (Collin, 1933) Ebejer 1998b ; Ebejer 2016b Gymnochiromyia mihalyii Soós, 1979 Ebejer 2016b , Rif , Oued Siflaou (281 m), Aïn Tissemlal (Azilane, 1255 m), AP , Larache (5 m) Gymnochiromyia tschirnhausi Ebejer, 2018 Ebejer 2018 , AA , S of village Sidi R'bat (10 m) Gymnochiromyia zernyi (Czerny, 1929) Ebejer 1998b ; Ebejer 2016b HELEOMYZIDAE K. Kettani, A.J. Woźnica Number of species: 19 . Expected: 30 Faunistic knowledge of the family in Morocco: moderate Heleomyzinae Gymnomus Loew, 1863 Gymnomus atlasicus Woźnica, 2011 Woźnica 2011 , HA , Tazzeka Gymnomus caesius (Meigen, 1830) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Neoleria Malloch, 1919 Neoleria sp.* Rif Heteromyzinae Tephrochlamys Loew, 1862 Tephrochlamys rufiventris (Meigen, 1830)* AA Suilliinae Suillia Robineau-Desvoidy, 1830 Suillia bicolor (Zetterstedt, 1838)* Rif Suillia bistrigata (Meigen, 1830) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Bouhachem, 1098 m), Jebel Lakraâ (Talassemtane, 1541 m), MA , 6 km S of Azrou (1610 m), 20 km S of Azrou (1720 m) Suillia humilis (Meigen, 1830) = Helomyza similis Meigen, in Mouna 1998 : 85 Mouna 1998 ; AP (Maâmora) – MISR Suillia notata (Meigen, 1830) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Bouhachem, 1180 m), Jebel Lakraâ (Talassemtane, 1541 m), Oued Kbir (Béni Ratene, 157 m) Suillia oxyphora (Mik, 1900)* Rif Suillia pallida (Fallén, 1820) Séguy 1941d , HA , Tizi-n'Test (2200 m); Mouna 1998 Suillia tuberiperda (Rondani, 1876) Ebejer et al. 2019 , MA , 6 km S of Azrou (1610 m) Suillia variegata (Loew, 1862) = Helomyza variegata Loew, in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AP , Sidi Yahia du Gharb; Mouna 1998 Trixoscelidinae Trixoscelis Rondani, 1856 Trixoscelis approximata (Loew, 1865) Cassar et al. 2008 , Rif , Smir lagoon; Rif , Tétouan Trixoscelis baliogastra (Černý, 1909) Hackman 1970 Trixoscelis canescens (Loew, 1865) Ebejer et al. 2019 , Rif , Aïn Tissemlal (Azilane, 1255 m) Trixoscelis curvata Carles-Tolrá, 1993 Ebejer et al. 2019 , AP , Larache (5 m) Trixoscelis laeta (Becker, 1907) Becker 1907 ; Séguy 1941d , AA , Agadir; Hackman 1970 ; Mouna 1998 ; Cassar et al. 2008 , Rif , Smir lagoon Trixoscelis mendizabali Hackman, 1970 Hackman 1970 , AA , Aït Melloul (Oued Souss) Trixoscelis pedestris (Loew, 1865) Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 76 m) New records for Morocco Neoleria sp. Rif: Tétouan, Jebel Bouhachem ( PNPB ), Amsemlil, 35.26234°N, 5.43341°W , 1067 m, 11.xii.15, forest ( Pinus pinaster ), K. Kettani leg. (1♀), A.J. Woźnica det. Tephrochlamys rufiventris (Meigen, 1830) Rif: Tétouan, Jebel Bouhachem ( PNPB ), Remla, 35.236865, -5.408025, 961 m, 22.iv.18, forest ( Pinus pinaster ), K. Kettani leg. (1♀), A.J. Woźnica det. Anti Atlas: Errachidia, 13 km E of Goulmima, 31°44.568N, 4°51.945W , dry stony steppe 1100 m, 3.v.2012, M.J. Ebejer leg. (1♂), A.J. Woźnica det. Suillia bicolor (Zetterstedt, 1838) Rif: Talassemtane, Jebel Lakraâ, 35°06.913N, 5°08.034W , 1541 m, 12.vi.2013, meadow in mixed forest, M.J. Ebejer leg. (1♀); Jebel Bouhachem, Taghzout, Adrou, 556 m, 35°22.39N, 5°32.28W , 25.iv.2015, M.J. Ebejer leg. (2♀♀), A.J. Woźnica det. Suillia oxyphora (Mik, 1900) Rif: Jebel Talassemtane, 35°07'N, 5°07'W , 1554 m, 13.iv.2009, Fir forest ( Abies maroccana ), A. Taheri leg. (1♂3♀♀); Azilane, Aïn Tissemlal, 35°11.67N, 5°15.20W , 1255 m, 4.vii.13–13.viii.2013, K. Kettani leg. (1♀), Fir forest ( Abies maroccana ), A.J. Woźnica det. Heleomyzinae Gymnomus Loew, 1863 Gymnomus atlasicus Woźnica, 2011 Woźnica 2011 , HA , Tazzeka Gymnomus caesius (Meigen, 1830) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Neoleria Malloch, 1919 Neoleria sp.* Rif Heteromyzinae Tephrochlamys Loew, 1862 Tephrochlamys rufiventris (Meigen, 1830)* AA Suilliinae Suillia Robineau-Desvoidy, 1830 Suillia bicolor (Zetterstedt, 1838)* Rif Suillia bistrigata (Meigen, 1830) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Bouhachem, 1098 m), Jebel Lakraâ (Talassemtane, 1541 m), MA , 6 km S of Azrou (1610 m), 20 km S of Azrou (1720 m) Suillia humilis (Meigen, 1830) = Helomyza similis Meigen, in Mouna 1998 : 85 Mouna 1998 ; AP (Maâmora) – MISR Suillia notata (Meigen, 1830) Ebejer et al. 2019 , Rif , Moulay Abdelsalam (Bouhachem, 1180 m), Jebel Lakraâ (Talassemtane, 1541 m), Oued Kbir (Béni Ratene, 157 m) Suillia oxyphora (Mik, 1900)* Rif Suillia pallida (Fallén, 1820) Séguy 1941d , HA , Tizi-n'Test (2200 m); Mouna 1998 Suillia tuberiperda (Rondani, 1876) Ebejer et al. 2019 , MA , 6 km S of Azrou (1610 m) Suillia variegata (Loew, 1862) = Helomyza variegata Loew, in Becker and Stein 1913 : 93 Becker and Stein 1913 , Rif , Tanger; Séguy 1953a , AP , Sidi Yahia du Gharb; Mouna 1998 Trixoscelidinae Trixoscelis Rondani, 1856 Trixoscelis approximata (Loew, 1865) Cassar et al. 2008 , Rif , Smir lagoon; Rif , Tétouan Trixoscelis baliogastra (Černý, 1909) Hackman 1970 Trixoscelis canescens (Loew, 1865) Ebejer et al. 2019 , Rif , Aïn Tissemlal (Azilane, 1255 m) Trixoscelis curvata Carles-Tolrá, 1993 Ebejer et al. 2019 , AP , Larache (5 m) Trixoscelis laeta (Becker, 1907) Becker 1907 ; Séguy 1941d , AA , Agadir; Hackman 1970 ; Mouna 1998 ; Cassar et al. 2008 , Rif , Smir lagoon Trixoscelis mendizabali Hackman, 1970 Hackman 1970 , AA , Aït Melloul (Oued Souss) Trixoscelis pedestris (Loew, 1865) Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 76 m) New records for Morocco Neoleria sp. Rif: Tétouan, Jebel Bouhachem ( PNPB ), Amsemlil, 35.26234°N, 5.43341°W , 1067 m, 11.xii.15, forest ( Pinus pinaster ), K. Kettani leg. (1♀), A.J. Woźnica det. Tephrochlamys rufiventris (Meigen, 1830) Rif: Tétouan, Jebel Bouhachem ( PNPB ), Remla, 35.236865, -5.408025, 961 m, 22.iv.18, forest ( Pinus pinaster ), K. Kettani leg. (1♀), A.J. Woźnica det. Anti Atlas: Errachidia, 13 km E of Goulmima, 31°44.568N, 4°51.945W , dry stony steppe 1100 m, 3.v.2012, M.J. Ebejer leg. (1♂), A.J. Woźnica det. Suillia bicolor (Zetterstedt, 1838) Rif: Talassemtane, Jebel Lakraâ, 35°06.913N, 5°08.034W , 1541 m, 12.vi.2013, meadow in mixed forest, M.J. Ebejer leg. (1♀); Jebel Bouhachem, Taghzout, Adrou, 556 m, 35°22.39N, 5°32.28W , 25.iv.2015, M.J. Ebejer leg. (2♀♀), A.J. Woźnica det. Suillia oxyphora (Mik, 1900) Rif: Jebel Talassemtane, 35°07'N, 5°07'W , 1554 m, 13.iv.2009, Fir forest ( Abies maroccana ), A. Taheri leg. (1♂3♀♀); Azilane, Aïn Tissemlal, 35°11.67N, 5°15.20W , 1255 m, 4.vii.13–13.viii.2013, K. Kettani leg. (1♀), Fir forest ( Abies maroccana ), A.J. Woźnica det. SPHAEROCERIDAE K. Kettani, P. Gatt Number of species: 67 . Expected: 130 Faunistic knowledge of the family in Morocco: poor Copromyzinae Borborillus Duda, 1923 Borborillus vitripennis (Meigen, 1830) Becker and Stein 1913 , Rif , Tanger; Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Copromyza Fallén, 1810 Copromyza equina Fallén, 1820 Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Crumomyia Macquart, 1835 Crumomyia glabrifrons (Meigen, 1830) Gatt et al. 2016 , Rif , Tariouma (El Anasser, 1383 m), MA , Lac Aguelmane Sidi Ali (2050 m) Lotophila Lioy, 1864 Lotophila atra (Meigen, 1830) Gatt 2006 , Rif , Tanger; Marshall et al. 2011 Norrbomia Papp, 1988 Norrbomia hispanica (Duda, 1923) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Norrbomia marginatis (Adams, 1905) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Norrbomia nilotica (Becker, 1903) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia niveipennis (Duda, 1923) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia somogyii (Papp, 1973) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia sordida (Zetterstedt, 1847) Gatt et al. 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), AP , Larache, Loukous Marsh Limosininae Bifronsina Roháček, 1983 Bifronsina bifrons (Stenhammar, 1855) Roháček et al. 2001 ; Marshall et al. 2011 Ceroptera Macquart, 1835 Ceroptera rufitarsis (Meigen, 1830) = Limosina picta Becker, in Becker and Stein 1913 : 94 Becker and Stein 1913 , Rif , Tanger; Papp 1984a ; Roháček et al. 2001 , Rif , Tanger Chaetopodella Duda, 1920 Chaetopodella scutellaris (Haliday, 1836) = Leptocera scutellaris (Haliday), in Séguy 1941a : 32 Séguy 1941a , HA , Imi-n'Ouaka (1500 m) Coproica Rondani, 1861 Coproica digitata (Duda, 1918) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Coproica ferruginata (Stenhammar, 1855) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Coproica hirticula Collin, 1956 Gatt et al. 2016 , Rif , Dardara (730 m), Oued Laou (30 m), Zarka waterfall (Yarghit, 135 m), Oued Ouara (Beni Zid, 440 m), Oued Jnane Azaghar (Bni Boufrah, 997 m), Oued Mhannech (125 m), Adrou (Taghzout, 556 m), Oued Jnane Niche (Jnane Niche, 27 m) Coproica hirtula (Rondani, 1880) Gatt et al. 2016 , Rif , Zarka waterfall (Yarghit, 135 m), AA , 14 km N Errachidia (Errachidia, 1214 m) Coproica lugubris (Haliday, 1836) Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Coproica pusio (Zetterstedt. 1847) Gatt et al. 2016 , Rif , Tamrabete (Oued Laou, 203 m), Smir lagoon (5 m) Coproica rohaceki Carles-Tolrá, 1990 Gatt et al. 2016 , Rif , Oued Amsa (Amsa, 14 m), Oued Moulay Bouchta (Dar Akobaâ, 285 m) Coproica rufifrons Hayashi, 1991 Gatt et al. 2016 , Rif , Issaguen (1543 m) Coproica vagans (Haliday, 1833) Gatt et al. 2016 , AA , 14 km N Errachidia (1214 m), AA , Oued Ziz (13 km N Erfoud, 800 m), SA , Merzouga (698 m) Eulimosina ochripes (Meigen, 1830) Gatt et al. 2016 , Rif , Issaguen (1543 m), Ksar el Kebir (20 m), MA , Lac Aguelamane Afenourrir (30 km SW Azrou, 1760 m) Leptocera Olivier, 1813 Leptocera caenosa (Rondani, 1880) Gatt et al. 2016 , Rif , Dardara (484 m) Leptocera fontinalis (Fallén, 1826) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Leptocera nigra Olivier, 1813 Munari 1993 , MA , Azrou; Roháček et al. 2001 ; Marshall et al. 2011 Limosina Macquart, 1835 Limosina silvatica (Meigen, 1830) Gatt et al. 2016 , Rif , Issaguen (1543 m), Talassemtane ( NPT , 1696 m), MA , 3,5 km S Azrou (1450 m), 17 km NW Zaida (Khénifra, 1878 m) Minilimosina Roháček, 1983 Minilimosina ( Svarciella ) vitripennis (Zetterstedt, 1847) Gatt et al. 2016 , MA , 3,5 km S Azrou (1450 m) Opacifrons Duda, 1918 Opacifrons coxata (Stenhammar, 1855) Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Opacifrons maculifrons (Becker, 1907) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Opalimosina Roháček, 1983 Opalimosina ( Opalimosina ) mirabilis (Collin, 1902) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Paralimosina Papp, 1973 Paralimosina fucata (Rondani, 1880) Gatt et al. 2016 , Rif , 5 km W Dardara (730 m), Dardara (484 m), Oued Azla (Mokdassen Oulya, 186 m), Oued Talembote (Talembote, 320 m), Oued El Khizana (El Khizana, 980 m), Jebel Kelaâ (Talassemtane, 1554 m), Adrou (Taghzout, 556 m) Phthitia Enderlein, 1938 Phthitia ( Kimosina ) sicana (Munari, 1988) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Phthitia ( Kimosina ) ciliata (Duda, 1918) Gatt et al. 2016 , SA , Bou Jarif (Goulimine) Phthitia ( Kimosina ) plumosula (Rondani, 1880) Gatt et al. 2016 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), Jebel Kelaâ (Talassemtane, 1554 m), Zaouiet and Habtiyène (Maggou, 1213 m), Oued Arozane (Bni Moussa, 317 m), Issaguen (1543 m), Zarka waterfall (Yarghit, 135 m), Taghramt (Bine El Ouidane, 276 m) Phthitia ( Kimosina ) pteremoides (Papp, 1973) Gatt et al. 2016 , SA , Bou Jarif (Goulimine) Poecilosomella Duda, 1925 Poecilosomella angulata (Thomson, 1869) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Pseudocollinella Duda, 1924 Pseudocollinella jorlii (Carles-Tolrá, 1990) Munari 1993 , MA , Aguelmane; Roháček et al. 2001 ; Marshall et al. 2011 Pullimosina Roháček, 1983 Pullimosina ( Pullimosina ) heteroneura (Haliday, 1836) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Pullimosina ( Pullimosina ) zayensis Marshall, 1986 Roháček et al. 2001 ; Marshall et al. 2011 Puncticorpus Duda, 1918 Puncticorpus lusitanicum (Richards, 1963) Papp 1984a ; Roháček et al. 2001 Rachispoda Lioy, 1864 Rachispoda acrosticalis (Becker, 1903) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda brevior (Roháček, 1991) Roháček 1991 , AP , Oued Bou-Regreg; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda duodecimseta (Papp, 1973) Roháček 1991 , EM , Guercif-Oued Moulouya; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda fuscipennis (Haliday, 1833) Roháček 1991 , EM , Oued Bou-Regreg; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda gel (Papp, 1978) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda kabuli (Papp, 1978) Roháček 1991 , MA , Taza, Oued Fès; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda lagura (Roháček, 1991) Gatt et al. 2016 , Rif , Ksar el Kebir (20 m), AA , Oued Ziz (12 km S Rissani, 737 m), Oued Ziz (13 km N Erfoud, 800 m), Oued Ziz (30 km N Erfoud, 894 m) Rachispoda lutosoidea (Duda, 1938) Roháček 1991 , MA , Azrou, Aguelmane, Oued Sebou; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda modesta (Duda, 1924) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda uniseta (Roháček, 1991) Roháček 1991 , MA , Taza, Oued Fès; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 ; Vaňhara and Rozkošný 1997 Rachispoda varicornis (Strobl, 1900) Roháček 1991 , AP , Oued Bou-Regreg, MA , Azrou; Munari 1993 , MA , Oued Sebou; Roháček et al. 2001 ; Marshall et al. 2011 Spelobia Spuler, 1924 Spelobia baezi (Papp, 1977) Roháček et al. 2001 ; Marshall et al. 2011 Spelobia clunipes (Meigen, 1830) Gatt et al. 2016 , Rif , 5 km W Dardara (730 m), Martil (beach and dunes), Oued Mhannech (18 m), Oued Afertane (Afertane, 56 m), Maggou waterfall (Maggou, 786 m), Onsar Akboul (NPB, 1315 m), Oued Ametrasse (Ametrasse, 841 m), Oued Tahaddart (Tahaddart, 87 m), Issaguen (1543 m), Lemtahane ( PNPB , 1088 m) Spelobia hungarica (Villeneuve, 1917) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Spelobia quaesita Roháček, 1983 Roháček et al. 2001 ; Marshall et al. 2011 Spinilimosina Roháček, 1983 Spinilimosina brevicostata (Duda, 1918) Roháček et al. 2001 ; Marshall et al. 2011 Telomerina Roháček, 1983 Telomerina pseudoleucoptera (Duda, 1924) Gatt et al. 2016 , Rif , Maggou waterfall (Maggou, 786 m) Thoracochaeta Duda, 1918 Thoracochaeta brachystoma (Stenhammar, 1855) Gatt 2006 ; Cassar et al. 2008 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Thoracochaeta erectiseta Carles-Tolrá, 1994 Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Trachyopella Duda, 1918 Trachyopella ( Trachyopella ) coprina (Duda, 1918) Gatt et al. 2016 , Rif , Oued Jnane Niche (Jnane Niche, 27 m) Trachyopella ( Trachyopella ) melania (Haliday, 1836) Gatt et al. 2016 , Rif , Oued Nwawel (Azla, 57 m), M'Diq (5 m) Sphaerocerinae Ischiolepta Lioy, 1864 Ischiolepta pusilla (Fallén, 1820) Gatt et al. 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), Aïn Tissemlal (Azilane, 1255 m) Ischiolepta vaporariorum (Haliday, 1836) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Lotobia Lioy, 1864 Lotobia africana (Becker, 1907) Roháček et al. 2001 ; Marshall et al. 2011 Lotobia pallidiventris (Meigen, 1830) Gatt 2006 , Rif , Tétouan, M'Diq, Oued Laou; Marshall et al. 2011 Sphaerocera Latreille, 1804 Sphaerocera curvipes Latreille, 1805 Roháček et al. 2001 ; Marshall et al. 2011 Copromyzinae Borborillus Duda, 1923 Borborillus vitripennis (Meigen, 1830) Becker and Stein 1913 , Rif , Tanger; Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Copromyza Fallén, 1810 Copromyza equina Fallén, 1820 Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Crumomyia Macquart, 1835 Crumomyia glabrifrons (Meigen, 1830) Gatt et al. 2016 , Rif , Tariouma (El Anasser, 1383 m), MA , Lac Aguelmane Sidi Ali (2050 m) Lotophila Lioy, 1864 Lotophila atra (Meigen, 1830) Gatt 2006 , Rif , Tanger; Marshall et al. 2011 Norrbomia Papp, 1988 Norrbomia hispanica (Duda, 1923) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Norrbomia marginatis (Adams, 1905) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Norrbomia nilotica (Becker, 1903) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia niveipennis (Duda, 1923) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia somogyii (Papp, 1973) Roháček et al. 2001 ; Marshall et al. 2011 Norrbomia sordida (Zetterstedt, 1847) Gatt et al. 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), AP , Larache, Loukous Marsh Limosininae Bifronsina Roháček, 1983 Bifronsina bifrons (Stenhammar, 1855) Roháček et al. 2001 ; Marshall et al. 2011 Ceroptera Macquart, 1835 Ceroptera rufitarsis (Meigen, 1830) = Limosina picta Becker, in Becker and Stein 1913 : 94 Becker and Stein 1913 , Rif , Tanger; Papp 1984a ; Roháček et al. 2001 , Rif , Tanger Chaetopodella Duda, 1920 Chaetopodella scutellaris (Haliday, 1836) = Leptocera scutellaris (Haliday), in Séguy 1941a : 32 Séguy 1941a , HA , Imi-n'Ouaka (1500 m) Coproica Rondani, 1861 Coproica digitata (Duda, 1918) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Coproica ferruginata (Stenhammar, 1855) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Coproica hirticula Collin, 1956 Gatt et al. 2016 , Rif , Dardara (730 m), Oued Laou (30 m), Zarka waterfall (Yarghit, 135 m), Oued Ouara (Beni Zid, 440 m), Oued Jnane Azaghar (Bni Boufrah, 997 m), Oued Mhannech (125 m), Adrou (Taghzout, 556 m), Oued Jnane Niche (Jnane Niche, 27 m) Coproica hirtula (Rondani, 1880) Gatt et al. 2016 , Rif , Zarka waterfall (Yarghit, 135 m), AA , 14 km N Errachidia (Errachidia, 1214 m) Coproica lugubris (Haliday, 1836) Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Coproica pusio (Zetterstedt. 1847) Gatt et al. 2016 , Rif , Tamrabete (Oued Laou, 203 m), Smir lagoon (5 m) Coproica rohaceki Carles-Tolrá, 1990 Gatt et al. 2016 , Rif , Oued Amsa (Amsa, 14 m), Oued Moulay Bouchta (Dar Akobaâ, 285 m) Coproica rufifrons Hayashi, 1991 Gatt et al. 2016 , Rif , Issaguen (1543 m) Coproica vagans (Haliday, 1833) Gatt et al. 2016 , AA , 14 km N Errachidia (1214 m), AA , Oued Ziz (13 km N Erfoud, 800 m), SA , Merzouga (698 m) Eulimosina ochripes (Meigen, 1830) Gatt et al. 2016 , Rif , Issaguen (1543 m), Ksar el Kebir (20 m), MA , Lac Aguelamane Afenourrir (30 km SW Azrou, 1760 m) Leptocera Olivier, 1813 Leptocera caenosa (Rondani, 1880) Gatt et al. 2016 , Rif , Dardara (484 m) Leptocera fontinalis (Fallén, 1826) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Leptocera nigra Olivier, 1813 Munari 1993 , MA , Azrou; Roháček et al. 2001 ; Marshall et al. 2011 Limosina Macquart, 1835 Limosina silvatica (Meigen, 1830) Gatt et al. 2016 , Rif , Issaguen (1543 m), Talassemtane ( NPT , 1696 m), MA , 3,5 km S Azrou (1450 m), 17 km NW Zaida (Khénifra, 1878 m) Minilimosina Roháček, 1983 Minilimosina ( Svarciella ) vitripennis (Zetterstedt, 1847) Gatt et al. 2016 , MA , 3,5 km S Azrou (1450 m) Opacifrons Duda, 1918 Opacifrons coxata (Stenhammar, 1855) Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Opacifrons maculifrons (Becker, 1907) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Opalimosina Roháček, 1983 Opalimosina ( Opalimosina ) mirabilis (Collin, 1902) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Paralimosina Papp, 1973 Paralimosina fucata (Rondani, 1880) Gatt et al. 2016 , Rif , 5 km W Dardara (730 m), Dardara (484 m), Oued Azla (Mokdassen Oulya, 186 m), Oued Talembote (Talembote, 320 m), Oued El Khizana (El Khizana, 980 m), Jebel Kelaâ (Talassemtane, 1554 m), Adrou (Taghzout, 556 m) Phthitia Enderlein, 1938 Phthitia ( Kimosina ) sicana (Munari, 1988) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Phthitia ( Kimosina ) ciliata (Duda, 1918) Gatt et al. 2016 , SA , Bou Jarif (Goulimine) Phthitia ( Kimosina ) plumosula (Rondani, 1880) Gatt et al. 2016 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), Jebel Kelaâ (Talassemtane, 1554 m), Zaouiet and Habtiyène (Maggou, 1213 m), Oued Arozane (Bni Moussa, 317 m), Issaguen (1543 m), Zarka waterfall (Yarghit, 135 m), Taghramt (Bine El Ouidane, 276 m) Phthitia ( Kimosina ) pteremoides (Papp, 1973) Gatt et al. 2016 , SA , Bou Jarif (Goulimine) Poecilosomella Duda, 1925 Poecilosomella angulata (Thomson, 1869) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Pseudocollinella Duda, 1924 Pseudocollinella jorlii (Carles-Tolrá, 1990) Munari 1993 , MA , Aguelmane; Roháček et al. 2001 ; Marshall et al. 2011 Pullimosina Roháček, 1983 Pullimosina ( Pullimosina ) heteroneura (Haliday, 1836) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Pullimosina ( Pullimosina ) zayensis Marshall, 1986 Roháček et al. 2001 ; Marshall et al. 2011 Puncticorpus Duda, 1918 Puncticorpus lusitanicum (Richards, 1963) Papp 1984a ; Roháček et al. 2001 Rachispoda Lioy, 1864 Rachispoda acrosticalis (Becker, 1903) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda brevior (Roháček, 1991) Roháček 1991 , AP , Oued Bou-Regreg; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda duodecimseta (Papp, 1973) Roháček 1991 , EM , Guercif-Oued Moulouya; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda fuscipennis (Haliday, 1833) Roháček 1991 , EM , Oued Bou-Regreg; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda gel (Papp, 1978) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda kabuli (Papp, 1978) Roháček 1991 , MA , Taza, Oued Fès; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda lagura (Roháček, 1991) Gatt et al. 2016 , Rif , Ksar el Kebir (20 m), AA , Oued Ziz (12 km S Rissani, 737 m), Oued Ziz (13 km N Erfoud, 800 m), Oued Ziz (30 km N Erfoud, 894 m) Rachispoda lutosoidea (Duda, 1938) Roháček 1991 , MA , Azrou, Aguelmane, Oued Sebou; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 Rachispoda modesta (Duda, 1924) Gatt 2006 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Rachispoda uniseta (Roháček, 1991) Roháček 1991 , MA , Taza, Oued Fès; Munari 1993 ; Roháček et al. 2001 ; Marshall et al. 2011 ; Vaňhara and Rozkošný 1997 Rachispoda varicornis (Strobl, 1900) Roháček 1991 , AP , Oued Bou-Regreg, MA , Azrou; Munari 1993 , MA , Oued Sebou; Roháček et al. 2001 ; Marshall et al. 2011 Spelobia Spuler, 1924 Spelobia baezi (Papp, 1977) Roháček et al. 2001 ; Marshall et al. 2011 Spelobia clunipes (Meigen, 1830) Gatt et al. 2016 , Rif , 5 km W Dardara (730 m), Martil (beach and dunes), Oued Mhannech (18 m), Oued Afertane (Afertane, 56 m), Maggou waterfall (Maggou, 786 m), Onsar Akboul (NPB, 1315 m), Oued Ametrasse (Ametrasse, 841 m), Oued Tahaddart (Tahaddart, 87 m), Issaguen (1543 m), Lemtahane ( PNPB , 1088 m) Spelobia hungarica (Villeneuve, 1917) Gatt 2006 , Rif , Tétouan, Martil; Marshall et al. 2011 Spelobia quaesita Roháček, 1983 Roháček et al. 2001 ; Marshall et al. 2011 Spinilimosina Roháček, 1983 Spinilimosina brevicostata (Duda, 1918) Roháček et al. 2001 ; Marshall et al. 2011 Telomerina Roháček, 1983 Telomerina pseudoleucoptera (Duda, 1924) Gatt et al. 2016 , Rif , Maggou waterfall (Maggou, 786 m) Thoracochaeta Duda, 1918 Thoracochaeta brachystoma (Stenhammar, 1855) Gatt 2006 ; Cassar et al. 2008 , Rif , Tétouan, M'Diq, Smir; Marshall et al. 2011 Thoracochaeta erectiseta Carles-Tolrá, 1994 Gatt 2006 , Rif , Tétouan, M'Diq, Smir, Oued Laou; Marshall et al. 2011 Trachyopella Duda, 1918 Trachyopella ( Trachyopella ) coprina (Duda, 1918) Gatt et al. 2016 , Rif , Oued Jnane Niche (Jnane Niche, 27 m) Trachyopella ( Trachyopella ) melania (Haliday, 1836) Gatt et al. 2016 , Rif , Oued Nwawel (Azla, 57 m), M'Diq (5 m) Sphaerocerinae Ischiolepta Lioy, 1864 Ischiolepta pusilla (Fallén, 1820) Gatt et al. 2016 , Rif , Oued Guallet (Bni Boufrah, 946 m), Aïn Tissemlal (Azilane, 1255 m) Ischiolepta vaporariorum (Haliday, 1836) Gatt 2006 , Rif , Tétouan, Oued Laou; Marshall et al. 2011 Lotobia Lioy, 1864 Lotobia africana (Becker, 1907) Roháček et al. 2001 ; Marshall et al. 2011 Lotobia pallidiventris (Meigen, 1830) Gatt 2006 , Rif , Tétouan, M'Diq, Oued Laou; Marshall et al. 2011 Sphaerocera Latreille, 1804 Sphaerocera curvipes Latreille, 1805 Roháček et al. 2001 ; Marshall et al. 2011 Ephydroidea BRAULIDAE K. Kettani Number of species: 1 . Expected: 1 Faunistic knowledge of the family in Morocco: good Braulinae Braula Nitzsch, 1818 Braula coeca Nitzsch, 1818 Séguy 1930a ; Mouna 1998 ; Howard et al. 2000 CAMILLIDAE K. Kettani, M.J. Ebejer Number of species: 4 . Expected: 5 Faunistic knowledge of the family in Morocco: poor Camilla Haliday in Curtis, 1837 Camilla acutipennis (Loew, 1865) Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 76 m), Chrabkha pond (Al Manzla, 58 m) Camilla flavicauda Duda, 1922 Mouna 1998 ; Pârvu et al. 2006 , AP , Cap Sim; Popescu-Mirceni 2011 Camilla glabra (Fallén, 1823) Séguy 1941d , HA , Tizi-n'Test; Mouna 1998 Camilla pruinosa Duda, 1934 Ebejer et al. 2019 , AP , Larache (5 m) CRYPTOCHETIDAE K. Kettani, E.P. Nartshuk Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Cryptochetum Rondani, 1875 Cryptochetum (as Cryptochaetum ) mimeuri Séguy, 1953 Séguy 1953c , MA , Ifrane; Nartshuk 1979 , MA , Ifrane DIASTATIDAE K. Kettani, M.J. Ebejer Number of species: 2 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Campichoetinae Campichoeta Macquart, 1835 Campichoeta obscuripennis (Meigen, 1830) Ebejer et al. 2019 , HA , Lalla Takrkoust (628 m) Diastatinae Diastata Meigen, 1830 Diastata adusta Meigen, 1830 = Diastata unipunctata Zetterstedt, 1847 Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 DROSOPHILIDAE K. Kettani, G. Bächli Number of species: 26 . Expected: 30 Faunistic knowledge of the family in Morocco: good Drosophilinae Drosophilini Drosophila Fallén, 1823 Drosophila ( Drosophila ) busckii Coquillett, 1901 = Drosophila rubrostriata Becker, 1908, in Séguy 1953a : 85 Séguy 1953a , AP , Rabat, Sidi Yahia du Gharb; Prevosti 1974 , HA , Asni; Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech – MISR Drosophila ( Drosophila ) buzzatii Patterson & Wheeler, 1942 Prevosti 1974 , HA , Asni (Admin forest); Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Drosophila ( Drosophila ) funebris (Fabricius, 1787) Prevosti 1974 , HA , Asni; Mouna 1998 ; AP (Maâmora) – MISR Drosophila ( Drosophila ) hydei Sturtevant, 1921 Staiger and Gloor 1952 , AP , Rabat; Gloor and Satiger 1954, AP , Rabat; Prevosti 1974 , HA , Asni (Admin forest); Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech Drosophila ( Drosophila ) immigrans Sturtevant, 1921 Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Drosophila ( Drosophila ) kuntzei Duda, 1924 Mouna 1998 ; Prevosti 1974 , AP , Essaouira, HA , Asni Drosophila ( Drosophila ) mercatorum Patterson & Wheeler, 1942 Prevosti 1974 , AA , Agadir (Admin forest near Agadir); Mouna 1998 Drosophila ( Drosophila ) phalerata Meigen, 1830 Prevosti 1974 , AP , Essaouira, HA , Asni; Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger), MA (Ifrane) – ZSM Drosophila ( Drosophila ) repleta Wollaston, 1858 Séguy 1953a , AP , Rabat, MA , Fès Drosophila ( Drosophila ) tsigana Burla & Gloor, 1952 Suwito et al. 2014 , MA , Ifrane Drosophila ( Sophophora ) ambigua Pomini, 1940 Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger), MA (Ifrane) – ZSM Drosophila ( Sophophora ) melanogaster Meigen, 1830 Medioni 1958 ; David and Bocquet 1973 , HA , Marrakech, AA , Agadir, Ouarzazate, Taroudant, AA , Zagora; Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Jousset and Plus 1975 ; Plus et al. 1975 , HA , Marrakech, AA , Agadir, Ouarzazate, Taroudant, AA , Zagora; Prevosti et al. 1975 , Rif , Tanger; Allemand and David 1976 , AA , Ouarzazate; Ashburner and Lemeunier 1976 , HA , Marrakech, AA , Agadir, Taroudant, AA , Zagora; Fleuriet 1976 , AA , Zagora; Plus et al. 1976 , AA , Ouarzazate, Zagora; Jousset 1976 , AA , Ouarzazate; Plus and Scotti 1984 , AA , Ouarzazate; Thomas-Orillard 1984 , AA , Ouarzazate; Afonso et al. 1985 , HA , Asni; Prevosti et al. 1985 , AP , Essaouira, AA , Agadir; David et al. 1986 , AP , Rabat, Casablanca; Ayala et al. 1989 , Rif , Chefchaouen; Boulétreau et al. 1992, AA , Agadir; Costa et al. 1992 , AP , Casablanca; Capy et al. 1993 , AP , Casablanca, Rabat; Ritchie et al. 1994 , AP , Casablanca; Mouna 1998 ; Bonnivard and Higuet 1999 , AA , Agadir; Chakir et al. 2002 , 2007 , 2008 , 2011 , HA , Marrakech; Ayrinhac et al. 2004 , HA , Marrakech; Catania et al. 2004 , HA , Marrakech AA , Agadir; Rohmer et al. 2004 , HA , Marrakech; Dieringer et al. 2005 , HA , Marrakech, AA , Agadir; Yassin and Orgogozo 2013 , HA , Marrakech; Bächli 2015 (TaxoDros) – HNHM , AP (Rabat), HA (Marrakech) – MISR , MA (Ifrane) – ZSM Drosophila ( Sophophora ) simulans Sturtevant, 1919 Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Baba-Aissa et al. 1988 , AP , Rabat; Nigro 1988 , AP , Larache; Capy et al. 1990, 1992 , 1993 , AP , Rabat, MA , Béni Mellal AA , Agadir; Chakrani et al. 1993 , AA , Agadir; Mouna 1998 ; Charlat et al. 2003 , AA , Agadir; Biémont et al. 2003 , HA , Marrakech; Chakir et al. 2002 , 2007 , 2008 , 2011 , HA , Marrakech; Nardon et al. 2005 , HA , Marrakech; Yassin and Orgogozo 2013 , HA , Marrakech; Bächli 2015 (TaxoDros); AA (Agadir) – ZSM Drosophila ( Sophophora ) subobscura Collin, 1936 Götz 1965 , Rif , Tanger; Prevosti 1971a , Rif , Tanger; Prevosti 1971b , AP , Essaouira, HA , Asni, AA , Ait-Melloul; Prevosti 1971c ; Gonzalez-Duarte et al. 1973, AP , Essaouira, HA , Asni AA , Agadir; Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Prevosti et al. 1975 , AA , Agadir; Duarte 1976, AP , Essaouira, HA , Asni AA , Agadir; Gonzalez 1976, AP , Essaouira, HA , Asni, AA , Agadir; Prevosti 1978 , Rif , Tanger, AP , Essaouira, HA , Asni AA , Agadir; Krimbas and Loukas 1980 , AA , Agadir; Cabrera et al. 1983 , Rif , Chefchaouen; Larruga et al. 1983 , Rif , Chefchaouen; Prevosti et al. 1985 , AA , Agadir; Pascual et al. 1986 , Rif , Chefchaouen; Latorre et al. 1986 , Rif , Chefchaouen; Constanti et al. 1986 , Rif , Chefchaouen; Ayala et al. 1989 , Rif , Chefchaouen; Afonso et al. 1990 , Rif , Chefchaouen; Pascual et al. 1990 , Rif , Chefchaouen; Paricio et al. 1991 , Rif , Chefchaouen; Menozzi and Krimbas 1992 , AA , Agadir; Latorre et al. 1992 , Rif , Chefchaouen; Fain et al. 1993 , AA , Agadir; Alberola and Frutos 1993; Alberola and Frutos 1996 ; Pinto et al. 1997 , HA , Asni, Marrakech AA , Agadir; Mouna 1998 ; David et al. 2003 , HA , Marrakech; Brehm et al. 2004 , AA , Agadir; Nardon et al. 2005 , HA , Marrakech; Bächli 2015 (TaxoDros); MA (Azrou) – NHMD Drosophila ( Sophophora ) suzukii (Matsumura, 1931) Landolt et al. 2012 , Rif , north-eastern Morocco Hirtodrosophila Duda, 1924 Hirtodrosophila cameraria (Haliday, 1833) Ebejer et al. 2019 , Rif , Aïn Ras el Ma, ruisseau maison forestière (Talassemtane) Lordiphosa Basden, 1961 Lordiphosa andalusiaca (Strobl, 1906) = Lordiphosa forcipata (Collin, 1952), in Hackman 1960 : 102 Hackman 1960 ; Bächli 2015 (TaxoDros); AP (Rabat) – NHMD Scaptomyza Hardy, 1849 Scaptomyza adusta (Loew, 1862) Ebejer et al. 2019 , Rif , Dardara (730 m), AP , Loukous marsh (2 m) Scaptomyza flava (Fallén, 1823) = Scaptomyza flaveola (Meigen, 1830), in Kozlowsky and Rungs 1932 : 66 Kozlowsky and Rungs 1932 , AP , Rabat Scaptomyza graminum (Fallén, 1823) Maarouf 2003 , HA , Chaouia; Bächli 2015 (TaxoDros); HA (Asni, Tinerhir) – NHMD Scaptomyza ( Parascaptomyza ) pallida (Zetterstedt, 1847) Ibn Jilali 1988 (agricultural areas); Maarouf 2003 , HA , Chaouia; Bächli 2015 (TaxoDros); AP (Essaouira) MHNNR, MA (Azrou) – NHMD Scaptodrosophila Duda, 1923 Scaptodrosophila rufifrons (Loew, 1873) Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Zaprionus Coquillett, 1901 Zaprionus indianus Gupta, 1970 Yassin and David 2010 Steganinae Gitonini Gitona Meigen, 1830 Gitona microchaeta Séguy, 1941 Séguy 1941d , AA , Agadir; Bächli 1982 , AA , Agadir; Bächli and Rocha Pité 1984 , AA , Agadir; Mouna 1998 Phortica Fallén, 1823 Phortica variegata (Fallén, 1823) Ebejer et al. 2019 , Rif , Bab Berred (1433 m), Jebel Lakraâ (Talassemtane, 1541 m) Steganini Leucophenga Mik, 1886 Leucophenga maculata (Dufour, 1839) Séguy 1934b , AP , Port-Liautey (Maâmora) EPHYDRIDAE K. Kettani, T. Zatwarnicki Number of species: 117 . Expected: 140 Faunistic knowledge of the family in Morocco: good Discomyzinae Discomyzini Actocetor Becker, 1903 Actocetor indicus (Wiedemann, 1824) = Actocetor margaritatus (Wiedemann, 1830), in Séguy 1934b : 162, 1953a : 86 Séguy 1934b , Rif , Béni Aross; Séguy 1953a , SA , Tindouf Discomyza Meigen, 1830 Discomyza incurva (Fallén, 1823) = Discomyza italica Séguy, 1929, in Vitte 1991 : 3; Dakki 1997 : 63 Cresson 1939 ; Vitte 1991 , AP , Atlantic coast and Plains; Dakki 1997 Psilopini Clanoneurum Becker, 1903 Clanoneurum cimiciforme (Haliday, 1855) Séguy 1941d , AA , Agadir; Dahl 1964 ; Vitte 1991 , AP , Rabat; Dakki 1997 Diasemocera Bezzi, 1895 Diasemocera aequalipes (Becker, 1907) = Psilopa aequalipes (Becker, 1907), in Ebejer et al. 2019 : 147 Zatwarnicki 2018 ; Ebejer et al. 2019 , AA , Lac Tiffert (4 km W of Merzouga, 702 m), Ziz river (13 km N of Erfoud, 800 m) Diasemocera biskrae (Becker, 1907) = Psilopa biskrae (Becker, 1907), in Vitte 1991 : 32 Vitte 1991 , AP , M'Diq; Dakki 1997 Diasemocera composita (Becker, 1903) = Psilopa composita (Becker, 1903), in Vitte 1991 : 32, Dakki 1997 : 63 Vitte 1991 , AP , Rabat; Dakki 1997 Diasemocera fratella (Becker, 1903) = Psilopa fratella (Becker, 1903), in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Errachidia (1 km N of Tarda, 1023 m), AA , Ziz river (13 km N of Erfoud, 800 m) Diasemocera glabricula (Fallén, 1813) = Psilopa nigritella Stenhammar, 1844, in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Diasemocera leucostoma (Meigen, 1830) = Psilopa leucostoma (Meigen, 1830), in Séguy 1941d : 18 Séguy 1941d , AA , Agadir Diasemocera maritima (Perris, 1847) = Psilopa maritima (Perris, 1847), in Cassar et al. 2008 : 25 Cassar et al. 2008 , Rif , Laou Basin Diasemocera nana (Loew, 1860) = Psilopa nana Loew, 1860, in Vitte 1991 : 32, Dakki 1997 : 63 Vitte 1991 , Rif , AP , Atlantic coast; Dakki 1997 Diasemocera rufithorax (Becker, 1903) = Psilopa rufithorax (Becker, 1903), in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Merzouga (714 m) Psilopa Fallén, 1823 Psilopa clara (Wollaston, 1858) = Psilopa rutilans Canzoneri & Meneghini, 1972, in Cassar et al. 2005 : 69 Cassar et al. 2005 , Rif , Smir lagoon; Zatwarnicki 2018 , AP , Larache (Lower Loukous), Safi, AA , Sidi Moussa, D'Agion (0–50 m) Psilopa meneghinii Canzoneri, 1986 Vitte 1991 , AP , Atlantic coast; Dakki 1997 Psilopa compta (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Psilopa nilotica (Becker, 1903) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m), 1 km N of Tarda (Errachidia, 1023 m), Merzouga (714 m), 2 km N Erfoud (818 m) Psilopa nitidula (Fallén, 1813) Becker and Stein 1913 , Rif , Tanger; Séguy 1941a , HA , Toubkal; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Zatwarnicki 1991 , Rif , Tanger, Tétouan, MA , Ifrane; Dakki 1997 Psilopa obscuripes Loew, 1860 Ebejer et al. 2019 , Rif , Oued Azla (near bridge, 80 m), AP , Larache (5 m), Lower Loukous saltmarsh (2 m), MA , Khénifra (28 km S of Timahdit, 2100 m) Psilopa polita (Macquart, 1835) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Risini Achaetorisa Papp, 1980 Achaetorisa brevicornis Papp, 1980 Papp 1980, HA , Ouirgane Ephydrinae Dagini Brachydeutera Leow, 1862 Brachydeutera meridionalis (Rondani, 1856) = Brachydeutera ibari Ninomyia, 1929, in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , Rif , Oued Martil (Taboula, 14 m) Parydrini Parydra Stenhammar, 1844 Parydra ( Chaetoapnaea ) fossarum (Haliday, 1833) Séguy 1930a , AP , Rabat, MA , Meknès, HA , Marrakech; Vitte 1991 ; Dakki 1997 ; Vitte 1988 , MA , lakes of Middle Atlas; MA (Jebel Khazzane) – MISR Parydra ( Chaetoapnaea ) hecate (Haliday, 1833) = Napaea hecate (Haliday), in Vaillant 1956b : 244 Vaillant 1956b , HA , Imi-N'Ifri Parydra ( Chaetoapnaea ) quadripunctata (Meigen, 1830) Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 , AP , Merja Zerga Parydra ( Paranapaea ) pubera Loew, 1860 Dahl 1964 , AA , Aït Melloul, Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Parydra ( Parydra ) aquila (Fallén, 1813) Vitte 1991 , Rif , Ouezzane, Ketama; Dakki 1997 Parydra ( Parydra ) coarctata (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Ksar el Kbir; Dakki 1997 Parydra ( Parydra ) cognata Loew, 1860 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Parydra ( Parydra ) flavitarsis Dahl, 1964 Vitte 1988 , Rif , MA , lakes of Middle Atlas; Vitte 1991 , Rif , MA , Fès; Dakki 1997 ; Gatt and Ebejer 2003 ; Dakki et al. 2003; Chillasse and Dakki 2004 , Rif , MA ; Dakki and Himmi 2008 , MA , Oued Sebou Parydra ( Parydra ) littoralis (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Tétouan, Ketama; Dakki 1997 Parydra ( Parydra ) nigritarsis Strobl, 1893 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Ketama; Dakki 1997 Parydra ( Parydra ) nubecula Becker, 1896 Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 , AP , Merja Zerga Parydra ( Parydra ) quinquemaculata Becker, 1896 Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Ephydrini Ephydra Fallén, 1823 Ephydra bivittata Loew, 1860 Vitte 1991 ; Dakki 1997 Ephydra flavipes (Macquart, 1843) Vitte 1991 , AP , Atlantic coast; Dakki 1997 Ephydra glauca Meigen, 1830 Vitte 1991 ; Dakki 1997 Ephydra macellaria Egger, 1862 Dahl 1964 , AA , Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 ; Vitte 1991 , AP , Atlantic coast Halmopota Haliday, 1856 Halmopota mediterranea Loew, 1860 Dahl 1964 , AA , Aït Melloul; Dakki 1997 ; Vitte 1991 , Rif , Asilah, Chefchaouen Paracoenia Cresson, 1935 Paracoenia fumosa (Stenhammar, 1844) Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Setacera Cresson, 1930 Setacera breviventris (Loew, 1860) Vitte 1991 , Rif , Ksar el Kbir; Dakki 1997 Scatellini Haloscatella Mathis, 1979 Haloscatella dichaeta (Loew, 1860) = Scatella dichaeta Loew, 1860, in Vitte 1988 : 392, 1991 : 26; Dakki 1997 : 62 Vitte 1988 , MA , Khemisset, Oued Beth, Dayat Aoua; Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Lamproscatella Hendel, 1917 Lamproscatella sibilans (Haliday, 1833) Ebejer et al. 2019 , AA , 1 km N of Tarda (Errachidia, 1023 m) Limnellia Malloch, 1925 Limnellia quadrata (Fallén, 1813) Vitte 1991 , Rif , Central Rif; Dakki 1997 Philotelma Becker, 1896 Philotelma nigripenne (Meigen, 1830) = Scatella nigripennis (Meigen, 1830), in Vitte 1988 : 391; Dakki 1997 : 62 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Scatella Robineau-Desvoidy, 1830 Scatella ( Neoscatella ) subguttata (Meigen, 1830) Vitte 1991 , AP , Atlantic and Mediterranean coast, Smir lagoon; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 Scatella ( Scatella ) ciliata Collin, 1930 Vitte 1991 , AP , Moulay Bousselham, Asilah; Dakki 1997 Scatella ( Scatella ) lacustris (Meigen, 1830) = Scatella ( Scatella ) tenuicosta Collin, 1930, in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Ziz river (13 km N of Erfoud, 800 m) Scatella ( Scatella ) lutosa (Haliday, 1833) Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Scatella ( Scatella ) obsoleta Loew, 1861 = Scatella callosicosta Bezzi, 1895, in Vitte 1988 : 392, 1991 : 26; Dakki 1997 : 62 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , M'Diq; Dakki 1997 Scatella ( Scatella ) paludum (Meigen, 1830) Dahl 1964 , AP , Oued Korifla; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatella ( Scatella ) rufipes Strobl, 1906 = Scatella rubida Becker, 1907, in Olafsson 1991 : 21; Vitte 1991 : 26; Dakki 1997 : 62 Olafsson 1991 , EM , Figuig, Defilia; Vitte 1991 , Rif , AP (Atlantic coast); Dakki 1997 ; Gatt and Ebejer 2003 Scatella ( Scatella ) stagnalis (Fallén, 1813) Séguy 1941a , HA , Toubkal; Dahl 1964 , AA , Aït Melloul, Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatophila Becker, 1896 Scatophila caviceps (Stenhammar, 1844) Vitte 1988 , AP , Rabat, Temara, MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatophila despecta (Haliday, 1839) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Khemisset, Oued Beth; Dakki 1997 Scatophila farinae Becker, 1903 Zatwarnicki 1987 , HA , Vallée de l'Ait Mizane; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Taounate; Dakki 1997 ; Gatt and Ebejer 2003 Scatophila modesta Becker, 1908 Vitte 1991 , Rif , Tétouan; Dakki 1997 Scatophila unicornis Czerny, 1900 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Gymnomyzinae Discocerinini Diclasiopa Hendel, 1917 Diclasiopa galactoptera (Becker, 1903) = Discocerina galactoptera Becker, in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 ; Kirk-Spriggs and McGregor 2009 Diclasiopa lacteipennis (Loew, 1862) = Discocerina lacteipennis Loew, 1862, in Vitte 1988 : 394, 1991 : 32; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Rabat; Vitte 1991 , MA , Khemisset, Taounate; Dakki 1997 Diclasiopa niveipennis (Becker, 1896) = Discocerina niveipennis (Becker, 1896), in Vitte 1988 : 394, 1991 : 32; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Discocerina Macquart, 1835 Discocerina obscurella (Fallén, 1813) Vitte 1988 , AP , MA , lakes of Middle Atlas; Vitte 1991 , MA , Fès, Taounate; Mathis 1997 ; Dakki 1997 ; Wolff et al. 2016 Ditrichophora Cresson, 1924 Ditrichophora calceata (Meigen, 1830) = Discocerina calceata (Meigen, 1830), in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Ditrichophora mauritanica (Vitte, 1991) = Discocerina mauritanica Vitte, 1991, in Vitte 1991 : 33 Vitte 1991 , Rif , MA , Azrou; Dakki 1997 ; Chillasse and Dakki 2004 , Rif , MA ; Dakki and Himmi 2008 , MA , Oued Sebou Gymnoclasiopa Hendel, 1930 Gymnoclasiopa plumosa (Fallén, 1823) = Discocerina plumosa (Fallén, 1823), in Vitte 1988 : 394, 1991 : 33; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Gymnoclasiopa pulchella (Meigen, 1830) = Discocerina pulchella (Meigen, 1830), in Vitte 1991 : 33; Dakki 1997 : 63 Vitte 1991 , Rif , Ketama, Ouezzane; Dakki 1997 Hecamedoides Hendel, 1917 Hecamedoides glaucellus (Stenhammar, 1844) = Discocerina glaucella (Stenhammar, 1844), in Vitte 1991 : 33 Vitte 1991 , Rif , AP Polytrichophora Cresson, 1924 Polytrichophora duplosetosa (Becker, 1896) AP (Rabat) – MISR Gymnomyzini Athyroglossa Loew, 1860 Athyroglossa ( Athyroglossa ) glabra (Meigen, 1830) Vitte 1988 , Rif , MA , lakes of Middle Atlas; Dakki 1997 Athyroglossa ( Athyroglossa ) nudiuscula Loew, 1860 Vitte 1991 , Rif ; Dakki 1997 Athyroglossa ( Parathyroglossa ) ordinata Becker, 1896 Vitte 1991 , Rif ; Vitte 1988 , MA , lakes of Middle Atlas; Mathis and Zatwarnicki 1990 , MA , Ifrane; Dakki 1997 Chlorichaeta Becker, 1922 Chlorichaeta albipennis (Loew, 1848) Vitte 1991 ; Dakki 1997 Mosillus Latreille, 1804 Mosillus subsultans (Fabricius, 1794) = Gymnopa subsultans Fabricius, in Séguy 1930a : 181 Séguy 1930a , MA , M'Rirt, HA , Imminen (Tachidirt); Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Mathis et al. 1993 , MA , Ifrane (1650 m), maison forestière (cedar forest: 2700 m), Oued Jaffar (N of source, 0–1500 m), HA , Mikdane (Jebel Ayachi); Dakki 1997 ; Koçak and Kemal 2010 Hecamedini Allotrichoma Becker, 1896 Allotrichoma laterale (Loew, 1860) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Allotrichoma leotoni Vitte, 1992 Vitte 1992 , Rif , Ouezzane, Boured Allotrichoma quadripectinatum (Becker, 1896) = Allotrichoma bellicosum Giordani Soika, 1956, in Vitte 1991 : 30; Dakki 1997 : 63 Vitte 1991 , Rif , AP ; Dakki 1997 Allotrichoma simplex (Loew, 1861) = Allotrichoma filiforme Becker, 1896, in Vitte 1988 : 393, 1991 : 30; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Khemisset, Oued Sebou; Dakki 1997 Elephantinosoma Becker, 1903 Elephantinosoma chnumi Becker, 1903 Gatt and Ebejer 2003 ; Kirk-Spriggs and McGregor 2009 Hecamede Haliday, 1837 Hecamede albicans (Meigen, 1830) Vitte 1991 , AP ; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Cassar et al. 2008 , Rif , Smir Lagoon; Popescu-Mirceni 2011 Lipochaetini Glenanthe Haliday, 1839 Glenanthe ripicola (Haliday, 1839) Vitte 1991 , AP ; Dakki 1997 ; Cassar et al. 2008 , Rif , Laou Basin; Zatwarnicki and Mathis 2011 , AA , Tarfaya – HNHM Homalometopus Becker, 1903 Homalometopus sp. Vitte 1991 Ochtherini Ochthera Latreille, 1802 Ochthera manicata (Fabricius, 1794) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Ochthera pilimana Becker, 1903 Dakki 1997 Ochthera schembrii Rondani, 1847 = Ochthera mantispa Loew, 1847, in Vitte 1988 : 394, 1991 : 28; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , AP ; Dakki 1997 Hydrelliinae Atissini Asmeringa Becker, 1903 Asmeringa inermis Becker, 1903 Vitte 1991 , AP , Rabat; Dakki 1997 ; Gatt and Ebejer 2003 Atissa Haliday, 1839 Atissa durrenbergensis Loew, 1864 Vitte 1991 , AP ; Dakki 1997 Atissa hepaticoloris Becker, 1903 Vitte 1991 , AP ; Dakki 1997 ; Gatt and Ebejer 2003 Atissa limosina Becker, 1896 Vitte 1991 , AP , Rabat, MA , Fès; Dakki 1997 Atissa pygmaea (Haliday, 1839) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 Ptilomyia Coquillett, 1900 Ptilomyia angustigenis (Becker, 1926) = Atissa angustigenis Becker, in Vitte 1988 : 393, 1991 : 30; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Gatt and Ebejer 2003 Dryxini Dryxo Robineau-Desvoidy, 1830 Dryxo ornata (Macquart, 1843) Mathis and Zatwarnicki 2002 , AA , Aoulouz Hydrelliini Hydrellia Robineau-Desvoidy, 1830 Hydrellia albifrons (Fallén, 1813) Vitte 1991 , AP , Rif ; Dakki 1997 Hydrellia argyrogenis Becker, 1896 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , Atlantic coast and Plains; Dakki 1997 Hydrellia armata Canzoneri & Meneghini, 1976 Vitte 1991 , Rif , Ksar el Kbir, MA , Fès; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou Hydrellia atlas Vitte, 1989 Vitte 1989 , MA , Dayat Aoua; Dakki 1997 ; Dakki et al. 2003; Chilasse and Dakki 2004, MA Hydrellia griseola (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Wolff et al. 2016 ; Rif (Oued Laou) – MISR Hydrellia maculiventris Becker, 1896 Vitte 1991 , Rif , AP , Atlantic coast; Dakki 1997 ; Gatt and Ebejer 2003 Hydrellia maura Meigen, 1838 = Hydrellia modesta Loew, 1860, in Vitte 1988 : 392, 1991 : 29; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Zatwarnicki 1988 , MA , Ifrane, HA , Vallée de l'Aït Mizane; Vitte 1991 ; Dakki 1997 Hydrellia nigricans (Stenhammar, 1844) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Hydrellia obscura (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 ; Rif (Aïn Jdioui) – MISR Hydrellia pubescen s Becker, 1926 = Hydrellia nasturtii Collin, 1928, in Vitte 1991 : 29; Dakki 1997 : 63 Vitte 1991 , MA , Fès; Dakki 1997 ; Gatt and Ebejer 2003 Hydrellia ranunculi (Haliday, 1838) Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Hydrellia rharbia Vitte, 1991 Vitte 1989 , AP , Merja Halloufa (near Moulay Bousselham); Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou Hydrellia subalbiceps Collin, 1966 Vitte 1991 , Rif , Ketama; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Notiphilini Notiphila Fallén, 1810 Notiphila ( Notiphila ) annulipes Stenhammar, 1844 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Notiphila ( Notiphila ) cinerea Fallén, 1830 Séguy 1930a , MA , Meknès; Séguy 1941a , HA , Imi-n'Ouaka; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Kirk-Spriggs and McGregor 2009 ; Rif (Talassemtane, Aïn Jdioui) – MISR Notiphila ( Notiphila ) cogani Canzoneri & Meneghini, 1979 Vitte 1988 , MA , lakes of Middle Atlas; Krivosheina 1998 ; Vitte 1991 ; Dakki 1997 ; Rif (Aïn Jdioui) – MISR Notiphila ( Notiphila ) dorsata Stenhammar, 1844 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , coastal lake areas; Dakki 1997 Notiphila ( Notiphila ) maculata Stenhammar, 1844 Vitte 1991 , Rif , AP , coastal plains; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Notiphila ( Notiphila ) riparia Meigen, 1830 Krivosheina 1998 , AA , Aït Melloul (Souss); Vitte 1988 , MA , lakes of Middle Atlas and reedbeds; Vitte 1991 ; Dakki 1997 Notiphila ( Notiphila ) stagnicola (Robineau-Desvoidy, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , AP , coastal plains; Dakki 1997 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 , AA , Ouarzazate Ilytheinae Hyadinini Hyadina Haliday, 1837 Hyadina guttata (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Hyadina pollinosa Oldenberg, 1923 Vitte 1991 , MA , Fès; Dakki 1997 Hyadina rufipes (Meigen, 1830) = Hyadina nitida (Macquart, 1835), in Vitte 1991 : 27; Dakki 1997 : 63 Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Nostima Coquillett, 1900 Nostima picta (Fallén, 1813) Vitte 1991 , AP ; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Pelina Haliday, 1837 Pelina aenea (Fallén, 1813) Vitte 1991 , Rif , Ouezzane; Dakki 1997 Pelina subpunctata Becker, 1896 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Philygria Stenhammar, 1844 Philygria posticata (Meigen, 1830) Ebejer et al. 2019 , MA , Khénifra (17 km SW of Midelt, 1940 m) Acknowledgements We gratefully acknowledge the assistance and cooperation of Martin J. Ebejer who contributed to the revision of this family. BRAULIDAE K. Kettani Number of species: 1 . Expected: 1 Faunistic knowledge of the family in Morocco: good Braulinae Braula Nitzsch, 1818 Braula coeca Nitzsch, 1818 Séguy 1930a ; Mouna 1998 ; Howard et al. 2000 Braulinae Braula Nitzsch, 1818 Braula coeca Nitzsch, 1818 Séguy 1930a ; Mouna 1998 ; Howard et al. 2000 CAMILLIDAE K. Kettani, M.J. Ebejer Number of species: 4 . Expected: 5 Faunistic knowledge of the family in Morocco: poor Camilla Haliday in Curtis, 1837 Camilla acutipennis (Loew, 1865) Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 76 m), Chrabkha pond (Al Manzla, 58 m) Camilla flavicauda Duda, 1922 Mouna 1998 ; Pârvu et al. 2006 , AP , Cap Sim; Popescu-Mirceni 2011 Camilla glabra (Fallén, 1823) Séguy 1941d , HA , Tizi-n'Test; Mouna 1998 Camilla pruinosa Duda, 1934 Ebejer et al. 2019 , AP , Larache (5 m) CRYPTOCHETIDAE K. Kettani, E.P. Nartshuk Number of species: 1 . Expected: 2 Faunistic knowledge of the family in Morocco: poor Cryptochetum Rondani, 1875 Cryptochetum (as Cryptochaetum ) mimeuri Séguy, 1953 Séguy 1953c , MA , Ifrane; Nartshuk 1979 , MA , Ifrane DIASTATIDAE K. Kettani, M.J. Ebejer Number of species: 2 . Expected: 3 Faunistic knowledge of the family in Morocco: poor Campichoetinae Campichoeta Macquart, 1835 Campichoeta obscuripennis (Meigen, 1830) Ebejer et al. 2019 , HA , Lalla Takrkoust (628 m) Diastatinae Diastata Meigen, 1830 Diastata adusta Meigen, 1830 = Diastata unipunctata Zetterstedt, 1847 Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 Campichoetinae Campichoeta Macquart, 1835 Campichoeta obscuripennis (Meigen, 1830) Ebejer et al. 2019 , HA , Lalla Takrkoust (628 m) Diastatinae Diastata Meigen, 1830 Diastata adusta Meigen, 1830 = Diastata unipunctata Zetterstedt, 1847 Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 DROSOPHILIDAE K. Kettani, G. Bächli Number of species: 26 . Expected: 30 Faunistic knowledge of the family in Morocco: good Drosophilinae Drosophilini Drosophila Fallén, 1823 Drosophila ( Drosophila ) busckii Coquillett, 1901 = Drosophila rubrostriata Becker, 1908, in Séguy 1953a : 85 Séguy 1953a , AP , Rabat, Sidi Yahia du Gharb; Prevosti 1974 , HA , Asni; Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech – MISR Drosophila ( Drosophila ) buzzatii Patterson & Wheeler, 1942 Prevosti 1974 , HA , Asni (Admin forest); Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Drosophila ( Drosophila ) funebris (Fabricius, 1787) Prevosti 1974 , HA , Asni; Mouna 1998 ; AP (Maâmora) – MISR Drosophila ( Drosophila ) hydei Sturtevant, 1921 Staiger and Gloor 1952 , AP , Rabat; Gloor and Satiger 1954, AP , Rabat; Prevosti 1974 , HA , Asni (Admin forest); Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech Drosophila ( Drosophila ) immigrans Sturtevant, 1921 Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Drosophila ( Drosophila ) kuntzei Duda, 1924 Mouna 1998 ; Prevosti 1974 , AP , Essaouira, HA , Asni Drosophila ( Drosophila ) mercatorum Patterson & Wheeler, 1942 Prevosti 1974 , AA , Agadir (Admin forest near Agadir); Mouna 1998 Drosophila ( Drosophila ) phalerata Meigen, 1830 Prevosti 1974 , AP , Essaouira, HA , Asni; Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger), MA (Ifrane) – ZSM Drosophila ( Drosophila ) repleta Wollaston, 1858 Séguy 1953a , AP , Rabat, MA , Fès Drosophila ( Drosophila ) tsigana Burla & Gloor, 1952 Suwito et al. 2014 , MA , Ifrane Drosophila ( Sophophora ) ambigua Pomini, 1940 Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger), MA (Ifrane) – ZSM Drosophila ( Sophophora ) melanogaster Meigen, 1830 Medioni 1958 ; David and Bocquet 1973 , HA , Marrakech, AA , Agadir, Ouarzazate, Taroudant, AA , Zagora; Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Jousset and Plus 1975 ; Plus et al. 1975 , HA , Marrakech, AA , Agadir, Ouarzazate, Taroudant, AA , Zagora; Prevosti et al. 1975 , Rif , Tanger; Allemand and David 1976 , AA , Ouarzazate; Ashburner and Lemeunier 1976 , HA , Marrakech, AA , Agadir, Taroudant, AA , Zagora; Fleuriet 1976 , AA , Zagora; Plus et al. 1976 , AA , Ouarzazate, Zagora; Jousset 1976 , AA , Ouarzazate; Plus and Scotti 1984 , AA , Ouarzazate; Thomas-Orillard 1984 , AA , Ouarzazate; Afonso et al. 1985 , HA , Asni; Prevosti et al. 1985 , AP , Essaouira, AA , Agadir; David et al. 1986 , AP , Rabat, Casablanca; Ayala et al. 1989 , Rif , Chefchaouen; Boulétreau et al. 1992, AA , Agadir; Costa et al. 1992 , AP , Casablanca; Capy et al. 1993 , AP , Casablanca, Rabat; Ritchie et al. 1994 , AP , Casablanca; Mouna 1998 ; Bonnivard and Higuet 1999 , AA , Agadir; Chakir et al. 2002 , 2007 , 2008 , 2011 , HA , Marrakech; Ayrinhac et al. 2004 , HA , Marrakech; Catania et al. 2004 , HA , Marrakech AA , Agadir; Rohmer et al. 2004 , HA , Marrakech; Dieringer et al. 2005 , HA , Marrakech, AA , Agadir; Yassin and Orgogozo 2013 , HA , Marrakech; Bächli 2015 (TaxoDros) – HNHM , AP (Rabat), HA (Marrakech) – MISR , MA (Ifrane) – ZSM Drosophila ( Sophophora ) simulans Sturtevant, 1919 Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Baba-Aissa et al. 1988 , AP , Rabat; Nigro 1988 , AP , Larache; Capy et al. 1990, 1992 , 1993 , AP , Rabat, MA , Béni Mellal AA , Agadir; Chakrani et al. 1993 , AA , Agadir; Mouna 1998 ; Charlat et al. 2003 , AA , Agadir; Biémont et al. 2003 , HA , Marrakech; Chakir et al. 2002 , 2007 , 2008 , 2011 , HA , Marrakech; Nardon et al. 2005 , HA , Marrakech; Yassin and Orgogozo 2013 , HA , Marrakech; Bächli 2015 (TaxoDros); AA (Agadir) – ZSM Drosophila ( Sophophora ) subobscura Collin, 1936 Götz 1965 , Rif , Tanger; Prevosti 1971a , Rif , Tanger; Prevosti 1971b , AP , Essaouira, HA , Asni, AA , Ait-Melloul; Prevosti 1971c ; Gonzalez-Duarte et al. 1973, AP , Essaouira, HA , Asni AA , Agadir; Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Prevosti et al. 1975 , AA , Agadir; Duarte 1976, AP , Essaouira, HA , Asni AA , Agadir; Gonzalez 1976, AP , Essaouira, HA , Asni, AA , Agadir; Prevosti 1978 , Rif , Tanger, AP , Essaouira, HA , Asni AA , Agadir; Krimbas and Loukas 1980 , AA , Agadir; Cabrera et al. 1983 , Rif , Chefchaouen; Larruga et al. 1983 , Rif , Chefchaouen; Prevosti et al. 1985 , AA , Agadir; Pascual et al. 1986 , Rif , Chefchaouen; Latorre et al. 1986 , Rif , Chefchaouen; Constanti et al. 1986 , Rif , Chefchaouen; Ayala et al. 1989 , Rif , Chefchaouen; Afonso et al. 1990 , Rif , Chefchaouen; Pascual et al. 1990 , Rif , Chefchaouen; Paricio et al. 1991 , Rif , Chefchaouen; Menozzi and Krimbas 1992 , AA , Agadir; Latorre et al. 1992 , Rif , Chefchaouen; Fain et al. 1993 , AA , Agadir; Alberola and Frutos 1993; Alberola and Frutos 1996 ; Pinto et al. 1997 , HA , Asni, Marrakech AA , Agadir; Mouna 1998 ; David et al. 2003 , HA , Marrakech; Brehm et al. 2004 , AA , Agadir; Nardon et al. 2005 , HA , Marrakech; Bächli 2015 (TaxoDros); MA (Azrou) – NHMD Drosophila ( Sophophora ) suzukii (Matsumura, 1931) Landolt et al. 2012 , Rif , north-eastern Morocco Hirtodrosophila Duda, 1924 Hirtodrosophila cameraria (Haliday, 1833) Ebejer et al. 2019 , Rif , Aïn Ras el Ma, ruisseau maison forestière (Talassemtane) Lordiphosa Basden, 1961 Lordiphosa andalusiaca (Strobl, 1906) = Lordiphosa forcipata (Collin, 1952), in Hackman 1960 : 102 Hackman 1960 ; Bächli 2015 (TaxoDros); AP (Rabat) – NHMD Scaptomyza Hardy, 1849 Scaptomyza adusta (Loew, 1862) Ebejer et al. 2019 , Rif , Dardara (730 m), AP , Loukous marsh (2 m) Scaptomyza flava (Fallén, 1823) = Scaptomyza flaveola (Meigen, 1830), in Kozlowsky and Rungs 1932 : 66 Kozlowsky and Rungs 1932 , AP , Rabat Scaptomyza graminum (Fallén, 1823) Maarouf 2003 , HA , Chaouia; Bächli 2015 (TaxoDros); HA (Asni, Tinerhir) – NHMD Scaptomyza ( Parascaptomyza ) pallida (Zetterstedt, 1847) Ibn Jilali 1988 (agricultural areas); Maarouf 2003 , HA , Chaouia; Bächli 2015 (TaxoDros); AP (Essaouira) MHNNR, MA (Azrou) – NHMD Scaptodrosophila Duda, 1923 Scaptodrosophila rufifrons (Loew, 1873) Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Zaprionus Coquillett, 1901 Zaprionus indianus Gupta, 1970 Yassin and David 2010 Steganinae Gitonini Gitona Meigen, 1830 Gitona microchaeta Séguy, 1941 Séguy 1941d , AA , Agadir; Bächli 1982 , AA , Agadir; Bächli and Rocha Pité 1984 , AA , Agadir; Mouna 1998 Phortica Fallén, 1823 Phortica variegata (Fallén, 1823) Ebejer et al. 2019 , Rif , Bab Berred (1433 m), Jebel Lakraâ (Talassemtane, 1541 m) Steganini Leucophenga Mik, 1886 Leucophenga maculata (Dufour, 1839) Séguy 1934b , AP , Port-Liautey (Maâmora) Drosophilinae Drosophilini Drosophila Fallén, 1823 Drosophila ( Drosophila ) busckii Coquillett, 1901 = Drosophila rubrostriata Becker, 1908, in Séguy 1953a : 85 Séguy 1953a , AP , Rabat, Sidi Yahia du Gharb; Prevosti 1974 , HA , Asni; Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech – MISR Drosophila ( Drosophila ) buzzatii Patterson & Wheeler, 1942 Prevosti 1974 , HA , Asni (Admin forest); Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Drosophila ( Drosophila ) funebris (Fabricius, 1787) Prevosti 1974 , HA , Asni; Mouna 1998 ; AP (Maâmora) – MISR Drosophila ( Drosophila ) hydei Sturtevant, 1921 Staiger and Gloor 1952 , AP , Rabat; Gloor and Satiger 1954, AP , Rabat; Prevosti 1974 , HA , Asni (Admin forest); Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech Drosophila ( Drosophila ) immigrans Sturtevant, 1921 Mouna 1998 ; Chakir et al. 2011 , HA , Marrakech; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Drosophila ( Drosophila ) kuntzei Duda, 1924 Mouna 1998 ; Prevosti 1974 , AP , Essaouira, HA , Asni Drosophila ( Drosophila ) mercatorum Patterson & Wheeler, 1942 Prevosti 1974 , AA , Agadir (Admin forest near Agadir); Mouna 1998 Drosophila ( Drosophila ) phalerata Meigen, 1830 Prevosti 1974 , AP , Essaouira, HA , Asni; Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger), MA (Ifrane) – ZSM Drosophila ( Drosophila ) repleta Wollaston, 1858 Séguy 1953a , AP , Rabat, MA , Fès Drosophila ( Drosophila ) tsigana Burla & Gloor, 1952 Suwito et al. 2014 , MA , Ifrane Drosophila ( Sophophora ) ambigua Pomini, 1940 Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger), MA (Ifrane) – ZSM Drosophila ( Sophophora ) melanogaster Meigen, 1830 Medioni 1958 ; David and Bocquet 1973 , HA , Marrakech, AA , Agadir, Ouarzazate, Taroudant, AA , Zagora; Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Jousset and Plus 1975 ; Plus et al. 1975 , HA , Marrakech, AA , Agadir, Ouarzazate, Taroudant, AA , Zagora; Prevosti et al. 1975 , Rif , Tanger; Allemand and David 1976 , AA , Ouarzazate; Ashburner and Lemeunier 1976 , HA , Marrakech, AA , Agadir, Taroudant, AA , Zagora; Fleuriet 1976 , AA , Zagora; Plus et al. 1976 , AA , Ouarzazate, Zagora; Jousset 1976 , AA , Ouarzazate; Plus and Scotti 1984 , AA , Ouarzazate; Thomas-Orillard 1984 , AA , Ouarzazate; Afonso et al. 1985 , HA , Asni; Prevosti et al. 1985 , AP , Essaouira, AA , Agadir; David et al. 1986 , AP , Rabat, Casablanca; Ayala et al. 1989 , Rif , Chefchaouen; Boulétreau et al. 1992, AA , Agadir; Costa et al. 1992 , AP , Casablanca; Capy et al. 1993 , AP , Casablanca, Rabat; Ritchie et al. 1994 , AP , Casablanca; Mouna 1998 ; Bonnivard and Higuet 1999 , AA , Agadir; Chakir et al. 2002 , 2007 , 2008 , 2011 , HA , Marrakech; Ayrinhac et al. 2004 , HA , Marrakech; Catania et al. 2004 , HA , Marrakech AA , Agadir; Rohmer et al. 2004 , HA , Marrakech; Dieringer et al. 2005 , HA , Marrakech, AA , Agadir; Yassin and Orgogozo 2013 , HA , Marrakech; Bächli 2015 (TaxoDros) – HNHM , AP (Rabat), HA (Marrakech) – MISR , MA (Ifrane) – ZSM Drosophila ( Sophophora ) simulans Sturtevant, 1919 Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Baba-Aissa et al. 1988 , AP , Rabat; Nigro 1988 , AP , Larache; Capy et al. 1990, 1992 , 1993 , AP , Rabat, MA , Béni Mellal AA , Agadir; Chakrani et al. 1993 , AA , Agadir; Mouna 1998 ; Charlat et al. 2003 , AA , Agadir; Biémont et al. 2003 , HA , Marrakech; Chakir et al. 2002 , 2007 , 2008 , 2011 , HA , Marrakech; Nardon et al. 2005 , HA , Marrakech; Yassin and Orgogozo 2013 , HA , Marrakech; Bächli 2015 (TaxoDros); AA (Agadir) – ZSM Drosophila ( Sophophora ) subobscura Collin, 1936 Götz 1965 , Rif , Tanger; Prevosti 1971a , Rif , Tanger; Prevosti 1971b , AP , Essaouira, HA , Asni, AA , Ait-Melloul; Prevosti 1971c ; Gonzalez-Duarte et al. 1973, AP , Essaouira, HA , Asni AA , Agadir; Prevosti 1974 , AP , Essaouira, HA , Asni (Admin forest); Prevosti et al. 1975 , AA , Agadir; Duarte 1976, AP , Essaouira, HA , Asni AA , Agadir; Gonzalez 1976, AP , Essaouira, HA , Asni, AA , Agadir; Prevosti 1978 , Rif , Tanger, AP , Essaouira, HA , Asni AA , Agadir; Krimbas and Loukas 1980 , AA , Agadir; Cabrera et al. 1983 , Rif , Chefchaouen; Larruga et al. 1983 , Rif , Chefchaouen; Prevosti et al. 1985 , AA , Agadir; Pascual et al. 1986 , Rif , Chefchaouen; Latorre et al. 1986 , Rif , Chefchaouen; Constanti et al. 1986 , Rif , Chefchaouen; Ayala et al. 1989 , Rif , Chefchaouen; Afonso et al. 1990 , Rif , Chefchaouen; Pascual et al. 1990 , Rif , Chefchaouen; Paricio et al. 1991 , Rif , Chefchaouen; Menozzi and Krimbas 1992 , AA , Agadir; Latorre et al. 1992 , Rif , Chefchaouen; Fain et al. 1993 , AA , Agadir; Alberola and Frutos 1993; Alberola and Frutos 1996 ; Pinto et al. 1997 , HA , Asni, Marrakech AA , Agadir; Mouna 1998 ; David et al. 2003 , HA , Marrakech; Brehm et al. 2004 , AA , Agadir; Nardon et al. 2005 , HA , Marrakech; Bächli 2015 (TaxoDros); MA (Azrou) – NHMD Drosophila ( Sophophora ) suzukii (Matsumura, 1931) Landolt et al. 2012 , Rif , north-eastern Morocco Hirtodrosophila Duda, 1924 Hirtodrosophila cameraria (Haliday, 1833) Ebejer et al. 2019 , Rif , Aïn Ras el Ma, ruisseau maison forestière (Talassemtane) Lordiphosa Basden, 1961 Lordiphosa andalusiaca (Strobl, 1906) = Lordiphosa forcipata (Collin, 1952), in Hackman 1960 : 102 Hackman 1960 ; Bächli 2015 (TaxoDros); AP (Rabat) – NHMD Scaptomyza Hardy, 1849 Scaptomyza adusta (Loew, 1862) Ebejer et al. 2019 , Rif , Dardara (730 m), AP , Loukous marsh (2 m) Scaptomyza flava (Fallén, 1823) = Scaptomyza flaveola (Meigen, 1830), in Kozlowsky and Rungs 1932 : 66 Kozlowsky and Rungs 1932 , AP , Rabat Scaptomyza graminum (Fallén, 1823) Maarouf 2003 , HA , Chaouia; Bächli 2015 (TaxoDros); HA (Asni, Tinerhir) – NHMD Scaptomyza ( Parascaptomyza ) pallida (Zetterstedt, 1847) Ibn Jilali 1988 (agricultural areas); Maarouf 2003 , HA , Chaouia; Bächli 2015 (TaxoDros); AP (Essaouira) MHNNR, MA (Azrou) – NHMD Scaptodrosophila Duda, 1923 Scaptodrosophila rufifrons (Loew, 1873) Mouna 1998 ; Bächli 2015 (TaxoDros); Rif (Tanger) – ZSM Zaprionus Coquillett, 1901 Zaprionus indianus Gupta, 1970 Yassin and David 2010 Steganinae Gitonini Gitona Meigen, 1830 Gitona microchaeta Séguy, 1941 Séguy 1941d , AA , Agadir; Bächli 1982 , AA , Agadir; Bächli and Rocha Pité 1984 , AA , Agadir; Mouna 1998 Phortica Fallén, 1823 Phortica variegata (Fallén, 1823) Ebejer et al. 2019 , Rif , Bab Berred (1433 m), Jebel Lakraâ (Talassemtane, 1541 m) Steganini Leucophenga Mik, 1886 Leucophenga maculata (Dufour, 1839) Séguy 1934b , AP , Port-Liautey (Maâmora) EPHYDRIDAE K. Kettani, T. Zatwarnicki Number of species: 117 . Expected: 140 Faunistic knowledge of the family in Morocco: good Discomyzinae Discomyzini Actocetor Becker, 1903 Actocetor indicus (Wiedemann, 1824) = Actocetor margaritatus (Wiedemann, 1830), in Séguy 1934b : 162, 1953a : 86 Séguy 1934b , Rif , Béni Aross; Séguy 1953a , SA , Tindouf Discomyza Meigen, 1830 Discomyza incurva (Fallén, 1823) = Discomyza italica Séguy, 1929, in Vitte 1991 : 3; Dakki 1997 : 63 Cresson 1939 ; Vitte 1991 , AP , Atlantic coast and Plains; Dakki 1997 Psilopini Clanoneurum Becker, 1903 Clanoneurum cimiciforme (Haliday, 1855) Séguy 1941d , AA , Agadir; Dahl 1964 ; Vitte 1991 , AP , Rabat; Dakki 1997 Diasemocera Bezzi, 1895 Diasemocera aequalipes (Becker, 1907) = Psilopa aequalipes (Becker, 1907), in Ebejer et al. 2019 : 147 Zatwarnicki 2018 ; Ebejer et al. 2019 , AA , Lac Tiffert (4 km W of Merzouga, 702 m), Ziz river (13 km N of Erfoud, 800 m) Diasemocera biskrae (Becker, 1907) = Psilopa biskrae (Becker, 1907), in Vitte 1991 : 32 Vitte 1991 , AP , M'Diq; Dakki 1997 Diasemocera composita (Becker, 1903) = Psilopa composita (Becker, 1903), in Vitte 1991 : 32, Dakki 1997 : 63 Vitte 1991 , AP , Rabat; Dakki 1997 Diasemocera fratella (Becker, 1903) = Psilopa fratella (Becker, 1903), in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Errachidia (1 km N of Tarda, 1023 m), AA , Ziz river (13 km N of Erfoud, 800 m) Diasemocera glabricula (Fallén, 1813) = Psilopa nigritella Stenhammar, 1844, in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Diasemocera leucostoma (Meigen, 1830) = Psilopa leucostoma (Meigen, 1830), in Séguy 1941d : 18 Séguy 1941d , AA , Agadir Diasemocera maritima (Perris, 1847) = Psilopa maritima (Perris, 1847), in Cassar et al. 2008 : 25 Cassar et al. 2008 , Rif , Laou Basin Diasemocera nana (Loew, 1860) = Psilopa nana Loew, 1860, in Vitte 1991 : 32, Dakki 1997 : 63 Vitte 1991 , Rif , AP , Atlantic coast; Dakki 1997 Diasemocera rufithorax (Becker, 1903) = Psilopa rufithorax (Becker, 1903), in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Merzouga (714 m) Psilopa Fallén, 1823 Psilopa clara (Wollaston, 1858) = Psilopa rutilans Canzoneri & Meneghini, 1972, in Cassar et al. 2005 : 69 Cassar et al. 2005 , Rif , Smir lagoon; Zatwarnicki 2018 , AP , Larache (Lower Loukous), Safi, AA , Sidi Moussa, D'Agion (0–50 m) Psilopa meneghinii Canzoneri, 1986 Vitte 1991 , AP , Atlantic coast; Dakki 1997 Psilopa compta (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Psilopa nilotica (Becker, 1903) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m), 1 km N of Tarda (Errachidia, 1023 m), Merzouga (714 m), 2 km N Erfoud (818 m) Psilopa nitidula (Fallén, 1813) Becker and Stein 1913 , Rif , Tanger; Séguy 1941a , HA , Toubkal; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Zatwarnicki 1991 , Rif , Tanger, Tétouan, MA , Ifrane; Dakki 1997 Psilopa obscuripes Loew, 1860 Ebejer et al. 2019 , Rif , Oued Azla (near bridge, 80 m), AP , Larache (5 m), Lower Loukous saltmarsh (2 m), MA , Khénifra (28 km S of Timahdit, 2100 m) Psilopa polita (Macquart, 1835) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Risini Achaetorisa Papp, 1980 Achaetorisa brevicornis Papp, 1980 Papp 1980, HA , Ouirgane Ephydrinae Dagini Brachydeutera Leow, 1862 Brachydeutera meridionalis (Rondani, 1856) = Brachydeutera ibari Ninomyia, 1929, in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , Rif , Oued Martil (Taboula, 14 m) Parydrini Parydra Stenhammar, 1844 Parydra ( Chaetoapnaea ) fossarum (Haliday, 1833) Séguy 1930a , AP , Rabat, MA , Meknès, HA , Marrakech; Vitte 1991 ; Dakki 1997 ; Vitte 1988 , MA , lakes of Middle Atlas; MA (Jebel Khazzane) – MISR Parydra ( Chaetoapnaea ) hecate (Haliday, 1833) = Napaea hecate (Haliday), in Vaillant 1956b : 244 Vaillant 1956b , HA , Imi-N'Ifri Parydra ( Chaetoapnaea ) quadripunctata (Meigen, 1830) Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 , AP , Merja Zerga Parydra ( Paranapaea ) pubera Loew, 1860 Dahl 1964 , AA , Aït Melloul, Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Parydra ( Parydra ) aquila (Fallén, 1813) Vitte 1991 , Rif , Ouezzane, Ketama; Dakki 1997 Parydra ( Parydra ) coarctata (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Ksar el Kbir; Dakki 1997 Parydra ( Parydra ) cognata Loew, 1860 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Parydra ( Parydra ) flavitarsis Dahl, 1964 Vitte 1988 , Rif , MA , lakes of Middle Atlas; Vitte 1991 , Rif , MA , Fès; Dakki 1997 ; Gatt and Ebejer 2003 ; Dakki et al. 2003; Chillasse and Dakki 2004 , Rif , MA ; Dakki and Himmi 2008 , MA , Oued Sebou Parydra ( Parydra ) littoralis (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Tétouan, Ketama; Dakki 1997 Parydra ( Parydra ) nigritarsis Strobl, 1893 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Ketama; Dakki 1997 Parydra ( Parydra ) nubecula Becker, 1896 Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 , AP , Merja Zerga Parydra ( Parydra ) quinquemaculata Becker, 1896 Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Ephydrini Ephydra Fallén, 1823 Ephydra bivittata Loew, 1860 Vitte 1991 ; Dakki 1997 Ephydra flavipes (Macquart, 1843) Vitte 1991 , AP , Atlantic coast; Dakki 1997 Ephydra glauca Meigen, 1830 Vitte 1991 ; Dakki 1997 Ephydra macellaria Egger, 1862 Dahl 1964 , AA , Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 ; Vitte 1991 , AP , Atlantic coast Halmopota Haliday, 1856 Halmopota mediterranea Loew, 1860 Dahl 1964 , AA , Aït Melloul; Dakki 1997 ; Vitte 1991 , Rif , Asilah, Chefchaouen Paracoenia Cresson, 1935 Paracoenia fumosa (Stenhammar, 1844) Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Setacera Cresson, 1930 Setacera breviventris (Loew, 1860) Vitte 1991 , Rif , Ksar el Kbir; Dakki 1997 Scatellini Haloscatella Mathis, 1979 Haloscatella dichaeta (Loew, 1860) = Scatella dichaeta Loew, 1860, in Vitte 1988 : 392, 1991 : 26; Dakki 1997 : 62 Vitte 1988 , MA , Khemisset, Oued Beth, Dayat Aoua; Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Lamproscatella Hendel, 1917 Lamproscatella sibilans (Haliday, 1833) Ebejer et al. 2019 , AA , 1 km N of Tarda (Errachidia, 1023 m) Limnellia Malloch, 1925 Limnellia quadrata (Fallén, 1813) Vitte 1991 , Rif , Central Rif; Dakki 1997 Philotelma Becker, 1896 Philotelma nigripenne (Meigen, 1830) = Scatella nigripennis (Meigen, 1830), in Vitte 1988 : 391; Dakki 1997 : 62 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Scatella Robineau-Desvoidy, 1830 Scatella ( Neoscatella ) subguttata (Meigen, 1830) Vitte 1991 , AP , Atlantic and Mediterranean coast, Smir lagoon; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 Scatella ( Scatella ) ciliata Collin, 1930 Vitte 1991 , AP , Moulay Bousselham, Asilah; Dakki 1997 Scatella ( Scatella ) lacustris (Meigen, 1830) = Scatella ( Scatella ) tenuicosta Collin, 1930, in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Ziz river (13 km N of Erfoud, 800 m) Scatella ( Scatella ) lutosa (Haliday, 1833) Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Scatella ( Scatella ) obsoleta Loew, 1861 = Scatella callosicosta Bezzi, 1895, in Vitte 1988 : 392, 1991 : 26; Dakki 1997 : 62 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , M'Diq; Dakki 1997 Scatella ( Scatella ) paludum (Meigen, 1830) Dahl 1964 , AP , Oued Korifla; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatella ( Scatella ) rufipes Strobl, 1906 = Scatella rubida Becker, 1907, in Olafsson 1991 : 21; Vitte 1991 : 26; Dakki 1997 : 62 Olafsson 1991 , EM , Figuig, Defilia; Vitte 1991 , Rif , AP (Atlantic coast); Dakki 1997 ; Gatt and Ebejer 2003 Scatella ( Scatella ) stagnalis (Fallén, 1813) Séguy 1941a , HA , Toubkal; Dahl 1964 , AA , Aït Melloul, Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatophila Becker, 1896 Scatophila caviceps (Stenhammar, 1844) Vitte 1988 , AP , Rabat, Temara, MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatophila despecta (Haliday, 1839) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Khemisset, Oued Beth; Dakki 1997 Scatophila farinae Becker, 1903 Zatwarnicki 1987 , HA , Vallée de l'Ait Mizane; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Taounate; Dakki 1997 ; Gatt and Ebejer 2003 Scatophila modesta Becker, 1908 Vitte 1991 , Rif , Tétouan; Dakki 1997 Scatophila unicornis Czerny, 1900 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Gymnomyzinae Discocerinini Diclasiopa Hendel, 1917 Diclasiopa galactoptera (Becker, 1903) = Discocerina galactoptera Becker, in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 ; Kirk-Spriggs and McGregor 2009 Diclasiopa lacteipennis (Loew, 1862) = Discocerina lacteipennis Loew, 1862, in Vitte 1988 : 394, 1991 : 32; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Rabat; Vitte 1991 , MA , Khemisset, Taounate; Dakki 1997 Diclasiopa niveipennis (Becker, 1896) = Discocerina niveipennis (Becker, 1896), in Vitte 1988 : 394, 1991 : 32; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Discocerina Macquart, 1835 Discocerina obscurella (Fallén, 1813) Vitte 1988 , AP , MA , lakes of Middle Atlas; Vitte 1991 , MA , Fès, Taounate; Mathis 1997 ; Dakki 1997 ; Wolff et al. 2016 Ditrichophora Cresson, 1924 Ditrichophora calceata (Meigen, 1830) = Discocerina calceata (Meigen, 1830), in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Ditrichophora mauritanica (Vitte, 1991) = Discocerina mauritanica Vitte, 1991, in Vitte 1991 : 33 Vitte 1991 , Rif , MA , Azrou; Dakki 1997 ; Chillasse and Dakki 2004 , Rif , MA ; Dakki and Himmi 2008 , MA , Oued Sebou Gymnoclasiopa Hendel, 1930 Gymnoclasiopa plumosa (Fallén, 1823) = Discocerina plumosa (Fallén, 1823), in Vitte 1988 : 394, 1991 : 33; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Gymnoclasiopa pulchella (Meigen, 1830) = Discocerina pulchella (Meigen, 1830), in Vitte 1991 : 33; Dakki 1997 : 63 Vitte 1991 , Rif , Ketama, Ouezzane; Dakki 1997 Hecamedoides Hendel, 1917 Hecamedoides glaucellus (Stenhammar, 1844) = Discocerina glaucella (Stenhammar, 1844), in Vitte 1991 : 33 Vitte 1991 , Rif , AP Polytrichophora Cresson, 1924 Polytrichophora duplosetosa (Becker, 1896) AP (Rabat) – MISR Gymnomyzini Athyroglossa Loew, 1860 Athyroglossa ( Athyroglossa ) glabra (Meigen, 1830) Vitte 1988 , Rif , MA , lakes of Middle Atlas; Dakki 1997 Athyroglossa ( Athyroglossa ) nudiuscula Loew, 1860 Vitte 1991 , Rif ; Dakki 1997 Athyroglossa ( Parathyroglossa ) ordinata Becker, 1896 Vitte 1991 , Rif ; Vitte 1988 , MA , lakes of Middle Atlas; Mathis and Zatwarnicki 1990 , MA , Ifrane; Dakki 1997 Chlorichaeta Becker, 1922 Chlorichaeta albipennis (Loew, 1848) Vitte 1991 ; Dakki 1997 Mosillus Latreille, 1804 Mosillus subsultans (Fabricius, 1794) = Gymnopa subsultans Fabricius, in Séguy 1930a : 181 Séguy 1930a , MA , M'Rirt, HA , Imminen (Tachidirt); Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Mathis et al. 1993 , MA , Ifrane (1650 m), maison forestière (cedar forest: 2700 m), Oued Jaffar (N of source, 0–1500 m), HA , Mikdane (Jebel Ayachi); Dakki 1997 ; Koçak and Kemal 2010 Hecamedini Allotrichoma Becker, 1896 Allotrichoma laterale (Loew, 1860) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Allotrichoma leotoni Vitte, 1992 Vitte 1992 , Rif , Ouezzane, Boured Allotrichoma quadripectinatum (Becker, 1896) = Allotrichoma bellicosum Giordani Soika, 1956, in Vitte 1991 : 30; Dakki 1997 : 63 Vitte 1991 , Rif , AP ; Dakki 1997 Allotrichoma simplex (Loew, 1861) = Allotrichoma filiforme Becker, 1896, in Vitte 1988 : 393, 1991 : 30; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Khemisset, Oued Sebou; Dakki 1997 Elephantinosoma Becker, 1903 Elephantinosoma chnumi Becker, 1903 Gatt and Ebejer 2003 ; Kirk-Spriggs and McGregor 2009 Hecamede Haliday, 1837 Hecamede albicans (Meigen, 1830) Vitte 1991 , AP ; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Cassar et al. 2008 , Rif , Smir Lagoon; Popescu-Mirceni 2011 Lipochaetini Glenanthe Haliday, 1839 Glenanthe ripicola (Haliday, 1839) Vitte 1991 , AP ; Dakki 1997 ; Cassar et al. 2008 , Rif , Laou Basin; Zatwarnicki and Mathis 2011 , AA , Tarfaya – HNHM Homalometopus Becker, 1903 Homalometopus sp. Vitte 1991 Ochtherini Ochthera Latreille, 1802 Ochthera manicata (Fabricius, 1794) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Ochthera pilimana Becker, 1903 Dakki 1997 Ochthera schembrii Rondani, 1847 = Ochthera mantispa Loew, 1847, in Vitte 1988 : 394, 1991 : 28; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , AP ; Dakki 1997 Hydrelliinae Atissini Asmeringa Becker, 1903 Asmeringa inermis Becker, 1903 Vitte 1991 , AP , Rabat; Dakki 1997 ; Gatt and Ebejer 2003 Atissa Haliday, 1839 Atissa durrenbergensis Loew, 1864 Vitte 1991 , AP ; Dakki 1997 Atissa hepaticoloris Becker, 1903 Vitte 1991 , AP ; Dakki 1997 ; Gatt and Ebejer 2003 Atissa limosina Becker, 1896 Vitte 1991 , AP , Rabat, MA , Fès; Dakki 1997 Atissa pygmaea (Haliday, 1839) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 Ptilomyia Coquillett, 1900 Ptilomyia angustigenis (Becker, 1926) = Atissa angustigenis Becker, in Vitte 1988 : 393, 1991 : 30; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Gatt and Ebejer 2003 Dryxini Dryxo Robineau-Desvoidy, 1830 Dryxo ornata (Macquart, 1843) Mathis and Zatwarnicki 2002 , AA , Aoulouz Hydrelliini Hydrellia Robineau-Desvoidy, 1830 Hydrellia albifrons (Fallén, 1813) Vitte 1991 , AP , Rif ; Dakki 1997 Hydrellia argyrogenis Becker, 1896 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , Atlantic coast and Plains; Dakki 1997 Hydrellia armata Canzoneri & Meneghini, 1976 Vitte 1991 , Rif , Ksar el Kbir, MA , Fès; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou Hydrellia atlas Vitte, 1989 Vitte 1989 , MA , Dayat Aoua; Dakki 1997 ; Dakki et al. 2003; Chilasse and Dakki 2004, MA Hydrellia griseola (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Wolff et al. 2016 ; Rif (Oued Laou) – MISR Hydrellia maculiventris Becker, 1896 Vitte 1991 , Rif , AP , Atlantic coast; Dakki 1997 ; Gatt and Ebejer 2003 Hydrellia maura Meigen, 1838 = Hydrellia modesta Loew, 1860, in Vitte 1988 : 392, 1991 : 29; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Zatwarnicki 1988 , MA , Ifrane, HA , Vallée de l'Aït Mizane; Vitte 1991 ; Dakki 1997 Hydrellia nigricans (Stenhammar, 1844) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Hydrellia obscura (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 ; Rif (Aïn Jdioui) – MISR Hydrellia pubescen s Becker, 1926 = Hydrellia nasturtii Collin, 1928, in Vitte 1991 : 29; Dakki 1997 : 63 Vitte 1991 , MA , Fès; Dakki 1997 ; Gatt and Ebejer 2003 Hydrellia ranunculi (Haliday, 1838) Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Hydrellia rharbia Vitte, 1991 Vitte 1989 , AP , Merja Halloufa (near Moulay Bousselham); Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou Hydrellia subalbiceps Collin, 1966 Vitte 1991 , Rif , Ketama; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Notiphilini Notiphila Fallén, 1810 Notiphila ( Notiphila ) annulipes Stenhammar, 1844 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Notiphila ( Notiphila ) cinerea Fallén, 1830 Séguy 1930a , MA , Meknès; Séguy 1941a , HA , Imi-n'Ouaka; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Kirk-Spriggs and McGregor 2009 ; Rif (Talassemtane, Aïn Jdioui) – MISR Notiphila ( Notiphila ) cogani Canzoneri & Meneghini, 1979 Vitte 1988 , MA , lakes of Middle Atlas; Krivosheina 1998 ; Vitte 1991 ; Dakki 1997 ; Rif (Aïn Jdioui) – MISR Notiphila ( Notiphila ) dorsata Stenhammar, 1844 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , coastal lake areas; Dakki 1997 Notiphila ( Notiphila ) maculata Stenhammar, 1844 Vitte 1991 , Rif , AP , coastal plains; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Notiphila ( Notiphila ) riparia Meigen, 1830 Krivosheina 1998 , AA , Aït Melloul (Souss); Vitte 1988 , MA , lakes of Middle Atlas and reedbeds; Vitte 1991 ; Dakki 1997 Notiphila ( Notiphila ) stagnicola (Robineau-Desvoidy, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , AP , coastal plains; Dakki 1997 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 , AA , Ouarzazate Ilytheinae Hyadinini Hyadina Haliday, 1837 Hyadina guttata (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Hyadina pollinosa Oldenberg, 1923 Vitte 1991 , MA , Fès; Dakki 1997 Hyadina rufipes (Meigen, 1830) = Hyadina nitida (Macquart, 1835), in Vitte 1991 : 27; Dakki 1997 : 63 Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Nostima Coquillett, 1900 Nostima picta (Fallén, 1813) Vitte 1991 , AP ; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Pelina Haliday, 1837 Pelina aenea (Fallén, 1813) Vitte 1991 , Rif , Ouezzane; Dakki 1997 Pelina subpunctata Becker, 1896 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Philygria Stenhammar, 1844 Philygria posticata (Meigen, 1830) Ebejer et al. 2019 , MA , Khénifra (17 km SW of Midelt, 1940 m) Acknowledgements We gratefully acknowledge the assistance and cooperation of Martin J. Ebejer who contributed to the revision of this family. Discomyzinae Discomyzini Actocetor Becker, 1903 Actocetor indicus (Wiedemann, 1824) = Actocetor margaritatus (Wiedemann, 1830), in Séguy 1934b : 162, 1953a : 86 Séguy 1934b , Rif , Béni Aross; Séguy 1953a , SA , Tindouf Discomyza Meigen, 1830 Discomyza incurva (Fallén, 1823) = Discomyza italica Séguy, 1929, in Vitte 1991 : 3; Dakki 1997 : 63 Cresson 1939 ; Vitte 1991 , AP , Atlantic coast and Plains; Dakki 1997 Psilopini Clanoneurum Becker, 1903 Clanoneurum cimiciforme (Haliday, 1855) Séguy 1941d , AA , Agadir; Dahl 1964 ; Vitte 1991 , AP , Rabat; Dakki 1997 Diasemocera Bezzi, 1895 Diasemocera aequalipes (Becker, 1907) = Psilopa aequalipes (Becker, 1907), in Ebejer et al. 2019 : 147 Zatwarnicki 2018 ; Ebejer et al. 2019 , AA , Lac Tiffert (4 km W of Merzouga, 702 m), Ziz river (13 km N of Erfoud, 800 m) Diasemocera biskrae (Becker, 1907) = Psilopa biskrae (Becker, 1907), in Vitte 1991 : 32 Vitte 1991 , AP , M'Diq; Dakki 1997 Diasemocera composita (Becker, 1903) = Psilopa composita (Becker, 1903), in Vitte 1991 : 32, Dakki 1997 : 63 Vitte 1991 , AP , Rabat; Dakki 1997 Diasemocera fratella (Becker, 1903) = Psilopa fratella (Becker, 1903), in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Errachidia (1 km N of Tarda, 1023 m), AA , Ziz river (13 km N of Erfoud, 800 m) Diasemocera glabricula (Fallén, 1813) = Psilopa nigritella Stenhammar, 1844, in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Diasemocera leucostoma (Meigen, 1830) = Psilopa leucostoma (Meigen, 1830), in Séguy 1941d : 18 Séguy 1941d , AA , Agadir Diasemocera maritima (Perris, 1847) = Psilopa maritima (Perris, 1847), in Cassar et al. 2008 : 25 Cassar et al. 2008 , Rif , Laou Basin Diasemocera nana (Loew, 1860) = Psilopa nana Loew, 1860, in Vitte 1991 : 32, Dakki 1997 : 63 Vitte 1991 , Rif , AP , Atlantic coast; Dakki 1997 Diasemocera rufithorax (Becker, 1903) = Psilopa rufithorax (Becker, 1903), in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Merzouga (714 m) Psilopa Fallén, 1823 Psilopa clara (Wollaston, 1858) = Psilopa rutilans Canzoneri & Meneghini, 1972, in Cassar et al. 2005 : 69 Cassar et al. 2005 , Rif , Smir lagoon; Zatwarnicki 2018 , AP , Larache (Lower Loukous), Safi, AA , Sidi Moussa, D'Agion (0–50 m) Psilopa meneghinii Canzoneri, 1986 Vitte 1991 , AP , Atlantic coast; Dakki 1997 Psilopa compta (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Psilopa nilotica (Becker, 1903) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m), 1 km N of Tarda (Errachidia, 1023 m), Merzouga (714 m), 2 km N Erfoud (818 m) Psilopa nitidula (Fallén, 1813) Becker and Stein 1913 , Rif , Tanger; Séguy 1941a , HA , Toubkal; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Zatwarnicki 1991 , Rif , Tanger, Tétouan, MA , Ifrane; Dakki 1997 Psilopa obscuripes Loew, 1860 Ebejer et al. 2019 , Rif , Oued Azla (near bridge, 80 m), AP , Larache (5 m), Lower Loukous saltmarsh (2 m), MA , Khénifra (28 km S of Timahdit, 2100 m) Psilopa polita (Macquart, 1835) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Risini Achaetorisa Papp, 1980 Achaetorisa brevicornis Papp, 1980 Papp 1980, HA , Ouirgane Ephydrinae Dagini Brachydeutera Leow, 1862 Brachydeutera meridionalis (Rondani, 1856) = Brachydeutera ibari Ninomyia, 1929, in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , Rif , Oued Martil (Taboula, 14 m) Parydrini Parydra Stenhammar, 1844 Parydra ( Chaetoapnaea ) fossarum (Haliday, 1833) Séguy 1930a , AP , Rabat, MA , Meknès, HA , Marrakech; Vitte 1991 ; Dakki 1997 ; Vitte 1988 , MA , lakes of Middle Atlas; MA (Jebel Khazzane) – MISR Parydra ( Chaetoapnaea ) hecate (Haliday, 1833) = Napaea hecate (Haliday), in Vaillant 1956b : 244 Vaillant 1956b , HA , Imi-N'Ifri Parydra ( Chaetoapnaea ) quadripunctata (Meigen, 1830) Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 , AP , Merja Zerga Parydra ( Paranapaea ) pubera Loew, 1860 Dahl 1964 , AA , Aït Melloul, Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Parydra ( Parydra ) aquila (Fallén, 1813) Vitte 1991 , Rif , Ouezzane, Ketama; Dakki 1997 Parydra ( Parydra ) coarctata (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Ksar el Kbir; Dakki 1997 Parydra ( Parydra ) cognata Loew, 1860 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Parydra ( Parydra ) flavitarsis Dahl, 1964 Vitte 1988 , Rif , MA , lakes of Middle Atlas; Vitte 1991 , Rif , MA , Fès; Dakki 1997 ; Gatt and Ebejer 2003 ; Dakki et al. 2003; Chillasse and Dakki 2004 , Rif , MA ; Dakki and Himmi 2008 , MA , Oued Sebou Parydra ( Parydra ) littoralis (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Tétouan, Ketama; Dakki 1997 Parydra ( Parydra ) nigritarsis Strobl, 1893 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , Ketama; Dakki 1997 Parydra ( Parydra ) nubecula Becker, 1896 Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 , AP , Merja Zerga Parydra ( Parydra ) quinquemaculata Becker, 1896 Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Ephydrini Ephydra Fallén, 1823 Ephydra bivittata Loew, 1860 Vitte 1991 ; Dakki 1997 Ephydra flavipes (Macquart, 1843) Vitte 1991 , AP , Atlantic coast; Dakki 1997 Ephydra glauca Meigen, 1830 Vitte 1991 ; Dakki 1997 Ephydra macellaria Egger, 1862 Dahl 1964 , AA , Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 ; Vitte 1991 , AP , Atlantic coast Halmopota Haliday, 1856 Halmopota mediterranea Loew, 1860 Dahl 1964 , AA , Aït Melloul; Dakki 1997 ; Vitte 1991 , Rif , Asilah, Chefchaouen Paracoenia Cresson, 1935 Paracoenia fumosa (Stenhammar, 1844) Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Setacera Cresson, 1930 Setacera breviventris (Loew, 1860) Vitte 1991 , Rif , Ksar el Kbir; Dakki 1997 Scatellini Haloscatella Mathis, 1979 Haloscatella dichaeta (Loew, 1860) = Scatella dichaeta Loew, 1860, in Vitte 1988 : 392, 1991 : 26; Dakki 1997 : 62 Vitte 1988 , MA , Khemisset, Oued Beth, Dayat Aoua; Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Lamproscatella Hendel, 1917 Lamproscatella sibilans (Haliday, 1833) Ebejer et al. 2019 , AA , 1 km N of Tarda (Errachidia, 1023 m) Limnellia Malloch, 1925 Limnellia quadrata (Fallén, 1813) Vitte 1991 , Rif , Central Rif; Dakki 1997 Philotelma Becker, 1896 Philotelma nigripenne (Meigen, 1830) = Scatella nigripennis (Meigen, 1830), in Vitte 1988 : 391; Dakki 1997 : 62 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Scatella Robineau-Desvoidy, 1830 Scatella ( Neoscatella ) subguttata (Meigen, 1830) Vitte 1991 , AP , Atlantic and Mediterranean coast, Smir lagoon; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Popescu-Mirceni 2011 Scatella ( Scatella ) ciliata Collin, 1930 Vitte 1991 , AP , Moulay Bousselham, Asilah; Dakki 1997 Scatella ( Scatella ) lacustris (Meigen, 1830) = Scatella ( Scatella ) tenuicosta Collin, 1930, in Ebejer et al. 2019 : 147 Ebejer et al. 2019 , AA , Ziz river (13 km N of Erfoud, 800 m) Scatella ( Scatella ) lutosa (Haliday, 1833) Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Scatella ( Scatella ) obsoleta Loew, 1861 = Scatella callosicosta Bezzi, 1895, in Vitte 1988 : 392, 1991 : 26; Dakki 1997 : 62 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , M'Diq; Dakki 1997 Scatella ( Scatella ) paludum (Meigen, 1830) Dahl 1964 , AP , Oued Korifla; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatella ( Scatella ) rufipes Strobl, 1906 = Scatella rubida Becker, 1907, in Olafsson 1991 : 21; Vitte 1991 : 26; Dakki 1997 : 62 Olafsson 1991 , EM , Figuig, Defilia; Vitte 1991 , Rif , AP (Atlantic coast); Dakki 1997 ; Gatt and Ebejer 2003 Scatella ( Scatella ) stagnalis (Fallén, 1813) Séguy 1941a , HA , Toubkal; Dahl 1964 , AA , Aït Melloul, Oued Souss; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatophila Becker, 1896 Scatophila caviceps (Stenhammar, 1844) Vitte 1988 , AP , Rabat, Temara, MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Scatophila despecta (Haliday, 1839) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Khemisset, Oued Beth; Dakki 1997 Scatophila farinae Becker, 1903 Zatwarnicki 1987 , HA , Vallée de l'Ait Mizane; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Taounate; Dakki 1997 ; Gatt and Ebejer 2003 Scatophila modesta Becker, 1908 Vitte 1991 , Rif , Tétouan; Dakki 1997 Scatophila unicornis Czerny, 1900 Ebejer et al. 2019 , AA , 14 km E of Rich (Errachidia, 1278 m) Gymnomyzinae Discocerinini Diclasiopa Hendel, 1917 Diclasiopa galactoptera (Becker, 1903) = Discocerina galactoptera Becker, in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 ; Kirk-Spriggs and McGregor 2009 Diclasiopa lacteipennis (Loew, 1862) = Discocerina lacteipennis Loew, 1862, in Vitte 1988 : 394, 1991 : 32; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Rabat; Vitte 1991 , MA , Khemisset, Taounate; Dakki 1997 Diclasiopa niveipennis (Becker, 1896) = Discocerina niveipennis (Becker, 1896), in Vitte 1988 : 394, 1991 : 32; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Discocerina Macquart, 1835 Discocerina obscurella (Fallén, 1813) Vitte 1988 , AP , MA , lakes of Middle Atlas; Vitte 1991 , MA , Fès, Taounate; Mathis 1997 ; Dakki 1997 ; Wolff et al. 2016 Ditrichophora Cresson, 1924 Ditrichophora calceata (Meigen, 1830) = Discocerina calceata (Meigen, 1830), in Vitte 1988 : 394; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Ditrichophora mauritanica (Vitte, 1991) = Discocerina mauritanica Vitte, 1991, in Vitte 1991 : 33 Vitte 1991 , Rif , MA , Azrou; Dakki 1997 ; Chillasse and Dakki 2004 , Rif , MA ; Dakki and Himmi 2008 , MA , Oued Sebou Gymnoclasiopa Hendel, 1930 Gymnoclasiopa plumosa (Fallén, 1823) = Discocerina plumosa (Fallén, 1823), in Vitte 1988 : 394, 1991 : 33; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Gymnoclasiopa pulchella (Meigen, 1830) = Discocerina pulchella (Meigen, 1830), in Vitte 1991 : 33; Dakki 1997 : 63 Vitte 1991 , Rif , Ketama, Ouezzane; Dakki 1997 Hecamedoides Hendel, 1917 Hecamedoides glaucellus (Stenhammar, 1844) = Discocerina glaucella (Stenhammar, 1844), in Vitte 1991 : 33 Vitte 1991 , Rif , AP Polytrichophora Cresson, 1924 Polytrichophora duplosetosa (Becker, 1896) AP (Rabat) – MISR Gymnomyzini Athyroglossa Loew, 1860 Athyroglossa ( Athyroglossa ) glabra (Meigen, 1830) Vitte 1988 , Rif , MA , lakes of Middle Atlas; Dakki 1997 Athyroglossa ( Athyroglossa ) nudiuscula Loew, 1860 Vitte 1991 , Rif ; Dakki 1997 Athyroglossa ( Parathyroglossa ) ordinata Becker, 1896 Vitte 1991 , Rif ; Vitte 1988 , MA , lakes of Middle Atlas; Mathis and Zatwarnicki 1990 , MA , Ifrane; Dakki 1997 Chlorichaeta Becker, 1922 Chlorichaeta albipennis (Loew, 1848) Vitte 1991 ; Dakki 1997 Mosillus Latreille, 1804 Mosillus subsultans (Fabricius, 1794) = Gymnopa subsultans Fabricius, in Séguy 1930a : 181 Séguy 1930a , MA , M'Rirt, HA , Imminen (Tachidirt); Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Mathis et al. 1993 , MA , Ifrane (1650 m), maison forestière (cedar forest: 2700 m), Oued Jaffar (N of source, 0–1500 m), HA , Mikdane (Jebel Ayachi); Dakki 1997 ; Koçak and Kemal 2010 Hecamedini Allotrichoma Becker, 1896 Allotrichoma laterale (Loew, 1860) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Allotrichoma leotoni Vitte, 1992 Vitte 1992 , Rif , Ouezzane, Boured Allotrichoma quadripectinatum (Becker, 1896) = Allotrichoma bellicosum Giordani Soika, 1956, in Vitte 1991 : 30; Dakki 1997 : 63 Vitte 1991 , Rif , AP ; Dakki 1997 Allotrichoma simplex (Loew, 1861) = Allotrichoma filiforme Becker, 1896, in Vitte 1988 : 393, 1991 : 30; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , MA , Khemisset, Oued Sebou; Dakki 1997 Elephantinosoma Becker, 1903 Elephantinosoma chnumi Becker, 1903 Gatt and Ebejer 2003 ; Kirk-Spriggs and McGregor 2009 Hecamede Haliday, 1837 Hecamede albicans (Meigen, 1830) Vitte 1991 , AP ; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Cassar et al. 2008 , Rif , Smir Lagoon; Popescu-Mirceni 2011 Lipochaetini Glenanthe Haliday, 1839 Glenanthe ripicola (Haliday, 1839) Vitte 1991 , AP ; Dakki 1997 ; Cassar et al. 2008 , Rif , Laou Basin; Zatwarnicki and Mathis 2011 , AA , Tarfaya – HNHM Homalometopus Becker, 1903 Homalometopus sp. Vitte 1991 Ochtherini Ochthera Latreille, 1802 Ochthera manicata (Fabricius, 1794) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Ochthera pilimana Becker, 1903 Dakki 1997 Ochthera schembrii Rondani, 1847 = Ochthera mantispa Loew, 1847, in Vitte 1988 : 394, 1991 : 28; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , AP ; Dakki 1997 Hydrelliinae Atissini Asmeringa Becker, 1903 Asmeringa inermis Becker, 1903 Vitte 1991 , AP , Rabat; Dakki 1997 ; Gatt and Ebejer 2003 Atissa Haliday, 1839 Atissa durrenbergensis Loew, 1864 Vitte 1991 , AP ; Dakki 1997 Atissa hepaticoloris Becker, 1903 Vitte 1991 , AP ; Dakki 1997 ; Gatt and Ebejer 2003 Atissa limosina Becker, 1896 Vitte 1991 , AP , Rabat, MA , Fès; Dakki 1997 Atissa pygmaea (Haliday, 1839) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 Ptilomyia Coquillett, 1900 Ptilomyia angustigenis (Becker, 1926) = Atissa angustigenis Becker, in Vitte 1988 : 393, 1991 : 30; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Gatt and Ebejer 2003 Dryxini Dryxo Robineau-Desvoidy, 1830 Dryxo ornata (Macquart, 1843) Mathis and Zatwarnicki 2002 , AA , Aoulouz Hydrelliini Hydrellia Robineau-Desvoidy, 1830 Hydrellia albifrons (Fallén, 1813) Vitte 1991 , AP , Rif ; Dakki 1997 Hydrellia argyrogenis Becker, 1896 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , Atlantic coast and Plains; Dakki 1997 Hydrellia armata Canzoneri & Meneghini, 1976 Vitte 1991 , Rif , Ksar el Kbir, MA , Fès; Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou Hydrellia atlas Vitte, 1989 Vitte 1989 , MA , Dayat Aoua; Dakki 1997 ; Dakki et al. 2003; Chilasse and Dakki 2004, MA Hydrellia griseola (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Wolff et al. 2016 ; Rif (Oued Laou) – MISR Hydrellia maculiventris Becker, 1896 Vitte 1991 , Rif , AP , Atlantic coast; Dakki 1997 ; Gatt and Ebejer 2003 Hydrellia maura Meigen, 1838 = Hydrellia modesta Loew, 1860, in Vitte 1988 : 392, 1991 : 29; Dakki 1997 : 63 Vitte 1988 , MA , lakes of Middle Atlas; Zatwarnicki 1988 , MA , Ifrane, HA , Vallée de l'Aït Mizane; Vitte 1991 ; Dakki 1997 Hydrellia nigricans (Stenhammar, 1844) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Hydrellia obscura (Meigen, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 ; Rif (Aïn Jdioui) – MISR Hydrellia pubescen s Becker, 1926 = Hydrellia nasturtii Collin, 1928, in Vitte 1991 : 29; Dakki 1997 : 63 Vitte 1991 , MA , Fès; Dakki 1997 ; Gatt and Ebejer 2003 Hydrellia ranunculi (Haliday, 1838) Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Hydrellia rharbia Vitte, 1991 Vitte 1989 , AP , Merja Halloufa (near Moulay Bousselham); Dakki 1997 ; Dakki and Himmi 2008 , MA , Oued Sebou Hydrellia subalbiceps Collin, 1966 Vitte 1991 , Rif , Ketama; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Notiphilini Notiphila Fallén, 1810 Notiphila ( Notiphila ) annulipes Stenhammar, 1844 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif ; Dakki 1997 Notiphila ( Notiphila ) cinerea Fallén, 1830 Séguy 1930a , MA , Meknès; Séguy 1941a , HA , Imi-n'Ouaka; Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 ; Pârvu et al. 2006 , AP , Merja Zerga; Kirk-Spriggs and McGregor 2009 ; Rif (Talassemtane, Aïn Jdioui) – MISR Notiphila ( Notiphila ) cogani Canzoneri & Meneghini, 1979 Vitte 1988 , MA , lakes of Middle Atlas; Krivosheina 1998 ; Vitte 1991 ; Dakki 1997 ; Rif (Aïn Jdioui) – MISR Notiphila ( Notiphila ) dorsata Stenhammar, 1844 Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , AP , coastal lake areas; Dakki 1997 Notiphila ( Notiphila ) maculata Stenhammar, 1844 Vitte 1991 , Rif , AP , coastal plains; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Notiphila ( Notiphila ) riparia Meigen, 1830 Krivosheina 1998 , AA , Aït Melloul (Souss); Vitte 1988 , MA , lakes of Middle Atlas and reedbeds; Vitte 1991 ; Dakki 1997 Notiphila ( Notiphila ) stagnicola (Robineau-Desvoidy, 1830) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 , Rif , AP , coastal plains; Dakki 1997 ; Pârvu et al. 2006 , AA , Lac Edehby, Ouarzazate; Popescu-Mirceni 2011 , AA , Ouarzazate Ilytheinae Hyadinini Hyadina Haliday, 1837 Hyadina guttata (Fallén, 1813) Vitte 1988 , MA , lakes of Middle Atlas; Vitte 1991 ; Dakki 1997 Hyadina pollinosa Oldenberg, 1923 Vitte 1991 , MA , Fès; Dakki 1997 Hyadina rufipes (Meigen, 1830) = Hyadina nitida (Macquart, 1835), in Vitte 1991 : 27; Dakki 1997 : 63 Vitte 1991 , AP , Moulay Bousselham; Dakki 1997 Nostima Coquillett, 1900 Nostima picta (Fallén, 1813) Vitte 1991 , AP ; Vitte 1988 , MA , lakes of Middle Atlas; Dakki 1997 Pelina Haliday, 1837 Pelina aenea (Fallén, 1813) Vitte 1991 , Rif , Ouezzane; Dakki 1997 Pelina subpunctata Becker, 1896 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Philygria Stenhammar, 1844 Philygria posticata (Meigen, 1830) Ebejer et al. 2019 , MA , Khénifra (17 km SW of Midelt, 1940 m) Acknowledgements We gratefully acknowledge the assistance and cooperation of Martin J. Ebejer who contributed to the revision of this family. Hippoboscoidea HIPPOBOSCIDAE K. Kettani, B. Droz Number of species: 17 . Expected: 25 Faunistic knowledge of the family in Morocco: moderate Hippoboscinae Hippoboscini Crataerina von Olfers, 1816 Crataerina acutipennis Austen, 1926 Mouna 1998 : 85 Crataerina pallida (Latreille, 1811) Maa 1969 ; Mouna 1998 ; AA (Tiznit) – MISR Hippobosca Linnaeus, 1758 Hippobosca camelina Leach, 1817 = Hippobosca dromedarina Speiser, in Séguy 1930a : 184 Séguy 1930a ; Bequaert 1939 , AP , Rabat, EM , Taourit, HA ; Séguy 1953a , AA , Zegdou, SA , Oued Agouidir; Mouna 1998 ; EM (Oued el Maa) – MISR Hippobosca equina (Linnaeus, 1758) Séguy 1930a , AP , Rabat, Settat, Mogador; Séguy 1953a , AA , Inzegane; Maa 1969 ; Bequaert 1939 , MA , Aguelmane, HA , Ijoukak – MISR Hippobosca fulva Austen, 1912 Bequaert 1939 , EM , Taourirt (Ebner), Tendrara (Ebner); Maa 1963 Hippobosca longipennis Fabricius, 1805 = Hippobosca capensis Olfers, in Bequaert 1939 : 78 Séguy 1930a , MA , Meknès; Bequaert 1939 , HA , Marrakech; Maa 1963 , 1969 ; Mouna 1998 ; AA (Agdz) – MISR Hippobosca variegata Megerle, 1803 = Hippobosca maculata Leach, in Séguy 1930a : 184 Séguy 1930a ; Moussiaux and Desmecht 2008 , HA (south) Icosta Speiser, 1905 Icosta minor (Bigot, 1858) = Lynchia minor Bigot, in Mouna 1998 : 85 Báez 1978 ; Mouna 1998 Ornithoica Rondani, 1878 Ornithoica turdi (Olivier in Latreille, 1811) Maa 1966 , EM , Figuig; Maa 1969 ; Mouna 1998 ; Droz and Haenni 2011 Ornithomyia Latreille, 1802 Ornithomyia avicularia (Linnaeus, 1758) Séguy 1930a ; Mouna 1998 Ornithomyia fringillina (Curtis, 1836) Séguy 1930a , MA , Meknès; Mouna 1998 Ornithophila Rondani, 1879 Ornithophila gestroi (Rondani, 1878) = Ornitheza gestroi Rondani, in Mouna 1998 : 85 Mouna 1998 ; Pape and Thompson 2019 Ornithophila metallica (Schiner, 1864) = Ornitheza metallica Schiner Mouna 1998 : 85 Pseudolynchia Bequaert, 1926 Pseudolynchia canariensis (Macquart, 1839) = Pseudolynchia maura Bigot, in Séguy 1930a : 184 Séguy 1930a ; Mouna 1998 Stenepteryx Leach, 1817 Stenepteryx hirundinis (Linnaeus, 1758) = Crataerina hirundinis Linnaeus, in Mouna 1998 : 85 Summer 1978 , MA , Midelt; Mouna 1998 Lipopteninae Lipoptena Nitzsch, 1818 Lipoptena capreoli Rondani, 1878 Mouna 1998 : 85 Melophagus Latreille, 1802 Melophagus ovinus (Linnaeus, 1758) = Melanophagus ovinus Linnaeus, in Mouna 1998 : 85 Séguy 1930a ; Maa 1969 ; Mouna 1998 NYCTERIBIIDAE K. Kettani, G. Graciolli Number of species: 8 . Expected: 18 Faunistic knowledge of the family in Morocco: poor Nycteribiinae Basilia Miranda-Ribeiro, 1903 Basilia italica Theodor, 1954 Aellen 1955 Nycteribia Latreille, 1796 Nycteribia ( Acrocholidia ) vexata Westwood, 1835 Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (hosts: Myotis oxygnathus Monticelli, 1885, Miniopterus shreibersii (Kuhl, 1817) and Rhinolophus mehelyi (Matschie, 1901)), MA , Grotte de Ras el Oued (host: Myotis oxygnathus and Miniopterus shreibersii ; Aellen 1963 ; Theodor 1967 , MA , Oued Mellah; Mouna 1998 Nycteribia ( Nycteribia ) latreillei (Leach, 1817) Séguy 1930a , Rif , Samsa (Tétouan); Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (host: Miniopterus shreibersii (Kuhl, 1817)), MA , Grotte de Ras el Oued (hosts: Myotis oxygnathus Monticelli, 1885 and Miniopterus shreibersii ); Aellen 1963 ; Theodor 1967 , AP , Mazagan (host: Myotis myotis (Bourhausen, 1797)); Mouna 1998 Nycteribia ( Nycteribia ) pedicularia Latreille, 1805 = Listropodia pedicularia Latreille, in Séguy 1930a : 185 Falcoz 1924 , Rif , Caverne d'Hercule; Séguy 1930a , Rif , Caverne d'Hercule; Mouna 1998 Nycteribia ( Nycteribia ) schmidtlii Schiner, 1853 = Listropodia schmidli Schiner, in Séguy 1930a : 186 Falcoz 1924 , Rif , Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne de Samsa, SA ; Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (undetermined bat), MA , Grotte de Ras el Ma (host: Rhinolophus ferrumequinum (Schreber, 1774)), Grotte de Ras el Oued (hosts: Miniopterus shreibersii (Kuhl, 1817) and Myotis oxygnathus Monticelli, 1885); Aellen 1963 ; Theodor 1967 (host: Rhinolophus ferrumequinum ); Mouna 1998 Penicillidia Kolenati, 1963 Penicillidia ( Penicillidia ) conspicua Speiser, 1901 Falcoz 1924 , Rif , Caverne d'Hercule, Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne d'Hercule, Caverne de Samsa; Aellen 1952 ; Aellen 1955 , MA , Grotte de Ras el Oued, AP , Grotte de Sidi Bou Knadel; Aellen 1963 ; Mouna 1998 ; Koçak and Kemal 2010 Penicillidia ( Penicillidia ) dufouri (Westwood, 1835) Falcoz 1924 , Rif , Caverne d'Hercule; Séguy 1930a , Rif , Caverne d'Hercule; Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel, MA , Grotte de Ras el Oued, AA , Oulad Teima; Aellen 1963 ; Theodor 1967 , AP , Mazagan ( Myotis myotis (Bourhausen, 1797)); Mouna 1998 ; Koçak and Kemal 2010 Phthiridium Hermann, 1804 Phthiridium biarticulatum Hermann, 1804 = Stylidia biarticulata Herman, in Falcoz 1924 : 310; Séguy 1930a : 185 Falcoz 1924 , Rif , Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne de Samsa; Aellen 1952 ; Aellen 1955 , MA , Grotte de Ras el Ma (host: Rhinolophus ferrumequinum (Schreber, 1774)), AA , Oulad Teima (host: Rhinolophus ferrumequinum ); Aellen 1963 ; Theodor 1967 (host: Rhinolophus ferrumequinum ); Mouna 1998 STREBLIDAE K. Kettani, G. Graciolli Number of species: 2 . Expected: 7 Faunistic knowledge of the family in Morocco: poor Brachytarsininae Brachytarsina Macquart, 1851 Brachytarsina flavipennis Macquart, 1851 = Nycteribosca kollari Frauenfeld, in Falcóz 1924: 226; Aellen 1955 : 100 Falcóz 1924, Rif , caverne d'Hercule, caverne de Samsa, près Tétouan (host: Rhinolophus ferrumequinum (Schreber, 1774); Séguy 1930a , Rif , caverne d'Hercule, caverne de Samsa; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (hosts: Rhinolophus mehelyi (Matschie, 1901) and Myotis oxygnathus Monticelli, 1885); Mouna 1998 ; Koçak and Kemal 2010 Raymondia Frauenfeld, 1855 Raymondia huberi Frauenfeld, 1855 = Raymondia setosa Jobling, 1930 Beaucournu et al. 1985 , AA , Assa (Bas Draa) (host: Asellia tridens (E. Geoffroy, 1813)) HIPPOBOSCIDAE K. Kettani, B. Droz Number of species: 17 . Expected: 25 Faunistic knowledge of the family in Morocco: moderate Hippoboscinae Hippoboscini Crataerina von Olfers, 1816 Crataerina acutipennis Austen, 1926 Mouna 1998 : 85 Crataerina pallida (Latreille, 1811) Maa 1969 ; Mouna 1998 ; AA (Tiznit) – MISR Hippobosca Linnaeus, 1758 Hippobosca camelina Leach, 1817 = Hippobosca dromedarina Speiser, in Séguy 1930a : 184 Séguy 1930a ; Bequaert 1939 , AP , Rabat, EM , Taourit, HA ; Séguy 1953a , AA , Zegdou, SA , Oued Agouidir; Mouna 1998 ; EM (Oued el Maa) – MISR Hippobosca equina (Linnaeus, 1758) Séguy 1930a , AP , Rabat, Settat, Mogador; Séguy 1953a , AA , Inzegane; Maa 1969 ; Bequaert 1939 , MA , Aguelmane, HA , Ijoukak – MISR Hippobosca fulva Austen, 1912 Bequaert 1939 , EM , Taourirt (Ebner), Tendrara (Ebner); Maa 1963 Hippobosca longipennis Fabricius, 1805 = Hippobosca capensis Olfers, in Bequaert 1939 : 78 Séguy 1930a , MA , Meknès; Bequaert 1939 , HA , Marrakech; Maa 1963 , 1969 ; Mouna 1998 ; AA (Agdz) – MISR Hippobosca variegata Megerle, 1803 = Hippobosca maculata Leach, in Séguy 1930a : 184 Séguy 1930a ; Moussiaux and Desmecht 2008 , HA (south) Icosta Speiser, 1905 Icosta minor (Bigot, 1858) = Lynchia minor Bigot, in Mouna 1998 : 85 Báez 1978 ; Mouna 1998 Ornithoica Rondani, 1878 Ornithoica turdi (Olivier in Latreille, 1811) Maa 1966 , EM , Figuig; Maa 1969 ; Mouna 1998 ; Droz and Haenni 2011 Ornithomyia Latreille, 1802 Ornithomyia avicularia (Linnaeus, 1758) Séguy 1930a ; Mouna 1998 Ornithomyia fringillina (Curtis, 1836) Séguy 1930a , MA , Meknès; Mouna 1998 Ornithophila Rondani, 1879 Ornithophila gestroi (Rondani, 1878) = Ornitheza gestroi Rondani, in Mouna 1998 : 85 Mouna 1998 ; Pape and Thompson 2019 Ornithophila metallica (Schiner, 1864) = Ornitheza metallica Schiner Mouna 1998 : 85 Pseudolynchia Bequaert, 1926 Pseudolynchia canariensis (Macquart, 1839) = Pseudolynchia maura Bigot, in Séguy 1930a : 184 Séguy 1930a ; Mouna 1998 Stenepteryx Leach, 1817 Stenepteryx hirundinis (Linnaeus, 1758) = Crataerina hirundinis Linnaeus, in Mouna 1998 : 85 Summer 1978 , MA , Midelt; Mouna 1998 Lipopteninae Lipoptena Nitzsch, 1818 Lipoptena capreoli Rondani, 1878 Mouna 1998 : 85 Melophagus Latreille, 1802 Melophagus ovinus (Linnaeus, 1758) = Melanophagus ovinus Linnaeus, in Mouna 1998 : 85 Séguy 1930a ; Maa 1969 ; Mouna 1998 Hippoboscinae Hippoboscini Crataerina von Olfers, 1816 Crataerina acutipennis Austen, 1926 Mouna 1998 : 85 Crataerina pallida (Latreille, 1811) Maa 1969 ; Mouna 1998 ; AA (Tiznit) – MISR Hippobosca Linnaeus, 1758 Hippobosca camelina Leach, 1817 = Hippobosca dromedarina Speiser, in Séguy 1930a : 184 Séguy 1930a ; Bequaert 1939 , AP , Rabat, EM , Taourit, HA ; Séguy 1953a , AA , Zegdou, SA , Oued Agouidir; Mouna 1998 ; EM (Oued el Maa) – MISR Hippobosca equina (Linnaeus, 1758) Séguy 1930a , AP , Rabat, Settat, Mogador; Séguy 1953a , AA , Inzegane; Maa 1969 ; Bequaert 1939 , MA , Aguelmane, HA , Ijoukak – MISR Hippobosca fulva Austen, 1912 Bequaert 1939 , EM , Taourirt (Ebner), Tendrara (Ebner); Maa 1963 Hippobosca longipennis Fabricius, 1805 = Hippobosca capensis Olfers, in Bequaert 1939 : 78 Séguy 1930a , MA , Meknès; Bequaert 1939 , HA , Marrakech; Maa 1963 , 1969 ; Mouna 1998 ; AA (Agdz) – MISR Hippobosca variegata Megerle, 1803 = Hippobosca maculata Leach, in Séguy 1930a : 184 Séguy 1930a ; Moussiaux and Desmecht 2008 , HA (south) Icosta Speiser, 1905 Icosta minor (Bigot, 1858) = Lynchia minor Bigot, in Mouna 1998 : 85 Báez 1978 ; Mouna 1998 Ornithoica Rondani, 1878 Ornithoica turdi (Olivier in Latreille, 1811) Maa 1966 , EM , Figuig; Maa 1969 ; Mouna 1998 ; Droz and Haenni 2011 Ornithomyia Latreille, 1802 Ornithomyia avicularia (Linnaeus, 1758) Séguy 1930a ; Mouna 1998 Ornithomyia fringillina (Curtis, 1836) Séguy 1930a , MA , Meknès; Mouna 1998 Ornithophila Rondani, 1879 Ornithophila gestroi (Rondani, 1878) = Ornitheza gestroi Rondani, in Mouna 1998 : 85 Mouna 1998 ; Pape and Thompson 2019 Ornithophila metallica (Schiner, 1864) = Ornitheza metallica Schiner Mouna 1998 : 85 Pseudolynchia Bequaert, 1926 Pseudolynchia canariensis (Macquart, 1839) = Pseudolynchia maura Bigot, in Séguy 1930a : 184 Séguy 1930a ; Mouna 1998 Stenepteryx Leach, 1817 Stenepteryx hirundinis (Linnaeus, 1758) = Crataerina hirundinis Linnaeus, in Mouna 1998 : 85 Summer 1978 , MA , Midelt; Mouna 1998 Lipopteninae Lipoptena Nitzsch, 1818 Lipoptena capreoli Rondani, 1878 Mouna 1998 : 85 Melophagus Latreille, 1802 Melophagus ovinus (Linnaeus, 1758) = Melanophagus ovinus Linnaeus, in Mouna 1998 : 85 Séguy 1930a ; Maa 1969 ; Mouna 1998 NYCTERIBIIDAE K. Kettani, G. Graciolli Number of species: 8 . Expected: 18 Faunistic knowledge of the family in Morocco: poor Nycteribiinae Basilia Miranda-Ribeiro, 1903 Basilia italica Theodor, 1954 Aellen 1955 Nycteribia Latreille, 1796 Nycteribia ( Acrocholidia ) vexata Westwood, 1835 Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (hosts: Myotis oxygnathus Monticelli, 1885, Miniopterus shreibersii (Kuhl, 1817) and Rhinolophus mehelyi (Matschie, 1901)), MA , Grotte de Ras el Oued (host: Myotis oxygnathus and Miniopterus shreibersii ; Aellen 1963 ; Theodor 1967 , MA , Oued Mellah; Mouna 1998 Nycteribia ( Nycteribia ) latreillei (Leach, 1817) Séguy 1930a , Rif , Samsa (Tétouan); Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (host: Miniopterus shreibersii (Kuhl, 1817)), MA , Grotte de Ras el Oued (hosts: Myotis oxygnathus Monticelli, 1885 and Miniopterus shreibersii ); Aellen 1963 ; Theodor 1967 , AP , Mazagan (host: Myotis myotis (Bourhausen, 1797)); Mouna 1998 Nycteribia ( Nycteribia ) pedicularia Latreille, 1805 = Listropodia pedicularia Latreille, in Séguy 1930a : 185 Falcoz 1924 , Rif , Caverne d'Hercule; Séguy 1930a , Rif , Caverne d'Hercule; Mouna 1998 Nycteribia ( Nycteribia ) schmidtlii Schiner, 1853 = Listropodia schmidli Schiner, in Séguy 1930a : 186 Falcoz 1924 , Rif , Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne de Samsa, SA ; Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (undetermined bat), MA , Grotte de Ras el Ma (host: Rhinolophus ferrumequinum (Schreber, 1774)), Grotte de Ras el Oued (hosts: Miniopterus shreibersii (Kuhl, 1817) and Myotis oxygnathus Monticelli, 1885); Aellen 1963 ; Theodor 1967 (host: Rhinolophus ferrumequinum ); Mouna 1998 Penicillidia Kolenati, 1963 Penicillidia ( Penicillidia ) conspicua Speiser, 1901 Falcoz 1924 , Rif , Caverne d'Hercule, Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne d'Hercule, Caverne de Samsa; Aellen 1952 ; Aellen 1955 , MA , Grotte de Ras el Oued, AP , Grotte de Sidi Bou Knadel; Aellen 1963 ; Mouna 1998 ; Koçak and Kemal 2010 Penicillidia ( Penicillidia ) dufouri (Westwood, 1835) Falcoz 1924 , Rif , Caverne d'Hercule; Séguy 1930a , Rif , Caverne d'Hercule; Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel, MA , Grotte de Ras el Oued, AA , Oulad Teima; Aellen 1963 ; Theodor 1967 , AP , Mazagan ( Myotis myotis (Bourhausen, 1797)); Mouna 1998 ; Koçak and Kemal 2010 Phthiridium Hermann, 1804 Phthiridium biarticulatum Hermann, 1804 = Stylidia biarticulata Herman, in Falcoz 1924 : 310; Séguy 1930a : 185 Falcoz 1924 , Rif , Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne de Samsa; Aellen 1952 ; Aellen 1955 , MA , Grotte de Ras el Ma (host: Rhinolophus ferrumequinum (Schreber, 1774)), AA , Oulad Teima (host: Rhinolophus ferrumequinum ); Aellen 1963 ; Theodor 1967 (host: Rhinolophus ferrumequinum ); Mouna 1998 Nycteribiinae Basilia Miranda-Ribeiro, 1903 Basilia italica Theodor, 1954 Aellen 1955 Nycteribia Latreille, 1796 Nycteribia ( Acrocholidia ) vexata Westwood, 1835 Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (hosts: Myotis oxygnathus Monticelli, 1885, Miniopterus shreibersii (Kuhl, 1817) and Rhinolophus mehelyi (Matschie, 1901)), MA , Grotte de Ras el Oued (host: Myotis oxygnathus and Miniopterus shreibersii ; Aellen 1963 ; Theodor 1967 , MA , Oued Mellah; Mouna 1998 Nycteribia ( Nycteribia ) latreillei (Leach, 1817) Séguy 1930a , Rif , Samsa (Tétouan); Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (host: Miniopterus shreibersii (Kuhl, 1817)), MA , Grotte de Ras el Oued (hosts: Myotis oxygnathus Monticelli, 1885 and Miniopterus shreibersii ); Aellen 1963 ; Theodor 1967 , AP , Mazagan (host: Myotis myotis (Bourhausen, 1797)); Mouna 1998 Nycteribia ( Nycteribia ) pedicularia Latreille, 1805 = Listropodia pedicularia Latreille, in Séguy 1930a : 185 Falcoz 1924 , Rif , Caverne d'Hercule; Séguy 1930a , Rif , Caverne d'Hercule; Mouna 1998 Nycteribia ( Nycteribia ) schmidtlii Schiner, 1853 = Listropodia schmidli Schiner, in Séguy 1930a : 186 Falcoz 1924 , Rif , Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne de Samsa, SA ; Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (undetermined bat), MA , Grotte de Ras el Ma (host: Rhinolophus ferrumequinum (Schreber, 1774)), Grotte de Ras el Oued (hosts: Miniopterus shreibersii (Kuhl, 1817) and Myotis oxygnathus Monticelli, 1885); Aellen 1963 ; Theodor 1967 (host: Rhinolophus ferrumequinum ); Mouna 1998 Penicillidia Kolenati, 1963 Penicillidia ( Penicillidia ) conspicua Speiser, 1901 Falcoz 1924 , Rif , Caverne d'Hercule, Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne d'Hercule, Caverne de Samsa; Aellen 1952 ; Aellen 1955 , MA , Grotte de Ras el Oued, AP , Grotte de Sidi Bou Knadel; Aellen 1963 ; Mouna 1998 ; Koçak and Kemal 2010 Penicillidia ( Penicillidia ) dufouri (Westwood, 1835) Falcoz 1924 , Rif , Caverne d'Hercule; Séguy 1930a , Rif , Caverne d'Hercule; Aellen 1952 ; Aellen 1955 , AP , Grotte de Sidi Bou Knadel, MA , Grotte de Ras el Oued, AA , Oulad Teima; Aellen 1963 ; Theodor 1967 , AP , Mazagan ( Myotis myotis (Bourhausen, 1797)); Mouna 1998 ; Koçak and Kemal 2010 Phthiridium Hermann, 1804 Phthiridium biarticulatum Hermann, 1804 = Stylidia biarticulata Herman, in Falcoz 1924 : 310; Séguy 1930a : 185 Falcoz 1924 , Rif , Caverne de Samsa (near Tétouan); Séguy 1930a , Rif , Caverne de Samsa; Aellen 1952 ; Aellen 1955 , MA , Grotte de Ras el Ma (host: Rhinolophus ferrumequinum (Schreber, 1774)), AA , Oulad Teima (host: Rhinolophus ferrumequinum ); Aellen 1963 ; Theodor 1967 (host: Rhinolophus ferrumequinum ); Mouna 1998 STREBLIDAE K. Kettani, G. Graciolli Number of species: 2 . Expected: 7 Faunistic knowledge of the family in Morocco: poor Brachytarsininae Brachytarsina Macquart, 1851 Brachytarsina flavipennis Macquart, 1851 = Nycteribosca kollari Frauenfeld, in Falcóz 1924: 226; Aellen 1955 : 100 Falcóz 1924, Rif , caverne d'Hercule, caverne de Samsa, près Tétouan (host: Rhinolophus ferrumequinum (Schreber, 1774); Séguy 1930a , Rif , caverne d'Hercule, caverne de Samsa; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (hosts: Rhinolophus mehelyi (Matschie, 1901) and Myotis oxygnathus Monticelli, 1885); Mouna 1998 ; Koçak and Kemal 2010 Raymondia Frauenfeld, 1855 Raymondia huberi Frauenfeld, 1855 = Raymondia setosa Jobling, 1930 Beaucournu et al. 1985 , AA , Assa (Bas Draa) (host: Asellia tridens (E. Geoffroy, 1813)) Brachytarsininae Brachytarsina Macquart, 1851 Brachytarsina flavipennis Macquart, 1851 = Nycteribosca kollari Frauenfeld, in Falcóz 1924: 226; Aellen 1955 : 100 Falcóz 1924, Rif , caverne d'Hercule, caverne de Samsa, près Tétouan (host: Rhinolophus ferrumequinum (Schreber, 1774); Séguy 1930a , Rif , caverne d'Hercule, caverne de Samsa; Aellen 1955 , AP , Grotte de Sidi Bou Knadel (hosts: Rhinolophus mehelyi (Matschie, 1901) and Myotis oxygnathus Monticelli, 1885); Mouna 1998 ; Koçak and Kemal 2010 Raymondia Frauenfeld, 1855 Raymondia huberi Frauenfeld, 1855 = Raymondia setosa Jobling, 1930 Beaucournu et al. 1985 , AA , Assa (Bas Draa) (host: Asellia tridens (E. Geoffroy, 1813)) Muscoidea ANTHOMYIIDAE K. Kettani, D.M. Ackland Number of species: 36 . Expected: Many more, especially in the mountains Faunistic knowledge of the family in Morocco: poor Anthomyiinae Adia Robineau-Desvoidy, 1830 Adia cinerella (Fallén, 1825) = Chortophila cinerella Fallén, in Séguy 1930a : 162 = Hylemyia cinerella Fallén, in Séguy 1941d : 18 Séguy 1941d , AA , Agadir; Séguy 1930a ; Mouna 1998 ; AP (Rabat), HA (Marrakech), AA (Tifnit (south of Agadir)) – MISR Anthomyia Meigen, 1803 Anthomyia imbrida Rondani, 1866 Séguy 1930a , MA , Meknès; Mouna 1998 Anthomyia liturata (Robineau-Desvoidy, 1830) = Hylemyia pullula Zetterstedt, in Séguy 1930a : 162 Séguy 1930a , MA , Ras el Ksar (1900 m), Tameghilt (1700–1800 m), Forêt Tiffert (2000–2200 m); Mouna 1998 Anthomyia quinquemaculata Macquart, 1839 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), MA , 3.5 km S of Azrou (1450 m) Anthomyia pluvialis (Linnaeus, 1758) Séguy 1929b ; Séguy 1930a , MA , Meknès; Michelsen 1980 , AP , Aïn Diab; Mouna 1998 ; Ackland 1987 , 2001 ; Pârvu et al. 2006 , AA , Foum Zguid (Tata); Popescu-Mirceni 2011 – MISR Anthomyia procellaris Rondani, 1866 Séguy 1929b ; Séguy 1930a , MA , Meknès, Berkane (1350–1400 m), Tlet n'Rhohr; Mouna 1998 Anthomyia tempestatum Wiedemann, 1830 Michelsen and Báez 1985 , HA ; Ackland 2001 ; Grabener 2017 Botanophila Lioy, 1864 Botanophila dissecta (Meigen, 1826) Mouna 1998 ; MA (Meknès) – MISR Botanophila varicolor (Meigen, 1826) Ebejer et al. 2019 , MA , Lac Aguelmane Sidi Ali (2052 m) Delia Robineau-Desvoidy, 1830 Delia antiqua (Meigen, 1826) Mouna 1998 Delia coarctata (Fallén, 1825) Mouna 1998 Delia flavibasis (Stein, 1903) = Hylemyia hordeacea Séguy, in Séguy 1941d : 18 Séguy 1934a , AP , Casablanca; Séguy 1936b , AP , Rabat; Séguy 1941d , AA , Taroudant; Mouna 1998 ; Ackland 2008 Delia flavogrisea (Ringdahl, 1926) 52 Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 Delia planipalpis (Stein, 1898) = Chortophila pilipyga Villeneuve, in Mouna 1998 : 85 Mouna 1998 ; AP (Mazagan) – MISR Delia platura (Meigen, 1826) = Chortophila cilicrura Rondani, in Séguy 1930a : 162 Séguy 1949a , SA , Goulimine; Mouna 1998 ; Singh et al. 2014 ; Grabener 2017 Delia radicum Linnaeus, 1758 = Chortophila brassicae Bouché, in Séguy 1934b : 162, Mouna 1998 : 85 Séguy 1934b , AP , Rabat; Mouna 1998 ; Biron et al. 2000 , AP , Rabat; Andreassen 2007 – MISR Fucellia Robineau-Desvoidy, 1842 Fucellia maritima (Haliday, 1838) Séguy 1930a , Rif , Agla near Cap Spartel (on Fucus ); Cassar et al. 2008 , Rif , Smir lagoon; Mouna 1998 Hylemya Robineau-Desvoidy, 1830 Hylemya vagans (Panzer, 1798) Mouna 1998 – MISR Leucophora Robineau-Desvoidy, 1830 Leucophora cinerea Robineau-Desvoidy, 1830 Ebejer et al. 2019 , MA , 17 km SW of Midelt (Khénifra, 1940 m) Leucophora dissimilis (Villeneuve, 1920) Ebejer et al. 2019 , MA , 17 km NW of Zaida (Khénifra, 1878 m) Paregle Schnabl, 1911 Paregle audacula (Harris, 1780) Pârvu and Zaharia 2007 Paregle pilipes (Stein, 1916) Mouna 1998 Phorbia Robineau-Desvoidy, 1830 Phorbia fumigata (Meigen, 1826) = Phorbia securis Tiensuu, in Maarouf et al. 1996 : 17 Balachowsky and Mesnil 1935 ; Bleuton 1938; Jourdan 1938 ; Maarouf and Chemseddine 1995 , AP , Chaouia, Doukkala, Abda; Maarouf and Chemseddine 1995 , HA , Chaouia, Doukkala, Abda; Maarouf et al. 1996 , AP , Safi, Settat, Sidi El Aydi, Jemaa Riah, Berrechid, Médiouna, Mohammédia, Bouznika, Skhirat, Kénitra, Sidi Allal Tazi, Souk Larbaa du Gharb, MA , Khernisset, Meknès, Douiyat, Fès, Sefrou, Annaceur, Oulad Saïd, El Aounate, Sidi Bennbur, Zemarnra, Chemmaïa, Jemaa, des Shaïm, Tlet Sidi Bouguedra, Khemisset Chaouïa, Béni Mellal, HA , Skhour Rehamna, Ben Guérir, Marrakech, Tamellalet, Kelaâ des Sraghna, Oulad Ayad, Afourér, Azilal, El Borouj, Guisser; Lhaloui et al. 1998 Phorbia sepia (Meigen, 1826) Bleuton 1938, MA , Fès, Meknès, Taza; Jourdan 1938 ; Mouna 1998 Subhylemyia Ringdahl, 1933 Subhylemyia longula (Fallén, 1824) Mouna 1998 ; AP (Cap Cantin) – MISR Pegomyinae Calythea Schnabl in Schnabl and Dziedzicki 1911 Calythea nigricans (Robineau-Desvoidy, 1830) = Calythea albicincta Fallén, in Séguy 1930a : 161 Séguy 1930a , MA , Meknès, Aïn Leuh; Mouna 1998 Mycophaga Rondani, 1856 Mycophaga testacea (Gimmerthal, 1834) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Pegomya Robineau-Desvoidy, 1830 Pegomya betae (Curtis, 1847) Rungs 1962 , AP ; Mouna 1998 Pegomya bicolor (Wiedemann, 1817) Séguy 1934b , AP , Rabat; Koçak and Kemal 2010 ; Pitkin et al. 2011 Pegomya hyoscyami (Panzer, 1809) Rungs 1962 , AP ; Mouna 1998 ; AP (Rabat) – MISR Pegomya rufina (Fallén, 1825) Mouna 1998 Pegomya testacea (De Geer, 1776) = Pegomya silacea Meigen, in Mouna 1998 : 85 Séguy 1930a , MA , Forêt Tiffert (2000–2200 m); Mouna 1998 Pegomya solennis (Meigen, 1826) = Pegomyia nigritarsis Fallén, in Séguy 1935a : 119 Séguy 1935a , AA , Oued Draa (Taffagount); Rungs 1962 ; Mouna 1998 Pegomya terminalis Rondani, 1866 Ebejer et al. 2019 , Rif , Adrou (556 m), Jebel Lakraâ (Talassemtane, 1541 m) Pegomya winthemi (Meigen, 1826) Mouna 1998 Pegoplata Schnabl & Dziedzicki, 1911 Pegoplata annulata (Pandellé, 1899) = Pegoplata virginea auctt, not Meigen AP (Rabat) – MISR FANNIIDAE K. Kettani, A.C. Pont Number of species: 10 . Expected: 17 Faunistic knowledge of the family in Morocco: poor Fannia Robineau-Desvoidy, 1830 Fannia canicularis (Linnaeus, 1761) Becker and Stein 1913 , Rif , Tanger; Charrier 1927 ; Séguy 1930a , HA , Tenfecht; Séguy 1932a ; Séguy 1941a ; Séguy 1941d , HA ; Séguy 1953a , AP , Rabat; Pont 1986a ; Mouna 1998 ; Pont pers. comm., MA , Azrou; AP (Dradek) – MISR Fannia cothurnata (Loew, 1873) 53 Mouna 1998 Fannia krimensis Ringdahl, 1934 Pont 1983 , HA , Jebel Ayachi; Pont 1986a ; Mouna 1998 Fannia lepida (Wiedemann, 1817) Pont pers. comm. Fannia leucosticta (Meigen, 1838) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA ; Pont 1986a ; Mouna 1998 Fannia monilis (Haliday, 1838) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), Dardara (484 m) Fannia norvegica Ringdahl, 1934 Pont 1986a ; Pont pers. comm., HA , Jebel Ayachi Fannia pallidibasis Pont, 1983 Pont 1983 , HA , Jebel Ayachi; Mouna 1998 Fannia scalaris (Fabricius, 1794) Charrier 1927 , Rif , Tanger; Séguy 1930a ; Séguy 1941a , HA ; Séguy 1953a , AP , Rabat; Pont 1986a ; Mouna 1998 ; Rif (Tanger): Caverne d'Hercule (Pont pers. comm.) – MHNP Fannia sociella (Zetterstedt, 1845) 54 Mouna 1998 MUSCIDAE K. Kettani, A.C. Pont Number of species: 115 . Expected: 140 Faunistic knowledge of the family in Morocco: moderate Atherigoninae Atherigona Rondani, 1856 Atherigona humeralis (Wiedemann, 1830) Pont pers. comm., AP , Casablanca Atherigona pulla (Wiedemann, 1830) Pont 1986b ; Pont pers. comm., AP , Larache, Rabat, HA , Asni, AA , Agadir, Taroudant Atherigona soccata Rondani, 1871 = Atherigona varia (Meigen, 1826) (misidentifications of authors) in Séguy 1930a : 159 Bléton and Fieuzet 1943 , MA , Fès; Séguy 1953a , AP , Rabat, Sidi Yahia du Gharb; Mouna 1998 ; Pont 1986b Atherigona varia (Meigen, 1826) = Atherigona quadripunctata Rossi, in Séguy 1949a : 159 Séguy 1930a , Rif , Tanger; Séguy 1941d ; Séguy 1949a , AA , Akka, Alnif; Bléton and Fieuzet 1943 , MA , Meknès, Gharb, Fouarat; Séguy 1953a , AP , Sidi Yahia du Gharb, Rabat; Pont 1986b ; Mouna 1998 ; Pont pers. comm., Rif , Meloussa, Tanger, AP , Larache, Rabat, HA , Jebel Ayachi, AA , Akka Atherigona ( Acritochaeta ) yorki Deeming, 1971 Pont 1986b , 1991a ; Dike 1990 ; Pont pers. comm., AP , Rabat Azeliinae Azeliini Azelia Robineau-Desvoidy, 1830 Azelia parva Rondani, 1866 Michelsen pers. comm., Rif , Ouezzane Hydrotaea Robineau-Desvoidy, 1830 Hydrotaea aenescens (Wiedemann, 1830) Morocco, first record 1989; Pont et al. 2007 Hydrotaea armipes (Fallén, 1825) Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi Hydrotaea capensis (Wiedemann, 1818) = Ophyra anthrax (Meigen, 1826), in Séguy 1941d : 20 Séguy 1934b , AP , Chellah; Séguy 1941d , AA , Agadir; Pont 1986b ; Hernández-Moreno and Peris 1989 , Rif , Tanger; Mouna 1998 ; Turchetto et al. 2003 ; Pont pers. comm., HA , Jebel Ayachi; Rif (Environ de Tanger (Pont pers. comm.)) – MHNP Hydrotaea cinerea Robineau-Desvoidy, 1830 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea dentipes (Fabricius, 1805) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea floccosa Macquart, 1835 = Hydrotaea armipes (Fallén, 1825) (misidentifications of authors) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger; Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi Hydrotaea glabricula (Fallén, 1825) Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora Hydrotaea ignava (Harris, 1780) = Ophyra leucostoma (Wiedemann, 1817), in Séguy 1953a : 87 Séguy 1953a , HA , Tadla; Pont 1986b ; Hernández-Moreno and Peris 1989 , Rif , Tanger; Pont pers. comm., AP , Casablanca Hydrotaea pellucens Portschinsky, 1879 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea tuberculata Rondani, 1866 Michelsen pers. comm., AP , Rabat Hydrotaea velutina Robineau-Desvoidy, 1830 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Thricops Rondani, 1856 Thricops simplex (Wiedemann, 1817) Pont 1986b , 1991b ; Pont pers. comm., HA , Jebel Ayachi Reinwardtiini Muscina Robineau-Desvoidy, 1830 Muscina levida (Harris, 1780) = Muscina assimilis (Fallén, 1823) Mouna 1998 – MHNP (no locality, on Boletus nigrescens (Pont pers. comm.)); AP (Rabat) – MISR Muscina prolapsa (Harris, 1780) = Muscina pabulorum (Fallén, 1817) Mouna 1998 ; Michelsen pers. comm., AP , Rabat, Larache; AP (Sidi Yahia) – MISR Muscina stabulans (Fallén, 1817) Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat, Mogador, MA , Meknès, HA , Aguerd El Had, Souss; Séguy 1932, HA , Taroudant; Séguy 1934b , AP , Rabat; Séguy 1953a , AP , Rabat, Salé, Forêt Maâmora, Salé; Pont 1986b ; Mouna 1998 ; Pont pers. comm., AP , Casablanca, MA , El Kebab, HA , Jebel Ayachi; AP (Casablanca, Rabat (Pont pers. comm.)) – MHNP; MISR Coenosiinae Coenosiini Coenosia Meigen, 1826 Coenosia antennata (Zetterstedt, 1849) Michelsen pers. comm., AP , Larache Coenosia atra Meigen, 1830 Pont 1986b ; Barták et al. 2004 ; Barták and Kubik 2005 ; Pont pers. comm., HA , Jebel Ayachi Coenosia attenuata Stein, 1903 Pont 1986b ; Pont pers. comm., EM , Figuig Coenosia humilis Meigen, 1826 Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Imlil, Asni, AA , Agadir Coenosia mixta Schnabl, 1911 Pont 1986b Coenosia nevadensis Lyneborg, 1970 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Coenosia pedella (Fallén, 1825) = Coenosia decipiens Meigen 1826 (certainly a misidentification) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger Coenosia praetexta Pandellé, 1899 Michelsen pers. comm., AP , Larache, Rabat Coenosia pumila (Fallén, 1825) 55 Pont 1986b ; Mouna 1998 ; Gregor et al. 2002 Coenosia testacea (Robineau-Desvoidy, 1830) Pont 1986b ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi Coenosia tigrina (Fabricius, 1775) Séguy 1930a , AP , Rabat, MA , Meknès; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi; MA (Ifrane) – MISR Lispocephala Pokorny, 1893 Lispocephala brachialis (Rondani, 1877) Pont 1986b ; Gregor et al. 2002 ; Barták and Kubik 2005 ; Pont pers. comm., HA , Jebel Ayachi Lispocephala mikii (Strobl, 1893) Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Jebel Ayachi, AA , Agadir Lispocephala ungulata (Rondani, 1866) Ackland and Pont 1966 , HA , Jebel Ayachi; Pont 1986b Orchisia Rondani, 1877 Orchisia costata (Meigen, 1826) Charrier 1927 , Rif , Tanger; Pont 1986b Schoenomyza Haliday, 1833 Schoenomyza litorella (Fallén, 1823) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Pont 1986b ; Mouna 1998 ; Pont pers. comm., EM , Figuig, HA , Asni, Imlil, Jebel Ayachi Limnophorini Limnophora Robineau-Desvoidy, 1830 Limnophora bipunctata Stein, 1908 Pont 1986b ; Pont pers. comm., EM , Figuig Limnophora flavitarsis Stein, 1908 Pont pers. comm., EM , Figuig, HA , Jebel Ayachi Limnophora mediterranea Pont, 2012 Pont pers. comm., HA , Jebel Ayachi Limnophora obsignata (Rondani, 1866) Séguy 1930a , MA , Aïn Leuh, HA , Aguerd El Had, Souss (Talekjount); Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi, AA , Agadir Limnophora olympiae Lyneborg, 1965 Pont 1986b ; Pont et al. 2012 a; Pont pers. comm., HA , Jebel Ayachi Limnophora pandellei Séguy, 1923 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Limnophora pollinifrons Stein, 1916 Pont 1986b ; Pont et al. 2012 a; Pont pers. comm., MA , Aïn El Orma Limnophora riparia (Fallén 1824) = Melanochelia riparia Fallén, in Séguy 1930a : 160 Séguy 1930a , AA , Souss (Tenfecht); Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Sidi Chamarouch; Pont 1986b ; Mouna 1998 Limnophora rufimana (Strobl, 1893) Séguy 1930a , MA , Aïn Leuh; Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Jebel Ayachi Limnophora tigrina (Am Stein, 1860) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Lispe Latreille, 1797 Lispe assimilis Wiedemann, 1824 = Lispe inexpectata Canzoneri & Meneghini, 1966 Canzoneri and Meneghini 1966 , MA , Oued Fès (Taza); Pont 1986b ; Vikhrev 2012b , AP , Essaouira, HA , Marrakech, SA , Tan-Tan Lispe apicalis Mik, 1869 Canzoneri and Meneghini 1966 , MA , Oued Sebou, EM , Guercif (Oued Moulouya); Canzoneri and Meneghini 1972 , MA , Taza, Oued Fès; Pont 1986b ; Koçak and Kemal 2010 Lispe bengalensis (Robineau-Desvoidy, 1830) = Lispe berlandi Séguy, in Séguy 1940 : 341 Séguy 1940 , AA , Rio de Oro (Oued Eddahab) (type locality of berlandi ) Lispe caesia Meigen, 1826 = Lispe microchaeta Séguy, in Séguy 1940 : 342 Séguy 1940 , AA , Rio de Oro (Oued Eddahab) (type locality of microchaeta ); Canzoneri and Meneghini 1966 , AP , Fedhala (Oued Nefifikh), Saline di Sète; Pont 1986b ; Mouna 1998 ; Koçak and Kemal 2010 ; Vikhrev et al. 2016 , AP , Oualidia lagoon, Essaouira, SA , Tan-Tan (salt lagoon); AP (Chellah) – MISR ; ZMUM Lispe candicans Kowarz, 1892 Séguy 1940 , AA , Rio de Oro (Villa Cisneros) Lispe cilitarsis Loew, 1856 Vikhrev 2012b , SA , Tan-Tan province Lispe draperi Séguy, 1930 Canzoneri and Meneghini 1966 , MA , Azrou, Aguelmane, Fès (Oued Sebou); Vikhrev 2011a , AP , Essaouira, HA , Oued N'fis (east of Marrakech) Lispe halophora Becker, 1903 Vikhrev pers. comm., SA , Tan-Tan province Lispe kowarzi Becker, 1903 Vikhrev 2012c , AP , Essaouira Lispe loewi Ringdahl, 1922 = Lispe litorea Fallén, 1825 (misidentification of authors) in Séguy 1930a : 160 Séguy 1930a , AP , saline mud in Mediterranean region; Canzoneri and Meneghini 1966 , AP , Fedhala; Pont 1986b ; Dakki 1997 ; Mouna 1998 Lispe marina Becker, 1913 Michelsen pers. comm., AP , Larache Lispe melaleuca Loew, 1847 Canzoneri and Meneghini 1966 , MA , Azrou (Aguelmane); Pont 1986b , 1991b Lispe modesta Stein, 1913 Vikhrev 2012b , AP , Essaouira, HA , Marrakech, SA , Tan-Tan Lispe nana Macquart, 1835 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès), Fès (Oued Sebou), Azrou (Aguelmane), EM , Guercif (Oued Moulouya); Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Aïn el Orma, EM , Figuig, HA , Jebel Ayachi; Rif (Oued Laou dunes), AP (Rabat) – MISR Lispe nivalis Wiedemann, 1830 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès); Pont 1986b , 1991; Vikhrev 2012c , AP , Essaouira, HA , Ouarzazate province, SA , Tan-Tan province; Pont pers. comm., MA , Aïn El Orma Lispe pectinipes Becker, 1903 = Lispa mixticia Séguy, in Séguy 1941d : 19 Séguy 1941d , HA , Taroudant (type locality of mixticia ); Pont 1986b ; Mouna 1998 ; Kirk-Spriggs and McGregor 2009 ; Vikhrev 2011b , AP , Essaouira; Pont pers. comm., HA , Jebel Ayachi Lispe pygmaea Fallén, 1825 Canzoneri and Meneghini 1966 , MA , Azrou (Aguelmane); Pont 1986b ; Vikhrev 2012a , AP , Essaouira Lispe rigida Becker, 1903 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès); Pont 1986b , 1991; Vikhrev 2012c , HA , Ouarzazate Lispe scalaris Loew, 1847 = Lispe maroccana Canzoneri & Meneghini, 1966 (as scalaris ssp.) = Lispe persica Becker, 1904 in Kirk-Spriggs and McGregor 2009 Canzoneri and Meneghini 1966 , AP , Fedhala, Dielfa (Oued Tadmid), EM , Guercif (Oued Moulouya), MA , Fès (Oued Sebou); Pont 1986b ; Kirk-Spriggs and McGregor 2009 ; Vikhrev 2012a , HA , Ouarzazate province Lispe tentaculata (De Geer, 1776) Séguy 1930a , HA , Kasba Taguendaft (Goundafa), Skoutana (Arround); Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Kirk-Spriggs and McGregor 2009 ; Pont pers. comm., MA , Aïn el Orma, EM , Figuig, HA , Jebel Ayachi; AP (Rabat), HA (Tizi-n'Tichka) – MISR Muscinae Muscini Dasyphora Robineau-Desvoidy, 1830 Dasyphora albofasciata (Macquart, 1839) = Dasiphora saltuum Rondani, 1862 Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Dasyphora penicillata (Egger, 1856) Pont 1986b ; Koçak and Kemal 2010 ; Pont pers. comm., HA , Jebel Ayachi Dasyphora cyanella (Meigen, 1826) Peris and Llorente 1963 , Rif , Tanger; Mouna 1998 Morellia Robineau-Desvoidy, 1830 Morellia asetosa Baranov, 1925 = Morellia simplex (Loew, 1857) (misidentification of authors) in Peris and Llorente 1963 , Rif , Tanger; Pont 1986b 56 Musca Linnaeus, 1758 Musca autumnalis De Geer, 1776 = Musca corvina Fabricius, 1781 in Séguy 1930a : 156 Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat, MA , Oued Korifla, Meknès; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; MA (Volubilis), AA (Tifnit) – MISR Musca biseta Hough, 1898 Pont 1986b ; Pont pers. comm., AP , Temara, MA , Timhadit, Meknès, EM , near Figuig Musca domestica Linnaeus, 1758 Charrier 1927 , Rif , Tanger; Séguy 1930a , 1932, 1934b , AP , Casablanca; Séguy 1934c , AP , Casablanca; Séguy 1941a , HA ; Peris and Llorente 1963 , Rif , Tanger, Melilla, Bab Taza, El Ajmas, Yebala; Pont 1986b ; Mouna 1998 ; Pârvu et al. 2006 , AA , Tiggane Tata; Popescu-Mirceni 2011 ; Pont pers. comm., AP , Temara, MA , Timhadit, Orionane, Lixus , Meknès, HA , Jebel Ayachi; Grabener 2017 ; HA (Jebel Tachdirt, 3100 m, Tachdirt (Bords Imminen), 2400–2600 m, Kasba Taguendaft (Goundafa), Andjera (Pont pers. comm.)) – MHNP Musca larvipara Portschinsky, 1910 Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora Musca osiris Wiedemann, 1830 = Musca vitripennis Meigen, 1826 (misidentifications of authors) in Séguy 1930a : 157 Séguy 1941d , AA , Agadir; Pont 1986b Musca sorbens Wiedemann, 1830 = Musca angustifrons Thomson, 1869 (misidentification of authors) in Séguy 1930a : 156, 1940 : 245, 1953a : 88 Séguy 1930a , MA , Oued Korifla, HA , Talingoult (Goundafa), Souss; Séguy 1940 , AA , Rio de Oro (Villa Cisneros); Séguy 1941d , AA , Agadir; Séguy 1949a , AA , from Foum Zguid to Zagora; Saccà 1952 ; Séguy 1953a , AP , Rabat; Peris and Llorente 1963 ; Pont 1986b , Rif , Melilla, Tanger, AP , Mogador; Mouna 1998 ; Koçak and Kemal 2010 ; Pont pers. comm., MA , Timhadit, HA ; Grabener 2017 – MISR Musca tempestiva Fallén, 1817 Séguy 1941d ; Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora, HA , Asni Musca vitripennis Meigen, 1926 = Plaxemyia vitripennis Meigen, 1826 in Becker and Stein 1913 : 91 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Ras el Ksar, Aïn Leuh, HA , Tinmel (Goundafa), Arround (Skoutana); Séguy 1941d , AA , Agadir; Peris and Llorent 1963, Rif , Tanger, Melilla, AP , Mogador; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; Rif (environs de Tanger, Sart. route de Spartel (Pont pers. comm.)) – MHNP; AP (Cap Cantin, Dradek), MA (Azrou) – MISR Neomyia Walker, 1859 Neomyia cornicina (Fabricius, 1781) = Cryptolucilia caesarion (Meigen, 1826) in Séguy 1930a : 156 Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger (Oued Judios), AP , Rabat, MA , M'Rirt, Aïn Leuh, Tizi-s'Tkrine, Forêt Zaers, Forêt Tiffert, HA , Arround (Skoutana), Tachdirt; Séguy 1941d (very common); Peris and Llorente 1963 , Rif , Tanger, AP , Mogador, Tzalatza, Reisana, Desembocadura del Lixus ; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi – MISR Neomyia viridescens (Robineau-Desvoidy, 1830) = Orthellia cornicina (Fabricius, 1781) (misidentifications of authors) Charrier 1927 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Polietes Rondani, 1866 Polietes meridionalis Peris & Llorente, 1963 Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Pyrellia Robineau-Desvoidy, 1830 Pyrellia vivida Robineau-Desvoidy, 1830 = Pyrellia cadaverina (Linnaeus, 1758) (misidentifications of authors) in Charrier 1927 : 620; Séguy 1930a : 156; Peris and Llorente 1963 : 252 = Pyrellia serena (Meigen, 1826) (misidentification of authors) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger; Séguy 1930a , MA , Aïn Leuh; Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Stomoxyini Haematobia Le Peletier & Serville, 1828 Haematobia irritans (Linnaeus, 1758) = Lyperosia irritans (Linnaeus, 1758) in Séguy 1930a : 157 Séguy 1930a , MA , Meknès; Pont 1986b ; Mouna 1998 Stomoxys Geoffroy, 1762 Stomoxys calcitrans (Linnaeus, 1758) Charrier 1927 , Rif , Tanger; Séguy 1930a ; Séguy 1941d ; Peris 1951 , Rif , Tanger; Pont 1986b ; Mouna 1998 ; Pârvu et al. 2006 , AA , Tiggane Tata; Dsouli 2009 ; Popescu-Mirceni 2011 ; Pont pers. comm., MA , Meknès, Timhadit, Azrou, Sidi Mjber, Neguerett, Tazekka, HA , Jebel Ayachi, El Kebab, AA , Figuig; HA (Haute Réghaya (Pont pers. comm.)) – MHNP; AP (Rabat), MA (Volubilis), HA – MISR Mydaeinae Graphomya Robineau-Desvoidy, 1830 Graphomya maculata (Scopoli, 1763) Séguy 1930a , MA , Forêt Zaers, Aïn Leuh; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; MA (Volubilis) – MISR Gymnodia Robineau-Desvoidy, 1863 Gymnodia eremophila (Brauer & Bergenstamm, 1894) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Gymnodia polystigma (Meigen, 1826) = Limnophora polystigma (Meigen, 1826) = Brontaea polystigma (Meigen, 1826) Mouna 1998 ; AP (Rabat) – MISR Gymnodia genurufa (Pandellé, 1899) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Gymnodia tonitrui (Wiedemann, 1824) = Limnophora tonitrui (Wiedemann, 1824) = Brontaea tonitrui (Wiedemann, 1824) Séguy 1949a , AA , Tata; Saccà 1952 , AP , Rabat; Pont 1986b ; Mouna 1998 Hebecnema Schnabl, 1889 Hebecnema fumosa (Meigen, 1826) Séguy 1930a , AP , Casablanca; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; Rif (Tanger), AP (Mogador (Pont pers. comm.)) – MHNP Hebecnema nigra (Robineau-Desvoidy, 1830) = Hebecnema vespertina (Fallén, 1823) (misidentifications of authors) in Séguy 1930a : 159 Séguy 1930a , AP , Casablanca; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Hebecnema umbratica (Meigen, 1826) Pont 1986b Myospila Rondani, 1856 Myospila meditabunda (Fabricius, 1781) Séguy 1941d , AA , Agadir; Pont 1970 ; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; AP (Cap Cantin) – MISR Phaoniinae Helina Robineau-Desvoidy, 1830 Helina clara (Meigen, 1826) = Mydaea clara (Meigen, 1826) in Séguy 1930a : 159 Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat; Pont 1986b ; Mouna 1998 ; AP (Rabat) – MISR Helina czernyi Lyneborg, 1970 Michelsen pers. comm., Rif , Chefchaouen, Ouezzane, MA , Azrou, HA , Asni Helina evecta (Harris, 1780) = Mydaea lucorum (Fallén, 1823) in Séguy 1930a : 159 Séguy 1930a ; Werner 1938 , EM , Oudjda-Berguent; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi Helina nevadensis Lyneborg, 1970 Pont 1986b ; Pont pers. comm., Rif , Talassemtane, HA , Jebel Ayachi Helina parcepilosa (Stein, 1907) Michelsen pers. comm., AP , Rabat, HA , Tinerhir Helina quadrum (Fabricius, 1805) = Mydaea quadrum "Fallén" in Séguy 1930a : 159 Séguy 1930a , AP , Rabat; Pont, 1986b; Mouna 1998 Helina reversio (Harris, 1780) Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; MA (Forêt Timelilt, 1650–1900 m (Pont pers. comm.)) – MNHN Helina richardi Pont, 2012 Pont 2012b , Rif , Ras el Ma, MA , Azrou, HA , Jebel Ayachi Helina sexmaculata (Preyssler, 1791) = Mydaea uliginosa (Fallén, 1825) Mouna 1998 ; Grabener 2017 ; MA (Aguelmane Azigza) – MISR Helina vockerothi Lyneborg, 1970 Michelsen pers. comm., HA , Tizi-n'Test (2100 m), Asni Phaonia Robineau-Desvoidy, 1830 Phaonia cincta (Zetterstedt, 1846) 57 Charrier 1927 , Rif , Tanger; Pont 1986b (record queried); Koçak and Kemal 2010 Phaonia errans (Meigen, 1826) Mouna 1998 ; Michelsen pers. comm., MA , Azrou; AP (Chellah), MA (Ifrane) – MISR Phaonia exoleta (Meigen, 1826) Michelsen pers. comm., AP , Larache Phaonia mediterranea Hennig, 1963 Pont 1973 , HA , Jebel Ayachi; Pont 1986b ; Mouna 1998 ; Gregor et al. 2002 Phaonia rufipalpis (Macquart, 1835) Michelsen pers. comm., Rif , Ouezzane Phaonia scutellata (Zetterstedt, 1845) Michelsen pers. comm., Rif , Ouezzane, HA , Asni, Tizi-n'Test, AA , Aoulouz Phaonia subventa (Harris, 1780) Vikhrev and Erofeeva 2018 , HA , Oukaimeden (2000 m); Rif (environs de Tanger (Pont pers. comm.)) – MHNP Phaonia trimaculata (Bouché, 1834) Séguy 1930a , AP , Casablanca; Séguy 1934b , AP , Maâmora; Séguy 1953a , AP , Port Lyautey, Maâmora, Rabat; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., AP , Forêt Maâmora, HA , Jebel Ayachi; AP (Rabat) – MISR Phaonia tuguriorum (Scopoli, 1763) = Phaonia signata (Meigen, 1826) in Séguy 1930a : 158 Séguy 1930a , MA , Forêt Timlilt; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Phaonia valida (Harris, 1780) = Phaonia erratica (Fallén, 1825) (misidentifications of authors) in Séguy 1953a : 87 Séguy 1953a , MA , Ifrane; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi; MA (Ifrane (Pont pers. comm.)) – MHNP Phaonia sp. near szelenyii Mihályi, 1974 HA (Haute Réghaya (Pont pers. comm.)) – MHNP SCATHOPHAGIDAE K. Kettani Number of species: 3 . Expected: 4 Faunistic knowledge of the family in Morocco: poor Scathophaginae Norellia Robineau-Desvoidy, 1830 Norellia tipularia (Fabricius, 1794) Ebejer et al. 2019 , Rif , Dardara (730 m) Scathophaga Meigen, 1803 Scathophaga stercoraria (Linnaeus, 1758) = Scathophaga merdaria Fabricius, 1794, in Séguy 1930a : 163 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, MA , Meknès; Mouna 1998 ; Koçak and Kemal 2010 ; AP (Azemour, Oued Yakem, Cap Cantin) – MISR Scathophaga lutaria (Fabricius, 1794) Ebejer et al. 2019 , Rif , Talassemtane (1554 m), Jebel Lakraâ (1541 m) ANTHOMYIIDAE K. Kettani, D.M. Ackland Number of species: 36 . Expected: Many more, especially in the mountains Faunistic knowledge of the family in Morocco: poor Anthomyiinae Adia Robineau-Desvoidy, 1830 Adia cinerella (Fallén, 1825) = Chortophila cinerella Fallén, in Séguy 1930a : 162 = Hylemyia cinerella Fallén, in Séguy 1941d : 18 Séguy 1941d , AA , Agadir; Séguy 1930a ; Mouna 1998 ; AP (Rabat), HA (Marrakech), AA (Tifnit (south of Agadir)) – MISR Anthomyia Meigen, 1803 Anthomyia imbrida Rondani, 1866 Séguy 1930a , MA , Meknès; Mouna 1998 Anthomyia liturata (Robineau-Desvoidy, 1830) = Hylemyia pullula Zetterstedt, in Séguy 1930a : 162 Séguy 1930a , MA , Ras el Ksar (1900 m), Tameghilt (1700–1800 m), Forêt Tiffert (2000–2200 m); Mouna 1998 Anthomyia quinquemaculata Macquart, 1839 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), MA , 3.5 km S of Azrou (1450 m) Anthomyia pluvialis (Linnaeus, 1758) Séguy 1929b ; Séguy 1930a , MA , Meknès; Michelsen 1980 , AP , Aïn Diab; Mouna 1998 ; Ackland 1987 , 2001 ; Pârvu et al. 2006 , AA , Foum Zguid (Tata); Popescu-Mirceni 2011 – MISR Anthomyia procellaris Rondani, 1866 Séguy 1929b ; Séguy 1930a , MA , Meknès, Berkane (1350–1400 m), Tlet n'Rhohr; Mouna 1998 Anthomyia tempestatum Wiedemann, 1830 Michelsen and Báez 1985 , HA ; Ackland 2001 ; Grabener 2017 Botanophila Lioy, 1864 Botanophila dissecta (Meigen, 1826) Mouna 1998 ; MA (Meknès) – MISR Botanophila varicolor (Meigen, 1826) Ebejer et al. 2019 , MA , Lac Aguelmane Sidi Ali (2052 m) Delia Robineau-Desvoidy, 1830 Delia antiqua (Meigen, 1826) Mouna 1998 Delia coarctata (Fallén, 1825) Mouna 1998 Delia flavibasis (Stein, 1903) = Hylemyia hordeacea Séguy, in Séguy 1941d : 18 Séguy 1934a , AP , Casablanca; Séguy 1936b , AP , Rabat; Séguy 1941d , AA , Taroudant; Mouna 1998 ; Ackland 2008 Delia flavogrisea (Ringdahl, 1926) 52 Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 Delia planipalpis (Stein, 1898) = Chortophila pilipyga Villeneuve, in Mouna 1998 : 85 Mouna 1998 ; AP (Mazagan) – MISR Delia platura (Meigen, 1826) = Chortophila cilicrura Rondani, in Séguy 1930a : 162 Séguy 1949a , SA , Goulimine; Mouna 1998 ; Singh et al. 2014 ; Grabener 2017 Delia radicum Linnaeus, 1758 = Chortophila brassicae Bouché, in Séguy 1934b : 162, Mouna 1998 : 85 Séguy 1934b , AP , Rabat; Mouna 1998 ; Biron et al. 2000 , AP , Rabat; Andreassen 2007 – MISR Fucellia Robineau-Desvoidy, 1842 Fucellia maritima (Haliday, 1838) Séguy 1930a , Rif , Agla near Cap Spartel (on Fucus ); Cassar et al. 2008 , Rif , Smir lagoon; Mouna 1998 Hylemya Robineau-Desvoidy, 1830 Hylemya vagans (Panzer, 1798) Mouna 1998 – MISR Leucophora Robineau-Desvoidy, 1830 Leucophora cinerea Robineau-Desvoidy, 1830 Ebejer et al. 2019 , MA , 17 km SW of Midelt (Khénifra, 1940 m) Leucophora dissimilis (Villeneuve, 1920) Ebejer et al. 2019 , MA , 17 km NW of Zaida (Khénifra, 1878 m) Paregle Schnabl, 1911 Paregle audacula (Harris, 1780) Pârvu and Zaharia 2007 Paregle pilipes (Stein, 1916) Mouna 1998 Phorbia Robineau-Desvoidy, 1830 Phorbia fumigata (Meigen, 1826) = Phorbia securis Tiensuu, in Maarouf et al. 1996 : 17 Balachowsky and Mesnil 1935 ; Bleuton 1938; Jourdan 1938 ; Maarouf and Chemseddine 1995 , AP , Chaouia, Doukkala, Abda; Maarouf and Chemseddine 1995 , HA , Chaouia, Doukkala, Abda; Maarouf et al. 1996 , AP , Safi, Settat, Sidi El Aydi, Jemaa Riah, Berrechid, Médiouna, Mohammédia, Bouznika, Skhirat, Kénitra, Sidi Allal Tazi, Souk Larbaa du Gharb, MA , Khernisset, Meknès, Douiyat, Fès, Sefrou, Annaceur, Oulad Saïd, El Aounate, Sidi Bennbur, Zemarnra, Chemmaïa, Jemaa, des Shaïm, Tlet Sidi Bouguedra, Khemisset Chaouïa, Béni Mellal, HA , Skhour Rehamna, Ben Guérir, Marrakech, Tamellalet, Kelaâ des Sraghna, Oulad Ayad, Afourér, Azilal, El Borouj, Guisser; Lhaloui et al. 1998 Phorbia sepia (Meigen, 1826) Bleuton 1938, MA , Fès, Meknès, Taza; Jourdan 1938 ; Mouna 1998 Subhylemyia Ringdahl, 1933 Subhylemyia longula (Fallén, 1824) Mouna 1998 ; AP (Cap Cantin) – MISR Pegomyinae Calythea Schnabl in Schnabl and Dziedzicki 1911 Calythea nigricans (Robineau-Desvoidy, 1830) = Calythea albicincta Fallén, in Séguy 1930a : 161 Séguy 1930a , MA , Meknès, Aïn Leuh; Mouna 1998 Mycophaga Rondani, 1856 Mycophaga testacea (Gimmerthal, 1834) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Pegomya Robineau-Desvoidy, 1830 Pegomya betae (Curtis, 1847) Rungs 1962 , AP ; Mouna 1998 Pegomya bicolor (Wiedemann, 1817) Séguy 1934b , AP , Rabat; Koçak and Kemal 2010 ; Pitkin et al. 2011 Pegomya hyoscyami (Panzer, 1809) Rungs 1962 , AP ; Mouna 1998 ; AP (Rabat) – MISR Pegomya rufina (Fallén, 1825) Mouna 1998 Pegomya testacea (De Geer, 1776) = Pegomya silacea Meigen, in Mouna 1998 : 85 Séguy 1930a , MA , Forêt Tiffert (2000–2200 m); Mouna 1998 Pegomya solennis (Meigen, 1826) = Pegomyia nigritarsis Fallén, in Séguy 1935a : 119 Séguy 1935a , AA , Oued Draa (Taffagount); Rungs 1962 ; Mouna 1998 Pegomya terminalis Rondani, 1866 Ebejer et al. 2019 , Rif , Adrou (556 m), Jebel Lakraâ (Talassemtane, 1541 m) Pegomya winthemi (Meigen, 1826) Mouna 1998 Pegoplata Schnabl & Dziedzicki, 1911 Pegoplata annulata (Pandellé, 1899) = Pegoplata virginea auctt, not Meigen AP (Rabat) – MISR Anthomyiinae Adia Robineau-Desvoidy, 1830 Adia cinerella (Fallén, 1825) = Chortophila cinerella Fallén, in Séguy 1930a : 162 = Hylemyia cinerella Fallén, in Séguy 1941d : 18 Séguy 1941d , AA , Agadir; Séguy 1930a ; Mouna 1998 ; AP (Rabat), HA (Marrakech), AA (Tifnit (south of Agadir)) – MISR Anthomyia Meigen, 1803 Anthomyia imbrida Rondani, 1866 Séguy 1930a , MA , Meknès; Mouna 1998 Anthomyia liturata (Robineau-Desvoidy, 1830) = Hylemyia pullula Zetterstedt, in Séguy 1930a : 162 Séguy 1930a , MA , Ras el Ksar (1900 m), Tameghilt (1700–1800 m), Forêt Tiffert (2000–2200 m); Mouna 1998 Anthomyia quinquemaculata Macquart, 1839 Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), MA , 3.5 km S of Azrou (1450 m) Anthomyia pluvialis (Linnaeus, 1758) Séguy 1929b ; Séguy 1930a , MA , Meknès; Michelsen 1980 , AP , Aïn Diab; Mouna 1998 ; Ackland 1987 , 2001 ; Pârvu et al. 2006 , AA , Foum Zguid (Tata); Popescu-Mirceni 2011 – MISR Anthomyia procellaris Rondani, 1866 Séguy 1929b ; Séguy 1930a , MA , Meknès, Berkane (1350–1400 m), Tlet n'Rhohr; Mouna 1998 Anthomyia tempestatum Wiedemann, 1830 Michelsen and Báez 1985 , HA ; Ackland 2001 ; Grabener 2017 Botanophila Lioy, 1864 Botanophila dissecta (Meigen, 1826) Mouna 1998 ; MA (Meknès) – MISR Botanophila varicolor (Meigen, 1826) Ebejer et al. 2019 , MA , Lac Aguelmane Sidi Ali (2052 m) Delia Robineau-Desvoidy, 1830 Delia antiqua (Meigen, 1826) Mouna 1998 Delia coarctata (Fallén, 1825) Mouna 1998 Delia flavibasis (Stein, 1903) = Hylemyia hordeacea Séguy, in Séguy 1941d : 18 Séguy 1934a , AP , Casablanca; Séguy 1936b , AP , Rabat; Séguy 1941d , AA , Taroudant; Mouna 1998 ; Ackland 2008 Delia flavogrisea (Ringdahl, 1926) 52 Pârvu et al. 2006 , AP , Merja Zerga; Pârvu and Zaharia 2007 ; Popescu-Mirceni 2011 Delia planipalpis (Stein, 1898) = Chortophila pilipyga Villeneuve, in Mouna 1998 : 85 Mouna 1998 ; AP (Mazagan) – MISR Delia platura (Meigen, 1826) = Chortophila cilicrura Rondani, in Séguy 1930a : 162 Séguy 1949a , SA , Goulimine; Mouna 1998 ; Singh et al. 2014 ; Grabener 2017 Delia radicum Linnaeus, 1758 = Chortophila brassicae Bouché, in Séguy 1934b : 162, Mouna 1998 : 85 Séguy 1934b , AP , Rabat; Mouna 1998 ; Biron et al. 2000 , AP , Rabat; Andreassen 2007 – MISR Fucellia Robineau-Desvoidy, 1842 Fucellia maritima (Haliday, 1838) Séguy 1930a , Rif , Agla near Cap Spartel (on Fucus ); Cassar et al. 2008 , Rif , Smir lagoon; Mouna 1998 Hylemya Robineau-Desvoidy, 1830 Hylemya vagans (Panzer, 1798) Mouna 1998 – MISR Leucophora Robineau-Desvoidy, 1830 Leucophora cinerea Robineau-Desvoidy, 1830 Ebejer et al. 2019 , MA , 17 km SW of Midelt (Khénifra, 1940 m) Leucophora dissimilis (Villeneuve, 1920) Ebejer et al. 2019 , MA , 17 km NW of Zaida (Khénifra, 1878 m) Paregle Schnabl, 1911 Paregle audacula (Harris, 1780) Pârvu and Zaharia 2007 Paregle pilipes (Stein, 1916) Mouna 1998 Phorbia Robineau-Desvoidy, 1830 Phorbia fumigata (Meigen, 1826) = Phorbia securis Tiensuu, in Maarouf et al. 1996 : 17 Balachowsky and Mesnil 1935 ; Bleuton 1938; Jourdan 1938 ; Maarouf and Chemseddine 1995 , AP , Chaouia, Doukkala, Abda; Maarouf and Chemseddine 1995 , HA , Chaouia, Doukkala, Abda; Maarouf et al. 1996 , AP , Safi, Settat, Sidi El Aydi, Jemaa Riah, Berrechid, Médiouna, Mohammédia, Bouznika, Skhirat, Kénitra, Sidi Allal Tazi, Souk Larbaa du Gharb, MA , Khernisset, Meknès, Douiyat, Fès, Sefrou, Annaceur, Oulad Saïd, El Aounate, Sidi Bennbur, Zemarnra, Chemmaïa, Jemaa, des Shaïm, Tlet Sidi Bouguedra, Khemisset Chaouïa, Béni Mellal, HA , Skhour Rehamna, Ben Guérir, Marrakech, Tamellalet, Kelaâ des Sraghna, Oulad Ayad, Afourér, Azilal, El Borouj, Guisser; Lhaloui et al. 1998 Phorbia sepia (Meigen, 1826) Bleuton 1938, MA , Fès, Meknès, Taza; Jourdan 1938 ; Mouna 1998 Subhylemyia Ringdahl, 1933 Subhylemyia longula (Fallén, 1824) Mouna 1998 ; AP (Cap Cantin) – MISR Pegomyinae Calythea Schnabl in Schnabl and Dziedzicki 1911 Calythea nigricans (Robineau-Desvoidy, 1830) = Calythea albicincta Fallén, in Séguy 1930a : 161 Séguy 1930a , MA , Meknès, Aïn Leuh; Mouna 1998 Mycophaga Rondani, 1856 Mycophaga testacea (Gimmerthal, 1834) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m) Pegomya Robineau-Desvoidy, 1830 Pegomya betae (Curtis, 1847) Rungs 1962 , AP ; Mouna 1998 Pegomya bicolor (Wiedemann, 1817) Séguy 1934b , AP , Rabat; Koçak and Kemal 2010 ; Pitkin et al. 2011 Pegomya hyoscyami (Panzer, 1809) Rungs 1962 , AP ; Mouna 1998 ; AP (Rabat) – MISR Pegomya rufina (Fallén, 1825) Mouna 1998 Pegomya testacea (De Geer, 1776) = Pegomya silacea Meigen, in Mouna 1998 : 85 Séguy 1930a , MA , Forêt Tiffert (2000–2200 m); Mouna 1998 Pegomya solennis (Meigen, 1826) = Pegomyia nigritarsis Fallén, in Séguy 1935a : 119 Séguy 1935a , AA , Oued Draa (Taffagount); Rungs 1962 ; Mouna 1998 Pegomya terminalis Rondani, 1866 Ebejer et al. 2019 , Rif , Adrou (556 m), Jebel Lakraâ (Talassemtane, 1541 m) Pegomya winthemi (Meigen, 1826) Mouna 1998 Pegoplata Schnabl & Dziedzicki, 1911 Pegoplata annulata (Pandellé, 1899) = Pegoplata virginea auctt, not Meigen AP (Rabat) – MISR FANNIIDAE K. Kettani, A.C. Pont Number of species: 10 . Expected: 17 Faunistic knowledge of the family in Morocco: poor Fannia Robineau-Desvoidy, 1830 Fannia canicularis (Linnaeus, 1761) Becker and Stein 1913 , Rif , Tanger; Charrier 1927 ; Séguy 1930a , HA , Tenfecht; Séguy 1932a ; Séguy 1941a ; Séguy 1941d , HA ; Séguy 1953a , AP , Rabat; Pont 1986a ; Mouna 1998 ; Pont pers. comm., MA , Azrou; AP (Dradek) – MISR Fannia cothurnata (Loew, 1873) 53 Mouna 1998 Fannia krimensis Ringdahl, 1934 Pont 1983 , HA , Jebel Ayachi; Pont 1986a ; Mouna 1998 Fannia lepida (Wiedemann, 1817) Pont pers. comm. Fannia leucosticta (Meigen, 1838) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA ; Pont 1986a ; Mouna 1998 Fannia monilis (Haliday, 1838) Ebejer et al. 2019 , Rif , Jebel Lakraâ (Talassemtane, 1541 m), Dardara (484 m) Fannia norvegica Ringdahl, 1934 Pont 1986a ; Pont pers. comm., HA , Jebel Ayachi Fannia pallidibasis Pont, 1983 Pont 1983 , HA , Jebel Ayachi; Mouna 1998 Fannia scalaris (Fabricius, 1794) Charrier 1927 , Rif , Tanger; Séguy 1930a ; Séguy 1941a , HA ; Séguy 1953a , AP , Rabat; Pont 1986a ; Mouna 1998 ; Rif (Tanger): Caverne d'Hercule (Pont pers. comm.) – MHNP Fannia sociella (Zetterstedt, 1845) 54 Mouna 1998 MUSCIDAE K. Kettani, A.C. Pont Number of species: 115 . Expected: 140 Faunistic knowledge of the family in Morocco: moderate Atherigoninae Atherigona Rondani, 1856 Atherigona humeralis (Wiedemann, 1830) Pont pers. comm., AP , Casablanca Atherigona pulla (Wiedemann, 1830) Pont 1986b ; Pont pers. comm., AP , Larache, Rabat, HA , Asni, AA , Agadir, Taroudant Atherigona soccata Rondani, 1871 = Atherigona varia (Meigen, 1826) (misidentifications of authors) in Séguy 1930a : 159 Bléton and Fieuzet 1943 , MA , Fès; Séguy 1953a , AP , Rabat, Sidi Yahia du Gharb; Mouna 1998 ; Pont 1986b Atherigona varia (Meigen, 1826) = Atherigona quadripunctata Rossi, in Séguy 1949a : 159 Séguy 1930a , Rif , Tanger; Séguy 1941d ; Séguy 1949a , AA , Akka, Alnif; Bléton and Fieuzet 1943 , MA , Meknès, Gharb, Fouarat; Séguy 1953a , AP , Sidi Yahia du Gharb, Rabat; Pont 1986b ; Mouna 1998 ; Pont pers. comm., Rif , Meloussa, Tanger, AP , Larache, Rabat, HA , Jebel Ayachi, AA , Akka Atherigona ( Acritochaeta ) yorki Deeming, 1971 Pont 1986b , 1991a ; Dike 1990 ; Pont pers. comm., AP , Rabat Azeliinae Azeliini Azelia Robineau-Desvoidy, 1830 Azelia parva Rondani, 1866 Michelsen pers. comm., Rif , Ouezzane Hydrotaea Robineau-Desvoidy, 1830 Hydrotaea aenescens (Wiedemann, 1830) Morocco, first record 1989; Pont et al. 2007 Hydrotaea armipes (Fallén, 1825) Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi Hydrotaea capensis (Wiedemann, 1818) = Ophyra anthrax (Meigen, 1826), in Séguy 1941d : 20 Séguy 1934b , AP , Chellah; Séguy 1941d , AA , Agadir; Pont 1986b ; Hernández-Moreno and Peris 1989 , Rif , Tanger; Mouna 1998 ; Turchetto et al. 2003 ; Pont pers. comm., HA , Jebel Ayachi; Rif (Environ de Tanger (Pont pers. comm.)) – MHNP Hydrotaea cinerea Robineau-Desvoidy, 1830 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea dentipes (Fabricius, 1805) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea floccosa Macquart, 1835 = Hydrotaea armipes (Fallén, 1825) (misidentifications of authors) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger; Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi Hydrotaea glabricula (Fallén, 1825) Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora Hydrotaea ignava (Harris, 1780) = Ophyra leucostoma (Wiedemann, 1817), in Séguy 1953a : 87 Séguy 1953a , HA , Tadla; Pont 1986b ; Hernández-Moreno and Peris 1989 , Rif , Tanger; Pont pers. comm., AP , Casablanca Hydrotaea pellucens Portschinsky, 1879 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea tuberculata Rondani, 1866 Michelsen pers. comm., AP , Rabat Hydrotaea velutina Robineau-Desvoidy, 1830 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Thricops Rondani, 1856 Thricops simplex (Wiedemann, 1817) Pont 1986b , 1991b ; Pont pers. comm., HA , Jebel Ayachi Reinwardtiini Muscina Robineau-Desvoidy, 1830 Muscina levida (Harris, 1780) = Muscina assimilis (Fallén, 1823) Mouna 1998 – MHNP (no locality, on Boletus nigrescens (Pont pers. comm.)); AP (Rabat) – MISR Muscina prolapsa (Harris, 1780) = Muscina pabulorum (Fallén, 1817) Mouna 1998 ; Michelsen pers. comm., AP , Rabat, Larache; AP (Sidi Yahia) – MISR Muscina stabulans (Fallén, 1817) Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat, Mogador, MA , Meknès, HA , Aguerd El Had, Souss; Séguy 1932, HA , Taroudant; Séguy 1934b , AP , Rabat; Séguy 1953a , AP , Rabat, Salé, Forêt Maâmora, Salé; Pont 1986b ; Mouna 1998 ; Pont pers. comm., AP , Casablanca, MA , El Kebab, HA , Jebel Ayachi; AP (Casablanca, Rabat (Pont pers. comm.)) – MHNP; MISR Coenosiinae Coenosiini Coenosia Meigen, 1826 Coenosia antennata (Zetterstedt, 1849) Michelsen pers. comm., AP , Larache Coenosia atra Meigen, 1830 Pont 1986b ; Barták et al. 2004 ; Barták and Kubik 2005 ; Pont pers. comm., HA , Jebel Ayachi Coenosia attenuata Stein, 1903 Pont 1986b ; Pont pers. comm., EM , Figuig Coenosia humilis Meigen, 1826 Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Imlil, Asni, AA , Agadir Coenosia mixta Schnabl, 1911 Pont 1986b Coenosia nevadensis Lyneborg, 1970 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Coenosia pedella (Fallén, 1825) = Coenosia decipiens Meigen 1826 (certainly a misidentification) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger Coenosia praetexta Pandellé, 1899 Michelsen pers. comm., AP , Larache, Rabat Coenosia pumila (Fallén, 1825) 55 Pont 1986b ; Mouna 1998 ; Gregor et al. 2002 Coenosia testacea (Robineau-Desvoidy, 1830) Pont 1986b ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi Coenosia tigrina (Fabricius, 1775) Séguy 1930a , AP , Rabat, MA , Meknès; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi; MA (Ifrane) – MISR Lispocephala Pokorny, 1893 Lispocephala brachialis (Rondani, 1877) Pont 1986b ; Gregor et al. 2002 ; Barták and Kubik 2005 ; Pont pers. comm., HA , Jebel Ayachi Lispocephala mikii (Strobl, 1893) Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Jebel Ayachi, AA , Agadir Lispocephala ungulata (Rondani, 1866) Ackland and Pont 1966 , HA , Jebel Ayachi; Pont 1986b Orchisia Rondani, 1877 Orchisia costata (Meigen, 1826) Charrier 1927 , Rif , Tanger; Pont 1986b Schoenomyza Haliday, 1833 Schoenomyza litorella (Fallén, 1823) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Pont 1986b ; Mouna 1998 ; Pont pers. comm., EM , Figuig, HA , Asni, Imlil, Jebel Ayachi Limnophorini Limnophora Robineau-Desvoidy, 1830 Limnophora bipunctata Stein, 1908 Pont 1986b ; Pont pers. comm., EM , Figuig Limnophora flavitarsis Stein, 1908 Pont pers. comm., EM , Figuig, HA , Jebel Ayachi Limnophora mediterranea Pont, 2012 Pont pers. comm., HA , Jebel Ayachi Limnophora obsignata (Rondani, 1866) Séguy 1930a , MA , Aïn Leuh, HA , Aguerd El Had, Souss (Talekjount); Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi, AA , Agadir Limnophora olympiae Lyneborg, 1965 Pont 1986b ; Pont et al. 2012 a; Pont pers. comm., HA , Jebel Ayachi Limnophora pandellei Séguy, 1923 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Limnophora pollinifrons Stein, 1916 Pont 1986b ; Pont et al. 2012 a; Pont pers. comm., MA , Aïn El Orma Limnophora riparia (Fallén 1824) = Melanochelia riparia Fallén, in Séguy 1930a : 160 Séguy 1930a , AA , Souss (Tenfecht); Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Sidi Chamarouch; Pont 1986b ; Mouna 1998 Limnophora rufimana (Strobl, 1893) Séguy 1930a , MA , Aïn Leuh; Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Jebel Ayachi Limnophora tigrina (Am Stein, 1860) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Lispe Latreille, 1797 Lispe assimilis Wiedemann, 1824 = Lispe inexpectata Canzoneri & Meneghini, 1966 Canzoneri and Meneghini 1966 , MA , Oued Fès (Taza); Pont 1986b ; Vikhrev 2012b , AP , Essaouira, HA , Marrakech, SA , Tan-Tan Lispe apicalis Mik, 1869 Canzoneri and Meneghini 1966 , MA , Oued Sebou, EM , Guercif (Oued Moulouya); Canzoneri and Meneghini 1972 , MA , Taza, Oued Fès; Pont 1986b ; Koçak and Kemal 2010 Lispe bengalensis (Robineau-Desvoidy, 1830) = Lispe berlandi Séguy, in Séguy 1940 : 341 Séguy 1940 , AA , Rio de Oro (Oued Eddahab) (type locality of berlandi ) Lispe caesia Meigen, 1826 = Lispe microchaeta Séguy, in Séguy 1940 : 342 Séguy 1940 , AA , Rio de Oro (Oued Eddahab) (type locality of microchaeta ); Canzoneri and Meneghini 1966 , AP , Fedhala (Oued Nefifikh), Saline di Sète; Pont 1986b ; Mouna 1998 ; Koçak and Kemal 2010 ; Vikhrev et al. 2016 , AP , Oualidia lagoon, Essaouira, SA , Tan-Tan (salt lagoon); AP (Chellah) – MISR ; ZMUM Lispe candicans Kowarz, 1892 Séguy 1940 , AA , Rio de Oro (Villa Cisneros) Lispe cilitarsis Loew, 1856 Vikhrev 2012b , SA , Tan-Tan province Lispe draperi Séguy, 1930 Canzoneri and Meneghini 1966 , MA , Azrou, Aguelmane, Fès (Oued Sebou); Vikhrev 2011a , AP , Essaouira, HA , Oued N'fis (east of Marrakech) Lispe halophora Becker, 1903 Vikhrev pers. comm., SA , Tan-Tan province Lispe kowarzi Becker, 1903 Vikhrev 2012c , AP , Essaouira Lispe loewi Ringdahl, 1922 = Lispe litorea Fallén, 1825 (misidentification of authors) in Séguy 1930a : 160 Séguy 1930a , AP , saline mud in Mediterranean region; Canzoneri and Meneghini 1966 , AP , Fedhala; Pont 1986b ; Dakki 1997 ; Mouna 1998 Lispe marina Becker, 1913 Michelsen pers. comm., AP , Larache Lispe melaleuca Loew, 1847 Canzoneri and Meneghini 1966 , MA , Azrou (Aguelmane); Pont 1986b , 1991b Lispe modesta Stein, 1913 Vikhrev 2012b , AP , Essaouira, HA , Marrakech, SA , Tan-Tan Lispe nana Macquart, 1835 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès), Fès (Oued Sebou), Azrou (Aguelmane), EM , Guercif (Oued Moulouya); Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Aïn el Orma, EM , Figuig, HA , Jebel Ayachi; Rif (Oued Laou dunes), AP (Rabat) – MISR Lispe nivalis Wiedemann, 1830 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès); Pont 1986b , 1991; Vikhrev 2012c , AP , Essaouira, HA , Ouarzazate province, SA , Tan-Tan province; Pont pers. comm., MA , Aïn El Orma Lispe pectinipes Becker, 1903 = Lispa mixticia Séguy, in Séguy 1941d : 19 Séguy 1941d , HA , Taroudant (type locality of mixticia ); Pont 1986b ; Mouna 1998 ; Kirk-Spriggs and McGregor 2009 ; Vikhrev 2011b , AP , Essaouira; Pont pers. comm., HA , Jebel Ayachi Lispe pygmaea Fallén, 1825 Canzoneri and Meneghini 1966 , MA , Azrou (Aguelmane); Pont 1986b ; Vikhrev 2012a , AP , Essaouira Lispe rigida Becker, 1903 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès); Pont 1986b , 1991; Vikhrev 2012c , HA , Ouarzazate Lispe scalaris Loew, 1847 = Lispe maroccana Canzoneri & Meneghini, 1966 (as scalaris ssp.) = Lispe persica Becker, 1904 in Kirk-Spriggs and McGregor 2009 Canzoneri and Meneghini 1966 , AP , Fedhala, Dielfa (Oued Tadmid), EM , Guercif (Oued Moulouya), MA , Fès (Oued Sebou); Pont 1986b ; Kirk-Spriggs and McGregor 2009 ; Vikhrev 2012a , HA , Ouarzazate province Lispe tentaculata (De Geer, 1776) Séguy 1930a , HA , Kasba Taguendaft (Goundafa), Skoutana (Arround); Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Kirk-Spriggs and McGregor 2009 ; Pont pers. comm., MA , Aïn el Orma, EM , Figuig, HA , Jebel Ayachi; AP (Rabat), HA (Tizi-n'Tichka) – MISR Muscinae Muscini Dasyphora Robineau-Desvoidy, 1830 Dasyphora albofasciata (Macquart, 1839) = Dasiphora saltuum Rondani, 1862 Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Dasyphora penicillata (Egger, 1856) Pont 1986b ; Koçak and Kemal 2010 ; Pont pers. comm., HA , Jebel Ayachi Dasyphora cyanella (Meigen, 1826) Peris and Llorente 1963 , Rif , Tanger; Mouna 1998 Morellia Robineau-Desvoidy, 1830 Morellia asetosa Baranov, 1925 = Morellia simplex (Loew, 1857) (misidentification of authors) in Peris and Llorente 1963 , Rif , Tanger; Pont 1986b 56 Musca Linnaeus, 1758 Musca autumnalis De Geer, 1776 = Musca corvina Fabricius, 1781 in Séguy 1930a : 156 Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat, MA , Oued Korifla, Meknès; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; MA (Volubilis), AA (Tifnit) – MISR Musca biseta Hough, 1898 Pont 1986b ; Pont pers. comm., AP , Temara, MA , Timhadit, Meknès, EM , near Figuig Musca domestica Linnaeus, 1758 Charrier 1927 , Rif , Tanger; Séguy 1930a , 1932, 1934b , AP , Casablanca; Séguy 1934c , AP , Casablanca; Séguy 1941a , HA ; Peris and Llorente 1963 , Rif , Tanger, Melilla, Bab Taza, El Ajmas, Yebala; Pont 1986b ; Mouna 1998 ; Pârvu et al. 2006 , AA , Tiggane Tata; Popescu-Mirceni 2011 ; Pont pers. comm., AP , Temara, MA , Timhadit, Orionane, Lixus , Meknès, HA , Jebel Ayachi; Grabener 2017 ; HA (Jebel Tachdirt, 3100 m, Tachdirt (Bords Imminen), 2400–2600 m, Kasba Taguendaft (Goundafa), Andjera (Pont pers. comm.)) – MHNP Musca larvipara Portschinsky, 1910 Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora Musca osiris Wiedemann, 1830 = Musca vitripennis Meigen, 1826 (misidentifications of authors) in Séguy 1930a : 157 Séguy 1941d , AA , Agadir; Pont 1986b Musca sorbens Wiedemann, 1830 = Musca angustifrons Thomson, 1869 (misidentification of authors) in Séguy 1930a : 156, 1940 : 245, 1953a : 88 Séguy 1930a , MA , Oued Korifla, HA , Talingoult (Goundafa), Souss; Séguy 1940 , AA , Rio de Oro (Villa Cisneros); Séguy 1941d , AA , Agadir; Séguy 1949a , AA , from Foum Zguid to Zagora; Saccà 1952 ; Séguy 1953a , AP , Rabat; Peris and Llorente 1963 ; Pont 1986b , Rif , Melilla, Tanger, AP , Mogador; Mouna 1998 ; Koçak and Kemal 2010 ; Pont pers. comm., MA , Timhadit, HA ; Grabener 2017 – MISR Musca tempestiva Fallén, 1817 Séguy 1941d ; Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora, HA , Asni Musca vitripennis Meigen, 1926 = Plaxemyia vitripennis Meigen, 1826 in Becker and Stein 1913 : 91 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Ras el Ksar, Aïn Leuh, HA , Tinmel (Goundafa), Arround (Skoutana); Séguy 1941d , AA , Agadir; Peris and Llorent 1963, Rif , Tanger, Melilla, AP , Mogador; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; Rif (environs de Tanger, Sart. route de Spartel (Pont pers. comm.)) – MHNP; AP (Cap Cantin, Dradek), MA (Azrou) – MISR Neomyia Walker, 1859 Neomyia cornicina (Fabricius, 1781) = Cryptolucilia caesarion (Meigen, 1826) in Séguy 1930a : 156 Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger (Oued Judios), AP , Rabat, MA , M'Rirt, Aïn Leuh, Tizi-s'Tkrine, Forêt Zaers, Forêt Tiffert, HA , Arround (Skoutana), Tachdirt; Séguy 1941d (very common); Peris and Llorente 1963 , Rif , Tanger, AP , Mogador, Tzalatza, Reisana, Desembocadura del Lixus ; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi – MISR Neomyia viridescens (Robineau-Desvoidy, 1830) = Orthellia cornicina (Fabricius, 1781) (misidentifications of authors) Charrier 1927 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Polietes Rondani, 1866 Polietes meridionalis Peris & Llorente, 1963 Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Pyrellia Robineau-Desvoidy, 1830 Pyrellia vivida Robineau-Desvoidy, 1830 = Pyrellia cadaverina (Linnaeus, 1758) (misidentifications of authors) in Charrier 1927 : 620; Séguy 1930a : 156; Peris and Llorente 1963 : 252 = Pyrellia serena (Meigen, 1826) (misidentification of authors) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger; Séguy 1930a , MA , Aïn Leuh; Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Stomoxyini Haematobia Le Peletier & Serville, 1828 Haematobia irritans (Linnaeus, 1758) = Lyperosia irritans (Linnaeus, 1758) in Séguy 1930a : 157 Séguy 1930a , MA , Meknès; Pont 1986b ; Mouna 1998 Stomoxys Geoffroy, 1762 Stomoxys calcitrans (Linnaeus, 1758) Charrier 1927 , Rif , Tanger; Séguy 1930a ; Séguy 1941d ; Peris 1951 , Rif , Tanger; Pont 1986b ; Mouna 1998 ; Pârvu et al. 2006 , AA , Tiggane Tata; Dsouli 2009 ; Popescu-Mirceni 2011 ; Pont pers. comm., MA , Meknès, Timhadit, Azrou, Sidi Mjber, Neguerett, Tazekka, HA , Jebel Ayachi, El Kebab, AA , Figuig; HA (Haute Réghaya (Pont pers. comm.)) – MHNP; AP (Rabat), MA (Volubilis), HA – MISR Mydaeinae Graphomya Robineau-Desvoidy, 1830 Graphomya maculata (Scopoli, 1763) Séguy 1930a , MA , Forêt Zaers, Aïn Leuh; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; MA (Volubilis) – MISR Gymnodia Robineau-Desvoidy, 1863 Gymnodia eremophila (Brauer & Bergenstamm, 1894) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Gymnodia polystigma (Meigen, 1826) = Limnophora polystigma (Meigen, 1826) = Brontaea polystigma (Meigen, 1826) Mouna 1998 ; AP (Rabat) – MISR Gymnodia genurufa (Pandellé, 1899) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Gymnodia tonitrui (Wiedemann, 1824) = Limnophora tonitrui (Wiedemann, 1824) = Brontaea tonitrui (Wiedemann, 1824) Séguy 1949a , AA , Tata; Saccà 1952 , AP , Rabat; Pont 1986b ; Mouna 1998 Hebecnema Schnabl, 1889 Hebecnema fumosa (Meigen, 1826) Séguy 1930a , AP , Casablanca; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; Rif (Tanger), AP (Mogador (Pont pers. comm.)) – MHNP Hebecnema nigra (Robineau-Desvoidy, 1830) = Hebecnema vespertina (Fallén, 1823) (misidentifications of authors) in Séguy 1930a : 159 Séguy 1930a , AP , Casablanca; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Hebecnema umbratica (Meigen, 1826) Pont 1986b Myospila Rondani, 1856 Myospila meditabunda (Fabricius, 1781) Séguy 1941d , AA , Agadir; Pont 1970 ; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; AP (Cap Cantin) – MISR Phaoniinae Helina Robineau-Desvoidy, 1830 Helina clara (Meigen, 1826) = Mydaea clara (Meigen, 1826) in Séguy 1930a : 159 Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat; Pont 1986b ; Mouna 1998 ; AP (Rabat) – MISR Helina czernyi Lyneborg, 1970 Michelsen pers. comm., Rif , Chefchaouen, Ouezzane, MA , Azrou, HA , Asni Helina evecta (Harris, 1780) = Mydaea lucorum (Fallén, 1823) in Séguy 1930a : 159 Séguy 1930a ; Werner 1938 , EM , Oudjda-Berguent; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi Helina nevadensis Lyneborg, 1970 Pont 1986b ; Pont pers. comm., Rif , Talassemtane, HA , Jebel Ayachi Helina parcepilosa (Stein, 1907) Michelsen pers. comm., AP , Rabat, HA , Tinerhir Helina quadrum (Fabricius, 1805) = Mydaea quadrum "Fallén" in Séguy 1930a : 159 Séguy 1930a , AP , Rabat; Pont, 1986b; Mouna 1998 Helina reversio (Harris, 1780) Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; MA (Forêt Timelilt, 1650–1900 m (Pont pers. comm.)) – MNHN Helina richardi Pont, 2012 Pont 2012b , Rif , Ras el Ma, MA , Azrou, HA , Jebel Ayachi Helina sexmaculata (Preyssler, 1791) = Mydaea uliginosa (Fallén, 1825) Mouna 1998 ; Grabener 2017 ; MA (Aguelmane Azigza) – MISR Helina vockerothi Lyneborg, 1970 Michelsen pers. comm., HA , Tizi-n'Test (2100 m), Asni Phaonia Robineau-Desvoidy, 1830 Phaonia cincta (Zetterstedt, 1846) 57 Charrier 1927 , Rif , Tanger; Pont 1986b (record queried); Koçak and Kemal 2010 Phaonia errans (Meigen, 1826) Mouna 1998 ; Michelsen pers. comm., MA , Azrou; AP (Chellah), MA (Ifrane) – MISR Phaonia exoleta (Meigen, 1826) Michelsen pers. comm., AP , Larache Phaonia mediterranea Hennig, 1963 Pont 1973 , HA , Jebel Ayachi; Pont 1986b ; Mouna 1998 ; Gregor et al. 2002 Phaonia rufipalpis (Macquart, 1835) Michelsen pers. comm., Rif , Ouezzane Phaonia scutellata (Zetterstedt, 1845) Michelsen pers. comm., Rif , Ouezzane, HA , Asni, Tizi-n'Test, AA , Aoulouz Phaonia subventa (Harris, 1780) Vikhrev and Erofeeva 2018 , HA , Oukaimeden (2000 m); Rif (environs de Tanger (Pont pers. comm.)) – MHNP Phaonia trimaculata (Bouché, 1834) Séguy 1930a , AP , Casablanca; Séguy 1934b , AP , Maâmora; Séguy 1953a , AP , Port Lyautey, Maâmora, Rabat; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., AP , Forêt Maâmora, HA , Jebel Ayachi; AP (Rabat) – MISR Phaonia tuguriorum (Scopoli, 1763) = Phaonia signata (Meigen, 1826) in Séguy 1930a : 158 Séguy 1930a , MA , Forêt Timlilt; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Phaonia valida (Harris, 1780) = Phaonia erratica (Fallén, 1825) (misidentifications of authors) in Séguy 1953a : 87 Séguy 1953a , MA , Ifrane; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi; MA (Ifrane (Pont pers. comm.)) – MHNP Phaonia sp. near szelenyii Mihályi, 1974 HA (Haute Réghaya (Pont pers. comm.)) – MHNP Atherigoninae Atherigona Rondani, 1856 Atherigona humeralis (Wiedemann, 1830) Pont pers. comm., AP , Casablanca Atherigona pulla (Wiedemann, 1830) Pont 1986b ; Pont pers. comm., AP , Larache, Rabat, HA , Asni, AA , Agadir, Taroudant Atherigona soccata Rondani, 1871 = Atherigona varia (Meigen, 1826) (misidentifications of authors) in Séguy 1930a : 159 Bléton and Fieuzet 1943 , MA , Fès; Séguy 1953a , AP , Rabat, Sidi Yahia du Gharb; Mouna 1998 ; Pont 1986b Atherigona varia (Meigen, 1826) = Atherigona quadripunctata Rossi, in Séguy 1949a : 159 Séguy 1930a , Rif , Tanger; Séguy 1941d ; Séguy 1949a , AA , Akka, Alnif; Bléton and Fieuzet 1943 , MA , Meknès, Gharb, Fouarat; Séguy 1953a , AP , Sidi Yahia du Gharb, Rabat; Pont 1986b ; Mouna 1998 ; Pont pers. comm., Rif , Meloussa, Tanger, AP , Larache, Rabat, HA , Jebel Ayachi, AA , Akka Atherigona ( Acritochaeta ) yorki Deeming, 1971 Pont 1986b , 1991a ; Dike 1990 ; Pont pers. comm., AP , Rabat Azeliinae Azeliini Azelia Robineau-Desvoidy, 1830 Azelia parva Rondani, 1866 Michelsen pers. comm., Rif , Ouezzane Hydrotaea Robineau-Desvoidy, 1830 Hydrotaea aenescens (Wiedemann, 1830) Morocco, first record 1989; Pont et al. 2007 Hydrotaea armipes (Fallén, 1825) Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi Hydrotaea capensis (Wiedemann, 1818) = Ophyra anthrax (Meigen, 1826), in Séguy 1941d : 20 Séguy 1934b , AP , Chellah; Séguy 1941d , AA , Agadir; Pont 1986b ; Hernández-Moreno and Peris 1989 , Rif , Tanger; Mouna 1998 ; Turchetto et al. 2003 ; Pont pers. comm., HA , Jebel Ayachi; Rif (Environ de Tanger (Pont pers. comm.)) – MHNP Hydrotaea cinerea Robineau-Desvoidy, 1830 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea dentipes (Fabricius, 1805) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea floccosa Macquart, 1835 = Hydrotaea armipes (Fallén, 1825) (misidentifications of authors) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger; Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi Hydrotaea glabricula (Fallén, 1825) Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora Hydrotaea ignava (Harris, 1780) = Ophyra leucostoma (Wiedemann, 1817), in Séguy 1953a : 87 Séguy 1953a , HA , Tadla; Pont 1986b ; Hernández-Moreno and Peris 1989 , Rif , Tanger; Pont pers. comm., AP , Casablanca Hydrotaea pellucens Portschinsky, 1879 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Hydrotaea tuberculata Rondani, 1866 Michelsen pers. comm., AP , Rabat Hydrotaea velutina Robineau-Desvoidy, 1830 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Thricops Rondani, 1856 Thricops simplex (Wiedemann, 1817) Pont 1986b , 1991b ; Pont pers. comm., HA , Jebel Ayachi Reinwardtiini Muscina Robineau-Desvoidy, 1830 Muscina levida (Harris, 1780) = Muscina assimilis (Fallén, 1823) Mouna 1998 – MHNP (no locality, on Boletus nigrescens (Pont pers. comm.)); AP (Rabat) – MISR Muscina prolapsa (Harris, 1780) = Muscina pabulorum (Fallén, 1817) Mouna 1998 ; Michelsen pers. comm., AP , Rabat, Larache; AP (Sidi Yahia) – MISR Muscina stabulans (Fallén, 1817) Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat, Mogador, MA , Meknès, HA , Aguerd El Had, Souss; Séguy 1932, HA , Taroudant; Séguy 1934b , AP , Rabat; Séguy 1953a , AP , Rabat, Salé, Forêt Maâmora, Salé; Pont 1986b ; Mouna 1998 ; Pont pers. comm., AP , Casablanca, MA , El Kebab, HA , Jebel Ayachi; AP (Casablanca, Rabat (Pont pers. comm.)) – MHNP; MISR Coenosiinae Coenosiini Coenosia Meigen, 1826 Coenosia antennata (Zetterstedt, 1849) Michelsen pers. comm., AP , Larache Coenosia atra Meigen, 1830 Pont 1986b ; Barták et al. 2004 ; Barták and Kubik 2005 ; Pont pers. comm., HA , Jebel Ayachi Coenosia attenuata Stein, 1903 Pont 1986b ; Pont pers. comm., EM , Figuig Coenosia humilis Meigen, 1826 Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Imlil, Asni, AA , Agadir Coenosia mixta Schnabl, 1911 Pont 1986b Coenosia nevadensis Lyneborg, 1970 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Coenosia pedella (Fallén, 1825) = Coenosia decipiens Meigen 1826 (certainly a misidentification) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger Coenosia praetexta Pandellé, 1899 Michelsen pers. comm., AP , Larache, Rabat Coenosia pumila (Fallén, 1825) 55 Pont 1986b ; Mouna 1998 ; Gregor et al. 2002 Coenosia testacea (Robineau-Desvoidy, 1830) Pont 1986b ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi Coenosia tigrina (Fabricius, 1775) Séguy 1930a , AP , Rabat, MA , Meknès; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi; MA (Ifrane) – MISR Lispocephala Pokorny, 1893 Lispocephala brachialis (Rondani, 1877) Pont 1986b ; Gregor et al. 2002 ; Barták and Kubik 2005 ; Pont pers. comm., HA , Jebel Ayachi Lispocephala mikii (Strobl, 1893) Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Jebel Ayachi, AA , Agadir Lispocephala ungulata (Rondani, 1866) Ackland and Pont 1966 , HA , Jebel Ayachi; Pont 1986b Orchisia Rondani, 1877 Orchisia costata (Meigen, 1826) Charrier 1927 , Rif , Tanger; Pont 1986b Schoenomyza Haliday, 1833 Schoenomyza litorella (Fallén, 1823) Séguy 1941a , HA , Tachdirt (Toubkal, 2500 m); Pont 1986b ; Mouna 1998 ; Pont pers. comm., EM , Figuig, HA , Asni, Imlil, Jebel Ayachi Limnophorini Limnophora Robineau-Desvoidy, 1830 Limnophora bipunctata Stein, 1908 Pont 1986b ; Pont pers. comm., EM , Figuig Limnophora flavitarsis Stein, 1908 Pont pers. comm., EM , Figuig, HA , Jebel Ayachi Limnophora mediterranea Pont, 2012 Pont pers. comm., HA , Jebel Ayachi Limnophora obsignata (Rondani, 1866) Séguy 1930a , MA , Aïn Leuh, HA , Aguerd El Had, Souss (Talekjount); Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi, AA , Agadir Limnophora olympiae Lyneborg, 1965 Pont 1986b ; Pont et al. 2012 a; Pont pers. comm., HA , Jebel Ayachi Limnophora pandellei Séguy, 1923 Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Limnophora pollinifrons Stein, 1916 Pont 1986b ; Pont et al. 2012 a; Pont pers. comm., MA , Aïn El Orma Limnophora riparia (Fallén 1824) = Melanochelia riparia Fallén, in Séguy 1930a : 160 Séguy 1930a , AA , Souss (Tenfecht); Vaillant 1956b , HA , Cascade Siroua, Lac Tamhda (Anremer), Sidi Chamarouch; Pont 1986b ; Mouna 1998 Limnophora rufimana (Strobl, 1893) Séguy 1930a , MA , Aïn Leuh; Pont 1986b ; Pont pers. comm., EM , Figuig, HA , Jebel Ayachi Limnophora tigrina (Am Stein, 1860) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Lispe Latreille, 1797 Lispe assimilis Wiedemann, 1824 = Lispe inexpectata Canzoneri & Meneghini, 1966 Canzoneri and Meneghini 1966 , MA , Oued Fès (Taza); Pont 1986b ; Vikhrev 2012b , AP , Essaouira, HA , Marrakech, SA , Tan-Tan Lispe apicalis Mik, 1869 Canzoneri and Meneghini 1966 , MA , Oued Sebou, EM , Guercif (Oued Moulouya); Canzoneri and Meneghini 1972 , MA , Taza, Oued Fès; Pont 1986b ; Koçak and Kemal 2010 Lispe bengalensis (Robineau-Desvoidy, 1830) = Lispe berlandi Séguy, in Séguy 1940 : 341 Séguy 1940 , AA , Rio de Oro (Oued Eddahab) (type locality of berlandi ) Lispe caesia Meigen, 1826 = Lispe microchaeta Séguy, in Séguy 1940 : 342 Séguy 1940 , AA , Rio de Oro (Oued Eddahab) (type locality of microchaeta ); Canzoneri and Meneghini 1966 , AP , Fedhala (Oued Nefifikh), Saline di Sète; Pont 1986b ; Mouna 1998 ; Koçak and Kemal 2010 ; Vikhrev et al. 2016 , AP , Oualidia lagoon, Essaouira, SA , Tan-Tan (salt lagoon); AP (Chellah) – MISR ; ZMUM Lispe candicans Kowarz, 1892 Séguy 1940 , AA , Rio de Oro (Villa Cisneros) Lispe cilitarsis Loew, 1856 Vikhrev 2012b , SA , Tan-Tan province Lispe draperi Séguy, 1930 Canzoneri and Meneghini 1966 , MA , Azrou, Aguelmane, Fès (Oued Sebou); Vikhrev 2011a , AP , Essaouira, HA , Oued N'fis (east of Marrakech) Lispe halophora Becker, 1903 Vikhrev pers. comm., SA , Tan-Tan province Lispe kowarzi Becker, 1903 Vikhrev 2012c , AP , Essaouira Lispe loewi Ringdahl, 1922 = Lispe litorea Fallén, 1825 (misidentification of authors) in Séguy 1930a : 160 Séguy 1930a , AP , saline mud in Mediterranean region; Canzoneri and Meneghini 1966 , AP , Fedhala; Pont 1986b ; Dakki 1997 ; Mouna 1998 Lispe marina Becker, 1913 Michelsen pers. comm., AP , Larache Lispe melaleuca Loew, 1847 Canzoneri and Meneghini 1966 , MA , Azrou (Aguelmane); Pont 1986b , 1991b Lispe modesta Stein, 1913 Vikhrev 2012b , AP , Essaouira, HA , Marrakech, SA , Tan-Tan Lispe nana Macquart, 1835 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès), Fès (Oued Sebou), Azrou (Aguelmane), EM , Guercif (Oued Moulouya); Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Aïn el Orma, EM , Figuig, HA , Jebel Ayachi; Rif (Oued Laou dunes), AP (Rabat) – MISR Lispe nivalis Wiedemann, 1830 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès); Pont 1986b , 1991; Vikhrev 2012c , AP , Essaouira, HA , Ouarzazate province, SA , Tan-Tan province; Pont pers. comm., MA , Aïn El Orma Lispe pectinipes Becker, 1903 = Lispa mixticia Séguy, in Séguy 1941d : 19 Séguy 1941d , HA , Taroudant (type locality of mixticia ); Pont 1986b ; Mouna 1998 ; Kirk-Spriggs and McGregor 2009 ; Vikhrev 2011b , AP , Essaouira; Pont pers. comm., HA , Jebel Ayachi Lispe pygmaea Fallén, 1825 Canzoneri and Meneghini 1966 , MA , Azrou (Aguelmane); Pont 1986b ; Vikhrev 2012a , AP , Essaouira Lispe rigida Becker, 1903 Canzoneri and Meneghini 1966 , MA , Taza (Oued Fès); Pont 1986b , 1991; Vikhrev 2012c , HA , Ouarzazate Lispe scalaris Loew, 1847 = Lispe maroccana Canzoneri & Meneghini, 1966 (as scalaris ssp.) = Lispe persica Becker, 1904 in Kirk-Spriggs and McGregor 2009 Canzoneri and Meneghini 1966 , AP , Fedhala, Dielfa (Oued Tadmid), EM , Guercif (Oued Moulouya), MA , Fès (Oued Sebou); Pont 1986b ; Kirk-Spriggs and McGregor 2009 ; Vikhrev 2012a , HA , Ouarzazate province Lispe tentaculata (De Geer, 1776) Séguy 1930a , HA , Kasba Taguendaft (Goundafa), Skoutana (Arround); Séguy 1941a , HA , Imi-n'Ouaka (1500 m); Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Kirk-Spriggs and McGregor 2009 ; Pont pers. comm., MA , Aïn el Orma, EM , Figuig, HA , Jebel Ayachi; AP (Rabat), HA (Tizi-n'Tichka) – MISR Muscinae Muscini Dasyphora Robineau-Desvoidy, 1830 Dasyphora albofasciata (Macquart, 1839) = Dasiphora saltuum Rondani, 1862 Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Dasyphora penicillata (Egger, 1856) Pont 1986b ; Koçak and Kemal 2010 ; Pont pers. comm., HA , Jebel Ayachi Dasyphora cyanella (Meigen, 1826) Peris and Llorente 1963 , Rif , Tanger; Mouna 1998 Morellia Robineau-Desvoidy, 1830 Morellia asetosa Baranov, 1925 = Morellia simplex (Loew, 1857) (misidentification of authors) in Peris and Llorente 1963 , Rif , Tanger; Pont 1986b 56 Musca Linnaeus, 1758 Musca autumnalis De Geer, 1776 = Musca corvina Fabricius, 1781 in Séguy 1930a : 156 Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat, MA , Oued Korifla, Meknès; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; MA (Volubilis), AA (Tifnit) – MISR Musca biseta Hough, 1898 Pont 1986b ; Pont pers. comm., AP , Temara, MA , Timhadit, Meknès, EM , near Figuig Musca domestica Linnaeus, 1758 Charrier 1927 , Rif , Tanger; Séguy 1930a , 1932, 1934b , AP , Casablanca; Séguy 1934c , AP , Casablanca; Séguy 1941a , HA ; Peris and Llorente 1963 , Rif , Tanger, Melilla, Bab Taza, El Ajmas, Yebala; Pont 1986b ; Mouna 1998 ; Pârvu et al. 2006 , AA , Tiggane Tata; Popescu-Mirceni 2011 ; Pont pers. comm., AP , Temara, MA , Timhadit, Orionane, Lixus , Meknès, HA , Jebel Ayachi; Grabener 2017 ; HA (Jebel Tachdirt, 3100 m, Tachdirt (Bords Imminen), 2400–2600 m, Kasba Taguendaft (Goundafa), Andjera (Pont pers. comm.)) – MHNP Musca larvipara Portschinsky, 1910 Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora Musca osiris Wiedemann, 1830 = Musca vitripennis Meigen, 1826 (misidentifications of authors) in Séguy 1930a : 157 Séguy 1941d , AA , Agadir; Pont 1986b Musca sorbens Wiedemann, 1830 = Musca angustifrons Thomson, 1869 (misidentification of authors) in Séguy 1930a : 156, 1940 : 245, 1953a : 88 Séguy 1930a , MA , Oued Korifla, HA , Talingoult (Goundafa), Souss; Séguy 1940 , AA , Rio de Oro (Villa Cisneros); Séguy 1941d , AA , Agadir; Séguy 1949a , AA , from Foum Zguid to Zagora; Saccà 1952 ; Séguy 1953a , AP , Rabat; Peris and Llorente 1963 ; Pont 1986b , Rif , Melilla, Tanger, AP , Mogador; Mouna 1998 ; Koçak and Kemal 2010 ; Pont pers. comm., MA , Timhadit, HA ; Grabener 2017 – MISR Musca tempestiva Fallén, 1817 Séguy 1941d ; Pont 1986b ; Pont pers. comm., AP , Forêt Maâmora, HA , Asni Musca vitripennis Meigen, 1926 = Plaxemyia vitripennis Meigen, 1826 in Becker and Stein 1913 : 91 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Ras el Ksar, Aïn Leuh, HA , Tinmel (Goundafa), Arround (Skoutana); Séguy 1941d , AA , Agadir; Peris and Llorent 1963, Rif , Tanger, Melilla, AP , Mogador; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; Rif (environs de Tanger, Sart. route de Spartel (Pont pers. comm.)) – MHNP; AP (Cap Cantin, Dradek), MA (Azrou) – MISR Neomyia Walker, 1859 Neomyia cornicina (Fabricius, 1781) = Cryptolucilia caesarion (Meigen, 1826) in Séguy 1930a : 156 Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , Rif , Tanger (Oued Judios), AP , Rabat, MA , M'Rirt, Aïn Leuh, Tizi-s'Tkrine, Forêt Zaers, Forêt Tiffert, HA , Arround (Skoutana), Tachdirt; Séguy 1941d (very common); Peris and Llorente 1963 , Rif , Tanger, AP , Mogador, Tzalatza, Reisana, Desembocadura del Lixus ; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi – MISR Neomyia viridescens (Robineau-Desvoidy, 1830) = Orthellia cornicina (Fabricius, 1781) (misidentifications of authors) Charrier 1927 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Polietes Rondani, 1866 Polietes meridionalis Peris & Llorente, 1963 Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Pyrellia Robineau-Desvoidy, 1830 Pyrellia vivida Robineau-Desvoidy, 1830 = Pyrellia cadaverina (Linnaeus, 1758) (misidentifications of authors) in Charrier 1927 : 620; Séguy 1930a : 156; Peris and Llorente 1963 : 252 = Pyrellia serena (Meigen, 1826) (misidentification of authors) in Charrier 1927 : 620 Charrier 1927 , Rif , Tanger; Séguy 1930a , MA , Aïn Leuh; Peris and Llorente 1963 , Rif , Tanger; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Stomoxyini Haematobia Le Peletier & Serville, 1828 Haematobia irritans (Linnaeus, 1758) = Lyperosia irritans (Linnaeus, 1758) in Séguy 1930a : 157 Séguy 1930a , MA , Meknès; Pont 1986b ; Mouna 1998 Stomoxys Geoffroy, 1762 Stomoxys calcitrans (Linnaeus, 1758) Charrier 1927 , Rif , Tanger; Séguy 1930a ; Séguy 1941d ; Peris 1951 , Rif , Tanger; Pont 1986b ; Mouna 1998 ; Pârvu et al. 2006 , AA , Tiggane Tata; Dsouli 2009 ; Popescu-Mirceni 2011 ; Pont pers. comm., MA , Meknès, Timhadit, Azrou, Sidi Mjber, Neguerett, Tazekka, HA , Jebel Ayachi, El Kebab, AA , Figuig; HA (Haute Réghaya (Pont pers. comm.)) – MHNP; AP (Rabat), MA (Volubilis), HA – MISR Mydaeinae Graphomya Robineau-Desvoidy, 1830 Graphomya maculata (Scopoli, 1763) Séguy 1930a , MA , Forêt Zaers, Aïn Leuh; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; MA (Volubilis) – MISR Gymnodia Robineau-Desvoidy, 1863 Gymnodia eremophila (Brauer & Bergenstamm, 1894) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Gymnodia polystigma (Meigen, 1826) = Limnophora polystigma (Meigen, 1826) = Brontaea polystigma (Meigen, 1826) Mouna 1998 ; AP (Rabat) – MISR Gymnodia genurufa (Pandellé, 1899) Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi Gymnodia tonitrui (Wiedemann, 1824) = Limnophora tonitrui (Wiedemann, 1824) = Brontaea tonitrui (Wiedemann, 1824) Séguy 1949a , AA , Tata; Saccà 1952 , AP , Rabat; Pont 1986b ; Mouna 1998 Hebecnema Schnabl, 1889 Hebecnema fumosa (Meigen, 1826) Séguy 1930a , AP , Casablanca; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; Rif (Tanger), AP (Mogador (Pont pers. comm.)) – MHNP Hebecnema nigra (Robineau-Desvoidy, 1830) = Hebecnema vespertina (Fallén, 1823) (misidentifications of authors) in Séguy 1930a : 159 Séguy 1930a , AP , Casablanca; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Hebecnema umbratica (Meigen, 1826) Pont 1986b Myospila Rondani, 1856 Myospila meditabunda (Fabricius, 1781) Séguy 1941d , AA , Agadir; Pont 1970 ; Pont 1986b ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi; AP (Cap Cantin) – MISR Phaoniinae Helina Robineau-Desvoidy, 1830 Helina clara (Meigen, 1826) = Mydaea clara (Meigen, 1826) in Séguy 1930a : 159 Becker and Stein 1913 , Rif , Tanger; Charrier 1927 , Rif , Tanger; Séguy 1930a , AP , Rabat; Pont 1986b ; Mouna 1998 ; AP (Rabat) – MISR Helina czernyi Lyneborg, 1970 Michelsen pers. comm., Rif , Chefchaouen, Ouezzane, MA , Azrou, HA , Asni Helina evecta (Harris, 1780) = Mydaea lucorum (Fallén, 1823) in Séguy 1930a : 159 Séguy 1930a ; Werner 1938 , EM , Oudjda-Berguent; Pont 1986b ; Mouna 1998 ; Pont pers. comm., MA , Ifrane, HA , Jebel Ayachi Helina nevadensis Lyneborg, 1970 Pont 1986b ; Pont pers. comm., Rif , Talassemtane, HA , Jebel Ayachi Helina parcepilosa (Stein, 1907) Michelsen pers. comm., AP , Rabat, HA , Tinerhir Helina quadrum (Fabricius, 1805) = Mydaea quadrum "Fallén" in Séguy 1930a : 159 Séguy 1930a , AP , Rabat; Pont, 1986b; Mouna 1998 Helina reversio (Harris, 1780) Pont 1986b ; Pont pers. comm., MA , Azrou, HA , Jebel Ayachi; MA (Forêt Timelilt, 1650–1900 m (Pont pers. comm.)) – MNHN Helina richardi Pont, 2012 Pont 2012b , Rif , Ras el Ma, MA , Azrou, HA , Jebel Ayachi Helina sexmaculata (Preyssler, 1791) = Mydaea uliginosa (Fallén, 1825) Mouna 1998 ; Grabener 2017 ; MA (Aguelmane Azigza) – MISR Helina vockerothi Lyneborg, 1970 Michelsen pers. comm., HA , Tizi-n'Test (2100 m), Asni Phaonia Robineau-Desvoidy, 1830 Phaonia cincta (Zetterstedt, 1846) 57 Charrier 1927 , Rif , Tanger; Pont 1986b (record queried); Koçak and Kemal 2010 Phaonia errans (Meigen, 1826) Mouna 1998 ; Michelsen pers. comm., MA , Azrou; AP (Chellah), MA (Ifrane) – MISR Phaonia exoleta (Meigen, 1826) Michelsen pers. comm., AP , Larache Phaonia mediterranea Hennig, 1963 Pont 1973 , HA , Jebel Ayachi; Pont 1986b ; Mouna 1998 ; Gregor et al. 2002 Phaonia rufipalpis (Macquart, 1835) Michelsen pers. comm., Rif , Ouezzane Phaonia scutellata (Zetterstedt, 1845) Michelsen pers. comm., Rif , Ouezzane, HA , Asni, Tizi-n'Test, AA , Aoulouz Phaonia subventa (Harris, 1780) Vikhrev and Erofeeva 2018 , HA , Oukaimeden (2000 m); Rif (environs de Tanger (Pont pers. comm.)) – MHNP Phaonia trimaculata (Bouché, 1834) Séguy 1930a , AP , Casablanca; Séguy 1934b , AP , Maâmora; Séguy 1953a , AP , Port Lyautey, Maâmora, Rabat; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., AP , Forêt Maâmora, HA , Jebel Ayachi; AP (Rabat) – MISR Phaonia tuguriorum (Scopoli, 1763) = Phaonia signata (Meigen, 1826) in Séguy 1930a : 158 Séguy 1930a , MA , Forêt Timlilt; Pont 1986b ; Dakki 1997 ; Mouna 1998 ; Pont pers. comm., HA , Jebel Ayachi Phaonia valida (Harris, 1780) = Phaonia erratica (Fallén, 1825) (misidentifications of authors) in Séguy 1953a : 87 Séguy 1953a , MA , Ifrane; Pont 1986b ; Pont pers. comm., HA , Jebel Ayachi; MA (Ifrane (Pont pers. comm.)) – MHNP Phaonia sp. near szelenyii Mihályi, 1974 HA (Haute Réghaya (Pont pers. comm.)) – MHNP SCATHOPHAGIDAE K. Kettani Number of species: 3 . Expected: 4 Faunistic knowledge of the family in Morocco: poor Scathophaginae Norellia Robineau-Desvoidy, 1830 Norellia tipularia (Fabricius, 1794) Ebejer et al. 2019 , Rif , Dardara (730 m) Scathophaga Meigen, 1803 Scathophaga stercoraria (Linnaeus, 1758) = Scathophaga merdaria Fabricius, 1794, in Séguy 1930a : 163 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, MA , Meknès; Mouna 1998 ; Koçak and Kemal 2010 ; AP (Azemour, Oued Yakem, Cap Cantin) – MISR Scathophaga lutaria (Fabricius, 1794) Ebejer et al. 2019 , Rif , Talassemtane (1554 m), Jebel Lakraâ (1541 m) Scathophaginae Norellia Robineau-Desvoidy, 1830 Norellia tipularia (Fabricius, 1794) Ebejer et al. 2019 , Rif , Dardara (730 m) Scathophaga Meigen, 1803 Scathophaga stercoraria (Linnaeus, 1758) = Scathophaga merdaria Fabricius, 1794, in Séguy 1930a : 163 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, MA , Meknès; Mouna 1998 ; Koçak and Kemal 2010 ; AP (Azemour, Oued Yakem, Cap Cantin) – MISR Scathophaga lutaria (Fabricius, 1794) Ebejer et al. 2019 , Rif , Talassemtane (1554 m), Jebel Lakraâ (1541 m) Oestroidea CALLIPHORIDAE K. Kettani, K. Rognes Number of species: 8 . Expected: 10 Faunistic knowledge of the family in Morocco: moderate Calliphorinae Bellardia Robineau-Desvoidy, 1863 Bellardia maroccana (Villeneuve, 1941) = Onesia maroccana Villeneuve, in Villeneuve 1932 : 123 Villeuneuve 1932b; Schumann 1974 , AP , Aïn Diab (Casablanca); Verves 2004 ; Koçak and Kemal 2010 Bellardia mascariensis (Villeneuve, 1926) Schumann 1974 , HA , Marrakech; Verves 2004 ; Koçak and Kemal 2010 Calliphora Robineau-Desvoidy, 1863 Calliphora vicina Robineau-Desvoidy, 1830 = Calliphora erythrocephala Meigen, in Séguy 1930a : 154 Séguy 1930a ; Maurice 1947 , Rif , Tanger; AP (Casablanca, Rabat), MA (Azrou, Ifrane) – MNHN , MISR Calliphora vomitoria (Linnaeus, 1758) Séguy 1930a ; Maurice 1947 , Rif , Tanger; Kurahashi 1971 ; Kurahashi and Magpayo 2000 ; Mouna 1998 ; AP (Rabat), HA – MISR , NHMD Chrysomyinae Chrysomya Robineau-Desvoidy, 1863 Chrysomya albiceps (Wiedemann, 1819) Maurice 1947 ; Séguy 1949a , HA , Alnif; Gonzales-Mora and Peris 1988; Mouna 1998 ; Verves 2003 ; Grabener 2017 ; Dawah et al. 2019 ; EM (Oujda) – MISR , AP (Rabat) – MNHN Luciliinae Lucilia Robineau-Desvoidy, 1863 Lucilia sericata (Meigen, 1824) Séguy 1930a , Rif , Tanger, EM , Berkane (1350–1400 m), MA , Meknès, Berrechid, HA , Tizi-n'Test, Goundafa (Jebel Imdress, 2000–2450 m); Séguy 1953a , AP , Rabat; Mouna 1998 ; Grabener 2017 ; AP (Dradek, Salé), MA (Azrou, Volubilis) – MISR , AP (Temara), MA (Ifrane) – MNHN Melanomyinae Melinda Robineau-Desvoidy, 1863 Melinda gentilis (Robineau-Desvoidy, 1830) MA (Ifrane) – MNHN Melinda viridicyanea (Robineau-Desvoidy, 1830) MA (Ifrane) – MNHN OESTRIDAE K. Kettani, T. Pape Number of species: 10 . Expected: 12 Faunistic knowledge of the family in Morocco: moderate Gastrophilinae Gasterophilus Leach, 1817 Gasterophilus flavipes (Olivier, 1811) Séguy 1928d , EM , Haute Moulouya; Séguy 1930a , EM , Itzer (Haute Moulouya); Mouna 1998 ; Li et al. 2019 Gasterophilus haemorrhoidalis (Linnaeus, 1758) Maurice 1947 ; Mouna 1998 ; AP (Rabat) – MISR Gasterophilus intestinalis (De Geer, 1776) Séguy 1930a ; Mouna 1998 ; Pandey et al. 1992 Gasterophilus nasalis (Linnaeus, 1758) = Gasterophilus veterinus Clark, in Mouna 1998 : 85 Maurice 1947 ; Pandey et al. 1992 ; Mouna 1998 Gasterophilus pecorum (Fabricius, 1794) Séguy 1930a ; Mouna 1998 Hypodermatinae Hypoderma Rondani, 1856 Hypoderma bovis (Linnaeus, 1758) Maurice 1947 ; Mouna 1998 Hypoderma lineatum (De Villers, 1789) Dakkok et al. 1978 , MA , Benslimane; Mouna 1998 Oestrinae Cephalopina Strand, 1928 Cephalopina titillator (Clark, 1797) = Cephalopsis titillator Clark, in Séguy 1930a : 139 Séguy 1930a , Sub- SA (camel breeding region); Mouna 1998 Oestrus Linnaeus, 1758 Oestrus ovis Linnaeus, 1761 Séguy 1930a , AP , Rabat (sheep breeding area), HA , Marrakech, Bou Tazzert; Maurice 1947 ; Mouna 1998 – MISR Rhinoestrus Brauer, 1886 Rhinoestrus purpureus (Brauer, 1858) Maurice 1947 ; Mouna 1998 POLLENIIDAE K. Kettani, K. Rognes Number of species: 12 . Expected: 15 Faunistic knowledge of the family in Morocco: moderate Pollenia Fabricius, 1794 Pollenia amentaria (Scopoli, 1763) Séguy 1949a , AA , Foum-el-Hassan; Séguy 1953a , MA , Sidi Allal Tazi; Mouna 1998 ; Pârvu et al. 2006 , SA , Foum Zghouig; Popescu-Mirceni 2011 ; Gisondi et al. 2020 Pollenia bicolor Robineau-Desvoidy, 1830 Rognes 1991a , HA , Mikdane (Jebel Ayachi); Rognes 1991b , HA , Mikdane (Jebel Ayachi); Gisondi et al. 2020 ; HA (Mikdane) – NHMUK Pollenia contempta Robineau-Desvoidy, 1863 Séguy 1930a , MA , Meknès, from M'Rirt to El Hajeb; Mouna 1998 ; Rognes 1992 Pollenia haeretica Séguy, 1928 Séguy 1934b , AP , Rabat; Rognes 2010 ; AP (40 km S Larache) – NHMD Pollenia ibalia Séguy, 1930 = Pollenia funebris Villeneuve, in Villeneuve 1932 : 284 = Pollenia rungsi Séguy, in Séguy 1953a : 88 Séguy 1930a , EM , Berkane, MA , Tlet n'Rhohr (in garden), Douar Ras el Ksar (900 m); Villeneuve 1932 , HA , Marrakech; Séguy 1953a , AP , Rabat; Séguy 1941a , HA , cañon Tessaout (M'Goum, 3000–3200 m); Mouna 1998 ; Rognes 2010 ; Grabener 2017 ; Gisondi et al. 2020 ; HA (Asni) – NHMUK ; HA (Ijoukak) – MNHN ; HA (15 km SW Tazenakht) – NHMD Pollenia leclercqiana (Lehrer, 1978) Rognes 2010 , 2011 ; Gisondi et al. 2020 Pollenia luteovillosa Rognes, 1987 Gisondi et al. 2020 , HA , Mikdane (Jbel Ayachi) Pollenia ponti Rognes, 1991 Rognes 1991b , HA , Jaffar river (Jebel Ayachi); Gisondi et al. 2020 ; HA (Jebel Ayachi) – NHMUK Pollenia rudis (Fabricius, 1794) Séguy 1930a , EM , Itzer (Haute Moulouya), Berkane (1350 m), AP , Casablanca, MA , Meknès, Aharmoumou (1100 m), Tlet n'Rhohr (in garden), Douar Ras el Ksar (1900 m), HA , Tizi-n'Test, Goundafa (Jebel Imdress, 2000–2450 m); Maurice 1947 , Rif , Tanger; Rognes 1987 , AP , Aïn Diab, Larache, HA , Asni, Jebel Ayachi, Mikdane; Grabener 2017 ; Gisondi et al. 2020 ; AP (Casablanca, Rabat), MA (Ifrane), HA (Haute Réghaya) – MNHN Pollenia ruficrura Rondani, 1862 Rognes 2011 ; Gisondi et al. 2020 Pollenia stigi (Rognes, 1992) Rognes 1992 , MA , Ifrane, Azrou; Gisondi et al. 2020 Pollenia vagabunda (Meigen, 1826) = Pollenia hasei Séguy, in Séguy 1928e : 370, Séguy 1930a : 147 Séguy 1928e ; Séguy 1930a , AP , Casablanca; Mouna 1998 ; Rognes 1992 ; Gisondi et al. 2020 RHINIIDAE K. Kettani, K. Rognes Number of species: 17 . Expected: ~20 Faunistic knowledge of the family in Morocco: moderate Cosmina Robineau-Desvoidy, 1830 Cosmina claripennis Robineau-Desvoidy, 1830 = Cosmina bezziana Villeneuve, in Villeneuve 1932 a: 285 Villeneuve 1932 a, AP , Mogador; Schumann 1986 ; Mouna 1998 ; Rognes 2002 Cosmina maroccana Séguy, 1949 Séguy 1949a , AA , Tenfecht, Vallée du Guir, SA , Guelmim; Séguy 1953a , AA , Tenfecht, Vallée du Guir, SA , Guelmim; Mouna 1998 ; Rognes 2002 ; Grabener 2017 Cosmina punctulata Robineau-Desvoidy, 1830 Séguy 1930a , AA , Tenfecht (Souss, 1000–1500 m) Cosmina viridis (Townsend, 1917) Ebejer et al. 2019 , AA , 13 km E of Goulmima (1100 m) Rhinia Robineau-Desvoidy, 1830 Rhinia apicalis (Wiedemann, 1830) Séguy 1930a , AP , Rabat, MA , Forêt Zaers; Zumpt 1956 ; Gonzales-Mora and Peris 1988; Mouna 1998 ; Verves 2003 ; Lehrer 2007 Rhyncomya Robineau-Desvoidy, 1830 Rhyncomya callopis (Loew, 1856) 58 Séguy 1941d , AA , Agadir; Séguy 1953a , AP , Sidi Ifni, SA , Tindouf, Amguilli Sguelma, Guelta des Zemmours; Schumann 1986 ; Mouna 1998 ; Rognes 1992 ; Dawah et al. 2019 ; El Hawagry and El-Azab 2019; MA (Tafilalt) – MISR Rhyncomya columbina (Meigen, 1824) Peris 1951 , Rif , Tanger, MA , Ifrane; Schumann 1986 ; Gonzalez-Mora and Peris 1988 Rhyncomya cyanescens (Loew, 1844) = Rhyncomya hemisia Séguy, in Séguy 1930a : 150 Séguy 1930a , EM , Berkane (1350–1400 m); Gonzales-Mora and Peris 1988; Rognes 2002 , MA Rhyncomya desertica (Peris, 1951) Grabener 2017 ; El Hawagry and El-Azab 2019 Rhyncomya impavida (Rossi, 1790) Séguy 1930a , Rif , Tanger, EM , Berkane (1350–1400 m), MA , Forêt Tiffert (2000–2200 m); Schumann 1986 ; Mouna 1998 Rhyncomya nigripes (Séguy, 1933) Grabener 2017 ; El Hawagry and El-Azab 2019 Rhyncomya ursina Séguy 1928 = Rhynchomyia ursina Séguy, in Séguy 1928b : 152 Séguy 1928b , AP , Atlantic coast of SA Rhyncomya yahavensis Rognes, 2002 Grabener 2017 ; Ebejer et al. 2019 , AA , 30 km W of Errachidia (1065 m) Rhyncomya zernyana Villeneuve, 1926 Zumpt 1956 ; Gonzales-Mora and Peris 1988 Stomorhina Rondani, 1861 Stomorhina lunata (Fabricius, 1805) Séguy 1930a ; Peris 1951 ; Gonzales-Mora and Peris 1988; Mouna 1998 ; AP (Rabat) – MISR Villeneuviella Austen, 1914 Villeneuviella icadion (Séguy, 1953) = Rhynchoestrus icadion Séguy, in Séguy 1953a : 89 Séguy 1953a , SA , Tindouf Villeneuviella weissi (Séguy, 1926) = Rhynchoestrus weissi Séguy, in Séguy 1934b : 162 Séguy 1934b , EM , Berkane Zobzit (1100 m); Séguy 1953a , SA , Mader Bergat RHINOPHORIDAE K. Kettani, T. Pape Number of species: 8 . Expected: 15 Faunistic knowledge of the family in Morocco: poor Rhinophorinae Melanophora Meigen, 1803 Melanophora roralis (Linnaeus, 1758) Peris 1963 , Rif , Tanger, AP , Larache; Mouna 1998 Oplisa Rondani, 1862 Oplisa aterrima (Strobl, 1899) = Hoplisa aterrima Strobl, in Peris 1963 : 602 Peris 1963 , Rif , Tanger, Zoco de Taleta, Ketama; Mouna 1998 ; Cerretti et al. 2020 Paykullia Robineau-Desvoidy, 1830 Paykullia carmela (Peris, 1963) = Chaetostevenia carmela Peris, in Peris 1963 : 606 Peris 1963 , Rif , Tanger; Cerretti et al. 2020 ; Pape and Thompson 2019 – MNCN Phyto Robineau-Desvoidy, 1830 Phyto atrior (Villeneuve, 1941) = Styloneuria atrior Villeneuve, in Villeneuve 1941 : 122 Villeneuve 1941 , AP , Rabat; Cerretti et al. 2020 ; Pape and Thompson 2019 – IRSNB Phyto discrepans Pandellé, 1896 Cerretti et al. 2020 , Rif , Chefchaouen (600 m), Ouezzane (300 m), MA , 40 km N Fès (1150 m) – NHMD Phyto melanocephala (Meigen, 1824) Ebejer et al. 2019 , Rif , Barrage Smir (145 m); Cerretti et al. 2020 Stevenia Robineau-Desvoidy, 1830 Stevenia deceptoria (Loew, 1847) Mulieri et al. 2010 ; Cerretti et al. 2020 Tricogena Rondani, 1856 Tricogena rubricosa (Meigen, 1824) = Frauenfeldia rubricosa Meigen, in Peris 1963 : 603 Peris 1963 , Rif , Tanger; Mouna 1998 ; Cerretti et al. 2020 – NHMD SARCOPHAGIDAE K. Kettani, D. Whitmore, T. Pape Number of species: 66 . Expected: ~150 Faunistic knowledge of the family in Morocco: poor Miltogramminae Amobia Robineau-Desvoidy, 1830 Amobia signata (Meigen, 1824) Pape 1996 ; Verves 2019 Apodacra Macquart, 1854 Apodacra africana Rohdendorf, 1930 Pape 1996 , Rif , Tanger; Verves 2019 Craticulina Pandellé, 1895 Craticulina antachates (Séguy, 1949) = Apodacra antachates Séguy, in Séguy 1949a : 160 Séguy 1949a , AA , Zagora; Pape 1996 , AA , Zagora; Mouna 1998 Craticulina tabaniformis (Fabricius, 1805) Fabricius 1805 , AP , Mogador; Séguy 1930a , AP , Mogador; Séguy 1935a , AP , beach of Rabat; Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Dolichotachina Villeneuve, 1913 Dolichotachina marginella (Wiedemann, 1930) Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Macronychia Rondani, 1859 Macronychia lemariei Jacentkovský, 1941* AP Macronychia polyodon (Meigen, 1824) Pape 1996 ; Verves 2019 Metopia Meigen, 1803 Metopia argyrocephala (Meigen, 1824) = Metopia leucocephala (Rossi), in Mouna 1998 : 86 Mouna 1998 Miltogramma Meigen, 1803 Miltogramma aurifrons Dufour, 1850 Séguy 1930a , AP , Rabat, MA , Meknès; Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019; Verves 2019 Miltogramma germari Meigen, 1824 Séguy 1930a , MA , Meknès, from M'Rirt to El Hajeb, Sidi Taibi; Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019; Verves 2019 Miltogramma maroccana (Séguy, 1941) = Sphecapatodes maroccana Séguy, in Séguy 1941d : 22 Séguy 1941d , AA , Taroudant; Pape and Szpila 2012 Miltogramma murina Meigen, 1824 Pape 1996 ; Verves 2019 Miltogramma oestracea (Fallén, 1820) Ebejer et al. 2019 , Rif , Belwazen (M'Diq, 200 m), AP , Lower Loukous saltmarsh (2 m) Miltogramma rutilans Meigen, 1824 Ebejer et al. 2019 , Rif , Oued Mhajrate (Ben Karrich, 180 m) Miltogramma testaceifrons (Roser, 1840) = Miltogramma pilitarsis Rondani, in Séguy 1930a : 145; Mouna 1998 : 86 Séguy 1930a , MA , Aïn Leuh; Pape 1996 ; Mouna 1998 Protomiltogramma Townsend, 1916 Protomiltogramma fasciata (Meigen, 1824) = Setulia fasciata (Meigen), in Mouna 1998 : 86 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Senotainia Macquart, 1846 Senotainia albifrons (Rondani, 1859) = Sphecapata albifrons Rondani, in Séguy 1930a : 145 Séguy 1930a Taxigramma Macquart, 1850 Taxigramma heteroneura (Meigen, 1830) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Taxigramma pluriseta (Pandellé, 1895) Ebejer et al. 2019 , Rif , Oued Mhajrate (Ben Karrich, 180 m), AP , Lower Loukous saltmarsh (2 m) Paramacronychiinae Nyctia Robineau-Desvoidy, 1830 Nyctia halterata (Panzer, 1798) = Musca maura Fabricius, in Fabricius 1805 : 302 Fabricius 1805 , Rif , Tanger; Pape 1996 , Rif , Tanger; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Nyctia lugubris (Macquart, 1834) Ebejer et al. 2019 , AP , Lower Loukous saltmarsh (2 m) Sarcophila Rondani, 1856 Sarcophila latifrons (Fallén, 1817) 59 Séguy 1930a , AP , Maâmora, HA , Skoutana (Arround, 2000–2400 m); Mouna 1998 Wohlfahrtia Brauer & Bergenstamm, 1889 Wohlfahrtia bella (Macquart, 1839) = Disjunctio bella (Macquart), in Séguy 1930a : 144 Séguy 1930a , MA , Aïn Leuh; Pape 1996 ; Mouna 1998 ; Hall et al. 2009 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia indigens Villeneuve, 1928 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia magnifica (Schiner, 1862) Delanoë 1922 , AP , Doukkala; Séguy 1930a , AP , Maâmora; Séguy 1941a , HA , Tizi-n'Icheden (3000 m); Maurice 1947 ; Pape 1996 ; Mouna 1998 ; Lmimouni et al. 2004 ; Tliqui et al. 2007 ; Farkas et al. 2009 , Rif , Al Hoceima, Taguidit, Tafensa, EM , Aghbal; Hall et al. 2009 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia nuba (Wiedemann, 1830) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia trina (Wiedemann, 1830) Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019 Sarcophaginae Blaesoxipha Loew, 1861 Blaesoxipha ( Blaesoxipha ) lapidosa Pape, 1994 Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019 Blaesoxipha ( Blaesoxipha ) litoralis (Villeneuve, 1911) Pape 1996 ; Verves 2019 Blaesoxipha ( Blaesoxipha ) pygmaea (Zetterstedt, 1844) Pape 1996 ; Verves 2019 Blaesoxipha ( Servaisia ) rossica Villeneuve, 1912 Pape 1996 ; Verves 2019 Ravinia Robineau-Desvoidy, 1863 Ravinia pernix (Harris, 1780) = Gesneriodes disjuncta Séguy, in Séguy 1938 : 43 = Sarcophaga striata (Fabricius), in Séguy 1941d : 22, Séguy 1949a : 159 Séguy 1938 , HA , Skoutana; Séguy 1941d , AA , Taroudant; Séguy 1949a , AA , Akka; Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga Meigen, 1826 Sarcophaga ( Bercaea ) africa (Wiedemann, 1824) Pape 1996 ; Abkari et al. 1998 ; Verves 2003 , 2019 ; Grabener 2017 ; El Hawagry and El-Azab 2019 Sarcophaga ( Helicophagella ) maculata Meigen, 1835 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Helicophagella ) melanura Meigen, 1826 El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Helicophagella ) novercoides Böttcher, 1913* Rif , HA , AA Sarcophaga ( Heteronychia ) balanina Pandellé, 1896 Whitmore et al. 2013 , AP , Larache; Fendane et al. 2018 , AP , Diabat (Essaouira), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Heteronychia ) cucullans Pandellé, 1896 Séguy 1930a , HA , Maharidja; Mouna 1998 Sarcophaga ( Heteronychia ) ferox Villeneuve, 1908 Whitmore 2011 , Rif , Ouezzane, AP , Larache, MA , Béni Mellal, Afourer, AA , Aoulouz; Whitmore et al. 2013 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Heteronychia ) filia Rondani, 1860 Whitmore 2011 , MA , Azrou, Timahdit; Whitmore et al. 2013 ; Verves 2019 Sarcophaga ( Heteronychia ) javita (Peris, González-Mora & Mingo, 1998)* AA Sarcophaga ( Heteronychia ) longestylata Strobl, 1906 Pape 1996 ; Whitmore et al. 2013 , MA , Ifrane, Azrou; Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Heteronychia ) minima Rondani, 1862 Whitmore 2011 , MA , Azrou, Ifrane, Afourer (Béni Mellal), HA , Ijoukak, Ouirgane (Marrakech), AA , Oulma (Agadir); Whitmore et al. 2013 ; Fendane et al. 2018 , AP , Smimou (Essaouira), El Akarta (Oualidia); Verves 2019 Sarcophaga ( Heteronychia ) obvia (Povolný, 2004) Whitmore et al. 2013 , MA , Afourer (Béni Mellal), HA , Ait Lekak (Marrakech), S Asni (Imlil, Marrakech), Tagadirt, Quirgane, AA , Oulma Ort (Agadir) Sarcophaga ( Heteronychia ) pandellei (Rohdendorf, 1937) Séguy 1930a , MA , Tizi-s'Tkrine; Mouna 1998 ; Whitmore et al. 2013 , MA , Azrou, Ifrane, Afourer (Béni Mellal) Sarcophaga ( Heteronychia ) tangerensis Whitmore, 2011 = Heteronychia ( Heteronychia ) amica Peris, González-Mora & Mingo, in Peris et al. 1998 : 173 Peris et al. 1998 , Rif , Tanger; Whitmore 2011 , Rif , Tanger Sarcophaga ( Heteronychia ) villeneuveana (Enderlein, 1928) = Pierretia ( Bercaea ) maroccana Rohdendorf, in Rohdendorf 1937 : 325 = Sarcophaga ( Heteronychia ) penicillata Villeneuve, in Coupland and Barker 2004 : 113 (misidentification) Rohdendorf 1937 , MA , Aïn Defali; Coupland and Barker 2004 ; Whitmore 2009 ; Fendane et al. 2018 , AP , Diabat (Essaouira), Ghabat Tansift (Souiria), Lalla Fatna (Safi), Laatoutate (Safi), El Akarta (Oualidia), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Heteronychia ) uncicurva Pandellé, 1896 Fendane et al. 2018 , AP , Smimou (Essaouira), Diabat (Essaouira), Lalla Fatna (Safi), Laatoutate (Safi), Bir Retma (Casablanca) Sarcophaga ( Liopygia ) argyrostoma (Robineau-Desvoidy, 1830)* HA Sarcophaga ( Liopygia ) crassipalpis Macquart, 1839 Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) aegyptica Salem, 1935 Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Liosarcophaga ) dux Thomson, 1869* SA Sarcophaga ( Liosarcophaga ) jacobsoni (Rohdendorf, 1937) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) marshalli Parker, 1923 Fendane et al. 2018 , AP , Smimou (Essaouira), Diabat (Essaouira), Ghabat Tansift (Souiria), Laatoutate (Safi); El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) pharaonis Rohdendorf, 1934 Carles-Tolrá 2002 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) tibialis Macquart, 1851 = Sarcophaga beckeri Villeneuve, in Maurice 1947 : 57; Mouna 1998 : 86 Maurice 1947 ; Mouna 1998 ; Fendane et al. 2018 , AP , Laatoutate (Safi) Sarcophaga ( Liosarcophaga ) teretirostris Pandellé, 1896 = Parasarcophaga decellei Lehrer, in Lehrer 1976 : 3 Lehrer 1976 , MA , Kandar, Imouzzer; Pape 1996 , MA , Kandar, Imouzzer; Verves 2019 Sarcophaga ( Liosarcophaga ) tuberosa Pandellé, 1896 60 Mouna 1998 Sarcophaga ( Myorhina ) nigriventris Meigen, 1826 Pape 1996 ; Mouna 1998 ; Fendane et al. 2018 , AP , Ghabat Tansift (Souiria), Laatoutate (Safi), El Akarta (Oualidia), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Myorhina ) soror Rondani, 1860 Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Pandelleana ) protuberans Pandellé, 1896 Séguy 1949a , AA , Agadir Tissint, SA , Guelmim, Foum-el-Hassan, Tata; Mouna 1998 Sarcophaga ( Parasarcophaga ) hirtipes Wiedemann, 1830 Pape 1996 ; Verves 2003 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Sarcophaga ) lehmanni Müller, 1922 Pape 1996 ; Cassar et al. 2005 , Rif , Smir lagoon; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Sarcophaga ) marcelleclercqi Lehrer, 1975 Lehrer 1975 ; Pape 1996 , MA , Azrou; Verves 2019 Sarcophaga ( Thyrsocnema ) belgiana (Lehrer, 1976) Lehrer 1976 ; Pape 1996 Sarcophaga ( Thyrsocnema ) sp. 61 Ebejer et al. 2019 [as incisilobata , misidentification], Rif , Tahaddart (8 m) New records for Morocco Macronychia lemariei Jacentkovský, 1941 Atlantic plain: Rabat, Forêt de Maâmora, 100 m, 25–26.iv.1989, 1♂1♀, Zoological Museum of Copenhagen Expedition (NMHD). Sarcophaga ( Helicophagella ) novercoides Böttcher, 1913 Rif: Ouezzane, 300 m, 21–22.iv.1989, 1♂, Zoological Museum of Copenhagen Expedition (NMHD). High Atlas: Marrakech, Ouirgane, 1000 m, 1–9.iv.1997 Mai, 1♂, C. Kassebeer leg. (NMHD). Anti Atlas: 30 km NW Aoulouz, 1400 m, 10.iv.1989, 1♂, Zoological Museum of Copenhagen Expedition (NMHD). Sarcophaga ( Heteronychia ) javita (Peris, González-Mora & Mingo, 1998) Anti Atlas: Agadir, S Oulma, 30°31'N, 9°09'W , 200 m, 21.iv.1997, 1♂, C. Kassebeer leg. (NMHD). Sarcophaga ( Liopygia ) argyrostoma (Robineau-Desvoidy, 1830) High Atlas: Marrakech, Tagadirt, Ouirgane, 1000 m, 1.x.1994, 1♀, C. Kassebeer leg. (NMHD). Sarcophaga ( Liosarcophaga ) dux Thomson, 1869 Sahara: Erfoud, Rissani area, 900 m, 13–14.iv.1989, Zoological Museum of Copenhagen Expedition ( NHMD ). TACHINIDAE K. Kettani, P. Cerretti, H.-P. Tschorsnig Number of species: 147 . Expected: 200 Faunistic knowledge of the family in Morocco: poor Dexiinae Dexiini Billaea Robineau-Desvoidy, 1830 Billaea lata (Macquart, 1849) = Rhynchodinera lata Macquart, in Séguy 1930a : 143 Séguy 1930a , MA , Aharmoumou, Camp Boulhout, Sidi Bettache, Aïn Sferguila, Meknès; Mouna 1998 ; AP (Mehdia) – MISR ; AP (Essaouira, 4 km E Ounara), HA (Marrakech, Lakhdar, N Demnate) – PCPT Estheria Robineau-Desvoidy, 1830 Estheria atripes Villeneuve, 1920 Cerretti and Tschorsnig 2012 Estheria iberica Tschorsnig, 2003* MA Estheria nigripes (Villeneuve, 1920) Cerretti and Tschorsnig 2012 ; MA (Béni Mellal, El Ksiba), AA (Agadir, Oulma) – PCPT Estheria picta (Meigen, 1826) 62 Moutia 1940 Zeuxia Meigen, 1826 Zeuxia aberrans (Loew, 1847) = Zeuxia nigripes Meigen, in Séguy 1941d : 23 Brémond 1938 ; Séguy 1941d , AP , Rabat, MA , Volubilis, AA , Agadir; IOBC-List 11; Mesnil 1980; Mouna 1998 ; Tschorsnig 2017; AA (10 km NW Aït-Baha) – PCPT Dufouriini Dufouria Robineau-Desvoidy, 1830 Dufouria nigrita (Fallén, 1810) Ebejer et al. 2019 , AP , Larache (Lower Loukous saltmarsh, 2 m); MA (Ouzoud) – PCPT Voriini Athrycia Robineau-Desvoidy, 1830 Athrycia trepida (Meigen, 1824)* MA Cyrtophloeba Rondani, 1856 Cyrtophloeba ruricola (Meigen, 1824) = Plagia ruricola Meigen, in Séguy 1935a : 120, in Rungs 1940 : 14 Séguy 1935a , MA , Ifrane; Rungs 1940 , MA (Cédraie); Mouna 1998 ; HA (Tizi-n'Test), AA (Taroudant) – PCPT Eriothrix Meigen, 1830 Eriothrix apennina (Rondani, 1862) Herting and Dely-Draskovits 1993 ; Koçak and Kemal 2010 Eriothrix rufomaculata (De Geer, 1776)* MA , HA Hypovoria Villeneuve, 1913 Hypovoria hilaris Villeneuve, 1912 Séguy 1935a , AP , Oued Beth; Séguy 1953a , AP , Sehoul; Mouna 1998 ; AA (10 km SE Aït-Ourir) – PCPT Hypovoria pilibasis (Villeneuve, 1922) Zeegers 2010; HA (Tizi-n'Test), AA (Taroudant) – PCPT Kirbya Robineau-Desvoidy, 1830 Kirbya moerens (Meigen, 1830)* MA Nanoplagia Villeneuve, 1929 Nanoplagia sinaica (Villeneuve in Hermann & Villeneuve 1909) Cerretti 2009; Grabener 2017 ; HA (Marrakech, 8 km N Ouirgane), AA (40 km SW Ouarzazate, 10 km SW Tazenakht, NE Agadir, 12 km W Oulma) – PCPT Periscepsia Gistel, 1848 Periscepsia meyeri (Villeneuve, 1930) Ebejer et al. 2019 , Rif , Adrou ( PNPB , 556 m) Stomina Robineau-Desvoidy, 1830 Stomina caliendrata (Rondani, 1862) = Morphomyia caliendrata Rondani, in Séguy 1930a : 143 Séguy 1930a , MA , from M'Rirt to Hajeb, HA , Kasba Taguendaft (Gounfada); Mouna 1998 ; HA (Massif Toubkal) – PCPT Thelaira Robineau-Desvoidy, 1830 Thelaira haematodes (Meigen, 1824) 63 = Phoenicella haematodes Meigen: Séguy 1930a : 142 Séguy 1930a , HA , Arround; Mouna 1998 Uclesia Girschner, 1901 Uclesia fumipennis Girschner, 1901 Séguy 1934b ; Herting and Dely-Draskovits 1993 ; Mouna 1998 ; HA (Marrakech) – MISR Voria Robineau-Desvoidy, 1830 Voria ruralis (Fallén, 1810) Jourdan 1935c Wagneria Robineau-Desvoidy, 1830 Wagneria dilatata Kugler, 1977 Kugler 1977 Exoristinae Acemyini Ceracia Rondani, 1865 Ceracia mucronifera Rondani, 1865 = Myothyria benoisti (Mesnil), in Mesnil 1959 : 20 Mesnil 1959 , MA , Forêt Maâmora near Tiflet; Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 Blondeliini Compsilura Bouché, 1834 Compsilura concinnata (Meigen, 1824) IOBC-list 1 (1956); Hérard and Fraval 1980 Istocheta Rondani, 1859 Istocheta cinerea (Macquart, 1850) Herting 1960 Istocheta longicornis (Fallén, 1810) = Latigena longicornis Fallén, in Séguy 1953a : 91 Séguy 1953a , AP , Forêt Zaers Lomachantha Rondani, 1859 Lomachantha parra Rondani, 1859 Efetov and Tarmann 1999 Robinaldia Herting, 1983 Robinaldia angustata (Villeneuve, 1933) Herting and Dely-Draskovits 1993 ; Tschorsnig and Herting 1994 Zaira Robineau-Desvoidy, 1830 Zaira cinerea (Fallén, 1820) Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 8 m) Eryciini Alsomyia Brauer & Bergenstamm, 1891 Alsomyia olfaciens (Pandellé, 1896) IOBC-List 12 (1993) Amphicestonia Villeneuve, 1939 Amphicestonia dispar (Villeneuve, 1922) Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 ; MA (Ifrane) – PCPT Aplomyia Robineau-Desvoidy, 1830 Aplomyia confinis (Fallén, 1820) Ebejer et al. 2019 , Rif , Dardara (484 m) Carcelia Robineau-Desvoidy, 1830 Carcelia dilaticornis Mesnil, 1950 Mesnil 1950 Carcelia iliaca (Ratzeburg, 1840)63 Mouna 1998 Carcelia lucorum (Meigen, 1824)* HA Drino Robineau-Desvoidy, 1863 Drino atropivora (Robineau-Desvoidy, 1830) = Sturmia atropivora Robineau-Desvoidy, in Bléton and Fieuzet 1939 : 64 De Lépiney and Mimeur 1932; Bléton and Fieuzet 1939 , MA , Fès; Mouna 1998 ; Tschorsnig 2017; AP (Rabat), MA (Bel Lakssiri) – MISR Drino galii (Brauer & Bergenstamm, 1891)* HA , AA Drino gilva (Hartig, 1838)63 = Sturmia gilva Hartig Mouna 1998 – MISR (no locality given) Drino imberbis (Wiedemann, 1830)63 Rungs 1954 ; Grabener 2017 Drino inconspicua (Meigen, 1830) Séguy 1935a , AP , Sehoul (Rabat); Bléton and Fieuzet 1939 , MA , Dayat Achleff; Mouna 1998 ; MA (Meknès, Béni Mellal) – MISR Drino maroccana Mesnil, 1951 De Lépiney 1930 ; De Lépiney and Mimeur 1932 (probably misidentified as Sturmia inconspicua ); Mesnil 1951 ; Herting and Dely-Draskovits 1993 ; Ziegler 2011 Drino triplaca Herting, 1979 Herting 1979 , AP , Rabat Drino vicina (Zetterstedt, 1849) = Sturmia vicina Zetterstedt, 1849 De Lépiney and Mimeur 1932; Bouclier-Maurin 1923 ; AP (Rabat) – MISR Gymnophryxe Villeneuve, 1922 Gymnophryxe carthaginiensis (Bischof, 1900) Mesnil 1956 Nilea Robineau-Desvoidy, 1863 Nilea innoxia Robineau-Desvoidy, 1863 Bléton and Fieuzet 1939 Phryxe Robineau-Desvoidy, 1830 Phryxe caudata (Rondani, 1859) Biliotti 1956 ; El Yousfi 1994 ; IOBC-list 11 (1989) Phryxe setifacies (Villeneuve, 1910) IOBC-list 11 (1989); IOBC-list 12 (1993) Phryxe vulgaris (Fallén, 1810) Séguy 1953a , MA , Tamrabta (1700 m) – PCPT ( HA , Marrakech, Imlil, S Asni) Ptesiomyia Brauer & Bergenstamm, 1893 Ptesiomyia microstoma Brauer & Bergenstamm, 1893 Séguy 1953a , AP , Rabat; EM (Mte des Béni Snassen, Taforalt), MA (Béni Mellal, Bin-el-Ouidane; Meknès, Ifrane ( NPI )), HA (Marrakech, Oukaimeden) – PCPT Senometopia Macquart, 1834 Senometopia separata (Rondani, 1859) Hérard and Fraval 1980 Tryphera Meigen, 1838 Tryphera lugubris (Meigen, 1824) IOBC-List 1 (1956) Ethillini Atylomyia Brauer, 1898 Atylomyia albifrons Villeneuve, 1911 = Atylomyia rungsi Mesnil, in Mesnil 1962: 778 Mesnil 1962, AA (near Agadir), Aït Melloul; Herting and Dely-Draskovits 1993 Exoristini Bessa Robineau-Desvoidy, 1863 Bessa parallela (Meigen, 1824) Séguy 1935a (probably misidentified as Bessa selecta ); Tschorsnig 2017 Chetogena Rondani, 1856 Chetogena filipalpis Rondani, 1859 Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 8 m); MA (Fès, Sidi Harazem, Ifrane, Forêt de Cèdres) – PCPT Chetogena mageritensis (Villeneuve & Mesnil, 1936) Herting and Dely-Draskovits 1993 Chetogena media Rondani, 1859* MA Chetogena nigrofasciata (Strobl, 1902) = Chetogena repanda (Mesnil, 1939), in Herting and Dely-Draskovits 1993 (type locality: Skel): 17 Gheibi et al. 2010 Chetogena obliquata (Fallén, 1810) De Lépiney and Mimeur 1932; Herting 1960; IOBC-list 12 (1993); HA (Marrakech, Oukaimeden) – PCPT Exorista Meigen, 1803 Exorista deligata Pandellé, 1896 Mesnil 1946 , AP , Sidi Taibi near Kénitra; Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 ; Gheibi et al. 2010 Exorista grandis (Zetterstedt, 1844) Ebejer et al. 2019 , Rif , Dardara (484 m) Exorista larvarum (Linnaeus, 1758) Mouna 1998 Exorista nova (Rondani, 1859) Tschorsnig 2017 Exorista rendina (Herting, 1975)* AA Exorista segregata (Rondani, 1859) Mouna 1998 ; AA (Agadir, Oulma) – PCPT Goniini Anurophylla Villeneuve, 1938 Anurophylla aprica (Villeneuve, 1912)* MA Baumhaueria Meigen, 1838 Baumhaueria goniaeformis (Meigen, 1824) De Lépiney and Mimeur 1932; AP (Maâmora) – MISR ; HA (Marrakech, Oukaimeden) – PCPT Blepharipa Rondani, 1856 Blepharipa pratensis (Meigen, 1824)* HA Ceratochaetops Mesnil, 1970 Ceratochaetops triseta (Villeneuve, 1922) = Ceratochoeta triseta Villeneuve, in Rungs 1940 : 15 Rungs 1940 , MA (Cédraie); Mouna 1998 ; MA (Khénifra, El-Herri, Ifrane ( NPI ), Meknès), HA (Marrakech, 8 km N Ouirgane) – PCPT Ceromasia Rondani, 1856 Ceromasia rubrifrons (Macquart, 1834) IOBC-list 11 (1989); IOBC-list 12 (1993); HA (Marrakech, Ouirgane, Tagadirt, S Asni) – PCPT Clemelis Robineau-Desvoidy, 1863 Clemelis pullata (Meigen, 1824) IOBC-list 13 (1997) Gaedia Meigen, 1838 Gaedia connexa Meigen, 1824 Séguy 1953a , EM , Berkane Gonia Meigen, 1803 Gonia aterrima Tschorsnig, 1991 Tschorsnig 1991 Gonia atra Meigen, 1826 Séguy 1930a , MA , Tizi-n'Bouftene, between Azrou and Ras el Ma, Forêt Azrou, HA , Arround (Skoutana), Jebel Likount, Asni; Mouna 1998 ; Grabener 2017 ; MA (Tighassaline, El-Herri, Aïn Leuh-Tagounit, Meknès, Ifrane ( NPI )), HA (40 km SW Ouarzazate, Marrakech, Ouirgane, Lakhdar, N Demnate, Oukaimeden), AA (Agadir, Oulma) – PCPT Gonia bimaculata Wiedemann, 1819 = Gonia cilipeda Rondani, in Séguy 1953a : 91 Séguy 1930a , AP , Rabat, MA , Tizi-s'Tkrine, Tizi-n'Bouftene, between Azrou and Ras el Ma, Forêt Azrou, Berkane, HA , Arround (Skoutana), Jebel Likount, Asni, Ouaouzert (Glaoua); Séguy 1953a , AP , Temara; AP (Rabat) – MISR ; HA (S Asni Ouirgane, Marrakech), AA (80 km N Taroudant, Aoulouz), AA (15 km NW Zagora) – PCPT Gonia capitata (De Geer, 1776) Mouna 1998 ; MA (Ifrane) – MISR Gonia maculipennis Egger, 1862* MA Gonia ornata Meigen, 1826 Séguy 1953a , AP , Rabat, MA , Ifrane, SA , Kelaâ M'Goum; MA (Ifrane, Forêt de Cèdres), HA (Oukaimeden (2600 m), Marrakech) – PCPT Gonia vacua Meigen, 1826* MA Pales Robineau-Desvoidy, 1830 Pales pavida (Meigen, 1824) IOBC-list 1 (1956); Cerretti 2005 ; MA (Fès, Sidi Harazem) – PCPT Platymya Robineau-Desvoidy, 1830 Platymya antennata (Brauer & Bergenstamm, 1891) Efetov and Tarmann 1999 Pseudogonia Brauer and von Bergenstamm, 1889 Pseudogonia rufifrons (Wiedemann, 1830) De Lépiney and Mimeur 1932; IOBC-list 1 (1956); Mouna 1998 ; Tschorsnig 2017; Grabener 2017 ; AA (S Tafraoute, Aït Mansur, Agadir, Oulma) – PCPT Sturmia Robineau-Desvoidy, 1830 Sturmia bella (Meigen, 1824) Stefanescu et al. 2012 ; Tschorsnig 2017 Winthemiini Nemorilla Rondani, 1856 Nemorilla maculosa (Meigen, 1824) Kozlovsky and Rungs 1933 ; Brémond and Rungs 1938 [as N. floralis ; probable misidentification]; IOBC list 1 (1956); Mouna 1998 ; EM (Oujda, Col de Jerada) – PCPT Phasiinae Cylindromyiini Besseria Robineau-Desvoidy, 1830 Besseria lateritia (Meigen, 1824)* HA Cylindromyia Meigen, 1803 Cylindromyia bicolor (Olivier, 1812) Séguy 1930a , AP , Rabat; Mouna 1998 Cylindromyia brassicaria (Fabricius, 1775) Séguy 1930a , EM , Soufouloud, MA , Aharmoumou, Berrechid, Meknès, Sidi Bettache, Berkane, HA , Tenfecht, Ouaounzert, Marrakech, Asni; Séguy 1935a , AP , Gharb; Séguy 1941a , HA , Jebel Ayachi; Séguy 1949, SA , Guelmim; Dupuis 1963 ; Mouna 1998 ; MA (Sefrou) – MISR ; AP (Essaouira, 4 km E Ounara), HA (10 km W Chichaoua, Oukaimeden), AA (140 km E Agadir, Aoulouz, Tizi-n'Tichka) – PCPT Cylindromyia intermedia Meigen, 1824 Becker and Stein 1913 , Rif , Tanger; HA (Ouirgane, Imlil, Tizi-n'Test), AA (10 km SE Ouarzazate (oasis), Taroudant) – PCPT Cylindromyia maroccana Tschorsnig, 1997 Tschorsnig 1997 , HA , Ouirgane, Tagadirt (1000 m) Cylindromyia pilipes Loew, 1844 Becker and Stein 1913 , Rif , Tanger; Herting and Dely-Draskovits 1993 ; Gilasian et al. 2013 Phania Meigen, 1824 Phania albisquama (Villeneuve, 1924)* MA Gymnosomatini Clytiomya Rondani, 1861 Clytiomya continua (Panzer, 1798) = Clytiomyia dalmatica Robineau-Desvoidy, in Séguy 1935a : 119 Séguy 1935a , AP , Gharb; Mouna 1998 ; AP (Rabat) – MISR Clytiomya sola (Rondani, 1861) Séguy 1935a ; Dupuis 1963 ; MA (Khénifra, Tighassaline) – PCPT Ectophasia Townsend, 1912 Ectophasia crassipennis (Fabricius, 1794) = Phasia crassipennis Fabricius, in Séguy 1930a : 141 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 Eliozeta Rondani, 1856 Eliozeta helluo (Fabricius, 1805) = Clytiomyia helluo Fabricius, in Séguy 1935: 119 Séguy 1930a , MA , Meknès (Aïn Sferguila); Jourdan 1935; Séguy 1935a , AP , Gharb; Thompson 1950 ; Dupuis 1963 ; Mouna 1998 ; Tschorsnig 2017 Gymnosoma Meigen, 1803 Gymnosoma carpocoridis Dupuis, 1961 Dupuis 1963 ; Herting and Dely-Draskovits 1993 Gymnosoma clavatum (Rohdendorf, 1947) Dupuis 1963 ; Grabener 2017 ; MA (Meknès, Ifrane ( NPI )), AA (140 km E Agadir, Aoulouz), AA (80 km S Zagora, Oued Draa, Mhamid) – PCPT Gymnosoma dolycoridis Dupuis, 1960 Dupuis 1963 ; MA (Fès, Sidi Harazem) – PCPT Gymnosoma rotundatum Linnaeus, 1758 64 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Mogador, MA , Tizi-s'Tkrine, Sidi Bettache, Moulay Aïn Djemine, HA , Asni; Séguy 1934b ; Séguy 1935a ; Thompson 1950 ; Mouna 1998 ; AP (Mogador), MA (Maghrawa) – MISR ; MA (Meknès, Ifrane ( NPI )) – PCPT Gymnosoma rungsi (Mesnil, 1952) = Rhodogyne rungsi (Mesnil), in Mesnil 1952 : 151 Mesnil 1952 , AP , Rabat; Dupuis 1963 ; Herting and Dely-Draskovits 1993 Leucostomatini Clairvillia Robineau-Desvoidy, 1830 Clairvillia biguttata (Meigen, 1824)* HA Dionomelia Kugler, 1978 Dionomelia hennigi Kugler, 1978* SA Leucostoma Meigen, 1803 Leucostoma abbreviatum Herting, 1971 Ziegler 2012 Leucostoma crassum Kugler, 1966* HA Leucostoma obsidianum (Wiedemann, 1830) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Leucostoma tetraptera (Meigen, 1824) Dupuis 1953 ; Ebejer et al. 2019 , Rif , Barrage Smir (27 m) Weberia Robineau-Desvoidy, 1830 Weberia digramma (Meigen, 1824)* AA Phasiini Elomya Robineau-Desvoidy, 1830 Elomya lateralis (Meigen, 1824) Séguy 1930a , AP , From Zarjoulea to Larache, MA , Berkane; Dupuis 1952 ; Dupuis 1963 ; Herting and Dely-Draskovits 1993 ; Mouna 1998 ; Cerretti and Ziegler 2004 ; MA (Khénifra, Tighassaline, Meknès, Ifrane ( NPI )), HA (Marrakech, Ouirgane) – PCPT Phasia Latreille, 1804 Phasia mesnili (Draber-Monko, 1965) Sun and Marshall 2003 , HA ; AP (10 km E Essaouira), AA (S Tafraoute, Aït Mansur, S Aït-Baha) – PCPT Phasia obesa (Fabricius, 1798) Sun and Marshall 2003 , HA , Asni Phasia pusilla Meigen, 1824 Dupuis 1963 ; Sun and Marshall 2003 , MA Phasia subcoleoptrata (Linnaeus, 1767) Dupuis 1963 ; Herting and Dely-Draskovits 1993 ; Sun and Marshall 2003 , MA ; Cerretti and Ziegler 2004 Phasia venturii (Draber-Monko, 1965) Sun and Marshall 2003 , HA , Asni; AP (Essaouira, 4 km E Ounara), AA (11 km NW Taliouine, 10 km SE Aït-Ourir) – PCPT Trichopodini Trichopoda Berthold, 1827 Trichopoda pennipes (Fabricius, 1794) Ebejer et al. 2019 , Rif , Tahaddart (8 m) Xystini Xysta Meigen, 1824 Xysta holosericea (Fabricius, 1805)* HA Tachininae Graphogastrini Graphogaster Rondani, 1868 Graphogaster vestita Rondani, 1868* MA Phytomyptera Rondani, 1845 Phytomyptera nigrina (Meigen, 1824) = Phytomyptera nitidiventris Rondani, in Bléton and Fieuzet 1939 : 64 Bléton and Fieuzet 1939 ; Mouna 1998 Leskiini Aphria Robineau-Desvoidy, 1830 Aphria longirostris (Meigen, 1824) Ebejer et al. 2019 , Rif , Jnane Niche (46 m) Bithia Robineau-Desvoidy, 1863 Bithia demotica (Egger, 1861) Tschorsnig and Bläsius 2001 ; IOBC-list 14 (2005); Tschorsnig 2017 Bithia modesta (Meigen, 1824) Tschorsnig and Bläsius 2001 ; Tschorsnig 2017 Linnaemyini + Ernestiini Gymnochaeta Robineau-Desvoidy, 1830 Gymnochaeta viridis Fallén, 1810 Séguy 1930a , HA , Arround (Skoutana); Mouna 1998 Linnaemya Robineau-Desvoidy, 1830 Linnaemya soror Zimin, 1954* MA , HA , AA Loewia Egger, 1856 Loewia setibarba Egger, 1856 Becker and Stein 1913 , Rif , Tanger Panzeria Robineau-Desvoidy, 1830 Panzeria castellana (Strobl, 1906)* HA Panzeria nemorum (Meigen, 1824)* MA Zophomyia Macquart, 1835 Zophomyia temula (Scopoli, 1763) Séguy 1930a , AP , Casablanca, MA , Meknès; Mouna 1998 ; MA (Khénifra, Tighassaline, Meknès, Ifrane ( NPI )) – PCPT Macquartiini Macquartia Robineau-Desvoidy, 1830 Macquartia chalconota (Meigen, 1824) Ebejer et al. 2019 , Rif , Smir lagoon; HA (Marrakech, Ouirgane, Tizi-n'Test), AA (Taroudant) – PCPT Macquartia macularis Villeneuve, 1926 Herting and Dely-Draskovits 1993 Macquartia tessellum (Meigen, 1824) = Macquartia brevicornis Macquart, in Séguy 1941d : 23 Séguy 1941d , MA , Meknès, HA , Tizi-n'Test; Mouna 1998 ; HA (Imlil, S Asni, Tizi-n'Test), AA (Taroudant) – PCPT Megaprosopini Microphthalma Macquart, 1844 Microphthalma europaea Egger, 1860 Ebejer et al. 2019 , AA , Ziz river (30 km N of Erfoud, 894 m) Minthoini Hyperaea Robineau-Desvoidy, 1863 Hyperaea femoralis (Meigen, 1824) Herting and Dely-Draskovits 1993 Mintho Robineau-Desvoidy, 1830 Mintho compressa (Fabricius, 1787) Walker 1849 ; Herting and Dely-Draskovits 1993 Mintho rufiventris (Fallén, 1817) = Mintho praeceps (Scopoli, 1763), in Séguy 1930a : 143; Séguy 1953a : 91 Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès; Séguy 1953a , SA , El Aïoun du Draa; Séguy 1949a , AA , Tata; Mouna 1998 ; Dawah 2011 ; AP (Rabat, Salé), MA (Meknès) – MISR ; MA (Meknès, Ifrane ( NPI )), AA (11 km NW Taliouine) – PCPT Minthodes Brauer & Bergenstamm, 1889 Minthodes numidica Villeneuve, 1932* AA Minthodes setifacies Mesnil, 1939 = Minthodes ( Myxominthodes ) setifacies Mesnil, in Mesnil 1939 : 211 Mesnil 1939 , MA , Forêt Azrou; Herting and Dely-Draskovits 1993 Plesina Meigen, 1838 Plesina phalerata (Meigen, 1824) Herting and Dely-Draskovits 1993 ; Cerretti and Tschorsnig 2008 Pseudomintho Brauer & Bergenstamm, 1889 Pseudomintho diversipes (Strobl, 1889) Ebejer et al. 2019 , Rif , Moulay Abdelsalam ( PNPB , 965 m); AP (Essaouira, 4 km E Ounara) – PCPT Siphonini Actia Robineau-Desvoidy, 1830 Actia infantula (Zetterstedt, 1844) Ebejer et al. 2019 , Rif , Tanger (Douar Dakchire forest, 320 m) Peribaea Robineau-Desvoidy, 1863 Peribaea apicalis Robineau-Desvoidy, 1863 Ebejer et al. 2019 , Rif , Dardara (484 m) Peribaea tibialis (Robineau-Desvoidy, 1851) Draber-Mońko 2011 Siphona Meigen, 1803 Siphona geniculata (De Geer, 1776) 65 Séguy 1930a , HA ; Mouna 1998 ; HA (Vallée Oued N'fis) – MISR Siphona maroccana Cerretti & Tschorsnig, 2007 Cerretti and Tshorsnig 2007, HA , Asif Mellah, W Tizi-n'Tichka Siphona variata Andersen, 1982 Ebejer et al. 2019 , Rif , Sidi Yahia Aârab (377 m), Oued Kbir ( PNPB , 157 m) Tachinini Germaria Robineau-Desvoidy, 1830 Germaria barbara Mesnil, 1963* HA Peleteria Robineau-Desvoidy, 1830 Peleteria ruficornis (Macquart, 1835)* HA , AA Tachina Meigen, 1803 Tachina corsicana (Villeneuve, 1931)* HA , AA Tachina fera (Linnaeus, 1761) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, Forêt Tiffert, Sidi Bettache, HA , Arround (Skoutana), Jebel Likount; Séguy 1953a , MA , Ifrane (1650 m); Mouna 1998 ; Rif (fir forest of Talassemtane), AP (Dradek) – MISR ( MA , Meknès); MA (Ifrane ( NPI )), Béni Mellal, Bin-el-Ouidane), HA (Marrakech, Ouirgane) – PCPT Tachina magnicornis (Zetterstedt, 1844) Séguy 1930a , MA , Ras el Ksar, Aïn Leuh, Sidi Bettache; Mouna 1998 ; MA (Béni Mellal, El Ksiba, 5 km N) – PCPT Tachina praeceps Meigen, 1824 HA , AA Triarthriini Lissoglossa Villeneuve, 1912 Lissoglossa bequaerti Villeneuve, 1912 Herting and Dely-Draskovits 1993 New records for Morocco The data added under the abbreviation "PCPT" (for "personal communication Hans-Peter Tschorsnig") are based on (unpublished) material which was identified by HPT for several collectors (M. Hauser, M. Hradský, C.F. Kassebeer, U. Koschwitz, J.A.W. Lucas, G. Miksch, H. and T. v. Oorschot, C. Schmid-Egger, M. Schwarz, K. Špatenka, V. Vrabec) during the last ~ 30 years. Usually only a few duplicate specimens were retained in the collection of SMNS . The main part was sent back to the collectors, but the data were noted by HPT on handwritten lists. Estheria iberica Tschorsnig, 2003 Middle Atlas: Ifrane, National Park of Ifrane, 19.ix.1989, K. Špatenka leg, 1 specimen, PCPT. Athrycia trepida (Meigen, 1824) Middle Atlas: Meknès; Ifrane, National Park of Ifrane, 22.v.1995, C. Kassebeer leg., 1 specimen, PCPT. Eriothrix rufomaculata (De Geer, 1776) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. High Atlas: Marrakech, Oukaimeden, 19.v.1995, C. Kassebeer leg., 7 specimens, PCPT. Kirbya moerens (Meigen, 1830) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. Carcelia lucorum (Meigen, 1824) High Atlas: Marrakech, Imlil, S Asni, 24.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Drino galii (Brauer & Bergenstamm, 1891) High Atlas: Marrakech, Ouirgane, 24.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Anti Atlas: 11 km NW Taliouine; Agadir, Ameskroud, 17.v.1995, C. Kassebeer leg., 2 specimens, PCPT. Chetogena media Rondani, 1859 Middle Atlas: Béni Mellal, El Ksiba, 30.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Exorista rendina (Herting, 1975) Anti Atlas: 11 km NW Taliouine, 15.iii.1997, M. Hauser leg., 1 male in SMNS ; 10 km NE Tafraoute, 14.iii.1997, G. Miksch leg., 1 male in SMNS , PCPT. Anurophylla aprica (Villeneuve, 1912) Middle Atlas: Béni Mellal, Afourer, 28.iii.1995, C. Kassebeer leg., 1 female in SMNS , PCPT. Blepharipa pratensis (Meigen, 1824) High Atlas: Tizi-n'Test, 2000 m, 21.v.1995, M. Hauser leg., 2 specimens, PCPT. Anti Atlas: Taroudant, PCPT. Gonia maculipennis Egger, 1862 Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 female in SMNS , PCPT. Gonia vacua Meigen, 1826 Middle Atlas: Meknès; Ifrane, National Park of Ifrane, 29.iii.1995 and 22.v.1995, C. Kassebeerleg., 2 specimens, PCPT. Besseria lateritia (Meigen, 1824) High Atlas: SE Asni, Imlil, 23.v.1995, M. Hauser leg., 2 specimens; Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Phania albisquama (Villeneuve, 1924) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec and L. Vrabcová leg., 1 specimen, PCPT. Clairvillia biguttata (Meigen, 1824) High Atlas: Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg, 1 specimen, PCPT. Dionomelia hennigi Kugler, 1978 SA : Boujdour, 8.v.1999, V. Vrabec leg, 1 male in SMNS , PCPT. Leucostoma crassum Kugler, 1966 High Atlas: Tizi-n'Test, pass 23.vi.1996, U. Koschwitz leg., 1 male in SMN, PCPT. Weberia digramma (Meigen, 1824) Anti Atlas: 10 km NW Aït-Baha, PCPT. Xysta holosericea (Fabricius, 1805) High Atlas: Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg, 1 specimen, PCPT. Linnaemya soror Zimin, 1954 Middle Atlas: Béni Mellal, El Ksiba, 5 km N; Béni Mellal, Afourer; Khénifra, Tighassaline; Meknès; National Park of Ifrane. High Atlas: Marrakech, Ouirgane; Marrakech, Tagaddirt, S Asni; Marrakech, Lakhdar, N Demnate. Anti Atlas: 11 km NW Taliouine, all C. Kassebeer leg., 57 specimens (collected between 25.iii. and 23.v.1995), PCPT. Panzeria castellana (Strobl, 1906) High Atlas: Marrakech, Ouirgane, 26.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Panzeria nemorum (Meigen, 1824) Middle Atlas: Meknès; National Park of Ifrane, 22.v.1995, C. Kassebeer leg., 1 specimen, PCPT. Graphogaster vestita Rondani, 1868 Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. Minthodes numidica Villeneuve, 1932 Anti Atlas: S Aït-Baha, PCPT. Germaria barbara Mesnil, 1963 High Atlas: S Tizi-n'Test, 1900 m, PCPT. Peleteria ruficornis (Macquart, 1835) High Atlas: Marrakech, Ouirgane; Marrakech, Tagaddirt, S Asni; Tizi-n'Test. Anti Atlas: Taroudant, all C. Kassebeer leg., 6 specimens (collected between 28.ix.1994 and 1.iv.1995), PCPT. Tachina corsicana (Villeneuve, 1931) High Atlas: Marrakech, Oukaimeden, 19.v.1995, C. Kassebeer leg., 1 specimen; Tizi-n'Test. Anti Atlas: Taroudant, 21.v.1995, M. Hauser leg., 2 specimens, PCPT. Tachina praeceps Meigen, 1824 High Atlas: Marrakech, Oukaimeden, 2500 m, 27.vi.1987, M. Schwarz leg., 1 specimen; Tizi-n'Test. Anti Atlas: Taroudant, 29.vi.1987, M. Schwarz leg., 1 specimen, PCPT. CALLIPHORIDAE K. Kettani, K. Rognes Number of species: 8 . Expected: 10 Faunistic knowledge of the family in Morocco: moderate Calliphorinae Bellardia Robineau-Desvoidy, 1863 Bellardia maroccana (Villeneuve, 1941) = Onesia maroccana Villeneuve, in Villeneuve 1932 : 123 Villeuneuve 1932b; Schumann 1974 , AP , Aïn Diab (Casablanca); Verves 2004 ; Koçak and Kemal 2010 Bellardia mascariensis (Villeneuve, 1926) Schumann 1974 , HA , Marrakech; Verves 2004 ; Koçak and Kemal 2010 Calliphora Robineau-Desvoidy, 1863 Calliphora vicina Robineau-Desvoidy, 1830 = Calliphora erythrocephala Meigen, in Séguy 1930a : 154 Séguy 1930a ; Maurice 1947 , Rif , Tanger; AP (Casablanca, Rabat), MA (Azrou, Ifrane) – MNHN , MISR Calliphora vomitoria (Linnaeus, 1758) Séguy 1930a ; Maurice 1947 , Rif , Tanger; Kurahashi 1971 ; Kurahashi and Magpayo 2000 ; Mouna 1998 ; AP (Rabat), HA – MISR , NHMD Chrysomyinae Chrysomya Robineau-Desvoidy, 1863 Chrysomya albiceps (Wiedemann, 1819) Maurice 1947 ; Séguy 1949a , HA , Alnif; Gonzales-Mora and Peris 1988; Mouna 1998 ; Verves 2003 ; Grabener 2017 ; Dawah et al. 2019 ; EM (Oujda) – MISR , AP (Rabat) – MNHN Luciliinae Lucilia Robineau-Desvoidy, 1863 Lucilia sericata (Meigen, 1824) Séguy 1930a , Rif , Tanger, EM , Berkane (1350–1400 m), MA , Meknès, Berrechid, HA , Tizi-n'Test, Goundafa (Jebel Imdress, 2000–2450 m); Séguy 1953a , AP , Rabat; Mouna 1998 ; Grabener 2017 ; AP (Dradek, Salé), MA (Azrou, Volubilis) – MISR , AP (Temara), MA (Ifrane) – MNHN Melanomyinae Melinda Robineau-Desvoidy, 1863 Melinda gentilis (Robineau-Desvoidy, 1830) MA (Ifrane) – MNHN Melinda viridicyanea (Robineau-Desvoidy, 1830) MA (Ifrane) – MNHN Calliphorinae Bellardia Robineau-Desvoidy, 1863 Bellardia maroccana (Villeneuve, 1941) = Onesia maroccana Villeneuve, in Villeneuve 1932 : 123 Villeuneuve 1932b; Schumann 1974 , AP , Aïn Diab (Casablanca); Verves 2004 ; Koçak and Kemal 2010 Bellardia mascariensis (Villeneuve, 1926) Schumann 1974 , HA , Marrakech; Verves 2004 ; Koçak and Kemal 2010 Calliphora Robineau-Desvoidy, 1863 Calliphora vicina Robineau-Desvoidy, 1830 = Calliphora erythrocephala Meigen, in Séguy 1930a : 154 Séguy 1930a ; Maurice 1947 , Rif , Tanger; AP (Casablanca, Rabat), MA (Azrou, Ifrane) – MNHN , MISR Calliphora vomitoria (Linnaeus, 1758) Séguy 1930a ; Maurice 1947 , Rif , Tanger; Kurahashi 1971 ; Kurahashi and Magpayo 2000 ; Mouna 1998 ; AP (Rabat), HA – MISR , NHMD Chrysomyinae Chrysomya Robineau-Desvoidy, 1863 Chrysomya albiceps (Wiedemann, 1819) Maurice 1947 ; Séguy 1949a , HA , Alnif; Gonzales-Mora and Peris 1988; Mouna 1998 ; Verves 2003 ; Grabener 2017 ; Dawah et al. 2019 ; EM (Oujda) – MISR , AP (Rabat) – MNHN Luciliinae Lucilia Robineau-Desvoidy, 1863 Lucilia sericata (Meigen, 1824) Séguy 1930a , Rif , Tanger, EM , Berkane (1350–1400 m), MA , Meknès, Berrechid, HA , Tizi-n'Test, Goundafa (Jebel Imdress, 2000–2450 m); Séguy 1953a , AP , Rabat; Mouna 1998 ; Grabener 2017 ; AP (Dradek, Salé), MA (Azrou, Volubilis) – MISR , AP (Temara), MA (Ifrane) – MNHN Melanomyinae Melinda Robineau-Desvoidy, 1863 Melinda gentilis (Robineau-Desvoidy, 1830) MA (Ifrane) – MNHN Melinda viridicyanea (Robineau-Desvoidy, 1830) MA (Ifrane) – MNHN OESTRIDAE K. Kettani, T. Pape Number of species: 10 . Expected: 12 Faunistic knowledge of the family in Morocco: moderate Gastrophilinae Gasterophilus Leach, 1817 Gasterophilus flavipes (Olivier, 1811) Séguy 1928d , EM , Haute Moulouya; Séguy 1930a , EM , Itzer (Haute Moulouya); Mouna 1998 ; Li et al. 2019 Gasterophilus haemorrhoidalis (Linnaeus, 1758) Maurice 1947 ; Mouna 1998 ; AP (Rabat) – MISR Gasterophilus intestinalis (De Geer, 1776) Séguy 1930a ; Mouna 1998 ; Pandey et al. 1992 Gasterophilus nasalis (Linnaeus, 1758) = Gasterophilus veterinus Clark, in Mouna 1998 : 85 Maurice 1947 ; Pandey et al. 1992 ; Mouna 1998 Gasterophilus pecorum (Fabricius, 1794) Séguy 1930a ; Mouna 1998 Hypodermatinae Hypoderma Rondani, 1856 Hypoderma bovis (Linnaeus, 1758) Maurice 1947 ; Mouna 1998 Hypoderma lineatum (De Villers, 1789) Dakkok et al. 1978 , MA , Benslimane; Mouna 1998 Oestrinae Cephalopina Strand, 1928 Cephalopina titillator (Clark, 1797) = Cephalopsis titillator Clark, in Séguy 1930a : 139 Séguy 1930a , Sub- SA (camel breeding region); Mouna 1998 Oestrus Linnaeus, 1758 Oestrus ovis Linnaeus, 1761 Séguy 1930a , AP , Rabat (sheep breeding area), HA , Marrakech, Bou Tazzert; Maurice 1947 ; Mouna 1998 – MISR Rhinoestrus Brauer, 1886 Rhinoestrus purpureus (Brauer, 1858) Maurice 1947 ; Mouna 1998 Gastrophilinae Gasterophilus Leach, 1817 Gasterophilus flavipes (Olivier, 1811) Séguy 1928d , EM , Haute Moulouya; Séguy 1930a , EM , Itzer (Haute Moulouya); Mouna 1998 ; Li et al. 2019 Gasterophilus haemorrhoidalis (Linnaeus, 1758) Maurice 1947 ; Mouna 1998 ; AP (Rabat) – MISR Gasterophilus intestinalis (De Geer, 1776) Séguy 1930a ; Mouna 1998 ; Pandey et al. 1992 Gasterophilus nasalis (Linnaeus, 1758) = Gasterophilus veterinus Clark, in Mouna 1998 : 85 Maurice 1947 ; Pandey et al. 1992 ; Mouna 1998 Gasterophilus pecorum (Fabricius, 1794) Séguy 1930a ; Mouna 1998 Hypodermatinae Hypoderma Rondani, 1856 Hypoderma bovis (Linnaeus, 1758) Maurice 1947 ; Mouna 1998 Hypoderma lineatum (De Villers, 1789) Dakkok et al. 1978 , MA , Benslimane; Mouna 1998 Oestrinae Cephalopina Strand, 1928 Cephalopina titillator (Clark, 1797) = Cephalopsis titillator Clark, in Séguy 1930a : 139 Séguy 1930a , Sub- SA (camel breeding region); Mouna 1998 Oestrus Linnaeus, 1758 Oestrus ovis Linnaeus, 1761 Séguy 1930a , AP , Rabat (sheep breeding area), HA , Marrakech, Bou Tazzert; Maurice 1947 ; Mouna 1998 – MISR Rhinoestrus Brauer, 1886 Rhinoestrus purpureus (Brauer, 1858) Maurice 1947 ; Mouna 1998 POLLENIIDAE K. Kettani, K. Rognes Number of species: 12 . Expected: 15 Faunistic knowledge of the family in Morocco: moderate Pollenia Fabricius, 1794 Pollenia amentaria (Scopoli, 1763) Séguy 1949a , AA , Foum-el-Hassan; Séguy 1953a , MA , Sidi Allal Tazi; Mouna 1998 ; Pârvu et al. 2006 , SA , Foum Zghouig; Popescu-Mirceni 2011 ; Gisondi et al. 2020 Pollenia bicolor Robineau-Desvoidy, 1830 Rognes 1991a , HA , Mikdane (Jebel Ayachi); Rognes 1991b , HA , Mikdane (Jebel Ayachi); Gisondi et al. 2020 ; HA (Mikdane) – NHMUK Pollenia contempta Robineau-Desvoidy, 1863 Séguy 1930a , MA , Meknès, from M'Rirt to El Hajeb; Mouna 1998 ; Rognes 1992 Pollenia haeretica Séguy, 1928 Séguy 1934b , AP , Rabat; Rognes 2010 ; AP (40 km S Larache) – NHMD Pollenia ibalia Séguy, 1930 = Pollenia funebris Villeneuve, in Villeneuve 1932 : 284 = Pollenia rungsi Séguy, in Séguy 1953a : 88 Séguy 1930a , EM , Berkane, MA , Tlet n'Rhohr (in garden), Douar Ras el Ksar (900 m); Villeneuve 1932 , HA , Marrakech; Séguy 1953a , AP , Rabat; Séguy 1941a , HA , cañon Tessaout (M'Goum, 3000–3200 m); Mouna 1998 ; Rognes 2010 ; Grabener 2017 ; Gisondi et al. 2020 ; HA (Asni) – NHMUK ; HA (Ijoukak) – MNHN ; HA (15 km SW Tazenakht) – NHMD Pollenia leclercqiana (Lehrer, 1978) Rognes 2010 , 2011 ; Gisondi et al. 2020 Pollenia luteovillosa Rognes, 1987 Gisondi et al. 2020 , HA , Mikdane (Jbel Ayachi) Pollenia ponti Rognes, 1991 Rognes 1991b , HA , Jaffar river (Jebel Ayachi); Gisondi et al. 2020 ; HA (Jebel Ayachi) – NHMUK Pollenia rudis (Fabricius, 1794) Séguy 1930a , EM , Itzer (Haute Moulouya), Berkane (1350 m), AP , Casablanca, MA , Meknès, Aharmoumou (1100 m), Tlet n'Rhohr (in garden), Douar Ras el Ksar (1900 m), HA , Tizi-n'Test, Goundafa (Jebel Imdress, 2000–2450 m); Maurice 1947 , Rif , Tanger; Rognes 1987 , AP , Aïn Diab, Larache, HA , Asni, Jebel Ayachi, Mikdane; Grabener 2017 ; Gisondi et al. 2020 ; AP (Casablanca, Rabat), MA (Ifrane), HA (Haute Réghaya) – MNHN Pollenia ruficrura Rondani, 1862 Rognes 2011 ; Gisondi et al. 2020 Pollenia stigi (Rognes, 1992) Rognes 1992 , MA , Ifrane, Azrou; Gisondi et al. 2020 Pollenia vagabunda (Meigen, 1826) = Pollenia hasei Séguy, in Séguy 1928e : 370, Séguy 1930a : 147 Séguy 1928e ; Séguy 1930a , AP , Casablanca; Mouna 1998 ; Rognes 1992 ; Gisondi et al. 2020 RHINIIDAE K. Kettani, K. Rognes Number of species: 17 . Expected: ~20 Faunistic knowledge of the family in Morocco: moderate Cosmina Robineau-Desvoidy, 1830 Cosmina claripennis Robineau-Desvoidy, 1830 = Cosmina bezziana Villeneuve, in Villeneuve 1932 a: 285 Villeneuve 1932 a, AP , Mogador; Schumann 1986 ; Mouna 1998 ; Rognes 2002 Cosmina maroccana Séguy, 1949 Séguy 1949a , AA , Tenfecht, Vallée du Guir, SA , Guelmim; Séguy 1953a , AA , Tenfecht, Vallée du Guir, SA , Guelmim; Mouna 1998 ; Rognes 2002 ; Grabener 2017 Cosmina punctulata Robineau-Desvoidy, 1830 Séguy 1930a , AA , Tenfecht (Souss, 1000–1500 m) Cosmina viridis (Townsend, 1917) Ebejer et al. 2019 , AA , 13 km E of Goulmima (1100 m) Rhinia Robineau-Desvoidy, 1830 Rhinia apicalis (Wiedemann, 1830) Séguy 1930a , AP , Rabat, MA , Forêt Zaers; Zumpt 1956 ; Gonzales-Mora and Peris 1988; Mouna 1998 ; Verves 2003 ; Lehrer 2007 Rhyncomya Robineau-Desvoidy, 1830 Rhyncomya callopis (Loew, 1856) 58 Séguy 1941d , AA , Agadir; Séguy 1953a , AP , Sidi Ifni, SA , Tindouf, Amguilli Sguelma, Guelta des Zemmours; Schumann 1986 ; Mouna 1998 ; Rognes 1992 ; Dawah et al. 2019 ; El Hawagry and El-Azab 2019; MA (Tafilalt) – MISR Rhyncomya columbina (Meigen, 1824) Peris 1951 , Rif , Tanger, MA , Ifrane; Schumann 1986 ; Gonzalez-Mora and Peris 1988 Rhyncomya cyanescens (Loew, 1844) = Rhyncomya hemisia Séguy, in Séguy 1930a : 150 Séguy 1930a , EM , Berkane (1350–1400 m); Gonzales-Mora and Peris 1988; Rognes 2002 , MA Rhyncomya desertica (Peris, 1951) Grabener 2017 ; El Hawagry and El-Azab 2019 Rhyncomya impavida (Rossi, 1790) Séguy 1930a , Rif , Tanger, EM , Berkane (1350–1400 m), MA , Forêt Tiffert (2000–2200 m); Schumann 1986 ; Mouna 1998 Rhyncomya nigripes (Séguy, 1933) Grabener 2017 ; El Hawagry and El-Azab 2019 Rhyncomya ursina Séguy 1928 = Rhynchomyia ursina Séguy, in Séguy 1928b : 152 Séguy 1928b , AP , Atlantic coast of SA Rhyncomya yahavensis Rognes, 2002 Grabener 2017 ; Ebejer et al. 2019 , AA , 30 km W of Errachidia (1065 m) Rhyncomya zernyana Villeneuve, 1926 Zumpt 1956 ; Gonzales-Mora and Peris 1988 Stomorhina Rondani, 1861 Stomorhina lunata (Fabricius, 1805) Séguy 1930a ; Peris 1951 ; Gonzales-Mora and Peris 1988; Mouna 1998 ; AP (Rabat) – MISR Villeneuviella Austen, 1914 Villeneuviella icadion (Séguy, 1953) = Rhynchoestrus icadion Séguy, in Séguy 1953a : 89 Séguy 1953a , SA , Tindouf Villeneuviella weissi (Séguy, 1926) = Rhynchoestrus weissi Séguy, in Séguy 1934b : 162 Séguy 1934b , EM , Berkane Zobzit (1100 m); Séguy 1953a , SA , Mader Bergat RHINOPHORIDAE K. Kettani, T. Pape Number of species: 8 . Expected: 15 Faunistic knowledge of the family in Morocco: poor Rhinophorinae Melanophora Meigen, 1803 Melanophora roralis (Linnaeus, 1758) Peris 1963 , Rif , Tanger, AP , Larache; Mouna 1998 Oplisa Rondani, 1862 Oplisa aterrima (Strobl, 1899) = Hoplisa aterrima Strobl, in Peris 1963 : 602 Peris 1963 , Rif , Tanger, Zoco de Taleta, Ketama; Mouna 1998 ; Cerretti et al. 2020 Paykullia Robineau-Desvoidy, 1830 Paykullia carmela (Peris, 1963) = Chaetostevenia carmela Peris, in Peris 1963 : 606 Peris 1963 , Rif , Tanger; Cerretti et al. 2020 ; Pape and Thompson 2019 – MNCN Phyto Robineau-Desvoidy, 1830 Phyto atrior (Villeneuve, 1941) = Styloneuria atrior Villeneuve, in Villeneuve 1941 : 122 Villeneuve 1941 , AP , Rabat; Cerretti et al. 2020 ; Pape and Thompson 2019 – IRSNB Phyto discrepans Pandellé, 1896 Cerretti et al. 2020 , Rif , Chefchaouen (600 m), Ouezzane (300 m), MA , 40 km N Fès (1150 m) – NHMD Phyto melanocephala (Meigen, 1824) Ebejer et al. 2019 , Rif , Barrage Smir (145 m); Cerretti et al. 2020 Stevenia Robineau-Desvoidy, 1830 Stevenia deceptoria (Loew, 1847) Mulieri et al. 2010 ; Cerretti et al. 2020 Tricogena Rondani, 1856 Tricogena rubricosa (Meigen, 1824) = Frauenfeldia rubricosa Meigen, in Peris 1963 : 603 Peris 1963 , Rif , Tanger; Mouna 1998 ; Cerretti et al. 2020 – NHMD Rhinophorinae Melanophora Meigen, 1803 Melanophora roralis (Linnaeus, 1758) Peris 1963 , Rif , Tanger, AP , Larache; Mouna 1998 Oplisa Rondani, 1862 Oplisa aterrima (Strobl, 1899) = Hoplisa aterrima Strobl, in Peris 1963 : 602 Peris 1963 , Rif , Tanger, Zoco de Taleta, Ketama; Mouna 1998 ; Cerretti et al. 2020 Paykullia Robineau-Desvoidy, 1830 Paykullia carmela (Peris, 1963) = Chaetostevenia carmela Peris, in Peris 1963 : 606 Peris 1963 , Rif , Tanger; Cerretti et al. 2020 ; Pape and Thompson 2019 – MNCN Phyto Robineau-Desvoidy, 1830 Phyto atrior (Villeneuve, 1941) = Styloneuria atrior Villeneuve, in Villeneuve 1941 : 122 Villeneuve 1941 , AP , Rabat; Cerretti et al. 2020 ; Pape and Thompson 2019 – IRSNB Phyto discrepans Pandellé, 1896 Cerretti et al. 2020 , Rif , Chefchaouen (600 m), Ouezzane (300 m), MA , 40 km N Fès (1150 m) – NHMD Phyto melanocephala (Meigen, 1824) Ebejer et al. 2019 , Rif , Barrage Smir (145 m); Cerretti et al. 2020 Stevenia Robineau-Desvoidy, 1830 Stevenia deceptoria (Loew, 1847) Mulieri et al. 2010 ; Cerretti et al. 2020 Tricogena Rondani, 1856 Tricogena rubricosa (Meigen, 1824) = Frauenfeldia rubricosa Meigen, in Peris 1963 : 603 Peris 1963 , Rif , Tanger; Mouna 1998 ; Cerretti et al. 2020 – NHMD SARCOPHAGIDAE K. Kettani, D. Whitmore, T. Pape Number of species: 66 . Expected: ~150 Faunistic knowledge of the family in Morocco: poor Miltogramminae Amobia Robineau-Desvoidy, 1830 Amobia signata (Meigen, 1824) Pape 1996 ; Verves 2019 Apodacra Macquart, 1854 Apodacra africana Rohdendorf, 1930 Pape 1996 , Rif , Tanger; Verves 2019 Craticulina Pandellé, 1895 Craticulina antachates (Séguy, 1949) = Apodacra antachates Séguy, in Séguy 1949a : 160 Séguy 1949a , AA , Zagora; Pape 1996 , AA , Zagora; Mouna 1998 Craticulina tabaniformis (Fabricius, 1805) Fabricius 1805 , AP , Mogador; Séguy 1930a , AP , Mogador; Séguy 1935a , AP , beach of Rabat; Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Dolichotachina Villeneuve, 1913 Dolichotachina marginella (Wiedemann, 1930) Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Macronychia Rondani, 1859 Macronychia lemariei Jacentkovský, 1941* AP Macronychia polyodon (Meigen, 1824) Pape 1996 ; Verves 2019 Metopia Meigen, 1803 Metopia argyrocephala (Meigen, 1824) = Metopia leucocephala (Rossi), in Mouna 1998 : 86 Mouna 1998 Miltogramma Meigen, 1803 Miltogramma aurifrons Dufour, 1850 Séguy 1930a , AP , Rabat, MA , Meknès; Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019; Verves 2019 Miltogramma germari Meigen, 1824 Séguy 1930a , MA , Meknès, from M'Rirt to El Hajeb, Sidi Taibi; Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019; Verves 2019 Miltogramma maroccana (Séguy, 1941) = Sphecapatodes maroccana Séguy, in Séguy 1941d : 22 Séguy 1941d , AA , Taroudant; Pape and Szpila 2012 Miltogramma murina Meigen, 1824 Pape 1996 ; Verves 2019 Miltogramma oestracea (Fallén, 1820) Ebejer et al. 2019 , Rif , Belwazen (M'Diq, 200 m), AP , Lower Loukous saltmarsh (2 m) Miltogramma rutilans Meigen, 1824 Ebejer et al. 2019 , Rif , Oued Mhajrate (Ben Karrich, 180 m) Miltogramma testaceifrons (Roser, 1840) = Miltogramma pilitarsis Rondani, in Séguy 1930a : 145; Mouna 1998 : 86 Séguy 1930a , MA , Aïn Leuh; Pape 1996 ; Mouna 1998 Protomiltogramma Townsend, 1916 Protomiltogramma fasciata (Meigen, 1824) = Setulia fasciata (Meigen), in Mouna 1998 : 86 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Senotainia Macquart, 1846 Senotainia albifrons (Rondani, 1859) = Sphecapata albifrons Rondani, in Séguy 1930a : 145 Séguy 1930a Taxigramma Macquart, 1850 Taxigramma heteroneura (Meigen, 1830) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Taxigramma pluriseta (Pandellé, 1895) Ebejer et al. 2019 , Rif , Oued Mhajrate (Ben Karrich, 180 m), AP , Lower Loukous saltmarsh (2 m) Paramacronychiinae Nyctia Robineau-Desvoidy, 1830 Nyctia halterata (Panzer, 1798) = Musca maura Fabricius, in Fabricius 1805 : 302 Fabricius 1805 , Rif , Tanger; Pape 1996 , Rif , Tanger; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Nyctia lugubris (Macquart, 1834) Ebejer et al. 2019 , AP , Lower Loukous saltmarsh (2 m) Sarcophila Rondani, 1856 Sarcophila latifrons (Fallén, 1817) 59 Séguy 1930a , AP , Maâmora, HA , Skoutana (Arround, 2000–2400 m); Mouna 1998 Wohlfahrtia Brauer & Bergenstamm, 1889 Wohlfahrtia bella (Macquart, 1839) = Disjunctio bella (Macquart), in Séguy 1930a : 144 Séguy 1930a , MA , Aïn Leuh; Pape 1996 ; Mouna 1998 ; Hall et al. 2009 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia indigens Villeneuve, 1928 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia magnifica (Schiner, 1862) Delanoë 1922 , AP , Doukkala; Séguy 1930a , AP , Maâmora; Séguy 1941a , HA , Tizi-n'Icheden (3000 m); Maurice 1947 ; Pape 1996 ; Mouna 1998 ; Lmimouni et al. 2004 ; Tliqui et al. 2007 ; Farkas et al. 2009 , Rif , Al Hoceima, Taguidit, Tafensa, EM , Aghbal; Hall et al. 2009 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia nuba (Wiedemann, 1830) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia trina (Wiedemann, 1830) Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019 Sarcophaginae Blaesoxipha Loew, 1861 Blaesoxipha ( Blaesoxipha ) lapidosa Pape, 1994 Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019 Blaesoxipha ( Blaesoxipha ) litoralis (Villeneuve, 1911) Pape 1996 ; Verves 2019 Blaesoxipha ( Blaesoxipha ) pygmaea (Zetterstedt, 1844) Pape 1996 ; Verves 2019 Blaesoxipha ( Servaisia ) rossica Villeneuve, 1912 Pape 1996 ; Verves 2019 Ravinia Robineau-Desvoidy, 1863 Ravinia pernix (Harris, 1780) = Gesneriodes disjuncta Séguy, in Séguy 1938 : 43 = Sarcophaga striata (Fabricius), in Séguy 1941d : 22, Séguy 1949a : 159 Séguy 1938 , HA , Skoutana; Séguy 1941d , AA , Taroudant; Séguy 1949a , AA , Akka; Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga Meigen, 1826 Sarcophaga ( Bercaea ) africa (Wiedemann, 1824) Pape 1996 ; Abkari et al. 1998 ; Verves 2003 , 2019 ; Grabener 2017 ; El Hawagry and El-Azab 2019 Sarcophaga ( Helicophagella ) maculata Meigen, 1835 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Helicophagella ) melanura Meigen, 1826 El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Helicophagella ) novercoides Böttcher, 1913* Rif , HA , AA Sarcophaga ( Heteronychia ) balanina Pandellé, 1896 Whitmore et al. 2013 , AP , Larache; Fendane et al. 2018 , AP , Diabat (Essaouira), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Heteronychia ) cucullans Pandellé, 1896 Séguy 1930a , HA , Maharidja; Mouna 1998 Sarcophaga ( Heteronychia ) ferox Villeneuve, 1908 Whitmore 2011 , Rif , Ouezzane, AP , Larache, MA , Béni Mellal, Afourer, AA , Aoulouz; Whitmore et al. 2013 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Heteronychia ) filia Rondani, 1860 Whitmore 2011 , MA , Azrou, Timahdit; Whitmore et al. 2013 ; Verves 2019 Sarcophaga ( Heteronychia ) javita (Peris, González-Mora & Mingo, 1998)* AA Sarcophaga ( Heteronychia ) longestylata Strobl, 1906 Pape 1996 ; Whitmore et al. 2013 , MA , Ifrane, Azrou; Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Heteronychia ) minima Rondani, 1862 Whitmore 2011 , MA , Azrou, Ifrane, Afourer (Béni Mellal), HA , Ijoukak, Ouirgane (Marrakech), AA , Oulma (Agadir); Whitmore et al. 2013 ; Fendane et al. 2018 , AP , Smimou (Essaouira), El Akarta (Oualidia); Verves 2019 Sarcophaga ( Heteronychia ) obvia (Povolný, 2004) Whitmore et al. 2013 , MA , Afourer (Béni Mellal), HA , Ait Lekak (Marrakech), S Asni (Imlil, Marrakech), Tagadirt, Quirgane, AA , Oulma Ort (Agadir) Sarcophaga ( Heteronychia ) pandellei (Rohdendorf, 1937) Séguy 1930a , MA , Tizi-s'Tkrine; Mouna 1998 ; Whitmore et al. 2013 , MA , Azrou, Ifrane, Afourer (Béni Mellal) Sarcophaga ( Heteronychia ) tangerensis Whitmore, 2011 = Heteronychia ( Heteronychia ) amica Peris, González-Mora & Mingo, in Peris et al. 1998 : 173 Peris et al. 1998 , Rif , Tanger; Whitmore 2011 , Rif , Tanger Sarcophaga ( Heteronychia ) villeneuveana (Enderlein, 1928) = Pierretia ( Bercaea ) maroccana Rohdendorf, in Rohdendorf 1937 : 325 = Sarcophaga ( Heteronychia ) penicillata Villeneuve, in Coupland and Barker 2004 : 113 (misidentification) Rohdendorf 1937 , MA , Aïn Defali; Coupland and Barker 2004 ; Whitmore 2009 ; Fendane et al. 2018 , AP , Diabat (Essaouira), Ghabat Tansift (Souiria), Lalla Fatna (Safi), Laatoutate (Safi), El Akarta (Oualidia), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Heteronychia ) uncicurva Pandellé, 1896 Fendane et al. 2018 , AP , Smimou (Essaouira), Diabat (Essaouira), Lalla Fatna (Safi), Laatoutate (Safi), Bir Retma (Casablanca) Sarcophaga ( Liopygia ) argyrostoma (Robineau-Desvoidy, 1830)* HA Sarcophaga ( Liopygia ) crassipalpis Macquart, 1839 Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) aegyptica Salem, 1935 Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Liosarcophaga ) dux Thomson, 1869* SA Sarcophaga ( Liosarcophaga ) jacobsoni (Rohdendorf, 1937) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) marshalli Parker, 1923 Fendane et al. 2018 , AP , Smimou (Essaouira), Diabat (Essaouira), Ghabat Tansift (Souiria), Laatoutate (Safi); El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) pharaonis Rohdendorf, 1934 Carles-Tolrá 2002 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) tibialis Macquart, 1851 = Sarcophaga beckeri Villeneuve, in Maurice 1947 : 57; Mouna 1998 : 86 Maurice 1947 ; Mouna 1998 ; Fendane et al. 2018 , AP , Laatoutate (Safi) Sarcophaga ( Liosarcophaga ) teretirostris Pandellé, 1896 = Parasarcophaga decellei Lehrer, in Lehrer 1976 : 3 Lehrer 1976 , MA , Kandar, Imouzzer; Pape 1996 , MA , Kandar, Imouzzer; Verves 2019 Sarcophaga ( Liosarcophaga ) tuberosa Pandellé, 1896 60 Mouna 1998 Sarcophaga ( Myorhina ) nigriventris Meigen, 1826 Pape 1996 ; Mouna 1998 ; Fendane et al. 2018 , AP , Ghabat Tansift (Souiria), Laatoutate (Safi), El Akarta (Oualidia), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Myorhina ) soror Rondani, 1860 Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Pandelleana ) protuberans Pandellé, 1896 Séguy 1949a , AA , Agadir Tissint, SA , Guelmim, Foum-el-Hassan, Tata; Mouna 1998 Sarcophaga ( Parasarcophaga ) hirtipes Wiedemann, 1830 Pape 1996 ; Verves 2003 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Sarcophaga ) lehmanni Müller, 1922 Pape 1996 ; Cassar et al. 2005 , Rif , Smir lagoon; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Sarcophaga ) marcelleclercqi Lehrer, 1975 Lehrer 1975 ; Pape 1996 , MA , Azrou; Verves 2019 Sarcophaga ( Thyrsocnema ) belgiana (Lehrer, 1976) Lehrer 1976 ; Pape 1996 Sarcophaga ( Thyrsocnema ) sp. 61 Ebejer et al. 2019 [as incisilobata , misidentification], Rif , Tahaddart (8 m) New records for Morocco Macronychia lemariei Jacentkovský, 1941 Atlantic plain: Rabat, Forêt de Maâmora, 100 m, 25–26.iv.1989, 1♂1♀, Zoological Museum of Copenhagen Expedition (NMHD). Sarcophaga ( Helicophagella ) novercoides Böttcher, 1913 Rif: Ouezzane, 300 m, 21–22.iv.1989, 1♂, Zoological Museum of Copenhagen Expedition (NMHD). High Atlas: Marrakech, Ouirgane, 1000 m, 1–9.iv.1997 Mai, 1♂, C. Kassebeer leg. (NMHD). Anti Atlas: 30 km NW Aoulouz, 1400 m, 10.iv.1989, 1♂, Zoological Museum of Copenhagen Expedition (NMHD). Sarcophaga ( Heteronychia ) javita (Peris, González-Mora & Mingo, 1998) Anti Atlas: Agadir, S Oulma, 30°31'N, 9°09'W , 200 m, 21.iv.1997, 1♂, C. Kassebeer leg. (NMHD). Sarcophaga ( Liopygia ) argyrostoma (Robineau-Desvoidy, 1830) High Atlas: Marrakech, Tagadirt, Ouirgane, 1000 m, 1.x.1994, 1♀, C. Kassebeer leg. (NMHD). Sarcophaga ( Liosarcophaga ) dux Thomson, 1869 Sahara: Erfoud, Rissani area, 900 m, 13–14.iv.1989, Zoological Museum of Copenhagen Expedition ( NHMD ). Miltogramminae Amobia Robineau-Desvoidy, 1830 Amobia signata (Meigen, 1824) Pape 1996 ; Verves 2019 Apodacra Macquart, 1854 Apodacra africana Rohdendorf, 1930 Pape 1996 , Rif , Tanger; Verves 2019 Craticulina Pandellé, 1895 Craticulina antachates (Séguy, 1949) = Apodacra antachates Séguy, in Séguy 1949a : 160 Séguy 1949a , AA , Zagora; Pape 1996 , AA , Zagora; Mouna 1998 Craticulina tabaniformis (Fabricius, 1805) Fabricius 1805 , AP , Mogador; Séguy 1930a , AP , Mogador; Séguy 1935a , AP , beach of Rabat; Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Dolichotachina Villeneuve, 1913 Dolichotachina marginella (Wiedemann, 1930) Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Macronychia Rondani, 1859 Macronychia lemariei Jacentkovský, 1941* AP Macronychia polyodon (Meigen, 1824) Pape 1996 ; Verves 2019 Metopia Meigen, 1803 Metopia argyrocephala (Meigen, 1824) = Metopia leucocephala (Rossi), in Mouna 1998 : 86 Mouna 1998 Miltogramma Meigen, 1803 Miltogramma aurifrons Dufour, 1850 Séguy 1930a , AP , Rabat, MA , Meknès; Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019; Verves 2019 Miltogramma germari Meigen, 1824 Séguy 1930a , MA , Meknès, from M'Rirt to El Hajeb, Sidi Taibi; Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019; Verves 2019 Miltogramma maroccana (Séguy, 1941) = Sphecapatodes maroccana Séguy, in Séguy 1941d : 22 Séguy 1941d , AA , Taroudant; Pape and Szpila 2012 Miltogramma murina Meigen, 1824 Pape 1996 ; Verves 2019 Miltogramma oestracea (Fallén, 1820) Ebejer et al. 2019 , Rif , Belwazen (M'Diq, 200 m), AP , Lower Loukous saltmarsh (2 m) Miltogramma rutilans Meigen, 1824 Ebejer et al. 2019 , Rif , Oued Mhajrate (Ben Karrich, 180 m) Miltogramma testaceifrons (Roser, 1840) = Miltogramma pilitarsis Rondani, in Séguy 1930a : 145; Mouna 1998 : 86 Séguy 1930a , MA , Aïn Leuh; Pape 1996 ; Mouna 1998 Protomiltogramma Townsend, 1916 Protomiltogramma fasciata (Meigen, 1824) = Setulia fasciata (Meigen), in Mouna 1998 : 86 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Senotainia Macquart, 1846 Senotainia albifrons (Rondani, 1859) = Sphecapata albifrons Rondani, in Séguy 1930a : 145 Séguy 1930a Taxigramma Macquart, 1850 Taxigramma heteroneura (Meigen, 1830) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Taxigramma pluriseta (Pandellé, 1895) Ebejer et al. 2019 , Rif , Oued Mhajrate (Ben Karrich, 180 m), AP , Lower Loukous saltmarsh (2 m) Paramacronychiinae Nyctia Robineau-Desvoidy, 1830 Nyctia halterata (Panzer, 1798) = Musca maura Fabricius, in Fabricius 1805 : 302 Fabricius 1805 , Rif , Tanger; Pape 1996 , Rif , Tanger; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Nyctia lugubris (Macquart, 1834) Ebejer et al. 2019 , AP , Lower Loukous saltmarsh (2 m) Sarcophila Rondani, 1856 Sarcophila latifrons (Fallén, 1817) 59 Séguy 1930a , AP , Maâmora, HA , Skoutana (Arround, 2000–2400 m); Mouna 1998 Wohlfahrtia Brauer & Bergenstamm, 1889 Wohlfahrtia bella (Macquart, 1839) = Disjunctio bella (Macquart), in Séguy 1930a : 144 Séguy 1930a , MA , Aïn Leuh; Pape 1996 ; Mouna 1998 ; Hall et al. 2009 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia indigens Villeneuve, 1928 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia magnifica (Schiner, 1862) Delanoë 1922 , AP , Doukkala; Séguy 1930a , AP , Maâmora; Séguy 1941a , HA , Tizi-n'Icheden (3000 m); Maurice 1947 ; Pape 1996 ; Mouna 1998 ; Lmimouni et al. 2004 ; Tliqui et al. 2007 ; Farkas et al. 2009 , Rif , Al Hoceima, Taguidit, Tafensa, EM , Aghbal; Hall et al. 2009 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia nuba (Wiedemann, 1830) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Wohlfahrtia trina (Wiedemann, 1830) Pape 1996 ; Mouna 1998 ; El Hawagry and El-Azab 2019 Sarcophaginae Blaesoxipha Loew, 1861 Blaesoxipha ( Blaesoxipha ) lapidosa Pape, 1994 Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019 Blaesoxipha ( Blaesoxipha ) litoralis (Villeneuve, 1911) Pape 1996 ; Verves 2019 Blaesoxipha ( Blaesoxipha ) pygmaea (Zetterstedt, 1844) Pape 1996 ; Verves 2019 Blaesoxipha ( Servaisia ) rossica Villeneuve, 1912 Pape 1996 ; Verves 2019 Ravinia Robineau-Desvoidy, 1863 Ravinia pernix (Harris, 1780) = Gesneriodes disjuncta Séguy, in Séguy 1938 : 43 = Sarcophaga striata (Fabricius), in Séguy 1941d : 22, Séguy 1949a : 159 Séguy 1938 , HA , Skoutana; Séguy 1941d , AA , Taroudant; Séguy 1949a , AA , Akka; Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga Meigen, 1826 Sarcophaga ( Bercaea ) africa (Wiedemann, 1824) Pape 1996 ; Abkari et al. 1998 ; Verves 2003 , 2019 ; Grabener 2017 ; El Hawagry and El-Azab 2019 Sarcophaga ( Helicophagella ) maculata Meigen, 1835 Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Helicophagella ) melanura Meigen, 1826 El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Helicophagella ) novercoides Böttcher, 1913* Rif , HA , AA Sarcophaga ( Heteronychia ) balanina Pandellé, 1896 Whitmore et al. 2013 , AP , Larache; Fendane et al. 2018 , AP , Diabat (Essaouira), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Heteronychia ) cucullans Pandellé, 1896 Séguy 1930a , HA , Maharidja; Mouna 1998 Sarcophaga ( Heteronychia ) ferox Villeneuve, 1908 Whitmore 2011 , Rif , Ouezzane, AP , Larache, MA , Béni Mellal, Afourer, AA , Aoulouz; Whitmore et al. 2013 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Heteronychia ) filia Rondani, 1860 Whitmore 2011 , MA , Azrou, Timahdit; Whitmore et al. 2013 ; Verves 2019 Sarcophaga ( Heteronychia ) javita (Peris, González-Mora & Mingo, 1998)* AA Sarcophaga ( Heteronychia ) longestylata Strobl, 1906 Pape 1996 ; Whitmore et al. 2013 , MA , Ifrane, Azrou; Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Heteronychia ) minima Rondani, 1862 Whitmore 2011 , MA , Azrou, Ifrane, Afourer (Béni Mellal), HA , Ijoukak, Ouirgane (Marrakech), AA , Oulma (Agadir); Whitmore et al. 2013 ; Fendane et al. 2018 , AP , Smimou (Essaouira), El Akarta (Oualidia); Verves 2019 Sarcophaga ( Heteronychia ) obvia (Povolný, 2004) Whitmore et al. 2013 , MA , Afourer (Béni Mellal), HA , Ait Lekak (Marrakech), S Asni (Imlil, Marrakech), Tagadirt, Quirgane, AA , Oulma Ort (Agadir) Sarcophaga ( Heteronychia ) pandellei (Rohdendorf, 1937) Séguy 1930a , MA , Tizi-s'Tkrine; Mouna 1998 ; Whitmore et al. 2013 , MA , Azrou, Ifrane, Afourer (Béni Mellal) Sarcophaga ( Heteronychia ) tangerensis Whitmore, 2011 = Heteronychia ( Heteronychia ) amica Peris, González-Mora & Mingo, in Peris et al. 1998 : 173 Peris et al. 1998 , Rif , Tanger; Whitmore 2011 , Rif , Tanger Sarcophaga ( Heteronychia ) villeneuveana (Enderlein, 1928) = Pierretia ( Bercaea ) maroccana Rohdendorf, in Rohdendorf 1937 : 325 = Sarcophaga ( Heteronychia ) penicillata Villeneuve, in Coupland and Barker 2004 : 113 (misidentification) Rohdendorf 1937 , MA , Aïn Defali; Coupland and Barker 2004 ; Whitmore 2009 ; Fendane et al. 2018 , AP , Diabat (Essaouira), Ghabat Tansift (Souiria), Lalla Fatna (Safi), Laatoutate (Safi), El Akarta (Oualidia), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Heteronychia ) uncicurva Pandellé, 1896 Fendane et al. 2018 , AP , Smimou (Essaouira), Diabat (Essaouira), Lalla Fatna (Safi), Laatoutate (Safi), Bir Retma (Casablanca) Sarcophaga ( Liopygia ) argyrostoma (Robineau-Desvoidy, 1830)* HA Sarcophaga ( Liopygia ) crassipalpis Macquart, 1839 Pape 1996 ; Grabener 2017 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) aegyptica Salem, 1935 Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Liosarcophaga ) dux Thomson, 1869* SA Sarcophaga ( Liosarcophaga ) jacobsoni (Rohdendorf, 1937) Pape 1996 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) marshalli Parker, 1923 Fendane et al. 2018 , AP , Smimou (Essaouira), Diabat (Essaouira), Ghabat Tansift (Souiria), Laatoutate (Safi); El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) pharaonis Rohdendorf, 1934 Carles-Tolrá 2002 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Liosarcophaga ) tibialis Macquart, 1851 = Sarcophaga beckeri Villeneuve, in Maurice 1947 : 57; Mouna 1998 : 86 Maurice 1947 ; Mouna 1998 ; Fendane et al. 2018 , AP , Laatoutate (Safi) Sarcophaga ( Liosarcophaga ) teretirostris Pandellé, 1896 = Parasarcophaga decellei Lehrer, in Lehrer 1976 : 3 Lehrer 1976 , MA , Kandar, Imouzzer; Pape 1996 , MA , Kandar, Imouzzer; Verves 2019 Sarcophaga ( Liosarcophaga ) tuberosa Pandellé, 1896 60 Mouna 1998 Sarcophaga ( Myorhina ) nigriventris Meigen, 1826 Pape 1996 ; Mouna 1998 ; Fendane et al. 2018 , AP , Ghabat Tansift (Souiria), Laatoutate (Safi), El Akarta (Oualidia), Sidi Abed (El Jadida), Bir Retma (Casablanca); Verves 2019 Sarcophaga ( Myorhina ) soror Rondani, 1860 Fendane et al. 2018 , AP , Sidi Abed (El Jadida) Sarcophaga ( Pandelleana ) protuberans Pandellé, 1896 Séguy 1949a , AA , Agadir Tissint, SA , Guelmim, Foum-el-Hassan, Tata; Mouna 1998 Sarcophaga ( Parasarcophaga ) hirtipes Wiedemann, 1830 Pape 1996 ; Verves 2003 ; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Sarcophaga ) lehmanni Müller, 1922 Pape 1996 ; Cassar et al. 2005 , Rif , Smir lagoon; El Hawagry and El-Azab 2019; Verves 2019 Sarcophaga ( Sarcophaga ) marcelleclercqi Lehrer, 1975 Lehrer 1975 ; Pape 1996 , MA , Azrou; Verves 2019 Sarcophaga ( Thyrsocnema ) belgiana (Lehrer, 1976) Lehrer 1976 ; Pape 1996 Sarcophaga ( Thyrsocnema ) sp. 61 Ebejer et al. 2019 [as incisilobata , misidentification], Rif , Tahaddart (8 m) New records for Morocco Macronychia lemariei Jacentkovský, 1941 Atlantic plain: Rabat, Forêt de Maâmora, 100 m, 25–26.iv.1989, 1♂1♀, Zoological Museum of Copenhagen Expedition (NMHD). Sarcophaga ( Helicophagella ) novercoides Böttcher, 1913 Rif: Ouezzane, 300 m, 21–22.iv.1989, 1♂, Zoological Museum of Copenhagen Expedition (NMHD). High Atlas: Marrakech, Ouirgane, 1000 m, 1–9.iv.1997 Mai, 1♂, C. Kassebeer leg. (NMHD). Anti Atlas: 30 km NW Aoulouz, 1400 m, 10.iv.1989, 1♂, Zoological Museum of Copenhagen Expedition (NMHD). Sarcophaga ( Heteronychia ) javita (Peris, González-Mora & Mingo, 1998) Anti Atlas: Agadir, S Oulma, 30°31'N, 9°09'W , 200 m, 21.iv.1997, 1♂, C. Kassebeer leg. (NMHD). Sarcophaga ( Liopygia ) argyrostoma (Robineau-Desvoidy, 1830) High Atlas: Marrakech, Tagadirt, Ouirgane, 1000 m, 1.x.1994, 1♀, C. Kassebeer leg. (NMHD). Sarcophaga ( Liosarcophaga ) dux Thomson, 1869 Sahara: Erfoud, Rissani area, 900 m, 13–14.iv.1989, Zoological Museum of Copenhagen Expedition ( NHMD ). TACHINIDAE K. Kettani, P. Cerretti, H.-P. Tschorsnig Number of species: 147 . Expected: 200 Faunistic knowledge of the family in Morocco: poor Dexiinae Dexiini Billaea Robineau-Desvoidy, 1830 Billaea lata (Macquart, 1849) = Rhynchodinera lata Macquart, in Séguy 1930a : 143 Séguy 1930a , MA , Aharmoumou, Camp Boulhout, Sidi Bettache, Aïn Sferguila, Meknès; Mouna 1998 ; AP (Mehdia) – MISR ; AP (Essaouira, 4 km E Ounara), HA (Marrakech, Lakhdar, N Demnate) – PCPT Estheria Robineau-Desvoidy, 1830 Estheria atripes Villeneuve, 1920 Cerretti and Tschorsnig 2012 Estheria iberica Tschorsnig, 2003* MA Estheria nigripes (Villeneuve, 1920) Cerretti and Tschorsnig 2012 ; MA (Béni Mellal, El Ksiba), AA (Agadir, Oulma) – PCPT Estheria picta (Meigen, 1826) 62 Moutia 1940 Zeuxia Meigen, 1826 Zeuxia aberrans (Loew, 1847) = Zeuxia nigripes Meigen, in Séguy 1941d : 23 Brémond 1938 ; Séguy 1941d , AP , Rabat, MA , Volubilis, AA , Agadir; IOBC-List 11; Mesnil 1980; Mouna 1998 ; Tschorsnig 2017; AA (10 km NW Aït-Baha) – PCPT Dufouriini Dufouria Robineau-Desvoidy, 1830 Dufouria nigrita (Fallén, 1810) Ebejer et al. 2019 , AP , Larache (Lower Loukous saltmarsh, 2 m); MA (Ouzoud) – PCPT Voriini Athrycia Robineau-Desvoidy, 1830 Athrycia trepida (Meigen, 1824)* MA Cyrtophloeba Rondani, 1856 Cyrtophloeba ruricola (Meigen, 1824) = Plagia ruricola Meigen, in Séguy 1935a : 120, in Rungs 1940 : 14 Séguy 1935a , MA , Ifrane; Rungs 1940 , MA (Cédraie); Mouna 1998 ; HA (Tizi-n'Test), AA (Taroudant) – PCPT Eriothrix Meigen, 1830 Eriothrix apennina (Rondani, 1862) Herting and Dely-Draskovits 1993 ; Koçak and Kemal 2010 Eriothrix rufomaculata (De Geer, 1776)* MA , HA Hypovoria Villeneuve, 1913 Hypovoria hilaris Villeneuve, 1912 Séguy 1935a , AP , Oued Beth; Séguy 1953a , AP , Sehoul; Mouna 1998 ; AA (10 km SE Aït-Ourir) – PCPT Hypovoria pilibasis (Villeneuve, 1922) Zeegers 2010; HA (Tizi-n'Test), AA (Taroudant) – PCPT Kirbya Robineau-Desvoidy, 1830 Kirbya moerens (Meigen, 1830)* MA Nanoplagia Villeneuve, 1929 Nanoplagia sinaica (Villeneuve in Hermann & Villeneuve 1909) Cerretti 2009; Grabener 2017 ; HA (Marrakech, 8 km N Ouirgane), AA (40 km SW Ouarzazate, 10 km SW Tazenakht, NE Agadir, 12 km W Oulma) – PCPT Periscepsia Gistel, 1848 Periscepsia meyeri (Villeneuve, 1930) Ebejer et al. 2019 , Rif , Adrou ( PNPB , 556 m) Stomina Robineau-Desvoidy, 1830 Stomina caliendrata (Rondani, 1862) = Morphomyia caliendrata Rondani, in Séguy 1930a : 143 Séguy 1930a , MA , from M'Rirt to Hajeb, HA , Kasba Taguendaft (Gounfada); Mouna 1998 ; HA (Massif Toubkal) – PCPT Thelaira Robineau-Desvoidy, 1830 Thelaira haematodes (Meigen, 1824) 63 = Phoenicella haematodes Meigen: Séguy 1930a : 142 Séguy 1930a , HA , Arround; Mouna 1998 Uclesia Girschner, 1901 Uclesia fumipennis Girschner, 1901 Séguy 1934b ; Herting and Dely-Draskovits 1993 ; Mouna 1998 ; HA (Marrakech) – MISR Voria Robineau-Desvoidy, 1830 Voria ruralis (Fallén, 1810) Jourdan 1935c Wagneria Robineau-Desvoidy, 1830 Wagneria dilatata Kugler, 1977 Kugler 1977 Exoristinae Acemyini Ceracia Rondani, 1865 Ceracia mucronifera Rondani, 1865 = Myothyria benoisti (Mesnil), in Mesnil 1959 : 20 Mesnil 1959 , MA , Forêt Maâmora near Tiflet; Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 Blondeliini Compsilura Bouché, 1834 Compsilura concinnata (Meigen, 1824) IOBC-list 1 (1956); Hérard and Fraval 1980 Istocheta Rondani, 1859 Istocheta cinerea (Macquart, 1850) Herting 1960 Istocheta longicornis (Fallén, 1810) = Latigena longicornis Fallén, in Séguy 1953a : 91 Séguy 1953a , AP , Forêt Zaers Lomachantha Rondani, 1859 Lomachantha parra Rondani, 1859 Efetov and Tarmann 1999 Robinaldia Herting, 1983 Robinaldia angustata (Villeneuve, 1933) Herting and Dely-Draskovits 1993 ; Tschorsnig and Herting 1994 Zaira Robineau-Desvoidy, 1830 Zaira cinerea (Fallén, 1820) Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 8 m) Eryciini Alsomyia Brauer & Bergenstamm, 1891 Alsomyia olfaciens (Pandellé, 1896) IOBC-List 12 (1993) Amphicestonia Villeneuve, 1939 Amphicestonia dispar (Villeneuve, 1922) Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 ; MA (Ifrane) – PCPT Aplomyia Robineau-Desvoidy, 1830 Aplomyia confinis (Fallén, 1820) Ebejer et al. 2019 , Rif , Dardara (484 m) Carcelia Robineau-Desvoidy, 1830 Carcelia dilaticornis Mesnil, 1950 Mesnil 1950 Carcelia iliaca (Ratzeburg, 1840)63 Mouna 1998 Carcelia lucorum (Meigen, 1824)* HA Drino Robineau-Desvoidy, 1863 Drino atropivora (Robineau-Desvoidy, 1830) = Sturmia atropivora Robineau-Desvoidy, in Bléton and Fieuzet 1939 : 64 De Lépiney and Mimeur 1932; Bléton and Fieuzet 1939 , MA , Fès; Mouna 1998 ; Tschorsnig 2017; AP (Rabat), MA (Bel Lakssiri) – MISR Drino galii (Brauer & Bergenstamm, 1891)* HA , AA Drino gilva (Hartig, 1838)63 = Sturmia gilva Hartig Mouna 1998 – MISR (no locality given) Drino imberbis (Wiedemann, 1830)63 Rungs 1954 ; Grabener 2017 Drino inconspicua (Meigen, 1830) Séguy 1935a , AP , Sehoul (Rabat); Bléton and Fieuzet 1939 , MA , Dayat Achleff; Mouna 1998 ; MA (Meknès, Béni Mellal) – MISR Drino maroccana Mesnil, 1951 De Lépiney 1930 ; De Lépiney and Mimeur 1932 (probably misidentified as Sturmia inconspicua ); Mesnil 1951 ; Herting and Dely-Draskovits 1993 ; Ziegler 2011 Drino triplaca Herting, 1979 Herting 1979 , AP , Rabat Drino vicina (Zetterstedt, 1849) = Sturmia vicina Zetterstedt, 1849 De Lépiney and Mimeur 1932; Bouclier-Maurin 1923 ; AP (Rabat) – MISR Gymnophryxe Villeneuve, 1922 Gymnophryxe carthaginiensis (Bischof, 1900) Mesnil 1956 Nilea Robineau-Desvoidy, 1863 Nilea innoxia Robineau-Desvoidy, 1863 Bléton and Fieuzet 1939 Phryxe Robineau-Desvoidy, 1830 Phryxe caudata (Rondani, 1859) Biliotti 1956 ; El Yousfi 1994 ; IOBC-list 11 (1989) Phryxe setifacies (Villeneuve, 1910) IOBC-list 11 (1989); IOBC-list 12 (1993) Phryxe vulgaris (Fallén, 1810) Séguy 1953a , MA , Tamrabta (1700 m) – PCPT ( HA , Marrakech, Imlil, S Asni) Ptesiomyia Brauer & Bergenstamm, 1893 Ptesiomyia microstoma Brauer & Bergenstamm, 1893 Séguy 1953a , AP , Rabat; EM (Mte des Béni Snassen, Taforalt), MA (Béni Mellal, Bin-el-Ouidane; Meknès, Ifrane ( NPI )), HA (Marrakech, Oukaimeden) – PCPT Senometopia Macquart, 1834 Senometopia separata (Rondani, 1859) Hérard and Fraval 1980 Tryphera Meigen, 1838 Tryphera lugubris (Meigen, 1824) IOBC-List 1 (1956) Ethillini Atylomyia Brauer, 1898 Atylomyia albifrons Villeneuve, 1911 = Atylomyia rungsi Mesnil, in Mesnil 1962: 778 Mesnil 1962, AA (near Agadir), Aït Melloul; Herting and Dely-Draskovits 1993 Exoristini Bessa Robineau-Desvoidy, 1863 Bessa parallela (Meigen, 1824) Séguy 1935a (probably misidentified as Bessa selecta ); Tschorsnig 2017 Chetogena Rondani, 1856 Chetogena filipalpis Rondani, 1859 Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 8 m); MA (Fès, Sidi Harazem, Ifrane, Forêt de Cèdres) – PCPT Chetogena mageritensis (Villeneuve & Mesnil, 1936) Herting and Dely-Draskovits 1993 Chetogena media Rondani, 1859* MA Chetogena nigrofasciata (Strobl, 1902) = Chetogena repanda (Mesnil, 1939), in Herting and Dely-Draskovits 1993 (type locality: Skel): 17 Gheibi et al. 2010 Chetogena obliquata (Fallén, 1810) De Lépiney and Mimeur 1932; Herting 1960; IOBC-list 12 (1993); HA (Marrakech, Oukaimeden) – PCPT Exorista Meigen, 1803 Exorista deligata Pandellé, 1896 Mesnil 1946 , AP , Sidi Taibi near Kénitra; Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 ; Gheibi et al. 2010 Exorista grandis (Zetterstedt, 1844) Ebejer et al. 2019 , Rif , Dardara (484 m) Exorista larvarum (Linnaeus, 1758) Mouna 1998 Exorista nova (Rondani, 1859) Tschorsnig 2017 Exorista rendina (Herting, 1975)* AA Exorista segregata (Rondani, 1859) Mouna 1998 ; AA (Agadir, Oulma) – PCPT Goniini Anurophylla Villeneuve, 1938 Anurophylla aprica (Villeneuve, 1912)* MA Baumhaueria Meigen, 1838 Baumhaueria goniaeformis (Meigen, 1824) De Lépiney and Mimeur 1932; AP (Maâmora) – MISR ; HA (Marrakech, Oukaimeden) – PCPT Blepharipa Rondani, 1856 Blepharipa pratensis (Meigen, 1824)* HA Ceratochaetops Mesnil, 1970 Ceratochaetops triseta (Villeneuve, 1922) = Ceratochoeta triseta Villeneuve, in Rungs 1940 : 15 Rungs 1940 , MA (Cédraie); Mouna 1998 ; MA (Khénifra, El-Herri, Ifrane ( NPI ), Meknès), HA (Marrakech, 8 km N Ouirgane) – PCPT Ceromasia Rondani, 1856 Ceromasia rubrifrons (Macquart, 1834) IOBC-list 11 (1989); IOBC-list 12 (1993); HA (Marrakech, Ouirgane, Tagadirt, S Asni) – PCPT Clemelis Robineau-Desvoidy, 1863 Clemelis pullata (Meigen, 1824) IOBC-list 13 (1997) Gaedia Meigen, 1838 Gaedia connexa Meigen, 1824 Séguy 1953a , EM , Berkane Gonia Meigen, 1803 Gonia aterrima Tschorsnig, 1991 Tschorsnig 1991 Gonia atra Meigen, 1826 Séguy 1930a , MA , Tizi-n'Bouftene, between Azrou and Ras el Ma, Forêt Azrou, HA , Arround (Skoutana), Jebel Likount, Asni; Mouna 1998 ; Grabener 2017 ; MA (Tighassaline, El-Herri, Aïn Leuh-Tagounit, Meknès, Ifrane ( NPI )), HA (40 km SW Ouarzazate, Marrakech, Ouirgane, Lakhdar, N Demnate, Oukaimeden), AA (Agadir, Oulma) – PCPT Gonia bimaculata Wiedemann, 1819 = Gonia cilipeda Rondani, in Séguy 1953a : 91 Séguy 1930a , AP , Rabat, MA , Tizi-s'Tkrine, Tizi-n'Bouftene, between Azrou and Ras el Ma, Forêt Azrou, Berkane, HA , Arround (Skoutana), Jebel Likount, Asni, Ouaouzert (Glaoua); Séguy 1953a , AP , Temara; AP (Rabat) – MISR ; HA (S Asni Ouirgane, Marrakech), AA (80 km N Taroudant, Aoulouz), AA (15 km NW Zagora) – PCPT Gonia capitata (De Geer, 1776) Mouna 1998 ; MA (Ifrane) – MISR Gonia maculipennis Egger, 1862* MA Gonia ornata Meigen, 1826 Séguy 1953a , AP , Rabat, MA , Ifrane, SA , Kelaâ M'Goum; MA (Ifrane, Forêt de Cèdres), HA (Oukaimeden (2600 m), Marrakech) – PCPT Gonia vacua Meigen, 1826* MA Pales Robineau-Desvoidy, 1830 Pales pavida (Meigen, 1824) IOBC-list 1 (1956); Cerretti 2005 ; MA (Fès, Sidi Harazem) – PCPT Platymya Robineau-Desvoidy, 1830 Platymya antennata (Brauer & Bergenstamm, 1891) Efetov and Tarmann 1999 Pseudogonia Brauer and von Bergenstamm, 1889 Pseudogonia rufifrons (Wiedemann, 1830) De Lépiney and Mimeur 1932; IOBC-list 1 (1956); Mouna 1998 ; Tschorsnig 2017; Grabener 2017 ; AA (S Tafraoute, Aït Mansur, Agadir, Oulma) – PCPT Sturmia Robineau-Desvoidy, 1830 Sturmia bella (Meigen, 1824) Stefanescu et al. 2012 ; Tschorsnig 2017 Winthemiini Nemorilla Rondani, 1856 Nemorilla maculosa (Meigen, 1824) Kozlovsky and Rungs 1933 ; Brémond and Rungs 1938 [as N. floralis ; probable misidentification]; IOBC list 1 (1956); Mouna 1998 ; EM (Oujda, Col de Jerada) – PCPT Phasiinae Cylindromyiini Besseria Robineau-Desvoidy, 1830 Besseria lateritia (Meigen, 1824)* HA Cylindromyia Meigen, 1803 Cylindromyia bicolor (Olivier, 1812) Séguy 1930a , AP , Rabat; Mouna 1998 Cylindromyia brassicaria (Fabricius, 1775) Séguy 1930a , EM , Soufouloud, MA , Aharmoumou, Berrechid, Meknès, Sidi Bettache, Berkane, HA , Tenfecht, Ouaounzert, Marrakech, Asni; Séguy 1935a , AP , Gharb; Séguy 1941a , HA , Jebel Ayachi; Séguy 1949, SA , Guelmim; Dupuis 1963 ; Mouna 1998 ; MA (Sefrou) – MISR ; AP (Essaouira, 4 km E Ounara), HA (10 km W Chichaoua, Oukaimeden), AA (140 km E Agadir, Aoulouz, Tizi-n'Tichka) – PCPT Cylindromyia intermedia Meigen, 1824 Becker and Stein 1913 , Rif , Tanger; HA (Ouirgane, Imlil, Tizi-n'Test), AA (10 km SE Ouarzazate (oasis), Taroudant) – PCPT Cylindromyia maroccana Tschorsnig, 1997 Tschorsnig 1997 , HA , Ouirgane, Tagadirt (1000 m) Cylindromyia pilipes Loew, 1844 Becker and Stein 1913 , Rif , Tanger; Herting and Dely-Draskovits 1993 ; Gilasian et al. 2013 Phania Meigen, 1824 Phania albisquama (Villeneuve, 1924)* MA Gymnosomatini Clytiomya Rondani, 1861 Clytiomya continua (Panzer, 1798) = Clytiomyia dalmatica Robineau-Desvoidy, in Séguy 1935a : 119 Séguy 1935a , AP , Gharb; Mouna 1998 ; AP (Rabat) – MISR Clytiomya sola (Rondani, 1861) Séguy 1935a ; Dupuis 1963 ; MA (Khénifra, Tighassaline) – PCPT Ectophasia Townsend, 1912 Ectophasia crassipennis (Fabricius, 1794) = Phasia crassipennis Fabricius, in Séguy 1930a : 141 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 Eliozeta Rondani, 1856 Eliozeta helluo (Fabricius, 1805) = Clytiomyia helluo Fabricius, in Séguy 1935: 119 Séguy 1930a , MA , Meknès (Aïn Sferguila); Jourdan 1935; Séguy 1935a , AP , Gharb; Thompson 1950 ; Dupuis 1963 ; Mouna 1998 ; Tschorsnig 2017 Gymnosoma Meigen, 1803 Gymnosoma carpocoridis Dupuis, 1961 Dupuis 1963 ; Herting and Dely-Draskovits 1993 Gymnosoma clavatum (Rohdendorf, 1947) Dupuis 1963 ; Grabener 2017 ; MA (Meknès, Ifrane ( NPI )), AA (140 km E Agadir, Aoulouz), AA (80 km S Zagora, Oued Draa, Mhamid) – PCPT Gymnosoma dolycoridis Dupuis, 1960 Dupuis 1963 ; MA (Fès, Sidi Harazem) – PCPT Gymnosoma rotundatum Linnaeus, 1758 64 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Mogador, MA , Tizi-s'Tkrine, Sidi Bettache, Moulay Aïn Djemine, HA , Asni; Séguy 1934b ; Séguy 1935a ; Thompson 1950 ; Mouna 1998 ; AP (Mogador), MA (Maghrawa) – MISR ; MA (Meknès, Ifrane ( NPI )) – PCPT Gymnosoma rungsi (Mesnil, 1952) = Rhodogyne rungsi (Mesnil), in Mesnil 1952 : 151 Mesnil 1952 , AP , Rabat; Dupuis 1963 ; Herting and Dely-Draskovits 1993 Leucostomatini Clairvillia Robineau-Desvoidy, 1830 Clairvillia biguttata (Meigen, 1824)* HA Dionomelia Kugler, 1978 Dionomelia hennigi Kugler, 1978* SA Leucostoma Meigen, 1803 Leucostoma abbreviatum Herting, 1971 Ziegler 2012 Leucostoma crassum Kugler, 1966* HA Leucostoma obsidianum (Wiedemann, 1830) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Leucostoma tetraptera (Meigen, 1824) Dupuis 1953 ; Ebejer et al. 2019 , Rif , Barrage Smir (27 m) Weberia Robineau-Desvoidy, 1830 Weberia digramma (Meigen, 1824)* AA Phasiini Elomya Robineau-Desvoidy, 1830 Elomya lateralis (Meigen, 1824) Séguy 1930a , AP , From Zarjoulea to Larache, MA , Berkane; Dupuis 1952 ; Dupuis 1963 ; Herting and Dely-Draskovits 1993 ; Mouna 1998 ; Cerretti and Ziegler 2004 ; MA (Khénifra, Tighassaline, Meknès, Ifrane ( NPI )), HA (Marrakech, Ouirgane) – PCPT Phasia Latreille, 1804 Phasia mesnili (Draber-Monko, 1965) Sun and Marshall 2003 , HA ; AP (10 km E Essaouira), AA (S Tafraoute, Aït Mansur, S Aït-Baha) – PCPT Phasia obesa (Fabricius, 1798) Sun and Marshall 2003 , HA , Asni Phasia pusilla Meigen, 1824 Dupuis 1963 ; Sun and Marshall 2003 , MA Phasia subcoleoptrata (Linnaeus, 1767) Dupuis 1963 ; Herting and Dely-Draskovits 1993 ; Sun and Marshall 2003 , MA ; Cerretti and Ziegler 2004 Phasia venturii (Draber-Monko, 1965) Sun and Marshall 2003 , HA , Asni; AP (Essaouira, 4 km E Ounara), AA (11 km NW Taliouine, 10 km SE Aït-Ourir) – PCPT Trichopodini Trichopoda Berthold, 1827 Trichopoda pennipes (Fabricius, 1794) Ebejer et al. 2019 , Rif , Tahaddart (8 m) Xystini Xysta Meigen, 1824 Xysta holosericea (Fabricius, 1805)* HA Tachininae Graphogastrini Graphogaster Rondani, 1868 Graphogaster vestita Rondani, 1868* MA Phytomyptera Rondani, 1845 Phytomyptera nigrina (Meigen, 1824) = Phytomyptera nitidiventris Rondani, in Bléton and Fieuzet 1939 : 64 Bléton and Fieuzet 1939 ; Mouna 1998 Leskiini Aphria Robineau-Desvoidy, 1830 Aphria longirostris (Meigen, 1824) Ebejer et al. 2019 , Rif , Jnane Niche (46 m) Bithia Robineau-Desvoidy, 1863 Bithia demotica (Egger, 1861) Tschorsnig and Bläsius 2001 ; IOBC-list 14 (2005); Tschorsnig 2017 Bithia modesta (Meigen, 1824) Tschorsnig and Bläsius 2001 ; Tschorsnig 2017 Linnaemyini + Ernestiini Gymnochaeta Robineau-Desvoidy, 1830 Gymnochaeta viridis Fallén, 1810 Séguy 1930a , HA , Arround (Skoutana); Mouna 1998 Linnaemya Robineau-Desvoidy, 1830 Linnaemya soror Zimin, 1954* MA , HA , AA Loewia Egger, 1856 Loewia setibarba Egger, 1856 Becker and Stein 1913 , Rif , Tanger Panzeria Robineau-Desvoidy, 1830 Panzeria castellana (Strobl, 1906)* HA Panzeria nemorum (Meigen, 1824)* MA Zophomyia Macquart, 1835 Zophomyia temula (Scopoli, 1763) Séguy 1930a , AP , Casablanca, MA , Meknès; Mouna 1998 ; MA (Khénifra, Tighassaline, Meknès, Ifrane ( NPI )) – PCPT Macquartiini Macquartia Robineau-Desvoidy, 1830 Macquartia chalconota (Meigen, 1824) Ebejer et al. 2019 , Rif , Smir lagoon; HA (Marrakech, Ouirgane, Tizi-n'Test), AA (Taroudant) – PCPT Macquartia macularis Villeneuve, 1926 Herting and Dely-Draskovits 1993 Macquartia tessellum (Meigen, 1824) = Macquartia brevicornis Macquart, in Séguy 1941d : 23 Séguy 1941d , MA , Meknès, HA , Tizi-n'Test; Mouna 1998 ; HA (Imlil, S Asni, Tizi-n'Test), AA (Taroudant) – PCPT Megaprosopini Microphthalma Macquart, 1844 Microphthalma europaea Egger, 1860 Ebejer et al. 2019 , AA , Ziz river (30 km N of Erfoud, 894 m) Minthoini Hyperaea Robineau-Desvoidy, 1863 Hyperaea femoralis (Meigen, 1824) Herting and Dely-Draskovits 1993 Mintho Robineau-Desvoidy, 1830 Mintho compressa (Fabricius, 1787) Walker 1849 ; Herting and Dely-Draskovits 1993 Mintho rufiventris (Fallén, 1817) = Mintho praeceps (Scopoli, 1763), in Séguy 1930a : 143; Séguy 1953a : 91 Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès; Séguy 1953a , SA , El Aïoun du Draa; Séguy 1949a , AA , Tata; Mouna 1998 ; Dawah 2011 ; AP (Rabat, Salé), MA (Meknès) – MISR ; MA (Meknès, Ifrane ( NPI )), AA (11 km NW Taliouine) – PCPT Minthodes Brauer & Bergenstamm, 1889 Minthodes numidica Villeneuve, 1932* AA Minthodes setifacies Mesnil, 1939 = Minthodes ( Myxominthodes ) setifacies Mesnil, in Mesnil 1939 : 211 Mesnil 1939 , MA , Forêt Azrou; Herting and Dely-Draskovits 1993 Plesina Meigen, 1838 Plesina phalerata (Meigen, 1824) Herting and Dely-Draskovits 1993 ; Cerretti and Tschorsnig 2008 Pseudomintho Brauer & Bergenstamm, 1889 Pseudomintho diversipes (Strobl, 1889) Ebejer et al. 2019 , Rif , Moulay Abdelsalam ( PNPB , 965 m); AP (Essaouira, 4 km E Ounara) – PCPT Siphonini Actia Robineau-Desvoidy, 1830 Actia infantula (Zetterstedt, 1844) Ebejer et al. 2019 , Rif , Tanger (Douar Dakchire forest, 320 m) Peribaea Robineau-Desvoidy, 1863 Peribaea apicalis Robineau-Desvoidy, 1863 Ebejer et al. 2019 , Rif , Dardara (484 m) Peribaea tibialis (Robineau-Desvoidy, 1851) Draber-Mońko 2011 Siphona Meigen, 1803 Siphona geniculata (De Geer, 1776) 65 Séguy 1930a , HA ; Mouna 1998 ; HA (Vallée Oued N'fis) – MISR Siphona maroccana Cerretti & Tschorsnig, 2007 Cerretti and Tshorsnig 2007, HA , Asif Mellah, W Tizi-n'Tichka Siphona variata Andersen, 1982 Ebejer et al. 2019 , Rif , Sidi Yahia Aârab (377 m), Oued Kbir ( PNPB , 157 m) Tachinini Germaria Robineau-Desvoidy, 1830 Germaria barbara Mesnil, 1963* HA Peleteria Robineau-Desvoidy, 1830 Peleteria ruficornis (Macquart, 1835)* HA , AA Tachina Meigen, 1803 Tachina corsicana (Villeneuve, 1931)* HA , AA Tachina fera (Linnaeus, 1761) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, Forêt Tiffert, Sidi Bettache, HA , Arround (Skoutana), Jebel Likount; Séguy 1953a , MA , Ifrane (1650 m); Mouna 1998 ; Rif (fir forest of Talassemtane), AP (Dradek) – MISR ( MA , Meknès); MA (Ifrane ( NPI )), Béni Mellal, Bin-el-Ouidane), HA (Marrakech, Ouirgane) – PCPT Tachina magnicornis (Zetterstedt, 1844) Séguy 1930a , MA , Ras el Ksar, Aïn Leuh, Sidi Bettache; Mouna 1998 ; MA (Béni Mellal, El Ksiba, 5 km N) – PCPT Tachina praeceps Meigen, 1824 HA , AA Triarthriini Lissoglossa Villeneuve, 1912 Lissoglossa bequaerti Villeneuve, 1912 Herting and Dely-Draskovits 1993 New records for Morocco The data added under the abbreviation "PCPT" (for "personal communication Hans-Peter Tschorsnig") are based on (unpublished) material which was identified by HPT for several collectors (M. Hauser, M. Hradský, C.F. Kassebeer, U. Koschwitz, J.A.W. Lucas, G. Miksch, H. and T. v. Oorschot, C. Schmid-Egger, M. Schwarz, K. Špatenka, V. Vrabec) during the last ~ 30 years. Usually only a few duplicate specimens were retained in the collection of SMNS . The main part was sent back to the collectors, but the data were noted by HPT on handwritten lists. Estheria iberica Tschorsnig, 2003 Middle Atlas: Ifrane, National Park of Ifrane, 19.ix.1989, K. Špatenka leg, 1 specimen, PCPT. Athrycia trepida (Meigen, 1824) Middle Atlas: Meknès; Ifrane, National Park of Ifrane, 22.v.1995, C. Kassebeer leg., 1 specimen, PCPT. Eriothrix rufomaculata (De Geer, 1776) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. High Atlas: Marrakech, Oukaimeden, 19.v.1995, C. Kassebeer leg., 7 specimens, PCPT. Kirbya moerens (Meigen, 1830) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. Carcelia lucorum (Meigen, 1824) High Atlas: Marrakech, Imlil, S Asni, 24.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Drino galii (Brauer & Bergenstamm, 1891) High Atlas: Marrakech, Ouirgane, 24.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Anti Atlas: 11 km NW Taliouine; Agadir, Ameskroud, 17.v.1995, C. Kassebeer leg., 2 specimens, PCPT. Chetogena media Rondani, 1859 Middle Atlas: Béni Mellal, El Ksiba, 30.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Exorista rendina (Herting, 1975) Anti Atlas: 11 km NW Taliouine, 15.iii.1997, M. Hauser leg., 1 male in SMNS ; 10 km NE Tafraoute, 14.iii.1997, G. Miksch leg., 1 male in SMNS , PCPT. Anurophylla aprica (Villeneuve, 1912) Middle Atlas: Béni Mellal, Afourer, 28.iii.1995, C. Kassebeer leg., 1 female in SMNS , PCPT. Blepharipa pratensis (Meigen, 1824) High Atlas: Tizi-n'Test, 2000 m, 21.v.1995, M. Hauser leg., 2 specimens, PCPT. Anti Atlas: Taroudant, PCPT. Gonia maculipennis Egger, 1862 Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 female in SMNS , PCPT. Gonia vacua Meigen, 1826 Middle Atlas: Meknès; Ifrane, National Park of Ifrane, 29.iii.1995 and 22.v.1995, C. Kassebeerleg., 2 specimens, PCPT. Besseria lateritia (Meigen, 1824) High Atlas: SE Asni, Imlil, 23.v.1995, M. Hauser leg., 2 specimens; Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Phania albisquama (Villeneuve, 1924) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec and L. Vrabcová leg., 1 specimen, PCPT. Clairvillia biguttata (Meigen, 1824) High Atlas: Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg, 1 specimen, PCPT. Dionomelia hennigi Kugler, 1978 SA : Boujdour, 8.v.1999, V. Vrabec leg, 1 male in SMNS , PCPT. Leucostoma crassum Kugler, 1966 High Atlas: Tizi-n'Test, pass 23.vi.1996, U. Koschwitz leg., 1 male in SMN, PCPT. Weberia digramma (Meigen, 1824) Anti Atlas: 10 km NW Aït-Baha, PCPT. Xysta holosericea (Fabricius, 1805) High Atlas: Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg, 1 specimen, PCPT. Linnaemya soror Zimin, 1954 Middle Atlas: Béni Mellal, El Ksiba, 5 km N; Béni Mellal, Afourer; Khénifra, Tighassaline; Meknès; National Park of Ifrane. High Atlas: Marrakech, Ouirgane; Marrakech, Tagaddirt, S Asni; Marrakech, Lakhdar, N Demnate. Anti Atlas: 11 km NW Taliouine, all C. Kassebeer leg., 57 specimens (collected between 25.iii. and 23.v.1995), PCPT. Panzeria castellana (Strobl, 1906) High Atlas: Marrakech, Ouirgane, 26.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Panzeria nemorum (Meigen, 1824) Middle Atlas: Meknès; National Park of Ifrane, 22.v.1995, C. Kassebeer leg., 1 specimen, PCPT. Graphogaster vestita Rondani, 1868 Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. Minthodes numidica Villeneuve, 1932 Anti Atlas: S Aït-Baha, PCPT. Germaria barbara Mesnil, 1963 High Atlas: S Tizi-n'Test, 1900 m, PCPT. Peleteria ruficornis (Macquart, 1835) High Atlas: Marrakech, Ouirgane; Marrakech, Tagaddirt, S Asni; Tizi-n'Test. Anti Atlas: Taroudant, all C. Kassebeer leg., 6 specimens (collected between 28.ix.1994 and 1.iv.1995), PCPT. Tachina corsicana (Villeneuve, 1931) High Atlas: Marrakech, Oukaimeden, 19.v.1995, C. Kassebeer leg., 1 specimen; Tizi-n'Test. Anti Atlas: Taroudant, 21.v.1995, M. Hauser leg., 2 specimens, PCPT. Tachina praeceps Meigen, 1824 High Atlas: Marrakech, Oukaimeden, 2500 m, 27.vi.1987, M. Schwarz leg., 1 specimen; Tizi-n'Test. Anti Atlas: Taroudant, 29.vi.1987, M. Schwarz leg., 1 specimen, PCPT. Dexiinae Dexiini Billaea Robineau-Desvoidy, 1830 Billaea lata (Macquart, 1849) = Rhynchodinera lata Macquart, in Séguy 1930a : 143 Séguy 1930a , MA , Aharmoumou, Camp Boulhout, Sidi Bettache, Aïn Sferguila, Meknès; Mouna 1998 ; AP (Mehdia) – MISR ; AP (Essaouira, 4 km E Ounara), HA (Marrakech, Lakhdar, N Demnate) – PCPT Estheria Robineau-Desvoidy, 1830 Estheria atripes Villeneuve, 1920 Cerretti and Tschorsnig 2012 Estheria iberica Tschorsnig, 2003* MA Estheria nigripes (Villeneuve, 1920) Cerretti and Tschorsnig 2012 ; MA (Béni Mellal, El Ksiba), AA (Agadir, Oulma) – PCPT Estheria picta (Meigen, 1826) 62 Moutia 1940 Zeuxia Meigen, 1826 Zeuxia aberrans (Loew, 1847) = Zeuxia nigripes Meigen, in Séguy 1941d : 23 Brémond 1938 ; Séguy 1941d , AP , Rabat, MA , Volubilis, AA , Agadir; IOBC-List 11; Mesnil 1980; Mouna 1998 ; Tschorsnig 2017; AA (10 km NW Aït-Baha) – PCPT Dufouriini Dufouria Robineau-Desvoidy, 1830 Dufouria nigrita (Fallén, 1810) Ebejer et al. 2019 , AP , Larache (Lower Loukous saltmarsh, 2 m); MA (Ouzoud) – PCPT Voriini Athrycia Robineau-Desvoidy, 1830 Athrycia trepida (Meigen, 1824)* MA Cyrtophloeba Rondani, 1856 Cyrtophloeba ruricola (Meigen, 1824) = Plagia ruricola Meigen, in Séguy 1935a : 120, in Rungs 1940 : 14 Séguy 1935a , MA , Ifrane; Rungs 1940 , MA (Cédraie); Mouna 1998 ; HA (Tizi-n'Test), AA (Taroudant) – PCPT Eriothrix Meigen, 1830 Eriothrix apennina (Rondani, 1862) Herting and Dely-Draskovits 1993 ; Koçak and Kemal 2010 Eriothrix rufomaculata (De Geer, 1776)* MA , HA Hypovoria Villeneuve, 1913 Hypovoria hilaris Villeneuve, 1912 Séguy 1935a , AP , Oued Beth; Séguy 1953a , AP , Sehoul; Mouna 1998 ; AA (10 km SE Aït-Ourir) – PCPT Hypovoria pilibasis (Villeneuve, 1922) Zeegers 2010; HA (Tizi-n'Test), AA (Taroudant) – PCPT Kirbya Robineau-Desvoidy, 1830 Kirbya moerens (Meigen, 1830)* MA Nanoplagia Villeneuve, 1929 Nanoplagia sinaica (Villeneuve in Hermann & Villeneuve 1909) Cerretti 2009; Grabener 2017 ; HA (Marrakech, 8 km N Ouirgane), AA (40 km SW Ouarzazate, 10 km SW Tazenakht, NE Agadir, 12 km W Oulma) – PCPT Periscepsia Gistel, 1848 Periscepsia meyeri (Villeneuve, 1930) Ebejer et al. 2019 , Rif , Adrou ( PNPB , 556 m) Stomina Robineau-Desvoidy, 1830 Stomina caliendrata (Rondani, 1862) = Morphomyia caliendrata Rondani, in Séguy 1930a : 143 Séguy 1930a , MA , from M'Rirt to Hajeb, HA , Kasba Taguendaft (Gounfada); Mouna 1998 ; HA (Massif Toubkal) – PCPT Thelaira Robineau-Desvoidy, 1830 Thelaira haematodes (Meigen, 1824) 63 = Phoenicella haematodes Meigen: Séguy 1930a : 142 Séguy 1930a , HA , Arround; Mouna 1998 Uclesia Girschner, 1901 Uclesia fumipennis Girschner, 1901 Séguy 1934b ; Herting and Dely-Draskovits 1993 ; Mouna 1998 ; HA (Marrakech) – MISR Voria Robineau-Desvoidy, 1830 Voria ruralis (Fallén, 1810) Jourdan 1935c Wagneria Robineau-Desvoidy, 1830 Wagneria dilatata Kugler, 1977 Kugler 1977 Exoristinae Acemyini Ceracia Rondani, 1865 Ceracia mucronifera Rondani, 1865 = Myothyria benoisti (Mesnil), in Mesnil 1959 : 20 Mesnil 1959 , MA , Forêt Maâmora near Tiflet; Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 Blondeliini Compsilura Bouché, 1834 Compsilura concinnata (Meigen, 1824) IOBC-list 1 (1956); Hérard and Fraval 1980 Istocheta Rondani, 1859 Istocheta cinerea (Macquart, 1850) Herting 1960 Istocheta longicornis (Fallén, 1810) = Latigena longicornis Fallén, in Séguy 1953a : 91 Séguy 1953a , AP , Forêt Zaers Lomachantha Rondani, 1859 Lomachantha parra Rondani, 1859 Efetov and Tarmann 1999 Robinaldia Herting, 1983 Robinaldia angustata (Villeneuve, 1933) Herting and Dely-Draskovits 1993 ; Tschorsnig and Herting 1994 Zaira Robineau-Desvoidy, 1830 Zaira cinerea (Fallén, 1820) Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 8 m) Eryciini Alsomyia Brauer & Bergenstamm, 1891 Alsomyia olfaciens (Pandellé, 1896) IOBC-List 12 (1993) Amphicestonia Villeneuve, 1939 Amphicestonia dispar (Villeneuve, 1922) Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 ; MA (Ifrane) – PCPT Aplomyia Robineau-Desvoidy, 1830 Aplomyia confinis (Fallén, 1820) Ebejer et al. 2019 , Rif , Dardara (484 m) Carcelia Robineau-Desvoidy, 1830 Carcelia dilaticornis Mesnil, 1950 Mesnil 1950 Carcelia iliaca (Ratzeburg, 1840)63 Mouna 1998 Carcelia lucorum (Meigen, 1824)* HA Drino Robineau-Desvoidy, 1863 Drino atropivora (Robineau-Desvoidy, 1830) = Sturmia atropivora Robineau-Desvoidy, in Bléton and Fieuzet 1939 : 64 De Lépiney and Mimeur 1932; Bléton and Fieuzet 1939 , MA , Fès; Mouna 1998 ; Tschorsnig 2017; AP (Rabat), MA (Bel Lakssiri) – MISR Drino galii (Brauer & Bergenstamm, 1891)* HA , AA Drino gilva (Hartig, 1838)63 = Sturmia gilva Hartig Mouna 1998 – MISR (no locality given) Drino imberbis (Wiedemann, 1830)63 Rungs 1954 ; Grabener 2017 Drino inconspicua (Meigen, 1830) Séguy 1935a , AP , Sehoul (Rabat); Bléton and Fieuzet 1939 , MA , Dayat Achleff; Mouna 1998 ; MA (Meknès, Béni Mellal) – MISR Drino maroccana Mesnil, 1951 De Lépiney 1930 ; De Lépiney and Mimeur 1932 (probably misidentified as Sturmia inconspicua ); Mesnil 1951 ; Herting and Dely-Draskovits 1993 ; Ziegler 2011 Drino triplaca Herting, 1979 Herting 1979 , AP , Rabat Drino vicina (Zetterstedt, 1849) = Sturmia vicina Zetterstedt, 1849 De Lépiney and Mimeur 1932; Bouclier-Maurin 1923 ; AP (Rabat) – MISR Gymnophryxe Villeneuve, 1922 Gymnophryxe carthaginiensis (Bischof, 1900) Mesnil 1956 Nilea Robineau-Desvoidy, 1863 Nilea innoxia Robineau-Desvoidy, 1863 Bléton and Fieuzet 1939 Phryxe Robineau-Desvoidy, 1830 Phryxe caudata (Rondani, 1859) Biliotti 1956 ; El Yousfi 1994 ; IOBC-list 11 (1989) Phryxe setifacies (Villeneuve, 1910) IOBC-list 11 (1989); IOBC-list 12 (1993) Phryxe vulgaris (Fallén, 1810) Séguy 1953a , MA , Tamrabta (1700 m) – PCPT ( HA , Marrakech, Imlil, S Asni) Ptesiomyia Brauer & Bergenstamm, 1893 Ptesiomyia microstoma Brauer & Bergenstamm, 1893 Séguy 1953a , AP , Rabat; EM (Mte des Béni Snassen, Taforalt), MA (Béni Mellal, Bin-el-Ouidane; Meknès, Ifrane ( NPI )), HA (Marrakech, Oukaimeden) – PCPT Senometopia Macquart, 1834 Senometopia separata (Rondani, 1859) Hérard and Fraval 1980 Tryphera Meigen, 1838 Tryphera lugubris (Meigen, 1824) IOBC-List 1 (1956) Ethillini Atylomyia Brauer, 1898 Atylomyia albifrons Villeneuve, 1911 = Atylomyia rungsi Mesnil, in Mesnil 1962: 778 Mesnil 1962, AA (near Agadir), Aït Melloul; Herting and Dely-Draskovits 1993 Exoristini Bessa Robineau-Desvoidy, 1863 Bessa parallela (Meigen, 1824) Séguy 1935a (probably misidentified as Bessa selecta ); Tschorsnig 2017 Chetogena Rondani, 1856 Chetogena filipalpis Rondani, 1859 Ebejer et al. 2019 , Rif , Aïn Jdioui (Tahaddart, 8 m); MA (Fès, Sidi Harazem, Ifrane, Forêt de Cèdres) – PCPT Chetogena mageritensis (Villeneuve & Mesnil, 1936) Herting and Dely-Draskovits 1993 Chetogena media Rondani, 1859* MA Chetogena nigrofasciata (Strobl, 1902) = Chetogena repanda (Mesnil, 1939), in Herting and Dely-Draskovits 1993 (type locality: Skel): 17 Gheibi et al. 2010 Chetogena obliquata (Fallén, 1810) De Lépiney and Mimeur 1932; Herting 1960; IOBC-list 12 (1993); HA (Marrakech, Oukaimeden) – PCPT Exorista Meigen, 1803 Exorista deligata Pandellé, 1896 Mesnil 1946 , AP , Sidi Taibi near Kénitra; Herting and Dely-Draskovits 1993 ; Cerretti and Ziegler 2004 ; Gheibi et al. 2010 Exorista grandis (Zetterstedt, 1844) Ebejer et al. 2019 , Rif , Dardara (484 m) Exorista larvarum (Linnaeus, 1758) Mouna 1998 Exorista nova (Rondani, 1859) Tschorsnig 2017 Exorista rendina (Herting, 1975)* AA Exorista segregata (Rondani, 1859) Mouna 1998 ; AA (Agadir, Oulma) – PCPT Goniini Anurophylla Villeneuve, 1938 Anurophylla aprica (Villeneuve, 1912)* MA Baumhaueria Meigen, 1838 Baumhaueria goniaeformis (Meigen, 1824) De Lépiney and Mimeur 1932; AP (Maâmora) – MISR ; HA (Marrakech, Oukaimeden) – PCPT Blepharipa Rondani, 1856 Blepharipa pratensis (Meigen, 1824)* HA Ceratochaetops Mesnil, 1970 Ceratochaetops triseta (Villeneuve, 1922) = Ceratochoeta triseta Villeneuve, in Rungs 1940 : 15 Rungs 1940 , MA (Cédraie); Mouna 1998 ; MA (Khénifra, El-Herri, Ifrane ( NPI ), Meknès), HA (Marrakech, 8 km N Ouirgane) – PCPT Ceromasia Rondani, 1856 Ceromasia rubrifrons (Macquart, 1834) IOBC-list 11 (1989); IOBC-list 12 (1993); HA (Marrakech, Ouirgane, Tagadirt, S Asni) – PCPT Clemelis Robineau-Desvoidy, 1863 Clemelis pullata (Meigen, 1824) IOBC-list 13 (1997) Gaedia Meigen, 1838 Gaedia connexa Meigen, 1824 Séguy 1953a , EM , Berkane Gonia Meigen, 1803 Gonia aterrima Tschorsnig, 1991 Tschorsnig 1991 Gonia atra Meigen, 1826 Séguy 1930a , MA , Tizi-n'Bouftene, between Azrou and Ras el Ma, Forêt Azrou, HA , Arround (Skoutana), Jebel Likount, Asni; Mouna 1998 ; Grabener 2017 ; MA (Tighassaline, El-Herri, Aïn Leuh-Tagounit, Meknès, Ifrane ( NPI )), HA (40 km SW Ouarzazate, Marrakech, Ouirgane, Lakhdar, N Demnate, Oukaimeden), AA (Agadir, Oulma) – PCPT Gonia bimaculata Wiedemann, 1819 = Gonia cilipeda Rondani, in Séguy 1953a : 91 Séguy 1930a , AP , Rabat, MA , Tizi-s'Tkrine, Tizi-n'Bouftene, between Azrou and Ras el Ma, Forêt Azrou, Berkane, HA , Arround (Skoutana), Jebel Likount, Asni, Ouaouzert (Glaoua); Séguy 1953a , AP , Temara; AP (Rabat) – MISR ; HA (S Asni Ouirgane, Marrakech), AA (80 km N Taroudant, Aoulouz), AA (15 km NW Zagora) – PCPT Gonia capitata (De Geer, 1776) Mouna 1998 ; MA (Ifrane) – MISR Gonia maculipennis Egger, 1862* MA Gonia ornata Meigen, 1826 Séguy 1953a , AP , Rabat, MA , Ifrane, SA , Kelaâ M'Goum; MA (Ifrane, Forêt de Cèdres), HA (Oukaimeden (2600 m), Marrakech) – PCPT Gonia vacua Meigen, 1826* MA Pales Robineau-Desvoidy, 1830 Pales pavida (Meigen, 1824) IOBC-list 1 (1956); Cerretti 2005 ; MA (Fès, Sidi Harazem) – PCPT Platymya Robineau-Desvoidy, 1830 Platymya antennata (Brauer & Bergenstamm, 1891) Efetov and Tarmann 1999 Pseudogonia Brauer and von Bergenstamm, 1889 Pseudogonia rufifrons (Wiedemann, 1830) De Lépiney and Mimeur 1932; IOBC-list 1 (1956); Mouna 1998 ; Tschorsnig 2017; Grabener 2017 ; AA (S Tafraoute, Aït Mansur, Agadir, Oulma) – PCPT Sturmia Robineau-Desvoidy, 1830 Sturmia bella (Meigen, 1824) Stefanescu et al. 2012 ; Tschorsnig 2017 Winthemiini Nemorilla Rondani, 1856 Nemorilla maculosa (Meigen, 1824) Kozlovsky and Rungs 1933 ; Brémond and Rungs 1938 [as N. floralis ; probable misidentification]; IOBC list 1 (1956); Mouna 1998 ; EM (Oujda, Col de Jerada) – PCPT Phasiinae Cylindromyiini Besseria Robineau-Desvoidy, 1830 Besseria lateritia (Meigen, 1824)* HA Cylindromyia Meigen, 1803 Cylindromyia bicolor (Olivier, 1812) Séguy 1930a , AP , Rabat; Mouna 1998 Cylindromyia brassicaria (Fabricius, 1775) Séguy 1930a , EM , Soufouloud, MA , Aharmoumou, Berrechid, Meknès, Sidi Bettache, Berkane, HA , Tenfecht, Ouaounzert, Marrakech, Asni; Séguy 1935a , AP , Gharb; Séguy 1941a , HA , Jebel Ayachi; Séguy 1949, SA , Guelmim; Dupuis 1963 ; Mouna 1998 ; MA (Sefrou) – MISR ; AP (Essaouira, 4 km E Ounara), HA (10 km W Chichaoua, Oukaimeden), AA (140 km E Agadir, Aoulouz, Tizi-n'Tichka) – PCPT Cylindromyia intermedia Meigen, 1824 Becker and Stein 1913 , Rif , Tanger; HA (Ouirgane, Imlil, Tizi-n'Test), AA (10 km SE Ouarzazate (oasis), Taroudant) – PCPT Cylindromyia maroccana Tschorsnig, 1997 Tschorsnig 1997 , HA , Ouirgane, Tagadirt (1000 m) Cylindromyia pilipes Loew, 1844 Becker and Stein 1913 , Rif , Tanger; Herting and Dely-Draskovits 1993 ; Gilasian et al. 2013 Phania Meigen, 1824 Phania albisquama (Villeneuve, 1924)* MA Gymnosomatini Clytiomya Rondani, 1861 Clytiomya continua (Panzer, 1798) = Clytiomyia dalmatica Robineau-Desvoidy, in Séguy 1935a : 119 Séguy 1935a , AP , Gharb; Mouna 1998 ; AP (Rabat) – MISR Clytiomya sola (Rondani, 1861) Séguy 1935a ; Dupuis 1963 ; MA (Khénifra, Tighassaline) – PCPT Ectophasia Townsend, 1912 Ectophasia crassipennis (Fabricius, 1794) = Phasia crassipennis Fabricius, in Séguy 1930a : 141 Séguy 1930a , MA , Aïn Leuh; Mouna 1998 Eliozeta Rondani, 1856 Eliozeta helluo (Fabricius, 1805) = Clytiomyia helluo Fabricius, in Séguy 1935: 119 Séguy 1930a , MA , Meknès (Aïn Sferguila); Jourdan 1935; Séguy 1935a , AP , Gharb; Thompson 1950 ; Dupuis 1963 ; Mouna 1998 ; Tschorsnig 2017 Gymnosoma Meigen, 1803 Gymnosoma carpocoridis Dupuis, 1961 Dupuis 1963 ; Herting and Dely-Draskovits 1993 Gymnosoma clavatum (Rohdendorf, 1947) Dupuis 1963 ; Grabener 2017 ; MA (Meknès, Ifrane ( NPI )), AA (140 km E Agadir, Aoulouz), AA (80 km S Zagora, Oued Draa, Mhamid) – PCPT Gymnosoma dolycoridis Dupuis, 1960 Dupuis 1963 ; MA (Fès, Sidi Harazem) – PCPT Gymnosoma rotundatum Linnaeus, 1758 64 Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , Rif , Tanger, AP , Mogador, MA , Tizi-s'Tkrine, Sidi Bettache, Moulay Aïn Djemine, HA , Asni; Séguy 1934b ; Séguy 1935a ; Thompson 1950 ; Mouna 1998 ; AP (Mogador), MA (Maghrawa) – MISR ; MA (Meknès, Ifrane ( NPI )) – PCPT Gymnosoma rungsi (Mesnil, 1952) = Rhodogyne rungsi (Mesnil), in Mesnil 1952 : 151 Mesnil 1952 , AP , Rabat; Dupuis 1963 ; Herting and Dely-Draskovits 1993 Leucostomatini Clairvillia Robineau-Desvoidy, 1830 Clairvillia biguttata (Meigen, 1824)* HA Dionomelia Kugler, 1978 Dionomelia hennigi Kugler, 1978* SA Leucostoma Meigen, 1803 Leucostoma abbreviatum Herting, 1971 Ziegler 2012 Leucostoma crassum Kugler, 1966* HA Leucostoma obsidianum (Wiedemann, 1830) Ebejer et al. 2019 , AA , Ziz river (10 km S of Errachidia, 1008 m) Leucostoma tetraptera (Meigen, 1824) Dupuis 1953 ; Ebejer et al. 2019 , Rif , Barrage Smir (27 m) Weberia Robineau-Desvoidy, 1830 Weberia digramma (Meigen, 1824)* AA Phasiini Elomya Robineau-Desvoidy, 1830 Elomya lateralis (Meigen, 1824) Séguy 1930a , AP , From Zarjoulea to Larache, MA , Berkane; Dupuis 1952 ; Dupuis 1963 ; Herting and Dely-Draskovits 1993 ; Mouna 1998 ; Cerretti and Ziegler 2004 ; MA (Khénifra, Tighassaline, Meknès, Ifrane ( NPI )), HA (Marrakech, Ouirgane) – PCPT Phasia Latreille, 1804 Phasia mesnili (Draber-Monko, 1965) Sun and Marshall 2003 , HA ; AP (10 km E Essaouira), AA (S Tafraoute, Aït Mansur, S Aït-Baha) – PCPT Phasia obesa (Fabricius, 1798) Sun and Marshall 2003 , HA , Asni Phasia pusilla Meigen, 1824 Dupuis 1963 ; Sun and Marshall 2003 , MA Phasia subcoleoptrata (Linnaeus, 1767) Dupuis 1963 ; Herting and Dely-Draskovits 1993 ; Sun and Marshall 2003 , MA ; Cerretti and Ziegler 2004 Phasia venturii (Draber-Monko, 1965) Sun and Marshall 2003 , HA , Asni; AP (Essaouira, 4 km E Ounara), AA (11 km NW Taliouine, 10 km SE Aït-Ourir) – PCPT Trichopodini Trichopoda Berthold, 1827 Trichopoda pennipes (Fabricius, 1794) Ebejer et al. 2019 , Rif , Tahaddart (8 m) Xystini Xysta Meigen, 1824 Xysta holosericea (Fabricius, 1805)* HA Tachininae Graphogastrini Graphogaster Rondani, 1868 Graphogaster vestita Rondani, 1868* MA Phytomyptera Rondani, 1845 Phytomyptera nigrina (Meigen, 1824) = Phytomyptera nitidiventris Rondani, in Bléton and Fieuzet 1939 : 64 Bléton and Fieuzet 1939 ; Mouna 1998 Leskiini Aphria Robineau-Desvoidy, 1830 Aphria longirostris (Meigen, 1824) Ebejer et al. 2019 , Rif , Jnane Niche (46 m) Bithia Robineau-Desvoidy, 1863 Bithia demotica (Egger, 1861) Tschorsnig and Bläsius 2001 ; IOBC-list 14 (2005); Tschorsnig 2017 Bithia modesta (Meigen, 1824) Tschorsnig and Bläsius 2001 ; Tschorsnig 2017 Linnaemyini + Ernestiini Gymnochaeta Robineau-Desvoidy, 1830 Gymnochaeta viridis Fallén, 1810 Séguy 1930a , HA , Arround (Skoutana); Mouna 1998 Linnaemya Robineau-Desvoidy, 1830 Linnaemya soror Zimin, 1954* MA , HA , AA Loewia Egger, 1856 Loewia setibarba Egger, 1856 Becker and Stein 1913 , Rif , Tanger Panzeria Robineau-Desvoidy, 1830 Panzeria castellana (Strobl, 1906)* HA Panzeria nemorum (Meigen, 1824)* MA Zophomyia Macquart, 1835 Zophomyia temula (Scopoli, 1763) Séguy 1930a , AP , Casablanca, MA , Meknès; Mouna 1998 ; MA (Khénifra, Tighassaline, Meknès, Ifrane ( NPI )) – PCPT Macquartiini Macquartia Robineau-Desvoidy, 1830 Macquartia chalconota (Meigen, 1824) Ebejer et al. 2019 , Rif , Smir lagoon; HA (Marrakech, Ouirgane, Tizi-n'Test), AA (Taroudant) – PCPT Macquartia macularis Villeneuve, 1926 Herting and Dely-Draskovits 1993 Macquartia tessellum (Meigen, 1824) = Macquartia brevicornis Macquart, in Séguy 1941d : 23 Séguy 1941d , MA , Meknès, HA , Tizi-n'Test; Mouna 1998 ; HA (Imlil, S Asni, Tizi-n'Test), AA (Taroudant) – PCPT Megaprosopini Microphthalma Macquart, 1844 Microphthalma europaea Egger, 1860 Ebejer et al. 2019 , AA , Ziz river (30 km N of Erfoud, 894 m) Minthoini Hyperaea Robineau-Desvoidy, 1863 Hyperaea femoralis (Meigen, 1824) Herting and Dely-Draskovits 1993 Mintho Robineau-Desvoidy, 1830 Mintho compressa (Fabricius, 1787) Walker 1849 ; Herting and Dely-Draskovits 1993 Mintho rufiventris (Fallén, 1817) = Mintho praeceps (Scopoli, 1763), in Séguy 1930a : 143; Séguy 1953a : 91 Séguy 1930a , AP , Rabat, Casablanca, MA , Meknès; Séguy 1953a , SA , El Aïoun du Draa; Séguy 1949a , AA , Tata; Mouna 1998 ; Dawah 2011 ; AP (Rabat, Salé), MA (Meknès) – MISR ; MA (Meknès, Ifrane ( NPI )), AA (11 km NW Taliouine) – PCPT Minthodes Brauer & Bergenstamm, 1889 Minthodes numidica Villeneuve, 1932* AA Minthodes setifacies Mesnil, 1939 = Minthodes ( Myxominthodes ) setifacies Mesnil, in Mesnil 1939 : 211 Mesnil 1939 , MA , Forêt Azrou; Herting and Dely-Draskovits 1993 Plesina Meigen, 1838 Plesina phalerata (Meigen, 1824) Herting and Dely-Draskovits 1993 ; Cerretti and Tschorsnig 2008 Pseudomintho Brauer & Bergenstamm, 1889 Pseudomintho diversipes (Strobl, 1889) Ebejer et al. 2019 , Rif , Moulay Abdelsalam ( PNPB , 965 m); AP (Essaouira, 4 km E Ounara) – PCPT Siphonini Actia Robineau-Desvoidy, 1830 Actia infantula (Zetterstedt, 1844) Ebejer et al. 2019 , Rif , Tanger (Douar Dakchire forest, 320 m) Peribaea Robineau-Desvoidy, 1863 Peribaea apicalis Robineau-Desvoidy, 1863 Ebejer et al. 2019 , Rif , Dardara (484 m) Peribaea tibialis (Robineau-Desvoidy, 1851) Draber-Mońko 2011 Siphona Meigen, 1803 Siphona geniculata (De Geer, 1776) 65 Séguy 1930a , HA ; Mouna 1998 ; HA (Vallée Oued N'fis) – MISR Siphona maroccana Cerretti & Tschorsnig, 2007 Cerretti and Tshorsnig 2007, HA , Asif Mellah, W Tizi-n'Tichka Siphona variata Andersen, 1982 Ebejer et al. 2019 , Rif , Sidi Yahia Aârab (377 m), Oued Kbir ( PNPB , 157 m) Tachinini Germaria Robineau-Desvoidy, 1830 Germaria barbara Mesnil, 1963* HA Peleteria Robineau-Desvoidy, 1830 Peleteria ruficornis (Macquart, 1835)* HA , AA Tachina Meigen, 1803 Tachina corsicana (Villeneuve, 1931)* HA , AA Tachina fera (Linnaeus, 1761) Becker and Stein 1913 , Rif , Tanger; Séguy 1930a , MA , Tizi-s'Tkrine, Forêt Tiffert, Sidi Bettache, HA , Arround (Skoutana), Jebel Likount; Séguy 1953a , MA , Ifrane (1650 m); Mouna 1998 ; Rif (fir forest of Talassemtane), AP (Dradek) – MISR ( MA , Meknès); MA (Ifrane ( NPI )), Béni Mellal, Bin-el-Ouidane), HA (Marrakech, Ouirgane) – PCPT Tachina magnicornis (Zetterstedt, 1844) Séguy 1930a , MA , Ras el Ksar, Aïn Leuh, Sidi Bettache; Mouna 1998 ; MA (Béni Mellal, El Ksiba, 5 km N) – PCPT Tachina praeceps Meigen, 1824 HA , AA Triarthriini Lissoglossa Villeneuve, 1912 Lissoglossa bequaerti Villeneuve, 1912 Herting and Dely-Draskovits 1993 New records for Morocco The data added under the abbreviation "PCPT" (for "personal communication Hans-Peter Tschorsnig") are based on (unpublished) material which was identified by HPT for several collectors (M. Hauser, M. Hradský, C.F. Kassebeer, U. Koschwitz, J.A.W. Lucas, G. Miksch, H. and T. v. Oorschot, C. Schmid-Egger, M. Schwarz, K. Špatenka, V. Vrabec) during the last ~ 30 years. Usually only a few duplicate specimens were retained in the collection of SMNS . The main part was sent back to the collectors, but the data were noted by HPT on handwritten lists. Estheria iberica Tschorsnig, 2003 Middle Atlas: Ifrane, National Park of Ifrane, 19.ix.1989, K. Špatenka leg, 1 specimen, PCPT. Athrycia trepida (Meigen, 1824) Middle Atlas: Meknès; Ifrane, National Park of Ifrane, 22.v.1995, C. Kassebeer leg., 1 specimen, PCPT. Eriothrix rufomaculata (De Geer, 1776) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. High Atlas: Marrakech, Oukaimeden, 19.v.1995, C. Kassebeer leg., 7 specimens, PCPT. Kirbya moerens (Meigen, 1830) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. Carcelia lucorum (Meigen, 1824) High Atlas: Marrakech, Imlil, S Asni, 24.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Drino galii (Brauer & Bergenstamm, 1891) High Atlas: Marrakech, Ouirgane, 24.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Anti Atlas: 11 km NW Taliouine; Agadir, Ameskroud, 17.v.1995, C. Kassebeer leg., 2 specimens, PCPT. Chetogena media Rondani, 1859 Middle Atlas: Béni Mellal, El Ksiba, 30.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Exorista rendina (Herting, 1975) Anti Atlas: 11 km NW Taliouine, 15.iii.1997, M. Hauser leg., 1 male in SMNS ; 10 km NE Tafraoute, 14.iii.1997, G. Miksch leg., 1 male in SMNS , PCPT. Anurophylla aprica (Villeneuve, 1912) Middle Atlas: Béni Mellal, Afourer, 28.iii.1995, C. Kassebeer leg., 1 female in SMNS , PCPT. Blepharipa pratensis (Meigen, 1824) High Atlas: Tizi-n'Test, 2000 m, 21.v.1995, M. Hauser leg., 2 specimens, PCPT. Anti Atlas: Taroudant, PCPT. Gonia maculipennis Egger, 1862 Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 female in SMNS , PCPT. Gonia vacua Meigen, 1826 Middle Atlas: Meknès; Ifrane, National Park of Ifrane, 29.iii.1995 and 22.v.1995, C. Kassebeerleg., 2 specimens, PCPT. Besseria lateritia (Meigen, 1824) High Atlas: SE Asni, Imlil, 23.v.1995, M. Hauser leg., 2 specimens; Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Phania albisquama (Villeneuve, 1924) Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec and L. Vrabcová leg., 1 specimen, PCPT. Clairvillia biguttata (Meigen, 1824) High Atlas: Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg, 1 specimen, PCPT. Dionomelia hennigi Kugler, 1978 SA : Boujdour, 8.v.1999, V. Vrabec leg, 1 male in SMNS , PCPT. Leucostoma crassum Kugler, 1966 High Atlas: Tizi-n'Test, pass 23.vi.1996, U. Koschwitz leg., 1 male in SMN, PCPT. Weberia digramma (Meigen, 1824) Anti Atlas: 10 km NW Aït-Baha, PCPT. Xysta holosericea (Fabricius, 1805) High Atlas: Marrakech, Lakhdar, N Demnate, 27.iii.1995, C. Kassebeer leg, 1 specimen, PCPT. Linnaemya soror Zimin, 1954 Middle Atlas: Béni Mellal, El Ksiba, 5 km N; Béni Mellal, Afourer; Khénifra, Tighassaline; Meknès; National Park of Ifrane. High Atlas: Marrakech, Ouirgane; Marrakech, Tagaddirt, S Asni; Marrakech, Lakhdar, N Demnate. Anti Atlas: 11 km NW Taliouine, all C. Kassebeer leg., 57 specimens (collected between 25.iii. and 23.v.1995), PCPT. Panzeria castellana (Strobl, 1906) High Atlas: Marrakech, Ouirgane, 26.iii.1995, C. Kassebeer leg., 1 specimen, PCPT. Panzeria nemorum (Meigen, 1824) Middle Atlas: Meknès; National Park of Ifrane, 22.v.1995, C. Kassebeer leg., 1 specimen, PCPT. Graphogaster vestita Rondani, 1868 Middle Atlas: Ifrane, Forêt de Cèdres, 29.iv.1999, V. Vrabec leg., 1 specimen, PCPT. Minthodes numidica Villeneuve, 1932 Anti Atlas: S Aït-Baha, PCPT. Germaria barbara Mesnil, 1963 High Atlas: S Tizi-n'Test, 1900 m, PCPT. Peleteria ruficornis (Macquart, 1835) High Atlas: Marrakech, Ouirgane; Marrakech, Tagaddirt, S Asni; Tizi-n'Test. Anti Atlas: Taroudant, all C. Kassebeer leg., 6 specimens (collected between 28.ix.1994 and 1.iv.1995), PCPT. Tachina corsicana (Villeneuve, 1931) High Atlas: Marrakech, Oukaimeden, 19.v.1995, C. Kassebeer leg., 1 specimen; Tizi-n'Test. Anti Atlas: Taroudant, 21.v.1995, M. Hauser leg., 2 specimens, PCPT. Tachina praeceps Meigen, 1824 High Atlas: Marrakech, Oukaimeden, 2500 m, 27.vi.1987, M. Schwarz leg., 1 specimen; Tizi-n'Test. Anti Atlas: Taroudant, 29.vi.1987, M. Schwarz leg., 1 specimen, PCPT.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868009/

Towards the application of Tc toxins as a universal protein translocation system

Tc toxins are bacterial protein complexes that inject cytotoxic enzymes into target cells using a syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigate whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, Ras-binding domain of CRAF kinase, and TEV protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to apply Tc toxins as a universal protein translocation system. Introduction Bacteria produce numerous pore-forming toxins that function by puncturing the plasma membrane of target cells. There, they either form a perforating pore that dissipates crucial electrochemical gradients or function as an injection device that translocates a toxic molecule into the cytoplasm. Tripartite toxin complexes (Tc) belong to the latter class and are widespread in insect and human pathogens 1 , 2 . Originally discovered in the insect pathogen Photorhabdus luminescens 3 , many gene loci encoding these proteins have since been found in other organisms. Tc toxins appear to be particularly well represented in enterobacteria, with examples being the insect pathogen Xenorhabdus nematophila 4 , 5 , the facultative human pathogen Photorhabdus asymbiotica 6 , and the deadly human and insect pathogens Yersinia spp . 7 , 8 . Tc toxins consist of three components: TcA, TcB, and TcC. The ~ 1.4 MDa TcA is a homopentameric bell-shaped molecule that mediates target cell association, membrane penetration, and toxin translocation 9 . TcA consists of a central, pre-formed α-helical channel connected to an enclosing outer shell by a linker that acts as an entropic spring during toxin injection 10 , 11 . The shell is composed of a structurally conserved α-helical domain that is decorated by a neuraminidase-like domain, as well as by variable immunoglobulin-fold receptor-binding domains. In some Tc toxins, the latter are functionally replaced by small soluble proteins that form a quaternary complex with the TcA subunit 12 , 13 . TcB and TcC together form a ~ 250 kDa cocoon that encapsulates the autoproteolytically cleaved ~ 30 kDa C-terminal hypervariable region (HVR) of TcC, the actual cytotoxic component of the Tc toxin 10 , 14 . The HVR resides in a partially or completely unfolded state in the cocoon 10 , 15 . Binding of TcB-TcC to TcA and the subsequent pH-dependent prepore-to-pore transition of the ABC holotoxin result in a continuous translocation channel from the TcB-TcC lumen across the target cell membrane into the cytoplasm that allows the translocation of the HVR 16 . Previously, we resolved several crucial steps of the Tc intoxication mechanism 17 . The first of these is holotoxin formation, where the key feature is a conformational transition of the TcB domain that binds to TcA. This domain is a six-bladed β-propeller, and upon contact of TcA with TcB, the closed blades of the β-propeller unfold and refold in an open form. Consequently, the HVR passes through the β-propeller and enters the translocation channel 16 . The assembled holotoxin binds to receptors on the target cell surface and is endocytosed 10 , 18 . Upon acidification of the late endosome, the bottom of the TcA shell opens and the prepore-to-pore transition of the Tc toxin occurs 9 . During this process, the compaction of the stretched linker between the shell and the channel drives the channel through the now open bottom of the shell and across the membrane 11 . The α-helical domain of the outer shell, which possesses a stabilizing protein knot, functions as a stator for the transition 19 . Subsequently, the tip of the channel opens and the HVR is translocated into the target cell cytoplasm, where it interferes with critical cellular processes, ultimately causing cell death 18 . Two HVRs from Photorhabdus luminescens TcC proteins have been found to function as ADP-ribosyltransferases targeting actin (TccC3HVR) and Rho GTPases such as RhoA and Cdc42 (TccC5HVR) 18 . However, no HVR structures have been solved so far, limiting our understanding of the structural requirements for proteins translocated by Tc toxins. Although previous studies on other bacterial ADP-ribosyltransferases have shown that these enzymes can in fact be structurally similar even without any significant sequence similarity 20 – 23 , we do not know if this also holds true for Tc toxin HVRs. In this study, we raise an interesting question related to this topic: can the sophisticated Tc toxin translocation system be hijacked and used to transport proteins other than the natural HVRs? Such a proof of concept has already been demonstrated for the anthrax toxin, which was used to transport proteins fused to anthrax lethal factor into cells, including the TccC3HVR of Tc toxins 24 . In fact, similar designs based on the diphtheria toxin and Pseudomonas aeruginosa exotoxin A have already been explored as anticancer drugs 25 , 26 . These systems however have the disadvantage that the fused cargo is exposed to the external environment during delivery, potentially causing the cargo to show premature and unspecific activity and limiting the usefulness of such constructs for both medical and research applications. This drawback could be avoided if the cargo were to be transported to its destination in an inactive form inside the TcB-TcC cocoon, and only activated after protein translocation through the TcA pentamer. To achieve this, the cargo protein to be translocated would have to be fused to the C-terminus of TcC instead of the native HVR in order to get encapsulated in the cocoon and subsequently translocated through TcA into the target cell (Fig. 1a, b ). We explore these aspects here by swapping the TccC3HVR to comparably sized proteins with diverse origins and functions (Supplementary Fig. 1a ), and then assess holotoxin formation and cargo translocation of these constructs. We find that no stable ABC holotoxin is formed for cargos below a total size of ~ 20 kDa, which is in accordance with our previous finding that an empty TcB-TcC cocoon does not form ABC with high-affinity either 16 . We then screen different cargos for their translocation after triggering the prepore-to-pore transition in vitro. Several, but not all small proteins are successfully translocated when fused with parts of TccC3HVR. Generally, the non-translocated cargos have a considerably lower isoelectric point (pI) than the translocated constructs. Furthermore, the crystal structures of two non-translocated constructs show us that the formation of structural elements inside the TcB-TcC cocoon, in particular those that interact stably with the inner surface of the cocoon, also prevent cargo translocation. Fig. 1 Tc toxin translocation mechanism and exchange of TccC HVRs. a Schematic of the Tc toxin translocation mechanism. The cocoon-like TcB-TcC component (blue-purple) encapsulates the autoproteolytically cleaved cytotoxic C-terminus of TcC known as the HVR (black). Upon binding of the TcB-TcC component to the pentameric TcA component (visible monomers in red, beige, orange, and green) via the TcA-binding domain of TcB (darker blue), the HVR is released into the central channel of the toxin (gray). Upon pH-induced prepore-to-pore transition at the cell membrane, the channel opens and the HVR is translocated into the cytoplasm. b Schematic of the experimental concept. The HVR in the cocoon is recombinantly replaced by an alternative protein to be translocated (green). c Effect of TccC3HVR to TccC5HVR replacement in the TcdB2-TccC3 cocoon on cytotoxicity. The ability of the TccC5HVR construct to kill HEK293T cells demonstrates that it is able to effectively translocate through the pore formed by the TcdA1 pentamer. A fourfold higher concentration of the TccC5HVR construct is needed to obtain a cytotoxic effect comparable to that of the original TccC3HVR. While this may indicate less-efficient translocation, the effect is more likely owing to TccC5HVR being a less-potent toxin than TccC3HVR, a finding confirmed by previous studies 18 . Experiments were performed in triplicates with qualitatively identical results. Scale bars: 100 μm Together, our results show that cargo proteins must fulfill three prerequisites to be successfully translocated by TcA. The first is the cargo size, which needs to be above a threshold of ~ 20 kDa to form a stable holotoxin complex. The second is the net charge, which must be positive at neutral pH values. The third is that the cargo must not form structural elements within TcB-TcC. Observing these guidelines is the key to creating functional Tc-based protein injection devices. Results TcC-HVRs are exchangeable in Tc toxins As an initial proof that different cargos fused to TcC can be translocated, we tested whether HVRs from different TcC proteins are exchangeable and result in functional, toxic ABC complexes. For this, we replaced the TccC3HVR sequence after the autoproteolytic cleavage site in TcdB2-TccC3 to that of TccC5HVR, resulting in the chimeric TcdB2-TccC3-TccC5HVR complex. After assembly of the ABC-TccC5HVR holotoxin, we assessed cytotoxicity on HEK293T cells. Complete cell death occurs upon addition of 2 nM ABC-TccC5HVR toxin, compared with cell death at 0.5 nM when exposed to the ABC-TccC3HVR holotoxin (Fig. 1c ). This is in accordance with previous findings that the cytotoxic effect of TccC5HVR is less pronounced than that of TccC3HVR 18 . This experiment shows that the cocoon formed by TcdB2-TccC3 is capable of also encapsulating and translocating other HVRs, such as TccC5HVR. We therefore chose TcdB2-TccC3 to function as a cocoon scaffold for translocation of other cargo proteins, which are not components of the Tc toxin system. Importantly, the two different HVRs do not show any pronounced sections of sequence identity (Supplementary Fig. 1b ), indicating that there is no conserved motif in Tcc-HVRs that is a general prerequisite for successful translocation. Replacement of TcC HVR by heterologous cargo proteins Our next step was to replace the HVR of TccC3 with unrelated heterologous proteins and test the capability of the holotoxin to translocate these. The criteria used to select replacement proteins were (i) a small size (11–34 kDa) to guarantee that they fit into the TcB-TcC cocoon, (ii) diverse folds to assess whether this influences the translocation capability, (iii) different oligomeric arrangements to see if this affects proper cocoon assembly, and (iv) various organismal origins to further reduce bias. The proteins selected according to these criteria were the small GTPase cell division control protein 42 homolog (Cdc42) from Homo sapiens 27 , the herpesviral infected cell protein 47 (ICP47) 28 , the light/oxygen/voltage-sensing domain of the Arabidopsis thaliana blue light receptor (iLOV) 29 , the multi-ligand binding enzyme dihydrofolate reductase from Escherichia coli (DHFR) 30 , the Ras-binding domain of CRAF kinase from Homo sapiens (RBD) 31 , and tobacco etch virus (TEV) protease 32 . Several of these proteins possess interesting properties that were hypothesized to provide additional information on requirements for translocation: Cdc42 is a homodimer in solution 33 , ICP47 is intrinsically disordered, iLOV has a flavin mononucleotide chromophore, and TEV contains two β-barrels, which represent stable folds 34 (Supplementary Fig. 1a ). Interestingly, all tested chimeric cocoons could be well expressed in E. coli and purified. We then mixed the cocoons with TcA and assessed holotoxin formation by negative stain EM after size exclusion chromatography (SEC). In case of the wild-type TccC3HVR, the affinity of TcB-TcC to TcA is in the picomolar range, resulting in almost complete holotoxin formation 16 . This was also the case for holotoxins composed of TcA and cocoons containing Cdc42 (20.3 kDa) and TEV (28.1 kDa) (Fig. 2a, b , Supplementary Fig. 2a ). This demonstrates that TcB-TcC complexes with non-native cargos are able to form holotoxin complexes. In contrast, holotoxin formation was tremendously reduced when cocoons with ICP47 (11.3 kDa), iLOV (13.2 kDa), and DHFR (18.4 kDa) as cargos were used, indicating that there is a size limit for the cargo (Fig. 2b , Supplementary Fig. 2a ). Previously, we demonstrated that the HVR inside the cocoon has an influence on the gatekeeper domain of the β-propeller and an empty cocoon has a much lower affinity to TcA than the wild-type 16 . We have proposed that this might be caused by steric pressure applied by the HVR and consequently no or a small HVR would result in reduced complex formation. Our results with the different cargos support this hypothesis and indicate that the minimal size requirement for the cargo is around 20 kDa in order to guarantee high-affinity holotoxin assembly. Fig. 2 ABC holotoxin formation requires a cargo size above a distinct threshold. a Comparison of analytical size exclusion chromatography profiles for ABC holotoxin formed by mixing TcA (600 nM) with TcB-TcC (1.2 μM), separate TcA (600 nM), and separate TcB-TcC(WT) (600 nM). The fractions indicated by the gray bar were pooled and analyzed by EM. b Negative stain electron micrographs of holotoxins formed by TcA and TcB-TcC with the indicated cargos (WT and ICP47 constructs). Insets: 2D class averages of representative TcA pentamers and holotoxins, with the percentages of particles in the class averages shown below. c Negative stain micrographs of holotoxins formed by TcA and TcB-TcC with RBD cargos, with 2D class averages like those described in b . While the cargo HVR45-RBD does not trigger holotoxin formation, the larger HVR128-RBD results in assembled holotoxins. Scale bars in b and c : 100 nm The size of the cargo determines holotoxin formation To further explore the influence of cargo size on holotoxin formation, we created a truncated version of the native TccC3HVR (residues 1–132) and increased the size of the shorter cargos by adding differently sized parts of TccC3HVR to the N- or C-termini (see Methods for details). To achieve this, we introduced an EcoRI restriction site in frame after the first 138 or 384 base pairs (bp) of the sequence coding for the HVR, resulting in two possible N-terminal extensions of the cargo proteins with 45 or 128 residues from the original HVR, respectively. In addition, we introduced a NotI restriction site 150 bp upstream of the stop codon, resulting in a C-terminal extension of the cargo with the C-terminal 50 residues of the HVR. Altogether, five different combinations of extensions of the cargos were possible: HVR45-cargo, HVR128-cargo, HVR45-cargo-HVR50, HVR128-cargo-HVR50, and cargo-HVR50. As ICP47 has almost no secondary structure that could influence the size dependency (Supplementary Fig. 1a ), it was a particularly compelling test case. Similarly to the cocoon with only ICP47 (11.3 kDa), cocoons containing the chimeras ICP47-HVR50 (17.1 kDa) or HVR45-ICP47-HVR50 (21.6 kDa) did not have a high affinity to TcA. The same was true for HVR45-RBD (14.2 kDa). However, the longer constructs, namely HVR128-RBD (22.4 kDa), HVR128-ICP47 (24.5 kDa) and HVR128-ICP47-HVR50 (30.3 kDa), resulted in high-affinity holotoxin formation (Fig. 2a–c ). Increasing the size of iLOV (HVR128-iLOV (26.4 kDa)) and DHFR (HVR128-DHFR (31.6 kDa)) had the same effect (Supplementary Fig. 2a ). In all chimeras that led to high-affinity holotoxin assembly, the cargo was fused to HVR128. Therefore, one might ask whether these first 128 residues of TccC3HVR contain a motif important for activating TcB-TcC, which the holotoxin-forming Cdc42 and TEV constructs coincidentally possess. However, since HVR(1–128) alone does not activate the cocoon, as can be seen with the HVR (1–132) truncation construct (13.8 kDa) of TccC3HVR (Supplementary Fig. 2b ), we believe that the presence of HVR128 is not a prerequisite for the activation mechanism. Taken together, these results confirm our hypothesis that the cargo has to have a certain size (at least ~ 20 kDa) in order to activate the cocoon and put it into an assembly-competent state. At the same time the variety of assembly-competent constructs suggests that the nature of the cargo is not important for this activation mechanism, supporting the idea that a general high steric 'pressure' is sufficient. Translocation of cargo proteins by TcA Having demonstrated that holotoxins containing heterologous cargos can be assembled, our next step towards using the Tc scaffold as a customized protein injection system was to show that the cargo can be successfully translocated. To assess the translocation of different cargo proteins without having to rely on protein-specific enzymatic activity readouts, we developed a cell-free in vitro translocation assay. First, the prepore-to-pore transition of ABC is triggered by shifting the pH to 11. If the cargo can be translocated, it will be ejected through the TcA translocation channel and dissociate from the holotoxin, as described for wild-type ABC (ABC(WT)) 15 . Successfully translocated 20–30 kDa cargos can then be easily separated from the 1.7 MDa ABC injection machinery by SEC (Supplementary Fig. 3a ), whereas non-translocated cargos will still be holotoxin-associated and therefore co-migrate with the 1.7 MDa peak (Supplementary Fig. 3b ). As proof of principle, we first assessed the release of TccC3HVR in ABC(WT) before moving on to test the heterologous cargos. Indeed, after 48 h of incubation at pH 11, a substantial fraction of TccC3HVR migrates much later than the ABC peak (Fig. 3a ), indicating that it has been successfully released and translocated through TcA. Fig. 3 Translocation of various non-natural cargos in vitro and activation of TccC3HVR. a Successful translocation of ICP47, RBD, and iLOV fused to the N- or C-terminus of TccC3HVR. After incubation at pH 11, which causes the toxin to undergo prepore-to-pore transition, Western blots show that these constructs migrate at higher retention volumes during size exclusion chromatography in comparison with pH 8-incubated controls. This corresponds to translocation and release of the cargo proteins from the holotoxin, as illustrated schematically in Supplementary Fig. 3 . b ADP-ribosylation of F-actin by translocated TccC3HVR in comparison with recombinantly purified TccC3HVR. Western blots show the increasing appearance of ADP-ribosylated actin dependent on the TccC3HVR concentration for both preparations. −: no TcCC3HVR, +: 100 n m recombinantly purified TccC3HVR. c No translocation was observed for Cdc42 and TEV protease cargo alone, or DHFR and Cdc42 fused to the N- or C-terminus of TccC3HVR. Western blots show the presence of the constructs at lower retention volumes during size exclusion chromatography both at pH 8 and pH 11, meaning they co-localize with the rest of the holotoxin even after prepore-to-pore transition and are not translocated. Uncropped images of the Western blots in panels a – c are provided as a Source Data file To test whether the TccC3HVR folds and adopts an active conformation after in vitro translocation, we compared the activity of the translocated ADP-ribosyltransferase with that of recombinantly purified TccC3HVR, using an actin-ribosylating assay 35 , 36 (Methods). Although the activity of the translocated ADP-ribosyltransferase was about half of that of the recombinantly purified enzyme, it clearly ADP-ribosylated F-actin (Fig. 3b ). This demonstrates that most of the translocated ADP-ribosyltransferase was folded to its active form after translocation. Importantly, this in vitro assay shows that folding and activity of the ADP-ribosyltransferase do not require additional cofactors such as chaperones and we believe that this is also the case for other translocated cargos. Next, we tested whether the various cargo proteins could be translocated. We first assessed Cdc42 and TEV, which do not need to be fused to TccC3HVR fragments in order to form a holotoxin. However, the proteins were not translocated by TcA (Fig. 3c ). To determine whether fusing sequences from TccC3HVR can restore translocation competence, we extended Cdc42 with either the C-terminus of TccC3HVR or with both of its termini. However, these cargos could also not be translocated by TcA (Fig. 3c ), indicating that neither the C-terminus nor both termini of the HVR are sufficient to enable translocation. However, the four other cargos that facilitated holotoxin formation only when fused to fragments of TccC3HVR were successfully translocated, namely HVR128-ICP47-HVR50, HVR128-ICP47, HVR128-iLOV, and HVR128-RBD (Fig. 3a ). As the C-terminal region that is translocated first in ABC(WT) 16 differs considerably between these cargos, we conclude that there is no specific sequence at the C-terminus that determines whether a cargo is translocated or not. In the case of HVR128-iLOV, iLOV did not produce fluorescence in the cocoon, whereas TcB-TcC-iLOV did (Supplementary Fig. 3d ), indicating that HVR128-iLOV is stored in an unfolded form. We conclude that extending the N-terminus by 128 additional residues prevents the folding of HVR128-iLOV inside the cocoon, whereas the fluorescence of TcB-TcC-iLOV shows that iLOV without the extension is able to fold inside the cocoon and has access to the cofactor FMN. In contrast to the other three HVR128-containing cargos, DHFR is not translocated when fused to the same HVR128 N-terminus (Fig. 3c ). Together with the non-translocated HVR45-Cdc42-HVR50 cargo, this shows that the sequence of the N-terminus is also not the determinant of cargo transport through TcA. Therefore, there must be another factor at work that establishes translocation competence. A comparison of the four translocated fusion proteins (HVR128-ICP47, HVR128-ICP47-HVR50, HVR128-RBD, and HVR128-iLOV) and the native cargos Tcc3HVR and Tcc5HVR shows that their common feature is a positive net charge at neutral pH, with isoelectric points of at least 7.9 (Fig. 3a ). In the case of the fusion constructs, this is mainly due to the highly positively charged HVR128 (pI 9.75), which is larger than the cargo protein in all cases (maximum size 13.2 kDa). We therefore conclude that besides being large enough, the cargo has to be positively charged (pI ≥ ~ 8) in order to be translocated. In line with this, the six constructs that formed holotoxin complexes but did not show translocation had mostly negatively charged cargos (Fig. 4 ). Only two non-translocated cargos, namely HVR45-Cdc42-HVR50 and TEV were positively charged. The TEV construct we used has a highly positively charged penta-arginine tail to allow purification by cation exchange chromatography without changing its activity 37 , a modification that raises the pI of TEV from 8.67 to 9.62. Although such a change in pI should favor translocation, it is possible that distributing the positive charges in such an uneven manner rather hinders it. Fig. 4 List of cargos tested in this study. The nomenclature of the chimeras, for example, HVR128-ICP47-HVR50, indicates how many N- or C-terminal TccC3HVR residues have been pre- or appended to the cargo protein. The colored bars indicate the composition of the constructs and are used consistently in all figures. n.a.: not applicable. *The pI of TccC5HVR is changed from 8.65 to 7.90 by the addition of four residues (MPEF) to the N-terminus, resulting from cloning. **Toxicity to HEK293T cells (see Fig. 1c ) TEV protease is not an exceptionally stable protein, as it unfolds at ~ 52 °C 38 . However, it contains two small β-barrels (Supplementary Fig. 1a ), which are known to be a notoriously stable fold 34 . Therefore, we believe that these domains are more rigid than the native Tcc3HVR or other translocated cargos such as HVR128-iLOV, which might prevent the protein from being translocated. To prove this, we determined the structure of TcB-TcC-TEV to a resolution of 3.7 à using X-ray crystallography (Supplementary Table 1 ). The cocoon of TcB-TcC-TEV shows an identical size and shape to the TcB-TcC(WT) cocoon 10 , with a C α RMSD of 0.552 à 2 . However, the electron density inside appears already at a higher sigma level, indicating that the encapsulated TEV is indeed more rigid and ordered than the native TccC3HVR (Supplementary Fig. 3c ). This is the likely cause for TEV not being translocated. As folded proteins do not fit through the narrow constriction site of the translocation channel 16 , the formation of defined structural elements such as β-barrels in the cocoon probably results in translocation arrest. Interaction with cocoon inhibits translocation of Cdc42 Although Cdc42 does not possess any folds that are immediately classifiable as very stable, we examined the Cdc42 constructs in more detail and solved the crystal structure of TcB-TcC-Cdc42 to 2.0 à to find out whether Cdc42 folding inside the cocoon is nonetheless a plausible explanation for its translocation incompetence (Fig. 5a , Supplementary Table 1 ). Fig. 5 Structure of TcB-TcC-Cdc42 alone and in complex with TcA. a Crystal structure of the TcB-TcC-Cdc42 complex. The C-terminal region of Cdc42 (N167–P179, green) forms a helix inside the cocoon. b Top view into TcB-TcC-Cdc42, with the TcC model removed for illustrative purposes. The C-terminal Cdc42 α-helix (green) can be seen attached to one side of the cocoon, whereas remaining Cdc42 density is too disordered to resolve. c Surface representation of the binding pocket of the hydrophobic C-terminal Cdc42 helix in the TcB section of the cocoon. The molecular surface of TcB is colored according to its hydrophobicity 52 , with hydrophobic regions shown in ochre and polar regions in white. d Model of the C-terminal Cdc42 α-helix in the binding pocket of TcB. Two orientations are shown. Side chains that face the Cdc42 α-helix are indicated. e Rigid-body fit of the crystal structure of TcB-TcC-Cdc42 into the cryo-EM density map of ABC-Cdc42. Density corresponding to the α-helix of Cdc42 is also present in the cryo-EM structure of the holotoxin at the same site. f Comparison of cryo-EM density reconstructions of ABC(WT) (PDB 6H6F) and ABC-Cdc42 (this work). The surfaces of the TcA, TcB, and TcC subunits are transparent, and the density corresponding to the ADP-ribosyltransferase TccC3HVR (WT, left) and Cdc42 (right) is dark gray. The latter is shown at a lower threshold and filtered to 15 à resolution The overall shape of the TcB-TcC-Cdc42 cocoon is identical to wild-type (TcB-TcC(WT)) and empty TcB-TcC 10 , 16 , with C α -RMSD values of 0.424 A 2 and 0.414 à 2 between TcB-TcC-Cdc42/TcB-TcC(WT) and TcB-TcC-Cdc42/empty TcB-TcC, respectively, indicating that a different cargo does not influence the RHS repeat structure of the cocoon. Similarly to TccC3HVR in the TcB-TcC(WT) cocoon, Cdc42 is not structured inside the cocoon. However, we found an ordered electron density in close spatial proximity to the β-propeller domain, which corresponds to an α-helix not present in other TcB-TcC structures obtained so far. The high-resolution of the map and availability of the Cdc42 structure 27 enabled us to identify the α-helix inside the cocoon as the C-terminus of Cdc42 (N167—P179) (Supplementary Fig. 4a, b ). Importantly, the helix is attached to the TcB protein in an orientation perpendicular to that expected from natural translocation (Fig. 5a, b ), and its amphipathic nature facilitates interaction with a hydrophobic pocket on the inner surface of the cocoon (Fig. 5c ). The side chains of F169, I173, L177, and P179 are rigidly oriented toward the hydrophobic cleft, where they are in close contact with L39, L41, P42, L366, L368, M702, N704, V708, H710, L1203, and F1349 of the cocoon (Fig. 5d ). In contrast, D170, E171, and E178 face the TcB-TcC lumen with more degrees of freedom. This is reflected in the quality of the crystallographic density, with only the residues pointing toward the cocoon surface being well resolved (Supplementary Fig. 4c ). Interestingly, the affinity of the Cdc42 α-helix for this part of the cocoon is strong enough to displace the N-terminus of TcB, which resides at this position in TcB-TcC(WT) (Supplementary Fig. 4d ). The N-terminus of TcB-TcC-Cdc42 is correspondingly not resolved, indicating that it protrudes into the cocoon lumen. The tight attachment of Cdc42 via its C-terminus in close spatial proximity to the TcB gatekeeper domain 16 could help this construct to form a holotoxin with high affinity comparable to the constructs with larger cargos, despite Cdc42 being at the lower limit of the cargo size prerequisite (Fig. 2 , Supplementary Fig. 2 ). The stable nature of the Cdc42 α-helix interaction with the cocoon raises the question of whether it remains bound even after holotoxin formation, in which case it would not be able to enter the translocation channel. We addressed this issue by determining the 5 à structure of the ABC-Cdc42 holotoxin using cryo-EM (Supplementary Fig. 5 ). Indeed, a small helix-shaped density appears at the interaction site even at a comparably high-map binarization threshold, despite the limited resolution of the 3D reconstruction. Fitting the entire crystal structure of TcB-TcC-Cdc42 into the cryo-EM map results in a very good match of the Cdc42 α-helix with the additional map density of ABC-Cdc42 (Fig. 5e ). In contrast, no comparable cryo-EM density is present at the same position in ABC(WT) and an aspartyl protease deficient variant 16 (Supplementary Fig. 6 ), indicating that the cargo does not form any structural elements at this location in functional holotoxins. The tight association of the C-terminal α-helix to the cocoon results in Cdc42 translocation arrest already at an early stage. If the C-terminus that would normally be translocated first through TcA 16 is bound elsewhere, then why does Cdc42 form a holotoxin? Analysis of the cryo-EM data shows that no additional density is present in the TcA translocation channel, unlike in ABC(WT) (Fig. 5f ). There is however a stretch of Cdc42 density that protrudes into the β-propeller domain of TcA but does not continue further into the channel. This indicates that although Cdc42 cannot be translocated, it applies a steric pressure on the TcB gatekeeper domain, resulting in the high-affinity binding of TcB to TcA. These results demonstrate that if the encapsulated cargo forms stable structural elements that strongly interact with the inner lumen of the cocoon, the cargo cannot be translocated by TcA even if it is positively charged and large enough. TcC-HVRs are exchangeable in Tc toxins As an initial proof that different cargos fused to TcC can be translocated, we tested whether HVRs from different TcC proteins are exchangeable and result in functional, toxic ABC complexes. For this, we replaced the TccC3HVR sequence after the autoproteolytic cleavage site in TcdB2-TccC3 to that of TccC5HVR, resulting in the chimeric TcdB2-TccC3-TccC5HVR complex. After assembly of the ABC-TccC5HVR holotoxin, we assessed cytotoxicity on HEK293T cells. Complete cell death occurs upon addition of 2 nM ABC-TccC5HVR toxin, compared with cell death at 0.5 nM when exposed to the ABC-TccC3HVR holotoxin (Fig. 1c ). This is in accordance with previous findings that the cytotoxic effect of TccC5HVR is less pronounced than that of TccC3HVR 18 . This experiment shows that the cocoon formed by TcdB2-TccC3 is capable of also encapsulating and translocating other HVRs, such as TccC5HVR. We therefore chose TcdB2-TccC3 to function as a cocoon scaffold for translocation of other cargo proteins, which are not components of the Tc toxin system. Importantly, the two different HVRs do not show any pronounced sections of sequence identity (Supplementary Fig. 1b ), indicating that there is no conserved motif in Tcc-HVRs that is a general prerequisite for successful translocation. Replacement of TcC HVR by heterologous cargo proteins Our next step was to replace the HVR of TccC3 with unrelated heterologous proteins and test the capability of the holotoxin to translocate these. The criteria used to select replacement proteins were (i) a small size (11–34 kDa) to guarantee that they fit into the TcB-TcC cocoon, (ii) diverse folds to assess whether this influences the translocation capability, (iii) different oligomeric arrangements to see if this affects proper cocoon assembly, and (iv) various organismal origins to further reduce bias. The proteins selected according to these criteria were the small GTPase cell division control protein 42 homolog (Cdc42) from Homo sapiens 27 , the herpesviral infected cell protein 47 (ICP47) 28 , the light/oxygen/voltage-sensing domain of the Arabidopsis thaliana blue light receptor (iLOV) 29 , the multi-ligand binding enzyme dihydrofolate reductase from Escherichia coli (DHFR) 30 , the Ras-binding domain of CRAF kinase from Homo sapiens (RBD) 31 , and tobacco etch virus (TEV) protease 32 . Several of these proteins possess interesting properties that were hypothesized to provide additional information on requirements for translocation: Cdc42 is a homodimer in solution 33 , ICP47 is intrinsically disordered, iLOV has a flavin mononucleotide chromophore, and TEV contains two β-barrels, which represent stable folds 34 (Supplementary Fig. 1a ). Interestingly, all tested chimeric cocoons could be well expressed in E. coli and purified. We then mixed the cocoons with TcA and assessed holotoxin formation by negative stain EM after size exclusion chromatography (SEC). In case of the wild-type TccC3HVR, the affinity of TcB-TcC to TcA is in the picomolar range, resulting in almost complete holotoxin formation 16 . This was also the case for holotoxins composed of TcA and cocoons containing Cdc42 (20.3 kDa) and TEV (28.1 kDa) (Fig. 2a, b , Supplementary Fig. 2a ). This demonstrates that TcB-TcC complexes with non-native cargos are able to form holotoxin complexes. In contrast, holotoxin formation was tremendously reduced when cocoons with ICP47 (11.3 kDa), iLOV (13.2 kDa), and DHFR (18.4 kDa) as cargos were used, indicating that there is a size limit for the cargo (Fig. 2b , Supplementary Fig. 2a ). Previously, we demonstrated that the HVR inside the cocoon has an influence on the gatekeeper domain of the β-propeller and an empty cocoon has a much lower affinity to TcA than the wild-type 16 . We have proposed that this might be caused by steric pressure applied by the HVR and consequently no or a small HVR would result in reduced complex formation. Our results with the different cargos support this hypothesis and indicate that the minimal size requirement for the cargo is around 20 kDa in order to guarantee high-affinity holotoxin assembly. Fig. 2 ABC holotoxin formation requires a cargo size above a distinct threshold. a Comparison of analytical size exclusion chromatography profiles for ABC holotoxin formed by mixing TcA (600 nM) with TcB-TcC (1.2 μM), separate TcA (600 nM), and separate TcB-TcC(WT) (600 nM). The fractions indicated by the gray bar were pooled and analyzed by EM. b Negative stain electron micrographs of holotoxins formed by TcA and TcB-TcC with the indicated cargos (WT and ICP47 constructs). Insets: 2D class averages of representative TcA pentamers and holotoxins, with the percentages of particles in the class averages shown below. c Negative stain micrographs of holotoxins formed by TcA and TcB-TcC with RBD cargos, with 2D class averages like those described in b . While the cargo HVR45-RBD does not trigger holotoxin formation, the larger HVR128-RBD results in assembled holotoxins. Scale bars in b and c : 100 nm The size of the cargo determines holotoxin formation To further explore the influence of cargo size on holotoxin formation, we created a truncated version of the native TccC3HVR (residues 1–132) and increased the size of the shorter cargos by adding differently sized parts of TccC3HVR to the N- or C-termini (see Methods for details). To achieve this, we introduced an EcoRI restriction site in frame after the first 138 or 384 base pairs (bp) of the sequence coding for the HVR, resulting in two possible N-terminal extensions of the cargo proteins with 45 or 128 residues from the original HVR, respectively. In addition, we introduced a NotI restriction site 150 bp upstream of the stop codon, resulting in a C-terminal extension of the cargo with the C-terminal 50 residues of the HVR. Altogether, five different combinations of extensions of the cargos were possible: HVR45-cargo, HVR128-cargo, HVR45-cargo-HVR50, HVR128-cargo-HVR50, and cargo-HVR50. As ICP47 has almost no secondary structure that could influence the size dependency (Supplementary Fig. 1a ), it was a particularly compelling test case. Similarly to the cocoon with only ICP47 (11.3 kDa), cocoons containing the chimeras ICP47-HVR50 (17.1 kDa) or HVR45-ICP47-HVR50 (21.6 kDa) did not have a high affinity to TcA. The same was true for HVR45-RBD (14.2 kDa). However, the longer constructs, namely HVR128-RBD (22.4 kDa), HVR128-ICP47 (24.5 kDa) and HVR128-ICP47-HVR50 (30.3 kDa), resulted in high-affinity holotoxin formation (Fig. 2a–c ). Increasing the size of iLOV (HVR128-iLOV (26.4 kDa)) and DHFR (HVR128-DHFR (31.6 kDa)) had the same effect (Supplementary Fig. 2a ). In all chimeras that led to high-affinity holotoxin assembly, the cargo was fused to HVR128. Therefore, one might ask whether these first 128 residues of TccC3HVR contain a motif important for activating TcB-TcC, which the holotoxin-forming Cdc42 and TEV constructs coincidentally possess. However, since HVR(1–128) alone does not activate the cocoon, as can be seen with the HVR (1–132) truncation construct (13.8 kDa) of TccC3HVR (Supplementary Fig. 2b ), we believe that the presence of HVR128 is not a prerequisite for the activation mechanism. Taken together, these results confirm our hypothesis that the cargo has to have a certain size (at least ~ 20 kDa) in order to activate the cocoon and put it into an assembly-competent state. At the same time the variety of assembly-competent constructs suggests that the nature of the cargo is not important for this activation mechanism, supporting the idea that a general high steric 'pressure' is sufficient. Translocation of cargo proteins by TcA Having demonstrated that holotoxins containing heterologous cargos can be assembled, our next step towards using the Tc scaffold as a customized protein injection system was to show that the cargo can be successfully translocated. To assess the translocation of different cargo proteins without having to rely on protein-specific enzymatic activity readouts, we developed a cell-free in vitro translocation assay. First, the prepore-to-pore transition of ABC is triggered by shifting the pH to 11. If the cargo can be translocated, it will be ejected through the TcA translocation channel and dissociate from the holotoxin, as described for wild-type ABC (ABC(WT)) 15 . Successfully translocated 20–30 kDa cargos can then be easily separated from the 1.7 MDa ABC injection machinery by SEC (Supplementary Fig. 3a ), whereas non-translocated cargos will still be holotoxin-associated and therefore co-migrate with the 1.7 MDa peak (Supplementary Fig. 3b ). As proof of principle, we first assessed the release of TccC3HVR in ABC(WT) before moving on to test the heterologous cargos. Indeed, after 48 h of incubation at pH 11, a substantial fraction of TccC3HVR migrates much later than the ABC peak (Fig. 3a ), indicating that it has been successfully released and translocated through TcA. Fig. 3 Translocation of various non-natural cargos in vitro and activation of TccC3HVR. a Successful translocation of ICP47, RBD, and iLOV fused to the N- or C-terminus of TccC3HVR. After incubation at pH 11, which causes the toxin to undergo prepore-to-pore transition, Western blots show that these constructs migrate at higher retention volumes during size exclusion chromatography in comparison with pH 8-incubated controls. This corresponds to translocation and release of the cargo proteins from the holotoxin, as illustrated schematically in Supplementary Fig. 3 . b ADP-ribosylation of F-actin by translocated TccC3HVR in comparison with recombinantly purified TccC3HVR. Western blots show the increasing appearance of ADP-ribosylated actin dependent on the TccC3HVR concentration for both preparations. −: no TcCC3HVR, +: 100 n m recombinantly purified TccC3HVR. c No translocation was observed for Cdc42 and TEV protease cargo alone, or DHFR and Cdc42 fused to the N- or C-terminus of TccC3HVR. Western blots show the presence of the constructs at lower retention volumes during size exclusion chromatography both at pH 8 and pH 11, meaning they co-localize with the rest of the holotoxin even after prepore-to-pore transition and are not translocated. Uncropped images of the Western blots in panels a – c are provided as a Source Data file To test whether the TccC3HVR folds and adopts an active conformation after in vitro translocation, we compared the activity of the translocated ADP-ribosyltransferase with that of recombinantly purified TccC3HVR, using an actin-ribosylating assay 35 , 36 (Methods). Although the activity of the translocated ADP-ribosyltransferase was about half of that of the recombinantly purified enzyme, it clearly ADP-ribosylated F-actin (Fig. 3b ). This demonstrates that most of the translocated ADP-ribosyltransferase was folded to its active form after translocation. Importantly, this in vitro assay shows that folding and activity of the ADP-ribosyltransferase do not require additional cofactors such as chaperones and we believe that this is also the case for other translocated cargos. Next, we tested whether the various cargo proteins could be translocated. We first assessed Cdc42 and TEV, which do not need to be fused to TccC3HVR fragments in order to form a holotoxin. However, the proteins were not translocated by TcA (Fig. 3c ). To determine whether fusing sequences from TccC3HVR can restore translocation competence, we extended Cdc42 with either the C-terminus of TccC3HVR or with both of its termini. However, these cargos could also not be translocated by TcA (Fig. 3c ), indicating that neither the C-terminus nor both termini of the HVR are sufficient to enable translocation. However, the four other cargos that facilitated holotoxin formation only when fused to fragments of TccC3HVR were successfully translocated, namely HVR128-ICP47-HVR50, HVR128-ICP47, HVR128-iLOV, and HVR128-RBD (Fig. 3a ). As the C-terminal region that is translocated first in ABC(WT) 16 differs considerably between these cargos, we conclude that there is no specific sequence at the C-terminus that determines whether a cargo is translocated or not. In the case of HVR128-iLOV, iLOV did not produce fluorescence in the cocoon, whereas TcB-TcC-iLOV did (Supplementary Fig. 3d ), indicating that HVR128-iLOV is stored in an unfolded form. We conclude that extending the N-terminus by 128 additional residues prevents the folding of HVR128-iLOV inside the cocoon, whereas the fluorescence of TcB-TcC-iLOV shows that iLOV without the extension is able to fold inside the cocoon and has access to the cofactor FMN. In contrast to the other three HVR128-containing cargos, DHFR is not translocated when fused to the same HVR128 N-terminus (Fig. 3c ). Together with the non-translocated HVR45-Cdc42-HVR50 cargo, this shows that the sequence of the N-terminus is also not the determinant of cargo transport through TcA. Therefore, there must be another factor at work that establishes translocation competence. A comparison of the four translocated fusion proteins (HVR128-ICP47, HVR128-ICP47-HVR50, HVR128-RBD, and HVR128-iLOV) and the native cargos Tcc3HVR and Tcc5HVR shows that their common feature is a positive net charge at neutral pH, with isoelectric points of at least 7.9 (Fig. 3a ). In the case of the fusion constructs, this is mainly due to the highly positively charged HVR128 (pI 9.75), which is larger than the cargo protein in all cases (maximum size 13.2 kDa). We therefore conclude that besides being large enough, the cargo has to be positively charged (pI ≥ ~ 8) in order to be translocated. In line with this, the six constructs that formed holotoxin complexes but did not show translocation had mostly negatively charged cargos (Fig. 4 ). Only two non-translocated cargos, namely HVR45-Cdc42-HVR50 and TEV were positively charged. The TEV construct we used has a highly positively charged penta-arginine tail to allow purification by cation exchange chromatography without changing its activity 37 , a modification that raises the pI of TEV from 8.67 to 9.62. Although such a change in pI should favor translocation, it is possible that distributing the positive charges in such an uneven manner rather hinders it. Fig. 4 List of cargos tested in this study. The nomenclature of the chimeras, for example, HVR128-ICP47-HVR50, indicates how many N- or C-terminal TccC3HVR residues have been pre- or appended to the cargo protein. The colored bars indicate the composition of the constructs and are used consistently in all figures. n.a.: not applicable. *The pI of TccC5HVR is changed from 8.65 to 7.90 by the addition of four residues (MPEF) to the N-terminus, resulting from cloning. **Toxicity to HEK293T cells (see Fig. 1c ) TEV protease is not an exceptionally stable protein, as it unfolds at ~ 52 °C 38 . However, it contains two small β-barrels (Supplementary Fig. 1a ), which are known to be a notoriously stable fold 34 . Therefore, we believe that these domains are more rigid than the native Tcc3HVR or other translocated cargos such as HVR128-iLOV, which might prevent the protein from being translocated. To prove this, we determined the structure of TcB-TcC-TEV to a resolution of 3.7 à using X-ray crystallography (Supplementary Table 1 ). The cocoon of TcB-TcC-TEV shows an identical size and shape to the TcB-TcC(WT) cocoon 10 , with a C α RMSD of 0.552 à 2 . However, the electron density inside appears already at a higher sigma level, indicating that the encapsulated TEV is indeed more rigid and ordered than the native TccC3HVR (Supplementary Fig. 3c ). This is the likely cause for TEV not being translocated. As folded proteins do not fit through the narrow constriction site of the translocation channel 16 , the formation of defined structural elements such as β-barrels in the cocoon probably results in translocation arrest. Interaction with cocoon inhibits translocation of Cdc42 Although Cdc42 does not possess any folds that are immediately classifiable as very stable, we examined the Cdc42 constructs in more detail and solved the crystal structure of TcB-TcC-Cdc42 to 2.0 à to find out whether Cdc42 folding inside the cocoon is nonetheless a plausible explanation for its translocation incompetence (Fig. 5a , Supplementary Table 1 ). Fig. 5 Structure of TcB-TcC-Cdc42 alone and in complex with TcA. a Crystal structure of the TcB-TcC-Cdc42 complex. The C-terminal region of Cdc42 (N167–P179, green) forms a helix inside the cocoon. b Top view into TcB-TcC-Cdc42, with the TcC model removed for illustrative purposes. The C-terminal Cdc42 α-helix (green) can be seen attached to one side of the cocoon, whereas remaining Cdc42 density is too disordered to resolve. c Surface representation of the binding pocket of the hydrophobic C-terminal Cdc42 helix in the TcB section of the cocoon. The molecular surface of TcB is colored according to its hydrophobicity 52 , with hydrophobic regions shown in ochre and polar regions in white. d Model of the C-terminal Cdc42 α-helix in the binding pocket of TcB. Two orientations are shown. Side chains that face the Cdc42 α-helix are indicated. e Rigid-body fit of the crystal structure of TcB-TcC-Cdc42 into the cryo-EM density map of ABC-Cdc42. Density corresponding to the α-helix of Cdc42 is also present in the cryo-EM structure of the holotoxin at the same site. f Comparison of cryo-EM density reconstructions of ABC(WT) (PDB 6H6F) and ABC-Cdc42 (this work). The surfaces of the TcA, TcB, and TcC subunits are transparent, and the density corresponding to the ADP-ribosyltransferase TccC3HVR (WT, left) and Cdc42 (right) is dark gray. The latter is shown at a lower threshold and filtered to 15 à resolution The overall shape of the TcB-TcC-Cdc42 cocoon is identical to wild-type (TcB-TcC(WT)) and empty TcB-TcC 10 , 16 , with C α -RMSD values of 0.424 A 2 and 0.414 à 2 between TcB-TcC-Cdc42/TcB-TcC(WT) and TcB-TcC-Cdc42/empty TcB-TcC, respectively, indicating that a different cargo does not influence the RHS repeat structure of the cocoon. Similarly to TccC3HVR in the TcB-TcC(WT) cocoon, Cdc42 is not structured inside the cocoon. However, we found an ordered electron density in close spatial proximity to the β-propeller domain, which corresponds to an α-helix not present in other TcB-TcC structures obtained so far. The high-resolution of the map and availability of the Cdc42 structure 27 enabled us to identify the α-helix inside the cocoon as the C-terminus of Cdc42 (N167—P179) (Supplementary Fig. 4a, b ). Importantly, the helix is attached to the TcB protein in an orientation perpendicular to that expected from natural translocation (Fig. 5a, b ), and its amphipathic nature facilitates interaction with a hydrophobic pocket on the inner surface of the cocoon (Fig. 5c ). The side chains of F169, I173, L177, and P179 are rigidly oriented toward the hydrophobic cleft, where they are in close contact with L39, L41, P42, L366, L368, M702, N704, V708, H710, L1203, and F1349 of the cocoon (Fig. 5d ). In contrast, D170, E171, and E178 face the TcB-TcC lumen with more degrees of freedom. This is reflected in the quality of the crystallographic density, with only the residues pointing toward the cocoon surface being well resolved (Supplementary Fig. 4c ). Interestingly, the affinity of the Cdc42 α-helix for this part of the cocoon is strong enough to displace the N-terminus of TcB, which resides at this position in TcB-TcC(WT) (Supplementary Fig. 4d ). The N-terminus of TcB-TcC-Cdc42 is correspondingly not resolved, indicating that it protrudes into the cocoon lumen. The tight attachment of Cdc42 via its C-terminus in close spatial proximity to the TcB gatekeeper domain 16 could help this construct to form a holotoxin with high affinity comparable to the constructs with larger cargos, despite Cdc42 being at the lower limit of the cargo size prerequisite (Fig. 2 , Supplementary Fig. 2 ). The stable nature of the Cdc42 α-helix interaction with the cocoon raises the question of whether it remains bound even after holotoxin formation, in which case it would not be able to enter the translocation channel. We addressed this issue by determining the 5 à structure of the ABC-Cdc42 holotoxin using cryo-EM (Supplementary Fig. 5 ). Indeed, a small helix-shaped density appears at the interaction site even at a comparably high-map binarization threshold, despite the limited resolution of the 3D reconstruction. Fitting the entire crystal structure of TcB-TcC-Cdc42 into the cryo-EM map results in a very good match of the Cdc42 α-helix with the additional map density of ABC-Cdc42 (Fig. 5e ). In contrast, no comparable cryo-EM density is present at the same position in ABC(WT) and an aspartyl protease deficient variant 16 (Supplementary Fig. 6 ), indicating that the cargo does not form any structural elements at this location in functional holotoxins. The tight association of the C-terminal α-helix to the cocoon results in Cdc42 translocation arrest already at an early stage. If the C-terminus that would normally be translocated first through TcA 16 is bound elsewhere, then why does Cdc42 form a holotoxin? Analysis of the cryo-EM data shows that no additional density is present in the TcA translocation channel, unlike in ABC(WT) (Fig. 5f ). There is however a stretch of Cdc42 density that protrudes into the β-propeller domain of TcA but does not continue further into the channel. This indicates that although Cdc42 cannot be translocated, it applies a steric pressure on the TcB gatekeeper domain, resulting in the high-affinity binding of TcB to TcA. These results demonstrate that if the encapsulated cargo forms stable structural elements that strongly interact with the inner lumen of the cocoon, the cargo cannot be translocated by TcA even if it is positively charged and large enough. Discussion Taken together, our results indicate that the P. luminescens Tc toxin can be successfully transformed into a universal protein translocation system as long as the cargo protein fulfills several prerequisites. The first of these is cargo size. While its upper limit is defined by the size of the cocoon, an exact value was not determined in this study. The largest cargo tested here was HVR128-DHFR (31.6 kDa), which is 1.2 kDa larger than the TccC3HVR natural cargo (Fig. 4 ). At the same time, the largest natural cargo of a P. luminescens Tc toxin is TccC1HVR at 35.1 kDa, providing a potential upper size limit and meaning that larger ADP-ribosyltransferases like the 49.4 kDa C2 toxin of Clostridium botulinum 21 would likely not fit into the cocoon. It remains to be explored whether the cocoon itself can be enlarged by adding further RHS repeats, thereby expanding the upper cargo size limit. Importantly, we discovered that the lower size limit of the cargo is in the 20–22 kDa range, and the ability to form the ABC holotoxin is drastically reduced if the cargo size is below this limit (Fig. 4 , Fig. 6 ). This finding echoes previous results showing that a comparable decrease in affinity between empty TcB-TcC and TcA occurs because there is no HVR present to apply steric pressure on the TcB gatekeeper domain that is essential for binding to TcA 16 . Similarly, small cargos will likely be too mobile inside the cocoon to cause gatekeeper destabilization. The lower size limit is slightly flexible, e.g. Cdc42 (20.3 kDa) forms holotoxin whereas HVR45-ICP47-HVR50 (21.6 kDa) does not (Fig. 4 ). This is potentially caused by the C-terminal helix of Cdc42 attaching the rest of the cargo to the vicinity of the cocoon exit, allowing the Cdc42 N-terminus to destabilize the gatekeeper more easily, which results in holotoxin formation but also prevents subsequent cargo transport. We therefore hypothesize that translocation will likely be impaired if a TcB-TcC cocoon loaded with a small cargo binds with high affinity to TcA. Fig. 6 Prerequisites for creating a functional customized Tc translocation system. TcA and TcB-TcC assemble into a holotoxin with high affinity only when the cargo is sufficiently large (≥ 20–22 kDa). Translocation upon prepore-to-pore transition only occurs if the cargo is unfolded in the cocoon and its pI is ≥ 8 The second prerequisite for successful transport is a net positive charge of the cargo protein. All naturally occurring HVRs of P. luminescens Tc toxins have a pI of at least 7.9 (TccC5HVR), and the protein with the lowest pI for which we observed translocation in our in vitro assay is HVR128-iLOV with a pI = 8.73 (Fig. 4 ). Since the gatekeeper constriction site that serves as the entry point into the TcA channel is formed by several acidic residues 16 , negatively charged constructs might be repelled. In addition, the translocation channel of TcA has several negatively charged bands 10 , 11 that facilitate the transport of cations but not anions 10 , 39 . It is unlikely that the translocation behavior is different when the cargo is translocated across a native membrane in comparison with the in vitro system, as TcA forms a translocation channel through the membrane that is laterally closed and does not allow the entry of lipids into the channel lumen 11 . The transport of the native TccC3HVR across lipid nanodiscs occurs without the need for a proton gradient or a chaperone 15 , although a facilitation of translocation by the latter two factors cannot be excluded in the context of a cellular environment. Furthermore, single channel conductivity experiments 9 and cell toxicity assays with TcA in the absence of a TcB-TcC cocoon 40 show that an open pore is formed in the membrane. Consequently, we believe cargo proteins can move unhindered through the transmembrane channel provided their pI is basic enough to do so. A sufficiently large size and a positive net charge are however not the only points that need to be considered, because some of the larger cargos with high pI were not translocated (TEV and HVR45-Cdc42-HVR50). Therefore, a third prerequisite needs to be fulfilled: the encapsulated cargo must not form any tertiary structures or stably interact with the cocoon. The two abovementioned cargos violate this prerequisite, with TEV containing two highly stable β-barrels, and Cdc42 possessing an amphipathic C-terminus that associates with a hydrophobic binding pocket in the cocoon. To summarize, we recommend adhering to the following guidelines in order to successfully turn a Tc toxin into a protein translocation device (Fig. 6 ): (i) Choose a protein between 20 and 35 kDa to insert after the autoproteolytic cleavage site of TcC. In case the cargo is too small, add N- or C-terminal extensions that do not interfere with protein function or link several copies of the protein together. (ii) Choose a protein with a pI of at least 8.0. Increase the pI by addition of positively charged residues or site-directed mutagenesis if necessary and possible. (iii) Avoid cargo proteins that might form highly stable structures already in the cocoon lumen. (iv) Avoid cargo proteins that contain amphipathic helices or extensive hydrophobic patches that might stably interact with the inner surface of the cocoon. Methods Source plasmids for cargo proteins pGEX-iLOV was a gift from John Christie (Addgene plasmid #26587) 29 . pcDXc3A-AN44-ICP47 containing the coding sequence (CDS) for ICP47 with a C-terminal HA epitope was a gift from Robert Tampé, Goethe University of Frankfurt, Germany. pGEX-RBD with the CDS for the Ras-binding domain (RBD) of CRAF kinase was a gift from Andreas Ernst, Goethe University of Frankfurt, Germany. A customized plasmid with the CDS for dihydrofolate reductase (DHFR) was a gift from Yaowen Wu, Max Planck Institute of Molecular Physiology, Dortmund, Germany. pOPIN-MBP-TEV with the CDS for TEV protease with a C-terminal penta-arginine tail was a gift from the Dortmund Protein Facility (DPF), Max Planck Institute of Molecular Physiology, Dortmund, Germany. Cloning of TcB-TcC with non-natural cargo proteins We used the fusion protein TcdB2-TccC3 10 as a carrier for heterologous cargo proteins to replace TccC3HVR. We therefore introduced an EcoRI restriction site by site-directed mutagenesis after the codon coding for the conserved P680 of TccC3, which is two residues after the aspartyl protease site 15 . Subsequently, we cloned the cargo proteins in frame via EcoRI and XhoI, resulting in an N-terminal extension of four residues (MPEF). In the case of TccC5HVR, the N-terminal extension results in a change of the pI from 8.65 to 7.90. In the case of Cdc42, the EcoRI site was inserted after the codon for L678, and the resulting point mutation P680F was reverted by site-directed mutagenesis. Cargo proteins with N-terminal extensions were generated by inserting the EcoRI site 135 or 384 base pairs after the aspartyl protease site and restriction cloning as described above, resulting in 45 or 128 N-terminal residues of TccC3HVR and two residues (EF) encoded by the EcoRI site being attached to the cargo. Cargo proteins with C-terminal extensions were generated by inserting a NotI restriction site 150 base pairs upstream of the stop codon, followed by restriction cloning via EcoRI and NotI. This resulted in a C-terminal extension of the cargo proteins containing 50 C-terminal residues of TccC3HVR and three residues (GGR) encoded by the NotI site. An overview of all resulting TcB-TcC cargos is listed in Fig. 4 . An overview of the primers used in this study is shown in Supplementary Table 3 . Protein production P. luminescens TcdA1 (TcA) was expressed and purified as described previously 16 . E. coli BL21-CodonPlus(DE3)-RIPL cells were transformed with pET19b containing the tcdA1 gene with an N-terminal hexahistidine tag and a pre-culture was inoculated from a freshly transformed colony. 10 L LB medium were inoculated to an OD 600 of 0.05 and incubated at 37 °C. At an OD 600 of 0.6, expression was induced by 25 μM IPTG and carried out overnight at 20 °C. Cells were disrupted in lysis buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 5 mM imidazole, 0.05% Tween-20) using a microfluidizer. After removal of cell debris by centrifugation, the supernatant was applied to a 5 mL Ni-NTA column (GE Healthcare Life Sciences) and washed with 10 column volumes (CVs) of washing buffer (lysis buffer with 50 mM imidazole). Subsequently, the protein was eluted with elution buffer (lysis buffer with 150 mM imidazole). Eluted TcdA1 was dialyzed against 20 mM HEPES-NaOH pH 8.0, 150 mM NaCl, 0.05% Tween-20 and SEC on a Sephacryl S400 16/60 column (GE Healthcare Life Sciences) equilibrated in the same buffer was performed as a final purification step. All TcB-TcC cargo variants were expressed and purified analogously to WT TcB-TcC as described previously 11 . E. coli BL21-CodonPlus(DE3)-RIPL cells were transformed with pET28a encoding the tcdB2-tccC3 genes (WT or cargo variants) with an N-terminal hexahistidine tag. In total, 5 or 10 L of LB medium containing 30 µM IPTG were directly inoculated with a freshly transformed colony. Cells were grown at 28 °C for 4 h, followed by 25 °C for 20 h and 20 °C for 24 h. Subsequently, cells were disrupted in lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol) using a microfluidizer. After removal of cell debris, the supernatant was applied to a 5 mL Ni-NTA column (GE Healthcare Life Sciences) and washed with 10 CVs of washing buffer (lysis buffer with 40 mM imidazole), followed by elution with a linear gradient from 40 mM to 250 mM imidazole over 10 CVs. The eluted protein was diluted with dilution buffer (20 mM Tris-HCl pH 8.0, 5% glycerol) to a final NaCl concentration of 20 mM and immediately loaded on a 5 mL HiTrapQ column (GE Healthcare Life Sciences) as a second purification step. After washing with 10 CVs of washing buffer 2 (20 mM Tris-HCl pH 8.0, 20 mM NaCl, 5% glycerol), the protein was eluted with a linear gradient from 20 to 500 mM NaCl over 20 CVs. Fractions containing TcB-TcC were subjected to SEC using a Superdex 200 10/300 or a Superdex 200 16/60 column (GE Healthcare Life Sciences) equilibrated in gel filtration buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol) as a final purification step. The gene coding for TccC3HVR (TccC3(679–960)) was cloned in pET19b in frame with a C-terminal 3C protease cleavage site and a hexahistidine tag. Expression was performed in E. coli BL21-CodonPlus(DE3)-RIPL cells. 2 L LB medium were inoculated to an OD 600 of 0.05 from an overnight pre-culture and incubated at 37 °C. At an OD 600 of 0.6, expression was induced by 100 μM IPTG and carried out overnight at 20 °C. Cells were disrupted in lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole) using a microfluidizer. After removal of cell debris by centrifugation, the supernatant was applied to a 5 mL Ni-NTA column (GE Healthcare Life Sciences) and washed with 10 CVs of lysis buffer, followed by elution with a gradient from 10 to 250 mM imidazole over 15 CVs. Eluted TccC3HVR was dialyzed against 20 mM Tris-HCl pH 8.0, 150 mM NaCl for 2 h before addition of recombinantly purified, hexahistidine-tagged Human Rhinovirus 3C protease (0.05 mg per mg of TccC3HVR), and dialysis was continued for 16 h at 4 °C. Subsequently, the protein solution was again applied to a 5 mL Ni-NTA column and the flow-through containing TccC3HVR without the histidine tag was subjected to SEC on a Superdex 200 16/60 column (GE Healthcare Life Sciences) using gel filtration buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl). ABC holotoxin formation Purified TcA (600 nM pentamer) and different TcB-TcC variants (1.2 μM) were mixed together and incubated for at least 1 h at 4 °C before removing the excess of free TcB-TcC by SEC on a Superose 6 Increase 5/150 column (GE Healthcare Life Sciences). For TcB-TcC variants containing HVR-RBD chimeras, 300 nM of TcA pentamer and 600 nM of TcB-TcC were used instead. The formation of the resulting ABC variants was verified by negative stain electron microscopy. Negative stain electron microscopy After SEC, 3 μL of each ABC variant at 0.1 mg/mL were incubated for 1 min on a glow-discharged 400-mesh copper grid (Agar Scientific) with an additional layer of thin carbon film. Subsequently, the sample was blotted with Whatman no. 4 filter paper and stained with 0.75% uranyl formate. Images were recorded on a FEI Tecnai Spirit TEM operating at 120 kV and equipped with a F416 CMOS detector (TVIPS). To quantify the ratio of holotoxin assembly, we picked at least 1000 particles from every ABC variant using crYOLO 41 and subjected them to 2D classification with ISAC 42 in SPHIRE 43 . Cell intoxication HEK293T cells were intoxicated with WT ABC or ABC-TccC5HVR. Cells (5 × 10 4 per well) were grown adherently overnight in 400 μL DMEM/F12 medium (Pan Biotech) and 0.5 or 2 nM holotoxin was subsequently added. Incubation was performed for 16 h at 37 °C before imaging. Experiments were performed in triplicate. Cells were not tested for Mycoplasma contamination. In vitro protein translocation assay After ABC formation and removal of unbound TcB-TcC via SEC, 200 nM ABC (WT or cargo variants) was mixed with DDM (final concentration: 0.1%) and dialyzed against 20 mM CAPS-NaOH pH 11.2, 150 mM NaCl, 0.1% DDM for 48 h at 4 °C. As a parallel control experiment, the same amount of ABC with 0.1% DDM was dialyzed against 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% DDM under the same conditions. Subsequently, the dialyzed proteins were subjected to SEC on a Superose 6 Increase 5/150 column equilibrated in the respective dialysis buffer. SEC fractions corresponding to the exclusion volume (aggregated holotoxin after dialysis), the major peak of the holotoxin, the tail of the holotoxin peak, and the peak after holotoxin were analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot for the presence of the cargo. In the cases where the cargo is translocated and released from the holotoxin, a Western blot signal appears in the fractions after the holotoxin peak at pH 11 (Supplementary Fig. 3a ). In the cases where the cargo is not translocated, Western blot signals are only found in fractions containing the holotoxin, both at pH 8 and pH 11 (Supplementary Fig. 3b ). Western blot and immunodetection After SDS-PAGE of the collected SEC fractions (10 μL per fraction) on a 4–15% acrylamide gradient gel, the proteins were transferred onto a PVDF membrane using a Trans-Blot Turbo semi-dry transfer system (Bio-Rad). In the cases where the cargo proteins were fusion constructs with N- or C-terminal parts of TccC3HVR, a custom-made anti-TccC3HVR rabbit polyclonal antibody (Cambridge Research Biochemicals) was used as the primary antibody at 1:1000 dilution. For Cdc42 without a TccC3HVR fusion, an anti-Cdc42 rabbit polyclonal antibody (Cell Signaling Technology, Cat. No. 2462) was used as the primary antibody at 1:1000 dilution. For TEV, an anti-TEV protease rabbit polyclonal antibody (Novus Biologicals, Cat. No. NBP1–97669) was used as the primary antibody at 1:500 dilution. An HRP-conjugated goat anti-rabbit antibody (Bio-Rad, Cat. No. 170–6515) was applied as the secondary antibody at 1:2000 dilution in all cases. Detection was performed with Western Lightning Plus ECL reagent (PerkinElmer, Cat. No. NEL104001EA) and imaged in a ChemiDoc MP imaging system (Bio-Rad). Isolation of translocated TccC3HVR To isolate translocated TccC3HVR, we assembled ABC wild-type holotoxin (700 nM) as described above, and triggered the prepore-to-pore transition by dialysis against 20 mM CAPS-NaOH pH 11.2, 150 mM NaCl in the presence of 9 μM Msp1D1 nanodisc scaffold protein (Cube Biotech, Cat. No. 26112) and 490 μM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Cat. No. 850457 C) dissolved in 1% sodium cholate. After 72 h of dialysis at 4 °C, we subjected the holotoxin to SEC on a Superose 6 increase 5/150 column equilibrated in dialysis buffer and collected the fractions after the holotoxin peak (see Supplementary Fig. 3a ). ADP-ribosylation assay For in vitro ADP-ribosylation, rabbit muscle actin was prepared as described previously 44 and filamentous actin (F-actin) was formed by overnight incubation in ADP-ribosylation buffer (50 mM HEPES-NaOH pH 7.3 100 mM KCl, 2 mM MgCl 2 ) at 4 °C. In total, 2 μM of F-actin were mixed with 1 mM NAD + and different concentrations (50–500 pM) of recombinantly purified or translocated TccC3HVR and incubated for 12 min at 22 °C. The reaction was stopped by addition of SDS-PAGE sample buffer and immediate heating to 95 °C. ADP-ribosylation of F-actin was assessed via SDS-PAGE, Western blot and immunodetection using anti-pan-ADP-ribose binding reagent (Millipore, Cat. No. MABE1016) at 1:2000 dilution and an HRP-conjugated goat anti-rabbit antibody as described above. Fluorescence spectroscopy Florescence emission spectra of TcB-TcC(WT), TcB-TcC-iLOV and TcB-TcC-HVR130-iLOV (500 nm protein concentration) were recorded in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween-20 using a FluoroMax-4 fluorescence spectrophotometer (Horiba). The excitation wavelength was 450 nm, and emission was recorded from 490 to 620 nm. Purified iLOV with a GST-tag 29 was used as positive control. X-ray crystallography of TcB-TcC-Cdc42 and TcB-TcC-Cdc42 TcB-TcC-Cdc42 and TcB-TcC-TEV were crystallized using the sitting-drop vapor diffusion method at 20 ˚C. For TcB-TcC-Cdc42, initially, 2D crystals formed after mixing 1 µL of 10 mg/mL protein solution with 1 µL reservoir solution containing 0.1 M sodium chloride, 0.1 M magnesium chloride, 0.1 M tri-sodium citrate pH 5.5 and 12% PEG 4000. The 2D crystals were used to prepare a seed solution. Final 3D crystals were obtained by mixing 1 µL of 10 mg/mL protein solution with 0.5 µL seed solution and 1.5 µL reservoir solution containing 0.1 m magnesium chloride, 0.1 M tri-sodium acetate pH 4.6 and 12 % PEG 6000. TcB-TcC-TEV crystallized in the same crystallization buffer without seeding. Prior to flash freezing in liquid nitrogen, the crystals were soaked in reservoir solution containing 20% glycerol as a cryoprotectant. X-ray diffraction data were collected at the PXII-X10SA beamline at the Swiss Light Source (Villigen, Switzerland) using a wavelength of 0.97958 à and 0.9998 à for TcB-TcC-Cdc42 and TcB-TcC-TEV, respectively. The X-ray data set was integrated and scaled using XDS 45 . Phases were determined by molecular replacement with PHASER implemented in PHENIX 46 using the crystal structure of WT TcdB2-TccC3 (PDB 4O9X) as a search model. For the TcB-TcC-TEV crystal two data sets were collected with 60˚ translation and merged together using XSCALE 45 . For phasing, the merged data set was extended to a resolution of 3.3 à and the refinement was performed with a resolution cutoff of 3.7 à in PHENIX 46 . TcB-TcC-Cdc42 crystallized in the orthorhombic space group P2 1 2 1 2 1 with unit cell dimensions of 96 × 156 × 179 à and one molecule per asymmetric unit. TcB-TcC-TEV crystallized in primitive hexagonal space group P3 2 21 with unit cell dimensions of 234 × 234 × 143 à and one molecule per AU. The structures were optimized by iteration of manual and automatic refinement using COOT 47 and PHENIX 48 to a final R free of 25% for TcB-TcC-Cdc42 and 34% for TcB-TcC-TEV. Data collection and refinement statistics are summarized in Supplementary Table 1 . Cryo-EM sample preparation and data acquisition In total, 3 µL of 2.1 mg/mL ABC-Cdc42 in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween-20 were applied to a glow-discharged holey carbon grid (Quantifoil, QF 2/1, 300 mesh). Subsequently, the sample was vitrified in liquid ethane with a Cryoplunge3 plunger (Cp3, Gatan) using 1.6 s blotting time at 90% humidity and 22 °C. A data set of ABC-Cdc42 was collected at the Max Planck Institute of Molecular Physiology, Dortmund using a Cs corrected Titan Krios equipped with an XFEG and a Falcon II direct electron detector. Images were recorded using the automated acquisition program EPU (FEI) at a magnification of 59,000× corresponding to a pixel size of 1.14 à /pixel on the specimen level. In total, 3024 movie-mode images were acquired in a defocus range of 1.0–3.2 μm. Each movie comprised 24 frames with a total cumulative dose of ~ 65 e − /à 2 . Image processing of ABC-Cdc42 After initial screening of all micrographs, 2754 images were selected for further processing. Movie frames were aligned, dose-corrected and averaged using MotionCor2 49 . The integrated images were also used to determine the contrast transfer function parameters with CTER 50 , implemented in the SPHIRE software package 43 . Initially, 2025 particles were manually picked and 2D class averages generated by Relion 51 were used as an autopicking template. In all, 99,980 particles were auto-picked from the images using the Relion 1.4 autopicker. Subsequently, reference-free 2D classification and cleaning of the dataset were performed with the iterative stable alignment and clustering approach ISAC 42 in SPHIRE. ISAC was executed with a pixel size of 7.2 à /pixel on the particle level. The 'Beautifier' tool of SPHIRE was then applied to obtain refined and sharpened 2D class averages at the original pixel size, showing high-resolution features (Supplementary Fig. 5b ). From the initial set of particles, the clean set used for 3D refinement contained 56,665 particles. We applied the previously obtained cryo-EM structure of ABC(WT) (EMDB-2551) as an initial model after scaling and filtering it to 12 à resolution and performed 3D refinement in SPHIRE. The resolution of the final density was estimated to be 7.02/5.11 à according to FSC 0.5/0.143 after applying a soft Gaussian mask. The B-factor was estimated to be −246.4 à 2 . Local FSC calculation was performed using the Local Resolution tool in SPHIRE. (Supplementary Fig. 5e ) and the electron density map was filtered according to its local resolution using the 3D Local Filter tool in SPHIRE. Cryo-EM data processing statistics are summarized in Supplementary Table 2 . Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. Source plasmids for cargo proteins pGEX-iLOV was a gift from John Christie (Addgene plasmid #26587) 29 . pcDXc3A-AN44-ICP47 containing the coding sequence (CDS) for ICP47 with a C-terminal HA epitope was a gift from Robert Tampé, Goethe University of Frankfurt, Germany. pGEX-RBD with the CDS for the Ras-binding domain (RBD) of CRAF kinase was a gift from Andreas Ernst, Goethe University of Frankfurt, Germany. A customized plasmid with the CDS for dihydrofolate reductase (DHFR) was a gift from Yaowen Wu, Max Planck Institute of Molecular Physiology, Dortmund, Germany. pOPIN-MBP-TEV with the CDS for TEV protease with a C-terminal penta-arginine tail was a gift from the Dortmund Protein Facility (DPF), Max Planck Institute of Molecular Physiology, Dortmund, Germany. Cloning of TcB-TcC with non-natural cargo proteins We used the fusion protein TcdB2-TccC3 10 as a carrier for heterologous cargo proteins to replace TccC3HVR. We therefore introduced an EcoRI restriction site by site-directed mutagenesis after the codon coding for the conserved P680 of TccC3, which is two residues after the aspartyl protease site 15 . Subsequently, we cloned the cargo proteins in frame via EcoRI and XhoI, resulting in an N-terminal extension of four residues (MPEF). In the case of TccC5HVR, the N-terminal extension results in a change of the pI from 8.65 to 7.90. In the case of Cdc42, the EcoRI site was inserted after the codon for L678, and the resulting point mutation P680F was reverted by site-directed mutagenesis. Cargo proteins with N-terminal extensions were generated by inserting the EcoRI site 135 or 384 base pairs after the aspartyl protease site and restriction cloning as described above, resulting in 45 or 128 N-terminal residues of TccC3HVR and two residues (EF) encoded by the EcoRI site being attached to the cargo. Cargo proteins with C-terminal extensions were generated by inserting a NotI restriction site 150 base pairs upstream of the stop codon, followed by restriction cloning via EcoRI and NotI. This resulted in a C-terminal extension of the cargo proteins containing 50 C-terminal residues of TccC3HVR and three residues (GGR) encoded by the NotI site. An overview of all resulting TcB-TcC cargos is listed in Fig. 4 . An overview of the primers used in this study is shown in Supplementary Table 3 . Protein production P. luminescens TcdA1 (TcA) was expressed and purified as described previously 16 . E. coli BL21-CodonPlus(DE3)-RIPL cells were transformed with pET19b containing the tcdA1 gene with an N-terminal hexahistidine tag and a pre-culture was inoculated from a freshly transformed colony. 10 L LB medium were inoculated to an OD 600 of 0.05 and incubated at 37 °C. At an OD 600 of 0.6, expression was induced by 25 μM IPTG and carried out overnight at 20 °C. Cells were disrupted in lysis buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 5 mM imidazole, 0.05% Tween-20) using a microfluidizer. After removal of cell debris by centrifugation, the supernatant was applied to a 5 mL Ni-NTA column (GE Healthcare Life Sciences) and washed with 10 column volumes (CVs) of washing buffer (lysis buffer with 50 mM imidazole). Subsequently, the protein was eluted with elution buffer (lysis buffer with 150 mM imidazole). Eluted TcdA1 was dialyzed against 20 mM HEPES-NaOH pH 8.0, 150 mM NaCl, 0.05% Tween-20 and SEC on a Sephacryl S400 16/60 column (GE Healthcare Life Sciences) equilibrated in the same buffer was performed as a final purification step. All TcB-TcC cargo variants were expressed and purified analogously to WT TcB-TcC as described previously 11 . E. coli BL21-CodonPlus(DE3)-RIPL cells were transformed with pET28a encoding the tcdB2-tccC3 genes (WT or cargo variants) with an N-terminal hexahistidine tag. In total, 5 or 10 L of LB medium containing 30 µM IPTG were directly inoculated with a freshly transformed colony. Cells were grown at 28 °C for 4 h, followed by 25 °C for 20 h and 20 °C for 24 h. Subsequently, cells were disrupted in lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol) using a microfluidizer. After removal of cell debris, the supernatant was applied to a 5 mL Ni-NTA column (GE Healthcare Life Sciences) and washed with 10 CVs of washing buffer (lysis buffer with 40 mM imidazole), followed by elution with a linear gradient from 40 mM to 250 mM imidazole over 10 CVs. The eluted protein was diluted with dilution buffer (20 mM Tris-HCl pH 8.0, 5% glycerol) to a final NaCl concentration of 20 mM and immediately loaded on a 5 mL HiTrapQ column (GE Healthcare Life Sciences) as a second purification step. After washing with 10 CVs of washing buffer 2 (20 mM Tris-HCl pH 8.0, 20 mM NaCl, 5% glycerol), the protein was eluted with a linear gradient from 20 to 500 mM NaCl over 20 CVs. Fractions containing TcB-TcC were subjected to SEC using a Superdex 200 10/300 or a Superdex 200 16/60 column (GE Healthcare Life Sciences) equilibrated in gel filtration buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol) as a final purification step. The gene coding for TccC3HVR (TccC3(679–960)) was cloned in pET19b in frame with a C-terminal 3C protease cleavage site and a hexahistidine tag. Expression was performed in E. coli BL21-CodonPlus(DE3)-RIPL cells. 2 L LB medium were inoculated to an OD 600 of 0.05 from an overnight pre-culture and incubated at 37 °C. At an OD 600 of 0.6, expression was induced by 100 μM IPTG and carried out overnight at 20 °C. Cells were disrupted in lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole) using a microfluidizer. After removal of cell debris by centrifugation, the supernatant was applied to a 5 mL Ni-NTA column (GE Healthcare Life Sciences) and washed with 10 CVs of lysis buffer, followed by elution with a gradient from 10 to 250 mM imidazole over 15 CVs. Eluted TccC3HVR was dialyzed against 20 mM Tris-HCl pH 8.0, 150 mM NaCl for 2 h before addition of recombinantly purified, hexahistidine-tagged Human Rhinovirus 3C protease (0.05 mg per mg of TccC3HVR), and dialysis was continued for 16 h at 4 °C. Subsequently, the protein solution was again applied to a 5 mL Ni-NTA column and the flow-through containing TccC3HVR without the histidine tag was subjected to SEC on a Superdex 200 16/60 column (GE Healthcare Life Sciences) using gel filtration buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl). ABC holotoxin formation Purified TcA (600 nM pentamer) and different TcB-TcC variants (1.2 μM) were mixed together and incubated for at least 1 h at 4 °C before removing the excess of free TcB-TcC by SEC on a Superose 6 Increase 5/150 column (GE Healthcare Life Sciences). For TcB-TcC variants containing HVR-RBD chimeras, 300 nM of TcA pentamer and 600 nM of TcB-TcC were used instead. The formation of the resulting ABC variants was verified by negative stain electron microscopy. Negative stain electron microscopy After SEC, 3 μL of each ABC variant at 0.1 mg/mL were incubated for 1 min on a glow-discharged 400-mesh copper grid (Agar Scientific) with an additional layer of thin carbon film. Subsequently, the sample was blotted with Whatman no. 4 filter paper and stained with 0.75% uranyl formate. Images were recorded on a FEI Tecnai Spirit TEM operating at 120 kV and equipped with a F416 CMOS detector (TVIPS). To quantify the ratio of holotoxin assembly, we picked at least 1000 particles from every ABC variant using crYOLO 41 and subjected them to 2D classification with ISAC 42 in SPHIRE 43 . Cell intoxication HEK293T cells were intoxicated with WT ABC or ABC-TccC5HVR. Cells (5 × 10 4 per well) were grown adherently overnight in 400 μL DMEM/F12 medium (Pan Biotech) and 0.5 or 2 nM holotoxin was subsequently added. Incubation was performed for 16 h at 37 °C before imaging. Experiments were performed in triplicate. Cells were not tested for Mycoplasma contamination. In vitro protein translocation assay After ABC formation and removal of unbound TcB-TcC via SEC, 200 nM ABC (WT or cargo variants) was mixed with DDM (final concentration: 0.1%) and dialyzed against 20 mM CAPS-NaOH pH 11.2, 150 mM NaCl, 0.1% DDM for 48 h at 4 °C. As a parallel control experiment, the same amount of ABC with 0.1% DDM was dialyzed against 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% DDM under the same conditions. Subsequently, the dialyzed proteins were subjected to SEC on a Superose 6 Increase 5/150 column equilibrated in the respective dialysis buffer. SEC fractions corresponding to the exclusion volume (aggregated holotoxin after dialysis), the major peak of the holotoxin, the tail of the holotoxin peak, and the peak after holotoxin were analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot for the presence of the cargo. In the cases where the cargo is translocated and released from the holotoxin, a Western blot signal appears in the fractions after the holotoxin peak at pH 11 (Supplementary Fig. 3a ). In the cases where the cargo is not translocated, Western blot signals are only found in fractions containing the holotoxin, both at pH 8 and pH 11 (Supplementary Fig. 3b ). Western blot and immunodetection After SDS-PAGE of the collected SEC fractions (10 μL per fraction) on a 4–15% acrylamide gradient gel, the proteins were transferred onto a PVDF membrane using a Trans-Blot Turbo semi-dry transfer system (Bio-Rad). In the cases where the cargo proteins were fusion constructs with N- or C-terminal parts of TccC3HVR, a custom-made anti-TccC3HVR rabbit polyclonal antibody (Cambridge Research Biochemicals) was used as the primary antibody at 1:1000 dilution. For Cdc42 without a TccC3HVR fusion, an anti-Cdc42 rabbit polyclonal antibody (Cell Signaling Technology, Cat. No. 2462) was used as the primary antibody at 1:1000 dilution. For TEV, an anti-TEV protease rabbit polyclonal antibody (Novus Biologicals, Cat. No. NBP1–97669) was used as the primary antibody at 1:500 dilution. An HRP-conjugated goat anti-rabbit antibody (Bio-Rad, Cat. No. 170–6515) was applied as the secondary antibody at 1:2000 dilution in all cases. Detection was performed with Western Lightning Plus ECL reagent (PerkinElmer, Cat. No. NEL104001EA) and imaged in a ChemiDoc MP imaging system (Bio-Rad). Isolation of translocated TccC3HVR To isolate translocated TccC3HVR, we assembled ABC wild-type holotoxin (700 nM) as described above, and triggered the prepore-to-pore transition by dialysis against 20 mM CAPS-NaOH pH 11.2, 150 mM NaCl in the presence of 9 μM Msp1D1 nanodisc scaffold protein (Cube Biotech, Cat. No. 26112) and 490 μM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Cat. No. 850457 C) dissolved in 1% sodium cholate. After 72 h of dialysis at 4 °C, we subjected the holotoxin to SEC on a Superose 6 increase 5/150 column equilibrated in dialysis buffer and collected the fractions after the holotoxin peak (see Supplementary Fig. 3a ). ADP-ribosylation assay For in vitro ADP-ribosylation, rabbit muscle actin was prepared as described previously 44 and filamentous actin (F-actin) was formed by overnight incubation in ADP-ribosylation buffer (50 mM HEPES-NaOH pH 7.3 100 mM KCl, 2 mM MgCl 2 ) at 4 °C. In total, 2 μM of F-actin were mixed with 1 mM NAD + and different concentrations (50–500 pM) of recombinantly purified or translocated TccC3HVR and incubated for 12 min at 22 °C. The reaction was stopped by addition of SDS-PAGE sample buffer and immediate heating to 95 °C. ADP-ribosylation of F-actin was assessed via SDS-PAGE, Western blot and immunodetection using anti-pan-ADP-ribose binding reagent (Millipore, Cat. No. MABE1016) at 1:2000 dilution and an HRP-conjugated goat anti-rabbit antibody as described above. Fluorescence spectroscopy Florescence emission spectra of TcB-TcC(WT), TcB-TcC-iLOV and TcB-TcC-HVR130-iLOV (500 nm protein concentration) were recorded in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween-20 using a FluoroMax-4 fluorescence spectrophotometer (Horiba). The excitation wavelength was 450 nm, and emission was recorded from 490 to 620 nm. Purified iLOV with a GST-tag 29 was used as positive control. X-ray crystallography of TcB-TcC-Cdc42 and TcB-TcC-Cdc42 TcB-TcC-Cdc42 and TcB-TcC-TEV were crystallized using the sitting-drop vapor diffusion method at 20 ˚C. For TcB-TcC-Cdc42, initially, 2D crystals formed after mixing 1 µL of 10 mg/mL protein solution with 1 µL reservoir solution containing 0.1 M sodium chloride, 0.1 M magnesium chloride, 0.1 M tri-sodium citrate pH 5.5 and 12% PEG 4000. The 2D crystals were used to prepare a seed solution. Final 3D crystals were obtained by mixing 1 µL of 10 mg/mL protein solution with 0.5 µL seed solution and 1.5 µL reservoir solution containing 0.1 m magnesium chloride, 0.1 M tri-sodium acetate pH 4.6 and 12 % PEG 6000. TcB-TcC-TEV crystallized in the same crystallization buffer without seeding. Prior to flash freezing in liquid nitrogen, the crystals were soaked in reservoir solution containing 20% glycerol as a cryoprotectant. X-ray diffraction data were collected at the PXII-X10SA beamline at the Swiss Light Source (Villigen, Switzerland) using a wavelength of 0.97958 à and 0.9998 à for TcB-TcC-Cdc42 and TcB-TcC-TEV, respectively. The X-ray data set was integrated and scaled using XDS 45 . Phases were determined by molecular replacement with PHASER implemented in PHENIX 46 using the crystal structure of WT TcdB2-TccC3 (PDB 4O9X) as a search model. For the TcB-TcC-TEV crystal two data sets were collected with 60˚ translation and merged together using XSCALE 45 . For phasing, the merged data set was extended to a resolution of 3.3 à and the refinement was performed with a resolution cutoff of 3.7 à in PHENIX 46 . TcB-TcC-Cdc42 crystallized in the orthorhombic space group P2 1 2 1 2 1 with unit cell dimensions of 96 × 156 × 179 à and one molecule per asymmetric unit. TcB-TcC-TEV crystallized in primitive hexagonal space group P3 2 21 with unit cell dimensions of 234 × 234 × 143 à and one molecule per AU. The structures were optimized by iteration of manual and automatic refinement using COOT 47 and PHENIX 48 to a final R free of 25% for TcB-TcC-Cdc42 and 34% for TcB-TcC-TEV. Data collection and refinement statistics are summarized in Supplementary Table 1 . Cryo-EM sample preparation and data acquisition In total, 3 µL of 2.1 mg/mL ABC-Cdc42 in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween-20 were applied to a glow-discharged holey carbon grid (Quantifoil, QF 2/1, 300 mesh). Subsequently, the sample was vitrified in liquid ethane with a Cryoplunge3 plunger (Cp3, Gatan) using 1.6 s blotting time at 90% humidity and 22 °C. A data set of ABC-Cdc42 was collected at the Max Planck Institute of Molecular Physiology, Dortmund using a Cs corrected Titan Krios equipped with an XFEG and a Falcon II direct electron detector. Images were recorded using the automated acquisition program EPU (FEI) at a magnification of 59,000× corresponding to a pixel size of 1.14 à /pixel on the specimen level. In total, 3024 movie-mode images were acquired in a defocus range of 1.0–3.2 μm. Each movie comprised 24 frames with a total cumulative dose of ~ 65 e − /à 2 . Image processing of ABC-Cdc42 After initial screening of all micrographs, 2754 images were selected for further processing. Movie frames were aligned, dose-corrected and averaged using MotionCor2 49 . The integrated images were also used to determine the contrast transfer function parameters with CTER 50 , implemented in the SPHIRE software package 43 . Initially, 2025 particles were manually picked and 2D class averages generated by Relion 51 were used as an autopicking template. In all, 99,980 particles were auto-picked from the images using the Relion 1.4 autopicker. Subsequently, reference-free 2D classification and cleaning of the dataset were performed with the iterative stable alignment and clustering approach ISAC 42 in SPHIRE. ISAC was executed with a pixel size of 7.2 à /pixel on the particle level. The 'Beautifier' tool of SPHIRE was then applied to obtain refined and sharpened 2D class averages at the original pixel size, showing high-resolution features (Supplementary Fig. 5b ). From the initial set of particles, the clean set used for 3D refinement contained 56,665 particles. We applied the previously obtained cryo-EM structure of ABC(WT) (EMDB-2551) as an initial model after scaling and filtering it to 12 à resolution and performed 3D refinement in SPHIRE. The resolution of the final density was estimated to be 7.02/5.11 à according to FSC 0.5/0.143 after applying a soft Gaussian mask. The B-factor was estimated to be −246.4 à 2 . Local FSC calculation was performed using the Local Resolution tool in SPHIRE. (Supplementary Fig. 5e ) and the electron density map was filtered according to its local resolution using the 3D Local Filter tool in SPHIRE. Cryo-EM data processing statistics are summarized in Supplementary Table 2 . Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. Supplementary information Supplementary Information Peer Review File Reporting Summary Source Data Supplementary Information Peer Review File Reporting Summary Source Data Supplementary information Supplementary information is available for this paper at 10.1038/s41467-019-13253-8.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10745416/

Antibiotics and Antibiotic Resistance—Mur Ligases as an Antibacterial Target

The emergence of Multidrug Resistance (MDR) strains of bacteria has accelerated the search for new antibacterials. The specific bacterial peptidoglycan biosynthetic pathway represents opportunities for the development of novel antibacterial agents. Among the enzymes involved, Mur ligases, described herein, and especially the amide ligases MurC-F are key targets for the discovery of multi-inhibitors, as they share common active sites and structural features. 1. Introduction Microorganisms have existed on Earth for centuries, but it was not until the 17th century that Antoni van Leeuwenhoek, a renowned Dutch scientist, made groundbreaking observations identifying various microorganisms, including yeasts and even red blood cells [ 1 ]. His work marked an important moment in the history of biology, laying the foundation for microbiology and bacteriology. Following Leeuwenhoek's discoveries, a series of events triggered a surge of interest in microbiology, leading to significant advancements in the field. In the years that followed, numerous scientists studied microorganisms, particularly those that caused major epidemics. In 1835, Agostino Bassi identified Beauveria bassiana as the microbial origin of the silkworm disease called muscardine [ 2 ]. In 1854, Filippo Pacini isolated and identified the Vibrio cholerae as a pathogen [ 3 ]; meanwhile, Casimir Davaine discovered the anthrax bacillus [ 4 ]. These discoveries propelled microbiology forward, driven by the efforts of figures like Robert Koch [ 5 ] and Louis Pasteur [ 6 ]. The early 20th century marked a significant period in the history of antibiotics. German scientist Paul Ehrlich created the first effective drugs ( Figure 1 ) against syphilis in 1910 [ 7 ], and, later, Alexander Fleming made a groundbreaking discovery in 1928, observing that Penicillium notatum had the ability to inhibit the growth of Staphylococcus aureus (S. aureus) . This discovery [ 8 ] laid the foundation for the development of penicillin G 3 and other antibiotics such as sulfanilamide 4 [ 9 , 10 ]. The golden age of antibiotic discovery occurred from the 1940s to the 1960s, with the identification of numerous families of antibiotics 5 – 15 ( Figure 2 ) [ 11 ]. However, bacteria developed resistance mechanisms in response to antibiotic treatments [ 12 , 13 ]. This resistance is a significant global health threat, and the World Health Organization (WHO) considers it one of the most serious challenges to global health. Infections caused by resistant bacteria, coupled with diminishing antibiotic effectiveness, have raised alarms [ 14 , 15 , 16 ]. Bacterial resistance can occur through different mechanisms [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ], including extracellular resistance (biofilms), natural or innate resistance (inherent to specific strains), and acquired resistance (mutations or acquisition of resistance genes). Resistance mechanisms involve alterations in genes affecting interactions between bacteria and antibiotics, modifications of antibiotic targets, enzymatic mechanisms to prevent antibiotic binding, the hindrance of antibiotic entry through bacterial membranes, and the efflux of antibiotics from bacterial cells. This modification involves changes in the target's genetic sequences, resulting in decreased efficacy of the antibiotic. For example, this phenomenon has been observed in a strain of Mycobacterium leprae resistant to rifampicin 16 , an antibiotic of the rifamycin family ( Figure 3 ) [ 27 ]. Enzymes produced by the bacteria can also inactivate the antibiotic by cleaving it. For example, enzymes known as " β-lactamase " are capable of hydrolyzing β-lactam rings found in antibiotics such as penicillins 17 ( Scheme 1 ) to inactive compounds 18 [ 28 ]. β-lactams target bacterial wall biosynthesis by inhibiting the transpeptidase activity of penicillin-binding proteins (PBPs), which are involved in the final steps of peptidoglycan synthesis. The open form of β-lactams results in the loss of their biological activity against PBP transpeptidases. The ability of an antibiotic to reach its target within bacteria can also occur via the bacterial membranes [ 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. Despite the challenges of bacterial resistance, antibiotics remain effective against certain pathogenic bacteria. These antibiotics target specific bacterial processes, such as DNA replication, RNA polymerase activity, protein synthesis, and bacterial wall synthesis. For example, antibiotics can inhibit DNA gyrase and RNA polymerase [ 38 , 39 , 40 , 41 , 42 ], interfere with the metabolism of folic acid [ 43 , 44 , 45 , 46 , 47 ], disrupt protein synthesis [ 48 , 49 , 50 , 51 , 52 , 53 ], and target the bacterial wall synthesis process [ 54 , 55 , 56 , 57 , 58 , 59 ]. Efforts to combat resistance involve the development of new antibiotics targeting novel bacterial processes and exploring new therapeutic targets. These ongoing efforts are crucial in the fight against antibiotic-resistant bacteria and the preservation of effective treatment options. 2. The Peptidoglycan Chain Peptidoglycan, also called murein, is a biopolymer present in all bacteria that provides protection from the external environment, especially osmotic pressure [ 60 ]. A notable difference between bacteria is that the cell wall is composed of about 95% peptidoglycan chains for Gram-positive bacteria and about 20% for Gram-negative bacteria, which explains the effectiveness of some antibiotics compared to others. Its complex chemical structure is defined by a cross-linked network in which a repeating unit contains an N -acetylglucosamine (NAG) and an N -acetylmuramic acid (NAM) linked by a β-1 bond → 4 ( Figure 4 ) [ 61 ]. This peptidoglycan forms a three-dimensional network linked by cross-links simply composed of peptides, a pentaglycine here. This cross-linking is linked on both sides with two small peptides that are grafted by the carboxylic acid function of MurNAc. The chemical nature of these peptides differs according to the bacteria; in Gram-positive bacteria, the unnatural amino acid called meso -diaminopimelic acid ( m-DAP ) is found, whereas in Gram-negative bacteria, L-Lysine is present. This structure presents an impressive chemical diversity and explains the strength of the peptidoglycan chain in bacteria. Indeed, several elements that compose it testify to this, such as the MurNAc, a saccharide not present in eukaryotic cells, amino acids of the d series, and m-DAP . The biosynthesis of the peptidoglycan chain ( Scheme 2 ) is a complex process that takes place in every part of the bacterium [ 62 ]. It is common to all Gram-positive or Gram-negative bacteria with some variations for Mycobacterium tuberculosis [ 63 ]. Each enzyme involved in this biosynthesis is important because the inhibition of one would lead to bacterial lysis and therefore cell death. During this biosynthesis, the formation of several key intermediates is obtained in each site of the bacterium (cytoplasm, membrane, and periplasm). The starting point of this synthesis is the conversion of fructose-6-phosphate (F6P, 19 ) to uridine diphosphate- N -acetylglucosamine (UDP-GlcNAc, 20 ). Then, a lactoyl function is introduced on this substrate, followed by a pentapeptide chain to form uridine diphosphate N -acetylmuramoyl-pentapeptide (UDP-MurNAc-pentapeptide, 21 ). This entity, which is nothing but a part of the peptidoglycan-repeating unit, binds to undecaprenylphosphate (C55P) of the plasma membrane and is then glycosylated to obtain Lipid II ( 22 ). An enzyme called flippase will allow Lipid II to pass from the internal face of the membrane to its external face [ 64 ]. Finally, this monomer is polymerized in the periplasm by transglycosylation and transpeptidation steps to end up with the mature peptidoglycan ( 23 ). Mur ligases are involved at several parts of this process; their role is to catalyze the formation of a peptide bond with the corresponding amino acid on the substrate, which is specific to each of these enzymes. These enzymes have all been identified as non-ribosomal ATP-dependent proteins in the cytoplasm, and MurA and MurB allow the biosynthesis of UDP-MurNAc ( 25 ) from UDP-GlcNAc ( 28 ) ( Figure 5 ). MurA or UDP-GlcNAc enolpyruvyltransferase catalyzes the transfer of an enolpyruvate moiety from phosphoenol pyruvate (PEP) to UDP-GlcNAc ( 20 ) with phosphate release. The crystallographic structure [ 65 ] of MurA as well as its mechanism [ 66 ] have been well identified ( Scheme 3 ). Its mode of action involves an addition–elimination mechanism: the anti- addition of PEP on UDP-GlcNAc ( 20 ) is catalyzed by Asp 305 (numbering of MurA from Escherichia coli ) and Cys 115 of MurA to form the corresponding tetrahedral intermediate, and then the syn-elimination allows UDP-GlcNAc enolpyruvate ( 24 ) to be obtained. Currently, the only antibiotic used that specifically targets MurA is fosfomycin ( 26 ) ( Figure 6 ). It is a PEP analog which binds irreversibly to Cys 115 (numbering of MurA from Escherichia coli) of the active site of MurA, rendering the enzyme inactive and causing cell death. However, there are many mechanisms of resistance to this compound: mutations of the enzyme, cellular permeability, or an enzymatic action that inactivates the antibiotic. Similarly, innate resistance of certain species such as Mycobacterium tuberculosis or Chlamydia trachomatis exist where an Asp [ 67 ] replaces the targeted Cys residue. Numerous covalent and noncovalent inhibitors have been developed [ 68 ]. Avenaciolide compounds [ 69 ] (isolated from Neosartorya fischeri ) are tetrahedral intermediate inhibitors, while quinazolinone analogs [ 70 ] are competitive inhibitors. Compound 27 is promising because it shows good biological activities with an MIC of 16 μg/mL −1 and 32 μg/mL −1 on methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus subtilis (B. subtilis), respectively. Furthermore, it was shown to be specific for MurA enzymes with an IC 50 of 2.8 μM on MurA from Escherichia coli (E. coli) and 7.9 μM on resistant MurA. Compound 28 is active on Escherichia coli and Staphylococcus aureus, respectively, with an MIC of 1 μg/mL −1 and 8 μg/mL −1 while showing an IC 50 on MurA of 47 μM. MurB or UDP-GlcNAc enolpyruvate reductase catalyzes the stereoselective reduction of UDP-GlcNAc enolpyruvate ( 24 ) to UDP-MurNAc ( 25 ) with the action of a co-enzyme called Nicotinamide Adenine Dinucleotide Phosphate (NADPH) [ 71 ]. This enzyme also contains an active site with the Flavin Adenine Dinucleotide cofactor (FAD). The crystallographic structure [ 72 , 73 ] of MurB and its mechanism [ 74 , 75 ] have been elucidated ( Scheme 4 ). The first step of the mechanism involves the formation of the FAD-MurB complex, which acts as a redox intermediate. Similarly, after the formation of the second NADPH-MurB complex, the reduction of FAD by NADPH leads to the reaction intermediate FADH 2 -MurB by transferring the H4 ( pro-S) from NADPH to the N of FAD. After the release of NADP+, UDP-GlcNAc enolpyruvate ( 24 ) binds to the enzyme. The second step is the reduction of the latter by the FADH 2 -MurB thanks to the transfer of hydrogen in C-3 on the enolpyruvate part. After the release of FAD, the enolate intermediate obtained is stabilized by the carboxylic acid of the substrate with the enzyme. Finally, the isomerization of the substrate to the final product UDP-MurNAc ( 25 ) requires the presence of water. Currently, there is no antibiotic used for the MurB of bacteria. However, few inhibitors have been developed ( Figure 7 ) such as the imidazolinone 29 , one of the first inhibitors targeting the MurB substrate site from Escherichia coli . It shows activity against Staphylococcus aureus strains [ 76 ]. Compounds 30 and 31 have been identified as multi-inhibitors of MurA/MurB targeting FAD, with interesting activities on Staphylococcus aureus strains [ 77 , 78 ]. Finally, the last cytoplasmic steps allowing the formation of UDP-MurNAc pentapeptide from the UDP-MurNAc ( 25 ) involve MurC-F, which are grouped into a family of four amino acid ligase enzymes that form a peptide bond in the presence of the corresponding amino acid [ 79 , 80 ]. Other enzymes at the plasma membrane are responsible for the continuation of this synthesis ( Scheme 5 ). Among them, MurG, a glycosyltransferase, catalyzes the glycosylation between Lipid I and a GlcNAc unit from UDP-GlcNAc ( 20 ) to form Lipid II. This essential enzyme is highly conserved in all bacterial species. However, glycosyltransferases are present in a large majority of cells in both prokaryotes and eukaryotes, which requires a high specificity of potential MurG inhibitors in drug design [ 81 ]. The crystallographic structure of MurG has been solved, as well as its mechanism, by Walker et al. [ 82 , 83 , 84 ]. Other teams have shown that the structure of MurG is able to interact with other proteins such as MraY [ 85 ], and with the MurE-MurF dimer complex [ 86 ], which allows the substrate to evolve with small distances within this protein–protein complex. Several inhibitors targeting MurG have been developed as the pentacyclic compound ( 32 ) ( Figure 8 ) [ 87 ]. In addition, the use of high-throughput screening has allowed the development of new inhibitors against the MurG, such as the pyrimidinetrione ( 33 ) [ 88 , 89 ]; more recently, the diazepanone analog ( 34 ) was found to inhibit both MraY and MurG [ 90 ]. 3. MurC-F Ligases as New Antibiotic Targets In the fight against bacterial resistance, another effective way is to go after seldom exploited targets that do not show any resistance mechanism. In this context, enzymes involved in bacterial wall biosynthesis meet this criterion. Among some of these enzymes, resistances have already appeared, such as in the periplasm where penicillin that targets PBPs has become ineffective with the appearance of β-lactamases or with glycopeptides that target Lipid II and has become less active. By contrast, Mur ligases have an activity further upstream in the biosynthesis. These enzymes have many advantages that make them an innovative therapeutic target: they are essential for bacterial survival, ubiquitous within prokaryotes with no equivalent in eukaryotes. To date, they are relevant targets in the development of multi-inhibitors and they do not present any resistance mechanism [ 91 ]. MurC-F ligases are responsible for the formation of the Lipid I precursor (UDP-MurNAc-pentapeptide, 38 ) from UDP-MurNAc ( 25 ) by the successive synthesis of tri-, tetra-, and penta-peptides by MurC, MurD, MurE, and MurF ( Scheme 6 ) [ 92 ]. The characterization of the protein sequences of MurC-F enzymes has been performed on different bacterial strains, both Gram-positive and Gram-negative [ 93 ]. Even if sequential differences between species can be observed, each of them shares the same structural topology, i.e., a protein divided into three different domains with a conserved active site [ 94 ]. The main differences in the protein sequences of the Mur ligases are that the amino acid binding site differs depending on which one is used. The MurC enzyme catalyzes the addition of the first amino acid of the peptide chain bound to the UDP-MurNAc ( 25 ). In most species, this amino acid is L-Ala, but in very rare cases, other amino acids such as glycine or L-Ser are used. MurD catalyzes the addition of the second amino acid to the substrate. Except for some variations in amino acids due to modifications in biosynthesis, the amino acid is a D-Glu in all species. Studies have shown the importance of having the D-enantiomer of glutamic acid because L-Glu is not a substrate for MurD. For MurE, which catalyzes the third amino acid addition, the amino acid is either a meso-diaminopimelic acid ( m-A2pm ) in most Gram-negative bacteria and in Bacilli species, while in Gram-positive bacteria, the amino acid is an L-Lys. Studies have shown that MurE contains a very specific active site for its own amino acid; if the wrong amino acid were to be incorporated, it would result in cell lysis. Finally, MurF catalyzes the addition of an unnatural dipeptide D-Ala-D-Ala. In some resistant bacterial species, a modification of this dipeptide by D-Ala-D-Ser or by D-Ala-D-Lac can be observed. To date, each of these enzymes has been purified and studied with the corresponding substrates and co-substrates [ 95 ]. This allowed the authors to propose the active sites of the Mur ligases as well as the reaction mechanism where we present these parameters. The stability parameters of Mur ligases are important to determine the specificity and kinetic parameters. A characterization study of these parameters was performed on MurC-F enzymes from Mycobacterium tuberculosis [ 96 ]. The specific activities of MurC-F are calculated by measuring the concentration of the released inorganic phosphate ( Scheme 6 B). They are estimated to be 1.2, 0.8, 1.3, and 0.9 μmol/min/mg/protein (μmol of phosphate formed per minute per milligram of enzyme) for MurC, MurD, MurE, and MurF, respectively. The stability of each of the proteins showed a great sensitivity according to the temperature and the pH of the medium. Indeed, the enzymatic activity remains stable under 40 °C with an optimal activity between 35 °C and 40 °C, while a higher temperature (between 45 and 70 °C) shows a significant decrease in the activity. Moreover, enzymes are sensitive to the pH of the buffered medium since the optimal pH is estimated between pH 8 and 8.5. Small variations with a more acidic or basic pH are enough to strongly decrease the activity. Mur ligases use the energy of the phosphate bond to catalyze the formation of the amino acid amide bond on the growing peptide chain ( Scheme 6 ). This mechanism, common to all four Mur ligases, starts with the elaboration of a complex with the enzyme and its substrates in the following way: first, the ATP binds to the enzyme, and then the substrate UDP sugar is added, as well as the amino acid at the end. In the active site, the first step is to activate the carboxylic acid of the substrate UDP sugar with ATP ( Scheme 6 A) to form the acyl phosphate intermediate. Two Mg ions 2+ will create a bridge between the negatively charged groups of ATP and the substrate UDP sugar to facilitate the phosphorylation of the carboxylate group of the substrate. Then, the second step involves the amino acid, which displaces the phosphate by a nucleophilic attack with the formation of the tetrahedral intermediate. Finally, the release of the phosphate P i allows for obtaining the peptide substrate. The catalytic activity of a base is necessary to recover the proton from the amine group. Other studies have shown that the ATP phosphate could replace this base or the enzyme itself since this would better fit the normal energy scale of the reaction [ 97 ]. With MurC-F, therefore, L-Ala, D-Glu, m-A2pm, and the dipeptide D-Ala-D-Ala can be introduced successively in the presence of ATP ( Scheme 6 B). In addition to the mechanistic data of these enzymes, X-ray structures of MurC-F ligases for several different species have been elucidated [ 98 , 99 , 100 , 101 ]. Inhibitors of MurC-F Escherichia coli were developed since they have the advantage of containing multiple active sites per enzyme, allowing researchers to target specific areas of interest to design molecules that may have biological activity benefits. Moreover, the increasing emergence of bacterial resistance challenges the conventional approach to developing antibacterials. Mur ligases are ideal targets for this purpose since they share similar three-dimensional structures, making them a set of targets for a single compound. 3.1. Inhibitors from "Medicinal Chemistry Approach" This follows a simple mono-therapeutic model aimed at designing a drug for a specific target in a particular region or event. This approach has proven effective in the development of numerous antibiotics. The identification of potential hits (molecules of interest) involves in silico, in vitro, and in vivo studies [ 102 ]. The first characterized inhibitors targeted domain 3 of Mur ligases and emerged in the 1990s, along with the initial elucidation of crystallographic structures. 3.1.1. Amino Acid Mimics of Mur Ligases The exploration of MurC inhibition using analogs of L-Alanine began early, even before the complete structure of the enzyme was known. Takahashi et al. showed that glycine could inhibit the addition of L-Alanine to the UDP-MurNAc substrate as a competitive inhibitor [ 103 ]. Several studies a few years later, including those by Parquet [ 104 ] et al. and VillaFranca et al. [ 105 ], demonstrated that various L-amino acids, without incorporation into the natural substrate, could inhibit MurC. D-configurations of amino acids, however, showed no inhibitory activity. For example, the three following amino acid analogs, 39 , 40 , and 41 ( Figure 9 ), were the most potent competitive inhibitors, ranging from millimolar to minimum activity for 76 . Inhibitors of MurD using analogs of D- or L-glutamic acid showed similar results as those for MurC. Van Heijenoort et al. demonstrated weakly active analogs, as seen with compounds 42 and 43 with residual activities of 58% and 47%, respectively, at a concentration of 10 mM ( Figure 10 ) [ 106 ]. This same team conducted similar studies on analogs of meso-diaminopimelic acid on the MurE enzyme [ 107 ]. Among synthetized products, compounds 44 and 45 were the best analogs in terms of MurE inhibition, although their activity was weak, with IC 50 values of 2.3 mM and 0.56 mM for 44 and 45 , respectively. For MurF, the literature does not show results containing derivatives with motifs from the D-Ala-D-Ala dipeptide. 3.1.2. Heterocyclic Inhibitors In 2001, Gobec et al. based their work on this mechanism with MurD and the tetrahedral transition state to design phosphino-alanine derivatives, but without significant inhibition [ 108 ]. In 2007, the same group published small compounds, such as sulfonamides of D-glutamic acid. They co-crystallized inhibitors like compound 46 ( Figure 11 ) with the MurD enzyme to better understand its binding site [ 109 ]. This allowed the group to develop other analogs with improved activity and the ability to crystallize the compounds, such as compound 47 with an IC 50 of 85 μM [ 110 ]. The modification of position 6 of the naphthalene in 46 allowed the crystallographic structure of the inhibitor/MurD complex to show that this position enables interactions between the substituents and the di-phosphate binding site of domain 1. Subsequently, Gobec et al., in collaboration with Mašič's group, performed ligand-docking studies, leading to the identification of a new interesting structure that could bind to the di-phosphate binding site. These structures 48 – 50 contained rhodanine-type heterocycles, such as compounds, which was active with an IC 50 of 45 μM (for 48 ). Subsequent SAR studies resulted in the discovery of analogs with improved biological activities, such as compounds 49 and 50 with IC 50 values in the micromolar range [ 111 , 112 , 113 ]. These recent studies enabled the synthesis of a large library of hybrid molecules capable of binding both to the active site of the amino acid in domain 3 and to the di-phosphate pocket in domain 1, using in silico analysis and considering that the D-glutamic acid part must remain. However, no molecule with significant in vivo bacterial activity has been identified. Additionally, some synthesized compounds, such as those containing the "ene-rhodanines" heterocycle, may be considered as PAINS (pan-assay interference compounds) that produce false positives in the biological experimentation used to determine IC 50 [ 114 ]. MurC presents a unique case where Lee et al. synthesized derivatives of benzylidene rhodanines [ 115 ]. Gobec et al. attempted to develop sulfonamide inhibitors of D-glutamic acid targeting MurE [ 116 ]. Subsequently, they synthesized a second generation of non-D-glutamic acid sulfonamide inhibitors that mimic it with rigid diacids. These inhibitors were found to be active on MurD, not MurE [ 117 ]. Compounds 51 and 52 ( Figure 12 ), exhibiting IC 50 values of 182 μM and 8.4 μM, respectively, were co-crystallized with the enzyme, and the structures showed that these inhibitors bind at the D-glutamic acid position, where the 2-cyano-4-fluoro-phenyl occupies the uracil pocket. 3.2. Inhibitors Mimicking the Natural Substrate UDP-MurNAc-(Peptide) 3.2.1. Phosphinic Inhibitors One of the first families of inhibitors is the "phosphinic" compounds that target the natural substrate-binding site in domain 1 of Mur ligases. The phosphinic acid in these molecules provides a tetrahedral geometry that blocks the enzymatic mechanism, and the uridine part of the compounds allows them to position themselves in the active site [ 118 , 119 ]. For MurC, AstraZeneca published the synthesis of two highly active inhibitors, 53 and 54 , with respective IC 50 values of 49 nM and 60 μM ( Figure 13 ) [ 120 ]. Compound 53 forms a stable enzyme/ATP/inhibitor and enzyme/ADP/inhibitor complex, which stops the enzymatic activity [ 121 ]. The first phosphinic inhibitor 55 was synthesized for MurD in 1996 by Blanot et al., which was active with an IC 50 in the micromolar range [ 122 ]. Similarly, Merck published other inhibitors in 1998 with similar structures. They showed that adding the glucosamine part to compound 56 improved its activity, reaching IC 50 values in the nanomolar range [ 123 ]. Later, Blanot et al., in collaboration with Gobec's team, published simplified inhibitors by retaining only the peptide-phosphinic part, based on compound 57 , with an IC 50 of 20 nM [ 124 ]. Even without improving this inhibitory activity, they still published active compounds like compound 58 , which was the most active with an IC 50 of 78 μM. For MurE, Merck's group synthesized compounds 59 and 60 with respective IC 50 values of 1.1 μM and 700 μM ( Figure 14 ), similar to compound 56 for MurC [ 125 ]. Similarly, Gobec et al. used 60 as a starting point to add various substituents, but without significant inhibition, with compound 61 showing the best residual activity at 71% at 1 mM [ 126 ]. Docking studies of an analog of 61 showed that this family of molecules is not transition-state inhibitors but rather competitive inhibitors of the substrate. Besides the good enzymatic activities of these phosphinic analogs, none have shown antibacterial activity, which could be explained by their low cell wall penetration. However, an important point to consider in the design of these derivatives is that the uridine part is beneficial for retaining good IC 50 values for each of the Mur ligases. Moreover, the total synthesis of phosphinic compounds proves to be challenging in terms of the number of steps. Several research groups have explored the laboratory synthesis of various natural substrates of Mur ligases, which has served as a synthetic tool, knowing that the nucleosidic synthesis of di-phosphate derivatives is a true challenge [ 127 , 128 , 129 , 130 , 131 ]. 3.2.2. Peptidic Inhibitors In 2003, Fishwick et al. published small peptides as analogs of the UDP sugar substrate targeting MurD [ 132 ]. This peptidic macrocycle 62 was designed based on the enzyme's crystallographic structure and the docking of this potential inhibitor in the active site. Several analogs were synthesized, with compound 63 showing the best activity with an IC 50 of 0.7 μM (5.1 μM for 60 ) ( Figure 15 ). There are also a few examples where authors developed peptidic derivatives targeting MurE [ 133 , 134 ]. On the other hand, several groups of biologists have been interested in peptide synthesis using the phage display technique with different peptide libraries. Several studies have identified peptides targeting MurC [ 135 ], MurD [ 136 ], MurE [ 137 , 138 ], and MurF [ 139 ], inhibiting with weak IC 50 values in the mM range. 3.2.3. Heterocyclic Inhibitors A very rapid technique to obtain inhibitors is to screen molecules through biological assays. AstraZeneca did this by screening its compound library targeting MurC [ 140 ]. Compound 64 was identified as an inhibitor with an IC 50 of 2.3 μM ( Figure 16 ). Its action seems to target the di-phosphate binding site, but the mode of inhibition has not been clearly elucidated. Moreover, it shows binding affinities to other proteins, such as bovine serum albumin. For MurD, Obreza's team synthesized sulfono-hydrazine derivatives that also mimic the UDP sugar substrate's di-phosphates [ 141 ]. Compound 65 showed the best IC 50 of 30 μM but did not exhibit antibacterial activity. Additionally, the same team developed other similar analogs, like compound 66 , but without improving its activity [ 142 ]. For the same enzyme, Gobec's team performed virtual screening of a molecule library, which led to the identification of inhibitors [ 143 ]. A research group from Abbott Laboratories conducted a high-throughput affinity screening of a molecule library targeting MurF, which highlighted molecule 67 as an attractive candidate [ 144 , 145 ]. Enzymatic studies revealed an IC 50 of 8 μM, and through SAR studies, this result was optimized with compound 68 , which had an IC 50 of 22 nM. Compound 67 was co-crystallized with MurF and bound to the active site in place of the natural UDP sugar substrate in a conformation called "closed" of the enzyme [ 146 , 147 ]. However, these compounds did not exhibit antibacterial activity, even in the presence of permeable strains, which may indicate non-specific interactions with other proteins. Based on these results, Gobec's team decided to take up the structural motif of compound 68 to improve its antibacterial activities [ 148 ]. This led to compound 69 , which was active against Staphylococcus pneumoniae strains with an MIC of 16 μg/mL. However, 69 and its analogs did not exhibit activity against other bacterial strains. The authors published a second generation of these compounds active against several strains [ 149 ], where their antibacterial action resulted from membrane degradation. Other heterocyclic analogs were synthesized by Gobec's laboratory with a few antibacterial activities [ 150 , 151 ]. In 2006, a research group from Johnson & Johnson developed pyrimidine derivatives that inhibited MurF but showed no in vivo activity [ 152 ]. Only from 2007 did the same group identify, through molecule screening, hydroxyquinoline-type derivatives of interest, such as compound 70 with an IC 50 of 29 μM. Although this compound exhibited activity against both Gram-positive and Gram-negative bacterial strains, it is possible that compound 70 interacted with other proteins other than MurF [ 153 , 154 ]. 3.3. Inhibitors Mimicking the Co-Substrate ATP There are very few inhibitors of this type in the literature targeting Mur ligases. This can be explained by the fact that there are many ATP-dependent enzymes, including kinases, resulting in a very severe lack of selectivity. Therefore, the few synthesized inhibitors were obtained through screening techniques of molecule libraries. In 2008, Dougherty's team from the pharmaceutical group Pfizer carried out a screening of its molecule library and managed to demonstrate that compound 71 is a competitive inhibitor of ATP, selectively targeting MurC from only a few enterobacterial strains closely related to Escherichia coli ( Figure 17 ). However, this compound did not show antibacterial activity [ 155 ]. 3.4. Natural Inhibitors Using the same molecule screening technique (in this case, from plants), a research group identified natural compounds like 72 and 73 that were found to be active against MurE ( Figure 18 ) [ 156 , 157 , 158 ]. Compound 72 showed an IC 50 of 67 μM against MurE from M. tuberculosis and weakly inhibited the growth of a mutant strain of Mycobacterium tuberculosis . As for compound 73 , it had an IC 50 of 75 μM against MurE and was active against a panel of Gram-positive and Gram-negative strains. It also acted on efflux by inhibiting a protein responsible for NorA. 3.5. Multi-Target Synthetic Approaches The synthesis of multi-inhibitor compounds involves a "multi-therapeutic" model, where a molecule is produced to inhibit multiple biological processes by targeting several enzymes simultaneously. Generally, the choice is made on similar enzymes with close activities, as demonstrated with the Mur ligases. The reason behind this concept is to combat bacterial resistance to the purported new antibacterial compound built on these criteria. The advantage of multi-inhibition is that it leaves no chance for bacteria to react or adapt when exposed to this toxic agent. In the literature, there are a few relatively recent examples of multi-targeted Mur ligases that we present. This principle poses a challenge in developing such compounds while retaining attractive biological properties. 3.5.1. Mimicking the Amino Acid of Mur Ligases Solmajer et al. and Gobec et al. developed new structures derived from D-glutamic acid using several in silico approaches, as they had done for classical inhibitors. Among these previous studies, they also identified multi-active compounds, and SAR studies were carried out based on these results [ 159 ]. The 1,3-phenyl-dicarboxylic acid, which is the cyclic mimic of D-glutamic acid, seems to play an important role in the binding to each of the Mur ligases. Initially, the team developed analogs of this type targeting MurD and MurE, as shown by compound 74 with an IC 50 of 270 μM on MurD and an IC 50 of 32 μM on MurE [ 160 ]. Subsequently, the group developed other analogs by modifying the heterocyclic part that fits into the di-phosphate pocket while retaining the 1,3-phenyl-dicarboxylic acid [ 161 ]. They synthesized compound 75 , which is the best in the series and capable of inhibiting all four Mur ligases with IC 50 values of 41 μM, 60 μM, 93 μM, and 89 μM for MurC, MurD, MurE, and MurF, respectively ( Figure 19 ). Although these molecules are multi-active, with compound 75 being one of the best multi-inhibitors synthesized to date, there is no revealed antibacterial activity. The group attempted to measure activities on different strains with analogs close to 75 (without the di-carboxylic acid groups), but the results only showed weak activities. 3.5.2. Mimicking the Natural Substrate UDP-MurNAc(-Peptide) Phosphonic Acid Inhibitors Gobec et al. developed a series of phosphoric acid analogs of hydroxyethylamines as a bioisostere of the tetrahedral intermediate of the UDP sugar substrate [ 162 ]. Compound 76 is the best analog in this series, active against all four enzymes with IC 50 values of 26 μM, 530 μM, 160 μM, and 150 μM for MurC, MurD, MurE, and MurF, respectively ( Figure 20 ). However, these compounds do not show antibacterial activities. These analogs, including 76 , are the only phosphorylated derivatives that are multi-active against Mur ligases. Heterocyclic Inhibitors After observing that the N -acyl hydrazone structure has potential in terms of biological activity in medicinal chemistry, Gobec's group designed analogs around this structure and showed that some are active, like compound 77 with IC 50 values of 123 μM and 230 μM for MurC and MurD, respectively ( Figure 21 ) [ 163 ]. Moreover, this compound showed weak antibacterial activities against Escherichia coli and Staphylococcus aureus . The group also designed other heterocyclic structures to adopt a more closed conformation than with the natural substrates of enzymes MurD and MurF but still show activities on different strains. Gobec et al., who synthesized a good number of benzylidenesulfonyl hydrazine analogs, showed that some of them were multi-inhibitors [ 164 ]. They performed SAR studies to optimize enzymatic activities but without antibacterial activities. Singh's laboratory identified several multi-active structures of Mur ligases through different screenings, including pulvinones [ 165 ] and phenyl dihydrothienopyrazolol [ 166 ] with activities against multiple strains. Recently, they identified a naphthyl-type tetronic acid structure capable of inhibiting not only all four Mur ligases but also MurA and MurB [ 167 ]. Compound 78 showed the best activities in the series with an IC 50 in the range of 20 μM for each enzyme, and it inhibited the bacterial growth of Escherichia coli and Staphylococcus aureus strains ( Figure 22 ). 3.5.3. Mimicking the Co-Substrate ATP Very recently, Zega et al. identified several compounds with different structures that exhibit multi-inhibitory activities against MurC to MurF. These compounds were identified through screening a collection of molecules originally designed to inhibit kinases by the competitive inhibition of ATP [ 168 ]. Compounds 79 and 80 are multi-inhibitors of MurC-F with IC 50 values ranging from 10 to 368 μM ( Figure 23 ). Because these compounds mimic ATP, the authors conducted kinetic and NMR studies on compound 79 and showed that it actually binds to the amino acid binding site. They also performed additional biological analyses with eukaryotic kinases and demonstrated that these molecules are specific and promising for the development of new antibacterials. 3.5.4. Natural Analogs Süssmuth et al. identified feglymycin ( 81 ), isolated from Streptomyces strains, as an active inhibitor of both MurA and MurC [ 169 ]. Feglymycin, a 13-mer peptide, exhibited activity with an IC 50 in the range of μM for MurA and in the range of mM for MurC ( Figure 24 ). 3.1. Inhibitors from "Medicinal Chemistry Approach" This follows a simple mono-therapeutic model aimed at designing a drug for a specific target in a particular region or event. This approach has proven effective in the development of numerous antibiotics. The identification of potential hits (molecules of interest) involves in silico, in vitro, and in vivo studies [ 102 ]. The first characterized inhibitors targeted domain 3 of Mur ligases and emerged in the 1990s, along with the initial elucidation of crystallographic structures. 3.1.1. Amino Acid Mimics of Mur Ligases The exploration of MurC inhibition using analogs of L-Alanine began early, even before the complete structure of the enzyme was known. Takahashi et al. showed that glycine could inhibit the addition of L-Alanine to the UDP-MurNAc substrate as a competitive inhibitor [ 103 ]. Several studies a few years later, including those by Parquet [ 104 ] et al. and VillaFranca et al. [ 105 ], demonstrated that various L-amino acids, without incorporation into the natural substrate, could inhibit MurC. D-configurations of amino acids, however, showed no inhibitory activity. For example, the three following amino acid analogs, 39 , 40 , and 41 ( Figure 9 ), were the most potent competitive inhibitors, ranging from millimolar to minimum activity for 76 . Inhibitors of MurD using analogs of D- or L-glutamic acid showed similar results as those for MurC. Van Heijenoort et al. demonstrated weakly active analogs, as seen with compounds 42 and 43 with residual activities of 58% and 47%, respectively, at a concentration of 10 mM ( Figure 10 ) [ 106 ]. This same team conducted similar studies on analogs of meso-diaminopimelic acid on the MurE enzyme [ 107 ]. Among synthetized products, compounds 44 and 45 were the best analogs in terms of MurE inhibition, although their activity was weak, with IC 50 values of 2.3 mM and 0.56 mM for 44 and 45 , respectively. For MurF, the literature does not show results containing derivatives with motifs from the D-Ala-D-Ala dipeptide. 3.1.2. Heterocyclic Inhibitors In 2001, Gobec et al. based their work on this mechanism with MurD and the tetrahedral transition state to design phosphino-alanine derivatives, but without significant inhibition [ 108 ]. In 2007, the same group published small compounds, such as sulfonamides of D-glutamic acid. They co-crystallized inhibitors like compound 46 ( Figure 11 ) with the MurD enzyme to better understand its binding site [ 109 ]. This allowed the group to develop other analogs with improved activity and the ability to crystallize the compounds, such as compound 47 with an IC 50 of 85 μM [ 110 ]. The modification of position 6 of the naphthalene in 46 allowed the crystallographic structure of the inhibitor/MurD complex to show that this position enables interactions between the substituents and the di-phosphate binding site of domain 1. Subsequently, Gobec et al., in collaboration with Mašič's group, performed ligand-docking studies, leading to the identification of a new interesting structure that could bind to the di-phosphate binding site. These structures 48 – 50 contained rhodanine-type heterocycles, such as compounds, which was active with an IC 50 of 45 μM (for 48 ). Subsequent SAR studies resulted in the discovery of analogs with improved biological activities, such as compounds 49 and 50 with IC 50 values in the micromolar range [ 111 , 112 , 113 ]. These recent studies enabled the synthesis of a large library of hybrid molecules capable of binding both to the active site of the amino acid in domain 3 and to the di-phosphate pocket in domain 1, using in silico analysis and considering that the D-glutamic acid part must remain. However, no molecule with significant in vivo bacterial activity has been identified. Additionally, some synthesized compounds, such as those containing the "ene-rhodanines" heterocycle, may be considered as PAINS (pan-assay interference compounds) that produce false positives in the biological experimentation used to determine IC 50 [ 114 ]. MurC presents a unique case where Lee et al. synthesized derivatives of benzylidene rhodanines [ 115 ]. Gobec et al. attempted to develop sulfonamide inhibitors of D-glutamic acid targeting MurE [ 116 ]. Subsequently, they synthesized a second generation of non-D-glutamic acid sulfonamide inhibitors that mimic it with rigid diacids. These inhibitors were found to be active on MurD, not MurE [ 117 ]. Compounds 51 and 52 ( Figure 12 ), exhibiting IC 50 values of 182 μM and 8.4 μM, respectively, were co-crystallized with the enzyme, and the structures showed that these inhibitors bind at the D-glutamic acid position, where the 2-cyano-4-fluoro-phenyl occupies the uracil pocket. 3.1.1. Amino Acid Mimics of Mur Ligases The exploration of MurC inhibition using analogs of L-Alanine began early, even before the complete structure of the enzyme was known. Takahashi et al. showed that glycine could inhibit the addition of L-Alanine to the UDP-MurNAc substrate as a competitive inhibitor [ 103 ]. Several studies a few years later, including those by Parquet [ 104 ] et al. and VillaFranca et al. [ 105 ], demonstrated that various L-amino acids, without incorporation into the natural substrate, could inhibit MurC. D-configurations of amino acids, however, showed no inhibitory activity. For example, the three following amino acid analogs, 39 , 40 , and 41 ( Figure 9 ), were the most potent competitive inhibitors, ranging from millimolar to minimum activity for 76 . Inhibitors of MurD using analogs of D- or L-glutamic acid showed similar results as those for MurC. Van Heijenoort et al. demonstrated weakly active analogs, as seen with compounds 42 and 43 with residual activities of 58% and 47%, respectively, at a concentration of 10 mM ( Figure 10 ) [ 106 ]. This same team conducted similar studies on analogs of meso-diaminopimelic acid on the MurE enzyme [ 107 ]. Among synthetized products, compounds 44 and 45 were the best analogs in terms of MurE inhibition, although their activity was weak, with IC 50 values of 2.3 mM and 0.56 mM for 44 and 45 , respectively. For MurF, the literature does not show results containing derivatives with motifs from the D-Ala-D-Ala dipeptide. 3.1.2. Heterocyclic Inhibitors In 2001, Gobec et al. based their work on this mechanism with MurD and the tetrahedral transition state to design phosphino-alanine derivatives, but without significant inhibition [ 108 ]. In 2007, the same group published small compounds, such as sulfonamides of D-glutamic acid. They co-crystallized inhibitors like compound 46 ( Figure 11 ) with the MurD enzyme to better understand its binding site [ 109 ]. This allowed the group to develop other analogs with improved activity and the ability to crystallize the compounds, such as compound 47 with an IC 50 of 85 μM [ 110 ]. The modification of position 6 of the naphthalene in 46 allowed the crystallographic structure of the inhibitor/MurD complex to show that this position enables interactions between the substituents and the di-phosphate binding site of domain 1. Subsequently, Gobec et al., in collaboration with Mašič's group, performed ligand-docking studies, leading to the identification of a new interesting structure that could bind to the di-phosphate binding site. These structures 48 – 50 contained rhodanine-type heterocycles, such as compounds, which was active with an IC 50 of 45 μM (for 48 ). Subsequent SAR studies resulted in the discovery of analogs with improved biological activities, such as compounds 49 and 50 with IC 50 values in the micromolar range [ 111 , 112 , 113 ]. These recent studies enabled the synthesis of a large library of hybrid molecules capable of binding both to the active site of the amino acid in domain 3 and to the di-phosphate pocket in domain 1, using in silico analysis and considering that the D-glutamic acid part must remain. However, no molecule with significant in vivo bacterial activity has been identified. Additionally, some synthesized compounds, such as those containing the "ene-rhodanines" heterocycle, may be considered as PAINS (pan-assay interference compounds) that produce false positives in the biological experimentation used to determine IC 50 [ 114 ]. MurC presents a unique case where Lee et al. synthesized derivatives of benzylidene rhodanines [ 115 ]. Gobec et al. attempted to develop sulfonamide inhibitors of D-glutamic acid targeting MurE [ 116 ]. Subsequently, they synthesized a second generation of non-D-glutamic acid sulfonamide inhibitors that mimic it with rigid diacids. These inhibitors were found to be active on MurD, not MurE [ 117 ]. Compounds 51 and 52 ( Figure 12 ), exhibiting IC 50 values of 182 μM and 8.4 μM, respectively, were co-crystallized with the enzyme, and the structures showed that these inhibitors bind at the D-glutamic acid position, where the 2-cyano-4-fluoro-phenyl occupies the uracil pocket. 3.2. Inhibitors Mimicking the Natural Substrate UDP-MurNAc-(Peptide) 3.2.1. Phosphinic Inhibitors One of the first families of inhibitors is the "phosphinic" compounds that target the natural substrate-binding site in domain 1 of Mur ligases. The phosphinic acid in these molecules provides a tetrahedral geometry that blocks the enzymatic mechanism, and the uridine part of the compounds allows them to position themselves in the active site [ 118 , 119 ]. For MurC, AstraZeneca published the synthesis of two highly active inhibitors, 53 and 54 , with respective IC 50 values of 49 nM and 60 μM ( Figure 13 ) [ 120 ]. Compound 53 forms a stable enzyme/ATP/inhibitor and enzyme/ADP/inhibitor complex, which stops the enzymatic activity [ 121 ]. The first phosphinic inhibitor 55 was synthesized for MurD in 1996 by Blanot et al., which was active with an IC 50 in the micromolar range [ 122 ]. Similarly, Merck published other inhibitors in 1998 with similar structures. They showed that adding the glucosamine part to compound 56 improved its activity, reaching IC 50 values in the nanomolar range [ 123 ]. Later, Blanot et al., in collaboration with Gobec's team, published simplified inhibitors by retaining only the peptide-phosphinic part, based on compound 57 , with an IC 50 of 20 nM [ 124 ]. Even without improving this inhibitory activity, they still published active compounds like compound 58 , which was the most active with an IC 50 of 78 μM. For MurE, Merck's group synthesized compounds 59 and 60 with respective IC 50 values of 1.1 μM and 700 μM ( Figure 14 ), similar to compound 56 for MurC [ 125 ]. Similarly, Gobec et al. used 60 as a starting point to add various substituents, but without significant inhibition, with compound 61 showing the best residual activity at 71% at 1 mM [ 126 ]. Docking studies of an analog of 61 showed that this family of molecules is not transition-state inhibitors but rather competitive inhibitors of the substrate. Besides the good enzymatic activities of these phosphinic analogs, none have shown antibacterial activity, which could be explained by their low cell wall penetration. However, an important point to consider in the design of these derivatives is that the uridine part is beneficial for retaining good IC 50 values for each of the Mur ligases. Moreover, the total synthesis of phosphinic compounds proves to be challenging in terms of the number of steps. Several research groups have explored the laboratory synthesis of various natural substrates of Mur ligases, which has served as a synthetic tool, knowing that the nucleosidic synthesis of di-phosphate derivatives is a true challenge [ 127 , 128 , 129 , 130 , 131 ]. 3.2.2. Peptidic Inhibitors In 2003, Fishwick et al. published small peptides as analogs of the UDP sugar substrate targeting MurD [ 132 ]. This peptidic macrocycle 62 was designed based on the enzyme's crystallographic structure and the docking of this potential inhibitor in the active site. Several analogs were synthesized, with compound 63 showing the best activity with an IC 50 of 0.7 μM (5.1 μM for 60 ) ( Figure 15 ). There are also a few examples where authors developed peptidic derivatives targeting MurE [ 133 , 134 ]. On the other hand, several groups of biologists have been interested in peptide synthesis using the phage display technique with different peptide libraries. Several studies have identified peptides targeting MurC [ 135 ], MurD [ 136 ], MurE [ 137 , 138 ], and MurF [ 139 ], inhibiting with weak IC 50 values in the mM range. 3.2.3. Heterocyclic Inhibitors A very rapid technique to obtain inhibitors is to screen molecules through biological assays. AstraZeneca did this by screening its compound library targeting MurC [ 140 ]. Compound 64 was identified as an inhibitor with an IC 50 of 2.3 μM ( Figure 16 ). Its action seems to target the di-phosphate binding site, but the mode of inhibition has not been clearly elucidated. Moreover, it shows binding affinities to other proteins, such as bovine serum albumin. For MurD, Obreza's team synthesized sulfono-hydrazine derivatives that also mimic the UDP sugar substrate's di-phosphates [ 141 ]. Compound 65 showed the best IC 50 of 30 μM but did not exhibit antibacterial activity. Additionally, the same team developed other similar analogs, like compound 66 , but without improving its activity [ 142 ]. For the same enzyme, Gobec's team performed virtual screening of a molecule library, which led to the identification of inhibitors [ 143 ]. A research group from Abbott Laboratories conducted a high-throughput affinity screening of a molecule library targeting MurF, which highlighted molecule 67 as an attractive candidate [ 144 , 145 ]. Enzymatic studies revealed an IC 50 of 8 μM, and through SAR studies, this result was optimized with compound 68 , which had an IC 50 of 22 nM. Compound 67 was co-crystallized with MurF and bound to the active site in place of the natural UDP sugar substrate in a conformation called "closed" of the enzyme [ 146 , 147 ]. However, these compounds did not exhibit antibacterial activity, even in the presence of permeable strains, which may indicate non-specific interactions with other proteins. Based on these results, Gobec's team decided to take up the structural motif of compound 68 to improve its antibacterial activities [ 148 ]. This led to compound 69 , which was active against Staphylococcus pneumoniae strains with an MIC of 16 μg/mL. However, 69 and its analogs did not exhibit activity against other bacterial strains. The authors published a second generation of these compounds active against several strains [ 149 ], where their antibacterial action resulted from membrane degradation. Other heterocyclic analogs were synthesized by Gobec's laboratory with a few antibacterial activities [ 150 , 151 ]. In 2006, a research group from Johnson & Johnson developed pyrimidine derivatives that inhibited MurF but showed no in vivo activity [ 152 ]. Only from 2007 did the same group identify, through molecule screening, hydroxyquinoline-type derivatives of interest, such as compound 70 with an IC 50 of 29 μM. Although this compound exhibited activity against both Gram-positive and Gram-negative bacterial strains, it is possible that compound 70 interacted with other proteins other than MurF [ 153 , 154 ]. 3.2.1. Phosphinic Inhibitors One of the first families of inhibitors is the "phosphinic" compounds that target the natural substrate-binding site in domain 1 of Mur ligases. The phosphinic acid in these molecules provides a tetrahedral geometry that blocks the enzymatic mechanism, and the uridine part of the compounds allows them to position themselves in the active site [ 118 , 119 ]. For MurC, AstraZeneca published the synthesis of two highly active inhibitors, 53 and 54 , with respective IC 50 values of 49 nM and 60 μM ( Figure 13 ) [ 120 ]. Compound 53 forms a stable enzyme/ATP/inhibitor and enzyme/ADP/inhibitor complex, which stops the enzymatic activity [ 121 ]. The first phosphinic inhibitor 55 was synthesized for MurD in 1996 by Blanot et al., which was active with an IC 50 in the micromolar range [ 122 ]. Similarly, Merck published other inhibitors in 1998 with similar structures. They showed that adding the glucosamine part to compound 56 improved its activity, reaching IC 50 values in the nanomolar range [ 123 ]. Later, Blanot et al., in collaboration with Gobec's team, published simplified inhibitors by retaining only the peptide-phosphinic part, based on compound 57 , with an IC 50 of 20 nM [ 124 ]. Even without improving this inhibitory activity, they still published active compounds like compound 58 , which was the most active with an IC 50 of 78 μM. For MurE, Merck's group synthesized compounds 59 and 60 with respective IC 50 values of 1.1 μM and 700 μM ( Figure 14 ), similar to compound 56 for MurC [ 125 ]. Similarly, Gobec et al. used 60 as a starting point to add various substituents, but without significant inhibition, with compound 61 showing the best residual activity at 71% at 1 mM [ 126 ]. Docking studies of an analog of 61 showed that this family of molecules is not transition-state inhibitors but rather competitive inhibitors of the substrate. Besides the good enzymatic activities of these phosphinic analogs, none have shown antibacterial activity, which could be explained by their low cell wall penetration. However, an important point to consider in the design of these derivatives is that the uridine part is beneficial for retaining good IC 50 values for each of the Mur ligases. Moreover, the total synthesis of phosphinic compounds proves to be challenging in terms of the number of steps. Several research groups have explored the laboratory synthesis of various natural substrates of Mur ligases, which has served as a synthetic tool, knowing that the nucleosidic synthesis of di-phosphate derivatives is a true challenge [ 127 , 128 , 129 , 130 , 131 ]. 3.2.2. Peptidic Inhibitors In 2003, Fishwick et al. published small peptides as analogs of the UDP sugar substrate targeting MurD [ 132 ]. This peptidic macrocycle 62 was designed based on the enzyme's crystallographic structure and the docking of this potential inhibitor in the active site. Several analogs were synthesized, with compound 63 showing the best activity with an IC 50 of 0.7 μM (5.1 μM for 60 ) ( Figure 15 ). There are also a few examples where authors developed peptidic derivatives targeting MurE [ 133 , 134 ]. On the other hand, several groups of biologists have been interested in peptide synthesis using the phage display technique with different peptide libraries. Several studies have identified peptides targeting MurC [ 135 ], MurD [ 136 ], MurE [ 137 , 138 ], and MurF [ 139 ], inhibiting with weak IC 50 values in the mM range. 3.2.3. Heterocyclic Inhibitors A very rapid technique to obtain inhibitors is to screen molecules through biological assays. AstraZeneca did this by screening its compound library targeting MurC [ 140 ]. Compound 64 was identified as an inhibitor with an IC 50 of 2.3 μM ( Figure 16 ). Its action seems to target the di-phosphate binding site, but the mode of inhibition has not been clearly elucidated. Moreover, it shows binding affinities to other proteins, such as bovine serum albumin. For MurD, Obreza's team synthesized sulfono-hydrazine derivatives that also mimic the UDP sugar substrate's di-phosphates [ 141 ]. Compound 65 showed the best IC 50 of 30 μM but did not exhibit antibacterial activity. Additionally, the same team developed other similar analogs, like compound 66 , but without improving its activity [ 142 ]. For the same enzyme, Gobec's team performed virtual screening of a molecule library, which led to the identification of inhibitors [ 143 ]. A research group from Abbott Laboratories conducted a high-throughput affinity screening of a molecule library targeting MurF, which highlighted molecule 67 as an attractive candidate [ 144 , 145 ]. Enzymatic studies revealed an IC 50 of 8 μM, and through SAR studies, this result was optimized with compound 68 , which had an IC 50 of 22 nM. Compound 67 was co-crystallized with MurF and bound to the active site in place of the natural UDP sugar substrate in a conformation called "closed" of the enzyme [ 146 , 147 ]. However, these compounds did not exhibit antibacterial activity, even in the presence of permeable strains, which may indicate non-specific interactions with other proteins. Based on these results, Gobec's team decided to take up the structural motif of compound 68 to improve its antibacterial activities [ 148 ]. This led to compound 69 , which was active against Staphylococcus pneumoniae strains with an MIC of 16 μg/mL. However, 69 and its analogs did not exhibit activity against other bacterial strains. The authors published a second generation of these compounds active against several strains [ 149 ], where their antibacterial action resulted from membrane degradation. Other heterocyclic analogs were synthesized by Gobec's laboratory with a few antibacterial activities [ 150 , 151 ]. In 2006, a research group from Johnson & Johnson developed pyrimidine derivatives that inhibited MurF but showed no in vivo activity [ 152 ]. Only from 2007 did the same group identify, through molecule screening, hydroxyquinoline-type derivatives of interest, such as compound 70 with an IC 50 of 29 μM. Although this compound exhibited activity against both Gram-positive and Gram-negative bacterial strains, it is possible that compound 70 interacted with other proteins other than MurF [ 153 , 154 ]. 3.3. Inhibitors Mimicking the Co-Substrate ATP There are very few inhibitors of this type in the literature targeting Mur ligases. This can be explained by the fact that there are many ATP-dependent enzymes, including kinases, resulting in a very severe lack of selectivity. Therefore, the few synthesized inhibitors were obtained through screening techniques of molecule libraries. In 2008, Dougherty's team from the pharmaceutical group Pfizer carried out a screening of its molecule library and managed to demonstrate that compound 71 is a competitive inhibitor of ATP, selectively targeting MurC from only a few enterobacterial strains closely related to Escherichia coli ( Figure 17 ). However, this compound did not show antibacterial activity [ 155 ]. 3.4. Natural Inhibitors Using the same molecule screening technique (in this case, from plants), a research group identified natural compounds like 72 and 73 that were found to be active against MurE ( Figure 18 ) [ 156 , 157 , 158 ]. Compound 72 showed an IC 50 of 67 μM against MurE from M. tuberculosis and weakly inhibited the growth of a mutant strain of Mycobacterium tuberculosis . As for compound 73 , it had an IC 50 of 75 μM against MurE and was active against a panel of Gram-positive and Gram-negative strains. It also acted on efflux by inhibiting a protein responsible for NorA. 3.5. Multi-Target Synthetic Approaches The synthesis of multi-inhibitor compounds involves a "multi-therapeutic" model, where a molecule is produced to inhibit multiple biological processes by targeting several enzymes simultaneously. Generally, the choice is made on similar enzymes with close activities, as demonstrated with the Mur ligases. The reason behind this concept is to combat bacterial resistance to the purported new antibacterial compound built on these criteria. The advantage of multi-inhibition is that it leaves no chance for bacteria to react or adapt when exposed to this toxic agent. In the literature, there are a few relatively recent examples of multi-targeted Mur ligases that we present. This principle poses a challenge in developing such compounds while retaining attractive biological properties. 3.5.1. Mimicking the Amino Acid of Mur Ligases Solmajer et al. and Gobec et al. developed new structures derived from D-glutamic acid using several in silico approaches, as they had done for classical inhibitors. Among these previous studies, they also identified multi-active compounds, and SAR studies were carried out based on these results [ 159 ]. The 1,3-phenyl-dicarboxylic acid, which is the cyclic mimic of D-glutamic acid, seems to play an important role in the binding to each of the Mur ligases. Initially, the team developed analogs of this type targeting MurD and MurE, as shown by compound 74 with an IC 50 of 270 μM on MurD and an IC 50 of 32 μM on MurE [ 160 ]. Subsequently, the group developed other analogs by modifying the heterocyclic part that fits into the di-phosphate pocket while retaining the 1,3-phenyl-dicarboxylic acid [ 161 ]. They synthesized compound 75 , which is the best in the series and capable of inhibiting all four Mur ligases with IC 50 values of 41 μM, 60 μM, 93 μM, and 89 μM for MurC, MurD, MurE, and MurF, respectively ( Figure 19 ). Although these molecules are multi-active, with compound 75 being one of the best multi-inhibitors synthesized to date, there is no revealed antibacterial activity. The group attempted to measure activities on different strains with analogs close to 75 (without the di-carboxylic acid groups), but the results only showed weak activities. 3.5.2. Mimicking the Natural Substrate UDP-MurNAc(-Peptide) Phosphonic Acid Inhibitors Gobec et al. developed a series of phosphoric acid analogs of hydroxyethylamines as a bioisostere of the tetrahedral intermediate of the UDP sugar substrate [ 162 ]. Compound 76 is the best analog in this series, active against all four enzymes with IC 50 values of 26 μM, 530 μM, 160 μM, and 150 μM for MurC, MurD, MurE, and MurF, respectively ( Figure 20 ). However, these compounds do not show antibacterial activities. These analogs, including 76 , are the only phosphorylated derivatives that are multi-active against Mur ligases. Heterocyclic Inhibitors After observing that the N -acyl hydrazone structure has potential in terms of biological activity in medicinal chemistry, Gobec's group designed analogs around this structure and showed that some are active, like compound 77 with IC 50 values of 123 μM and 230 μM for MurC and MurD, respectively ( Figure 21 ) [ 163 ]. Moreover, this compound showed weak antibacterial activities against Escherichia coli and Staphylococcus aureus . The group also designed other heterocyclic structures to adopt a more closed conformation than with the natural substrates of enzymes MurD and MurF but still show activities on different strains. Gobec et al., who synthesized a good number of benzylidenesulfonyl hydrazine analogs, showed that some of them were multi-inhibitors [ 164 ]. They performed SAR studies to optimize enzymatic activities but without antibacterial activities. Singh's laboratory identified several multi-active structures of Mur ligases through different screenings, including pulvinones [ 165 ] and phenyl dihydrothienopyrazolol [ 166 ] with activities against multiple strains. Recently, they identified a naphthyl-type tetronic acid structure capable of inhibiting not only all four Mur ligases but also MurA and MurB [ 167 ]. Compound 78 showed the best activities in the series with an IC 50 in the range of 20 μM for each enzyme, and it inhibited the bacterial growth of Escherichia coli and Staphylococcus aureus strains ( Figure 22 ). 3.5.3. Mimicking the Co-Substrate ATP Very recently, Zega et al. identified several compounds with different structures that exhibit multi-inhibitory activities against MurC to MurF. These compounds were identified through screening a collection of molecules originally designed to inhibit kinases by the competitive inhibition of ATP [ 168 ]. Compounds 79 and 80 are multi-inhibitors of MurC-F with IC 50 values ranging from 10 to 368 μM ( Figure 23 ). Because these compounds mimic ATP, the authors conducted kinetic and NMR studies on compound 79 and showed that it actually binds to the amino acid binding site. They also performed additional biological analyses with eukaryotic kinases and demonstrated that these molecules are specific and promising for the development of new antibacterials. 3.5.4. Natural Analogs Süssmuth et al. identified feglymycin ( 81 ), isolated from Streptomyces strains, as an active inhibitor of both MurA and MurC [ 169 ]. Feglymycin, a 13-mer peptide, exhibited activity with an IC 50 in the range of μM for MurA and in the range of mM for MurC ( Figure 24 ). 3.5.1. Mimicking the Amino Acid of Mur Ligases Solmajer et al. and Gobec et al. developed new structures derived from D-glutamic acid using several in silico approaches, as they had done for classical inhibitors. Among these previous studies, they also identified multi-active compounds, and SAR studies were carried out based on these results [ 159 ]. The 1,3-phenyl-dicarboxylic acid, which is the cyclic mimic of D-glutamic acid, seems to play an important role in the binding to each of the Mur ligases. Initially, the team developed analogs of this type targeting MurD and MurE, as shown by compound 74 with an IC 50 of 270 μM on MurD and an IC 50 of 32 μM on MurE [ 160 ]. Subsequently, the group developed other analogs by modifying the heterocyclic part that fits into the di-phosphate pocket while retaining the 1,3-phenyl-dicarboxylic acid [ 161 ]. They synthesized compound 75 , which is the best in the series and capable of inhibiting all four Mur ligases with IC 50 values of 41 μM, 60 μM, 93 μM, and 89 μM for MurC, MurD, MurE, and MurF, respectively ( Figure 19 ). Although these molecules are multi-active, with compound 75 being one of the best multi-inhibitors synthesized to date, there is no revealed antibacterial activity. The group attempted to measure activities on different strains with analogs close to 75 (without the di-carboxylic acid groups), but the results only showed weak activities. 3.5.2. Mimicking the Natural Substrate UDP-MurNAc(-Peptide) Phosphonic Acid Inhibitors Gobec et al. developed a series of phosphoric acid analogs of hydroxyethylamines as a bioisostere of the tetrahedral intermediate of the UDP sugar substrate [ 162 ]. Compound 76 is the best analog in this series, active against all four enzymes with IC 50 values of 26 μM, 530 μM, 160 μM, and 150 μM for MurC, MurD, MurE, and MurF, respectively ( Figure 20 ). However, these compounds do not show antibacterial activities. These analogs, including 76 , are the only phosphorylated derivatives that are multi-active against Mur ligases. Heterocyclic Inhibitors After observing that the N -acyl hydrazone structure has potential in terms of biological activity in medicinal chemistry, Gobec's group designed analogs around this structure and showed that some are active, like compound 77 with IC 50 values of 123 μM and 230 μM for MurC and MurD, respectively ( Figure 21 ) [ 163 ]. Moreover, this compound showed weak antibacterial activities against Escherichia coli and Staphylococcus aureus . The group also designed other heterocyclic structures to adopt a more closed conformation than with the natural substrates of enzymes MurD and MurF but still show activities on different strains. Gobec et al., who synthesized a good number of benzylidenesulfonyl hydrazine analogs, showed that some of them were multi-inhibitors [ 164 ]. They performed SAR studies to optimize enzymatic activities but without antibacterial activities. Singh's laboratory identified several multi-active structures of Mur ligases through different screenings, including pulvinones [ 165 ] and phenyl dihydrothienopyrazolol [ 166 ] with activities against multiple strains. Recently, they identified a naphthyl-type tetronic acid structure capable of inhibiting not only all four Mur ligases but also MurA and MurB [ 167 ]. Compound 78 showed the best activities in the series with an IC 50 in the range of 20 μM for each enzyme, and it inhibited the bacterial growth of Escherichia coli and Staphylococcus aureus strains ( Figure 22 ). Phosphonic Acid Inhibitors Gobec et al. developed a series of phosphoric acid analogs of hydroxyethylamines as a bioisostere of the tetrahedral intermediate of the UDP sugar substrate [ 162 ]. Compound 76 is the best analog in this series, active against all four enzymes with IC 50 values of 26 μM, 530 μM, 160 μM, and 150 μM for MurC, MurD, MurE, and MurF, respectively ( Figure 20 ). However, these compounds do not show antibacterial activities. These analogs, including 76 , are the only phosphorylated derivatives that are multi-active against Mur ligases. Heterocyclic Inhibitors After observing that the N -acyl hydrazone structure has potential in terms of biological activity in medicinal chemistry, Gobec's group designed analogs around this structure and showed that some are active, like compound 77 with IC 50 values of 123 μM and 230 μM for MurC and MurD, respectively ( Figure 21 ) [ 163 ]. Moreover, this compound showed weak antibacterial activities against Escherichia coli and Staphylococcus aureus . The group also designed other heterocyclic structures to adopt a more closed conformation than with the natural substrates of enzymes MurD and MurF but still show activities on different strains. Gobec et al., who synthesized a good number of benzylidenesulfonyl hydrazine analogs, showed that some of them were multi-inhibitors [ 164 ]. They performed SAR studies to optimize enzymatic activities but without antibacterial activities. Singh's laboratory identified several multi-active structures of Mur ligases through different screenings, including pulvinones [ 165 ] and phenyl dihydrothienopyrazolol [ 166 ] with activities against multiple strains. Recently, they identified a naphthyl-type tetronic acid structure capable of inhibiting not only all four Mur ligases but also MurA and MurB [ 167 ]. Compound 78 showed the best activities in the series with an IC 50 in the range of 20 μM for each enzyme, and it inhibited the bacterial growth of Escherichia coli and Staphylococcus aureus strains ( Figure 22 ). 3.5.3. Mimicking the Co-Substrate ATP Very recently, Zega et al. identified several compounds with different structures that exhibit multi-inhibitory activities against MurC to MurF. These compounds were identified through screening a collection of molecules originally designed to inhibit kinases by the competitive inhibition of ATP [ 168 ]. Compounds 79 and 80 are multi-inhibitors of MurC-F with IC 50 values ranging from 10 to 368 μM ( Figure 23 ). Because these compounds mimic ATP, the authors conducted kinetic and NMR studies on compound 79 and showed that it actually binds to the amino acid binding site. They also performed additional biological analyses with eukaryotic kinases and demonstrated that these molecules are specific and promising for the development of new antibacterials. 3.5.4. Natural Analogs Süssmuth et al. identified feglymycin ( 81 ), isolated from Streptomyces strains, as an active inhibitor of both MurA and MurC [ 169 ]. Feglymycin, a 13-mer peptide, exhibited activity with an IC 50 in the range of μM for MurA and in the range of mM for MurC ( Figure 24 ). 4. Conclusions Over the last few decades, the peptidoglycan biosynthetic pathway has emerged as a promising and attractive target for antibacterial drug discovery. Among the various enzymes involved, the Mur ligase family has drawn attention due to its exclusive presence in bacteria (not found in human cells) and as they are essential for bacterial cell wall biosynthesis. An in-depth understanding of their structures has led to the development of powerful new antibacterial agents. While each Mur ligase can be considered a unique antibacterial target, MurC-F ligases have highly conserved amino acid regions in their active sites. This characteristic can be used in the design of promising Mur ligase multi-inhibitors. A number of inhibitors from different chemical families were developed against Mur ligases, with significant inhibitory activity. Modern techniques remain highly utilized in the design of potentially active molecules, particularly with virtual screening methods [ 170 , 171 ]. However, none has yet been shown to have antibacterial activity. One reason could be the difficulty for these compounds to cross the bacterial membrane and reach the cytoplasm where Mur ligases activities are located. Another possibility explaining the lack of antibacterial activity may be the complexity of the Mur ligase pathway, which is high, making it difficult for the inhibitor to reach the various sites. In the future, a better understanding of the protein–protein interactions of the Mur ligase pathway, combined with the consideration of factors enabling better penetration of the bacterial wall, will enable the design of Mur ligase inhibitors with proven antibacterial activity. Figures and Schemes Figure 1 Structure of Salvarsan ( 1 ), Neosalvarsan ( 2 ), penicillin G ( 3 ), and sulfanilamide ( 4 ). Figure 2 Some examples of families of antibiotics. Figure 3 Structure of rifampicin ( 16 ). molecules-28-08076-sch001_Scheme 1 Scheme 1 Hydrolysis by β-lactamase. Figure 4 Peptidoglycan structures. molecules-28-08076-sch002_Scheme 2 Scheme 2 Key steps in peptidoglycan biosynthesis. Figure 5 MurA- and MurB-catalyzed formation of UDP-MurNAc. molecules-28-08076-sch003_Scheme 3 Scheme 3 Mechanism of MurA−catalyzed UDP-MurNAc enolpyruvate (adapted from Ref. [ 47 ]). Figure 6 Structure of fosfomycin ( 26 ) and compounds 27 and 28 . molecules-28-08076-sch004_Scheme 4 Scheme 4 MurB-catalyzed mechanism of UDP-MurNAc formation (adapted from ref. [ 72 ]). Figure 7 Structure of some MurB inhibitors. molecules-28-08076-sch005_Scheme 5 Scheme 5 Enzymatic synthesis of Lipid II and its translocation. Figure 8 Selected MurG inhibitors. molecules-28-08076-sch006_Scheme 6 Scheme 6 ( A ) General mechanism; ( B ) general scheme of UDP-MurNAc-pentapeptide formation. Figure 9 Structure of L-Alanine analogs. Figure 10 Structure of D-glutamic acid analogs and of meso-diaminopimelic acid analogs. Figure 11 Structure of sulfonamide and heterocyclic analogs of D-glutamic acid. Figure 12 Structure of analogs 51 and 52 targeting MurD. Figure 13 Structure of phosphinic analogs of MurC and MurD. Figure 14 Structure of phosphinic analogs of MurE. Figure 15 Structure of peptides 62 and 63 targeting MurD. Figure 16 Structure of compound 64 targeting MurC, compounds 65 and 66 targeting MurD, and compounds 67 – 70 targeting MurF. Figure 17 Structure of compound 71 targeting MurC. Figure 18 Structure of natural compounds 72 and 73 targeting MurE. Figure 19 Structure of multi-inhibitors 74 and 75 targeting MurC-F. Figure 20 Structure of multi-inhibitor 76 targeting MurC-F. Figure 21 Structure of multi-inhibitor 77 targeting MurC and D. Figure 22 Structure of multi-inhibitor 78 targeting MurA-F. Figure 23 Structure of multi-inhibitor compounds 79 and 80 targeting MurC-F. Figure 24 Structure of feglymycin ( 81 ) targeting MurA and C.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4488766/

A Novel Chimeric Anti-PA Neutralizing Antibody for Postexposure Prophylaxis and Treatment of Anthrax

Anthrax is a highly lethal infectious disease caused by the bacterium Bacillus anthracis , and the associated shock is closely related to the lethal toxin (LeTx) produced by the bacterium. The central role played by the 63 kDa protective antigen (PA63) region of LeTx in the pathophysiology of anthrax makes it an excellent therapeutic target. In the present study, a human/murine chimeric IgG mAb, hmPA6, was developed by inserting murine antibody variable regions into human constant regions using antibody engineering technology. hmPA6 expressed in 293F cells could neutralize LeTx both in vitro and in vivo . At a dose of 0.3 mg/kg, it could protect all tested rats from a lethal dose of LeTx. Even administration of 0.6 mg/kg hmPA6 48 h before LeTx challenge protected all tested rats. The results indicate that hmPA6 is a potential candidate for clinical application in anthrax treatment. Results ELISA ELISA was performed to test the binding sensitivity of hmPA6 to PA63. hmPA6 recognized rPA63 in a dose-dependent manner, and the graph of hmPA6 concentration and absorbance at 450 nm was a hyperbolic curve ( Fig. 1 ). Western blot Western blot analysis showed that hmPA6 could specifically recognize rPA63 ( Fig. 2 ). No reaction was seen with the negative control. Immunoprecipitation Immunoprecipitation was performed using PA83, which could be split to active PA63 using trypsin. A protein of about 63 kDa was detected on SDS-PAGE, and its sequence matched that of the B. anthracis protective antigen in the Swiss-Prot database ( Fig. 3A,C,D ). A 63 kDa membrane protein was also detected using a commercial anti-PA antibody ( Fig. 3B ), and this protein did not reaction with any other antibodies. Kinetics of binding The equilibrium dissociation constant (Kd) for hmPA6 was determined by BiaCoreX100 analysis. The rate constants kon and koff were evaluated directly from the BiaCoreX100 sensogram. The Kd was also determined using the BiaCoreX100. One striking feature of hmPA6 is its very slow off rate, which may explain its high affinity of 1.438 × 10 −10 M ( Fig. 4 ). In vitro LeTx neutralization assay The ability of hmPA6 to protect against LeTx was assessed in J774A.1 cells. hmPA6, PA83, and different concentrations of LF were simultaneously added to cells. Cell viability test results indicated that hmPA6 could completely neutralize LeTx. At 10 μg/mL LF and 0.1 μg/mL PA83, >80% of the hmPA6-treated cells remained viable, while only 26% of the control IgG antibody-treated cells remained viable. At 0.01 μg/mL LF and 0.1 μg/mL PA83, 100% of the hmPA6-treated cells were viable, while only 50% ( Fig. 5 ) of the control cells were. Protection of F344 rats F344 rats were injected hmPA6 antibody via the tail vein either before or after LeTx injection. The survival time of group III was significantly ( P 80% of the hmPA6-treated cells remained viable, while only 26% of the control IgG antibody-treated cells remained viable. At 0.01 μg/mL LF and 0.1 μg/mL PA83, 100% of the hmPA6-treated cells were viable, while only 50% ( Fig. 5 ) of the control cells were. Protection of F344 rats F344 rats were injected hmPA6 antibody via the tail vein either before or after LeTx injection. The survival time of group III was significantly ( P 80% cell viability, while the control antibody maintained only 26%. Moreover, in previous study, they often used F344 rats challenged with LeTx ( Table 1 ) before tested with B. anthracis spores. Therefore F344 rats was injected with LeTx vial tail vain. The hmPA6 with a Kd of 0.14 nM protected all rats from death at a concentration of 0.3 mg/kg (45 μg per rat). However, in a previous study, 1.5 mg/kg raxibacumab (of human origin) with a Kd of 2.78 nM was administered 24 h before anthrax lethal toxin administration in F344 rats 24 . We also tested an hmPA6 concentration of 0.6 mg/kg (90 μg per rat) administered 48 h before LeTx injection; this dose also protected all rats. Our findings indicate that hmPA6 may be used as a prophylactic. Other humanized or chimeric mAbs have been examined in other animal model, but the affinity found in the present study is the same as or better than those found previously 32 33 . Murine mAbs against PA have been developed, but our chimeric mAb maintains a balance between the high-affinity murine component and the low-immunogenicity human component. Other mAbs of human origin are available but are difficult to produce and are expensive 32 . The chimeric mAb hmPA6 is produced in 293F cells, and the method of production is very convenient. In the in vivo test in the present study, irrespective of whether LF was injected before or after hmPA6, the antibody protected the rats from death provided that it was administered before or simultaneously with PA. This finding indicated that hmPA6 could not prevent LF from binding PA63. Further, no morphological changes were observed in the rats of only injection LeTx, probably because the time between injection and sacrifice was very short (only about 90 mins), and no histological changes occurred in this period. The IHC analysis showed a strong positive result in the group injected only LeTx. However, the group that received LeTx + 45 μg hmPA6 showed a weakly positive reaction. These findings indicate that hmPA6 prevents PA from binding to cell receptors. However, further experiments are required to validate this hypothesis. Future studies should focus on detailed characterization of this mAb (specificity, toxicity studies, autoantigen testing, etc.). Second, epitope mapping and structure function analysis of hmPA6 should be performed. In the in vivo experiment in the present study, we demonstrated that hmPA6 could not interfere with LF binding to PA. Further experimentation is needed to determine the exact mechanism by which hmPA6 neutralizes LeTx. Lastly, more animal tests are required, for example, in which animals are challenged with anthrax spores. In summary, we reported a human/murine chimeric IgG, namely, hmPA6, which can specifically identify PA with high affinity, neutralize LeTx, and protect macrophages and F344 rats from anthrax-related death. We also showed that PA63 is a good immunogen. From our findings, we believe that once hmPA6 is further characterized, it can be used alone or in combination with other neutralizing mAbs for treatment of anthrax. Materials and Methods Mouse mAb development Recombinant PA63 protein (rPA63) was expressed in Escherichia coli BL21 by using the pColdII vector and purified by affinity chromatography. Six BALB/c mice aged 7 weeks were intraperitoneally (ip) immunized with rPA63 and adjuvant as described previously 34 . After immunization, the mouse spleen showing the best titer was removed, and splenocytes were extracted and fused with SP2/0 myeloma cells using hybridoma technology 35 . Positive clones were screened by ELISA using active PA63-coated 96-well plates, and subcloning was conducted based on standard protocols. Clonal expansion was conducted with Hybridoma-SFM (Gibco,USA). The cell supernatant was then removed and purified by affinity chromatography with protein G (GE, USA) according to the manufacturer's purification system. All experiments involving animals were performed in accordance with the protocols approved by the Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA. Construction of human/mouse chimeric antibody expression vector Total RNA was extracted from the PA6 hybridoma cells using the TRIzol reagent (Invitrogen), and cDNA was synthesized using reverse transcriptase SuperScript II according to the manufacturer's instructions. Eukaryotic vectors were constructed by separately cloning PA6 heavy and light variable regions into pTH and pTL, which respectively include constant regions of IgG1 heavy and light chains. Murine variable regions of the heavy (V H ) and light chains (V L ) were first amplified by PCR using PA6 cDNA as the template. To obtain V H and V L nucleotide sequences, these chains were cloned into the pMD-18T vector. PCR primers were designed using the In-FusionR HD Cloning Kit (Clontech), and V H and V L were amplified from right-sequenced pMD-18T vectors by using these primers. Finally, V H and V L were separately cloned into linearized pTH and pTL vectors, respectively, by infusion PCR using the In-FusionR HD Cloning Kit. The recombinant pTH/PA6 V H and pTH/PA6 V L vectors were sequenced by Genescript. Sequences were further analyzed using the VBASE2 database ( http://www.vbase2.org/ ). Antibody expression and purification The recombinant vectors were simultaneously transfected into FreeStyle™ 293-F Cells (293F) using 293fectin with the FreeStyle™ 293 Expression System (Invitrogen). Six days after transient transfection, the cell supernatant was harvested and purified by affinity chromatography with protein A (GE, USA) in accordance with the manufacturer's purification system. The purity of the chimeric antibody (hmPA6) was examined by 10% SDS-PAGE and Coomassie blue staining. ELISA Ninety-six-well enzyme immunoassay plates were coated overnight at 4 °C with 50 μL of rPA63 antigen (2 μg/mL) diluted in 50 mM sodium carbonate buffer (pH 9.6). The plates were blocked and serial two-fold dilutions of hmPA6 were added to the wells (3 wells for each concentration) as the primary antibody. The plates were incubated at 37 °C for 1 h and then washed 3 times with 300 μL of PBS containing 0.05% Tween 20 (PBST). Subsequently, goat anti-human IgG–HRP conjugate (Sigma) was added as the secondary antibody and incubated at 37 °C for 30 min. After color development, the absorbance values of the wells were detected at 450 nm. Non-correlated IgG1 was used as the control. The absorbance values at 450 nm of hmPA6 were plotted using GraphPad Prism software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Western blot analysis The cell lysates of rPA63 recombinant bacteria and E. coli BL21 were separately run on a 10% SDS-PAGE gel and then transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked with PBS containing 5% dry milk at 4 °C overnight and then incubated for 1 h at RT with 1:2000 diluted hmPA6 from 1 mg/mL stock. After it was washed 3 times with PBST, the membrane was incubated with a 1:4000 diluted secondary HRP-conjugated goat anti-human antibody (Sigma) for an additional 30 min at RT. Following the same washing procedure, the signal was detected using ECL Western Blot Substrate (Pierce) according to the manufacturer's instructions. Immunoprecipitation A mixture of PA63 and PA83 was prepared by incubating PA83 [20 mM Tris (pH 8.0) and 150 mM NaCl] with 0.5 μg/mL trypsin (Sigma) for 30 min at 22 °C, followed by addition of 10 μg/mL soybean trypsin inhibitor (Sigma) 14 . Then, the mixture was incubated with 5 μg of hmPA6 at 4 °C and rotated for 3 h. Next, 50 μl protein-A Sepharose (Invitrogen, USA) was added and incubated at 4 °C. The immune complexes that formed were washed 3 times with PBST. Subsequently, 50 μL elution buffer was added to separate these antibody-antigen complexes from protein-A Sepharose. As a negative control, another anti-TLR4 chimeric antibody (generated by our lab) was created using the same protocol. The protein complexes were isolated by running two 10% SDS-PAGE gels; one was transferred onto a nitrocellulose membrane, and the other was stained with Coomassie blue. The nitrocellulose membrane was blocked at 4 °C overnight, incubated with 1:5000 diluted rabbit polyclonal anti-PA antibody (Pierce, USA) for 1 h at RT, washed with PBST 3 times, and reacted with 1:4000 diluted goat anti-rabbit IgG-HRP conjugate (Sigma) for an additional 30 min at RT. The membrane was washed 3 times with PBST, and the hybridization signal was detected using ECL Western Blot substrate. The target bands on SDS-PAGE gel were subjected to mass spectra identification with an ABI 4700 proteomics analyzer and MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA). The mass spectra were then searched within the Swiss-Prot database using the MASCOT search engine ( http://www.matrix science.com ; Matrix Science, UK). Affinity and kinetic assay of antibody The Biacore X100 System (GE, USA) was used to analyze the affinity and kinetics of the hmPA6 antibody. PA83 was diluted to 25 μg/mL with acetate buffer (10 mM NaAc, pH 4.5) and immobilized on the surface of a CM5 sensor chip (GE, USA) to capture purified mAb, which was diluted in running buffer (10 mM HEPES, 150 mM NaCl, 5 mM EDTA-Na 2 , 0.05% P20; pH 7.4) to achieve different concentrations ranging from 5 to 80 nmol/L. The association time was set up at 180 s and the dissociation time, at 600 s, followed by regeneration with 50 mM glycine–HCl (pH 2.2). Sensograms were evaluated using the Biacore X100 evaluation software. In vitro LeTx neutralization assay The in vitro LeTx neutralization assay was performed as described previously 29 . Briefly, murine macrophage J774A.1 cells cultured in DMEM containing 10% fetal bovine seru and 1% penicillin/streptomycin were seeded in 96-well plates to 70% confluence. LF was diluted serially in complete medium containing PA and hmPA6. This mixture was applied to the cells (3 wells for each dilution) at the following final concentrations: LF, 0.01 ~ 10,000 ng/mL; PA, 0.1 μg/mL; and hmPA6, 4 μg/mL. The plates were then incubated for 3 h at 37 °C. Untreated cells and cells treated with only LeTx acted as the controls. Cell viability was determined using the AQ assay (Promega, MI) according to the manufacturer's instructions. In vivo LeTx neutralization assay The in vivo LeTx neutralization assay was performed using female Fischer 344 (F344) rats weighing between 130 and 160 g. Every rats of each group (n = 6) were injected via the tail vein with a mixture of PA + LF (LeTx) and different amounts of hmPA6 antibody prepared in sterile PBS. Each rat was administered 300 μL of the mixture. Further, the rats were also treated with different concentrations of the antibody 5 min before exposure to LeTx. For this experiment, they were injected intravenously with PBS or 15, 30, or 45 μg of the antibody before receiving an intravenous injection of LeTx (30 μg PA + 30 μg LF). Additionally, double the complete protection dose of antibody (90 μg) was injected to test its prophylactic ability. The rats were inoculated with 90 μg antibody followed by LeTx administration after different times, from 5 min to 48 h. Two additional experiments were conducted with F344 rats. One group received PA (30 μg) injection 5 min after LF + hmPA6 (30 μg + 45 μg, respectively), while the other received 30 μg PA 5 min before LF + hmPA6 (at the same doses). After injection of LeTx, signs of malaise and death were checked for every 30 min for the first 8 h and then at 16 h and 24 h, followed by twice-daily checks for 1 week. Tissue pathology and immunohistochemical examination The lungs of the F344 rats were embedded in paraffin wax at the Department of Pathology, Nanjing Medical University (Jiangsu, China), using routine methods. Sections (5 μm) were deparaffinized with xylene and then dehydrated in decreasing concentrations of alcohol. Some sections were treated with H&E staining and examined by light microscopy to determine the pathological features of the lung tissues. For the remaining sections, endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxidase in Tris-buffered saline. Some of these tissue sections were then incubated with rabbit polyclonal anti-PA primary antibody (Pierce, USA), followed by the EnVision HRP complex (DAKO, Carpinteria, CA). They were then counterstained with hematoxylin QS (Vector Laboratories, Burlingame, CA). The results were analyzed according to the IHC score (IHS) as described previously 36 . Briefly, the IHS was determined by evaluation of both staining density and intensity. Multiplication of the intensity and percentage scores yielded the final IHS. Samples with IHS ≤3 were considered weakly positive, while those with IHS ≥6 were considered strongly positive. The IHC results were evaluated by two independent investigators blinded to the rat groups. In cases of conflict, a pathologist reviewed the cases, and a consensus was reached. Statistical analysis of survival data Kaplan Meier analysis was used for evaluation of survival. Survival data were analyzed using the GraphPad Prism version 4 statistical analysis software (San Diego, CA). A t -test was used to compare the mean survival time between groups. A two-tailed log rank test was used to determine the statistical significance of differences between groups. A P value of <0.05 was considered statistically significant. Mouse mAb development Recombinant PA63 protein (rPA63) was expressed in Escherichia coli BL21 by using the pColdII vector and purified by affinity chromatography. Six BALB/c mice aged 7 weeks were intraperitoneally (ip) immunized with rPA63 and adjuvant as described previously 34 . After immunization, the mouse spleen showing the best titer was removed, and splenocytes were extracted and fused with SP2/0 myeloma cells using hybridoma technology 35 . Positive clones were screened by ELISA using active PA63-coated 96-well plates, and subcloning was conducted based on standard protocols. Clonal expansion was conducted with Hybridoma-SFM (Gibco,USA). The cell supernatant was then removed and purified by affinity chromatography with protein G (GE, USA) according to the manufacturer's purification system. All experiments involving animals were performed in accordance with the protocols approved by the Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA. Construction of human/mouse chimeric antibody expression vector Total RNA was extracted from the PA6 hybridoma cells using the TRIzol reagent (Invitrogen), and cDNA was synthesized using reverse transcriptase SuperScript II according to the manufacturer's instructions. Eukaryotic vectors were constructed by separately cloning PA6 heavy and light variable regions into pTH and pTL, which respectively include constant regions of IgG1 heavy and light chains. Murine variable regions of the heavy (V H ) and light chains (V L ) were first amplified by PCR using PA6 cDNA as the template. To obtain V H and V L nucleotide sequences, these chains were cloned into the pMD-18T vector. PCR primers were designed using the In-FusionR HD Cloning Kit (Clontech), and V H and V L were amplified from right-sequenced pMD-18T vectors by using these primers. Finally, V H and V L were separately cloned into linearized pTH and pTL vectors, respectively, by infusion PCR using the In-FusionR HD Cloning Kit. The recombinant pTH/PA6 V H and pTH/PA6 V L vectors were sequenced by Genescript. Sequences were further analyzed using the VBASE2 database ( http://www.vbase2.org/ ). Antibody expression and purification The recombinant vectors were simultaneously transfected into FreeStyle™ 293-F Cells (293F) using 293fectin with the FreeStyle™ 293 Expression System (Invitrogen). Six days after transient transfection, the cell supernatant was harvested and purified by affinity chromatography with protein A (GE, USA) in accordance with the manufacturer's purification system. The purity of the chimeric antibody (hmPA6) was examined by 10% SDS-PAGE and Coomassie blue staining. ELISA Ninety-six-well enzyme immunoassay plates were coated overnight at 4 °C with 50 μL of rPA63 antigen (2 μg/mL) diluted in 50 mM sodium carbonate buffer (pH 9.6). The plates were blocked and serial two-fold dilutions of hmPA6 were added to the wells (3 wells for each concentration) as the primary antibody. The plates were incubated at 37 °C for 1 h and then washed 3 times with 300 μL of PBS containing 0.05% Tween 20 (PBST). Subsequently, goat anti-human IgG–HRP conjugate (Sigma) was added as the secondary antibody and incubated at 37 °C for 30 min. After color development, the absorbance values of the wells were detected at 450 nm. Non-correlated IgG1 was used as the control. The absorbance values at 450 nm of hmPA6 were plotted using GraphPad Prism software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Western blot analysis The cell lysates of rPA63 recombinant bacteria and E. coli BL21 were separately run on a 10% SDS-PAGE gel and then transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked with PBS containing 5% dry milk at 4 °C overnight and then incubated for 1 h at RT with 1:2000 diluted hmPA6 from 1 mg/mL stock. After it was washed 3 times with PBST, the membrane was incubated with a 1:4000 diluted secondary HRP-conjugated goat anti-human antibody (Sigma) for an additional 30 min at RT. Following the same washing procedure, the signal was detected using ECL Western Blot Substrate (Pierce) according to the manufacturer's instructions. Immunoprecipitation A mixture of PA63 and PA83 was prepared by incubating PA83 [20 mM Tris (pH 8.0) and 150 mM NaCl] with 0.5 μg/mL trypsin (Sigma) for 30 min at 22 °C, followed by addition of 10 μg/mL soybean trypsin inhibitor (Sigma) 14 . Then, the mixture was incubated with 5 μg of hmPA6 at 4 °C and rotated for 3 h. Next, 50 μl protein-A Sepharose (Invitrogen, USA) was added and incubated at 4 °C. The immune complexes that formed were washed 3 times with PBST. Subsequently, 50 μL elution buffer was added to separate these antibody-antigen complexes from protein-A Sepharose. As a negative control, another anti-TLR4 chimeric antibody (generated by our lab) was created using the same protocol. The protein complexes were isolated by running two 10% SDS-PAGE gels; one was transferred onto a nitrocellulose membrane, and the other was stained with Coomassie blue. The nitrocellulose membrane was blocked at 4 °C overnight, incubated with 1:5000 diluted rabbit polyclonal anti-PA antibody (Pierce, USA) for 1 h at RT, washed with PBST 3 times, and reacted with 1:4000 diluted goat anti-rabbit IgG-HRP conjugate (Sigma) for an additional 30 min at RT. The membrane was washed 3 times with PBST, and the hybridization signal was detected using ECL Western Blot substrate. The target bands on SDS-PAGE gel were subjected to mass spectra identification with an ABI 4700 proteomics analyzer and MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA). The mass spectra were then searched within the Swiss-Prot database using the MASCOT search engine ( http://www.matrix science.com ; Matrix Science, UK). Affinity and kinetic assay of antibody The Biacore X100 System (GE, USA) was used to analyze the affinity and kinetics of the hmPA6 antibody. PA83 was diluted to 25 μg/mL with acetate buffer (10 mM NaAc, pH 4.5) and immobilized on the surface of a CM5 sensor chip (GE, USA) to capture purified mAb, which was diluted in running buffer (10 mM HEPES, 150 mM NaCl, 5 mM EDTA-Na 2 , 0.05% P20; pH 7.4) to achieve different concentrations ranging from 5 to 80 nmol/L. The association time was set up at 180 s and the dissociation time, at 600 s, followed by regeneration with 50 mM glycine–HCl (pH 2.2). Sensograms were evaluated using the Biacore X100 evaluation software. In vitro LeTx neutralization assay The in vitro LeTx neutralization assay was performed as described previously 29 . Briefly, murine macrophage J774A.1 cells cultured in DMEM containing 10% fetal bovine seru and 1% penicillin/streptomycin were seeded in 96-well plates to 70% confluence. LF was diluted serially in complete medium containing PA and hmPA6. This mixture was applied to the cells (3 wells for each dilution) at the following final concentrations: LF, 0.01 ~ 10,000 ng/mL; PA, 0.1 μg/mL; and hmPA6, 4 μg/mL. The plates were then incubated for 3 h at 37 °C. Untreated cells and cells treated with only LeTx acted as the controls. Cell viability was determined using the AQ assay (Promega, MI) according to the manufacturer's instructions. In vivo LeTx neutralization assay The in vivo LeTx neutralization assay was performed using female Fischer 344 (F344) rats weighing between 130 and 160 g. Every rats of each group (n = 6) were injected via the tail vein with a mixture of PA + LF (LeTx) and different amounts of hmPA6 antibody prepared in sterile PBS. Each rat was administered 300 μL of the mixture. Further, the rats were also treated with different concentrations of the antibody 5 min before exposure to LeTx. For this experiment, they were injected intravenously with PBS or 15, 30, or 45 μg of the antibody before receiving an intravenous injection of LeTx (30 μg PA + 30 μg LF). Additionally, double the complete protection dose of antibody (90 μg) was injected to test its prophylactic ability. The rats were inoculated with 90 μg antibody followed by LeTx administration after different times, from 5 min to 48 h. Two additional experiments were conducted with F344 rats. One group received PA (30 μg) injection 5 min after LF + hmPA6 (30 μg + 45 μg, respectively), while the other received 30 μg PA 5 min before LF + hmPA6 (at the same doses). After injection of LeTx, signs of malaise and death were checked for every 30 min for the first 8 h and then at 16 h and 24 h, followed by twice-daily checks for 1 week. Tissue pathology and immunohistochemical examination The lungs of the F344 rats were embedded in paraffin wax at the Department of Pathology, Nanjing Medical University (Jiangsu, China), using routine methods. Sections (5 μm) were deparaffinized with xylene and then dehydrated in decreasing concentrations of alcohol. Some sections were treated with H&E staining and examined by light microscopy to determine the pathological features of the lung tissues. For the remaining sections, endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxidase in Tris-buffered saline. Some of these tissue sections were then incubated with rabbit polyclonal anti-PA primary antibody (Pierce, USA), followed by the EnVision HRP complex (DAKO, Carpinteria, CA). They were then counterstained with hematoxylin QS (Vector Laboratories, Burlingame, CA). The results were analyzed according to the IHC score (IHS) as described previously 36 . Briefly, the IHS was determined by evaluation of both staining density and intensity. Multiplication of the intensity and percentage scores yielded the final IHS. Samples with IHS ≤3 were considered weakly positive, while those with IHS ≥6 were considered strongly positive. The IHC results were evaluated by two independent investigators blinded to the rat groups. In cases of conflict, a pathologist reviewed the cases, and a consensus was reached. Statistical analysis of survival data Kaplan Meier analysis was used for evaluation of survival. Survival data were analyzed using the GraphPad Prism version 4 statistical analysis software (San Diego, CA). A t -test was used to compare the mean survival time between groups. A two-tailed log rank test was used to determine the statistical significance of differences between groups. A P value of <0.05 was considered statistically significant. Additional Information How to cite this article : Xiong, S. et al. A Novel Chimeric Anti-PA Neutralizing Antibody for Postexposure Prophylaxis and Treatment of Anthrax. Sci. Rep. 5 , 11776; doi: 10.1038/srep11776 (2015).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851783/

Identification of a conserved membrane localization domain within numerous large bacterial protein toxins

Vibrio cholerae is the causative agent of the diarrheal disease cholera. Many virulence factors contribute to intestinal colonization and disease including the Multifunctional Autoprocessing RTX toxin (MARTX Vc ). The Rho-inactivation domain (RID) of MARTX Vc is responsible for inactivating the Rho-family of small GTPases, which leads to depolymerization of the actin cytoskeleton. Based on a deletion analysis of RID to determine the minimal functional domain, we have identified a subdomain at the N terminus of RID that is homologous to the membrane targeting C1 domain of Pasteurella multocida toxin. A GFP fusion to this subdomain from RID colocalized with a plasma membrane marker when transiently expressed within HeLa cells and can be found in the membrane fraction following subcellular fractionation. This C1-like subdomain is present in multiple families of bacterial toxins, including all of the clostridial glucosyltransferase toxins and various MARTX toxins. GFP-fusions to these homologous domains are also membrane associated, indicating that this is a conserved membrane localization domain (MLD). We have identified three residues (Y23, S68, R70) as necessary for proper localization of one but not all MLDs. In addition, we found that substitution of the RID MLD with the MLDs from two different effector domains from the Vibrio vulnificus MARTX toxin restored RID activity, indicating that there is functional overlap between these MLDs. This study describes the initial recognition of a family of conserved plasma membrane-targeting domains found in multiple large bacterial toxins.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679989/

Function of Nod-like Receptors in Microbial Recognition and Host Defense

Summary Nucleotide oligomerization domain (NOD)-like receptors (NLRs) are a specialized group of intracellular proteins that play a critical role in the regulation of the host innate immune response. NLRs act as scaffolding proteins that assemble signaling platforms that trigger nuclear factor-κB and mitogen-activated protein kinase signaling pathways and control the activation of inflammatory caspases. Importantly, mutations in several members of the NLR family have been linked to a variety of inflammatory diseases consistent with these molecules playing an important role in host-pathogen interactions and the inflammatory response. In this review, we focus on the role of Nod1 and Nod2 in host defense and in particular discuss recent finding regarding the role of Nlrc4, Nlpr1, and Nlrp3 inflammasomes in caspase-1 activation and subsequent release of proinflammatory cytokines such as interleukin-1β.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2747959/

The Global Regulator CodY Regulates Toxin Gene Expression in Bacillus anthracis and Is Required for Full Virulence ▿ †

In gram-positive bacteria, CodY is an important regulator of genes whose expression changes upon nutrient limitation and acts as a repressor of virulence gene expression in some pathogenic species. Here, we report the role of CodY in Bacillus anthracis , the etiologic agent of anthrax. Disruption of codY completely abolished virulence in a toxinogenic, noncapsulated strain, indicating that the activity of CodY is required for full virulence of B. anthracis . Global transcriptome analysis of a codY mutant and the parental strain revealed extensive differences. These differences could reflect direct control for some genes, as suggested by the presence of CodY binding sequences in their promoter regions, or indirect effects via the CodY-dependent control of other regulatory proteins or metabolic rearrangements in the codY mutant strain. The differences included reduced expression of the anthrax toxin genes in the mutant strain, which was confirmed by lacZ reporter fusions and immunoblotting. The accumulation of the global virulence regulator AtxA protein was strongly reduced in the mutant strain. However, in agreement with the microarray data, expression of atxA , as measured using an atxA-lacZ transcriptional fusion and by assaying atxA mRNA, was not significantly affected in the codY mutant. An atxA-lacZ translational fusion was also unaffected. Overexpression of atxA restored toxin component synthesis in the codY mutant strain. These results suggest that CodY controls toxin gene expression by regulating AtxA accumulation posttranslationally.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123439/

Branching Out: Alterations in Bacterial Physiology and Virulence Due to Branched-Chain Amino Acid Deprivation

The branched-chain amino acids (BCAAs [Ile, Leu, and Val]) represent important nutrients in bacterial physiology, with roles that range from supporting protein synthesis to signaling and fine-tuning the adaptation to amino acid starvation. In some pathogenic bacteria, the adaptation to amino acid starvation includes induction of virulence gene expression: thus, BCAAs support not only proliferation during infection, but also the evasion of host defenses. BACTERIAL ADAPTATION TO NUTRIENT AVAILABILITY Bacteria sense a variety of physical and chemical signals in their environment and use this information to coordinate an adaptive response that promotes their growth and survival. These signals include nutrients such as sugars, lipids, metals, and amino acids, and their depletion triggers upregulation of high-affinity nutrient acquisition systems and/or biosynthesis pathways. Nutrient availability is also an important environmental cue during infection, as pathogens encounter fluctuating nutrient access in a host and must adapt their metabolism accordingly to proliferate and avoid being cleared. Host environments impose an added challenge by actively sequestering nutrients to limit pathogen growth. To cope, pathogens release toxins that act to liberate nutrients from host tissues. In this way, virulence determinant production is tied to the nutrient composition of a given host niche. Several transcriptional regulators are positioned at the intersection of metabolism and pathogenesis, such as CcpA (responds to a preferred carbon source), CodY (responds to GTP and branched-chain amino acids [BCAAs]), and RpiR (responds to pentose phosphate pathway intermediates) ( 1 , 2 ). The relevance of such regulators to gross bacterial physiology is typically evidenced by mutating the regulator and measuring transcriptional changes to identify shifts in metabolism that presumably allow the pathogen to adapt to restriction of a given nutritional signal. This approach assumes that adaptive responses are binary, with each category defined by a completely active or inactive regulator. This methodology, however, fails to capture the intermediate responses that occur along a gradient of nutrient concentrations, which are more likely to represent the conditions pathogens encounter in vivo . It has remained challenging to capture the adaptive responses along a spectrum of nutrient availability because the mechanisms pathogens use to obtain nutrients, which therefore dictate this spectrum, are often unknown. Without a good understanding of the mechanisms that influence intracellular levels of regulatory nutrients, it remains largely unanswered how pathogens fine-tune intracellular nutrient pools at levels that are sufficient to promote growth in host tissues, while still allowing the bacteria to maintain sensitivity to changes in their external environment. Several recent studies have applied a novel approach to address these gaps and have characterized the adaptive responses that correspond to graded nutritional levels ( 3 – 5 ). This approach, applied to the CodY regulator, involves mutating the effector binding site to mimic graded nutrient depletion, resulting in protein variants with fixed activation states along the full spectrum of CodY activity. Readers are directed to a recent review by Brinsmade ( 6 ) for a detailed summary of these studies. Comparison of the transcriptomes of cells bearing each protein variant has revealed that CodY-regulated genes are expressed as a hierarchy, whereby some target genes are derepressed upon a slight reduction of CodY activity, whereas other target genes remain repressed under the same conditions and are derepressed only upon a more significant reduction of CodY activity ( 3 – 5 ). These studies reveal the sensitivity of important physiological responses, including virulence gene expression, to changes in intracellular nutrient availability, namely BCAA concentrations. This has renewed interest in characterizing the factors that dictate intracellular BCAA concentrations, such as endogenous synthesis and acquisition mechanisms, and in investigating the relative contribution of these mechanisms to supplying BCAAs at key threshold concentrations that define a microorganism's physiological state. In this review, we will discuss the roles of BCAAs in bacterial physiology, the mechanisms of biosynthesis and transport, and the recent advancements made in understanding how depletion of intracellular sources impacts pathogen proliferation and adaptation in host niches, with a focus on the role of BCAAs in regulating CodY. Although CodY is found only in Gram-positive bacteria, we will nonetheless discuss BCAA metabolism in both Gram-negative and Gram-positive bacteria to highlight shared physiological roles and mechanisms of acquisition and biosynthesis. MULTIFACETED ROLE FOR BRANCHED-CHAIN AMINO ACIDS IN BACTERIAL PHYSIOLOGY The BCAAs are small nonpolar amino acids with branched alkyl side chains that make them hydrophobic and confer unique properties in proteins. Leu is a strong stabilizer of α-helical structures and, as such, is typically found in the inner helical core of proteins ( 7 ), whereas the substitution of the β-carbon with a methyl group on Ile and Val creates bulkiness that destabilizes α-helical structures; thus, Ile and Val are preferentially located in β-sheets ( 8 , 9 ). Bacteria synthesize BCAAs through a conserved pathway that is present in fungi and plants, but absent in mammals. The level of synthesis is dependent on the availability of metabolites linked to central metabolism, including pyruvate, acetyl coenzyme A (acetyl-CoA), and oxaloacetate ( Fig. 1 ). The biosynthetic pathway also provides intermediates for the synthesis of vitamin B 5 (pantothenate) ( 10 ) and branched-chain fatty acids (BCFAs) ( Fig. 1 ). BCFAs are the predominant fatty acids in Gram-positive bacterial membranes, and the nature and abundance of specific BCFAs determine the biophysical properties of the membrane ( 11 ). This contrasts with Gram-negative bacteria, where the predominant fatty acids are straight-chain fatty acids (SCFAs) and the biophysical properties of the membrane are determined by the degree of saturation ( 11 ). SCFA and BCFA synthesis proceeds through the same multienzyme fatty acid synthesis (FAS-II) pathway; however, the substrates that initiate the pathway differ. Acetyl-CoA serves as the substrate for SCFA synthesis, whereas branched-chain acyl-CoA serves as the substrate for BCFA synthesis ( 12 ). When initiated with branched-chain acyl-CoA substrates, the pathway produces even-chain anteiso-FAs derived from Ile, even-chain iso-FAs derived from Leu, and odd-chain iso-FAs derived from Val. Regulation of the ratio of iso to anteiso fatty acids and/or SCFA to BCFA facilitates adaptation to changes in temperature, pH, salinity, and CO 2 ( 13 – 20 ). The positioning of the methyl group on the acyl chain of anteiso-FAs disrupts close packing of membrane lipids, promoting a more fluid membrane. This property is critical to adaptation to growth at low temperatures, during which the anteiso-FA (namely, a15:0) content is increased ( 13 , 17 , 18 , 20 – 22 ). Thus, the importance of BCAAs for bacterial physiology stems from their integration with central metabolism, their requirement for protein synthesis, and their requirement for environmental adaptation via BCFA synthesis in Gram-positive bacteria. FIG 1 Integration of the BCAA biosynthetic pathway with cellular metabolism. Metabolites highlighted in blue are connected to BCAA biosynthesis. BCAAs AS INDICATORS OF CELLULAR METABOLIC STATUS In addition to their physiological roles, the BCAAs are effectors of the global transcriptional regulators leucine-responsive regulatory protein (Lrp) in Gram-negative bacteria and CodY in Gram-positive bacteria ( 23 , 24 ). These global regulators coordinate the response to nutrient availability and regulate metabolic reprogramming to sustain growth upon nutrient exhaustion, as exemplified by the characteristic metabolic shift to stationary phase under laboratory growth conditions ( Fig. 2 ). This transition coincides with accumulation of (p)ppGpp, a metabolite synthesized from GTP during the stringent response, a response provoked by amino acid starvation ( 25 ). FIG 2 Involvement of BCAAs in the regulatory response to amino acid deprivation. Exponential growth is associated with nutrient consumption and subsequent depletion. Amino acid depletion triggers the synthesis of ppGpp from GTP, correlating with the entrance to stationary phase. Accumulation of ppGpp induces lrp expression in Gram-negative bacteria. (i) Leu binds to Lrp, genes involved in amino acid synthesis and transport are activated, and genes involved in amino acid catabolism are repressed. (ii) Depletion of GTP and BCAAs trigger a decrease in CodY DNA-binding activity in Gram-positive bacteria, and CodY target genes involved in amino acid biosynthesis and transport are expressed. (iii) BCAAs promote the hydrolysis activity of the enzyme that converts ppGpp to GTP, limiting either induction of lrp expression or inactivation of CodY. Lrp is a highly conserved transcriptional regulator in enteric bacteria and regulates gene expression upon entry into stationary phase ( 23 ). Lrp DNA-binding activity is enhanced, antagonized, or not affected by Leu availability, depending on the target gene, and binding can lead to either transcriptional repression or activation ( 26 ). Its target genes include those involved in glutamate, glutamine, BCAA, and serine biosynthesis, glycine degradation, BCAA and oligopeptide transport, and pilus formation ( 26 ). CodY, a conserved transcriptional regulator in low-GC Gram-positive bacteria (i.e., Firmicutes ), senses the metabolic status of the cell to promote adaptation to nutrient limitation ( 27 , 28 ). CodY is activated through direct interaction with BCAAs and GTP ( 27 – 30 ), with the exception of Lactococcus lactis and Streptococcus pneumoniae , where CodY responds to only BCAAs ( 31 – 33 ). In its active state, CodY binds to DNA, typically leading to transcriptional repression, although direct activation has been observed involving an unknown mechanism ( 34 , 35 ). Thus, in general, CodY target genes are repressed during rapid growth and expressed upon nutrient deprivation. CodY regulates metabolic genes involved in amino acid synthesis, purine biosynthesis, sugar and amino acid transport, and the Krebs cycle ( 31 , 36 – 39 ), as well as, in some species, sporulation ( 27 , 40 – 42 ) and biofilm formation ( 43 – 45 ). CodY also regulates virulence gene expression in Gram-positive pathogens, including Bacillus anthracis ( 46 – 48 ), Bacillus cereus ( 43 , 49 – 51 ), Clostridium difficile ( 40 , 52 , 53 ), Clostridium perfringens ( 41 , 54 , 55 ), Listeria monocytogenes ( 35 , 39 , 56 , 57 ), Staphylococcus aureus ( 5 , 37 , 38 , 58 – 60 ), S. pneumoniae ( 32 ), and Streptococcus pyogenes ( 45 , 61 – 63 ). The role of CodY as a regulator of metabolism and virulence has been comprehensively reviewed elsewhere; thus, readers are directed to several recent reviews for more information ( 6 , 24 , 64 , 65 ). The accumulation of (p)ppGpp and consequential depletion of GTP impact the regulatory responses of Lrp and CodY, respectively, thereby linking these regulators to the stringent response ( Fig. 2 ). Accumulation of (p)ppGpp induces lrp expression, and depletion of GTP decreases CodY DNA-binding activity ( 27 – 30 , 66 ). In Betaproteobacteria and Gammaproteobacteria , the relative levels of (p)ppGpp and GTP are controlled by the synthetase RelA, which converts GTP to (p)ppGpp, and the hydrolase SpoT, which reverses the reaction ( 67 ). In other genera, a single bifunctional enzyme, RSH (RelA/SpoT homologue [or Rel]), interconverts (p)ppGpp and GTP ( 67 ). Recently, BCAAs were found to regulate the accumulation of (p)ppGpp ( 68 ). In Alphaproteobacteria , Val and Ile bind and activate the domain of the RSH that is responsible for hydrolyzing (p)ppGpp to GTP ( 68 ). Thus, the stringent response is countered under conditions of high BCAAs and promoted under conditions of BCAA limitation. This regulatory mechanism might also occur in other bacteria, as Val binds to the RelA enzyme in Gammaproteobacteria and Leu binds to the RSH enzyme in Gram-positive bacteria ( 68 ). Thus, BCAAs influence Lrp and CodY regulatory responses directly by binding and regulating their DNA-binding activity, and indirectly by regulating (p)ppGpp hydrolysis to GTP. An emerging role for isoleucine in regulating Gram-positive environmental adaptation. Historically, all three BCAAs have been considered equal CodY effectors. Indeed, all three bind to CodY and activate its DNA-binding activity ( 29 , 30 , 69 , 70 ), and yet, some studies have observed a stronger effect of Ile on CodY DNA-binding activity than either Leu or Val ( 31 , 71 – 73 ). A predominant role for Ile in regulating CodY activity during growth has also emerged. Depletion of Ile and not Leu or Val relieves repression of CodY-regulated genes in B. subtilis ( 29 ), L. monocytogenes ( 57 ), and S. aureus ( 38 , 69 ). Furthermore, as CodY target genes tend to comprise metabolic pathways, such as amino acid biosynthesis, the levels of Ile in the growth medium consequentially impact growth rate. Addition of excess Ile to the growth medium impairs growth of L. lactis ( 71 ), Streptococcus mutans ( 73 ), and S. aureus ( 38 ), whereas Ile depletion has the opposite effect. S. aureus growth is significantly impaired in media lacking Leu; however, its growth is restored upon simultaneous Ile and Leu depletion ( 69 ). An S. mutans mutant that is unable to synthesize (p)ppGpp (i.e., mimicking a strain with constitutively active CodY) is not able to grow in media lacking Leu or Val, but is able to grow in media lacking Ile, Leu, and Val ( 74 ). These growth restoration phenotypes are most likely CodY dependent, requiring Ile depletion to relieve CodY repression of amino acid biosynthesis. Given that the results of DNA-binding assays are influenced by the conditions tested (i.e., pH or salt [ 75 ]), the growth assays are likely a more accurate representation of intracellular conditions. As such, this would suggest that, inside the cell, Ile is the predominant BCAA to affect CodY activity. When considered in the context of Gram-positive pathogens, in which CodY also regulates virulence gene expression, this suggests that Ile could serve as a host metabolic cue. Indeed, Ile availability influences virulence gene expression in L. monocytogenes and S. aureus (discussed in more detail below) ( 38 , 57 , 69 , 76 ). These advances have renewed interest in the factors that influence intracellular availability of Ile (and BCAAs in general) to better understand how pathogens scavenge BCAAs and where in the host they encounter BCAA limitation. An emerging role for isoleucine in regulating Gram-positive environmental adaptation. Historically, all three BCAAs have been considered equal CodY effectors. Indeed, all three bind to CodY and activate its DNA-binding activity ( 29 , 30 , 69 , 70 ), and yet, some studies have observed a stronger effect of Ile on CodY DNA-binding activity than either Leu or Val ( 31 , 71 – 73 ). A predominant role for Ile in regulating CodY activity during growth has also emerged. Depletion of Ile and not Leu or Val relieves repression of CodY-regulated genes in B. subtilis ( 29 ), L. monocytogenes ( 57 ), and S. aureus ( 38 , 69 ). Furthermore, as CodY target genes tend to comprise metabolic pathways, such as amino acid biosynthesis, the levels of Ile in the growth medium consequentially impact growth rate. Addition of excess Ile to the growth medium impairs growth of L. lactis ( 71 ), Streptococcus mutans ( 73 ), and S. aureus ( 38 ), whereas Ile depletion has the opposite effect. S. aureus growth is significantly impaired in media lacking Leu; however, its growth is restored upon simultaneous Ile and Leu depletion ( 69 ). An S. mutans mutant that is unable to synthesize (p)ppGpp (i.e., mimicking a strain with constitutively active CodY) is not able to grow in media lacking Leu or Val, but is able to grow in media lacking Ile, Leu, and Val ( 74 ). These growth restoration phenotypes are most likely CodY dependent, requiring Ile depletion to relieve CodY repression of amino acid biosynthesis. Given that the results of DNA-binding assays are influenced by the conditions tested (i.e., pH or salt [ 75 ]), the growth assays are likely a more accurate representation of intracellular conditions. As such, this would suggest that, inside the cell, Ile is the predominant BCAA to affect CodY activity. When considered in the context of Gram-positive pathogens, in which CodY also regulates virulence gene expression, this suggests that Ile could serve as a host metabolic cue. Indeed, Ile availability influences virulence gene expression in L. monocytogenes and S. aureus (discussed in more detail below) ( 38 , 57 , 69 , 76 ). These advances have renewed interest in the factors that influence intracellular availability of Ile (and BCAAs in general) to better understand how pathogens scavenge BCAAs and where in the host they encounter BCAA limitation. FACTORS THAT INFLUENCE INTRACELLULAR BCAA AVAILABILITY BCAA biosynthesis. BCAAs are synthesized through a conserved pathway in Gram-negative and Gram-positive bacteria ( Fig. 1 ), with the exception of the BCAA auxotrophs Erysipelothrix rhusiopathiae , Mycoplasma spp., Ureaplasma spp., Peptostreptococcus anaerobius , Streptococcus pyogenes , and Streptococcus agalactiae ( 1 ). For detailed biochemical descriptions of each of the biosynthetic enzymes and regulation of their activity, readers are directed to a recent review by Amorim Franco and Blanchard ( 77 ). The Leu biosynthetic genes are typically clustered in an operon ( leuABCD ) that may be either separate from the ilv genes or organized in a single ilv - leu operon, depending on the species. Several mechanisms of transcriptional regulation of these genes have been described, many involving transcriptional repression in response to BCAA availability. In Gram-negative bacteria, this is primarily mediated by attenuation. The attenuator region precedes the operon and is transcribed with the biosynthetic operon. It encodes a small BCAA-rich peptide, and as such, its rate of translation is determined by BCAA availability. When BCAA levels are high, the peptide is readily translated, allowing for a terminator secondary structure to form, which terminates transcription. When BCAA levels are low, the peptide is translated slowly, promoting an antiterminator secondary structure to form, allowing transcription to proceed. In Escherichia coli , the expression of many of the BCAA biosynthetic genes (organized as ilvGMEDA , leuABCD , ilvC , ilvIH , and ilvBN ) is controlled by attenuation. The ilvGMEDA operon is preceded by a 32-amino-acid (aa) peptide that contains 15 BCAAs ( 78 , 79 ), ilvBN is preceded by a 32-aa peptide with 11 BCAAs ( 80 ), and the leuABCD operon is preceded by a 28-aa peptide containing 4 Leu ( 81 ). Attenuation is also the primary mechanism of regulating the leu operon in Salmonella enterica serovar Typhimurium ( 82 , 83 ) and putative leader peptides and terminator hairpins are found upstream of BCAA biosynthesis genes across various Gram-negative species ( 84 ), suggesting that BCAA-dependent attenuation is a conserved mechanism. The biosynthetic genes in E. coli are also regulated by Lrp, which binds to the ilvIH and ilvG promoters in the absence of Leu to activate and repress transcription, respectively ( 85 – 87 ). In Gram-positive bacteria, multiple global regulators coordinate expression of the BCAA biosynthetic operon in response to not only BCAA availability but also carbon and nitrogen availability ( 88 ). In B. subtilis , the single ilv-leu operon is positively regulated by the carbon catabolite protein A (CcpA) ( 88 , 89 ), a global transcriptional regulator that regulates carbon utilization in response to a preferred carbon source ( 90 ). This positive regulation is antagonized by either TnrA, a regulator that responds to nitrogen limitation, or CodY ( 91 ). Together, this allows for conservation of carbon and nitrogen when exogenous BCAA sources are present. Additional fine-turning of ilv-leu expression is mediated by a Leu-responsive T-box riboswitch ( 88 , 92 – 94 ), as well as mRNA processing ( 95 ). CodY-dependent repression of BCAA biosynthesis is common across Gram-positive bacteria ( 32 , 37 , 38 , 51 , 52 , 56 ), whereas TnrA homologues are less conserved ( 96 ). In contrast to the tRNA-mediated attenuation observed in B. subtilis , recent experimental evidence implicates ribosome-mediated attenuation as an important mechanism regulating BCAA biosynthesis in S. aureus and L. monocytogenes ( 69 , 76 ). Furthermore, bioinformatics analysis of the leader sequence of the BCAA biosynthetic genes in L. lactis ( 97 ), Corynebacterium glutamicum ( 98 , 99 ), and Streptococcus spp. ( 100 ) have revealed BCAA-rich peptides and terminator hairpins consistent with attenuation, suggesting that ribosome-mediated attenuation is a common mechanism controlling transcription of the BCAA biosynthetic operon in Gram-positive bacteria. In S. aureus , an additional regulatory mechanism governing BCAA biosynthesis has been described involving repression by the essential Gcp/YeaZ complex ( 101 , 102 ). YeaZ binds upstream of the ilv-leu operon, suggesting possible direct repression of the operon ( 102 ), although the conditions that regulate its binding are currently unknown. The BCAA auxotrophy paradox. Even in the presence of intact biosynthesis genes, some Gram-positive bacteria synthesize little to no BCAAs in the absence of an exogenous source, with some even being misclassified as auxotrophs. L. monocytogenes synthesizes very little Ile, Leu, and Val despite possessing intact and functional biosynthetic genes ( 103 , 104 ). Similarly, S. pneumoniae is unable to grow in a chemically defined medium when Ile, Leu, or Val is omitted ( 105 ). Streptococcus suis exhibits a Leu auxotrophy despite possessing intact Leu biosynthesis genes and synthesizes moderate levels of Val but no detectable Ile ( 106 ). Despite possessing an intact BCAA biosynthetic operon, S. aureus exhibits a significant growth delay in the absence of Leu, and growth in the absence of Val occurs only upon accumulation of suppressor mutations ( 69 , 107 ). One possible explanation for an observed "auxotrophy" could be the availability of biosynthetic precursors. L. monocytogenes lacks a 2-oxoglutarate dehydrogenase enzyme and is therefore unable to derive oxaloacetate from the tricarboxylic acid (TCA) cycle ( Fig. 1 ). Instead, L. monocytogenes carboxylates pyruvate to form oxaloacetate in environments where glucose is the sole carbon source ( 103 ). Shortage of this indirect BCAA precursor might explain the limited amounts of BCAAs synthesized in this species ( 103 , 104 ). Another possible factor could be tight transcriptional repression of the biosynthetic operon. S. aureus is able to grow in the absence of Val only upon selection of strains with mutations that relieve either CodY-dependent repression or attenuator-dependent repression ( 69 ), indicating that the ilv-leu operon remains under tight repressive control even in the absence of Val. Repression is relieved upon Ile depletion via CodY and to a lesser extent, upon Leu depletion via transcriptional attenuation ( 69 ). Such tight control is also observed in L. monocytogenes , which also regulates BCAA biosynthesis in response to Ile via CodY-dependent repression and a BCAA-rich attenuator peptide ( 76 ). Specialized BCAA transporters. The repression of the BCAA biosynthetic genes in response to BCAA availability allows for conservation of carbon and nitrogen when an exogenous source can be acquired. Active transporters specific to BCAAs are common across bacteria and require either ATP or the proton motive force to import BCAAs. BCAA transporters in Gram-negative bacteria include the high-affinity LIV-I system and the low-affinity LIV-II and LIV-III systems ( Table 1 ). LIV-I is an ATP-binding cassette (ABC) transporter encoded by livJKHMGF ( 108 ). Two substrate binding proteins mediate BCAA transport through this system; LIV-B ( livJ ), which can bind all three BCAAs, and LS-B ( livK ), which is Leu specific ( 109 , 110 ). LIV-II, also known as BrnQ, is a permease with 12 transmembrane helices and belongs to the major facilitator superfamily (MFS). LIV-III is a permease homologous to LIV-II, with transport activity that is obviated only in a LIV-II deficient background in Salmonella Typhimurium and Pseudomonas aeruginosa ( 111 , 112 ). LIV-II and LIV-III use energy from the proton motive force to couple BCAA transport with Na + across an energy gradient ( 113 ). An additional MFS transporter specific to Ile transport and unique to Francisella tularensis has 12 transmembrane helices and belongs to the phagosomal nutrient transporter family of MFS transporters identified in Legionella pneumophila ( 114 , 115 ). TABLE 1 BCAA transporters Organism Transporter Energy source Specificity a Reference(s) Gram-negative bacteria Chlamydia trachomatis LIV-II ( brnQ ) PMF b ILV 156 Escherichia coli LIV-I ( livKHMGF ) ATP L 108–110 , 157 LIV-I ( livJHMGF ) ATP ILV LIV-II ( brnQ ) PMF ILV Francisella tularensis ileP PMF I 114 Pseudomonas aeruginosa LIV-I ATP ILV 158–162 LIV-II ( braB ) PMF ILV LIV-III ( braZ ) PMF ILV Salmonella Typhimurium LIV-I ATP ILV 111 , 163–166 LIV-II ( brnQ ) PMF ILV LIV-II PMF ILV Gram-positive bacteria Bacillus subtilis bcaP PMF ILV 118 braB PMF ILV brnQ PMF ILV Corynebacterium glutamicum brnQ PMF ILV 122 Lactobacillus delbrueckii brnQ PMF ILV 121 Lactococcus lactis brnQ PMF ILV 116 , 117 bcaP PMF ILV Staphylococcus aureus brnQ1 PMF ILV 107 , 119 brnQ2 PMF I brnQ3 NA c NA bcaP PMF ILV Streptococcus pneumoniae livAJHMGF ATP ILV 120 a I, isoleucine; L, leucine; V, valine. b PMF, proton motive force. c NA, not applicable. BrnQ also functions as a BCAA transporter in Gram-positive bacteria, along with a second nonhomologous permease, BcaP ( Table 1 ). L. lactis acquires BCAAs via both BcaP and BrnQ, with BcaP playing a more predominant role ( 116 , 117 ). Similarly, BcaP is the predominant transporter in B. subtilis , with two additional transporters, BrnQ and BraB (a BrnQ homologue), contributing to Ile and Val uptake and an unidentified transporter contributing to Leu uptake ( 118 ). In contrast, BrnQ1 serves as the predominant transporter for S. aureus growth, with BcaP playing a secondary role ( 107 ). S. aureus encodes two additional brnQ homologues: BrnQ2, an Ile-dedicated transporter, and BrnQ3, which has no observed BCAA transport function ( 107 , 119 ). A LIV-I system with a substrate binding protein able to bind BCAAs has been described in S. pneumoniae , although no transport function has yet been ascribed to this system ( 120 ). BrnQ also directs BCAA transport in Lactobacillus delbrueckii and C. glutamicum ( 121 , 122 ). BCAA biosynthesis. BCAAs are synthesized through a conserved pathway in Gram-negative and Gram-positive bacteria ( Fig. 1 ), with the exception of the BCAA auxotrophs Erysipelothrix rhusiopathiae , Mycoplasma spp., Ureaplasma spp., Peptostreptococcus anaerobius , Streptococcus pyogenes , and Streptococcus agalactiae ( 1 ). For detailed biochemical descriptions of each of the biosynthetic enzymes and regulation of their activity, readers are directed to a recent review by Amorim Franco and Blanchard ( 77 ). The Leu biosynthetic genes are typically clustered in an operon ( leuABCD ) that may be either separate from the ilv genes or organized in a single ilv - leu operon, depending on the species. Several mechanisms of transcriptional regulation of these genes have been described, many involving transcriptional repression in response to BCAA availability. In Gram-negative bacteria, this is primarily mediated by attenuation. The attenuator region precedes the operon and is transcribed with the biosynthetic operon. It encodes a small BCAA-rich peptide, and as such, its rate of translation is determined by BCAA availability. When BCAA levels are high, the peptide is readily translated, allowing for a terminator secondary structure to form, which terminates transcription. When BCAA levels are low, the peptide is translated slowly, promoting an antiterminator secondary structure to form, allowing transcription to proceed. In Escherichia coli , the expression of many of the BCAA biosynthetic genes (organized as ilvGMEDA , leuABCD , ilvC , ilvIH , and ilvBN ) is controlled by attenuation. The ilvGMEDA operon is preceded by a 32-amino-acid (aa) peptide that contains 15 BCAAs ( 78 , 79 ), ilvBN is preceded by a 32-aa peptide with 11 BCAAs ( 80 ), and the leuABCD operon is preceded by a 28-aa peptide containing 4 Leu ( 81 ). Attenuation is also the primary mechanism of regulating the leu operon in Salmonella enterica serovar Typhimurium ( 82 , 83 ) and putative leader peptides and terminator hairpins are found upstream of BCAA biosynthesis genes across various Gram-negative species ( 84 ), suggesting that BCAA-dependent attenuation is a conserved mechanism. The biosynthetic genes in E. coli are also regulated by Lrp, which binds to the ilvIH and ilvG promoters in the absence of Leu to activate and repress transcription, respectively ( 85 – 87 ). In Gram-positive bacteria, multiple global regulators coordinate expression of the BCAA biosynthetic operon in response to not only BCAA availability but also carbon and nitrogen availability ( 88 ). In B. subtilis , the single ilv-leu operon is positively regulated by the carbon catabolite protein A (CcpA) ( 88 , 89 ), a global transcriptional regulator that regulates carbon utilization in response to a preferred carbon source ( 90 ). This positive regulation is antagonized by either TnrA, a regulator that responds to nitrogen limitation, or CodY ( 91 ). Together, this allows for conservation of carbon and nitrogen when exogenous BCAA sources are present. Additional fine-turning of ilv-leu expression is mediated by a Leu-responsive T-box riboswitch ( 88 , 92 – 94 ), as well as mRNA processing ( 95 ). CodY-dependent repression of BCAA biosynthesis is common across Gram-positive bacteria ( 32 , 37 , 38 , 51 , 52 , 56 ), whereas TnrA homologues are less conserved ( 96 ). In contrast to the tRNA-mediated attenuation observed in B. subtilis , recent experimental evidence implicates ribosome-mediated attenuation as an important mechanism regulating BCAA biosynthesis in S. aureus and L. monocytogenes ( 69 , 76 ). Furthermore, bioinformatics analysis of the leader sequence of the BCAA biosynthetic genes in L. lactis ( 97 ), Corynebacterium glutamicum ( 98 , 99 ), and Streptococcus spp. ( 100 ) have revealed BCAA-rich peptides and terminator hairpins consistent with attenuation, suggesting that ribosome-mediated attenuation is a common mechanism controlling transcription of the BCAA biosynthetic operon in Gram-positive bacteria. In S. aureus , an additional regulatory mechanism governing BCAA biosynthesis has been described involving repression by the essential Gcp/YeaZ complex ( 101 , 102 ). YeaZ binds upstream of the ilv-leu operon, suggesting possible direct repression of the operon ( 102 ), although the conditions that regulate its binding are currently unknown. The BCAA auxotrophy paradox. Even in the presence of intact biosynthesis genes, some Gram-positive bacteria synthesize little to no BCAAs in the absence of an exogenous source, with some even being misclassified as auxotrophs. L. monocytogenes synthesizes very little Ile, Leu, and Val despite possessing intact and functional biosynthetic genes ( 103 , 104 ). Similarly, S. pneumoniae is unable to grow in a chemically defined medium when Ile, Leu, or Val is omitted ( 105 ). Streptococcus suis exhibits a Leu auxotrophy despite possessing intact Leu biosynthesis genes and synthesizes moderate levels of Val but no detectable Ile ( 106 ). Despite possessing an intact BCAA biosynthetic operon, S. aureus exhibits a significant growth delay in the absence of Leu, and growth in the absence of Val occurs only upon accumulation of suppressor mutations ( 69 , 107 ). One possible explanation for an observed "auxotrophy" could be the availability of biosynthetic precursors. L. monocytogenes lacks a 2-oxoglutarate dehydrogenase enzyme and is therefore unable to derive oxaloacetate from the tricarboxylic acid (TCA) cycle ( Fig. 1 ). Instead, L. monocytogenes carboxylates pyruvate to form oxaloacetate in environments where glucose is the sole carbon source ( 103 ). Shortage of this indirect BCAA precursor might explain the limited amounts of BCAAs synthesized in this species ( 103 , 104 ). Another possible factor could be tight transcriptional repression of the biosynthetic operon. S. aureus is able to grow in the absence of Val only upon selection of strains with mutations that relieve either CodY-dependent repression or attenuator-dependent repression ( 69 ), indicating that the ilv-leu operon remains under tight repressive control even in the absence of Val. Repression is relieved upon Ile depletion via CodY and to a lesser extent, upon Leu depletion via transcriptional attenuation ( 69 ). Such tight control is also observed in L. monocytogenes , which also regulates BCAA biosynthesis in response to Ile via CodY-dependent repression and a BCAA-rich attenuator peptide ( 76 ). Specialized BCAA transporters. The repression of the BCAA biosynthetic genes in response to BCAA availability allows for conservation of carbon and nitrogen when an exogenous source can be acquired. Active transporters specific to BCAAs are common across bacteria and require either ATP or the proton motive force to import BCAAs. BCAA transporters in Gram-negative bacteria include the high-affinity LIV-I system and the low-affinity LIV-II and LIV-III systems ( Table 1 ). LIV-I is an ATP-binding cassette (ABC) transporter encoded by livJKHMGF ( 108 ). Two substrate binding proteins mediate BCAA transport through this system; LIV-B ( livJ ), which can bind all three BCAAs, and LS-B ( livK ), which is Leu specific ( 109 , 110 ). LIV-II, also known as BrnQ, is a permease with 12 transmembrane helices and belongs to the major facilitator superfamily (MFS). LIV-III is a permease homologous to LIV-II, with transport activity that is obviated only in a LIV-II deficient background in Salmonella Typhimurium and Pseudomonas aeruginosa ( 111 , 112 ). LIV-II and LIV-III use energy from the proton motive force to couple BCAA transport with Na + across an energy gradient ( 113 ). An additional MFS transporter specific to Ile transport and unique to Francisella tularensis has 12 transmembrane helices and belongs to the phagosomal nutrient transporter family of MFS transporters identified in Legionella pneumophila ( 114 , 115 ). TABLE 1 BCAA transporters Organism Transporter Energy source Specificity a Reference(s) Gram-negative bacteria Chlamydia trachomatis LIV-II ( brnQ ) PMF b ILV 156 Escherichia coli LIV-I ( livKHMGF ) ATP L 108–110 , 157 LIV-I ( livJHMGF ) ATP ILV LIV-II ( brnQ ) PMF ILV Francisella tularensis ileP PMF I 114 Pseudomonas aeruginosa LIV-I ATP ILV 158–162 LIV-II ( braB ) PMF ILV LIV-III ( braZ ) PMF ILV Salmonella Typhimurium LIV-I ATP ILV 111 , 163–166 LIV-II ( brnQ ) PMF ILV LIV-II PMF ILV Gram-positive bacteria Bacillus subtilis bcaP PMF ILV 118 braB PMF ILV brnQ PMF ILV Corynebacterium glutamicum brnQ PMF ILV 122 Lactobacillus delbrueckii brnQ PMF ILV 121 Lactococcus lactis brnQ PMF ILV 116 , 117 bcaP PMF ILV Staphylococcus aureus brnQ1 PMF ILV 107 , 119 brnQ2 PMF I brnQ3 NA c NA bcaP PMF ILV Streptococcus pneumoniae livAJHMGF ATP ILV 120 a I, isoleucine; L, leucine; V, valine. b PMF, proton motive force. c NA, not applicable. BrnQ also functions as a BCAA transporter in Gram-positive bacteria, along with a second nonhomologous permease, BcaP ( Table 1 ). L. lactis acquires BCAAs via both BcaP and BrnQ, with BcaP playing a more predominant role ( 116 , 117 ). Similarly, BcaP is the predominant transporter in B. subtilis , with two additional transporters, BrnQ and BraB (a BrnQ homologue), contributing to Ile and Val uptake and an unidentified transporter contributing to Leu uptake ( 118 ). In contrast, BrnQ1 serves as the predominant transporter for S. aureus growth, with BcaP playing a secondary role ( 107 ). S. aureus encodes two additional brnQ homologues: BrnQ2, an Ile-dedicated transporter, and BrnQ3, which has no observed BCAA transport function ( 107 , 119 ). A LIV-I system with a substrate binding protein able to bind BCAAs has been described in S. pneumoniae , although no transport function has yet been ascribed to this system ( 120 ). BrnQ also directs BCAA transport in Lactobacillus delbrueckii and C. glutamicum ( 121 , 122 ). BCAAS AT THE CROSSROADS OF METABOLISM AND VIRULENCE Mechanisms that support growth during infection. BCAA availability at various infection sites remains undefined; however, both BCAA biosynthesis and transport have been linked to promoting the virulence of pathogens during infection, suggesting that pathogens encounter BCAA limitation in vivo ( 120 , 123 – 129 ). Concentrations of BCAAs have been estimated in some host environments relevant to pathogens. Levels of BCAAs in the bloodstream range from 20 to 92 µM for Ile, 40 to 250 µM for Leu, and 65 to 266 µM for Val ( 130 ). Human nasal secretions contain Leu levels in the range of 130 to 287 µM, Val levels in the range of 13 to 156 µM, and very little or no Ile ( 131 ). Indeed, some pathogens exploit these extracellular BCAA sources during infection. S. aureus requires both the BrnQ1 and BcaP transporters for optimal fitness during systemic infection and nasal colonization ( 107 , 119 ). BCAA acquisition also likely contributes to S. aureus lung infection, as transport genes are upregulated in a pneumonia model ( 132 ). The contribution of BCAA biosynthesis to S. aureus growth in vivo remains to be determined, although biosynthesis likely plays a role in maintaining BCAA levels, as the biosynthetic genes are upregulated when S. aureus is grown in blood, the lung environment, and in nasal secretions ( 131 – 133 ). In S. pneumoniae , BCAA transport supports growth in a systemic infection model, pneumonia model, and meningitis model, but not in a colonization model ( 120 , 129 ), whereas BCAA biosynthesis is required for invasion of host tissue following intranasal colonization, but is not required for systemic infection ( 134 ). BCAA transport also contributes to growth of Yersinia pestis during systemic infection ( 128 ). Despite some pathogens being able to exploit extracellular BCAAs, some pathogens require BCAA biosynthesis for infection, including Klebsiella pneumoniae , Neisseria meningitidis , and P. aeruginosa ( 123 , 124 , 135 ). Intracellular pathogens face the challenge of direct competition with the host for intracellular BCAAs since they are essential nutrients in humans. Indeed, several intracellular pathogens, including Burkholderia pseudomallei , Mycobacterium bovis , Mycobacterium tuberculosis , and L. monocytogenes rely on BCAA biosynthesis for replication inside host cells ( 136 – 140 ). Yet, some intracellular pathogens are auxotrophic for BCAAs and therefore necessitate transporters to obtain BCAAs. The BCAA auxotroph Legionella pneumophila requires the Val transporter PhtJ for intracellular growth ( 115 , 141 ), and pathogenic subspecies of Francisella , which have lost the capacity to synthesize BCAAs, require the Ile transporter IleP for intracellular replication and infection in vivo ( 114 ). Interestingly, F. tularensis has been observed to induce a transient increase in cytosolic BCAA concentrations following infection, suggesting that it might manipulate host metabolism to support its own growth during infection ( 114 ). If deprived of BCAAs, Gram-positive pathogens face challenges not only in supporting protein synthesis and growth, but also in maintaining the appropriate BCFA content to protect against host defenses that target the bacterial membrane. A role for BCFA synthesis in promoting resistance to host defenses is best highlighted in L. monocytogenes , where BCFAs comprise 75 to 98% of the membrane ( 13 , 14 , 16 , 21 , 142 ). BCFA-deficient strains have increased susceptibility to antimicrobial peptide killing and lysozyme digestion and decreased production of the virulence factor listeriolysin O ( 143 ), all of which likely contribute to the decreased intracellular growth and virulence of a strain deficient of BCFAs ( 143 , 144 ). In S. aureus , BCFAs comprise 44 to 63% of the membrane and a BCFA-deficient strain exhibits reduced adherence to host cells and is attenuated in vivo ( 107 , 145 , 146 ). While these studies reveal that BCAA deprivation limits pathogen metabolism and physiology in vivo , more research is needed to elucidate the relative importance of biosynthesis versus acquisition in various host niches and how this source preference might be regulated. One mechanism governing source preference involves positioning of the gene in the hierarchy of the CodY regulon. The BCAA biosynthetic operon and the transporter genes in B. subtilis are both controlled by CodY. CodY binds to a 15-nucleotide (nt) binding motif, AATTTTCWGAAAATT ( 71 , 147 ), and nucleotide substitutions in the motif that deviate from the consensus sequence decrease the binding affinity of CodY ( 147 , 148 ). The binding strength of the motif thus correlates with the extent of repression and dictates the positioning of the gene within the hierarchy of graded target gene derepression ( 3 ). In B. subtilis , BCAA biosynthesis and transport are derepressed at similar points along the spectrum of CodY activity ( 3 ), but in S. aureus , in which this hierarchical response is also observed, the BCAA transporter brnQ2 is derepressed upon modest decreases in CodY activity, whereas BCAA biosynthesis remains repressed at this same level of CodY inactivation ( 5 ), suggesting that S. aureus prioritizes nutrient scavenging upon modest nutrient depletion to prevent unnecessary divergence of carbon and nitrogen to nutrient synthesis. The positioning of other transcriptional regulators in the CodY hierarchy adds an additional layer of metabolic fine-tuning. For example, CodY is a direct repressor of braB , a BCAA transporter in B. subtilis , but it is a stronger repressor of scoC , which also represses braB expression. braB expression is therefore optimal at intermediate levels of CodY activity, when CodY-dependent braB repression is partially relieved, and scoC remains repressed ( 149 ). Such precise sensitivity to CodY activity represents one way by which pathogens can fine-tune the coordination of nutrient acquisition strategies. BCAAs as host cues to regulate virulence. The predominant role of CodY as a negative regulator of virulence and the hierarchical regulation of CodY target genes in S. aureus is but one example of how pathogens respond to BCAA starvation and adapt to their environment. In some pathogens, it has been shown that CodY can also function as a positive regulator. Table 2 outlines the positive and/or negative regulatory role of CodY on notable virulence factors in several Gram-positive pathogens. To fully appreciate the complexity of pathogen-specific CodY responses, this section will contrast the predominant repressive role of CodY in S. aureus to the complex role of CodY as both a positive and negative regulator of virulence in L. monocytogenes (summarized in Fig. 3 ). The advancements made in these pathogens reveal that pathogen lifestyle might have influenced the regulatory response coordinated in response to BCAA availability. TABLE 2 Regulation of virulence by CodY in Gram-positive pathogens Organism Phenotype of codY mutant in vivo Notable virulence gene regulation Reference(s) Staphylococcus aureus Hypervirulent in murine skin abscess and pneumonia; no effect on systemic infection Indirect repression of delta-toxin/RNAIII via repression of agr activator; direct repression of biofilm synthesis ( icaADBC ), alpha-toxin ( hla ), hyaluronidase ( hysA ), Panton-Valentine leucocidin ( lukSF-PV ) 37 , 59 , 167 , 168 Streptococcus pneumoniae Reduced colonization; no effect on systemic infection Direct activation of adhesion protein choline-binding protein ( pcpA ) 32 Bacillus anthracis Attenuated virulence in murine toxinogenic model Indirect activation of anthrax toxin components ( cya , lef , pagA ) and direct repression of S layer proteins ( sap , eag ) via AtxA; activation of iron scavenging systems 46–48 Clostridium perfringens Type D NT a Direct and indirect activation of epsilon toxin (ETX); repression of sporulation 41 , 54 Type A Activation of sporulation and enterotoxin (CPE) 55 Bacillus cereus (F4810/72) Attenuated virulence in Galleria mellonella infection model Indirect activation of cytotoxin ( cytK ), enterotoxin ( nhe ), and hemolysin ( hbl ) via direct activation of regulator plcR ; direct repression of cereulide ( cesPTABCD ) and inhibitor metalloprotease 1 ( inhA1 ) 50 Clostridium difficile NT Indirect repression of toxin A ( tcdA ) and B ( tcdB ) via direct repression of tcdR 53 Listeria monocytogenes Attenuated virulence in murine systemic infection model Indirect activation of listeriolysin O ( hyl ) via direct activation of regulator prfA ; direct activation of flagellar biosynthesis and ActA 35 , 39 , 56 , 57 Streptococcus pyogenes NT Indirect activation of surface proteins via activation of regulator mga ; activation of regulators fasX and pel / sagA 61 , 62 a NT, not tested. FIG 3 CodY regulation of virulence genes in Staphylococcus aureus and Listeria monocytogenes . CodY functions primarily as a repressor in S. aureus , and its target genes are repressed in the presence of Ile and expressed as a hierarchy upon Ile depletion (black line). Some target genes are activated by CodY in S. aureus and are expressed in the presence of Ile and repressed upon Ile depletion (red line). In L. monocytogenes , CodY functions as both an activator and a repressor under both high- and low-Ile conditions. Under high-Ile conditions, CodY acts as an activator (red line). CodY also functions as an activator under low-Ile conditions (red line) and induces expression of virulence genes. Black lines indicate genes that are repressed by CodY, and red lines indicate genes that are activated by CodY. The thickness of the line corresponds to the relative proportion of genes in that category. In S. aureus , CodY binds to DNA under BCAA-replete conditions and primarily acts as a repressor of virulence genes ( 5 , 37 , 38 ). CodY regulates approximately 5% of the S. aureus genome, with the majority of its targets (85%) subject to repression by CodY ( 37 , 38 ). These targets include virulence genes, such as the capsule genes, α-hemolysin and adhesion protein genes, as well as regulators of virulence gene expression, including the agr locus and saeRS two-component system ( 60 , 150 ). Most of the virulence genes are directly repressed by CodY, whereas others, including the capsule genes and hemolysin genes, are indirectly activated through agr ( 37 , 38 ). The genes activated by CodY, which fall into the categories of nucleotide transport/metabolism and adhesion proteins, do not have a CodY binding sequence, suggesting an indirect mechanism of regulation ( 37 ). The coordination of virulence gene expression with the environment is crucial for S. aureus to limit unwanted host damage, as exemplified by the hypervirulence of a codY mutant in a skin abscess and pneumonia model of infection ( 59 ). To ensure the appropriate expression of virulence genes, the CodY regulon is expressed as a hierarchy that depends on the extent of CodY activation and therefore nutrient (e.g., Ile) availability ( Fig. 3 ) ( 5 ). The graded response prioritizes expression of amino acid and peptide transport over synthesis upon modest nutrient limitation and reserves expression of hydrolytic enzyme and toxin production for more severe nutrient limitation ( Fig. 3 ) ( 5 ). Also within this spectrum are other virulence gene regulators, including the agr and sae loci, which together form a regulatory cascade that integrates several environmental cues, such as growth phase and host defenses ( 5 , 150 , 151 ). Together, the graded response and regulatory cascade are thought to maximize nutrient acquisition while limiting host toxicity ( 5 ). In contrast to S. aureus , CodY in L. monocytogenes can bind to DNA under both BCAA-replete and BCAA-depleted conditions and can function as both a repressor and activator ( 35 , 39 ). CodY directly or indirectly regulates approximately 14% of L. monocytogenes genes. Approximately 66% of these genes are upregulated in a codY mutant compared to the wild-type strain when grown in nutrient-rich medium (i.e., BCAA replete), consistent with the role of CodY as a repressor ( Fig. 3 ) ( 39 ). The repressed genes are primarily involved in nutrient metabolism and transport and stress responses and include some virulence factors. The remaining 33% of differentially regulated genes that are downregulated in a codY mutant revealed an underappreciated role for CodY as an activator in this organism ( 39 ). The genes that are activated by CodY under these conditions include the arginine biosynthesis pathway and flagellar biosynthesis genes. Interestingly, CodY also acts as an activator under BCAA-depleted conditions, with approximately 30% of differentially regulated genes downregulated in a codY mutant compared to the wild-type strain when grown in BCAA-limited growth medium ( Fig. 3 ). Under BCAA-depleted conditions, CodY is a direct activator of prfA , a global virulence gene regulator in L. monocytogenes ( 35 ). This leads to activation of PrfA-regulated virulence factors, including listeriolysin O and the surface protein ActA, which are important for intracellular replication and cell-to-cell spread, respectively ( 57 , 152 , 153 ). Consequently, an L. monocytogenes codY mutant is impaired in motility and intracellular replication ( 35 , 39 ). The role of CodY during L. monocytogenes infection is more difficult to discern, because although a codY mutant is attenuated in vivo in comparison to a wild-type (WT) strain, implicating CodY as an activator of virulence, a codY mutation rescues the virulence of a strain where CodY is constitutively active (i.e., a relA mutant), also demonstrating its role as a repressor of virulence ( 35 , 56 ). It is therefore challenging to classify CodY as either a repressor or activator of virulence in this organism. Rather its overall impact on virulence will depend on the resulting gene expression profile at any given CodY activation state. The advancements made in these pathogens highlight how BCAA (namely, Ile) limitation is linked to promoting virulence of both organisms but via distinct mechanisms: that is, via CodY-dependent repression of virulence genes in S. aureus and CodY-dependent activation and/or repression of virulence genes in L. monocytogenes ( 5 , 39 ). As such, virulence is significantly influenced by CodY activity, such that two distinct lifestyles (i.e., nontoxic versus toxic for S. aureus and motile versus cytosolic replication for L. monocytogenes ) are displayed at either end of the spectrum of BCAA concentrations, suggesting that even modest changes in intracellular Ile concentrations can have drastic consequences for virulence. It is not surprising, then, that two recent studies have uncovered that both pathogens tightly regulate BCAA biosynthesis via a shared mechanism resulting in a BCAA "auxotrophy" phenotype, which might allow them to increase their capacity to respond to a wider range of BCAA levels to reduce the likelihood of untimely virulence determinant expression ( 69 , 76 ). BCAA auxotrophy: a metabolic strategy to promote environmental adaptation? As discussed in a previous section, both S. aureus and L. monocytogenes require the addition of BCAAs to the growth medium to support growth due to minimal levels of BCAA biosynthesis ( 69 , 104 , 107 , 119 ). Two mechanisms of repression control this: (i) Ile-dependent CodY repression and (ii) a cis- acting BCAA-dependent attenuator ( 69 , 76 ). CodY represses the attenuator and the ilv-leu operon under high-Ile conditions to limit BCAA biosynthesis. As Ile is depleted, repression is relieved; however, the attenuator further regulates the levels of ilv-leu transcripts in response to Leu and Ile availability in S. aureus and all three BCAAs in L. monocytogenes ( 69 , 76 ). This additional "checkpoint" in repression is thought to delay the repletion of BCAAs, extending the range of BCAA starvation and therefore the range of CodY activation states. Indeed, an L. monocytogenes strain lacking the attenuator-dependent repression synthesizes more BCAAs and exhibits reduced expression of the virulence gene regulator prfA in comparison to the wild-type strain ( 76 ); that is to say, prompt repletion of BCAAs via endogenous synthesis prevents the cells from reaching a state of BCAA deprivation necessary for CodY to bind to the prfA promoter and activate its transcription. As discussed previously, several Gram-positive bacteria display little to no BCAA biosynthesis, suggesting that this might represent a metabolic strategy to better coordinate virulence gene expression in response to nutritional cues in the environment. Mechanisms that support growth during infection. BCAA availability at various infection sites remains undefined; however, both BCAA biosynthesis and transport have been linked to promoting the virulence of pathogens during infection, suggesting that pathogens encounter BCAA limitation in vivo ( 120 , 123 – 129 ). Concentrations of BCAAs have been estimated in some host environments relevant to pathogens. Levels of BCAAs in the bloodstream range from 20 to 92 µM for Ile, 40 to 250 µM for Leu, and 65 to 266 µM for Val ( 130 ). Human nasal secretions contain Leu levels in the range of 130 to 287 µM, Val levels in the range of 13 to 156 µM, and very little or no Ile ( 131 ). Indeed, some pathogens exploit these extracellular BCAA sources during infection. S. aureus requires both the BrnQ1 and BcaP transporters for optimal fitness during systemic infection and nasal colonization ( 107 , 119 ). BCAA acquisition also likely contributes to S. aureus lung infection, as transport genes are upregulated in a pneumonia model ( 132 ). The contribution of BCAA biosynthesis to S. aureus growth in vivo remains to be determined, although biosynthesis likely plays a role in maintaining BCAA levels, as the biosynthetic genes are upregulated when S. aureus is grown in blood, the lung environment, and in nasal secretions ( 131 – 133 ). In S. pneumoniae , BCAA transport supports growth in a systemic infection model, pneumonia model, and meningitis model, but not in a colonization model ( 120 , 129 ), whereas BCAA biosynthesis is required for invasion of host tissue following intranasal colonization, but is not required for systemic infection ( 134 ). BCAA transport also contributes to growth of Yersinia pestis during systemic infection ( 128 ). Despite some pathogens being able to exploit extracellular BCAAs, some pathogens require BCAA biosynthesis for infection, including Klebsiella pneumoniae , Neisseria meningitidis , and P. aeruginosa ( 123 , 124 , 135 ). Intracellular pathogens face the challenge of direct competition with the host for intracellular BCAAs since they are essential nutrients in humans. Indeed, several intracellular pathogens, including Burkholderia pseudomallei , Mycobacterium bovis , Mycobacterium tuberculosis , and L. monocytogenes rely on BCAA biosynthesis for replication inside host cells ( 136 – 140 ). Yet, some intracellular pathogens are auxotrophic for BCAAs and therefore necessitate transporters to obtain BCAAs. The BCAA auxotroph Legionella pneumophila requires the Val transporter PhtJ for intracellular growth ( 115 , 141 ), and pathogenic subspecies of Francisella , which have lost the capacity to synthesize BCAAs, require the Ile transporter IleP for intracellular replication and infection in vivo ( 114 ). Interestingly, F. tularensis has been observed to induce a transient increase in cytosolic BCAA concentrations following infection, suggesting that it might manipulate host metabolism to support its own growth during infection ( 114 ). If deprived of BCAAs, Gram-positive pathogens face challenges not only in supporting protein synthesis and growth, but also in maintaining the appropriate BCFA content to protect against host defenses that target the bacterial membrane. A role for BCFA synthesis in promoting resistance to host defenses is best highlighted in L. monocytogenes , where BCFAs comprise 75 to 98% of the membrane ( 13 , 14 , 16 , 21 , 142 ). BCFA-deficient strains have increased susceptibility to antimicrobial peptide killing and lysozyme digestion and decreased production of the virulence factor listeriolysin O ( 143 ), all of which likely contribute to the decreased intracellular growth and virulence of a strain deficient of BCFAs ( 143 , 144 ). In S. aureus , BCFAs comprise 44 to 63% of the membrane and a BCFA-deficient strain exhibits reduced adherence to host cells and is attenuated in vivo ( 107 , 145 , 146 ). While these studies reveal that BCAA deprivation limits pathogen metabolism and physiology in vivo , more research is needed to elucidate the relative importance of biosynthesis versus acquisition in various host niches and how this source preference might be regulated. One mechanism governing source preference involves positioning of the gene in the hierarchy of the CodY regulon. The BCAA biosynthetic operon and the transporter genes in B. subtilis are both controlled by CodY. CodY binds to a 15-nucleotide (nt) binding motif, AATTTTCWGAAAATT ( 71 , 147 ), and nucleotide substitutions in the motif that deviate from the consensus sequence decrease the binding affinity of CodY ( 147 , 148 ). The binding strength of the motif thus correlates with the extent of repression and dictates the positioning of the gene within the hierarchy of graded target gene derepression ( 3 ). In B. subtilis , BCAA biosynthesis and transport are derepressed at similar points along the spectrum of CodY activity ( 3 ), but in S. aureus , in which this hierarchical response is also observed, the BCAA transporter brnQ2 is derepressed upon modest decreases in CodY activity, whereas BCAA biosynthesis remains repressed at this same level of CodY inactivation ( 5 ), suggesting that S. aureus prioritizes nutrient scavenging upon modest nutrient depletion to prevent unnecessary divergence of carbon and nitrogen to nutrient synthesis. The positioning of other transcriptional regulators in the CodY hierarchy adds an additional layer of metabolic fine-tuning. For example, CodY is a direct repressor of braB , a BCAA transporter in B. subtilis , but it is a stronger repressor of scoC , which also represses braB expression. braB expression is therefore optimal at intermediate levels of CodY activity, when CodY-dependent braB repression is partially relieved, and scoC remains repressed ( 149 ). Such precise sensitivity to CodY activity represents one way by which pathogens can fine-tune the coordination of nutrient acquisition strategies. BCAAs as host cues to regulate virulence. The predominant role of CodY as a negative regulator of virulence and the hierarchical regulation of CodY target genes in S. aureus is but one example of how pathogens respond to BCAA starvation and adapt to their environment. In some pathogens, it has been shown that CodY can also function as a positive regulator. Table 2 outlines the positive and/or negative regulatory role of CodY on notable virulence factors in several Gram-positive pathogens. To fully appreciate the complexity of pathogen-specific CodY responses, this section will contrast the predominant repressive role of CodY in S. aureus to the complex role of CodY as both a positive and negative regulator of virulence in L. monocytogenes (summarized in Fig. 3 ). The advancements made in these pathogens reveal that pathogen lifestyle might have influenced the regulatory response coordinated in response to BCAA availability. TABLE 2 Regulation of virulence by CodY in Gram-positive pathogens Organism Phenotype of codY mutant in vivo Notable virulence gene regulation Reference(s) Staphylococcus aureus Hypervirulent in murine skin abscess and pneumonia; no effect on systemic infection Indirect repression of delta-toxin/RNAIII via repression of agr activator; direct repression of biofilm synthesis ( icaADBC ), alpha-toxin ( hla ), hyaluronidase ( hysA ), Panton-Valentine leucocidin ( lukSF-PV ) 37 , 59 , 167 , 168 Streptococcus pneumoniae Reduced colonization; no effect on systemic infection Direct activation of adhesion protein choline-binding protein ( pcpA ) 32 Bacillus anthracis Attenuated virulence in murine toxinogenic model Indirect activation of anthrax toxin components ( cya , lef , pagA ) and direct repression of S layer proteins ( sap , eag ) via AtxA; activation of iron scavenging systems 46–48 Clostridium perfringens Type D NT a Direct and indirect activation of epsilon toxin (ETX); repression of sporulation 41 , 54 Type A Activation of sporulation and enterotoxin (CPE) 55 Bacillus cereus (F4810/72) Attenuated virulence in Galleria mellonella infection model Indirect activation of cytotoxin ( cytK ), enterotoxin ( nhe ), and hemolysin ( hbl ) via direct activation of regulator plcR ; direct repression of cereulide ( cesPTABCD ) and inhibitor metalloprotease 1 ( inhA1 ) 50 Clostridium difficile NT Indirect repression of toxin A ( tcdA ) and B ( tcdB ) via direct repression of tcdR 53 Listeria monocytogenes Attenuated virulence in murine systemic infection model Indirect activation of listeriolysin O ( hyl ) via direct activation of regulator prfA ; direct activation of flagellar biosynthesis and ActA 35 , 39 , 56 , 57 Streptococcus pyogenes NT Indirect activation of surface proteins via activation of regulator mga ; activation of regulators fasX and pel / sagA 61 , 62 a NT, not tested. FIG 3 CodY regulation of virulence genes in Staphylococcus aureus and Listeria monocytogenes . CodY functions primarily as a repressor in S. aureus , and its target genes are repressed in the presence of Ile and expressed as a hierarchy upon Ile depletion (black line). Some target genes are activated by CodY in S. aureus and are expressed in the presence of Ile and repressed upon Ile depletion (red line). In L. monocytogenes , CodY functions as both an activator and a repressor under both high- and low-Ile conditions. Under high-Ile conditions, CodY acts as an activator (red line). CodY also functions as an activator under low-Ile conditions (red line) and induces expression of virulence genes. Black lines indicate genes that are repressed by CodY, and red lines indicate genes that are activated by CodY. The thickness of the line corresponds to the relative proportion of genes in that category. In S. aureus , CodY binds to DNA under BCAA-replete conditions and primarily acts as a repressor of virulence genes ( 5 , 37 , 38 ). CodY regulates approximately 5% of the S. aureus genome, with the majority of its targets (85%) subject to repression by CodY ( 37 , 38 ). These targets include virulence genes, such as the capsule genes, α-hemolysin and adhesion protein genes, as well as regulators of virulence gene expression, including the agr locus and saeRS two-component system ( 60 , 150 ). Most of the virulence genes are directly repressed by CodY, whereas others, including the capsule genes and hemolysin genes, are indirectly activated through agr ( 37 , 38 ). The genes activated by CodY, which fall into the categories of nucleotide transport/metabolism and adhesion proteins, do not have a CodY binding sequence, suggesting an indirect mechanism of regulation ( 37 ). The coordination of virulence gene expression with the environment is crucial for S. aureus to limit unwanted host damage, as exemplified by the hypervirulence of a codY mutant in a skin abscess and pneumonia model of infection ( 59 ). To ensure the appropriate expression of virulence genes, the CodY regulon is expressed as a hierarchy that depends on the extent of CodY activation and therefore nutrient (e.g., Ile) availability ( Fig. 3 ) ( 5 ). The graded response prioritizes expression of amino acid and peptide transport over synthesis upon modest nutrient limitation and reserves expression of hydrolytic enzyme and toxin production for more severe nutrient limitation ( Fig. 3 ) ( 5 ). Also within this spectrum are other virulence gene regulators, including the agr and sae loci, which together form a regulatory cascade that integrates several environmental cues, such as growth phase and host defenses ( 5 , 150 , 151 ). Together, the graded response and regulatory cascade are thought to maximize nutrient acquisition while limiting host toxicity ( 5 ). In contrast to S. aureus , CodY in L. monocytogenes can bind to DNA under both BCAA-replete and BCAA-depleted conditions and can function as both a repressor and activator ( 35 , 39 ). CodY directly or indirectly regulates approximately 14% of L. monocytogenes genes. Approximately 66% of these genes are upregulated in a codY mutant compared to the wild-type strain when grown in nutrient-rich medium (i.e., BCAA replete), consistent with the role of CodY as a repressor ( Fig. 3 ) ( 39 ). The repressed genes are primarily involved in nutrient metabolism and transport and stress responses and include some virulence factors. The remaining 33% of differentially regulated genes that are downregulated in a codY mutant revealed an underappreciated role for CodY as an activator in this organism ( 39 ). The genes that are activated by CodY under these conditions include the arginine biosynthesis pathway and flagellar biosynthesis genes. Interestingly, CodY also acts as an activator under BCAA-depleted conditions, with approximately 30% of differentially regulated genes downregulated in a codY mutant compared to the wild-type strain when grown in BCAA-limited growth medium ( Fig. 3 ). Under BCAA-depleted conditions, CodY is a direct activator of prfA , a global virulence gene regulator in L. monocytogenes ( 35 ). This leads to activation of PrfA-regulated virulence factors, including listeriolysin O and the surface protein ActA, which are important for intracellular replication and cell-to-cell spread, respectively ( 57 , 152 , 153 ). Consequently, an L. monocytogenes codY mutant is impaired in motility and intracellular replication ( 35 , 39 ). The role of CodY during L. monocytogenes infection is more difficult to discern, because although a codY mutant is attenuated in vivo in comparison to a wild-type (WT) strain, implicating CodY as an activator of virulence, a codY mutation rescues the virulence of a strain where CodY is constitutively active (i.e., a relA mutant), also demonstrating its role as a repressor of virulence ( 35 , 56 ). It is therefore challenging to classify CodY as either a repressor or activator of virulence in this organism. Rather its overall impact on virulence will depend on the resulting gene expression profile at any given CodY activation state. The advancements made in these pathogens highlight how BCAA (namely, Ile) limitation is linked to promoting virulence of both organisms but via distinct mechanisms: that is, via CodY-dependent repression of virulence genes in S. aureus and CodY-dependent activation and/or repression of virulence genes in L. monocytogenes ( 5 , 39 ). As such, virulence is significantly influenced by CodY activity, such that two distinct lifestyles (i.e., nontoxic versus toxic for S. aureus and motile versus cytosolic replication for L. monocytogenes ) are displayed at either end of the spectrum of BCAA concentrations, suggesting that even modest changes in intracellular Ile concentrations can have drastic consequences for virulence. It is not surprising, then, that two recent studies have uncovered that both pathogens tightly regulate BCAA biosynthesis via a shared mechanism resulting in a BCAA "auxotrophy" phenotype, which might allow them to increase their capacity to respond to a wider range of BCAA levels to reduce the likelihood of untimely virulence determinant expression ( 69 , 76 ). BCAA auxotrophy: a metabolic strategy to promote environmental adaptation? As discussed in a previous section, both S. aureus and L. monocytogenes require the addition of BCAAs to the growth medium to support growth due to minimal levels of BCAA biosynthesis ( 69 , 104 , 107 , 119 ). Two mechanisms of repression control this: (i) Ile-dependent CodY repression and (ii) a cis- acting BCAA-dependent attenuator ( 69 , 76 ). CodY represses the attenuator and the ilv-leu operon under high-Ile conditions to limit BCAA biosynthesis. As Ile is depleted, repression is relieved; however, the attenuator further regulates the levels of ilv-leu transcripts in response to Leu and Ile availability in S. aureus and all three BCAAs in L. monocytogenes ( 69 , 76 ). This additional "checkpoint" in repression is thought to delay the repletion of BCAAs, extending the range of BCAA starvation and therefore the range of CodY activation states. Indeed, an L. monocytogenes strain lacking the attenuator-dependent repression synthesizes more BCAAs and exhibits reduced expression of the virulence gene regulator prfA in comparison to the wild-type strain ( 76 ); that is to say, prompt repletion of BCAAs via endogenous synthesis prevents the cells from reaching a state of BCAA deprivation necessary for CodY to bind to the prfA promoter and activate its transcription. As discussed previously, several Gram-positive bacteria display little to no BCAA biosynthesis, suggesting that this might represent a metabolic strategy to better coordinate virulence gene expression in response to nutritional cues in the environment. CONCLUSIONS AND FUTURE DIRECTIONS Recent advancements in the area of BCAA metabolism have identified the importance of BCAA acquisition and synthesis for pathogen growth in vivo and have revealed that a pathogen's preferred strategy reflects its unique physiological needs and host tissue preferences. An emerging theme is that regulation of nutrient source preference is critical to maintaining tight control over intracellular concentrations of BCAAs and ensures that pathogens are responsive to fluctuations in these levels and therefore are able to initiate the appropriate adaptive response, which can have significant consequences for virulence. Therefore, future studies should continue to focus on identifying how each of these mechanisms influences intracellular pools of BCAAs. In Gram-positive bacteria, this includes evaluating how manipulation of BCAA transporters and/or biosynthesis influences intracellular levels of Ile and, subsequently, CodY activity. In S. aureus , depletion of exogenous Ile has a significant impact on CodY activity, and therefore CodY target genes are derepressed in a strain lacking the Ile transporter BrnQ2 ( 69 ). Similarly, mutation of BCAA transporters in B. subtilis leads to a decrease in CodY activation, although the sole contributions of each of the three transporters remain to be determined ( 118 ). Levels of endogenous synthesis, too, impact CodY activity in L. monocytogenes and B. subtilis ( 29 , 76 ). In Gram-negative bacteria, the role of BCAA deprivation in regulating virulence remains to be explored. Evidence in the Gram-negative swine pathogen Actinobacillus pleuropneumoniae suggests the existence of a parallel response. A. pleuropneumoniae encounters BCAA limitation in the porcine lung and requires BCAA biosynthesis for full virulence in this environment ( 154 ). Furthermore, BCAA deprivation triggers upregulation of not only BCAA biosynthesis, but also several genes that are upregulated during infection, some of which have putative Lrp binding sites ( 155 ). This suggests that BCAA deprivation, sensed via Lrp, might also act as an important environmental cue for Gram-negative pathogens. Future studies will provide more insight into how pathogens obtain BCAAs and how they regulate their intracellular levels to promote their survival and ability to cause disease.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194065/

A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site containing multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention. Introduction It is believed that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously termed nCoV-2019) was introduced into the human population from a poorly characterized animal reservoir in late 2019 ( Ge et al., 2013 , Wang et al., 2020 , Zhou et al., 2020b , Zhu et al., 2020 ). The epicenter of the subsequent SARS-CoV-2 spread was Wuhan, Hubei province, China, with more than 65,000 cases occurring in this area ( WHO, 2020a ). However, infections have now been detected in more than 110 countries and massive outbreaks are currently ongoing in the United States, Italy, and Spain ( WHO, 2020a , WHO, 2020b ). Understanding which features of SARS-CoV-2 are essential for infection of human cells should provide insights into viral transmissibility and pathogenesis and might reveal targets for intervention. The spike protein of coronaviruses is incorporated into the viral envelope and facilitates viral entry into target cells. For this, the surface unit S1 binds to a cellular receptor while the transmembrane unit S2 facilitates fusion of the viral membrane with a cellular membrane ( Hoffmann et al., 2018 , Hulswit et al., 2016 , Millet and Whittaker, 2018 ). Membrane fusion depends on S protein cleavage by host cell proteases at the S1/S2 and the S2′ site ( Figure 1 A), which results in S protein activation ( Hoffmann et al., 2018 , Hulswit et al., 2016 , Millet and Whittaker, 2018 ). Cleavage of the S protein can occur in the constitutive secretory pathway of infected cells or during viral entry into target cells and is essential for viral infectivity. Therefore, the responsible enzymes constitute potential targets for antiviral intervention. Figure 1 The Multibasic Motif at the S1/S2 Cleavage Site of SARS-2-S Is Unique among Related Group 2b Betacoronaviruses (A) Schematic illustration of a coronavirus spike glycoprotein in which functional domains and cleavage sites are highlighted (RBD, receptor-binding domain; RBM, receptor-binding motif; TD, transmembrane domain). (B) Protein models for SARS-S and SARS-2-S based on the PDB: 5X5B structure ( Yuan et al., 2017 ) as a template. Colored in red are the S1/S2 and S2′ cleavage sites. Further, the S1 subunit (blue), including the RBD (purple), and the S2 subunit (gray) are depicted. (C and D) Amino acid sequence alignment of residues around the S1/S2 and S2′ cleavage sites of group 2b betacoronaviruses found in humans, civet cats, raccoon dog, pangolin, and bats (C) or coronaviruses that are able to infect humans (D). Basic amino acid residues are highlighted in red, while gray boxes mark the presence of multibasic motifs. Numbers refer to amino acid residues (n/a, no information available). The symbol " ∗ " refers to amino acid residues that are conserved among all tested sequences, while the symbols ":" and "." indicate positions with heterogeneous amino acid residues that share highly similar or similar biochemical properties. Our previous work revealed that the activity of the cellular serine protease TMPRSS2, which activates several coronaviruses ( Bertram et al., 2013 , Gierer et al., 2013 , Glowacka et al., 2011 , Matsuyama et al., 2010 , Shirato et al., 2013 , Shirato et al., 2016 , Shulla et al., 2011 ), is also required for robust SARS-CoV-2 infection of human lung cells ( Hoffmann et al., 2020 ). However, it is conceivable that the activity of other cellular proteases is also necessary. Thus, the Middle East respiratory syndrome coronavirus spike protein (MERS-S) is activated by a two-step process: MERS-S is first cleaved by furin at the S1/S2 site in infected cells, which is required for subsequent TMPRSS2-mediated cleavage at the S2′ site ( Figure 1 A) during viral entry into lung cells ( Kleine-Weber et al., 2018 , Park et al., 2016 , Millet and Whittaker, 2014 ). A cathepsin B/L-dependent auxiliary activation pathway is operative in many TMPRSS2 − cell lines but seems not to be available in viral target cells in the lung because TMPRSS2-dependent activation of the S protein is essential for robust MERS-CoV and SARS-CoV spread and pathogenesis in the infected host ( Iwata-Yoshikawa et al., 2019 , Simmons et al., 2005 , Zhou et al., 2015 ). The S1/S2 site in SARS-CoV-2 forms an exposed loop ( Figure 1 B) that harbors multiple arginine residues (multibasic) ( Walls et al., 2020 , Wrapp et al., 2020 ) that are not found in SARS-CoV-related coronaviruses (SARSr-CoV) but are present in the human coronaviruses OC43, HKU1, and MERS-CoV ( Figure 1 C). However, the contribution of this multibasic cleavage site to SARS-CoV-2 infection of human cells is unknown and was in the focus of the present study. Results The Multibasic S1/S2 Site in the Spike Protein of SARS-CoV-2 Is Required for Efficient Proteolytic Cleavage of the Spike Protein In order to address the role of the multibasic S1/S2 cleavage site in SARS-CoV-2 infection, we generated S protein mutants with altered S1/S2 cleavage sites ( Figure 2 A). In particular, we exchanged the multibasic cleavage site against its monobasic counterparts present in SARS-S or RaTG13-S ( Figure 2 A, RaTG13 is a bat coronavirus closely related to SARS-CoV-2 [ Zhou et al., 2020b ]). This resulted in mutants SARS-2-S (SARS) and SARS-2-S (RaTG). Moreover, we either deleted all arginines in the S1/S2 site of SARS-2-S or inserted an additional arginine residue (jointly with an alanine to lysine exchange), giving rise to mutants SARS-2-S (delta) and SARS-2-S (opt), respectively. Finally, we introduced the S1/S2 sites of SARS-2-S and RaTG13-S into the background of SARS-S ( Figure 2 A), which yielded the mutants SARS-S (SARS-2) and SARS-S (RaTG). Figure 2 The Multibasic S1/S2 Site of SARS-2-S Is Cleaved by Furin, and Cleavage Is Required for Syncytium Formation and Entry into Human Lung Cells (A) Overview of the SARS-S and SARS-2-S S1/S2 mutants analyzed. (B) Analysis of furin-mediated S protein priming. Rhabdoviral particles harboring the indicated S proteins containing a C-terminal V5 tag for detection were lysed and subjected to western blot analysis. Detection of vesicular stomatitis virus matrix protein (VSV-M) served as control. (C) Rhabdoviral particles bearing MERS-S, SARS-S, or SARS-2-S equipped with a V5 or HA epitope tag at their C terminus (or no glycoprotein at all, control) were produced in the absence or presence of furin inhibitor (FI, decanoyl-RVKR-CMK; 1 μM or 10 μM) and analyzed for S protein processing by western blot analysis. Detection of VSV-M served as control. (D) Syncytium formation assay: Vero or Vero-TMPRSS2 cells were transfected to express the indicated S proteins (or no S protein, empty vector, control). At 24 h post transfection, cells were incubated in the presence or absence of trypsin (1 μg/mL) for an additional 24 h before they were fixed, stained with May-Gruenwald and Giemsa solution, and analyzed by bright field microscopy (scale bars, 200 μm). White arrowheads indicate syncytia. For (B)–(D), representative data from three (B and C) or four (D) independent experiments are shown. (E) Transduction of Vero (TMPRSS2 − ) and Calu-3 (TMPRSS2 + ) cells with rhabdoviral particles bearing the indicated S proteins or vesicular stomatitis virus glycoprotein (VSV-G). At 16 h post transduction, virus-encoded firefly luciferase was quantified in cell lysates. Presented are the mean data from three independent experiments. Transduction efficiency is shown relative to that measured for particles not bearing a viral glycoprotein. Error bars indicate the standard error of the mean. Statistical significance was tested by one-way analysis of variance with Dunnett's post test (p > 0.05, ns; ∗∗∗ p ≤ 0.001). The effects of the above described S1/S2 mutations on viral entry were examined using vesicular stomatitis virus (VSV) particles bearing S proteins because these particles are safe and adequately reflect coronavirus entry into target cells. Immunoblot of VSV particles bearing S proteins with a C-terminal antigenic tag revealed that all S proteins were readily incorporated into VSV particles. SARS-2-S WT (wild type) was efficiently cleaved at the S1/S2 site ( Figure 2 B), in keeping with published data ( Hoffmann et al., 2020 , Walls et al., 2020 ). Exchange of the S1/S2 site of SARS-2-S against those of SARS-S and RaTG13-S abrogated cleavage, and this effect was also seen when the multibasic motif was deleted ( Figure 2 B). Moreover, insertion of an additional arginine residue jointly with an alanine-to-lysine exchange at the S1/S2 site did not appreciably increase cleavability. Finally, insertion of the S1/S2 site of SARS-2-S into SARS-S increased S protein cleavability while insertion of the RaTG13 S1/S2 site did not ( Figure 2 B). These results indicate that the presence of several arginine residues at the S1/S2 site is required for efficient SARS-2-S proteolytic processing in human cells and also confers high cleavability to SARS-S. Furin Cleaves the SARS-CoV-2 Spike Protein at the S1/S2 Site, and Cleavage Is Required for Efficient Cell-Cell Fusion We next investigated which protease is required for S protein processing at the S1/S2 site. The S1/S2 motif matches the minimal furin sequence RXXR and is closely related to the furin consensus sequence RX[K/R]R. Therefore, we analyzed whether decanoyl-RVKR-CMK, a furin inhibitor, blocks SARS-2-S processing at the S1/S2 site. Decanoyl-RVKR-CMK inhibited processing of MERS-S, which is known to depend on furin ( Gierer et al., 2015 , Millet and Whittaker, 2014 ), in a concentration-dependent manner and had no effect on SARS-S expression ( Figure 2 C), as expected. Processing of SARS-2-S was also inhibited, indicating that furin cleaves SARS-2-S at the S1/S2 site. In order to determine whether cleavage at the S1/S2 site is required for SARS-2-S-driven cell-cell fusion, we studied S-protein-dependent formation of multinucleated giant cells (syncytia). No syncytia were observed in the absence of S protein expression while MERS-S WT expression resulted in syncytium formation, which was increased upon addition of trypsin or expression of TMPRSS2 ( Figure 2 D). Expression of SARS-S WT or SARS-S harboring the S1/S2 site of RaTG13-S did not induce syncytium formation in the absence of protease, but modest multikaryon formation was detected in the presence of trypsin or TMPRSS2. In contrast, SARS-S harboring the SARS-2-S S1/S2 site induced syncytia in the absence of protease, and syncytium formation was markedly increased by trypsin and, particularly, TMPRSS2. SARS-2-S expression triggered syncytium formation that was strongly increased by trypsin and TMPRSS2. Syncytium formation was clearly less prominent and required the presence of trypsin or TMPRSS2 when the SARS-2-S S1/S2 site was replaced by that of SARS-S or RaTG13-S. Moreover, deletion of the multibasic motif resulted in a spike protein that was no longer able to induce syncytium formation even in the presence of trypsin or TMPRSS2. Finally, the addition of an arginine residue to the S1/S2 site of SARS-2-S jointly with alanine-to-lysine exchange strongly increased syncytium formation, indicating that viral variants with optimized S1/S2 sites might show augmented cell-cell spread and potentially altered pathogenicity. Thus, the S1/S2 site of SARS-2-S is required for cell-cell fusion, and this process can be augmented by adding basic residues to the S1/S2 site. Cleavage of the SARS-CoV-2 Spike Protein at the S1/S2 Site Is Required for Viral Entry into Human Lung Cells We finally examined the importance of the S1/S2 site for S-protein-mediated virus-cell fusion. Blockade of SARS-2-S cleavage at the S1/S2 site (mutants SARS-2-S (SARS), SARS-2-S (RaTG), and SARS-2-S (delta)) abrogated entry into the TMPRSS2 + human lung cell line Calu-3 ( Figure 2 E), in which the cathepsin B/L-dependent S protein activation pathway is not sufficiently available ( Park et al., 2016 ). In contrast, entry into TMPRSS2 − Vero cells, which is known to be cathepsin B/L dependent, was not affected by these mutations ( Figure 1 E), in keeping with results reported by Walls and colleagues ( Walls et al., 2020 ). Optimization of the S1/S2 site did not increase entry into the cell lines tested; it slightly decreased entry into both Vero and Calu-3 cells, for, at present, unclear reasons. Finally, alterations of the S1/S2 site of SARS-S did not augment entry efficiency. Collectively, these results demonstrate that a multibasic S1/S2 site is essential for SARS-2-S-driven entry into human lung cells while a monobasic site is sufficient for SARS-S. The Multibasic S1/S2 Site in the Spike Protein of SARS-CoV-2 Is Required for Efficient Proteolytic Cleavage of the Spike Protein In order to address the role of the multibasic S1/S2 cleavage site in SARS-CoV-2 infection, we generated S protein mutants with altered S1/S2 cleavage sites ( Figure 2 A). In particular, we exchanged the multibasic cleavage site against its monobasic counterparts present in SARS-S or RaTG13-S ( Figure 2 A, RaTG13 is a bat coronavirus closely related to SARS-CoV-2 [ Zhou et al., 2020b ]). This resulted in mutants SARS-2-S (SARS) and SARS-2-S (RaTG). Moreover, we either deleted all arginines in the S1/S2 site of SARS-2-S or inserted an additional arginine residue (jointly with an alanine to lysine exchange), giving rise to mutants SARS-2-S (delta) and SARS-2-S (opt), respectively. Finally, we introduced the S1/S2 sites of SARS-2-S and RaTG13-S into the background of SARS-S ( Figure 2 A), which yielded the mutants SARS-S (SARS-2) and SARS-S (RaTG). Figure 2 The Multibasic S1/S2 Site of SARS-2-S Is Cleaved by Furin, and Cleavage Is Required for Syncytium Formation and Entry into Human Lung Cells (A) Overview of the SARS-S and SARS-2-S S1/S2 mutants analyzed. (B) Analysis of furin-mediated S protein priming. Rhabdoviral particles harboring the indicated S proteins containing a C-terminal V5 tag for detection were lysed and subjected to western blot analysis. Detection of vesicular stomatitis virus matrix protein (VSV-M) served as control. (C) Rhabdoviral particles bearing MERS-S, SARS-S, or SARS-2-S equipped with a V5 or HA epitope tag at their C terminus (or no glycoprotein at all, control) were produced in the absence or presence of furin inhibitor (FI, decanoyl-RVKR-CMK; 1 μM or 10 μM) and analyzed for S protein processing by western blot analysis. Detection of VSV-M served as control. (D) Syncytium formation assay: Vero or Vero-TMPRSS2 cells were transfected to express the indicated S proteins (or no S protein, empty vector, control). At 24 h post transfection, cells were incubated in the presence or absence of trypsin (1 μg/mL) for an additional 24 h before they were fixed, stained with May-Gruenwald and Giemsa solution, and analyzed by bright field microscopy (scale bars, 200 μm). White arrowheads indicate syncytia. For (B)–(D), representative data from three (B and C) or four (D) independent experiments are shown. (E) Transduction of Vero (TMPRSS2 − ) and Calu-3 (TMPRSS2 + ) cells with rhabdoviral particles bearing the indicated S proteins or vesicular stomatitis virus glycoprotein (VSV-G). At 16 h post transduction, virus-encoded firefly luciferase was quantified in cell lysates. Presented are the mean data from three independent experiments. Transduction efficiency is shown relative to that measured for particles not bearing a viral glycoprotein. Error bars indicate the standard error of the mean. Statistical significance was tested by one-way analysis of variance with Dunnett's post test (p > 0.05, ns; ∗∗∗ p ≤ 0.001). The effects of the above described S1/S2 mutations on viral entry were examined using vesicular stomatitis virus (VSV) particles bearing S proteins because these particles are safe and adequately reflect coronavirus entry into target cells. Immunoblot of VSV particles bearing S proteins with a C-terminal antigenic tag revealed that all S proteins were readily incorporated into VSV particles. SARS-2-S WT (wild type) was efficiently cleaved at the S1/S2 site ( Figure 2 B), in keeping with published data ( Hoffmann et al., 2020 , Walls et al., 2020 ). Exchange of the S1/S2 site of SARS-2-S against those of SARS-S and RaTG13-S abrogated cleavage, and this effect was also seen when the multibasic motif was deleted ( Figure 2 B). Moreover, insertion of an additional arginine residue jointly with an alanine-to-lysine exchange at the S1/S2 site did not appreciably increase cleavability. Finally, insertion of the S1/S2 site of SARS-2-S into SARS-S increased S protein cleavability while insertion of the RaTG13 S1/S2 site did not ( Figure 2 B). These results indicate that the presence of several arginine residues at the S1/S2 site is required for efficient SARS-2-S proteolytic processing in human cells and also confers high cleavability to SARS-S. Furin Cleaves the SARS-CoV-2 Spike Protein at the S1/S2 Site, and Cleavage Is Required for Efficient Cell-Cell Fusion We next investigated which protease is required for S protein processing at the S1/S2 site. The S1/S2 motif matches the minimal furin sequence RXXR and is closely related to the furin consensus sequence RX[K/R]R. Therefore, we analyzed whether decanoyl-RVKR-CMK, a furin inhibitor, blocks SARS-2-S processing at the S1/S2 site. Decanoyl-RVKR-CMK inhibited processing of MERS-S, which is known to depend on furin ( Gierer et al., 2015 , Millet and Whittaker, 2014 ), in a concentration-dependent manner and had no effect on SARS-S expression ( Figure 2 C), as expected. Processing of SARS-2-S was also inhibited, indicating that furin cleaves SARS-2-S at the S1/S2 site. In order to determine whether cleavage at the S1/S2 site is required for SARS-2-S-driven cell-cell fusion, we studied S-protein-dependent formation of multinucleated giant cells (syncytia). No syncytia were observed in the absence of S protein expression while MERS-S WT expression resulted in syncytium formation, which was increased upon addition of trypsin or expression of TMPRSS2 ( Figure 2 D). Expression of SARS-S WT or SARS-S harboring the S1/S2 site of RaTG13-S did not induce syncytium formation in the absence of protease, but modest multikaryon formation was detected in the presence of trypsin or TMPRSS2. In contrast, SARS-S harboring the SARS-2-S S1/S2 site induced syncytia in the absence of protease, and syncytium formation was markedly increased by trypsin and, particularly, TMPRSS2. SARS-2-S expression triggered syncytium formation that was strongly increased by trypsin and TMPRSS2. Syncytium formation was clearly less prominent and required the presence of trypsin or TMPRSS2 when the SARS-2-S S1/S2 site was replaced by that of SARS-S or RaTG13-S. Moreover, deletion of the multibasic motif resulted in a spike protein that was no longer able to induce syncytium formation even in the presence of trypsin or TMPRSS2. Finally, the addition of an arginine residue to the S1/S2 site of SARS-2-S jointly with alanine-to-lysine exchange strongly increased syncytium formation, indicating that viral variants with optimized S1/S2 sites might show augmented cell-cell spread and potentially altered pathogenicity. Thus, the S1/S2 site of SARS-2-S is required for cell-cell fusion, and this process can be augmented by adding basic residues to the S1/S2 site. Cleavage of the SARS-CoV-2 Spike Protein at the S1/S2 Site Is Required for Viral Entry into Human Lung Cells We finally examined the importance of the S1/S2 site for S-protein-mediated virus-cell fusion. Blockade of SARS-2-S cleavage at the S1/S2 site (mutants SARS-2-S (SARS), SARS-2-S (RaTG), and SARS-2-S (delta)) abrogated entry into the TMPRSS2 + human lung cell line Calu-3 ( Figure 2 E), in which the cathepsin B/L-dependent S protein activation pathway is not sufficiently available ( Park et al., 2016 ). In contrast, entry into TMPRSS2 − Vero cells, which is known to be cathepsin B/L dependent, was not affected by these mutations ( Figure 1 E), in keeping with results reported by Walls and colleagues ( Walls et al., 2020 ). Optimization of the S1/S2 site did not increase entry into the cell lines tested; it slightly decreased entry into both Vero and Calu-3 cells, for, at present, unclear reasons. Finally, alterations of the S1/S2 site of SARS-S did not augment entry efficiency. Collectively, these results demonstrate that a multibasic S1/S2 site is essential for SARS-2-S-driven entry into human lung cells while a monobasic site is sufficient for SARS-S. Discussion Our results reveal commonalities between the proteolytic activation of SARS-CoV-2 and MERS-CoV. Both viruses depend on furin-mediated pre-cleavage of their S proteins at the S1/S2 site for subsequent S protein activation by TMPRSS2 in lung cells, which fail to express robust levels of cathepsin L ( Park et al., 2016 ). Thus, inhibitors of furin and TMPRSS2 might be considered as a treatment option for COVID-19, and a TMPRSS2 inhibitor that blocks SARS-CoV-2 infection has recently been described ( Hoffmann et al., 2020 ). Regarding furin inhibition, it must be taken into account that furin, unlike TMPRSS2, is required for normal development ( Roebroek et al., 1998 ). Blockade of this enzyme for prolonged time periods might thus be associated with unwanted toxic effects. In contrast, a brief treatment might be well tolerated and still associated with a therapeutic benefit ( Sarac et al., 2002 , Sarac et al., 2004 ). For avian influenza A viruses, a multibasic cleavage site in the viral hemagglutinin protein is a central virulence factor ( Luczo et al., 2015 ). Thus, viruses with a monobasic cleavage site are activated by TMPRSS2 or related proteases with an expression profile confined to the aerodigestive tract. As a consequence, viral replication is limited to these organs and does not result in severe disease. In contrast, viruses with a multibasic cleavage site are activated by ubiquitously expressed proprotein convertases, including furin, and can thus spread systemically and cause massive disease. In the context of coronavirus infection, S protein cleavability has been identified as a determinant of zoonotic potential ( Menachery et al., 2020 , Yang et al., 2014 ). The presence of a highly cleavable S1/S2 site in SARS-2-S may therefore not have been unexpected. However, it is noteworthy that all SARS-CoV-2-related coronaviruses of bats and pangolins identified today harbor a monobasic cleavage site ( Lam et al., 2020 , Li et al., 2020 , Zhang et al., 2020 ). It will thus be interesting to determine how the multibasic motif was acquired by SARS-CoV-2, and a recent study suggested that a recombination event might have been responsible ( Zhang et al., 2020 , Zhou et al., 2020a ). Limitations of the Study Our results demonstrate that the multibasic S1/S2 cleavage site is essential for SARS-2-S-driven entry into TMPRSS2 + lung cells. It will be interesting to extend these studies to primary human respiratory epithelial cells and to authentic SARS-CoV-2, which requires a reverse genetics system not available to the present study. Limitations of the Study Our results demonstrate that the multibasic S1/S2 cleavage site is essential for SARS-2-S-driven entry into TMPRSS2 + lung cells. It will be interesting to extend these studies to primary human respiratory epithelial cells and to authentic SARS-CoV-2, which requires a reverse genetics system not available to the present study. STAR☠Methods Key Resources Table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Monoclonal anti-HA antibody produced in mouse Sigma-Aldrich Cat.#: H3663; RRID: AB_262051 Monoclonal anti-β-actin antibody produced in mouse Sigma-Aldrich Cat.#: A5441; RRID: AB_476744 Monoclonal anti-VSV-M (23H12) antibody KeraFast Cat.#: EB0011; RRID: AB_2734773 Monoclonal anti-mouse, peroxidase-coupled Dianova Cat.#: 115-035-003; RRID: AB_10015289 Anti-VSV-G antibody (I1, produced from CRL-2700 mouse hybridoma cells) ATCC Cat.# CRL-2700; RRID: CVCL_G654 Bacterial and Virus Strains VSV ∗ ΔG-FLuc Berger Rentsch and Zimmer, 2011 N/A One Shot™ OmniMAX™ 2 T1R Chemically Competent E. coli Thermo Fisher Scientific Cat.#: C854003 Chemicals, Peptides, and Recombinant Proteins Lipofectamine LTX with Plus Reagent Thermo Fisher Scientific Cat.#: 15338100 Furin inhibitor, decanoyl-RVKR-CMK Tocris Cat.#: 3501 May-Grünwald solution Sigma-Aldrich Cat.#: 63590 Giemsa solution Sigma-Aldrich Cat.#: GS500 Critical Commercial Assays Beetle-Juice Kit PJK Cat.#: 102511 Experimental Models: Cell Lines 293T DSMZ Cat.#: ACC-635; RRID: CVCL_0063 Calu-3 Laboratory of Stephan Ludwig ATCC Cat# HTB-55; RRID: CVCL_0609 Vero Laboratory of Andrea Maisner ATCC Cat# CRL-1586; RRID: CVCL_0574 Vero-TMPRSS2 Hoffmann et al., 2020 N/A Oligonucleotides SARS-S (BamHI) F CTTGGATCCGCCACCATGTTTATTTTC TTATTATTTC Sigma-Aldrich N/A SARS-SΔ18 (XbaI) R CTTTCTAGACTACTTGCAGCAAGAA CCACAAGAGC Sigma-Aldrich N/A SARS-SΔ18 (-)STOP (XbaI) R CTTTCTAGACTTGCAGCAAG AACCACAAGAGC Sigma-Aldrich N/A SARS-2-S (BamHI) F GAATTCGGATCCGCCACCATGTTCGT GTTTCTGGTGCTGC Sigma-Aldrich N/A SARS-2-SΔ18 (XbaI) R AAGGCCTCTAGACTACTTGCAGCA GCTGCCACAGC Sigma-Aldrich N/A SARS-2-SΔ18 (-)STOP (XbaI) R AAGGCCTCTAGACTTGCA GCAGCTGCCACAGC Sigma-Aldrich N/A SARS-S (SARS) F CAGACAAACAGCCCCAGACGGGCCAG AAGTACTAGCCAAAAATCTATTG Sigma-Aldrich N/A SARS-S (SARS) R TCTGGCCCGTCTGGGGCTGTTTGTCT GTGTATGGTAACTAGCACAAATGC Sigma-Aldrich N/A SARS-S (RaTG) F CAGACAAACAGCAGAAGTACTAGCCA AAAATC Sigma-Aldrich N/A SARS-S (RaTG) R TCTGCTGTTTGTCTGTGTATGGTAACTA GCACAAATGC Sigma-Aldrich N/A SARS-2-S (SARS) F GTTTCTTTATTACGTTCTGTGGCCAGC CAGAGCATC Sigma-Aldrich N/A SARS-2-S (SARS) R ACGTAATAAAGAAACTGTCTGGTAGC TGGCACAGATG Sigma-Aldrich N/A SARS-2-S (RaTG) F CAGACAAACAGCAGATCTGTGGCCAGC CAGAGCATC Sigma-Aldrich N/A SARS-2-S (RaTG) R GCTGGCCACAGATCTGCTGTTTGTCTG TGTCTGGTAGC Sigma-Aldrich N/A SARS-2-S (delta) F CAAACAGCCCCGCATCTGTGGCCAGCC AGAGCATC Sigma-Aldrich N/A SARS-2-S (delta) R GCTGGCCACAGATGCGGGGCTGTTTGTC TGTGTCTGGTAGC Sigma-Aldrich N/A SARS-2-S (opt) F CGAAGACGAAAAAGATCTGTGGCCAGCCA GAGCATC Sigma-Aldrich N/A SARS-2-S (opt) R TCTTTTTCGTCTTCGGCTGTTTGTCTGTGT CTGG Sigma-Aldrich N/A pCG1 Seq F CCTGGGCAACGTGCTGGT Sigma-Aldrich N/A pCG1 Seq R GTCAGATGCTCAAGGGGCTTCA Sigma-Aldrich N/A SARS-S 387F TGTTATACGAGCATGTAAC Sigma-Aldrich N/A SARS-S 790F AAGCCAACTACATTTATGC Sigma-Aldrich N/A SARS S 1194F TGATGTAAGACAAATAGCG Sigma-Aldrich N/A SARS S 1575F TATTAAGAACCAGTGTGTC Sigma-Aldrich N/A SARS S 1987F GTGCTAGTTACCATACAG Sigma-Aldrich N/A SARS S 2391F CTAAAGCCAACTAAGAGG Sigma-Aldrich N/A SARS S 2787F TCAACTGCATTGGGCAAG Sigma-Aldrich N/A SARS-2-S 651F CAAGATCTACAGCAAGCACACC Sigma-Aldrich N/A SARS-2-S 1380F GTCGGCGGCAACTACAATTAC Sigma-Aldrich N/A SARS-2-S 1992F CTGTCTGATCGGAGCCGAGCAC Sigma-Aldrich N/A SARS-2-S 2648F TGAGATGATCGCCCAGTACAC Sigma-Aldrich N/A SARS-2-S 3286F GCCATCTGCCACGACGGCAAAG Sigma-Aldrich N/A pCG1-V5 F TCCCTAACCCTCTCCTCGGTCTCGATTCTACGTG AAAGCTGATCTTTTTCCCTCTGCC Sigma-Aldrich N/A pCG1-V5 R GACCGAGGAGAGGGTTAGGGATAGGCTTACCG CATGCCTGCAGGTTTAAACAGTCG Sigma-Aldrich N/A pCG1-XhoI R CTCCTCGAGTTCATAAGAGAAGAGGG Sigma-Aldrich N/A Recombinant DNA Plasmid: pCG1-SARS-S Hoffmann et al., 2013 N/A Plasmid: pCG1-SARS-S-HA Hoffmann et al., 2020 N/A Plasmid: pCG1-SARS-2-S Hoffmann et al., 2020 N/A Plasmid: pCG1-SARS-2-S-HA Hoffmann et al., 2020 N/A Plasmid: pCG1-SARS-SΔ18 Hoffmann et al., 2013 N/A Plasmid: pCG1-SARS-SΔ18-V5 This paper N/A Plasmid: pCG1-SARS-2-SΔ18 This paper N/A Plasmid: pCG1-SARS-2-SΔ18-V5 This paper N/A Plasmid: pCG1-SARS-SΔ18 (SARS-2) This paper N/A Plasmid: pCG1-SARS-SΔ18-V5 (SARS-2) This paper N/A Plasmid: pCG1-SARS-SΔ18 (RaTG) This paper N/A Plasmid: pCG1-SARS-SΔ18-V5 (RaTG) This paper N/A Plasmid: pCG1-SARS-2-SΔ18 (SARS) This paper N/A Plasmid: pCG1-SARS-2-SΔ18-V5 (SARS) This paper N/A Plasmid: pCG1-SARS-2-SΔ18 (RaTG) This paper N/A Plasmid: pCG1-SARS-2-SΔ18-V5 (RaTG) This paper N/A Plasmid: pCG1-SARS-2-SΔ18 (delta) This paper N/A Plasmid: pCG1-SARS-2-SΔ18-V5 (delta) This paper N/A Plasmid: pCG1-SARS-2-SΔ18 (opt) This paper N/A Plasmid: pCG1-SARS-2-SΔ18-V5 (opt) This paper N/A Plasmid: pCAGGS-MERS-S-V5 Gierer et al., 2013 N/A Plasmid: pCAGGS-VSV-G Brinkmann et al., 2017 N/A Plasmid: pCAGGS-DsRed Hoffmann et al., 2013 N/A Plasmid: pCG1 Laboratory of Roberto Cattaneo N/A Plasmid: pCG1-V5 This paper N/A Software and Algorithms Hidex Sense Microplate Reader Software Hidex Deutschland Vertrieb GmbH https://www.hidex.de/ ChemoStar Imager Software (version v.0.3.23) Intas Science Imaging Instruments GmbH https://www.intas.de/ ZEN imaging software Carl Zeiss https://www.zeiss.com/ Clustal Omega European Molecular Biology Laboratory – European Bioinformatics Institute (EMBL-EBI) https://www.ebi.ac.uk/Tools/msa/clustalo/ ; Madeira et al., 2019 Adobe Photoshop CS5 Extended (version 12.0 3 32) Adobe https://www.adobe.com/ GraphPad Prism (version 8.3.0(538)) GraphPad Software https://www.graphpad.com/ YASARA (version 19.1.27) YASARA Biosciences GmbH http://www.yasara.org/ ; Krieger and Vriend, 2014 Microsoft Office Standard 2010 (version 14.0.7232.5000) Microsoft Corporation https://products.office.com/home Other Prefusion structure of SARS-CoV spike glycoprotein (5X5B) Yuan et al., 2017 https://www.rcsb.org/structure/5X5B Resource Availability Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Stefan Pöhlmann ( [email protected] ). Materials Availability All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement. Data and Code Availability The study did not generate unique datasets or code. Method Details Cell cultures 293T (human, kidney) and Vero (African green monkey, kidney) cells were cultivated in Dulbecco's Modified Eagle Medium (PAN-Biotech) supplemented with 10% fetal bovine serum (Biochrom), 100 U/mL of penicillin and 0.1 mg/mL of streptomycin (PAN-Biotech). Vero cells that stably express human TMPRSS2 have been described previously ( Hoffmann et al., 2020 ) and were cultivated in the presence of 10 μg/mL blasticidin (Invivogen). Calu-3 (human, lung; kindly provided by Stephan Ludwig, Westfälische Wilhelms-Universität, Muenster/Germany) cells were cultivated in Minimum Essential Medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Biochrom), 100 U/mL of penicillin and 0.1 mg/mL of streptomycin (PAN-Biotech), 1x non-essential amino acid solution (from 100x stock, PAA) and 10 mM sodium pyruvate (Thermo Fisher Scientific). All cell lines were incubated at 37°C and 5% CO 2 in a humidified atmosphere. Plasmids Expression plasmids for full-length vesicular stomatitis virus (VSV) glycoprotein (VSV-G), Middle-East respiratory syndrome coronavirus spike glycoprotein (MERS-S) containing a C-terminal V5 epitope tag, severe acute respiratory syndrome coronavirus spike glycoprotein (SARS-S) and severe acute respiratory syndrome coronavirus 2 spike glycoprotein (SARS-2-S) both equipped with a C-terminal hemagglutinin (HA) epitope tag have been described previously ( Brinkmann et al., 2017 , Hoffmann et al., 2020 ). Empty pCG1 expression vector was kindly provided by Roberto Cattaneo, Mayo Clinic, Rochester, MN/USA). Based on the SARS-S and SARS-2-S expression plasmids we cloned mutated versions with alterations at the S1/S2 cleavage site: We generated SARS-S containing the cleavage site of SARS-2-S, SARS-S (SARS-2), or BetaCoV/bat/Yunnan/RaTG13/2013 (RaTG; GISAID: EPI_ISL_402131), SARS-S (RaTG). Further, we generated SARS-2-S harboring the S1/S2 cleavage site of SARS-S, SARS-2-S (SARS) or RaTG-S, SARS-2-S (RaTG). Finally, we constructed SARS-2-S variants in which either the multibasic motif was deleted, SARS-2-S (delta), or in which the proline residue preceding the multibasic motif was mutated to arginine and the alanine residue within the minimal furin motif was changed to lysine in order to increase the basic environment at the S1/S2 site, SARS-2-S (opt). All newly cloned spike protein constructs further contained a deletion of 18 amino acids at their respective C terminus as this has been shown to improve coronavirus spike protein incorporation into VSV particles and thus transduction ( Schwegmann-Weßels et al., 2009 ). Further, for each construct an untagged variant as well as a version containing a C-terminal V5 epitope tag was constructed. Preparation of pseudotyped particles and transduction experiments A previously published protocol was employed to produce VSV pseudotype particles (VSVpp) carrying foreign viral glycoproteins in their envelope ( Berger Rentsch and Zimmer, 2011 , Kleine-Weber et al., 2019 ). First, 293T cells were transfected with expression plasmid for the respective spike glycoprotein or VSV-G or empty expression vector by calcium-phosphate precipitation. At 16 h posttransfection, the cells were inoculated with VSV ∗ ΔG-fLuc (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern/Switzerland), a replication-deficient VSV vector that lacks the genetic information for VSV-G and encodes for eGFP and firefly luciferase (fLuc), at a multiplicity of infection of 3. After 1 h of incubation, the inoculum was removed and cells were washed with phosphate-buffered saline (PBS) before medium containing anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) was added to all cells except for those expressing VSV-G (here, medium without antibody was added). Cells were further incubated for 16 h, before the VSVpp containing supernatants were harvested, freed from cellular debris by centrifugation and used for experiments. For transduction, target cells were grown in 96-well plates until they reached 50%–80% confluency. The culture supernatant was removed by aspiration and 100 μl/well of the respective pseudotype were added (quadruplicate samples). At 16 h posttransduction, culture supernatants were aspirated and cells lysed in 1x cell culture lysis reagent (prepared from 5x stock, Promega) for 20 min at room temperature. The lysates were then transferred to white, opaque-walled 96-well plates and luciferase activity was quantified by measuring luminescence upon addition of a substrate (PJK) using a Hidex Sense plate luminometer (Hidex). Western blot analysis For the analysis of S protein processing, we subjected VSVpp harboring V5- or HA-tagged S proteins to SDS-PAGE and western blot analysis. For this, we loaded 1 mL VSVpp onto 50 μl of a 20% (w/v) sucrose cushion and performed high-speed centrifugation (25.000 g for 120 min at 4°C). Next, we removed 1 mL of supernatant, added 50 μl of 2x SDS-sample buffer and incubated the samples for 15 min at 96°C. Thereafter, the samples were subjected to SDS-PAGE and protein transfer to nitrocellulose membranes by western blot. The membranes were subsequently blocked in 5% skim milk solution (PBS containing 0.05% Tween-20 [PBS-T] and 5% skim milk powder) for 1 h at room temperature. The blots were then incubated over night at 4°C with primary antibody solution (all antibodies were diluted in PBS-T containing 5% skim milk; mouse anti-HA tag [Sigma-Aldrich, H3663, 1:2,500], mouse anti-V5 tag [Thermo Fisher Scientific, R960-25, 1:2,500] or VSV matrix protein [Kerafast, EB0011, 1:2,500]). Following this incubation, the blots were washed 3x with PBS-T before they were incubated for 1 h at room temperature with peroxidase-coupled goat anti-mouse antibody (Dianova, 115-035-003, 1:10,000). Finally, the blots were again washed and imaged. For this, an in house-prepared enhanced chemiluminescent solution (0.1 M Tris-HCl [pH 8.6], 250 μg/mL luminol, 1 mg/mL para-hydroxycoumaric acid, 0.3% H 2 O 2 ) and the ChemoCam imaging system along with the ChemoStar Professional software (Intas Science Imaging Instruments GmbH) were used. Syncytium formation assay Vero or Vero-TMPRSS2 cells were grown on coverslips seeded in 24-well plates and transfected with S protein expression plasmids (1 μg/well) using Lipofectamine 2000 LTX with Plus reagent (Thermo Fisher Scientific) and OptiMEM medium (GIBCO). After 6 h the transfection solutions were aspirated and the cells further incubated for 24 h in standard culture medium. Next, the medium was changed to serum free medium ± 1 μg/mL bovine trypsin (Sigma-Aldrich) and the cells were incubated for additional 24 h. Then, the cells were washed with PBS, fixed with 4% paraformaldehyde solution for 20 min at room temperature, washed again, air-dried and incubated for 30 min with May-Gruenwald solution (Sigma-Aldrich). Thereafter, the cells were washed three times with deionized water, air-dried and incubated for 30 min with 1:10 diluted Giemsa solution (Sigma-Aldrich). After an additional washing interval with deionized water, the samples were air-dried and analyzed by bright-field microscopy using a Zeiss LSM800 confocal laser scanning microscope and the ZEN imaging software (both from Zeiss). Sequence analysis and protein models Sequence alignments were performed using the Clustal Omega online tool ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). Protein models were designed using the YASARA software ( http://www.yasara.org/index.html ). For the generation of the SARS-2-S protein model the protein sequence was first modeled on a SARS-S template (PDB: 5X5B, ( Yuan et al., 2017 )) using the SWISS-MODEL online tool ( https://swissmodel.expasy.org/ ). The following sequences information were obtained from National Center for Biotechnology Information (NCBI) database: SARS-CoV BJ01 (GenBank: AY278488.2), SARS-CoV CUHK-W1 (GenBank: AY278554.2), SARS-CoV Frankfurt-1 (GenBank: AY291315.1), SARS-CoV Tor2 (GenBank: CS050815.1), SARS-CoV Urbani (GenBank: AY278741.1), civet SARS-CoV SZ16 (GenBank: AY304488.1), civet SARS-CoV civet020 (GenBank: AY572038.1), raccoon dog SARS-CoV A030 (GenBank: AY687357.1), bat SARSr-CoV BtKY72/KEN (GenBank: KY352407.1), bat SARSr-CoV BM48-31/BGR/2008 (GenBank: GU190215.1), bat SARSr-CoV Rs4231 (GenBank: KY417146.1), bat SARSr-CoV WIV16 (GenBank: KT444582.1), bat SARSr-CoV Rs4874 (GenBank: KY417150.1), bat SARSr-CoV SL-CoVZC45 (GenBank: MG772933.1), bat SARSr-CoV SL-CoVZXC21 (GenBank: MG772934.1), bat SARSr-CoV LYRa11 (GenBank: KF569996.1), bat SARSr-CoV LYRa3 (GenBank: KF569997.1), bat SARSr-CoV WIV1 (GenBank: KF367457.1), bat SARSr-CoV RsSHC014 (GenBank: KC881005.1), bat SARSr-CoV Rs3367 (GenBank: KC881006.1), bat SARSr-CoV Cp/Yunnan2011 (GenBank: JX993988.1), bat SARSr-CoV Rp/Shaanxi2011 (GenBank: JX993987.1), bat SARSr-CoV HKU3-1 (GenBank: DQ022305.2), bat SARSr-CoV Rm1 (GenBank: DQ412043.1), bat SARSr-CoV Rp3 (GenBank: DQ071615.1), bat SARSr-CoV Rf1 (GenBank: DQ412042.1), bat SARSr-CoV 279 (GenBank: DQ648857.1), bat SARSr-CoV 273 (GenBank: DQ648856.1), bat SARSr-CoV YN2013 (GenBank: KJ473816.1), bat SARSr-CoV Rs/HuB2013 (GenBank: KJ473814.1), bat SARSr-CoV Rs/GX2013 (GenBank: KJ473815.1), bat SARSr-CoV Rf/SX2013 (GenBank: KJ473813.1), bat SARSr-CoV Rf/JL2012 (GenBank: KJ473811.1), bat SARSr-CoV Rf/HeB2013 (GenBank: KJ473812.1), bat SARSr-CoV YNLF/34C (GenBank: KP886809.1), bat SARSr-CoV YNLF/31C (GenBank: KP886808.1), bat SARSr-CoV Rs672 (GenBank: FJ588686.1), bat SARSr-CoV Rs7327 (GenBank: KY417151.1), bat SARSr-CoV Rs4084 (GenBank: KY417144.1), bat SARSr-CoV Rs9401 (GenBank: KY417152.1), bat SARSr-CoV Rs4247 (GenBank: KY417148.1), bat SARSr-CoV Rs4255 (GenBank: KY417149.1), bat SARSr-CoV Rs4081 (GenBank: KY417143.1), bat SARSr-CoV Rs4237 (GenBank: KY417147.1), bat SARSr-CoV As6526 (GenBank: KY417142.1), bat SARSr-CoV Rf4092 (GenBank: KY417145.1), bat SARSr-CoV Longquan-140 (GenBank: KF294457.1), bat SARSr-CoV Rs806 (GenBank: FJ588692.1), bat SARSr-CoV Anlong-103 (GenBank: KY770858.1), bat SARSr-CoV JTMC15 (GenBank: KU182964.1), bat SARSr-CoV 16BO133 (GenBank: KY938558.1), bat SARSr-CoV B15-21 (GenBank: KU528591.1), pangolin coronavirus MP789 (GenBank: MT084071.1). In addition the following sequences information were obtained from the Global Initiative on Sharing All Influenza Data (GISAID) database: SARS-CoV-2 (GISAID: EPI_ISL_404895), bat SARSr-CoV RaTG13 (GISAID: EPI_ISL_402131). Quantification and Statistical Analysis If not stated otherwise, statistical significance was tested by one-way analysis of variance with Dunnet's posttest (GraphPad Prism 7.03). Only p values of 0.05 or lower were considered statistically significant (p > 0.05 [ns, not significant], p ≤ 0.05 [ ∗ ], p ≤ 0.01 [ ∗∗ ], p ≤ 0.001 [ ∗∗∗ ]). For all statistical analyses, the GraphPad Prism 7 software package was used (GraphPad Software). Key Resources Table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Monoclonal anti-HA antibody produced in mouse Sigma-Aldrich Cat.#: H3663; RRID: AB_262051 Monoclonal anti-β-actin antibody produced in mouse Sigma-Aldrich Cat.#: A5441; RRID: AB_476744 Monoclonal anti-VSV-M (23H12) antibody KeraFast Cat.#: EB0011; RRID: AB_2734773 Monoclonal anti-mouse, peroxidase-coupled Dianova Cat.#: 115-035-003; RRID: AB_10015289 Anti-VSV-G antibody (I1, produced from CRL-2700 mouse hybridoma cells) ATCC Cat.# CRL-2700; RRID: CVCL_G654 Bacterial and Virus Strains VSV ∗ ΔG-FLuc Berger Rentsch and Zimmer, 2011 N/A One Shot™ OmniMAX™ 2 T1R Chemically Competent E. coli Thermo Fisher Scientific Cat.#: C854003 Chemicals, Peptides, and Recombinant Proteins Lipofectamine LTX with Plus Reagent Thermo Fisher Scientific Cat.#: 15338100 Furin inhibitor, decanoyl-RVKR-CMK Tocris Cat.#: 3501 May-Grünwald solution Sigma-Aldrich Cat.#: 63590 Giemsa solution Sigma-Aldrich Cat.#: GS500 Critical Commercial Assays Beetle-Juice Kit PJK Cat.#: 102511 Experimental Models: Cell Lines 293T DSMZ Cat.#: ACC-635; RRID: CVCL_0063 Calu-3 Laboratory of Stephan Ludwig ATCC Cat# HTB-55; RRID: CVCL_0609 Vero Laboratory of Andrea Maisner ATCC Cat# CRL-1586; RRID: CVCL_0574 Vero-TMPRSS2 Hoffmann et al., 2020 N/A Oligonucleotides SARS-S (BamHI) F CTTGGATCCGCCACCATGTTTATTTTC TTATTATTTC Sigma-Aldrich N/A SARS-SΔ18 (XbaI) R CTTTCTAGACTACTTGCAGCAAGAA CCACAAGAGC Sigma-Aldrich N/A SARS-SΔ18 (-)STOP (XbaI) R CTTTCTAGACTTGCAGCAAG AACCACAAGAGC Sigma-Aldrich N/A SARS-2-S (BamHI) F GAATTCGGATCCGCCACCATGTTCGT GTTTCTGGTGCTGC Sigma-Aldrich N/A SARS-2-SΔ18 (XbaI) R AAGGCCTCTAGACTACTTGCAGCA GCTGCCACAGC Sigma-Aldrich N/A SARS-2-SΔ18 (-)STOP (XbaI) R AAGGCCTCTAGACTTGCA GCAGCTGCCACAGC Sigma-Aldrich N/A SARS-S (SARS) F CAGACAAACAGCCCCAGACGGGCCAG AAGTACTAGCCAAAAATCTATTG Sigma-Aldrich N/A SARS-S (SARS) R TCTGGCCCGTCTGGGGCTGTTTGTCT GTGTATGGTAACTAGCACAAATGC Sigma-Aldrich N/A SARS-S (RaTG) F CAGACAAACAGCAGAAGTACTAGCCA AAAATC Sigma-Aldrich N/A SARS-S (RaTG) R TCTGCTGTTTGTCTGTGTATGGTAACTA GCACAAATGC Sigma-Aldrich N/A SARS-2-S (SARS) F GTTTCTTTATTACGTTCTGTGGCCAGC CAGAGCATC Sigma-Aldrich N/A SARS-2-S (SARS) R ACGTAATAAAGAAACTGTCTGGTAGC TGGCACAGATG Sigma-Aldrich N/A SARS-2-S (RaTG) F CAGACAAACAGCAGATCTGTGGCCAGC CAGAGCATC Sigma-Aldrich N/A SARS-2-S (RaTG) R GCTGGCCACAGATCTGCTGTTTGTCTG TGTCTGGTAGC Sigma-Aldrich N/A SARS-2-S (delta) F CAAACAGCCCCGCATCTGTGGCCAGCC AGAGCATC Sigma-Aldrich N/A SARS-2-S (delta) R GCTGGCCACAGATGCGGGGCTGTTTGTC TGTGTCTGGTAGC Sigma-Aldrich N/A SARS-2-S (opt) F CGAAGACGAAAAAGATCTGTGGCCAGCCA GAGCATC Sigma-Aldrich N/A SARS-2-S (opt) R TCTTTTTCGTCTTCGGCTGTTTGTCTGTGT CTGG Sigma-Aldrich N/A pCG1 Seq F CCTGGGCAACGTGCTGGT Sigma-Aldrich N/A pCG1 Seq R GTCAGATGCTCAAGGGGCTTCA Sigma-Aldrich N/A SARS-S 387F TGTTATACGAGCATGTAAC Sigma-Aldrich N/A SARS-S 790F AAGCCAACTACATTTATGC Sigma-Aldrich N/A SARS S 1194F TGATGTAAGACAAATAGCG Sigma-Aldrich N/A SARS S 1575F TATTAAGAACCAGTGTGTC Sigma-Aldrich N/A SARS S 1987F GTGCTAGTTACCATACAG Sigma-Aldrich N/A SARS S 2391F CTAAAGCCAACTAAGAGG Sigma-Aldrich N/A SARS S 2787F TCAACTGCATTGGGCAAG Sigma-Aldrich N/A SARS-2-S 651F CAAGATCTACAGCAAGCACACC Sigma-Aldrich N/A SARS-2-S 1380F GTCGGCGGCAACTACAATTAC Sigma-Aldrich N/A SARS-2-S 1992F CTGTCTGATCGGAGCCGAGCAC Sigma-Aldrich N/A SARS-2-S 2648F TGAGATGATCGCCCAGTACAC Sigma-Aldrich N/A SARS-2-S 3286F GCCATCTGCCACGACGGCAAAG Sigma-Aldrich N/A pCG1-V5 F TCCCTAACCCTCTCCTCGGTCTCGATTCTACGTG AAAGCTGATCTTTTTCCCTCTGCC Sigma-Aldrich N/A pCG1-V5 R GACCGAGGAGAGGGTTAGGGATAGGCTTACCG CATGCCTGCAGGTTTAAACAGTCG Sigma-Aldrich N/A pCG1-XhoI R CTCCTCGAGTTCATAAGAGAAGAGGG Sigma-Aldrich N/A Recombinant DNA Plasmid: pCG1-SARS-S Hoffmann et al., 2013 N/A Plasmid: pCG1-SARS-S-HA Hoffmann et al., 2020 N/A Plasmid: pCG1-SARS-2-S Hoffmann et al., 2020 N/A Plasmid: pCG1-SARS-2-S-HA Hoffmann et al., 2020 N/A Plasmid: pCG1-SARS-SΔ18 Hoffmann et al., 2013 N/A Plasmid: pCG1-SARS-SΔ18-V5 This paper N/A Plasmid: pCG1-SARS-2-SΔ18 This paper N/A Plasmid: pCG1-SARS-2-SΔ18-V5 This paper N/A Plasmid: pCG1-SARS-SΔ18 (SARS-2) This paper N/A Plasmid: pCG1-SARS-SΔ18-V5 (SARS-2) This paper N/A Plasmid: pCG1-SARS-SΔ18 (RaTG) This paper N/A Plasmid: pCG1-SARS-SΔ18-V5 (RaTG) This paper N/A Plasmid: pCG1-SARS-2-SΔ18 (SARS) This paper N/A Plasmid: pCG1-SARS-2-SΔ18-V5 (SARS) This paper N/A Plasmid: pCG1-SARS-2-SΔ18 (RaTG) This paper N/A Plasmid: pCG1-SARS-2-SΔ18-V5 (RaTG) This paper N/A Plasmid: pCG1-SARS-2-SΔ18 (delta) This paper N/A Plasmid: pCG1-SARS-2-SΔ18-V5 (delta) This paper N/A Plasmid: pCG1-SARS-2-SΔ18 (opt) This paper N/A Plasmid: pCG1-SARS-2-SΔ18-V5 (opt) This paper N/A Plasmid: pCAGGS-MERS-S-V5 Gierer et al., 2013 N/A Plasmid: pCAGGS-VSV-G Brinkmann et al., 2017 N/A Plasmid: pCAGGS-DsRed Hoffmann et al., 2013 N/A Plasmid: pCG1 Laboratory of Roberto Cattaneo N/A Plasmid: pCG1-V5 This paper N/A Software and Algorithms Hidex Sense Microplate Reader Software Hidex Deutschland Vertrieb GmbH https://www.hidex.de/ ChemoStar Imager Software (version v.0.3.23) Intas Science Imaging Instruments GmbH https://www.intas.de/ ZEN imaging software Carl Zeiss https://www.zeiss.com/ Clustal Omega European Molecular Biology Laboratory – European Bioinformatics Institute (EMBL-EBI) https://www.ebi.ac.uk/Tools/msa/clustalo/ ; Madeira et al., 2019 Adobe Photoshop CS5 Extended (version 12.0 3 32) Adobe https://www.adobe.com/ GraphPad Prism (version 8.3.0(538)) GraphPad Software https://www.graphpad.com/ YASARA (version 19.1.27) YASARA Biosciences GmbH http://www.yasara.org/ ; Krieger and Vriend, 2014 Microsoft Office Standard 2010 (version 14.0.7232.5000) Microsoft Corporation https://products.office.com/home Other Prefusion structure of SARS-CoV spike glycoprotein (5X5B) Yuan et al., 2017 https://www.rcsb.org/structure/5X5B Resource Availability Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Stefan Pöhlmann ( [email protected] ). Materials Availability All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement. Data and Code Availability The study did not generate unique datasets or code. Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Stefan Pöhlmann ( [email protected] ). Materials Availability All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement. Data and Code Availability The study did not generate unique datasets or code. Method Details Cell cultures 293T (human, kidney) and Vero (African green monkey, kidney) cells were cultivated in Dulbecco's Modified Eagle Medium (PAN-Biotech) supplemented with 10% fetal bovine serum (Biochrom), 100 U/mL of penicillin and 0.1 mg/mL of streptomycin (PAN-Biotech). Vero cells that stably express human TMPRSS2 have been described previously ( Hoffmann et al., 2020 ) and were cultivated in the presence of 10 μg/mL blasticidin (Invivogen). Calu-3 (human, lung; kindly provided by Stephan Ludwig, Westfälische Wilhelms-Universität, Muenster/Germany) cells were cultivated in Minimum Essential Medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Biochrom), 100 U/mL of penicillin and 0.1 mg/mL of streptomycin (PAN-Biotech), 1x non-essential amino acid solution (from 100x stock, PAA) and 10 mM sodium pyruvate (Thermo Fisher Scientific). All cell lines were incubated at 37°C and 5% CO 2 in a humidified atmosphere. Plasmids Expression plasmids for full-length vesicular stomatitis virus (VSV) glycoprotein (VSV-G), Middle-East respiratory syndrome coronavirus spike glycoprotein (MERS-S) containing a C-terminal V5 epitope tag, severe acute respiratory syndrome coronavirus spike glycoprotein (SARS-S) and severe acute respiratory syndrome coronavirus 2 spike glycoprotein (SARS-2-S) both equipped with a C-terminal hemagglutinin (HA) epitope tag have been described previously ( Brinkmann et al., 2017 , Hoffmann et al., 2020 ). Empty pCG1 expression vector was kindly provided by Roberto Cattaneo, Mayo Clinic, Rochester, MN/USA). Based on the SARS-S and SARS-2-S expression plasmids we cloned mutated versions with alterations at the S1/S2 cleavage site: We generated SARS-S containing the cleavage site of SARS-2-S, SARS-S (SARS-2), or BetaCoV/bat/Yunnan/RaTG13/2013 (RaTG; GISAID: EPI_ISL_402131), SARS-S (RaTG). Further, we generated SARS-2-S harboring the S1/S2 cleavage site of SARS-S, SARS-2-S (SARS) or RaTG-S, SARS-2-S (RaTG). Finally, we constructed SARS-2-S variants in which either the multibasic motif was deleted, SARS-2-S (delta), or in which the proline residue preceding the multibasic motif was mutated to arginine and the alanine residue within the minimal furin motif was changed to lysine in order to increase the basic environment at the S1/S2 site, SARS-2-S (opt). All newly cloned spike protein constructs further contained a deletion of 18 amino acids at their respective C terminus as this has been shown to improve coronavirus spike protein incorporation into VSV particles and thus transduction ( Schwegmann-Weßels et al., 2009 ). Further, for each construct an untagged variant as well as a version containing a C-terminal V5 epitope tag was constructed. Preparation of pseudotyped particles and transduction experiments A previously published protocol was employed to produce VSV pseudotype particles (VSVpp) carrying foreign viral glycoproteins in their envelope ( Berger Rentsch and Zimmer, 2011 , Kleine-Weber et al., 2019 ). First, 293T cells were transfected with expression plasmid for the respective spike glycoprotein or VSV-G or empty expression vector by calcium-phosphate precipitation. At 16 h posttransfection, the cells were inoculated with VSV ∗ ΔG-fLuc (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern/Switzerland), a replication-deficient VSV vector that lacks the genetic information for VSV-G and encodes for eGFP and firefly luciferase (fLuc), at a multiplicity of infection of 3. After 1 h of incubation, the inoculum was removed and cells were washed with phosphate-buffered saline (PBS) before medium containing anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) was added to all cells except for those expressing VSV-G (here, medium without antibody was added). Cells were further incubated for 16 h, before the VSVpp containing supernatants were harvested, freed from cellular debris by centrifugation and used for experiments. For transduction, target cells were grown in 96-well plates until they reached 50%–80% confluency. The culture supernatant was removed by aspiration and 100 μl/well of the respective pseudotype were added (quadruplicate samples). At 16 h posttransduction, culture supernatants were aspirated and cells lysed in 1x cell culture lysis reagent (prepared from 5x stock, Promega) for 20 min at room temperature. The lysates were then transferred to white, opaque-walled 96-well plates and luciferase activity was quantified by measuring luminescence upon addition of a substrate (PJK) using a Hidex Sense plate luminometer (Hidex). Western blot analysis For the analysis of S protein processing, we subjected VSVpp harboring V5- or HA-tagged S proteins to SDS-PAGE and western blot analysis. For this, we loaded 1 mL VSVpp onto 50 μl of a 20% (w/v) sucrose cushion and performed high-speed centrifugation (25.000 g for 120 min at 4°C). Next, we removed 1 mL of supernatant, added 50 μl of 2x SDS-sample buffer and incubated the samples for 15 min at 96°C. Thereafter, the samples were subjected to SDS-PAGE and protein transfer to nitrocellulose membranes by western blot. The membranes were subsequently blocked in 5% skim milk solution (PBS containing 0.05% Tween-20 [PBS-T] and 5% skim milk powder) for 1 h at room temperature. The blots were then incubated over night at 4°C with primary antibody solution (all antibodies were diluted in PBS-T containing 5% skim milk; mouse anti-HA tag [Sigma-Aldrich, H3663, 1:2,500], mouse anti-V5 tag [Thermo Fisher Scientific, R960-25, 1:2,500] or VSV matrix protein [Kerafast, EB0011, 1:2,500]). Following this incubation, the blots were washed 3x with PBS-T before they were incubated for 1 h at room temperature with peroxidase-coupled goat anti-mouse antibody (Dianova, 115-035-003, 1:10,000). Finally, the blots were again washed and imaged. For this, an in house-prepared enhanced chemiluminescent solution (0.1 M Tris-HCl [pH 8.6], 250 μg/mL luminol, 1 mg/mL para-hydroxycoumaric acid, 0.3% H 2 O 2 ) and the ChemoCam imaging system along with the ChemoStar Professional software (Intas Science Imaging Instruments GmbH) were used. Syncytium formation assay Vero or Vero-TMPRSS2 cells were grown on coverslips seeded in 24-well plates and transfected with S protein expression plasmids (1 μg/well) using Lipofectamine 2000 LTX with Plus reagent (Thermo Fisher Scientific) and OptiMEM medium (GIBCO). After 6 h the transfection solutions were aspirated and the cells further incubated for 24 h in standard culture medium. Next, the medium was changed to serum free medium ± 1 μg/mL bovine trypsin (Sigma-Aldrich) and the cells were incubated for additional 24 h. Then, the cells were washed with PBS, fixed with 4% paraformaldehyde solution for 20 min at room temperature, washed again, air-dried and incubated for 30 min with May-Gruenwald solution (Sigma-Aldrich). Thereafter, the cells were washed three times with deionized water, air-dried and incubated for 30 min with 1:10 diluted Giemsa solution (Sigma-Aldrich). After an additional washing interval with deionized water, the samples were air-dried and analyzed by bright-field microscopy using a Zeiss LSM800 confocal laser scanning microscope and the ZEN imaging software (both from Zeiss). Sequence analysis and protein models Sequence alignments were performed using the Clustal Omega online tool ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). Protein models were designed using the YASARA software ( http://www.yasara.org/index.html ). For the generation of the SARS-2-S protein model the protein sequence was first modeled on a SARS-S template (PDB: 5X5B, ( Yuan et al., 2017 )) using the SWISS-MODEL online tool ( https://swissmodel.expasy.org/ ). The following sequences information were obtained from National Center for Biotechnology Information (NCBI) database: SARS-CoV BJ01 (GenBank: AY278488.2), SARS-CoV CUHK-W1 (GenBank: AY278554.2), SARS-CoV Frankfurt-1 (GenBank: AY291315.1), SARS-CoV Tor2 (GenBank: CS050815.1), SARS-CoV Urbani (GenBank: AY278741.1), civet SARS-CoV SZ16 (GenBank: AY304488.1), civet SARS-CoV civet020 (GenBank: AY572038.1), raccoon dog SARS-CoV A030 (GenBank: AY687357.1), bat SARSr-CoV BtKY72/KEN (GenBank: KY352407.1), bat SARSr-CoV BM48-31/BGR/2008 (GenBank: GU190215.1), bat SARSr-CoV Rs4231 (GenBank: KY417146.1), bat SARSr-CoV WIV16 (GenBank: KT444582.1), bat SARSr-CoV Rs4874 (GenBank: KY417150.1), bat SARSr-CoV SL-CoVZC45 (GenBank: MG772933.1), bat SARSr-CoV SL-CoVZXC21 (GenBank: MG772934.1), bat SARSr-CoV LYRa11 (GenBank: KF569996.1), bat SARSr-CoV LYRa3 (GenBank: KF569997.1), bat SARSr-CoV WIV1 (GenBank: KF367457.1), bat SARSr-CoV RsSHC014 (GenBank: KC881005.1), bat SARSr-CoV Rs3367 (GenBank: KC881006.1), bat SARSr-CoV Cp/Yunnan2011 (GenBank: JX993988.1), bat SARSr-CoV Rp/Shaanxi2011 (GenBank: JX993987.1), bat SARSr-CoV HKU3-1 (GenBank: DQ022305.2), bat SARSr-CoV Rm1 (GenBank: DQ412043.1), bat SARSr-CoV Rp3 (GenBank: DQ071615.1), bat SARSr-CoV Rf1 (GenBank: DQ412042.1), bat SARSr-CoV 279 (GenBank: DQ648857.1), bat SARSr-CoV 273 (GenBank: DQ648856.1), bat SARSr-CoV YN2013 (GenBank: KJ473816.1), bat SARSr-CoV Rs/HuB2013 (GenBank: KJ473814.1), bat SARSr-CoV Rs/GX2013 (GenBank: KJ473815.1), bat SARSr-CoV Rf/SX2013 (GenBank: KJ473813.1), bat SARSr-CoV Rf/JL2012 (GenBank: KJ473811.1), bat SARSr-CoV Rf/HeB2013 (GenBank: KJ473812.1), bat SARSr-CoV YNLF/34C (GenBank: KP886809.1), bat SARSr-CoV YNLF/31C (GenBank: KP886808.1), bat SARSr-CoV Rs672 (GenBank: FJ588686.1), bat SARSr-CoV Rs7327 (GenBank: KY417151.1), bat SARSr-CoV Rs4084 (GenBank: KY417144.1), bat SARSr-CoV Rs9401 (GenBank: KY417152.1), bat SARSr-CoV Rs4247 (GenBank: KY417148.1), bat SARSr-CoV Rs4255 (GenBank: KY417149.1), bat SARSr-CoV Rs4081 (GenBank: KY417143.1), bat SARSr-CoV Rs4237 (GenBank: KY417147.1), bat SARSr-CoV As6526 (GenBank: KY417142.1), bat SARSr-CoV Rf4092 (GenBank: KY417145.1), bat SARSr-CoV Longquan-140 (GenBank: KF294457.1), bat SARSr-CoV Rs806 (GenBank: FJ588692.1), bat SARSr-CoV Anlong-103 (GenBank: KY770858.1), bat SARSr-CoV JTMC15 (GenBank: KU182964.1), bat SARSr-CoV 16BO133 (GenBank: KY938558.1), bat SARSr-CoV B15-21 (GenBank: KU528591.1), pangolin coronavirus MP789 (GenBank: MT084071.1). In addition the following sequences information were obtained from the Global Initiative on Sharing All Influenza Data (GISAID) database: SARS-CoV-2 (GISAID: EPI_ISL_404895), bat SARSr-CoV RaTG13 (GISAID: EPI_ISL_402131). Cell cultures 293T (human, kidney) and Vero (African green monkey, kidney) cells were cultivated in Dulbecco's Modified Eagle Medium (PAN-Biotech) supplemented with 10% fetal bovine serum (Biochrom), 100 U/mL of penicillin and 0.1 mg/mL of streptomycin (PAN-Biotech). Vero cells that stably express human TMPRSS2 have been described previously ( Hoffmann et al., 2020 ) and were cultivated in the presence of 10 μg/mL blasticidin (Invivogen). Calu-3 (human, lung; kindly provided by Stephan Ludwig, Westfälische Wilhelms-Universität, Muenster/Germany) cells were cultivated in Minimum Essential Medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Biochrom), 100 U/mL of penicillin and 0.1 mg/mL of streptomycin (PAN-Biotech), 1x non-essential amino acid solution (from 100x stock, PAA) and 10 mM sodium pyruvate (Thermo Fisher Scientific). All cell lines were incubated at 37°C and 5% CO 2 in a humidified atmosphere. Plasmids Expression plasmids for full-length vesicular stomatitis virus (VSV) glycoprotein (VSV-G), Middle-East respiratory syndrome coronavirus spike glycoprotein (MERS-S) containing a C-terminal V5 epitope tag, severe acute respiratory syndrome coronavirus spike glycoprotein (SARS-S) and severe acute respiratory syndrome coronavirus 2 spike glycoprotein (SARS-2-S) both equipped with a C-terminal hemagglutinin (HA) epitope tag have been described previously ( Brinkmann et al., 2017 , Hoffmann et al., 2020 ). Empty pCG1 expression vector was kindly provided by Roberto Cattaneo, Mayo Clinic, Rochester, MN/USA). Based on the SARS-S and SARS-2-S expression plasmids we cloned mutated versions with alterations at the S1/S2 cleavage site: We generated SARS-S containing the cleavage site of SARS-2-S, SARS-S (SARS-2), or BetaCoV/bat/Yunnan/RaTG13/2013 (RaTG; GISAID: EPI_ISL_402131), SARS-S (RaTG). Further, we generated SARS-2-S harboring the S1/S2 cleavage site of SARS-S, SARS-2-S (SARS) or RaTG-S, SARS-2-S (RaTG). Finally, we constructed SARS-2-S variants in which either the multibasic motif was deleted, SARS-2-S (delta), or in which the proline residue preceding the multibasic motif was mutated to arginine and the alanine residue within the minimal furin motif was changed to lysine in order to increase the basic environment at the S1/S2 site, SARS-2-S (opt). All newly cloned spike protein constructs further contained a deletion of 18 amino acids at their respective C terminus as this has been shown to improve coronavirus spike protein incorporation into VSV particles and thus transduction ( Schwegmann-Weßels et al., 2009 ). Further, for each construct an untagged variant as well as a version containing a C-terminal V5 epitope tag was constructed. Preparation of pseudotyped particles and transduction experiments A previously published protocol was employed to produce VSV pseudotype particles (VSVpp) carrying foreign viral glycoproteins in their envelope ( Berger Rentsch and Zimmer, 2011 , Kleine-Weber et al., 2019 ). First, 293T cells were transfected with expression plasmid for the respective spike glycoprotein or VSV-G or empty expression vector by calcium-phosphate precipitation. At 16 h posttransfection, the cells were inoculated with VSV ∗ ΔG-fLuc (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern/Switzerland), a replication-deficient VSV vector that lacks the genetic information for VSV-G and encodes for eGFP and firefly luciferase (fLuc), at a multiplicity of infection of 3. After 1 h of incubation, the inoculum was removed and cells were washed with phosphate-buffered saline (PBS) before medium containing anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) was added to all cells except for those expressing VSV-G (here, medium without antibody was added). Cells were further incubated for 16 h, before the VSVpp containing supernatants were harvested, freed from cellular debris by centrifugation and used for experiments. For transduction, target cells were grown in 96-well plates until they reached 50%–80% confluency. The culture supernatant was removed by aspiration and 100 μl/well of the respective pseudotype were added (quadruplicate samples). At 16 h posttransduction, culture supernatants were aspirated and cells lysed in 1x cell culture lysis reagent (prepared from 5x stock, Promega) for 20 min at room temperature. The lysates were then transferred to white, opaque-walled 96-well plates and luciferase activity was quantified by measuring luminescence upon addition of a substrate (PJK) using a Hidex Sense plate luminometer (Hidex). Western blot analysis For the analysis of S protein processing, we subjected VSVpp harboring V5- or HA-tagged S proteins to SDS-PAGE and western blot analysis. For this, we loaded 1 mL VSVpp onto 50 μl of a 20% (w/v) sucrose cushion and performed high-speed centrifugation (25.000 g for 120 min at 4°C). Next, we removed 1 mL of supernatant, added 50 μl of 2x SDS-sample buffer and incubated the samples for 15 min at 96°C. Thereafter, the samples were subjected to SDS-PAGE and protein transfer to nitrocellulose membranes by western blot. The membranes were subsequently blocked in 5% skim milk solution (PBS containing 0.05% Tween-20 [PBS-T] and 5% skim milk powder) for 1 h at room temperature. The blots were then incubated over night at 4°C with primary antibody solution (all antibodies were diluted in PBS-T containing 5% skim milk; mouse anti-HA tag [Sigma-Aldrich, H3663, 1:2,500], mouse anti-V5 tag [Thermo Fisher Scientific, R960-25, 1:2,500] or VSV matrix protein [Kerafast, EB0011, 1:2,500]). Following this incubation, the blots were washed 3x with PBS-T before they were incubated for 1 h at room temperature with peroxidase-coupled goat anti-mouse antibody (Dianova, 115-035-003, 1:10,000). Finally, the blots were again washed and imaged. For this, an in house-prepared enhanced chemiluminescent solution (0.1 M Tris-HCl [pH 8.6], 250 μg/mL luminol, 1 mg/mL para-hydroxycoumaric acid, 0.3% H 2 O 2 ) and the ChemoCam imaging system along with the ChemoStar Professional software (Intas Science Imaging Instruments GmbH) were used. Syncytium formation assay Vero or Vero-TMPRSS2 cells were grown on coverslips seeded in 24-well plates and transfected with S protein expression plasmids (1 μg/well) using Lipofectamine 2000 LTX with Plus reagent (Thermo Fisher Scientific) and OptiMEM medium (GIBCO). After 6 h the transfection solutions were aspirated and the cells further incubated for 24 h in standard culture medium. Next, the medium was changed to serum free medium ± 1 μg/mL bovine trypsin (Sigma-Aldrich) and the cells were incubated for additional 24 h. Then, the cells were washed with PBS, fixed with 4% paraformaldehyde solution for 20 min at room temperature, washed again, air-dried and incubated for 30 min with May-Gruenwald solution (Sigma-Aldrich). Thereafter, the cells were washed three times with deionized water, air-dried and incubated for 30 min with 1:10 diluted Giemsa solution (Sigma-Aldrich). After an additional washing interval with deionized water, the samples were air-dried and analyzed by bright-field microscopy using a Zeiss LSM800 confocal laser scanning microscope and the ZEN imaging software (both from Zeiss). Sequence analysis and protein models Sequence alignments were performed using the Clustal Omega online tool ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). Protein models were designed using the YASARA software ( http://www.yasara.org/index.html ). For the generation of the SARS-2-S protein model the protein sequence was first modeled on a SARS-S template (PDB: 5X5B, ( Yuan et al., 2017 )) using the SWISS-MODEL online tool ( https://swissmodel.expasy.org/ ). The following sequences information were obtained from National Center for Biotechnology Information (NCBI) database: SARS-CoV BJ01 (GenBank: AY278488.2), SARS-CoV CUHK-W1 (GenBank: AY278554.2), SARS-CoV Frankfurt-1 (GenBank: AY291315.1), SARS-CoV Tor2 (GenBank: CS050815.1), SARS-CoV Urbani (GenBank: AY278741.1), civet SARS-CoV SZ16 (GenBank: AY304488.1), civet SARS-CoV civet020 (GenBank: AY572038.1), raccoon dog SARS-CoV A030 (GenBank: AY687357.1), bat SARSr-CoV BtKY72/KEN (GenBank: KY352407.1), bat SARSr-CoV BM48-31/BGR/2008 (GenBank: GU190215.1), bat SARSr-CoV Rs4231 (GenBank: KY417146.1), bat SARSr-CoV WIV16 (GenBank: KT444582.1), bat SARSr-CoV Rs4874 (GenBank: KY417150.1), bat SARSr-CoV SL-CoVZC45 (GenBank: MG772933.1), bat SARSr-CoV SL-CoVZXC21 (GenBank: MG772934.1), bat SARSr-CoV LYRa11 (GenBank: KF569996.1), bat SARSr-CoV LYRa3 (GenBank: KF569997.1), bat SARSr-CoV WIV1 (GenBank: KF367457.1), bat SARSr-CoV RsSHC014 (GenBank: KC881005.1), bat SARSr-CoV Rs3367 (GenBank: KC881006.1), bat SARSr-CoV Cp/Yunnan2011 (GenBank: JX993988.1), bat SARSr-CoV Rp/Shaanxi2011 (GenBank: JX993987.1), bat SARSr-CoV HKU3-1 (GenBank: DQ022305.2), bat SARSr-CoV Rm1 (GenBank: DQ412043.1), bat SARSr-CoV Rp3 (GenBank: DQ071615.1), bat SARSr-CoV Rf1 (GenBank: DQ412042.1), bat SARSr-CoV 279 (GenBank: DQ648857.1), bat SARSr-CoV 273 (GenBank: DQ648856.1), bat SARSr-CoV YN2013 (GenBank: KJ473816.1), bat SARSr-CoV Rs/HuB2013 (GenBank: KJ473814.1), bat SARSr-CoV Rs/GX2013 (GenBank: KJ473815.1), bat SARSr-CoV Rf/SX2013 (GenBank: KJ473813.1), bat SARSr-CoV Rf/JL2012 (GenBank: KJ473811.1), bat SARSr-CoV Rf/HeB2013 (GenBank: KJ473812.1), bat SARSr-CoV YNLF/34C (GenBank: KP886809.1), bat SARSr-CoV YNLF/31C (GenBank: KP886808.1), bat SARSr-CoV Rs672 (GenBank: FJ588686.1), bat SARSr-CoV Rs7327 (GenBank: KY417151.1), bat SARSr-CoV Rs4084 (GenBank: KY417144.1), bat SARSr-CoV Rs9401 (GenBank: KY417152.1), bat SARSr-CoV Rs4247 (GenBank: KY417148.1), bat SARSr-CoV Rs4255 (GenBank: KY417149.1), bat SARSr-CoV Rs4081 (GenBank: KY417143.1), bat SARSr-CoV Rs4237 (GenBank: KY417147.1), bat SARSr-CoV As6526 (GenBank: KY417142.1), bat SARSr-CoV Rf4092 (GenBank: KY417145.1), bat SARSr-CoV Longquan-140 (GenBank: KF294457.1), bat SARSr-CoV Rs806 (GenBank: FJ588692.1), bat SARSr-CoV Anlong-103 (GenBank: KY770858.1), bat SARSr-CoV JTMC15 (GenBank: KU182964.1), bat SARSr-CoV 16BO133 (GenBank: KY938558.1), bat SARSr-CoV B15-21 (GenBank: KU528591.1), pangolin coronavirus MP789 (GenBank: MT084071.1). In addition the following sequences information were obtained from the Global Initiative on Sharing All Influenza Data (GISAID) database: SARS-CoV-2 (GISAID: EPI_ISL_404895), bat SARSr-CoV RaTG13 (GISAID: EPI_ISL_402131). Quantification and Statistical Analysis If not stated otherwise, statistical significance was tested by one-way analysis of variance with Dunnet's posttest (GraphPad Prism 7.03). Only p values of 0.05 or lower were considered statistically significant (p > 0.05 [ns, not significant], p ≤ 0.05 [ ∗ ], p ≤ 0.01 [ ∗∗ ], p ≤ 0.001 [ ∗∗∗ ]). For all statistical analyses, the GraphPad Prism 7 software package was used (GraphPad Software). Author Contributions Conceptualization, M.H. and S.P.; Formal Analysis, M.H. and S.P.; Investigation, M.H. and H.K.-W; Writing – Original Draft, M.H. and S.P.; Writing – Review & Editing, all authors; Funding Acquisition, S.P. Declaration of Interest The authors declare not competing interests

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